@article {pmid40627177, year = {2025}, author = {Azani, A and Sharafi, M and Doachi, R and Akbarzadeh, S and Lorestani, P and Haji Kamanaj Olia, A and Zahed, Z and Gharedaghi, H and Ghasrsaz, H and Foroozand, H and Rahimi, H and Tahmasebi, S and Behfar, Q}, title = {Applications of CRISPR-Cas9 in mitigating cellular senescence and age-related disease progression.}, journal = {Clinical and experimental medicine}, volume = {25}, number = {1}, pages = {237}, pmid = {40627177}, issn = {1591-9528}, mesh = {Humans ; *Cellular Senescence/genetics ; *CRISPR-Cas Systems ; *Aging/genetics ; Epigenesis, Genetic ; *Gene Editing/methods ; Disease Progression ; Animals ; Telomerase/genetics/metabolism ; }, abstract = {Aging is a multifaceted process influenced by many elements. During cell division, the repetitive DNA sequences at the ends of chromosomes called telomeres protect them from degradation. Telomeres shorten alongside each cell division, eventually contributing to cellular senescence and aging. Telomerase as an enzyme has a role in the maintenance of telomere length. Reduced function of telomerase is linked to acceleration of aging and age-related diseases. By affecting cellular function, mutations in particular genes can cause aging. Genes involved in DNA repair, cellular metabolism, and inflammation play the key roles in this process. Accumulated mutations result in cellular dysfunction and age-related diseases over time. Epigenetic changes are the modifications that impact gene expression without altering the DNA sequence. Lifestyle factors (diet, exercise, stress) and environmental influences (toxins, trauma) can cause epigenetic alterations. DNA methylation as well as histone modifications are examples of epigenetic alterations. They influence how cells work and are essential to the aging process. Understanding these molecular mechanisms is essential for developing interventions to promote healthy aging and prevent age-related diseases. This paper explores the potential of CRISPR/Cas9 as a gene-editing tool to target these mechanisms and mitigate age-related conditions, ultimately enhancing longevity and quality of life.}, }
@article {pmid40627029, year = {2025}, author = {M, KR and C, SD}, title = {Recent insights into actinobacteria research in antimicrobial resistance: a review.}, journal = {Molecular biology reports}, volume = {52}, number = {1}, pages = {683}, pmid = {40627029}, issn = {1573-4978}, mesh = {*Actinobacteria/genetics/metabolism/drug effects ; *Anti-Bacterial Agents/pharmacology ; Multigene Family ; Humans ; *Drug Resistance, Multiple, Bacterial/genetics ; Streptomyces/genetics/metabolism ; *Drug Resistance, Bacterial/genetics ; Biosynthetic Pathways/genetics ; }, abstract = {Antimicrobial resistance (AMR) has emerged as a global health crisis, taking 4.71 million lives in the year 2021 and posing significant challenges to healthcare systems. Actinobacteria, particularly Streptomyces sp., are a well-established source of bioactive secondary metabolites, including antibiotics such as polyketides, aminoglycosides, and macrolides with activity against multidrug-resistant (MDR) bacteria. However, only 10% of the antibiotic genes are expressed, and others are silent in cryptic biosynthetic gene clusters (BGCs) that remain inactive under standard laboratory conditions. Advances in genome mining, bioinformatics tools like antiSMASH, and molecular techniques such as CRISPR-Cas have facilitated the identification of these clusters. Furthermore, innovative strategies such as co-culturing and HDAC inhibitors have shown promise in activating cryptic biosynthetic pathways to combat emerging antimicrobial resistance. Despite these advancements, the rapid evolution of resistance requires continuous research and global collaboration to ensure a sustainable pipeline of effective antibiotics. This review provides insight into actinobacteria-derived antibiotics, resistance mechanisms, and emerging biotechnological interventions to address the AMR crisis, underscoring the urgent need for multidisciplinary antibiotic discovery and stewardship efforts.}, }
@article {pmid40626824, year = {2025}, author = {Özdemir, BH}, title = {Navigating Immunological Barriers in Xenotransplantation: Recent Advances and Promising Strides.}, journal = {Experimental and clinical transplantation : official journal of the Middle East Society for Organ Transplantation}, volume = {23}, number = {6}, pages = {421-430}, doi = {10.6002/ect.2023.0351}, pmid = {40626824}, issn = {2146-8427}, mesh = {Humans ; Animals ; *Transplantation, Heterologous/adverse effects ; *Graft Rejection/immunology/prevention & control/genetics ; Animals, Genetically Modified ; Swine ; *Immunity, Humoral ; *Graft Survival ; *Antibodies, Heterophile/immunology/genetics/blood ; *Antigens, Heterophile/immunology/genetics ; Immunity, Cellular ; Treatment Outcome ; CRISPR-Cas Systems ; Gene Editing ; }, abstract = {The review introduces the challenges and potential solutions in xenotransplantation, focusing on pig-to-human organ transplant. Xenotransplantation, mainly with the use of pig organs, is a promising solution because of the reproductive capacity, size, and physiological resemblance of pigs to humans. However, immunological barriers, especially humoral and cellular immune responses, pose substantial challenges. The humoral immune response, involving antibodies targeting xenoantigens, is a substantial barrier. Anti-α-galactose antibodies, targeting α-Gal epitopes, are crucial in hyperacute rejection and acute humoral xenograft rejection. Genetic modifications, including CRISPR/Cas9 technology, aim to eliminate xenoantigens like α-Gal, potentially overcoming these challenges. This review discusses the use of genetically modified pigs for xenotransplantation, emphasizing the removal of xenoantigens, expression of human complement regulatory proteins, and transgenic expres-sion of human regulatory factors. Recent advancements, such as the world's first porcine-to-human heart transplant, highlight the potential of genetic manipu-lation in overcoming immune rejection barriers.}, }
@article {pmid40626243, year = {2025}, author = {Mitousis, L and Musiol-Kroll, E and Wohlleben, W}, title = {CRISPR-Cas in actinomycetes: still a lot to be discovered.}, journal = {microLife}, volume = {6}, number = {}, pages = {uqaf010}, pmid = {40626243}, issn = {2633-6693}, abstract = {Actinomycetes are important producers of valuable natural products that are applied in medicine or industry. The enzymes necessary for the synthesis of those compounds are encoded in biosynthetic gene clusters (BGCs) in the genome. However, the discovery of new natural products or the improvement of production levels can be hindered by difficulties in genetic manipulation, since standard methods often do not or not efficiently work in actinomycetes. One possible explanation for this could be the presence of nucleic acid defense systems such as CRISPR-Cas. Even though there is a lot of research published about CRISPR-Cas systems in general, the knowledge about the function of CRISPR-Cas in actinomycetes is very limited. Based on sequence data it is known that CRISPR-Cas systems occur in around half of all sequenced actinobacterial genomes. Moreover, in silico analyses of those systems have led to the discovery of new subtypes. The few examples of experimental evidence of CRISPR-Cas activity in vivo or in vitro, however, point to some special features, regarding crRNA maturation or life-cycle dependent CRISPR-Cas activity. This short review draws attention to this neglected research area and highlights the available data about CRISPR-Cas in actinomycetes.}, }
@article {pmid40364716, year = {2025}, author = {Cahn, JKB and Ludwicki, H and Shingler, J and Gulvin, S and Zhang, Y and Kristopeit, A and Ton, C and Winters, MA and Wagner, JM and Moore, J}, title = {CRISPR-Editing of the Vero Cell Line Improves Processability of Live Virus Vaccines by Enabling Targeted Proteolysis of Fibronectin.}, journal = {Biotechnology and bioengineering}, volume = {122}, number = {8}, pages = {2082-2092}, doi = {10.1002/bit.29028}, pmid = {40364716}, issn = {1097-0290}, mesh = {Animals ; Chlorocebus aethiops ; Vero Cells ; *Gene Editing/methods ; *Fibronectins/genetics/metabolism ; *CRISPR-Cas Systems ; *Viral Vaccines/genetics/metabolism ; Proteolysis ; Vaccines, Attenuated/genetics ; }, abstract = {Removal of host cell components is a significant cost driver in the production of live virus vaccines. Filtration processes such as tangential flow filtration can be effective in this capacity by leveraging the relative size difference between viral particles and host proteins; however, filtration membranes can be fouled by larger proteins, particularly those of the extracellular matrix. In this study, we used CRISPR editing to insert the recognition element of the highly-selective TEV protease into various positions of the gene encoding fibronectin in the genome of the Vero cell line, a common platform for viral production. By screening edited cell lines, we identified a promising candidate line in which fibronectin could be effectively removed by treating with the protease during processing, eliminating filter fouling and allowing for viral purification without the need for costly chromatography steps.}, }
@article {pmid40345583, year = {2025}, author = {Flores-Arenas, C and Malekos, E and Montano, C and Covarrubias, S and Sudek, L and Dempsey, V and Huynh, V and Katzman, S and Carpenter, S}, title = {CRISPRi screen uncovers lncRNA regulators of human monocyte growth.}, journal = {The Journal of biological chemistry}, volume = {301}, number = {6}, pages = {110204}, pmid = {40345583}, issn = {1083-351X}, mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Monocytes/cytology/metabolism ; *Cell Proliferation ; *CRISPR-Cas Systems ; THP-1 Cells ; Interferon Regulatory Factors/genetics/metabolism ; Apoptosis ; }, abstract = {Long noncoding RNAs are emerging as critical regulators of biological processes. While there are over 36,000 lncRNAs annotated in the human genome, we do not know the function of the majority. Here, we performed a high-throughput CRISPRi screen to identify those lncRNAs that are important for viability in human monocytes using the cell line THP1. We identified a total of 38 hits from the screen and validated and characterized two of the top intergenic hits. The first is a lncRNA neighboring the macrophage viability transcription factor IRF8 (RP11-542M13.2 hereafter referred to as long noncoding RNA regulator of monocyte proliferation, LNCRMP), and the second is a lncRNA called OLMALINC (oligodendrocyte maturation-associated long intervening non-coding RNA) that was previously found to be important in oligodendrocyte maturation. Transcriptional repression of LNCRMP and OLMALINC from monocytes severely limited their proliferation capabilities. RNA-seq analysis of knockdown lines showed that LNCRMP regulated proapoptotic pathways, while knockdown of OLMALINC impacted genes associated with the cell cycle. Data support both LNCRMP and OLMALINC functioning in cis to regulate their neighboring proteins that are also essential for THP1 cell growth. This research highlights the importance of high-throughput screening as a powerful tool for quickly discovering functional long non-coding RNAs (lncRNAs) that play a vital role in regulating monocyte viability.}, }
@article {pmid40193304, year = {2025}, author = {Florindi, C and Jang, Y and Shani, K and Moretti, P and Bertarelli, C and Lanzani, G and Parker, KK and Lodola, F and Vurro, V}, title = {A Cardiac Microphysiological System for Studying Ca2+ Propagation via Non-genetic Optical Stimulation.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {217}, pages = {}, doi = {10.3791/67823}, pmid = {40193304}, issn = {1940-087X}, mesh = {*Photic Stimulation ; *Light Signal Transduction ; *Calcium/metabolism ; Induced Pluripotent Stem Cells ; Humans ; *Myocytes, Cardiac/metabolism ; CRISPR-Cas Systems ; CRISPR-Associated Protein 9 ; Cations, Divalent/metabolism ; }, abstract = {In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely accepted by the scientific community, these systems are still limited in longevity due to the absence of non-invasive stimulation techniques. Phototransducers provide an efficient stimulation method, offering a wireless approach with high temporal and spatial resolution while minimizing invasiveness in stimulation processes. In this manuscript, we present a fully optical method for stimulating and detecting the activity of an in vitro cardiac microphysiological model. Specifically, we fabricated engineered laminar anisotropic tissues by seeding human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated in a 3D bioreactor suspension culture. We employed a phototransducer, an amphiphilic azobenzene derivative, named Ziapin2, for stimulation and a Ca[2+] dye (X-Rhod 1) for monitoring the system's response. The results demonstrate that Ziapin2 can photomodulate Ca[2+] responses in the employed system without compromising tissue integrity, viability, or behavior. Furthermore, we showed that the light-based stimulation approach offers a similar resolution compared to electrical stimulation, the current gold standard. Overall, this protocol opens promising perspectives for the application of Ziapin2 and material-based photostimulation in cardiac research.}, }
@article {pmid40624545, year = {2025}, author = {Ahmed, HMM and Zheng, L and Hunnekuhl, VS}, title = {Transferable approaches to CRISPR-Cas9 induced genome editing in non-model insects: a brief guide.}, journal = {Frontiers in zoology}, volume = {22}, number = {1}, pages = {13}, pmid = {40624545}, issn = {1742-9994}, support = {TE1380/1//Deutsche Forschungsgemeinschaft/ ; }, abstract = {Despite the large variety of insect species with divergent morphological, developmental and physiological features questions on gene function could for a long time only be addressed in few model species. The adaption of the bacterial CRISPR-Cas system for genome editing in eukaryotic cells widened the scope of the field of functional genetics: for the first time the creation of heritable genetic changes had become possible in a very broad range of organisms. Since then, targeted genome editing using the CRISPR-Cas technology has greatly increased the possibilities for genetic manipulation in non-model insects where molecular genetic tools were little established. The technology allows for site-specific mutagenesis and germline transformation. Importantly, it can be used for the generation of gene knock-outs, and for the knock-in of transgenes and generation of gene-reporter fusions. CRISPR-Cas induced genome editing can thus be applied to address questions in basic research in various insect species and other study organisms. Notably, it can also be used in applied insect biotechnology to design new pest and vector control strategies such as gene drives and precision guided Sterile Insect Technique. However, establishing CRISPR in a new model requires several practical considerations that depend on the scientific questions and on the characteristics of the respective study organism. Therefore, this review is intended to give a literature overview on different CRISPR-Cas9 based methods that have already been established in diverse insects. After discussing some required pre-conditions of the study organism, we provide a guide through experimental considerations when planning to conduct CRISPR-Cas9 genome editing, such as the design and delivery of guide RNAs, and of Cas9 endonuclease. We discuss the use of different repair mechanisms including homology directed repair (HDR) for a defined insertion of genetic elements. Furthermore, we describe different molecular methods for genetic screening and the use of visible markers. We focus our review on experimental work in insects, but due to the ubiquitous functionality of the CRISPR-Cas system many considerations are transferable to other non-model organisms.}, }
@article {pmid40623912, year = {2025}, author = {Wang, YW and Tang, YM}, title = {[Advances in the application strategies of CRISPR/Cas9 technology in chimeric antigen receptor T cell therapy for hematological malignancies].}, journal = {Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi}, volume = {46}, number = {5}, pages = {481-488}, doi = {10.3760/cma.j.cn121090-20240911-00343}, pmid = {40623912}, issn = {0253-2727}, mesh = {Humans ; *CRISPR-Cas Systems ; *Hematologic Neoplasms/therapy ; *Receptors, Chimeric Antigen ; *Immunotherapy, Adoptive/methods ; T-Lymphocytes/immunology ; Gene Editing ; }, abstract = {Chimeric antigen receptor (CAR) T-cell therapy has achieved breakthroughs in treating relapsed/refractory B-cell malignancies. However, it still faces challenges, including complex manufacturing processes, limited indications, T-cell exhaustion, and insufficient durability of therapeutic efficacy. CRISPR/Cas9, a highly efficient and relatively simple gene-editing technology, offers new avenues for overcoming these limitations. This review briefly outlines the working mechanism of CRISPR/Cas9 and focuses on its recent applications and clinical practices in developing universal CAR T-cells, enhancing T-cell function, and extending CAR T-cell therapy to T-cell and myeloid leukemias. Furthermore, this review highlights optimization strategies developed over the past two years to enhance the editing precision, delivery efficiency, and safety of the CRISPR/Cas9 system, aiming to provide insights for the optimal design and clinical application of CAR T-cell therapy.}, }
@article {pmid40623181, year = {2025}, author = {Aumann, RA and Gouvi, G and Gregoriou, ME and Rehling, T and Sollazzo, G and Bourtzis, K and Schetelig, MF}, title = {Decoding and engineering temperature-sensitive lethality in Ceratitis capitata for pest control.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {122}, number = {28}, pages = {e2503604122}, doi = {10.1073/pnas.2503604122}, pmid = {40623181}, issn = {1091-6490}, support = {FF17000//SITplus, Hort Innovation/ ; SCHE 1833/7-1//Deutsche Forschungsgemeinschaft (DFG)/ ; 101059523//Horizon Europe Research and Innovation Action/ ; }, mesh = {Animals ; *Ceratitis capitata/genetics ; *Pest Control, Biological/methods ; Male ; Female ; Temperature ; Mutation ; Phenotype ; }, abstract = {The Sterile Insect Technique (SIT) is a species-specific and environmentally friendly method for effectively controlling pest insect populations based on releasing reared, sterile insects into infested areas. Sex sorting in rearing facilities, enabling male-only releases, is necessary to ensure SIT programs are efficient, cost-effective and, in case of mosquito control, also safe. This can be greatly facilitated by genetic sexing strains (GSS), exhibiting sex-specific phenotypic markers. However, the development of GSS remains challenging. The construction of a temperature-sensitive lethal (tsl)-based GSS in the Mediterranean fruit fly (Ceratitis capitata) over three decades ago was considered a major breakthrough for SIT programs but was never successfully replicated in other pests. After over 30 y of research, we have pinpointed a specific mutation in the C. capitata lysine--tRNA ligase (Lysyl-tRNA synthetase, LysRS) gene responsible for the tsl phenotype. Introducing this specific mutation into a wild-type strain produced full embryonic lethality under heat stress, replicating the original mutant phenotype. The random integration of a LysRS minigene reversed this effect. The high conservation of LysRS among insects suggests that tsl-based GSS could be expanded to multiple pest species and extend applications of SIT programs for disease prevention and the protection of agriculture.}, }
@article {pmid40622878, year = {2025}, author = {Perry, JK and Schwartzberg, PL and Golec, DP}, title = {Investigating Murine CD4 T Cell Differentiation Using CRISPR-Cas9 Ribonucleoprotein Complex-mediated Gene Ablation.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {220}, pages = {}, doi = {10.3791/67380}, pmid = {40622878}, issn = {1940-087X}, mesh = {Animals ; Mice ; *Ribonucleoproteins/genetics ; *CRISPR-Cas Systems ; *CD4-Positive T-Lymphocytes/cytology/immunology ; Cell Differentiation/genetics ; *Gene Knockout Techniques/methods ; Mice, Knockout ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {The widespread accessibility of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technology has made gene targeting in primary cells a routine method for evaluating gene function in T cells. Given the cost and limited availability of knockout (KO) mouse strains, testing preliminary hypotheses involving gene function in T cells can be prohibitive using gene-targeted animal models. However, using commercially available resources, including predesigned guide RNAs (gRNAs), researchers can conveniently generate gene-targeted naïve T cells that can be used for T cell activation and differentiation studies. Here we outline a protocol for using nucleofection-delivered CRISPR-Cas9 ribonucleoprotein complexes (RNPs) to efficiently generate gene KO murine naïve CD4 T cells that can be used to evaluate gene function in CD4 T cell differentiation, in vitro. Isolation of naïve CD4 T cells from mouse secondary lymphoid organs, followed by nucleofection with Cas9-gRNA complexes ensures gene KO is initiated before downstream T cell activation, offering a strategic advantage over retroviral-mediated gRNA delivery, which typically requires preactivation of T cells, preventing the evaluation of effects in naïve T cells. Furthermore, this nucleofection-based method bypasses potential developmental issues associated with gene KO animals. Following Cas9-gRNA delivery, we describe protocols for studying CD4 T cell differentiation into Th1, Th2, Th17, and Treg lineages using in vitro polarization. In addition, this protocol is adaptable to using gene-targeted CD4 or CD8 T cells for numerous downstream applications, including other T cell activation studies in vitro and adoptive transfer studies in vivo. The use of CRISPR-Cas9 methods has streamlined our ability to evaluate gene function in T cells and allows for the routine KO of many genes of interest, freeing researchers from limitations associated with studying gene KO animals.}, }
@article {pmid40622492, year = {2025}, author = {Parmar, H and Goel, A and Gelaw, TA and Reddy, MK}, title = {Enhancing drought resilience in crops: mechanistic approaches in the face of climate challenge.}, journal = {Plant molecular biology}, volume = {115}, number = {4}, pages = {82}, pmid = {40622492}, issn = {1573-5028}, support = {09/512(0244)/2019-EMR-I//Human Resource Development Centre, Council of Scientific And Industrial Research/ ; }, mesh = {*Crops, Agricultural/genetics/physiology ; *Droughts ; Climate Change ; Quantitative Trait Loci/genetics ; Stress, Physiological ; Plant Breeding ; Gene Editing ; Gene Expression Regulation, Plant ; }, abstract = {Enhancing drought resilience in crops has become a critical challenge in the face of global climate change, which is exacerbating the frequency and severity of drought events. This review explores mechanistic approaches aimed to improve crop drought tolerance, focusing on physiological, biochemical, and molecular mechanisms. We examine the key molecular pathways involved in drought stress responses, including the Mitogen-Activated Protein Kinase (MAPKs) signaling pathway, hormonal regulation, transcriptional control, and post-translational modifications such as ubiquitination-mediated protein degradation, and plant-microbe interaction. The review also delves into the mechanisms of drought stress tolerance, including drought escape, avoidance, and tolerance. It highlights significant traits contributing to drought resilience, such as stomatal regulation and root architecture. Furthermore, we discuss genomics and breeding approaches, including quantitative trait loci (QTL) mapping, marker-assisted selection (MAS), and cutting-edge CRISPR-Cas-based genome editing technologies. These advanced techniques, such as base editing, prime editing, and multiplexing, transform crop improvement strategies by facilitating precise and efficient modifications for enhanced drought resilience, with the success stories in crops such as rice, maize, wheat, and others. Integrating these mechanistic and technological approaches offers promising avenues for developing drought-resilient crops, ensuring food security under increasingly unpredictable climate conditions.}, }
@article {pmid40619763, year = {2025}, author = {Fritz, B and Lapp, CJ and Gescher, J}, title = {Influence of Different Transposon Families on Genomic Stability of Shewanella oneidensis MR1.}, journal = {Microbial biotechnology}, volume = {18}, number = {7}, pages = {e70188}, doi = {10.1111/1751-7915.70188}, pmid = {40619763}, issn = {1751-7915}, support = {031B0847A//Bundesministerium für Bildung und Forschung/ ; }, mesh = {*Shewanella/genetics ; *DNA Transposable Elements/genetics ; *Genomic Instability ; Transposases/genetics/metabolism ; Genome, Bacterial ; Plasmids/genetics ; Whole Genome Sequencing ; }, abstract = {Shewanella oneidensis, recognised as an important model organism for exoelectrogenic electron transport, has been extensively studied for its potential applications in bioelectrochemical systems. To date, the activity of transposable elements in this organism has not been conclusively investigated. This study focused on transposases, specifically insertion sequences (IS), which make up approximately 4.7% of the organism's genome, and evaluated their impact on genome stability under stress conditions. Using whole genome sequencing, two IS families, ISSOD1 and ISSOD2, were identified as the most active, both showing similar transposition patterns across all tested stressors. A CRISPR/dCas9 cytosine deaminase system was used to introduce stop codons in the ISSOD2 transposase genes, resulting in a significant reduction of transposition events under stress conditions. Analysis of transposition patterns revealed a high frequency of insertions occurring on the megaplasmid, which predominantly carries non-essential genes. Experiments performed here to delete the megaplasmid resulted in the elimination of approximately 35% of its sequence, including an unexpected complete loss of the ori/repA region. Therefore, it was hypothesised that the megaplasmid either exists in a metastable state, possibly representing a cointegrated intermediate within the ISSOD9 (Tn3 member) transposition mechanism, or consists of two replicons that have been combined in previous assemblies due to long overlapping homologies resulting from the presence of ISSOD9. These findings highlight the dynamics of transposable elements in S. oneidensis and suggest strategies to improve strain stability by inactivating these elements and at least reducing megaplasmid sequences. Such approaches could improve the suitability of the organism for industrial applications.}, }
@article {pmid40384567, year = {2025}, author = {Schneider, PG and Liu, S and Bullinger, L and Ostendorf, BN}, title = {BEscreen: a versatile toolkit to design base editing libraries.}, journal = {Nucleic acids research}, volume = {53}, number = {W1}, pages = {W68-W72}, doi = {10.1093/nar/gkaf406}, pmid = {40384567}, issn = {1362-4962}, support = {70114327//German Cancer Aid/ ; 10.24.1.010MN//Fritz Thyssen/ ; //Guangzhou/ ; //Charité-BIH Digital Clinician/ ; //German Research Foundation/ ; }, mesh = {*Software ; *Gene Editing/methods ; *Gene Library ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Internet ; CRISPR-Cas Systems ; }, abstract = {Base editing enables the high-throughput screening of genetic variants for phenotypic effects. Base editing screens require the design of single guide RNA (sgRNA) libraries to enable either gene- or variant-centric approaches. While computational tools supporting the design of sgRNAs exist, no solution offers versatile and scalable library design enabling all major use cases. Here, we introduce BEscreen, a comprehensive base editing guide design tool provided as a web server (bescreen.ostendorflab.org) and as a command line tool. BEscreen provides variant-, gene-, and region-centric modes to accommodate various screening approaches. The variant mode accepts genomic coordinates, amino acid changes, or rsIDs as input. The gene mode designs near-saturation libraries covering the entire coding sequence of given genes or transcripts, and the region mode designs all possible guides for given genomic regions. BEscreen enables selection of guides by biological consequence, it features comprehensive customization of base editor characteristics, and it offers optional annotation using Ensembl's Variant Effect Predictor. In sum, BEscreen is a highly versatile tool to design base editing screens for a wide range of use cases with seamless scalability from individual variants to large, near-saturation libraries.}, }
@article {pmid40377102, year = {2025}, author = {Chapdelaine-Trépanier, V and Shenoy, S and Masud, W and Minju-Op, A and Bérubé, MA and Schönherr, S and Forer, L and Fradet-Turcotte, A and Taliun, D and Cuella-Martin, R}, title = {CRISPR-BEasy: a free web-based service for designing sgRNA tiling libraries for CRISPR-dependent base editing screens.}, journal = {Nucleic acids research}, volume = {53}, number = {W1}, pages = {W193-W202}, doi = {10.1093/nar/gkaf382}, pmid = {40377102}, issn = {1362-4962}, support = {FRN:DV2-197674/CAPMC/CIHR/Canada ; //Canada Excellence Chair in Genomic Medicine/ ; //McGill University/ ; //Fonds de Recherche du Québec-Santé/ ; //Doctoral Training Scholarships/ ; //Canada Research Chair Tier II in Molecular Biology and Genomic Instability/ ; FRN:DV2-197674/CAPMC/CIHR/Canada ; }, mesh = {Internet ; *Gene Editing/methods ; *Software ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Library ; Humans ; }, abstract = {CRISPR-dependent base editing (BE) enables the modeling and correction of genetic mutations at single-base resolution. Base editing screens, where point mutations are queried en masse, are powerful tools to systematically draw genotype-phenotype associations and characterise the function of genes and other genomic elements. However, the lack of user-friendly web-based tools for designing base editing screens can hinder broad technology adoption. Here, we introduce CRISPR-BEasy (https://crispr-beasy.cerc-genomic-medicine.ca), a free, automated web-based server that streamlines the creation of single guide (sg)RNA tiling libraries for base editing screens. Researchers can provide their genes or genomic features of interest, their base editors of choice, and target sequences to act as positive and negative controls. The server designs and annotates sgRNA libraries by integrating custom code with publicly available tools such as crisprVerse and Ensembl's Variant Effect Predictor. CRISPR-BEasy provides downloadable results, including sgRNA on/off-target scores, predicted mutational outcomes per base editor, and intuitive interactive visualizations for data quality assessment. CRISPR-BEasy also provides a separate tool that assembles sgRNA libraries into oligonucleotides for cloning following the detailed protocol documented in the searchable web server manual. Together, CRISPR-BEasy ensures the seamless design of cloning-ready sgRNA libraries, seeking to democratise access to base editing screening technologies.}, }
@article {pmid40337925, year = {2025}, author = {Mekonnen, AM and Seong, K and Kim, H and Park, J}, title = {Variant-aware Cas-OFFinder: web-based in silico variant-aware potential off-target site identification for genome editing applications.}, journal = {Nucleic acids research}, volume = {53}, number = {W1}, pages = {W118-W124}, doi = {10.1093/nar/gkaf389}, pmid = {40337925}, issn = {1362-4962}, support = {RS-2024-00322053//Rural Development Administration/ ; //National Research Foundation of Korea/ ; //Korean government/ ; RS-2023-00278658//MSIT/ ; //Bio & Medical Technology Development/ ; RS-2024-00439078//Korean government (MSIT)/ ; BK21 Four//Korean government (MSIT)/ ; }, mesh = {*Gene Editing/methods ; *Software ; Humans ; Internet ; *CRISPR-Cas Systems ; Genome, Human ; Genetic Variation ; Haplotypes ; Computer Simulation ; }, abstract = {Genome editing based on CRISPR systems has been widely used in the vast areas of biomedical and agricultural applications. However, identifying the potential off-target sites remains challenging, particularly in individuals with diverse genetic variations. Several in silico tools have been developed to predict potential off-target sites, but they have limitations on their performance and scalability. In this paper, we present "Variant-aware Cas-OFFinder," a novel pipeline based on Cas-OFFinder for identifying potential off-target sites by accounting for individual genetic variants. We benchmarked the pipeline's improved scalability and performance with the human genome and pepper cultivars, having unique potential off-target sites on each allele at the haplotype level. The web tool is open to all users without a login requirement and is freely available online at https://rgetoolkit.com/var-cas-offinder.}, }
@article {pmid40334276, year = {2025}, author = {Liu, H and Dong, J and Wu, R and Dai, J and Lou, X and Xia, F and Willner, I and Huang, F}, title = {Light-Triggered CRISPR/Cas12a for Genomic Editing and Tumor Regression.}, journal = {Angewandte Chemie (International ed. in English)}, volume = {64}, number = {28}, pages = {e202502892}, doi = {10.1002/anie.202502892}, pmid = {40334276}, issn = {1521-3773}, support = {22377112//National Natural Science Foundation of China/ ; 22090050//National Natural Science Foundation of China/ ; 2021YFA1200400//National Key R&D Program of China/ ; 22161142020//Joint NSFC-ISF Research Grant Program/ ; }, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Light ; Animals ; Hep G2 Cells ; Mice ; Hepatocyte Growth Factor/genetics ; Cell Proliferation ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {A photo-triggered CRISPR/Cas12a machinery for in vitro and in vivo gene editing is introduced. The system consists of a caged, inactive ortho-nitrobenzyl phosphate ester photo-responsive crRNA, which, upon light-induced deprotection, yields the active CRISPR/Cas12a gene editing machinery (LAC12aGE). The LAC12aGE system induces specific thymidine-rich (TTTN) protospacer-adjacent motif (PAM)-guided double-stranded breaks in genomic DNA, which upon non-homologous end-joining lead to gene repair. The LAC12aGE machinery is applied for gene editing of an exogenous dual fluorescent reporter gene in living cells, as well as the endogenous gene encoding DNA methyltransferase 1. In addition, the LAC12aGE is applied for in vitro gene editing and disruption of the hepatocyte growth factor (HGF) gene in HepG2 cells, where knockout of the HGF gene results in inhibited cell proliferation and migration, as well as enhanced apoptosis. Moreover, the in vivo knockout and disruption of the HGF gene in HepG2 tumors by the LAC12aGE machinery is demonstrated. The cyclic temporal development of the LAC12aGE system in tumors shows effective inhibition of tumor growth and enhanced apoptosis/necrosis of tumor tissues compared to control systems.}, }
@article {pmid40308216, year = {2025}, author = {Marino, GB and Evangelista, JE and Clarke, DJB and Ma'ayan, A}, title = {L2S2: chemical perturbation and CRISPR KO LINCS L1000 signature search engine.}, journal = {Nucleic acids research}, volume = {53}, number = {W1}, pages = {W338-W350}, doi = {10.1093/nar/gkaf373}, pmid = {40308216}, issn = {1362-4962}, support = {OT2OD036435/GF/NIH HHS/United States ; OT2OD030160/GF/NIH HHS/United States ; U24CA264250/GF/NIH HHS/United States ; U24CA271114/GF/NIH HHS/United States ; R01DK131525/GF/NIH HHS/United States ; RC2DK131995/GF/NIH HHS/United States ; }, mesh = {Humans ; *CRISPR-Cas Systems ; Gene Knockout Techniques ; *Software ; Internet ; *Search Engine ; Gene Expression Profiling ; Small Molecule Libraries/pharmacology ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Transcriptome ; }, abstract = {As part of the Library of Integrated Network-Based Cellular Signatures (LINCS) NIH initiative, 248 human cell lines were profiled with the L1000 assay to measure the effect of 33 621 small molecules and 7508 single-gene CRISPR knockouts. From this massive dataset, we computed 1.678 million sets of up- and down-regulated genes. These gene sets are served for search by the LINCS L1000 Signature Search (L2S2) web server application. With L2S2, users can identify small molecules and single gene CRISPR KOs that produce gene expression profiles similar or opposite to their submitted single or up/down gene sets. L2S2 also includes a consensus search feature that ranks perturbations across all cellular contexts, time points, and concentrations. To demonstrate the utility of L2S2, we crossed the L2S2 gene sets with gene sets collected for the RummaGEO resource. The analysis identified clusters of differentially expressed genes that match drug classes, tissues, and diseases, pointing to many opportunities for drug repurposing and drug discovery. Overall, the L2S2 web server application can be used to further the development of personalized therapeutics while expanding our understanding of complex human diseases. The L2S2 web server application is available at https://l2s2.maayanlab.cloud.}, }
@article {pmid39729054, year = {2025}, author = {Ballantine, J and Tisdale, JF}, title = {Gene therapy for sickle cell disease: recent advances, clinical trials and future directions.}, journal = {Cytotherapy}, volume = {27}, number = {7}, pages = {826-834}, pmid = {39729054}, issn = {1477-2566}, support = {T32 CA060441/CA/NCI NIH HHS/United States ; ZIA HL006008/ImNIH/Intramural NIH HHS/United States ; }, mesh = {Humans ; *Anemia, Sickle Cell/therapy/genetics ; *Genetic Therapy/methods ; Gene Editing ; Clinical Trials as Topic ; Hematopoietic Stem Cell Transplantation ; CRISPR-Cas Systems/genetics ; Genetic Vectors/genetics ; Animals ; beta-Thalassemia/therapy/genetics ; }, abstract = {Sickle cell disease (SCD) is the most common inherited blood disorder worldwide, impacting millions and imposing severe healthcare challenges, particularly in resource-limited regions. Current treatments have variable efficacy and require lifelong adherence. Allogeneic Hematopoietic Stem Cell Transplantation can be curative but comes with significant side effects and limited donor availability limits its widespread applicability. Gene therapy, by addressing the root genetic causes, offers a revolutionary alternative. This article discusses the molecular mechanisms of SCD and β-thalassemia and highlights advancements in gene therapy, such as gene addition via lentiviral vectors and gene editing with CRISPR/Cas9 technology. Clinical trials have brought about significant progress but challenges remain, including leukemogenesis, delivery efficiency and cost. Future efforts must focus on enhancing efficiency, reducing costs, developing nongenotoxic conditioning regimens and methods for in vivo application.}, }
@article {pmid40619482, year = {2025}, author = {Bodmer, N and Uth, K and Mehmeti, R and Demir, CS and Stroka, D and Ghaffari-Tabrizi-Wizsy, N and Lugli, A and de Wever, O and Zlobec, I and Tschan, MP}, title = {CDX2 loss in colorectal cancer cells is associated with invasive properties and tumor budding.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {24113}, pmid = {40619482}, issn = {2045-2322}, support = {ESKAS, nr. 2021.0019 / Kosovo / OP//Swiss Government Excellence Scholarship/ ; 31003A_166578/1//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung/ ; 31003A_166578/1//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung/ ; }, mesh = {*CDX2 Transcription Factor/genetics/metabolism ; *Colorectal Neoplasms/pathology/genetics/metabolism ; Humans ; Animals ; Cell Movement/genetics ; Cell Line, Tumor ; Neoplasm Invasiveness ; Mice ; Cell Proliferation ; Epithelial-Mesenchymal Transition/genetics ; Cadherins/metabolism ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; }, abstract = {In colorectal cancer (CRC), tumor buds (TB) are observed histologically as single tumor cell or small tumor cell clusters located mainly at the advancing tumor edge. TB are a marker of poor prognosis and correlate with metastatic disease in CRC patients. They often lack expression of CDX2 and overexpress markers involved in epithelial-mesenchymal transition (EMT). We evaluated the function of CDX2 in CRC proliferation and migration using CRISPR/Cas9 technology and demonstrated a possible link to tumor dissociation and tumor budding. Knocking out CDX2 in CRC cell lines significantly increased migration. Importantly, the observed phenotypes could be rescued by re-expressing CDX2 and by specific CRISPR synergistic activation mediator (SAM) of endogenous CDX2 in CDX2 low expressing CRC cell lines. Multiplex immunofluorescence (mIF) analysis of primary tumor regions compared to TB in a CDX2-positive CRC patient sample as well as patient derived xenografts (PDX) revealed significantly lower CDX2 expression and correlating E-cadherin levels in TB compared to primary tumor regions, in both models. Accordingly, increased invasiveness of CRC CDX2 knockout cells was seen in ex ovo xenografts. Taken together, our results provide further insight into the function of CDX2 in preventing CRC cell migration, tumor budding and tumor aggressiveness.}, }
@article {pmid40617436, year = {2025}, author = {Marpaung, DSS and Chen, YY and Singuru, MMR and Chuang, MC}, title = {Structure-transformable poly (thymine) activators of CRISPR/Cas12a for highly sensitive detection of mercury (II) ions.}, journal = {International journal of biological macromolecules}, volume = {319}, number = {Pt 4}, pages = {145748}, doi = {10.1016/j.ijbiomac.2025.145748}, pmid = {40617436}, issn = {1879-0003}, abstract = {Off-target effects of the CRISPR/Cas system refer to suboptimal activity triggered by activators containing unintended modifications beyond the intended target, which can lead to unwanted genetic changes-particularly problematic in therapeutic applications. In this study, the off-target effects of Cas12a are strategically repurposed for advanced Hg[2+] detection by leveraging a structure-transformable activator. The proposed approach integrates a two-segment poly-T DNA activator with the CRISPR/Cas12a system, where the activator forms hairpin structures through thymine-Hg[2+]-thymine base pairing in response to mercury concentrations. Increasing Hg[2+] concentrations promote the formation and stability of hairpin DNA, significantly suppressing Cas12a activity and resulting in an ON-OFF fluorescence signal modulated by trans-cleavage activity. The effect of varying thymine segment lengths in the poly-T activator was investigated to increase the hybridization burden for crRNA, ultimately enhancing detection sensitivity. The Hg[2+]-dependent structural transformation of the activator was characterized using circular dichroism (CD), while urea and native PAGE analyses confirmed the catalytic behavior of both cis- and trans-cleavage activities, respectively, as a function of Hg[2+] concentration. The trans-cleavage efficiency (kcat/KM) decreased by 3.03-fold as Hg[2+] concentration increased from 0 to 40 nM. Under optimized conditions, the biosensor demonstrated a linear detection range of 0-30 nM, a detection limit as low as 0.372 nM, and high selectivity for Hg[2+] over other metal ions. Furthermore, the biosensor was successfully applied for Hg[2+] detection in field water samples. These findings establish a robust foundation for exploiting the off-target effects of the CRISPR/Cas12a system for the efficient detection of heavy metals in environmental monitoring.}, }
@article {pmid40617152, year = {2025}, author = {Rangu, SS and Misra, CS and Shaikh, S and Singh, M and Bindal, G and Singh, S and Purageri, S and Shettigar, S and Warke, R and Cherian, S and Rath, D}, title = {RNA extraction-free CRISPR-based SARS-CoV-2 detection in viral transport medium and dry swab-a comparative analysis: Short title: Extraction-free CRISPR-based Covid detection.}, journal = {Diagnostic microbiology and infectious disease}, volume = {113}, number = {3}, pages = {116982}, doi = {10.1016/j.diagmicrobio.2025.116982}, pmid = {40617152}, issn = {1879-0070}, abstract = {CRISPR-Cas-based methods have shown high efficacy in detecting SARS-CoV-2. Amidst the Covid-19 pandemic, numerous studies have explored SARS-CoV-2 detection methods without the need for RNA extraction, aiming to reduce cost and processing time. Here, we assessed a CRISPR-based SARS-CoV-2 detection method's ability to detect the virus in viral transport medium (VTM). Swabs obtained from Covid-19 positive patients and stored in two Indian brands of VTM were examined alongside dry swab samples. The samples underwent proteinase -K treatment followed by heat incubation. The released nucleic acids were tested by RT-LAMP and CRISPR-based detection. We conclude that SARS-CoV-2 can be detected in VTM of two commercial preparations as well as dry swab samples without RNA extraction and purification. COVID detection was found to be more efficient for dry swab samples compared to VTM samples. The work flow described in this paper can be extended to other respiratory diseases.}, }
@article {pmid40057303, year = {2025}, author = {Wang, L and Hu, Y and Qiu, Y and Lin, H and Li, X and Fu, S and Zeng, YY and Ghouse, M and Long, C and Liu, Y and Fei, JF}, title = {Establishing a semi-homology-directed recombination method for precision gene integration in axolotls.}, journal = {Journal of genetics and genomics = Yi chuan xue bao}, volume = {52}, number = {7}, pages = {942-953}, doi = {10.1016/j.jgg.2025.03.001}, pmid = {40057303}, issn = {1673-8527}, mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; *Ambystoma mexicanum/genetics ; Gene Editing/methods ; SOXB1 Transcription Factors/genetics ; *Homologous Recombination ; }, abstract = {The axolotl is broadly used in regenerative, developmental, and evolutionary biology research. Targeted gene knock-in is crucial for precision transgenesis, enabling disease modeling, visualization, tracking, and functional manipulation of specific cells or genes of interest (GOIs). Existing CRISPR/Cas9-mediated homology-independent method for gene knock-in often causes "scars/indels" at integration junctions. Here, we develop a CRISPR/Cas9-mediated semi-homology-directed recombination (HDR) knock-in method using a donor construct containing a single homology arm for the precise integration of GOIs. This semi-HDR approach achieves seamless single-end integration of the Cherry reporter gene and a large inducible Cre cassette into intronless genes like Sox2 and Neurod6 in axolotls, which are challenging to modify with the homology-independent method. Additionally, we integrate the inducible Cre cassette into intron-containing loci (e.g., Nkx2.2 and FoxA2) without introducing indels via semi-HDR. GOIs are properly expressed in F0 founders, with approximately 5%-10% showing precise integration confirmed by genotyping. Furthermore, using the Nkx2.2:CreER[T2] line, we fate-map spinal cord p3 neural progenitor cells, revealing that Nkx2.2[+] cells adopt different lineages in development and regeneration, preferentially generating motoneurons over oligodendrocytes during regeneration. Overall, this semi-HDR method balances efficiency and precision in the integration of GOIs, providing a valuable tool for generating knock-in axolotls and potentially extending to other species.}, }
@article {pmid40616579, year = {2025}, author = {Urnov, F and Kassim, S and Musunuru, K and Liu, D and Lee, A and Barrera, L and Stetkiewicz, P and Bruno, J and Hewitt, M and Lister, T and Malech, H and Gasch, L and Diver, M and Gertler, N and Grignon, F and Le, A and Lehmicke, M and Almendro-Navarro, V and Lembong, J}, title = {Advancing gene-editing platforms to improve the viability of rare-disease therapeutics: key insights from a 2024 Scientific Exchange hosted by ARM, ISCT, and Danaher.}, journal = {Cytotherapy}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.jcyt.2025.06.010}, pmid = {40616579}, issn = {1477-2566}, abstract = {Rare-disease therapeutics face viability challenges due to small patient populations and drug-development and regulatory frameworks that were not developed to address rapidly progressive or quickly fatal conditions. Because the majority of rare diseases are genetic in nature, gene-editing modalities offer substantial promise. This Scientific Exchange, co-hosted by the Alliance for Regenerative Medicine, the International Society for Cell and Gene Therapy, and Danaher Corporation in November 2024, set out to address the challenge of realizing the full promise of gene editing for rare-disease therapies by advancing platforms that leverage stable and reusable processes or components to develop multiple therapies. Through multi-stakeholder engagement and discussions of case studies in CRISPR/Cas nuclease, base, and prime editing, 4 key opportunities emerged that deliver value by holding platform elements constant and/or streamlining development steps: (1) consistent delivery vehicle; (2) consistent manufacturing; (3) benefit-risk appropriate quality requirements; and (4) expansive clinical trial designs. Together, these opportunities could yield up to 5-fold efficiency gains and result in substantial value creation for patients, regulators, and developers, potentially decreasing the time required to dose patients with a new gene-editing therapy from years down to 6 months.}, }
@article {pmid40615444, year = {2025}, author = {Novotná, M and Tinti, M and Faria, JRC and Horn, D}, title = {Precision-edited histone tails disrupt polycistronic gene expression controls in trypanosomes.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {6194}, pmid = {40615444}, issn = {2041-1723}, support = {217105/Z/19/Z//Wellcome Trust (Wellcome)/ ; }, mesh = {*Histones/genetics/metabolism ; *Trypanosoma brucei brucei/genetics/metabolism ; Protozoan Proteins/genetics/metabolism ; RNA Polymerase II/metabolism/genetics ; Promoter Regions, Genetic/genetics ; CRISPR-Cas Systems ; *Gene Expression Regulation ; Gene Editing ; Mutation ; Acetylation ; Lysine/metabolism ; Protein Processing, Post-Translational ; Methylation ; Gene Expression Profiling ; Chromatin/metabolism ; }, abstract = {Transcription of protein coding genes in trypanosomatids is atypical and almost exclusively polycistronic. In Trypanosoma brucei, for example, approximately 150 polycistrons, and 8000 genes, are constitutively transcribed by RNA polymerase II. The RNA pol-II promoters are also unconventional and characterised by regions of chromatin enriched for histones with specific patterns of post-translational modification on their divergent N-terminal tails. To investigate the roles of histone tail-residues in gene expression control in T. brucei, we engineered strains exclusively expressing mutant histones. We used an inducible CRISPR-Cas9 system to delete >40 histone H4 genes, complementing the defect with a single ectopic H4 gene. The resulting "hist[one]H4" strains were validated using whole-genome sequencing and transcriptome analysis. We then performed saturation mutagenesis of six histone H4 N-terminal tail lysine residues, that are either acetylated or methylated, and profiled relative fitness of 384 distinct precision-edited mutants. H4[lys10] mutations were not tolerated, but we derived nineteen strains exclusively expressing distinct H4[lys4] or H4[lys14] mutants. Proteomic and transcriptomic analysis of H4[lys4] glutamine mutants revealed significantly reduced expression of genes adjacent to RNA pol-II promoters, where glutamine mimics abnormally elevated acetylation. Thus, we present direct evidence for polycistronic expression control by modified histone H4 N-terminal tail residues in trypanosomes.}, }
@article {pmid40615387, year = {2025}, author = {Lou, S and DJiake Tihagam, R and Wasko, UN and Equbal, Z and Venkatesan, S and Braczyk, K and Przanowski, P and Il Koo, B and Saltani, I and Singh, AT and Likhite, S and Powers, S and Souza, GMPR and Maxwell, RA and Yu, J and Zhu, LJ and Beenhakker, M and Abbott, SBG and Lu, Z and Green, MR and Meyer, KC and Tushir-Singh, J and Bhatnagar, S}, title = {Targeting microRNA-dependent control of X chromosome inactivation improves the Rett Syndrome phenotype.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {6169}, pmid = {40615387}, issn = {2041-1723}, mesh = {*Rett Syndrome/genetics/therapy ; *MicroRNAs/genetics/metabolism ; *X Chromosome Inactivation/genetics ; Animals ; Female ; Methyl-CpG-Binding Protein 2/genetics/metabolism ; Humans ; Mice ; RNA, Long Noncoding/genetics/metabolism ; Phenotype ; Fibroblasts/metabolism ; CRISPR-Cas Systems ; Disease Models, Animal ; Male ; }, abstract = {X chromosome inactivation (XCI) is induced by Xist long non-coding RNA and protein-coding genes. However, the role of small non-coding RNA function in XCI remains unidentified. Our genome-wide, loss-of-function CRISPR/Cas9 screen in female fibroblasts identified microRNAs (miRNAs) as regulators of XCI. A striking finding is the identification of miR106a among the top candidates from the screen. Loss of miR106a is accompanied by altered Xist interactome, leading to dissociation and destabilization of Xist. XCI interference via miR106a inhibition has therapeutic implications for Rett syndrome (RTT) girls with a defective X-linked MECP2 gene. Here, we discovered that the inhibition of miR106a significantly improves several facets of RTT pathology: it increases the life span, enhances locomotor activity and exploratory behavior, and diminishes breathing variabilities. Our results suggest that miR106a targeting offers a feasible therapeutic strategy for RTT and other monogenic X-linked neurodevelopmental disorders.}, }
@article {pmid40613815, year = {2025}, author = {Pinto, D and Mendes, I and Cunha, MV}, title = {Genomic Survey Reveals no Detectable Bacteriophage Activity in Mycobacterium bovis Across a Large Population.}, journal = {FEMS microbiology ecology}, volume = {}, number = {}, pages = {}, doi = {10.1093/femsec/fiaf072}, pmid = {40613815}, issn = {1574-6941}, abstract = {Phages are major drivers of bacterial evolution, yet their ecological and evolutionary interactions with Mycobacterium bovis, a key member of the Mycobacterium tuberculosis complex (MTBC), remain understudied. In this work, we investigate the elusive phage-bacterium interface in M. bovis by integrating comparative genomics of 200 isolates from infected animals with molecular analyses of M. bovis-positive environmental samples. Despite employing diverse and complementary approaches, we found no evidence of active or recent phage infections: no novel prophages beyond the conserved phiRv1, no expansion of CRISPR arrays, and no co-occurrence of M. bovis and mycobacteriophages in host tissues or environmental matrices. Intriguingly, we identified multiple independent excision events of phiRv1 across closely related lineages, suggesting recent prophage mobilization driven by unidentified ecological or genomic triggers. These findings echo previous observations in M. tuberculosis and point toward a stable, phage-scarce landscape across MTBC members. Our results raise compelling questions about the barriers to phage predation in M. bovis, the functionality of its CRISPR-Cas system, and the selective pressures underlying prophage retention and loss. By shedding light on these underexplored dynamics, our study reveals critical gaps in the ecological understanding of M. bovis and highlights opportunities for phage-based innovation in TB control.}, }
@article {pmid40613714, year = {2025}, author = {Wu, D and Snead, S and Ganguly, C and Babu, K and Li, Y and Kathiresan, V and Shen, R and Zhang, X and Rajan, R and Qin, PZ}, title = {Structural integrity and side-chain interaction at the loop region of the bridge helix modulate Cas9 substrate discrimination.}, journal = {Nucleic acids research}, volume = {53}, number = {12}, pages = {}, doi = {10.1093/nar/gkaf557}, pmid = {40613714}, issn = {1362-4962}, support = {MCB-1716744//National Science Foundation/ ; MCB-1818107//National Science Foundation/ ; MCB-1716423//National Science Foundation/ ; MCB-2424888//National Science Foundation/ ; R35-GM145341/NH/NIH HHS/United States ; //Arnold and Mabel Beckman Foundation/ ; }, mesh = {*CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics ; DNA/metabolism/chemistry/genetics ; Substrate Specificity ; CRISPR-Cas Systems ; Models, Molecular ; Protein Binding ; DNA Cleavage ; Protein Conformation ; }, abstract = {CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) has been revolutionizing genome engineering, and in-depth understanding of mechanisms governing its DNA discrimination is critical for continuing technology advances. An arginine-rich bridge helix (BH) connecting the nuclease lobe and the recognition lobe, which is conserved across the Cas9 family, exists in a helix-loop-helix conformation in the apo wild-type protein but converts to a long contiguous helix in the Cas9/RNA binary complex. In this work, distances measured with spin labels were utilized to investigate BH's conformational transitions in the solution state upon single-guide RNA (sgRNA) binding, which is a critical early event preceding DNA binding and cleavage. Analyses show that sgRNA binding drives BH conformational changes in the wild-type SpyCas9 (SpyCas9WT) as well as in two BH-loop variants, SpyCas92Pro and SpyCas92Ala. Each Cas9-sgRNA binary complex, however, exhibits distinct BH features that reveal mutation-specific effects on helical integrity versus side-chain interactions. In addition, the BH conformational variations can be correlated to the observed changes in the mismatch cleavage profiles of the Cas9 variants. The work represents the first use of distances measured by site-directed spin labeling to investigate Cas9 protein conformational changes in the solution state and advances our understanding on the structure-dynamic-function relationship governing DNA target discrimination by Cas9.}, }
@article {pmid40613710, year = {2025}, author = {Hibshman, GN and Taylor, DW}, title = {Structural basis of a dual-function type II-B CRISPR-Cas9.}, journal = {Nucleic acids research}, volume = {53}, number = {12}, pages = {}, doi = {10.1093/nar/gkaf585}, pmid = {40613710}, issn = {1362-4962}, support = {R35GM138348/NH/NIH HHS/United States ; F-1938//Welch Foundation/ ; }, mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; *Francisella/genetics/enzymology ; Gene Editing ; *Bacterial Proteins/chemistry/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Streptococcus pyogenes/genetics/enzymology ; DNA/genetics/chemistry/metabolism ; DNA Cleavage ; }, abstract = {Cas9 from Streptococcus pyogenes (SpCas9) revolutionized genome editing by enabling programmable DNA cleavage guided by an RNA. However, SpCas9 tolerates mismatches in the DNA-RNA duplex, which can lead to deleterious off-target editing. Here, we reveal that Cas9 from Francisella novicida (FnCas9) possesses a unique structural feature-the REC3 clamp-that underlies its intrinsic high-fidelity DNA targeting. Through kinetic and structural analyses, we show that the REC3 clamp forms critical contacts with the PAM-distal region of the R-loop, thereby imposing a novel checkpoint during enzyme activation. Notably, F. novicida encodes a noncanonical small CRISPR-associated RNA (scaRNA) that enables FnCas9 to repress an endogenous bacterial lipoprotein gene, subverting host immune detection. Structures of FnCas9 with scaRNA illustrate how partial R-loop complementarity hinders REC3 clamp docking and prevents cleavage in favor of transcriptional repression. The REC3 clamp is conserved across type II-B CRISPR-Cas9 systems, pointing to a potential path for engineering precise genome editors or developing novel antibacterial strategies. These findings reveal the molecular basis of heightened specificity and virulence enabled by FnCas9, with broad implications for biotechnology and therapeutic development.}, }
@article {pmid40613705, year = {2025}, author = {Schwartz, CI and Abell, NS and Li, A and Aradhana,  and Tycko, J and Truong, A and Montgomery, SB and Hess, GT}, title = {Toward optimizing diversifying base editors for high-throughput mutational scanning studies.}, journal = {Nucleic acids research}, volume = {53}, number = {12}, pages = {}, doi = {10.1093/nar/gkaf620}, pmid = {40613705}, issn = {1362-4962}, support = {R35GM150462/NH/NIH HHS/United States ; NIH-1F99DK126120-01/NH/NIH HHS/United States ; NIH-4K00DK126120-03/NH/NIH HHS/United States ; S10RR025518-01/NH/NIH HHS/United States ; }, mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; Cytidine Deaminase/genetics/metabolism ; HEK293 Cells ; CRISPR-Associated Protein 9/genetics/metabolism ; Point Mutation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mutation ; DNA Mutational Analysis/methods ; AICDA (Activation-Induced Cytidine Deaminase) ; }, abstract = {Base editors, including diversifying base editors that create C>N mutations, are potent tools for systematically installing point mutations in mammalian genomes and studying their effect on cellular function. Numerous base editor options are available for such studies, but little information exists on how the composition of the editor (deaminase, recruitment method, and fusion architecture) affects editing. To address this knowledge gap, the effect of various design features, such as deaminase recruitment and delivery method (electroporation or lentiviral transduction), on editing was assessed across ∼200 synthetic target sites. The direct fusion of a hyperactive variant of activation-induced cytidine deaminase to the N-terminus of dCas9 (DivA-BE) produced the highest editing efficiency, ∼4-fold better than the previous CRISPR-X method. Additionally, DivA-BE mutagenized the DNA strand that anneals to the targeting sgRNA (target strand) to create complementary C>N mutations, which were absent when the deaminase was fused to the C-terminus of dCas9. Based on these studies that comprehensively analyze the editing patterns of several popular base editors, DivA-BE editors efficiently diversified their target sites, albeit with increased indel frequencies. Overall, the improved editing efficiency makes the DivA-BE editors ideal for discovering functional variants in mutational scanning assays.}, }
@article {pmid40613203, year = {2025}, author = {Criollo Delgado, L and Zamalutdinov, A and Potokina, E}, title = {Identification of Soybean E1-E4 Gene Orthologs in the Guar Genome Using Comprehensive Transcriptome Assembly and Annotation.}, journal = {Frontiers in bioscience (Scholar edition)}, volume = {17}, number = {2}, pages = {26548}, doi = {10.31083/FBS26548}, pmid = {40613203}, issn = {1945-0524}, support = {24-26-00073//Russian Science Foundation/ ; }, mesh = {*Glycine max/genetics ; *Cyamopsis/genetics ; *Transcriptome/genetics ; *Genome, Plant ; Molecular Sequence Annotation ; Photoperiod ; Gene Expression Regulation, Plant ; Plant Proteins/genetics ; *Genes, Plant ; Gene Expression Profiling ; Galactans ; Mannans ; Plant Gums ; }, abstract = {BACKGROUND: We publish the first available transcriptome assembly of guar (Cyamopsis tetragonoloba (L.) Taub.), a well-known source of guar gum (food additive E 412). At high latitudes, e.g., in Russia, the main challenge for guar cultivation is the long photoperiod during summer, which delays flowering and maturation of guar plants. Meanwhile, identifying of genes affecting the photoperiod sensitivity of guar would have a major impact on the development of marker-assisted breeding of this valuable food crop.<br><br>METHODS: RNA isolated from leaves of early and late flowering guar plants grown under long-day conditions were used to generate de novo transcriptome assembly. A similarity search was conducted using BLASTN 2.2.31+ with default settings to identify homologous sequences of soybean maturity genes E1-E4 in guar transcriptome and genome assembly. Gene prediction tools such as AUGUSTUS and FGENESH+ were used to predict the exon-intron structure of the candidate genes. Functional annotation of the amino acid sequence was performed using InterProScan v. 5.68-100.<br><br>RESULTS: The transcriptome assembly contained sequences of 96,447 clustered transcript isoforms in the leaves of guar plants grown under long-day conditions. The transcriptome assembly was annotated using BLAST against the Glycine max genome, and 42,615 guar transcripts (44.2%) were found to be similar to soybean genes. We used the developed transcriptome assembly to discover orthologs of the E1-E4 soybean loci in the guar genome that have the greatest impact on the flowering and maturation of this closely related, short-day legume crop. A high level of identity was detected between peptide sequences encoding by orthologous genes E1 and CtE1 (80%), E2 and CtE2 (93%), E3 and CtE3 (83%), and E4 and CtE4 (91%). The sequences and the intron-exon structure of the genes in soybean and guar were similar, suggesting that the genetic pathways underlying basic flowering mechanisms are conserved between these two legume crops.<br><br>CONCLUSIONS: The revealed intron-exon structure of the guar genes CtE1-CtE4 creates possibilities for their targeted mutagenesis, e.g., using CRISPR-Cas and developing new guar germplasm with low sensitivity to photoperiod.}, }
@article {pmid40611477, year = {2025}, author = {Bulle, M and Abbagani, S and Raza, A}, title = {Genome blaze: engineering chilli pepper chloroplasts for sustainable production of capsaicinoids through organellar genome editing.}, journal = {Plant biology (Stuttgart, Germany)}, volume = {}, number = {}, pages = {}, doi = {10.1111/plb.70067}, pmid = {40611477}, issn = {1438-8677}, support = {PDF/2018/001226//Science and Engineering Research Board/ ; }, abstract = {The quest for superior parental lines of chilli pepper, enriched with heightened levels of phytochemicals, such as capsinoids (CATs) and capsaicinoids (CAPs), has recently gained momentum. Plant scientists now pay more attention to generating pepper cultivars that offer both increased yield and richness in these crucial ingredients. Indeed, the average pepper yield per hectare, as well as richness of these ingredients, are affected by multiple environmental stimuli. However, expression of specific genes is crucial to stimulate the CAT and CAP levels in response to environmental conditions. Recently, new technologies, like genome editing using CRISPR/Cas and engineering of chloroplasts, have been tested in chilli pepper. The advances in genome editing tools, such as the prime editor and base editor methods, have tremendous potential for plant organelle genome engineering. In this article, we highlight recent advancements in CRISPR/Cas and plastid engineering within Capsicum, coupled with application of base editing approaches for editing plant organelle DNA. We also assess the challenges and opportunities in the pursuit of commercial and sustainable production of bioactive compounds specific to Capsicum species.}, }
@article {pmid40611420, year = {2025}, author = {Rezaei, M and Jalali, A and Sadah Al-Azzawi, DH}, title = {Engineered Bacteriophages: Advances in Phage Genome Redesign Strategies for Therapeutic and Environmental Applications.}, journal = {Protein and peptide letters}, volume = {}, number = {}, pages = {}, doi = {10.2174/0109298665372719250616085616}, pmid = {40611420}, issn = {1875-5305}, abstract = {Bacteriophages, or phages, have emerged as powerful platforms in synthetic biology, offering innovative solutions for therapeutic and environmental challenges through advanced genome redesign strategies. This review explores a wide range of phage engineering techniques, including CRISPR (clustered regularly-interspaced short palindromic repeats)-Cas systems, phage display, random and site-directed mutagenesis, retrons, and rebooting approaches, highlighting their potential to create phages with tailored functionalities. CRISPR-Cas systems enable precise genome editing, allowing the development of phages with expanded host ranges, biofilm degradation capabilities, and targeted antimicrobial activity. Phage display facilitates the presentation of peptides on phage surfaces, enabling applications in targeted drug delivery, tumor imaging, and bioremediation. Beyond these, techniques like retron-mediated recombination and homologous recombination offer additional avenues for precise phage genome modification. In the therapeutic realm, engineered phages show promise in combating drug-resistant infections, modulating the microbiome, and delivering targeted therapies for cancer and other diseases. Environmentally, phage-based strategies, such as the use of phage-displayed metal-binding peptides, provide innovative solutions for bioremediation and reducing exposure to toxic heavy metals. This review also addresses challenges, such as phage resistance, immune responses, and the limitations of current engineering methods, while exploring future directions, including the development of improved CRISPR systems, phage-based biosensors, and high-throughput screening platforms. By integrating cutting-edge genome redesign strategies with diverse applications, this review underscores the transformative potential of engineered bacteriophages in addressing global healthcare and environmental sustainability challenges.}, }
@article {pmid40611351, year = {2025}, author = {Smidler, AL and Akbari, OS}, title = {CRISPR technologies for the control and study of malaria-transmitting anopheline mosquitoes.}, journal = {Parasites &amp; vectors}, volume = {18}, number = {1}, pages = {252}, pmid = {40611351}, issn = {1756-3305}, support = {R01AI151004/GF/NIH HHS/United States ; R01AI151004/GF/NIH HHS/United States ; }, mesh = {*Anopheles/genetics/parasitology/physiology ; Animals ; *Mosquito Control/methods ; *Mosquito Vectors/genetics/parasitology/physiology ; *CRISPR-Cas Systems ; *Malaria/transmission/prevention &amp; control ; Gene Editing ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Female ; Animals, Genetically Modified ; }, abstract = {Malaria is one of the deadliest diseases on the planet, killing approximately 600,000 people annually, and is transmitted by the bite of an anopheline mosquito. Anophelines, and the diseases they transmit, have changed the course of history and the fate of nations, and their successful control promises to end the transmission of malaria. With the advent of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technologies, the study and control of these deadly pests have been revolutionized. As the release of genetically modified anophelines is being considered, here we outline the advances in CRISPR/Cas9 technologies and how they have revolutionized the study of anopheline basic biology and the development of innovative vector control strategies. We outline the major findings of CRISPR-based basic biological research into traits relevant for vector control including, but not limited to, olfaction, chemosensation, neurobiology, and reproduction. Further, we summarize the advancements in CRISPR-based innovative vector control strategies, such as the precision-guided sterile insect technique (pgSIT), inherited female elimination by genetically encoded nucleases to interrupt alleles (IFEGENIA), X-shredder, Y-linked editors, and gene drives. All in all, this review summarizes the basic biological and vector control research undertaken using CRISPR since its advent approximately a decade ago.}, }
@article {pmid40516528, year = {2025}, author = {Matthews, RE and Danac, JMC and Naden, EL and Farleigh Smith, LE and Lestari, S and Gungi, A and Appert, A and Buttress, T and Verma, A and Sinclair, O and Chong, F and Suberu, J and Antrobus, R and Bonev, B and Dawson, MA and Reid, AJ and Timms, RT and Ahringer, J and Tchasovnikarova, IA}, title = {CRAMP1 drives linker histone expression to enable Polycomb repression.}, journal = {Molecular cell}, volume = {85}, number = {13}, pages = {2503-2516.e8}, doi = {10.1016/j.molcel.2025.05.031}, pmid = {40516528}, issn = {1097-4164}, mesh = {*Histones/genetics/metabolism ; *Polycomb Repressive Complex 2/genetics/metabolism ; Animals ; Humans ; Mice ; Promoter Regions, Genetic ; Heterochromatin/genetics/metabolism ; CRISPR-Cas Systems ; *Epigenetic Repression ; }, abstract = {In contrast to the well-understood role of core histones in DNA packaging, the function of the linker histone (H1) remains enigmatic. Challenging the prevailing view that linker histones are a general feature of heterochromatin, here we show a critical requirement for H1 in Polycomb repressive complex 2 (PRC2) function. A CRISPR-Cas9 genetic screen using a fluorescent PRC2 reporter identified an essential role for the poorly characterized gene CRAMP1 in PRC2-mediated repression. CRAMP1 localizes to the promoters of expressed H1 genes and positively regulates their transcription. CRAMP1 ablation simultaneously depletes all linker histones, which results in selective decompaction of H3K27me3-marked loci and derepression of PRC2 target genes without concomitant loss of PRC2 occupancy or enzymatic activity. Strikingly, we find that linker histones preferentially localize to genomic loci marked by H3K27me3 across diverse cell types and organisms. Altogether, these data demonstrate a prominent role for linker histones in epigenetic repression by PRC2.}, }
@article {pmid40359943, year = {2025}, author = {Wang, Y and Wu, J and Yang, S and Li, X and Wang, J and Lv, Q and Zhu, X and Lu, G and Zhang, J and Shen, WH and Liu, B and Lin, J and Dong, A}, title = {Structural and functional interrelationships of histone H2A with its variants H2A.Z and H2A.W in Arabidopsis.}, journal = {Structure (London, England : 1993)}, volume = {33}, number = {7}, pages = {1240-1249.e5}, doi = {10.1016/j.str.2025.04.015}, pmid = {40359943}, issn = {1878-4186}, mesh = {*Histones/chemistry/genetics/metabolism ; *Arabidopsis/genetics/metabolism ; *Arabidopsis Proteins/chemistry/metabolism/genetics ; Nucleosomes/metabolism/chemistry ; Cryoelectron Microscopy ; CRISPR-Cas Systems ; Mutation ; Protein Isoforms/chemistry/metabolism/genetics ; }, abstract = {Multiple histone H2A variants are known in eukaryotes. However, the functional relationship between H2A and its variants in plants remains largely obscure. Using CRISPR-Cas9 editing, we generated a mutant lacking four H2A isoforms in Arabidopsis and analyzed the functional and structural relationships between H2A, H2A.Z, and H2A.W. RNA sequencing and phenotype analyses revealed mild changes in gene transcription and plant development in mutants lacking H2A, H2A.Z, or H2A.W compared with the wild-type plants. Chromatin immunoprecipitation sequencing analysis showed that H2A can substitute for both H2A.Z and H2A.W across the genome, including in euchromatin and heterochromatin regions. However, H2A.Z replaced both H2A and H2A.W primarily within the euchromatin regions. By using DNA and histones from Arabidopsis, we constructed nucleosomes containing H2A, H2A.Z, or H2A.W and resolved their cryogenic electron microscopy (cryo-EM) structures at near-atomic resolution. Collectively, the results reveal the structural similarity and functional redundancy of H2A and its variants in Arabidopsis.}, }
@article {pmid38944889, year = {2025}, author = {Kang, ES and Kim, NH and Lim, HK and Jeon, H and Han, K and No, YH and Kim, K and Khaleel, ZH and Shin, D and Eom, K and Nam, J and Lee, BS and Kim, HJ and Suh, M and Lee, J and Thach, TT and Hyun, J and Kim, YH}, title = {Structure-Guided Engineering of Thermodynamically Enhanced SaCas9 for Improved Gene Suppression.}, journal = {Advanced materials (Deerfield Beach, Fla.)}, volume = {37}, number = {26}, pages = {e2404680}, doi = {10.1002/adma.202404680}, pmid = {38944889}, issn = {1521-4095}, support = {//National Research Foundation of Korea/ ; NRF-2023R1A2C3005731//Korean government/ ; RS-2023-00302458)//Korean government/ ; IBS-R015-D1//Institute for Basic Science/ ; }, mesh = {Animals ; Mice ; *Protein Engineering/methods ; Thermodynamics ; Humans ; Molecular Dynamics Simulation ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; CRISPR-Cas Systems ; HEK293 Cells ; DNA/metabolism ; }, abstract = {Proteins with multiple domains play pivotal roles in various biological processes, necessitating a thorough understanding of their structural stability and functional interplay. Here, a structure-guided protein engineering approach is proposed to develop thermostable Cas9 (CRISPR-associated protein 9) variant for CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference applications. By employing thermodynamic analysis, combining distance mapping and molecular dynamics simulations, deletable domains are identified to enhance stability while preserving the DNA recognition function of Cas9. The resulting engineered Cas9, termed small and dead form Cas9, exhibits improved thermostability and maintains target DNA recognition function. Cryo-electron microscopy analysis reveals structural integrity with reduced atomic density in the deleted domain. Fusion with functional elements enables intracellular delivery and nuclear localization, demonstrating efficient gene suppression in diverse cell types. Direct delivery in the mouse brain shows enhanced knockdown efficiency, highlighting the potential of structure-guided engineering to develop functional CRISPR systems tailored for specific applications. This study underscores the significance of integrating computational and experimental approaches for protein engineering, offering insights into designing tailored molecular tools for precise biological interventions.}, }
@article {pmid40612691, year = {2024}, author = {Lin, J and Yang, J}, title = {CRISPR-Cas systems: A revolution in genome editing and its diverse applications.}, journal = {Journal of biomed research}, volume = {5}, number = {1}, pages = {108-114}, pmid = {40612691}, issn = {2693-5910}, abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) Cas (CRISPR6 associated protein) system is an advanced adaptive immune system found in prokaryotes. First discovered in1987, CRISPR Cas has revolutionized genetic research in the past two decades. CRISPR-Cas9 the most widespread system enables precise gene editing by creating double strand breaks. Its ease of use and cost-effectiveness has lowered the barrier to entry for genetic research. CRISPR holds immense potential in many fields from agriculture to medicine. In agriculture, CRISPR has accelerated crop improvement by enabling precise gene edits for desirable traits. In medicine, CRISPR holds promise in xenotransplant, cancers and infectious diseases (HIV) treatment. This review traces the historical development of CRISPR-Cas systems, explores their unique applications, and discusses future advancements aimed at enhancing CRISPR's precision and expanding its applications through technologies like prime and base editing.}, }
@article {pmid40610423, year = {2025}, author = {Fuhrmeister, ER and Kim, S and Mairal, SA and McCormack, C and Chieng, B and Swarthout, JM and Paulos, AH and Njenga, SM and Pickering, AJ}, title = {Context-Seq: CRISPR-Cas9 targeted nanopore sequencing for transmission dynamics of antimicrobial resistance.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {5898}, pmid = {40610423}, issn = {2041-1723}, support = {OPP1129535//Bill and Melinda Gates Foundation (Bill &amp; Melinda Gates Foundation)/ ; UL1TR002544//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 1906957//National Science Foundation (NSF)/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; Animals ; Humans ; *Nanopore Sequencing/methods ; *Drug Resistance, Bacterial/genetics ; Kenya ; Anti-Bacterial Agents/pharmacology ; Dogs ; *Bacteria/genetics/drug effects ; Klebsiella pneumoniae/genetics/drug effects ; Child ; Adult ; Escherichia coli/genetics/drug effects ; Poultry/microbiology ; beta-Lactamases/genetics ; Genome, Bacterial ; Haemophilus influenzae/genetics/drug effects ; }, abstract = {Precisely understanding how and to what extent antimicrobial resistance (AMR) is exchanged between animals and humans is needed to inform control strategies. Metagenomic sequencing has low detection for rare targets such as antibiotic resistance genes, while whole genome sequencing of isolates misses exchange between uncultured bacterial species. We introduce Context-Seq, CRISPR-Cas9 targeted sequencing of ARGs and their genomic context with long-reads. Using Context-Seq, we investigate genetically similar AMR elements containing the ARGs blaCTX-M and blaTEM between adults, children, poultry, and dogs in Nairobi, Kenya. We identify genetically distinct clusters containing blaTEM and blaCTX-M that are shared between animals and humans within and between households. We also uncover potentially pathogenic hosts of ARGs including Escherichia coli, Klebsiella pneumoniae, and Haemophilus influenzae in this study context. Context-Seq complements conventional methods to obtain an additional view of bacterial and mammalian hosts in the proliferation of AMR.}, }
@article {pmid40610421, year = {2025}, author = {Sek, K and Chen, AXY and Cole, T and Armitage, JD and Tong, J and Yap, KM and Munoz, I and Dunbar, PA and Wu, S and van Elsas, MJ and Hidajat, O and Scheffler, C and Giuffrida, L and Henderson, MA and Meyran, D and Souza-Fonseca-Guimaraes, F and Nguyen, D and Huang, YK and de Menezes, MN and Derrick, EB and Chan, CW and Todd, KL and Chan, JD and Li, J and Lai, J and Petley, EV and Mardiana, S and Bosco, A and Waithman, J and Parish, IA and Mølck, C and Stewart, GD and Kats, L and House, IG and Darcy, PK and Beavis, PA}, title = {Tumor site-directed A1R expression enhances CAR T cell function and improves efficacy against solid tumors.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {6123}, pmid = {40610421}, issn = {2041-1723}, support = {836379//Cancer Council Victoria/ ; }, mesh = {Animals ; Humans ; Mice ; *Receptors, Chimeric Antigen/genetics/metabolism/immunology ; *Neoplasms/therapy/immunology/genetics ; *Immunotherapy, Adoptive/methods ; *T-Lymphocytes/immunology/metabolism ; CRISPR-Cas Systems ; Tumor Microenvironment/immunology ; Cell Line, Tumor ; Female ; Receptor, Adenosine A2A/genetics/metabolism ; Mice, Inbred C57BL ; }, abstract = {The efficacy of Chimeric Antigen Receptor T cells against solid tumors is limited by immunosuppressive factors in the tumor microenvironment including adenosine, which suppresses Chimeric Antigen Receptor T cells through activation of the A2A receptor. To overcome this, Chimeric Antigen Receptor T cells are engineered to express A1 receptor, a receptor that signals inversely to A2A receptor. Using murine and human Chimeric Antigen Receptor T cells, constitutive A1 receptor overexpression significantly enhances Chimeric Antigen Receptor T cell effector function albeit at the expense of Chimeric Antigen Receptor T cell persistence. Through a CRISPR/Cas9 homology directed repair "knock-in" approach we demonstrate that Chimeric Antigen Receptor T cells engineered to express A1 receptor in a tumor-localized manner, enhances anti-tumor therapeutic efficacy. This is dependent on the transcription factor IRF8 and is transcriptionally unique when compared to A2A receptor deletion. This data provides a novel approach for enhancing Chimeric Antigen Receptor T cell efficacy in solid tumors and provides proof of principle for site-directed expression of factors that promote effector T cell differentiation.}, }
@article {pmid40610153, year = {2025}, author = {Ren, W and Li, M and Liu, X and You, W and You, Q and Li, B and Ye, H and Zhang, R}, title = {Specific detection of DNA and RNA by the CRISPR-Cas12a system containing spacer split crRNA.}, journal = {Analytica chimica acta}, volume = {1367}, number = {}, pages = {344204}, doi = {10.1016/j.aca.2025.344204}, pmid = {40610153}, issn = {1873-4324}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *DNA/analysis/genetics ; *RNA/analysis/genetics ; MicroRNAs/analysis/genetics ; *CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {BACKGROUND: The CRISPR/Cas12a system has emerged as a versatile molecular diagnostic tool due to its dual cis- and trans-cleavage activities. However, two key limitations hinder its broad application: high tolerance to single-base mismatches in DNA targets and strict reliance on DNA activators. To address these challenges, we hypothesized that structural reengineering of crRNA could enhance specificity and functional versatility. This study aimed to develop a modified Cas12a system capable of detecting DNA and RNA targets with improved single-base resolution, thereby expanding its utility in molecular diagnostics and clinical subclassification.<br><br>RESULTS: We engineered split crRNAs by introducing a split site within the spacer region, creating a spacer-split crRNA-activated Cas12a system (SPCas12a). This system exhibited three key advantages: First, SPCas12a demonstrated significantly enhanced specificity in discriminating single-base mutations compared to conventional full-sized crRNA systems. Second, it bypassed the DNA activator requirement, enabling direct detection of miRNA targets without reverse transcription. In addition, AlphaFold Server predictive structural modeling analysis showed that the split site selected by SPCas12a gives the Cas12a complex an open structural domain, which is conducive to the stable function of Cas12a. Third, integration with isothermal amplification enabled constructing an "AND" logic gate detection platform that processes multiple inputs within 40&#xa0;min. As a proof-of-concept, SPCas12a successfully distinguished triple-negative breast cancer (TNBC) subtype cell lines by analyzing miRNA-210 and miRNA-21 biomarkers in different cell lines.<br><br>SIGNIFICANCE: SPCas12a overcomes fundamental limitations of current CRISPR diagnostics by unifying high-specificity DNA mutation detection and direct RNA sensing in a single platform. The split-crRNA design principle provides a universally adaptable strategy to enhance CRISPR-Cas systems, with immediate applications in precision oncology and infectious disease stratification where base-level discrimination and multi-target detection are critical.}, }
@article {pmid40609997, year = {2025}, author = {Kalinina, NO and Spechenkova, NA and Taliansky, ME}, title = {Biotechnological Approaches to Plant Antiviral Resistance: CRISPR-Cas or RNA Interference?.}, journal = {Biochemistry. Biokhimiia}, volume = {90}, number = {6}, pages = {804-817}, doi = {10.1134/S0006297925600139}, pmid = {40609997}, issn = {1608-3040}, mesh = {*RNA Interference ; *CRISPR-Cas Systems ; *Plant Diseases/virology/genetics/prevention &amp; control ; Gene Editing/methods ; *Plants/virology/genetics ; *Disease Resistance/genetics ; *Biotechnology/methods ; Plant Viruses/genetics ; }, abstract = {Established genome editing technologies, such as CRISPR-Cas and RNA interference (RNAi), have significantly advanced research studies in nearly all fields of life sciences, including biotechnology and medicine, and have become increasingly in demand in plant biology. In the review, we present the main principles of the CRISPR-Cas and RNAi technologies and their application in model plants and crops for the control of viral diseases. The review explores the antiviral effects they provide, including direct suppression of genomes of DNA- and RNA-containing viruses and inhibition of activity of host genes that increase plant susceptibility to viruses. We also provide a detailed comparison of the effectiveness of CRISPR-Cas and RNAi methods in plant protection, as well as discuss their advantages and disadvantages, factors limiting their application in practice, and possible approaches to overcome such limitations.}, }
@article {pmid40609996, year = {2025}, author = {Kazakova, AA and Leonova, EI and Sopova, JV and Chirinskaite, AV and Minskaya, ES and Kukushkin, IS and Ivanov, RA and Reshetnikov, VV}, title = {Progress in CRISPR/CAS13-Mediated Suppression of Influenza A and SARS-CoV-2 Virus Infection in in vitro and in vivo Models.}, journal = {Biochemistry. Biokhimiia}, volume = {90}, number = {6}, pages = {786-803}, doi = {10.1134/S0006297925601212}, pmid = {40609996}, issn = {1608-3040}, mesh = {*CRISPR-Cas Systems ; Animals ; *SARS-CoV-2/genetics ; Humans ; *COVID-19/therapy/virology ; *Influenza A virus/genetics ; *Influenza, Human/therapy/virology/genetics ; Disease Models, Animal ; RNA, Viral/genetics/metabolism ; Antiviral Agents ; }, abstract = {The worldwide number of deaths from complications caused by severe influenza and COVID-19 is about 1 million cases annually. Development of the effective antiviral therapy strategies for the disease treatment is one of the most important tasks. Use of the CRISPR/Cas13 system, which specifically degrades viral RNA and significantly reduces titer of the virus, could be a solution of this problem. Despite the fact that Cas13 nucleases have been discovered only recently, they already have shown high efficiency in suppressing viral transcripts in cell cultures. The recent advances in mRNA technology and improvements in non-viral delivery systems have made it possible to effectively use CRISPR/Cas13 in animal models as well. In this review, we analyzed experimental in vitro and in vivo studies on the use of CRISPR/Cas13 systems as an antiviral agent in cell cultures and animal models and discussed main directions for improving the CRISPR/Cas13 system. These data allow us to understand prospects and limitations of the further use of CRISPR/Cas13 in the treatment of viral diseases.}, }
@article {pmid40609995, year = {2025}, author = {Volodina, OV and Demchenko, AG and Anuchina, AA and Ryzhkova, OP and Kovalskaya, VA and Kondrateva, EV and Tabakov, VY and Lavrov, AV and Smirnikhina, SA}, title = {Selection of Optimal pegRNAs to Enhance Efficiency of Prime Editing in AT-Rich Genome Regions.}, journal = {Biochemistry. Biokhimiia}, volume = {90}, number = {6}, pages = {773-785}, doi = {10.1134/S0006297924604672}, pmid = {40609995}, issn = {1608-3040}, mesh = {*Gene Editing/methods ; Humans ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; *Cystic Fibrosis/genetics/therapy ; CRISPR-Cas Systems ; }, abstract = {Prime editing is a highly promising strategy for treating hereditary disorders due to its superior efficiency and safety profile compared to the conventional CRISPR-Cas9 systems. This study is dedicated to development of a causal therapy for cystic fibrosis by targeting the F508del variant of the CFTR gene using prime editing, as this specific deletion accounts for a substantial proportion of cystic fibrosis cases. While prime editing has shown remarkable precision in introducing targeted genetic modifications, its application in AT-rich genomic regions, such as the one containing the F508del variant, remains challenging. To overcome this limitation, we systematically evaluated 24 pegRNAs designed for two distinct prime editing systems, PEmax and PE2-NG. Efficiency of the F508del variant correction reached 2.81% (without normalization for transfection efficiency) in the airway basal cells from the patients with homozygous F508del mutation. However, the average transfection efficiency was only 11.9%, emphasizing critical need for the advancements in delivery methodologies. These findings highlight potential of prime editing as an approach for treating cystic fibrosis, while also underscoring necessity for further optimization of both editing constructs and delivery vectors to achieve clinically relevant correction levels.}, }
@article {pmid40609268, year = {2025}, author = {Allan, AC and Scott, B and Tate, W and Jameson, PE and MacRae, E and Conner, AJ and Drummond, R and Foster, A and Martin, C and Caradus, J}, title = {Human-mediated outdoor genome editing is not possible so therefore poses no risk to the environment.}, journal = {Ecotoxicology and environmental safety}, volume = {302}, number = {}, pages = {118609}, doi = {10.1016/j.ecoenv.2025.118609}, pmid = {40609268}, issn = {1090-2414}, abstract = {There is a world-wide re-examination of the regulations that surround genetic technologies, including gene edited organisms. In many countries, crop plants with small gene edits and where no foreign DNA is introduced are exempt from detailed regulatory assessment. This will allow these types of plants to be released, after assessment for benefit and risk, by plant breeders or plant scientists. The full regulatory risk assessment and risk management of novel (including transgenic) plants is well established and focusses on five key criteria (weediness, gene flow, plant pests, non-target impact, biodiversity). However, plants produced by "traditional" plant breeding technologies, many of which have been subject to random mutagenesis or wide crosses that may introduce enormous numbers of DNA changes, are almost never considered novel so are not assessed for risk through a regulatory system. The most targeted, versatile and widely used gene editing technique involves the enzyme-RNA complex, CRISPR-Cas. This method can produce far more precise and targeted changes than "traditional" mutagenesis techniques. Getting the CRISPR-Cas machinery into plant or animal cells requires highly sterile tissue culture and sophisticated delivery tools. Therefore, gene editing, in the open environment by "field spraying", is not currently possible. While other uses of nucleotide chemistry - such as double stranded RNA - have been applied to plants to knock down gene expression, this is not gene editing and produces no DNA change. Suggestions that gene editing using CRISPR-Cas can occur through spraying directly on to plants in the outside environment is fanciful, incorrect and misleading.}, }
@article {pmid40608406, year = {2025}, author = {Ito, K and Ito, Y}, title = {Comparative genomic analysis of Latilactobacillus sakei strains provides new insights into their association with different niche adaptations.}, journal = {Microbiology (Reading, England)}, volume = {171}, number = {7}, pages = {}, doi = {10.1099/mic.0.001578}, pmid = {40608406}, issn = {1465-2080}, mesh = {*Genome, Bacterial ; Phylogeny ; *Adaptation, Physiological/genetics ; Plasmids/genetics ; *Latilactobacillus sakei/genetics/classification/physiology/growth &amp; development/metabolism ; Genomics ; Carbohydrate Metabolism/genetics ; Fermentation ; Genetic Variation ; }, abstract = {Latilactobacillus sakei, a lactic acid bacterium in diverse environments such as fermented foods, meat and the human gastrointestinal tract, exhibits significant genetic diversity and niche-specific adaptations. This study conducts a comprehensive comparative genomic analysis of 29 complete L. sakei genomes to uncover the genetic mechanisms underlying these adaptations. Phylogenetic analysis divided the species into three distinct clades that did not correlate with the source of isolation and did not suggest any niche-specific evolutionary direction. The pan-genome analysis revealed a substantial core genome alongside a diverse genetic repertoire, indicating both high genetic conservation and adaptability. Predicted growth rates based on codon use bias analysis suggest that L. sakei strains have an overall faster growth rate and may be able to efficiently dominate in competitive environments. Plasmid analysis revealed a variety of plasmids carrying genes essential for carbohydrate metabolism, enhancing L. sakei's ability to thrive in various fermentation substrates. It was also found that the number of genes belonging to the GH1 family amongst sugar metabolism-related genes present on chromosomes and plasmids varies between strains and that AA1, which is involved in alcohol oxidation, has been acquired from plasmids. blast analysis revealed that some strains have environmental adaptation gene clusters of cell surface polysaccharides that may mediate attachment to food and mucosa. The knowledge gleaned from this study lays a solid foundation for future research aimed at harnessing the genetic traits of L. sakei strains for industrial and health-related applications.}, }
@article {pmid40608358, year = {2025}, author = {Li, YG and Haeusser, D and Margolin, W and Christie, PJ}, title = {Conjugative delivery of toxin genes ccdB and kil confers synergistic killing of bacterial recipients.}, journal = {Journal of bacteriology}, volume = {}, number = {}, pages = {e0016825}, doi = {10.1128/jb.00168-25}, pmid = {40608358}, issn = {1098-5530}, abstract = {The bacterial type IV secretion systems (T4SS) are medically problematic for their roles in the dissemination of mobile genetic elements or effector proteins, but they also have great potential for new antimicrobial therapies. Recent studies have deployed the T4SS subfamily of conjugation systems to deliver gene editing CRISPR/Cas systems to disrupt drug resistance genes or kill targeted bacterial recipients. However, the therapeutic potential of conjugative CRISPR/Cas delivery is compromised by mutations or host repair systems that diminish the efficiency with which CRISPR/Cas induces double-strand breaks in new transconjugants. Here, we compared the efficiencies of conjugation-based killing systems based on the delivery of CRISPR-Cas9 elements or toxin genes encoding the bacteriophage lambda Kil peptide or the F plasmid-encoded CcdB. Escherichia coli equipped with one of two efficient conjugation systems, pKM101 (IncN) or F (IncF), served as donors to mobilize plasmids carrying the cognate oriT sequence and one or more toxic elements. Overall, toxin gene delivery proved significantly more effective than CRISPR-Cas9 in killing of transconjugant population, but the highest levels of growth suppression of both E. coli and Klebsiella pneumoniae recipients were achieved by a combination of CRISPR-Cas9 plus one or two toxin genes. By contrast, capsule production conferred no or very slight protective effects on plasmid acquisition and killing of either species. We propose that the conjugative co-transfer of two or more toxic elements with distinct mechanisms of action has strong potential for growth suppression of targeted species in environmental or clinical settings.IMPORTANCEThe prevalence of antibiotic resistance emphasizes the need for alternative antimicrobial intervention strategies. We engineered Escherichia coli for conjugative transmission of plasmids encoding CRISPR-Cas9 elements or genes encoding the cell division inhibitor Kil or gyrase poisoner CcdB. Delivery of toxin genes more effectively suppressed the growth of E. coli recipients than CRISPR-Cas9, but the combinatorial delivery of CRISPR-Cas9 and a toxin gene or two toxin genes elicited the strongest killing effects. Capsule production by E. coli or Klebsiella pneumoniae recipient cells had no or little protective effect on plasmid acquisition or growth suppression. Our findings suggest that probiotic donor strains equipped for conjugative delivery of two or more toxic elements may prove effective as an alternative or adjunct to traditional antimicrobials.}, }
@article {pmid40607431, year = {2025}, author = {Wang, JW and Liu, JH and Xun, JJ}, title = {CCR5 gene editing and HIV immunotherapy: current understandings, challenges, and future directions.}, journal = {Frontiers in immunology}, volume = {16}, number = {}, pages = {1590690}, pmid = {40607431}, issn = {1664-3224}, mesh = {*Receptors, CCR5/genetics/immunology ; Humans ; *Gene Editing/methods ; *HIV Infections/therapy/immunology/genetics ; *Immunotherapy/methods ; *HIV-1/immunology ; CRISPR-Cas Systems ; Genetic Therapy/methods ; }, abstract = {Human immunodeficiency virus (HIV) infection remains a major global public health challenge. Although highly active antiretroviral therapy (HAART or ART) can effectively control viral replication, it fails to eradicate latent viral reservoirs and poses limitations such as lifelong medication and cumulative drug toxicity. This study focuses on the pivotal role of C-C chemokine receptor 5 (CCR5) gene editing in HIV immunotherapy, particularly highlighting the natural resistance to R5-tropic HIV strains observed in the "Berlin" and "London" patients carrying the homozygous CCR5-Δ32 mutation. We further explore the synergistic potential of multiplex gene editing strategies-including CCR5, CXCR4, and HIV LTR loci-and the combinatorial mechanisms between gene editing technologies and immunotherapy. A personalized treatment framework is proposed to address the clinical heterogeneity among people living with HIV. In addition, we assess the balance between long-term safety and global accessibility of gene-editing approaches such as CRISPR/Cas9, emphasizing strategies to enhance therapeutic efficacy while reducing cost and off-target effects. Our findings suggest that the integration of CCR5-targeted gene editing with immune-based interventions holds great promise for overcoming current therapeutic limitations and achieving functional HIV cure. However, key challenges-such as immune rejection, viral tropism switching, and economic feasibility-must be resolved. This integrative approach provides a robust theoretical and technical foundation for the next generation of HIV treatment paradigms.}, }
@article {pmid40605043, year = {2025}, author = {Salazar-García, LM and Damas-Ramos, LC and Trejo-Alarcón, LM and Rago, D and Ahonen, L and Cruz-Morales, P and Ponce-Noyola, P and Licona-Cassani, C}, title = {CRISPR-driven enhanced hydrocarbon emulsification in an environmental Pseudomonas aeruginosa strain.}, journal = {Microbial cell factories}, volume = {24}, number = {1}, pages = {151}, pmid = {40605043}, issn = {1475-2859}, support = {NNF20CC0035580//Novo Nordisk Fonden/ ; }, mesh = {*Pseudomonas aeruginosa/metabolism/genetics/isolation &amp; purification ; Glycolipids/metabolism/biosynthesis ; *CRISPR-Cas Systems ; *Hydrocarbons/metabolism/chemistry ; Bacterial Proteins/genetics/metabolism ; Pyocyanine/metabolism/biosynthesis ; Emulsions ; Gene Editing ; Sigma Factor/genetics ; Petroleum Pollution ; }, abstract = {BACKGROUND: Oil spills are a major concern due to the economic impact and severe effects on the ecosystem. To mitigate oil spills, hydrocarbon dispersion through emulsification is a promising approach, as it makes oil more susceptible to degradation by microorganisms. Environmental strains of Pseudomonas aeruginosa have demonstrated significant potential for producing rhamnolipids (RMLs) and pyocyanin (PYO), secondary metabolites associated to hydrocarbon emulsification. In this study, we isolated and characterized an environmental strain from an oil-contaminated site in the Gulf of Mexico. Upon genome sequencing and taxonomic classification, we developed genetic engineering tools and assessed their capacity to produce PYO and RMLs, molecules relevant for hydrocarbon emulsification.<br><br>RESULTS: Using the CRISPR/Cas9-APOBEC1-UGI system, we generated a targeted cytosine to thymine transition in the rpoS gene to generate a premature STOP codon. The resulting mutant exhibited increased production of PYO and RMLs, along with enhanced gasoline emulsification in cell-free supernatants, demonstrating successful modulation of a key regulatory gene. While the strain IGLPR01 retains certain virulence-associated features, this study contributes to the exploration of environmental isolates as future candidate chassis for biosurfactant production, emphasizing the need for further safety evaluation and rational attenuation strategies.<br><br>CONCLUSION: This study provides a successful example of implementing CRISPR-based editing in an environmental P. aeruginosa strain. Despite the technical challenges, a genetic editing system was established and validated through a proof of concept to increase production of relevant metabolites. Our work demonstrates the applicability of genetic engineering tools in non-model environmental isolates, facilitating further developments. Importantly, the presence of virulence-associated features highlights the need for in-depth evaluation of pathogenicity and containment strategies before considering any future biotechnological applications.}, }
@article {pmid40605002, year = {2025}, author = {Chou, J and Esmaeili Anvar, N and Elghaish, R and Chen, J and Hart, T}, title = {Z-scores outperform similar methods for analyzing CRISPR paralog synthetic lethality screens.}, journal = {Genome biology}, volume = {26}, number = {1}, pages = {188}, pmid = {40605002}, issn = {1474-760X}, support = {R35GM130119/GM/NIGMS NIH HHS/United States ; R35GM130119/GM/NIGMS NIH HHS/United States ; R35GM130119/GM/NIGMS NIH HHS/United States ; P30CA16672/CA/NCI NIH HHS/United States ; P30CA16672/CA/NCI NIH HHS/United States ; R35CA274234/CA/NCI NIH HHS/United States ; P30CA16672/CA/NCI NIH HHS/United States ; }, mesh = {Humans ; *CRISPR-Cas Systems ; *Synthetic Lethal Mutations ; Cell Line, Tumor ; }, abstract = {Genetic screens offer a promising strategy for identifying tumor-specific therapeutic targets, but single-gene knockout screens often miss functionally redundant paralogs. Multiplex Cas9 and Cas12a CRISPR systems have been deployed to assay genetic interactions, but analysis pipelines vary considerably. Here we evaluate data from four in4mer CRISPR/Cas12a screens in cancer cell lines, using delta log fold change, Z-transformed dLFC, and rescaled dLFC approaches to identify synthetic lethal interactions. Both ZdLFC and RdLFC provide more consistent identification of synthetic lethal pairs across cell lines compared to the unscaled dLFC method, while ZdLFC benefits from not requiring a training set of known interactors.}, }
@article {pmid40577485, year = {2025}, author = {Rissone, A and La Spina, M and Bresciani, E and Syed, ZA and Combs, CA and Kirby, M and Elkahloun, A and Chen, V and Sood, R and Burgess, SM and Puertollano, R}, title = {The transcription factors Tfeb and Tfe3 are required for survival and embryonic development of pancreas and liver in zebrafish.}, journal = {PLoS genetics}, volume = {21}, number = {6}, pages = {e1011754}, doi = {10.1371/journal.pgen.1011754}, pmid = {40577485}, issn = {1553-7404}, mesh = {Animals ; *Zebrafish/genetics/embryology/growth &amp; development ; *Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics/metabolism ; *Zebrafish Proteins/genetics/metabolism ; *Pancreas/embryology/metabolism/growth &amp; development ; *Liver/embryology/metabolism/growth &amp; development ; *Embryonic Development/genetics ; Autophagy/genetics ; Gene Expression Regulation, Developmental ; Apoptosis/genetics ; Oxidative Stress/genetics ; CRISPR-Cas Systems ; }, abstract = {The transcription factors TFEB and TFE3 modulate expression of lysosomal, autophagic, and metabolic genes to restore energy and cellular homeostasis in response to a variety of stress conditions. Since their role during vertebrate development is less characterized, we used CRISPR/Cas9 to deplete tfeb, tfe3a, and tfe3b in zebrafish. The simultaneous lack of these genes compromised embryo survival during early development, with an almost complete lethality of the larvae by 8-10 dpf. The knockout animals showed apoptosis in brain and retina and alterations in pancreas, liver, and gut. Exocrine pancreas presented the most severe defects, with accumulation of abnormal zymogen granules leading to acinar atrophy in embryos and pancreatitis-like phenotypes in adults; likely due to a block of the autophagy machinery implicated in removal of damaged granules. Knockout animals displayed increased susceptibility to oxidative and heat-shock stress. Our work reveals an essential role of Tfeb and Tfe3 in maintaining cellular and tissue homeostasis during development.}, }
@article {pmid40399675, year = {2025}, author = {Han, M and Fu, ML and Zhu, Y and Choi, AA and Li, E and Bezney, J and Cai, S and Miles, L and Ma, Y and Qi, LS}, title = {Programmable control of spatial transcriptome in live cells and neurons.}, journal = {Nature}, volume = {643}, number = {8070}, pages = {241-251}, pmid = {40399675}, issn = {1476-4687}, mesh = {Animals ; *Neurons/metabolism/cytology ; *Transcriptome/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Messenger/metabolism/genetics ; Mice ; Neurites/metabolism ; Humans ; RNA Transport/genetics ; Ribosomes/metabolism ; Neuronal Outgrowth/genetics ; Actins/genetics/metabolism ; Rats ; Protein Biosynthesis ; Cells, Cultured ; Microtubules/metabolism ; Female ; Axons/metabolism ; }, abstract = {Spatial RNA organization has a pivotal role in diverse cellular processes and diseases[1-4]. However, functional implications of the spatial transcriptome remain largely unexplored due to limited technologies for perturbing endogenous RNA within specific subcellular regions[1,5]. Here we present CRISPR-mediated transcriptome organization (CRISPR-TO), a system that harnesses RNA-guided, nuclease-dead dCas13 for programmable control of endogenous RNA localization in live cells. CRISPR-TO enables targeted localization of endogenous RNAs to diverse subcellular compartments, including the outer mitochondrial membrane, p-bodies, stress granules, telomeres and nuclear stress bodies, across various cell types. It allows for inducible and reversible bidirectional RNA transport along microtubules via motor proteins, facilitating real-time manipulation and monitoring of RNA localization dynamics in living cells. In primary cortical neurons, we demonstrate that repositioned mRNAs undergo local translation along neurites and at neurite tips, and co-transport with ribosomes, with β-actin mRNA localization enhancing the formation of dynamic filopodial protrusions and inhibiting axonal regeneration. CRISPR-TO-enabled screening in primary neurons identifies Stmn2 mRNA localization as a driver of neurite outgrowth. By enabling large-scale perturbation of the spatial transcriptome, CRISPR-TO bridges a critical gap left by sequencing and imaging technologies, offering a versatile platform for high-throughput functional interrogation of RNA localization in living cells and organisms.}, }
@article {pmid40344384, year = {2025}, author = {Yao, Z and Li, W and He, K and Wang, H and Xu, Y and Wu, Q and Wang, L and Nie, Y}, title = {Facilitating crRNA Design by Integrating DNA Interaction Features of CRISPR-Cas12a System.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {12}, number = {25}, pages = {e2501269}, doi = {10.1002/advs.202501269}, pmid = {40344384}, issn = {2198-3844}, support = {32172175//National Natural Science Foundation of China/ ; 32172307//National Natural Science Foundation of China/ ; 2023C02006//Key R&amp;D Program of Zhejiang/ ; KYCX24_2592//Postgraduate Research &amp; Practice Innovation Program of Jiangsu Province/ ; NO.111-2-06//Priority Academic Program Development of Jiangsu Higher Education Institutions, the 111 Project/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *DNA/genetics/metabolism ; Molecular Dynamics Simulation ; *CRISPR-Associated Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {The CRISPR-Cas12a system has gained significant attention as a rapid nucleic acid diagnostic tool due to its crRNA-guided trans-cleavage activity. Accurately predicting the activity of different targets is significant to facilitate the crRNA availability but remains challenging. In this study, a novel approach is presented that combines molecular dynamics simulations and neural network modeling to predict the trans-cleavage activity. Unlike conventional tools that rely solely on the base sequences, our method integrated sequence features and molecular interaction features of DNA in the CRISPR-Cas12a system, significantly improving prediction accuracy. Through feature importance analysis, key sequence features that influence Cas12a trans-cleavage activity are identified. Additionally, a crRNA-DNA library with over 23 456 feature sequences from representative viruses and bacteria is established, and validated the high predictive accuracy of the model (Pearson's r = 0.9328) by screening crRNAs from reference targets. This study offers new insights into the molecular interactions of Cas12a/crRNA-DNA and provides a reliable framework for optimizing crRNA design, facilitating the application of the CRISPR-Cas12a in rapid nucleic acid diagnostics.}, }
@article {pmid40302201, year = {2025}, author = {Hao, JH and Kang, X and Zhang, L and Chen, J and Wang, D and Dong, S and Li, X and Gao, L and Yang, G and Yuan, X and Chu, X and Wang, JG}, title = {CRISPR/Cas9-Mediated SiEPF2 Mutagenesis Attenuates Drought Tolerance and Yield in Foxtail Millet (Setaria italica).}, journal = {Plant, cell &amp; environment}, volume = {48}, number = {8}, pages = {6043-6046}, doi = {10.1111/pce.15597}, pmid = {40302201}, issn = {1365-3040}, support = {//This study was supported by the National Key Research and Development Programme of China (2023YFD1202702), National Natural Science Foundation (32400217 and 32200222) and Start-up Fund of Shanxi Agricultural University (2021BQ84)./ ; }, mesh = {*Setaria Plant/genetics/physiology/growth &amp; development ; Droughts ; *Plant Proteins/genetics/metabolism/physiology ; *CRISPR-Cas Systems/genetics ; Mutagenesis ; Plant Stomata/physiology ; Gene Expression Regulation, Plant ; Drought Resistance ; }, abstract = {Plants employ peptide ligands to coordinate development and integrate environmental signals via dedicated cascades (#ref-0013). Epidermal patterning factor (EPF), plays a significant role in regulating stomatal density, seed germination and panicle development (#ref-0008). EPF/EPFL enhance drought tolerance by reducing stomatal density have been reported in multiple species, including Hordeum vulgare, Arabidopsis thaliana, Populus spp., Vitis vinifera, Sorghum bicolor and Brassica napus (#ref-0001). Our previous study has shown that EPF can mediate drought resistance in foxtail millet by regulating stomatal density (#ref-0004). Furthermore, evidence suggests that plants can regulate photosynthesis through stomatal modification, ultimately enhancing yield (#ref-0005). Beyond stomatal density regulation, the EPF/EPFL gene family modulates seed germination through phytohormone signalling and regulates inflorescence development via ligand-receptor interactions (#ref-0007). OsEPFLs act as upstream ligands for the OsER1 receptor, activating the MAPK signalling cascade to regulate panicle morphogenesis (#ref-0002). In this study, we sought to elucidate how SiEPF2 balances drought resistance and yield in foxtail millet by modulating stomatal density and panicle morphology. Our findings not only provide novel insights into SiEPF2's role in abiotic stress responses but also contribute valuable genetic resources for high-yield breeding programmes in millet crops.}, }
@article {pmid40280813, year = {2025}, author = {Hu, N and Tian, H and Li, Y and Li, X and Li, D and Li, L and Wang, S and Zhang, Y and Shi, X and Huang, B and Lu, Q and Wang, T and Pan, X and Tu, L and Dai, D and Zhang, B and Peng, R and Yan, F}, title = {pHNRhCas9NG, single expression cassette-based dual-component dual-transcription unit CRISPR/Cas9 system for plant genome editing.}, journal = {Trends in biotechnology}, volume = {43}, number = {7}, pages = {1788-1808}, doi = {10.1016/j.tibtech.2025.03.016}, pmid = {40280813}, issn = {1879-3096}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genome, Plant/genetics ; Nicotiana/genetics ; Plants, Genetically Modified/genetics ; Solanum lycopersicum/genetics ; Arabidopsis/genetics ; DNA, Bacterial/genetics ; Promoter Regions, Genetic ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome-editing (GEd) technology has revolutionized plant science, facilitating gene function studies and crop improvement. Despite its success, plant-specific CRISPR/Cas9 systems require further optimization. This study aims to boost plant GEd efficiency by revamping the CRISPR/Cas9 system. We addressed large fragment deletions in T-DNA (transfer DNA) postgenomic insertion by developing a binary expression vector, pHNR, which maintains T-DNA integrity using protective sequences. We discovered an artificial promoter, P35SIC47, effective in tobacco, Arabidopsis, and tomato transformation, and designed a dual-component dual-transcription unit CRISPR/Cas9 system (DDS) with optimal gene expression at a poly(A) length of ~150 base pairs. Enhancing the poly(A) tail length of Cas9 mRNA significantly boosted plant GEd efficiency. We also identified compatible hCas9 versions through transitory expression in tobacco leaves. Utilizing pHNRhCas9NG, we efficiently knocked out ten genes in tomato, achieving almost 100% gene-editing efficiency. Our system offers a novel, scalable tool for plant GEd, advancing CRISPR/Cas9 capabilities.}, }
@article {pmid40263591, year = {2025}, author = {Rohde, T and Demirtas, TY and Süsser, S and Shaw, AH and Kaulich, M and Billmann, M}, title = {BaCoN (Balanced Correlation Network) improves prediction of gene buffering.}, journal = {Molecular systems biology}, volume = {21}, number = {7}, pages = {807-824}, pmid = {40263591}, issn = {1744-4292}, support = {397484323 - TRR259//Deutsche Forschungsgemeinschaft (DFG)/ ; }, mesh = {Humans ; CRISPR-Cas Systems ; *Gene Regulatory Networks ; Cell Line, Tumor ; Algorithms ; Transcriptome ; *Computational Biology/methods ; Neoplasms/genetics ; }, abstract = {Buffering between genes, where one gene can compensate for the loss of another gene, is fundamental for robust cellular functions. While experimentally testing all possible gene pairs is infeasible, gene buffering can be predicted genome-wide under the assumption that a gene's buffering capacity depends on its expression level and its absence primes a severe fitness phenotype of the buffered gene. We developed BaCoN (Balanced Correlation Network), a post hoc unsupervised correction method that amplifies specific signals in expression-vs-fitness correlation networks. We quantified 147 million potential buffering relationships by associating CRISPR-Cas9-screening fitness effects with transcriptomic data across 1019 Cancer Dependency Map (DepMap) cell lines. BaCoN outperformed state-of-the-art methods, including multiple linear regression based on our compiled gene buffering prediction metrics. Combining BaCoN with batch correction or Cholesky data whitening further boosts predictive performance. We characterized 808 high-confidence buffering predictions and found that in contrast to buffering gene pairs overall, buffering paralogs were on different chromosomes. BaCoN performance increases with more screens and genes considered, making it a valuable tool for gene buffering predictions from the growing DepMap.}, }
@article {pmid40205048, year = {2025}, author = {Fell, CW and Villiger, L and Lim, J and Hiraizumi, M and Tagliaferri, D and Yarnall, MTN and Lee, A and Jiang, K and Kayabolen, A and Krajeski, RN and Schmitt-Ulms, C and Ramani, H and Yousef, SM and Roberts, N and Vakulskas, CA and Nishimasu, H and Abudayyeh, OO and Gootenberg, JS}, title = {Reprogramming site-specific retrotransposon activity to new DNA sites.}, journal = {Nature}, volume = {642}, number = {8069}, pages = {1080-1089}, pmid = {40205048}, issn = {1476-4687}, mesh = {*Retroelements/genetics ; Animals ; Humans ; CRISPR-Cas Systems/genetics ; Finches/genetics ; *Gene Editing/methods ; *DNA/genetics ; Genome/genetics ; *Mutagenesis, Insertional/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; Mice ; Reverse Transcription ; }, abstract = {Retroelements have a critical role in shaping eukaryotic genomes. For instance, site-specific non-long terminal repeat retrotransposons have spread widely through preferential integration into repetitive genomic sequences, such as microsatellite regions and ribosomal DNA genes[1-6]. Despite the widespread occurrence of these systems, their targeting constraints remain unclear. Here we use a computational pipeline to discover multiple new site-specific retrotransposon families, profile members both biochemically and in mammalian cells, find previously undescribed insertion preferences and chart potential evolutionary paths for retrotransposon retargeting. We identify R2Tg, an R2 retrotransposon from the zebra finch, Taeniopygia guttata, as an orthologue that can be retargeted by payload engineering for target cleavage, reverse transcription and scarless insertion of heterologous payloads at new genomic sites. We enhance this activity by fusing R2Tg to CRISPR-Cas9 nickases for efficient insertion at new genomic sites. Through further screening of R2 orthologues, we select an orthologue, R2Tocc, with natural reprogrammability and minimal insertion at its natural 28S site, to engineer SpCas9[H840A]-R2Tocc, a system we name site-specific target-primed insertion through targeted CRISPR homing of retroelements (STITCHR). STITCHR enables the scarless, efficient installation of edits, ranging from a single base to 12.7 kilobases, gene replacement and use of in vitro transcribed or synthetic RNA templates. Inspired by the prevalence of nLTR retrotransposons across eukaryotic genomes, we anticipate that STITCHR will serve as a platform for scarless programmable integration in dividing and non-dividing cells, with both research and therapeutic applications.}, }
@article {pmid40199626, year = {2025}, author = {Lu, Y and Wang, J and Xu, Y and Xu, M and Li, B and Fan, Z and Liu, J and Li, X and Cai, Z and Zheng, Y and Wang, W and Yang, J and Zhang, Z and Liu, Z}, title = {Long-offset paired nicking-based efficient and precise strategy for in vivo targeted insertion.}, journal = {Trends in biotechnology}, volume = {43}, number = {7}, pages = {1743-1764}, doi = {10.1016/j.tibtech.2025.02.020}, pmid = {40199626}, issn = {1879-3096}, mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; Genetic Therapy/methods ; Gene Knock-In Techniques/methods ; *Gene Targeting/methods ; DNA Repair ; Hepatocytes/metabolism ; Deoxyribonuclease I/metabolism/genetics ; }, abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-based targeted insertion of DNA fragments holds great promise for gene therapy. However, designing highly efficient and precise integration of large DNA segments in somatic cells while avoiding unpredictable products remains challenging. Here, we devised a novel long-offset paired nicking target integration (LOTI) strategy, which enhances the capacity of Cas9 nickase (Cas9n) in targeted gene integration in somatic cells, yielding higher knock-in (KI) efficiency compared with classical nickase-based approaches. The underlying repair mechanism involves the DNA repair proteins Rad51 and Rad52, and Ligase I/III. Moreover, we achieved efficient KI of at least 1.5-kb gene fragments in hepatocytes and recovery 55% FIX activity in a hemophilia B mouse model using only one-dose plasmid DNA delivery. Compared with the Cas9-based strategy, LOTI reduces off-target activity and restricts the formulation of unwanted insertions and deletions (indels) at the target site. Thus, LOTI provides a precise and efficient strategy for gene integration in somatic cells in vivo.}, }
@article {pmid40192038, year = {2025}, author = {Shi, K and Huang, W and Zhu, M and Teng, S and Zhang, J and Duan, Z and Zhu, C and Hu, T and Wang, K and Wang, Z}, title = {Efficient genetic transformation and genome editing via an Agrobacterium-mediated in commercial oat (Avena sativa L.) cultivars.}, journal = {Journal of integrative plant biology}, volume = {67}, number = {7}, pages = {1697-1699}, doi = {10.1111/jipb.13915}, pmid = {40192038}, issn = {1744-7909}, support = {2005DKA21003//the National Center for Forestry and Grassland Genetic Resources/ ; }, mesh = {*Avena/genetics/microbiology ; *Gene Editing/methods ; *Transformation, Genetic ; *Agrobacterium/genetics ; Plants, Genetically Modified ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; }, abstract = {An optimized Agrobacterium-mediated transformation protocol for immature and mature oat embryos increased transformation efficiencies and the number of transformable cultivars and enabled highly efficient CRISPR/Cas9 and CRISPR/Cas12i genome editing to accelerate oat biotechnology breeding.}, }
@article {pmid40187931, year = {2025}, author = {Jiang, Y and Xiao, Z and Luo, Z and Zhou, S and Tong, C and Jin, S and Liu, X and Qin, R and Xu, R and Pan, L and Li, J and Wei, P}, title = {Improving plant C-to-G base editors with a cold-adapted glycosylase and TadA-8e variants.}, journal = {Trends in biotechnology}, volume = {43}, number = {7}, pages = {1765-1787}, doi = {10.1016/j.tibtech.2025.03.001}, pmid = {40187931}, issn = {1879-3096}, mesh = {*Gene Editing/methods ; Oryza/genetics ; Plants, Genetically Modified/genetics ; *Cytidine Deaminase/genetics/metabolism ; *Uracil-DNA Glycosidase/genetics/metabolism ; Cytosine/metabolism ; Guanine/metabolism ; Humans ; CRISPR-Cas Systems ; Animals ; Cold Temperature ; }, abstract = {Plant cytosine (C)-to-guanine (G) base editors (CGBEs) have been established but suffer from limited editing efficiencies and low outcome purities. This study engineered a cod uracil DNA glycosylase (cod UNG, coUNG) from the cold-adapted fish Gadus morhua for plant CGBE, demonstrating 1.71- to 2.54-fold increases in C-to-G editing efficiency compared with the CGBE using human UNG (hUNG). Further engineering took advantage of TadA-8e-derived cytidine deaminases (TadA-CDs). These variants induced C substitutions with efficiencies ranging from 26.28% to 30.82% in rice cells, whereas adenine (A) conversion was negligible. By integrating coUNG and TadA-CDc elements with SpCas9 nickase, the resulting CDc-CGBEco achieved pure C-to-G editing without byproducts in up to 52.08% of transgenic lines. Whole-genome sequencing (WGS) analysis revealed no significant off-target effects of the CDc-BEs in rice. In addition, CDc-CGBEco enabled precise C-to-G editing in soybean and tobacco. These engineered CGBEs enhanced editing efficiency, purity, and specificity, suggesting their broad potential for applications in scientific research and crop breeding.}, }
@article {pmid40119518, year = {2025}, author = {He, X and Yan, T and Song, Z and Xiang, L and Xiang, J and Yang, Y and Ren, K and Bu, J and Xu, X and Li, Z and Guo, X and Lin, B and Zhou, Q and Lin, G and Gu, F}, title = {Correcting a patient-specific Rhodopsin mutation with adenine base editor in a mouse model.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {33}, number = {7}, pages = {3101-3113}, doi = {10.1016/j.ymthe.2025.03.021}, pmid = {40119518}, issn = {1525-0024}, mesh = {Animals ; Disease Models, Animal ; Mice ; *Gene Editing/methods ; *Adenine/metabolism ; Humans ; *Retinitis Pigmentosa/genetics/therapy ; *Mutation ; *Rhodopsin/genetics ; Gene Knock-In Techniques ; Dependovirus/genetics ; Genetic Vectors/genetics ; Electroretinography ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Retina/metabolism/pathology ; Genetic Therapy/methods ; }, abstract = {Genome editing offers a great promise to treating human genetic diseases. To assess genome-editing-mediated therapeutic effects in vivo, an animal model is indispensable. The genomic disparities between mice and humans often impede the direct clinical application of genome-editing-mediated treatments using conventional mouse models. Thus, the generation of a mouse model with a humanized genomic segment containing a patient-specific mutation is highly sought after for translational research. In this study, we successfully developed a knockin mouse model for autosomal-dominant retinitis pigmentosa (adRP), designated as hT17M knockin, which incorporates a 75-nucleotide DNA segment with the T17M mutation (Rhodopsin-c.C50T; p.T17M). This model demonstrated significant reductions in electroretinogram amplitudes and exhibited disruptions in retinal structure. Subsequently, we administered an adeno-associated virus vectors carrying an adenine base editor (ABE) and a single-guide RNA specifically targeting the T17M mutation, achieving a peak correction rate of 39.7% at the RNA level and significantly improving retinal function in ABE-injected mice. These findings underscore that the hT17M knockin mouse model recapitulates the clinical features of adRP patients and exhibits therapeutic effects with ABE-mediated treatments. It offers a promising avenue for the development of gene-editing therapies for RP.}, }
@article {pmid40603034, year = {2025}, author = {Sugo, T and Shirasago, Y and Yoshimoto, S and Kitajima, M}, title = {[GenAhead Bio: your partner for extensive support of genome editing and co-‍development of nucleic acid delivery].}, journal = {Nihon yakurigaku zasshi. Folia pharmacologica Japonica}, volume = {160}, number = {4}, pages = {274-278}, doi = {10.1254/fpj.25004}, pmid = {40603034}, issn = {0015-5691}, mesh = {*Gene Editing ; Humans ; *Nucleic Acids/administration &amp; dosage ; CRISPR-Cas Systems ; Animals ; RNA, Small Interfering ; }, abstract = {Inspired by my experiences working in research at an overseas biotech venture, I founded GenAhead Bio Inc. in 2018. GenAhead Bio adopts a unique dual-business structure, providing contract services for generating genetically modified cells using highly efficient CRISPR/Cas9 genome editing technology for researchers, while simultaneously pursuing a nucleic acid drug business aiming to develop nucleic acid drugs such as antisense oligonucleotides and siRNAs. Based on the emerging delivery system called Antibody-Nucleic acid Conjugate, where an antibody is covalently linked to a nucleic acid as a targeting ligand, we are conducting drug developmental research by delivering nucleic acids to the organs where antibodies accumulate. Our ultimate goal is to apply this technology to genome editing for gene modification in specific cell types. In this review, we will introduce some case studies of genome editing, including single nucleotide substitutions, as well as the delivery of siRNA to the skeletal muscle using anti-transferrin receptor (CD71) antibody and its therapeutic effects on muscular diseases.}, }
@article {pmid40601773, year = {2025}, author = {Chafe, SC and Zhai, K and Aghaei, N and Miletic, P and Huang, Z and Brown, KR and Mobilio, D and Young, D and Suk, Y and Grewal, S and McKenna, D and Alizada, Z and Kieliszek, AM and Lam, FC and Escudero, L and Huang, Q and Huebner, A and Lu, J and Ang, P and Anand, A and Custers, S and Apel, E and Slassi, S and Brakel, B and Kim, J and Liu, JKC and Bassey-Archibong, BI and Abdo, R and Shargall, Y and Lu, JQ and Cutz, JC and Zhang, Q and Li, SS and Venugopal, C and Hynds, RE and Dufour, A and Moffat, J and Swanton, C and Bao, S and Singh, SK}, title = {A genome-wide in vivo CRISPR activation screen identifies BACE1 as a therapeutic vulnerability of lung cancer brain metastasis.}, journal = {Science translational medicine}, volume = {17}, number = {805}, pages = {eadu2459}, doi = {10.1126/scitranslmed.adu2459}, pmid = {40601773}, issn = {1946-6242}, mesh = {Humans ; *Brain Neoplasms/secondary/genetics ; *Lung Neoplasms/pathology/genetics ; *Amyloid Precursor Protein Secretases/metabolism/genetics ; *Aspartic Acid Endopeptidases/metabolism/genetics ; Animals ; *Carcinoma, Non-Small-Cell Lung/genetics/pathology ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mice ; ErbB Receptors/metabolism ; *CRISPR-Cas Systems/genetics ; Xenograft Model Antitumor Assays ; Gene Expression Regulation, Neoplastic ; }, abstract = {Brain metastasis occurs in up to 40% of patients with non-small cell lung cancer (NSCLC). Considerable genomic heterogeneity exists between the primary lung tumor and respective brain metastasis; however, the identity of the genes capable of driving brain metastasis is incompletely understood. Here, we carried out an in vivo genome-wide CRISPR activation screen to identify molecular drivers of brain metastasis from an orthotopic xenograft model derived from a patient with NSCLC. We found that activating expression of the Alzheimer's disease-associated beta-secretase 1 (BACE1) led to a substantial increase in brain metastases. Furthermore, genetic and pharmacological inhibition of BACE1 blocked NSCLC brain metastasis. Mechanistically, we identified that BACE1 acts through epidermal growth factor receptor to drive this metastatic phenotype. Together, our data highlight the power of in vivo CRISPR activation screening to unveil molecular drivers and potential therapeutic targets of NSCLC brain metastasis.}, }
@article {pmid40599263, year = {2025}, author = {Yang, R and Tang, T and Wulae,  and Liu, P and Wang, X and Jiang, Y and Zhang, S and Wang, M and Yang, L and Wang, C}, title = {[One-Step Detection of Human Influenza B Virus Through Recombinase Polymerase Amplification and CRISPR/Cas12a Protein].}, journal = {Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition}, volume = {56}, number = {2}, pages = {549-555}, pmid = {40599263}, issn = {1672-173X}, mesh = {*Influenza B virus/isolation &amp; purification/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Recombinases/genetics/metabolism ; Sensitivity and Specificity ; *Influenza, Human/diagnosis/virology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {OBJECTIVE: To establish a one-step detection method based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a protein for the rapid and sensitive detection of human influenza B virus.<br><br>METHODS: RPA amplification primers were designed according to the conserved gene (NS1 gene) of human influenza B virus (Victoria lineage). The reaction system was established using the standard plasmid as the template. First of all, the reaction system was incubated at 37 &#x2103; for 15 minutes for RPA amplification. Then, the CRISPR/Cas12a system on the tube cap was thoroughly mixed with the RPA amplification product at the bottom of the tube through fast centrifugation, and real-time fluorescence detection was carried out at 37 &#x2103;. The reaction conditions were optimized to establish a one-step RPA-CRISPR/Cas12a detection method for human influenza B virus. The sensitivity of the testing method was evaluated using standard plasmids and pseudoviruses, and the specificity was evaluated using other viruses that may cause febrile respiratory syndrome. The consistency between the results of the one-step detection method and those of RT-qPCR detection was evaluated by testing real samples.<br><br>RESULTS: A one-step detection method based on RPA-CRISPR/Cas12a was successfully established. The optimal reaction conditions included a reaction temperature of 37 &#x2103;, a Cas12a/crRNA concentation ratio of 1&#x2236;1, a Cas12a concentration of 120 nmol/L, a single-stranded DNA (ssDNA) probe concentration of 300 nmol/L, and a primer concentration of 480 nmol/L. The method could detect standard plasmid DNA as low as 2.8 copies/&#x3bc;L within 25 minutes and pseudoviruses as low as 2.77 copies/&#x3bc;L within 30 minutes. The testing method showed high specificity, and no cross-reaction was observed with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), influenza A (H1N1) virus, or respiratory syncytial virus subgroup A. When testing clinical samples, the sensitivity and the specificity for examining clinical samples were 93.33% and 100%, respectively, and consistency with RT-qPCR results was 97.14%.<br><br>CONCLUSION: With the one-step detection method based on RPA-CRISPR/Cas12a established in this study, the whole sample detection process, including nucleic acid release, reverse transcription, isothermal amplification, CRISPR/Cas12a system cleavage, and fluorescence signal output, can be completed within 30 minutes. Its high sensitivity, specificity, and successful application in clinical samples highlight its potential for rapid point-of-care testing in clinical settings.}, }
@article {pmid40598996, year = {2025}, author = {Han, HG and Nandre, R and Eom, H and Choi, YJ and Ro, HS}, title = {Development of a CRISPR/Cas9 RNP-mediated genetic engineering system in Paecilomyces variotii.}, journal = {Journal of microbiology (Seoul, Korea)}, volume = {63}, number = {6}, pages = {e2502011}, doi = {10.71150/jm.2502011}, pmid = {40598996}, issn = {1976-3794}, support = {2023R1A2C1007213//National Research Foundation of Korea/ ; //Ministry of Science and ICT/ ; RS2024-00322425//Rural Development Administration/ ; }, mesh = {*CRISPR-Cas Systems ; *Paecilomyces/genetics/metabolism ; Promoter Regions, Genetic ; *Gene Editing/methods ; *Ribonucleoproteins/genetics/metabolism ; *Genetic Engineering/methods ; Green Fluorescent Proteins/genetics/metabolism ; Fungal Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {A thermophilic strain of Paecilomyces variotii (MR1), capable of surviving temperatures above 40°C, was isolated from a paper mill and investigated as a host for heterologous protein production. To prevent environmental dissemination of spores, UV mutagenesis was employed to create a conidia-deficient strain, UM7. This strain underwent gene editing using Cas9-gRNA ribonucleoprotein (RNP) with HR donor DNA fragments, incorporating promoter sequences amplified from the genomic DNA of P. variotii (PH4, PP2, PS8, Ptub, Ptef1, and PgpdA), along with a signal sequence-tagged eGFP, flanked by 5'-upstream (336 bp) and 3'-downstream (363 bp) regions of pyrG. Co-transformation of HR donor DNA with RNP into protoplasts yielded 48 mutant pyrG transformants capable of surviving in the presence of 5-fluoroorotic acid (5-FOA). Sequence analysis identified 16 of the 48 pyrG-disrupted mutants carrying complete HR donor DNAs with the six different promoter sequences, indicating successful homology-directed repair (HDR). Evaluation of promoter strength revealed that PgpdA was the most effective for intracellular GFP production; however, it resulted in negligible extracellular GFP signal under all promoter conditions. A newly edited strain with an HDR integration module connecting PgpdA directly to eGFP, without the signal sequence, exhibited enhanced GFP expression in both mycelial cells and culture broth, suggesting that the signal peptide negatively affect protein expression and secretion. This work represents the first successful RNP-mediated gene editing in P. variotii, contributing to the application of this thermophilic fungus in protein production.}, }
@article {pmid40598890, year = {2025}, author = {Wei, W and Gao, CH and Jiang, X and Qiao, J and Zhang, L and Yan, Y and Zhao, G and Yang, K and Yan, J and Yang, M}, title = {CARF-dependent preferential RNA cleavage by Csm6 increases drug susceptibility of mycobacteria.}, journal = {Nucleic acids research}, volume = {53}, number = {12}, pages = {}, doi = {10.1093/nar/gkaf622}, pmid = {40598890}, issn = {1362-4962}, support = {32070079//National Natural Science Foundation of China/ ; 32370127//National Natural Science Foundation of China/ ; 2662025ZHPY001//Central Universities/ ; 2020kfyXJJS119//Central Universities/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Mycobacterium smegmatis/genetics/drug effects/metabolism ; *RNA Cleavage ; *Mycobacterium tuberculosis/genetics/drug effects ; *Bacterial Proteins/genetics/metabolism/chemistry ; Drug Resistance, Bacterial/genetics ; Mycolic Acids/metabolism ; Gene Expression Regulation, Bacterial ; }, abstract = {CRISPR-Cas systems are prokaryotic adaptive immune systems that defend against invading mobile genetic elements. The type III-A CRISPR-Cas system has been studied in the evolutionary and epidemiological context of Mycobacterium tuberculosis, the causative agent of tuberculosis. However, its biological function remains poorly understood. Here, we demonstrate that heterologous expression of csm6, a single-stranded RNA ribonuclease of the CRISPR-Cas system, exhibits preferential RNA cleavage activity targeting host transcripts. This activity significantly downregulates ribosomal and mycolic acid biosynthesis pathway genes, leading to a global reduction in translation levels and an increased drug susceptibility of Mycobacterium smegmatis. Furthermore, mutagenesis analysis revealed that Csm6's biological function critically depends on its CARF domain rather than its HEPN domain. In conclusion, our study elucidates the biological role of the Csm6 protein in the CRISPR-Cas system, both in vitro and in vivo, highlighting how preferential RNA cleavage impacts multiple mycobacterial processes. These findings provide novel insights into the functional diversity of CRISPR-Cas systems in mycobacteria.}, }
@article {pmid40598087, year = {2025}, author = {Garimella, SS and Minami, SA and Khanchandani, AN and Abad Santos, JC and Schaffer, SR and Shah, PS}, title = {A simplified two-plasmid system for orthogonal control of mammalian gene expression using light-activated CRISPR effector.}, journal = {BMC biotechnology}, volume = {25}, number = {1}, pages = {58}, pmid = {40598087}, issn = {1472-6750}, support = {n/a//AMPAC Fine Chemicals Summer Research Internship/ ; n/a//Hellman Family Foundation/ ; n/a//Good Food Institute/ ; }, mesh = {*Plasmids/genetics ; HEK293 Cells ; Humans ; *Optogenetics/methods ; Green Fluorescent Proteins/genetics/metabolism ; Light ; *CRISPR-Cas Systems/genetics ; Animals ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Expression Regulation ; Gene Expression ; }, abstract = {BACKGROUND: Optogenetic systems use light-responsive proteins to control gene expression, ion channels, protein localization, and signaling with the "flip of a switch". One such tool is the light activated CRISPR effector (LACE) system. Its ability to regulate gene expression in a tunable, reversible, and spatially resolved manner makes it attractive for many applications. However, LACE relies on delivery of four separate components on individual plasmids, which can limit its use. Here, we optimize LACE to reduce the number of plasmids needed to deliver all four components.<br><br>RESULTS: The two-plasmid LACE (2pLACE) system combines the four components of the original LACE system into two plasmids. Following construction, the behavior of 2pLACE was rigorously tested using optogenetic control of enhanced green fluorescent protein (eGFP) expression as a reporter. Using human HEK293T cells, we optimized the ratio of the two plasmids, measured activation as a function of light intensity, and determined the frequency of the light to activate the maximum fluorescence. Overall, the 2pLACE system showed a similar dynamic range, tunability, and activation kinetics as the original four plasmid LACE (4pLACE) system. Interestingly, 2pLACE also had less variability in activation signal compared to 4pLACE. We also demonstrate the optimal LACE system also depends on cell type. In mouse myoblast C2C12 cells, 2pLACE displayed less variability compared to 4pLACE, similar to HEK293T cells. However, 2pLACE also had a smaller dynamic range in C2C12 cells compared to 4pLACE.<br><br>CONCLUSIONS: This simplified system for optogenetics will be more amenable to biotechnology applications where variability needs to be minimized. By optimizing the LACE system to use fewer plasmids, 2pLACE becomes a flexible tool in multiple research applications. However, the optimal system may depend on cell type and application.}, }
@article {pmid40598062, year = {2025}, author = {Ajdanian, L and Torkamaneh, D}, title = {Mother transformer: A High-Throughput, Cost-Effective in Planta Hairy Root Transformation Method for Cannabis.}, journal = {BMC biotechnology}, volume = {25}, number = {1}, pages = {60}, pmid = {40598062}, issn = {1472-6750}, mesh = {*Plant Roots/genetics/microbiology ; *Plants, Genetically Modified/genetics ; *Agrobacterium/genetics ; *Transformation, Genetic ; *Cannabis/genetics ; Cost-Benefit Analysis ; *High-Throughput Screening Assays/methods ; }, abstract = {BACKGROUND: Hairy root (HR) transformation assays mediated by Agrobacterium rhizogenes, both in vitro and ex vitro, are essential tools in plant biotechnology and functional genomics. These assays can be significantly influenced by various factors, which ultimately can enhance the efficiency. In this study, we optimized a two-step ex vitro HR transformation method using the actual mother plant combined with the RUBY system and compared with existing methods.<br><br>RESULTS: The two-step ex vitro method proved more efficient than both the one-step ex vitro and in vitro methods, with the highest transformation efficiency of 90% observed in the actual plant. This technique also demonstrated a faster and less complicated approach, reducing time to achieve massive transgenic HR formation by 9-29 days compared to other methods.<br><br>CONCLUSIONS: A novel, quicker, less complicated, and more efficient two-step transformation method for cannabis has been established, presenting a significantly lower risk of contamination. This protocol is particularly interesting to produce secondary metabolites using the CRISPR/Cas system in cannabis. We anticipate that this method will facilitate substantial time savings by rapidly producing hundreds of transformed samples.}, }
@article {pmid40597657, year = {2025}, author = {Malekos, E and Montano, C and Carpenter, S}, title = {CRISPRware: a software package for contextual gRNA library design.}, journal = {BMC genomics}, volume = {26}, number = {1}, pages = {607}, pmid = {40597657}, issn = {1471-2164}, support = {F31AI179201//National Institute of Allergy and Infectious Diseases/ ; R35GM137801/GM/NIGMS NIH HHS/United States ; }, mesh = {*Software ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Library ; *CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing ; Humans ; Animals ; }, abstract = {We present CRISPRware, an efficient method for generating guide RNA (gRNA) libraries against transcribed, translated, and noncoding regions. CRISPRware leverages next-generation sequencing data to design context-specific gRNAs and can account for genetic variation, which allows allele-specific guide design on a genome-wide scale. As a demonstration of use and to create a publicly available resource, we use CRISPRware to identify and score gRNAs against coding sequences in six model organisms for Cas9 and Cas12A and host these in a publicly available session on the UCSC Genome Browser.}, }
@article {pmid40596971, year = {2025}, author = {Martí-Díaz, R and Sánchez-Del-Campo, L and Montenegro, MF and Hernández-Caselles, T and Piñero-Madrona, A and Cabezas-Herrera, J and Rodríguez-López, JN}, title = {Ex vivo engineering of phagocytic signals in breast cancer cells for a whole tumor cell-based vaccine.}, journal = {BMC cancer}, volume = {25}, number = {1}, pages = {1029}, pmid = {40596971}, issn = {1471-2407}, support = {21407/FPI/20//Fundaci&#xf3;n S&#xe9;neca/ ; FSRM/10.13039/100007801(22544/PI/24)//Fundaci&#xf3;n S&#xe9;neca/ ; FSRM/10.13039/100007801(22544/PI/24)//Fundaci&#xf3;n S&#xe9;neca/ ; FSRM/10.13039/100007801(22544/PI/24)//Fundaci&#xf3;n S&#xe9;neca/ ; FSRM/10.13039/100007801(22544/PI/24)//Fundaci&#xf3;n S&#xe9;neca/ ; FSRM/10.13039/100007801(22544/PI/24)//Fundaci&#xf3;n S&#xe9;neca/ ; PID2023-149281OB-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; CPP2023-010510//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; PID2023-149281OB-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; CPP2023-010510//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; PID2023-149281OB-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; PID2023-149281OB-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; }, mesh = {Animals ; Female ; Mice ; *Phagocytosis/immunology ; *Cancer Vaccines/immunology ; *Breast Neoplasms/immunology/therapy/pathology ; Humans ; CD47 Antigen/genetics ; Cell Line, Tumor ; CRISPR-Cas Systems ; Macrophages/immunology ; Mice, Inbred BALB C ; }, abstract = {BACKGROUND: Today, cell therapies are constantly evolving and providing new options for cancer patients. These therapies are mostly based on the inoculation of immune cells extracted from a person's own tumor; however, some studies using whole tumor cell-based vaccines are approaching the level of maturity required for clinical use. Although these latest therapies will have to be developed further and adapted to overcome many ethical barriers, there is no doubt that therapeutic cancer vaccines are the next frontier of immunotherapy.<br><br>METHODS: Ionizing radiation and CD47 knockout via CRISPR-Cas9 genome editing were used to optimize the macrophage-mediated phagocytosis of breast cancer cells. These cells were subsequently used in several mouse models to determine their potential as novel whole-cell-based vaccines to drive antitumor immunity. To improve the recognition of tumor cells by activated immune cells, this cellular therapy was combined with anti-PD-1 antibody treatments.<br><br>RESULTS: Here, we showed that irradiation of 4T1 breast cancer cells increases their immunogenicity and, when injected into the blood of immunocompetent mice, elicits a complete antitumor immune response mediated, in part, by the adaptive immune system. Next, to improve the macrophage-mediated phagocytosis of breast cancer cells, we knocked out CD47 in 4T1 cells. When injected in the bloodstream, irradiated CD47 knockout cells activated both the adaptive and the innate immune systems. Therefore, we used these ex vivo engineered cells as a whole tumor cell-based vaccine to treat breast tumors in immunocompetent mice. A better response was obtained when these cells were combined with an anti-PD-1 antibody.<br><br>CONCLUSION: These results suggest that tumor cells obtained from surgical samples of a breast cancer patient could be engineered ex vivo and used as a novel cell therapy to drive antitumor immunity.}, }
@article {pmid40596718, year = {2025}, author = {Schuster, B and Dobiášovská, I and Ćurčić, J and Pajer, P and Borna, &#x160; and Bartůněk, P}, title = {SWITCHER, a CRISPR-inducible floxed wild-type Cre regulating CRISPR activity.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {982}, pmid = {40596718}, issn = {2399-3642}, mesh = {*CRISPR-Cas Systems/genetics ; *Integrases/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/methods ; Humans ; CRISPR-Associated Proteins/genetics/metabolism ; Endodeoxyribonucleases/genetics/metabolism ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems/genetics ; Bacterial Proteins ; }, abstract = {Although several Cre-regulated CRISPR/Cas platforms exist, a CRISPR/Cas-controlled Cre-system remains a challenge. Here, we present a genetic switch we term SWITCHER based on a floxed wild-type Cre-construct representing a CRISPR-inducible and self-limiting kill switch. By leveraging CRISPR/Cas12a-mediated crRNA-array maturation, we showcase SWITCHER's dual role-not just as a recombinase but as a CRISPR switch, capable of orchestrating distinct Cas12a/crRNA-encoded programs.}, }
@article {pmid40595661, year = {2025}, author = {Boswell, CW and Hoppe, C and Sherrard, A and Miao, L and Kojima, ML and Martino, P and Zhao, N and Stasevich, TJ and Nicoli, S and Giraldez, AJ}, title = {Genetically encoded affinity reagents are a toolkit for visualizing and manipulating endogenous protein function in vivo.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {5503}, pmid = {40595661}, issn = {2041-1723}, support = {R01 HD100035/HD/NICHD NIH HHS/United States ; R35 GM122580/GM/NIGMS NIH HHS/United States ; }, mesh = {Animals ; Zebrafish/genetics/embryology/metabolism ; CRISPR-Cas Systems/genetics ; Zebrafish Proteins/metabolism/genetics ; Single-Domain Antibodies/genetics/metabolism ; Epitopes/genetics/metabolism ; Gene Knock-In Techniques/methods ; Humans ; Single-Chain Antibodies/genetics/metabolism ; }, abstract = {Probing endogenous protein localization and function in vivo remains challenging due to laborious gene targeting and monofunctional alleles. Here, we develop a multifunctional and adaptable toolkit based on genetically encoded affinity reagents (GEARs). GEARs use small epitopes recognized by nanobodies and single chain variable fragments to enable fluorescent visualization, manipulation and degradation of protein targets in vivo. Furthermore, we outline a CRISPR/Cas9-based epitope tagging pipeline to demonstrate its utility for producing knock-in alleles that have broad applications. We use GEARs to examine the native behavior of the pioneer transcription factor Nanog and the planar cell polarity protein Vangl2 during early zebrafish development. Together, this toolkit provides a versatile system for probing and perturbing endogenous protein function while circumventing challenges associated with conventional gene targeting and is broadly available to the model organism community.}, }
@article {pmid40595633, year = {2025}, author = {Fu, W and Ma, J and Wang, Z and Tang, N and Pan, D and Su, M and Wu, Z and Gan, J and Ji, Q}, title = {Mechanisms and engineering of a miniature type V-N CRISPR-Cas12 effector enzyme.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {5667}, pmid = {40595633}, issn = {2041-1723}, support = {22277078//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Cryoelectron Microscopy ; Gene Editing/methods ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry/ultrastructure ; Humans ; DNA/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics/chemistry/ultrastructure ; Protein Engineering ; }, abstract = {Type V CRISPR-Cas12 systems are highly diverse in their functionality and molecular compositions, including miniature Cas12f1 and Cas12n genome editors that provide advantages for efficient in vivo therapeutic delivery due to their small size. In contrast to Cas12f1 nucleases that utilize a homodimer structure for DNA targeting and cleavage with a preference for T- or C-rich PAMs, Cas12n nucleases are likely monomeric proteins and uniquely recognize rare A-rich PAMs. However, the molecular mechanisms behind RNA-guided genome targeting and cleavage by Cas12n remain unclear. Here, we present the cryo-electron microscopy (cryo-EM) structure of Rothia dentocariosa Cas12n (RdCas12n) bound to a single guide RNA (sgRNA) and target DNA, illuminating the intricate molecular architecture of Cas12n and its sgRNA, as well as PAM recognition and nucleic-acid binding mechanisms. Through structural comparisons with other Cas12 nucleases and the ancestral precursor TnpB, we provide insights into the evolutionary significance of Cas12n in the progression from TnpB to various Cas12 nucleases. Additionally, we extensively modify the sgRNA and convert RdCas12n into an effective genome editor in human cells. Our findings enhance the understanding of the evolutionary mechanisms of type V CRISPR-Cas12 systems and offer a molecular foundation for engineering Cas12n genome editors.}, }
@article {pmid40595632, year = {2025}, author = {Zhang, Y and Zhang, T and Xiao, X and Wang, Y and Kawalek, A and Ou, J and Ren, A and Sun, W and de Bakker, V and Liu, Y and Li, Y and Yang, L and Ye, L and Jia, N and Veening, JW and Liu, X}, title = {CRISPRi screen identifies FprB as a synergistic target for gallium therapy in Pseudomonas aeruginosa.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {5870}, pmid = {40595632}, issn = {2041-1723}, support = {82270012//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82200047//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {*Pseudomonas aeruginosa/drug effects/genetics ; *Gallium/pharmacology/therapeutic use ; Animals ; *Pseudomonas Infections/drug therapy/microbiology ; Mice ; Biofilms/drug effects/growth &amp; development ; *Anti-Bacterial Agents/pharmacology ; Humans ; Microbial Sensitivity Tests ; Reactive Oxygen Species/metabolism ; *Bacterial Proteins/genetics/metabolism ; Oxidative Stress/drug effects ; CRISPR-Cas Systems ; Female ; Iron/metabolism ; }, abstract = {With the rise of antibiotic-resistant bacteria, non-antibiotic therapies like gallium gain increasing attention. Intravenous gallium nitrate is under Phase II clinical trials to treat chronic Pseudomonas aeruginosa infections in cystic fibrosis patients. However, its clinical efficacy is constrained by the achievable peak concentration in human tissue. To address this limitation, we apply a genome-wide CRISPR interference approach (CRISPRi-seq) to identify potential synergistic targets with gallium. We classify the essential genes by response time and growth reduction, pinpointing the most vulnerable therapeutic targets in this species. In addition, we identify a highly conserved gene, fprB, encoding a ferredoxin-NADP[+] reductase, whose deletion sensitizes P. aeruginosa to gallium, lowering its MIC by 32-fold and shifting mode of action from bacteriostatic to bactericidal. Further investigation reveals that FprB plays a critical role in modulating oxidative stress induced by gallium, via control of iron homeostasis and reactive oxygen species accumulation. Deleting fprB enhances gallium's efficacy against biofilm formation and improves outcomes in a murine lung infection model of P. aeruginosa, suggesting FprB is a promising drug target in combination with gallium. Overall, our data show CRISPRi-seq as a powerful tool for systematic genetic analysis of P. aeruginosa, advancing the identification of novel therapeutic targets.}, }
@article {pmid40595611, year = {2025}, author = {Irie, N and Takeda, N and Satou, Y and Araki, K and Ono, M}, title = {Machine learning-assisted decoding of temporal transcriptional dynamics via fluorescent timer.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {5720}, pmid = {40595611}, issn = {2041-1723}, support = {DCRPGF\100007//Cancer Research UK (CRUK)/ ; JP21K07082, JP21H00433, JP24K10259, JPJSCCA2020008//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JPJSCCA2020008//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP16H0627601, JP22H04922//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 24gm1810001s0103//Japan Agency for Medical Research and Development (AMED)/ ; }, mesh = {Animals ; *Machine Learning ; *Forkhead Transcription Factors/genetics/metabolism ; Mice ; *Transcription, Genetic ; Flow Cytometry/methods ; Gene Expression Regulation ; Mice, Inbred C57BL ; Single-Cell Analysis/methods ; Enhancer Elements, Genetic ; T-Lymphocytes, Regulatory/metabolism ; Genes, Reporter ; Neural Networks, Computer ; Spleen/metabolism/cytology ; CRISPR-Cas Systems ; }, abstract = {Investigating the temporal dynamics of gene expression is crucial for understanding gene regulation across various biological processes. Using the Fluorescent Timer protein, the Timer-of-cell-kinetics-and-activity system enables analysis of transcriptional dynamics at the single-cell level. However, the complexity of Timer fluorescence data has limited its broader application. Here, we introduce an integrative approach combining molecular biology and machine learning to elucidate Foxp3 transcriptional dynamics through flow cytometric Timer analysis. We have developed a convolutional neural network-based method that incorporates image conversion and class-specific feature visualisation for class-specific feature identification at the single-cell level. Biologically, we developed a novel CRISPR mutant of Foxp3 fluorescent Timer reporter mice lacking the enhancer Conserved Non-coding Sequence 2, which revealed new roles of this enhancer in regulating Foxp3 transcription frequency under specific conditions. Furthermore, analysis of wild-type Foxp3 fluorescent Timer reporter mice at different ages uncovered distinct patterns of Foxp3 expression from neonatal to aged mice, highlighting prominent thymus-like features of neonatal splenic Foxp3[+] T cells. In conclusion, our study uncovers previously unrecognised Foxp3 transcriptional dynamics, establishing a proof-of-concept for integrating CRISPR, single-cell dynamics analysis, and machine learning methods as advanced techniques to understand transcriptional dynamics in vivo.}, }
@article {pmid40595537, year = {2025}, author = {Hofmann, R and Herman, C and Mo, CY and Mathai, J and Marraffini, LA}, title = {Deep mutational scanning identifies Cas1 and Cas2 variants that enhance type II-A CRISPR-Cas spacer acquisition.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {5730}, pmid = {40595537}, issn = {2041-1723}, mesh = {*CRISPR-Cas Systems/genetics ; *Streptococcus pyogenes/genetics/virology ; *CRISPR-Associated Proteins/genetics/metabolism ; Mutation ; *Bacterial Proteins/genetics/metabolism ; Bacteriophages/genetics ; *Endonucleases/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {A remarkable feature of CRISPR-Cas systems is their ability to acquire short sequences from invading viruses to create a molecular record of infection. These sequences, called spacers, are inserted into the CRISPR locus and mediate sequence-specific immunity in prokaryotes. In type II-A CRISPR systems, Cas1, Cas2 and Csn2 form a supercomplex with Cas9 to integrate viral sequences. While the structure of the integrase complex has been described, a detailed functional analysis of the spacer acquisition machinery is lacking. We developed a genetic system that combines deep mutational scanning (DMS) of Streptococcus pyogenes cas genes with a method to select bacteria that acquire new spacers. Here, we show that this procedure reveals key interactions at the Cas1-Cas2 interface critical for spacer integration, identifies Cas variants with enhanced spacer acquisition and immunity against phage infection, and provides insights into the molecular determinants of spacer acquisition, offering a platform to improve CRISPR-Cas-based applications.}, }
@article {pmid40595093, year = {2025}, author = {Wei, T and Yan, Y and Niu, M and Dong, X and Li, H and Sun, Y and Fa, Y}, title = {A rapid LASV detection method based on CRISPR-Cas13a and recombinase aided amplification with special lateral-flow test strips.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {20640}, pmid = {40595093}, issn = {2045-2322}, support = {2023YFC2605100//National Key Research and Development Program of China/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism ; *Lassa virus/isolation &amp; purification/genetics ; Humans ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; *Lassa Fever/diagnosis/virology ; Reagent Strips ; Animals ; }, abstract = {Lassa virus (LASV) is a high-risk pathogen associated with severe viral hemorrhagic fever in both humans and animals. Owing to its significant treatment challenges and high infectivity, LASV is classified as a biosafety level 4 (BSL-4) pathogen. It is essential to establish a rapid LASV detection method to prevent and control the disease. To address the biosecurity threats caused by LASV, in this study, we developed a new test method for LASV detection by combining the recombinase-mediated isothermal amplification (RAA) and CRISPR-Cas13a detection technology. The detection efficiency of this method was evaluated and compared with existing methods. The results demonstrate that this new detection maintains relatively high sensitivity and specificity, while having excellent simplicity and rapidity. The sensitivity of the method for detecting the LASV can achieve a threshold of 10[1] copies/µL using fluorescence detection in 90 min and 10[2] copies/µL with lateral flow strip detection in just an hour, which only needs a simple constant temperature equipment to achieve. The application of this detection method holds substantial biosecurity significance for underdeveloped regions (e.g., West Africa), as well as for countries like China, which have a vast territory and uneven development of medical testing levels in various regions.}, }
@article {pmid40594904, year = {2025}, author = {Sayem, M and Rafi, MA and Mishu, ID and Mahmud, Z}, title = {Comprehensive genomic analysis reveals virulence and antibiotic resistance genes in a multidrug-resistant Bacillus cereus isolated from hospital wastewater in Bangladesh.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {22915}, pmid = {40594904}, issn = {2045-2322}, mesh = {*Bacillus cereus/genetics/pathogenicity/isolation &amp; purification/drug effects ; *Wastewater/microbiology ; Bangladesh ; *Drug Resistance, Multiple, Bacterial/genetics ; Phylogeny ; Hospitals ; Virulence/genetics ; Genome, Bacterial ; Whole Genome Sequencing ; Genomics/methods ; Anti-Bacterial Agents/pharmacology ; Virulence Factors/genetics ; Humans ; }, abstract = {Hospital wastewater represents a significant reservoir for antimicrobial-resistant bacteria, including multidrug-resistant (MDR) Bacillus cereus, a pathogen of growing concern due to its potential impact on public health and environmental safety. This study characterizes the genomic features, antimicrobial resistance (AMR) mechanisms, and virulence potential of Bacillus cereus MBC, isolated from hospital wastewater in Dhaka, Bangladesh. Using whole-genome sequencing (WGS) and advanced bioinformatics, we analyzed the isolate's taxonomy, phylogenetics, functional annotation, and biosynthetic potential. The genome, spanning 5.6 Mb with a GC content of 34.84%, contained 5,881 protein-coding sequences, including 1,424 hypothetical proteins, and 28 genes associated with AMR. Phylogenetic analysis revealed a close genetic relationship with Bacillus cereus ATCC 14579, sharing virulence factors such as hemolysin BL (HBL), non-hemolytic enterotoxin (NHE), and cytotoxin K (CytK), all contributing to its pathogenicity. The ability to form biofilms further enhances the strain's persistence and resistance in hospital environments. AMR profiling identified genes conferring resistance to beta-lactams (e.g., BcI, BcII, BcIII), tetracyclines (tetB(P)), glycopeptides (vanY), and fosfomycin, highlighting the bacterium's capacity to resist a wide array of antibiotics. Functional annotation revealed metabolic pathways involved in iron acquisition and the biosynthesis of siderophores such as petrobactin and bacillibactin, reinforcing the bacterium's adaptability in nutrient-limited environments. Mobile genetic elements, including prophages, CRISPR-Cas systems, and transposable elements, suggest significant horizontal gene transfer (HGT), enhancing genetic plasticity and resistance spread. Pangenomic analysis, involving 125 B. cereus strains, revealed a high degree of genetic diversity and close relationships with strains from clinical, food, and agricultural environments, emphasizing the overlap between clinical and environmental reservoirs of resistance. The strain's isolation from hospital wastewater underscores the complex interplay between environmental contaminants and bacterial evolution, which fosters MDR traits. Our findings underscore the urgent need for enhanced genomic surveillance and wastewater management strategies to mitigate the spread of MDR B. cereus and AMR genes in hospital environments.}, }
@article {pmid40594722, year = {2025}, author = {Ren, X and Yao, XR and Chen, K and Xiao, WT and He, JY}, title = {CRISPR-Cas9 screening identifies a gene signature predictive of prognosis in glioblastoma.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {21077}, pmid = {40594722}, issn = {2045-2322}, support = {82304083//National Natural Science Foundation of China/ ; 2023JJ40584//Natural Science Foundation of Hunan Province/ ; }, mesh = {Humans ; *Glioblastoma/genetics/pathology/mortality ; Prognosis ; *CRISPR-Cas Systems/genetics ; *Brain Neoplasms/genetics/pathology/mortality ; Gene Expression Regulation, Neoplastic ; Biomarkers, Tumor/genetics ; Cell Proliferation/genetics ; Gene Expression Profiling ; Gene Regulatory Networks ; *Transcriptome ; }, abstract = {Glioblastoma (GBM) is the most aggressive primary brain malignancy, characterized by a poor prognosis and limited therapeutic options. Identifying essential genes and pathways involved in GBM proliferation is important for developing prognostic biomarkers and potential therapeutic targets. In this study, genome-wide CRISPR-Cas9 screening data from the dependency map (DepMap) database were analyzed to explore proliferation-related essential genes and pathways in GBM. A five-gene prognostic signature-CLSPN, HSP90B1, MED10, SAMM50, and TOMM20-was constructed using univariate, LASSO, and multivariate Cox regression analyses, and its prognostic value was evaluated in independent cohorts. Weighted gene co-expression network analysis (WGCNA) and gene set enrichment analysis (GSEA) suggested that the E2F targets pathway may be involved in GBM proliferation, consistent with the CRISPR screening results. Among the identified genes, MED10 was preliminarily implicated in regulating GBM cell proliferation and migration, as supported by functional assays. These findings propose a proliferation-related gene signature with potential prognostic relevance in GBM and indicate the E2F targets pathway as a biological process potentially associated with tumor progression. MED10 warrants further investigation as a candidate gene in the context of GBM biology and therapy.}, }
@article {pmid40593724, year = {2025}, author = {Hu, M and Zhang, B and Shan, Y and Cao, F and Wang, Y and Qi, W and Wang, X and Shen, Y and Guo, X and Zhang, M and Tian, T and Xie, W and Zhang, M and Liang, F and Pei, D and Zhou, X}, title = {Scalable modulation of CRISPR‒Cas enzyme activity using photocleavable phosphorothioate DNA.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {5939}, pmid = {40593724}, issn = {2041-1723}, support = {32150019//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32300692//National Natural Science Foundation of China (National Science Foundation of China)/ ; 92068201//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2024M760985//China Postdoctoral Science Foundation/ ; GZB20240235//China Postdoctoral Science Foundation/ ; 2024T170292//China Postdoctoral Science Foundation/ ; 2023M741235//China Postdoctoral Science Foundation/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Phosphorothioate Oligonucleotides/metabolism/chemistry/genetics ; *DNA/metabolism/chemistry/genetics ; Light ; Humans ; }, abstract = {The regulation of CRISPR‒Cas activity is critical for developing advanced biotechnologies. Optical control of CRISPR‒Cas system activity can be achieved by modulation of Cas proteins or guide RNA (gRNA), but these approaches either require complex protein engineering modifications or customization of the optically modulated gRNAs according to the target. Here, we present a method, termed photocleavable phosphorothioate DNA (PC&amp;PS DNA)-mediated regulation of CRISPR‒Cas activity (DNACas), that is versatile and overcomes the limitations of conventional methods. In DNACas, CRISPR‒Cas activity is silenced by the affinity binding of PC&amp;PS DNA and restored through light-triggered chemical bond breakage of PC&amp;PS DNA. The universality of DNACas is demonstrated by adopting the PC&amp;PS DNA to regulate various CRISPR‒Cas enzymes, achieving robust light-switching performance. DNACas is further adopted to develop a light-controlled one-pot LAMP-BrCas12b detection method and a spatiotemporal gene editing strategy. We anticipate that DNACas could be employed to drive various biotechnological advances.}, }
@article {pmid40593661, year = {2025}, author = {Hwang, HY and Lee, M and Yi, H and Seok, C and Lim, K and Na, YR and Kang, JS and Park, JH and Kim, D}, title = {Engineered Sdd7 cytosine base editors with enhanced specificity.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {5881}, pmid = {40593661}, issn = {2041-1723}, support = {2020R1A2C2101714//National Research Foundation of Korea (NRF)/ ; RS-2025-00521074//National Research Foundation of Korea (NRF)/ ; HI21C1314, HR22C1363, RS-2024-02507183,21A0202L1-12//Korea Health Industry Development Institute (KHIDI)/ ; }, mesh = {*Gene Editing/methods ; *Cytosine/metabolism ; Humans ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; *Cytosine Deaminase/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {Cytosine base editors (CBEs) revolutionize genome editing by enabling precise C-to-T conversions without double-strand breaks. Sdd7, a recently developed cytosine deaminase, exhibits high activity across a broad protospacer range but induces unintended off-target effects, including bystander mutations within and upstream of the protospacer and both gRNA-dependent and independent deamination. Here, we report that BE4max and Sdd7 induce bystander editing upstream of the protospacer. To overcome this, we engineer two Sdd7 variants, Sdd7e1 and Sdd7e2, enhancing specificity while preserving on-target efficiency. These variants display reduced bystander editing, narrowed editing windows, and significantly lower off-target activity. Delivery as ribonucleoproteins via engineered virus-like particles (eVLPs) further improves specificity, nearly eliminating bystander edits and increasing precise single-point mutations. Our findings establish Sdd7e1 and Sdd7e2, especially when delivered via eVLP, as high-fidelity CBEs poised for safe, precise therapeutic genome editing.}, }
@article {pmid40593653, year = {2025}, author = {Pastuszka, A and Mazzuoli, MV and Crestani, C and Deborde, L and Sismeiro, O and Lemaire, C and Rong, V and Gominet, M and Jacquemet, E and Legendre, R and Lanotte, P and Firon, A}, title = {The virulence regulator CovR boosts CRISPR-Cas9 immunity in Group B Streptococcus.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {5678}, pmid = {40593653}, issn = {2041-1723}, support = {ANR-22-CE15-0024//Agence Nationale de la Recherche (French National Research Agency)/ ; LabEx IBEID, ANR-10-LABX-62-IBEID//Agence Nationale de la Recherche (French National Research Agency)/ ; }, mesh = {*Streptococcus agalactiae/genetics/immunology/pathogenicity ; *CRISPR-Cas Systems/genetics/immunology ; *Bacterial Proteins/genetics/metabolism/immunology ; Virulence/genetics ; Gene Expression Regulation, Bacterial ; Streptococcal Infections/immunology/microbiology ; Promoter Regions, Genetic ; *CRISPR-Associated Protein 9/genetics/metabolism ; Operon ; Host-Pathogen Interactions/immunology/genetics ; Mice ; Animals ; Humans ; }, abstract = {CRISPR-Cas9 immune systems protect bacteria from foreign DNA. However, immune efficiency is constrained by Cas9 off-target cleavages and toxicity. How bacteria regulate Cas9 to maximize protection while preventing autoimmunity is not understood. Here, we show that the master regulator of virulence, CovR, regulates CRISPR-Cas9 immunity against mobile genetic elements in Streptococcus agalactiae, a pathobiont responsible for invasive neonatal infections. We show that CovR binds to and represses a distal promoter of the cas operon, integrating immunity within the virulence regulatory network. The CovR-regulated promoter provides a controlled increase in off-target cleavages to counteract mutations in the target DNA, restores the potency of old immune memory, and stimulates the acquisition of new memory in response to recent infections. Regulation of Cas9 by CovR is conserved at the species level, with lineage specificities suggesting different adaptive trajectories. Altogether, we describe the coordinated regulation of immunity and virulence that enhances the bacterial immune repertoire during host-pathogen interaction.}, }
@article {pmid40593649, year = {2025}, author = {Thorpe, C and Luo, W and Ji, Q and Eggenberger, AL and Chicowski, AS and Xu, W and Sandhu, R and Lee, K and Whitham, SA and Qi, Y and Wang, K and Jiang, S}, title = {Enhancing biolistic plant transformation and genome editing with a flow guiding barrel.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {5624}, pmid = {40593649}, issn = {2041-1723}, support = {2019-67013-29016//United States Department of Agriculture | Agricultural Research Service (USDA Agricultural Research Service)/ ; IOW04714//United States Department of Agriculture | Agricultural Research Service (USDA Agricultural Research Service)/ ; IOW04308//United States Department of Agriculture | Agricultural Research Service (USDA Agricultural Research Service)/ ; IOS-2132693//NSF | BIO | Division of Biological Infrastructure (DBI)/ ; }, mesh = {*Gene Editing/methods ; *Biolistics/methods/instrumentation ; Zea mays/genetics ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Triticum/genetics ; *Transformation, Genetic ; Onions/genetics ; Genome, Plant ; Seedlings/genetics ; Genetic Engineering/methods ; }, abstract = {The biolistic delivery system is an essential tool in plant genetic engineering, capable of delivering DNAs, RNAs, and proteins independent of tissue type, genotype, or species. However, its efficiency and consistency remain longstanding challenges despite decades of widespread use. Here, through advanced simulations, we identify gas and particle flow barriers as the root cause of these limitations. We show that a flow guiding barrel (FGB) achieves a 22-fold enhancement in transient transfection efficiency, a 4.5-fold increase in CRISPR-Cas9 ribonucleoprotein editing efficiency in onion epidermis, and a 17-fold improvement in viral infection efficiency in maize seedlings. Furthermore, stable transformation frequency in maize using B104 immature embryos increases over 10-fold, while in planta CRISPR-Cas12a-mediated genome editing efficiency in wheat meristems doubles in both T0 and T1 generations. This study provides insights into the fundamental mechanisms underlying biolistic inefficiency and demonstrates a practical solution that enables broader and more reliable applications in plant genetic engineering.}, }
@article {pmid40593618, year = {2025}, author = {Lin, M and Qiu, Z and Hao, M and Qi, W and Zhang, T and Shen, Y and Xiao, H and Liang, C and Xie, L and Jiang, Y and Cheng, M and Tian, T and Zhou, X}, title = {Cas12a Cis-cleavage mediated lateral flow assay enables multiplex and ultra-specific nucleic acid detection.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {5597}, pmid = {40593618}, issn = {2041-1723}, support = {2023M741238//China Postdoctoral Science Foundation/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/metabolism/genetics ; Sensitivity and Specificity ; *Bacterial Proteins/metabolism/genetics ; *Nucleic Acids/genetics/analysis ; Nucleic Acid Hybridization ; }, abstract = {CRISPR technology holds significant promise for advancing nucleic acid assays. However, current CRISPR diagnostic techniques, reliant on indiscriminate trans-cleavage mechanisms, face challenges in developing multiplex detection formats. Moreover, chaotic trans-cleavage activity often results from mismatched targets, leading to specificity issues. To address these limitations, here we exploit a double-key recognition mechanism based on CRISPR-Cas12a cis-cleavage and invasive hybridization identification of released sticky-end DNA products. By integrating multiplexed nucleic acid amplification, the double-key Cas12a detection mechanism, and a lateral flow detection platform, we develop a method termed Cas12a cis-cleavage mediated lateral flow assay (cc-LFA). We demonstrate that the cc-LFA exhibited superior specificity compared to three mainstream trans-cleavage-based CRISPR diagnostic techniques, achieving single-base resolution detection free from high-concentration wild-type DNA background interference. cc-LFA is also applied for highly specific detection of multiple respiratory pathogen samples and precise multiplexed detection of nine high-risk human papillomavirus (HPV) subtypes, achieving over 90% sensitivity and 100% specificity, respectively. Additionally, we present a portable device to automate nucleic acid amplification and strip detection procedures, showcasing the potential of cc-LFA for future applications in decentralized laboratory scenarios.}, }
@article {pmid40593576, year = {2025}, author = {Kiernan, KA and Taylor, DW}, title = {Visualization of a multi-turnover Cas9 after product release.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {5681}, pmid = {40593576}, issn = {2041-1723}, support = {R35GM138348//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; }, mesh = {RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry/ultrastructure ; Gene Editing/methods ; Streptococcus pyogenes/enzymology/genetics ; Cryoelectron Microscopy ; Kinetics ; DNA/metabolism/chemistry/genetics ; Bacterial Proteins/metabolism/genetics ; }, abstract = {While the most widely used CRISPR-Cas enzyme is the Cas9 endonuclease from Streptococcus pyogenes (Cas9), it exhibits single-turnover enzyme kinetics which leads to long residence times on product DNA. This blocks access to DNA repair machinery and acts as a major bottleneck during CRISPR-Cas9 gene editing. Cas9 can eventually be removed from the product by extrinsic factors, such as translocating polymerases, but the mechanisms contributing to Cas9 dissociation following cleavage remain poorly understood. Here, we employ truncated guide RNAs as a strategy to weaken PAM-distal nucleic acid interactions and promote faster enzyme turnover. Using kinetics-guided cryo-EM, we examine the conformational landscape of a multi-turnover Cas9, including the first detailed snapshots of Cas9 dissociating from product DNA. We discovered that while the PAM-distal product dissociates from Cas9 following cleavage, tight binding of the PAM-proximal product directly inhibits re-binding of new targets. Our work provides direct evidence as to why Cas9 acts as a single-turnover enzyme and will guide future Cas9 engineering efforts.}, }
@article {pmid40593519, year = {2025}, author = {Abe, I and Ohno, H and Mochizuki, M and Hayashi, K and Saito, H}, title = {Split RNA switch orchestrates pre- and post-translational control to enable cell type-specific gene expression.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {5362}, pmid = {40593519}, issn = {2041-1723}, support = {JP20H05626//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP25H00970//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP20H05701//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP20K12644//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP25K03464//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP22bm0104001//Japan Agency for Medical Research and Development (AMED)/ ; JP23bm1223002h0002//Japan Agency for Medical Research and Development (AMED)/ ; JP23bm1123040//Japan Agency for Medical Research and Development (AMED)/ ; }, mesh = {Humans ; MicroRNAs/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Gene Expression Regulation ; Gene Editing/methods ; HEK293 Cells ; *Protein Processing, Post-Translational ; RNA, Messenger/genetics/metabolism ; }, abstract = {RNA switch is a synthetic RNA-based technology that controls gene expression in response to cellular RNAs and proteins, thus enabling cell type-specific gene regulation and holding promise for gene therapy, regenerative medicine, and cell therapy. However, individual RNA switches often lack the specificity required for practical applications due to low ON/OFF ratios and difficulty in finding distinct and single biomolecule targets. To address these issues, we present "split RNA switches" that integrate outputs from multiple RNA switches by exploiting protein splicing. We show that split RNA switches significantly improve the ON/OFF ratio of microRNA-responsive ON switch system by canceling leaky OFF level in human cells. Using this approach, we achieve efficient cell purification using drug-resistance genes based on endogenous microRNA profiles and CRISPR-mediated genome editing with minimal off-target-cell effects. Additionally, we demonstrate RNA-based synthetic circuits using split RNA switches to enable the detection of multiple microRNAs and proteins with logical operations. Split RNA switches highlight the potential of post-translational processing as a versatile and comprehensive strategy for advancing mRNA-based therapeutic technologies.}, }
@article {pmid40570069, year = {2025}, author = {Speth, ZJ and Rehard, DG and Norton, PJ and Franz, AWE}, title = {Performance of two low-threshold population replacement gene drives in cage populations of the yellow fever mosquito, Aedes aegypti.}, journal = {PLoS genetics}, volume = {21}, number = {6}, pages = {e1011757}, doi = {10.1371/journal.pgen.1011757}, pmid = {40570069}, issn = {1553-7404}, mesh = {Animals ; *Aedes/genetics/virology ; CRISPR-Cas Systems/genetics ; *Gene Drive Technology/methods ; *Mosquito Vectors/genetics/virology ; Male ; Female ; *Yellow Fever/virology/transmission/genetics ; Promoter Regions, Genetic ; Mosquito Control/methods ; Animals, Genetically Modified ; }, abstract = {Aedes aegypti is the predominant vector for arboviruses including dengue, Zika, and chikungunya viruses, which infect over 100 million people annually. Mosquito population replacement in which arbovirus-susceptible mosquitoes in the field are replaced by laboratory-engineered refractory mosquitoes represents a novel genetic control measure to interrupt arboviral disease cycles. For this approach, the engineered mosquitoes need to harbor two genetic components: an antiviral effector construct which is linked to a gene drive (GD). We tested the performance of two single-locus CRISPR/Cas9 based GD for Ae. aegypti population replacement in small cage populations for up to 16 generations. Starting from a low release threshold of 1:9 GD bearing males to wild-type males, we observed two GD constructs in which Cas9 was expressed from two different germline promoters, nanos and zpg, to increase in frequency in all cage populations. By G16, an average of 72% and 82% of individuals from the zpg-GD and nanos-GD populations, respectively, harbored at least one GD copy with corresponding increases in allele frequencies. This indicated that the two single-locus, CRISPR/Cas9-based homing GD exhibited continuous super-Mendelian inheritance in populations of Ae. aegypti. Gene drive blocking indel (GDBI, a.k.a. "resistant alleles") frequency was measured for each discrete generation in pooled samples from the six populations harboring GD. We found that populations with Cas9 expression under control of the nanos-promoter accumulated GDBI at more than twice the rate of those populations harboring the zpg-promoter driven GD. Based on preexisting data sets for homing and GDBI frequencies in addition to the cage trial observations, the relative contributions of sex-specific homing rates, maternal Cas9 deposition and potential fitness effects were modeled in MGDrivE for both GD, further explaining their divergent performance. Our study demonstrates the feasibility of low-threshold, single-locus CRISPR/Cas9 based GD for Ae. aegypti population replacement.}, }
@article {pmid40488470, year = {2025}, author = {Deng, Z and Weng, X and Tang, H and Zou, T and Zhou, X and Liu, H and Wen, P and Luo, G and Gan, T and He, J}, title = {Rapid and sensitive detection of Mycobacterium tuberculosis using the RPA/Cas12f1_ge4.1 system with fluorescence and lateral flow readouts.}, journal = {Microbiology spectrum}, volume = {13}, number = {7}, pages = {e0265224}, pmid = {40488470}, issn = {2165-0497}, mesh = {*Mycobacterium tuberculosis/isolation &amp; purification/genetics ; Humans ; *Tuberculosis/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Fluorescence ; CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Recombinases/genetics/metabolism ; DNA, Bacterial/genetics ; Limit of Detection ; }, abstract = {UNLABELLED: Tuberculosis remains a major global health threat, with existing detection methods often limited by efficiency and resource demands. Our previous PAM-dependent dsDNA Target-activated Cas12f1 Trans Reporter (PDTCTR) fluorescence sensing platform, while effective for PAM-dependent pathogen detection, was constrained by its reliance on specialized fluorescence equipment and lack of visual output, limiting its use in resource-limited settings. To overcome these limitations, we introduce an innovative RPA/CRISPR-Cas12f1_ge4.1 dual-mode system for rapid Mycobacterium tuberculosis detection. This system combines engineered Cas12f_ge4.1 with recombinase polymerase amplification (RPA), offering both fluorescent and lateral flow detection. It achieves high sensitivity with detection limits of 10 copies/µL (fluorescence) and 100 copies/µL (lateral flow), alongside 100% specificity. In clinical validation, compared with a commercial qPCR kit, the fluorescent and lateral flow approaches demonstrate sensitivities of 94.52% (69/73, 95% confidence interval [CI]: 85.84%-98.23%) and 90.41% (66/73, 95% CI: 80.67%-95.73%), respectively, while maintaining 100% (40/40, 95% CI: 89.09%-100%) specificity and high concordance (kappa values: 0.924 and 0.878). Detection is completed within 1 h, providing a rapid, sensitive, and specific solution for M. tuberculosis identification. This dual-mode capability represents a significant advancement in current tuberculosis diagnostics, enabling both sensitive laboratory confirmation and rapid point-of-care screening. Our versatile and efficient method promises to transform tuberculosis diagnostics, particularly in resource-constrained environments.<br><br>IMPORTANCE: Tuberculosis (TB) remains a significant global health challenge, demanding rapid and accurate detection for effective management. The innovative RPA/CRISPR-Cas12f1_ge4.1 dual-mode system represents a major advancement in TB diagnostics, offering highly sensitive and specific detection of Mycobacterium tuberculosis DNA. This adaptable system, incorporating both fluorescent and lateral flow detection modes, is designed for use in both advanced laboratories and resource-limited settings. Its high performance, rigorously validated through clinical trials, holds the potential to revolutionize TB diagnosis, particularly in high-burden, low-resource areas. By facilitating earlier treatment and enhancing control of TB transmission, this system could significantly contribute to global efforts in combating this persistent public health threat.}, }
@article {pmid40422178, year = {2025}, author = {Siddiqui, FA and Chim-Ong, A and Wang, C and Miao, J and Cui, L}, title = {The PfK13 G533S mutation confers artemisinin partial resistance in multiple genetic backgrounds of Plasmodium falciparum.}, journal = {Antimicrobial agents and chemotherapy}, volume = {69}, number = {7}, pages = {e0016225}, doi = {10.1128/aac.00162-25}, pmid = {40422178}, issn = {1098-6596}, support = {U19AI181593/NH/NIH HHS/United States ; }, mesh = {*Plasmodium falciparum/genetics/drug effects ; *Artemisinins/pharmacology ; *Antimalarials/pharmacology ; *Drug Resistance/genetics ; *Protozoan Proteins/genetics ; Mutation/genetics ; Malaria, Falciparum/drug therapy/parasitology ; Humans ; CRISPR-Cas Systems ; }, abstract = {Mutations in the Plasmodium falciparum Kelch 13 (PfK13) protein are the key determinant of artemisinin partial resistance. While more than 200 PfK13 mutations have been identified in global parasite populations, only 13 have been validated to confer in vivo or in vitro artemisinin partial resistance. In the western Greater Mekong Subregion, the prevalence of the PfK13 G533S mutation has significantly increased in recent years. Field isolates carrying the PfK13 G533S mutation showed slower parasite clearance and higher day-3 positivity rates after artemisinin treatment, while culture-adapted isolates displayed significantly elevated ring-stage survival rates. Here, the PfK13 G533S mutation was introduced using CRISPR/Cas9 into four parasite strains: Dd2, 3D7, GB4, and F09N25 (a recent culture-adapted field isolate from the China-Myanmar border area). Across all four genetic backgrounds, the PfK13 G533S mutation conferred ring-stage survival rates of 12%-23% with a minimal fitness cost, explaining its rising prevalence in the region. In contrast, the PfK13 G533A mutation, sporadically detected in world P. falciparum populations, did not increase ring-stage survival rates when engineered into the 3D7 and Dd2 strains. These findings validate the PfK13 G533S mutation as a critical marker for artemisinin resistance surveillance and underscore the importance of monitoring its spread.}, }
@article {pmid40592986, year = {2025}, author = {Elhamouly, NA and Atta, N and Liu, S and Tu, Y and Peng, D}, title = {A novel assay incorporating CRISPR with RPA in a single pot for visual and accurate detection of Aphelenchoides besseyi in soybean.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {21217}, pmid = {40592986}, issn = {2045-2322}, mesh = {*Glycine max/parasitology ; *Nucleic Acid Amplification Techniques/methods ; Animals ; *Plant Diseases/parasitology ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Tylenchida/genetics/isolation &amp; purification ; }, abstract = {Aphelenchoides besseyi is a highly prevalent plant parasitic nematode which has a substantial impact and poses an economic risk to soybean cultivation, with a reported 2017 outbreak resulting in significant yield losses of up to 60%. Therefore, more effective control of this nematode depends on early and accurate nucleic acid detection. One of the promising detection approaches is to combine the CRISPR technology with the isothermal RPA. However, incorporating the RPA amplicon with the CRISPR ingredients in a single pot remains a significant challenge due to their incompatibility. In the current research, we propose a visual nucleic acid detection technique that takes less than thirty minutes and is highly sensitive for detecting A. besseyi. First, we conduct the RPA amplification, then we perform the CRISPR reaction using either a portable thermal cup or our body heat temperature. We tested this new assay on forty-four soybean samples exhibiting GSFR syndrome symptoms, and it effectively detected samples containing the A. besseyi. We designed three different ways for data collection and visualization to suit the requirements of various environments. Our findings confirm that the suggested new low-instrumentation portable single-pot RPA-CRISPR assay is durable, specific, and has strong nucleic acid sensitivity in the open field.}, }
@article {pmid40591069, year = {2025}, author = {Anari, E and Anari, R and Pazoki-Toroudi, H}, title = {A decade of scientific advancements and collaborations on CRISPR-Cas9 application in cancer research: a bibliometric review.}, journal = {Discover oncology}, volume = {16}, number = {1}, pages = {1232}, pmid = {40591069}, issn = {2730-6011}, support = {25550//Iran University of Medical Sciences/ ; }, abstract = {The CRISPR-Cas system, a novel gene-editing technique, was a focus of research on cancer diagnosis and treatment during the last decade. A systematic review of the research trends and collaborations of the literature published in PubMed database between 2014 and 2024 resulted in 3241 papers. Bibliometric analysis and data visualization were performed using Excel, RStudio, and VOSviewer software. An increasing trend in the number of publications and citations has been observed since 2014, with a moderate fall after 2023. China and the USA have contributed the most publications and international collaborations, and high-income nations involved the highest contributions in the literature. The most productive institutes were Harvard Medical School and Dana-Farber Cancer Institute. Wang and Li contributed the maximum number of publications in this field. The prominent sources were Cancer Research, and Cancers. The application of CRISPR technique in breast and colorectal cancers has been frequently studied during the last decade. Future trials should explore the specificity and safety of CRISPR in various cancers.}, }
@article {pmid40590986, year = {2025}, author = {K, MMP and Pagariya, MC and Jadhav, PR and Gawade, NS and Sarode, DK and Karkute, SG and Kardile, HB and Deshmukh, R and Penna, S and Kawar, PG}, title = {Advancing ornamental plant breeding through genomic technologies: opportunities, challenges, and future directions.}, journal = {Functional &amp; integrative genomics}, volume = {25}, number = {1}, pages = {140}, pmid = {40590986}, issn = {1438-7948}, mesh = {*Plant Breeding/methods ; *Genome, Plant ; Gene Editing ; *Genomics/methods ; CRISPR-Cas Systems ; *Plants/genetics ; }, abstract = {The ornamental plants constitute an important sector of horticulture industry, which are worth billions of dollars worldwide. There is a growing demand for new and improved cultivars and hence, breeders employ new tools and methods to address the problem of plant improvement. Recent advancements in Ornamental plant genomics have seen a great revolution due to new technologies of whole genome sequencing which have created previously unheard-of breeding program prospects. Research into gene regulation, genomic variations, genome evolution, and other biological processes are now aided by the use of complete genome sequencing data. The assembly of high-quality genomes for various ornamental species has facilitated the identification of genes controlling desirable traits such as flower color, shape, fragrance, biotic and abiotic stress resistance. The CRISPR/Cas9 based genome editing technology has offered immense scope for ornamental plant improvement through the enhancement of floral characteristics. Herein, we discuss how these genomic resources can be leveraged to improve breeding efficiency, accelerate the development of novel cultivars to augment the sustainability of the ornamental plant industry. This review aims to provide a viewpoint for the application of whole genome sequencing in ornamental plant breeding, highlighting the opportunities, challenges, and future prospects.}, }
@article {pmid40589866, year = {2025}, author = {Chen, X and Zhang, S and Lin, S and Wang, S and Huang, M and Chen, S and Chen, S}, title = {A combination of recombinase polymerase amplification with CRISPR technology rapidly detects goose parvovirus with high accuracy and sensitivity.}, journal = {Frontiers in cellular and infection microbiology}, volume = {15}, number = {}, pages = {1566603}, pmid = {40589866}, issn = {2235-2988}, mesh = {Animals ; Sensitivity and Specificity ; *Parvoviridae Infections/diagnosis/veterinary/virology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism/genetics ; *Poultry Diseases/diagnosis/virology ; Geese/virology ; Ducks/virology ; *Parvovirinae/genetics/isolation &amp; purification ; *Parvovirus/genetics/isolation &amp; purification ; *Molecular Diagnostic Techniques/methods ; }, abstract = {BACKGROUND: Goose parvovirus (GPV) poses a significant threat to the waterfowl industry, necessitating reliable detection methods. However, conventional techniques are often time-consuming, equipment-dependent, or lack sufficient sensitivity for detecting early-stage infection. In contrast, emerging CRISPR/Cas12a-based systems offer a promising alternative for rapid, sensitive, and on-site diagnostics.<br><br>METHODS: We developed and optimized a recombinase polymerase amplification (RPA)-CRISPR/Cas12a assay targeting the conserved VP3 gene of GPV. The analytical and diagnostic performance of this assay was rigorously validated using plasmid standards and clinical specimens from both experimentally infected and field-collected ducklings.<br><br>RESULTS: Our developed assay combines RPA with CRISPR/Cas12a technology for rapid GPV nucleic acids detection. This method achieves a detection limit of 10 copies/&#x3bc;L of the VP3 gene within one hour, demonstrating high sensitivity and rapid turnaround. The assay exhibited exceptional specificity, with no cross-reactivity against other waterfowl viruses, and showed robust reproducibility, with intra- and inter-assay coefficients of variation consistently below 5.0%. Clinical validation using 42 field samples confirmed a diagnostic sensitivity of 100% and 95.5% specificity, showing superior performance to real-time quantitative PCR (qPCR) in both metrics. Furthermore, the assay supports flexible visual readouts using portable blue light transilluminators, facilitating on-site interpretation.<br><br>CONCLUSIONS: This study established a highly field-deployable RPA-CRISPR/Cas12a assay for rapid, visual detection of GPV with outstanding sensitivity and specificity. Its capability for instrument-free on-site diagnosis via blue light transillumination makes this approach particularly promising for resource-limited settings.}, }
@article {pmid40588792, year = {2025}, author = {Ma, Q and Zhang, Y and Chen, L and Bao, Y and Guo, W and Feng, K and Huang, T and Cai, YD}, title = {Machine Learning-Driven Discovery of Essential Binding Preference in Anti-CRISPR Proteins.}, journal = {Proteomics. Clinical applications}, volume = {}, number = {}, pages = {e70013}, doi = {10.1002/prca.70013}, pmid = {40588792}, issn = {1862-8354}, support = {XDB38050200//Strategic Priority Research Program of Chinese Academy of Sciences/ ; XDA26040304//Strategic Priority Research Program of Chinese Academy of Sciences/ ; 2022YFF1203202//National Key R&amp;D Program of China/ ; GZNL2024A01003//Major Project of Guangzhou National Laboratory/ ; SRPG22-007//Self-Supporting Program of Guangzhou Laboratory/ ; 202002//Key Laboratory of Tissue Microenvironment and Tumor of Chinese Academy of Sciences/ ; ZR2022MC072//Shandong Provincial Natural Science Foundation/ ; 2024KCXTD081//Key Scientific Research Project of General Universities in Guangdong Province/ ; }, abstract = {PURPOSE: Anti-CRISPR (Acr) proteins can evade CRISPR-Cas immunity, yet their molecular determinants remain poorly understood. This study aimed to uncover key features driving Acr activity, thereby advancing both fundamental knowledge and the rational design of robust CRISPR-based tools.<br><br>EXPERIMENTAL DESIGN: We compiled a binary-encoded matrix of 761 InterPro-annotated domains and binding-site features for known Acr proteins. Seven feature ranking algorithms were applied to prioritize determinant features, and an incremental feature selection strategy, coupled with four distinct classifiers, was used to identify optimal subsets. Consensus key features were defined by intersecting the top subsets across all methods.<br><br>RESULTS: Key identified features include the DUF2829 domain, the Lambda repressor-like domain and Sulfolobus islandicus virus proteins, the Cro/C1-type helix-turn-helix domain, phage protein, and replication initiator A. These findings illuminate novel structural modules and regulatory motifs that underpin Acr inhibition.<br><br>CONCLUSIONS: This study provides critical theoretical support for deciphering Acr mechanisms and offers actionable insights for engineering next-generation CRISPR-Cas applications in clinical and biotechnological settings.<br><br>SUMMARY: The CRISPR system is a part of the antiviral immune defense initially discovered in bacteria and archaea. At present, the CRISPR system has become the cornerstone of genome editing technologies such as CRISPR-Cas9, widely used in clinical, agricultural, and biological research. Anti-CRISPR proteins are a group of proteins that inhibit the normal activity of CRISPR-Cas system in certain bacteria or archaea and avoid having the phages' genomes destroyed by the prokaryotic cells. The anti-CRISPR protein family has various components, but with similar functions to help exogenous DNA escape from the immune system. This study tried to uncover molecular mechanisms for anti-CRISPR proteins.}, }
@article {pmid40588529, year = {2025}, author = {Park, BS and Lee, M and Kim, J and Kim, T}, title = {Perturbomics: CRISPR-Cas screening-based functional genomics approach for drug target discovery.}, journal = {Experimental &amp; molecular medicine}, volume = {}, number = {}, pages = {}, pmid = {40588529}, issn = {2092-6413}, support = {CAP23011-000//National Research Council of Science and Technology (National Research Council of Science &amp; Technology)/ ; 2E33131//Korea Institute of Science and Technology (KIST)/ ; RS-2024-00452719//National Research Foundation of Korea (NRF)/ ; }, abstract = {Despite more than two decades since the completion of the first draft of the Human Genome Project, a substantial proportion of human genes remain poorly characterized in terms of their functions. Functional genomics aims to elucidate the roles and interactions of genes and genetic elements, providing insights into their involvement in various biological processes. In this context, the perturbomics approach-a systematic analysis of phenotypic changes resulting from gene function modulation-offers valuable insights into the function of unannotated genes. With the advent of CRISPR-Cas-based genome and epigenome editing, CRISPR screens have become the method of choice for perturbomics studies, enabling the identification of target genes whose modulation may hold therapeutic potential for diseases such as cancer, cardiovascular disorders and neurodegeneration. These findings contribute to the development of targeted drug therapies and the design of gene and cell therapies for regenerative medicine. Here we highlight recent technical advances in CRISPR-based perturbomics, focusing on more physiologically relevant, single-cell-level analyses and their successful applications in discovering novel therapeutic strategies.}, }
@article {pmid40588516, year = {2025}, author = {Malong, L and Roskosch, J and Hager, C and Fortin, JP and Schmucki, R and Callow, MG and Weile, C and Romeo, V and Patsch, C and Martin, S and Costa, M and Modrusan, Z and Villaseñor, R and Koller, E and Haley, B and Spang, A and Roudnicky, F}, title = {A CRISPR/Cas9 screen reveals proteins at the endosome-Golgi interface that modulate cellular anti-sense oligonucleotide activity.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {5378}, pmid = {40588516}, issn = {2041-1723}, mesh = {*Endosomes/metabolism ; *CRISPR-Cas Systems/genetics ; Humans ; *Golgi Apparatus/metabolism ; Animals ; Lysosomes/metabolism ; *Oligonucleotides, Antisense/metabolism/genetics ; Mice ; GTPase-Activating Proteins/metabolism/genetics ; HeLa Cells ; HEK293 Cells ; Adaptor Protein Complex 1/metabolism/genetics ; }, abstract = {Anti-sense oligonucleotides (ASOs) are modified synthetic single-stranded molecules with enhanced stability, activity, and bioavailability. They associate with RNA through sequence complementarity and can reduce or alter mRNA expression upon binding of splice site positions. To target RNA in the nucleus or cytoplasm, ASOs must cross membranes, a poorly understood process. We performed an unbiased CRISPR/Cas9 knockout screen with a genetic splice reporter to identify genes that can increase or decrease ASO activity, resulting in the most comprehensive catalog of ASO-activity modifier genes. Here we reveal distinct targets, including AP1M1 and TBC1D23, linking ASO activity to transport of cargo between the Golgi and endosomes. AP1M1 absence strongly increases ASO activity by delaying endosome-to-lysosome transport in vitro and in vivo. Prolonged ASO residence time in the endosomal system may increase the likelihood of ASO escape. This insight into AP1M1 role in ASO trafficking suggests a way for enhancing the therapeutic efficacy of ASOs by manipulating the endolysosomal pathways.}, }
@article {pmid40588042, year = {2025}, author = {Long, R and Tang, D and Yang, T and Li, M and Lu, Y and Liu, W and Jiang, L}, title = {Advanced research and exploration of CRISPR technology in the field of directed evolution.}, journal = {Biotechnology advances}, volume = {}, number = {}, pages = {108633}, doi = {10.1016/j.biotechadv.2025.108633}, pmid = {40588042}, issn = {1873-1899}, abstract = {Directed molecular evolution is the key technology for obtaining enzymes, proteins, metabolic pathways, and other components of living organisms that have specific functions or desirable properties, which are indispensable in a variety of industrial and medical applications. Despite the success of traditional methods, they are often limited by low efficiency and the high cost of obtaining desired mutants. The advent of CRISPR technology has significantly advanced the field by enabling precise and efficient gene targeting, offering new prospects for directed evolution. This review provides a comprehensive overview of CRISPR tools and their applications in directed evolution, highlighting the principles, technological advancements, and specific applications of CRISPR-based mutation and screening platforms. We discuss the key findings from the use of CRISPR in enzyme and genome evolution, showcasing its ability to generate genetic diversity and select for improved phenotypes. The study underscores the unique value of CRISPR in directed evolution, particularly in its flexibility to target and edit various species' genomes, and its potential to accelerate the discovery of novel biomolecules with enhanced properties.}, }
@article {pmid40587425, year = {2025}, author = {Li, X and Peng, Y and Xue, W and Liu, X and Luo, S and Wei, P and Wang, J}, title = {CRISPR-based Shuttle Cloning: A High-throughput Cloning Method.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {220}, pages = {}, doi = {10.3791/68503}, pmid = {40587425}, issn = {1940-087X}, mesh = {*Cloning, Molecular/methods ; Plasmids/genetics ; *Genetic Vectors/genetics ; *CRISPR-Cas Systems ; DNA/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {The development of genome-wide plasmid libraries using existing genomic repositories serves as a pivotal prerequisite for systematic functional characterization of genes across diverse biological processes. Current high-throughput methodologies for inter-vector DNA fragment transfer, however, necessitate PCR amplification of target sequences prior to cloning, rendering the generation of genome-scale plasmid collections technically demanding and time-intensive. By leveraging a CRISPRshuttle cassette, we developed a new high-throughput cloning method, CRISPR-based shuttle cloning (CRISPRshuttle cloning), which facilitates the transfer of many DNA fragments from donor plasmids sharing identical backbone sequences to a CRISPRshuttle-compatible vector without PCR amplification of the DNA fragments. Here, we present a protocol for CRISPRshuttle. This protocol involves two sequential test tube reactions prior to bacterial transformation. First, target DNA fragments are excised from donor plasmids by Cas9-mediated cleavage of their shared vector backbone sequence. Second, the excised DNA fragments are inserted into linearized CRISPRshuttle-compatible vectors through Gibson assembly. Our results demonstrate that the efficiency of CRISPRshuttle exceeds 94% and that two researchers can generate about 300 plasmids in 7 days using CRISPRshuttle. CRISPRshuttle facilitates efficient, adaptable, and cost-effective DNA fragment transfer between vectors, significantly streamlining genome-wide plasmid library generation.}, }
@article {pmid40587016, year = {2025}, author = {Choudhary, A and Kumar, A and Munshi, A}, title = {Genetic variants in oncogenic miRNA and 3' untranslated region of tumor suppressor genes: emerging insight into cancer genetics.}, journal = {Medical oncology (Northwood, London, England)}, volume = {42}, number = {8}, pages = {303}, pmid = {40587016}, issn = {1559-131X}, support = {211610155703//University Grants Commission/ ; BT/PR45460/MED/12/952/2022//Department of Biotechnology, Ministry of Science and Technology, India/ ; }, mesh = {Humans ; *MicroRNAs/genetics ; *Genes, Tumor Suppressor ; *3' Untranslated Regions/genetics ; *Neoplasms/genetics ; *Breast Neoplasms/genetics ; *Genetic Variation ; Female ; Gene Expression Regulation, Neoplastic ; Oncogenes ; }, abstract = {The miRNAs are key regulators of post-transcriptional gene expression. These are associated with the different molecular mechanisms which are engaged in the pathogenesis of various cancers, including breast cancer. The tumor suppressor and oncogenic miRNAs have a significant impact on cell proliferation, metastasis, angiogenesis, and apoptotic pathways. Variation in oncogenic miRNA encoding and the 3' UTR of the tumor suppressor genes associated with the development and prognosis of the cancer is being explored. These genetic variants alter oncogenic miRNAs' stability, target recognition, and binding ability, thereby resulting in the dysregulation of their target tumor suppressor gene, leading to uncontrolled cell division, a significant hallmark of cancer. The current review has been compiled to explore the genetic variation reported in the oncogenic miRNA encoding and 3' UTR of their target tumor suppressor genes associated with the development of breast cancer and its progression, focusing on the associated molecular mechanisms. Further, an effort has been made to discuss the possible therapeutic strategies, especially anti-miR, RNA interference, CRISPR/Cas, and ASOs, that have the potential to restore the function of the dysregulated tumor suppressor as well as oncogenic miRNA encoding genes.}, }
@article {pmid40586829, year = {2025}, author = {Hussain, MS and Maqbool, M and Arab, MM and Rana, AJ and Ashique, S and Khan, Y and Jakhmola, V and Gupta, G}, title = {Transforming Hemophilia Management: Lessons from Gene Therapy Clinical Trials.}, journal = {Molecular biotechnology}, volume = {}, number = {}, pages = {}, pmid = {40586829}, issn = {1559-0305}, abstract = {Gene therapy signifies a transformative revolution in hemophilia care, providing the possibility for sustained endogenous synthesis of coagulation factors and limiting the need for external factor supplementation. Preliminary experiments in hemophilia B via adeno-associated viral (AAV) vectors encountered constraints owing to immunological reactions and temporary translation. Progress in vector technology, particularly via self-complementary AAV innovation and codon-optimized mini-factor IX (FIX) concepts, has markedly improved transduction performance and prolonged FIX activity. Initial investigations have shown encouraging outcomes, with certain individuals sustaining consistent FIX expressions for more than 8 years; hence, decreasing yearly bleeding incidents and requiring preventive therapy. The development of gene therapy for hemophilia A has encountered substantial obstacles owing to the enormous size of the factor VIII (FVIII) gene. The recent experiments using AAV serotypes 5 (AAV5) vectors that encode B-domain-deleted FVIII constructs have shown sustained levels along with substantial decreases in hemorrhage incidents. Research has shown prolonged FVIII expression, with some individuals attaining almost normal coagulation efficiency. Phase III studies have validated long-term effectiveness and safety, with transient transaminase elevations being the most common adverse event. Notwithstanding these advancements, difficulties persist, including immunological reactions to vector capsids, hepatotoxicity, and unpredictability in translation levels. Innovative approaches including lentiviral vectors, gene-editing technologies, and novel customized connection strategies demonstrate possibilities for enhancing the effectiveness of gene therapy. Continuous clinical research and improvement in delivery systems will be crucial in substantiating gene therapy as a definitive approach for hemophilia.}, }
@article {pmid40586312, year = {2025}, author = {Soderholm, A and Vunjak, M and de Almeida, M and Popitsch, N and Podvalnaya, N and Araguas-Rodriguez, P and Scinicariello, S and Nischwitz, E and Butter, F and Ketting, RF and Ameres, SL and Müller-McNicoll, M and Zuber, J and Versteeg, GA}, title = {ERH regulates type II interferon immune signaling through post-transcriptional regulation of JAK2 mRNA.}, journal = {Nucleic acids research}, volume = {53}, number = {12}, pages = {}, doi = {10.1093/nar/gkaf545}, pmid = {40586312}, issn = {1362-4962}, support = {10.55776/P36572//Austrian Science Fund/ ; 10.55776/P30415//Austrian Science Fund/ ; 10.55776/P30231//Austrian Science Fund/ ; 10.55776/P36945//Austrian Science Fund/ ; 10.55776/F79//Austrian Science Fund/ ; 10.55776/W1261//Austrian Science Fund/ ; //Austrian Academy of Sciences/ ; FFG-852936//Austrian Research Promotion Agency/ ; FFG-852936//&#xd6;sterreichische Forschungsf&#xf6;rderungsgesellschaft/ ; }, mesh = {*Janus Kinase 2/genetics/metabolism ; Humans ; *Interferon-gamma/metabolism/immunology/genetics ; Signal Transduction/genetics ; *RNA, Messenger/genetics/metabolism ; *RNA Processing, Post-Transcriptional ; Introns ; Immunity, Innate/genetics ; CRISPR-Cas Systems ; Serine-Arginine Splicing Factors/genetics ; HEK293 Cells ; RNA Splicing ; Animals ; }, abstract = {Type II interferon (IFNγ) signaling is essential for innate immunity and critical for effective immunological checkpoint blockade in cancer immunotherapy. Genetic screen identification of post-transcriptional regulators of this pathway has been challenging since such factors are often essential for cell viability. Here, we utilize our inducible CRISPR/Cas9 approach to screen for key post-transcriptional regulators of IFNγ signaling, and in this way, we identify ERH and the ERH-associated splicing and RNA export factors MAGOH, SRSF1, and ALYREF. Loss of these factors impairs post-transcriptional mRNA maturation of JAK2, a crucial kinase for IFNγ signaling, resulting in abrogated JAK2 protein levels and diminished IFNγ signaling. Further analysis highlights a critical role for ERH in preventing intron retention in AU-rich regions in specific transcripts, such as JAK2. This regulation is markedly different from previously described retention of GC-rich introns. Overall, these findings reveal that post-transcriptional JAK2 processing is a critical rate-limiting step for the IFNγ-driven innate immune response.}, }
@article {pmid40585259, year = {2025}, author = {Zheng, Z and Yu, M and Ai, L and Wang, B and Lian, S and Liu, J and Li, L and Tsai, S and Kleinstiver, B and Ip, L}, title = {GenomePAM directs PAM characterization and engineering of CRISPR-Cas nucleases using mammalian genome repeats.}, journal = {Research square}, volume = {}, number = {}, pages = {}, doi = {10.21203/rs.3.rs-4552906/v1}, pmid = {40585259}, issn = {2693-5015}, abstract = {Characterizing the protospacer adjacent motif (PAM) requirements of different Cas enzymes is a bottleneck in the discovery of Cas proteins and their engineered variants in mammalian cell contexts. To overcome this challenge and to enable more scalable characterization of PAM preferences, we develop a method named GenomePAM that allows for direct PAM characterization in mammalian cells. GenomePAM leverages genomic repetitive sequences as target sites and does not require protein purification or synthetic oligos. GenomePAM uses a 20-nt protospacer that occurs ~16,942 times in every human diploid cell and is flanked by nearly random sequences. We demonstrate that GenomePAM can accurately characterize the PAM requirement of type II and type V nucleases, including the minimal PAM requirement of the near-PAMless SpRY and extended PAM for CjCas9. Beyond PAM characterization, GenomePAM allows for simultaneous comparison of activities and fidelities among different Cas nucleases on thousands of match and mismatch sites across the genome using a single gRNA and provides insight into the genome-wide chromatin accessibility profiles in different cell types.}, }
@article {pmid40585234, year = {2025}, author = {Jain, P and Orosco, C and Rananaware, S and Huang, B and Hanna, M and Ahmadimashhadi, MR and Lewis, J and Baugh, M and Bodin, A and Flannery, S and Langue, I and Fang, Z and Karalkar, V and Meister, K}, title = {DNA-guided CRISPR/Cas12 for RNA targeting.}, journal = {Research square}, volume = {}, number = {}, pages = {}, pmid = {40585234}, issn = {2693-5015}, abstract = {CRISPR-Cas nucleases are transforming genome editing, RNA editing, and diagnostics but have been limited to RNA-guided systems. We present ΨDNA, a DNA-based guide for Cas12 enzymes, engineered for specific and efficient RNA targeting. ΨDNA mimics a crRNA but with a reverse orientation, enabling stable Cas12-RNA assembly and activating trans-cleavage without RNA components. ΨDNAs are effective in sensing short and long RNAs and demonstrated 100% accuracy for detecting HCV RNA in clinical samples. We discovered that ΨDNAs can guide certain Cas12 enzymes for RNA targeting in cells, enhancing mRNA degradation via ribosome stalling and enabling multiplex knockdown of multiple RNA transcripts. This study establishes ΨDNA as a robust alternative to RNA guides, augmenting CRISPR-Cas12's potential for diagnostic applications and for targeted RNA modulation in cellular environments.}, }
@article {pmid40581000, year = {2025}, author = {Fraikin, N and Samuel, B and Burstein, D and Lesterlin, C}, title = {Strategies for zygotic gene expression during plasmid establishment.}, journal = {Plasmid}, volume = {}, number = {}, pages = {102754}, doi = {10.1016/j.plasmid.2025.102754}, pmid = {40581000}, issn = {1095-9890}, abstract = {Conjugative plasmids are key drivers of horizontal gene transfer and the spread of antimicrobial resistance. Their successful establishment in new hosts requires overcoming diverse bacterial defence mechanisms, such as restriction-modification systems, CRISPR-Cas systems, and the SOS response. Plasmids achieve this through a leading region-encoded zygotic program of anti-defence genes expressed early in conjugation. This program employs diverse strategies, including single-stranded promoters, repressed double-stranded promoters, and protein translocation. This review explores the diversity of these zygotic programs, the mechanisms underlying their timely regulation, and the array of anti-defence functions they encode. Further investigation of leading region genes is crucial for discovering novel counter-defence strategies and understanding their tailored regulation across diverse plasmid and bacterial species, ultimately enabling us to better understand and potentially manipulate plasmid transfer.}, }
@article {pmid40580284, year = {2025}, author = {Manjunatha, C and Aditya, K and Prasannakumar, MK and Pramesh, D and Buella, P and Patil, SS and Venkateshbabu, G and Harish, J and Karan, R and Ramegowda, HV}, title = {Isothermal Amplification Techniques: An Emerging Tool for On-Site Detection of Phytopathogens in Field Conditions.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2943}, number = {}, pages = {47-64}, pmid = {40580284}, issn = {1940-6029}, mesh = {*Nucleic Acid Amplification Techniques/methods ; *Plant Diseases/microbiology/virology ; *Molecular Diagnostic Techniques/methods ; Point-of-Care Systems ; *Plants/microbiology ; CRISPR-Cas Systems ; }, abstract = {This chapter reviews various isothermal amplification techniques, which are alternative to PCR for detecting plant pathogens. These methods, including NASBA, SDA, LAMP, HDA, and RPA, amplify nucleic acids at a constant temperature, making them potentially more suitable for point-of-care (POC) applications. The review compares these techniques regarding their mechanisms, advantages, and limitations, highlighting their potential for rapid, cost-effective, and on-site plant disease diagnosis, including the integration of these techniques with CRISPR-Cas systems and lab-on-a-chip technologies.}, }
@article {pmid40580153, year = {2025}, author = {Sharan, P and Kumar, BK and Kumar, A and Rai, P}, title = {Isothermal amplification for rapid and sensitive detection of hepatitis B virus: what we know so far? and way forward.}, journal = {Expert review of molecular diagnostics}, volume = {}, number = {}, pages = {}, doi = {10.1080/14737159.2025.2527634}, pmid = {40580153}, issn = {1744-8352}, abstract = {INTRODUCTION: Despite vaccine availability, Hepatitis B Virus (HBV) remains a major global health threat, especially in areas with low vaccination coverage and poor healthcare. Around 250 million people are chronically infected. Achieving the World Health Organisation's 2030 eradication goal is difficult, particularly due to diagnostic challenges in low-resource settings. While HBsAg detection is standard, low antigen levels and mutations hinder its reliability. Though molecular methods for HBV DNA offer high specificity, their cost and complexity limit use in under-resourced areas. Isothermal amplification emerges as a promising alternative, offering a more affordable, effective, and simplified approach to HBV detection, potentially improving access to timely diagnosis and care.<br><br>AREAS COVERED: This review evaluates the efficacy of various isothermal techniques to give insights into their benefits and limits, guiding researchers and clinicians in selecting the most effective assays for HBV molecular diagnostics.<br><br>EXPERT OPINION: RPA and PSR are the most promising isothermal assays for HBV detection in field settings. RPA is faster (&#x223c;20 min), works at low temperatures (37-42&#x202f;&#xb0;C), and uses stable lyophilized reagents, while PSR is simple, can be clubbed with visual detection, making both ideal for a low-resource setup.}, }
@article {pmid40580034, year = {2025}, author = {Lee, H and Rashid, F and Hwang, J and London, JA and Fishel, R and Berger, JM and Myong, S and Ha, T}, title = {A high-throughput single-molecule platform to study DNA supercoiling effect on protein-DNA interactions.}, journal = {Nucleic acids research}, volume = {53}, number = {12}, pages = {}, doi = {10.1093/nar/gkaf581}, pmid = {40580034}, issn = {1362-4962}, support = {R35 GM122569/NH/NIH HHS/United States ; R35 CA263778/NH/NIH HHS/United States ; R37 GM071747/NH/NIH HHS/United States ; R35 CA263778/NH/NIH HHS/United States ; R01 GM149729/NH/NIH HHS/United States ; R01 CA067007/NH/NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; }, mesh = {*DNA, Superhelical/metabolism/chemistry/genetics ; DNA Gyrase/metabolism ; Plasmids/genetics/chemistry/metabolism ; Protein Binding ; *MutS DNA Mismatch-Binding Protein/metabolism/genetics ; *Single Molecule Imaging/methods ; Escherichia coli Proteins/metabolism ; Adenosine Triphosphate/metabolism ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; DNA/metabolism/chemistry ; }, abstract = {DNA supercoiling significantly influences DNA metabolic pathways. To examine its impact on DNA-protein interactions at the single-molecule level, we developed a highly efficient and reliable protocol to modify plasmid DNA at specific sites, allowing us to label plasmids with fluorophores and biotin. We then induced physiological levels of negative or positive supercoiling in these plasmids using gyrase or reverse gyrase, respectively. By comparing supercoiled DNA with relaxed circular DNA, we assessed the effects of supercoiling on CRISPR-Cas9 and the mismatch repair protein MutS. We found that negative DNA supercoiling exacerbates off-target effects in DNA unwinding by Cas9. For MutS, we observed that both negative and positive DNA supercoiling enhance the binding interaction between MutS and a mismatched base pair but do not affect the rate of ATP-induced sliding clamp formation. These findings not only underscore the versatility of our protocol but also open new avenues for exploring the intricate dynamics of protein-DNA interactions under the influence of supercoiling.}, }
@article {pmid40532220, year = {2025}, author = {Lu, Q and Ye, C and Mao, W and Zeng, F and Liu, Z and Chen, R and Cheng, X and Gao, Y and Wang, M and Liu, M and Tang, S and Song, Y}, title = {Targeting Senescent Alveolar Type 2 Cells with a Gene-Editable FePt Dual-Atom Catalyst for Mitigating Idiopathic Pulmonary Fibrosis.}, journal = {ACS nano}, volume = {19}, number = {25}, pages = {23162-23176}, doi = {10.1021/acsnano.5c04686}, pmid = {40532220}, issn = {1936-086X}, mesh = {*Idiopathic Pulmonary Fibrosis/drug therapy/pathology/metabolism/genetics ; *Cellular Senescence/drug effects ; Animals ; Mice ; Humans ; Hyaluronic Acid/chemistry ; Gene Editing ; *Alveolar Epithelial Cells/drug effects/metabolism/pathology ; *Platinum/chemistry/pharmacology ; CRISPR-Cas Systems ; Catalysis ; *Iron/chemistry ; Mice, Inbred C57BL ; }, abstract = {Idiopathic pulmonary fibrosis (IPF) remains an age-related, fatal, incurable, epithelial-driven fibrotic lung disease despite the availability of approved antifibrotic drugs. The medical need for effective antipulmonary fibrotic therapies is thus very high. A promising therapeutic intervention for IPF is to target key cellular senescence processes in alveolar type 2 (AT2) cells. Herein, we introduce an inhalable gene-editable nanoplatform, comprising a CRISPR-Cas9 gene-editing system linked to a core FePt diatomic catalyst, encapsulated within a biocompatible hyaluronic acid (HA) surface layer (FePtR@HA). The FePt diatomic site facilitates H2O2 bridge adsorption, enabling efficient O-O bond cleavage and rapid catalytic conversion. The strong Fe-Pt interaction modulates the metal's d-band center, optimizing the adsorption of oxygen-containing intermediates. This precise regulation efficiently clears ROS, delivering robust antioxidant and antisenescence effects to AT2 cells. Simultaneously, the CRISPR-Cas9 gene editing system knocks down the pro-aging gene KAT7, reducing senescence-associated secretory phenotype (SASP) factors and further reversing AT2 cell senescence. Additionally, we demonstrated the antifibrotic efficacy of FePtR@HA in a lung-on-a-chip model, where it reprogrammed the fibrotic microenvironment, prevented myofibroblast recruitment to AT2 cells. Moreover, FePtR@HA showed satisfactory results in IPF mouse models, alleviating fibrosis and presenting a highly promising approach to combat the progression of IPF.}, }
@article {pmid40517335, year = {2025}, author = {Wang, Q and Yang, S and Chen, X and Song, S and Wu, Y and Li, Y and Meng, X and Dai, L and Yang, Z and Wu, H and Xia, J and Xu, J}, title = {Mutually Activated Dual-Exponential Amplification DNA Machine Enhances Robust CRISPR/Cas12a Feedback Propagation for Ultrasensitive miRNA Detection.}, journal = {Analytical chemistry}, volume = {97}, number = {25}, pages = {13504-13513}, doi = {10.1021/acs.analchem.5c01953}, pmid = {40517335}, issn = {1520-6882}, mesh = {*MicroRNAs/analysis/genetics/blood ; Humans ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *DNA/genetics/chemistry ; *Breast Neoplasms/diagnosis/genetics ; Limit of Detection ; Female ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Breast cancer (BC) remains a critical global health challenge, necessitating ultrasensitive methods for detecting biomarkers such as miR-155, a key regulator in BC progression. Here, we present a mutually activated dual-exponential amplification DNA machine (MADEA-DNA machine) for ultrasensitive miR-155 detection. This system integrates exponential rolling circle amplification (E-RCA) and autocatalytic incremental strand displacement amplification (AI-SDA), driven by a bidirectional activation mechanism. Target miR-155 initiates E-RCA via a functional primer probe (FPP) or AI-SDA through a functional hairpin probe (FHP), with amplification products cross-activating the counterpart system to establish a self-reinforcing loop. The resultant amplicons further activate CRISPR/Cas12a, enabling the trans-cleavage of fluorescent reporters for signal amplification. The MADEA-DNA machine achieves a detection limit of 1.26 fM, with a dynamic range spanning 5 fM-10 nM, and demonstrates exceptional specificity against mismatched and nontarget miRNAs. Validation in human serum revealed significantly elevated miR-155 levels in BC patients versus healthy donors, corroborated by qRT-PCR. This system combines machine-like operational efficiency, dual-amplification synergy, and CRISPR-enhanced sensitivity, offering a robust platform for liquid biopsy applications in early BC diagnostics.}, }
@article {pmid40511702, year = {2025}, author = {Sun, X and Li, X and Jiang, H and Shan, Y and Zhou, S and Wang, Z}, title = {Accurate Diagnosis of Colorectal Cancer Using a Combination of Lectin-Induced Recombinase Polymerase Amplification and CRISPR/Cas12a Assay on a Point-of-Care Testing Platform with Deep Learning Assistant.}, journal = {Analytical chemistry}, volume = {97}, number = {25}, pages = {13086-13094}, doi = {10.1021/acs.analchem.5c00468}, pmid = {40511702}, issn = {1520-6882}, mesh = {*Colorectal Neoplasms/diagnosis ; Humans ; *CRISPR-Cas Systems ; Animals ; *Point-of-Care Testing ; Mice ; *Deep Learning ; *Nucleic Acid Amplification Techniques/methods ; *Lectins/metabolism/chemistry ; *Recombinases/metabolism ; Exosomes/metabolism ; Cell Line, Tumor ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Specific glycosylation patterns on exosome surfaces represent novel diagnostic biomarkers for cancer liquid biopsy. Lectins can induce exosome aggregation through multiple bindings with exosomal glycoproteins. In this work, we developed a one-pot lectin-induced recombinase polymerase amplification (RPA) and CRISPR/Cas12a-mediated cleavage assay (LI-RPA-CRISPR/Cas12a) for diagnosing colorectal cancer (CRC) through the interactions of abundant α-fucose residues on CRC cell-derived exosome surfaces with Ulex Europaeus Agglutinin I (UEA-I). The combination of a homemade portable isothermal amplification device, the as-proposed LI-RPA-CRISPR/Cas12a exhibits a wide detection range from 2 × 10[6] to 1 × 10[2] extracellular vehicles (EVs) μL[-1] with a visual limit of detection (LOD) as low as 1.0 × 10[2] EVs μL[-1], and has been successfully utilized to dynamically monitor the progression of tumors in mice-bearing SW480 CRC subtype at an early stage. After integration with a long short-term memory (LSTM) deep learning model, the LI-RPA-CRISPR/Cas12a achieves accurate diagnosis of primary colorectal cancer with a drop of blood through a smartphone-based data analysis application, reaching an accuracy of 95% in 100 clinical samples. This rapid, sensitive, and user-friendly approach provides a promising platform for point-of-care testing (POCT) diagnosis of CRC, enabling early detection and monitoring of disease progression through a minimally invasive liquid biopsy.}, }
@article {pmid40491322, year = {2025}, author = {Cao, J and Guo, Z and Xu, X and Li, P and Fang, Y and Deng, S}, title = {Advances in CRISPR-Cas9 in lineage tracing of model animals.}, journal = {Animal models and experimental medicine}, volume = {8}, number = {6}, pages = {1004-1022}, pmid = {40491322}, issn = {2576-2095}, support = {22SH19//Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences and Comparative Medicine Center, Peking Union Medical College, Collaborative Innovation Program of the Chinese Academy of Sciences/ ; 2023-PT180-01//Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences/ ; }, mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *Cell Lineage/genetics ; *Models, Animal ; Mice ; Swine ; Zebrafish ; *Cell Tracking/methods ; }, abstract = {Cell lineage tracing is a key technology for describing the developmental history of individual progenitor cells and assembling them to form a lineage development tree. However, traditional methods have limitations of poor stability and insufficient resolution. As an efficient and flexible gene editing tool, CRISPR-Cas9 system has been widely used in biological research. Furthermore, CRISPR-Cas9 gene editing-based tracing methods can introduce fluorescent proteins, reporter genes, or DNA barcodes for high-throughput sequencing, enabling precise lineage analysis, significantly improving precision and resolution, and expanding its application range. In this review, we summarize applications of CRISPR-Cas9 system in cell lineage tracing, with special emphasis on its successful applications in traditional model animals (e.g., zebrafish and mice), large animal models (pigs), and human cells or organoids. We also discussed its potential prospects and challenges in xenotransplantation and regenerative medicine.}, }
@article {pmid40481182, year = {2025}, author = {Yu, L and Liu, Y and Lin, X}, title = {Transitioning from native to synthetic receptors: broadening T-cell engineering and beyond.}, journal = {Cellular &amp; molecular immunology}, volume = {22}, number = {7}, pages = {712-729}, pmid = {40481182}, issn = {2042-0226}, support = {82341212//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82293664//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {Humans ; *T-Lymphocytes/immunology ; Animals ; *Receptors, Antigen, T-Cell/genetics/immunology ; Gene Editing ; *Immunotherapy, Adoptive/methods ; *Cell Engineering/methods ; *Receptors, Chimeric Antigen/genetics/immunology ; CRISPR-Cas Systems ; *Receptors, Artificial/genetics/immunology ; }, abstract = {T-cell immunotherapy has progressed rapidly, evolving from native T-cell receptor biology to the development of innovative synthetic receptors that extend therapeutic applications beyond cancer. This review explores engineering strategies, ranging from natural TCRs to synthetic receptors, that increase T-cell activation and therapeutic potential. We begin by highlighting the foundational role of native receptors in the T-cell response, emphasizing how these structural and functional insights inform the design of next-generation synthetic receptors. Comparisons between CAR and TCR-like synthetic receptors underscore their respective advantages in specificity, efficacy, and safety, as well as potential areas for further improvement. In addition, gene editing technologies such as CRISPR-Cas9 enable precise modifications to the T-cell genome, enhancing receptor performance and minimizing immunogenic risks. In addition to tumors, these engineered T cells can be directed against viral infections, autoimmune disorders, and other diseases. We also explore advanced strategies that engage multiple immune cell types to achieve synergistic, durable responses. By demonstrating how native and synthetic receptors collectively drive innovation, this review aims to inspire new research directions and ultimately expand the scope of T-cell engineering for universal therapeutic applications.}, }
@article {pmid40434897, year = {2025}, author = {Chen, L and Chen, X and Kashina, A}, title = {Amino acid-level differences in alpha-tubulin sequences are uniquely required for meiosis.}, journal = {Molecular biology of the cell}, volume = {36}, number = {7}, pages = {ar89}, doi = {10.1091/mbc.E24-11-0529}, pmid = {40434897}, issn = {1939-4586}, mesh = {Schizosaccharomyces/metabolism/genetics ; *Meiosis/physiology/genetics ; *Tubulin/metabolism/genetics ; Schizosaccharomyces pombe Proteins/metabolism/genetics ; Amino Acid Sequence ; CRISPR-Cas Systems ; Protein Isoforms/metabolism/genetics ; Amino Acids/metabolism/genetics ; Spores, Fungal/metabolism ; Spindle Apparatus/metabolism ; }, abstract = {Members of the tubulin gene family members are essential components of the cytoskeleton; however, functional diversity of tubulin isoforms is poorly understood. Here, we addressed this question using Schizosaccharomyces pombe as a model system. These yeast encode two α-tubulins, nda2 and atb2, that are very similar at the amino acid level but differ in their roles in organism's survival: nda2 deletion is lethal, while lack of atb2 does not interfere with cell viability. Using CRISPR-Cas9 gene editing, we generated a yeast strain expressing atb2 amino acid sequence utilizing nda2 codon usage in the native nda2 locus. Such nda2-coded atb2 (NCA) yeast, unlike nda2 knockout, were viable and displayed no visible abnormalities in vegetative life cycle. Instead, they displayed strong impairments in sporulation and meiosis, linked to altered balance of several spindle proteins. Our data indicate that nda2 protein is uniquely required for normal meiosis, and identify novel protein- and nucleotide-level determinants driving functional distinction between closely related tubulin isoforms.}, }
@article {pmid40378737, year = {2025}, author = {Zheng, H and Guo, J and Wang, H and Wang, Y and Zhou, W and Liu, K and Ma, Q and Qi, Y}, title = {A zeolitic imidazolate framework-90 enhanced ultrasensitive ATP sensing platform with HCR and CRISPR-Cas12a dual signal amplification for live bacteria detection.}, journal = {Journal of hazardous materials}, volume = {494}, number = {}, pages = {138612}, doi = {10.1016/j.jhazmat.2025.138612}, pmid = {40378737}, issn = {1873-3336}, mesh = {*Adenosine Triphosphate/analysis ; *Zeolites/chemistry ; *Imidazoles/chemistry ; CRISPR-Cas Systems ; *Staphylococcus aureus/isolation &amp; purification ; *Biosensing Techniques/methods ; Aptamers, Nucleotide/chemistry ; Limit of Detection ; Nucleic Acid Hybridization ; *Metal-Organic Frameworks/chemistry ; }, abstract = {Bacteria are prevalent environmental pollutants. Live bacteria can proliferate and spread under appropriate conditions, presenting higher risks compared to non-viable counterparts. However, detecting live bacteria remains a challenge. Adenosine triphosphate (ATP), an energy currency in organisms, offers a reliable biomarker for the live bacteria sensing. Herein, we developed a zeolitic imidazolate framework-90 (ZIF-90) enhanced ATP sensing platform to detect live bacteria. The nanosystem which based on ZIF-90, encapsulating the DNA decorated magnetic beads (MB@S1) through self-assembly. When the S. aureus aptamers on ZIF-90 bonded to bacteria, the skeleton structure of ZIF-90 was disrupted by ATP leakage from live bacteria, leading to the release of MB@S1. Then, the MB@S1 initiated the hybridization chain reaction (HCR) and they were transduced by CRISPR-Cas12a to amplify the signal twice. The proposed ZIF-90 blocking sensing strategy (ZIF-90 strategy) exhibited remarkable sensitivity with the limit of detection (LOD) down to 0.223 pM for ATP detection, about 500 folds lower than the traditional ATP aptamer blocking sensing strategy (aptamer strategy). Furthermore, we used a smartphone for on-site analysis, realizing the quantification of live S. aureus with the LOD of 2.0 CFU/mL. Therefore, the approach possessed great application potential for public health, environment monitoring, bioanalysis and food safety.}, }
@article {pmid40299756, year = {2025}, author = {An, Y and Wang, SQ and Jia, XY and Jiao, X and Qu, MQ and Dong, Y and Wang, ZY and Ma, ZY and Yang, S and Han, X and Huang, LC and Chen, NN and Jiang, C and Lu, MZ and Dai, JF and Zhang, J}, title = {Bioengineered poplar fibres via PagGLR2.8 editing: A synergistic design for high-performance biocomposites.}, journal = {Plant biotechnology journal}, volume = {23}, number = {7}, pages = {2824-2838}, pmid = {40299756}, issn = {1467-7652}, support = {32371902//National Natural Science Foundation of China/ ; 51903222//National Natural Science Foundation of China/ ; 2021YFD2200205//National Key Research and Development Program of China/ ; 2021C02070-1//the Key Scientific and Technological Grant of Zhejiang for Breeding New Agricultural Varieties/ ; 2018FR013//the research foundation of Zhejiang A&amp;F University/ ; //Zhejiang students' technology and innovation program/ ; }, mesh = {*Populus/genetics/metabolism ; Plants, Genetically Modified/genetics ; Polyesters ; Gene Editing ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Bioengineering ; }, abstract = {The urgent need to replace petroleum-derived materials with sustainable alternatives drives innovation at the nexus of plant biotechnology and materials science. Here, we engineered Populus alba × P. glandulosa '84 K' through CRISPR-Cas9-mediated knockout of PagGLR2.8, a glutamate receptor gene regulating vascular development, to investigate its role in fibre biosynthesis and composite performance. Knockout of PagGLR2.8 improved the quality of poplar fibre by altering the structure and development mode of poplar vascular tissue. Our study established the relationship between fibre quantity and structure and the performance of polylactic acid (PLA) composites. The mechanical and fire-resistance properties of these transgenic plant fibres/PLA composites significantly outperformed those of pure PLA, demonstrating the potential of phloem fibres to reinforce toughened composites. Notably, we also evaluated flammability and dripping behaviours, with findings indicating that our optimised fibre/PLA composites exhibit superior strengths, modulus, fire resistance, and anti-dripping, surpassing those of PLA. This research unveils a groundbreaking approach to regulating composite properties through genetic manipulation and highlights the promising potential of plant-derived materials in enriching forest resources and advancing the sustainable utilisation of poplar fibres and polymers.}, }
@article {pmid39397560, year = {2025}, author = {Gou, S and Liu, Y and Li, Q and Yang, J and Qiu, L and Zhao, Y}, title = {CRISPR/Cas12 System-Based Assay for Rapid, Sensitive Detection of Rotavirus in Food Samples.}, journal = {Foodborne pathogens and disease}, volume = {22}, number = {7}, pages = {459-466}, doi = {10.1089/fpd.2024.0078}, pmid = {39397560}, issn = {1556-7125}, mesh = {*Rotavirus/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Food Microbiology/methods ; *Molecular Diagnostic Techniques/methods ; Humans ; *Food Contamination/analysis ; RNA, Viral/genetics ; }, abstract = {Foodborne viruses have become an important threat to food safety and human health. Among the foodborne viruses, group A rotavirus is the most important pathogen of diarrhea in autumn and winter. The field detection of rotavirus is crucial for the early control of infection and patient management. Quantitative real-time reverse transcription-polymerase chain reaction is the most widely used in virus detection. However, the technique relies on high-cost instruments and trained personnel, which limit its use in field detection. In this study, we developed accurate, realizable, and simple detection methods by combining optimized CRISPR (clustered regularly interspaced short palindromic repeats) Cas12 and reverse transcription loop-mediated isothermal amplification (RT-LAMP) (reverse transcription loop-mediated isothermal amplification) to reduce the requirements for temperature control and costly real-time fluorescence polymerase chain reaction instruments. We investigated two nucleic acid detection systems combining RT-LAMP with CRISPR Cas12a and RT-LAMP with CRISPR Cas12b and compared them with reverse transcription-quantitative polymerase chain reaction. The resulting detection system only needs a reaction temperature and in single tube to react for 60 min with the detection sensitivity of 38 copies/μL. Overall, this study developed an innovative method for the rapid detection of rotavirus in food samples, which will help to effectively identify food contaminated by pathogens and prevent human infections and economic losses caused by disease outbreaks.}, }
@article {pmid40579649, year = {2025}, author = {Liu, ZG and Wang, N and Wang, WJ and Huang, L and Chi, SS and Nie, YX and Yuan, MS and Sun, YR and Mu, YL and Li, K}, title = {Transcriptome analysis reveals no obvious unintended effects in the spleen of CRISPR/Cas9-mediated CD163 and pAPN double-knockout pigs.}, journal = {Functional &amp; integrative genomics}, volume = {25}, number = {1}, pages = {139}, pmid = {40579649}, issn = {1438-7948}, support = {2022ZD04020//Biological Breeding-National Science and Technology Major Project/ ; CAAS-CSAB-202401//Innovation Program of Chinese Academy of Agricultural Sciences/ ; 202102//Outstanding Talents Training Fund in Shenzhen/ ; }, mesh = {Animals ; *CRISPR-Cas Systems ; CD163 Antigen ; *Antigens, Differentiation, Myelomonocytic/genetics/metabolism ; *Receptors, Cell Surface/genetics/metabolism ; *Spleen/metabolism ; Swine/genetics ; *Antigens, CD/genetics/metabolism ; *Transcriptome ; Gene Editing ; Gene Knockout Techniques ; Gene Expression Profiling ; Membrane Proteins ; }, abstract = {Gene editing provides powerful tools for farm animal breeding. Our group previously obtained CD163/pAPN double-knockout (DKO) pigs via CRISPR/Cas9 and somatic cell nuclear transfer. These pigs are not only resistant to three infectious viruses but also maintain normal production performance. However, unintended effects of CRISPR/Cas9 tools may pose potential risks to animal well-being or safety. This study aimed to characterize the differences in splenic gene expression between wild-type (WT) and DKO pigs, providing a basis for safety evaluation of gene-edited animals. A comprehensive transcriptional panorama reflected considerable congruence in the aggregate gene expression profiles of the DKO and WT pigs. Comparisons between 35-day-old and 11-month-old DKO pigs and their WT equivalents revealed 225 and 242 differentially expressed genes (DEGs), respectively, a count significantly lower than that of the DEGs in the disparate developmental stage comparison groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that the majority of DEGs between DKO and WT pigs correlated with the biological functions of CD163 and pAPN, without any alterations in the expression of tumor suppressor genes in DKO pigs. This revealed a less pronounced effect of dual gene editing on the gene expression profile of porcine spleens than the effect of animal maturation, with no evident unanticipated consequences observed in DKO pigs.}, }
@article {pmid40579133, year = {2025}, author = {Mo, Y and Shu, Y and Mo, Y and Liu, J and Xu, O and Deng, H and Wang, Q}, title = {[CRISPR-Cas9-mediated CDC20 gene knockout inhibits cervical cancer cell proliferation, invasion and metastasis].}, journal = {Nan fang yi ke da xue xue bao = Journal of Southern Medical University}, volume = {45}, number = {6}, pages = {1200-1211}, doi = {10.12122/j.issn.1673-4254.2025.06.09}, pmid = {40579133}, issn = {1673-4254}, mesh = {Humans ; *Uterine Cervical Neoplasms/pathology/genetics/metabolism ; Female ; *Cdc20 Proteins/genetics ; *Cell Proliferation ; Animals ; Cell Movement ; Neoplasm Invasiveness ; Apoptosis ; Mice, Nude ; beta Catenin/metabolism ; *CRISPR-Cas Systems ; Mice ; Cell Line, Tumor ; Gene Knockout Techniques ; Neoplasm Metastasis ; }, abstract = {OBJECTIVES: To study the effect of CDC20 knockdown on proliferation, migration and invasion of cervical cancer cells and its underlying mechanism.<br><br>METHODS: CDC20 expression in cervical cancer tissues was analyzed using the TCGA database, and the protein expressions of CDC20 and &#x3b2;-Catenin in clinical specimens of cervical cancer and adjacent tissues were detected using immunohistochemistry. A dual target sgRNA2&amp;7 sequence for CDC20 gene was designed for CDC20 gene knockdown in cervical cancer C33A cells using CRISPR/Cas9 technology, and CDC20 mRNA and protein expression levels in the transfected cells were detected using qRT-PCR and Western blotting. The changes in proliferation, cell cycle, apoptosis, migration and invasiveness of the transfected cells were evaluated using colony-forming assay, fluorescence activated cell sorting (FACS) and Transwell assay. In the animal experiment, na&#xef;ve C33A cells and the cells with CDC20 knockdown were injected subcutaneously into the left and right axillae of nude mice (n=5) to observe tumor growth. The expressions of CDC20 and &#x3b2;-Catenin proteins in transfected cells and the xenograft were analyzed using Western blotting, and their interaction was confirmed by co-immunoprecipitation (CoIP) and immunofluorescence co-localization assays.<br><br>RESULTS: Cervical cancer tissues expressed significantly higher CDC20 and &#x3b2;&#x2011;Catenin levels than the adjacent tissues. C33A cells with CDC20 knockdown showed reduced proliferation, increased apoptosis, and lowered migration and invasion abilities. CDC20 knockdown significantly suppressed the growth of C33A cell xenograft in nude mice, and the tumor-bearing mice did not exhibit obvious body mass changes. CDC20 and &#x3b2;-Catenin levels were both significantly lowered in C33A cells with CDC20 knockdown. Co-immunoprecipitation and co-localization assays confirmed the interaction between CDC20 and &#x3b2;&#x2011;Catenin.<br><br>CONCLUSIONS: CDC20 is highly expressed in cervical cancer tissues, and CDC20 knockdown can suppress proliferation, invasion, and metastasis while enhancing apoptosis of C33A cells, which is closely related with the regulation of the Wnt/&#x3b2;-Catenin signaling pathway.}, }
@article {pmid40578341, year = {2025}, author = {Mahler, M and Cui, L and Smith, LM and Wandera, KG and Dietrich, O and Mayo-Muñoz, D and Balamkundu, S and Lee, ME and Ye, H and Liu, CF and Wu, J and Mathew, J and Dubrulle, J and Malone, LM and Jackson, SA and Fairbanks, AJ and Dedon, PC and Brouns, SJJ and Fineran, PC}, title = {Phage arabinosyl-hydroxy-cytosine DNA modifications result in distinct evasion and sensitivity responses to phage defense systems.}, journal = {Cell host &amp; microbe}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.chom.2025.06.005}, pmid = {40578341}, issn = {1934-6069}, abstract = {Bacteria encode diverse anti-phage systems, such as CRISPR-Cas and restriction modification (RM), which limit infection by targeting phage DNA. We identified a DNA modification in phages, i.e., 5-arabinosyl-hydroxy-cytosine (5ara-hC), which adds arabinose to cytosines via a hydroxy linkage and protects phage from DNA targeting. The hydroxy linkage was common among arabinoslyated phages, with some arabinosylated phages encoding arabinose-5ara-hC transferases (Aat) that add a second or third arabinose to DNA. DNA arabinosylation enables evasion from DNA-targeting type I CRISPR-Cas and type II RM systems. However, arabinosylated phages remain sensitive to RNA-targeting CRISPR-Cas (type III and VI) and promiscuous type IV restriction endonucleases. 5ara-hC enables evasion of glycosylase defenses that target phages with glucosylated hydroxymethyl cytosines, and 5ara-ara-hC protects against some defenses capable of targeting 5ara-hC-modified phages. Collectively, this work identifies DNA modifications that enable phages to evade multiple defenses yet remain vulnerable to some systems that target RNA or modified nucleobases.}, }
@article {pmid40575705, year = {2025}, author = {Chen, S and Liu, Z and Lo, CH and Wang, Q and Ning, K and Zhang, Q and Zhao, J and Shen, Y and Sun, Y}, title = {Gene therapy for ocular hypertension using hfCas13d-mediated mRNA targeting.}, journal = {PNAS nexus}, volume = {4}, number = {6}, pages = {pgaf168}, pmid = {40575705}, issn = {2752-6542}, abstract = {Glaucoma is a major global cause of irreversible vision loss. It is marked by elevated intraocular pressure (IOP) and the loss of retinal ganglion cells (RGC). While there are medical and surgical therapies for glaucoma aiming to reduce aqueous humor production or enhance its drainage, these treatments are often inadequate for effectively managing the disease. In this study, we developed a targeted therapy for glaucoma by knocking down two genes associated with aqueous humor production (aquaporin 1 [AQP1] and carbonic anhydrase type 2 [CA2]) using Cas13 RNA editing systems. We demonstrate that hfCas13d-mediated knockdown of AQP1 and CA2 significantly lowers IOP in wild-type mice and in a corticosteroid-induced glaucoma mouse model. We show that the lowered IOP results from decreasing aqueous production without affecting the outflow facility; this treatment also significantly promotes RGC survival as compared with untreated control groups. Therefore, CRISPR-Cas-based gene editing may be an effective treatment to lower IOP for glaucomatous optic neuropathy.}, }
@article {pmid40575544, year = {2025}, author = {Choi, JH and Yoon, J and Chen, M and Shin, M and Goldston, LL and Lee, KB and Choi, JW}, title = {CRISPR/Cas-Based Nanobiosensor Using Plasmonic Nanomaterials to Detect Disease Biomarkers.}, journal = {Biochip journal}, volume = {19}, number = {2}, pages = {167-181}, pmid = {40575544}, issn = {1976-0280}, abstract = {The development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) technology (CRISPR/Cas) as a gene-editing tool has the potential to revolutionize nucleic acid analysis. Recently, CRISPR/Cas systems have demonstrated considerable promise in the development of biosensors for the detection of essential disease biomarkers because they exhibit nonspecific collateral cleavage properties upon target sequence recognition. However, the CRISPR/Cas-based biosensors developed thus far have limitations, such as complicated steps, low sensitivity, low selectivity, and low signal-to-noise ratios. These limitations can be overcome by incorporating the unique characteristics of plasmonic nanomaterials into CRISPR/Cas systems to enhance the signal and improve the sensitivity of these biosensors. From this perspective, current interdisciplinary studies on CRISPR/Cas-based nanobiosensors comprising plasmonic nanomaterials can contribute to the development of highly sensitive CRISPR/Cas-based nanobiosensors. These nanobiosensors can detect attractive disease biomarkers, such as viral nucleic acids, small molecules, and proteins. This review article provides a thorough overview of nanobiosensors that incorporate CRISPR/Cas systems combined with plasmonic nanomaterials to enhance biosensing performance. We believe this review will inspire novel approaches and further innovation in the fields of molecular diagnostics and biomedicine aimed at using CRISPR/Cas systems and plasmonic nanomaterials for more personalized and effective medical treatments.}, }
@article {pmid40573878, year = {2025}, author = {Ullah, F and Ali, S and Siraj, M and Akhtar, MS and Zaman, W}, title = {Plant Microbiomes Alleviate Abiotic Stress-Associated Damage in Crops and Enhance Climate-Resilient Agriculture.}, journal = {Plants (Basel, Switzerland)}, volume = {14}, number = {12}, pages = {}, doi = {10.3390/plants14121890}, pmid = {40573878}, issn = {2223-7747}, abstract = {Plant microbiomes, composed of a diverse array of microorganisms such as bacteria, fungi, archaea, and microalgae, are critical to plant health and resilience, playing key roles in nutrient cycling, stress mitigation, and disease resistance. Climate change is expected to intensify various abiotic stressors, such as drought, salinity, temperature extremes, nutrient deficiencies, and heavy metal toxicity. Plant-associated microbiomes have emerged as a promising natural solution to help mitigate these stresses and enhance agricultural resilience. However, translating laboratory findings into real-world agricultural benefits remains a significant challenge due to the complexity of plant-microbe interactions under field conditions. We explore the roles of plant microbiomes in combating abiotic stress and discuss advances in microbiome engineering strategies, including synthetic biology, microbial consortia design, metagenomics, and CRISPR-Cas, with a focus on enhancing their practical application in agriculture. Integrating microbiome-based solutions into climate-smart agricultural practices may contribute to long-term sustainability. Finally, we underscore the importance of interdisciplinary collaboration in overcoming existing challenges. Microbiome-based solutions hold promise for improving global food security and promoting sustainable agricultural practices in the face of climate change.}, }
@article {pmid40573432, year = {2025}, author = {Hu, G and Wei, Z and Guo, J and Zhao, K and Qiao, Q and Zhu, X and Wu, T and Rong, H and Ning, S and Hao, Z and Chi, Y and Cui, L and Ge, Y}, title = {A Single-Tube Two-Step MIRA-CRISPR/Cas12b Assay for the Rapid Detection of Mpox Virus.}, journal = {Viruses}, volume = {17}, number = {6}, pages = {}, doi = {10.3390/v17060841}, pmid = {40573432}, issn = {1999-4915}, support = {2023YFC2605100, 2023YFC2605104//the National Key R&amp;D Program of China/ ; BK20231374, BK20221413//the natural science foundation of Jiangsu Province/ ; }, mesh = {*CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Animals ; Humans ; Limit of Detection ; *Capripoxvirus/isolation &amp; purification/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Associated Proteins/genetics ; }, abstract = {Mpox is a zoonotic disease caused by the Mpox virus (MPXV). The rapid and accurate diagnosis of MPXV is essential for the timely and effective prevention, control, and treatment of the disease. In this study, we combined Multienzyme Isothermal Rapid Amplification (MIRA) (at 42 °C) and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 12b(CRISPR/Cas12b) (at 60 °C) to develop a single-tube two-step assay for rapid MPXV detection, leveraging the distinct physical states of tricosane at these temperatures. MIRA amplification primers and CRISPR/cas12b SgRNA were designed based on the MPXV F3L gene. After screening the primers and sgRNAs, the reaction conditions were optimized, and the performances of the assay were evaluated. The detection limit (LOD) of this single-tube two-step MIRA-CRISPR/Cas12b assay for MPXV is four copies of DNA molecules. No cross-reactivity with other pathogens (herpes simplex virus (HSV), Epstein-Barr virus (EBV), Coxsackievirus A16 (CVA16), Enterovirus A71 (EV-A71), and measles virus (MeV)) was found. The assay also showed good consistency with quantitative real-time PCR (qPCR) (Kappa = 0.9547, p < 0.05, n = 100) in the detection of clinical samples, with a sensitivity of 98.5% and a specificity of 97.0%. The single-tube two-step MIRA-CRISPR/Cas12b assay permits the rapid (within 45 min), sensitive, and specific detection of MPXV. The lack of need for opening the reaction tube eliminates the risk of product contamination.}, }
@article {pmid40573367, year = {2025}, author = {Workman, AM and Heaton, MP and Vander Ley, BL}, title = {CD46 Gene Editing Confers Ex Vivo BVDV Resistance in Fibroblasts from Cloned Angus Calves.}, journal = {Viruses}, volume = {17}, number = {6}, pages = {}, doi = {10.3390/v17060775}, pmid = {40573367}, issn = {1999-4915}, support = {3040-32000-036-00D//USDA-ARS appropriated funds/ ; }, mesh = {Animals ; Cattle ; *Gene Editing ; *Fibroblasts/virology/immunology ; *Bovine Virus Diarrhea-Mucosal Disease/genetics/virology/immunology/prevention &amp; control ; *Membrane Cofactor Protein/genetics ; Cloning, Organism ; *Diarrhea Viruses, Bovine Viral ; CRISPR-Cas Systems ; *Disease Resistance/genetics ; Female ; }, abstract = {A previous study demonstrated that a 19-nucleotide edit, encoding a six amino acid substitution in the bovine CD46 gene, dramatically reduced bovine viral diarrhea virus (BVDV) susceptibility in a cloned Gir (Bos indicus) heifer. The present study aimed to replicate this result in American Angus (Bos taurus) using genetically matched controls and larger sample sizes. CRISPR/Cas9-mediated homology-directed repair introduced the identical CD46 edit, encoding the A82LPTFS amino acid sequence, into exon 2 of CD46 in primary Angus fibroblasts. Thirty-three cloned embryos (22 CD46-edited and 11 unedited) were transferred to recipient cows. However, all pregnancies resulted in pre- and perinatal losses due to cloning-related abnormalities, preventing in vivo BVDV challenge. Consequently, ex vivo BVDV susceptibility assays were performed on primary fibroblast cell lines rescued from deceased cloned Angus calves. Infection studies revealed significantly reduced susceptibility in the edited lines, comparable to the resistance previously observed from the edited Gir heifer. These studies extend the applicability of this finding from Gir to the most common US beef breed, Angus, suggesting the potential for broad application of CD46 editing in BVDV control. Continued advancements in cloning technology will enhance the potential of gene-editing for producing disease-resistant livestock.}, }
@article {pmid40572275, year = {2025}, author = {Kalu, MC and Acharya, A and Jorth, P and Wong-Beringer, A}, title = {ESBL-Producing Escherichia coli and Klebsiella pneumoniae Exhibit Divergent Paths During In-Human Evolution Towards Carbapenem Resistance.}, journal = {Microorganisms}, volume = {13}, number = {6}, pages = {}, pmid = {40572275}, issn = {2076-2607}, support = {not applicable//Merck &amp; Co., Inc., Rahway, NJ, USA (United States)/ ; }, abstract = {Treatment of infections caused by ESBL-producing Escherichia coli (EC) and Klebsiella pneumoniae (KP) with carbapenem antibiotics can lead to the development of carbapenem resistance over time through the acquisition of porin mutations and plasmids bearing blaKPC. However, the impact of genetic background and the presence of CRISPR-Cas systems on the evolutionary path towards carbapenem resistance in EC and KP has yet to be investigated. The in-human evolution following repeated carbapenem treatment among ESBL-producing Escherichia coli (EC) and Klebsiella pneumoniae (KP) clinical pairs (n = 45 pairs) was examined to determine the relationship between strain genetic background (MLST, CRISPR-Cas) and the evolved genetic mutations related to resistance, virulence, and metabolism by whole genome sequencing. ST131 and ST258 were predominant among seven distinct STs in EC (70%, 19/27) and 11 STs in KP (33%, 6/18), respectively. Complete CRISPR-Cas systems were present in 22% EC (6/27) and 27.8% (5/18) KP pairs, but none in strains belonging to ST131 or ST258; partial loss of CRISPR-Cas was associated with increased carbapenem resistance. Porin, virulence, and metabolism-related genetic mutations were present on the chromosome in both the EC and KP evolved strains, but their presence was differentially associated with the CRISPR-Cas system. Future research on the role of antibiotic exposure in the species-specific resistance evolution of the Enterobacterales could guide antimicrobial stewardship efforts.}, }
@article {pmid40572209, year = {2025}, author = {Antequera-Zambrano, L and Parra-Sánchez, &#xc1; and González-Paz, L and Fernandez, E and Martinez-Navarrete, G}, title = {Distribution of Genetic Determinants Associated with CRISPR-Cas Systems and Resistance to Antibiotics in the Genomes of Archaea and Bacteria.}, journal = {Microorganisms}, volume = {13}, number = {6}, pages = {}, pmid = {40572209}, issn = {2076-2607}, abstract = {The CRISPR-Cas system represents an adaptive immune mechanism found across diverse Archaea and Bacteria, allowing them to defend against invading genetic elements such as viruses and plasmids. Despite its broad distribution, the prevalence and complexity of CRISPR-Cas systems differ significantly between these domains. This study aimed to characterize and compare the genomic distribution, structural features, and functional implications of CRISPR-Cas systems and associated antibiotic resistance genes in 30 archaeal and 30 bacterial genomes. Through bioinformatic analyses of CRISPR arrays, cas gene architectures, direct repeats (DRs), and thermodynamic properties, we observed that Archaea exhibit a higher number and greater complexity of CRISPR loci, with more diverse cas gene subtypes exclusively of Class 1. Bacteria, in contrast, showed fewer CRISPR loci, comprising a mix of Class 1 and Class 2 systems, with Class 1 representing the majority (~75%) of the detected systems. Notably, Bacteria lacking CRISPR-Cas systems displayed a higher prevalence of antibiotic resistance genes, suggesting a possible inverse correlation between the presence of these immune systems and the acquisition of such genes. Phylogenetic and thermodynamic analyses further highlighted domain-specific adaptations and conservation patterns. These findings support the hypothesis that CRISPR-Cas systems play a dual role: first, as a defense mechanism preventing the integration of foreign genetic material-reflected in the higher complexity and diversity of CRISPR loci in Archaea-and second, as a regulator of horizontal gene transfer, evidenced by the lower frequency of antibiotic resistance genes in organisms with active CRISPR-Cas systems. Together, these results underscore the evolutionary and functional diversification of CRISPR-Cas systems in response to environmental and selective pressures.}, }
@article {pmid40493452, year = {2025}, author = {Xue, H and Mishra, MK and Liu, Y and Liu, P and Grzybowski, M and Pandey, R and Usa, K and Vanden Avond, MA and Bala, N and Alli, AA and Cowley, AW and Qiu, Q and Greene, AS and Rao, S and O'Meara, CC and Geurts, AM and Liang, M}, title = {Physiological role and mechanisms of action for a long noncoding haplotype region.}, journal = {Cell reports}, volume = {44}, number = {6}, pages = {115805}, doi = {10.1016/j.celrep.2025.115805}, pmid = {40493452}, issn = {2211-1247}, mesh = {Humans ; *Haplotypes/genetics ; Polymorphism, Single Nucleotide/genetics ; Animals ; Induced Pluripotent Stem Cells/metabolism ; Blood Pressure/genetics ; Gene Editing/methods ; Mice ; CRISPR-Cas Systems ; }, abstract = {Direct targeting of noncoding genomic regions harboring common sequence variants associated with human traits through in vivo animal model studies and precise genome editing in human cells is essential for closing the critical gap between genetic discoveries and physiological understanding. However, such investigation has been impractical for many of these variants as they are in haplotypes containing multiple single-nucleotide polymorphisms (SNPs) spanning thousands of base pairs and have small effect sizes. We developed an integrated approach to address this challenge, combining an efficient two-step technique to precisely edit large haplotypes in human induced pluripotent stem cells and orthologous region deletion in phenotypically permissive animal models. As proof of principle, we applied this approach to examine a blood pressure-associated locus with a noncoding haplotype containing 11 SNPs spanning 17.4 kbp. We found a robust blood pressure effect of nearly 10 mmHg and identified the physiological and molecular mechanisms involved.}, }
@article {pmid40434888, year = {2025}, author = {Guo, J and Zhou, Z and Li, R and Xing, Z and Zhang, L and Zhao, S and Wei, W and Wang, J and Deng, T}, title = {A genome-wide base-editing screen uncovers a pivotal role of paxillin δ ubiquitination in influenza virus infection.}, journal = {Cell reports}, volume = {44}, number = {6}, pages = {115748}, doi = {10.1016/j.celrep.2025.115748}, pmid = {40434888}, issn = {2211-1247}, mesh = {Animals ; *Ubiquitination ; *Paxillin/metabolism/genetics ; Humans ; Mice ; Virus Replication ; *Influenza A virus/physiology ; *Gene Editing ; *Orthomyxoviridae Infections/virology/genetics/metabolism ; Madin Darby Canine Kidney Cells ; CRISPR-Cas Systems ; Virus Internalization ; Dogs ; Protein Isoforms/metabolism ; }, abstract = {Dissecting host factors critical for viral infection and understanding their mechanisms of action is critical for identifying drug targets. Here, we leverage a genome-wide CRISPR base-editing screen to identify functional lysine residues in host factors required for influenza A virus (IAV) replication. Multiple host genes, including GSTM4, FLNC, HMGB1, ZNF236, GRIP1, and PXN, along with regulatory lysine codons, are identified. Among these, paxillin (encoded by PXN) is identified as an important host entry factor. Depletion of paxillin significantly reduces IAV infection in both cell cultures and mice. Further analysis suggests that the δ isoform of paxillin, rather than the canonical β isoform, plays the key role. Additionally, our data indicate that lysine 68 of paxillin δ undergoes K6-linked ubiquitination and regulates influenza virus replication via modulating endosome-dependent viral entry. These observations contribute to understanding how influenza viruses interact with host factors and may inform therapeutic development.}, }
@article {pmid40384639, year = {2025}, author = {Ye, J and Shen, Y and Lin, Z and Xu, L and Wang, L and Lin, X and Huang, B and Ma, Z and Yu, Z and Lin, D and Chen, W and Feng, S}, title = {A CRISPR/Cas12a-Assisted SERS Nanosensor for Highly Sensitive Detection of HPV DNA.}, journal = {ACS sensors}, volume = {10}, number = {6}, pages = {4286-4296}, doi = {10.1021/acssensors.5c00547}, pmid = {40384639}, issn = {2379-3694}, mesh = {*Spectrum Analysis, Raman/methods ; *CRISPR-Cas Systems ; *DNA, Viral/analysis/genetics ; *Human papillomavirus 16/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; Humans ; *Human papillomavirus 18/genetics/isolation &amp; purification ; Biotin/chemistry ; Limit of Detection ; }, abstract = {The lack of timely and effective screening and diagnosis is a major contributing factor to the high mortality rate of cervical cancer in low-income countries and resource-limited regions. Therefore, the development of a rapid, sensitive, and easily deployable diagnostic tool for HPV DNA is of critical importance. In this study, we present a novel high-sensitivity and high-specificity detection method for HPV16 and HPV18 by integrating the CRISPR/Cas12a system with surface-enhanced Raman scattering (SERS) technology. This method leverages the trans-cleavage activity of the CRISPR/Cas12a system, which cleaves biotin-modified spherical nucleic acids (Biotin-SNA) in the presence of target DNA, releasing free Biotin-DNA. The released Biotin-DNA preferentially binds to streptavidin-modified magnetic beads (SAV-MB), reducing the capture of Biotin-SNA by SAV-MB and thereby significantly enhancing detection sensitivity. This method offers the potential for point-of-care diagnostics as it operates efficiently at 37 °C without the need for thermal cycling. Using standard DNA samples, we demonstrated that this biosensor achieved detection limits as low as 209 copies/μL and 444 copies/μL within 95 min. When combined with recombinase polymerase amplification (RPA), the sensor demonstrated enhanced sensitivity, enabling detection of target DNA at concentrations as low as 1 copy/μL within approximately 50 min. Furthermore, validation with clinical samples confirmed the feasibility and practical applicability of this method. This novel SERS-based sensor offers a new and effective tool in the prevention and detection of cervical cancer.}, }
@article {pmid40300601, year = {2025}, author = {Kang, X and Li, X and Zhou, J and Zhang, Y and Qiu, L and Tian, C and Deng, Z and Liang, X and Zhang, Z and Du, S and Hu, S and Wang, N and Yue, Z and Xu, Y and Gao, Y and Dai, J and Wang, Z and Yu, C and Chen, J and Wu, Y and Chen, L and Yao, Y and Yao, S and Yang, X and Yan, L and Wen, Q and Depies, OM and Chan, K and Liang, X and Li, G and Zi, Z and Liu, X and Gan, H}, title = {Extrachromosomal DNA replication and maintenance couple with DNA damage pathway in tumors.}, journal = {Cell}, volume = {188}, number = {13}, pages = {3405-3421.e27}, doi = {10.1016/j.cell.2025.04.012}, pmid = {40300601}, issn = {1097-4172}, mesh = {Humans ; *DNA Replication ; Ataxia Telangiectasia Mutated Proteins/metabolism ; DNA End-Joining Repair ; DNA Breaks, Double-Stranded ; *Neoplasms/genetics/metabolism/pathology ; *DNA Damage ; Cell Line, Tumor ; DNA Ligase ATP/metabolism ; DNA-Directed DNA Polymerase/metabolism ; DNA, Circular/metabolism/genetics ; DNA Polymerase theta ; CRISPR-Cas Systems ; }, abstract = {Extrachromosomal DNA (ecDNA) drives the evolution of cancer cells. However, the functional significance of ecDNA and the molecular components involved in its replication and maintenance remain largely unknown. Here, using CRISPR-C technology, we generated ecDNA-carrying (ecDNA+) cell models. By leveraging these models alongside other well-established systems, we demonstrated that ecDNA can replicate and be maintained in ecDNA+ cells. The replication of ecDNA activates the ataxia telangiectasia mutated (ATM)-mediated DNA damage response (DDR) pathway. Topoisomerases, such as TOP1 and TOP2B, play a role in ecDNA replication-induced DNA double-strand breaks (DSBs). A subset of these elevated DSBs persists into the mitotic phase and is primarily repaired by the alternative non-homologous end joining (alt-NHEJ) pathway, which involves POLθ and LIG3. Correspondingly, ecDNA maintenance requires DDR, and inhibiting DDR impairs the circularization of ecDNA. In summary, we demonstrate reciprocal interactions between ecDNA maintenance and DDR, providing new insights into the detection and treatment of ecDNA+ tumors.}, }
@article {pmid40571317, year = {2025}, author = {Karinen, S and Forero-Rodríguez, J and Järvinen, A and Eklund, KK and Barreto, G and Salem, A}, title = {CRISPR/Cas9-mediated Knockout of LYVE1 In Human Tongue Cancer Cells Reveals Transcriptomic Changes in Metastasis-associated Pathways.}, journal = {Cancer genomics &amp; proteomics}, volume = {22}, number = {4}, pages = {525-537}, doi = {10.21873/cgp.20519}, pmid = {40571317}, issn = {1790-6245}, mesh = {Humans ; *Tongue Neoplasms/genetics/pathology/metabolism ; *CRISPR-Cas Systems ; *Transcriptome ; *Vesicular Transport Proteins/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; Epithelial-Mesenchymal Transition ; Cell Line, Tumor ; Gene Knockout Techniques ; Neoplasm Metastasis ; *Squamous Cell Carcinoma of Head and Neck/genetics ; }, abstract = {BACKGROUND/AIM: Tongue squamous cell carcinoma (TSCC), a highly aggressive subtype of head and neck cancers, is characterized by frequent lymphatic metastasis and poor prognosis. Recently, we showed that lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) is involved in TSCC progression, yet the underlying molecular mechanisms remain unclear.<br><br>MATERIALS AND METHODS: CRISPR/Cas9 gene editing was employed to generate LYVE1 knockout (KO) TSCC cell lines. Single-cell clones were isolated, screened, and validated through sequencing and Inference of CRISPR Edits (ICE) analysis and qRT-PCR. RNA sequencing was performed on LYVE1 KO and wild-type (WT) cells to identify differentially expressed genes (DEGs). Bioinformatic analyses, including Gene Ontology (GO) enrichment and protein-protein interaction (PPI) network mapping, were conducted to explore affected pathways. Finally, network topology was examined using NetworkAnalyzer and cytoHubba plugins.<br><br>RESULTS: Transcriptomic analysis revealed significant down-regulation of pro-metastatic pathways, including epithelial-mesenchymal transition (EMT), extracellular matrix remodeling, and immune modulation. DEG analysis identified 263 genes, with key down-regulated targets such as WNT5A, TGFB2, and MMP2, and up-regulation of tumor-suppressive genes including PTGS2. GO and PPI analyses highlighted LYVE1's pivotal role in regulating cell adhesion, migration, and immune response.<br><br>CONCLUSION: LYVE1 KO reduces TSCC invasive potential by disrupting EMT and tumor-stroma interactions, aligning with previous experimental findings. These results suggest LYVE1 as a critical driver of metastasis, highlighting its potential as a therapeutic target.}, }
@article {pmid40571168, year = {2025}, author = {Jeong, JH and Kim, SH and Kim, JY}, title = {Empowering Agrobacterium: Ternary vector systems as a new arsenal for plant transformation and genome editing.}, journal = {Biotechnology advances}, volume = {}, number = {}, pages = {108631}, doi = {10.1016/j.biotechadv.2025.108631}, pmid = {40571168}, issn = {1873-1899}, abstract = {The continuous evolution of plant transformation technologies is pivotal for accelerating genetic advancements in agriculture. Among these, ternary vector systems have emerged as a transformative innovation, significantly enhancing Agrobacterium-mediated plant transformation by overcoming critical biological barriers. Unlike traditional binary vectors, ternary vector systems incorporate accessory virulence genes and immune suppressors that overcome the intrinsic transformation barriers of recalcitrant crops. This has enabled 1.5- to 21.5-fold increases in stable transformation efficiency in species previously resistant to Agrobacterium-mediated transformation, such as maize, sorghum, and soybean, thereby expanding the effective host range of plant genetic engineering. Furthermore, the fusion of ternary vectors with advanced genome editing technologies like CRISPR/Cas is revolutionizing precision breeding, facilitating unprecedented control over genetic modifications. Future innovations are likely to focus on expanding the capabilities of ternary vectors, including transient delivery of morphogenic factors to enhance regeneration and organelle-targeted transformation for broader genetic modifications. Additionally, refining Agrobacterium engineering, such as developing auxotrophic strains for improved biosafety and optimizing secretion systems for enhanced protein delivery, presents exciting opportunities for further advancements. This review highlights the recent breakthroughs in ternary vector engineering, including its potential to optimize regeneration pathways via morphogenic factors and extend genetic transformation to previously unamenable plant species. By bridging the gap between transformation efficiency and targeted genome modifications, these advancements are reshaping the landscape of plant biotechnology, driving more resilient and high-performing crops in an era of global agricultural challenges.}, }
@article {pmid40569097, year = {2025}, author = {Jenkins, K and Layton, D and Gough, T and O'Neil, T and Malaver Otega, L and Mishra, K and Bruce, K and Morris, K and Wise, T and Challagulla, A and Doran, T and Bean, A}, title = {Production of immune receptor knockout chickens via direct in vivo transfection of primordial germ cells.}, journal = {Animal biotechnology}, volume = {36}, number = {1}, pages = {2523027}, doi = {10.1080/10495398.2025.2523027}, pmid = {40569097}, issn = {1532-2378}, mesh = {Animals ; *Chickens/genetics ; *Receptor, Interferon alpha-beta/genetics ; *Gene Knockout Techniques/veterinary/methods ; *Transfection/veterinary/methods ; *Animals, Genetically Modified/genetics ; *Germ Cells ; *Receptors, Interleukin-1 Type I/genetics ; CRISPR-Cas Systems ; Chick Embryo ; Male ; Female ; }, abstract = {The advancement of genetic engineering in chickens has enabled significant advancement in developmental biology, bioreactors, and disease resilience. The development of CRISPR/Cas9 genome engineering technology has further expanded the potential applications of genetic engineering in poultry. In this study we aimed to evaluate the efficacy of a direct in vivo transfection method, previously demonstrated to produce transgenic chickens, in generating gene knockout (KO) chickens. Specifically, we targeted the Interferon-α/β Receptor 1 (IFNAR1) and Interleukin 1 receptor, type I (IL1R1), both critical pathways in the inflammatory and antiviral responses. We designed guide RNAs targeting the genes and validated their efficiency in vivo via microinjection into the developing embryos. PCR analysis confirmed the presence of gene deletions in chimeric roosters, which were subsequently bred to produce G1 germline heterozygote KO offspring. Homozygous KO chickens were generated and subjected to phenotypic and functional analyses. Our results demonstrated successful generation of functional knockouts of both IFNAR1 and IL1R1 using a direct in vivo transfection. Overall, this study demonstrates that direct in vivo transfection provides a robust and predictable method for generating KO chickens, facilitating further research into avian immune responses and the development of antiviral strategies.}, }
@article {pmid40568938, year = {2025}, author = {Liu, X and Tan, H and Wang, J and Cao, Y and Li, P and Fan, X and Wang, Q and Zhang, H and Zhang, J and Yang, T and Zhao, G and Zhang, X and Duan, X and Zi, L and Liu, L and Ma, L and Chen, Z and Liang, L and Liu, R}, title = {SELECT: high-precision genome editing strategy via integration of CRISPR-Cas and DNA damage response for cross-species applications.}, journal = {Nucleic acids research}, volume = {53}, number = {12}, pages = {}, doi = {10.1093/nar/gkaf595}, pmid = {40568938}, issn = {1362-4962}, support = {2023YFC3402300//National Key R&amp;D Program of China/ ; 22208044//National Natural Science Foundation of China/ ; 22278058//National Natural Science Foundation of China/ ; XLYC2203075//Xingliao Talent Plan/ ; 2023JJ12SN030//Science and Technology Innovation Foundation of Dalian/ ; 2024-MSBA-09//Natural Science Foundation of Liaoning Province/ ; DUT24YG131//Fundamental Research Funds for the Central Universities/ ; DUT23YG219//Fundamental Research Funds for the Central Universities/ ; }, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Saccharomyces cerevisiae/genetics ; Escherichia coli/genetics ; *DNA Damage ; Promoter Regions, Genetic ; DNA Breaks, Double-Stranded ; }, abstract = {CRISPR-based methods enable genome modifications for diverse applications but often face challenges, such as inconsistent efficiencies, reduced performance in iterative modifications, and difficulties generating high-quality datasets for high-throughput genome engineering. Here, we present SELECT (SOS Enhanced programmabLE CRISPR-Cas ediTing), a novel strategy integrating the CRISPR-Cas system with the DNA damage response. By employing designed and optimized double-strand break induced promoters that are activated upon genome editing, SELECT enables a counter-selection process to eliminate unedited cells, ensuring high-fidelity editing. This approach achieves up to 100% efficiency for point mutations, iterative knockouts, and insertions. In high-throughput library editing, SELECT achieved up to 94.2% efficiency and preserved higher library diversity compared with conventional methods. Application of SELECT in flaviolin biosynthesis resulted in a 3.97-fold increase in production. Furthermore, integration with machine learning tools allowed rapid mapping of genotype-phenotype relationships. SELECT provides a versatile platform for precision genome engineering in Escherichia coli and Saccharomyces cerevisiae.}, }
@article {pmid40567005, year = {2025}, author = {Zeng, Z and Wang, H and Luo, Y and Chen, W and Xu, M and Wei, H and Chen, Z and Xiang, T and Wang, L and Han, N and Huang, X and Bian, H}, title = {CRISPR/Cas9-mediated editing of barley lipoxygenase genes promotes grain fatty acid accumulation and storability.}, journal = {GM crops &amp; food}, volume = {16}, number = {1}, pages = {482-497}, doi = {10.1080/21645698.2025.2523069}, pmid = {40567005}, issn = {2164-5701}, mesh = {*Hordeum/genetics/metabolism/enzymology ; *CRISPR-Cas Systems ; Gene Editing ; *Lipoxygenase/genetics/metabolism ; *Fatty Acids/metabolism ; *Edible Grain/genetics/metabolism ; Plants, Genetically Modified/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Seeds/genetics/metabolism ; Gene Expression Regulation, Plant ; }, abstract = {Plant lipoxygenases (LOXs) catalyze the oxidation of polyunsaturated fatty acids, which can adversely affect grain storability. Although the genetic engineering of LOXs holds great potential for improving grain storage quality, this approach remains largely unexplored in barley. In this study, we identified five LOX genes in the barley genome: HvLOXA, HvLOXB, and HvLOXC1-3. HvLOXC1 exhibited the highest expression in early developing grains, roots, and shoots; HvLOXA was predominantly expressed in embryos, whereas HvLOXB and HvLOXC3 were weakly expressed across tissues. Transgene-free homozygous barley mutants of loxB, loxC1, and loxAloxC1 were generated using CRISPR/Cas9-mediated genome editing. Compared to the wild-type, all mutants displayed normal plant height, tiller number, and grain size, although the loxC1 and loxAloxC1 mutants exhibited significantly lower thousand grain weights. Notably, the total LOX activity in mature grains decreased by 36-42% in loxC1 mutants and by 94% in loxAloxC1 mutants, with no significant change observed in loxB mutants. Additionally, the loxAloxC1 double mutants had a significantly lower malondialdehyde content and accumulated 10-21% more fatty acids than the wild-type. Artificial aging treatment experiments revealed that loxAloxC1 mutants had enhanced grain storability, demonstrated by significantly higher germination rates, reduced lipid peroxidation, and improved seedling growth. Our findings highlight that the targeted knockout of LOX genes, particularly the double mutation of HvLOXA and HvLOXC1, represents a promising genetic strategy for improving grain storability and nutritional value in barley.}, }
@article {pmid40566742, year = {2025}, author = {Ni, H and Kelley, K and Xie, N and Zou, H and Friedel, RH}, title = {Generation of Plexin-B1 Conditional Knockout Mouse With CRISPR/Cas9 Technology.}, journal = {Genesis (New York, N.Y. : 2000)}, volume = {63}, number = {3}, pages = {e70019}, doi = {10.1002/dvg.70019}, pmid = {40566742}, issn = {1526-968X}, support = {NS092735/NS/NINDS NIH HHS/United States ; NS134159/NS/NINDS NIH HHS/United States ; AG077828/AG/NIA NIH HHS/United States ; CA196521/CA/NCI NIH HHS/United States ; DOH01-C39068GG//New York State Department of Health/ ; DOH01-C38330GG//New York State Department of Health/ ; }, mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; Mice, Knockout ; *Nerve Tissue Proteins/genetics ; Alleles ; Gene Targeting ; *Receptors, Cell Surface/genetics ; Gene Editing/methods ; Female ; }, abstract = {Plexins are axon guidance transmembrane receptors that control cytoskeleton and membrane dynamics in development and adult physiology. As plexins are expressed in multiple cell types in various tissues, floxed alleles that enable conditional deletion are needed to facilitate cell type-specific functional analysis. We report here the generation of a conditional floxed allele of Plexin-B1 (gene symbol Plxnb1) in mouse using CRISPR/Cas9 technology to insert two loxP sites flanking critical exons. Targeting reagents (Cas9 protein, sgRNAs, ssODNs) were delivered into single-cell embryos by electroporation. After screening a total of 128 mouse pups by PCR and Sanger sequencing, two mice were identified carrying both loxP sites in the targeted Plxnb1 locus (success rate ~ 1.6%). The usage of Alt-R modified ssODNs increased targeting frequencies at one loxP site, but not the other. We also tested homology directed repair (HDR) enhancer V2 reagent, but addition of the enhancer reduced the viability of mouse embryos. The Plxnb1[flox] allele was successfully transmitted through the germline in Mendelian ratios, and effective excision of the floxed region was confirmed by breeding with Cre recombinase strains.}, }
@article {pmid40566685, year = {2025}, author = {Liu, J and Zhang, R and Chai, N and Su, L and Zheng, Z and Liu, T and Guo, Z and Ma, Y and Xie, Y and Xie, X and Lin, Q and Chen, L and Liu, YG and Zhu, Q}, title = {Programmable genome engineering and gene modifications for plant biodesign.}, journal = {Plant communications}, volume = {}, number = {}, pages = {101427}, doi = {10.1016/j.xplc.2025.101427}, pmid = {40566685}, issn = {2590-3462}, abstract = {Plant science has entered a transformative era with genome editing by enabling precise DNA alterations to address global challenges such as climate adaptability and food safety. These alterations are primarily driven by the integration of three modular components that can be activated or suppressed: DNA-targeting modules, effector modules, and control modules. The field has evolved from protein-centric systems (zinc finger nucleases and transcription activator-like effector nucleases) to RNA-focused platforms (CRISPR-Cas and other nucleases), which facilitate diverse control over genetic and epigenetic contexts. The modular design of DNA-targeting modules paired with effector domains, with or without inducible systems, provides scientists with superior precision in regulating transcription and altering chromatin states. The present review article examines these three modules and highlights various optimization methods. Additionally, it outlines innovative tools such as optogenetic systems and receptor-integrated systems that enable spatiotemporal control of genome editor expression. These modular instruments overcome traditional boundaries and allow scientists to create plants with favorable characteristics, decipher complex gene networks, and adopt sustainable farming practices.}, }
@article {pmid40565548, year = {2025}, author = {Xavier, KVM and Silva, AMA and Luz, ACO and da Silva, FSC and de Melo, BST and Pitta, JLLP and Leal-Balbino, TC}, title = {Diversity and Role of Prophages in Pseudomonas aeruginosa: Resistance Genes and Bacterial Interactions.}, journal = {Genes}, volume = {16}, number = {6}, pages = {}, doi = {10.3390/genes16060656}, pmid = {40565548}, issn = {2073-4425}, mesh = {*Pseudomonas aeruginosa/genetics/virology/drug effects/pathogenicity ; *Prophages/genetics ; CRISPR-Cas Systems/genetics ; *Drug Resistance, Bacterial/genetics ; Humans ; Pseudomonas Infections/microbiology ; Genome, Bacterial ; Brazil ; }, abstract = {Pseudomonas aeruginosa is a major pathogen associated with hospital-acquired infections, and the spread of carbapenem-resistant isolates highlights the urgency of developing non-conventional therapies, such as phage therapy. For this alternative to be effective, understanding phage-host interactions is crucial for the selection of candidate phages and offers new insights into these dynamics. Background/Objectives: This study aimed to characterize prophage diversity in clinical P. aeruginosa genomes, assess the relationship between phages and the CRISPR/Cas system, and investigate the potential role of prophages in disseminating resistance genes. Methods: A total of 141 genomes from Brazilian hospitals were analyzed. Prophage detection was performed using VIBRANT, and in silico analyses were conducted to evaluate taxonomic diversity, the presence of resistance genes, phage life cycle, genomic distribution, and the presence of the CRISPR/Cas system. Results: A total of 841 viral sequences were identified by the VIBRANT tool, of which 498 were confirmed by CheckV, with a predominance of the class Caudoviricetes and high overall phage diversity. No statistically significant difference was observed in the number of prophages between isolates with and without CRISPR/Cas systems. Prophages carrying resistance genes such as rsmA, OXA-56, SPM-1, and others were detected in isolates harboring the type I-C CRISPR/Cas system. Additionally, prophages showed no preference for specific insertion sites along the bacterial genome. Conclusions: These findings provide evidence of a well-established phage-host relationship. The dual role of prophages-as vectors of antimicrobial resistance and as potential therapeutic agents-reflects their dynamic impact on bacterial communities and reinforces their importance in developing new strategies to combat antimicrobial resistance.}, }
@article {pmid40565267, year = {2025}, author = {Chakraborty, M and Nielsen, L and Nash, D and Bruder, MR and Nissimov, JI and Charles, TC and Aucoin, MG}, title = {Baculovirus Variant Detection from Transient CRISPR-Cas9-Mediated Disruption of gp64 at Different Gene Locations.}, journal = {International journal of molecular sciences}, volume = {26}, number = {12}, pages = {}, doi = {10.3390/ijms26125805}, pmid = {40565267}, issn = {1422-0067}, support = {RGPIN 355513-2017/RGPIN-2023-03666//Natural Sciences and Engineering Research Council/ ; }, mesh = {*CRISPR-Cas Systems ; Animals ; *Baculoviridae/genetics ; *Viral Envelope Proteins/genetics ; Genetic Vectors/genetics ; Genome, Viral ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; Sf9 Cells ; Mutation ; }, abstract = {The Baculovirus Expression Vector System (BEVS) is an important protein and complex biologics production platform. The baculovirus GP64 protein is the major envelope glycoprotein that aids in virus entry and is required for cell-to-cell transmission in cell culture. Several studies have developed strategies around gp64 gene disruption in an attempt to minimize baculovirus co-production. Here, we investigate the result of transiently targeting the baculovirus gp64 gene with CRISPR-Cas9 during infection. Because not all genomes are effectively disrupted, we describe a variant calling methodology that allows the detection of the targeted mutations in gp64 even though these mutations are not the dominant sequences. Using a transfection-infection assay (T-I assay), the AcMNPV gp64 gene was targeted at six different locations to evaluate the effects of single and multiple targeting sites, and we demonstrated a reduction in the levels of baculovirus vectors while maintaining or enhancing foreign protein production when protein was driven by a p6.9 promoter. Viral genomes were subsequently isolated from the supernatant and cell pellet fractions, and our sequencing pipeline successfully detected indel mutations within gp64 for most of the single-guide RNA (sgRNA) targets. We also observed that 68.8% of variants found in the virus stock were conserved upon virus propagation in cell culture, thus indicating that they are not detrimental to viral fitness. This work provides a comprehensive assessment of CRISPR-Cas9 genome editing of baculovirus vectors, with potential applications in enhancing the efficiency of the BEVS.}, }
@article {pmid40565111, year = {2025}, author = {Bouchard, C and Rousseau, J and Lamothe, G and Dubost, M and Bastrenta, L and Ramezani, S and Tremblay, JP}, title = {In Vitro Correction of Point Mutations in the DYSF Gene Using Prime Editing.}, journal = {International journal of molecular sciences}, volume = {26}, number = {12}, pages = {}, doi = {10.3390/ijms26125647}, pmid = {40565111}, issn = {1422-0067}, support = {TREM-01//Jain Foundation/ ; }, mesh = {Humans ; *Point Mutation ; *Gene Editing/methods ; HEK293 Cells ; *Dysferlin/genetics ; Genetic Therapy/methods ; Myoblasts/metabolism ; Fibroblasts/metabolism ; CRISPR-Cas Systems ; }, abstract = {Dysferlinopathy is caused by over 500 mutations in the gene encoding dysferlin, including close to 300 point mutations. One option to cure the disease is to use a gene therapy to correct these mutations at the root. Prime editing is a technique which can replace the mutated nucleotide with the wild-type nucleotide. In this article, prime editing is used to correct several point mutations in the DYSF gene responsible for dysferlinopathy. In vitro editing of HEK293T cells reaches up to 31%. Notably, editing was more efficient in myoblasts than in patient-derived fibroblasts. The prime editing technique was also used to create a new myoblast clone containing a patient mutation from a healthy myoblast cell line.}, }
@article {pmid40521923, year = {2025}, author = {Cao, Y and Huang, C and Li, K and Zhang, X and Tong, M and Tu, J and Yang, K and Yuan, S and Zhang, H}, title = {CRISPR/Cas12a-assisted electrochemiluminescent detection of ochratoxin A based on COF@Ru coupled with a DNA tetrahedral scaffold.}, journal = {Analytical methods : advancing methods and applications}, volume = {17}, number = {25}, pages = {5298-5307}, doi = {10.1039/d5ay00427f}, pmid = {40521923}, issn = {1759-9679}, mesh = {*Ochratoxins/analysis ; *CRISPR-Cas Systems ; *Electrochemical Techniques/methods ; *Biosensing Techniques/methods ; *Luminescent Measurements/methods ; *Metal-Organic Frameworks/chemistry ; Limit of Detection ; Gold/chemistry ; *DNA/chemistry ; Metal Nanoparticles/chemistry ; Aptamers, Nucleotide/chemistry ; DNA Probes/chemistry ; }, abstract = {To sensitively detect ochratoxin A (OTA), a CRISPR/Cas12a-assisted electrochemiluminescence (ECL) aptasensor based on COF@Ru coupled with a DNA tetrahedral scaffold (DTS) was successfully fabricated. The covalent organic framework (COF) acted as a confined micro-reactor for the tris(2,2'-bipyridyl)ruthenium(II)/tri-n-propylamine (Ru(bpy)3[2+]/TPrA) system and hence, evidently enhanced ECL signals. Au nanoparticles (AuNPs) coupled with DTS caused more ferrocene (Fc)-modified quenching DNA probe molecules to be attached to the surface of the sensing electrode, which could reduce the detection background. Activated CRISPR/Cas12a could effectively amplify the ECL signal since it could cut off a considerable amount of quenching DNA probe molecules and make them move away from the electrode. Owing to these strategies, the fabricated ECL aptasensor could detect OTA as low as 3.5 fg mL[-1] in a linear detection range of 10[-5]-100 ng mL[-1]. Furthermore, the CRISPR/Cas12a and COF@Ru coupled with DTS-based ECL biosensor possessed high stability and specificity. More importantly, the ECL biosensor could effectively detect OTA in food samples, indicating that it can play a role in monitoring OTA levels in the field of food safety.}, }
@article {pmid40518166, year = {2025}, author = {Ge, L and Xiong, H}, title = {[Advances in CRISPR-Cas9 genome editing for the treatment of muscular dystrophies].}, journal = {Zhonghua er ke za zhi = Chinese journal of pediatrics}, volume = {63}, number = {7}, pages = {808-811}, doi = {10.3760/cma.j.cn112140-20250421-00356}, pmid = {40518166}, issn = {0578-1310}, mesh = {Humans ; *Genetic Therapy/methods ; *CRISPR-Cas Systems ; *Muscular Dystrophies/therapy/genetics ; Animals ; *Gene Editing/methods ; Muscular Dystrophy, Duchenne/therapy/genetics ; Disease Models, Animal ; }, }
@article {pmid40501421, year = {2025}, author = {Zheng, Y and Liu, B and Zuo, Q and Deng, F and Lan, C}, title = {Detection of Streptococcus anginosus in fecal samples using PCR-CRISPR /Cas12a system.}, journal = {Bioanalysis}, volume = {17}, number = {11}, pages = {737-745}, doi = {10.1080/17576180.2025.2518042}, pmid = {40501421}, issn = {1757-6199}, mesh = {*Feces/microbiology ; Humans ; *CRISPR-Cas Systems/genetics ; *Polymerase Chain Reaction/methods ; *Streptococcus anginosus/genetics/isolation &amp; purification ; DNA, Bacterial/genetics ; RNA, Ribosomal, 16S/genetics ; }, abstract = {OBJECTIVE: To develop a highly sensitive and specific detection method based on PCR-CRISPR/Cas12a for the detection of Streptococcus anginosus (S. anginosus) in feces and to evaluate its detection rate in the general population as well as its potential as a gastrointestinal tumor marker.<br><br>MATERIALS AND METHODS: Specific primers and crRNA targeting the 16S rDNA of S. anginosus were designed to construct a PCR-CRISPR/Cas12a detection system. A total of 230 fecal samples were collected from the general population, and bacterial DNA was extracted. The target gene was detected using this system to verify its sensitivity, specificity, and stability.<br><br>RESULTS: The established detection system demonstrated strong specificity, with stable recognition of S. anginosus, and a minimum detection limit of 10[-7] ng/&#x3bc;L. The detection rate of S. anginosus in fecal samples from the general population was 51.7%.<br><br>CONCLUSION: The PCR-CRISPR/Cas12a system can efficiently detect S. anginosus in feces, providing a reliable technical tool for exploring its association with gastrointestinal tumors.}, }
@article {pmid40490360, year = {2025}, author = {Kagawa, N and Umesono, Y and Suzuki, KT and Mochii, M}, title = {Step-by-Step Protocol for Making a Knock-In Xenopus laevis to Visualize Endogenous Gene Expression.}, journal = {Development, growth &amp; differentiation}, volume = {67}, number = {5}, pages = {293-302}, doi = {10.1111/dgd.70011}, pmid = {40490360}, issn = {1440-169X}, support = {//University of Hyogo/ ; JP18K06266//Japan Society for the Promotion of Science/ ; JP21H03829//Japan Society for the Promotion of Science/ ; 22NIBB331//NIBB Collaborative Research Program/ ; 23NIBB340//NIBB Collaborative Research Program/ ; JPMJCR 2025//JST, CREST/ ; }, mesh = {Animals ; *Xenopus laevis/genetics ; *Gene Knock-In Techniques/methods ; CRISPR-Cas Systems/genetics ; Green Fluorescent Proteins/genetics/metabolism ; }, abstract = {We established a novel knock-in technique, New and Easy Xenopus Targeted integration (NEXTi), to recapitulate endogenous gene expression by reporter expression. NEXTi is a CRISPR-Cas9-based method to integrate a donor DNA containing a reporter gene (egfp) into the target 5' untranslated region (UTR) of the Xenopus laevis genome. It enables us to track eGFP expression under the regulation of endogenous promoter/enhancer activities. We obtained about 2% to 13% of knock-in vector-injected embryos showing eGFP signal in a tissue-specific manner, targeting krt.12.2.L, myod1.S, sox2.L, and bcan.S loci, as previously reported. In addition, F1 embryos which show stable eGFP signals were obtained by outcrossing the matured injected frogs with wild-type animals. Integrations of donor DNAs into target 5' UTRs were confirmed by PCR amplification and sequencing. Here, we describe the step-by-step protocol for preparation of donor DNA and single guide RNA, microinjection, and genotyping of F1 animals for the NEXTi procedure.}, }
@article {pmid39883121, year = {2025}, author = {Paryani, M and Gupta, N and Jain, SK and Butani, S}, title = {Lowering LDL cholesterol by PCSK9 inhibition: a new era of gene silencing, RNA, and alternative therapies.}, journal = {Naunyn-Schmiedeberg's archives of pharmacology}, volume = {398}, number = {6}, pages = {6597-6615}, pmid = {39883121}, issn = {1432-1912}, mesh = {Humans ; *Antibodies, Monoclonal/pharmacology/therapeutic use ; Cholesterol, LDL/blood ; CRISPR-Cas Systems ; Gene Silencing ; Hypercholesterolemia/drug therapy/economics ; *Oligonucleotides, Antisense/pharmacology/therapeutic use ; *Plant Extracts/pharmacology/therapeutic use ; Proprotein Convertase 9/genetics/metabolism ; *Proprotein Convertases/antagonists &amp; inhibitors ; Protein Subunit Vaccines/pharmacology/therapeutic use ; *RNA, Small Interfering/pharmacology/therapeutic use ; *Anticholesteremic Agents/pharmacology/therapeutic use ; Fibronectin Type III Domain ; Recombinant Proteins ; }, abstract = {Proprotein convertase subtilisin/kexin type 9 (PCSK9) discovery has added a new paradigm to our understanding of cholesterol homeostasis and lipid metabolism. Since its discovery, PCSK9 inhibitors have become a widely investigated therapeutic class for lipid management in cardiovascular diseases and hypercholesterolemia. Scientists have explored different approaches for PCSK9 inhibition, such as monoclonal antibodies (mAbs), gene silencing and gene editing techniques, vaccines, mimetic peptides, and small molecules. European Medicines Agency (EMA) and United States Food and Drug Administration (US FDA) have approved only three PCSK9 inhibitors, including two monoclonal antibodies and one small interfering ribonucleic acid (siRNA). Despite the efficacy of approved large molecules, high costs and the need for regular injection have limited their adherence to the patient. This review aims to provide an understanding of PCSK9's function in Low-Density Lipoprotein Cholesterol (LDL-C) management, its current treatment, recent advancements, and potential future development of small molecules in the class of PCSK9 inhibitors.}, }
@article {pmid30965672, year = {2019}, author = {Song, HY and Chien, CS and Yarmishyn, AA and Chou, SJ and Yang, YP and Wang, ML and Wang, CY and Leu, HB and Yu, WC and Chang, YL and Chiou, SH}, title = {Generation of GLA-Knockout Human Embryonic Stem Cell Lines to Model Autophagic Dysfunction and Exosome Secretion in Fabry Disease-Associated Hypertrophic Cardiomyopathy.}, journal = {Cells}, volume = {8}, number = {4}, pages = {}, pmid = {30965672}, issn = {2073-4409}, mesh = {Apoptosis ; *Autophagy ; CRISPR-Cas Systems ; Cardiomyopathy, Hypertrophic/etiology/*genetics/*pathology ; Cell Line ; Exosomes ; Fabry Disease/*complications ; Gene Knockout Techniques ; Human Embryonic Stem Cells/metabolism ; Humans ; *Models, Biological ; Myocytes, Cardiac/metabolism ; Reactive Oxygen Species/metabolism ; Trihexosylceramides/metabolism ; alpha-Galactosidase/*genetics ; }, abstract = {Fabry disease (FD) is a rare inherited disorder characterized by a wide range of systemic symptoms; it is particularly associated with cardiovascular and renal problems. Enzyme replacement therapy and pharmacological chaperone migalastat are the only approved and effective treatment strategies for FD patients. It is well documented that alpha-galactosidase A (GLA) enzyme activity deficiency causes globotriaosylceramide (Gb3) accumulation, which plays a crucial role in the etiology of FD. However, the detailed mechanisms remain unclear, and the lack of a reliable and powerful disease model is an obstacle. In this study, we created such a model by using CRISPR/Cas9-mediated editing of GLA gene to knockout its expression in human embryonic stem cells (hESCs). The cardiomyocytes differentiated from these hESCs (GLA-null CMs) were characterized by the accumulation of Gb3 and significant increases of cell surface area, the landmarks of FD-associated cardiomyopathy. Furthermore, we used mass spectrometry to compare the proteomes of GLA-null CMs and parental wild type CMs and found that the Rab GTPases involved in exocytotic vesicle release were significantly downregulated. This caused impairment of autophagic flux and protein turnover, resulting in an increase of reactive oxygen species and apoptosis. To summarize, we established a FD model which can be used as a promising tool to study human hypertrophic cardiomyopathy in a physiologically and pathologically relevant manner and to develop new therapies by targeting Rab GTPases signaling-related exosomal vesicles transportation.}, }
@article {pmid28565744, year = {2017}, author = {Cheng, LH and Liu, Y and Niu, T}, title = {[Chromosomal large fragment deletion induced by CRISPR/Cas9 gene editing system].}, journal = {Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi}, volume = {38}, number = {5}, pages = {427-431}, pmid = {28565744}, issn = {0253-2727}, mesh = {Animals ; *CRISPR-Cas Systems ; Chromosome Deletion ; DNA ; Gene Deletion ; *Gene Editing ; HEK293 Cells ; Humans ; Mice ; Multigene Family ; Mutagenesis, Insertional ; NIH 3T3 Cells ; Plasmids ; }, abstract = {Objective: Using CRISPR-Cas9 gene editing technology to achieve a number of genes co-deletion on the same chromosome. Methods: CRISPR-Cas9 lentiviral plasmid that could induce deletion of Aloxe3-Alox12b-Alox8 cluster genes located on mouse 11B3 chromosome was constructed via molecular clone. HEK293T cells were transfected to package lentivirus of CRISPR or Cas9 cDNA, then mouse NIH3T3 cells were infected by lentivirus and genomic DNA of these cells was extracted. The deleted fragment was amplified by PCR, TA clone, Sanger sequencing and other techniques were used to confirm the deletion of Aloxe3-Alox12b-Alox8 cluster genes. Results: The CRISPR-Cas9 lentiviral plasmid, which could induce deletion of Aloxe3-Alox12b-Alox8 cluster genes, was successfully constructed. Deletion of target chromosome fragment (Aloxe3-Alox12b-Alox8 cluster genes) was verified by PCR. The deletion of Aloxe3-Alox12b-Alox8 cluster genes was affirmed by TA clone, Sanger sequencing, and the breakpoint junctions of the CRISPR-Cas9 system mediate cutting events were accurately recombined, insertion mutation did not occur between two cleavage sites at all. Conclusion: Large fragment deletion of Aloxe3-Alox12b-Alox8 cluster genes located on mouse chromosome 11B3 was successfully induced by CRISPR-Cas9 gene editing system.}, }
@article {pmid28549865, year = {2017}, author = {Powell, SK and Gregory, J and Akbarian, S and Brennand, KJ}, title = {Application of CRISPR/Cas9 to the study of brain development and neuropsychiatric disease.}, journal = {Molecular and cellular neurosciences}, volume = {82}, number = {}, pages = {157-166}, pmid = {28549865}, issn = {1095-9327}, support = {P50 MH096890/MH/NIMH NIH HHS/United States ; R01 MH101454/MH/NIMH NIH HHS/United States ; R01 MH106056/MH/NIMH NIH HHS/United States ; }, mesh = {Brain/*growth &amp; development ; Brain Diseases/therapy ; CRISPR-Cas Systems/*genetics ; *Gene Editing/methods ; Gene Expression/*genetics ; Humans ; Induced Pluripotent Stem Cells/*cytology ; }, abstract = {CRISPR/Cas9 technology has transformed our ability to manipulate the genome and epigenome, from efficient genomic editing to targeted localization of effectors to specific loci. Through the manipulation of DNA- and histone-modifying enzyme activities, activation or repression of gene expression, and targeting of transcriptional regulators, the role of gene-regulatory and epigenetic pathways in basic biology and disease processes can be directly queried. Here, we discuss emerging CRISPR-based methodologies, with specific consideration of neurobiological applications of human induced pluripotent stem cell (hiPSC)-based models.}, }
@article {pmid28549739, year = {2017}, author = {Deng, H and Gao, R and Liao, X and Cai, Y}, title = {Characterization of a major facilitator superfamily transporter in Shiraia bambusicola.}, journal = {Research in microbiology}, volume = {168}, number = {7}, pages = {664-672}, doi = {10.1016/j.resmic.2017.05.002}, pmid = {28549739}, issn = {1769-7123}, mesh = {Ascomycota/chemistry/drug effects/*genetics/pathogenicity ; CRISPR-Cas Systems ; Membrane Transport Proteins/*genetics/*metabolism ; Mutation ; Perylene/*analogs &amp; derivatives/metabolism/pharmacology ; Phenol ; Quinones/*metabolism/pharmacology ; Reactive Oxygen Species/metabolism ; Sasa/microbiology ; }, abstract = {Reactive oxygen species (ROS) generated by photo-activated hypocrellin from Shiraia bambusicola are detrimental to cellular macromolecules. However, S. bambusicola can still maintain excellent morphology during continuous hypocrellin production, indicating an extraordinary autoresistance system that protects against the harmful ROS. In this study, a major facilitator superfamily transporter (MFS) was isolated from S. bambusicola and deleted using the clustered regularly interspaced short palindromic repeat sequences (CRISPR)/Cas9 system. The ΔMFS mutant abolished hypocrellin production and was slightly sensitive to 40-μM hypocrellin, while the ΔMFS compliment strain restored hypocrellin production and resistance. Hypocrellin treatment also enhanced the relative expression of MFS in wild-type S. bambusicola. Subsequent pathogenicity assays showed that MFS deletion reduced damage to bamboo leaves. By contrast, restoration of hypocrellin production in the MFS compliment strain generated similar necrotic lesions on bamboo leaves to those observed with the wild-type strain. These results revealed that the identified MFS is involved in efflux of hypocrellin from cells, which reduces the hypocrellin toxicity. Furthermore, hypocrellin contributed to the virulence of S. bambusicola on bamboo leaves. These findings could help to reduce plant loss by disrupting hypocrellin biosynthesis in S. bambusicola, or overexpressing the associated resistance gene in transgenic plants.}, }
@article {pmid40562814, year = {2025}, author = {Qiao, JH and Zang, Y and Gao, Q and Liu, S and Zhang, XW and Hu, W and Wang, Y and Han, C and Li, D and Wang, XB}, title = {Transgene- and tissue culture-free heritable genome editing using RNA virus-based delivery in wheat.}, journal = {Nature plants}, volume = {}, number = {}, pages = {}, pmid = {40562814}, issn = {2055-0278}, support = {32425046//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, abstract = {CRISPR-Cas genome editing technology is a cutting-edge strategy for crop breeding. However, the delivery of genome-editing reagents remains to be a technological bottleneck in monocot plants[1]. Here we engineered barley yellow striate mosaic virus (BYSMV) into a negative-strand RNA virus-based vector system[2] for delivery of both Cas9 and single guide RNA to achieve heritable gene editing in different wheat cultivars. We found that fusion of a mobile transfer RNA sequence[3] to the Cas9 messenger RNA and single guide RNAs could deliver them into the growth points of axillary meristems to achieve gene editing before tiller generation. The resulting nascent tillers contained simultaneous mutations in the three homoeoalleles. Moreover, the progeny seedlings are virus-free and harbour bi-allelic or homozygous mutations. Given BYSMV infects 26 monocot species[4], the BYSMV delivery system could have wide applicability for achieving highly efficient, non-transgenic and less genotype-dependent heritable genome editing, thereby facilitating genomic studies and crops breeding.}, }
@article {pmid40560955, year = {2025}, author = {Leprince, A and Moineau, S}, title = {CRISPR-Cas in the Cheese Industry.}, journal = {Annual review of virology}, volume = {}, number = {}, pages = {}, doi = {10.1146/annurev-virology-092623-111016}, pmid = {40560955}, issn = {2327-0578}, abstract = {Bacteria have evolved a wide range of defense systems to combat phage infections. In the cheese industry, lactic acid bacteria (LAB) used for milk fermentation continuously face threats from phages. Therefore, selecting or developing industrial strains with enhanced phage resistance requires a focus on robust defense systems. Among these systems, the clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) are notably prevalent in LAB. The early characterization of this adaptive immune system was closely tied to the cheese industry, particularly with Streptococcus thermophilus in which CRISPR-Cas systems are ubiquitous and highly active. This review underscores the contributions of S. thermophilus and its virulent phages to our understanding of the function and mechanisms of CRISPR-Cas systems. Additionally, we review the diversity of CRISPR-Cas systems in LAB used in the cheese industry, the counter-defense strategies employed by dairy phages, and the applications of CRISPR-Cas systems within this sector.}, }
@article {pmid40560862, year = {2025}, author = {Nakamura, K and Ito, S and Ohguchi, Y and Jimbo, T and Wada, Y and Nakajima, R and Kanou, M and Yamana, K and Ueda, H}, title = {Establishment of Cre/LoxP-mediated multifunctional reporter knock-in rats with the CRISPR system.}, journal = {PloS one}, volume = {20}, number = {6}, pages = {e0325444}, doi = {10.1371/journal.pone.0325444}, pmid = {40560862}, issn = {1932-6203}, mesh = {Animals ; Rats ; *Integrases/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; *Genes, Reporter ; Rats, Transgenic ; Male ; }, abstract = {Rats and mice are essential experimental animals in preclinical research, serving as models for various human diseases and contributing significantly to drug development. Rats offer distinct advantages over mice due to their larger size, which allows for more complex surgical procedures, repeated blood sampling, or sophisticated behavioral analysis. However, unlike the case with mice, genetically modified rat lines for achieving complex experimental objectives-such as tissue-specific gene knockout or visualization of specific cell populations-are still limited. We here established LoxP-mediated multifunctional reporter KI rats, enabling us to evaluate fluorescence, bioluminescence, and cell-killing assays simultaneously with only one gene-modified rat line. CRISPR/Cas12a, also known as CRISPR/Cpf1, was successfully used to insert the Cre sequence into a target locus to generate Cre driver rats. These results will contribute to the application of gene-modified rats for a more comprehensive understanding of physiology, and for extrapolation of their capabilities in preclinical research.}, }
@article {pmid40560492, year = {2025}, author = {Lv, D and Xu, Y and Wang, ZX and Zhang, QL and Yan, JP and Xu, JW}, title = {CRISPR/Cas9-mediated genome editing in Ganoderma lucidum: recent advances and biotechnological opportunities.}, journal = {World journal of microbiology &amp; biotechnology}, volume = {41}, number = {7}, pages = {223}, pmid = {40560492}, issn = {1573-0972}, support = {32360014//National Natural Science Foundation of China/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Reishi/genetics/metabolism ; *Biotechnology/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Fungal ; }, abstract = {Ganoderma lucidum is a well-known traditional medicinal mushroom that has attracted considerable attention due to its potential as a promising cell factory for producing high-value bioactive compounds. However, conventional methods for the genetic manipulation of G. lucidum are often time-consuming and labor-intensive, hindering research into the biosynthesis and regulatory mechanisms of its valuable natural products. In recent years, the clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) system has emerged as a powerful genome editing tool, extensively utilized in life sciences research due to its high efficiency and user-friendliness. This review provides a structured overview of advancements in CRISPR/Cas9-mediated genome editing technology in G. lucidum. We first discuss the development and optimization of the CRISPR system, focusing on the various expression strategies for Cas9 and guide RNA established in G. lucidum. Furthermore, we highlight the application of this system for targeted gene deletion, insertion, and replacement in genome editing, as well as its use in the functional analysis of genes in G. lucidum. In addition, we discuss the limitations and challenges associated with employing CRISPR/Cas9 tools in G. lucidum and provide an outlook on the future development of the CRISPR/Cas9 system and its applications in this organism.}, }
@article {pmid40560319, year = {2025}, author = {Peer, LA and Mir, BA}, title = {Molecular mechanisms and genetic regulation of self-incompatibility in flowering plants: implications for crop improvement and evolutionary biology.}, journal = {Plant molecular biology}, volume = {115}, number = {4}, pages = {76}, pmid = {40560319}, issn = {1573-5028}, mesh = {*Self-Incompatibility in Flowering Plants/genetics ; *Crops, Agricultural/genetics ; Biological Evolution ; *Gene Expression Regulation, Plant ; Evolution, Molecular ; Pollination/genetics ; Self-Fertilization/genetics ; Plant Proteins/genetics/metabolism ; }, abstract = {Self-incompatibility is a fundamental biological mechanism in flowering plants that prevents self-fertilization, thereby promoting outcrossing and enhancing genetic diversity. This complex system has independently evolved across multiple angiosperm lineages and is crucial in maintaining plant reproductive success. Recent research has expanded our understanding of self-incompatibility's molecular basis and uncovered key genes and signaling pathways involved in self-incompatibility responses, such as S-RNase in Solanaceae and PrsS-PrpS in Papaveraceae, as well as the SRK-SCR interaction in Brassicaceae. However, despite significant advances, many aspects of self-incompatibility, such as the interplay between gene duplications, polyploidization, and the evolution of novel self-incompatibility mechanisms, remain underexplored. This review integrates findings from various plant families, including Solanaceae, Rosaceae, Papaveraceae, and Brassicaceae, and discusses the evolutionary dynamics of self-incompatibility systems, highlighting the role of gene duplication, recombination, and translocation events in shaping self-incompatibility diversity. Special emphasis is placed on understanding how modern molecular techniques, such as CRISPR/Cas9 and marker-assisted selection, can be employed to transition self-incompatibility to self-compatibility in economically significant crops. Additionally, the role of epigenetic changes and modifier genes in mediating transitions from self-incompatibility to self-compatibility is addressed, offering insights into how these mechanisms can be leveraged for crop breeding and hybrid seed production. Future research should focus on elucidating the molecular mechanisms underlying self-incompatibility responses, exploring the potential of targeted gene editing to overcome reproductive barriers, and understanding the evolutionary resilience of self-incompatibility systems to environmental changes.}, }
@article {pmid40559612, year = {2025}, author = {Jurić, I and Jelić, M and Markanović, M and Kanižaj, L and Bošnjak, Z and Budimir, A and Kuliš, T and Tambić-Andrašević, A and Ivančić-Baće, I and Mareković, I}, title = {CRISPR-Cas Dynamics in Carbapenem-Resistant and Carbapenem-Susceptible Klebsiella pneumoniae Clinical Isolates from a Croatian Tertiary Hospital.}, journal = {Pathogens (Basel, Switzerland)}, volume = {14}, number = {6}, pages = {}, doi = {10.3390/pathogens14060604}, pmid = {40559612}, issn = {2076-0817}, support = {10106-24-1295 to I.M. and 20286539 to I.I.B.//School od Medicine, University of Zagreb and Faculty of Science, University of Zagreb/ ; }, mesh = {*Klebsiella pneumoniae/genetics/drug effects/isolation &amp; purification/classification ; Humans ; *CRISPR-Cas Systems/genetics ; *Carbapenems/pharmacology ; *Klebsiella Infections/microbiology ; beta-Lactamases/genetics ; Croatia ; Tertiary Care Centers ; Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests ; Bacterial Proteins/genetics ; Multilocus Sequence Typing ; Drug Resistance, Multiple, Bacterial/genetics ; *Carbapenem-Resistant Enterobacteriaceae/genetics/drug effects/isolation &amp; purification ; }, abstract = {(1) Background: CRISPR-Cas systems provide adaptive immunity against mobile genetic elements (MGEs) carrying antimicrobial resistance (AMR) genes. Carbapenem-resistant (CR) Klebsiella pneumoniae is a major public health concern, and the role of CRISPR-Cas in its resistance is understudied. This study explored CRISPR-Cas associations with multidrug resistance in clinical K. pneumoniae. (2) Methods: 400 K. pneumoniae isolates (200 CR and 200 carbapenem susceptible (CS)) were analyzed. Carbapenemase genes (blaOXA-48, blaNDM-1, blaKPC-2), cas1, rpoB, and CRISPR1-3 loci were identified by PCR, while only CRISPR loci were sequenced. Genetic relatedness was assessed via PFGE, MLST, and spacer analysis. Statistical analysis utilized chi-squared and Fisher's exact tests. (3) Results: CRISPR-Cas was present in 15.8% of isolates, mainly subtypes I-E and I-E* (93.3%), with CRISPR3 loci showing the greatest spacer diversity. Clonal complexes ST14/15/101 (CR) and ST35 (CS) were identified. blaOXA-48 was linked to CRISPR-Cas-negative strains, while blaNDM-1 and blaKPC-2 were more frequent in CRISPR-Cas-positive strains (p < 0.0001). Imipenem/relebactam resistance was higher in CRISPR-Cas-negative isolates. (4) Conclusions: K. pneumoniae CRISPR-Cas systems correlate with specific carbapenemase profiles, suggesting pressure against blaOXA-48 acquisition. The coexistence of I-E and I-E* subtypes highlight synergies in targeting MGEs. CRISPR loci could be tools for subtyping organisms following MLST.}, }
@article {pmid40558493, year = {2025}, author = {Parikh, SJ and Terron, HM and Burgard, LA and Butler, DD and LaFerla, FM and Lane, S and Leissring, MA}, title = {5' DREDGE: Direct Repeat-Enabled Downregulation of Gene Expression via the 5' UTR of Target Genes.}, journal = {Cells}, volume = {14}, number = {12}, pages = {}, doi = {10.3390/cells14120866}, pmid = {40558493}, issn = {2073-4409}, support = {1R01AG066928-04A1/NH/NIH HHS/United States ; }, mesh = {*5' Untranslated Regions/genetics ; *Down-Regulation/genetics ; Humans ; CRISPR-Cas Systems/genetics ; RNA, Messenger/genetics/metabolism ; }, abstract = {Despite the availability of numerous methods for controlling gene expression, there remains a strong need for technologies that maximize two key properties: selectivity and reversibility. To this end, we developed a novel approach that exploits the highly sequence-specific nature of CRISPR-associated endoribonucleases (Cas RNases), which recognize and cleave short RNA sequences known as direct repeats (DRs). In this approach, referred to as DREDGE (direct repeat-enabled downregulation of gene expression), selective control of gene expression is enabled by introducing one or more DRs into the untranslated regions (UTRs) of target mRNAs, which can then be cleaved upon expression of the cognate Cas RNase. We previously demonstrated that the expression of target genes with DRs in their 3' UTRs are efficiently controlled by the DNase-dead version of Cas12a (dCas12a) with a high degree of selectivity and complete reversibility. Here, we assess the feasibility of using DREDGE to regulate the expression of genes with DRs inserted in their 5' UTRs. Among the five different Cas RNases tested, Csy4 was found to be the most efficient in this context, yielding robust downregulation with rapid onset in doxycycline-regulatable systems targeting either a stably expressed fluorescent protein or an endogenous gene, both in a fully reversible manner. Unexpectedly, dCas12a was also found to be modestly effective despite binding essentially irreversibly to the cut mRNA on its 5' end and thereby boosting mRNA levels. Our results expand the utility of DREDGE as an attractive method for regulating gene expression in a targeted, highly selective, and fully reversible manner.}, }
@article {pmid40558461, year = {2025}, author = {Jeddoub, O and Touil, N and Nyabi, O and El Fahime, E and Ennibi, K and Gala, JL and Benjouad, A and Belayachi, L}, title = {The Use of CRISPR-Cas Systems for Viral Detection: A Bibliometric Analysis and Systematic Review.}, journal = {Biosensors}, volume = {15}, number = {6}, pages = {}, doi = {10.3390/bios15060379}, pmid = {40558461}, issn = {2079-6374}, mesh = {*CRISPR-Cas Systems ; Bibliometrics ; Humans ; *Viruses/isolation &amp; purification/genetics ; *Virus Diseases/diagnosis ; }, abstract = {Viral infections impose a significant burden on global public health and the economy. This study examines the current state of CRISPR-Cas system research, focusing on their applications in viral detection and their evolution over recent years. A bibliometric analysis and systematic review were conducted using articles published between 2019 and 2024, retrieved from Web of Science, Scopus, and PubMed databases. Out of 2713 identified articles, 194 were included in the analysis. The findings reveal substantial growth in scientific output related to CRISPR-Cas systems, with the United States leading in research and development in this field. The rapid increase in CRISPR-Cas research during this period underscores its immense potential to transform viral diagnostics. With advantages such as speed, precision, and suitability for deployment in resource-limited settings, CRISPR-Cas systems outperform many traditional diagnostic methods. The concerted efforts of scientists worldwide further highlight the promising future of this technology. CRISPR-Cas systems are emerging as a powerful alternative, offering the possibility of expedited and accessible point-of-care testing and paving the way for more equitable and effective diagnostics on a global scale.}, }
@article {pmid40558442, year = {2025}, author = {Du, K and Zeng, Q and Jiang, M and Hu, Z and Zhou, M and Xia, K}, title = {CRISPR/Cas12a-Based Biosensing: Advances in Mechanisms and Applications for Nucleic Acid Detection.}, journal = {Biosensors}, volume = {15}, number = {6}, pages = {}, doi = {10.3390/bios15060360}, pmid = {40558442}, issn = {2079-6374}, support = {No. 82330035//National Natural Science Foundation of China/ ; No. 82130043//National Natural Science Foundation of China/ ; No. 82361138573//National Natural Science Foundation of China/ ; No. 2021SK1010//National Natural Science Foundation of Hunan Province/ ; No. 2024JJ5340//Natural Science Foundation of Hunan Province/ ; No. 2024RC3207//Science and Technology innovation Program of Hunan Province/ ; No. 23B0437//Scientific Research Fund of Hunan Provincial Education Department/ ; }, mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Humans ; *Nucleic Acids/analysis ; DNA/analysis ; }, abstract = {Nucleic acid detection technology is crucial for molecular diagnosis. The advent of CRISPR/Cas12a-based nucleic acid detection has considerably broadened its scope, from the identification of infectious disease-causing microorganisms to the detection of disease-associated biomarkers. This innovative system capitalizes on the non-specific single-strand cleavage activity of Cas12a upon target DNA recognition. By employing a fluorescent probe in the form of a single-stranded DNA/RNA, this technology enables the observation of fluorescence changes resulting from nonspecific cleavage, thereby facilitating detection. CRISPR/Cas12a-based detection systems can be regarded as a new type of biosensor, offering a practical and efficient approach for nucleic acid analysis in various diagnostic settings. CRISPR/Cas12a-based biosensors outperform conventional nucleic acid detection methods in terms of portability, simplicity, speed, and efficiency. In this review, we elucidate the detection principle of CRISPR/Cas12a-based biosensors and their application in disease diagnostics and discuss recent innovations and technological challenges, aiming to provide insights for the research and further development of CRISPR/Cas12a-based biosensors in personalized medicine. Our findings show that although CRISPR/Cas12a-based biosensors have considerable potential for various applications and theoretical research, certain challenges remain. These include simplifying the reaction process, enhancing precision, broadening the scope of disease detection, and facilitating the translation of research findings into clinical practice. We anticipate that ongoing advancements in CRISPR/Cas12a-based biosensors will address these challenges.}, }
@article {pmid40558428, year = {2025}, author = {De Silva, PIT and Hiniduma, K and Canete, R and Bhalerao, KS and Shawky, SM and Gunathilaka, H and Rouge, JL and Mosa, IM and Steffens, DC and Manning, K and Diniz, BS and Rusling, JF}, title = {Multiplexed CRISPR Assay for Amplification-Free Detection of miRNAs.}, journal = {Biosensors}, volume = {15}, number = {6}, pages = {}, doi = {10.3390/bios15060346}, pmid = {40558428}, issn = {2079-6374}, support = {2231490//U.S. National Science Foundation/ ; }, mesh = {*MicroRNAs/blood/analysis ; Humans ; *Biosensing Techniques ; *CRISPR-Cas Systems ; Limit of Detection ; }, abstract = {CRISPR-Cas proteins from bacteria are powerful tools for gene editing and molecular diagnostics. Expanding capacity of CRISPR to low cost, multiplexed assays of biomarkers is a key to future disease diagnostics, since multiple biomarker detection is essential for reliable diagnostics. Herein we describe a multiplexed assay in a 3D-printed 96-well plate with CRISPR-Cas13a immobilized in each well to target three circulating blood biomarker microRNAs (miRNAs 34c-5p, 200c-3p, and 30e-5p) for Alzheimer's disease (ALZ). Immobilized Cas13a is equipped with different crRNAs complementary to each miRNA target. MiRNA binding to crRNA complements activates the collateral RNase activity of Cas13a, cleaving a quenched fluorescent reporter (RNaseAlert) with fluorophore and quencher connected by an RNA oligonucleotide to enable fluorescence measurements. We achieved ultralow limits of detection (LOD) of 0.74 fg/mL for miRNA 34c-5p, 0.70 fg/mL for miRNA 30e-5p, and 7.4 fg/mL for miRNA 200c-3p, with dynamic ranges from LODs up to about 1800 pg/mL. The accuracy of the assay was validated by spike-recovery studies and good correlation of levels of patient plasma samples vs. a referee method. This new approach provides selective, sensitive multiplex miRNA biosensing, and simultaneously accommodates analysis of standards and controls.}, }
@article {pmid40558037, year = {2025}, author = {Taran, JA and Mintaev, RR and Glazkova, DV and Belugin, BV and Bogoslovskaya, EV and Shipulin, GA}, title = {[Influence of Homology Arm Length and Structure on the Efficiency of Long Transgene Integration into a Cleavage Site Induced by SpCas9 or AsCpf1].}, journal = {Molekuliarnaia biologiia}, volume = {59}, number = {2}, pages = {255-265}, pmid = {40558037}, issn = {0026-8984}, mesh = {Humans ; *Receptors, CCR5/genetics/metabolism ; Dependovirus/genetics ; *CRISPR-Cas Systems ; *Transgenes ; Green Fluorescent Proteins/genetics/metabolism ; *CRISPR-Associated Protein 9/genetics/metabolism ; Genetic Vectors/genetics ; *Bacterial Proteins/genetics/metabolism ; }, abstract = {One of the promising new approaches to the treatment of HIV infection is CRISPR/Cas-mediated knockout of the CCR5 receptor gene followed by the integration of an anti-HIV gene into the break site. Numerous studies have focused on the knockout of the CCR5 gene; however, the efficiency of subsequent targeted integration of long fragments remains poorly studied. To evaluate the efficiency of this approach, we used HT1080 cells and investigated the integration of a cassette expressing the EGFP gene into the CCR5 locus using two different nucleases (SpCas9 and AsCpf1) and various donor DNA constructs delivered by recombinant adeno-associated viral vectors (rAAV). For each nuclease, we designed five variants of donor DNA differing in the length (ranging from 150 to 1000 bp) or structure of the homology arms. The efficiency of transgene integration with 150 bp homology arms was the lowest for both nucleases and differed significantly from constructs with longer homology arms. Furthermore, it was shown that the presence of nuclease cleavage sites in the donor DNA flanking the cassette with homology arms did not affect the efficiency of transgene integration during AAV delivery. We demonstrated that the AsCpf1 nuclease provided higher efficiency of EGFP transgene integration than SpCas9, despite the lower efficiency of CCR5 knockout. The maximum percentage of cells with the integrated transgene was achieved using the AsCpf1 nuclease and an expression cassette with 600 bp homology arms, reaching 59 ± 6%.}, }
@article {pmid40558035, year = {2025}, author = {Mintaev, RR and Glazkova, DV and Taran, JA and Bogoslovskaya, EV and Shipulin, GA}, title = {[Improving the Efficiency and Safety of Human CCR5 Gene Editing by Selection of Optimal Guide RNAs for SpCAS9 and CAS12A].}, journal = {Molekuliarnaia biologiia}, volume = {59}, number = {2}, pages = {234-243}, pmid = {40558035}, issn = {0026-8984}, mesh = {*Receptors, CCR5/genetics/metabolism ; Humans ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism ; *HIV Infections/genetics/therapy ; *CRISPR-Associated Proteins/genetics/metabolism ; HEK293 Cells ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; }, abstract = {Advances in CRISPR/Cas-mediated genome editing have opened up treatment alternatives for many human diseases, including HIV infection. Knockout of the CCR5 gene as a potential way to treat HIV infection has long been studied. Here we analyzed guide RNAs for SpCas9 and AsCas12a nucleases targeting CCR5 gene which had been previously studied and selected the most effective among them. We also designed novel guide RNAs for the same nucleases using bioinformatics resources. We compared the efficiency of target site cleavage for all selected gRNAs using three nucleases: wt SpCas9, SpCas9-HF1-plus, and AsCas12a, as well as their off- target activities. We demonstrated that among the tested guide RNAs two for SpCas9- HF1-plus and three for AsCas12a exhibited high cleavage activity, cutting CCR5 gene in 60-72% of cells, and had off-target activities below the limit of detection. Thus, these guide RNAs may be candidates for future development of gene therapies against HIV infection.}, }
@article {pmid40557869, year = {2025}, author = {Li, X and Zhu, Z and Wu, J and Li, C and Liu, Z and Wang, J and Li, P and Zhang, Z and Huang, Y and Hong, J and Wu, T}, title = {PAM-free hairpin target binding activates trans-cleavage activity of Cas12a.}, journal = {Nucleic acids research}, volume = {53}, number = {12}, pages = {}, doi = {10.1093/nar/gkaf596}, pmid = {40557869}, issn = {1362-4962}, support = {22474045//National Natural Science Foundation of China/ ; 82172372//National Natural Science Foundation of China/ ; 82303589//National Natural Science Foundation of China/ ; 82000512//National Natural Science Foundation of China/ ; 2024JYCXJJ010//Central Universities, HUST/ ; }, mesh = {*CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *CRISPR-Cas Systems ; Molecular Docking Simulation ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; *Bacterial Proteins/metabolism/chemistry/genetics ; *DNA/chemistry/metabolism/genetics ; Calcium/analysis ; Nucleic Acid Conformation ; Protein Binding ; }, abstract = {CRISPR-Cas12a has been demonstrated to be activated for its trans-cleavage activity by single- and double-stranded DNA containing a protospacer adjacent motif (PAM), but other types of activators have remained undiscovered. In this work, we found that a hairpin-structured substrate can activate the trans-cleavage activity of Cas12a without a PAM, and the parameters of the hairpin loop obviously affect the activity. Cas12a exhibits sequence preference for proximal loops, preferring to recognize polyadenine hairpin loop activators. Molecular docking and dynamic calculations provide a theoretical basis for the activation of Cas12a by hairpin activators. Leveraging the efficient activation capability of the hairpin activator, we constructed an allosteric detection platform for non-nucleic acid targets, capable of sensitively and specifically detecting hypochlorous acid and calcium ions. This novel activator of Cas12a holds enormous potential for the development of multi-functional biological platforms.}, }
@article {pmid40482035, year = {2025}, author = {Dai, P and Liu, T and Yang, D and Luo, Y and Liu, LD and Xie, X and Shang, Y and Liu, X and Meng, FL}, title = {Protocol for genetic dissection of class switch recombination using genome-editing tools.}, journal = {STAR protocols}, volume = {6}, number = {2}, pages = {103882}, pmid = {40482035}, issn = {2666-1667}, mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Immunoglobulin Class Switching/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Animals ; Electroporation/methods ; *Recombination, Genetic ; Mice ; }, abstract = {Antibody class switch recombination is achieved through programmed DNA damage, and the processing of programmed DNA lesions requires the coordinated action of many DNA metabolic factors. Here, we present a protocol for inducing class switch recombination using base editors or CRISPR-Cas9. We provide optimized guide RNA (gRNA) sequences and describe steps for cytokine activation, electroporation, surface immunoglobulin detection, and data analysis. This method allows researchers to investigate the involvement of specific factors in antibody diversification and elucidate their functional roles. For complete details on the use and execution of this protocol, please refer to Dai et al.[1].}, }
@article {pmid40436039, year = {2025}, author = {Tang, S and Žedaveinytė, R and Burman, N and Pandey, S and Ramirez, JL and Kulber, LM and Wiegand, T and Wilkinson, RA and Ma, Y and Zhang, DJ and Lampe, GD and Berisa, M and Jovanovic, M and Wiedenheft, B and Sternberg, SH}, title = {Protein-primed homopolymer synthesis by an antiviral reverse transcriptase.}, journal = {Nature}, volume = {}, number = {}, pages = {}, pmid = {40436039}, issn = {1476-4687}, abstract = {Bacteria defend themselves from viral predation using diverse immune systems, many of which target foreign DNA for degradation[1]. Defence-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this strategy by using DNA synthesis instead[2,3]. We and others recently showed that DRT2 systems use an RNA template to assemble a de novo gene that encodes the antiviral effector protein Neo[4,5]. It remains unclear whether similar mechanisms of defence are used by other related DRT families. Here, we show that DRT9 systems defend against phage using DNA homopolymer synthesis. Viral infection triggers polydeoxyadenylate (poly-dA) accumulation in the cell, driving abortive infection and population-level immunity. Cryo-electron microscopy structures reveal how a non-coding RNA serves as both a structural scaffold and reverse transcription template to direct hexameric complex assembly and poly-dA synthesis. Notably, biochemical and functional experiments identify tyrosine residues within the reverse transcriptase itself that probably prime DNA synthesis, leading to the formation of protein-DNA covalent adducts. Synthesis of poly-dA by DRT9 in vivo is regulated by the competing activities of phage-encoded triggers and host-encoded silencers. Collectively, our study identifies a nucleic-acid-driven defence system that expands the paradigm of bacterial immunity and broadens the known functions of reverse transcriptases.}, }
@article {pmid40418630, year = {2025}, author = {Biar, CG and Bodkin, N and Carvill, GL and Calhoun, JD}, title = {Protocol to perform multiplexed assays of variant effect using curated loci prime editing.}, journal = {STAR protocols}, volume = {6}, number = {2}, pages = {103851}, pmid = {40418630}, issn = {2666-1667}, mesh = {*Gene Editing/methods ; Humans ; *Genetic Variation/genetics ; CRISPR-Cas Systems/genetics ; High-Throughput Nucleotide Sequencing/methods ; }, abstract = {Multiplexed assays of variant effect (MAVEs) perform simultaneous characterization of many variants. Here, we present a protocol to perform MAVEs using curated loci prime editing (cliPE), an accessible experimental pipeline that enables prime editing of a target gene. We describe steps for designing prime editing reagents, screening for genome editing efficiency, selecting a pool of cells edited to harbor different genetic variants, and sequencing. Lastly, we detail procedures for performing enrichment analysis to identify variants with normal or aberrant activity.}, }
@article {pmid40397576, year = {2025}, author = {Zhong, X and Wu, H and Wang, G and Sun, Z}, title = {Protocol for CRISPR-mediated deletion of cis-regulatory element in murine Th17 cells for in vivo assessment of effector function.}, journal = {STAR protocols}, volume = {6}, number = {2}, pages = {103831}, pmid = {40397576}, issn = {2666-1667}, support = {P30 CA033572/CA/NCI NIH HHS/United States ; R01 AI109644/AI/NIAID NIH HHS/United States ; R21 AI163256/AI/NIAID NIH HHS/United States ; }, mesh = {Animals ; Mice ; *Th17 Cells/metabolism/immunology/cytology ; Encephalomyelitis, Autoimmune, Experimental/genetics/immunology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Cell Differentiation ; *Regulatory Sequences, Nucleic Acid/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mice, Inbred C57BL ; }, abstract = {Studying the cis-regulatory elements (CREs) of genes in Th17 cells during autoimmune disease progression, such as experimental autoimmune encephalomyelitis (EAE), is often limited by the availability of gene-edited mice. Here, we present a protocol for CRISPR-mediated deletion of a CRE in murine Th17 cells for in vivo assessment of effector function in EAE. We describe steps for dual U6gRNA construction, preparation of retroviruses, viral delivery, and Th17 differentiation. We then detail procedures for in vivo functionality analysis. For complete details on the use and execution of this protocol, please refer to Zhong et al.[1][,][2].}, }
@article {pmid40310725, year = {2025}, author = {Hao, M and Lu, P and Guan, JL}, title = {Protocol for in vivo CRISPR knockout screening of autophagy genes to identify breast cancer metastasis suppressors.}, journal = {STAR protocols}, volume = {6}, number = {2}, pages = {103798}, pmid = {40310725}, issn = {2666-1667}, mesh = {Animals ; *Autophagy/genetics ; *Breast Neoplasms/genetics/pathology ; Mice ; Female ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; Humans ; Neoplasm Metastasis/genetics ; Lung Neoplasms/secondary/genetics ; }, abstract = {Metastasis represents the primary cause of mortality among patients with breast cancer. Here, we present a protocol for utilizing an in vivo custom CRISPR-Cas9 knockout library to systematically investigate autophagy regulatory genes implicated in breast cancer metastasis to the lung in mice. We describe steps for library synthesis, cloning, and virus packaging and transfection. We then detail procedures for genome DNA collection and transplantation, followed by analysis of screening data. This protocol enables efficient identification of potential suppressors of breast cancer metastasis in vivo. For complete details on the use and execution of this protocol, please refer to Hao et al.[1].}, }
@article {pmid40215169, year = {2025}, author = {Ortega, P and Sanchez, A and Seldin, M and Buisson, R}, title = {Oligo-seq protocol for mapping DNA motifs targeted by base editors.}, journal = {STAR protocols}, volume = {6}, number = {2}, pages = {103758}, pmid = {40215169}, issn = {2666-1667}, mesh = {*Gene Editing/methods ; *Nucleotide Motifs/genetics ; CRISPR-Cas Systems/genetics ; *Sequence Analysis, DNA/methods ; *DNA/genetics/chemistry ; Humans ; High-Throughput Nucleotide Sequencing/methods ; }, abstract = {Determining which DNA sequences are preferentially targeted by base editors is critical for understanding how APOBECs, AID, and other CRISPR-Cas9 base editors edit DNA in cells or improve their editing efficiency. We have developed Oligo-seq, an in vitro sequencing-based method to identify the preferred sequence motifs targeted by these enzymes. This assay monitors DNA deaminase activity on DNA oligonucleotides containing random nucleotides and/or DNA structures and determines by sequencing which sequences are preferentially deaminated. For complete details on the use and execution of this protocol, please refer to Sanchez et al.[1].}, }
@article {pmid40198219, year = {2025}, author = {Obolenski, S and Olvera-León, R and Sun, D and Adams, DJ and Waters, AJ}, title = {Protocol for the functional evaluation of genetic variants using saturation genome editing.}, journal = {STAR protocols}, volume = {6}, number = {2}, pages = {103710}, pmid = {40198219}, issn = {2666-1667}, mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Humans ; *Genetic Variation/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; High-Throughput Nucleotide Sequencing/methods ; Gene Library ; }, abstract = {Saturation genome editing (SGE) employs CRISPR-Cas9 and homology-directed repair (HDR) to introduce exhaustive nucleotide modifications at specific genomic sites in multiplex, enabling the functional analysis of genetic variants while preserving their native genomic context. Here, we present a protocol for SGE-based variant evaluation in HAP1-A5 cells. We describe the steps for designing variant libraries, single-guide RNAs (sgRNAs), and oligonucleotide primers for PCR. We also detail the sample preparation before the SGE screen, the cellular screening process, and subsequent next-generation sequencing (NGS) library preparation. For complete details on the use and execution of this protocol, please refer to Radford et al.,[1] Waters et al.,[2] and Olvera-León et al.[3].}, }
@article {pmid40173037, year = {2025}, author = {Della Volpe, L and Vacca, R and Di Micco, R}, title = {Protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human hematopoietic stem and progenitor cells.}, journal = {STAR protocols}, volume = {6}, number = {2}, pages = {103722}, pmid = {40173037}, issn = {2666-1667}, mesh = {Humans ; *Gene Editing/methods ; *Hematopoietic Stem Cells/cytology/metabolism ; *CRISPR-Cas Systems/genetics ; *Cell Culture Techniques/methods ; }, abstract = {Long-range correction strategies require ex vivo activation of hematopoietic stem and progenitor cells (HSPCs) to engage the homology-directed repair (HDR) mechanism, but prolonged culture causes harmful cellular responses, reducing the long-term functionality of gene-edited (GE) HSPCs. Here, we present a protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human HSPCs. We describe steps for HSPC thawing, ex vivo treatments, gene editing, and downstream in vitro and in vivo analyses to assess the functionality of GE-HSPCs. For complete details on the use and execution of this protocol, please refer to della Volpe et al.[1].}, }
@article {pmid40120111, year = {2025}, author = {Liang, Z and Wu, X and Ye, Z and She, L and Ma, X and Huo, YX}, title = {Protocol for identification of sgRNA mutants using high-throughput screening technique and multiplex genome editing.}, journal = {STAR protocols}, volume = {6}, number = {2}, pages = {103690}, pmid = {40120111}, issn = {2666-1667}, mesh = {*Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; *High-Throughput Screening Assays/methods ; *Mutation ; Humans ; Gene Library ; }, abstract = {In the CRISPR-Cas9 system, tandem expression of multiple identical single-guide RNAs (sgRNAs) often triggers homologous sequences loss, which affects multiplex genome editing efficiencies. Here, we present a protocol for high-throughput screening of functional sgRNAs with nonrepetitive mutants. We describe steps for constructing the screening platform, designing and constructing sgRNA libraries, and screening sgRNA mutants. These mutants can interact with the Cas9 protein, enabling multiplex genome editing. For complete details on the use and execution of this protocol, please refer to Liang et al.[1].}, }
@article {pmid40088451, year = {2025}, author = {Kohabir, KAV and Linthorst, J and Wolthuis, RMF and Sistermans, EA}, title = {Protocol for high-precision CRISPR-Cas12a-based SNV detection on synthetic DNA, cell line cfDNA models, and liquid biopsies.}, journal = {STAR protocols}, volume = {6}, number = {2}, pages = {103696}, pmid = {40088451}, issn = {2666-1667}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Liquid Biopsy/methods ; *Cell-Free Nucleic Acids/genetics ; *Polymorphism, Single Nucleotide/genetics ; *DNA/genetics ; Cell Line ; Gene Editing/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {CRISPR-based diagnostics (CRISPRdx) offer promising tools for rapid and cost-effective genetic testing, but achieving single-nucleotide fidelity remains a challenge. Here, we present a protocol for high-precision detection of single-nucleotide variants (SNVs) using a Cas12a-based approach. We describe how to apply our publicly available ARTEMIS algorithm to identify targetable SNVs, design optimized CRISPR RNAs (crRNAs), and perform fluorescence-based CRISPRdx assays on synthetic DNA, cell line-derived cell-free DNA (cfDNA), and liquid biopsy samples. For complete details on the use and execution of this protocol, please refer to Kohabir et al.[1].}, }
@article {pmid40555885, year = {2025}, author = {Asah-Asante, R and Tang, L and Gong, X and Fan, S and Yan, C and Asante, JO and Zeng, Q}, title = {Exploring pigment-producing Streptomyces as an alternative source to synthetic pigments: diversity, biosynthesis, and biotechnological applications. A review.}, journal = {World journal of microbiology &amp; biotechnology}, volume = {41}, number = {7}, pages = {211}, pmid = {40555885}, issn = {1573-0972}, mesh = {*Streptomyces/metabolism/genetics/classification ; *Pigments, Biological/biosynthesis/chemistry ; Biosynthetic Pathways/genetics ; *Biotechnology/methods ; Multigene Family ; CRISPR-Cas Systems ; Melanins/biosynthesis ; Gene Editing ; Anthraquinones/metabolism ; Prodigiosin/analogs &amp; derivatives/biosynthesis ; Synthetic Biology ; Fermentation ; Benzoisochromanequinones ; }, abstract = {The increasing health and environmental concerns associated with synthetic pigments have intensified the global search for natural, eco-friendly alternatives. Among microbial sources, Streptomyces, a genus within the class Actinomycetia, has emerged as a prolific source of bioactive pigments with wide-ranging industrial applications. The review provides a comprehensive synthesis of pigment-producing Streptomyces, focusing on their ecological diversity, biosynthetic pathways, and taxonomic relevance. This review has discussed key pigment classes, including melanin, prodiginine, quinone, and actinorhodin, with their bioactive properties, such as antioxidants, antimicrobials, and anticancer. The review further emphasizes recent advancements in synthetic biology, including clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas)-based gene editing, which has facilitated the activation of silent biosynthetic gene clusters (BGCs) that can enhance pigment yield. Additionally, this review discusses the optimization and fermentation protocols, industrial scalability, and the application of pigments in food, pharmaceutical, cosmetic, textile, and environmental sectors. Despite these advancements, critical research gaps persist, particularly in toxicological evaluation, pigment stability under industrial conditions, high-throughput screening of unexplored Streptomyces strains, and the integration of AI-based predictive tools for pathway optimization. Addressing these gaps is essential to unlocking the full potential of Streptomyces-derived pigments to replace harmful synthetic colorants.}, }
@article {pmid40554662, year = {2025}, author = {Li, C and Liu, X and Li, Y and Peng, Q and Miao, J and Liu, X}, title = {The Tetracycline-Inducible/CRISPR-Cas9 System is an Efficient Tool for Studying Gene Function in Phytophthora sojae.}, journal = {Molecular plant pathology}, volume = {26}, number = {6}, pages = {e70114}, doi = {10.1111/mpp.70114}, pmid = {40554662}, issn = {1364-3703}, support = {32372602//National Natural Science Foundation of China/ ; 32272599//National Natural Science Foundation of China/ ; 2024ZC-KJXX-062//the Project of Shaanxi Young Stars in Science and Technology/ ; }, mesh = {*Phytophthora/genetics/drug effects ; *CRISPR-Cas Systems/genetics/drug effects ; *Tetracycline/pharmacology ; Promoter Regions, Genetic/genetics ; }, abstract = {The present study presents a novel approach combining a tetracycline-inducible system (Tet-On) and CRISPR-Cas9 techniques to investigate the function of two essential genes in Phytophthora sojae. We constructed a donor vector in which the reverse tetracycline transactivator (rtTA) is driven by an oomycete promoter. Additionally, it contains a fused TetR binding site and the minimum oomycete promoter, as well as 1000-bp homologous arms of the promoter upstream and downstream sequences. The promoter of the target gene was replaced with a tetracycline-responsive promoter (Ptet) using a CRISPR-Cas9 system. In the native transformants, the target gene was induced by the administration of tetracycline and repressed in its absence. Using the Tet-On/CRISPR-Cas9 system, we obtained inducible transformants of PsAF5 and PsCesA3. The phenotype of PsAF5 inducible transformants without doxycycline was consistent with that of ΔPsAF5 transformants, specifically characterised by an increase in oospore production and heightened sensitivity to H2O2. PsCesA3 inducible transformants could not grow in the absence of doxycycline, which means PsCesA3 is an essential protein for P. sojae. In conclusion, the Tet-On/CRISPR-Cas9 system represents an effective approach for investigating crucial genes in P. sojae.}, }
@article {pmid40554553, year = {2025}, author = {Wei, SC and Cantor, AJ and Walleshauser, J and Mepani, R and Melton, K and Bans, A and Khekare, P and Gupta, S and Wang, J and Soares, C and Kiwan, R and Lee, J and McCawley, S and Jani, V and Leong, WI and Shahi, PK and Chan, J and Boivin, P and Otoupal, P and Pattnaik, BR and Gamm, DM and Saha, K and Gowen, BG and Haak-Frendscho, M and Janatpour, MJ and Silverman, AP}, title = {Evaluation of subretinally delivered Cas9 ribonucleoproteins in murine and porcine animal models highlights key considerations for therapeutic translation of genetic medicines.}, journal = {PloS one}, volume = {20}, number = {6}, pages = {e0317387}, doi = {10.1371/journal.pone.0317387}, pmid = {40554553}, issn = {1932-6203}, mesh = {Animals ; Mice ; *Gene Editing/methods ; *Ribonucleoproteins/genetics/administration &amp; dosage ; Swine ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/administration &amp; dosage ; Retina/metabolism ; *Genetic Therapy/methods ; Retinal Pigment Epithelium/metabolism ; Disease Models, Animal ; }, abstract = {Genetic medicines, including CRISPR/Cas technologies, extend tremendous promise for addressing unmet medical need in inherited retinal disorders and other indications; however, there remain challenges for the development of therapeutics. Herein, we evaluate genome editing by engineered Cas9 ribonucleoproteins (eRNP) in vivo via subretinal administration using mouse and pig animal models. Subretinal administration of adenine base editor and double strand break-inducing Cas9 nuclease eRNPs mediate genome editing in both species. Editing occurs in retinal pigmented epithelium (RPE) and photoreceptor cells, with favorable tolerability in both species. Using transgenic reporter strains, we determine that editing primarily occurs close to the site of administration, within the bleb region associated with subretinal injection. Our results show that subretinal administration of BE-eRNPs in mice mediates base editing of up to 12% of the total neural retina, with an average rate of 7% observed at the highest dose tested. In contrast, a substantially lower editing efficiency was observed in minipigs; even with direct quantification of only the treated region, a maximum base editing rate of 1.5%, with an average rate of <1%, was observed. Our data highlight the importance of species consideration in preclinical studies for the development of genetic medicines targeting the eye and provide an example of a lack of translation between small and larger animal models in the context of subretinal administration of Cas9 eRNPs.}, }
@article {pmid40553877, year = {2025}, author = {Saleem, S and Amin, W and Bhatti, F and Majid, M and Fazal, A}, title = {Nucleic acid-based strategies to mitigate stripe rust disease of wheat for achieving global food security - A review.}, journal = {International journal of biological macromolecules}, volume = {}, number = {}, pages = {145353}, doi = {10.1016/j.ijbiomac.2025.145353}, pmid = {40553877}, issn = {1879-0003}, abstract = {Wheat (Triticum aestivum), being a global staple crop, is critical in ensuring food security due to its significant nutritional value. However, it faces numerous challenges from both biotic and abiotic stresses, with fungal diseases being particularly detrimental to yield. Among these, wheat stripe rust, caused by the fungal pathogen Puccinia striiformis, poses a severe threat to wheat. Globally, 5.47 million tons of grains are lost due to the stripe rust pathogen, equivalent to a loss of USD 979 million annually; almost 88 % of the world's wheat production is susceptible to stripe rust. This review accentuates the global extensive distribution of stripe rust, detailing its causes and impact on crop productivity and mitigating approaches following traditional, genomic, and post-genomics. The mitigation approaches to wheat stripe rust have been mainly categorized into primitive (pre-genomic), modern (genomic), and next-generation (post-genomic) approaches. The primitive approaches include traditional breeding, phenotypic selection, and exotic germplasm to introduce resistance leads to early success in disease management. The advanced genomic era, with tools like QTL mapping, GWAS, marker-assisted selection, and high-throughput sequencing to deploy resistance genes, helps in precise mapping and developing high-throughput genotyping for large-scale screening and introgression of multiple resistant genes. The gene-editing approaches, including CRISPR/Cas9, RNAi, and epigenomics, now enable precise gene editing and regulation for durable resistance, together with multi-omics techniques, to identify resistant pathways and biomarkers with enhanced understanding of host-pathogen interactions and resistance mechanisms. Climate change events like shifts in rainfall patterns and rising temperatures expand the rust-prone area and pose more challenges in developing durable rust-resistant cultivars. Furthermore, the review explores using wheat's valuable genetic resources and integrating AI-based technologies to enhance stripe rust resistance by analyzing large datasets, including pathogen evolution and growth stages, allowing for timely interventions of the stripe rust epidemic. The role of multiomics approaches, particularly genomics and transcriptomics, in unraveling the genetic basis of stress tolerance is highlighted. A forward-looking framework is proposed, emphasizing the use of interdisciplinary methodologies, including big data, multi-omics, and AI-driven approaches, that hold immense promise to revolutionize wheat protection with the development of climate-resilient wheat genotypes and ensure real-time disease monitoring and precision-resistant strategies against the evolving rust pathogen.}, }
@article {pmid40553564, year = {2025}, author = {Kratzmeier, C and Taheri, M and Mei, Z and Lim, I and Khalil, MA and Carter-Cooper, B and Fanaroff, RE and Ong, CS and Schneider, EB and Chang, S and Leyder, E and Li, D and Luzina, IG and Banerjee, A and Krupnick, AS}, title = {Lung adenocarcinoma-derived IFN-γ promotes growth by modulating CD8+ T cell production of CCR5 chemokines.}, journal = {The Journal of clinical investigation}, volume = {}, number = {}, pages = {}, doi = {10.1172/JCI191070}, pmid = {40553564}, issn = {1558-8238}, abstract = {Since the lung is a mucosal barrier organ with a unique immunologic environment, mechanisms of immunoregulation in lung cancer may differ from those of other malignancies. Consistent with this notion, we found that CD8+ T cells play a paradoxical role in facilitating, rather than ameliorating, the growth of multiple lung adenocarcinoma models. These include spontaneous, carcinogen-induced, and transplantable tumor cell line models. Specifically, we found that CD8+ T cells promote homing of CD4+Foxp3+ T regulatory cells to the tumor bed by increasing levels of CCR5 chemokines in the tumor microenvironment in an IFN-γ and TNF-α dependent manner. Contrary to their canonical role, these Th1 cytokines contributed to accelerated growth of murine lung adenocarcinomas while suppressing the growth of other malignancies. Surprisingly, lung cancer cells themselves can serve as a dominant source of IFN-γ, and deletion of this cytokine from cancer cells using CRISPR/Cas-9 decreases tumor growth. Importantly for translational applications, a high level of IFN-γ was also found in human lung cancer patients at both the mRNA and protein level. Our data outlines what we deem a novel and previously undefined lung cancer specific immunoregulatory pathway that may be harnessed to tailor immune based therapy specifically for this malignancy.}, }
@article {pmid40553349, year = {2025}, author = {Mirchandani, I and Khandhediya, Y and Chauhan, K}, title = {Review on Advancement of AI in Synthetic Biology.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2952}, number = {}, pages = {483-490}, pmid = {40553349}, issn = {1940-6029}, mesh = {*Synthetic Biology/methods ; *Artificial Intelligence ; Gene Editing/methods ; CRISPR-Cas Systems ; Machine Learning ; Humans ; }, abstract = {The way biological systems are built and designed has been revolutionized by synthetic biology. Further enhancements like predictive modeling, optimization and systematic design of complex biological systems, is now possible due to integration of Artificial Intelligence into synthetic biology. This review shares insights on the role of AI in advancement of synthetic biology, including genome editing, metabolic pathway optimization and biological circuit design etc. AI-driven tools contribute to the increased efficiency and precision. Application of deep learning and machine learning has made it possible to make CRISPR-cas9, de novo protein design and gene circuit development more precise. However, there are still some persistent challenges, especially in curating high-quality biological datasets and bridging interdisciplinary gaps between computational and experimental scientists. Future perspectives focus on causal reasoning in AI models, integration of physics based algorithms, and promoting collaboration across disciplines to achieve breakthroughs in both synthetic biology and AI. By joining these fields, the transformative power of synthetic biology and AI can be unlocked and applied in the fields of medicine, biotechnology and environmental sustainability, pioneering a way for a new era of bioengineering.}, }
@article {pmid40553339, year = {2025}, author = {Pandey, S and Choudhari, JK and Tripathi, A and Singh, A and Antony, A and Chouhan, U}, title = {Artificial Intelligence-Based Genome Editing in CRISPR/Cas9.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2952}, number = {}, pages = {273-282}, pmid = {40553339}, issn = {1940-6029}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Artificial Intelligence ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Precision Medicine ; Genomics/methods ; }, abstract = {Artificial intelligence (AI) plays a critical role in predicting and improving genome editing methods, including CRISPR/Cas9. Recently, several AI models, such as DeepCRISPR, CRISTA, and Deep High Fidelity (DeepHF), have been utilized to design guide RNAs (gRNAs) for CRISPR-Cas systems. These models assess genomic context, desired mutation type, on-target and off-target scores, and potential off-target locations. AI models help improve various genome editing methods, such as base, prime, and epigenome editing, which allow for precise and intentional changes to DNA sequences without the need for donor DNA templates. Furthermore, integrating AI with genome editing and precision medicine enables the creation of personalized treatments tailored to each individual's unique genetic profile. Examining genomic data enables the identification of mutations, variations, and biomarkers linked to diseases like cancer, diabetes, and Alzheimer's disease. Integrating AI with genome editing can potentially enhance genetic modification techniques' precision, efficiency, and cost-effectiveness. Furthermore, it presents novel prospects for fields like genetics, biomedicine, and healthcare, which could significantly impact human health. However, several challenges still exist, including high costs, inaccurate edits, and effective delivery methods for CRISPR components, improved editing performance, and safety in clinical applications.}, }
@article {pmid40553337, year = {2025}, author = {Khammampalli, S and Vindal, V}, title = {Artificial Intelligence in CRISPR-Cas Systems: A Review of Tool Applications.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2952}, number = {}, pages = {243-257}, pmid = {40553337}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems ; *Artificial Intelligence ; *Gene Editing/methods ; Humans ; Machine Learning ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA Editing ; }, abstract = {Genetic engineering is a method used to alter an organism's DNA, which could entail altering a base pair, removing a section of DNA, or introducing a new DNA segment. Over time, genetic engineering has progressed from basic cloning for research purposes to advanced synthetic biology, leading to new biomedical applications. Targeted genomic editing is one method of cellular reprogramming that aims to change the state of a cell. The invention of CRISPR Cas systems has greatly simplified gene editing. These systems use a unique RNA-guided DNA endonuclease, a protein that can cut DNA and be trained to target new places by changing the sequence of its guide RNA. Integrating CRISPR-Cas systems with artificial intelligence opens new insights into the study of genetic engineering and its applications. Extensive research utilizing deep learning and machine learning has been conducted to predict the outcomes of CRISPR-Cas9 editing. Artificial intelligence also predicts RNA editing events and CRISPR off-target cleavage sites. Scientists often struggle to identify the ideal perturbation for their specific application because of the ample search space and expensive genetic trials. The algorithmic method using artificial intelligence utilizes the cause-and-effect link between variables in a complicated system like genome regulation to determine which perturbation is most effective in each successive round of testing, thereby making artificial intelligence an effective technique in gene editing.}, }
@article {pmid40553335, year = {2025}, author = {Johnson, SJS}, title = {AI Revolutionizing Cell and Genetic Engineering: Innovations and Applications.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2952}, number = {}, pages = {219-232}, pmid = {40553335}, issn = {1940-6029}, mesh = {Humans ; *Artificial Intelligence ; *Genetic Engineering/methods ; Gene Editing/methods ; CRISPR-Cas Systems ; Precision Medicine/methods ; *Cell Engineering/methods ; Drug Discovery/methods ; Genomics/methods ; }, abstract = {The integration of Artificial Intelligence (AI) into the realms of cell and genetic engineering has ushered in a transformative era of innovation and discovery. This chapter explores the myriad ways in which AI technologies are revolutionizing these fields, from data analysis and drug discovery to genomic sequencing and gene editing. AI algorithms are adept at analyzing vast datasets, uncovering intricate patterns, and predicting biological phenomena with unparalleled accuracy. In drug discovery, AI-driven platforms expedite the identification of potential therapeutics by simulating molecular interactions and predicting their efficacy. Genomic sequencing efforts benefit from AI's ability to interpret genetic variations and their implications for health and disease. Furthermore, AI-guided gene editing techniques, such as CRISPR-Cas9, enable precise and targeted modifications of the genome. Beyond the laboratory, AI facilitates personalized medicine by analyzing genetic data to tailor treatments to individual patients. This chapter underscores the pivotal role of AI in advancing cell and genetic engineering, promising a future of unprecedented scientific breakthroughs and personalized healthcare solutions.}, }
@article {pmid40553283, year = {2025}, author = {Suk, Y and Apel, E and Custers, S and Miletic, P and Zhai, K and Chokshi, C and Venugopal, C and Moffat, J and Singh, SK}, title = {CRISPR-Cas9-Guided Genetic Manipulation of Patient-Derived Brain Tumor Cells.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2944}, number = {}, pages = {173-184}, pmid = {40553283}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems ; Humans ; *Brain Neoplasms/genetics/pathology ; *Gene Editing/methods ; Gene Knockout Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; }, abstract = {CRISPR-Cas9 technology has revolutionized scientific research and has provided scientists with the ability to change DNA bases specifically and precisely at predetermined sites. The CRISPR-Cas9 knockout (KO) and activation (a) platforms developed by Hart et al. and Sanson et al. allow for RNA-directed genome editing to both decrease or increase gene expression, respectively [1, 2]. In this chapter the two techniques, CRISPR KO and CRISPRa, will be discussed and explained in further detail for optimized application against patient derived brain tumor cells.}, }
@article {pmid40553057, year = {2025}, author = {Long, W and Li, Q and Jin, T and Lu, Z and Hu, F and Zhang, H and Xu, Y}, title = {Dual detection of hypervirulent genes of Klebsiella pneumoniae using a single CRISPR-Cas12a system modulated using entropy-driven circuits.}, journal = {Analytical methods : advancing methods and applications}, volume = {}, number = {}, pages = {}, doi = {10.1039/d5ay00563a}, pmid = {40553057}, issn = {1759-9679}, abstract = {A set of different CRISPR-based analyses have been extensively used to detect nucleic acids owing to their characteristics of being sensitive, specific, rapid and easy to operate. However, one of its major challenges is application to multiplex detection (https://pubmed.ncbi.nlm.nih.gov/31460243/) using a single CRISPR-Cas system because the detection relies on the indiscriminate trans-cleavage activity of the Cas protein. Here we developed a CRISPR-based dual gene assay system for hypervirulent Klebsiella pneumoniae (hvKp) by integrating two parallel entropy-driven circuits (EDCs) and the trans-cleavage activity of the Cas12a protein towards fluorescent substrate reporters. The EDC reaction released two different fluorescent reporters and the same trigger probe according to different targets. The trigger probe activated the trans-cleavage activity of Cas12a towards reporter probes to achieve the purpose of the dual detection of different genes with a single CRISPR-Cas12a system in one tube. Furthermore, the assay system was designed to detect different mRNA sequences (rmpA and peg-344) of hvKp, requiring no reverse transcription procedure and providing technical support for the identification of hvKp and classical Klebsiella pneumoniae (cKp). The detection limits for rmpA and peg-344 were as low as 0.10 fmol L[-1] and 0.17 fmol L[-1], respectively, within 25 minutes under isothermal conditions. It enabled rapid dual detection of hvKp in a single tube using only a CRISPR-Cas12a system, demonstrating great potential for point-of-care testing due to its high sensitivity, low cost, and robust ability to detect various mRNA biomarkers.}, }
@article {pmid40551276, year = {2025}, author = {Zhang, Q and Yu, Y and Yin, B and Xu, L and Chen, H and Qiao, R and Chen, A and Zhu, N and Wu, X}, title = {An ultrasensitive and specific CRISPR-Cas13a-mediated point-of-care assay for monkeypox detection and PCR-based clade detection.}, journal = {Infectious diseases of poverty}, volume = {14}, number = {1}, pages = {56}, pmid = {40551276}, issn = {2049-9957}, support = {202308003//Nanjing Science and technology plan project/ ; BE2022669//Social Development Project of Jiangsu Provincial Science and Technology Department/ ; SJCX23_0855//Graduate Research and Innovation Projects of Jiangsu Province/ ; RCMS24008//Talent Lift Program of The Second Hospital of Nanjing/ ; }, mesh = {Sensitivity and Specificity ; Humans ; *CRISPR-Cas Systems ; *Point-of-Care Systems ; *Mpox, Monkeypox/diagnosis/virology ; *Monkeypox virus/isolation &amp; purification/genetics/classification ; *Polymerase Chain Reaction/methods ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; }, abstract = {BACKGROUND: The rapid increase in the number of monkeypox cases poses a considerable threat to the international community, necessitating sensitive, fast, and available diagnostic methods. Therefore, the objective of this study was to develop a rapid, sensitive and simple method with high clinical applicability.<br><br>METHODS: We developed a simple, rapid point-of-care assay to detect monkeypox virus (MPXV) using multienzyme isothermal rapid amplification (MIRA) coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a system. The detection system was optimized by synthesizing plasmids, and the detection sensitivity was explored by the continuous dilution of the plasmid. We validated the accuracy of this assay on 202 clinical MPXV samples and 104 interference samples through the kappa test. The visual interpretation of the results was realized by combining the assay with lateral flow strips. In addition, we developed a PCR-based method to identify MPXV Clades I and II, and the accuracy was tested through a kappa test on 202 clinical monkeypox samples and 104 interference samples.<br><br>RESULTS: Our assay achieved an analytical sensitivity of 14.4 copies/ml and high selectivity, as it differentiated MPXV from three other Orthopoxvirus species. The clinical testing results for 202 monkeypox samples and 104 interference samples demonstrated 100% sensitivity and specificity. Compared with quantitative PCR (qPCR), three samples tested as positive using our assay, which showed that the performance of this assay was superior to that of the qPCR assay. Combined with lateral flow strips, its availability and simplicity provide an alternative point-of-care diagnostic method for MPXV testing in remote settings and resource-poor areas. The results of 32 clinical samples showed that lateral flow strips had a high detection sensitivity and could identify samples with Ct value of 39 as positive. The clade identification assay detected as few as 200 copies/ml within 40&#xa0;min and no cross-reaction was observed between Clades I and II. The clinical samples tested were all Clade II, which was consistent with the circulating clade in the Chinese mainland.<br><br>CONCLUSIONS: The MIRA-CRISPR-Cas13a-MPXV system offers a rapid, sensitive and specific approach for monkeypox diagnosis, with significance for&#xa0;monitoring monkeypox epidemics. The clade identification assay based on PCR could accurately distinguish Clade I from Clade II within 40&#xa0;min and can be implemented for high-throughput operation.}, }
@article {pmid40551141, year = {2025}, author = {Kim, MS and Jeong, DE and Choi, SK}, title = {Harnessing an anti-CRISPR protein for powering CRISPR/Cas9-mediated genome editing in undomesticated Bacillus strains.}, journal = {Microbial cell factories}, volume = {24}, number = {1}, pages = {143}, pmid = {40551141}, issn = {1475-2859}, support = {KGM1192511//Korea Research Institute of Bioscience and Biotechnology/ ; IGM042411//HLB Genex/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Bacillus/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Plasmids/genetics ; CRISPR-Associated Protein 9/antagonists &amp; inhibitors ; RNA, Guide, CRISPR-Cas Systems/genetics ; Bacillus subtilis/genetics ; }, abstract = {BACKGROUND: Wild-type Bacillus strains have significant industrial and medical value, but their effective utilization often requires strain improvement. The CRISPR/Cas9 system has become the primary tool for genome editing, allowing precise introduction of desired mutations at specific chromosomal locations. However, the practical application of CRISPR/Cas9 in most wild-type Bacillus strains remains challenging due to cellular toxicity resulting from Cas9/sgRNA activity. Therefore, controlling Cas9 toxicity is essential for the widespread application of the CRISPR/Cas9 system in wild-type Bacillus strains.<br><br>RESULTS: We employed AcrIIA4, an anti-CRISPR protein that inhibits the Cas9/sgRNA ribonucleoprotein complex from interacting with DNA, to mitigate Cas9/sgRNA-mediated toxicity, thereby enabling CRISPR/Cas9-based genome editing in wild-type strains. The newly constructed CRISPR/anti-CRISPR (CAC) plasmids harbor both cas9 and acrIIA4 genes controlled by the Pspac and Pxyl promoters, respectively, along with the repressor genes lacI and xylR. This design allows precise control of Cas9 activity through inducers. Xylose, which induces AcrIIA4 expression, effectively alleviated Cas9/sgRNA-mediated toxicity during transformation. Under xylose induction, the CAC plasmid led to a remarkable 139-fold increase in the transformation efficiency of wild-type Bacillus subtilis compared to a plasmid lacking anti-CRISPR. Meanwhile, IPTG induction promoted Cas9 expression, facilitating efficient genome editing. Upon IPTG induction, the genome editing efficiency in wild-type B. subtilis increased from 0 to 95.8% in transformants carrying the CAC plasmid. Importantly, our findings extend beyond B. subtilis, revealing that the anti-CRISPR protein dramatically enhanced transformation and genome editing efficiencies in Bacillus pumilus. Moreover, we demonstrated that the CAC system successfully enabled the generation of spo0A mutants in Bacillus mojavensis, Bacillus tequilensis, and Paenibacillus polymyxa.<br><br>CONCLUSIONS: In this study, we developed a CAC system that utilizes the anti-CRISPR protein AcrIIA4 to reduce Cas9/sgRNA-mediated toxicity in Bacillus strains. This system enables precise control of AcrIIA4 and Cas9 expression through inducers, significantly enhancing the efficiency of transformation and genome editing in wild-type Bacillus strains. Therefore, the CAC system stands as a powerful tool to facilitate genome editing in diverse wild-type Bacillus species.}, }
@article {pmid40551124, year = {2025}, author = {Shu, J and Tan, Q and Huang, Z and Zhang, T and Ye, L and Fu, S and Mao, Z}, title = {One-pot one-step detection platform for severe fever with thrombocytopenia syndrome virus via the CRISPR/Cas12a detection system.}, journal = {Virology journal}, volume = {22}, number = {1}, pages = {203}, pmid = {40551124}, issn = {1743-422X}, support = {2025KY435//Medical and Health Research Project of Zhejiang Province/ ; 2022CZBJ088//the Top Talent of Changzhou "The 14th Five-Year Plan" High Level Health Talents Training Project/ ; }, mesh = {*Phlebovirus/genetics/isolation &amp; purification ; *Severe Fever with Thrombocytopenia Syndrome/diagnosis/virology ; Humans ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Limit of Detection ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne virus that primarily causes SFTS. Although a common testing route is available, a timely, conventional and accurate method for SFTSV detection is urgently needed. In the present study, we established a platform that combines the recombinase polymerase amplification (RPA) assay with the clustered regularly interspaced short palindromic repeats-CRISPR associated proteins (CRISPR/Cas) 12a technique in one step in one pot. The procedure can be completed within 45 min at a constant temperature without a sophisticated instrument. This method targets the S gene of SFTSV, with a detection limit (LoD) of 11.7 copies per reaction and high specificity, without cross reactivity with other pathogens. Furthermore, across 46 test samples, this method achieved 89.13% consistency with the PCR method (41/46). Together, the reaction system developed in the present study provides not only a novel method for SFTSV detection but also an alternative method for detecting RNA viruses.}, }
@article {pmid40550676, year = {2025}, author = {Zhan, Y and Zheng, L and Shen, J and Hu, Y and Luo, X and Dai, L}, title = {[Development of a miniaturized CRISPR/Cas gene editing tool for human gut Bacteroides].}, journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology}, volume = {41}, number = {6}, pages = {2360-2372}, doi = {10.13345/j.cjb.240959}, pmid = {40550676}, issn = {1872-2075}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Bacteroides/genetics ; Humans ; *Gastrointestinal Microbiome/genetics ; Bacteroides fragilis/genetics ; }, abstract = {: Bacteroides, as one of the most abundant and diverse genera in the human gut, is regarded as a window into the study of gut microbiota-host interactions. Currently, CRISPR/Cas-based gene editing systems targeting Bacteroides have been widely applied, while the large size of Cas nucleases limits their potential application scenarios (such as in situ gut Bacteroides editing based on phage delivery). Therefore, this study aims to develop a compact and highly efficient genetic editing tool in Bacteroides., We developed a miniaturized CRISPR/Cas gene editing system for human gut Bacteroides. First, the editing capabilities of different miniaturized CRISPR/Cas systems, including AsCas12f, CasΦ2, and ISDge10, were evaluated in Bacteroides fragilis. Subsequently, the editing capability of AsCas12f was assessed across various Bacteroides species, and the size of this system was further optimized. The results demonstrated that the CRISPR/AsCas12f genome editing system exhibited the highest editing efficiency in B. fragilis. The CRISPR/AsCas12f system achieved efficient genome editing in B. fragilis, Bacteroides thetaiotaomicron, and Phocaeicola vulgatus. Furthermore, with a repair template of 500 bp homologous arms, the editing efficiency remained as high as 94.7%. In conclusion, CRISPR/AsCas12f can serve as a chassis tool enzyme for the development of Bacteroides-based miniature gene editors and derivative technologies, laying a foundation for the further development of gene editing technology for Bacteroides.}, }
@article {pmid40549893, year = {2025}, author = {Wei, Y and Yue, T and Wang, Y and Yang, Y}, title = {Fertile androgenetic mice generated by targeted epigenetic editing of imprinting control regions.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {122}, number = {27}, pages = {e2425307122}, doi = {10.1073/pnas.2425307122}, pmid = {40549893}, issn = {1091-6490}, support = {2018YFC1004500//National Key Research and Developmental Program of China/ ; 2017YFC1001300//National Key Research and Developmental Program of China/ ; }, mesh = {Animals ; *Genomic Imprinting/genetics ; Male ; Mice ; DNA Methylation/genetics ; *Gene Editing/methods ; Female ; *Epigenesis, Genetic ; *Fertility/genetics ; Spermatozoa/metabolism ; CRISPR-Cas Systems ; Alleles ; Epigenome Editing ; }, abstract = {Each new mammalian life begins with the fusion of an oocyte and a sperm to produce a fertilized egg containing two sets of genomes, one from the mother and one from the father. Androgenesis, a way for producing offspring solely from male genetic material, is limited in mammals, presumably due to barriers arising from genomic imprinting, an epigenetic mechanism leading to monoallelic gene expression. Here, we report adult mammalian offspring derived from the genetic material of two sperm cells. These mice, which we refer to as androgenetic mice, were produced via targeted DNA methylation editing of seven imprinting control regions (ICRs) through CRISPR-based epigenome engineering. Two sperm cells were injected into an enucleated oocyte to form putatively diploid embryos. Allele-specific epigenetic editing was achieved by injecting guide RNAs with protospacer adjacent motif (PAM) sequences designed to match one allele but not the other. The birth of androgenetic mice that were able to develop to adulthood demonstrates that mammalian androgenesis is achievable by targeted epigenetic remodeling of a few defined ICRs.}, }
@article {pmid40549156, year = {2025}, author = {Sui, M and Zhou, M and Cui, M and Liu, H and Zhang, X and Hu, N and Li, Y and Wang, B and Yang, G and Gui, P and Zhu, L and Wan, F and Zhang, B}, title = {Novel drug-inducible CRISPRa/i systems for rapid and reversible manipulation of gene transcription.}, journal = {Cellular and molecular life sciences : CMLS}, volume = {82}, number = {1}, pages = {249}, pmid = {40549156}, issn = {1420-9071}, support = {82371443//National Natural Science Foundation of China grant/ ; 2023YFE0117600//National Key R&amp;D Program of China grant/ ; }, mesh = {Humans ; Tamoxifen/pharmacology/analogs &amp; derivatives ; *CRISPR-Cas Systems/genetics ; *Transcription, Genetic/drug effects ; Animals ; Gene Expression Regulation/drug effects ; Mice ; HEK293 Cells ; Receptors, Estrogen/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {CRISPR activation and interference (CRISPRa/i) are highly effective tools to regulate transcription by fusing dead Cas9 (dCas9) with transcriptional regulatory factors guided by small guide RNA (sgRNA) in mammalian cells and mice. Still, a controllable gene regulation system is desired to investigate and manipulate dynamic biological processes. Here, we reported flexible drug-responsive CRISPRa/i systems by fusing mutated human estrogen receptor (ERT2) domains, which responded to estrogen analogue tamoxifen or its active metabolite 4-hydroxy-tamoxifen (4OHT), to CRISPRa/i components for transcriptional regulation. Upon 4OHT treatment, the optimal variants, ERT2-ERT2-CRISPRa/i-ERT2 (iCRISPRa/i), showed rapid protein translocation of iCRISPRa/i from cytoplasm to nucleus and subsequent transcriptional response. The inducible transcriptional manipulation could be restored to its original level when 4OHT was withdrawn. Moreover, the efficiencies of gene expression regulation of iCRISPRa/i were comparable to those of non-inducible and doxycycline-inducible counterparts, with a lower leakage and a faster drug response activity. The iCRISPRa/i systems successfully induced phenotypic changes in various cell lines. These results highlight that iCRISPRa/i systems could achieve fast and flexible drug-responsive transcriptional modulation and phenotypic changes, and thus provide better options for gain- and loss-of-function model construction and gene therapy.}, }
@article {pmid40548936, year = {2025}, author = {Li, Y and Zhao, C and Cao, Y and Chen, X and Tang, Y and Zhou, X and Ingmer, H and Jiao, X and Li, Q}, title = {Oxidative stress elicited by phage infection induces Staphylococcal type III-A CRISPR-Cas system.}, journal = {Nucleic acids research}, volume = {53}, number = {12}, pages = {}, doi = {10.1093/nar/gkaf541}, pmid = {40548936}, issn = {1362-4962}, support = {32430103//National Natural Science Foundation of China/ ; B2302024//National Natural Science Foundation of China/ ; 2023YFD1800503//National Key Research and Development Program of China/ ; 2022YFC2604200//National Key Research and Development Program of China/ ; RZZ202302//Jiangsu Key Laboratory of Zoonosis Major Independent Research/ ; 2024ZB284//Jiangsu Funding Program for Excellent Postdoctoral Talent/ ; 2023B1515120016//Guangdong Basic and Applied Basic Research Foundation/ ; JCYJ20220818100616034//Shenzhen Science and Technology Innovation Commission/ ; KYCX24_3850//Yangzhou University/ ; 32430103//Priority Academic Program Development of Jiangsu Higher Education Institution (PAPD)/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Oxidative Stress/genetics ; *Staphylococcus aureus/virology/genetics/metabolism/immunology ; Promoter Regions, Genetic ; Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; *Bacteriophages ; *Staphylococcus Phages ; CRISPR-Associated Proteins/genetics/metabolism ; }, abstract = {In prokaryotes, the CRISPR-Cas system provides immunity to invading mobile genetic elements, but its expression is commonly repressed in the absence of phage infection to prevent autoimmunity. How bacteria senses phage infection and activates CRISPR-Cas system are poorly understood. Here, we demonstrate that an essential promoter Pcas, located within the cas1 gene, is the primary promoter driving expression of cas genes encoding the Cas10-Csm interference complex in Staphylococcus aureus type III-A CRISPR-Cas system during phage infection. As a conserved promoter in Staphylococci type III-A CRISPR-Cas system, the Pcas loses its ability to activate cas genes expression when mutated at the C186 site. Importantly, we find that the transcriptional regulator MgrA directly represses type III-A CRISPR-Cas system by interacting with Pcas to prevent autoimmunity. Upon phage infection, MgrA senses oxidative stress and dissociates from the Pcas, alleviating the transcriptional repression and subsequently triggering a robust immunity against phages. Our work provides evidence for the requirement of Pcas within cas1 during type III-A CRISPR-Cas interference stage, and reveals that MgrA-mediated regulation provides an effective mechanism for bacteria to balance avoiding autoimmunityand defending against phages.}, }
@article {pmid40548934, year = {2025}, author = {Flores-Fernández, CN and Lin, D and Robins, K and O'Callaghan, CA}, title = {UniClo: scarless hierarchical DNA assembly without sequence constraint.}, journal = {Nucleic acids research}, volume = {53}, number = {12}, pages = {}, doi = {10.1093/nar/gkaf548}, pmid = {40548934}, issn = {1362-4962}, support = {BB/X511122/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; //University of Oxford/ ; }, mesh = {DNA Methylation ; *DNA/genetics/metabolism/chemistry ; Plasmids/genetics ; CRISPR-Cas Systems ; *Deoxyribonucleases, Type II Site-Specific/metabolism/genetics ; Genetic Vectors/genetics ; }, abstract = {Type IIS restriction enzyme-mediated DNA assembly is efficient but has sequence constraints and can result in unwanted sequence scars. To overcome these drawbacks, we developed UniClo, a type IIS restriction enzyme-mediated method for universal and flexible DNA assembly. This is achieved through a combination of vector engineering, DNA methylation using recombinant methylases, and steric blockade using CRISPR-dCas9 technology to regulate this methylation. Type IIS restriction enzyme sites within fragments to be assembled are methylated using recombinant methylases, while the fragment-flanking outer sites are protected from methylation by a recombinant dCas9-sgRNA complex. During the subsequent assembly reaction, only the protected flanking sites are cut as only they are unmethylated. Fragments are correctly assembled, despite containing internal sites for the single type IIS restriction enzyme used for the one-pot assembly. The assembled plasmid can be used as a donor plasmid in a subsequent assembly round with the same type IIS restriction enzyme and the assembly vector engineering ensures removal of potential scars by a trimming process. This simplifies assembly design and only three vectors are required for any multi-round assembly. These vectors all use the same pair of overhangs. UniClo provides a simple scarless approach for hierarchical assembly of any sequence and has wide potential application.}, }
@article {pmid40548648, year = {2025}, author = {Alariqi, M and Ramadan, M and Yu, L and Hui, F and Hussain, A and Zhou, X and Yu, Y and Zhang, X and Jin, S}, title = {Enhancing Specificity, Precision, Accessibility, Flexibility, and Safety to Overcome Traditional CRISPR/Cas Editing Challenges and Shape Future Innovations.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {}, number = {}, pages = {e2416331}, doi = {10.1002/advs.202416331}, pmid = {40548648}, issn = {2198-3844}, support = {2023ZD04074//Biological Breeding-Major Projects/ ; 32325039//National Natural Science Fund of China for Distinguished Young Scholars/ ; 2021hszd013//Hubei Hongshan Laboratory/ ; W2433067//National Natural Science Foundation of China for International Young Scientists/ ; 32272128//National Natural Science Foundation of China/ ; }, abstract = {Derived from the bacterial immune system, CRISPR/Cas9 induces DSBs at specific DNA sequences, which are repaired by the cell's endogenous mechanisms, leading to gene insertions, deletions, or substitutions. Despite its transformative potential, several challenges remain in optimizing of CRISPR/Cas systems, including off-target effects, delivery methods, PAM restrictions, and the limitations of traditional editing approaches. This review focuses on the interplay between these challenges and their contributions to gene editing precision, specificity, accessibility, flexibility, and safety. How reducing off-target effects enhances specificity and safety is explored, while discussing the role of HDR-based editing in achieving precise gene modifications, alongside alternative methods such as base editing and prime editing. Improved delivery mechanisms are examined for their impact on accessibility and efficiency, while the reduction of PAM restrictions is highlighted for its contributions to flexibility. Lastly, emerging cleavage-free editing technologies are evaluated as they relate to safety and accessibility. This focused review aims to clarify the connections among these aspects and outline future research directions for advancing CRISPR-based applications.}, }
@article {pmid40548119, year = {2025}, author = {Kumbhakar, DV and Thakkar, L and Akhand, C and Sharaf, S and Vemuganti, GK}, title = {Nanomaterials targeting cancer stem cells to overcome drug resistance and tumor recurrence.}, journal = {Frontiers in oncology}, volume = {15}, number = {}, pages = {1499283}, pmid = {40548119}, issn = {2234-943X}, abstract = {A cancer stem cell (CSC) is an immortal cell that is capable of self-renewal, continuous proliferation, differentiation into various cancer cell lineages, metastatic dissemination, tumorigenesis, maintaining tumor heterogeneity, and resistance to conventional treatments. Targeted therapies have made huge advances in the past few years, but resistance is still a major roadblock to their success, in addition to their life-threatening side effects. Progressive treatments are now available, including immunotherapies, CRISPR-Cas 9, sonodynamic therapy, chemodynamic therapy, antibody-drug nanoconjugates, cell-based therapies, gene therapy, and ferroptosis-based therapy, which have replaced surgery, chemotherapy, and radiotherapy for cancer treatment. The challenge is to develop targeted treatment strategies that are effective in eradicating CSCs, as they are resistant to anticancer drugs, causing treatment failure, relapse, and recurrence of cancer. An overview of the fundamental characteristics of CSCs, drug resistance, tumor recurrence, and signaling pathways as well as biomarkers associated with their metastatic potential of CSC is elucidated in this review. The regulatory frameworks for manufacturing and conducting clinical trials on cancer therapy are explicated. Furthermore, we summarize a variety of promising nanocarriers (NCs) that have been used directly and/or synergistic therapies coupled with the therapeutic drug of choice for the detection, targeting, and imaging of CSCs to surmount therapeutic resistance and stemness-related signaling pathways and eradicate CSCs, hence alleviating the limitation of conventional therapies. Nanoparticle-mediated ablation therapies (NMATs) are also being argued as a method for burning or freezing cancer cells without undergoing open surgery. Additionally, we discuss the recent clinical trials testing exosomes, CRISPR/Cas9, and nanodrugs, which have already received approval for several new technologies, while others are still in the early stages of testing. The objective of this review is to elucidate the advantages of nanocarriers in conquering cancer drug resistance and to discuss the most recent developments in this field.}, }
@article {pmid40544217, year = {2025}, author = {Ossio, A and Merino-Mascorro, A and Leon, JS and Heredia, N and Garcia, S}, title = {Detection of Murine Norovirus on Fresh Produce Through a CRISPR/Cas13a RNase-Based Capsid Integrity Assay.}, journal = {Food and environmental virology}, volume = {17}, number = {3}, pages = {35}, pmid = {40544217}, issn = {1867-0342}, support = {2019-67017-29642//National Institute of Food and Agriculture/ ; PROVERICYT//Universidad Autonoma de Nuevo Leon/ ; PROVERICYT//Universidad Autonoma de Nuevo Leon/ ; }, mesh = {*Norovirus/genetics/isolation &amp; purification ; CRISPR-Cas Systems ; *Ribonucleases/genetics/metabolism ; Animals ; *Capsid/chemistry/metabolism ; *Food Contamination/analysis ; Mice ; *Capsid Proteins/genetics/metabolism ; Food Microbiology/methods ; Fruit/virology ; Humans ; }, abstract = {Standard food detection methods do not distinguish between infectious and non-infectious human norovirus leading to uncertainty in the management of a norovirus positive food sample. These methods also require expensive RT-qPCR-based equipment and reagents. In contrast, CRISPR-based, compared to RT-qPCR-based, detection methods are generally less expensive and yield similar sensitivity and specificity. Our goal was to detect norovirus with an intact capsid, a proxy for infectivity, through a CRISPR-Cas13a-based detection method together with an RNase-capsid integrity assay. We termed this assay: Foodborne RNA-virus Enzymatic Sensing for High-throughput on fresh produce (CRISPR FRESH) reflecting its potential to detect infectious or potentially infectious virus particles. Our CRISPR FRESH method detected murine norovirus (MNV-1), with an intact capsid, at a limit of detection of 2.59 log10 gc/25 g (5 gc/rx). This method did not cross-react with other targets (synthetic DNA targets for hepatitis A virus; human norovirus GI, GII; rotavirus). Compared with RT-qPCR, CRISPR FRESH showed an increased sensitivity when detecting low copy numbers of RNase-pre-treated MNV-1 in lettuce and blueberries samples. Viral detection with the RT-qPCR assay is quantifiable while the CRISPR assay is present/absent. This report describes a CRISPR-based detection of potentially infectious viruses in food samples.}, }
@article {pmid40540504, year = {2025}, author = {Yamashita, K and Muramoto, T}, title = {Efficient endogenous protein labelling in Dictyostelium using CRISPR/Cas9 knock-in and split fluorescent proteins.}, journal = {PloS one}, volume = {20}, number = {6}, pages = {e0326577}, pmid = {40540504}, issn = {1932-6203}, mesh = {*Dictyostelium/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; *Luminescent Proteins/genetics/metabolism ; *Protozoan Proteins/genetics/metabolism ; Green Fluorescent Proteins/genetics/metabolism ; Histones/genetics/metabolism ; }, abstract = {Fluorescent protein tagging is a powerful technique for visualising protein dynamics; however, full-length fluorescent protein knock-in can be inefficient at certain genomic loci, making it challenging to achieve stable and uniform expression. To address this issue, we used CRISPR/Cas9-mediated knock-in strategies with split fluorescent proteins in Dictyostelium discoideum. This approach enabled efficient integration of the short mNeonGreen2 (mNG2) fragment, mNG211, particularly at functionally critical loci such as major histone h2bv3, where full-length tagging was unsuccessful. Our analysis revealed that inserting tandem repeats of mNG211 at the h2bv3 locus progressively impaired cell proliferation, indicating that functional disruption depends on insert size. These findings suggest that using short tags like mNG211 minimises functional interference and facilitates knock-in at sensitive loci. We further optimised the fluorescence intensity by fine-tuning the expression of the long fragment, mNG21-10, and introducing tandem repeats of mNG211. This approach provides a reliable method for precise and stable endogenous protein labelling, facilitating live-cell imaging and functional studies in D. discoideum.}, }
@article {pmid40539512, year = {2025}, author = {Sung, K and Jung, Y and Kim, N and Kim, YW and Kim, HH and Kim, SK and Bae, S}, title = {A rational engineering strategy for structural dynamics modulation enables target specificity enhancement of the Cas9 nuclease.}, journal = {Nucleic acids research}, volume = {53}, number = {12}, pages = {}, doi = {10.1093/nar/gkaf535}, pmid = {40539512}, issn = {1362-4962}, support = {2018R1A2B2001422//National Research Foundation of Korea/ ; 2021M3A9H3015389//National Research Foundation of Korea/ ; RS-2024-00451880//National Research Foundation of Korea/ ; RS-2024-00455559//National Research Foundation of Korea/ ; SRC-NRF2022R1A5A102641311//National Research Foundation of Korea/ ; RS-2022-NR070713//MSIT/ ; RS-2023-00260968//MSIT/ ; RS-2024-00357556//MSIT/ ; RS-2024-00338871//MSIT/ ; RS-2024-00332601//Korean Fund for Regenerative Medicine (KFRM)/ ; }, mesh = {Substrate Specificity ; *Protein Engineering/methods ; Humans ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; DNA/metabolism/chemistry ; Mutation ; Gene Editing ; Models, Molecular ; *Endonucleases/metabolism/genetics/chemistry ; }, abstract = {Structural dynamics of an enzyme plays a crucial role in enzymatic activity and substrate specificity, yet rational engineering of the dynamics for improved enzymatic properties remains a challenge. Here, we present a new biochemical strategy of intermediate state stabilization that modulates the multistep dynamic mechanisms of enzyme reactions to improve substrate specificity. We employ this strategy to enhance CRISPR-Cas9 nuclease specificity. By incorporating positively charged residues into the noncatalytic REC2 domain of Cas9, we stabilize the REC2-DNA interaction that forms exclusively in a catalytically inactive intermediate conformation of the Cas9 complex. This enables off-target trapping in the inactive conformation and thus reduces off-target cleavage in human cells. Furthermore, we combine the REC2 modification with mutations in previous rational variants, leading to the development of a combinational variant named Correct-Cas9, which connotes "combined with rationally engineered REC-Two" Cas9. Assessed by high-throughput analysis at thousands of target sequences, Correct-Cas9 exhibits increased target specificity compared to its parental variants, demonstrating a synergy between our strategy and previous rational approaches. Our method of intermediate state stabilization, either alone or combined with conventional approaches, could be applied to various nucleic acid-processing enzymes that undergo conformational changes upon target binding, to enhance their target specificity effectively.}, }
@article {pmid40539281, year = {2025}, author = {Glenthøj, A and Carlsen, SBIS and Hoffmann, M and Haastrup, EK and Andersen, LP and Toft, N and Kornblit, BT and Petersen, JB and Hasle, H and Ifversen, MRS}, title = {[CRISPR as a functional cure for hemoglobinopathies].}, journal = {Ugeskrift for laeger}, volume = {187}, number = {22}, pages = {}, doi = {10.61409/V12240888}, pmid = {40539281}, issn = {1603-6824}, mesh = {Humans ; *Gene Editing/methods ; *Hemoglobinopathies/therapy/genetics ; *Genetic Therapy/methods ; Anemia, Sickle Cell/therapy/genetics ; *CRISPR-Cas Systems ; beta-Thalassemia/therapy/genetics ; Fetal Hemoglobin/genetics/biosynthesis ; }, abstract = {Severe haemoglobinopathies, including sickle cell disease and β-thalassaemia, represent significant global health burdens. CRISPR technology enables precise genetic editing of haematopoietic stem cells, with current therapies focused on boosting fetal haemoglobin production for a functional cure. This review finds that, while promising, ex vivo approaches require advanced facilities and substantial resources, limiting accessibility where the need is highest. Future development of in vivo methods may expand global access, addressing the urgent need for scalable and affordable treatments for these debilitating diseases.}, }
@article {pmid40538650, year = {2025}, author = {Song, Y and Hu, Q and Han, Y and Liu, H and Huang, Z and Niu, M and Dong, X and Yan, K and Jin, L and Li, H and Sun, Y}, title = {Detection assay of polymyxin resistance coding mcr-1 gene based on CRISPR/Cas13a system.}, journal = {Frontiers in cellular and infection microbiology}, volume = {15}, number = {}, pages = {1553681}, pmid = {40538650}, issn = {2235-2988}, mesh = {*CRISPR-Cas Systems ; *Escherichia coli Proteins/genetics ; *Polymyxins/pharmacology ; *Escherichia coli/genetics/drug effects/isolation &amp; purification ; *Anti-Bacterial Agents/pharmacology ; *Drug Resistance, Bacterial/genetics ; Humans ; Sensitivity and Specificity ; Real-Time Polymerase Chain Reaction/methods ; Microbial Sensitivity Tests ; Escherichia coli Infections/microbiology ; }, abstract = {INTRODUCTION: Polymyxins are reserved as an ultimate defense against multidrug-resistant bacteria. The emergence of the polymyxin resistance gene mcr-1 poses a potential risk for the treatment of severe infections caused by Gram-negative bacteria. Timely detection and monitoring the mcr-1 gene are essential for guiding anti-infective therapy and controlling the spread of polymyxin resistance. Quantitative real-time PCR (qPCR) is one of the common methods for detecting resistance genes. However, qPCR has equipment dependency, and is not feasible in primary healthcare settings. Currently, there remains a lack of a highly sensitive and portable method for detecting the mcr-1 gene.<br><br>METHODS: We established and optimized detection assays of the mcr-1 gene based on CRISPR/Cas13a system and lateral flow strips. The detection method was preliminarily evaluated using clinical isolates from Escherichia coli, compared with qPCR.<br><br>RESULTS: The method for detecting the mcr-1 gene based on the CRISPR/Cas13a system and lateral flow strips was established, with a detection limit of 100 copies/mL. This method demonstrated high analytical specificity, with no cross-reactivity detected in non-mcr-1 and non-resistant strains. Among 36 clinical isolates, the method identified 31 strains as positive for the mcr-1 gene, and had a 100% concordance rate with the results of qPCR.<br><br>CONCLUSIONS: We established a detection method for the polymyxin resistance mcr-1 gene based on the CRISPR/Cas13a system. This method enables visual readouts without instruments, making it potentially applicable to primary healthcare settings and field surveillance.}, }
@article {pmid40537891, year = {2025}, author = {Gao, Q and Zhang, T and Yuan, Y and Li, G and Li, B and Xiong, C}, title = {Detection of KPC-Producing Carbapenem-Resistant Klebsiella pneumoniae Based on CRISPR Cas12a.}, journal = {Journal of microbiology and biotechnology}, volume = {35}, number = {}, pages = {e2502042}, doi = {10.4014/jmb.2502.02042}, pmid = {40537891}, issn = {1738-8872}, mesh = {*Klebsiella pneumoniae/genetics/drug effects/isolation &amp; purification/enzymology ; *Bacterial Proteins/genetics/metabolism ; *beta-Lactamases/genetics/metabolism ; *Carbapenems/pharmacology ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; Humans ; Klebsiella Infections/microbiology ; Anti-Bacterial Agents/pharmacology ; *Carbapenem-Resistant Enterobacteriaceae/genetics/isolation &amp; purification/enzymology ; *CRISPR-Associated Proteins/genetics ; *Endodeoxyribonucleases/genetics ; Polymerase Chain Reaction/methods ; ROC Curve ; }, abstract = {To develop a detection system for Klebsiella pneumoniae carbapenemase (KPC) and provide a reference for clinical prevention and control of nosocomial infections caused by multidrug-resistant K. pneumoniae. The KPC resistance gene was amplified by PCR. Guided by crRNA, Cas12a specifically identified the resistance gene and activated its trans-cleavage activity. In the detection system, a fluorescence probe was cleaved by activated Cas12a, and the fluorescence signal was measured using a microplate reader. Under optimized conditions, the fluorescence signal appeared within 12 min, peaked at 40 min and completed detection within 60 min. sensitivity: 91.2%, specificity: 84.1%, detection limit: 0.01 ng/μl. The samples were examined by fluorescence-CRISPR Cas12a and PCR. The coincidence rate was 85.9%, Kappa value was 0.8. The ROC curve analysis revealed an AUC of 0.916, with an optimal cutoff value of 1.55, sensitivity of 91.2%, and specificity of 84.1%. The CRISPR Cas12a detection of carbapenem-resistant K. pneumoniae (CRKP) demonstrates high sensitivity, specificity, and broad applicability. This method requires standard molecular biology equipment but does not rely on sequencing-based platforms.}, }
@article {pmid40537846, year = {2025}, author = {Sarno, F and Jacob, JJ and Eilers, RE and Nebbioso, A and Altucci, L and Rots, MG}, title = {Epigenetic editing and epi-drugs: a combination strategy to simultaneously target KDM4 as a novel anticancer approach.}, journal = {Clinical epigenetics}, volume = {17}, number = {1}, pages = {105}, pmid = {40537846}, issn = {1868-7083}, mesh = {Humans ; *Jumonji Domain-Containing Histone Demethylases/genetics/antagonists &amp; inhibitors ; *Epigenesis, Genetic/drug effects ; DNA Methylation/drug effects ; *Gene Editing/methods ; Cell Line, Tumor ; *Antineoplastic Agents/pharmacology ; Gene Expression Regulation, Neoplastic/drug effects ; *Neoplasms/genetics/drug therapy ; CRISPR-Cas Systems ; Epigenome Editing ; }, abstract = {KDM4-A/B/C, preferentially demethylating di- and tri-methylated lysine 9 on histone H3, are overexpressed in cancers and considered interesting therapeutic targets. Consequently, KDM4 inhibitors have been developed to block their enzymatic activity. However, the potential lack of specificity of such small molecules (epi-drugs) may contribute to dose-limiting toxicities. In the pursuit of more specific interventions, epigenetic editing (epi-editing) has emerged as a powerful tool to modulate gene expression by modifying the epigenetic profile of specific genomic locations. The recently developed CRISPRoff (dCas9 fused to DNMT3A/3L and KRAB), guided by sgRNAs, is successfully used for gene repression by introducing methylation of DNA and (indirectly) of histones at the targeted genomic region. We propose that combining epi-editing (here to prevent the expression of KDM4) with epi-drugs (to inhibit the KDM4 protein activity) may represent a novel path for synergistic anticancer effects through simultaneous inhibition of gene expression and protein activity. Upon validating the downregulation of KDM4A in HEK293T cells through epi-editing, we demonstrated its repression in colon, breast and hepatocellular carcinomas which was effective in preventing (breast, MCF7) or inhibiting (colon, HCT116) cancer cell growth. Anticancer effect was also confirmed for these cell lines using the KDM4 inhibitor QC6352. In parallel, our studies demonstrate a previously unnoticed increase in the expression of KDM4-A/B/C genes following the inhibition of protein activity using the pan-KDM4 inhibitors QC6352 and JIB-04. Importantly, this induction of gene expression was fully prevented or even further inhibited by epi-editing. Then, we assessed the efficacy of our dual-targeted silencing approach in cancer cells and demonstrated that the inhibition in cancer cell growth by epi-drug or epigenetic editing could be further improved by combining the treatments. Building upon these findings, we introduce a novel, potentially synergistic, therapeutic strategy that combines epi-drug administration with epi-editing. This innovative approach aims to reduce drug toxicity and the potential development of resistance by preventing drug-induced upregulation of target enzyme expression, thereby further increasing anticancer effects.}, }
@article {pmid40537532, year = {2025}, author = {Norota, K and Ishizuka, S and Hirose, M and Sato, Y and Maeki, M and Tokeshi, M and Ibrahim, SM and Harashima, H and Yamada, Y}, title = {Lipid nanoparticle delivery of the CRISPR/Cas9 system directly into the mitochondria of cells carrying m.7778G&gt;T mutation in MtDNA (mt-Atp8).}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {18717}, pmid = {40537532}, issn = {2045-2322}, support = {HI1813/7-1//Deutsche Forschungsgemeinschaft/ ; 23H00541//Ministry of Education, Culture, Sports, Science and Technology/ ; JPMJFR203X//Japan Science and Technology Corporation/ ; }, mesh = {*CRISPR-Cas Systems ; *DNA, Mitochondrial/genetics ; Humans ; Animals ; *Nanoparticles/chemistry/administration &amp; dosage ; HeLa Cells ; Mice ; *Point Mutation ; *Mitochondria/genetics/metabolism ; *Lipids/chemistry ; Mitochondrial Diseases/genetics/therapy ; DNA Breaks, Double-Stranded ; Ribonucleoproteins/genetics ; Liposomes ; }, abstract = {Mitochondrial genome mutations are associated with various diseases and gene therapy targeted to mitochondria has the potential to effectively treat such diseases. Here, we targeted a point mutation in mitochondrial DNA (mtDNA) that can cause mitochondrial diseases via delivery of the clustered, regularly interspaced, short palindromic repeats/Cas9 (CRISPR/Cas9) system to mitochondria using an innovative lipid nanoparticle (LNP) delivery system. To overcome the major barrier of the mitochondrial membrane structure, we investigated a strategy to deliver ribonucleoprotein (RNP) directly to mitochondria via membrane fusion using MITO-Porter, a mitochondria-targeting lipid nanoparticle. First, we constructed RNP-MITO-Porter, in which an RNP was loaded into MITO-Porter using a microfluidic device. Sequence-specific double-strand breaks were confirmed when the constructed RNP-MITO-Porter was applied to isolated mitochondria. Next, the RNP-MITO-Porter was applied to HeLa cells, and a portion of the RNP-MITO-Porter was colocalized with mitochondria and caused sequence-specific double-strand breaks in mtDNA. Finally, RNP-MITO-Porter was successfully delivered to mitochondria of cells derived from a mouse carrying a point mutation (m.7778G > T) in mtDNA (mt-Atp8) (LMSF-N-MTFVB cells), and created double-strand breaks at the target sequence. RNP-MITO-Porter is expected to contribute significantly to the clinical application of mitochondrion-targeted gene therapy.}, }
@article {pmid40480225, year = {2025}, author = {Zou, S and Sun, Y and Tang, W}, title = {Charting the development and engineering of CRISPR base editors: lessons and inspirations.}, journal = {Cell chemical biology}, volume = {32}, number = {6}, pages = {789-808}, doi = {10.1016/j.chembiol.2025.05.003}, pmid = {40480225}, issn = {2451-9448}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cytosine/metabolism/chemistry ; Adenine/chemistry/metabolism ; }, abstract = {CRISPR base editors (BEs) have introduced a new chapter in precise genome editing. The brief but fruitful history of BE development documents many case studies that not only lay the foundation of base-editing technology but are also instrumental to future protein engineering efforts. In this review, we summarize the development and engineering of various BEs with a focus on recent progress. These include traditional cytosine and adenine base editors (CBEs and ABEs), novel TadA-derived CBEs, transversion BEs, dual BEs, and CRISPR-free BEs. We discuss each aspect of the workflow and highlight the successes and challenges encountered in the engineering process.}, }
@article {pmid40456614, year = {2025}, author = {Zetterdahl, OG and Crowe, JA and Reyhani, S and Güra, MA and Labastida-Botey, O and Girard, AS and Froese, DS and Ahlenius, H and Canals, I}, title = {Generation of iPSC Lines with Tagged α-Synuclein for Visualization of Endogenous Protein in Human Cellular Models of Neurodegenerative Disorders.}, journal = {eNeuro}, volume = {12}, number = {6}, pages = {}, doi = {10.1523/ENEURO.0093-25.2025}, pmid = {40456614}, issn = {2373-2822}, mesh = {Humans ; *alpha-Synuclein/metabolism/genetics ; *Induced Pluripotent Stem Cells/metabolism ; *Neurodegenerative Diseases/metabolism/pathology ; Neurons/metabolism ; Cell Line ; CRISPR-Cas Systems ; Gene Editing ; Cell Differentiation ; }, abstract = {α-Synuclein is a synaptic protein that accumulates primarily in synucleinopathies and secondarily in certain lysosomal storage disorders. However, its physiological roles in health and disease are not fully understood. In part, this has been hampered by the inability to visualize α-synuclein and its cellular localization, due to the lack of specific antibodies and faithful reporters. Here, we used CRISPR/Cas9-based genome editing to generate human-induced pluripotent stem cell (iPSC) lines in which the α-synuclein (SNCA) gene has been tagged with the short HA peptide either at the N-terminus or C-terminus or with the fluorescent protein mCherry at the C-terminus of the protein. These diverse strategies revealed the C-terminus HA-tag as the best option. C-Terminus HA-tagged α-synuclein had unchanged protein expression and did not generate degradation by-products. Importantly, we show that following differentiation to neurons, the C-terminus HA-tagged iPSC line had unaffected electrophysiological properties and could be used to visualize accumulation of α-synuclein upon inhibition of lysosomal function and under physiological protein levels. It is our expectation that this line and tagging approach will be very useful in further studies examining α-synuclein aggregation and its role in cellular dysfunction and neurodegeneration.}, }
@article {pmid40349174, year = {2025}, author = {Cui, Z and Huang, F and Fang, K and Yan, J and Zhang, Y and Kang, DD and Zhou, Y and Zhao, Y and Everitt, JI and Hankey, W and Armstrong, AJ and Huang, J and Wang, H and Jin, VX and Dong, Y and Wang, Q}, title = {SCORT-Cas13d Nanotherapy Precisely Targets the 'Undruggable' Transcription Factor HoxB13 in Metastatic Prostate Cancer In Vivo.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {12}, number = {23}, pages = {e2417605}, doi = {10.1002/advs.202417605}, pmid = {40349174}, issn = {2198-3844}, support = {R01CA287510/CA/NCI NIH HHS/United States ; R35GM144117/GM/NIGMS NIH HHS/United States ; //Department of Pathology/ ; //Duke University School of Medicine/ ; 2023-TRG-0006//North Carolina Biotechnology Center/ ; R01CA287510/CA/NCI NIH HHS/United States ; R35GM144117/GM/NIGMS NIH HHS/United States ; }, mesh = {Male ; *Homeodomain Proteins/genetics/metabolism ; *Prostatic Neoplasms/genetics/pathology/metabolism/therapy ; Animals ; Mice ; Humans ; Cell Line, Tumor ; *Nanoparticles ; CRISPR-Cas Systems/genetics ; Neoplasm Metastasis ; Cell Proliferation ; }, abstract = {Metastatic cancer, the primary cause of cancer mortality, frequently exhibits heightened dependence on certain transcription factors (TFs), which serve as master regulators of oncogenic signaling yet are often untargetable by small molecules. Selective Cell in ORgan Targeting (SCORT) nanoparticles are developed for precise CRISPR/Cas13d mRNA and gRNA delivery to metastatic cancer cells in vivo, aiming to knock down the undruggable oncogenic TF HoxB13. In prostate cancer liver metastasis models driven by HoxB13, repeated systemic SCORT-Cas13d-gHoxB13 treatment significantly decreases HoxB13 expression, reduces metastasis, and extends mouse survival. Prolonged treatment shows no significant impact on major organ function, histology or immune markers. Mechanistically, SCORT-Cas13d-gHoxB13 treatment suppresses metastatic tumor proliferation and angiogenesis while promoting apoptosis by regulating multiple gene pathways. Unexpectedly, it inhibits the non-canonical, EMT-independent oncogenic function of Snail. These findings suggest that SCORT-Cas13d-gHoxB13 can effectively and safely target the undruggable HoxB13 in metastatic prostate cancer, positioning CRISPR/Cas13d as a potential treatment.}, }
@article {pmid40325913, year = {2025}, author = {Sugita, K and Kurata, M}, title = {Identification of Target Genes Using Innovative Screening Systems.}, journal = {Pathology international}, volume = {75}, number = {6}, pages = {257-266}, pmid = {40325913}, issn = {1440-1827}, mesh = {Humans ; *Neoplasms/genetics ; *Genetic Testing/methods ; Oncogenes/genetics ; High-Throughput Nucleotide Sequencing ; Single-Cell Analysis/methods ; CRISPR-Cas Systems ; Mutation ; Animals ; }, abstract = {Advances in cancer biology have been achieved by the identification of oncogenes and tumor suppressor genes through the remarkable progression of next-generation sequencing. New techniques, such as single-cell analysis, help uncover cancer progression and heterogeneity. Reverse genetic screenings, including methods like random mutagenesis via retroviruses, transposons, RNA interference, and CRISPR, are useful for exploring gene functions and their roles in cancer. Especially in random mutagenesis, CRISPR screening and its modifications have recently emerged as powerful tools due to their comprehensiveness and simplicity in inducing genetic mutations. Initially, CRISPR screening focused on analyzing biological phenotypes in a cell population. It has since evolved to incorporate advanced techniques, such as combining single-cell and spatial analyses. These developments enable the investigation of cell-cell and spatial interactions, which more closely mimic In Vivo microenvironments, offering deeper insights into complex biological processes. These approaches allow for the identification of essential genes involved in cancer survival, drug resistance, and tumorigenesis. Together, these technologies are advancing cancer research and therapeutic development.}, }
@article {pmid40301572, year = {2025}, author = {Zamora-Dorta, M and Laine-Menéndez, S and Abia, D and González-García, P and López, LC and Fernández-Montes, P and Calvo, E and Vázquez, J and Enríquez, JA and Balsa, E}, title = {Time-resolved mitochondrial screen identifies regulatory components of oxidative metabolism.}, journal = {EMBO reports}, volume = {26}, number = {12}, pages = {3045-3074}, pmid = {40301572}, issn = {1469-3178}, support = {PID2019-110766GA-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades (MCIU)/ ; PID2022-137404OB-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades (MCIU)/ ; PID2021-1279880B//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades (MCIU)/ ; FPU21/06416//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades (MCIU)/ ; CEX2021-001154-S//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades (MCIU)/ ; CEX2020-001041-S//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades (MCIU)/ ; Margarita Salas-UAM postdoctoral fellowship CA4/RSUE/2022-00037//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades (MCIU)/ ; FPI-UAM PhD fellowship//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades (MCIU)/ ; ERC-2020-STG grant agreement n{degree sign} 948478//EC | ERC | HORIZON EUROPE European Research Council (ERC)/ ; PR_EX_2022-01//CRIS Cancer Foundation (CRIS Foundation)/ ; LCF/PR/H23/52430010//'la Caixa' Foundation ('la Caixa')/ ; PRTR//EC | NextGenerationEU (NGEU)/ ; CB16/10/00282//Centro de Investigaci&#xf3;n Biom&#xe9;dica en Red Fragilidad y Envejecimiento Saludable (CIBERFES)/ ; }, mesh = {Humans ; *Mitochondria/metabolism/genetics ; *Oxidative Phosphorylation ; CRISPR-Cas Systems ; Mitochondrial Proteins/metabolism/genetics ; Oxidation-Reduction ; Amino Acids, Branched-Chain/metabolism ; Mitochondrial Diseases/genetics/metabolism ; }, abstract = {Defects in mitochondrial oxidative metabolism underlie many genetic disorders with limited treatment options. The incomplete annotation of mitochondrial proteins highlights the need for a comprehensive gene inventory, particularly for Oxidative Phosphorylation (OXPHOS). To address this, we developed a CRISPR/Cas9 loss-of-function library targeting nuclear-encoded mitochondrial genes and conducted galactose-based screenings to identify novel regulators of mitochondrial function. Our study generates a gene catalog essential for mitochondrial metabolism and maps a dynamic network of mitochondrial pathways, focusing on OXPHOS complexes. Computational analysis identifies RTN4IP1 and ECHS1 as key OXPHOS genes linked to mitochondrial diseases in humans. RTN4IP1 is found to be crucial for mitochondrial respiration, with complexome profiling revealing its role as an assembly factor required for the complete assembly of complex I. Furthermore, we discovered that ECHS1 controls oxidative metabolism independently of its canonical function in fatty acid oxidation. Its deletion impairs branched-chain amino acids (BCAA) catabolism, disrupting lipoic acid-dependent enzymes such as pyruvate dehydrogenase (PDH). This deleterious phenotype can be rescued by restricting valine intake or catabolism in ECHS1-deficient cells.}, }
@article {pmid40268165, year = {2025}, author = {Jerke, U and Eulenberg-Gustavus, C and Wagner, DL and Schreiber, A and Kettritz, R}, title = {CRISPR-Cas9 mediated proteinase 3 autoantigen deletion as a treatment strategy for anti-neutrophil cytoplasmic autoantibody-associated vasculitis.}, journal = {Kidney international}, volume = {108}, number = {1}, pages = {145-149}, doi = {10.1016/j.kint.2025.03.020}, pmid = {40268165}, issn = {1523-1755}, mesh = {Humans ; *Myeloblastin/genetics/immunology/metabolism ; *CRISPR-Cas Systems ; *Autoantigens/genetics/immunology ; Neutrophils/immunology ; *Gene Editing/methods ; *Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/therapy/immunology/genetics ; Hematopoietic Stem Cells/immunology ; Cell Differentiation ; Proof of Concept Study ; Antibodies, Antineutrophil Cytoplasmic/immunology ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cells, Cultured ; *Genetic Therapy/methods ; Leukocyte Elastase ; }, abstract = {INTRODUCTION: Proteinase 3 (PR3) is a major autoantigen in patients with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV). Here, we performed a proof-of-principle study using ex vivo CRISPR-Cas9 guided gene editing to eliminate the PR3 autoantigen as an alternative to suppressing the autoimmune response to PR3.<br><br>METHODS: A ribonucleoprotein (RNP) complex of Cas9 protein and a PR3-specific single guide-RNA was transfected into human CD34[+] hematopoietic stem and progenitor cells (HSPC) by electroporation. Effects on PR3 protein abundance, neutrophil differentiation, and ANCA-dependent and -independent neutrophil responses were assessed.<br><br>RESULTS: Gene editing introduced a frame shift in exon 2 of PRTN3. Consequently, PR3 protein was efficiently reduced in neutrophil-differentiated HSPCs as demonstrated by immunoblotting, ELISA, microscopy, and the complete absence of PR3-specific proteolytic activity. Human neutrophil elastase served as control and was not affected. PR3-deleted (PR3[KO])- and PR3 wild-type (PR3[WT])-HSPCs showed similar neutrophil differentiation. Importantly, general neutrophil defense functions to non-ANCA receptor-independent and -dependent stimuli were similar in PR3[KO]- and PR3[WT]-neutrophils as was constitutive apoptosis. Flow cytometry showed that cell membrane-PR3 was significantly reduced on PR3[KO]-neutrophils and consequent neutrophil activation to either monoclonal antibodies to PR3 or human PR3-ANCA was attenuated. In contrast, myeloperoxidase-ANCA stimulation was not affected.<br><br>CONCLUSIONS: We show the feasibility and efficacy of depleting the PR3 autoantigen in human CD34[+] HSPCs using CRISPR-Cas9. Depleting the PR3 autoantigen instead of suppressing the autoimmune response to PR3 could potentially lead to drug-free remission, particularly in patients with refractory or relapsing disease.}, }
@article {pmid40172759, year = {2025}, author = {Yang, H and Ji, X and Zhong, H and Yang, X and Hu, D and Cai, G and Wu, Z}, title = {CRISPR screening identifies protein methylation and ubiquitination modifications that modulate aflatoxin B1 cytotoxicity.}, journal = {Science China. Life sciences}, volume = {68}, number = {7}, pages = {2121-2136}, pmid = {40172759}, issn = {1869-1889}, mesh = {*Aflatoxin B1/toxicity ; *Ubiquitination/drug effects ; Humans ; Protein Processing, Post-Translational/drug effects ; Methylation/drug effects ; *Cystathionine beta-Synthase/metabolism/genetics ; S-Adenosylmethionine/metabolism ; CRISPR-Cas Systems ; Mitochondria/metabolism/drug effects ; Cell Survival/drug effects ; Gene Knockout Techniques ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Death/drug effects ; }, abstract = {Aflatoxin B1 (AFB1) is one of the most potent mycotoxins affecting human health and animal production. To deeply understand the host-toxin interaction, we performed CRISPR screening and identified cystathionine β-synthase (CBS) as a critical host gene affecting AFB1 cytotoxicity. Mechanistic studies revealed that CBS affects AFB1-induced cell death by regulating the abundance of protein post-translational modifications (PTMs) in host cells. First, AFB1 disrupted the transfer of S-adenosylmethionine (SAM) from the cytoplasm to the mitochondria, thereby reducing the intra-mitochondrial protein methylation level. Deficient intra-mitochondrial protein methylation impaired mitochondrial function and caused cell death. CBS knockout (KO) can enhance SAM generation and mobilization to restore intra-mitochondrial SAM levels by rescuing the perturbed methionine cycle after AFB1 exposure, thereby alleviating AFB1-induced cell death. Second, AFB1 decreased global protein ubiquitination levels by affecting gene expression of ubiquitin-modified enzymes. CBS-KO and pharmaceutical treatment correcting gene expression of ubiquitin-modified enzymes can rescue AFB1-induced cell death. We also investigated two PTM-regulating small molecules, SAM and PR-619, which can increase cell viability in AFB1-exposed cells.}, }
@article {pmid40100189, year = {2025}, author = {Džafo, E and Hafezi, M and Attianese, GMPG and Reichenbach, P and Grillet, S and Garcia, H and Cribioli, E and Voize, C and Tissot, S and de Silly, RV and Coukos, G and Scholten, K and Irving, M and Gentner, B}, title = {DNA-dependent protein kinase inhibitors PI-103 and samotolisib augment CRISPR/Cas9 knock-in efficiency in human T cells.}, journal = {Cytotherapy}, volume = {27}, number = {6}, pages = {766-773}, doi = {10.1016/j.jcyt.2025.02.001}, pmid = {40100189}, issn = {1477-2566}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *T-Lymphocytes/drug effects/metabolism/immunology ; *DNA-Activated Protein Kinase/antagonists &amp; inhibitors ; *Gene Knock-In Techniques/methods ; *Protein Kinase Inhibitors/pharmacology ; *Pyrimidines/pharmacology ; }, abstract = {The adoptive transfer of autologous peripheral blood T cells gene-modified to express preselected, tumor antigen-specific T-cell receptors (TCRs) is a promising treatment for solid cancers. While gene-transfer by viral transduction is highly efficient, the insertional site is not targeted and persistence of the T cells is oftentimes limited. In contrast, site-specific integration of the TCR into the TCR α chain (TRAC) locus by CRISPR/Cas9 has been shown to enable more consistent and physiologic levels of exogenous TCR expression coupled with superior persistence and tumor control in preclinical studies. Here, we sought to improve the efficiency of CRISPR/Cas9 mediated TCR knock-in (KI) into the TRAC locus of primary human T cells. In addition to the previously reported DNA-dependent protein kinase (DNA-PK) inhibitor M3814, we demonstrated that PI-103 and samotolisib markedly increase KI efficiency in a process that is good manufacturing process (GMP)-compatible. Importantly, samotolisib enabled the generation of a potent T-cell product, having no negative impact on T-cell viability, phenotype, expansion, effector function, and tumor control. Overall, we conclude that our GMP-compatible CRISPR/Cas9 protocol comprising samotolisib to augment TCR KI efficiency is suitable for the generation of genetically modified T cells for clinical use.}, }
@article {pmid40035969, year = {2025}, author = {Feng, Y and Liu, G and Li, H and Cheng, L}, title = {The landscape of cell lineage tracing.}, journal = {Science China. Life sciences}, volume = {68}, number = {7}, pages = {1941-1963}, pmid = {40035969}, issn = {1869-1889}, mesh = {*Cell Lineage/genetics ; Humans ; Animals ; *Cell Tracking/methods ; CRISPR-Cas Systems ; Cell Differentiation ; }, abstract = {Cell fate changes play a crucial role in the processes of natural development, disease progression, and the efficacy of therapeutic interventions. The definition of the various types of cell fate changes, including cell expansion, differentiation, transdifferentiation, dedifferentiation, reprogramming, and state transitions, represents a complex and evolving field of research known as cell lineage tracing. This review will systematically introduce the research history and progress in this field, which can be broadly divided into two parts: prospective tracing and retrospective tracing. The initial section encompasses an array of methodologies pertaining to isotope labeling, transient fluorescent tracers, non-fluorescent transient tracers, non-fluorescent genetic markers, fluorescent protein, genetic marker delivery, genetic recombination, exogenous DNA barcodes, CRISPR-Cas9 mediated DNA barcodes, and base editor-mediated DNA barcodes. The second part of the review covers genetic mosaicism, genomic DNA alteration, TCR/BCR, DNA methylation, and mitochondrial DNA mutation. In the final section, we will address the principal challenges and prospective avenues of enquiry in the field of cell lineage tracing, with a particular focus on the sequencing techniques and mathematical models pertinent to single-cell genetic lineage tracing, and the value of pursuing a more comprehensive investigation at both the spatial and temporal levels in the study of cell lineage tracing.}, }
@article {pmid39828625, year = {2025}, author = {Yang, J and Xu, X and Yang, L and Tian, Y and Wang, J and Han, D}, title = {Dynamic Genomic Imaging and Tracking in Living Cells by a DNA Origami-Based CRISPR‒dCas9 System.}, journal = {Small methods}, volume = {9}, number = {6}, pages = {e2401559}, doi = {10.1002/smtd.202401559}, pmid = {39828625}, issn = {2366-9608}, support = {2021YFA0909400//National Key Research and Development Program of China/ ; 2020YFA0211100//National Key Research and Development Program of China/ ; 22225402//National Natural Science Foundation of China/ ; 22204099//National Natural Science Foundation of China/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *DNA/chemistry/genetics ; *Genomics/methods ; Nanostructures/chemistry ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Green Fluorescent Proteins/genetics/metabolism ; }, abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)-associated system has displayed promise in visualizing the dynamics of target loci in living cells, which is important for studying genome regulation. However, developing a cell-friendly and rapid transfection method for achieving dynamic and long-term genomic imaging in living cells with high specificity and accuracy is still challenging. Herein, a robust and versatile method is presented that employs a barrel-shaped DNA nanostructure (TUBE) modified with aptamers for loading, protecting, and delivering CRISPR-Cas9 to visualize specific genomic loci in living cells. This approach enables dynamic tracking of target genomic regions (Chr3q29, a repetitive region of chromosome 3) throughout the mitotic process and captures variations in their spatial distribution and quantity accurately. Distinct dynamic behaviors between the Chr3q29 and telomeres are observed, which are linked to their unique chromosomal positions and levels of mobility. High-resolution multicolor labeling of the target genes is achieved, with a high degree of colocalization between the enhanced green fluorescent protein and cyanine-5 channels, facilitating precise imaging of target loci. This method not only supports dynamic genomic imaging but also enables multiplexed tracking, providing a powerful visualization tool for studying cellular processes and genetic interactions in real time within living cells.}, }
@article {pmid39718905, year = {2025}, author = {Yao, ZY and Yu, MJ and Li, QQ and Gong, JS and Zhang, P and Jiang, JY and Su, C and Xu, G and Jia, BY and Xu, ZH and Shi, JS}, title = {Unlocking Green Biomanufacturing Potential: Superior Heterologous Gene Expression with a T7 Integration Overexpression System in Bacillus subtilis.}, journal = {ACS synthetic biology}, volume = {14}, number = {6}, pages = {1977-1987}, doi = {10.1021/acssynbio.4c00694}, pmid = {39718905}, issn = {2161-5063}, mesh = {*Bacillus subtilis/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *DNA-Directed RNA Polymerases/genetics/metabolism ; *Viral Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Gene Expression ; }, abstract = {Industrial biotechnology employs cells for producing valuable products and serving as biocatalysts sustainably, addressing resource, energy, and environmental issues. Bacillus subtilis is a preferred host for creating microbial chassis cells and producing industrial enzymes and functional nutritional products. In this study, a dual-module T7 integration expression system in B. subtilis was established. The first module, driven by the T7 RNA polymerase, was integrated into the genome via the CRISPR/Cas9 system. Another module responsible for expression control was systematically integrated into 28 discrete chromosomal loci and the impact of different genomic positions on gene expression was explored, resulting in a high-intensity integrated expression system. Furthermore, by modifying the LacI repressor factor for biological regulation, we achieved a strong expression intensity without the inducer addition. This system was successfully used to express phospholipase D and hyaluronic acid lyase, resulting in extracellular enzyme activities of 339.12 U/mL and 2.60 × 10[4] U/mL, respectively. Additionally, by exclusively targeting the HA gene cluster for expression, a production yield of 6.86 g/L was achieved on a 5 L fermentation scale. The system eliminates the use of antibiotics and inducers, offering a controllable, efficient, and promising gene expression regulation tool in B. subtilis, enhancing its potential for biomanufacturing applications.}, }
@article {pmid39236976, year = {2025}, author = {Hou, ZH and Gao, Y and Zheng, JC and Zhao, MJ and Liu, Y and Cui, XY and Li, ZY and Wei, JT and Yu, TF and Zheng, L and Jiao, YC and Yang, SH and Hao, JM and Chen, J and Zhou, YB and Chen, M and Qiu, L and Ma, YZ and Xu, ZS}, title = {GmBSK1-GmGSK1-GmBES1.5 regulatory module controls heat tolerance in soybean.}, journal = {Journal of advanced research}, volume = {73}, number = {}, pages = {187-198}, doi = {10.1016/j.jare.2024.09.004}, pmid = {39236976}, issn = {2090-1224}, mesh = {*Glycine max/genetics/physiology/metabolism ; Plants, Genetically Modified/genetics ; Gene Expression Regulation, Plant ; *Thermotolerance/genetics ; CRISPR-Cas Systems ; *Plant Proteins/genetics/metabolism ; *Heat-Shock Response/genetics ; Reactive Oxygen Species/metabolism ; Signal Transduction/genetics ; Brassinosteroids/metabolism ; }, abstract = {INTRODUCTION: Heat stress poses a severe threat to the growth and production of soybean (Glycine max). Brassinosteroids (BRs) actively participate in plant responses to abiotic stresses, however, the role of BR signaling pathway genes in response to heat stress in soybean remains poorly understood.<br><br>OBJECTIVES: In this study, we investigate the regulatory mechanisms of GmBSK1 and GmBES1.5 in response to heat stress and the physiological characteristics and yield performance under heat stress conditions.<br><br>METHODS: Transgenic technology and CRISPR/Cas9 technology were used to generated GmBSK1-OE, GmBES1.5-OE and gmbsk1 transgenic soybean plants, and transcriptome analysis, LUC activity assay and EMSA assay were carried out to elucidate the potential molecular mechanism underlying GmBSK1-GmBES1.5-mediated heat stress tolerance in soybean.<br><br>RESULTS: CRISPR/Cas9-generated gmbsk1 knockout mutants exhibited increased sensitivity to heat stress due to a reduction in their ability to scavenge reactive oxygen species (ROS). The expression of GmBES1.5 was up-regulated in GmBSK1-OE plants under heat stress conditions, and it directly binds to the E-box motif present in the promoters of abiotic stress-related genes, thereby enhancing heat stress tolerance in soybean plants. Furthermore, we identified an interaction between GmGSK1 and GmBES1.5, while GmGSK1 inhibits the transcriptional activity of GmBES1.5. Interestingly, the interaction between GmBSK1 and GmGSK1 promotes the localization of GmGSK1 to the plasma membrane and releases the transcriptional activity of GmBES1.5.<br><br>CONCLUSION: Our findings suggest that both GmBSK1 and GmBES1.5 play crucial roles in conferring heat stress tolerance, highlighting a potential strategy for breeding heat-tolerant soybean crops involving the regulatory module consisting of GmBSK1-GmGSK1-GmBES1.5.}, }
@article {pmid39097089, year = {2025}, author = {Tan, S and Yuan, C and Zhu, Y and Chang, S and Li, Q and Ding, J and Gao, X and Tian, R and Han, Z and Hu, Z}, title = {Glutathione hybrid poly (beta-amino ester)-plasmid nanoparticles for enhancing gene delivery and biosafety.}, journal = {Journal of advanced research}, volume = {73}, number = {}, pages = {697-711}, doi = {10.1016/j.jare.2024.07.038}, pmid = {39097089}, issn = {2090-1224}, mesh = {*Glutathione/chemistry ; *Plasmids/chemistry/genetics ; Animals ; *Nanoparticles/chemistry ; Humans ; Mice ; Reactive Oxygen Species/metabolism ; *Gene Transfer Techniques ; *Polymers/chemistry ; Gene Editing/methods ; Genetic Therapy/methods ; Transfection/methods ; CRISPR-Cas Systems ; }, abstract = {INTRODUCTION: CRISPR/Cas9 gene editing technology has significantly advanced gene therapy, with gene vectors being one of the key factors for its success. Poly (beta-amino ester) (PBAE), a distinguished non-viral cationic gene vector, is known to elevate intracellular reactive oxygen species (ROS) levels, which may cause cytotoxicity and, consequently, impact gene transfection efficacy (T.E.).<br><br>OBJECTIVES: To develop a simple but efficient strategy to improve the gene delivery ability and biosafety of PBAE both in vivo and in vitro.<br><br>METHODS: We used glutathione (GSH), a clinically utilized drug with capability to modulating intracellular ROS level, to prepare a hybrid system with PBAE-plasmid nanoparticles (NPs). This system was characterized by flow cytometry, RNA-seq, Polymerase Chain Reaction (PCR) and Sanger sequencing in vitro, and its safety and efficacy in vivo was evaluated by imaging, PCR, Sanger sequencing and histology analysis.<br><br>RESULTS: The particle size of GSH-PBAE-plasmid NPs were 168.31&#xa0;nm with a &#x3b6;-potential of 15.21&#xa0;mV. An enhancement in T.E. and gene editing efficiency, ranging from 10&#xa0;% to 100&#xa0;%, was observed compared to GSH-free PBAE-plasmid NPs in various cell lines. In vitro results proved that GSH-PBAE-plasmid NPs reduced intracellular ROS levels by 25&#xa0;%-40&#xa0;%, decreased the total number of upregulated/downregulated genes from 4,952 to 789, and significantly avoided the disturbance in gene expression related to cellular oxidative stress-response and cell growth regulation signaling pathway compared to PBAE-plasmid NPs. They also demonstrated lower impact on the cell cycle, slighter hemolysis, and higher cell viability after gene transfection. Furthermore, GSH hybrid PBAE-plasmid NPs exhibited superior safety and improved tumor suppression ability in an Epstein-Barr virus (EBV)-infected murine tumor model, via targeting cleavage the EBV related oncogene by delivering CRISPR/Cas9 gene editing system and down-regulating the expression levels. This simple but effective strategy is expected to promote clinical applications of non-viral vector gene delivery.}, }
@article {pmid38084666, year = {2025}, author = {Ogawa, Y and Lu, Y and Kiyozumi, D and Chang, HY and Ikawa, M}, title = {CRISPR/Cas9-mediated genome editing reveals seven testis-enriched transmembrane glycoproteins dispensable for male fertility in mice.}, journal = {Andrology}, volume = {13}, number = {5}, pages = {1251-1260}, pmid = {38084666}, issn = {2047-2927}, support = {R01 HD088412/HD/NICHD NIH HHS/United States ; JP22K15103//Japan Society for the Promotion of Science/ ; JP21H02487//Japan Society for the Promotion of Science/ ; JP21H00231//Japan Society for the Promotion of Science/ ; JP21K19263//Japan Society for the Promotion of Science/ ; JP21H05033//Japan Society for the Promotion of Science/ ; JPMJPR2143//Japan Science and Technology Agency/ ; R01HD088412//Eunice Kennedy Shriver National Institute of Child Health and Human Development/ ; INV-001902/GATES/Gates Foundation/United States ; }, mesh = {Animals ; Male ; *Testis/metabolism ; *Fertility/genetics ; Mice ; *CRISPR-Cas Systems ; Mice, Knockout ; *Gene Editing ; *Membrane Glycoproteins/genetics/metabolism ; Female ; Sperm Motility/genetics ; Spermatozoa/metabolism ; Spermatogenesis/genetics ; }, abstract = {BACKGROUND: Mammalian fertilization is mediated by multiple sperm acrosomal proteins, many of which are testis-enriched transmembrane glycoproteins expressed during spermiogenesis (e.g., Izumo sperm-egg fusion 1, Sperm acrosome associated 6, and Transmembrane protein 95).<br><br>METHODS: We hypothesized that proteins with these features might have a role in sperm-egg interaction and thus carried out an in-silico screen based on multiple public databases. We generated knockout mouse lines lacking seven candidate proteins by the CRISPR/Cas9 system and conducted detailed analyses on the fecundity of the knockout males, as well as their testis appearance and weight, testis and epididymis histology, and sperm motility and morphology.<br><br>RESULTS: Through the in-silico screen, we identified 4932438H23Rik, A disintegrin and metalloproteinase domain-containing protein 29, SAYSvFN domain-containing protein 1, Sel-1 suppressor of lin-12-like 2 (C. elegans), Testis-expressed protein 2, Transmembrane and immunoglobulin domain-containing 3, and Zinc and ring finger 4. Phenotypic analyses unveiled that the knockout males showed normal testis gross appearance, normal testis and epididymis histology, and normal sperm morphology and motility. Fertility tests further indicated that the knockout male mice could sire pups with normal litter sizes when paired with wild-type females.<br><br>DISCUSSION AND CONCLUSION: These findings suggest that these seven proteins are individually dispensable for male reproduction and fertilization. Future studies are warranted to devise advanced in-silico screening approaches that permit effective identification of gamete fusion-required sperm proteins.}, }
@article {pmid40545778, year = {2025}, author = {Li, Y and Jin, L and Li, W and Wang, K and Su, H and Mao, H and Chen, W and Lan, C and Li, Q and Kaufmann, K and Yan, W}, title = {Modulation of an evolutionarily conserved epigenetic regulon controlling abscisic acid catabolism enhances drought tolerance in wheat.}, journal = {The New phytologist}, volume = {}, number = {}, pages = {}, doi = {10.1111/nph.70302}, pmid = {40545778}, issn = {1469-8137}, support = {32272127//National Natural Science Foundation of China/ ; }, abstract = {Drought stress significantly reduces crop yield by triggering abscisic acid (ABA) accumulation in plants. It involves the suppression of CYP707A genes, which encode enzymes that catalyze ABA. However, little is known about epigenetic control in the CYP707A gene-mediated drought stress response in wheat. In this study, we reported that TaCYP707A-6A/6B/6D but not TaCYP707A-5A/5B/5D participates in drought response in common wheat. Disruption of TaCYP707A-6B showed enhanced drought tolerance but also decreased fertility. Expression of TaCYP707A-6B is negatively associated with H3K27me3 level. An evolutionarily conserved CTCTGYTY motif cluster (binding site for a Jumonji H3K27me3 demethylase) was found in the intron of TaCYP707A-6B as well as the intron of CYP707A homologs in other plant species. Blocking the CTCTGYTY motif by dead Cas9 (dCas9) maintained a high level of H3K27me3 on the CYP707A gene, while decreasing its expression level leading to enhanced drought tolerance in both wheat and Arabidopsis. In particular, the mutant in which the intron bound by H3K27me3 demethylase was cut out without change of splicing pattern showed enhanced drought tolerance. Therefore, our study provides a novel approach to improve plant drought tolerance by manipulating an evolutionarily conserved cis-element bound by histone demethylases in the intron of CYP707A genes.}, }
@article {pmid40545632, year = {2025}, author = {Swinnen, G and Lizé, E and Loera Sánchez, M and Stolz, S and Soyk, S}, title = {Application of a GRF-GIF chimera enhances plant regeneration for genome editing in tomato.}, journal = {Plant biotechnology journal}, volume = {}, number = {}, pages = {}, doi = {10.1111/pbi.70212}, pmid = {40545632}, issn = {1467-7652}, support = {802008//European Research Council/International ; 310030_212218//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung/ ; }, abstract = {Genome editing has become a routine tool for functionally characterizing plant and animal genomes. However, stable genome editing in plants remains limited by the time- and labour-intensive process of generating transgenic plants, as well as by the efficient isolation of desired heritable edits. In this study, we evaluated the impact of the morphogenic regulator GRF-GIF on plant regeneration and genome editing outcomes in tomato. We demonstrate that expressing a tomato GRF-GIF chimera reliably accelerates the onset of shoot regeneration from callus tissue culture by approximately one month and nearly doubles the number of recovered transgenic plants. Consequently, the GRF-GIF chimera enables the recovery of a broader range of edited haplotypes and simplifies the isolation of mutants harbouring heritable edits, but without markedly interfering with plant growth and development. Based on these findings, we outline strategies that employ basic or advanced diagnostic pipelines for efficient isolation of single- and higher-order mutants in tomato. Our work represents a technical advantage for tomato transformation and genome editing, with potential applications across other Solanaceae species.}, }
@article {pmid40545185, year = {2025}, author = {Mondal, AR and Misra, A}, title = {'Emerging cell-specific therapies in cardiovascular disease'.}, journal = {Vascular pharmacology}, volume = {}, number = {}, pages = {107516}, doi = {10.1016/j.vph.2025.107516}, pmid = {40545185}, issn = {1879-3649}, abstract = {Atherosclerosis is a leading cause of cardiovascular morbidity and mortality worldwide, driven by complex interactions among various plaque cell types, including endothelial cells, macrophages, and smooth muscle cells. Traditional therapies targeting systemic risk factors such as cholesterol and blood pressure fail to directly address the underlying mechanisms governing plaque formation and progression. Recent advances in cell-specific therapies offer new avenues for targeting the cellular and molecular processes driving atherosclerosis. This Review explores innovative strategies including nanoparticles, viral vectors and CRISPR-Cas9 technology, which have the potential to modulate gene expression and behaviour within plaques cells to alleviate disease. By focusing on the specific roles of key cell types in atherosclerosis, these emerging therapies promise to provide more precise, effective, and personalised treatment options without inducing off-target effects. Moreover, insights gained from successful applications of these technologies in oncology are considered for potential repurposing in atherosclerosis-related disease. As these cell-specific approaches advance through preclinical and clinical development, they may significantly enhance our ability to treat atherosclerosis at its cellular roots, offering new hope for reducing the burden of cardiovascular disease.}, }
@article {pmid40544537, year = {2025}, author = {Sinha, S and Upadhyay, LSB}, title = {Understanding antimicrobial resistance (AMR) mechanisms and advancements in AMR diagnostics.}, journal = {Diagnostic microbiology and infectious disease}, volume = {113}, number = {2}, pages = {116949}, doi = {10.1016/j.diagmicrobio.2025.116949}, pmid = {40544537}, issn = {1879-0070}, abstract = {The overuse and abuse of antibiotics, which results in the evolution of resistant microorganisms, is the primary cause of the global health catastrophe known as antimicrobial resistance (AMR). The enzymatic breakdown of antibiotics, target site modification, efflux pump overexpression, and the formation of biofilm are some of the mechanisms responsible for acquiring antimicrobial resistance (AMR). These mechanisms enable bacteria to evade or neutralize the effects of antimicrobial agents, complicating treatment options and increasing mortality rates. The rapid dissemination of resistance genes via horizontal gene transfer further exacerbates the problem, necessitating urgent intervention. Advanced AMR diagnostics are transforming the fight against antimicrobial resistance. Biosensors enable rapid, point-of-care detection; Cluster regularly interspaced short palindromic repeat (CRISPR) technologies offer precise identification of resistance genes; and mass spectrometry provides fast, accurate profiling. Automated systems streamline workflows and boost throughput, while flow cytometry delivers real-time, single-cell analysis of phenotypic resistance. Together, these innovations accelerate detection and support targeted antimicrobial stewardship, essential for combating the global AMR threat. This review covers the mechanisms underlying antimicrobial resistance (AMR) and recent advancements in AMR diagnostic technologies.}, }
@article {pmid40544408, year = {2025}, author = {Yu, D and Xie, Z and Zhang, Y and Xie, Z and Fan, Q and Luo, S and Xie, L and Li, M and Zeng, T and Zhang, M and Li, X and Wei, Y and Wu, A and Wan, L}, title = {A dual fluorescence channel RAA-based CRISPR-Cas12a/Cas13a system for highly sensitive detection of Gyrovirus galga1 and Gyrovirus homsa1.}, journal = {Virulence}, volume = {16}, number = {1}, pages = {2521012}, doi = {10.1080/21505594.2025.2521012}, pmid = {40544408}, issn = {2150-5608}, abstract = {Gyrovirus galga1 (GyG1) and Gyrovirus homsa1 (GyH1) are the second and third most common gyroviruses identified, respectively, after chicken anaemia virus. They were first reported in 2011 and are currently prevalent worldwide. However, limited research on these pathogens and a lack of prevention and control strategies have necessitated the establishment of a rapid diagnostic technique to address new challenges in infectious diseases. Recombinase acid amplification (RAA) combined with CRISPR - Cas12a or CRISPR - Cas13a technology has major advantages for highly sensitive and rapid diagnosis. Specific targets can activate CRISPR-Cas trans-cleavage activity, resulting in non-specific cleavage of single-stranded DNA by the CRISPR - Cas12a complex and RNA cleavage by the CRISPR - Cas13a complex. In this study, for the first time, we combined RAA-based CRISPR - Cas12a and CRISPR - Cas13a systems for simultaneous differential diagnosis of GyG1 and GyH1 infection. The results showed that dual fluorescence channel RAA-based CRISPR - Cas12a/Cas13a technology could detect GyG1 and GyH1 within one hour, with a minimum detection limit of 1.5 copies of the target DNA standard/µL and no cross-reactivity with other avian pathogens. In addition, this method could be used for clinical detection, with the results exhibiting high consistency with those obtained by qPCR. These findings demonstrate that our RAA-based CRISPR - Cas12a/Cas13a dual-channel detection system can detect two different subtypes of gyrovirus in a sample with good specificity and high sensitivity, improving the detection efficiency and providing a new technique for the study of viral infection dynamics.}, }
@article {pmid40544221, year = {2025}, author = {Ajibode, ET and Bender, AR and Yehl, K}, title = {Profiling crRNA architectures for enhanced Cas12 biosensing.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {947}, pmid = {40544221}, issn = {2399-3642}, abstract = {CRISPR-Cas diagnostics are revolutionizing point-of-care molecular testing due to the programmability, simplicity, and sensitivity of Cas systems with trans-cleavage activity. CRISPR-Cas12 assays are promising for detecting single nucleotide polymorphisms (SNPs). However, reports vary widely describing Cas12 SNP sensitivity, and an underlying mechanism is lacking. We systematically varied crRNA length and valency to investigate the role of crRNA architectures on Cas12 biosensing in the context of speed-of-detection, sensitivity, and selectivity. Our results demonstrate that crRNAs complementary to 20 base pairs of the target DNA is optimal for rapid and sensitive detection, while a complementary length of 15 base pairs is ideal for robust SNP detection. Additionally, we uncovered a unique periodicity in SNP sensitivity based on nucleotide position and developed a structural model explaining what drives Cas12 SNP sensitivity. Lastly, we showed that bivalent CRISPR-Cas sensors have synergistic and enhanced activity that is distance dependent.}, }
@article {pmid40542569, year = {2025}, author = {Han, R and Gao, X and Qi, Y and Lu, X and Wang, X and Tang, X}, title = {Spacer-Complementary Single-Stranded DNA Oligonucleotides Can Serve as Target-Specific Inhibitors in CRISPR/Cas9 Systems.}, journal = {Cell biochemistry and function}, volume = {43}, number = {6}, pages = {e70088}, doi = {10.1002/cbf.70088}, pmid = {40542569}, issn = {1099-0844}, support = {//This research was supported by Guangxi Natural Science Foundation Project (2025GXNSFAA069363, 2023GXNSFAA026260)./ ; }, abstract = {The continuous expression of the CRISPR/Cas system in organisms can lead to various potential issues. Some anti-CRISPR strategies have been developed to achieve precise control over CRISPR/Cas, yet these strategies are predominantly protein-based, with the most commonly used anti-CRISPR (Acr) proteins lacking sufficient target specificity. However, in this study, we designed a single-stranded DNA (ssDNA) inhibitor that was complementary to the spacer region on the guide RNA, operating at the nucleic acid level. We demonstrated that this method effectively inhibits the cleavage activity of Cas9-sgRNA ribonucleoprotein (RNP) in a target-specific manner through in vitro cleavage experiments. Furthermore, we explored the binding position and effective length of this inhibitory ssDNA, finding that its inhibitory effect was significantly reduced when the length of continuous complementarity with the 5' end of the spacer was less than 7nt. The truncated ssDNA also showed potential in reducing off-target effects. Moreover, we applied nucleic acid inhibitors to embryos via microinjection, and gene editing activity was significantly reduced, as evidenced by a decrease in the mosaicism rate of mouse embryos undergoing normal gene editing from (84.4 ± 4.4) % to 0%. Our study introduces a convenient and target-specific nucleic acid inhibitor capable of achieving precise regulation of gene editing.}, }
@article {pmid40540289, year = {2025}, author = {Han, JH and Lee, SY and Park, HH}, title = {AcrVIA6 Is a Monomeric DNA-Binding Protein That Does Not Directly Inhibit Cas13a.}, journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology}, volume = {39}, number = {12}, pages = {e70753}, pmid = {40540289}, issn = {1530-6860}, support = {RS-(2025-02316334)//National Research Foundation of Korea (NRF)/ ; }, abstract = {The CRISPR-Cas system is a crucial adaptive immune mechanism in prokaryotes, providing defense against invading genetic elements. Among various CRISPR-Cas systems, the type VI-A system, employing RNA-guided RNase Cas13a, has been extensively studied for its ability to target and degrade single-stranded RNA. Anti-CRISPR (Acr) proteins have evolved as natural inhibitors of these systems, with AcrVIA proteins specifically targeting the Cas13a enzyme. However, there is currently conflicting debate regarding the anti-CRISPR function of AcrVIA6. This study reveals that AcrVIA6 functions as a DNA-binding protein rather than a Cas13a inhibitor, as it does not block RNA cleavage. These findings challenge its role in CRISPR-Cas regulation.}, }
@article {pmid40540136, year = {2025}, author = {Ponnurangan, V and Namachivayam, R and Pradeep, RKM and Jesudoss, D and Eswaran, K and Loganathan, A and Kumar, KK and Vaikuntavasan, P and Maduraimuthu, D and Shanmugam, V}, title = {Biotechnological breakthroughs in rice disease management: an overview from transgenics to CRISPR.}, journal = {Molecular biology reports}, volume = {52}, number = {1}, pages = {616}, pmid = {40540136}, issn = {1573-4978}, support = {BT/PR40456/ AGIII/103/1248/2020//Department of Biotechnology, New Delhi, India/ ; }, abstract = {Rice production faces persistent challenges due to a plethora of infectious agents, which cause substantial economic losses and pose significant threats to food security. Effective management of rice diseases is hindered by the lack of resistant sources and the slow pace of conventional breeding, which often fails to keep up with the rapid evolution of pathogens. Although transgenic approaches have contributed to disease resistance, they are limited by regulatory hurdles and public concern over the use of genetically modified organisms. Emerging genome-editing technologies, particularly CRISPR/Cas systems, offer promising alternatives by enabling the precise modification of native genes involved in plant susceptibility. CRISPR/Cas9-mediated inactivation of negative defense regulators, such as SWEET and ERF family genes, conferred broad-spectrum disease resistance. This review discusses the use of CRISPR/Cas technologies in developing disease-resistant rice varieties, along with emerging approaches and databases relevant to genome editing in plant research. Furthermore, it also highlights recent developments in CRISPR-based diagnostics for pathogen detection and the regulatory frameworks for the commercialization of edited crops. Overall, it underscores the transformative potential of CRISPR technologies in developing elite, climate-resilient rice cultivars, thereby supporting sustainable disease management and global food security.}, }
@article {pmid40539514, year = {2025}, author = {Li, Y and Li, J and Pei, X and Wei, J and Gan, J and Lin, J}, title = {Catalytic-state structure of Candidatus Hydrogenedentes Cas12b revealed by cryo-EM studies.}, journal = {Nucleic acids research}, volume = {53}, number = {12}, pages = {}, doi = {10.1093/nar/gkaf519}, pmid = {40539514}, issn = {1362-4962}, support = {2017YFA0504602//National Key Research and Development Program of China/ ; 32130063//National Key Research and Development Program of China/ ; 31770784//National Natural Science Foundation of China/ ; 32371252//National Natural Science Foundation of China/ ; 2021-01-07-00-07-E00074//Shanghai Municipal Education Commission/ ; SN-ZJU-SIAS009//Zhejiang University/ ; 31770784//National Natural Science Foundation of China/ ; 32371252//National Natural Science Foundation of China/ ; }, abstract = {The CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) systems are adaptive immune mechanisms that play critical roles in defending archaea and bacteria against invading entities. These systems can be divided into two classes, with class 2 comprising three types (II, V, and VI). Because of their ability to cleave double-stranded DNA, many class 2 CRISPR-Cas proteins have been harnessed as genome editing tools. Unlike the well-studied type II Cas9 proteins, the structural studies of the type V-B Cas12b proteins are limited, hindering their engineering and broader application. Here, we report four complex structures of ChCas12b, which reveal many unique structural features. The folding of the single guide RNA (sgRNA) of ChCas12b is distinct from that of AacCas12b and BthCas12b. Notably, many of these unique features are involved in ChCas12b-sgRNA interaction, suggesting that they are co-evolved. While ChCas12b shares a conserved two-cation-assisted catalytic mechanism with its homologs, it recognizes a longer guide:target heteroduplex, potentially offering higher fidelity in DNA editing. Altogether, our studies suggested that Cas12b family proteins exhibit significant diversity in their folding, sgRNA and target DNA binding. In the future, it is worth characterizing more representative proteins to identify CRISPR-Cas proteins with higher gene editing ability and fidelity.}, }
@article {pmid40539280, year = {2025}, author = {Bak, RO and Holm, M and Møller, B and Mikkelsen, JG and Mogensen, TH}, title = {[CRISPR/Cas gene editing of haematopoietic stem cells for curing primary immunodeficiency].}, journal = {Ugeskrift for laeger}, volume = {187}, number = {22}, pages = {}, doi = {10.61409/V02250083}, pmid = {40539280}, issn = {1603-6824}, abstract = {Primary immunodeficiencies are rare monogenic inborn errors of immunity and can involve any combination of infection, autoimmunity, inflammation, and malignancy. While increased use of whole genome sequencing has vastly improved diagnosis, curative treatment options beyond haematopoietic stem cell transplantation are still lacking behind. In this review, we present and discuss the promising avenues of CRISPR/Cas gene editing of patient stem cells for curing these diseases through homology-directed repair, base- or prime editing and delivery by nanoparticles or viral derivatives. However, technological, regulatory, and economic challenges exist on the road to safe and broad implementation of this technology for personalized medicine in the clinic.}, }
@article {pmid40538711, year = {2025}, author = {Lathakumari, RH and Vajravelu, LK and Gopinathan, A and Vimala, PB and Panneerselvam, V and Ravi, SSS and Thulukanam, J}, title = {The gut virome and human health: From diversity to personalized medicine.}, journal = {Engineering microbiology}, volume = {5}, number = {1}, pages = {100191}, pmid = {40538711}, issn = {2667-3703}, abstract = {The human gut virome plays a crucial role in the gut and overall health; its diversity and regulatory functions influence bacterial populations, metabolism, and immune responses. Bacteriophages (phages) and eukaryotic viruses within the gut microbiome contribute to these processes, and recent advancements in sequencing technologies and bioinformatics have greatly expanded our understanding of the gut virome. These advances have led to the development of phage-based therapeutics, diagnostics, and artificial intelligence-driven precision medicine. The emerging field of phageomics shows promise for delivering personalized phage therapies that combat antimicrobial resistance by specifically targeting pathogenic bacteria while preserving beneficial microbes. Moreover, CRISPR-Cas systems delivered via phages have shown success in selectively targeting antibiotic resistance genes and enhancing treatment effectiveness. Phage-based diagnostics are highly sensitive in detecting bacterial pathogens, offering significant benefits for human health and zoonotic disease surveillance. This synthesis of the current knowledge highlights the pivotal role of the gut virome in regulating microbial communities and its transformative potential in personalized medicine, emphasizing its importance in advancing therapeutic and diagnostic strategies for improving health outcomes.}, }
@article {pmid40536889, year = {2025}, author = {Wall, LA and Wall, D}, title = {Acquired CRISPR spacers and rhamnose-glucose polysaccharide defects confer resistance to Streptococcus mutans phage ɸAPCM01.}, journal = {Microbiology (Reading, England)}, volume = {171}, number = {6}, pages = {}, pmid = {40536889}, issn = {1465-2080}, mesh = {*Streptococcus mutans/virology/genetics/metabolism ; *Rhamnose/metabolism/genetics ; Biofilms/growth &amp; development ; *Streptococcus Phages/genetics/physiology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Glucose/metabolism ; Mutation ; *Polysaccharides/metabolism ; Bacterial Proteins/genetics/metabolism ; *Polysaccharides, Bacterial/genetics ; }, abstract = {Streptococcus mutans is commonly associated with the development of dental caries worldwide. Due to their specificity for S. mutans, phage represents a promising avenue for future targeted therapeutic strategies. In this study, we investigated how phage resistance develops in S. mutans. As a model phage, we used ɸAPCM01, which is known to infect a serotype e strain. We isolated and sequenced the genomes of 15 spontaneous resistant mutants and found that 10 had acquired novel clustered regularly interspaced short palindromic repeats (CRIPSR) spacers targeting the phage, with a total of 18 new spacers identified. Additionally, eight strains contained mutations in rhamnose-glucose polysaccharide biosynthetic genes, three of which also acquired spacers. Only the rgp mutants exhibited defects in phage adsorption, supporting the role of these cell surface glycans as the phage receptor. Mutations in rgpF and the newly identified gene rgpX led to severe cell division defects and impaired biofilm formation, the latter of which was also shared by an rgpD mutant. Thus, rgp mutations confer phage resistance but impose severe fitness costs, limiting pathogenic potential. Surprisingly, we found that ɸAPCM01 was capable of binding to and injecting its genome into UA159, a model serotype c strain. However, UA159 was resistant to infection due to an unknown post-entry defence mechanism. Consequently, ɸAPCM01 has the potential to infect both major serotypes associated with dental caries.}, }
@article {pmid40536816, year = {2025}, author = {Liao, X and Liu, Q and Chuai, G}, title = {PrimeNet: rational design of Prime editing pegRNAs by deep learning.}, journal = {Briefings in bioinformatics}, volume = {26}, number = {3}, pages = {}, doi = {10.1093/bib/bbaf293}, pmid = {40536816}, issn = {1477-4054}, support = {T24250193//National Natural Science Foundation of China/ ; 32341008//National Natural Science Foundation of China/ ; 62002265//National Natural Science Foundation of China/ ; 2021YFF1201200//National Key Research and Development Program of China/ ; 2021YFF1200900//National Key Research and Development Program of China/ ; //Shanghai Pilot Program for Basic Research/ ; //Shanghai Science and Technology Innovation Action Plan-Key Specialization in Computational Biology/ ; //Shanghai Shuguang Scholars Project/ ; //Shanghai Excellent Academic Leader Project/ ; 2021SHZDZX0100//Shanghai Municipal Science and Technology Major Project/ ; //Fundamental Research Funds for the Central Universities/ ; 2025080107//Tongji University "Medicine + X" Cross Research Program/ ; }, mesh = {Humans ; *Gene Editing/methods ; *Deep Learning ; HEK293 Cells ; K562 Cells ; CRISPR-Cas Systems ; DNA Methylation ; Epigenesis, Genetic ; }, abstract = {The rapid development of gene editing technology has revolutionized life science research and biotechnology applications. Prime editing, a precise gene editing tool, has shown promise in various applications, including disease research and therapeutic interventions. However, its suboptimal editing efficiency for extensive fragments and lack of predictive models have hindered its widespread adoption. Existing models exhibit low prediction accuracy and limitations, such as neglecting epigenetic factors that impact gene editing effects. To address these challenges, we developed PrimeNet, a novel prediction model that integrates significant epigenetic factors, including chromatin accessibility and DNA methylation. By incorporating data from multiple cell lines and introducing multiscale convolution and attention mechanisms, PrimeNet enhances the accuracy of predictions and generalization performance. Our results show that PrimeNet achieves a Spearman correlation coefficient of 0.94 and 0.82 on two datasets originated from HEK293T and K562 cell lines, respectively, outperforming existing models. This novel model has the potential to guide experimental design, enhance the success rate of gene editing, and reduce unnecessary experimental costs, thereby advancing the application of gene editing technology in genetic disease treatment and related fields.}, }
@article {pmid40536049, year = {2025}, author = {Ullah, M and Naeem, M and Andoh, V and Khan, MN and Han, J and Rizwan, M and Hussain, N and Saeed, M and Chen, Y and Chen, H}, title = {Lactic Acid Bacteria as Biofactories: Mechanistic Insights, Engineering Strategies, and Future Horizons for Heterologous Enzyme Expression.}, journal = {Journal of agricultural and food chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1021/acs.jafc.5c02311}, pmid = {40536049}, issn = {1520-5118}, abstract = {Lactic acid bacteria (LAB) constitute a genetically heterogeneous group that is uniquely capable of converting soluble carbohydrates into lactic acid. Such LAB, with a long history of safe consumption in fermented foods, are considered food-grade microorganisms and are highly sought after for a variety of biotechnological applications. Due to their unique properties, LAB can be genetically engineered to produce industrially significant enzymes. LAB act as an expression host for these enzymes by combining already existing engineering systems with techniques such as CRISPR-Cas. This review outlines the progress achieved to date on genome manipulation methods for LAB engineering and future perspectives of genetic tools. These strategies contribute greatly to fully unleashing the potential of LAB, and we further elaborate on how genome editing tools can enhance the capacity of heterologous expression in LAB.}, }
@article {pmid40533429, year = {2025}, author = {Avaro, AS and Griffiths, AD and Santiago, JG}, title = {Degradation of Reporter Molecules Imposes a Fundamental Limit of Detection on CRISPR Diagnostics.}, journal = {Analytical chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1021/acs.analchem.5c01060}, pmid = {40533429}, issn = {1520-6882}, abstract = {The sensitivity of CRISPR-Cas systems used for molecular diagnostics remains a major bottleneck in the adoption of this technology. The vast majority of CRISPR-based assays use dually labeled, single-stranded reporters and fluorescence signal readouts to infer enzymatic activity and the presence (or absence) of target nucleic acid. The limit of detection of such assays is set by the kinetics of the Cas enzymes and a slow yet measurable increase in the fluorescence signal. We demonstrate here that the background signal and limits of detection of most assays are very likely limited by the degradation of reporter molecules. This degradation is dynamic and is not associated with enzymatic activity. We present theory and experiments to design and calibrate CRISPR assays. We introduce a new kinetic framework to account for the degradation of reporter molecules and derive a fundamental limit of detection for CRISPR-based assays. Our data show that Michaelis-Menten kinetics alone are insufficient to describe reporter (substrate) cleavage rates. The framework and techniques presented here should help reduce the frequency and magnitude of errors currently routinely made in quantifying CRISPR kinetics and interpreting CRISPR diagnostic fluorescence signals.}, }
@article {pmid40531937, year = {2025}, author = {Poudel, B and Sathe, A and Bede, JC and Kushalappa, AC}, title = {Editing metacaspase (StMC7) gene enhances late blight resistance in Russet Burbank potato.}, journal = {PloS one}, volume = {20}, number = {6}, pages = {e0325702}, pmid = {40531937}, issn = {1932-6203}, mesh = {*Solanum tuberosum/genetics/microbiology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics ; Phytophthora infestans/pathogenicity ; CRISPR-Cas Systems ; *Gene Editing ; *Plant Proteins/genetics ; *Caspases/genetics ; Gene Expression Regulation, Plant ; Gene Silencing ; Alternaria/pathogenicity ; Plants, Genetically Modified ; }, abstract = {Plants induce hypersensitive response programmed cell death (HR-PCD), upon biotrophic pathogen infection, to contain the pathogen to the point of infection. Apoptotic-like PCD (AL-PCD) has been reported upon prolonged hemibiotrophic and necrotrophic pathogen infection in potato, to feed on the dead cells for their growth. In potato, silencing of the gene StHRC lead to the suppression of AL-PCD, thus increasing resistance to blights in potato. This was also associated with a significant reduction in the expression of the metacaspase gene StMC7. Accordingly, the gene StMC7 was silenced in potato cultivar 'Russet Burbank' using CRISPR-Cas9 to improve disease resistance against late blight of potato caused by Phytophthora infestans. Following pathogen infection, the disease severity, pathogen biomass and StMC7 gene expression was lower in Stmc7 mutants as compared to wild type. Disease severity was also decreased in Alternaria solani inoculated Stmc7 mutants, compared to the wild type, suggesting possible multiple disease resistance in the Stmc7 knockdown mutants. This confirms that the silencing of StMC7 improves late blight disease resistance in potato.}, }
@article {pmid40531391, year = {2025}, author = {Wang, Y and Li, H and Luo, S and Zhong, M and Liu, J and Li, B}, title = {Research Progress on Signal Conversion Based on Aptamer Combined CRISPR/Cas System in Biosensors.}, journal = {Molecular diagnosis &amp; therapy}, volume = {}, number = {}, pages = {}, pmid = {40531391}, issn = {1179-2000}, support = {2025YFHZ0329//Sichuan Province Science and Technology Support Program/ ; 2024JYJ014//Sichuan Province Science and Technology Support Program/ ; 24CGZH10//Sichuan Province Science and Technology Support Program/ ; }, abstract = {The CRISPR/Cas system has been extensively used in the fields of biology, food safety, and environmental monitoring. This is in part because its can to be used in combination with isothermal amplification-mediated signal amplification technology along with its extraordinary trans-cleavage ability, which has initiated a new era of biosensing applications. The popularity of functional nucleic acids has enabled aptamers to convert non-nucleic acid substances into programmable nucleic acid sequences through methods such as direct detection, lock activation, sandwich design, induction of conformations, and split aptamers. Additionally, CRISPR/Cas systems have been extended beyond nucleic acid detection to include ions, small molecules, proteins, cells, bacteria, viruses, and other non-nucleic acid-based target substances. This article provides a brief overview of the mechanisms of action of four Cas proteins, the generation of aptamers, and their combined applications. Moreover, we focus on the research progress of biosensors based on aptamer-based signal conversion combined with the CRISPR/Cas system.}, }
@article {pmid40531235, year = {2025}, author = {Wang, Y and Yang, H and Wang, K and Zhu, W and Wang, X}, title = {Establishment of a method for the rapid detection of carbapenem-resistant Pseudomonas aeruginosa based on bla_NDM with one-tube RPA-CRISPR/Cas12a visualization.}, journal = {Archives of microbiology}, volume = {207}, number = {8}, pages = {179}, pmid = {40531235}, issn = {1432-072X}, support = {No. LYG06521202376//the Lianyungang City Sixth 521 High-level Talent Training Project Scientific Research Project/ ; No. Z2022070//the 2022 Medical Research guiding Project of Jiangsu Provincial Health Commission/ ; No. SJCX23_1838//the 2023 Jiangsu Graduate Research Innovation Plan project/ ; }, mesh = {*Pseudomonas aeruginosa/genetics/drug effects/isolation &amp; purification/enzymology ; *Carbapenems/pharmacology ; *Pseudomonas Infections/microbiology/diagnosis ; Humans ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; Anti-Bacterial Agents/pharmacology ; *beta-Lactamases/genetics ; Bacterial Proteins/genetics ; China ; Drug Resistance, Bacterial/genetics ; Microbial Sensitivity Tests ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Pseudomonas aeruginosa (PA), which is a common Gram-negative bacterium, can become carbapenem-resistant Pseudomonas aeruginosa (CRPA) upon the development of antibiotic resistance, making clinical treatment difficult. CRPA with antibiotic resistance genes (ARGs), such as bla_NDM and bla_KPC, is common in China. The development of tests for ARGs would facilitate the more rapid identification of CRPA in China. Isothermal amplification research has improved, but limitations remain, including a lack of specialized equipment, the difficulty of developing sophisticated primers, and aerosol pollution. Thus, clinical examination methods must improve. We successfully integrated RPA with CRISPR/Cas12a, and we identified bla_NDM as our institution's primary CRPA. RPA-CRISPR/Cas12a could accurately detect target DNA within 40 min without cross-reacting with other bacteria. The results showed high concordance with conventional culture-based methods, including 100% agreement in clinical sample validation. The method reliably identified standard PA strains and 29 clinical isolates, detecting PA at concentrations as low as 10[-1] CFU. In addition, the closed-tube format effectively minimized the risk of aerosol contamination. This platform offers a rapid and user-friendly tool for identifying bla_NDM-positive CRPA, and this tool is particularly suitable for early screening and clinical management in resource-limited settings. The simplicity and accuracy of this approach make it a promising option for infection control and public health surveillance.}, }
@article {pmid40530883, year = {2025}, author = {Ganote, CL and Caesar, L and Rice, DW and Whitaker, RJ and Newton, ILG}, title = {Evolutionary trends in Bombella apis CRISPR-Cas systems.}, journal = {mSystems}, volume = {}, number = {}, pages = {e0016625}, doi = {10.1128/msystems.00166-25}, pmid = {40530883}, issn = {2379-5077}, abstract = {UNLABELLED: Bacteria and archaea employ a rudimentary immune system, CRISPR-Cas, to protect against foreign genetic elements such as bacteriophage. CRISPR-Cas systems are found in Bombella apis. B. apis is an important honey bee symbiont, found primarily in larvae, queens, and hive compartments. B. apis is found in the worker bee gut but is not considered a core member of the bee microbiome and has therefore been understudied with regard to its importance in the honey bee colony. However, B. apis appears to play beneficial roles in the colony, by protecting developing brood from fungal pathogens and by bolstering their development under nutritional stress. Previously, we identified CRISPR-Cas systems as being acquired by B. apis in its transition to bee association, as they are absent in a sister clade. Here, we assess the variation and distribution of CRISPR-Cas types across B. apis strains. We found multiple CRISPR-Cas types, some of which have multiple arrays, within the same B. apis genomes and also in the honey bee queen gut metagenomes. We analyzed the spacers between strains to identify the history of mobile element interaction for each B. apis strain. Finally, we predict interactions between viral sequences and CRISPR systems from different honey bee microbiome members. Our analyses show that the B. apis CRISPR-Cas systems are dynamic; that microbes in the same niche have unique spacers, which supports the functionality of these CRISPR-Cas systems; and that acquisition of new spacers may be occurring in multiple locations in the genome, allowing for a flexible antiviral arsenal for the microbe.<br><br>IMPORTANCE: Honey bee worker gut microbes have been implicated in everything from protection from pathogens to breakdown of complex polysaccharides in the diet. However, there are multiple niches within a honey bee colony that host different groups of microbes, including the acetic acid bacterium Bombella apis. B. apis is found in the colony food stores, in association with brood, in worker hypopharyngeal glands, and in the queen's digestive tract. The roles that B. apis may serve in these environments are just beginning to be discovered and include the production of a potent antifungal that protects developing bees and supplementation of dietary lysine to young larvae, bolstering their nutrition. Niche specificity in B. apis may be affected by the pressures of bacteriophage and other mobile elements, which may target different strains in each specific bee environment. Studying the interplay between B. apis and its mobile genetic elements (MGEs) may help us better understand microbial community dynamics within the colony and the potential ramifications for the honey bee host.}, }
@article {pmid40530826, year = {2025}, author = {Carolak, E and Czajkowska, J and Stypułkowska, A and Waszczuk, W and Dutkiewicz, A and Grzymajlo, K}, title = {Being a better version of yourself: genetically engineered probiotic bacteria as host defense enhancers in the control of intestinal pathogens.}, journal = {Gut microbes}, volume = {17}, number = {1}, pages = {2519696}, doi = {10.1080/19490976.2025.2519696}, pmid = {40530826}, issn = {1949-0984}, mesh = {*Probiotics ; Humans ; *Gastrointestinal Microbiome ; Genetic Engineering ; Animals ; *Microorganisms, Genetically-Modified/genetics ; *Bacteria/genetics ; }, abstract = {Intestinal pathogens pose a significant global health burden, and traditional antibiotic treatments often disrupt the beneficial gut microbiota that plays a crucial role in maintaining host health through pathogen prevention and immune regulation. Although probiotics have emerged as promising therapeutic agents, their efficacy is limited by strain-dependent variations, survival challenges in the gastrointestinal tract, and inconsistent immune responses. Recent advances in genetic engineering, particularly CRISPR-Cas systems and their combinations with complementary technologies, such as Cre-lox and RecE/T, have enabled the precise modification of probiotic strains to enhance their therapeutic potential. These enhanced probiotics demonstrate improved functionality through multiple mechanisms, including increased adhesion via the expression of specific proteins (InlA, FnBPA, and LAP), targeted antimicrobial activity through engineered sensing systems (Lactococcus lactis detecting Vibrio cholerae CAI-1), and enhanced immunomodulation through cytokine production. Results have demonstrated the potential of genetically modified probiotics in preventing and treating gastrointestinal infections through mechanisms that include competitive exclusion, bacteriocin production, intestinal barrier reinforcement, and immune modulation. However, challenges remain in ensuring genetic stability and preventing horizontal gene transfer. Future research should focus on optimizing probiotic strains for targeted applications while addressing biosafety concerns. By understanding the complex interplay between probiotics, pathogens, and host immunity, innovative strategies can be developed to harness the full therapeutic potential of probiotic interventions in maintaining gut health.}, }
@article {pmid40530689, year = {2025}, author = {Budzak, J and Siegel, TN}, title = {Next generation genetic screens in kinetoplastids.}, journal = {Nucleic acids research}, volume = {53}, number = {11}, pages = {}, doi = {10.1093/nar/gkaf515}, pmid = {40530689}, issn = {1362-4962}, support = {ALTF 764-2022//EMBO Postdoctoral Fellowship/ ; 101105761 - VSG-SWITCH//Marie Sk&#x142;odowska-Curie Actions Postdoctoral Fellowship/ ; SI 1610/2-2//German Research Foundation/ ; SI 1610/4-1//German Research Foundation/ ; 101044320//ERC-CoG/ ; }, mesh = {*Kinetoplastida/genetics ; *Genetic Testing/methods ; Single-Cell Analysis/methods ; Genome, Protozoan ; CRISPR-Cas Systems ; Genomics/methods ; High-Throughput Nucleotide Sequencing ; }, abstract = {The genomes of all organisms encode diverse functional elements, including thousands of genes and essential noncoding regions for gene regulation and genome organization. Systematic perturbation of these elements is crucial to understanding their roles and how their disruption impacts cellular function. Genetic perturbation approaches, which disrupt gene expression or function, provide valuable insights by linking genetic changes to observable phenotypes. However, perturbing individual genomic elements one at a time is impractical. Genetic screens overcome this limitation by enabling the simultaneous perturbation of numerous genomic elements within a single experiment. Traditionally, these screens relied on simple, high-throughput readouts such as cell fitness, differentiation, or one-dimensional fluorescence. However, recent advancements have introduced powerful technologies that combine genetic screens with image-based and single-cell sequencing readouts, allowing researchers to study how perturbations affect complex cellular phenotypes on a genome-wide scale. These innovations, alongside the development of CRISPR-Cas technologies, have significantly enhanced the precision, efficiency, and scalability of genetic screening approaches. In this review, we discuss the genetic screens performed in kinetoplastid parasites to date, emphasizing their application to both coding and noncoding regions of the genome. Furthermore, we explore how integrating image-based and single-cell sequencing technologies with genetic screens holds the potential to deliver unprecedented insights into cellular function and regulatory mechanisms.}, }
@article {pmid40529587, year = {2025}, author = {Lee, CK and Lee, HJ and Jeong, SH and Lee, SJ}, title = {Precision targeting of genetic variations in mixed bacterial cultures using CRISPR-Cas12a-programmed λ phages.}, journal = {Frontiers in microbiology}, volume = {16}, number = {}, pages = {1575339}, pmid = {40529587}, issn = {1664-302X}, abstract = {The CRISPR-Cas system, an adaptive immune mechanism in prokaryotes against bacteriophages, has been developed into a versatile tool for recognizing and cleaving target nucleic acid sequences. In this study, we developed a model system by integrating CRISPR-Cas12a into the genome of temperate bacteriophage λ, enabling precise regulation of lysogeny and lysis in Escherichia coli. We confirmed that λ phage, armed with Cas12a nuclease and CRISPR RNA (crRNA) targeting specific sequences, could inhibit the lysogenic cycle of E. coli cells. We demonstrated that the CRISPR-Cas12a-loaded temperate λ phage mimicked a lytic phage by selectively killing cells carrying the target genomic sequence. Furthermore, by employing truncated crRNA to enhance target recognition specificity, we found that the synthetic phage could distinguish single nucleotide variations in the genomic target DNA, enabling precise targeting and selective elimination of target cells in homogeneous bacterial cultures. To further validate its specificity, we tested this system in mixed bacterial cultures, wherein Cas12a nuclease and truncated crRNA-loaded bacteriophages selectively eliminated only those cells carrying the target sequences perfectly matching the crRNA. These results highlight the potential of this approach for advancing precision microbiome modulation.}, }
@article {pmid40528015, year = {2025}, author = {Ottaviani, L and Lefeuvre, R and Montes, E and Widiez, T and Giorni, P and Mithöfer, A and Marocco, A and Lanubile, A}, title = {A loss-of-function of ZmWRKY125 induced by CRISPR/Cas9 improves resistance against Fusarium verticillioides in maize kernels.}, journal = {Plant cell reports}, volume = {44}, number = {7}, pages = {144}, pmid = {40528015}, issn = {1432-203X}, mesh = {*Zea mays/microbiology/genetics/immunology/metabolism ; *Fusarium/physiology/pathogenicity ; *Plant Diseases/microbiology/genetics/immunology ; *Disease Resistance/genetics ; *Plant Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant ; Cyclopentanes/metabolism ; Oxylipins/metabolism ; Abscisic Acid/metabolism ; Plant Growth Regulators/metabolism ; Loss of Function Mutation ; Transcription Factors/genetics/metabolism ; }, abstract = {ZmWRKY125 negatively regulates maize resistance to Fusarium verticillioides infection through modulating phytohormone, ROS scavenging and secondary metabolite gene expression as well as jasmonic and abscisic acid biosynthetic pathway activity. Fusarium verticillioides causes heavy damage to maize growth and yield and is responsible for mycotoxin contamination. Despite its widespread occurrence, few resistant genes have been identified and functionally validated for their role in the defense mechanisms against this fungus in maize. WRKY transcription factors are known to be crucial in regulating the expression of defense-responsive genes towards pathogen attack. In this context, in our previous genome-wide association study one SNP in the gene ZmWRKY125 was found significantly associated with the responses to F. verticillioides infection in maize seedlings. Here, loss-of-function mutant lines of ZmWRKY125 were obtained by the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system. The zmwrky125 edited lines were phenotypically evaluated showing a decrease by about 5 and 4 times of Fusarium ear rot (FER) severity and fumonisin contamination, respectively, compared to the wild-type genotype. The transient expression of ZmWRKY125 in maize protoplasts confirmed a nuclear localization as expected for a transcription factor. RNA-seq analysis comparison using two zmwrky125 edited lines and the wild-type genotype highlighted an enhanced modulation of the jasmonic acid (JA) and abscisic acid (ABA) hormones, redox state, cell wall modification, and secondary metabolism-associated genes after fungal infection. Moreover, the increased expression of JA- and ABA-related genes correlated with a wider accumulation of these two phytohormones in the mutant background in contrast to wild-type. This data provided new information for understanding the function of ZmWRKY125, despite further field evaluations will be required for validation of the resistance against FER.}, }
@article {pmid40527733, year = {2025}, author = {Obuchi, W and Zargani-Piccardi, A and Leandro, K and Rufino-Ramos, D and Di Lanni, E and Frederick, DM and Maalouf, K and Nieland, L and Xiao, T and Repiton, P and Vaine, CA and Kleinstiver, BP and Bragg, DC and Lee, H and Miller, MA and Breakefield, XO and Breyne, K}, title = {Engineering of CD63 Enables Selective Extracellular Vesicle Cargo Loading and Enhanced Payload Delivery.}, journal = {Journal of extracellular vesicles}, volume = {14}, number = {6}, pages = {e70094}, pmid = {40527733}, issn = {2001-3078}, support = {//Ionis Pharmaceuticals/ ; NCI-CA232103/BC/NCI NIH HHS/United States ; //Friedreich's Ataxia Research Alliance Australia/ ; //Daiichi Sankyo Company/ ; NIH DP2-CA259675//National Science Foundation/ ; NIH DP2-CA281401//National Science Foundation/ ; NIH K22-CA2802019-01//National Science Foundation/ ; NIH R01-GM138790//National Science Foundation/ ; //Kayden-Lambert MGH Research Scholar Award/ ; }, mesh = {*Extracellular Vesicles/metabolism ; *Tetraspanin 30/metabolism/genetics ; Animals ; Humans ; Mice ; HEK293 Cells ; Protein Engineering/methods ; }, abstract = {Extracellular vesicles (EVs) are mediators of intercellular communication through the transfer of nucleic acids, lipids and proteins between cells. This property makes bioengineered EVs promising therapeutic vectors. However, it remains challenging to isolate EVs with a therapeutic payload due to the heterogeneous nature of cargo loading into EVs. In this study, enrichment of EVs with a desired cargo was possible through engineering of the hallmark CD63 transmembrane protein. E-NoMi refers to engineered CD63 with mCherry on the inside of the EV membrane and a tag (3xFLAG) exposed on the outside of the EV membrane. To facilitate EV loading during biogenesis, cargo proteins, such as EGFP, Cre recombinase and the CRISPR-Cas nuclease (SaCas9), were fused to a nanobody (Nb) protein with a high affinity for mCherry. FLAG-tag-based immunocapture from cell conditioned media allowed selection of cargo-loaded E-NoMi-EVs, and tobacco etch virus (TEV) protease cleavage sites were used to remove the 3xFLAG-tag from the surface of E-NoMi-EVs after capture. For functional payload delivery to recipient cells, the vesicular stomatitis virus G (VSV-G) fusogenic protein was incorporated into E-NoMi-EVs to form fusogenic EV-based vectors (EVVs) and proved to be 10-fold more effective at cargo delivery than EVs generated by size-exclusion chromatography. Functional delivery of cargo with E-NoMi-EVVs was validated in two mouse brain models in vivo.}, }
@article {pmid40527417, year = {2025}, author = {Yaikhan, T and Singkhamanan, K and Suwannasin, S and Dechathai, T and Yingkajorn, M and Chusri, S and Surachat, K}, title = {Genomic insights into Metapseudomonas otitidis PA-NS83: The first clinical isolate from Thailand and its comparative genomic analysis.}, journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases}, volume = {}, number = {}, pages = {105786}, doi = {10.1016/j.meegid.2025.105786}, pmid = {40527417}, issn = {1567-7257}, abstract = {Metapseudomonas otitidis was first isolated from human middle ear fluid and has since been detected in both environmental and clinical samples, emerging as an opportunistic pathogen linked to chronic otitis media and other infections. This study reports the first clinical isolate of M. otitidis from Thailand, PA-NS83, and presents a comprehensive genomic characterization. Whole-genome sequencing and comparative analysis with 37 publicly available M. otitidis genomes revealed a diverse antimicrobial resistance (AMR) profile, with PA-NS83 carrying AMR genes commonly found in environmental isolates. Virulence gene analysis identified key determinants associated with biofilm formation, motility, secretion systems, and iron acquisition, highlighting its potential pathogenicity. Pan-genome analysis demonstrated substantial genomic diversity, with PA-NS83 clustering closely with M. otitidis CSMC7, an environmental isolate from polystyrene waste. However, PA-NS83 harbored 419 unique genes, including virulence-associated genes and a CRISPR-Cas system, suggesting adaptation to clinical settings. These findings underscore the genetic plasticity of M. otitidis and its potential role in human infections. Continued genomic surveillance and functional studies are essential to further assess its clinical significance and antimicrobial resistance mechanisms.}, }
@article {pmid40527291, year = {2025}, author = {Wang, J and Zhang, W and Li, W and Xie, Q and Zang, Z and Liu, C}, title = {Enhancement of CRISPR-Cas12a system through universal circular RNA design.}, journal = {Cell reports methods}, volume = {5}, number = {6}, pages = {101076}, doi = {10.1016/j.crmeth.2025.101076}, pmid = {40527291}, issn = {2667-2375}, mesh = {*RNA, Circular/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Humans ; Gene Editing/methods ; *Endodeoxyribonucleases/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; DNA Repair ; Bacterial Proteins ; CRISPR-Associated Proteins ; }, abstract = {Precise control of Cas12a activity is crucial to address incompatibility in isothermal amplification-CRISPR-Cas12a one-pot nucleic acid detection. We developed a light-triggerable circular RNA system for dynamic LbCas12a regulation. By employing circular CRISPR guide RNA (crRNA) or a split circular universal direct repeat region with a replaceable spacer, we resolved the incompatibility between isothermal amplification and CRISPR detection. This system demonstrated robust performance in detecting trace nucleic acids in clinical samples. Furthermore, DNA modifications on circular crRNA enabled CRISPR-Cas12a regulation via base excision repair (BER) enzymes, offering potential for BER enzyme detection and modulation of LbCas12a cleavage activity by BER enzymes. This programmable strategy holds promise for selective gene editing in cells with elevated BER enzyme expression, such as uracil DNA glycosylase (UDG) in colon cancer cells. The circular RNA-assisted approach represents a resource-efficient method with significant potential for medical diagnostics and future clinical gene therapy applications.}, }
@article {pmid40526416, year = {2025}, author = {Ortega-Sanz, I and Rubio, A and Pérez-Pulido, AJ}, title = {Bacteriophages in Pseudomonas aeruginosa evade the CRISPR-Cas I-F system by depletion of PAM sequences.}, journal = {Microbial genomics}, volume = {11}, number = {6}, pages = {}, pmid = {40526416}, issn = {2057-5858}, mesh = {*Pseudomonas aeruginosa/virology/genetics ; *CRISPR-Cas Systems/genetics ; *Bacteriophages/genetics ; Genome, Viral ; Base Composition ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins systems provide bacteria with an adaptive immune system against exogenous sequences, such as plasmids and bacteriophages (viruses of prokaryotes). To avoid autoimmunity, the recognition of a very short sequence called the protospacer adjacent motif (PAM) is essential for efficient immune response triggering. This raises the question of whether viruses targeted by certain CRISPR-Cas systems have a higher or lower frequency of their PAM sequences than non-targeted viruses. This was tested here in the opportunistic human pathogen Pseudomonas aeruginosa that presents five different types of CRISPR-Cas systems. The most frequent of them is the subtype I-F (present in 36% of the strains), which has the PAM 5'-CC sequence. When the viral genomes targeted by this system were analysed, their PAM frequency was found to be lower than that of non-targeted viruses. Remarkably, targeted viruses have comparatively lower G+C content. All this could be the result of selection pressure on the genome of these viruses to escape from the CRISPR-Cas I-F system.}, }
@article {pmid40526281, year = {2025}, author = {Bulle, M and Rahman, MM and Islam, MR and Abbagani, S}, title = {Strategies to develop climate-resilient chili peppers: transcription factor optimization through genome editing.}, journal = {Planta}, volume = {262}, number = {2}, pages = {30}, pmid = {40526281}, issn = {1432-2048}, mesh = {*Capsicum/genetics/physiology ; *Gene Editing/methods ; *Transcription Factors/genetics/metabolism ; Stress, Physiological ; Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Climate Change ; Genome, Plant ; }, abstract = {Chili peppers (Capsicum spp.), a globally significant crop revered for their nutritional, economic, and cultural importance, are increasingly imperiled by the converging burdens of climate-induced abiotic stresses, including drought, heat, and salinity, and relentless biotic assaults from pathogens and insect herbivores. These overlapping stressors not only destabilize yield but also compromise the metabolic intricacy responsible for the accumulation of health-promoting secondary metabolites. Although Capsicum exhibits remarkable genetic and phytochemical diversity, the integrated transcriptional, metabolic, and epigenetic frameworks that underpin its stress resilience remain poorly delineated. This review synthesizes recent advances in decoding core transcription factor families, such as CaNAC, CaWRKY, and CaMYB, that serve as pivotal regulators of osmotic adjustment, reactive oxygen species detoxification, hormonal crosstalk, and secondary metabolite biosynthesis under stress conditions. We further highlight how multi-omics-guided gene discovery, when paired with CRISPR/Cas-mediated genome editing, enables precise reprogramming of key regulatory loci to enhance adaptive responses. Emerging innovations, including base editing, prime editing, and novel nucleases like Cas12a and Cas13d, are expanding the functional genome-editing landscape, while the integration of morphogenic regulators and genotype-independent transformation platforms is beginning to circumvent long-standing obstacles in Capsicum genetic engineering. Lastly, we propose a transformative framework that converges transcription factor modulation, multi-omics strategies, precision phenotyping, and next-generation genome editing to accelerate the development of climate-resilient Capsicum cultivars with optimized metabolic traits. This strategic convergence of molecular insight and biotechnological innovation offers a robust foundation for building next-generation chili pepper varieties capable of withstanding intensifying environmental and pathogenic pressures, ultimately safeguarding yield, nutritional quality, and agricultural sustainability in the face of global climate change.}, }
@article {pmid40525624, year = {2025}, author = {Zhang, Y and Lin, Y and Li, R and Jiang, D and Cai, R and Su, G and Yu, Y and Qu, G}, title = {Split G-Quadruplex-Integrated CRISPR-Cas Biosensor for One-Pot, Signal-On Visual Detection of Pathogen Nucleic Acids.}, journal = {Analytical chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1021/acs.analchem.5c02037}, pmid = {40525624}, issn = {1520-6882}, abstract = {Pathogen nucleic acid analysis has emerged as an indispensable component of contemporary healthcare systems, serving dual roles in personalized clinical management and population-level disease surveillance. Herein, we present a novel G-quadruplex-integrated CRISPR-Cas biosensing platform that performs in a signal-on mechanism for colorimetric detection of pathogen nucleic acids in one-pot. By harnessing the unique properties of split G4 structures, we develop a universal visual probe that generates a distinct green colorimetric signal upon target recognition, which effectively couples with both Cas12 and Cas13 systems. Using the monkeypox virus (MPXV) B7R gene and a conserved respiratory syncytial virus (RSV) sequence as model targets for Cas12a and Cas13, respectively, we establish a detection workflow combining recombinase polymerase amplification (RPA) with CRISPR-mediated cleavage, visualized through enzymatic mediated color conversion. The tube-in-tube cartridge architecture adopted in this work enables seamless integration of RPA and CRISPR-based detection within a single closed-tube system, effectively eliminating cross-contamination risks. We successfully validate the platform for detection of MPXV in environmental samples and RSV in clinical specimens, achieving a detection limit of 1 copy per test and perfect concordance with PCR methods (40/40 agreement). The colorimetric biosensing platform developed herein demonstrates rapid (<60 min) and facial performance, establishing a novel molecular diagnostic paradigm that achieves laboratory-comparable accuracy for real-time surveillance and point-of-care applications.}, }
@article {pmid40524239, year = {2025}, author = {Villora, SA and Zhao, Y and Silva, PC and Hahn, AA and Olanin, V and Groll, D and Maurer, S and Roetzer, V and Szymanski, W and Procida-Kowalski, T and Philipp, N and Koch, A and Bartkuhn, M and Graumann, J and Volckmann, R and Koster, J and Rossbach, O and Salzig, D and Dammann, R and Sigges, C and Halbritter, J and Haerteis, S and Richter, AM}, title = {Epigenetic silencing and CRISPR-mediated reactivation of tight junction protein claudin10b (CLDN10B) in renal cancer.}, journal = {Clinical epigenetics}, volume = {17}, number = {1}, pages = {102}, pmid = {40524239}, issn = {1868-7083}, support = {2023_1_01//FCMH Forschungscampus Mittelhessen/ ; 2023_1_01//FCMH Forschungscampus Mittelhessen/ ; 2023_1_01//FCMH Forschungscampus Mittelhessen/ ; 2024_1_02//FCMH Forschungscampus Mittelhessen,/ ; 2024_1_02//FCMH Forschungscampus Mittelhessen,/ ; 2024_1_02//FCMH Forschungscampus Mittelhessen,/ ; 031B1232B//BMBF/ ; 031B1232B//BMBF/ ; 031B1232B//BMBF/ ; }, mesh = {Humans ; *Claudins/genetics ; DNA Methylation ; *Kidney Neoplasms/genetics ; *Carcinoma, Renal Cell/genetics ; Epigenesis, Genetic ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Gene Silencing ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Male ; Female ; Tight Junctions ; Mixed Function Oxygenases ; Proto-Oncogene Proteins ; }, abstract = {BACKGROUND: The kidney's tubular system relies on cell polarity and tight junctions to maintain structure and function and disruptions contribute to diseases like cancer. Loss of tight junction proteins such as Claudins can actively contribute to tumorigenesis.<br><br>RESULTS: We aimed to identify biomarkers for renal carcinoma, after kidney transplantation and conventional kidney tumors. We identified the epigenetic silencing of the Claudin 10 gene isoform B (CLDN10B) through DNA hypermethylation in renal cancers, including clear cell (ccRCC), papillary (pRCC) and post-transplantation renal carcinoma (PT-ccRCC). In contrast, CLDN10A was hypomethylated in ccRCC and pRCC. Differential methylation of the isoforms discriminates RCC from other malignancies. The epigenetic alteration of CLDN10B significantly correlated with reduced patient survival and advanced tumor staging. CLDN10B overexpression or induction significantly inhibited migration, cell cycle progression, and cellular growth. Using a CRISPR-based epigenetic editing tool&#xa0;reactivated CLDN10B to its endogenous level using VP160 and TET1 by promoter demethylation and significantly demonstrated its tumor-suppressive effects in 2D and 3D cell models.<br><br>CONCLUSION: Our findings suggest that CLDN10B acts as a tumor suppressor, and its epigenetic regulation may represent a therapeutic target for RCC. Ultimately, understanding CLDN10B's regulation and function could provide new insights into renal cancer treatment.}, }
@article {pmid40524010, year = {2025}, author = {Levengood, H and Smith, L and Gillis, S and Zhou, Y and Zhang, C}, title = {Plantago species are emerging model organisms for functional genomics and stress biology.}, journal = {Plant cell reports}, volume = {44}, number = {7}, pages = {142}, pmid = {40524010}, issn = {1432-203X}, support = {1923557//National Science Foundation/ ; }, mesh = {*Genomics/methods ; *Plantago/genetics/physiology ; *Stress, Physiological/genetics ; Genome, Plant/genetics ; CRISPR-Cas Systems ; }, abstract = {Species in the Plantago genus are emerging model organisms to multiple research disciplines. The genus Plantago has long been recognized for its significance in various research fields, yet it remains underutilized as a model organism in scientific studies. Several Plantago species possess unique traits, including easily accessible vascular tissues, medicinal properties, gynodieocity, and remarkable adaptability to diverse environmental conditions. These characteristics position Plantago as a promising model for research in areas such as plant vascular biology, stress physiology, reproductive biology, ecology, and medicinal biochemistry. Recent advancements, including the development of genetic transformation systems, the availability of sequenced genomes, and the application of CRISPR-Cas9 technology, have significantly enhanced the capability of using Plantago as a model system. This review discusses the research potential of Plantago species, highlighting key historical discoveries and recent breakthroughs that demonstrate their value across multiple scientific disciplines.}, }
@article {pmid40523955, year = {2025}, author = {Rong, C and Zhang, R and Xie, J and Li, J and Yan, T and Liu, Z and Liu, Y and Xu, R and Shi, X and Zhao, X and Song, J and Meng, Y and Chang, Z and Ding, Y and Ding, C}, title = {Type-A and -C response regulator genes positively impact rice plant height and panicle architecture.}, journal = {Plant cell reports}, volume = {44}, number = {7}, pages = {141}, pmid = {40523955}, issn = {1432-203X}, support = {No BK20231470//Basic Research Program of Jiangsu Province/ ; }, mesh = {*Oryza/genetics/growth &amp; development/metabolism/anatomy &amp; histology ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; Cytokinins/metabolism ; Signal Transduction/genetics ; Mutation ; Plant Growth Regulators/metabolism ; CRISPR-Cas Systems ; Genes, Plant ; }, abstract = {Thirteen type -A and two type -C RR genes exhibited distinct expression patterns and unique functions. Notably, RR2 and RR4 had the most significant positive effects impact on panicle development. Cytokinin signal transduction occurs through a "two-component system". Type-A and -C response regulators (RRs) are groups of proteins of similar structures constituting significant components of cytokinin signal transduction. In rice, 13 (Type-A) and 2 (Type-C) RRs have been identified to date; however, their functions remain partially known. In this study, we examined the expression patterns of Type-A and Type-C RRs in rice using RNA-Seq and confirmed their functions by constructing mutants of the 15 genes with CRISPR/Cas9. Almost all Type-A RRs played positive roles in the development of secondary branches and secondary spikelets, particularly RR2 and RR4. Notably, rr1 rr2 and rr8 rr12 rr13 higher-order mutants displayed small panicle sizes and decreased plant height. Additionally, both Type-C RRs played positive roles in regulating heading date. RNA-seq revealed several genes with significantly altered expression in the rr2 and rr4 mutants, with almost half of the differentially expressed genes (DEGs) overlapping between the two mutants. Many of the DEGs were associated with the cytokinin and abscisic acid pathways. Our findings provide new insights into the functions of Type-A and -C RRs in rice growth and may serve as a foundation for future studies focusing on cytokinin signaling.}, }
@article {pmid40522908, year = {2025}, author = {Schloßhauer, JL and Shamloo, S and Schamrin, K and Imig, J}, title = {Dual CRISPR-Interference Strategy for Targeting Synthetic Lethal Interactions Between Non-Coding RNAs in Cancer Cells.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {219}, pages = {}, doi = {10.3791/67752}, pmid = {40522908}, issn = {1940-087X}, mesh = {Humans ; *RNA, Long Noncoding/genetics ; Cell Line, Tumor ; *CRISPR-Cas Systems ; Streptococcus pyogenes/genetics/enzymology ; *Melanoma/genetics/pathology ; Staphylococcus aureus/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Long non-coding RNAs (lncRNAs) represent a vast and functionally diverse class of RNA molecules, with over 100,000 predicted in the human genome. Although lncRNAs are less conserved across species compared to protein-coding genes, they play critical roles in gene regulation, chromatin interactions, and cancer progression. Their involvement in cancer makes them promising therapeutic targets. CRISPR interference (CRISPRi), utilizing catalytically inactive Cas9 fused with a transcriptional repressor such as KRAB-MeCP2, offers a precise method for targeting nuclear lncRNAs and assessing their functions. This study introduces a dual CRISPRi system using orthogonal CRISPRi technologies from Staphylococcus aureus and Streptococcus pyogenes based on dCas9-KRAB-MeCP2, optimized for combinatorial targeting of lncRNAs in human melanoma cells. The protocol facilitates combinatorial gene knockdown or synthetic lethal screening of lncRNA pairs, providing a novel tool for cancer research. By exploring synthetic lethality between lncRNAs, this approach can help identify lncRNA interactions critical for cancer cell survival, offering new therapeutic strategies. The dual system's functionality is demonstrated, highlighting its potential in identifying critical cancer-specific lncRNA interactions.}, }
@article {pmid40522884, year = {2025}, author = {Christenson, AE and Divekar, NS and Hernández Zamora, MG and Nguyen, CD and Nuñez, JK}, title = {CRISPR Epigenome Editing in Human Cells using Plasmid DNA Transfection and mRNA Nucleofection Delivery.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {219}, pages = {}, doi = {10.3791/68089}, pmid = {40522884}, issn = {1940-087X}, mesh = {Humans ; *Plasmids/genetics/administration &amp; dosage ; *RNA, Messenger/genetics/administration &amp; dosage ; *Transfection/methods ; *DNA/genetics/administration &amp; dosage ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Epigenome/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Epigenome Editing ; }, abstract = {Epigenetics refers to chemical modifications of histone proteins and DNA that can regulate the expression of genes. The human epigenome is altered dynamically during cell differentiation and aging, and many diseases are associated with aberrant epigenome patterning. Recent advances in CRISPR have led to the development of programmable tools to edit epigenetic modifications at targeted genomic loci, enabling precise rewriting of epigenetic modifications in human cells. CRISPR-based epigenome editors rely on catalytically dead Cas9 coupled with epigenetic modifiers that ultimately result in programmed repression or activation of targeted genes in mammalian genomes. Unlike traditional genome editing methods, epigenome editing does not require DNA breaks or changes in the human genome sequence and thus serves as a safer alternative to control gene expression. In this protocol, we highlight two different methods to perform dCas9-mediated epigenome editing in human cell lines using plasmid DNA transfections and nucleofection of mRNAs encoding CRISPR epigenome editors. We demonstrate programmable epigenome editing to transiently repress genes using CRISPR interference (CRISPRi) and for silencing genes durably for many weeks using CRISPRoff, a fusion of dCas9 to the KRAB domain and de novo DNA methyltransferase complex. We also provide guidance on quantitative methods to measure successful epigenome editing of target genes and key considerations on which epigenome editing tool to use, depending on experimental criteria.}, }
@article {pmid40522066, year = {2025}, author = {Zhang, J and Zhu, A and Wei, S and Shi, Y and Chen, C and Huang, T and Tang, T and Zhao, J and Cai, Y and Han, C and Zhao, J and Wang, Y}, title = {Identification of Pan-Coronavirus Neutralizing Aptamers Through CRISmers Targeting the Conserved Fusion Peptide.}, journal = {Advanced healthcare materials}, volume = {}, number = {}, pages = {e2501869}, doi = {10.1002/adhm.202501869}, pmid = {40522066}, issn = {2192-2659}, support = {2023YFA0915000//National Key Research and Development Program of China/ ; 2022YFC2303700//National Key Research and Development Program of China/ ; 82273967//National Natural Science Foundation of China/ ; 82025001//National Natural Science Foundation of China/ ; 2021QN020576//Department of Science and Technology of Guangdong Province/ ; 2021B1515130005//Guangdong Basic and Applied Research Projects/ ; JSGG20200807171801013//Shenzhen Science and Technology Innovation Committee/ ; //Shenzhen Institute of Synthetic Biology Scientific Research Program/ ; 2023M742397//China Postdoctoral Science Foundation/ ; GZC20231724//Postdoctoral Fellowship Program of CPSF/ ; }, abstract = {A series of coronavirus outbreaks emphasize the necessity to develop pan-coronavirus therapeutics. Previously, the CRISmers system is developed, a Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins (CRISPR/Cas)-mediated cellular screening platform for the identification of RNA aptamers. In this study, CRISmers are applied to target the fusion peptide (FP) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), a conserved region across coronavirus genera, with E.coli serving as the screening host. From this approach, a lead aptamer, #FP-10 is identified, which demonstrates potent pan-coronavirus neutralization activity against multiple SARS-CoV-2 variants and alphacoronaviruses (HCoV-229E and HCoV-NL63), demonstrating its potential for development into a broad-spectrum therapeutic candidate.}, }
@article {pmid40521185, year = {2025}, author = {Wu, D and Shu, H and Zhang, M and Wei, X and Ji, J and Shen, H and Zhang, H and Xie, L and Zhou, L and Yang, L and Jiang, J and Chen, C and Tian, S and Zhang, X and Long, X and He, X and Wang, H}, title = {Mannosylated neutrophil vesicles targeting macrophages alleviate liver inflammation by delivering CRISPR/Cas9 RNPs.}, journal = {Theranostics}, volume = {15}, number = {13}, pages = {6221-6235}, pmid = {40521185}, issn = {1838-7640}, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Mice ; *Macrophages/metabolism ; *Neutrophils/metabolism ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics/metabolism ; *Ribonucleoproteins/genetics/administration &amp; dosage/metabolism ; Gene Editing/methods ; Liver/pathology ; Disease Models, Animal ; Mice, Inbred C57BL ; Mannose/metabolism/chemistry ; Inflammation/therapy ; Humans ; CRISPR-Associated Protein 9 ; Male ; Hepatitis/therapy ; Inflammasomes/metabolism ; }, abstract = {Background: Inflammation is a key driver of various liver diseases. NLRP3 inflammasome in hepatic macrophages is a key mediator of inflammation and has emerged as a promising target. Genome editing presents a powerful approach to modulate inflammation by directly disrupting genes such as NLRP3 directly. However, efficient and cell-specific delivery of CRISPR/Cas9 ribonucleoproteins (RNPs) remains challenging. Methods: We developed a novel delivery system by encapsulating CRISPR/Cas9 RNPs within mannosylated neutrophil membranes vesicles (Cas9/gNLRP3@M-N) to enhance targeting hepatic macrophages. Results: Cas9/gNLRP3@M-N selectively accumulated in hepatic macrophages, effectively disrupted the NLRP3 gene, attenuated inflammation in acute fulminant hepatitis, and improved disease outcomes in chronic steatohepatitis model. Conclusions: Cas9/gNLRP3@M-N represents a promising targeted gene-editing approach for the treatment of inflammatory liver diseases.}, }
@article {pmid40521034, year = {2025}, author = {Al-Ouqaili, MTS and Ahmad, A and Jwair, NA and Al-Marzooq, F}, title = {Harnessing bacterial immunity: CRISPR-Cas system as a versatile tool in combating pathogens and revolutionizing medicine.}, journal = {Frontiers in cellular and infection microbiology}, volume = {15}, number = {}, pages = {1588446}, pmid = {40521034}, issn = {2235-2988}, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *Bacteria/genetics/immunology ; *Bacterial Infections/therapy/immunology ; Animals ; Genetic Therapy/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has emerged as an adaptable instrument for several uses. The CRISPR-Cas system employs Cas proteins and programmable RNA molecules to guide the recognition and cleavage of specific DNA regions, permitting accurate genome editing. It is derived from the bacterial immune system and allows for accurate and efficient modification of DNA sequences. This technique provides unparalleled gene editing, control, and precise alteration opportunities. This review aims to offer a comprehensive update of the core concepts of the CRISPR-Cas system and recent progress, while also providing an overview of the significant applications in diverse fields such as microbiology and medicine. The CRISPR-Cas9 gene editing technique has facilitated substantial advancements in comprehending gene function, simulating diseases, and creating innovative therapeutics. CRISPR-based therapeutics present a hopeful prospect for addressing intricate ailments, including genetic disorders, malignancies, and infectious diseases, as they serve as viable substitutes for conventional pharmaceuticals. In microbiology, this method serves as a diagnostic and therapeutic tool that proves highly efficient in eliminating bacteria that have developed resistance to various antibiotics. Despite its significant potential, CRISPR encounters ethical, safety, and regulatory obstacles that necessitate meticulous deliberation. Concerns regarding off-target effects, poor delivery to target tissues, and unwanted side effects emphasize the necessity to thoroughly examine the technology. It is necessary to balance the advantages and difficulties CRISPR presents. Consequently, more rigorous preclinical and clinical experiments are essential before using it in humans.}, }
@article {pmid40520364, year = {2025}, author = {Nitulescu, AM and Du, W and Glaser, V and Kath, J and Aird, EJ and Cullot, G and Greensmith, R and Mikkelsen, NS and Stein, M and Bak, RO and Kaminski, M and Corn, JE and Wagner, DL}, title = {Single-stranded HDR templates with truncated Cas12a-binding sequences improve knock-in efficiencies in primary human T cells.}, journal = {Molecular therapy. Nucleic acids}, volume = {36}, number = {2}, pages = {102568}, pmid = {40520364}, issn = {2162-2531}, abstract = {CRISPR-Cas12a gene editing offers an alternative to Cas9-based methods, providing better targeting of AT-rich regions, simplified guide RNA manufacturing, and high specificity. However, the efficacy of donor-based editing is subject to various factors, with template format playing a crucial role. Currently, the predominant non-viral template format for homology-directed repair (HDR) after nuclease-induced DNA breaks is double-stranded DNA, which is toxic when transfected at high doses. Others have demonstrated that using single-stranded DNA (ssDNA) with flanking double-stranded Cas-target-sequences (CTS) as a template for Cas9-mediated gene editing can mitigate this toxicity and increase knock-in efficiency. Here, we investigate CTS design for AsCas12a Ultra by exploring PAM orientation and binding requirements. Additionally, we rule out ssDNase activity of AsCas12a under cell-physiological Mg[2+] conditions. Finally, we showcase the advantage of ssDNA donors with CTS (ssCTS) at high doses for delivering clinically relevant transgenes of varying sizes into three TCR-CD3 complex genes (TRAC, CD3ζ, CD3ε), achieving up to 90% knock-in rates for a 0.8kb-insert at the CD3ε locus. Long-read sequencing confirmed higher HDR rates and revealed that CTS reduced partial integration events compared to unmodified ssDNA. Overall, AsCas12a and ssCTS represent a platform for highly efficient knock-in in primary human T cells with minimal toxicity.}, }
@article {pmid40518326, year = {2025}, author = {Zhao, Y and Tan, J and Fang, L and Jiang, L}, title = {Leveraging gene editing to combat methane emissions in ruminant agriculture.}, journal = {Trends in biotechnology}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.tibtech.2025.05.020}, pmid = {40518326}, issn = {1879-3096}, abstract = {Methane emissions from ruminants represent a major contributor to global greenhouse gases (GHGs), posing challenges for sustainable agriculture and climate change mitigation. Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas)-based gene editing offer transformative approaches to address this issue by targeting both forage crops and rumen methanogens. Enhancing lipid content and secondary metabolites in forage crops can suppress methanogenesis and improve feed efficiency, while precise genome editing in methanogenic Archaea can disrupt pathways critical to methane production. However, challenges remain regarding delivery methods, gene targeting specificity, ecological impacts, and regulatory acceptance. In this review, we explore current progress, identify key knowledge gaps, and highlight the need for interdisciplinary strategies that integrate biotechnology, synthetic biology, and regulatory frameworks to develop effective and scalable methane mitigation solutions.}, }
@article {pmid40517395, year = {2025}, author = {Marín-Sanz, M and Sánchez-León, S and Guzmán-López, MH and Starker, CG and Voytas, DF and Barro, F}, title = {Cas9- and Cas12a-mediated excision and replacement of the celiac disease-related α-gliadin immunogenic complex in hexaploid wheat.}, journal = {Plant biotechnology journal}, volume = {}, number = {}, pages = {}, doi = {10.1111/pbi.70200}, pmid = {40517395}, issn = {1467-7652}, support = {//European Union "NextGenerationEU"/PRTR/ ; QUAL21_023 IAS//Junta de Andaluc&#xed;a/ ; //Conexi&#xf3;n TRIGO/WheatNet/ ; PID2022-142139OB-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; TED2021-129733B-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; }, abstract = {Celiac disease (CD) is a chronic enteropathy affecting approximately 1% of the global population. Wheat α-gliadins are a major contributor to the autoimmune response, as they contain one of the most immunogenic peptides, the 33-mer, along with numerous variants. In this study, we used CRISPR/Cas technology to mutate genes encoding α-gliadins. This approach employed paired sgRNAs to precisely excise immunogenic regions while preserving non-immunogenic sequences within the α-gliadins. Furthermore, we replaced the excised region with an α-gliadin-based double-stranded oligodeoxynucleotide (dsODN) designed with nucleotide changes to reduce immunoreactivity and increase peptidase cleavage sites. Two different CRISPR systems, Cas9 and Cas12a, were applied to generate wheat protoplasts and plants with non-immunogenic regions. Cas9 demonstrated superior performance in terms of editing frequency, excision and replacement of immunogenic fragments. However, the Cas12a nuclease (Cpf1) showed promising editing efficiency, offering the potential for future wheat editing applications. Using the Cas9 system, we achieved a 74.2% excision rate of the 33-mer in wheat plants. Subsequent analyses showed a significant reduction in the reactivity to the G12 monoclonal antibody, capable of identifying the 33-mer peptide and a decrease in the prolamin levels compared to the wild-type. Additionally, we developed a high-throughput sequencing-based software specifically designed to identify mutations in multi-copy gene families. This innovative tool enabled fast, parallel screening of the samples in this study and facilitated the identification of the specific editing patterns produced by the designed constructs.}, }
@article {pmid40516885, year = {2025}, author = {Cunha da Silva, G and Rossi, CC}, title = {Defense systems and mobile elements in Staphylococcus haemolyticus: a genomic view of resistance dissemination.}, journal = {Microbial pathogenesis}, volume = {206}, number = {}, pages = {107808}, doi = {10.1016/j.micpath.2025.107808}, pmid = {40516885}, issn = {1096-1208}, abstract = {Staphylococcus haemolyticus is a multidrug-resistant opportunistic pathogen and a major reservoir of antimicrobial resistance (AMR) genes within the Staphylococcaceae family. Its high genomic plasticity, frequent association with mobile genetic elements (MGEs), and prevalence in clinical settings underscore its relevance as both a threat and a conduit for resistance dissemination. In this study, we performed a comprehensive pan-genomic analysis of the S. haemolyticus defensome - including restriction-modification (RM), abortive infection (Abi), and CRISPR-Cas systems - across 692 high-quality genomes. Our results reveal a highly diverse and modular repertoire of immune systems, often organized in physical clusters and frequently associated with MGEs. We identified evidence of antagonistic interactions, with both defense and anti-defense elements encoded on plasmids and prophages. CRISPR spacer analysis showed a predominant targeting of phages, and genomes encoding CRISPR-Cas systems exhibited a lower abundance of MGEs and AMR genes, suggesting a trade-off between defense and gene acquisition. RNA-seq data from one reference strain indicate that only a fraction of the defensome is actively transcribed under standard conditions, hinting at environment-responsive regulation. Together, these findings provide new insights into the genomic strategies sustaining the persistence and adaptability of S. haemolyticus in clinical environments. The interplay between its immune systems and mobilome likely contributes not only to its evolutionary trajectory, but also to its role in the horizontal transfer of resistance determinants among pathogenic staphylococci. A deeper understanding of this immune-mobilome interface may help inform future strategies to limit the spread of resistance.}, }
@article {pmid40516718, year = {2025}, author = {Bu, G and Ma, X and Jin, H and Wang, L}, title = {CRISPR-Cas and RNA sequencing reveal nutrient enhancement pathways in quinoa for plant-based athlete recovery diets and future food security.}, journal = {International journal of biological macromolecules}, volume = {318}, number = {Pt 4}, pages = {144785}, doi = {10.1016/j.ijbiomac.2025.144785}, pmid = {40516718}, issn = {1879-0003}, abstract = {Quinoa (Chenopodium quinoa Willd.) is a nutrient-dense, climate-resilient pseudocereal with growing relevance to food and nutritional security. However, its potential remains underutilized due to suboptimal levels of key micronutrients. In this study, to apply CRISPR-Cas9 for the simultaneous enhancement of multiple nutrients in quinoa, marking a significant advancement in crop biofortification, we employed multiplexed CRISPR-Cas9 genome editing to target five genes involved in lysine transport, phytic acid biosynthesis, and vitamin C and E biosynthetic pathways. Both knockout and homology-directed knock-in strategies were applied to induce heritable mutations in genes such as CqAAP1, CqIPK1, CqGGP, and CqHPT. Homology-directed knock-in refers to a precise gene-editing method that uses a template to insert specific genetic changes at targeted sites in the genome. Edited lines exhibited significant improvements in seed nutrient concentrations, including lysine (+35 %), zinc (+43 %), vitamin C (+114 %), and vitamin E (+45 %), without yield or growth penalties. Whole-transcriptome profiling via RNA sequencing (RNA-Seq) identified 1284 differentially expressed genes (FDR < 0.05), predominantly associated with amino acid metabolism, redox regulation, and vitamin biosynthesis. Gene Ontology (GO) and KEGG enrichment analyses confirmed the transcriptional activation of nutrient assimilation and antioxidant pathways. Integration of qRT-PCR with RNA-Seq confirmed the reliability of expression data. Structural validations of edits were performed using PCR, gel electrophoresis, and Sanger sequencing. Phenotypic assessments, including seed weight, morphology, and exploratory herbivory assays, confirmed agronomic stability. This study presents a technically validated framework for simultaneous nutritional trait stacking in quinoa using nucleic acid engineering tools. Our findings offer critical insights into the transcriptional plasticity of quinoa and establish a functional genomics platform for developing multi-nutrient biofortified crops to address hidden hunger and advance sustainable food systems.}, }
@article {pmid40515942, year = {2025}, author = {Saha, T and Saha, RP and Singh, MK and Priya, K and Singh, S and Rajeev, M and Bhattacharya, D and Nag, M and Lahiri, D}, title = {An overview on in-vivo generation of CAR-T cells using CRISPR-loaded functionalized nanocarriers for treating B-cell lineage acute lymphoblastic leukemia.}, journal = {Molecular biology reports}, volume = {52}, number = {1}, pages = {596}, pmid = {40515942}, issn = {1573-4978}, mesh = {Humans ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Immunotherapy, Adoptive/methods ; Animals ; *Receptors, Chimeric Antigen/genetics/metabolism/immunology ; T-Lymphocytes/immunology/metabolism ; Mice ; Nanoparticles/chemistry ; *Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy/immunology/genetics ; *Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/therapy/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; B-Lymphocytes ; }, abstract = {Chimeric antigen receptor T (CAR-T) cell therapy has become a milestone in the management of B cell lineage acute lymphoblastic leukemia. Yet, the traditional method-dependent on ex vivo manipulation, amplification, and reinfusion of autologous T cells-is high-cost, low-scalability, and severely immune-related toxicity. Here, we report a new nano-immunoengineering platform that allows in vivo production of chimeric antigen receptor T cells through the use of functionalized nanoparticles carrying clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) gene editing elements. These nanoparticles are engineered to specifically target blood circulating T lymphocytes and deliver CRISPR/Cas9 complexes that have the ability to integrate chimeric antigen receptor constructs into the TRAC locus and knock out immune checkpoint genes like programmed cell death protein 1 (PD-1) simultaneously. Targeted delivery, endosomal escape, and efficient genome editing with minimal off-target effects are ensured through gold-based and DNA nanostructure-based carriers. Preclinical models show effective in vivo programming of functional chimeric antigen receptor T cells with vigorous antitumor efficacy, improved persistence, and decreased cytokine release syndrome. This method is a revolutionary breakthrough in cancer immunotherapy that provides a scalable, economical, and clinically flexible replacement for conventional chimeric antigen receptor T cell production.}, }
@article {pmid40515919, year = {2025}, author = {Ye, Y and Zhang, M and Nie, H and Duan, Y and Ye, Z and Zheng, C}, title = {Screening for Host Proteins with Pro- and Antiviral Activity via High-Throughput CRISPR.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2940}, number = {}, pages = {273-281}, pmid = {40515919}, issn = {1940-6029}, mesh = {Humans ; *CRISPR-Cas Systems ; Antiviral Agents/pharmacology ; *High-Throughput Screening Assays/methods ; *Host-Pathogen Interactions/genetics ; Animals ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Virus Diseases/virology/genetics ; Viruses/genetics ; Virus Replication ; }, abstract = {The whole-genome library for CRISPR screening serves as an important biotechnological tool aimed at probing gene function in mammalian cells, providing a foundation for the systematic discovery of essential genes corresponding to biological effects. Research indicates that whole-genome library cell lines can be used to identify host factors and potential drug targets associated with viral infections effectively at high throughput, providing crucial evidence for the development of novel antiviral drugs. Here, we primarily discuss the methods for constructing genome-scale CRISPR screens in cell lines and their applications in virology research. By cloning and expressing the whole genomic DNA of specific organisms, stable cell lines, which can be utilized for functional validation, drug screening, and gene function studies, can be established. Through gene knockout or overexpression techniques, in-depth analyses of the key roles that genes play in the viral life cycle could be conducted, revealing how viruses exploit the biological mechanisms of host cells for replication and evasion of immune responses. These findings not only enhance our understanding of the interactions between viruses and host cells but also yield new targets for the development of antiviral drugs and vaccines.}, }
@article {pmid40515918, year = {2025}, author = {Wang, J and Zheng, C and Xiao, W}, title = {Zebrafish as a Model for Studying Virus-Host Interactions.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2940}, number = {}, pages = {261-272}, pmid = {40515918}, issn = {1940-6029}, mesh = {Animals ; *Zebrafish/virology/genetics ; *Host-Pathogen Interactions/genetics ; *Rhabdoviridae/physiology/genetics ; Disease Models, Animal ; CRISPR-Cas Systems ; *Rhabdoviridae Infections/virology/genetics ; *Fish Diseases/virology/genetics ; }, abstract = {During the long evolutionary process, viruses and hosts have engaged in a game of immune escape and viral clearance, resulting in an exquisite model of virus-host interaction. An appropriate animal model can help unravel the mysteries of this process. Owing to their susceptibility to multiple viruses, small size, high fecundity, low cost, ease of visualization, and efficient gene editing, zebrafish (Danio rerio) have recently emerged as a powerful vertebrate model system for studying virus-host interactions. In this chapter, we describe in detail the experimental protocols for analyzing virus-host interactions in a zebrafish model in which spring viremia of carp virus (SVCV) is used as an example. These protocols include the generation of knockout zebrafish via CRISPR/Cas9 technology, methods for viral infection of zebrafish larvae or adults, analysis of in vivo viral resistance of host genes, analysis of host and viral gene expression in zebrafish larvae via qRT-PCR and Western blotting, and detection of host protein binding to viral proteins via immunoprecipitation. These experimental protocols provide an effective reference for studying virus-host interactions in a zebrafish model.}, }
@article {pmid40515917, year = {2025}, author = {Zhou, Q and Wu, T and Zheng, C and Zou, X}, title = {The Application of Base-Editing Technology to Investigate Virus-Host Interactions and Antiviral Therapeutic Strategies.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2940}, number = {}, pages = {251-260}, pmid = {40515917}, issn = {1940-6029}, mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; *Host-Pathogen Interactions/genetics ; *Antiviral Agents/therapeutic use/pharmacology ; *Virus Diseases/virology/genetics/drug therapy/therapy ; *Viruses/genetics/drug effects ; Animals ; }, abstract = {Understanding viral pathogenesis and developing effective countermeasures hinge on deciphering the complex relationships between viruses and their hosts. While conventional approaches have shown limitations in unraveling these intricate interactions, researchers are now turning to cutting-edge genetic tools, particularly base-editing technology. This section explores how base editing can be innovatively applied to investigate virus-host dynamics and to create novel antiviral therapeutic approaches.}, }
@article {pmid40515905, year = {2025}, author = {Huang, W and Zhao, X and Zheng, C and Shen, Y}, title = {CRISPR-Mediated Viral Gene Knock-In for Studying Viral-Host Interactions.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2940}, number = {}, pages = {109-119}, pmid = {40515905}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems ; *Herpesvirus 1, Human/genetics/physiology ; Red Fluorescent Protein ; Humans ; Luminescent Proteins/genetics/metabolism ; Virus Replication/genetics ; *Host-Pathogen Interactions/genetics ; *Gene Knock-In Techniques/methods ; Animals ; Capsid Proteins/genetics/metabolism ; }, abstract = {CRISPR-mediated gene knock-in is a powerful tool for studying virus-host interactions. In this protocol, we describe the methodology for the knock-in of a red fluorescent protein, mCherry, fused to the viral protein VP26, a component of the herpesvirus capsid. By integrating the mCherry gene into the HSV-1 genome, we enable detailed studies of viral protein dynamics, viral replication, and virus-host interactions in living cells. In particular, this approach allows real-time monitoring of the subcellular localization of the viral capsid. The protocol provides a step-by-step guide for designing the gene knock-in vectors, transfection, selection, and validation of the mCherry fusion to the VP26 protein in the HSV-1 genome.}, }
@article {pmid40514654, year = {2025}, author = {Li, M and Zhou, S and Zhang, S and Xie, X and Nie, J and Wang, Q and Ma, L and Cheng, Y and Luo, J}, title = {Transdermal delivery of CRISPR/Cas9-mediated melanoma gene therapy via polyamines-modified thermosensitive hydrogels.}, journal = {Journal of nanobiotechnology}, volume = {23}, number = {1}, pages = {441}, pmid = {40514654}, issn = {1477-3155}, support = {2024BCA001//Project of Technological Innovation Plan in Hubei Province/ ; 2024BCA001//Project of Technological Innovation Plan in Hubei Province/ ; 2024040701010046//Natural Science Foundation of Wuhan City/ ; 2025AFB729//Hubei Provincial Natural Science Foundation of China/ ; }, mesh = {*Hydrogels/chemistry ; Animals ; *CRISPR-Cas Systems/genetics ; Doxorubicin/pharmacology/administration &amp; dosage/chemistry ; *Melanoma/therapy/genetics/drug therapy ; Humans ; Cell Line, Tumor ; *Genetic Therapy/methods ; *Polyamines/chemistry ; Mice ; Y-Box-Binding Protein 1/genetics/metabolism ; Gene Editing ; Administration, Cutaneous ; Drug Delivery Systems ; Mice, Nude ; }, abstract = {The main obstacles to the clinical application of the CRISPR/Cas9 system are off-target effects and low delivery efficiency. There is an urgent need to develop new delivery strategies and technologies. Three types of in situ injectable hydrogels with different electrical properties were created to find the most secure and efficient sustained-release drug delivery system. After in vitro and in vivo comparisons, we found that the positively charged hydrogels had higher cellular uptake, stronger gene editing efficiency, greater cytotoxicity, longer tumor accumulation, and better anti-tumor efficacy than negatively charged and neutral hydrogels. We designed single guide RNA targeting the Y-box binding protein 1 (YB-1) gene and then used it to create a ribonucleoprotein complex with Cas9 protein. Doxorubicin was co-encapsulated into this positively charged hydrogel to create a co-delivery system. By knocking down YB-1, the expression of YB-1 was reduced, inhibiting the growth and migration of melanoma cells. The strategy of combining YB-1 gene editing and intratumoral injection enhanced the therapeutic effect of doxorubicin while reducing side effects.}, }
@article {pmid40514251, year = {2025}, author = {Qurtam, AA}, title = {Epigenetic reprogramming in breast cancer: The role of CRISPR-Cas 9.}, journal = {Bulletin du cancer}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.bulcan.2025.03.007}, pmid = {40514251}, issn = {1769-6917}, abstract = {Epigenetic alterations are known to be a significant factor in the development and advancement of breast cancer (BC), which continues to be a substantial cause of illness and death in women globally. The emergence of CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9) technology has fundamentally transformed our capacity to edit the genome with unparalleled accuracy, providing novel opportunities for therapeutic intervention. This review examines the utilization of CRISPR-Cas9 to alter epigenetic landscapes to combat BC. We examine the fundamental processes of CRISPR-Cas9 and its derivatives, including dCas9, in their ability to specifically target DNA methylation and histone alterations. The highlighted review showcases the potential of CRISPR-Cas9 in reactivating silenced tumor suppressor genes and inhibiting oncogenes. In addition, we analyze the incorporation of CRISPR-based epigenetic editing into current medicines, offering valuable knowledge on the use of combination therapies to improve treatment effectiveness and overcome resistance. This review intends to highlight the revolutionary potential of CRISPR-Cas9 in generating targeted, personalized therapeutics for BC by explaining the present advancements and future applications. The incorporation of this state-of-the-art technology with conventional and developing therapies holds the potential to establish more efficient and long-lasting remedies in the battle against BC, ultimately enhancing patient results and rates of survival.}, }
@article {pmid40512855, year = {2025}, author = {Boardman, DA and Mangat, S and Gillies, JK and Leon, L and Fung, VCW and Haque, M and Mojibian, M and Halvorson, T and Huang, Q and Tuomela, K and Wardell, CM and Brown, A and Lam, AJ and Levings, MK}, title = {Armored human CAR Treg cells with PD1 promoter-driven IL-10 have enhanced suppressive function.}, journal = {Science advances}, volume = {11}, number = {24}, pages = {eadx7845}, pmid = {40512855}, issn = {2375-2548}, mesh = {Humans ; *Interleukin-10/genetics/metabolism ; *T-Lymphocytes, Regulatory/immunology/metabolism ; *Programmed Cell Death 1 Receptor/genetics ; *Promoter Regions, Genetic ; Animals ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; Mice ; Dendritic Cells/immunology ; CRISPR-Cas Systems ; Immunotherapy, Adoptive/methods ; }, abstract = {Regulatory T cell (Treg cell) therapy has been transformed through the use of chimeric antigen receptors (CARs). We previously found that human Treg cells minimally produce IL-10 and have a limited capacity to control innate immunity compared to type 1 regulatory T cells (Tr1 cells). To create "hybrid" CAR Treg cells with Tr1 cell-like properties, we examined whether the PDCD1 locus could be exploited to endow Treg cells with CAR-regulated IL-10 expression. CRISPR-mediated PD1 deletion increased CAR Treg cell activation, and knock-in of IL10 under control of the PD1 promoter resulted in CAR-induced IL-10 secretion. IL10 knock-in improved CAR Treg cell function, as determined by increased suppression of dendritic cells and alloantigen- and islet autoantigen-specific T cells. In vivo, IL10 knock-in CAR Treg cells were stable, safe, and suppressed dendritic cells and xenogeneic graft-versus-host disease. CRISPR-mediated engineering to simultaneously remove an inhibitory signal and enhance a suppressive mechanism is a previously unexplored approach to improve CAR Treg cell potency.}, }
@article {pmid40510793, year = {2025}, author = {Zhang, Y and Lv, L and Xu, S and Chen, Y}, title = {Innovative nucleic acid detection of Clostridioides difficile utilizing the PAM-unconventional, one-step LAMP/CRISPR-Cas12b detection platforms.}, journal = {Frontiers in cellular and infection microbiology}, volume = {15}, number = {}, pages = {1594271}, pmid = {40510793}, issn = {2235-2988}, mesh = {*Clostridioides difficile/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; *Clostridium Infections/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Humans ; Bacterial Proteins/genetics ; Feces/microbiology ; Bacterial Toxins/genetics ; }, abstract = {INTRODUCTION: Clostridioides difficile (C. difficile), a human pathogen that causes diarrhea and colon lesions, has garnered widespread attention. Rapid and accurate detection of bacterial virulence factors is essential for the diagnosis of C. difficile infection (CDI). To date, numerous laboratory tests have been developed; however, none fully meet the combined requirements of speed, cost-effectiveness, portability, sensitivity, and specificity. Molecular diagnostic technologies based on CRISPR-Cas systems have provided a promising solution to this challenge. Nonetheless, the limited compatibility between pre-amplification and CRISPR cleavage, coupled with the inherent selectivity of CRISPR systems for protospacer adjacent motif (PAM) sequences near the target site, poses additional constraints on the broader adoption of this approach.<br><br>METHODS: Here, we developed PAM-unconventional, One-step LAMP/CRISPR-Cas12b (POLC) detection platforms for the toxin-encoding genes tcdA and tcdB of C. difficile.<br><br>RESULTS: The POLC platforms operated at 60 &#xb0;C, enabling result interpretation either through fluorescence intensity measurements or direct visualization under UV light. The limits of detection (LoDs) ranged from 3 to 14 copies/&#x3bc;L using a fluorescence reader and from 6 to 18 copies/&#x3bc;L via direct observation. Compared to qPCR, which typically requires over an hour, the POLC platforms reduced the detection time to approximately 40 minutes. Each reaction cost approximately USD 6.5, offering a substantial cost saving compared to qPCR-based commercial kits (over USD 10 per test). In clinical validation with 55 fecal samples, the tcdA POLC assay achieved 86.4% sensitivity and 84.8% specificity, while the tcdB POLC assay demonstrated 96.6% sensitivity and 100% specificity, using qPCR as the reference standard.<br><br>DISCUSSION: Our research presents innovative CRISPR-based one-step nucleic acid detection platforms that eliminate canonical PAM sequence requirements. These platforms exhibit high sensitivity and specificity while achieving rapid detection under simple conditions, making them promising candidates for clinical diagnostics and point-of-care testing (POCT).}, }
@article {pmid40507794, year = {2025}, author = {Ho, CH and Tsai, CY and Chang, CC and Hu, CJ and Huang, CY and Lu, YC and Lin, PH and Lin, CH and Lin, HI and OuYang, CH and Hsu, CJ and Liu, TC and Chen, YT and Chan, YH and Cheng, YF and Wu, CC}, title = {Low Efficiency of Homology-Independent Targeted Integration for CRISPR/Cas9 Correction in the Vicinity of the SLC26A4 c.919-2A&gt;G Variant.}, journal = {International journal of molecular sciences}, volume = {26}, number = {11}, pages = {}, pmid = {40507794}, issn = {1422-0067}, support = {106-T04//National Taiwan University Hospital/ ; 107-2622-B-002-008-CC2//National Science and Technology Council of Taiwan/ ; }, mesh = {*Sulfate Transporters/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Hearing Loss, Sensorineural/genetics ; Goiter, Nodular/genetics ; *Membrane Transport Proteins/genetics ; }, abstract = {Recessive variants of SLC26A4 are a common cause of hereditary hearing impairment and are responsible for non-syndromic enlarged vestibular aqueducts and Pendred syndrome. Patients with bi-allelic SLC26A4 variants often suffer from fluctuating hearing loss and recurrent vertigo, ultimately leading to severe to profound hearing impairment. However, there are currently no satisfactory prevention or treatment options for this condition. The CRISPR/Cas9 genome-editing technique is a well-known tool for correcting point mutations or manipulating genes and shows potential therapeutic applications for hereditary disorders. In this study, we used the homology-independent targeted integration (HITI) strategy to correct the SLC26A4 c.919-2A>G variant, the most common SLC26A4 variant in the Han Chinese population. Next-generation sequencing was performed to evaluate the editing efficiency of the HITI strategy. The results showed that only 0.15% of the reads successfully exhibited HITI integration, indicating that the c.919-2 region may not be a suitable region for HITI selection. This suggests that other site selection or insertion strategies may be needed to improve the efficiency of correcting the SLC26A4 c.919-2A>G variant. This experience may serve as a valuable reference for other researchers considering CRISPR target design in this region.}, }
@article {pmid40506744, year = {2025}, author = {Kristof, A and Karunakaran, K and Allen, C and Mizote, P and Briggs, S and Jian, Z and Nash, P and Blazeck, J}, title = {Engineering novel CRISPRi repressors for highly efficient mammalian gene regulation.}, journal = {Genome biology}, volume = {26}, number = {1}, pages = {164}, pmid = {40506744}, issn = {1474-760X}, support = {DP2 CA280622/CA/NCI NIH HHS/United States ; NSF GRFP//National Science Foundation/ ; 1DP2CA280622-01//NIH Office of the Director/ ; }, mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Expression Regulation ; *Repressor Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; Animals ; Gene Knockdown Techniques ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Associated Protein 9 ; }, abstract = {BACKGROUND: CRISPR interference (CRISPRi), the repurposing of the RNA-guided endonuclease dCas9 as a programmable transcriptional repressor, allows highly specific repression (knockdown) of gene expression. CRISPRi platforms can often have incomplete knockdown, performance variability across cell lines and gene targets, and inconsistencies dependent on the guide RNA sequence employed.<br><br>RESULTS: Here, we explore the combination of novel repressor domains with strong Kr&#xfc;ppel-associated box (KRAB) repressors, screening >&#x2009;100 bipartite and tripartite fusion proteins for their ability to reduce gene expression as CRISPRi effectors. We show that these novel repressor fusions have reduced dependence on guide RNA sequences, better slow cell growth when used to knock down expression of essential genes, and function in either fusion or scaffold modalities. Furthermore, we isolate and characterize a particularly effective CRISPRi platform, dCas9-ZIM3(KRAB)-MeCP2(t), which shows improved gene repression of endogenous targets both at the transcript and protein level across several cell lines and when deployed in genome-wide screens.<br><br>CONCLUSIONS: We posit that these novel repressor fusions can enhance the reproducibility and utility of CRISPRi in mammalian cells.}, }
@article {pmid40506725, year = {2025}, author = {Wang, T and Jiang, W and Huang, Z and Yuan, Z and Chen, Z and Lin, J}, title = {Multiplex detection of respiratory RNA viruses without amplification based on CRISPR-Cas13a immunochromatographic test strips.}, journal = {Virology journal}, volume = {22}, number = {1}, pages = {192}, pmid = {40506725}, issn = {1743-422X}, support = {2024T021//Fuzhou University Testing Fund of Precious Apparatus/ ; 2022-S-wr4//Fuzhou Health Science and Technology Plan Soft Science Research Project/ ; 2022-S-rc5//Young and Middle-aged Talent Research Project of Fuzhou City/ ; 2020J05279//Fujian Provincial Natural Science Foundation of China/ ; 2020Y9140//Joint Funds for the Innovation of Sience and Technology, Fujian Province/ ; 2021Y9107//Joint Funds for the Innovation of Science and Technology, Fujian Province/ ; 2021S263//Fuzhou Science and Technology Project/ ; 2021ZQNZD010//Major Research Projects for Young and Middle-aged Researchers of Fujian Provincial Health Commission/ ; 2022J01521//Natural Science Foundation of Fujian Province/ ; 2022Y4003//Fujian Provincial Science and Technology Plan Project/ ; 2022ZD01001//Fujian Provincial Major Health Research Project/ ; 2021GGA053//Fujian Provincial Health Technology Project/ ; 2023YFC3304304//National Key Research &amp; Development Plan/ ; }, mesh = {Humans ; *SARS-CoV-2/isolation &amp; purification/genetics/immunology ; Sensitivity and Specificity ; *Chromatography, Affinity/methods ; *COVID-19/diagnosis/virology ; *RNA Viruses/isolation &amp; purification/genetics ; CRISPR-Cas Systems ; *Respiratory Tract Infections/diagnosis/virology ; Reagent Strips ; Influenza, Human/diagnosis/virology ; }, abstract = {Acute respiratory infections, caused by RNA viruses like respiratory syncytial virus, influenza, rhinovirus, and coronavirus, are major global health threats. Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) is the gold standard for detecting these viruses but is time-consuming, complex, and requires specialized equipment. There is a need for rapid, convenient, and multi-target detection methods to improve disease prevention and control. This study developed a multi-target immunochromatographic detection method using LbuCas13a protein and "band elimination" test strips for detecting SARS-CoV-2 and influenza virus. The method's performance was evaluated by testing known 5 positive and 4 negative samples for SARS-CoV-2 and comparing results with fluorescent PCR and colloidal gold methods. Detection sensitivity was quantified using digital PCR and qPCR. The immunochromatographic test strips showed 100% concordance with fluorescent PCR and colloidal gold methods in initial clinical SARS-CoV-2 detection. Subsequently, we used dual-target immunochromatographic test strips to detect 9 SARS-CoV-2 positive samples and 9 H3N2 positive samples. However, false negatives were observed in dual-target detection of SARS-CoV-2 and H3N2 samples, likely due to low sample concentration or sample degradation. The method had a minimum detection limit of 381.75 copies/µL, as determined by digital PCR and qPCR. The developed multi-target immunochromatographic detection method offers a rapid, low-cost, and simple approach for detecting both SARS-CoV-2 and influenza viruses. With high sensitivity, specificity, and reliability, this method holds promise as a practical tool for RNA virus diagnosis and improving public health response to respiratory infections.}, }
@article {pmid40504161, year = {2025}, author = {Tien, JC and Zhai, Y and Wu, R and Zhang, Y and Chang, Y and Cheng, Y and Todd, AJ and Wheeler, CE and Li, S and Mannan, R and Cheng, C and Magnuson, B and Cruz, G and Cao, Y and Mahapatra, S and Stolfi, C and Cao, X and Su, F and Wang, R and Yang, J and Zhou, L and Qiao, Y and Xiao, L and Cieslik, M and Wang, X and Wang, Z and Chou, J and Fearon, ER and Ding, K and Cho, KR and Chinnaiyan, AM}, title = {Defining CDK12 as a tumor suppressor and therapeutic target in mouse models of tubo-ovarian high-grade serous carcinoma.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {122}, number = {24}, pages = {e2426909122}, doi = {10.1073/pnas.2426909122}, pmid = {40504161}, issn = {1091-6490}, support = {P50-CA186786//HHS | NIH | National Cancer Institute (NCI)/ ; R35-CA231996//HHS | NIH | National Cancer Institute (NCI)/ ; U2C-CA271854//HHS | NIH | National Cancer Institute (NCI)/ ; 22037003//MOST | National Natural Science Foundation of China (NSFC)/ ; W81XWH-21-1-0458//U.S. Department of Defense (DOD)/ ; }, mesh = {Animals ; Female ; Mice ; *Ovarian Neoplasms/genetics/pathology/drug therapy/metabolism ; *Cyclin-Dependent Kinases/genetics/metabolism ; *Cystadenocarcinoma, Serous/genetics/pathology/drug therapy/metabolism ; Disease Models, Animal ; Humans ; CRISPR-Cas Systems ; Tumor Suppressor Protein p53/genetics/metabolism ; Mice, Knockout ; Cell Line, Tumor ; }, abstract = {Ovarian cancer is the sixth leading cause of cancer death among American women, with most fatalities attributable to tubo-ovarian high-grade serous carcinoma (HGSC). This malignancy usually develops resistance to conventional chemotherapy, underscoring the need for robust preclinical models to guide the development of novel therapies. Here, we introduce an HGSC mouse model generated via Ovgp1-driven Cre recombinase effecting CRISPR/Cas9-mediated deletion of Trp53, Rb1, and Nf1 tumor suppressors in mouse oviductal epithelium (m-sgPRN model). Cyclin-dependent kinase 12 (CDK12) inactivation-frequently observed in human HGSC-is associated with poorer outcomes, DNA damage accumulation (including tandem duplications), and increased tumor immunogenicity. In our system, coablation of Cdk12 (m-sgPRN;Cdk12KO) recapitulated hallmark features of HGSC, while accelerating tumor progression and reducing survival. In a conventional (Cre-lox-mediated) Trp53/Nf1/Rb1 triple knockout model with concurrent Cdk12 ablation (PRN;Cdk12KO mice), we observed T cell-rich immune infiltrates mirroring those seen clinically. We established both models as subcutaneous or intraperitoneal syngeneic allografts of CDK12-inactivated HGSC that exhibited sensitivity to immune checkpoint blockade. Furthermore, a CRISPR/Cas9 synthetic lethality screen in PRN;Cdk12KO-derived cell lines identified CDK13-an essential paralog of CDK12-as the most depleted candidate, confirming a previously reported synthetic lethal interaction. Pharmacologic CDK13/12 degradation (employing YJ1206) demonstrated enhanced efficacy in cell lines derived from both m-sgPRN;Cdk12KO and PRN;Cdk12KO models. Our results define CDK12 as a key tumor suppressor in tubo-ovarian HGSC and highlight CDK13 targeting as a promising therapeutic approach in CDK12-inactive disease. Additionally, we have established valuable in vivo resources to facilitate further investigation and drug development in this challenging malignancy.}, }
@article {pmid40503054, year = {2025}, author = {Nawaz, A and Ariffin, NS and Wong, TW}, title = {Functionalized chitosan as nano-delivery platform for CRISPR-Cas9 in cancer treatment.}, journal = {Asian journal of pharmaceutical sciences}, volume = {20}, number = {3}, pages = {101041}, pmid = {40503054}, issn = {2221-285X}, abstract = {CRISPR-Cas system permanently deletes any harmful gene-of-interest to combat cancer growth. Chitosan (CS) is a potential cancer therapeutic that mediates via PI3K/Akt/mTOR, MAPK and NF-kβ signaling pathway modulation. CS and its covalent derivatives have been designed as nanocarrier of CRISPR-Cas9 alone (plasmid or ribonucleoprotein) or in combination with chemical drug for cancer treatment. The nanocarrier was functionalized with polyethylene glycol (PEG), targeting ligand, cell penetrating ligand and its inherent positive zeta potential to mitigate premature clearance and particulate aggregation, and promote cancer cell/nucleus targeting and permeabilization to enable CRISPR-Cas9 acting on the host DNA. Different physicochemical attributes are required for the CS-based nanocarrier to survive from the administration site, through the systemic circulation-extracellular matrix-mucus-mucosa axis, to the nucleus target. CRISPR-Cas9 delivery is met with heterogeneous uptake by the cancer cells. Choice of excipients such as targeting ligand and PEG may be inappropriate due to lacking overexpressed cancer receptor or availability of excessive metabolizing enzyme and immunoglobulin that defies the survival and action of these excipients rendering nanocarrier fails to reach the target site. Cancer omics analysis should be implied to select excipients which meet the pathophysiological needs, and chitosan nanocarrier with a "transformative physicochemical behavior" is essential to succeed CRISPR-Cas9 delivery.}, }
@article {pmid40501577, year = {2025}, author = {Babajanyan, SG and Garushyants, SK and Wolf, YI and Koonin, EV}, title = {Evolution of antivirus defense in prokaryotes depending on the environmental virus prevalence and virome dynamics.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, doi = {10.1101/2025.05.27.656525}, pmid = {40501577}, issn = {2692-8205}, abstract = {UNLABELLED: Prokaryotes can acquire antivirus immunity via two fundamentally distinct types of processes: direct interaction with the virus as in CRISPR-Cas adaptive immunity systems and horizontal gene transfer (HGT) which is the main route of transmission of innate immunity systems. These routes of defense evolution are not mutually exclusive and can operate simultaneously, but empirical observations suggest that at least in some bacterial and archaeal species, one or the other route dominates the defense landscape. We hypothesized that the observed dichotomy stems from different life-history tradeoffs characteristic of these organisms. To test this hypothesis, we analyzed a mathematical model of a well-mixed prokaryote population under a stochastically changing viral prevalence. Optimization of the long-term population growth rate reveals two contrasting modes of defense evolution. In stable, predictable and fluctuating, unpredictable environments with a moderate viral prevalence, direct interaction with the virus and horizontal transfer of defense genes become the optimal routes of immunity acquisition, respectively. In the HGT-dominant mode, we observed a universal distribution of the fraction of microbes with different immune repertoires. Under very low virus prevalence, the cost of immunity exceeds the benefits such that the optimal state of a prokaryote is complete defense systems. By contrast, under very high virus prevalence, horizontal spread of defense systems dominates regardless of the stability of the virome. These findings might explain consistent but enigmatic patterns in the spread of antivirus defense systems among prokaryotes such as the ubiquity of adaptive immunity in hyperthermophiles contrasting their patchy distribution among mesophiles.<br><br>IMPORTANCE: The virus-host arms race is a major component of the evolutionary process in all organisms that drove the evolution of a broad variety of immune mechanisms. In the last few years, over 200 distinct antivirus defense systems have been discovered in prokaryotes. There are two major modes of immunity acquisition: innate immune systems spread through microbial populations via horizontal gene transfer (HGT) whereas adaptive-type immune systems acquire immunity via direct interaction with the virus. We developed a mathematical model to explore the short term evolution of prokaryotic immunity and show that in stable environments with predictable viral repertoires, adaptive-type immunity is the optimal defense strategy whereas in fluctuating environments with unpredictable virus composition, HGT dominates the immune landscape.}, }
@article {pmid40501453, year = {2025}, author = {Kamoen, L and de Bruin, DA and Kralemann, LEM and Roos, K and Wildhagen, MMDA and van Schendel, R and Hooykaas, PJJ and de Pater, S and Tijsterman, M}, title = {Division of labor within polymerase theta in repair of CRISPR-induced DNA breaks in Arabidopsis thaliana.}, journal = {PNAS nexus}, volume = {4}, number = {6}, pages = {pgaf183}, pmid = {40501453}, issn = {2752-6542}, abstract = {To develop efficient strategies for precise mutagenesis in plants, it is crucial to characterize the mechanisms involved in the repair of CRISPR-induced double strand breaks (DSBs). Polymerase theta (Polθ)-mediated end joining (TMEJ) and classical nonhomologous end joining are key pathways that generate a wide array of mutations during DSB repair. To direct repair towards more predictable outcomes, we examined the impact of direct repeats flanking DSBs, which may trigger extended microhomology-mediated end joining (eMMEJ). Unexpectedly, we found that eMMEJ in Arabidopsis thaliana requires Polθ, in contrast to eMMEJ in animals. By reintroducing mutated versions of Polθ into Polθ-deficient plants we discovered that only the helicase activity of Polθ is needed for eMMEJ; we demonstrate that plants lacking Polθ's polymerase domain are incapable of TMEJ and are resistant to TMEJ-dependent T-DNA integration but still support extended microhomology-guided DSB repair at genomic sites with direct repeats. These findings reveal species-specific functionality of Polθ and point to functional divergence in TMEJ across species. Additionally, these insights provide new opportunities to direct targeted mutagenesis in plants toward single, predictable outcomes, paving the way for more efficient crop engineering. Classification: Biological, Health, and Medical Sciences.}, }
@article {pmid40500518, year = {2025}, author = {Song, D and Xu, W and Zhuo, Y and Zhu, A and Long, F}, title = {Orthogonal CRISPR/Cas system facilitated dual-color fluorescence fiber-embedded optofluidic nano-biochip for parallel amplification-free on-site detection of bacterium and virus.}, journal = {Mikrochimica acta}, volume = {192}, number = {7}, pages = {417}, pmid = {40500518}, issn = {1436-5073}, support = {8242031//Natural Science Foundation of Beijing/ ; 2022YFF0609102//National Key R&amp;D Program of China/ ; }, mesh = {*CRISPR-Cas Systems ; *Escherichia coli O157/isolation &amp; purification/genetics ; *SARS-CoV-2/isolation &amp; purification/genetics ; Limit of Detection ; *Lab-On-A-Chip Devices ; COVID-19/diagnosis/virology ; Humans ; Optical Fibers ; Fluorescence ; Biosensing Techniques/methods ; }, abstract = {Bacterial and viral co-infections significantly exacerbate morbidity and mortality. Rapid, sensitive, and parallel detection of these pathogens remains a critical challenge. Here, an orthogonal CRISPR/Cas system facilitated dual-color fluorescence fiber-embedded optofluidic nano-biochip (CD-FOB) was fabricated. Leveraging the time-resolved effect, the CD-FOB achieved ultrasensitive parallel detection of Escherichia coli O157:H7 (E. coli O157:H7) and SARS-CoV-2 based on a multiple signal enhancement strategy, including the collateral cleavage activity of CRISPR/Cas, evanescent wave fluorescence enhancement, DNA-mediated signal amplification, and air-displacement fluorescence enhancement. Without the need for amplification, the CD-FOB system has a detection limit of 643 CFU/mL for E. coli O157:H7 and 3.48 copies/μL for SARS-CoV-2 within 50 min analysis time. To enable rapid on-site detection, a lyophilized CRISPR/Cas assay was prepared using stabilized freeze-dried reagents for detecting E. coli O157:H7 and SARS-CoV-2 in actual samples, achieving recoveries ranging from 70.5% to 200.5%. The unique combination of technical simplicity, multiplexing capability, and operational robustness positions CD-FOB as a versatile solution for combating current and future pathogen threats.}, }
@article {pmid40500390, year = {2025}, author = {Chen, S and Chen, Q and You, X and Zhou, Z and Kong, N and Ambrosio, F and Cao, Y and Abdi, R and Tao, W}, title = {Using RNA therapeutics to promote healthy aging.}, journal = {Nature aging}, volume = {5}, number = {6}, pages = {968-983}, pmid = {40500390}, issn = {2662-8465}, mesh = {Humans ; *Healthy Aging/genetics ; Animals ; *Aging/genetics ; *RNA/therapeutic use ; RNA Interference ; Aptamers, Nucleotide/therapeutic use ; *Genetic Therapy/methods ; Neurodegenerative Diseases/therapy/genetics ; }, abstract = {Aging is characterized by a gradual decline of cellular and physiological functions over time and an increased risk of different diseases. RNA therapeutics constitute an emerging approach to target the molecular mechanisms of aging and age-related diseases via rational design and have several advantages over traditional drug therapies, including high specificity, low toxicity and the potential for rapid development and production. Here, we discuss the latest developments in RNA therapeutics designed to promote healthy aging, including RNA activation, messenger RNA therapy, RNA interference, antisense oligonucleotides, aptamers and CRISPR-Cas-mediated RNA editing. We also review the latest preclinical and clinical studies of RNA technology for treating age-related diseases, including neurodegenerative, cardiovascular and musculoskeletal diseases. Finally, we discuss the challenges of RNA technology aimed at supporting healthy aging. We anticipate that the fusion of RNA therapeutics and aging biology will have an important effect on the development of new medicines and maximization of their efficacy.}, }
@article {pmid40500074, year = {2025}, author = {Lv, J and Yang, Y and Fabrick, JA and Wu, Y}, title = {Gene editing to enhance pesticide resistance in a beneficial predatory mite.}, journal = {Pesticide biochemistry and physiology}, volume = {212}, number = {}, pages = {106466}, doi = {10.1016/j.pestbp.2025.106466}, pmid = {40500074}, issn = {1095-9939}, mesh = {Animals ; *Gene Editing ; *Mites/genetics/drug effects ; CRISPR-Cas Systems ; Receptors, Nicotinic/genetics ; Drug Resistance/genetics ; Female ; Pest Control, Biological ; *Pesticides/pharmacology ; Acaricides/pharmacology ; Benzamides/pharmacology ; Predatory Behavior ; }, abstract = {Successful integrated pest management (IPM) often depends on a suite of control strategies that are compatible with one another. The predatory mite Neoseiulus californicus (Acari: Phytoseiidae) is a key biological control agent of spider mites (especially Tetranychus spp.) and other diminutive, yet important arthropod pests of agriculture. However, like many natural enemies, N. californicus is highly sensitive to chemical pesticides, limiting its overall effectiveness in the field. Here, we used CRISPR/Cas9 gene editing and Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) to create N. californicus harboring loss-of-function mutations in the nicotinic acetylcholine receptor α6 subunit (nAChRα6), the target of the pesticide, spinetoram. The resulting knockout strain (FZ-α6KO) exhibited a 23-fold increase in resistance to spinetoram compared to its parental strain. Inheritance of resistance to spinetoram in FZ-α6KO was autosomal, recessive, and tightly linked with the nAChRα6 gene. We demonstrate that by pairing gene editing with ReMOT Control, extremely small and fragile beneficial arthropods can be genetically manipulated. These results suggest that genetic modification to enhance pesticide resistance in beneficial predatory mites could improve the compatibility between the use of pesticides and biological control organisms.}, }
@article {pmid40500063, year = {2025}, author = {Mocchetti, A and Nikoloudi, AA and Vontas, J and De Rouck, S and Van Leeuwen, T}, title = {CRISPR/Cas9 knock-out of nAChR α6 confers resistance to spinosyns in Frankliniella occidentalis and is associated with a higher fitness cost than target-site mutation G275E.}, journal = {Pesticide biochemistry and physiology}, volume = {212}, number = {}, pages = {106455}, doi = {10.1016/j.pestbp.2025.106455}, pmid = {40500063}, issn = {1095-9939}, mesh = {Animals ; *Receptors, Nicotinic/genetics ; *Insecticides/pharmacology ; *CRISPR-Cas Systems/genetics ; *Insecticide Resistance/genetics ; *Macrolides/pharmacology/toxicity ; *Thysanoptera/genetics/drug effects ; Drug Combinations ; Mutation ; Gene Knockout Techniques ; }, abstract = {Thrips are major agricultural pests globally and spinosyn insecticides like spinosad and spinetoram are commonly used for their control. However, numerous cases of resistance have emerged, often linked to mutations in the nicotinic acetylcholine receptor (nAChR) α6 subunit, the main molecular target of spinosyns. In this study, toxicological data for spinosad and spinetoram were obtained from a susceptible strain of Frankliniella occidentalis, as well as two field-collected resistant strains carrying the G275E resistance mutation. Notably, a new candidate resistance mutation never reported before, T202A, was identified in one of the field collected populations and its possible role in resistance is discussed. Further, CRISPR/Cas9-mediated knockout (KO) of α6 was performed in the susceptible strain to shed light on the phenotypic strength of this resistance mechanism previously observed in the field. The KO conferred complete insensitivity to spinosad and significant resistance to spinetoram, although higher doses of spinetoram remained lethal, suggesting potential interaction with a secondary target. Finally, in an experimental evolution approach, the α6 KO allele rapidly disappeared, indicating a substantial fitness cost. In contrast, G275E alleles persisted.}, }
@article {pmid40499559, year = {2025}, author = {Vaz, M and Watson, KL and Moorehead, RA}, title = {Reduced JAG1 Expression Through miR-200 Overexpression or Crispr-Cas Mediated Knockout Impairs TNBC Growth and Metastasis.}, journal = {Molecular carcinogenesis}, volume = {}, number = {}, pages = {}, doi = {10.1002/mc.23937}, pmid = {40499559}, issn = {1098-2744}, support = {//This study was funded by a CIHR project grant PJT-162218 and a CIHR Priority grant PLL-192130/ ; }, abstract = {Studies from our lab demonstrated that increasing miR-200 expression in human triple negative breast cancer (TNBC) reduced tumor growth and metastasis In Vivo. In this study, we found that overexpression of miR-200s in TNBC cells significantly reduced the expression of JAG1. When JAG1 was knocked out in MDA-MB-231 cells proliferation and invasion were significantly reduced In Vitro. Moreover, loss of JAG1 inhibited mammary tumor growth and metastasis In Vivo. RNA sequencing revealed that loss of JAG1 altered the expression of genes associated with the ECM, angiogenesis, and EMT. These results imply that miR-200s may mediate some of their antitumor actions through reducing JAG1 expression and suggest that agents targeting JAG1 should be further evaluated as a therapeutic strategy for TNBC.}, }
@article {pmid40499557, year = {2025}, author = {Kang, B and Lee, S and Ko, DH and Venkatesh, J and Kwon, JK and Kim, H and Kang, BC}, title = {Virus-induced systemic and heritable gene editing in pepper (Capsicum annuum L.).}, journal = {The Plant journal : for cell and molecular biology}, volume = {122}, number = {5}, pages = {e70257}, pmid = {40499557}, issn = {1365-313X}, support = {RS-2024-00322053//Rural Development Administration, Republic of Korea/ ; RS-2021-NR059217//the National Research Foundation of Korea/ ; }, mesh = {*Capsicum/genetics/virology ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; *Plant Viruses/genetics ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Oxidoreductases/genetics ; }, abstract = {Genome editing using the CRISPR/Cas system enables rapid and efficient plant breeding by directly introducing desired traits into elite lines within a short time frame. However, challenges associated with conventional Agrobacterium tumefaciens-mediated transformation and regeneration have limited gene editing in pepper (Capsicum annuum L.). In this study, we applied and optimized a virus-induced gene editing (VIGE) system to overcome these limitations. We inoculated transgenic pepper seedlings already expressing Cas9 with vectors based on tobacco rattle virus 2 (TRV2) expressing single guide RNAs (sgRNAs) targeting Phytoene desaturase (PDS); shoots regenerated from inoculated cotyledons displayed photobleaching phenotypes. To promote sgRNA mobility and maintain its integrity, we modified the pTRV2-sgRNA vector by incorporating a self-cleaving hammerhead ribozyme (HH) sequence to produce an intact sgRNA fused to part of the mobile RNA of FLOWERING LOCUS T. Additionally, we tested alternative mobile elements, such as tRNA[Ile] and tRNA[Met]. Furthermore, we cultivated plants at the low temperature of 20°C following TRV inoculation to increase TRV persistence and spread. These optimizations, including vector modifications and cultivation conditions, resulted in a systemic editing efficiency of 36.3%, as evidenced by systemic leaves showing photobleaching phenotypes. We determined that 8.5% of progeny from plants inoculated with the pTRV-HH-CaPDS-sgRNA-FT construct were mutated at the CaPDS locus. In addition, we used our VIGE system to successfully edit FASCICULATE, producing mutants whose inflorescences showed a fasciculate phenotype. Direct inoculation with a TRV-based vector expressing a mobile sgRNA to bypass tissue culture, therefore, offers an effective tool for molecular studies and breeding in pepper.}, }
@article {pmid40499551, year = {2025}, author = {Stadager, J and Bernardini, C and Hartmann, L and May, H and Wiepcke, J and Kuban, M and Najafova, Z and Johnsen, SA and Legewie, S and Traube, FR and Jude, J and Rathert, P}, title = {CRISPR GENome and epigenome engineering improves loss-of-function genetic-screening approaches.}, journal = {Cell reports methods}, volume = {5}, number = {6}, pages = {101078}, doi = {10.1016/j.crmeth.2025.101078}, pmid = {40499551}, issn = {2667-2375}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Epigenome/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genetic Testing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Epithelial-Mesenchymal Transition/genetics ; *Loss of Function Mutation/genetics ; Gene Knockout Techniques ; Cell Differentiation/genetics ; Epigenome Editing ; }, abstract = {CRISPR-Cas9 technology has revolutionized genotype-to-phenotype assignments through large-scale loss-of-function (LOF) screens. However, limitations like editing inefficiencies and unperturbed genes cause significant noise in data collection. To address this, we introduce CRISPR gene and epigenome engineering (CRISPRgenee), which uses two specific single guide RNAs (sgRNAs) to simultaneously repress and cleave the target gene within the same cell, increasing LOF efficiencies and reproducibility. CRISPRgenee outperforms conventional CRISPR knockout (CRISPRko), CRISPR interference (CRISPRi), and CRISPRoff systems in suppressing challenging targets and regulators of cell proliferation. Additionally, it efficiently suppresses modulators of epithelial-to-mesenchymal transition (EMT) and impairs neuronal differentiation in a human induced pluripotent stem cell (iPSC) model. CRISPRgenee exhibits improved depletion efficiency, reduced sgRNA performance variance, and accelerated gene depletion compared to individual CRISPRi or CRISPRko screens, ensuring consistency in phenotypic effects and identifying more significant gene hits. By combining CRISPRko and CRISPRi, CRISPRgenee increases LOF rates without increasing genotoxic stress, facilitating library size reduction for advanced LOF screens.}, }
@article {pmid40499189, year = {2025}, author = {Fernandes, PMB and Fernandes, AAR and Maurastoni, M and Rodrigues, SP}, title = {Lab Legends and Field Phantoms: The Tale of Virus-Resistant Plants.}, journal = {Annual review of virology}, volume = {}, number = {}, pages = {}, doi = {10.1146/annurev-virology-092623-101850}, pmid = {40499189}, issn = {2327-0578}, abstract = {Plant viruses present significant challenges to global agriculture, causing crop losses, threatening food security, and imposing economic burdens. Advances in biotechnology have revolutionized strategies to attack these threats, with genetically modified and genome-edited virus-resistant plants, developed using precision tools such as RNA interference and CRISPR/Cas technology, playing pivotal roles. Despite these breakthroughs, fragmented regulatory frameworks and divergent policies across regions including the European Union and the Global South hinder the global adoption of such innovations. Multifaceted approaches, including gene pyramiding, microbiome-based strategies, and pathogen-targeted defenses, show promise for enhancing plant resilience. This review explores the biological, regulatory, and ethical dimensions of deploying virus-resistant crops, emphasizing the need for harmonization of international regulation to maximize biotechnological benefits. By addressing these challenges, biotechnology can advance sustainable agriculture, secure food systems, and mitigate the effect of plant viral diseases.}, }
@article {pmid40499044, year = {2025}, author = {Zhan, X and Jiang, Y and Li, Z and Hu, X and Lan, F and Ying, B and Wu, Y}, title = {Split DNA Tetrahedron-Mediated Spatiotemporal-Hierarchy CRISPR Cascade Integrated with Au@Pt Nanolabels and Artificial Intelligence for a Cervical Cancer MicroRNA Bioassay.}, journal = {ACS nano}, volume = {}, number = {}, pages = {}, doi = {10.1021/acsnano.5c01376}, pmid = {40499044}, issn = {1936-086X}, abstract = {The screening and monitoring of microRNAs as cancer molecular biomarkers is clinically significant, but traditional methods lack sufficient sensitivity, accuracy, and convenience. The CRISPR-colorimetric lateral flow assay (CLFA) integration offers a promising and efficient solution; however, cumbersome preamplification and poor quantification hinder clinical adoption. In this study, we developed a one-step isothermal CRISPR-Cas cascaded sensing system that is preamplification-free. At its core is a designed and selected split DNA tetrahedron activator, employing spatiotemporal-hierarchy mechanisms to precisely bidirectionally drive the kinetics of two Cas enzymes, accelerating the activation of Cas13a while delaying the initiation of Cas12a, to achieve optimal balance. This system enables ultrasensitive, single-step, single-tube, and rapid detection of a cervical cancer relative biomarker, microRNA-21, achieving a limit of detection of 38 aM with a broad linear range. The CRISPR system is further integrated with CLFA enhanced by ultrathin platinum-protected gold nanolabels (Au@Pt, also named Au@s-Pt), along with a smartphone equipped with dual convolutional neural network models (YOLO v5 and MobileNet v3), enabling more precise, rapid quantification of target miRNA. Using this integrated platform, miRNA-21 levels in cervical cancer and precancerous samples can be accurately quantified with approximately 30 min at low cost and without the need for large, sophisticated instruments, with results showing good concordance with quantitative real-time polymerase chain reaction. This platform provides an efficient, highly sensitive, user-friendly, and quantifiable point-of-care testing solution.}, }
@article {pmid40498336, year = {2025}, author = {Patel, E and Das, P and Hazra, S and Sharma, M and Chhabra, G and Gill, BS and Sharma, S and Kaur, A and Singla, D and Sandhu, JS}, title = {Mutation in soybean Lox-2 PLAT/LH2 domain through CRISPR/Cas9 reduces seed lipoxygenase activity: responsible for undesirable flavour.}, journal = {Transgenic research}, volume = {34}, number = {1}, pages = {29}, pmid = {40498336}, issn = {1573-9368}, mesh = {*Glycine max/genetics/enzymology/growth &amp; development ; *Seeds/genetics/enzymology ; *CRISPR-Cas Systems/genetics ; *Lipoxygenase/genetics/metabolism ; Plants, Genetically Modified/genetics/growth &amp; development ; Mutation ; *Plant Proteins/genetics/metabolism ; }, abstract = {Soybean, a protein and oil rich legume is primarily used as livestock feed and to a lesser extent for human consumption due to undesirable flavour in the seeds caused by L-2 isozyme of lipoxygenase. Herein, soybean with reduced isozyme activity was developed through CRISPR/Cas9 targeted mutation in L-2 encoding Lox-2 gene. sgRNA designed from PLAT/LH2 domain in second exon of Lox-2 (Lox-2 E2) was validated by in vitro cleavage assay; inserted in CRISPR/Cas9 binary vector and used for genetic transformation of SL1074 cultivar hypocotyl segments. A total of 12 T0 putative plants were identified through PCR. Amongst these, four revealed mutation at the target sgRNA site by CEL1 assay and substitution of a base A with G six bp upstream of PAM converting lysine to glutamic acid at 119 position. T1 and T2 seeds derived from mutant T0-37 plant showed upto 25.49% reduction in isozyme activity as compared to SL1074. The base substitution was confirmed in T1 progeny; segregation analysis revealed homozygosity and heritability of mutation in T2 plants. The interaction between structural models of SL1074, mutant domains and negatively charged substrates revealed strong binding affinity of the substrates with positively charged lysine in SL1074 domain due to formation of two hydrogen bonds. On the contrary, weak binding of the substrates with negatively charged glutamic acid in mutant domain and absence of hydrogen bond explained reduction of isozyme activity in T2 seeds. The mutant soybean with reduced isozyme activity is an important source for introgressing the trait in plant breeding programs.}, }
@article {pmid40498069, year = {2025}, author = {Chen, P and Li, X and Zhou, Q and Chen, J and Lu, L and Wang, P and Zhang, G and Sun, D and Huang, X and Liu, J and Wang, X}, title = {Configuration of adaptable template RNA architectures to unfold the editable space of a nuclease prime editor.}, journal = {Nucleic acids research}, volume = {53}, number = {11}, pages = {}, pmid = {40498069}, issn = {1362-4962}, support = {2021YFF1000700//National Key R&amp;D Program of China/ ; 32272848//National Natural Science Foundation of China/ ; CARS-39-03//China Agricultural Research System/ ; 2022GDTSLD-46//China Agricultural Research System/ ; }, mesh = {*Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; *RNA/chemistry/genetics ; Humans ; DNA Breaks, Double-Stranded ; CRISPR-Cas Systems ; }, abstract = {The nuclease prime editor (PEn) combines double-strand break (DSB) induction with reverse transcription for editing. Recently, high-activity PEn forms (e.g. uPEn) have been developed via the concomitant application of DNA repair regulator(s). While the standard uPEn introduces edits only downstream of the nuclease-induced DNA break, we seek innovative designs to enable upstream-directed editing by re-configuring guide/template RNAs to drive prime edits into the target strand (TS), instead of the conventional non-TS. We first devise a dual-RNA uPEn strategy by supplementing a cleavage-competent sgRNA with an accessory template RNA for modifying target strand (ActRNA:t). Characterization of the dual-RNA system allows us to next develop a bifunctional target strand-programming pegRNA (tsp-pegRNA). Both the dual- and single-RNA upstream-modifying uPEn forms (versions 3.1/3.2) successfully drive diverse types of accurate edits into a panel of locations refractory to the standard uPEn and the latest nickase PE. Moreover, we provide insights on the role of uPEn's helper module (i.e. i53) in driving TS prime edits. Additional co-administration of a DNA-dependent protein kinase inhibitor with uPEn3.2 leads to further optimization of editing purities. Together, these advances transform uPEn into a highly applicable tool with much-expanded editable space, and lay a strong foundation for future development of PEn/PE platforms.}, }
@article {pmid40498068, year = {2025}, author = {Qiao, J and Zhang, J and Jiang, Q and Jin, S and He, R and Qiao, B and Liu, Y}, title = {Boosting CRISPR/Cas12a intrinsic RNA detection capability through pseudo hybrid DNA-RNA substrate design.}, journal = {Nucleic acids research}, volume = {53}, number = {11}, pages = {}, pmid = {40498068}, issn = {1362-4962}, support = {2022YFC2304304//National Key Research and Development Program of China/ ; 2023DJC136//Science and Technology Innovation Talent Plan of Hubei Province/ ; 2025AFB825//Natural Science Foundation of Hubei Province/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics ; MicroRNAs/genetics/analysis ; *DNA/chemistry/genetics ; *RNA/analysis/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; Nucleic Acid Hybridization ; Limit of Detection ; }, abstract = {The CRISPR/Cas12a [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 12a] system is known for its intrinsic RNA-guided trans-cleavage activity; however, its RNA detection sensitivity is limited, with conventional methods typically achieving detection limits in the nanomolar range. Here, we report the development of a "pseudo hybrid DNA-RNA" (PHD) assay that significantly enhances the RNA detection capability of Cas12a. The PHD assay achieves a striking detection limit of 7.7 pM using single CRISPR RNA (crRNA) and 33.8 fM using pooled crRNAs. Importantly, this assay exhibits ultra-high specificity, capable of distinguishing mutated RNA target sequences at the protospacer adjacent motif (PAM)-distal region. It can also detect ultrashort RNA sequences as short as 6-8 nt and long RNAs with complex secondary structures. Additionally, the PHD assay enables PAM-free attomolar-level DNA detection. We further demonstrate the practical utility of the PHD assay by successfully detecting miR-155 biomarkers and human pappilloma virus 16 DNA in clinical samples. We anticipate that the design principles established in this study can be extended to other CRISPR/Cas enzymes, thereby accelerating the development of powerful nucleic acid testing tools for various applications.}, }
@article {pmid40497031, year = {2025}, author = {Galli, C}, title = {Current Techniques of Gene Editing in Pigs for Xenotransplantation.}, journal = {Transplant international : official journal of the European Society for Organ Transplantation}, volume = {38}, number = {}, pages = {13807}, pmid = {40497031}, issn = {1432-2277}, mesh = {Animals ; *Transplantation, Heterologous/methods ; *Gene Editing/methods ; Swine/genetics ; Humans ; CRISPR-Cas Systems ; Animals, Genetically Modified ; Nuclear Transfer Techniques ; Endogenous Retroviruses ; }, abstract = {Shortage of human organs for transplantation has created a demand for alternative solutions of which xenotransplantation is amongst the most promising one in the short term. However, the immune reaction following transplantation of a pig organ is greater than the one elicited during allotransplantation. Genetic engineering of the pig is required so that pig organs or tissues are made less immunogenic to humans by eliminating some antigens and by expressing human proteins that can reduce the damage by the host immune system. To generate founder animals with the desired mutations genetic engineering of somatic cells with multiplexed mutations combined with somatic cell nuclear transfer (SCNT) is the best solution with the technology available today. Safety concerns include potential zoonosis, primarily porcine endogenous retroviruses (PERVs). Ethical considerations might arise from the use animals involved in research. Genome editing techniques based CRISPR-Cas9, have greatly facilitated the modification of pig's genome to address coagulation and inflammation issues, to mention just a few, arising after the pig organ is transplanted into a human. However, further research is needed to ensure safety and efficacy of the genome edits introduced in the pig genome are compatible with the health and welfare of the pigs.}, }
@article {pmid40496602, year = {2025}, author = {Jangra, S and Seal, DR and Ghosh, A}, title = {Biotechnological advancements for sustainable management of thrips.}, journal = {3 Biotech}, volume = {15}, number = {7}, pages = {204}, pmid = {40496602}, issn = {2190-572X}, abstract = {Thrips are minute, slender, polyphagous, thysanopteran insect pests that cause huge economic losses to crops by directly feeding on the phloem sap and transmitting several plant viruses. Thrips have emerged as a major threat to global agriculture and food security. Current management options mainly rely on chemical insecticides; however, thrips have evolved resistance to most commonly used insecticides, making management extremely difficult. The availability of host-plant resistance is limited in the case of thrips. Biotechnological approaches such as genetic engineering, RNA interference (RNAi), antisense oligonucleotides (ASOs), artificial microRNA (amiRNA), and genome editing have paved the way for the development of environmentally sustainable thrips management options. However, the adoption of these biotechnological approaches needs further refinement and validation. Transgenic plants with anti-herbivory proteins hold promise to be successful in managing thrips. Spray-induced gene silencing (SIGS) and gene editing would be novel alternatives to hazardous pesticides. This review discusses the progress made towards using modern biotechnological interventions in functional genomics, emphasizing their application in sustainable thrips management.}, }
@article {pmid40494869, year = {2025}, author = {Basu, M and Xiao, JF and Kailasam Mani, SK and Qu, F and Lin, Y and Duex, J and Ye, H and Neang, V and Theodorescu, D}, title = {Genes driving three-dimensional growth of immortalized cells and cancer.}, journal = {Cell death &amp; disease}, volume = {16}, number = {1}, pages = {442}, pmid = {40494869}, issn = {2041-4889}, support = {R01 CA075115/CA/NCI NIH HHS/United States ; R29 CA075115/CA/NCI NIH HHS/United States ; CA075115//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; }, mesh = {Humans ; Animals ; *Urinary Bladder Neoplasms/genetics/pathology ; Mice ; Cell Proliferation/genetics ; Mitogen-Activated Protein Kinase 1/genetics/metabolism/antagonists &amp; inhibitors ; Cell Line, Tumor ; Cell Transformation, Neoplastic/genetics ; CRISPR-Cas Systems ; }, abstract = {Unchecked growth in three-dimensions (3D) in culture is a key feature of immortalized cells on the path to malignant transformation and hence a potential target phenotype for prevention. Also, expression of genes driving this process, but not that of 2D growth, would likely be more specific to cancer development and their inhibition would be less toxic to normal cells, many of which can grow in 2D but rarely in 3D culture. To define such genes, we compared CRISPR depletion screens performed in HBLAK, a spontaneously immortalized, non-tumorigenic human urothelial cell line, grown in 2D to those in 3D. Using the CRISPR Bassik DTKP (drug target kinase phosphatase) deletion library targeting 2,333 genes, we identified 85 genes which were specifically lost in 3D cultures. Prioritizing hits to those associated with bladder cancer in patients provided us with a set of 11 genes. Only one gene, MAPK1 remained relevant if a human pan-cancer criteria was applied. Single gene in vitro validation confirmed that MAPK1 inhibition was specific to 3D growth. We also found that MAPK1 depletion led to significant growth reductions in human tumor xenografts in vivo. Inhibition of MAPK1 by Ulixertinib, an orally active MAPK1 inhibitor, led to human bladder cancer growth inhibition in both 3D in vitro and in vivo models. In summary, screening for genes specifically driving 3D growth in immortalized cells may provide targets for both prevention and early therapy in bladder and other cancers while potentially limiting therapeutic toxicity.}, }
@article {pmid40494841, year = {2025}, author = {Liu, H and Zhang, M and Sun, L and Peng, Y and Sun, Y and Fan, Y and Li, H and Liu, D and Lu, H}, title = {Synergistic optimization enhancing the precision and efficiency of cytosine base editors in poplar.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {904}, pmid = {40494841}, issn = {2399-3642}, mesh = {*Populus/genetics/drug effects ; *Gene Editing/methods ; *Cytosine/metabolism ; *CRISPR-Cas Systems ; Genome, Plant ; Plants, Genetically Modified/genetics ; }, abstract = {CRISPR/Cas9 genome editing technology, particularly cytosine base editing (CBE) systems, emerges as a powerful tool for precise genomic modification in plants, offering transformative applications across agricultural and forestry research and breeding programs. However, current CBE systems in poplar exhibit low efficiency and imprecise base substitutions, and optimization of base editing systems specifically for poplar remains a significant challenge. To address these limitations, we engineer a high-efficiency poplar CBE system (hyPopCBE) by integrating the MS2-UGI system, fusing Rad51 DNA-binding domain, and modifying the nuclear localization signal. Through stepwise optimization, we develop hyPopCBE-V4, which exhibits a synergistic effect in woody plants. Compared to the original hyPopCBE-V1, hyPopCBE-V4 improves C to T editing efficiency while reducing byproducts and exhibiting a narrower editing window. The proportion of plants with clean C to T edits (without byproducts) increases from 20.93% to 40.48%, and the efficiency of clean homozygous C to T editing rises from 4.65% to 21.43%. Using hyPopCBE-V1 and its variants, we induce Pro197Leu mutation in the herbicide target gene PagALS. Poplar lines with edits in all four PagALS homologues exhibit high resistance to tribenuron and nicosulfuron. This study employs a multi-component synergistic optimization strategy that specifically enhances the efficiency and precision of CBE editing in poplar while improving synchronous editing of alleles. Through editing the herbicide resistance gene PagALS, we obtain the herbicide-resistant poplar germplasm. Our research provides a more precise and efficient CBE tool for genetic modification in poplar that can also be applied to other forestry species, demonstrating its potential for advancing forestry research and breeding programs.}, }
@article {pmid40493278, year = {2025}, author = {Kaur, J and Viswanathan P, A and Bari, VK}, title = {CRISPR/Cas9-mediated editing of jasmonic acid pathways to enhance biotic &amp; abiotic stress tolerance: An overview &amp; prospects.}, journal = {Functional &amp; integrative genomics}, volume = {25}, number = {1}, pages = {125}, pmid = {40493278}, issn = {1438-7948}, mesh = {*Cyclopentanes/metabolism ; *Oxylipins/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Stress, Physiological/genetics ; Crops, Agricultural/genetics ; Plant Growth Regulators/metabolism ; Signal Transduction ; }, abstract = {Food security is becoming increasingly important as the world's population grows, and the likelihood that climate change could impair agricultural supply complicates matters further. However, plants actively suppress growth as an adaptation strategy to enhance survival under stress conditions. Phytohormone jasmonates (JAs) regulate various physiological processes, including plant growth, development, and senescence. Plant biotic and abiotic stress responses cause dynamic shifts in the metabolism and signaling of JAs, suggesting that JAs response impacts plant development and resistance to various stresses. The JAs-associated responses depend on core components of JAs -signaling, including the transcriptional repressors protein JAZ and the transcription factor MYC2. While traditional breeding has greatly benefited the world, this approach has several disadvantages, such as the emergence of undesirable traits and species barriers. Genome editing technology has revolutionized plant biology research and has significant ramifications for agriculture and global food security, particularly in light of climate change and population growth. CRISPR/Cas9 and its derivative tools have been used for genome editing in numerous crops to improve or alter desired plant phenotypes. This review summarizes JA's role in plant stress and defense and how CRISPR/Cas9-editing technology modifies plant JA's responses, especially against biotic and abiotic stress.}, }
@article {pmid40493189, year = {2025}, author = {Gorelik, A and Paulo, JA and Schroeter, CB and Lad, M and Shurr, A and Mastrokalou, C and Siddiqi, S and Suyari, O and Brognard, J and Walter, D and Matthews, J and Palmer, TM and Gygi, SP and Ahel, I}, title = {CRISPR screens and quantitative proteomics reveal remodeling of the aryl hydrocarbon receptor-driven proteome through PARP7 activity.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {122}, number = {24}, pages = {e2424985122}, doi = {10.1073/pnas.2424985122}, pmid = {40493189}, issn = {1091-6490}, support = {224095/Z/21/Z//Wellcome Trust (WT)/ ; 210634 223107 302632//Wellcome Trust (WT)/ ; BB/R007195/1 BB/W016613/1//UKRI | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; C35050/A22284//Cancer Research UK (CRUK)/ ; GM67945//HHS | NIH (NIH)/ ; GM132129//HHS | NIH (NIH)/ ; }, mesh = {*Receptors, Aryl Hydrocarbon/metabolism/genetics ; Humans ; *Proteome/metabolism/genetics ; *Poly(ADP-ribose) Polymerases/metabolism/genetics ; Proteomics/methods ; Suppressor of Cytokine Signaling 3 Protein/metabolism/genetics ; Cell Line, Tumor ; Signal Transduction ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Lung Neoplasms/metabolism/genetics ; Nucleoside Transport Proteins ; Basic Helix-Loop-Helix Transcription Factors ; }, abstract = {PARP7 is an enzyme that uses donor substrate NAD[+] to attach a single ADP-ribose moiety onto proteins related to immunity, transcription, and cell growth and motility. Despite the importance of PARP7 in these processes, PARP7 signaling networks remain underresearched. Here, we used genome-wide CRISPR screens and multiplex quantitative proteomics in distinct lung cancer cell lines treated with a PARP7 inhibitor to better understand PARP7 molecular functions. We find that manipulating the aryl hydrocarbon receptor (AHR) transcriptional activity mediates PARP7 inhibitor sensitivity and triggers robust changes to the AHR-controlled proteome (AHR-ome). One of the striking features of such AHR-ome remodeling was the downregulation of filamins A and B concurrent with the induction of the corresponding E3 ubiquitin ligase ASB2. We also show that suppressor of cytokine signaling 3 (SOCS3) crosstalks to AHR. Inhibition of PARP7 in SOCS3 knockout cells leads to reduced viability compared to wild-type cells treated with a PARP7 inhibitor. Our results reveal signaling interplay between PARP7, AHR, and SOCS3 and establish an invaluable resource to study the role of PARP7 in the regulation of AHR signaling and innate immunity through its ADP-ribosyl transferase activity.}, }
@article {pmid40491435, year = {2025}, author = {Santarossa, BA and Mariani, &#xc9; and Corrêa, ADP and Costa, FC and Taylor, MC and Kelly, JM and Elias, MC and Calderano, SG}, title = {Stage-specific MCM protein expression in Trypanosoma cruzi: insights into metacyclogenesis and G1 arrested epimastigotes.}, journal = {Frontiers in cellular and infection microbiology}, volume = {15}, number = {}, pages = {1584812}, pmid = {40491435}, issn = {2235-2988}, mesh = {*Trypanosoma cruzi/growth &amp; development/genetics/metabolism ; *Protozoan Proteins/genetics/metabolism ; *Life Cycle Stages ; *Minichromosome Maintenance Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *G1 Phase Cell Cycle Checkpoints ; }, abstract = {Trypanosoma cruzi is a protozoan parasite that is the etiological agent of Chagas disease, which is endemic to Latin America with reported cases in non-endemic regions such as Europe, Asia, and Oceania due to migration. During its lifecycle, T. cruzi alternates between replicative and non-replicative infective lifeforms. Metacyclogenesis is the most studied transition of the T. cruzi life cycle, where replicative epimastigotes differentiate into infective metacyclic trypomastigotes inside the gut of the triatomine vector. This early-branching organism expresses a divergent pre-replication complex (pre-RC) where the only conserved component is the MCM2-7 protein family. Given the role of pre-RC components in cell cycle regulation, we investigated whether MCM expression and location could be involved in proliferation control in epimastigotes and during metacyclogenesis. Using CRISPR/Cas9, we tagged MCM subunits and tracked their expression and subcellular localization. Our findings reveal that MCM subunits are consistently expressed and localized to the nucleus throughout the epimastigote cell cycle, including in G1/G0-arrested cells. However, MCM subunits are degraded during metacyclogenesis as cells enter the G0 state, marking the transition to replication arrest. Therefore, epimastigotes arrested in G1/G0 can either maintain MCM complex expression and resume the cell cycle when conditions become favorable, or they can undergo metacyclogenesis, exiting the cell cycle and entering a G0 state, where MCM subunits are degraded as part of the replication repression mechanism.}, }
@article {pmid40491004, year = {2025}, author = {Capelli, L and Marzari, S and Spezzani, E and Bertucci, A}, title = {Synthetic CRISPR Networks Driven by Transcription Factors via Structure-Switching DNA Translators.}, journal = {Journal of the American Chemical Society}, volume = {147}, number = {24}, pages = {21184-21193}, doi = {10.1021/jacs.5c06913}, pmid = {40491004}, issn = {1520-5126}, mesh = {*CRISPR-Cas Systems ; *Transcription Factors/metabolism/chemistry ; *DNA/chemistry/metabolism/genetics ; Synthetic Biology ; CRISPR-Associated Proteins/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; }, abstract = {CRISPR-Cas systems have advanced many domains in life sciences, enabling diverse applications in gene editing, diagnostics, and biosensing. Here, we introduce a platform that leverages transcription factors (TFs) to regulate CRISPR-Cas12a trans-cleavage activity via engineered DNA translators. These dynamic DNA structures respond to TF binding by switching conformations, modulating Cas12a activity. Using TATA-binding protein and Myc-Max as TF models, we optimized DNA translators for precise and tunable control with rapid response kinetics. We demonstrated the platform's specificity and versatility by integrating TF-induced regulation into synthetic biology networks, including the activation of a fluorogenic RNA aptamer (Mango III) and the creation of an artificial multimolecular communication pathway between Cas12a and Cas13a. This work establishes TFs as effective regulators of CRISPR-Cas systems, enabling novel protein-nucleic acid communication channels, showing potential for novel synthetic biology applications.}, }
@article {pmid40490981, year = {2025}, author = {Zheng, Y and Du, F and Hang, Y and Ma, W and Yang, G and Wu, N and Sun, X}, title = {Recent Advances in Genome Base Editing Technology and Its Applications in Industrial Microorganism.}, journal = {Biotechnology journal}, volume = {20}, number = {6}, pages = {e70052}, doi = {10.1002/biot.70052}, pmid = {40490981}, issn = {1860-7314}, support = {2023YFA0914400//National Key Research and Development Program of China/ ; BK20230059//Natural Science Foundation of Jiangsu Province/ ; 32422068//National Natural Science Foundation of China/ ; 22378209//National Natural Science Foundation of China/ ; 22038007//National Natural Science Foundation of China/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Industrial Microbiology/methods ; Metabolic Engineering ; Synthetic Biology ; Saccharomyces cerevisiae/genetics ; }, abstract = {Base editing technology is a novel gene-editing approach derived from the CRISPR/Cas9 system, enabling precise and efficient base conversion. Due to operational simplicity, strong target specificity, high editing efficiency, and minimal editing byproducts, base editing has been widely applied in gene therapy, crop breeding, construction of model organisms, and microbial metabolic engineering. In this review, we systematically summarize the development history and recent advancements of several major base editors, including cytosine base editors (CBEs), adenine base editors (ABEs), CRISPR-free base editors, C-to-G base editors (CGBEs), glycosylase base editors (GBEs), and IscB-derived base editors. Furthermore, we comprehensively summarize optimization strategies for base editors and its application in constructing efficient industrial microorganism, such as Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, Yarrowia lipolytica, and Aspergillus niger. This review aims to facilitate the broader application of base editing technologies in synthetic biology and accelerate their translational potential.}, }
@article {pmid40490955, year = {2025}, author = {Ruan, S and Wang, A and Zou, H and Lin, Y and Ye, L and Liang, S}, title = {Synthetic Genetic Circuits Enabled in Komagataella phaffii Through T7 RNAP/CRISPRa System.}, journal = {Biotechnology journal}, volume = {20}, number = {6}, pages = {e70036}, doi = {10.1002/biot.70036}, pmid = {40490955}, issn = {1860-7314}, support = {2021YFC2104000//National Key Research and Development Program/ ; 32272276//National Natural Science Foundation of China/ ; }, mesh = {*Gene Regulatory Networks/genetics ; *DNA-Directed RNA Polymerases/genetics/metabolism ; *Synthetic Biology/methods ; *Saccharomycetales/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Viral Proteins/genetics/metabolism ; Promoter Regions, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {The CRISPR activation (CRISPRa) transcriptional system has become a powerful synthetic biology tool for the regulation of endogenous gene expression, allowing for precise fine-tuning of target genes through the simple modification of sgRNA sequences. In this study, we demonstrate that sgRNAs can be effectively expressed using the T7 transcription system. The insertion of tRNA sequences between PT7 and sgRNAs significantly enhances the efficiency of transcriptional activation. Furthermore, the design of PT7-tRNA-sgRNA arrays facilitates the multiplexed activation of genes. sgRNA expression was regulated by the Tet-on induction system, split-T7 system, and RNA cleavage processing by HH-HDV, resulting in the creation of a Boolean logic gene circuit capable of performing both AND and OR logic operations. Finally, we developed a UPR self-responsive system by utilizing endogenous promoters that are responsive to UPR signals to control the expression of T7 RNAP. This system dynamically regulates the expression of the endogenous HAC1 transcription factor, thus enhancing the secretion of heterologous proteins. The findings from this study highlight the potential of utilizing the T7 transcription system for the construction of genetic circuits, providing a practical toolkit for gene regulation in the industrial Komagataella phaffii strain.}, }
@article {pmid40490752, year = {2025}, author = {Masson, JD and Taglietti, V and Ruby, F and Ono, H and Mouri, N and Jorge, A and Guillaud, L and Tiret, L and Relaix, F}, title = {Extensive striated muscle damage in a rat model of Duchenne muscular dystrophy with Dmd exons 10-17 duplication.}, journal = {Skeletal muscle}, volume = {15}, number = {1}, pages = {16}, pmid = {40490752}, issn = {2044-5040}, support = {19507, 22946//AFM-T&#xe9;l&#xe9;thon/ ; EQU20200301021//Fondation pour la Recherche M&#xe9;dicale/ ; 101080690//European MAGIC Project consortium/ ; }, mesh = {Animals ; *Muscular Dystrophy, Duchenne/genetics/pathology ; Exons ; Disease Models, Animal ; Rats ; *Dystrophin/genetics ; Rats, Sprague-Dawley ; *Gene Duplication ; Male ; *Muscle, Striated/pathology ; CRISPR-Cas Systems ; Gene Editing ; }, abstract = {BACKGROUND: Duchenne muscular dystrophy (DMD) mainly affects young boys with out-of-frame mutations in the DMD gene, leading to dystrophin deficiency. This loss disrupts the assembly of the sarcolemmal dystrophin-associated glycoprotein complex, resulting in membrane fragility and damage during muscle contraction-relaxation cycles. Consequently, patients experience progressive muscle weakness, loss of ambulation and cardiorespiratory failure. Gene therapy represents one of the most promising therapeutic approaches, requiring rigorous preclinical validation of candidate strategies. While several preclinical models of dystrophin deficiency mimic point mutations or exon deletions, no existing rat model accurately replicates DMD gene duplications, which account for approximately 10% of DMD cases.<br><br>METHODS: Using CRISPR/Cas9 genome editing, we generated a&#x2009;~&#x2009;125 kbp duplication encompassing exons 10-17 of the Dmd gene in Sprague Dawley rats. To characterise disease progression in these rats, we assessed biochemical, histological and functional biomarkers at 6 and 10 months of age, comparing them to their healthy littermates.<br><br>RESULTS: We established the R-DMDdup10-17 line. The microstructure of limb, diaphragm and cardiac muscles of R-DMDdup10-17 (DMD) rats exhibited dystrophic changes at 6 and 10 months, including loss of myofibres and fibrosis. These alterations led to a significant body mass reduction, muscle weakness (including diaphragm deficiency) and cardiac electrical defects. Premature lethality was observed between 10 and 13 months.<br><br>CONCLUSION: Duplication of the Dmd genomic region encompassing exons 10 to 17 in rats results in dystrophin deficiency, severe striated muscle dystrophy, and premature death. The R-DMDdup10-17 line represents the first reported genetic model of a severe and early lethal duplication variant in the Dmd gene. It provides a critical tool for assessing targeted gene therapies aimed to correct such mutations.}, }
@article {pmid40489621, year = {2025}, author = {Zheng, X and Wu, S and Qiu, J and Li, A and Li, L and Yan, G and Li, M and Meng, F and Zhang, K and Lin, S}, title = {Induction of a neurotoxin in diatoms by iron limitation via cysteine synthase.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {122}, number = {24}, pages = {e2424843122}, doi = {10.1073/pnas.2424843122}, pmid = {40489621}, issn = {1091-6490}, support = {U2106205//MOST | NSFC | National Natural Science Foundation of China-Shandong Joint Fund (-)/ ; tstp20231216//Special Foundation for Taishan Scholar of Shandong Province/ ; }, mesh = {*Iron/metabolism ; *Diatoms/metabolism/genetics ; *Neurotoxins/biosynthesis/metabolism ; *Amino Acids, Diamino/biosynthesis/metabolism ; *Cysteine Synthase/metabolism/genetics ; Humans ; Cyanobacteria Toxins ; CRISPR-Cas Systems ; }, abstract = {The β-N-methylamino-L-alanine (BMAA) is an emerging neurotoxin associated with human neurodegenerative diseases such as Alzheimer's disease. Here, we report the prevalence of BMAA synthesis in protein forms by marine diatoms and reconstruct its tentative biosynthesis pathway. Remarkably, the BMAA production is strongly induced by iron limitation. Transcriptomic analyses suggest that cysteine synthase (CysK) is involved in BMAA synthesis. This is verified as CRISPR/Cas9-based CysK knockout abolished BMAA production and addition of the recombinant CysK to the mutant restored BMAA synthesis. As diatoms are the most abundant primary producers in ocean, the prevalence of BMAA in diatoms has significant public health implications. The biosynthesis pathway provides biomarkers for further investigation of BMAA production in marine diatoms and insights for understanding the pathological mechanism for human neurodegenerative diseases.}, }
@article {pmid40489409, year = {2025}, author = {Yuan, Y and Jiang, Z and Zeng, Y and Tang, J and Luo, J and Xie, C and Gong, Y}, title = {Genome-Wide CRISPR Screen for Unveiling Radiosensitive and Radioresistant Genes.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {219}, pages = {}, doi = {10.3791/67982}, pmid = {40489409}, issn = {1940-087X}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Radiation Tolerance/genetics ; Lung Neoplasms/genetics/radiotherapy ; Gene Editing/methods ; Cell Line, Tumor ; }, abstract = {The CRISPR-Cas9 system has been harnessed and repurposed into a powerful genome editing tool. By leveraging this technology, researchers can precisely cut, paste, and even rewrite DNA sequences within living cells. Nevertheless, the application of CRISPR screen technology goes far beyond mere experimentation. It serves as a pivotal tool in the fight against genetic diseases, systematically dissecting complex genetic landscapes, empowering researchers to unravel the molecular mechanisms underlying biological phenomena, and enabling scientists to identify and target the root causes of illnesses such as cancer, cystic fibrosis, and sickle cell anemia. Among all, cancer poses a formidable challenge for medicine, spurring eradication efforts. Radiotherapy, as a traditional treatment, yields results but has limitations. It eradicates cancer cells but also damages healthy tissues, causing adverse effects that reduce quality of life. Additionally, not all cancer cells respond to radiotherapy, and some may develop resistance, worsening the condition. To address this, a comprehensive whole-genome CRISPR screen technology is introduced, as it enables the efficient identification of radiosensitive and radioresistant genes, thereby advancing the field of cancer research and treatment. A genome-wide CRISPR screen was conducted in lung adenocarcinoma cells exposed to irradiation following the described protocol, through which both radioresistance- and radiosensitivity-associated genes were identified.}, }
@article {pmid40487915, year = {2025}, author = {Wu, X and Liu, Y and He, Z and Zhou, X and Liesack, W and Peng, J}, title = {Coevolution and cross-infection patterns between viruses and their host methanogens in paddy soils.}, journal = {ISME communications}, volume = {5}, number = {1}, pages = {ycaf088}, pmid = {40487915}, issn = {2730-6151}, abstract = {Methanogens play a critical role in global methane (CH4) emissions from rice paddy ecosystems. Through the integration of metagenomic analysis and meta-analysis, we constructed a CRISPR spacer database comprising 14 475 spacers derived from 351 methanogenic genomes. This enabled the identification of viruses targeting key methanogenic families prevalent in rice paddies, including Methanosarcinaceae, Methanotrichaceae, Methanobacteriaceae, Methanocellaceae, and Methanomassiliicoccaceae. We identified 419 virus-host linkages involving 56 methanogenic host species and 189 viruses, spanning the families Straboviridae, Salasmaviridae, Kyanoviridae, Herelleviridae, and Demerecviridae, along with 126 unclassified viral entities. These findings highlight a virome composition that is markedly distinct from those observed in gut environments. Cross-infection patterns were supported by the presence of specific viruses predicted to infect multiple closely related methanogenic species. Evidence for potential virus-host coevolution was observed in 24 viruses encoding anti-CRISPR proteins, likely facilitating evasion of host CRISPR-mediated immunity. Collectively, this study reveals a complex and dynamic network of virus-host interactions shaping methanogen communities in rice paddy ecosystems.}, }
@article {pmid40483517, year = {2025}, author = {Takahashi, G and Maeda, M and Shinozaki, K and Harada, G and Ito, S and Miyaoka, Y}, title = {High-throughput robotic isolation of human iPS cell clones reveals frequent homozygous induction of identical genetic manipulations by CRISPR-Cas9.}, journal = {Stem cell research &amp; therapy}, volume = {16}, number = {1}, pages = {295}, pmid = {40483517}, issn = {1757-6512}, support = {20K21409//Japan Society for the Promotion of Science/ ; 20H03442//Japan Society for the Promotion of Science/ ; 24K02028//Japan Society for the Promotion of Science/ ; 18K15054//Japan Society for the Promotion of Science/ ; 22K15386//Japan Society for the Promotion of Science/ ; 23jm0610092h0001//Japan Agency for Medical Research and Development (AMED)/ ; Takeda Science Foundation//Takeda Science Foundation/ ; Sumitomo Foundation//Sumitomo Foundation/ ; Ichiro Kanehara Foundation//Ichiro Kanehara Foundation/ ; }, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Homozygote ; Clone Cells/cytology ; *Robotics ; }, abstract = {BACKGROUND: Genome editing in human iPS cells is a powerful approach in regenerative medicine. CRISPR-Cas9 is the most common genome editing tool, but it often induces byproduct insertions and deletions in addition to the desired edits. Therefore, genome editing of iPS cells produces diverse genotypes. Existing assays mostly analyze genome editing results in cell populations, but not in single cells. However, systematic profiling of genome editing outcomes in single iPS cells was lacking. Due to the high mortality of human iPS cells as isolated single cells, it has been difficult to analyze genome-edited iPS cell clones in a high-throughput manner.<br><br>METHODS: In this study, we developed a method for high-throughput iPS cell clone isolation based on the precise robotic picking of cell clumps derived from single cells grown in extracellular matrices. We first introduced point mutations into human iPS cell pools by CRISPR-Cas9. These genome-edited human iPS cells were dissociated and cultured as single cells in extracellular matrices to form cell clumps, which were then isolated using a cell-handling robot to establish genome-edited human iPS cell clones. Genome editing outcomes in these clones were analyzed by amplicon sequencing to determine the genotypes of individual iPS cell clones. We identified and distinguished the sequences of different insertions and deletions induced by CRISPR-Cas9 while determining their genotypes. We also cryopreserved the established iPS cell clones and recovered them after determining their genotypes.<br><br>RESULTS: We analyzed over 1,000 genome-edited iPS cell clones and found that homozygous editing was much more frequent than heterozygous editing. We also observed frequent homozygous induction of identical genetic manipulations, including insertions and deletions, such as 1-bp insertions and 8-bp deletions. Moreover, we successfully cryopreserved and then recovered genome-edited iPS cell clones, demonstrating that our cell-handling robot-based method is valuable in establishing genome-edited iPS cell clones.<br><br>CONCLUSIONS: This study revealed a previously unknown property of genome editing in human iPS cells that identical sequence manipulations tend to be induced in both copies of the target sequence in individual cells. Our new cloning method and findings will facilitate the application of genome editing to human iPS cells.}, }
@article {pmid40481308, year = {2025}, author = {Du, W and Zhao, L and Diao, K and Zheng, Y and Yang, Q and Zhu, Z and Zhu, X and Tang, D}, title = {A versatile CRISPR/Cas9 system off-target prediction tool using language model.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {882}, pmid = {40481308}, issn = {2399-3642}, support = {82070199//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Deep Learning ; Software ; Computational Biology/methods ; }, abstract = {Genome editing with the CRISPR/Cas9 system has revolutionized life and medical sciences, particularly in treating monogenic genetic diseases by enabling long-term therapeutic effects from a single intervention. However, the CRISPR/Cas9 system can tolerate mismatches and DNA/RNA bulges at target sites, leading to unintended off-target effects that pose challenges for gene-editing therapy development. Existing high-throughput detection and in silico prediction methods are often limited to specifically designed single guide RNAs (sgRNAs) and perform poorly on unseen sequences. To address these limitations, we introduce CCLMoff, a deep learning framework for off-target prediction that incorporates a pretrained RNA language model from RNAcentral. CCLMoff captures mutual sequence information between sgRNAs and target sites and is trained on a comprehensive, updated dataset. This approach enables accurate off-target identification and strong generalization across diverse NGS-based detection datasets. Model interpretation reveals the biological importance of the seed region, underscoring CCLMoff's analytical capabilities. The development of CCLMoff lays the foundation for a comprehensive, end-to-end sgRNA design platform, enhancing both the precision and efficiency of CRISPR/Cas9-based therapeutics. CCLMoff is a versatile tool and is publicly available at github.com/duwa2/CCLMoff .}, }
@article {pmid40480713, year = {2025}, author = {Zhou, C and Zhang, Y and Yang, X and Zhao, Z and Xia, M and Wei, C and Wu, Q and Chang, Z and Ma, L and Yin, L}, title = {A magnetic CRISPR/Cas12a-SERS nanobiosensor for amplification-free and ultrasensitive detection of norovirus in water and food samples.}, journal = {Analytica chimica acta}, volume = {1363}, number = {}, pages = {344133}, doi = {10.1016/j.aca.2025.344133}, pmid = {40480713}, issn = {1873-4324}, mesh = {*Norovirus/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Spectrum Analysis, Raman/methods ; *Food Contamination/analysis ; Limit of Detection ; DNA, Single-Stranded/chemistry ; *Water Microbiology ; *Food Microbiology ; Magnetic Phenomena ; *CRISPR-Associated Proteins/metabolism ; }, abstract = {BACKGROUND: Norovirus (NoV) is the leading cause of foodborne disease outbreaks worldwide, typically spreading via contaminated food and water. Rapid, sensitive, and portable detection of NoV is crucial.<br><br>RESULTS: Here, we presented a magnetic CRISPR/Cas12a-SERS nanobiosensor capable of detecting NoV with high sensitivity, accuracy, speed, and portability. In this nanobiosensor, SERS nanoprobes linked to magnetic nanoprobes via linker single-stranded DNAs (ssDNAs). The presence of NoV nucleic acid triggered Cas12a's trans-cleavage activity, degrading the linker ssDNA. After magnetic separation, the dissociated SERS nanoprobes were efficiently separated from the magnetic nanoprobes. This enhanced the SERS signal in the supernatant, detectable using a portable Raman spectrometer. The detection limit for NoV is 100 copies/mL within 60&#xa0;min. The nanobiosensor was further assessed in real-world settings, demonstrating excellent sensitivity and selectivity for detecting trace NoV in complex food samples.<br><br>SIGNIFICANCE: This approach not only broadens CRISPR-based pathogen detection but also provides a reliable tool for monitoring foodborne viruses. Its potential extends beyond NoV, promising enhanced surveillance of various pathogens in food safety, environmental monitoring, and public health sectors.}, }
@article {pmid40480203, year = {2025}, author = {McNamara, HM and Sozen, B}, title = {From genes to geometry: Controlling embryo models by programming genomic activation.}, journal = {Cell stem cell}, volume = {32}, number = {6}, pages = {857-858}, doi = {10.1016/j.stem.2025.04.013}, pmid = {40480203}, issn = {1875-9777}, mesh = {Animals ; Mice ; *Embryo, Mammalian/metabolism/cytology ; Mouse Embryonic Stem Cells/metabolism/cytology ; CRISPR-Cas Systems/genetics ; Embryonic Stem Cells/metabolism/cytology ; *Genome ; *Models, Biological ; }, abstract = {Embryo-like models derived from stem cells have emerged as powerful tools to study early development. In this issue, Lodewijk et al.[1] demonstrate that activating just two enhancers via CRISPR activation (CRISPRa) in mouse embryonic stem cells (ESCs) can drive self-organization into structured embryo-like models, offering a genome-driven approach in stem cell and developmental biology.}, }
@article {pmid40478881, year = {2025}, author = {Ratajczyk, EJ and Bath, J and Šulc, P and Doye, JPK and Louis, AA and Turberfield, AJ}, title = {Controlling DNA-RNA strand displacement kinetics with base distribution.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {122}, number = {23}, pages = {e2416988122}, pmid = {40478881}, issn = {1091-6490}, support = {EP/W524311/1//UKRI | Engineering and Physical Sciences Research Council (EPSRC)/ ; CCF 2211794//National Science Foundation (NSF)/ ; }, mesh = {Kinetics ; *DNA/chemistry/genetics/metabolism ; *RNA/chemistry/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Thermodynamics ; Gene Editing ; }, abstract = {DNA-RNA hybrid strand displacement underpins the function of many natural and engineered systems. Understanding and controlling factors affecting DNA-RNA strand displacement reactions is necessary to enable control of processes such as CRISPR-Cas9 gene editing. By combining multiscale modeling with strand displacement experiments, we show that the distribution of bases within the displacement domain has a very strong effect on reaction kinetics, a feature unique to DNA-RNA hybrid strand displacement. Merely by redistributing bases within a displacement domain of fixed base composition, we are able to design sequences whose reaction rates span more than four orders of magnitude. We extensively characterize this effect in reactions involving the invasion of dsDNA by an RNA strand, as well as the invasion of a hybrid duplex by a DNA strand. In all-DNA strand displacement reactions, we find a predictable but relatively weak sequence dependence, confirming that DNA-RNA strand displacement permits far more thermodynamic and kinetic control than its all-DNA counterpart. We show that oxNA, a recently introduced coarse-grained model of DNA-RNA hybrids, can reproduce trends in experimentally observed reaction rates. We also develop a simple kinetic model for predicting strand displacement rates. On the basis of these results, we argue that base distribution effects may play an important role in natural R-loop formation and in the function of the guide RNAs that direct CRISPR-Cas systems.}, }
@article {pmid40478482, year = {2025}, author = {Syahrani, RA and Wanandi, SI and Nihayah, S and Arumsari, S and Watanabe, Y and Mizuno, S and Louisa, M and Wuyung, PE}, title = {Survivin knockout attenuates the progressiveness of BT549 triple negative-breast cancer cells: an in vitro study highlighting stemness and cellular stress response mechanisms.}, journal = {Molecular biology reports}, volume = {52}, number = {1}, pages = {560}, pmid = {40478482}, issn = {1573-4978}, mesh = {Humans ; *Survivin/genetics/metabolism ; *Triple Negative Breast Neoplasms/genetics/metabolism/pathology ; Cell Line, Tumor ; Cell Proliferation/genetics ; *Neoplastic Stem Cells/metabolism/pathology ; Apoptosis/genetics ; Female ; Gene Knockout Techniques ; Gene Expression Regulation, Neoplastic/genetics ; CRISPR-Cas Systems ; Stress, Physiological/genetics ; Cell Cycle/genetics ; }, abstract = {BACKGROUND: Survivin, an inhibitor of apoptosis proteins (IAPs), is more strongly expressed in triple negative-breast cancer (TNBC) than other subtypes of breast cancer and closely associated with aggressiveness characterized by rapid progression and poor prognosis. The main function of survivin is to regulate cell division and prevent apoptosis. However, other survivin mechanisms are complex and not fully understood. The aim of this study was to evaluate the effects of survivin knockout on TNBC progressiveness relating to proliferation, apoptosis, stemness, cellular stress response, and metastasis mechanisms.<br><br>METHODS AND RESULTS: The CRISPR/Cas9 (clustered regularly interspaced short palindrom repeat-associated Cas9) system was utilized to establish survivin knockout in the BT549 TNBC cell line. Both knockout and wild-type cells were used to study the role of survivin in various biological mechanisms. Apoptosis-, pluripotency-, and cellular stress-related proteins were examined via proteomic arrays. The cell cycle, apoptosis, and expression of breast cancer stem cell markers were analyzed via flow cytometry. Metastasis-related markers were evaluated via qRT&#x2012;PCR. Here, we report that survivin knockout inhibits proliferation and induces apoptosis in TNBC cells. Moreover, survivin knockout suppressed the expression of pluripotent markers and promoted a shift toward activation response to cellular stress, such as genotoxic, hypoxic, and oxidative stress. Additionally, survivin knockout suppressed metastasis.<br><br>CONCLUSION: The loss of survivin leads to attenuation of TNBC progression by altering various mechanisms, particularly by suppressing stemness and altering the cellular stress response. We propose that knocking out survivin could be a potential strategy for breast cancer therapy, especially TNBC.}, }
@article {pmid40474742, year = {2025}, author = {Hung, YH and Wang, E and Agtarap, T and Klaas, G and Slotkin, RK}, title = {Engineering an RNA/protein-binding module for higher transgene protein production and improved long-term durability.}, journal = {The Plant journal : for cell and molecular biology}, volume = {122}, number = {5}, pages = {e70254}, doi = {10.1111/tpj.70254}, pmid = {40474742}, issn = {1365-313X}, support = {DBI-2050394//National Science Foundation/ ; MCB-1904326//National Science Foundation/ ; MCB-2230587//National Science Foundation/ ; }, mesh = {*Arabidopsis/genetics/metabolism ; *Transgenes/genetics ; Plants, Genetically Modified/genetics/metabolism ; *RNA-Binding Proteins/metabolism/genetics ; *Arabidopsis Proteins/genetics/metabolism ; Gene Editing ; RNA Interference ; Gene Expression Regulation, Plant ; RNA, Plant/genetics/metabolism ; CRISPR-Cas Systems ; DNA Methylation ; RNA, Messenger/metabolism/genetics ; }, abstract = {Improvement and research of plants depends on the long-term expression of transgenes. However, the durability of transgene expression is routinely hampered by silencing pathways that start as the post-transcriptional process of mRNA degradation by RNA interference (RNAi). To avoid transgene silencing, we aimed to inhibit the sorting of transgene mRNAs into RNAi. We manipulated a well-studied protein/RNA-binding module from Arabidopsis into a transgene transcript, where the transcript is now bound by an engineered RNA-binding protein that preferentially sorts the RNA into translation. We used the Cas9 transcript as a proof-of-principle and demonstrated higher Cas9 protein production and gene editing rates. In addition, transgenes with the engineered protein/RNA-binding module had improved long-term durability of transgene expression, as after several inbred generations these plants had higher Cas9 protein accumulation and lower levels of DNA methylation, a hallmark of transgene silencing. Our engineered system represents a successful manipulation of post-transcriptional RNA sorting for improved transgene performance, and could be applied to any transgene transcript.}, }
@article {pmid40473912, year = {2025}, author = {Omura, SN and Alfonse, LE and Ornstein, A and Morinaga, H and Hirano, H and Itoh, Y and Munoz, G and Garrity, AJ and Hoffman, GR and DiTommaso, T and Yan, WX and Cheng, DR and Scott, DA and Maben, Z and Nureki, O}, title = {Structural basis for target DNA cleavage and guide RNA processing by CRISPR-Casλ2.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {876}, pmid = {40473912}, issn = {2399-3642}, support = {JP23fa627001//Japan Agency for Medical Research and Development (AMED)/ ; }, mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Cryoelectron Microscopy ; *DNA Cleavage ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; Bacteriophages/genetics/enzymology ; Gene Editing ; }, abstract = {RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Among the diverse CRISPR-Cas effectors, CRISPR-Casλ-also referred to as Cas12n-is a recently identified miniature type V nuclease encoded in phage genomes. Given its demonstrated nuclease activity in both mammalian and plant cells, Casλ has emerged as a promising candidate for genome-editing applications. However, the precise molecular mechanisms of Casλ family enzymes remain poorly understood. In this study, we report the identification and detailed biochemical and structural characterizations of CRISPR-Casλ2. The cryo-electron microscopy structures of Casλ2 in five different functional states unveiled the dynamic domain rearrangements during its activation. Our biochemical analyses indicated that Casλ2 processes its precursor crRNA to a mature crRNA using the RuvC active site through a unique ruler mechanism, in which Casλ2 defines the spacer length of the mature crRNA. Furthermore, structural comparisons of Casλ2 with Casλ1 and CasΦ highlighted the diversity and conservation of phage-encoded type V CRISPR-Cas enzymes. Collectively, our findings augment the mechanistic understanding of diverse CRISPR-Cas nucleases and establish a framework for rational engineering of the CRISPR-Casλ-based genome-editing platform.}, }
@article {pmid40473600, year = {2025}, author = {Zhou, J and Hepperla, A and Simon, JM and Kim, K and Hu, Q and Zhang, C and Dong, L and Hu, L and Zhang, C and Liao, C and Fang, A and Adachi, Y and Fu, H and Wang, T and Liang, Q and Zhao, F and Liu, H and Takeda, M and Fang, J and Zhong, H and Ly, P and Wang, L and Kapur, P and Xu, L and Jia, L and Malladi, S and Brugarolas, J and Simon, MC and Li, B and Zhang, Q}, title = {SWI/SNF ATPase silenced HLF potentiates lung metastasis in solid cancers.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {5226}, pmid = {40473600}, issn = {2041-1723}, support = {R35 CA220483/CA/NCI NIH HHS/United States ; R01 CA294133/CA/NCI NIH HHS/United States ; RR190058//Cancer Prevention and Research Institute of Texas (Cancer Prevention Research Institute of Texas)/ ; P30 CA142543/CA/NCI NIH HHS/United States ; R01CA211732//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R35 GM146979/GM/NIGMS NIH HHS/United States ; R01 CA211732/CA/NCI NIH HHS/United States ; P50 CA196516/CA/NCI NIH HHS/United States ; P50CA196516//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; }, mesh = {Humans ; *Lung Neoplasms/secondary/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; Animals ; *DNA Helicases/metabolism/genetics ; Cell Line, Tumor ; *Nuclear Proteins/metabolism/genetics ; Mice ; Cell Movement/genetics ; Gene Expression Regulation, Neoplastic ; *Carcinoma, Renal Cell/genetics/pathology/metabolism ; *Kidney Neoplasms/pathology/genetics/metabolism ; Female ; Gene Silencing ; CRISPR-Cas Systems ; }, abstract = {Metastasis is the main cause of cancer-related deaths, yet the underlying mechanisms remain elusive. Here, using clear cell renal cell carcinoma (ccRCC), a tumor type with frequent lung metastases, we conduct an in vivo genome-wide CRISPR-Cas9 screen and identify HLF as a potent suppressor of lung metastasis. HLF depletion enhances ccRCC cell migration and lung metastasis, whereas HLF overexpression abrogates these effects. In ccRCC patients, HLF expression is reduced at metastatic sites and associates with epigenetic silencing mediated by the SWI/SNF ATPase subunit BRG1. HLF levels negatively correlate with migration potential in collagen. Mechanistically, HLF regulates LPXN expression, modulating the integration of collagen's mechanical cues with the actin cytoskeleton through Paxillin, thereby suppressing cancer cell migration and lung metastasis. Overexpression of HLF or pharmacological inhibition of BRG1 reduces cell invasion across multiple cancer types. Our findings suggest that targeting the BRG1-HLF axis offers a promising therapeutic strategy for combating metastatic cancers.}, }
@article {pmid40472130, year = {2025}, author = {Ghdayer Al Kaabi, NA and Kandhan, K and Hayat, F and Matar Al Blooshi, SA and Sheteiwy, MS and Alyafei, M}, title = {Shaping the future of date palm (Phoenix dactylifera) through new genetic improvement strategies.}, journal = {Functional plant biology : FPB}, volume = {52}, number = {}, pages = {}, doi = {10.1071/FP25021}, pmid = {40472130}, issn = {1445-4416}, mesh = {*Phoeniceae/genetics/metabolism ; *Plant Breeding/methods ; Gene Editing ; CRISPR-Cas Systems ; Genome-Wide Association Study ; Polymorphism, Single Nucleotide ; }, abstract = {Conventional breeding of date palm (Phoenix dactylifera) is inherently challenging due to its long generation time, dioecious nature, and high genetic heterogeneity. However, current developments in genomics and molecular biology offer promising avenues for accelerating breeding programs, particularly through high-throughput technologies including functional genomics. This article reviews genomic tools such as like CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein 9) that may bring significant changes in date palm breeding. The CRISPR-Cas9 system enables scientists to accurately target genomic regions, which helps enhance breeding accuracy by adding advantageous traits and eliminating unfavorable genes through precision editing. Transcriptome and metabolome analyses have also explained the regulation of thousands of differentially expressed genes (DEGs) and metabolic pathways under environmental stress. These studies contribute to enhance the knowledge of stress tolerance mechanisms, which include the secondary metabolic process of flavonoids. Genomic studies illustrating single nucleotide polymorphism (SNP)-based diversity between cultivars from north African and the Arabian Gulf provide new genetic resources for selective breeding. The work relates genome-wide association studies (GWAS) and miRNA profiling to elucidate key regulatory networks involved in fruit development and stress resilience. The integration of such advanced technologies, especially the CRISPR-Cas9 system, is revolutionizing the landscape of date palm breeding, opening new avenues for accelerated development of superior cultivars that meet the needs of modern agriculture.}, }
@article {pmid40472129, year = {2025}, author = {Sarfraz, Z and Zarlashat, Y and Ambreen, A and Mujahid, M and Iqbal, MS}, title = {Advanced gene editing techniques for enhancing disease resistance and climate resilience in crops.}, journal = {Functional plant biology : FPB}, volume = {52}, number = {}, pages = {}, doi = {10.1071/FP24357}, pmid = {40472129}, issn = {1445-4416}, mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics ; *Disease Resistance/genetics ; Climate Change ; CRISPR-Cas Systems ; Plants, Genetically Modified ; }, abstract = {Ensuring food security and solving the issues brought on by climate change require breeding and engineering of climate-resilient crops. Despite its contributions to reducing agricultural diseases, genetic engineering has several limitations, including high labor costs, lengthy processing times, and poor productivity. Genome editing has become a potential method to provide notable opportunities to explain complex biological processes, genetically solve the causes of diseases, and improve crops for disease resistance by effectively modifying multiple traits. Genome editing techniques including TALENs, ZFNs, and CRISPR/Cas9 increase agricultural productivity by developing climate-resistant crops and promoting climate-resilient agriculture. Among these approaches, CRISPR/Cas9 shows exceptional efficacy, minimal chance of off-target effects, and improved traits such as drought tolerance and disease resistance. This study explores advanced gene editing techniques for improving disease resistance in crops and developing climate-resilient varieties to reduce food insecurity and hunger. It demonstrates that these techniques have enhanced the nutritional content and resilience of many crops by fighting abiotic and biotic stresses. Future agricultural practices could alter the genes and improve disease-resistant crops by genome editing techniques.}, }
@article {pmid40471011, year = {2025}, author = {Li, H and Shen, J and Zhao, X and Ji, J and Wang, Y and Yang, L and Zhuang, M and Liu, L and Zhang, Y and Lv, H}, title = {Rapid design of transgene-free cabbage with desired anthocyanin contents via HI-Edit.}, journal = {Journal of integrative plant biology}, volume = {}, number = {}, pages = {}, doi = {10.1111/jipb.13943}, pmid = {40471011}, issn = {1744-7909}, support = {CAAS-ASTIP-IVFCAAS//the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences/ ; CARS-23//the China Agriculture Research System of MOF and MARA/ ; 2023YFD1201501//The National Key R&amp;D Program of China/ ; BE2023366//The Key Technology R&amp;D Program of Jiangsu Province/ ; }, abstract = {The HI-Edit system combines haploid induction and CRISPR/Cas-based genome editing to provide a promising way to design crops with desired traits in a rapid, precise and transgene-free manner. HI-Edit was applied to produce cabbages with desired anthocyanin contents.}, }
@article {pmid40470110, year = {2025}, author = {Bao, H and Chen, Y and Zong, Y and Jin, K and Lan, H}, title = {Recent Advances in the Delivery of Bone Morphogenetic Proteins for Targeting Glioma: An Updated Review.}, journal = {International journal of nanomedicine}, volume = {20}, number = {}, pages = {7093-7112}, pmid = {40470110}, issn = {1178-2013}, mesh = {Humans ; *Glioma/drug therapy/therapy ; Animals ; *Brain Neoplasms/drug therapy ; *Bone Morphogenetic Proteins/administration &amp; dosage/therapeutic use/pharmacokinetics ; Genetic Therapy/methods ; *Drug Delivery Systems/methods ; Nanoparticles/chemistry ; Blood-Brain Barrier/metabolism ; Hydrogels/chemistry ; }, abstract = {Bone Morphogenetic Proteins might be the most prospective in glioma treatment because of the facts that they can differentiate glioma cells, inhibit tumor growth and manage glioma stem cells. Its clinical application is hindered by several challenges, including limited permeability across the blood-brain barrier, which impedes effective delivery to the central nervous system; high susceptibility to enzymatic degradation, which compromises stability and therapeutic efficacy; and nonselective binding, which reduces specificity and may result in unintended off-target effects. This review systematically covers the advanced BMP delivery systems such as nanoparticles, smart carriers, gene therapy, and exosome-based system. Hydrogels, scaffolds, and microspheres' local delivery methods are also discussed as prospective options. The in vitro studies reveal that BMPs are effective and using in vivo glioma models there is also evidence of the effectiveness of BMPs. In addition, new clinical trials reveal concern with safety, tolerability, and therapeutic effects of BMPs, especially their combination with chemotherapy and immunotherapy. BMP specificity and therapeutic performance are further optimized by Personalized medicine and CRISPR/Cas engineering. However, regulatory barriers and product commercialization are challenging issues. This review highlights the need for novel approaches and advanced technologies to address the challenges associated with BMP delivery, aiming to establish BMP-based therapies as an effective treatment strategy for glioma.}, }
@article {pmid40469526, year = {2025}, author = {Isaacson, J and Bhanap, P and Putnam, N and Padilla, J and Fatima, N and Dotson, M and Hayoun, D and Ahmadi, M and Nonterah, G and Ji, Y}, title = {Enhancing CAR T-cell therapy manufacturing efficiency through semi-automated bioprocessing.}, journal = {Clinical &amp; translational immunology}, volume = {14}, number = {6}, pages = {e70025}, pmid = {40469526}, issn = {2050-0068}, abstract = {OBJECTIVES: Chimeric antigen receptor (CAR) T-cell therapies have revolutionised the treatment of blood-based malignancies. The use of manual CAR T-cell manufacturing methods is one of the challenges that contributes to these delays. As CAR T therapy emerges as a potential first- or second-line treatment option for these cancers, the demand for these therapies continues to rise. However, challenges persist in ensuring that the patients who need these therapies receive them in a timely manner. Automated CAR T-cell manufacturing methods that use software to control the equipment used in the process can help overcome the roadblocks associated with manual manufacturing, ultimately enabling a reduction in variability, increased efficiency, improved product quality and better data management. This paper aims to present an end-to-end semi-automated methodology for manufacturing CAR T cells using the Cell Therapy Systems (CTS™) Cellmation software - an off-the-shelf software solution - to control physically connected modular cell therapy instruments that eliminates the roadblocks associated with manual manufacturing.<br><br>METHODS: T&#xa0;cells from healthy donors were isolated and processed into CAR T&#xa0;cells using a semi-automated, connected, multi-instrument setup that leveraged electroporation and a CRISPR/Cas system for delivering the CD19-CAR construct to the T&#xa0;cells. Flow cytometry was used to assess cell type composition, cell viability and expression of T-cell activation markers throughout the process. We also measured exhaustion marker expression on T&#xa0;cells, T-cell receptor (TCR) knock-out, CAR knock-in and cytotoxic activity against NALM6 cells.<br><br>RESULTS: The results demonstrated the successful generation of functional CAR T&#xa0;cells using a semi-automated instrument workflow. The results were similar to the results from CAR T&#xa0;cells manufactured using non-automated processes; however, the successful connection and control of the instruments using automated software present an exciting opportunity for process developers and manufacturers who want to reduce manual touchpoints in their cell therapy manufacturing process.<br><br>CONCLUSION: The method that we describe in this paper could be beneficial to process development and manufacturing teams that might require flexibility in their CAR T&#xa0;cell manufacturing workflow and want to take advantage of modular systems that can be automated using the Cellmation software to reduce the problems associated with manual handling.}, }
@article {pmid40469284, year = {2025}, author = {Lin, Z and Song, Z and Yu, H and Zhou, Y and Liu, D and Zhang, P and Wei, L and Dai, G and Liang, G and He, Z and Hu, X and Chen, Y and Zhao, P and Lu, H and Zheng, M}, title = {Ultra-sensitive in situ detection of intracellular Mycobacterium tuberculosis with CRISPR/Cas12a.}, journal = {Frontiers in immunology}, volume = {16}, number = {}, pages = {1597654}, pmid = {40469284}, issn = {1664-3224}, mesh = {*Mycobacterium tuberculosis/genetics/isolation &amp; purification ; Humans ; Animals ; *CRISPR-Cas Systems ; Mice ; *Macrophages/microbiology ; RAW 264.7 Cells ; *Tuberculosis/diagnosis/microbiology ; Bronchoalveolar Lavage Fluid/microbiology ; Sensitivity and Specificity ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Mycobacterium tuberculosis (Mtb) invades and survives inside macrophages, evading detection and resisting antibiotic treatment, which results in severe clinical consequences such as fatal respiratory failure and systemic inflammation. Rapid and specific detection of intracellular Mtb is crucial for accurate diagnosis and optimizing treatment strategies. In this study, we developed a one-step CRISPR/Cas12a assay targeting the IS6110 gene for the specific and rapid detection of intracellular Mtb. Upon efficient delivery into RAW264.7 macrophages, the assay enabled direct visualization of Mtb IS6110 nucleic acid, generating detectable fluorescence signals. The diagnostic performance was further validated using bronchoalveolar lavage fluid (BALF) samples from clinical participants, achieving a sensitivity of 94%, which surpassed conventional methods such as culture (67%) and Xpert (78%), while maintaining a specificity of 100%. This CRISPR/Cas12a-based assay offers a highly sensitive, rapid, and innovative approach for intracellular Mtb detection, with significant potential to enhance tuberculosis diagnostic methodologies and improve clinical outcomes.}, }
@article {pmid40469012, year = {2025}, author = {Zhu, F and Zhao, Q}, title = {Spherical Nucleic Acids as Modulators of CRISPR/Cas12a by a Steric Barrier Effect for Designing Versatile Biosensors.}, journal = {Analytical chemistry}, volume = {97}, number = {23}, pages = {12215-12222}, doi = {10.1021/acs.analchem.5c00937}, pmid = {40469012}, issn = {1520-6882}, mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Gold/chemistry ; Metal Nanoparticles/chemistry ; *CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism ; *Bacterial Proteins/metabolism ; Humans ; *Nucleic Acids/chemistry ; }, abstract = {Discovery of CRISPR/Cas12a has revolutionized broad fields, including gene editing, molecular diagnosis, and biosensing. Flexible regulation of Cas12a activity is important for diverse CRISPR/Cas12a applications, especially for biosensing, but it still faces limitations and challenges. We find gold nanoparticles (AuNPs) modified with a single-stranded DNA activator create a huge steric barrier that strongly locks activators in a one-to-many manner and inhibits Cas12a activity. This finding offers a new way to modulate the activity of Cas12a for applications, such as designing versatile biosensors. We report a spherical nucleic acid (SNA)-modulating CRISPR/Cas12a (SNA-Cas) platform using SNAs as signal translators for target sensing. A stimuli-responsive SNA was constructed by modifying AuNPs with a DNA activator containing a specific trigger element, and the target triggers specific reactions (e.g., thiol-exchange chemical reaction and RNA-cleaving by DNAzymes) to release activators into solution. A free activator initiates trans-cleavage activity of CRISPR/Cas12a, scissoring fluorescent DNA reporters to produce amplified signals. To show proof of concept, we demonstrate SNA-Cas to sensitively detect diverse non-nucleic acid targets, including biological thiol cysteine, heavy metal Pb[2+], and biomarkers of O[6]-methylguanine-DNA-methyltransferases (MGMT) and fat mass and obesity-related protein (FTO) demethylases. This work opens one door for SNA modulating CRISPR/Cas activity and shows great potential in designing versatile biosensors for detecting diverse targets.}, }
@article {pmid40468657, year = {2025}, author = {Jun, JS and Kang, SJ and Hong, KW}, title = {Time-resolved analysis of Bacillus subtilis DB104 Spo0A-mutant transcriptome profile and enhancement of recombinant protein release.}, journal = {Journal of microbiology (Seoul, Korea)}, volume = {63}, number = {5}, pages = {e2411032}, doi = {10.71150/jm.2411032}, pmid = {40468657}, issn = {1976-3794}, support = {2021R1I1A2046284//National Research Foundation of Korea/ ; //Ministry of Education/ ; }, mesh = {*Bacillus subtilis/genetics/metabolism/growth &amp; development ; *Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; *Transcriptome ; Spores, Bacterial/genetics/growth &amp; development ; *Recombinant Proteins/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; Gene Expression Profiling ; Biofilms/growth &amp; development ; Mutation ; CRISPR-Cas Systems ; Quorum Sensing/genetics ; Gene Editing ; }, abstract = {Spo0A, the master regulator of sporulation initiation in Bacillus subtilis, controls over 500 genes directly or indirectly in early sporulation stages. Although the effects of Spo0A disruption on sporulation have been extensively studied, a comprehensive understanding of the genomic response throughout growth phases remain elusive. Here, we examined the transcriptomic changes in Spo0A mutant strain, R211E, and wild-type across a time-course RNA-seq to identify impacted biological processes and pathways. The R211E strain, which exhibits sporulation deficiency, was constructed using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas)9 system, highlighting the critical role of proper Cas9 dosing in gene editing. Functional analysis of 3,010 differentially expressed genes (DEGs) showed significant alterations in sporulation, quorum sensing, metabolism, and biofilm formation. The R211E disrupted the Spo0A-AbrB regulatory pathway, reducing biofilm formation and enhancing flagellar gene expression. Up-regulated metabolic pathways, including glycolysis, histidine, and purine biosynthesis, increased cell numbers during vegetative growth. Further, the mutant displayed elevated vegetative autolysin expression, resulting in reduced cell viability in the stationary phase. We also introduce the novel potential of R211E in a recombinant protein expression system that facilitated protein release into the supernatant, providing valuable insight for future research in metabolic engineering and efficient production systems in B. subtilis.}, }
@article {pmid40468633, year = {2025}, author = {Cao, M and Brennan, A and Lee, CM and Park, SH and Bao, G}, title = {Deep Learning Based Models for CRISPR/Cas Off-Target Prediction.}, journal = {Small methods}, volume = {}, number = {}, pages = {e2500122}, doi = {10.1002/smtd.202500122}, pmid = {40468633}, issn = {2366-9608}, support = {RP210116//Cancer Prevention and Research Institute of Texas/ ; R01HG011459/HG/NHGRI NIH HHS/United States ; R01HL169761/HL/NHLBI NIH HHS/United States ; }, abstract = {CRISPR/Cas genome editing technologies enable effective and controlled genetic modifications; however, off-target effects remain a significant concern, particularly in clinical applications. Experimental and in silico methods are developed to predict potential off-target sites (OTS), including deep learning based methods, which can automatically and comprehensively learn sequence features, offer a promising tool for OTS prediction. Here, this work reviews the existing OTS prediction tools with an emphasis on deep learning methods, characterizes datasets used for deep learning training and testing, and evaluates six deep learning models -CRISPR-Net, CRISPR-IP, R-CRISPR, CRISPR-M, CrisprDNT, and Crispr-SGRU -using six public datasets and validates OTS data from the CRISPRoffT database. Performance of these models is assessed using standardized metrics, such as Precision, Recall, F1 score, MCC, AUROC and PRAUC. This work finds that incorporating validated OTS datasets into model training enhanced overall model performance, and improved robustness of prediction, particularly with highly imbalanced datasets. While no model consistently outperforms other models across all scenarios, CRISPR-Net, R-CRISPR, and Crispr-SGRU show strong overall performance. This analysis demonstrates the importance of integrating high-quality validated OTS data with advanced deep learning architectures to improve CRISPR/Cas off-target site predictions, ensuring safer genome editing applications.}, }
@article {pmid40466745, year = {2025}, author = {Yang, P and Zeng, J and Li, L and Ma, R and Peng, J and Zhou, W and Fu, W and Wu, Y and Zhang, Y}, title = {From Cas proteins to cutting-edge biosensors: A new era in clinical pathogen diagnostics.}, journal = {The Journal of infection}, volume = {91}, number = {1}, pages = {106526}, doi = {10.1016/j.jinf.2025.106526}, pmid = {40466745}, issn = {1532-2742}, mesh = {*Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; *Communicable Diseases/diagnosis ; }, abstract = {Infectious pathogens exert a profound impact on global health and socio-economic stability, positioning them as a critical focus of scientific inquiry. To safeguard public health, propel advancements in medical diagnostics, and ensure food safety, the development of efficient technologies for rapid, onsite detection of pathogens is imperative. In light of recent research breakthroughs, CRISPR/Cas-based technologies for pathogen biosafety and molecular diagnostics have emerged as particularly promising in the realm of infectious disease detection. This review succinctly introduces the working principles of CRISPR/Cas systems and thoroughly discusses the design and development of various CRISPR/Cas-based biosensors. Importantly, this paper explores the robust applications of CRISPR/Cas-assisted biosensing for emerging infectious diseases, highlighting its potential in pathogen diagnostics with features like cost-effectiveness, multiplex detection and POCT applications. Furthermore, challenges and future developments of CRISPR/Cas-based biosensors for rapid and accurate pathogen detection in specialized settings are also summarized, integrating CRISPR detection with portable POCT biosensors, nanomaterials and novel colorimetric materials. As it builds on a lot of foundational work and offers new insights and detailed reference to advance the development and application of CRISPR technologies in clinical pathogen diagnostics, opening new avenues in medical diagnostics and the prevention and control of infectious diseases.}, }
@article {pmid40466639, year = {2025}, author = {Conti, A and Giannetti, K and Midena, F and Beretta, S and Gualandi, N and De Marco, R and Carsana, E and Varesi, A and Tavella, T and Alessandrini, L and Zarghamian, P and Weber, A and Ferrari, S and Brombin, C and Gilioli, D and Della Volpe, L and Xie, SZ and Merelli, I and Cathomen, T and Naldini, L and Di Micco, R}, title = {Senescence and inflammation are unintended adverse consequences of CRISPR-Cas9/AAV6-mediated gene editing in hematopoietic stem cells.}, journal = {Cell reports. Medicine}, volume = {6}, number = {6}, pages = {102157}, doi = {10.1016/j.xcrm.2025.102157}, pmid = {40466639}, issn = {2666-3791}, mesh = {*Gene Editing/methods ; *Hematopoietic Stem Cells/metabolism ; *CRISPR-Cas Systems/genetics ; *Inflammation/pathology/genetics ; *Cellular Senescence/genetics ; Animals ; Humans ; Tumor Suppressor Protein p53/metabolism ; NF-kappa B/metabolism ; Mice ; Interleukin 1 Receptor Antagonist Protein/pharmacology ; Interleukin-1/metabolism ; }, abstract = {Gene editing (GE) using homology-directed repair (HDR) in hematopoietic stem and progenitor cells (HSPCs) offers promise for long-range gene correction of inherited genetic disorders. However, cellular responses induced by CRISPR-Cas9/AAV6 engineering impair the long-term repopulating potential of HDR-edited HSPCs, adversely impacting the safety and efficacy of clinical translation. Our study uncovers a durable senescence-like response in genetically engineered HSPCs triggered by p53 and interleukin (IL)-1/nuclear factor κB (NF-κB) activation, which restricts graft size and clonal diversity in long-term transplantation assays. We show that transient p53 inhibition or blocking inflammatory pathways mitigates senescence-associated responses, improving the repopulating capacity of edited HSPCs. Importantly, we identify treatment with Anakinra, an IL-1 signaling antagonist, as a promising strategy to enhance polyclonal output in HDR-edited cells while minimizing genotoxicity risks associated with the editing procedure. Overall, our findings present strategies to overcome key hurdles in HDR-based HSPC gene therapies, providing a framework for enhancing their efficacy and safety in clinical applications.}, }
@article {pmid40466420, year = {2025}, author = {Jiang, J and Kan, X}, title = {A smartphone-enabled colorimetric tumor-derived exosomes sensing based on multi-enzyme catalysis and dual-recognition triggered CRISPR/Cas12a trans-cleavage.}, journal = {Biosensors &amp; bioelectronics}, volume = {286}, number = {}, pages = {117644}, doi = {10.1016/j.bios.2025.117644}, pmid = {40466420}, issn = {1873-4235}, mesh = {Humans ; *Exosomes/chemistry ; *Biosensing Techniques/instrumentation/methods ; *Colorimetry/instrumentation/methods ; *Smartphone ; CRISPR-Cas Systems ; A549 Cells ; Aptamers, Nucleotide/chemistry ; Limit of Detection ; *Lung Neoplasms/diagnosis ; Tetraspanin 30/chemistry ; Biomarkers, Tumor ; Epithelial Cell Adhesion Molecule/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Detection of protein profiling on exosomes exhibits great promise for early non-invasive and accurate diagnosis of tumor in clinical diagnostics. However, it still faces multiple challenges, such as expensive instruments requirement and weak specificity by single biomarker. Herein, based on a dual-recognition strategy, a ternary hybrid of a trigger DNA (TDNA), EpCAM aptamer, and CD63 aptamer was used to capture A549 cells-derived exosomes to release TDNA, which initiated the trans-cleavage activity of CRISPR/Cas12a to nonspecifically cleave single-stranded DNA (ssNDA) and then resulted in the isolating of ssDNA linked nanozyme of Zr/Fe-CeO2@Ir@CaO2@HA (ZFCIrCH). ZFCIrCH not only achieved H2O2/O2 self-supply, but also possessed high peroxidase-like, oxidase-like, and superoxide dismutase-like activities, thereby generating a sensitive colorimetric signal for A549 cells-derived exosomes detection with a low limit of detection (LOD) of 31 particles/mL. Using a smartphone to analyze colorimetric images, exosome concentration also can be precisely quantified with a LOD of 29 particles/mL, which could also successfully distinguish healthy people from lung cancer patients. With the advantages of high sensitivity, good specificity, low cost, and convenient on-site detection of tumor-derived exosomes, the present colorimetric sensor has great promise in the accurate diagnosis of diseases.}, }
@article {pmid40465697, year = {2025}, author = {Vo, QD}, title = {Gene editing therapy as a therapeutic approach for cardiovascular diseases in animal models: A scoping review.}, journal = {PloS one}, volume = {20}, number = {6}, pages = {e0325330}, pmid = {40465697}, issn = {1932-6203}, mesh = {Animals ; *Gene Editing/methods ; *Cardiovascular Diseases/therapy/genetics ; Disease Models, Animal ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Mice ; Humans ; Dependovirus/genetics ; }, abstract = {BACKGROUND: Cardiovascular diseases (CVDs) are the leading cause of mortality worldwide, with hereditary genetic factors contributing substantially to disease burden. Current treatments, including lifestyle modifications, pharmacotherapy, and surgical interventions, focus primarily on symptom management but fail to address underlying genetic causes, often resulting in disease progression or recurrence. Gene therapy has emerged as a transformative approach, offering a potential treatment. This review explores its efficacy and safety in animal models, identifying opportunities for future advancements.<br><br>METHODS: This review investigated studies on gene editing interventions in animal models of CVDs, retrieved from PubMed, ScienceDirect, and Web of Science up to December 2024.<br><br>RESULT: A total of 57 studies were included in this review. Mice (86%) were the predominant model, with CRISPR-Cas9 (53%) and AAV vectors (80%) as the most used tools. Key targets included PCSK9 (32%), LDLR (9%), and MYH6/7 (7%), achieving 25-85% editing efficiency in liver/heart tissues. Base editors (ABE/CBE) showed superior safety, with <1% off-targets versus CRISPR-Cas9's 2-5 off-targets per guide. Reported toxicity risks included liver injury (AAVs, 23%) and transient cytokine elevation (LNPs, 14%).<br><br>CONCLUSION: Gene editing therapy shows great potential for treating CVDs, with high efficiency, strong therapeutic outcomes, and favorable safety in animal models. Continued innovation and rigorous evaluation could transform cardiovascular treatment, benefiting patients with untreatable conditions.}, }
@article {pmid40465692, year = {2025}, author = {Cai, Y and Zhao, P and Wu, F and Zhao, H and Shao, H and Marra, A and Patel, P and O'Connell, E and Fink, E and Miele, MM and Li, Z and De Stanchina, E and Cocco, E and Razavi, P and Toska, E and Fanning, SW and Xu, G and Sablina, AA and Scaltriti, M and Chandarlapaty, S}, title = {Inhibition of NR2F2 restores hormone therapy response to endocrine refractory breast cancers.}, journal = {Science translational medicine}, volume = {17}, number = {801}, pages = {eadk7786}, doi = {10.1126/scitranslmed.adk7786}, pmid = {40465692}, issn = {1946-6242}, mesh = {Humans ; *Breast Neoplasms/drug therapy/genetics/metabolism/pathology ; Female ; *COUP Transcription Factor II/metabolism/antagonists &amp; inhibitors/genetics ; Animals ; Cell Line, Tumor ; *Drug Resistance, Neoplasm/drug effects/genetics ; Neurofibromin 1/metabolism/genetics ; Gene Expression Regulation, Neoplastic/drug effects ; Mice ; Receptors, Estrogen/metabolism ; CRISPR-Cas Systems/genetics ; }, abstract = {Endocrine resistance is frequently encountered in estrogen receptor-positive (ER+) breast cancer, often because of somatic mutations such as neurofibromin 1 (NF1) loss. The mechanisms by which ER-directed proliferation is lost in such cases are unknown, limiting the potential use of additional endocrine treatments. Here, we performed CRISPR-Cas9 knockout (KO) screens and found that nuclear receptor subfamily 2 group F member 2 (NR2F2), an orphan nuclear receptor, was essential for NF1 loss-induced endocrine resistance. Induction of NR2F2 was observed in ER+ cell line models and patient samples and occurred via activation of the mitogen-activated protein kinase (MAPK) pathway upon NF1 loss or other MAPK pathway genetic alterations. Mechanistically, increased NR2F2 orchestrated a repressed ER transcriptional program by repartitioning the ER cistrome, altering the balance of its associated transcriptional coregulators, and modifying global chromatin accessibility. Accordingly, genetic depletion or pharmacologic inhibition of NR2F2 restored sensitivity to hormone therapies in multiple models, including ER+ cell lines, patient-derived xenografts, and patient-derived organoid-based xenografts harboring diverse endocrine-resistance mechanisms such as NF1, AT-rich interactive domain-containing protein 1A (ARID1A), phoshatase and tensin homolog (PTEN) loss, or Kirsten rat sarcoma virus (KRAS) overexpression. Together, these findings underscore NR2F2 as a critical modulator of the hormone response pathway and suggest its inhibition as a promising strategy to overcome endocrine resistance in breast cancer.}, }
@article {pmid40465008, year = {2025}, author = {Jin, Y and Liang, X and Wang, X}, title = {Alternative splicing in stem cells and development: research progress and emerging technologies.}, journal = {Cell regeneration (London, England)}, volume = {14}, number = {1}, pages = {20}, pmid = {40465008}, issn = {2045-9769}, support = {YD9110002078//USTC Research Funds of the Double First-Class Initiative/ ; 32170557//National Natural Science Foundation of China/ ; 32400436//National Natural Science Foundation of China/ ; 2023IHM01034//Research Funds of Centre for Leading Medicine and Advanced Technologies of IHM/ ; 2024C879//Anhui Postdoctoral Scientific Research Program Foundation/ ; }, abstract = {Alternative splicing is a key regulatory mechanism that generates transcriptomic diversity by selectively splicing pre-RNA molecules in different ways, leading to the production of multiple RNA isoforms from a single gene. This process is crucial for the fine-tuning of gene expression and is tightly regulated during various biological processes. Recent studies have highlighted how alternative splicing contributes to stem cells self-renewal and differentiation, as well as how dysregulation of splicing factors can impact stem cells behavior and lead to developmental abnormalities or diseases. This review summarizes the current understanding of alternative splicing in stem cells and development, focusing on the molecular mechanisms that govern alternative splicing regulation, the role of splicing factors, and the impact of splicing isoforms on stem cell fate determination and developmental processes. We also discuss emerging technologies, such as CRISPR/Cas-based tools, single-cell long-read RNA sequencing, imaging technologies and 3D culture systems, which are advancing our ability to study alternative splicing in vitro and in vivo. Overall, this field is rapidly evolving, revealing new insights into how alternative splicing shapes the molecular landscape and functions of stem cells and developmental processes.}, }
@article {pmid40464988, year = {2025}, author = {Waqas, MAB and Awan, MJA and Amin, I and Arif, M and Mukhtar, Z and Mansoor, S}, title = {Engineering high yield basmati rice by editing multiple negative regulators of yield.}, journal = {Molecular biology reports}, volume = {52}, number = {1}, pages = {545}, pmid = {40464988}, issn = {1573-4978}, mesh = {*Oryza/genetics/growth &amp; development ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; Plant Proteins/genetics ; Genes, Plant/genetics ; Quantitative Trait Loci/genetics ; Edible Grain/genetics ; Gene Expression Regulation, Plant ; }, abstract = {BACKGROUND: Yield improvement in Basmati rice is important to meet the growing demand of Basmati and pivotal to world food security. Yield in rice is a complex quantitative trait controlled by many genes and yield-determining factors. Although dominant genes introgression through breeding have been extensively used but it is a laborious and time-consuming process. Yield genes OsD27, OsGW2, OsTGW6 and OsGN1a have been shown to negatively regulate an increase in the number of tillers, grain width, grain weight, and number of grains per panicle respectively in different genetic backgrounds of rice separately. The aim of our study is to establish a multiplex system in Super Basmati to check if it acts similarly and what is their combinatorial effect on yield enhancement.<br><br>METHODS: Here we generated Super Basmati quadruple edited lines for all four genes OsD27, OsGW2, OsTGW6 and OsGN1a through CRISPR-Cas9 polycistronic tRNA-gRNA (PTG) endogenous processing efficient multiplex editing system. Quadruple edited lines were characterized for key yield parameters such as number of tillers, number of grains, grain weight, overall yield increase, lodging resistance and cooking quality related traits.<br><br>RESULTS: We found that all quadruple edited plants produced more tillers, a greater number of grains with high grain weight in field evaluations along with decreased plant height. Overall, the contribution of all four genes was additive resulting in boosting the yield in Super Basmati remarkably. Our data hence demonstrated a promising multiplex genome editing approach for rapid generation of superior alleles for all four yield contributing factor genes in elite Basmati variety with 30% co-editing efficiency.<br><br>CONCLUSIONS: We conclude that multiplex CRISPR-Cas9 genome editing of yield-negative regulators may boost the yield of elite Basmati rice in a short time without causing any drastic effect on other agronomic and quality traits such as aroma, chalkiness, and cooking qualities. Data suggests that the tRNA-gRNA multiplexing CRISPR-Cas9 system is efficient in the Basmati rice background for simultaneously editing multiple genes. Due to its capability of rapid generation of beneficial alleles, this multiplex system is well suited for pyramiding of multiple alleles in Basmati rice and may prove a promising approach to enhance yield in Basmati rice while it may provide useful germplasm resource for high-yielding future rice breeding programs.}, }
@article {pmid40464976, year = {2025}, author = {Sharma, SS and Pandey, A and Kashyap, A and Goyal, L and Garg, P and Kushwaha, R and Sharma, J and Tripathi, S and Kumari, S and Thomas, G and Verma, M and Gupta, NC and Gupta, AK and Bhattacharya, R and Sharma, S and Rao, M}, title = {CRISPR/Cas9: efficient and emerging scope for Brassica crop improvement.}, journal = {Planta}, volume = {262}, number = {1}, pages = {14}, pmid = {40464976}, issn = {1432-2048}, support = {NASF/BGAM-9021/2022-23/NIPB//ICAR - National Agricultural Science Fund/ ; 2001-2019//Indian Council of Agricultural Research/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Brassica/genetics ; *Gene Editing/methods ; *Crops, Agricultural/genetics ; Genome, Plant ; Plant Breeding/methods ; Plants, Genetically Modified ; }, abstract = {CRISPR/Cas9 revolutionizes Brassica crop improvement by enhancing yield, quality, and stress resistance, providing a precise and versatile tool for genetic and agronomic advancements. The rapidly advancing CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) technologies are being employed in both diploid and polyploid species of Brassica for gene functions and precise genetic improvements. CRISPR/Cas technology has sparked significant attention among the scientific community due to its affordability, precision, and effectiveness compared to other genome editing techniques. The recent discoveries highlight the diverse applications of the CRISPR/Cas9 genome editing tool in enhancing agriculturally important traits in Brassica species. This technology has been utilized to improve yield, quality, and resistance to both biotic and abiotic stresses globally. Here, we present an overview that encourages researchers to explore and improve the functionality and genetic progress of Brassica U-triangle species utilizing genome editing technologies. In addition, ethical considerations and concerns associated with CRISPR technologies are addressed, providing valuable insight into how CRISPR/Cas9 tools and have revolutionized crop improvement with special emphasis on Brassica for various agronomically and nutritionally important traits.}, }
@article {pmid40464068, year = {2025}, author = {Liu, Y and Zhang, S and Hu, C}, title = {Cas7 meets Cas14: a strategic partnership in the type VII CRISPR-Cas.}, journal = {Protein &amp; cell}, volume = {16}, number = {2}, pages = {79-82}, pmid = {40464068}, issn = {1674-8018}, support = {23-0178-A0001//National University of Singapore Presidential Young Professorship (PYP)/ ; 23-1065-P0001//Singapore Ministry of Education Tier 1/ ; }, }
@article {pmid40461565, year = {2025}, author = {Yang, C and Fang, Q and Li, M and Zhang, J and Li, R and Zhou, T and Wang, K and Deng, J and Wang, X and Huang, C and Feng, Y and Zhang, X and Shi, L and Bi, C and Zhang, X and Yu, J and Hao, J}, title = {Prime editor with rational design and AI-driven optimization for reverse editing window and enhanced fidelity.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {5144}, pmid = {40461565}, issn = {2041-1723}, support = {32471478//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82203238//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; *Artificial Intelligence ; }, abstract = {Prime editing (PE) is a precise tool for introducing genetic mutations in eukaryotes. Extending the efficient editing scope and mitigating undesired byproducts are possible. We introduce reverse PE (rPE), a SpCas9-directed variant that enabled DNA editing at the 3' direction of HNH-mediated nick site. The rPE leveraging nCas9-D10A and rPE gRNA targeting the 5' direction of HNH-mediated nick site inscribes genetic alterations, achieving a reverse editing window and potentially high fidelity. HNH and reverse transcriptase engineered using protein language models in conjunction with La facilitate circular erPEmax and erPE7max, achieving editing efficiency up to 44.41% without nick gRNA or positive selection. Furthermore, our findings underscore the capability of rPE in inserting functionally enhanced variant (PIK3CD[E527G]) for cell therapy. By expanding the editing scope and enhancing genomic manipulability, rPE represents a meaningful advancement in prime editing, improving its utility for research and therapeutic applications.}, }
@article {pmid40461259, year = {2025}, author = {Esser, SP and Turzynski, V and Plewka, J and Nuy, J and Moore, CJ and Banas, I and Soares, AR and Lee, J and Woyke, T and Probst, AJ}, title = {Differential Expression of Core Metabolic Functions in Candidatus Altiarchaeum Inhabiting Distinct Subsurface Ecosystems.}, journal = {Environmental microbiology reports}, volume = {17}, number = {3}, pages = {e70096}, pmid = {40461259}, issn = {1758-2229}, support = {DFG PR1603/2-1//Ministerium f&#xfc;r Kultur und Wissenschaft des Landes Nordrhein- Westfalen ("Nachwuchsgruppe Dr. Alexander Probst") and the German Research Foundation/ ; //Aker B.P.: GeneOil Project/ ; //U.S. Department of Energy Joint Genome Institute/ ; //DOE Office of Science User Facility/ ; //the Office of Science of the U.S. Department of Energy operated/ ; }, mesh = {*Ecosystem ; Germany ; Groundwater/microbiology ; Symbiosis ; Transcriptome ; Gene Expression Profiling ; Genome, Archaeal ; Multigene Family ; }, abstract = {Candidatus Altiarchaea are widespread across aquatic subsurface ecosystems and possess a highly conserved core genome, yet adaptations of this core genome to different biotic and abiotic factors based on gene expression remain unknown. Here, we investigated the metatranscriptome of two Ca. Altiarchaeum populations that thrive in two substantially different subsurface ecosystems. In Crystal Geyser, a high-CO2 groundwater system in the USA, Ca. Altiarchaeum crystalense co-occurs with the symbiont Ca. Huberiarchaeum crystalense, while in the Muehlbacher sulfidic spring in Germany, an artesian spring high in sulfide concentration, Ca. A. hamiconexum is heavily infected with viruses. We here mapped metatranscriptome reads against their genomes to analyse the in situ expression profile of their core genomes. Out of 537 shared gene clusters, 331 were functionally annotated and 130 differed significantly in expression between the two sites. Main differences were related to genes involved in cell defence like CRISPR-Cas, virus defence, replication, transcription and energy and carbon metabolism. Our results demonstrate that altiarchaeal populations in the subsurface are likely adapted to their environment while influenced by other biological entities that tamper with their core metabolism. We consequently posit that viruses and symbiotic interactions can be major energy sinks for organisms in the deep biosphere.}, }
@article {pmid40460174, year = {2025}, author = {Tan, YS and Mo, BT and Li, GC and Guo, YR and Zhang, JZ and Wang, CZ}, title = {HarmGR13 mediates myo-inositol taste perception in Helicoverpa armigera larvae.}, journal = {PLoS genetics}, volume = {21}, number = {6}, pages = {e1011744}, pmid = {40460174}, issn = {1553-7404}, mesh = {Animals ; *Inositol/metabolism ; Larva/genetics/physiology ; *Taste Perception/genetics ; *Insect Proteins/genetics/metabolism/chemistry ; Sensilla/metabolism/physiology ; *Moths/genetics/physiology ; CRISPR-Cas Systems ; Phylogeny ; Ribose/metabolism ; *Receptors, Cell Surface/genetics/metabolism ; Taste/genetics ; Helicoverpa armigera ; }, abstract = {Myo-inositol, a sugar alcohol produced by most plants, serves as a nutrient and feeding stimulant for many phytophagous insects. Inositol-sensitive taste sensilla have been characterized in many Lepidoptera larvae, but their molecular bases remain unclear. In this study, we determined the gustatory receptors (GRs) for myo-inositol in larva of Helicoverpa armigera, a worldwide crop pest. First, electrophysiological analyses revealed that medial sensilla styloconica strongly responded to myo-inositol and ribose, with weaker responses to xylose, and one GRN inside sensillum may mediate the response to these three chemicals. Based on phylogenetic analysis of sugar GRs of Lepidoptera insects and previous results on Bombyx mori, we then selected two candidate GRs, HarmGR13 and HarmGR11. Using CRISPR-Cas9, we generated knockout mutants for the two GR genes. Knocking out HarmGR13 abolished the responses of the sensilla to myo-inositol, ribose, and xylose, while knocking out HarmGR11 showed no changes. Behavioral assays confirmed that larvae of HarmGR13 homozygous mutant lost the feeding preference to myo-inositol which the wild-type larvae had. Further functional analysis with Xenopus oocytes expressing system and two-electrode voltage-clamping demonstrated that myo-inositol and ribose specifically induced concentration-dependent currents in HarmGR13-expressing oocytes. Structural predictions and molecular docking of HarmGR13 revealed three amino acid residues might be involved in ligand binding. Mutation of these residues resulted in loss of oocyte responses to myo-inositol and ribose. We reveal that HarmGR13 is a receptor that mediates the activity of the cells sensitive to inositol and ribose in larvae, providing new molecular targets for the strategy of regulating the feeding behavior of pests by modifying taste.}, }
@article {pmid40460129, year = {2025}, author = {Jiang, W and Wang, S and Ahlheit, D and Fumagalli, T and Yang, Z and Ramanathan, S and Jiang, X and Weber, T and Dahlin, J and Borodina, I}, title = {High-throughput metabolic engineering of Yarrowia lipolytica through gene expression tuning.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {122}, number = {23}, pages = {e2426686122}, pmid = {40460129}, issn = {1091-6490}, support = {NNF20CC0035580//Novo Nordisk Fonden (NNF)/ ; NNF20OC0060809//Novo Nordisk Fonden (NNF)/ ; NNF21OC0072559//Novo Nordisk Fonden (NNF)/ ; No. 101123257//EC | H2020 | PRIORITY 'Excellent science' | H2020 European Research Council (ERC)/ ; }, mesh = {*Yarrowia/genetics/metabolism ; *Metabolic Engineering/methods ; Transcription Factors/genetics/metabolism ; Promoter Regions, Genetic ; *Gene Expression Regulation, Fungal ; CRISPR-Cas Systems ; Plasmids/genetics ; Gene Library ; Fungal Proteins/genetics/metabolism ; }, abstract = {The challenge of accurately predicting which genetic alternations lead to the desired phenotype necessitates high-throughput metabolic engineering approaches where numerous hypotheses can be tested simultaneously. We describe the CRISPR-Cas9-based method TUNE[YALI] that enables high-throughput tuning of gene expression in the common industrial yeast Yarrowia lipolytica. The method is based on replacing the promoters of the target genes with native Y. lipolytica promoters of varying strengths or removing the promoters entirely. To demonstrate the method's capabilities, we created a plasmid library that targets 56 transcription factors (TFs) and changes the expression of each TF to seven different levels. We transformed this library into reference and betanin-producing strains of Y. lipolytica and screened the resulting clones for changes in morphology, thermotolerance, or improved betanin production. The genetic markup of the yeast clones with the desired phenotypic changes was determined by sequencing the inserted plasmids. We identified multiple TFs whose regulatory changes increased thermotolerance, two TFs that eliminated pseudohyphal growth, and several TFs that increased betanin production. Analogous libraries can be designed to target any chosen group of genes and even all the genes. The libraries can be shared and reused, accelerating applied strain development projects and fundamental functional genomics research (TUNE[YALI]-TF kit and TUNE[YALI]-TF library are available via AddGene under catalog numbers #1000000255 and #217744).}, }
@article {pmid40459995, year = {2025}, author = {Wu, JL and Zheng, SS and Wang, L and Xiang, X and Li, F and Lv, ML and Wu, Q and Huang, ZX and Miao, HB}, title = {CRISPR-Cas System-Mediated Genetic Modification in Bacillus spp.: Current Status and Future.}, journal = {Journal of agricultural and food chemistry}, volume = {73}, number = {24}, pages = {14760-14775}, doi = {10.1021/acs.jafc.5c03140}, pmid = {40459995}, issn = {1520-5118}, mesh = {*CRISPR-Cas Systems ; *Bacillus/genetics/metabolism ; *Gene Editing/methods ; Bacterial Proteins/genetics/metabolism ; }, abstract = {Bacillus spp. are a group of Gram-positive bacteria that have shown significant potential for development in recent years. It is capable of utilizing low-cost substrates to produce various high-value-added compounds, making it widely applicable in fields such as feed, pharmaceuticals, and food. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system-mediated genetic modification is recognized as one of the most efficient technologies. The application of this technology for the genetic improvement of Bacillus spp. greatly enhanced the production performance of these strains. In this review, we summarize the various CRISPR-Cas systems that have been applied to Bacillus spp., with a particular focus on systematically outlining the strategies for implementing CRISPR-Cas-mediated genetic modification in these bacteria. Notably, homologous recombination is the most widely used strategy, while base editing is emerging as a novel and precise approach. Additionally, we discuss the importance of expression regulation strategies in establishing Bacillus spp. as a cell factory. Finally, we propose potential solutions to current technical challenges, providing insights for the development of high-performance genetically modified Bacillus spp. production strains.}, }
@article {pmid40459840, year = {2025}, author = {Keerthana, R and Rakshana, P and Salunkhe, SR and Sakthi, AR and Kokiladevi, E and Saraswathi, T and Pushpam, R and Raveendran, M and Sudha, M}, title = {CRISPR-Cas9 mediated enhancement of abiotic stress resilience in tomato: a comprehensive review of target genes.}, journal = {Molecular biology reports}, volume = {52}, number = {1}, pages = {538}, pmid = {40459840}, issn = {1573-4978}, mesh = {*Solanum lycopersicum/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Stress, Physiological/genetics ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; }, abstract = {Tomato (Solanum lycopersicum L.), a major vegetable crop grown worldwide, is consumed as both fresh and processed products. Concerns about the impact of abiotic stresses on tomato production are growing worldwide as climate change alters global weather patterns, adversely affecting crop yield and produce quality due to stresses like salt, heat, cold, and drought. Conventional breeding approaches such as hybridization, marker-assisted selection (MAS), and mutation breeding, have long been utilized to improve tomato resilience against abiotic stresses. These approaches are often hampered by extensive field trials, and require multiple generations limiting their efficiency in rapidly developing stress-tolerant cultivars. The efforts of traditional breeding systems are hindered by the narrow genetic base of tomatoes which poses a major bottleneck. Researchers have utilized CRISPR-Cas genome-editing technology to address this challenge to offer a precise and accelerated alternative for enhancing stress resilience in tomato. This versatile tool has gained attention for its simple, precise, and effective gene-editing capabilities. CRISPR-Cas based genome editing has successfully modified key genes related to stress-response pathways, enhancing abiotic stress resilience. Developing resistant cultivars help mitigate the impact of abiotic stress, thereby contributing to increased food production and food security. This review highlights recent progress in use of CRISPR-Cas9 gene editing to enhance tomato resilience to abiotic stresses.}, }
@article {pmid40459690, year = {2025}, author = {Lakhani, H and Kumar, N and Jangra, A and Negi, S and Dholariya, T and Tiwari, S}, title = {Streamlined protoplast transfection system for in-vivo validation and transgene-free genome editing in Banana.}, journal = {Transgenic research}, volume = {34}, number = {1}, pages = {28}, pmid = {40459690}, issn = {1573-9368}, support = {BT/PR25789/GET/119/97/2017//Department of Biotechnology, Ministry of Science and Technology, India/ ; BIRAC/Tech Transfer/08/I2/QUT-BBF//Biotechnology Industry Research Assistance Council/ ; }, mesh = {*Musa/genetics/growth &amp; development ; *Protoplasts/metabolism ; *Gene Editing/methods ; *Transfection/methods ; *Plants, Genetically Modified/genetics/growth &amp; development ; CRISPR-Cas Systems/genetics ; Transgenes/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Plant ; }, abstract = {The advancement in the CRISPR/Cas system has significantly streamlined genome editing in plants, rendering it simple, reliable, and efficient. However, the development of transgene-free crops is a challenging task for vegetatively propagated plants like banana. In the present study, we established banana protoplasts-based versatile and efficient platform for genome editing to overcome this limitation. Herein, a protocol has been optimized for protoplast isolation by considering leaf and embryogenic cell suspension (ECS) of banana cultivar Grand Naine. Freshly prepared ECS was identified as the best source for protoplast isolation. The protoplast viability and competency were checked by transfection with plasmid and RNP complex. Polyethylene glycol (PEG)-mediated protoplast transfection using pCAMBIA1302 and pJL50TRBO vectors showed GFP expression with 30 and 70% efficiency, respectively, eventually proving the protocol's efficacy. Further, gRNAs targeting banana β-carotene hydroxylase gene are validated by in-vitro cleavage test and subsequently used for RNP complex formation with varied ratios (1:1, 1:2, 1:5, and 1:10) of SpCas9 to gRNA1. Among these, a 1:2 molar ratio proved best to generate indel frequency with 7%. Sequencing analysis of the target amplicon revealed mutations upstream of the PAM region, specifically with gRNA1, among the three in-vitro validated gRNAs. This study evaluated the effectiveness of gRNAs in-vitro and in-vivo, yielding inconsistent results that highlight the need for comprehensive in-vivo validation of their functionality. Conclusively, the optimized protocol for banana transfection has the potential to be harnessed for the generation of transgene-free genetically improved banana.}, }
@article {pmid40457455, year = {2025}, author = {Zhang, T and Meng, Z and Yu, H and Zhang, Z and Liu, G and Qiu, A and Zheng, W and Ding, P and Kai, T}, title = {An integrated multi-mode detection platform based on CRISPR/Cas 12a and aptamers for ultra-sensitive identification of sulfamethazine and genes associated with sulfonamide resistance.}, journal = {Journal of nanobiotechnology}, volume = {23}, number = {1}, pages = {408}, pmid = {40457455}, issn = {1477-3155}, support = {2025ZZTS0167//Graduate Research Innovation Program of Central South University/ ; 82373635//the National Natural Science Foundation of China/ ; 2022RC1206//the Science and Technology Innovation Program of Hunan Province/ ; 2023QYJC013//the Central South University Research Program of Advanced Interdisciplinary Studies/ ; }, mesh = {*Sulfamethazine/analysis/pharmacology ; *Aptamers, Nucleotide/chemistry ; *CRISPR-Cas Systems/genetics ; *Sulfonamides/pharmacology ; Biosensing Techniques/methods ; *Anti-Bacterial Agents/analysis/pharmacology ; Electrochemical Techniques ; *Drug Resistance, Bacterial/genetics ; Metal-Organic Frameworks/chemistry ; Benzidines/chemistry ; Limit of Detection ; }, abstract = {The production and buildup of sulfamethazine (SMZ) and resistance genes for sulfonamide antibiotics (sul1) pose a serious risk to environmental and public health safety. Creating advanced sensing systems that are both highly sensitive and selective for the prolonged observation of SMZ concentrations in the environment, along with the quantification of sul1 gene prevalence, aims to identify trends in resistance, posing a considerable challenge. Here, we devised a platform (SMZ-sul1 multi-mode detection platform) that allows for the fluorescence detection of SMZ in environmental samples. This is achieved through the competition for the aptamer between the complementary base and SMZ, along with the colorimetric, photothermal, and electrochemical tracking of sul1, using a magnetic separation unit (FP@cDNA). MOF-818@PtPd (MPP) nanozymes with high peroxide mimetic enzyme activity were linked to FP@cDNA through Zr-O-P bond and employed as a catalyst for the 3,3',5,5'-tetramethylbenzidine (TMB) oxidation, as well as for electrocatalytic hydrogen peroxide (H2O2) reduction. The ability of Cas12a to perform trans cleavage was activated by its precise identification of the sul1, leading to the non-selective cutting of single-stranded DNA (ssDNA). Thereafter, the MPP nanoparticles were released into the supernatant, where they catalyzed the oxidation of TMB. Alternatively, the functioning CRISPR/Cas12a system specifically targeted and cleaved ssDNA present on the electrode, resulting in altered loading of MPP nanozymes and a decrease in the current associated with the catalytic reduction of H2O2. The remarkable magnetic separation capabilities of FP@cDNA, combined with the superior target recognition features of CRISPR/Cas12a and aptamer, facilitated the creation of a highly sensitive detection system, achieving detection limits of 0.67 pM for SMZ and 7.6 fM for sul1, and exhibit great potential for monitoring and prediction in the field of public health.}, }
@article {pmid40456823, year = {2025}, author = {Hong, D and Shu, M and Liu, J and Liu, L and Cheng, H and Zhu, M and Du, Y and Xu, B and Hu, D and Liu, Z and Zhao, Y and Dai, J and Lu, F and Huang, J}, title = {Divergent combinations of enhancers encode spatial gene expression.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {5091}, pmid = {40456823}, issn = {2041-1723}, support = {92474104//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32370586//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {Animals ; *Enhancer Elements, Genetic/genetics ; Mice ; Humans ; Brain/metabolism/embryology ; Chromatin/metabolism/genetics ; Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism ; Breast Neoplasms/genetics ; Spinal Cord/metabolism/embryology ; Gene Expression Regulation, Developmental ; Melanoma/genetics ; CRISPR-Cas Systems ; }, abstract = {Spatial transcriptomics and epigenomics have enabled mapping gene regulation in the tissue context. However, it remains poorly understood how spatial gene expression patterns are orchestrated by enhancers. Here we build eSpatial, a computational framework that deciphers spatially resolved enhancer regulation of gene expression by integrating spatial profiles of gene expression and chromatin accessibility. Applying eSpatial to diverse spatial datasets, including mouse embryo and brain, as well as human melanoma and breast cancer, we reveal a "spatial enhancer code", in which divergent combinations of enhancers regulate the same gene in spatially segregated domains. We validate the spatial enhancer code using public spatial datasets such as VISTA, Allen in situ hybridization (ISH), and H3K27ac MERFISH. Moreover, we conduct transgenic reporter assays and in vivo CRISPR/Cas9-mediated perturbation experiments to confirm the Atoh1 spatial enhancer code in determining Atoh1 spatial expression in mouse embryonic spinal cord and brain. Our study establishes the spatial enhancer code concept, revealing how combinations of enhancers dynamically shape gene expression across diverse biological contexts, providing insights into tissue-specific regulatory mechanisms and tumor heterogeneity.}, }
@article {pmid40456709, year = {2025}, author = {Hanlon, MB and Shohet, JM and Wolfe, SA}, title = {Selective targeting of genome amplifications and repeat elements by CRISPR-Cas9 nickases to promote cancer cell death.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {5126}, pmid = {40456709}, issn = {2041-1723}, support = {R01 CA275945/CA/NCI NIH HHS/United States ; R01CA275945//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; }, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Amplification ; *Neuroblastoma/genetics/pathology ; Cell Line, Tumor ; N-Myc Proto-Oncogene Protein/genetics ; Cell Death/genetics ; *Deoxyribonuclease I/metabolism/genetics ; DNA Breaks, Double-Stranded ; Neoplasms/genetics ; }, abstract = {Focal gene amplification serves as an oncogenic driver during tumorigenesis and is a hallmark of many forms of cancer. Oncogene amplifications promote genomic instability, which is integral to cancer cell survival and evolution. However, focal gene amplification potentially affords an opportunity for therapeutic exploitation. As a proof-of-concept, we leverage CRISPR-Cas9 nickase to selectively promote cancer cell death in MYCN-amplified neuroblastoma in a gene amplification-dependent manner. Our analysis demonstrates that CRISPR-Cas9 nickase can generate a lethal number of highly toxic, replication-dependent double-strand breaks in cells harboring amplified loci. Furthermore, we demonstrate that Cas9 nickase-mediated toxicity can be modulated in combination with small molecule inhibitors targeting key regulators of the DNA-damage response or cell death pathways. Importantly, our findings in MYCN-amplified neuroblastoma translate to other cancer types with distinct oncogene amplifications.}, }
@article {pmid40456230, year = {2025}, author = {Huda, NU and Hasan, KA and Saleem, F and Naz, S and Khan, S and Mirani, ZA and Noman Syed, M and Alswat, AS and Sohail, M}, title = {Coriander borne Salmonella superbug: genomic assessments of chromosomal and plasmid-associated resistance, virulence and MGEs of XDR Salmonella enterica Typhi NH1.}, journal = {Pathogens and global health}, volume = {}, number = {}, pages = {1-15}, doi = {10.1080/20477724.2025.2513769}, pmid = {40456230}, issn = {2047-7732}, abstract = {The drug-resistant strains of Salmonella enterica serovar Typhi (S. Typhi) are serious threats to health sectors worldwide. This study focuses on genomic characterization of environmentally derived an extensively drug resistant (XDR) strain of Salmonella Typhi, highlighting its potential to cause significant outbreak. The XDR strain (labeled as NH1) was isolated from fresh coriander, and characterized through whole-genome sequencing to investigate its lineage diversity, niche adaptation, sources and resistant mechanism. The NH1 strain exhibits phylogenomic association with clinical lineages S. Typhi NCTC8385 and ASM3025476. Its genome, assembled in 67 contigs, exhibited expected coverage and GC content, harboring a mega-plasmid, virulence factors, and intrinsic and extrinsic antimicrobial resistance genes. Plasmid annotation revealed IncQ1 and IncY plasmids responsible for the acquired resistance. SPIFinder identified SPI1-10 pathogenicity islands, and the CRISPR-Cas locus, associated with bacterial defense, bile salt resistance and biofilm formation, was present. NH1 strain also possesses numerous mobile elements (proteins and transposases), 12 prophages, and secretion systems (types I-IV, VI), aiding its survival in environment like vegetables. The genomic characterization of S. Typhi NH1 highlights the emergence of coriander-borne XDR strain, underscoring the need for targeted prevention to mitigate its public health impact.}, }
@article {pmid40455764, year = {2025}, author = {De Pablo-Moreno, JA and González-Brusi, L and Miguel-Batuecas, A and Bermejo-Álvarez, P and Revuelta, L and Liras, A}, title = {Development of a novel and viable knock-in factor V deficiency murine model: Utility for an ultra-rare disease.}, journal = {PloS one}, volume = {20}, number = {6}, pages = {e0321864}, pmid = {40455764}, issn = {1932-6203}, mesh = {Animals ; Mice ; *Disease Models, Animal ; *Factor V Deficiency/genetics/pathology ; *Factor V/genetics/metabolism/chemistry ; *Gene Knock-In Techniques ; Humans ; Female ; Male ; CRISPR-Cas Systems ; Phenotype ; Homozygote ; Mutation, Missense ; *Rare Diseases/genetics ; Partial Thromboplastin Time ; Prothrombin Time ; }, abstract = {Factor V deficiency is a congenital coagulation disorder characterized by the absence or malfunction of factor V (FV). The purpose of this study was to develop a viable FV-deficient mouse model using CRISPR/Cas9 technology. A viable pathological model of the disease was not available to develop new therapies. A previous in silico study was performed to select a mutation causing a mild disease phenotype in humans (Thr1898Met missense). Such mutation was replicated in mice by CRISPR-mediated homology directed repair. Following crossing, homozygous individuals were subjected to coagulometry assays, including FV levels, prothrombin time (PT), and activated partial thromboplastin time (aPTT). The in silico study suggested that the mutation destabilizes FV structure of both mouse and human variants, putatively producing a mild phenotype of the disease in mice. Mendelian inheritance was observed in the offspring. No spontaneous signs of blood clotting disturbances, premature deaths or gestational dysfunctions were observed. FV levels in homozygous animals were 24.5% ± 5.1; 39.7 sec ± 2.8; PT was 61.8% ± 6.3; 23.4 sec ± 1.6 (INR = 1.47 ± 0.12); and aPTT was 46.9 sec ± 3.2. A viable FV-deficient mouse model was generated by introducing a missense mutation in FV. The model exhibits a mild phenotype of the disease, akin to that observed in humans.}, }
@article {pmid40455288, year = {2025}, author = {Hu, J and Yu, W and Cui, J and Zhang, L and Yu, W}, title = {Recent advances in diagnostic technologies for postoperative central nervous system infections: a review.}, journal = {Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology}, volume = {}, number = {}, pages = {}, pmid = {40455288}, issn = {1590-3478}, abstract = {Postoperative central nervous system infections (PCNSIs), including meningitis, cerebral abscesses, and implant-associated infections, represent critical complications following neurosurgical procedures. These infections pose significant risks to patient outcomes due to delayed diagnosis, escalating antimicrobial resistance, and limited therapeutic efficacy. Conventional diagnostic approaches, such as cerebrospinal fluid (CSF) analysis, microbial cultures, and neuroimaging, exhibit notable limitations in sensitivity, specificity, and rapidity. This review highlights transformative technologies reshaping PCNSI diagnostics, including molecular assays (e.g., quantitative PCR, digital droplet PCR), metagenomic next-generation sequencing (mNGS), CRISPR-based pathogen detection platforms, metabolomics, and advanced molecular imaging modalities. Furthermore, we address translational challenges in clinical adoption, including cost barriers, standardization gaps, and the need for interdisciplinary collaboration. Emerging artificial intelligence (AI)-driven strategies are proposed to optimize pathogen identification, predict antimicrobial resistance profiles, and tailor personalized therapeutic regimens.}, }
@article {pmid40455261, year = {2025}, author = {Zhang, M and Zhou, L and Afridi, M and Guo, H and Cheng, H}, title = {An efficient hairy root system for validation of CRISPR/Cas system activities in cotton.}, journal = {Functional &amp; integrative genomics}, volume = {25}, number = {1}, pages = {116}, pmid = {40455261}, issn = {1438-7948}, support = {NK2022010303//the Ministry of Agriculture/ ; B23CJ0208//Hainan Seed Industry Laboratory/ ; YSPTZX202502//Hainan Province Academician Innovation Platform/ ; }, mesh = {*Gossypium/genetics/growth &amp; development/microbiology ; *Plant Roots/genetics/growth &amp; development ; *CRISPR-Cas Systems ; Agrobacterium/genetics ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; }, abstract = {The hairy root induction system has been widely applied in studying gene expression and function in plant species due to its rapidity and efficiency. The hairy root system is an efficient tool for evaluating the activities of CRISPR/Cas systems. Cotton hairy roots were primarily induced through cotton tissue culture under aseptic conditions and by injecting cotton stem tips under non-aseptic conditions. However, both methods are lab-intensive and time-consuming. In this study, an efficient cotton hairy root induction procedure was established via infecting cotton hypocotyls with Agrobacterium rhizogenes under non-sterile conditions. Cotton seedlings with expanded cotyledons were decapitated with a slanted cut, and the residual hypocotyl (maintained 1 cm apical portion) was inoculated with A. rhizogenes. Over 90% of the infected explants from all three tested varieties could produce hairy roots after 8 days of inoculation. The effictiveness of the method was tested by overexpressing two reporter genes (eGFP and GUS). The transformation efficiency of the GUS and eGFP were ranged from50-68.18% and 40.9-68.18%. In addition, the editing efficiency of target sites in different CRISPR/Cas systems were also tested in hairy root. This method provided technical support for screening suitable target sites for cotton gene editing.}, }
@article {pmid40454797, year = {2025}, author = {Wu, J and Yan, Q and Qiu, H and Gao, EB}, title = {Integrating Enzyme-DNA Complex and CRISPR/Cas12a for Robust Norovirus Detection.}, journal = {Journal of medical virology}, volume = {97}, number = {6}, pages = {e70426}, doi = {10.1002/jmv.70426}, pmid = {40454797}, issn = {1096-9071}, support = {//The Ningbo Clinical Research Center for Children's Health and Diseases (no. 2019A21002), the Ningbo public welfare project (no. 2022S127), the Ningbo key discipline Pediatrics (no. 2022-B17), the Innovation Project of Distinguished Medical Team in Ningbo (no. 2022020405). Ningbo Medical and Health Brand Discipline (PPXK2024-06)./ ; }, mesh = {*Norovirus/isolation &amp; purification/genetics ; Humans ; *Caliciviridae Infections/diagnosis/virology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Gastroenteritis/virology/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; RNA, Viral/genetics ; *Molecular Diagnostic Techniques/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Human norovirus (NoV) is a primary cause of acute gastroenteritis in children, making accurate and rapid detection essential for effective disease prevention and control. In this study, we developed a sensitive and efficient platform for pathogen nucleic acid detection by integrating asymmetric nucleic acid sequence-based amplification (asymmetric NASBA), enzyme-DNA molecular complex, and the clustered regularly interspaced short palindromic repeats (CRISPR) system, namely an A-enDMC platform. The target recognition capability of the enzyme-DNA complex operates independently from the signal amplification function of the CRISPR system. By decoupling the CRISPR reaction from the dependence on specific target sequences, the platform's universality and modularity are enhanced. The assay is fast (< 1.5 h), highly sensitive (< 5 copies/µL), and demonstrates no cross-reactivity with other common viruses. Compared to the widely used RT-qPCR method, the platform demonstrates high consistency in detection results, with the detection coincidence rate of 96.77% and a kappa value of 0.87. This platform provides a versatile technological tool for highly sensitive and specific RNA detection, demonstrating its extensive potential in real sample analysis.}, }
@article {pmid40453079, year = {2025}, author = {Lund, S and Gong, C and Yu, X and Staudt, LM and Hodson, DJ and Scheich, S}, title = {Strategies for CRISPR-based knock-ins in primary human B cells and lymphoma cell lines.}, journal = {Frontiers in immunology}, volume = {16}, number = {}, pages = {1589729}, pmid = {40453079}, issn = {1664-3224}, mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Knock-In Techniques/methods ; *Gene Editing/methods ; *B-Lymphocytes/metabolism/immunology ; Cell Line, Tumor ; *Lymphoma, Large B-Cell, Diffuse/genetics ; }, abstract = {Since its advent about ten years ago, the CRISPR-Cas9 system has been frequently used in biomedical applications. It has advanced various fields, and CRISPR-Cas9-based therapeutics have shown promising results in the treatment of specific hematological diseases. Furthermore, CRISPR gene editing technologies have revolutionized cancer research by enabling a broad range of genetic perturbations, including genetic knockouts and precise single nucleotide changes. This perspective focuses on the state-of-the-art methodology of CRISPR knock-ins to engineer immune cells. Since this technique relies on homology-directed repair (HDR) of double-strand breaks (DSBs) induced by the Cas9 enzyme, it can be used to introduce specific mutations into the target genome. Therefore, this methodology offers a valuable opportunity to functionally study specific mutations and to uncover their impacts not only on overall cell functions but also on the mechanisms behind cancer-related alterations in common signaling pathways. This article highlights CRISPR knock-in strategies, protocols, and applications in cancer and immune research, with a focus on diffuse large B cell lymphoma.}, }
@article {pmid40451886, year = {2025}, author = {Okamoto, M and Sasamoto, K and Takahashi-Nakaguchi, A and Zhao, F and Yamaguchi, M and Chibana, H}, title = {CRISPR-Cas9 RNP-Mediated Deletion of ERG25 in Non-albicans Candida Species, Including Candida auris.}, journal = {Medical mycology journal}, volume = {66}, number = {2}, pages = {35-43}, doi = {10.3314/mmj.24-00023}, pmid = {40451886}, issn = {2186-165X}, mesh = {*CRISPR-Cas Systems ; Candida glabrata/genetics ; *Candida auris/genetics ; *Fungal Proteins/genetics ; *Gene Deletion ; *Candida/genetics ; Candidiasis/microbiology ; Antifungal Agents/pharmacology ; }, abstract = {The incidence of infections caused by non-albicans Candida (NAC) species, including Candida glabrata and Candida tropicalis, has recently increased. Furthermore, Candida auris, a multidrug-resistant yeast, poses a serious threat to global health. The development of novel antifungal agents with alternative mechanisms of action is necessary to combat these fungi. However, genetic analyses of the virulence factors in these NAC species are insufficient for this purpose. Recent advancements in the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system have facilitated enhanced the genetic analysis of NAC species. The RNP-based system, in which the Cas9-gRNA complex is assembled in vitro and introduced into cells, offers a simplified approach to genetic modification, eliminating the need for species-specific plasmids. Previous our research identified the ERG25 gene, which encodes C-4 sterol methyl oxidase, as a promising antifungal target in C. glabrata. This study demonstrated deletion of the ERG25 homolog in C. glabrata and C. auris using an RNP-based CRISPR-Cas9 system. The deletion of ERG25 in C. auris and C. glabrata indicated that Erg25 is crucial for the survival of these pathogenic yeasts within the host. Furthermore, we have successfully deleted the ERG25 alleles in C. tropicalis and Candida parapsilosis, demonstrating the effectiveness of using both the CRISPR-Cas9 and Cre-loxP systems in these species for the first time.}, }
@article {pmid40451196, year = {2025}, author = {Smirnov, AV and Yunusova, AM}, title = {Novel CRISPR/Cas9-Based Approaches for Quantitative Study of DSB Repair Mechanics.}, journal = {Biochemistry. Biokhimiia}, volume = {90}, number = {4}, pages = {437-456}, doi = {10.1134/S0006297924601813}, pmid = {40451196}, issn = {1608-3040}, mesh = {*CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; *DNA Repair ; Humans ; Animals ; }, abstract = {This review examines modern approaches to studying double-strand break (DSB) DNA repair in mammalian cells, employing the CRISPR/Cas9 system. Due to its flexibility and efficacy, the Cas9 nuclease is used in numerous genetic reporters. We discuss various fluorescence-based genetic reporters used to monitor the repair process. In addition, among the innovative Cas9-based methods, special attention is given to the techniques that examine both single and multiple DSBs, including approaches such as DSB-TRIP and ddXR. These methods open new possibilities for investigating structural rearrangements or analyzing random genomic sites. Additionally, the review considers how DSBs induced by Cas9 differ from those made by other nucleases and how these peculiarities could impact DNA repair mechanisms. Understanding these differences is crucial for planning experiments aimed at studying DSB repair.}, }
@article {pmid40451118, year = {2025}, author = {Piñeiro-Silva, C and Gadea, J}, title = {Optimizing gene editing in pigs: The role of electroporation and lipofection.}, journal = {Animal reproduction science}, volume = {278}, number = {}, pages = {107874}, doi = {10.1016/j.anireprosci.2025.107874}, pmid = {40451118}, issn = {1873-2232}, mesh = {Animals ; *Electroporation/veterinary/methods ; Swine/genetics ; *Gene Editing/veterinary/methods ; *Animals, Genetically Modified ; }, abstract = {The production of genetically modified pigs is becoming increasingly important in both the agricultural and biomedical fields. Optimization of these processes is a key objective to improve the precision, scalability and viability of genetically modified animals for research and commercial applications. Among the available techniques, electroporation and lipofection have emerged as promising alternatives to traditional methods such as microinjection and somatic cell nuclear transfer (SCNT) due to their simplicity, cost-effectiveness, and potential for high-throughput applications. These methods allow the direct delivery of CRISPR/Cas components into zygotes and embryos, reducing the technical expertise required and bypassing some of the challenges associated with cloning. This review examines the application, efficacy, and outcomes of electroporation and lipofection as gene editing techniques in porcine gametes and embryos. We provide a comprehensive synthesis of recent advances, compare their efficacy, and discuss their potential in agricultural and biomedical research. The principles and mechanisms of both methods are reviewed, highlighting their advantages, such as cost-effectiveness and ease of implementation, over traditional approaches such as microinjection. In addition, we address their limitations, including variability in efficiency, and discuss recent protocol optimizations aimed at improving reproducibility and applicability. By analyzing these developments, this review provides valuable insights into the evolving role of electroporation and lipofection in porcine genetic modification strategies.}, }
@article {pmid40450609, year = {2025}, author = {Pal, S and Krishna, R and Dedhia, L and Panwar, HS and Karkute, SG and Rai, N and Kumar, R and Pandey, S and Singh, AK}, title = {CRISPR mediated gene editing for economically important traits in horticultural crops: progress and prospects.}, journal = {Transgenic research}, volume = {34}, number = {1}, pages = {26}, pmid = {40450609}, issn = {1573-9368}, mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics/growth &amp; development ; *CRISPR-Cas Systems/genetics ; *Plants, Genetically Modified/genetics/growth &amp; development ; Plant Breeding ; Genome, Plant ; }, abstract = {Horticultural crops, with their cost-effectiveness, rich mineral and vitamin content, and high yield potential, have become indispensable worldwide for ensuring food and nutritional security. With the world's population on the rise and climate change becoming more prominent, it is crucial to focus on creating resilient, high-yielding crop varieties that can withstand the changing climate. Genetic improvement of different horticultural crops using conventional tools is both time-consuming and labourious. However, the breeding period can be cut short by adopting modern breeding techniques, including CRISPR/Cas-mediated genome editing. In the present review, we discuss the progress made so far through genome editing to improve several horticultural crops for various traits like stress resistance, morphology, nutritional attributes, quality, shelf life, male sterility, architecture and economic yield. We have also discussed the emerging CRISPR technologies like base editing, epigenome editing, CRE editing, transposon-based editing, prime editing etc., along with their pros and cons and the future prospects. The ethical considerations for commercialization and current regulatory frameworks for gene-edited products have also been discussed.}, }
@article {pmid40450056, year = {2025}, author = {Ortiz-Rodríguez, LA and Cabanzo, R and Jaimes-Dueñez, J and Mendez-Sanchez, SC and Duque, JE}, title = {TropD-detector a CRISPR/LbCas12a-based system for rapid screening of Trypanosoma cruzi in Chagas vectors and reservoirs.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {19107}, pmid = {40450056}, issn = {2045-2322}, mesh = {*Trypanosoma cruzi/genetics/isolation &amp; purification ; *Chagas Disease/parasitology/diagnosis ; Animals ; *CRISPR-Cas Systems ; *Disease Reservoirs/parasitology ; Rhodnius/parasitology ; Cytochromes b/genetics ; Endodeoxyribonucleases/genetics ; Histones/genetics ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; }, abstract = {Chagas disease, also known as American Trypanosomiasis, is a zoonosis with global distribution caused by the parasite Trypanosoma cruzi, primarily transmitted through the feces of infected triatomines. The emergence of new cases highlights the importance of early pathogen detection in vectors and reservoirs to generate effective control strategies and establish preventive policies. The objective of this study was to design and validate a detection system of T. cruzi based on specific DNA cleavage, activation of Cas12a and trans-cleavage, targeting the genes Cytochrome B (Cytb), 18 S ribosomal subunit (SR18 s), and histone (H2 A). This system was validated for their uses in both vectors and reservoirs of the parasite. The initial step involved performing a bioinformatic analysis of the target genes, followed by the design of RNA guides specific to each cleavage site, along with primers for amplifying the target region through PCR and RPA. Subsequently, we sequenced the amplified DNA target and validated the detection system using T. cruzi DNA extracted from naturally infected Rhodnius pallescens in the metropolitan area of Bucaramanga, Colombia. After standardizing the method, we tested the CRISPR/Cas system with Silvio X10 laboratory strain of T. cruzi and scaled up to blood samples of naturally infected Didelphis marsupialis. As a result, we observed DNA cleavage using the CRISPR/Cas system with the Cytb guide, achieving a detection sensitivity of 118 parasite equivalents/mL in PCR and 116 parasite equivalents/mL with RPA amplification. Sequencing of the Cytb gene showed no mutations in the cleavage site. However, point mutations and indels were found in SR18S and H2 A, avoiding the formation of the CRISPR/LbCas12 complex. Furthermore, we introduce the design of a fluorescent detection prototype with CRISPR/LbCas12a called "Tropical Diseases Detector" (TropD-Detector). This device operates with an excitation wavelength of 480 nm emitted by an LED and a high-pass light filter with a cutoff wavelength of 500 nm. We detected positive samples using any photographic camera system. The TropD-Detector provides a visual, viable, and sensitive method for detecting T. cruzi in both vectors and reservoirs from endemic areas.}, }
@article {pmid40449999, year = {2025}, author = {Schweitzer, AY and Adams, EW and Nguyen, MTA and Lek, M and Isaacs, FJ}, title = {Precision multiplexed base editing in human cells using Cas12a-derived base editors.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {5061}, pmid = {40449999}, issn = {2041-1723}, support = {R01 GM117230/GM/NIGMS NIH HHS/United States ; GM117230//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; CF22-1046//Carlsbergfondet (Carlsberg Foundation)/ ; }, mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/metabolism/genetics ; Genome, Human ; HEK293 Cells ; Cell Line ; Bacterial Proteins ; }, abstract = {Base editors enable the direct conversion of target nucleotides without introducing DNA double strand breaks, making them a powerful tool for creating point mutations in a human genome. However, current Cas9-derived base editing technologies have limited ability to simultaneously edit multiple loci with base-pair level precision, hindering the generation of polygenic phenotypes. Here, we test the ability of six Cas12a-derived base editing systems to process multiple gRNAs from a single transcript. We identify base editor variants capable of multiplexed base editing and improve the design of the respective gRNA array expression cassette, enabling multiplexed editing of 15 target sites in multiple human cell lines, increasing state-of-the-art in multiplexing by three-fold in the field of mammalian genome engineering. To reduce bystander mutations, we also develop a Cas12a gRNA engineering approach that directs editing outcomes towards a single base-pair conversion. We combine these advances to demonstrate that both strategies can be combined to drive multiplex base editing with greater precision and reduced bystander mutation rates. Overcoming these key obstacles of mammalian genome engineering technologies will be critical for their use in studying single nucleotide variant-associated diseases and engineering synthetic mammalian genomes.}, }
@article {pmid40449852, year = {2025}, author = {Kolesova, E and Pulone, S and Kostyushev, D and Tasciotti, E}, title = {CRISPR/Cas bioimaging: From whole body biodistribution to single-cell dynamics.}, journal = {Advanced drug delivery reviews}, volume = {224}, number = {}, pages = {115619}, doi = {10.1016/j.addr.2025.115619}, pmid = {40449852}, issn = {1872-8294}, abstract = {This review explores the transformative role of CRISPR/Cas systems in optical bioimaging, emphasizing how advancements in nanoparticle (NP) technologies are revolutionizing the visualization of gene-editing processes both in vitro and in vivo. Optical imaging techniques, such as near-infrared (NIR) and fluorescence imaging, have greatly benefited from the integration of nanoformulated contrast agents, improving resolution, sensitivity, and specificity. CRISPR/Cas systems, originally developed just for gene editing, are now being coupled with these imaging modalities to enable real-time monitoring and quantitative measurements of metabolites, vitamins, proteins, nucleic acids and other entities in specific areas of the body, as well as tracking of CRISPR/Cas delivery, editing efficiency, and potential off-target effects. The development of CRISPR/Cas-loaded NPs allows for enhanced imaging and precise monitoring across multiple scales with multiplexed and multicolor imaging in complex settings, including potential in vivo diagnostics. CRISPR/Cas therapeutics as well as diagnostics are hindered by the lack of efficient and targeted delivery tools. Biomimetic NPs have emerged as promising tools for improving biocompatibility, enhancing targeting capabilities, and overcoming biological barriers, facilitating more efficient delivery and bioimaging of CRISPR/Cas systems in vivo. As the design of these NPs and delivery mechanisms improves, alongside advancements in endolysosomal escape, CRISPR/Cas-based bioimaging will continue to advance, offering unprecedented possibilities in precision medicine and theranostic applications.}, }
@article {pmid40448764, year = {2025}, author = {Jin, H and Sophocleous, A and Azfer, A and Ralston, SH}, title = {Analysis of Transcriptional Regulation in Bone Cells.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2885}, number = {}, pages = {247-269}, pmid = {40448764}, issn = {1940-6029}, mesh = {CRISPR-Cas Systems ; *Transcription, Genetic ; Promoter Regions, Genetic ; *Gene Expression Regulation ; Chromatin Immunoprecipitation/methods ; Humans ; Electrophoretic Mobility Shift Assay/methods ; Animals ; *Bone and Bones/cytology/metabolism ; *Osteoblasts/metabolism ; Mice ; Genes, Reporter ; }, abstract = {Transcription is a process by which the rate of RNA synthesis is regulated. Here we describe the techniques for carrying out promoter-reporter assays: electrophoretic mobility shift assays, chromosome conformation capture (3C) assays, chromatin immunoprecipitation assays, and CRISPR-Cas9 assay-five commonly used methods for studying and altering gene transcription.}, }
@article {pmid40448746, year = {2025}, author = {Kamli, H and Khan, NU}, title = {Revolutionising cancer intervention: the repercussions of CAR-T cell therapy on modern oncology practices.}, journal = {Medical oncology (Northwood, London, England)}, volume = {42}, number = {7}, pages = {228}, pmid = {40448746}, issn = {1559-131X}, mesh = {Humans ; *Neoplasms/therapy/immunology ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/immunology ; Gene Editing/methods ; Tumor Microenvironment/immunology ; CRISPR-Cas Systems ; Medical Oncology/methods/trends ; }, abstract = {Chimeric Antigen Receptor T-cell (CAR-T) therapy represents a groundbreaking advance in oncology, leveraging patient-specific immune cells to target malignant tumours precisely. By equipping T cells with synthetic receptors, CAR-T therapy achieves remarkable antitumor effects and offers hope for durable cancer control. However, several limitations persist, including antigen scarcity, immunosuppressive tumour microenvironments, and T-cell exhaustion. CRISPR-Cas9 gene editing has enhanced CAR-T potency by knocking out immune checkpoints (PD-1, CTLA-4) and improving persistence, while RNA interference (RNAi) silences immune-evasion genes (e.g. SOCS1). Nanozyme-based delivery systems enable precise CRISPR-Cas9 delivery (> 70% editing efficiency) and tumour targeting, overcoming instability and off-target effects. Innovations like SUPRA CARs, armoured CAR-T cells (e.g. IL-12/IL-21-secreting TRUCKs), and dual checkpoint inhibition synergize to reprogram the tumour microenvironment, reducing relapse by 40% in trials. Despite progress, high costs, manufacturing hurdles, and ethical concerns (e.g. germline editing risks) remain critical barriers. Emerging solutions include universal off-the-shelf CAR-Ts, hybrid nano-CRISPR systems, and AI-driven design, paving the way for scalable, personalised immunotherapy. This review highlights breakthroughs in CRISPR, RNAi, and nanotechnology, underscoring CAR-T therapy's transformative potential while addressing translational challenges for broader clinical adoption.}, }
@article {pmid40447846, year = {2025}, author = {Blaskovich, MAT and Cooper, MA}, title = {Antibiotics re-booted-time to kick back against drug resistance.}, journal = {npj antimicrobials and resistance}, volume = {3}, number = {1}, pages = {47}, pmid = {40447846}, issn = {2731-8745}, abstract = {After decades of neglect and a decline in antibiotic research and development, we are now finally witnessing the advent of new funding programs dedicated to new therapies. In addition to traditional new chemical entities that directly kill or arrest the growth of bacteria, alternative approaches are being identified and advanced towards proof-of-concept trials in the clinic. We briefly review the current pipeline of conventional new antibiotics and highlight in more depth promising alternatives, including potentiators of antibiotic action, bacteriophage, lysins and microbiome modulation. More innovative approaches, such as adaptive and innate immune modulators, CRISPR-Cas and diagnostic guided 'theranostics' are discussed and contrasted. Such exploratory therapies may require the development of alternative regulatory and clinical development pathways, but represent a potential circuit breaker from the current 'arms race' between bacteria and traditional antibiotics.}, }
@article {pmid40447637, year = {2025}, author = {Schwaemmle, H and Soldati, H and Lykoskoufis, NMR and Docquier, M and Hainard, A and Braun, SMG}, title = {CRISPR screen decodes SWI/SNF chromatin remodeling complex assembly.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {5011}, pmid = {40447637}, issn = {2041-1723}, support = {PCEFP3_194305//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; }, mesh = {Animals ; Mice ; *Chromatin Assembly and Disassembly/genetics ; *Chromosomal Proteins, Non-Histone/metabolism/genetics ; *Transcription Factors/metabolism/genetics ; Chromatin/metabolism/genetics ; RNA-Binding Proteins/metabolism/genetics ; Mouse Embryonic Stem Cells/metabolism ; *CRISPR-Cas Systems ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {The SWI/SNF (or BAF) complex is an essential chromatin remodeler, which is frequently mutated in cancer and neurodevelopmental disorders. These are often heterozygous loss-of-function mutations, indicating a dosage-sensitive role for SWI/SNF subunits. However, the molecular mechanisms regulating SWI/SNF subunit dosage to ensure complex assembly remain largely unexplored. We performed a CRISPR KO screen, using epigenome editing in mouse embryonic stem cells, and identified Mlf2 and Rbm15 as regulators of SWI/SNF complex activity. First, we show that MLF2, a poorly characterized chaperone protein, promotes SWI/SNF assembly and binding to chromatin. Rapid degradation of MLF2 reduces chromatin accessibility at sites that depend on high levels of SWI/SNF binding to maintain open chromatin. Next, we find that RBM15, part of the m[6]A writer complex, controls m[6]A modifications on specific SWI/SNF mRNAs to regulate subunit protein levels. Misregulation of m[6]A methylation causes overexpression of core SWI/SNF subunits leading to the assembly of incomplete complexes lacking the catalytic ATPase/ARP subunits. These data indicate that targeting modulators of SWI/SNF complex assembly may offer a potent therapeutic strategy for diseases associated with impaired chromatin remodeling.}, }
@article {pmid40447601, year = {2025}, author = {Hu, H and Guo, S and Li, Y and Dong, K and Lu, Y and Ye, K and Li, L and Zhou, X and Cheng, L and Xiao, X}, title = {Spatially blocked split CRISPR-Cas12a system for ultra-sensitive and versatile small molecule activation and detection.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {5035}, pmid = {40447601}, issn = {2041-1723}, mesh = {*CRISPR-Cas Systems/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; }, abstract = {Detecting small molecules is pivotal across fields like clinical diagnostics, environmental monitoring, and food safety. The CRISPR-Cas12a system, known for its simplicity and sensitivity, offers a promising basis for small molecule detection. However, current CRISPR-based detection methods face challenges, including complex design requirements, high background noise, and limited adaptability to different targets. In our study, we introduce the SBS-Cas system, leveraging a split crRNA mode to induce spatial hindrance on the scaffold strand through molecular binding. This approach prevents the assembly with Cas12a, effectively masking its trans-cleavage activity. By introducing small molecules that competitively bind to the macromolecule, we eliminate this spatial hindrance, activating Cas12a. Our results demonstrate high sensitivity, versatility, and adaptability in small molecule detection across multiple reactions, with successful intracellular imaging and responsive fluctuations in complex environments underscoring the system's robustness. This innovative CRISPR-Cas12a-based approach establishes a low-background, highly sensitive platform for small molecule detection. SBS-Cas promises not only to enhance tools for clinical, environmental, and food safety applications but also to advance CRISPR research, providing insights and expanding possibilities in molecular detection science.}, }
@article {pmid40447589, year = {2025}, author = {Liang, R and Wang, S and Cai, Y and Li, Z and Li, KM and Wei, J and Sun, C and Zhu, H and Chen, K and Gao, C}, title = {Circular RNA-mediated inverse prime editing in human cells.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {5057}, pmid = {40447589}, issn = {2041-1723}, mesh = {Humans ; *Gene Editing/methods ; *RNA, Circular/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; CRISPR-Associated Protein 9/genetics/metabolism ; Genome, Human ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {Prime editors are restricted to performing precise edits downstream of cleavage sites, thereby limiting their editing scope. Therefore, we develop inverse prime editors (iPEs) that act upstream of the nickase cleavage site by replacing nCas9-H840A with nCas9-D10A, but the editing efficiencies are limited. To address this limitation, we develop circular RNA-mediated iPEs (ciPEs), achieving editing efficiencies ranging from 0.1% to 24.7%. Further optimization using Rep-X helicase increases editing efficiencies to a range of 2.7%-55.4%. The Rep-X-assisted ciPE system thus expands the scope of editing and improves efficiencies at genomic sites that are previously difficult to target. The Rep-X-assisted ciPE system will complement canonical PE system in enabling more extensive and efficient editing across a wider range of the human genome.}, }
@article {pmid40447514, year = {2025}, author = {Yu, T and Zou, S and Long, Y and Ou, Y and Liu, S and Kang, T and Song, L and Sun, C and Liu, G}, title = {Glass fiber-interfaced CRISPR/Cas biosensing adaptable for diverse biomarker detection.}, journal = {Trends in biotechnology}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.tibtech.2025.05.001}, pmid = {40447514}, issn = {1879-3096}, abstract = {Developing a generic sensitive platform for detecting diverse biomarkers is essential for a comprehensive understanding of disease states, guiding precision medicine. Herein, we introduce a versatile platform based on glass fiber interfaced CRISPR/Cas with a universal reagent setting (g-CURS), which used a fixed pair of CRISPR RNA (crRNA) and a single-stranded DNA (ssDNA) activator to enable detection of multiple nucleic acids or proteins with ultrahigh sensitivity. The fixed ssDNA activator was labeled on multiple specific ligation products or detection antibodies conjugated on glass fiber to initiate CRISPR/Cas12a-assisted rapid and exponential cascade amplification through circular reporters (CRs), generating fluorescence signals readable by a portable detector. g-CURS was able to detect viral nucleic acids with attomolar sensitivity within 30 min and multiple low-abundance proteins in extracellular vesicles of Parkinson's disease (PD) serum with subpicomolar sensitivity within 80 min. g-CURS simplifies CRISPR/Cas biosensing using a standard reagent setting, holding promise for biomarker discovery free from bulky instruments.}, }
@article {pmid40447397, year = {2025}, author = {Gao, Y and Li, Y and Gao, Q and Zhang, T and Zhang, Y and Liu, T and Han, K}, title = {Multi-functional dumbbell DNA probe design and its application in signal amplification cascade -based assay of human immunodeficiency virus.}, journal = {Analytica chimica acta}, volume = {1362}, number = {}, pages = {344141}, doi = {10.1016/j.aca.2025.344141}, pmid = {40447397}, issn = {1873-4324}, mesh = {*DNA Probes/chemistry/genetics ; Humans ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; *DNA, Viral/analysis/genetics ; Limit of Detection ; *HIV/isolation &amp; purification/genetics ; }, abstract = {Sensitive assay of Human immunodeficiency virus (HIV) is the premise of accurate prevention and con-trol of AIDS. Dumbbell DNA showed promising potential in biosensing, imaging applications. This study presents a highly sensitive biosensing platform integrating dumbbell-shaped DNA probes with rolling circle transcription (RCT)-regulated CRISPR-Cas12a to achieve cascade signal amplification. The arrangement of the functional regions in the same unclosed dumbbell probes (UDPs) might affect final performance in the biosensor. Three different types UDPs (A/B/C) were specifically designed and the performances of the UDPs were evaluated in the RCT-CRISPR based cascade platform. Among these, type A UDP shows the best performance with a detection limit of 44.8 aM targeting HIV-DNA as well as high sensitivity and specificity. Also, type A UDP had the best behavior in the clinical sample assay. The difference of UDPs' performance might attribute to the variation of opening form of the UDPs considering the arrangement of various functional regions. The platform's modular design supports customization for diverse nucleic acid targets, making it adaptable for early disease detection and precision diagnostics.}, }
@article {pmid40446796, year = {2025}, author = {Yan, J and Luo, R and Rosen, BP and Liu, D and Wong, W and Leslie, CS and Huangfu, D}, title = {Discovery of NANOG enhancers and their essential roles in self-renewal and differentiation in human embryonic stem cells.}, journal = {Stem cell reports}, volume = {20}, number = {6}, pages = {102511}, pmid = {40446796}, issn = {2213-6711}, mesh = {Humans ; *Human Embryonic Stem Cells/cytology/metabolism ; *Cell Differentiation/genetics ; *Enhancer Elements, Genetic ; *Nanog Homeobox Protein/genetics/metabolism ; *Cell Self Renewal/genetics ; Animals ; Mice ; Cell Line ; CRISPR-Cas Systems ; }, abstract = {Human embryonic stem cells (hESCs) are notable for their ability to self-renew and to differentiate into all tissue types in the body. NANOG is a core regulator of hESC identity, and dynamic control of its expression is crucial to maintain the balance between self-renewal and differentiation. Transcriptional regulation depends on enhancers, but NANOG enhancers in hESCs are not well characterized. Here, we report two NANOG enhancers discovered from a CRISPR interference screen in hESCs. Deletion of a single copy of either enhancer significantly reduced NANOG expression, compromising self-renewal and increasing differentiation propensity. Interestingly, these two NANOG enhancers are involved in a tandem duplication event found in certain primates including humans but not in mice. However, the duplicated counterparts do not regulate NANOG expression. This work expands our knowledge of functional enhancers in hESCs and highlights the sensitivity of the hESC state to the dosage of core regulators and their enhancers.}, }
@article {pmid40446748, year = {2025}, author = {Zuo, Z and Liang, R and Fan, S and Shan, H and Zhang, H and Wang, X and Fei, T}, title = {Genome-wide CRISPR screens identify key regulators of adipogenesis and glucose uptake in beige adipocytes.}, journal = {Biochemical and biophysical research communications}, volume = {774}, number = {}, pages = {152093}, doi = {10.1016/j.bbrc.2025.152093}, pmid = {40446748}, issn = {1090-2104}, mesh = {*Adipogenesis/genetics ; *Glucose/metabolism ; *Adipocytes, Beige/metabolism/cytology ; Animals ; Humans ; Mice ; *CRISPR-Cas Systems ; Insulin Resistance ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {The beiging of white adipocytes enhances energy expenditure by utilizing fatty acids and glucose, offering therapeutic potential against obesity and type 2 diabetes. However, the genetic mechanisms driving this process remain unclear. Here, we performed multiple fluorescence-activated cell sorting (FACS)-based genome-wide CRISPR loss-of-function screens in beige adipocytes with or without insulin resistance (IR) induction, and systematically identified functional regulators of beige adipocyte adipogenesis and glucose metabolism. We further integrated transcriptomics and human genetics data to pinpoint key genes for adipogenesis and glucose metabolism in beige adipocytes. Moreover, we validated SULT2B1 and ATP1B2 as key adipogenesis genes for beige adipocytes, and COMMD7 gene as important regulator for glucose uptake against IR. These findings not only provide a comprehensive and valuable resource for cataloguing candidate functional genes underlying lipid and glucose homeostasis in beige adipocytes, but also offer potential therapeutic targets against metabolic disorders.}, }
@article {pmid40446205, year = {2025}, author = {Oikemus, S and Hu, K and Shin, M and Idrizi, F and Goodman-Khan, A and Kolb, A and Ghanta, KS and Lee, J and Wagh, A and Wolfe, SA and Zhu, LJ and Watts, JK and Lawson, ND}, title = {Identifying optimal conditions for precise knock-in of exogenous DNA into the zebrafish genome.}, journal = {Development (Cambridge, England)}, volume = {152}, number = {12}, pages = {}, doi = {10.1242/dev.204571}, pmid = {40446205}, issn = {1477-9129}, support = {R21OD030004//NIH Office of the Director/ ; R35HL171534/HL/NHLBI NIH HHS/United States ; 1UG3TR002668/NH/NIH HHS/United States ; R21OD030004/NH/NIH HHS/United States ; 1UG3TR002668/NH/NIH HHS/United States ; R35HL171534/NH/NIH HHS/United States ; //University of Massachusetts Medical School/ ; }, mesh = {Animals ; *Zebrafish/genetics ; *Genome/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; *DNA/genetics ; DNA Breaks, Double-Stranded ; Recombinational DNA Repair/genetics ; }, abstract = {CRISPR nucleases can be used to insert exogenous DNA into the zebrafish genome by homology-dependent repair (HDR), although germline transmission rates for precise edits remain quite low. Comparative studies to optimize HDR parameters for introducing base pair changes using short-read deep sequencing have been successful, but similar analysis for insertions is challenging due to read-length constraints. Here, we quantified editing outcomes using long-read sequencing to identify optimal template and CRISPR parameters for precise targeted insertion in zebrafish. Through side-by-side comparisons, we found that chemically modified templates out-perform those released in vivo from a plasmid, while Cas9 and Cas12a nucleases performed similarly for targeted insertion. Consistent with previous studies, precise editing rates were dependent on the distance between a double-strand break and the inserted sequence. We further found that non-homologous base pairs in homology templates significantly reduced precise editing rates. Using optimized parameters, we consistently achieved germline founder rates of greater than 20% for precise insertions across four loci. Together, our quantitative analyses identified optimal conditions for precise insertion of exogenous DNA into the zebrafish genome.}, }
@article {pmid40446005, year = {2025}, author = {Shi, L and Zhang, Y and Duan, Y and Sun, M and Yuan, C and Cao, L and Kong, X and Zhang, W and Zheng, H and Wang, Q}, title = {Genome-scale CRISPR screen identifies TMEM198 driving double membrane vesicle formation in swine alphacoronavirus and murine betacoronavirus infected cells.}, journal = {PLoS pathogens}, volume = {21}, number = {5}, pages = {e1013211}, pmid = {40446005}, issn = {1553-7374}, mesh = {Animals ; Mice ; Swine ; *Membrane Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Virus Replication ; *Alphacoronavirus/physiology/genetics ; Humans ; *Coronavirus Infections/virology/metabolism/genetics ; SARS-CoV-2 ; COVID-19/virology ; }, abstract = {COVID-19 pandemic caused by the SARS-CoV-2 which is well-publicized cross-species transmissibility. SARS-CoV-2 belongs to genus Betacoronavirus, several pathogenic alphacoronaviruses have shown similar patterns of emergence. Much less attention paid to host factors required for alphacoronavirus replication compared to those of betacoronaviruses. Here, we utilized a genome-wide CRISPR-Cas9-based screen to identify TMEM198 as a critical host protein for double-membrane vesicle (DMVs) formation during the replication of swine alphacoronavirus. Gene deletion of TMEM198 led to a reduction in the levels of viral infection in cells, whereas the ectopic expression of TMEM198 correspondingly resulted in an increase in infection levels. At the mechanistic level, TMEM198 directly binds to the C-terminal of nonstructural protein 3 (nsp3c) and nonstructural protein 4 (nsp4) to participate in the formation of DMVs. The first 35 amino acids at the N-terminal of TMEM198 are critical for the formation of DMVs and viral replication. Moreover, mice with a gene deletion of TMEM198 exhibit reduced susceptibility to the Betacoronavirus MHV. These results identify the function of TMEM198 in the formation of DMVs during the replication of swine alphacoronavirus and murine betacoronavirus.}, }
@article {pmid40445760, year = {2025}, author = {Lamperis, SM and McMahon, KM and Calvert, AE and Rink, JS and Vasan, K and Pandkar, MR and Crentsil, EU and Chalmers, ZR and McDonald, NR and Kosmala, CJ and Bonini, MG and Matei, D and Gordon, LI and Chandel, NS and Thaxton, CS}, title = {CRISPR screen reveals a simultaneous targeted mechanism to reduce cancer cell selenium and increase lipid oxidation to induce ferroptosis.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {122}, number = {22}, pages = {e2502876122}, pmid = {40445760}, issn = {1091-6490}, support = {T32GM142604//HHS | NIH (NIH)/ ; R01 ES035353/ES/NIEHS NIH HHS/United States ; T2022-018//V Foundation for Cancer Research (VFCR)/ ; T32 GM142604/GM/NIGMS NIH HHS/United States ; R01ES035353//HHS | NIH (NIH)/ ; P30 CA060553/CA/NCI NIH HHS/United States ; R01 CA216882/CA/NCI NIH HHS/United States ; R01CA216882//HHS | NIH (NIH)/ ; }, mesh = {*Ferroptosis/genetics/drug effects ; Humans ; Cell Line, Tumor ; Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism/genetics ; *Selenium/metabolism ; Oxidation-Reduction ; Female ; CRISPR-Cas Systems ; Lipid Peroxidation ; Scavenger Receptors, Class B/metabolism/genetics ; Lipoproteins, HDL/metabolism ; *Ovarian Neoplasms/metabolism/genetics/pathology ; Lipid Metabolism ; Nanoparticles/chemistry ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Ferroptosis is a cell death mechanism distinguished by its dependence on iron-mediated lipid oxidation. Cancer cells highly resistant to conventional therapies often demonstrate lipid metabolic and redox vulnerabilities that sensitize them to cell death by ferroptosis. These include a unique dependency on the lipid antioxidant selenoenzyme, glutathione peroxidase 4 (GPx4), that acts as a ferroptosis inhibitor. Synthetic high-density lipoprotein-like nanoparticle (HDL NP) targets the high-affinity HDL receptor scavenger receptor class B type 1 (SR-B1) and regulates cell and cell membrane lipid metabolism. Recently, we reported that targeting cancer cell SR-B1 with HDL NP depleted cell GPx4, which is accompanied by increased cell membrane lipid peroxidation and cancer cell death. These data suggest that HDL NP may induce ferroptosis. Thus, we conducted an unbiased CRISPR-based positive selection screen and target validation studies in ovarian clear cell carcinoma (OCCC) cell lines to ascertain the mechanism through which HDL NP regulates GPx4 and kills cancer cells. The screen revealed two genes, acyl-CoA synthetase long chain family member 4 (ACSL4) and thioredoxin reductase 1 (TXNRD1), whose loss conferred resistance to HDL NP. Validation of ACSL4 supports that HDL NP induces ferroptosis as the predominant mechanism of cell death, while validation of TXNRD1 revealed that HDL NP reduces cellular selenium and selenoprotein production, most notably, GPx4. Accordingly, we define cancer cell metabolic targets that can be simultaneously actuated by a multifunctional, synthetic HDL NP ligand of SR-B1 to kill cancer cells by ferroptosis.}, }
@article {pmid40445465, year = {2025}, author = {Hashemi, M and Khanaghah, XM and Nahand, JS}, title = {The CRISPR-Cas revolution in head and neck cancer: a new era of targeted therapy.}, journal = {Functional &amp; integrative genomics}, volume = {25}, number = {1}, pages = {113}, pmid = {40445465}, issn = {1438-7948}, mesh = {Humans ; *Head and Neck Neoplasms/therapy/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Therapy/methods ; Molecular Targeted Therapy ; Signal Transduction ; }, abstract = {Head and neck cancer (HNC) encompasses a diverse array of malignancies impacting the anatomical structures of the head and neck region, ranking as the seventh most prevalent cancer type globally. The occurrence and advancement of HNC are intricately linked to mutations and disruptions within critical signaling pathways, accentuating the imperative for targeted therapeutic interventions to rectify these genetic aberrations. Traditional treatment modalities, including surgical intervention and adjuvant chemotherapy or radiotherapy, frequently culminate in considerable morbidity and suboptimal prognoses. Recently, the CRISPR-Cas system has emerged as a revolutionary gene-editing platform, poised to redefine therapeutic approaches in gene therapy and oncological research. Despite its potential, CRISPR-Cas faces challenges such as off-target effects, delivery inefficiencies, and immunogenicity, which must be addressed for clinical success. This review meticulously evaluates the progress in CRISPR-Cas technologies aimed at targeting essential signaling pathways implicated in HNC, addressing current challenges while highlighting optimal targets, Cas nucleases, and innovative delivery mechanisms, thereby elucidating the therapeutic potential and clinical applicability of the CRISPR-Cas paradigm in the management of HNC.}, }
@article {pmid40442371, year = {2025}, author = {Nugent, PJ and Park, H and Wladyka, CL and Yelland, JN and Sinha, S and Chen, KY and Bynum, C and Quarterman, G and Lee, SC and Hsieh, AC and Subramaniam, AR}, title = {Decoding post-transcriptional regulatory networks by RNA-linked CRISPR screening in human cells.}, journal = {Nature methods}, volume = {22}, number = {6}, pages = {1237-1246}, pmid = {40442371}, issn = {1548-7105}, support = {GM119835//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; GM008268//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; CA230617//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; CA276308//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; GM135362//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 1846521//National Science Foundation (NSF)/ ; }, mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Regulatory Networks ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; HEK293 Cells ; Ribosomes/metabolism ; RNA, Messenger/genetics/metabolism ; *RNA/genetics/metabolism ; *RNA Processing, Post-Transcriptional ; }, abstract = {RNAs undergo a complex choreography of metabolic processes that are regulated by thousands of RNA-associated proteins. Here we introduce ReLiC, a scalable and high-throughput RNA-linked CRISPR approach to measure the responses of diverse RNA metabolic processes to knockout of 2,092 human genes encoding all known RNA-associated proteins. ReLiC relies on an iterative strategy to integrate genes encoding Cas9, single-guide RNAs (sgRNAs) and barcoded reporter libraries into a defined genomic locus. Combining ReLiC with polysome fractionation reveals key regulators of ribosome occupancy, uncovering links between translation and proteostasis. Isoform-specific ReLiC captures differential regulation of intron retention and exon skipping by SF3B complex subunits. Chemogenomic ReLiC screens decipher translational regulators upstream of messenger RNA (mRNA) decay and identify a role for the ribosome collision sensor GCN1 during treatment with the anti-leukemic drug homoharringtonine. Our work demonstrates ReLiC as a powerful framework for discovering and dissecting post-transcriptional regulatory networks in human cells.}, }
@article {pmid40442121, year = {2025}, author = {Ma, S and Liao, K and Chen, K and Cheng, T and Yang, X and Chen, P and Li, S and Li, M and Zhang, X and Zhang, Y and Huang, T and Wang, X and Wang, L and Lin, Y and Rong, Z}, title = {hpCasMINI: An engineered hypercompact CRISPR-Cas12f system with boosted gene editing activity.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {5001}, pmid = {40442121}, issn = {2041-1723}, support = {82370078//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82200072//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82304008//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82303995//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82370003//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals ; Humans ; Mice ; HEK293 Cells ; Fibroblast Growth Factors/genetics/metabolism ; Genetic Therapy/methods ; DNA Cleavage ; Tumor Suppressor Protein p53/genetics ; Dependovirus/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; Liver/metabolism ; PTEN Phosphohydrolase/genetics ; }, abstract = {Compact CRISPR-Cas systems have demonstrated potential for effective packaging into adeno-associated viruses (AAVs) for use in gene therapy. However, their applications are currently limited due to modest gene-editing activity. Here we introduce an engineered compact CRISPR-Cas12f (hpCasMINI, 554 aa), with hyper editing efficiency in mammalian cells via adding an α-helix structure to the N-terminus of an Un1Cas12f1 variant CasMINI (529 aa). The hpCasMINI system boosts gene activation and DNA cleavage activity with about 1.4-3.0-fold and 1.1-19.5-fold, respectively, and maintains the high specificity when compared to CasMINI. In addition, the system can activate luciferase reporter gene and endogenous Fgf21 gene in adult mouse liver, as well as construct liver tumorigenesis model via disrupting Trp53 and Pten genes and inserting oncogenic Kras[G12D] into the Trp53 locus. When compared to SpCas9 and LbCas12a, hpCasMINI displays higher gene activation and exhibits higher DNA cleavage specificity, although with lower activity, at the tested sites. Moreover, with a similar strategy, we engineer compact versions of hpOsCas12f1 (458 aa) from enOsCas12f1 and hpAsCas12f1 (447 aa) from AsCas12f1-HKRA, both of which display increased DNA cleavage activity, with hpAsCas12f1 also showing improved gene activation capability. Therefore, we develop activity-increased miniature hpCasMINI, hpOsCas12f1 and hpAsCas12f1 nucleases, which hold great potential for gene therapy in the future.}, }
@article {pmid40440869, year = {2025}, author = {Zhang, R and Zhou, Q and Huang, S and Zhang, N and Sun, D}, title = {Advancements in CRISPR-Cas-based strategies for combating antimicrobial resistance.}, journal = {Microbiological research}, volume = {298}, number = {}, pages = {128232}, doi = {10.1016/j.micres.2025.128232}, pmid = {40440869}, issn = {1618-0623}, mesh = {*CRISPR-Cas Systems ; *Bacteria/genetics/drug effects ; Gene Editing/methods ; *Anti-Bacterial Agents/pharmacology ; *Drug Resistance, Multiple, Bacterial/genetics ; Humans ; *Drug Resistance, Bacterial/genetics ; Bacterial Infections/drug therapy/microbiology ; Plasmids/genetics ; Genome, Bacterial ; }, abstract = {Multidrug resistance (MDR) in bacteria presents a significant global health threat, driven by the widespread dissemination of antibiotic-resistant genes (ARGs). The CRISPR-Cas system, known for its precision and adaptability, holds promise as a tool to combat antimicrobial resistance (AMR). Although previous studies have explored the use of CRISPR-Cas to target bacterial genomes or plasmids harboring resistance genes, the application of CRISPR-Cas-based antimicrobial therapies is still in its early stages. Challenges such as low efficiency and difficulties in delivering CRISPR to bacterial cells remain. This review provides an overview of the CRISPR-Cas system, highlights recent advancements in CRISPR-Cas-based antimicrobials and delivery strategies for combating AMR. The review also discusses potential challenges for the future development of CRISPR-Cas-based antimicrobials. Addressing these challenges would enable CRISPR therapies to become a practical solution for treating AMR infections in the future.}, }
@article {pmid40440223, year = {2025}, author = {Bachler, A and Padovan, A and Anderson, CJ and Wei, Y and Wu, Y and Pearce, S and Downes, S and James, B and Tessnow, AE and Sword, GA and Williams, M and Tay, WT and Gordon, KHJ and Walsh, TK}, title = {Disruption of HaVipR1 confers Vip3Aa resistance in the moth crop pest Helicoverpa armigera.}, journal = {PLoS biology}, volume = {23}, number = {5}, pages = {e3003165}, pmid = {40440223}, issn = {1545-7885}, mesh = {Animals ; *Moths/genetics/drug effects ; *Insecticide Resistance/genetics ; *Bacterial Proteins/pharmacology/genetics ; Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Crops, Agricultural/genetics/parasitology ; Pest Control, Biological/methods ; Plants, Genetically Modified/genetics ; Bacillus thuringiensis/genetics ; Helicoverpa armigera ; }, abstract = {The global reliance on Bacillus thuringiensis (Bt) proteins for controlling lepidopteran pests in cotton, corn, and soybean crops underscores the critical need to understand resistance mechanisms. Vip3Aa, one of the most widely deployed and currently effective Bt proteins in genetically modified crops, plays a pivotal role in pest management. This study investigates the molecular basis of Vip3Aa resistance in Australian Helicoverpa armigera through genetic crosses, and integrated genomic and transcriptomic analyses. We identified a previously uncharacterized gene, LOC110373801 (designated HaVipR1), as potentially important in Vip3Aa resistance in two field-derived resistant lines. Functional validation using CRISPR/Cas9 knockout in susceptible lines confirmed the gene's role in conferring high-level resistance to Vip3Aa. Despite extensive laboratory selection of Vip3Aa-resistant colonies in Lepidoptera, the biochemical mechanisms underlying resistance have remained elusive. Our research identifies HaVipR1 as a potential contributor to resistance, adding to our understanding of how insects may develop resistance to this important Bt protein. The identification of HaVipR1 contributes to our understanding of potential resistance mechanisms and may inform future resistance management strategies. Future work should explore the biochemical pathways influenced by HaVipR1 and assess its interactions with other resistance mechanisms. The approach utilized here underscores the value of field-derived resistant lines for understanding resistance in agricultural pests and highlights the need for targeted approaches to manage resistance sustainably.}, }
@article {pmid40439284, year = {2025}, author = {Ukita, Y and Suzuki, R and Miyoshi, K and Saito, K and Okumura, M and Chihara, T}, title = {Generation of Odorant Receptor-QF2 Knock-In Drivers for Improved Analysis of Olfactory Circuits in Drosophila.}, journal = {Genes to cells : devoted to molecular &amp; cellular mechanisms}, volume = {30}, number = {4}, pages = {e70028}, pmid = {40439284}, issn = {1365-2443}, support = {21H02479//Japan Society for the Promotion of Science/ ; 24K02062//Japan Society for the Promotion of Science/ ; 20K15903//Japan Society for the Promotion of Science/ ; JP23KJ1645//Japan Society for the Promotion of Science/ ; }, mesh = {Animals ; *Receptors, Odorant/genetics/metabolism ; *Drosophila melanogaster/genetics/metabolism ; *Drosophila Proteins/genetics/metabolism ; Gene Knock-In Techniques/methods ; CRISPR-Cas Systems ; Animals, Genetically Modified ; Olfactory Receptor Neurons/metabolism ; *Olfactory Pathways/metabolism ; }, abstract = {Drosophila melanogaster has provided numerous insights into the olfactory system, primarily relying on a series of transgenic Gal4 drivers. The combined use of Gal4/UAS and a second binary expression system, such as the QF/QUAS system, provides the opportunity to manipulate the two distinct cell populations, thereby accelerating the elucidation of the olfactory neural mechanisms. However, resources apart from the Gal4/UAS system have been poorly developed. In this study, we generated a series of odorant receptor (Or)-QF2 knock-in driver (Or-QF2[KI]) lines for 23 Ors using the CRISPR/Cas9 knock-in method. In these lines, the QF2 protein is cotranslated with each Or product. The expression pattern of the Or-QF2[KI] drivers mostly corresponded to that of the Or-Gal4 drivers. In addition, the Or42a-QF2[KI] driver identified the additional expression pattern of Or42a, which is consistent with the data of single-nucleus RNA sequencing and is attributed to the Or-QF2[KI] drivers' ability to reflect the endogenous expression of the Or genes. Thus, these Or-QF2[KI] drivers can be used as valuable genetic tools for olfactory research in Drosophila.}, }
@article {pmid40438494, year = {2025}, author = {Shao, Q and Ndzie Noah, ML and Golubnitschaja, O and Zhan, X}, title = {Mitochondrial medicine: "from bench to bedside" 3PM-guided concept.}, journal = {The EPMA journal}, volume = {16}, number = {2}, pages = {239-264}, pmid = {40438494}, issn = {1878-5077}, abstract = {Mitochondria are the primary sites for aerobic respiration and play a vital role in maintaining physiologic function at the cellular and organismal levels. Physiologic mitochondrial homeostasis, functions, health, and any kind of mitochondrial impairments are associated with systemic effects that are linked to the human health and pathologies. Contextually, mitochondria are acting as a natural vital biosensor in humans controlling status of physical and mental health in a holistic manner. So far, no any disorder is known as happening to humans independently from a compromised mitochondrial health as the cause (primary mitochondrial dysfunction) or a target of collateral damage (secondary mitochondrial injury). This certainty makes mitochondrial medicine be the superior instrument to reach highly ambitious objectives of predictive, preventive, and personalized medicine (PPPM/3PM). 3PM effectively implements the paradigm change from the economically ineffective reactive medical services to a predictive approach, targeted prevention and treatments tailored to individualized patient profiles in primary (protection against health-to-disease transition) and secondary (protection against disease progression) healthcare. Mitochondrial DNA (mtDNA) properties differ significantly from those of nuclear DNA (nDNA). For example, mtDNA as the cell-free DNA molecule is much more stable compared to nDNA, which makes mtDNA be an attractive diagnostic target circulating in human body fluids such as blood and tear fluid. Further, genetic variations in mtDNA contribute to substantial individual differences in disease susceptibility and treatment response. To this end, the current gene editing technologies, such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas, are still immature in mtDNA modification, and cannot be effectively applied in clinical practice posing a challenge for mtDNA-based therapies. In contrast, comprehensive multiomics technologies offer new insights into mitochondrial homeostasis, health, and functions, which enables to develop more effective multi-level diagnostics and targeted treatment strategies. This review article highlights health- and disease-relevant mitochondrial particularities and assesses involvement of mitochondrial medicine into implementing the 3PM objectives. By discussing the interrelationship between 3PM and mitochondrial medicine, we aim to provide a foundation for advancing early and predictive diagnostics, cost-effective targeted prevention in primary and secondary care, and exemplify personalized treatments creating proof-of-concept approaches for 3PM-guided clinical applications.}, }
@article {pmid40437542, year = {2025}, author = {Li, C and Zha, H and Jiao, Z and Wei, K and Gao, H and Lai, F and Zhou, Z and Luo, H and Li, P}, title = {Genetic engineering of E. coli K-12 for heterologous carbohydrate antigen production.}, journal = {Microbial cell factories}, volume = {24}, number = {1}, pages = {126}, pmid = {40437542}, issn = {1475-2859}, support = {CSTB2024NSCQ-MSX1157//Natural Science Foundation of Chongqing, China/ ; SWU-KT23010//Fundamental Research Funds for the Central Universities/ ; 32300736//National Natural Science Foundation of China/ ; }, mesh = {*Genetic Engineering/methods ; Animals ; *Escherichia coli K12/genetics/metabolism ; Mice ; Multigene Family ; Mice, Inbred BALB C ; }, abstract = {BACKGROUND: Carbohydrate-based vaccines have made a remarkable impact on public health over the past three decades. Efficient production of carbohydrate antigens is a crucial prerequisite for the development of such vaccines. The enzymes involved in the synthesis of bacterial surface carbohydrate antigens are usually encoded by large, uninterrupted gene clusters. Non-pathogenic E. coli glycoengineering starts with the genetic manipulation of these clusters. Heterologous gene cluster recombination through an expression plasmid has several drawbacks, including continuous antibiotic selection pressure, genetic instability, and metabolic burdens. In contrast, chromosome-level gene cluster expression can minimize the metabolic effects on the host and reduce industrial costs.<br><br>RESULTS: In this study, we employed the suicide vector-mediated allelic exchange method to directly replace the native polysaccharide gene clusters in E. coli with heterologous ones. Unlike previously strategies, this method does not rely on I-SceI endonuclease or CRISPR/Cas system to release the linearized DNA insert and &#x3bb;-red recombinase to promote its homologous recombination. Meanwhile, the vectors could be conveniently constructed by assembling multiple large DNA fragments in order in vitro. The scarless chromosomal insertions were confirmed by whole-genome sequencing and the polysaccharide phenotypes of all glycoengineered E. coli mutants were evaluated through growth curves, silver staining, western blot, and flow cytometry. The data indicated that there was no obvious metabolic burden associated with the insertion of large gene clusters into the E. coli W3110 O-antigen locus, and the glycoengineered E. coli can produce LPS with a recovery rate around 1% of the bacterial dry weight. Moreover, the immunogenicity of the heterologously expressed carbohydrate antigens was analyzed by mice immunization experiments. The ELISA data demonstrated the successful induction of anti-polysaccharide IgM or IgG antibodies.<br><br>CONCLUSIONS: We have provided a convenient and reliable genomic glycoengineering method to produce efficacious, durable, and cost-effective carbohydrate antigens in non-pathogenic E. coli. Non-pathogenic E. coli glycoengineering has great potential for the highly efficient synthesis of heterologous polysaccharides and can serve as a versatile platform to produce next-generation biomedical agents, including glycoconjugate vaccines, glycoengineered minicells or outer membrane vesicles (OMVs), polysaccharide-based diagnostic reagents, and more.}, }
@article {pmid40437234, year = {2025}, author = {Vanhooydonck, M and De Neef, E and De Saffel, H and Boel, A and Willaert, A and Callewaert, B and Claes, KBM}, title = {Prime editing outperforms homology-directed repair as a tool for CRISPR-mediated variant knock-in in zebrafish.}, journal = {Lab animal}, volume = {54}, number = {6}, pages = {165-172}, pmid = {40437234}, issn = {1548-4475}, support = {BOF21/DOC/242//Universiteit Gent (UGent)/ ; BOF GOA019-21//Universiteit Gent (UGent)/ ; }, mesh = {Animals ; *Zebrafish/genetics ; *Gene Knock-In Techniques/methods ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Recombinational DNA Repair ; Animals, Genetically Modified ; }, abstract = {Zebrafish serve as a valuable model organism for studying human genetic diseases. While generating knockout lines is relatively straightforward, introducing precise disease-specific genetic variants by knock-in (KI) remains challenging. KI lines, however, enable more accurate studies of molecular and physiological consequences of genetic diseases. Their generation is often hampered by low editing efficiency (EE) and potential off-target effects. Here, we optimized conventional CRISPR-Cas9-mediated homology-directed repair (HDR) strategies for precise KI of genetic variants in zebrafish and compared their efficacy with prime editing, a recently developed technique that is not yet commonly used. Using next-generation sequencing, we determined KI EE by HDR for six unique base-pair substitutions in three different zebrafish genes. We assessed the effect of (1) varying Cas9 amounts, (2) HDR templates with chemical modifications to improve integration efficiency, (3) different microinjection procedures and (4) introduction of additional synonymous guide-blocking variants in the HDR template. Increasing Cas9 amounts augmented KI EE, with optimal injected amounts of Cas9 between 200 pg and 800 pg. The use of Alt-R HDR templates further increased KI EE, while guide-blocking modifications did not. Injecting components directly into the cell was not superior to injections into the yolk. Prime editing, however, increased EE up to fourfold and expanded the F0 founder pool for four targets compared with conventional HDR editing, with fewer off-target effects. Therefore, prime editing is a very promising methodology for improving the creation of precise genomic edits in zebrafish, facilitating the modeling of human diseases.}, }
@article {pmid40436684, year = {2025}, author = {Jin, YY and Zhang, P and Liu, DP}, title = {Optimizing homology-directed repair for gene editing: the potential of single-stranded DNA donors.}, journal = {Trends in genetics : TIG}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.tig.2025.04.014}, pmid = {40436684}, issn = {0168-9525}, abstract = {CRISPR (clustered regularly interspaced short palindromic repeat) system-based precise genome editing remarkably impacts both scientific investigation and therapeutic practices. Among various techniques, DNA donor-mediated homology-directed repair (HDR) represents a promising method for precise gene editing. Although efficiency constraints have previously limited HDR, recent advancements have significantly enhanced its effectiveness. Therefore, it is essential to highlight the progress made in this field and to reassess the potential of the HDR approach. In this review, we explore the fundamental principles of HDR-dependent gene editing and evaluate current strategies to enhance HDR efficiency, with particular emphasis on single-stranded DNA (ssDNA) donor-mediated HDR. Finally, we discuss the prospects of high-efficiency ssDNA donor-mediated precise gene editing in laboratory research and clinical therapies.}, }
@article {pmid40436222, year = {2025}, author = {Amiri, M and Shojaei, A and Mashayekhan, S and Saeedi, S and Kamali, B and Biabanaki, ZS and Mousazadeh, S and Kiani, J and Molabashi, ZA and Karimi, M}, title = {Synthesis of low molecular weight polyethylene imine-polyamidoamine hybrid and its modified derivatives as efficient nanocarriers for pDNA and CRISPR/Cas9 delivery.}, journal = {International journal of pharmaceutics}, volume = {680}, number = {}, pages = {125778}, doi = {10.1016/j.ijpharm.2025.125778}, pmid = {40436222}, issn = {1873-3476}, mesh = {Humans ; Animals ; HEK293 Cells ; *DNA/administration &amp; dosage ; *Dendrimers/chemistry/administration &amp; dosage ; CRISPR-Cas Systems ; *Polyethyleneimine/chemistry ; *Polyamines/chemistry ; Mice ; Molecular Weight ; *Gene Transfer Techniques ; *Nanoparticles/chemistry/administration &amp; dosage ; MCF-7 Cells ; *Plasmids/administration &amp; dosage ; HCT116 Cells ; Mice, Inbred C57BL ; *Polyethylenes/chemistry ; *Imines/chemistry ; Histidine/chemistry ; Mesenchymal Stem Cells/metabolism ; *Drug Carriers/chemistry ; Cell Survival/drug effects ; }, abstract = {A branched copolymer based on polyethylene imine (PEI) 1.8 kDa and the first generation of polyamidoamine dendrimer (PAMAM G1) was synthesized as a low toxicity nanocarrier. This hybrid material, designated here by G1PEI, was used as a parent nanostructure for the next step to synthesize a series of new nanocarriers by attaching L-arginine (Arg), L-histidine (His), and heptafluorobutyric anhydride (F7) on G1PEI; in both cases of single modifier or in combination together. Positive zeta accompanied by gel retardation assay confirmed the suitable gene loading capacity of the nanocarriers with a minimum of 0.8:1 wt ratio. For the sake of screening, the biological performance of the nanocarriers was first evaluated in HEK-293 T cell line at various weight ratios. Accordingly, the best nanovectors were selected for pDNA delivery in MCF-7 and HCT-116 cell lines, as well as for CRISPR/Cas9 delivery in mesenchymal stem cells derived from the bone marrow of C57BL/6 green laboratory mice. Flow cytometry and fluorescence microscopic imaging revealed that G1PEI grafted with 10 groups of F7 (G1PEI-10F7), 5 groups of ArgF7 (G1PEI-5ArgF7), and 3 groups of ArgF7 accompanied with 2 groups of His (G1PEI-3ArgF7-2His) exhibited higher gene transfection efficiency than PEI 25 kDa (known as a golden standard in biological systems) in plasmid delivery and knockout, with observed knockout efficiencies ranging between 54 % to 90 %. Moreover, MTT tests demonstrated the lower toxicity of the nanocarriers compared to PEI 25 kDa.}, }
@article {pmid40435597, year = {2025}, author = {Kim, HJ and Park, DG and Choi, SJ and Cho, SD}, title = {PCSK9 is a passenger gene in head and neck cancer with minimal pathological influence.}, journal = {Archives of oral biology}, volume = {176}, number = {}, pages = {106302}, doi = {10.1016/j.archoralbio.2025.106302}, pmid = {40435597}, issn = {1879-1506}, mesh = {Humans ; *Proprotein Convertase 9/genetics/metabolism ; *Head and Neck Neoplasms/genetics/pathology ; Cell Proliferation/genetics ; Cell Line, Tumor ; Apoptosis/genetics ; Cell Movement ; CRISPR-Cas Systems ; Blotting, Western ; Gene Expression Regulation, Neoplastic ; }, abstract = {OBJECTIVES: In this study, we aimed to explore the pathological effects of PCSK9 in head and neck cancer (HNC) by ablating its expression using the CRISPR-Cas9 knockout system.<br><br>DESIGN: To investigate the clinical importance of PCSK9 in HNC, in silico analysis was performed using datasets from the GEO database and the UALCAN database. To evaluate the role of PCSK9 in HNC pathogenesis, both CRISPR-Cas9 knockout system and pharmacological inhibition were employed to ablate PCSK9 expression in HNC cell lines. The impact of PCSK9 on cellular growth and proliferation was assessed using Cell Counting Kit-8, soft agar, and clonogenic assays in a 2D culture model, as well as a hanging drop spheroid formation assay with live/dead staining in a 3D culture model. The involvement of PCSK9 in apoptosis induction was evaluated by detecting c-PARP expression through Western blotting, measuring the sub-G1 population via cell cycle assay, and verifying the Annexin V-positive population. Finally, changes in metastatic profiles associated with fluctuations in PCSK9 expression were examined using wound healing and transwell migration/invasion assays.<br><br>RESULTS: In in silico analysis results, PCSK9 appeared to be related to the progression of HNC. However, experimental results demonstrated that PCSK9 plays a minimal role in cancer cell proliferation, anchorage-independent growth, colony formation capacity, in 2D cultures, as well as spheroidal growth in 3D cultures, and apoptosis induction. Furthermore, PCSK9 marginally influenced wound closure, metastatic potential, and invasive ability.<br><br>CONCLUSION: Collectively, these data suggest that PCSK9 serves a neutral role in HNC, functioning as a passenger gene.}, }
@article {pmid40435438, year = {2025}, author = {Shu, F and Huang, Y and Yang, F and Guo, Y and Xu, R and Xiao, L and Feng, Y and Li, N}, title = {Calcium-dependent protein kinases 2A involved in the growth of both asexual and sexual stages of Cryptosporidium parvum.}, journal = {PLoS neglected tropical diseases}, volume = {19}, number = {5}, pages = {e0013107}, pmid = {40435438}, issn = {1935-2735}, mesh = {*Cryptosporidium parvum/growth &amp; development/enzymology/genetics ; Animals ; Cryptosporidiosis/parasitology ; Mice ; *Protein Kinases/genetics/metabolism ; CRISPR-Cas Systems ; *Protozoan Proteins/genetics/metabolism ; Oocysts/growth &amp; development ; Female ; Humans ; Life Cycle Stages ; Sporozoites/growth &amp; development ; }, abstract = {BACKGROUND: Cryptosporidium parvum is a protozoan pathogen that causes moderate to severe diarrhea in both humans and animals. Calcium-dependent protein kinases (CDPKs) are attractive drug targets against cryptosporidiosis given their critical role in the life cycle of Cryptosporidium spp. and their absence in human and animal hosts.<br><br>We used CRISPR-Cas9 technology to endogenously tag the CpCDPK2A gene in C. parvum IIdA20G1-HLJ strain with the hemagglutinin (HA) epitope and to delete the CpCDPK2A gene. An immunofluorescence assay was performed to localize the CpCDPK2A expression in the tagged strain and a luciferase assay was performed to compare growth rates of the tagged and deletion strains in vitro. Oocyst shedding, parasite load, villus length/crypt height ratio and survival of infected mice were used to evaluate the function of CpCDPK2A in vivo. The results revealed that CpCDPK2A was expressed in all the intracellular developmental stages, especially in the motile stages of sporozoites and merozoites. While CpCDPK2A is dispensable, deletion of the gene significantly reduced the growth of late asexual and sexual stages in vitro. In an interferon-&#x3b3; knockout mouse model, gene deletion of CpCDPK2A reduced oocyst shedding by 25-fold and increased survival of infected mice.<br><br>CONCLUSIONS/SIGNIFICANCE: These observations suggest that CpCDPK2A may contribute to both asexual and sexual replication of C. parvum and may be a potential target to block the transmission of this important zoonotic pathogen.}, }
@article {pmid40434364, year = {2025}, author = {Hecht, AD and Igoshin, OA}, title = {Kinetic Mechanism for Fidelity of CRISPR-Cas9 Variants.}, journal = {The journal of physical chemistry letters}, volume = {16}, number = {22}, pages = {5570-5578}, doi = {10.1021/acs.jpclett.5c01154}, pmid = {40434364}, issn = {1948-7185}, mesh = {Kinetics ; *CRISPR-Cas Systems ; Fluorescence Resonance Energy Transfer ; Substrate Specificity ; *CRISPR-Associated Protein 9/metabolism/chemistry/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; DNA/chemistry/metabolism ; }, abstract = {CRISPR-Cas9 is a nuclease creating DNA breaks at sites with sufficient complementarity to the RNA guide. Notably, Cas9 does not require exact RNA-DNA complementarity and can cleave off-target sequences. Various high-accuracy Cas9 variants have been developed, but the precise mechanism of how these variants achieve higher accuracy remains unclear. Here, we develop a kinetic model of Cas9 substrate selection and cleavage parametrized by data from the literature, including single-molecule Förster resonance energy transfer (FRET) measurements. Based on observed FRET transition statistics, we predict that the Cas9 substrate recognition and cleavage mechanism must allow for HNH domain transitions independent of substrate binding. Additionally, we show that the enhancement in Cas9 substrate specificity must be due to changes in kinetics rather than changes in substrate binding affinities. Finally, we use our model to identify kinetic parameters for HNH domain transitions that can be perturbed to enable high-accuracy cleavage while maintaining cleavage speeds.}, }
@article {pmid40432879, year = {2025}, author = {Melchior, F and Angelidou, IA and Chorianopoulou, M and Teichmann, B}, title = {The genetic technologies questionnaire in the Greek-speaking population: the moral judgement of the lay public.}, journal = {Frontiers in genetics}, volume = {16}, number = {}, pages = {1594724}, pmid = {40432879}, issn = {1664-8021}, abstract = {INTRODUCTION: Advancements in life sciences have significantly boosted biomedical capabilities. Genetic testing forecasts hereditary traits and disease susceptibility, while CRISPR/Cas allows permanent genome alterations. However, ethical considerations arise regarding the morality of these capabilities, particularly concerning the moral status, autonomy, and privacy of living beings. The lack of valid instruments to assess moral judgment in genetic technologies highlights the need for this study, aiming to translate and validate the "Genetic Technologies Questionnaire" (GTQ) and the short version of the "Conventional Technologies Questionnaire" (CTQ5) into Greek. As the full version of the GTQ with 30 questions could be too extensive for some studies, we also tested other versions: The short versions GTQ20-GR and GTQ5-GR which were already presented in the original study, as well as a version which included questions solely about humans (GTQ-H-GR) and is intended for use in human research and therapy, and the GTQ-Moral Status (GTQ-MS-GR), which included questions about genetic testing and gene editing in different living beings to investigate differences in moral status.<br><br>METHODS: A cross-sectional study involved 250 participants who completed an online questionnaire, assessing internal consistency, structural validity, known-groups validity, floor/ceiling effects, and retest reliability (subset of 50 participants). Correlational analyses explored relationships with education, age, genetic knowledge, religiosity, and genetic testing experience. The study followed the STROBE checklist for reporting.<br><br>RESULTS: The GTQ-GR (Cronbach's &#x3b1; = 0.929) and GTQ20-GR (&#x3b1; = 0.935) exhibit high reliability and stability in assessing moral judgment among lay people, whereas the GTQ5-GR (&#x3b1; = 0.866) and CTQ5-GR (&#x3b1; = 0.758) displayed some weaknesses. Participants tended to rate conventional technologies more favorably than genetic technologies, with genetic testing perceived more positively than genome editing. The two additional derived versions, GTQ-H-GR (&#x3b1; = 0.859) and GTQ-MS-GR (&#x3b1; = 0.787), also demonstrated solid psychometric characteristics.<br><br>CONCLUSION: The GTQ-GR is a valid and reliable questionnaire with strong psychometric properties and is now available in Greek.}, }
@article {pmid40431732, year = {2025}, author = {Meng, P and Ni, B and Li, C and Sha, Z and Liu, C and Ren, W and Wei, R and Liu, F and Li, J and Wang, Z}, title = {Establishment and Implementation of the Point-of-Care RT-RAA-CRISPR/Cas13a Diagnostic Test for Foot-And-Mouth Disease Virus Serotype O in Pigs.}, journal = {Viruses}, volume = {17}, number = {5}, pages = {}, pmid = {40431732}, issn = {1999-4915}, support = {2021YFD1800300//National Key R&amp;D Program of China/ ; }, mesh = {Animals ; Swine ; *Foot-and-Mouth Disease Virus/genetics/isolation &amp; purification/classification ; *Swine Diseases/diagnosis/virology ; *Foot-and-Mouth Disease/diagnosis/virology ; Serogroup ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Point-of-Care Systems ; RNA, Viral/genetics ; *Molecular Diagnostic Techniques/methods ; }, abstract = {Foot and mouth disease virus (FMDV) is a highly pathogenic virus that mainly infects cloven hooved animals, such as pigs. The establishment of a rapid, sensitive and accurate point-of-care detection method is critical for the timely identification and elimination of infected pigs for controlling this disease. In this study, a RT-RAA-CRISPR/Cas13a method was developed for the detection of FMDV serotype O in pigs. Six pairs of RT-RAA primers were designed based on the conserved gene sequence of FMDV serotype O, and the optimal amplification primers and reaction temperatures were screened. The CRISPR-derived RNA (crRNA) was further designed based on the optimal target band sequence and the most efficient crRNA was screened. The results revealed that FMDV-O-F4/R4 was the optimal primer set, and the optimal temperature for the RT-RAA reaction was 37 °C. Moreover, crRNA4 exhibited the strongest detection signal among the six crRNAs. The established RT-RAA-CRISPR/Cas13a method demonstrated high specificity and no cross-reactivity with other common swine pathogens such as Senecavirus A (SVA), porcine reproductive and respiratory virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), and pseudorabies virus (PRV), additionally, it was observed to be highly sensitive, with a detection limit of 19.1 copies/µL. The repeatability of this method was also observed to be good. This method could produce stable fluorescence and exhibited good repeatability when three independent experiments yielded the same results. A validation test using three types of simulated clinical samples (including swab, tissue, and serum samples) revealed a 100% concordance rate. The detection results could be visualized via a fluorescence reader or lateral flow strips (LFSs). Thus, a highly specific and sensitive RT-RAA-CRISPR/Cas13a detection method was developed and is expected to be applied for the rapid detection of FMDV serotype O in situ.}, }
@article {pmid40429982, year = {2025}, author = {Leal, K and Machuca, J and Gajardo, H and Palma, M and Contreras, MJ and Nuñez-Montero, K and Gutiérrez, &#xc1; and Barrientos, L}, title = {Structural Characterisation of TetR/AcrR Regulators in Streptomyces fildesensis So13.3: An In Silico CRISPR-Based Strategy to Influence the Suppression of Actinomycin D Production.}, journal = {International journal of molecular sciences}, volume = {26}, number = {10}, pages = {}, pmid = {40429982}, issn = {1422-0067}, support = {1210563//Fondecyt Regular/ ; 11230475//Fondecyt Iniciaci&#xf3;n/ ; 11240632//Fondecyt Iniciaci&#xf3;n/ ; }, mesh = {*Streptomyces/genetics/metabolism ; *Dactinomycin/biosynthesis ; *Bacterial Proteins/genetics/metabolism/chemistry ; Gene Expression Regulation, Bacterial ; Multigene Family ; Molecular Dynamics Simulation ; *Transcription Factors/genetics/metabolism/chemistry ; Anti-Bacterial Agents/biosynthesis ; *CRISPR-Cas Systems ; Biosynthetic Pathways/genetics ; }, abstract = {The growing threat of antimicrobial resistance has intensified the search for new bioactive compounds, particularly in extreme environments such as Antarctica. Streptomyces fildesensis So13.3, isolated from Antarctic soil, harbours a biosynthetic gene cluster (BGC) associated with actinomycin D production, an antibiotic with biomedical relevance. This study investigates the regulatory role of TetR/AcrR transcription factors encoded within this biosynthetic gene cluster (BGC), focusing on their structural features and expression under different nutritional conditions. Additionally, we propose that repressing an active pathway could lead to the activation of silent biosynthetic routes, and our in-silico analysis provides a foundation for selecting key mutations and experimentally validating this strategy. Expression analysis revealed that TetR-279, in particular, was upregulated in ISP4 and IMA media, suggesting its participation in nutrient-dependent BGC regulation. Structural modelling identified key differences between TetR-206 and TetR-279, with the latter containing a tetracycline-repressor-like domain. Molecular dynamics simulations confirmed TetR-279's structural stability but showed that the S166P CRISPy-web-guided mutation considerably affected its flexibility, while V167A and V167I had modest effects. These results underscore the importance of integrating omics, structural prediction, and gene editing to evaluate and manipulate transcriptional regulation in non-model bacteria. Targeted disruption of TetR-279 may derepress actinomycin biosynthesis, enabling access to silent or cryptic secondary metabolites with potential pharmaceutical applications.}, }
@article {pmid40429922, year = {2025}, author = {Sheng, H and Gao, P and Yang, C and Quilichini, TD and Kochian, LV and Datla, R and Xiang, D}, title = {Advances in Genome Editing Through Haploid Induction Systems.}, journal = {International journal of molecular sciences}, volume = {26}, number = {10}, pages = {}, pmid = {40429922}, issn = {1422-0067}, mesh = {*Gene Editing/methods ; *Haploidy ; CRISPR-Cas Systems ; *Crops, Agricultural/genetics ; Plant Breeding/methods ; *Genome, Plant ; Diploidy ; Plants, Genetically Modified/genetics ; }, abstract = {Groundbreaking advances in gene editing technologies are transforming modern plant breeding by enabling precise genetic modifications that dramatically accelerate crop improvement. Haploid and diploid induction systems have emerged as particularly powerful tools in this landscape, offering both efficient gene editing capabilities and rapid production of homozygous lines while seamlessly integrating with the advanced genome-editing platforms such as CRISPR-Cas systems. This review synthesizes the current state of knowledge regarding the mechanisms, applications, and recent progress in haploid and diploid induction systems for gene editing. We examine their transformative potential for enhancing genetic gains and compressing breeding timelines, with significant implications for global food security. Additionally, we provide a critical analysis of emerging challenges of genome editing in crops and outline promising future directions for research and development.}, }
@article {pmid40429745, year = {2025}, author = {Nilsri, N and Mekchaaum, R and Kalasin, S and Jongjitwimol, J and Daowtak, K}, title = {CRISPR/Cas9-Based Modeling of JAK2 V617F Mutation in K562 Cells Reveals Enhanced Proliferation and Sensitivity to Therapeutic Agents.}, journal = {International journal of molecular sciences}, volume = {26}, number = {10}, pages = {}, pmid = {40429745}, issn = {1422-0067}, support = {R2564C032 and AH-64-01-002//Naresuan University research funding and Faculty of Allied Health Sciences, Naresuan University research funding/ ; }, mesh = {Humans ; *Janus Kinase 2/genetics/metabolism ; Cell Proliferation/drug effects/genetics ; *CRISPR-Cas Systems/genetics ; K562 Cells ; Interferon-alpha/pharmacology ; *Mutation ; Arsenic Trioxide/pharmacology ; Myeloproliferative Disorders/genetics/drug therapy ; Antineoplastic Agents/pharmacology ; Point Mutation ; }, abstract = {The Janus kinase 2 (JAK2) protein fulfills an important role in hematopoiesis via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, as it provides the genetic driver of BCR::ABL1-negative myeloproliferative neoplasms (MPNs), which are clinically manifested as polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The most common cause of MPNs is the mutation of JAK2 V617F in the JAK2 gene, which results in increased cell proliferation. However, both the pathogenesis and treatment regimen of BCR::ABL1-negative MPNs remain poorly understood. The aim of the present study was to establish K562 cell lines with a point mutation in exon 14 (JAK2p.V617F) using CRISPR/Cas9 technology. The modified JAK2 V617F cell lines were examined for the gene mutation using droplet digital PCR (DDPCR), and the presence of the mutation was confirmed by DNA sequencing. Modified cells were characterized by measuring JAK2 gene expression and the extent of cell proliferation. Interferon α2a (IFN-α2a) and arsenic trioxide were also administered to the cells to explore their potential effects. The JAK2 V617F-mutated cells were found to exhibit a higher level of JAK2 gene expression compared with the wild type. Interestingly, a significant increase in the proliferation rate was observed with the modified cells compared with the wild type cells (p < 0.001), as assessed from the JAK2 gene expression levels. Furthermore, the treatments with IFN-α2a and arsenic trioxide led to the preferential suppression of the cell proliferation rate of the K562 expressing mutant JAK2 cells compared with the wild type cells, and this suppression occurred in a dose-dependent manner(p < 0.01). Moreover, the modified cells were able to differentiate into megakaryocyte-like cells following stimulation with phorbol 12 myristate 13 acetate (PMA). Taken together, the results of the present study have shown that the CRISPR/Cas9-modified JAK2 V617F model may be used as a disease model in the search of novel therapies for MPNs.}, }
@article {pmid40429744, year = {2025}, author = {Mikhaylova, E}, title = {Virus-Induced Genome Editing (VIGE): One Step Away from an Agricultural Revolution.}, journal = {International journal of molecular sciences}, volume = {26}, number = {10}, pages = {}, pmid = {40429744}, issn = {1422-0067}, support = {24-76-10065//Russian Science Foundation/ ; }, mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Plant Viruses/genetics ; *Genome, Plant ; Agriculture/methods ; *Crops, Agricultural/genetics/virology ; Plants, Genetically Modified/genetics ; Genetic Vectors/genetics ; }, abstract = {There is currently a worldwide trend towards deregulating the use of genome-edited plants. Virus-induced genome editing (VIGE) is a novel technique that utilizes viral vectors to transiently deliver clustered regularly interspaced short palindromic repeat (CRISPR) components into plant cells. It potentially allows us to obtain transgene-free events in any plant species in a single generation without in vitro tissue culture. This technology has great potential for agriculture and is already being applied to more than 14 plant species using more than 20 viruses. The main limitations of VIGE include insufficient vector capacity, unstable expression of CRISPR-associated (Cas) protein, plant immune reaction, host specificity, and reduced viral activity in meristem. Various solutions to these problems have been proposed, such as fusion of mobile elements, RNAi suppressors, novel miniature Cas proteins, and seed-borne viruses, but the final goal has not yet been achieved. In this review, the mechanism underlying the ability of different classes of plant viruses to transiently edit genomes is explained. It not only focuses on the latest achievements in virus-induced editing of crops but also provides suggestions for improving the technology. This review may serve as a source of new ideas for those planning to develop new approaches in VIGE.}, }
@article {pmid40429730, year = {2025}, author = {Fu, L and Li, P and Rui, Z and Sun, J and Yang, J and Wang, Y and Jia, D and Hu, J and Li, X and Ma, R}, title = {CRISPR/Cas9-Mediated Knockout of the White Gene in Agasicles hygrophila.}, journal = {International journal of molecular sciences}, volume = {26}, number = {10}, pages = {}, pmid = {40429730}, issn = {1422-0067}, support = {202304051001006//Science and Technology Innovation Teams of Shanxi Province/ ; 20210302123386//Fundamental Research Program of Shanxi Province/ ; 2016YJ03//Science and Technology Innovation Fund of Shanxi Agricultural University/ ; ZBXY23A-10//Cultivation and Innovation Program for Scientific research, College of Plant Protection, Shanxi Agricultural University/ ; }, mesh = {*CRISPR-Cas Systems ; Gene Editing/methods ; *Gene Knockout Techniques ; *Amaranthaceae/genetics ; }, abstract = {Agasicles hygrophila is the most effective natural enemy for the control of the invasive weed Alternanthera philoxeroides (Mart.) Griseb. However, research on the gene function and potential genetic improvement of A. hygrophila is limited due to a lack of effective genetic tools. In this study, we employed the A. hygrophila white (AhW) gene as a target gene to develop a CRISPR/Cas9-based gene editing method applicable to A. hygrophila. We showed that injection of Cas9/sgRNA ribonucleoprotein complexes (RNPs) of the AhW gene into pre-blastoderm eggs induced genetic insertion and deletion mutations, leading to white eyes. Our results demonstrate that CRISPR/Cas9-mediated gene editing is possible in A. hygrophila, offering a valuable tool for studies of functional genomics and genetic improvement of A. hygrophila, which could potentially lead to more effective control of invasive weeds through the development of improved strains of this biocontrol agent. In addition, the white-eyed mutant strain we developed could potentially be useful for other transgenic research studies on this species.}, }
@article {pmid40428391, year = {2025}, author = {Ziółkowska-Suchanek, I and Rozwadowska, N}, title = {Advancements in Gene Therapy for Non-Small Cell Lung Cancer: Current Approaches and Future Prospects.}, journal = {Genes}, volume = {16}, number = {5}, pages = {}, pmid = {40428391}, issn = {2073-4425}, mesh = {Humans ; *Carcinoma, Non-Small-Cell Lung/therapy/genetics ; *Genetic Therapy/methods/trends ; *Lung Neoplasms/therapy/genetics ; Gene Editing/methods ; CRISPR-Cas Systems ; Immunotherapy, Adoptive/methods ; }, abstract = {Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, characterized by late diagnosis and resistance to conventional therapies. Gene therapy has emerged as a promising alternative for NSCLC therapy, especially for patients with advanced disease who have exhausted conventional treatments. This article delved into the current developments in gene therapy for NSCLC, including gene replacement and tumor suppressor gene therapy, gene silencing, CRISPR/Cas9 gene editing, and immune modulation with CAR-T cell therapy. In addition, the challenges and future prospects of gene-based therapies for NSCLC were discussed.}, }
@article {pmid40427606, year = {2025}, author = {Gutiérrez-Hurtado, IA and García-Acéves, ME and Puga-Carrillo, Y and Guardado-Estrada, M and Becerra-Loaiza, DS and Carrillo-Rodríguez, VD and Plazola-Zamora, R and Godínez-Rubí, JM and Rangel-Villalobos, H and Aguilar-Velázquez, JA}, title = {Past, Present and Future Perspectives of Forensic Genetics.}, journal = {Biomolecules}, volume = {15}, number = {5}, pages = {}, pmid = {40427606}, issn = {2218-273X}, mesh = {Humans ; *Forensic Genetics/trends/methods/history ; Machine Learning ; CRISPR-Cas Systems ; }, abstract = {Forensic genetics has experienced remarkable advancements over the past decades, evolving from the analysis of a limited number of DNA segments to comprehensive genome-wide investigations. This progression has significantly improved the ability to establish genetic profiles under diverse conditions and scenarios. Beyond individual identification, forensic genetics now enables the inference of physical traits (e.g., eye, hair, and skin color, as well as body composition), biogeographic ancestry, lifestyle habits such as alcohol and tobacco use, and even the transfer of genital microbiomes post-coitus, among other characteristics. Emerging trends point to a future shaped by the integration of cutting-edge technologies, including CRISPR-Cas systems, artificial intelligence, and machine learning, which promise to further revolutionize the field. This review provides a thorough exploration of forensic genetics, tracing its evolution from its foundational methods (past) to its diverse modern applications (present) and offering insights into its potential future directions.}, }
@article {pmid40427601, year = {2025}, author = {Marin-Quilez, A and García-Tuñón, I and Benito, R and Ordoñez, JL and Díaz-Ajenjo, L and Lama-Villanueva, A and Guerrero, C and Pérez-Losada, J and González-Porras, JR and Hernández-Rivas, JM and Del Rey, M and Bastida, JM}, title = {Examining the Effects of the RUNX1 p.Leu43Ser Variant on FPD/AML Phenotypes Using a CRISPR/Cas9-Generated Knock-In Murine Model.}, journal = {Biomolecules}, volume = {15}, number = {5}, pages = {}, pmid = {40427601}, issn = {2218-273X}, mesh = {Animals ; *Core Binding Factor Alpha 2 Subunit/genetics/metabolism ; Mice ; *CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Gene Knock-In Techniques ; *Leukemia, Myeloid, Acute/genetics/pathology ; Phenotype ; *Blood Platelet Disorders/genetics/pathology ; Male ; Homozygote ; }, abstract = {Germline heterozygous variants in RUNX1 lead to Familial Platelet Disorder with Myeloid Leukemia Predisposition (FPD/AML). Cellular and/or animal models are helpful to uncovering the role of a variant in disease progression. Twenty-five mice per genotype (RUNX1[WT/WT], RUNX1[WT/L43S], RUNX1[L43S/L43S]), previously generated by CRISPR/Cas9, and nine sub-lethally irradiated mice per genotype were investigated. Peripheral blood (PB), bone marrow (BM), and spleen samples were analyzed by flow cytometry and histopathology. Deregulated genes were analyzed by RNA-seq in BM. An aberrant myeloid Mac1[+]Sca1[+]ckit[-] population in the PB, BM, and spleen of two homozygous and one heterozygous mouse was observed, as well as BM hypercellularity. No Mac1[+]Sca1[+]ckit[-] cells were detected in any RUNX1[WT/WT] mice. Moreover, the spleen of both homozygous mice showed destruction of the white/red pulp and the presence of apoptotic cells. The aberrant population was also detected in four irradiated mice, two heterozygous and two homozygous, in their PB, BM, and spleen. RNA-seq studies showed 698 genes significantly deregulated in the three non-irradiated Mac1[+]Sca1[+]ckit[-] mice vs. six healthy mice, highlighting the alteration of genes involved in apoptosis and DNA repair. These results indicate that the homozygous form of the variant p.Leu43Ser may contribute to the pathogenesis of aberrant cells.}, }
@article {pmid40427577, year = {2025}, author = {Palit, P and Minkara, M and Abida, M and Marwa, S and Sen, C and Roy, A and Pasha, MR and Mosae, PS and Saha, A and Ferdoush, J}, title = {PlastiCRISPR: Genome Editing-Based Plastic Waste Management with Implications in Polyethylene Terephthalate (PET) Degradation.}, journal = {Biomolecules}, volume = {15}, number = {5}, pages = {}, pmid = {40427577}, issn = {2218-273X}, support = {Start-up fund//University of Tennessee at Chattanooga/ ; }, mesh = {*Polyethylene Terephthalates/metabolism/chemistry ; *Gene Editing/methods ; *Waste Management/methods ; *Plastics/metabolism/chemistry ; Biodegradation, Environmental ; CRISPR-Cas Systems ; Humans ; }, abstract = {Plastic pollution has become a significant environmental issue worldwide, with global plastic production expected to reach 1800 million tons by 2050. One of the most commonly used plastics in the world is polyethylene terephthalate (PET), a synthetic polymer that is extremely durable but difficult to degrade. Thus, PET is dangerous to human health. To address this crisis, innovative approaches are being developed, including genome editing technologies. One of the recently advanced genome editing technologies is PlastiCRISPR, a novel concept that applies CRISPR-based genome editing to transform plastic waste management. PlastiCRISPR utilizes microorganisms to degrade plastic, generating valuable bioproducts like biofuels and biochemicals. Thus, this technology offers a sustainable solution because of its simple design, adequacy, and low cost, which can be integrated into existing waste management systems. Importantly, this review focuses on the PlastiCRISPR-based management of PET because it could drastically lower plastic waste, sustain natural resources by decreasing the requirement for plastic production, minimize energy intake, etc. Overall, this review provides an overview of the principles, applications, challenges, and future prospects of PlastiCRISPR in combating plastic pollution and shaping a more sustainable future.}, }
@article {pmid40426207, year = {2025}, author = {Wang, ZC and Stegall, H and Miyazawa, T and Keatinge-Clay, AT}, title = {A CRISPR-Cas9 system for knock-out and knock-in of high molecular weight DNA enables module-swapping of the pikromycin synthase in its native host.}, journal = {Microbial cell factories}, volume = {24}, number = {1}, pages = {125}, pmid = {40426207}, issn = {1475-2859}, support = {R01 GM145992/GM/NIGMS NIH HHS/United States ; GM145992/GM/NIGMS NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems ; *Streptomyces/genetics/metabolism/enzymology ; *Polyketide Synthases/genetics/metabolism ; Macrolides/metabolism ; Gene Editing/methods ; Gene Knockout Techniques ; Anti-Bacterial Agents/biosynthesis ; *Gene Knock-In Techniques ; Molecular Weight ; }, abstract = {BACKGROUND: Engineers seeking to generate natural product analogs through altering modular polyketide synthases (PKSs) face significant challenges when genomically editing large stretches of DNA.<br><br>RESULTS: We describe a CRISPR-Cas9 system that was employed to reprogram the PKS in Streptomyces venezuelae ATCC 15439 that helps biosynthesize the macrolide antibiotic pikromycin. We first demonstrate its precise editing ability by generating strains that lack megasynthase genes pikAI-pikAIV or the entire pikromycin biosynthetic gene cluster but produce pikromycin upon complementation. We then employ it to replace 4.4-kb modules in the pikromycin synthase with those of other synthases to yield two new macrolide antibiotics with activities similar to pikromycin.<br><br>CONCLUSION: Our gene-editing tool has enabled the efficient replacement of extensive and repetitive DNA regions within streptomycetes.}, }
@article {pmid40425955, year = {2025}, author = {Chen, Y and Zhang, X and Huang, W and Jin, Z and Wei, X and Li, J}, title = {Ultrasensitive detection of atrazine by Schottky junction photoelectrochemical aptamer sensor based on signal amplification by cascade catalysis of CRISPR/Cas12a and G-quadruplex/hemin DNAzyme.}, journal = {Mikrochimica acta}, volume = {192}, number = {6}, pages = {376}, pmid = {40425955}, issn = {1436-5073}, mesh = {*G-Quadruplexes ; *DNA, Catalytic/chemistry/metabolism ; *Aptamers, Nucleotide/chemistry ; Hemin/chemistry/metabolism ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; Limit of Detection ; *Atrazine/analysis/chemistry ; CRISPR-Cas Systems ; Electrodes ; CRISPR-Associated Proteins/metabolism/chemistry ; Photochemical Processes ; Catalysis ; Indoles/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {Atrazine (ATZ) is used extensively, resulting in residues in food and the environment, posing a serious threat to human health. Herein, Cd0.5Zn0.5S/Ti3C2 photoelectric material was synthesized and immobilized on a FTO electrode as a photoanode. A photoelectrochemical (PEC) aptamer sensor was constructed for the highly sensitive and selective determination of ATZ based on signal amplification via cascade catalysis of CRISPR/Cas12a and G-quadruplex/hemin DNAzyme (G4/hemin DNAzyme). G4/hemin DNAzyme catalyses the oxidation reaction between H2O2 and dopamine (DA) to form polydopamine (PDA) deposit. This process, in turn, inhibits the photocurrent at the photoanode, leading to a decrease in photocurrent. Concurrently, the depletion of DA as an electron donor for the PEC reaction at the photoelectrode further contributes to the decrease in photocurrent. ATZ can hybridize with ATZ aptamer (Apt) in Apt/cDNA to release activation strand (cDNA), which activates the activity of CRISPR/Cas12a and triggers cleavage of G4, causing the cleaving of G4/hemin DNAzyme immobilized on the electrode surface. This process leads to a decrease of G4/hemin DNAzymes amount on the electrode, consequently reducing both the PDA generation and the DA consumption. As a result, the photocurrent is restored. The cascade catalysis of CRISPR/Cas12a and G4/hemin DNAzyme has been demonstrated to result in photocurrent amplification. The photocurrent change was linear with the logarithmic value of ATZ concentration in the range 1.00 × 10[-12] to 1.00 × 10[-5] mol/L. The limit of detection was 3.47 × 10[-13] mol/L. The sensor has been successfully applied to the determination of trace ATZ in environmental and food samples.}, }
@article {pmid40425635, year = {2025}, author = {Imai, Y and Ozaki, S and Noda, T and Kobayashi, I and Sugitani, K and Kasashima, S and Morishita, E and Araiso, Y}, title = {Real-time imaging of blood coagulation and angiogenesis during development in a zebrafish model of type I antithrombin deficiency.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {18538}, pmid = {40425635}, issn = {2045-2322}, support = {JPMJSP2135//Japan Science and Technology Agency/ ; JPMJFR200F//Japan Science and Technology Agency/ ; }, mesh = {Animals ; *Zebrafish/genetics/embryology ; *Blood Coagulation/genetics ; Disease Models, Animal ; *Neovascularization, Physiologic ; Zebrafish Proteins/genetics ; Animals, Genetically Modified ; Thrombosis/genetics ; CRISPR-Cas Systems ; Angiogenesis ; }, abstract = {Severe type I antithrombin (AT) deficiency is considered to cause embryonic lethality. Although several pathological analyses using mice or zebrafish have been attempted, the previous studies did not unveil the detailed mechanism leading to lethality in the early developmental stage. In order to solve this problem, we established type I AT deficient zebrafish by the CRISPR/Cas9 system into Tg(gata1:dsRed) and Tg(fli1a:GFP) lines, so that we could conduct real-time imaging of thrombosis and angiogenesis using fluorescence stereo zoom microscopy. The established zebrafish AT (zAT) mutants harbored frameshift mutations which resulted to be type I AT deficient, unable to secrete zAT protein into blood. Both heterozygous (zAT[+/-]) and homozygous (zAT[-/-]) mutants showed reduced survival rate and diverse thrombosis up to 9 days post fertilization. In addition, blood vessel formation was delayed at 30 hpf in zAT[-/-], which was recovered normally by 5 dpf and had little effect on survival. Notably, we analyzed the differences in gene expression profiles under AT-depleted conditions by real-time quantitative PCR, and zAT[-/-] juvenile zebrafish showed increased PLG gene expression and decreased F2 gene expression. Our in vivo study revealed the effects of AT deficiency on embryos during development from the aspects of coagulation and vascular formation.}, }
@article {pmid40425580, year = {2025}, author = {Sharma, P and Kim, CY and Keys, HR and Imada, S and Joseph, AB and Ferro, L and Kunchok, T and Anderson, R and Sun, Y and Yilmaz, &#xd6;H and Weng, JK and Jain, A}, title = {Genetically encoded fluorescent reporter for polyamines.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {4921}, pmid = {40425580}, issn = {2041-1723}, support = {R35 GM151111/GM/NIGMS NIH HHS/United States ; R35GM151111//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; }, mesh = {*Polyamines/metabolism/analysis ; Humans ; *Genes, Reporter ; Mitochondria/metabolism ; Parkinson Disease/metabolism/genetics ; CRISPR-Cas Systems ; HEK293 Cells ; }, abstract = {Polyamines are abundant and evolutionarily conserved metabolites that are essential for life. Dietary polyamine supplementation extends life-span and health-span. Dysregulation of polyamine homeostasis is linked to Parkinson's disease and cancer, driving interest in therapeutically targeting this pathway. However, measuring cellular polyamine levels, which vary across cell types and states, remains challenging. We introduce a genetically encoded polyamine reporter for real-time measurement of polyamine concentrations in single living cells. This reporter utilizes the polyamine-responsive ribosomal frameshift motif from the OAZ1 gene. We demonstrate broad applicability of this approach and reveal dynamic changes in polyamine levels in response to genetic and pharmacological perturbations. Using this reporter, we conduct a genome-wide CRISPR screen and uncover an unexpected link between mitochondrial respiration and polyamine import, which are both risk factors for Parkinson's disease. By offering a lens to examine polyamine biology, this reporter may advance our understanding of these ubiquitous metabolites and accelerate therapy development.}, }
@article {pmid40425554, year = {2025}, author = {Borot, F and Humbert, O and Ehmsen, JT and Fields, E and Kohli, S and Radtke, S and Swing, K and Pande, D and Enstrom, MR and Laszlo, GS and Mayuranathan, T and Ali, AM and Weiss, MJ and Yen, JS and Newby, GA and Walter, RB and Liu, DR and Mukherjee, S and Kiem, HP}, title = {Multiplex base editing to protect from CD33 directed drugs for immune and gene therapy.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {4899}, pmid = {40425554}, issn = {2041-1723}, support = {R01 HL156647/HL/NHLBI NIH HHS/United States ; K99 HL163805/HL/NHLBI NIH HHS/United States ; P51 OD010425/OD/NIH HHS/United States ; R00 HL163805/HL/NHLBI NIH HHS/United States ; R50 CA274319/CA/NCI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; R01 CA266556/CA/NCI NIH HHS/United States ; P01 HL053749/HL/NHLBI NIH HHS/United States ; R01 HL136135/HL/NHLBI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R01 HL151765/HL/NHLBI NIH HHS/United States ; U42 OD011123/OD/NIH HHS/United States ; }, mesh = {Humans ; Animals ; *Gene Editing/methods ; *Sialic Acid Binding Ig-like Lectin 3/genetics/metabolism/immunology ; *Genetic Therapy/methods ; Hematopoietic Stem Cells/metabolism/drug effects ; Gemtuzumab/pharmacology ; *Immunotherapy/methods ; Polymorphism, Single Nucleotide ; CRISPR-Cas Systems ; Mice ; }, abstract = {The selection of genetically engineered immune or hematopoietic cells in vivo after gene editing remains a clinical problem and requires a method to spare on-target toxicity to normal cells. Here, we develop a base editing approach exploiting a naturally occurring CD33 single nucleotide polymorphism leading to removal of full-length CD33 surface expression on edited cells. CD33 editing in human and nonhuman primate hematopoietic stem and progenitor cells protects myeloid progeny from CD33-targeted therapeutics without affecting normal hematopoiesis in vivo, thus demonstrating potential for improved immunotherapies with reduced off-leukemia toxicity. For broader application to gene therapies, we demonstrate highly efficient (>70%) multiplexed adenine base editing of the CD33 and gamma globin genes, resulting in long-term persistence of dual gene-edited cells with HbF reactivation in nonhuman primates. Using the CD33 antibody-drug conjugate Gemtuzumab Ozogamicin, we show resistance of engrafted, multiplex edited human cells in vivo, and a 2-fold enrichment for edited cells in vitro. Together, our results highlight the potential of adenine base editors for improved immune and gene therapies.}, }
@article {pmid40425072, year = {2025}, author = {Nair, A and Rao, AS and Surabhi, MA and Gnanika, M and More, SS}, title = {Unravelling fungal pathogenesis: Advances in CRISPR-Cas9 for understanding virulence and adaptation.}, journal = {Fungal genetics and biology : FG &amp; B}, volume = {179}, number = {}, pages = {104006}, doi = {10.1016/j.fgb.2025.104006}, pmid = {40425072}, issn = {1096-0937}, mesh = {*CRISPR-Cas Systems ; *Fungi/pathogenicity/genetics ; Virulence/genetics ; Gene Editing ; Genome, Fungal ; *Adaptation, Physiological/genetics ; Humans ; Mycoses/microbiology ; Virulence Factors/genetics ; }, abstract = {Fungi, with their billion-year evolutionary history, have adapted to diverse ecological niches, including pathogenic roles that threaten global health, agriculture, and ecosystems. Fungal pathogenicity is shaped by the dynamic evolution of genetic traits that enable fungi to infect hosts, evade immune defenses, and develop resistance to antifungal treatments. Despite their significant clinical and ecological impact, the evolutionary processes underlying fungal virulence and adaptation remain incompletely understood. This review emphasizes the transformative role of CRISPR-Cas9 genome editing in revealing these mechanisms. By allowing precise manipulation of fungal genomes, CRISPR technologies have provided key insights into virulence factors, stress response mechanisms, immune evasion, and antifungal resistance pathways. These advances demonstrate how fungi adapt to selective pressures, repurpose conserved genetic pathways, and exploit genomic plasticity to thrive in host environments. This review explores the intersection of CRISPR technology and fungal biology, shedding light on its implications for understanding fungal pathogenesis and the potential to develop innovative therapeutic strategies against fungal infections. The integration of CRISPR applications into mycology holds promise for furthering our understanding of fungal evolutionary trajectories and enhancing the development of novel therapeutic approaches.}, }
@article {pmid40425009, year = {2025}, author = {Shang, K and Huang, D and Liu, J and Yu, Z and Bian, W and Chen, J and Zhao, Y and Liu, L and Jiang, J and Wang, Y and Duan, Y and Ge, J and Zhang, S and Zhou, C and Han, Y and Hu, Y and Zheng, W and Sun, J and Huang, H and Pei, S and Qian, P and Sun, J}, title = {CD97-directed CAR-T cells with enhanced persistence eradicate acute myeloid leukemia in diverse xenograft models.}, journal = {Cell reports. Medicine}, volume = {6}, number = {6}, pages = {102148}, doi = {10.1016/j.xcrm.2025.102148}, pmid = {40425009}, issn = {2666-3791}, mesh = {*Leukemia, Myeloid, Acute/therapy/immunology/pathology ; Humans ; Animals ; Xenograft Model Antitumor Assays ; *Receptors, Chimeric Antigen/metabolism/immunology ; Mice ; *Immunotherapy, Adoptive/methods ; Cell Line, Tumor ; *Antigens, CD/metabolism/immunology/genetics ; *T-Lymphocytes/immunology ; Mice, Inbred NOD ; CRISPR-Cas Systems ; Hematopoietic Stem Cells ; Mice, SCID ; }, abstract = {Chimeric antigen receptor (CAR)-T therapy on acute myeloid leukemia (AML) is hindered by the absence of a suitable tumor-specific antigen. Here, we propose CD97 as a potential target for CAR-T therapy against AML based on its broader and higher expression on AML cells compared to normal hematopoietic stem and progenitor cells (HSPCs). To resolve the fratricide problem caused by CD97 expression on T cells, we knock out CD97 in CAR-T cells using CRISPR-Cas9. Our CD97[KO] CAR-T cells eliminate both AML cell lines and primary AML cells effectively while showing tolerable toxicity to HSPCs. Furthermore, we mutate the CD3ζ domain of the CAR and find that the optimized CD97 CAR-T cells exhibit persistent anti-tumor activity both in vitro and in multiple xenograft models. Mechanistically, transcriptional profiles reveal that the optimized CAR-T cells delay differentiation and resist exhaustion. Collectively, our study supports CD97 as a promising target for CAR-T therapy against AML.}, }
@article {pmid40424180, year = {2025}, author = {Krishnaprasad, VH and Nayak, V and Kumar, S}, title = {World Health Organisation's Bacterial Pathogen Priority List (BPPL) 2017 and BPPL 2024 to combat global antimicrobial resistance crisis: 'challenges and opportunities'.}, journal = {The Journal of antimicrobial chemotherapy}, volume = {}, number = {}, pages = {}, doi = {10.1093/jac/dkaf167}, pmid = {40424180}, issn = {1460-2091}, abstract = {Antibiotic resistance, in a broader perspective, antimicrobial resistance (AMR) presents a formidable global health challenge, threatening the effectiveness of antibiotics and other antimicrobial agents. As a result, AMR has become more challenging or even impossible to treat, leading to increased morbidity and mortality. The World Health Organisation (WHO) has been at the forefront of international efforts to combat AMR by sensitizing the world about the pressing need to tackle AMR to save the future of the human race. This article analyses WHO's efforts to combat AMR, including creating the Bacterial Pathogen Priority List (BPPL), developing a global action plan to address AMR and promoting surveillance and stewardship programmes. This article also examines the progress achieved by BPPL 2017 and the challenges ahead for BPPL 2024. Additionally, this article explores various efforts to combat AMR through two major approaches, like 'research and development' and 'the policy and regulation-based' approach. This article underscores various emerging strategies to tackle AMR, for example, biofilm disruption, nanotechnology, antibiotic resistance breakers, antibody-antibiotic conjugates, rapid detection tools and alternative therapies like phage therapy, antimicrobial peptides, CRISPR-Cas system, probiotics and microbiota modulations. This article also highlights the importance of coordinated actions and sustained commitment to safeguarding public health and ensuring the continued effectiveness of antimicrobial therapies.}, }
@article {pmid40424009, year = {2025}, author = {Wei, L and Wang, Z and She, Y and Fu, H}, title = {CRISPR/Cas Multiplexed Biosensing: Advances, Challenges, and Perspectives.}, journal = {Analytical chemistry}, volume = {97}, number = {23}, pages = {11943-11958}, doi = {10.1021/acs.analchem.4c04428}, pmid = {40424009}, issn = {1520-6882}, mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems are renowned for their high sensitivity and specificity, enabling them as a powerful diagnostic toolbox. Multiplexed detection of panels of targets, as opposed to single targets, is imperative for reliable and conclusive disease diagnostics. However, multiplex application of the CRISPR/Cas system has long been hindered by indistinguishable signals from specific targets due to nonspecific chaotic trans-cleavage. To make a breakthrough, substantial efforts have been devoted to CRISPR/Cas-powered multiplexed biosensing strategies, which consequently experienced rapid development over the past five years. This review systematically summarizes recent advances in CRISPR/Cas multiplexed detection encompassing Cas9, Cas12, and Cas13. Key focus issues include multiplex biosensing strategies and their respective advantages and limitations, sensing mechanisms, and detection performance of novel validated examples. Finally, the status and challenges of CRISPR/Cas multiplexed biosensing are critically discussed, and future outlooks are proposed for their potential practical application. This Perspective aims to inspire significant research and promote the development of the next generation of CRISPR/Cas multiplexed biosensing.}, }
@article {pmid40422684, year = {2025}, author = {Ma, B and Li, Y and Wang, T and Li, D and Jia, S}, title = {Advances in CRISPR/Cas9-Based Gene Editing in Filamentous Fungi.}, journal = {Journal of fungi (Basel, Switzerland)}, volume = {11}, number = {5}, pages = {}, pmid = {40422684}, issn = {2309-608X}, support = {32160143//National Science Foundation of China/ ; }, abstract = {As an important class of microorganisms, filamentous fungi have crucial roles in protein secretion, secondary metabolite production and environmental pollution control. However, characteristics such as apical growth, heterokaryon, low homologous recombination (HR) efficiency and the scarcity of genetic markers mean that the application of traditional gene editing technology in filamentous fungi faces great challenges. The introduction of the RNA-mediated CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRlSPR-associated protein) system in filamentous fungi in recent years has revolutionized gene editing in filamentous fungi. In addition, the continuously expressed CRISPR system has significantly improved the editing efficiency, while the optimized sgRNA design and reduced cas9 concentration have effectively reduced the off-target effect, further enhancing the safety and reliability of the technology. In this review, we systematically analyze the molecular mechanism and regulatory factors of CRISPR/Cas9, focus on the optimization of its expression system and the improvement of the transformation efficiency in filamentous fungi, and reveal the core regulatory roles of HR and non-homologous end-joining (NHEJ) pathways in gene editing. Based on the analysis of various filamentous fungi applications, this review reveals the outstanding advantages of CRISPR/Cas9 in the enhancement of protein secretion, addresses the reconstruction of secondary metabolic pathways and pollutant degradation in the past decade, and provides a theoretical basis and practical guidance for the optimization of the technology and engineering applications.}, }
@article {pmid40422214, year = {2025}, author = {Gibson, J and Dhungana, A and Pokhrel, M and Arthur, B and Suresh, P and Adebayo, O and Cottle, RN}, title = {Validation of Clinical-Grade Electroporation Systems for CRISPR-Cas9-Mediated Gene Therapy in Primary Hepatocytes for the Correction of Inherited Metabolic Liver Disease.}, journal = {Cells}, volume = {14}, number = {10}, pages = {}, pmid = {40422214}, issn = {2073-4409}, support = {P20 GM146584/GM/NIGMS NIH HHS/United States ; R01 HL168093/HL/NHLBI NIH HHS/United States ; 2021000920//American Association for the Study of Liver Diseases Foundation/ ; 1R01HL168093-01A1/GF/NIH HHS/United States ; }, mesh = {*Hepatocytes/transplantation/metabolism ; *Electroporation/methods ; Animals ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; Mice ; Gene Editing/methods ; *Tyrosinemias/therapy/genetics ; Humans ; *Liver Diseases/therapy/genetics ; Disease Models, Animal ; Male ; Hydrolases ; }, abstract = {Hepatocyte transplantation (HTx) combined with ex vivo gene therapy has garnered significant interest due to its potential for treating many inherited metabolic liver diseases. The biggest obstacle for HTx is achieving sufficient engraftment levels to rescue diseased phenotypes, which becomes more challenging when combined with ex vivo gene editing techniques. However, recent technological advancements have improved electroporation delivery efficiency, cell viability, and scalability for cell therapy. We recently demonstrated the impacts of electroporation for cell-based gene therapy in a mouse model of hereditary tyrosinemia type 1 (HT1). Here, we explore the use of the clinical-grade electroporator, the MaxCyte ExPERT GTx, utilized in the first FDA-approved CRISPR therapy, Casgevy, and evaluate its potential in primary hepatocytes in terms of delivery efficiency and cell viability. We assessed the gene editing efficiency and post-transplantation engraftment of hepatocytes from mTmG mice electroporated with CRISPR-Cas9-ribonucleoproteins (RNPs) targeting 4-hydroxyphenylpyruvate dioxygenase (Hpd) in a fumarylacetoacetate hydrolase (Fah)-deficient mouse model of HT1. After surgery, Fah[-/-] graft recipients were cycled off and on nitisinone to achieve independence from drug-induced Hpd inhibition, an indicator of HT1 disease correction. Transplanted hepatocytes subjected to electroporation using the GTx system had a cell viability of 89.9% and 100% on-target gene editing efficiency. Recipients transplanted with GTx-electroporated cells showed a smaller weight reduction than controls transplanted with untransfected cells (7.9% and 13.8%, respectively). Further, there were no mortalities in the GTx-recipient mice, whereas there was 25% mortality in the control recipients. Mean donor cell engraftment was significantly higher in GTx-recipient mice compared to untransfected control recipients (97.9% and 81.6%, respectively). Our results indicate that the GTx system does not negatively impact hepatocyte functionality and engraftment potential, thereby demonstrating the promise of GTx electroporation in hepatocytes as a viable cell therapy for treating genetic diseases that affect the liver.}, }
@article {pmid40422195, year = {2025}, author = {Orlova, NA and Sinegubova, MV and Kolesov, DE and Khodak, YA and Tatarskiy, VV and Vorobiev, II}, title = {Genomic and Phenotypic Characterization of CHO 4BGD Cells with Quad Knockout and Overexpression of Two Housekeeping Genes That Allow for Metabolic Selection and Extended Fed-Batch Culturing.}, journal = {Cells}, volume = {14}, number = {10}, pages = {}, pmid = {40422195}, issn = {2073-4409}, support = {25-24-00140//Russian Science Foundation/ ; }, mesh = {CHO Cells ; Animals ; Cricetulus ; *Batch Cell Culture Techniques/methods ; CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques ; Gene Editing ; Apoptosis/genetics ; *Genes, Essential/genetics ; Phenotype ; *Genomics ; }, abstract = {Re-engineering of CHO cells using genome editing and the overexpression of multiple helper genes is the central track for obtaining better cell lines for the production of biopharmaceuticals. Using two subsequent rounds of genome editing of the CHO S cells, we have developed the cell line CHO 4BGD with four knockouts of two pro-apoptotic genes bak1 and bax, and two common selection markers genes-glul (GS) and dhfr, and additional copies of genes bcl-2 and beclin-1 used for enhancement of macroautophagy. The NGS sequencing of 4BGD cells revealed that all eight targeted alleles were successfully disrupted. Two edited loci out of eight contained large inserts of non-relevant DNA. Further data analysis shows that cells have no off-target DNA editing events, and all known CHO genes are preserved. The cells obtained are completely resistant to the induction of apoptosis, and they are suitable for the generation of stably transfected cell lines with the dhfr selection marker. They also properly undergo the target gene amplification. The 4BGD-derived clonal cell line that secretes the monoclonal antibody retains the ability for prolonged fed-batch culturing. The method of obtaining multiply edited CHO cells using the multiplex CRISPR/Cas9 editing and simultaneous stable transfection of plasmids, coding for the housekeeping genes, is suitable for the rapid generation of massively edited CHO cells.}, }
@article {pmid40422075, year = {2025}, author = {Guo, L and Duan, X and Li, J and Hao, Z and Bi, Y and Chen, Y and Du, X and Wu, S}, title = {YIPF5 is an essential host factor for porcine epidemic diarrhea virus double-membrane vesicle formation.}, journal = {Journal of virology}, volume = {99}, number = {6}, pages = {e0032025}, pmid = {40422075}, issn = {1098-5514}, support = {2023YFC3404300, 2023YFF0724700, 2023YFF1000200, 2021YFA0805900//National Key Research and Development Program of China/ ; 323QN261//the Natural Science Foundation for Youths of Hainan province, China/ ; 32425053//the National Natural Science Foundation of China (NSFC) Distinguished Young Scholar/ ; }, mesh = {Animals ; *Porcine epidemic diarrhea virus/physiology ; Swine ; Virus Replication ; *Coronavirus Infections/virology/metabolism/veterinary ; Cell Line ; *Swine Diseases/virology/metabolism ; CRISPR-Cas Systems ; *Host-Pathogen Interactions ; Viral Nonstructural Proteins/metabolism/genetics ; }, abstract = {UNLABELLED: Porcine epidemic diarrhea virus (PEDV) is a major coronavirus in swine, causing substantial economic losses in the industry. To deepen our understanding of the PEDV-host cell interactions, we performed whole-genome CRISPR/Cas9 screens on porcine IPEC-J2 and IPI-2I cell lines to identify key host factors essential for PEDV infection. Our study identified the Yip family 5 (YIPF5) protein as a critical host factor, where its knockout suppressed PEDV infection by specifically affecting the virus replication stage. YIPF5 interacts with viral non-structural protein (nsp) 3, 4, and 6, facilitating the formation of double-membrane vesicles (DMVs), essential for replication organelle biogenesis. The knockout of YIPF5 interferes with the interaction between nsp3 and nsp4, consequently impacting the formation of DMVs mediated by these proteins. These findings establish YIPF5 as a key host factor involved in DMV formation during PEDV infection, highlighting its potential as a therapeutic target.<br><br>IMPORTANCE: Coronaviruses pose serious health threats to both humans and animals. Identifying host genes critical for porcine epidemic diarrhea virus (PEDV) infection can uncover new therapeutic targets and enhance our understanding of coronavirus pathogenesis. In this study, we conducted genome-scale CRISPR/Cas9 screens in two porcine cell lines (IPEC-J2 and IPI-2I) and identified YIPF5 as an essential host factor for PEDV replication. Our results demonstrate that YIPF5 plays a pivotal role in the formation of PEDV-induced double-membrane vesicles (DMVs), which are crucial for viral replication. These findings shed new light on the molecular mechanisms of PEDV and suggest YIPF5 as a therapeutic target.}, }
@article {pmid40419487, year = {2025}, author = {Byrne, SM and Burleigh, SM and Fragoza, R and Jiang, Y and Savva, Y and Pabon, R and Kania, E and Rainaldi, J and Portell, A and Mali, P and Briggs, AW}, title = {An engineered U7 small nuclear RNA scaffold greatly increases ADAR-mediated programmable RNA base editing.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {4860}, pmid = {40419487}, issn = {2041-1723}, mesh = {*Adenosine Deaminase/metabolism/genetics ; Animals ; *RNA Editing/genetics ; Humans ; Mice ; *RNA, Small Nuclear/genetics/metabolism/chemistry ; *Gene Editing/methods ; *RNA-Binding Proteins/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; }, abstract = {Custom RNA base editing exploiting the human Adenosine Deaminase Acting on RNA (ADAR) enzyme may enable therapeutic gene editing without DNA damage or use of foreign proteins. ADAR's adenosine-to-inosine (effectively A-to-G) deamination activity can be targeted to transcripts using an antisense guide RNA (gRNA), but efficacy is challenged by limits of in vivo delivery. Embedding gRNAs into a U7 small nuclear RNA (snRNA) framework greatly enhances RNA editing with endogenous ADAR, and a 750-plex single-cell mutagenesis screen further improved the framework. An optimized scaffold with a stronger synthetic U7 promoter enables 76% RNA editing in vitro from a single DNA construct per cell, and 75% editing in a Hurler syndrome mouse brain after one systemic AAV injection, surpassing circular gRNA approaches. The technology also improves published DMD exon-skipping designs 25-fold in differentiated myoblasts. Our engineered U7 framework represents a universal scaffold for ADAR-based RNA editing and other antisense RNA therapies.}, }
@article {pmid40419416, year = {2025}, author = {Wachholz Junior, D and Pontes, RG and Hryniewicz, BM and Kubota, LT}, title = {Exploring a CRISPR/Cas12a-powered impedimetric biosensor for amplification-free detection of a pathogenic bacterial DNA.}, journal = {Biosensors &amp; bioelectronics}, volume = {285}, number = {}, pages = {117607}, doi = {10.1016/j.bios.2025.117607}, pmid = {40419416}, issn = {1873-4235}, mesh = {*Biosensing Techniques/methods ; *Staphylococcus aureus/genetics/isolation &amp; purification/pathogenicity ; *CRISPR-Cas Systems/genetics ; *DNA, Bacterial/genetics/isolation &amp; purification ; Humans ; Limit of Detection ; *Staphylococcal Infections/microbiology/diagnosis ; }, abstract = {Timely and precise detection of bacterial infections is essential for improving patient outcomes and reducing healthcare costs, especially for sepsis, where delayed diagnosis increases mortality. Traditional culture- and PCR-based methods are time consuming and require complex sample processing, making them unsuitable for rapid diagnostics in resource-limited settings. CRISPR/Cas-based methods, particularly when combined with electrochemical sensing, offer a promising alternative for rapid point-of-care (POC) diagnostics of bacterial infections due to their simplicity and specificity. This study proposes a label-free impedimetric biosensor using the CRISPR/Cas12a system for rapid and amplification-free detection of Staphylococcus aureus DNA, a primary pathogen responsible for sepsis. By leveraging CRISPR/Cas12a's target-activated collateral cleavage on non-specific DNA reporters we investigated the impact of using a protospacer adjacent motif (PAM) sequence on detection sensitivity and specificity. Our biosensor demonstrated ultra-sensitive detection, with limit of detection as low as 20 aM for dsDNA targets in buffer and without any pre-amplification steps. The study also confirmed CRISPR specificity's dependence on the PAM sequence, showing that mismatches on targeting sequences reduces cleavage efficiency, with a drastic reduction in trans-cleavage activity for single mismatch in PAM-containing sequences. Additionally, we examined how the DNA reporter affects performance, noting reduced cleavage efficiency when a ssDNA target was paired with a dsDNA reporter. Furthermore, validation experiments using human serum samples confirmed the biosensor's accuracy for bacterial DNA detection in clinical settings. This work advances CRISPR-powered electrochemical biosensors, providing a detailed discussion on developing a highly sensitive, fast and amplification-free tool for early detection of sepsis-causing bacteria.}, }
@article {pmid40418657, year = {2025}, author = {Johnson, MJ and DeFeo, AP and Slipek, NJ and Folsom, TD and Henley, T and Choudhry, MS and Webber, BR and Moriarity, BS}, title = {Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {219}, pages = {}, doi = {10.3791/68150}, pmid = {40418657}, issn = {1940-087X}, mesh = {Humans ; *T-Lymphocytes/cytology/immunology ; CRISPR-Cas Systems ; *DNA/genetics ; Animals ; *Cell Engineering/methods ; Mice ; *DNA End-Joining Repair ; *Genetic Engineering/methods ; }, abstract = {Many current adoptive cellular therapies rely on lenti- or retroviral vectors to engineer T cells for the expression of a chimeric antigen receptor (CAR) or exogenous T cell receptor (TCR) to target a specific tumor-associated antigen. Reliance on viral vectors for the production of therapeutic T cells significantly increases the timeline, cost, and complexity of manufacturing while limiting the translation of new therapies, particularly in the academic setting. A process is presented for efficient non-viral engineering of T cells using CRISPR/Cas9 and homology-mediated end joining to achieve targeted integration of large, multicistronic DNA cargo. This approach has achieved integration frequencies comparable to those of viral vectors while yielding highly functional T cells capable of potent anti-tumor efficacy both in vitro and in vivo. Notably, this method is rapidly adaptable to current good manufacturing practices (cGMP) and clinical scale-up, providing a near-term option for the manufacturing of therapeutic T cells for use in clinical trials.}, }
@article {pmid40417002, year = {2025}, author = {Gallala, M}, title = {Application of CRISPR/Cas gene editing for infectious disease control in poultry.}, journal = {Open life sciences}, volume = {20}, number = {1}, pages = {20251095}, pmid = {40417002}, issn = {2391-5412}, abstract = {The poultry industry faces multifaceted challenges, including escalating demand for poultry products, climate change impacting feed availability, emergence of novel avian pathogens, and antimicrobial resistance. Traditional disease control measures are costly and not always effective, prompting the need for complementary methods. Gene editing (GE, also called genome editing) technologies, particularly CRISPR/Cas9, offer promising solutions. This article summarizes recent advancements in utilizing CRISPR/Cas GE to enhance infectious disease control in poultry. It begins with an overview of modern GE techniques, highlighting CRISPR/Cas9's advantages over other methods. The potential applications of CRISPR/Cas in poultry infectious disease prevention and control are explored, including the engineering of innovative vaccines, the generation of disease-resilient birds, and in vivo pathogen targeting. Additionally, insights are provided regarding regulatory frameworks and future perspectives in this rapidly evolving field.}, }
@article {pmid40415627, year = {2025}, author = {Li, H and Zhu, Q and Zhu, J and Min, J}, title = {[Research progress in the developmental process of non-viral CAR-T technology].}, journal = {Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology}, volume = {41}, number = {5}, pages = {461-467}, pmid = {40415627}, issn = {1007-8738}, mesh = {Humans ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/genetics/immunology ; Animals ; Gene Transfer Techniques ; Genetic Vectors/genetics ; Gene Editing ; CRISPR-Cas Systems/genetics ; DNA Transposable Elements/genetics ; *T-Lymphocytes/immunology ; Neoplasms/therapy/immunology ; }, abstract = {Chimeric antigen receptor T (CAR-T) lymphocytes are at the forefront of adoptive immunotherapy research, and this technology has significantly advanced the prospects of tumor immunotherapy. CAR-T therapy has demonstrated remarkable efficacy in haematological tumours of lymphoid origin and provided therapeutic possibility for solid tumours. Currently, CAR-T cell preparation predominantly involves transfection of T cells with viral vectors. However, the production of viral vectors is time-consuming, expensive, and the vectors have low loading capacity, along with insertion instability. Consequently, there is a pressing need to develop more convenient and precise non-viral gene delivery methods. This paper reviews the most promising non-viral gene delivery technologies, including CRISPR/Cas9 gene editing, transposon systems such as Sleeping Beauty (SB) and PiggyBac (PB), and mRNA, and anticipates the future development of non-viral vector-based CAR-T therapies.}, }
@article {pmid40414878, year = {2025}, author = {Lobel, JH and Ingolia, NT}, title = {Precise measurement of molecular phenotypes with barcode-based CRISPRi systems.}, journal = {Genome biology}, volume = {26}, number = {1}, pages = {142}, pmid = {40414878}, issn = {1474-760X}, support = {F32 GM148044/GM/NIGMS NIH HHS/United States ; R01 GM135233/GM/NIGMS NIH HHS/United States ; R21 HG012991/HG/NHGRI NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems ; Phenotype ; Humans ; Promoter Regions, Genetic ; }, abstract = {Genome-wide CRISPR-Cas9 screens have untangled regulatory networks driving diverse biological processes. Their success relies on interrogating specific molecular phenotypes and distinguishing key regulators from background effects. Here, we realize these goals by optimizing CRISPR interference with barcoded expression reporter sequencing (CiBER-seq) to dramatically improve the sensitivity and scope of genome-wide screens. We systematically address technical factors that distort phenotypic measurements by normalizing expression reporters against closely matched promoters. We use our improved CiBER-seq to accurately capture known components of well-studied RNA and protein quality control systems. These results demonstrate the precision and versatility of CiBER-seq for dissecting cellular pathways.}, }
@article {pmid40414079, year = {2025}, author = {Lyu, H and Haag, C and Schwarz, N and Löffler, H and Mau-Holzmann, UA and Lerche, H and Rosa, F}, title = {Generation of an induced pluripotent stem cell (iPSC) line carrying a KCNA2 homozygous (p.Arg294His, R294H) mutation related to hereditary spastic paraplegia.}, journal = {Stem cell research}, volume = {86}, number = {}, pages = {103737}, doi = {10.1016/j.scr.2025.103737}, pmid = {40414079}, issn = {1876-7753}, mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Spastic Paraplegia, Hereditary/genetics/pathology ; Homozygote ; Cell Line ; *Mutation ; CRISPR-Cas Systems ; Kv1.2 Potassium Channel ; }, abstract = {The KCNA2 gene encodes the voltage-gated potassium channel Kv1.2, which is essential for repolarization of action potential. The R294H variant in KCNA2 represents the only potassium channel gene variant linked to hereditary spastic paraplegia (HSP) to date. However, this variant has been observed exclusively in heterozygous individuals. Here, we generated a homozygous KCNA2 R294H (c.881G > A) induced pluripotent stem cell (iPSC) line from a healthy individual iPSC line. The variant was introduced into both alleles using the CRISPR/Cas9 system. The resulting iPSC line has a normal karyotype, expresses key pluripotency markers, and is able to differentiate into all three germ layers.}, }
@article {pmid40414010, year = {2025}, author = {Cao, X and Gao, Z and Yin, P and Wang, H and Yang, L}, title = {Crystal structure and inhibition mechanism of AcrIIA11.}, journal = {Biochemical and biophysical research communications}, volume = {772}, number = {}, pages = {152073}, doi = {10.1016/j.bbrc.2025.152073}, pmid = {40414010}, issn = {1090-2104}, mesh = {Streptococcus pyogenes/enzymology/genetics ; Crystallography, X-Ray ; Staphylococcus aureus/enzymology ; *Bacterial Proteins/chemistry/metabolism/antagonists &amp; inhibitors ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/antagonists &amp; inhibitors/chemistry/metabolism ; Models, Molecular ; Protein Conformation ; Protein Binding ; }, abstract = {Anti-CRISPR (Acr) proteins are naturally evolved inhibitors that precisely target and suppress CRISPR-Cas systems, representing a sophisticated molecular arms race between bacteriophages and their bacterial hosts. While Class 1 systems dominate among sequenced prokaryotic genomes, Class 2 systems remain primary sources of editing tools. Here, we report the structural and mechanistic characterization of AcrIIA11, an anti-CRISPR protein that simultaneously inhibits Streptococcus pyogenes (SpyCas9) and Staphylococcus aureus Cas9 (SauCas9). The 3.2 Å crystal structure reveals a compact α/β fold with distinct electropositive clefts implicated in DNA binding. While DALI analysis identified structural homology to transcriptional regulators and the RecA inhibitor PsiB (RMSD 3.3 Å), functional studies established that AcrIIA11 forms stable ternary complexes with both Cas9 orthologs and sgRNA. Biochemical assays demonstrated stronger inhibition of SauCas9 compared to SpyCas9, with EMSA revealing a critical dichotomy: AcrIIA11 maintains SauCas9-sgRNA binding to specific target DNA while completely blocking cleavage activity. Computational docking localizes AcrIIA11 at the HNH-RuvC interface without obstructing DNA-binding channels in SauCas9, suggesting allosteric inhibition through HNH domain displacement. This work establishes AcrIIA11 as a dual-purpose Cas9 inhibitor that preserves target recognition while inactivating nuclease function-a mechanism with potential applications in precision CRISPR control.}, }
@article {pmid40413996, year = {2025}, author = {Duan, M and Li, G and Shen, J and Dai, R and Li, X and Liu, Z and Jia, F}, title = {A CRISPR/Cas12a biosensor for portable and accessible detection of Salmonella typhimurium via multi-indicator pH millidisc colorimetry and smartphone imaging platform.}, journal = {Biosensors &amp; bioelectronics}, volume = {286}, number = {}, pages = {117611}, doi = {10.1016/j.bios.2025.117611}, pmid = {40413996}, issn = {1873-4235}, mesh = {*Biosensing Techniques/instrumentation ; *Salmonella typhimurium/isolation &amp; purification/genetics ; Smartphone ; *Colorimetry/instrumentation ; CRISPR-Cas Systems/genetics ; Animals ; Chickens/microbiology ; Hydrogen-Ion Concentration ; Food Microbiology ; Limit of Detection ; }, abstract = {Conventional colorimetric CRISPR/Cas methods rely on a single chromogenic substrate and bulky and specialized signal detection instrument, which hinder their practical application. Herein, a portable and accessible CRISPR/Cas12a biosensor was for the first time reported to sensitively quantify Salmonella enterica serovar typhimurium (S. typhimurium), utilizing a multi-indicator pH millimeter disc (millidisc) for signal visualization, combined with a smartphone-based imaging platform for signal readout. The pH millidisc, composed of multiple indicators, possessed sensitive pH responsiveness and exhibited diverse color changes. The self-developed RGB mini-program, named DeepFood, was designed for portable smartphone use, featuring user-friendly operation and trend visualization for preliminary result analysis. Based on the RGB signal variation pattern with S. typhimurium, a distinct Senh signal type was designed, enhancing the signal-to-noise ratio from 3.38 to 7.11. Compared to the R signal type, the Senh signal type improved detection sensitivity by 36.23-fold (7.26 CFU/mL) in 0.01 M PBS buffer and 15.53-fold (1.41 × 10[2] CFU/mL) in chicken. The proposed biosensor offers significant improvements in detection sensitivity and practical applicability, with potential in food safety and environmental protection.}, }
@article {pmid40413994, year = {2025}, author = {Liu, Y and Liu, Y and Wu, S and Cao, R and Pan, Y and Zhou, F}, title = {Engineered Cas12a-based one-tube detection of DNMT3A R882 H/C mutation in acute myeloid leukemia.}, journal = {Biosensors &amp; bioelectronics}, volume = {286}, number = {}, pages = {117609}, doi = {10.1016/j.bios.2025.117609}, pmid = {40413994}, issn = {1873-4235}, mesh = {DNA Methyltransferase 3A ; *Leukemia, Myeloid, Acute/genetics/diagnosis ; Humans ; *DNA (Cytosine-5-)-Methyltransferases/genetics ; *CRISPR-Cas Systems/genetics ; Mutation ; *Biosensing Techniques/methods ; *Endodeoxyribonucleases/genetics/chemistry ; Bacterial Proteins ; CRISPR-Associated Proteins ; }, abstract = {Advances in sequencing technologies have identified numerous genetic alterations associated with acute myeloid leukemia (AML), many of which play critical roles in diagnosis, classification, and prognosis. Among these, mutations in the DNA methyltransferase 3 alpha (DNMT3A) gene are particularly prevalent, with the R882H and R882C variants being the most common. Accurate and sensitive detection of DNMT3A mutations is crucial for prognosis, treatment guidance, and early intervention in AML. However, existing detection methods often fail to achieve an optimal balance among sensitivity, turnaround time, and operational simplicity. To address this limitation, we aimed to develop a rapid and highly sensitive method for detecting DNMT3A mutations. The CRISPR/Cas12a system shows promise for genetic detection due to its high sensitivity and single-base specificity. Here, we established a Cas12a-based one-tube assay for the detection of DNMT3A R882 H/C mutations. We utilized the mismatch tolerance of enAsU-R Cas12a to design crRNA for DNMT3A R882 H/C mutation and integrated CRISPR/Cas12a system with ERA. The entire detection process can be completed within 1 h at 37 °C. The optimized detection system demonstrated a sensitivity of 0.1 % when analyzing genomic DNA. To validate its clinical applicability, we tested samples from 49 AML patients and successfully identified all DNMT3A R882H/C-positive cases, including one with a mutation rate as low as 0.24 %. These results highlight the potential of our Cas12a-based one-tube detection system as a rapid, sensitive, and cost-effective method for detecting DNMT3A R882 H/C mutation. This approach could serve as a valuable tool for both diagnostic and therapeutic monitoring.}, }
@article {pmid40413357, year = {2025}, author = {Jiang, B and An, Z and Niu, L and Qin, D}, title = {Precise genome editing process and its applications in plants driven by AI.}, journal = {Functional &amp; integrative genomics}, volume = {25}, number = {1}, pages = {109}, pmid = {40413357}, issn = {1438-7948}, support = {BLX202316//Fundamental Research Funds for the Central Universities/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Genome, Plant ; *Plants/genetics ; *Artificial Intelligence ; Plant Breeding ; }, abstract = {Genome editing technologies have emerged as the keystone of biotechnological research, enabling precise gene modification. The field has evolved rapidly through revolutionary advancements, transitioning from early explorations to the breakthrough of the CRISPR-Cas system. The emergence of the CRISPR-Cas system represents a huge leap in genome editing, prompting the development of advanced tools such as base and prime editors, thereby enhancing precise genomic engineering capabilities. The rapid integration of AI across disciplines is now driving another transformative phase in genome editing, streamlining workflows and enhancing precision. The application prospects of genome editing technology are extensive, particularly in plant breeding, where it has already presented unparalleled opportunities for improving plant traits. Here, we review early genome editing technologies, including meganucleases, ZFNs, TALENs, and CRISPR-Cas systems. We also provide a detailed introduction to next-generation editing tools-such as base editors and prime editors-and their latest applications in plants. At the same time, we summarize and prospect the cutting-edge developments and future trends of genome editing technologies in combination with the rapidly rising AI technology, including optimizing editing systems, predicting the efficiency of editing sites and designing editing strategies. We are convinced that as these technologies progress and their utilization expands, they will provide pioneering solutions to global challenges, ushering in an era of health, prosperity, and sustainability.}, }
@article {pmid40412199, year = {2025}, author = {Shen, Y and Tang, X and Wang, J and Dai, H and Cui, Y and Hu, Q and Wu, Y and Jia, F and Hao, G}, title = {Dual nuclease-amplified sensitive biosensor for enrofloxacin detection using a DNase I-assisted CRISPR/Cas12a (CRISPR-DNase I) system.}, journal = {Talanta}, volume = {295}, number = {}, pages = {128367}, doi = {10.1016/j.talanta.2025.128367}, pmid = {40412199}, issn = {1873-3573}, abstract = {Recent years have witnessed the flourishing of CRISPR/Cas-based biosensors in various fields. However, most of them were developed for nucleic acid detection because non-nucleic acid targets are unable to unleash the cleavage activity of the CRISPR/Cas system directly. To circumvent this problem, activator DNA and deoxyribonuclease I (DNase I) were introduced in this research to render the CRISPR/Cas12a system as a new powerful tool for the detection of enrofloxacin (ENR), a common veterinary drug. In this biosensor, target ENR competed with DNase I- and bovine serum albumin-ENR composite-modified gold nanoparticles (DNase I-AuNPs-BSA-ENR) for the binding sites on the surface of antibody-modified magnetic nanoparticles (immuno-MNPs). Then, the captured DNase I-AuNPs-BSA-ENR degraded the activator DNA in the solution, which inhibited the activation of the CRISPR/Cas12a system. Finally, the fluorescence released by the activated CRISPR/Cas12a system was measured for the quantitative detection of ENR. The ingenious use of activator DNA and DNase I helped transduce the target recognition event into the cleavage activity of the CRISPR/Cas12a system. Moreover, the dual enzymatic amplification from DNase I and the CRISPR/Cas12a system guaranteed the sensitivity of this method with a low detection limit of 0.04 ng/mL. The developed biosensor extended the application of the CRISPR/Cas12a system for the sensitive detection of non-nucleic acid targets, providing a powerful tool in various fields such as environmental monitoring, food safety and clinical diagnosis.}, }
@article {pmid40412122, year = {2025}, author = {Wang, R and Yang, Y and Wang, Z and Ma, C and Wu, M and Du, Y and Zhang, X and Cao, M and Xu, H}, title = {Stimuli-responsive peptide nanocarriers for tumor-specific CRISPR/Cas9 delivery and precision genome editing.}, journal = {Journal of colloid and interface science}, volume = {697}, number = {}, pages = {137932}, doi = {10.1016/j.jcis.2025.137932}, pmid = {40412122}, issn = {1095-7103}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; *Nanoparticles/chemistry ; *Peptides/chemistry ; HeLa Cells ; *Drug Carriers/chemistry ; Particle Size ; *Ribonucleoproteins/chemistry/genetics ; Cell Survival/drug effects ; *Neoplasms/genetics ; Surface Properties ; }, abstract = {CRISPR/Cas9 ribonucleoprotein (RNP) delivery remains a critical challenge due to its large size, instability, and off-target effects. Here, we report a stimuli-responsive cationic amphiphilic peptide, (CR3)3C, designed for cancer-targeted delivery of CRISPR/Cas9 RNP. The peptide integrates three functional domains: (1) a naphthyl-diphenylalanine (Nap-FF) motif enabling self-assembly into stable nanoparticles via aromatic interactions, (2) a matrix metalloproteinase-7 (MMP7)-cleavable linker (GPLGLA) for tumor microenvironment-specific activation, and (3) a redox-responsive cationic domain ((CRRR)3-C) for electrostatic RNP binding and glutathione (GSH)-triggered intracellular release. The (CR3)3C/RNP nanocomplexes (108.8 nm diameter, ζ = +10.89 mV) demonstrate exceptional stability and cellular uptake efficiency. Mechanistic studies reveal caveolae-mediated endocytosis and lipid raft-associated pathways, proton sponge effect-driven endosomal escape, and nuclear localization facilitated by Cas9's nuclear localization signal. In HeLa-EGFP cells, (CR3)3C/RNP shows 33.8 % gene editing efficiency at 100 nM RNP with >90 % cell viability. This work establishes a programmable, non-viral platform that synergizes enzymatic and redox responsiveness for tumor-targeted genome editing, addressing critical barriers in CRISPR therapeutics.}, }
@article {pmid40411834, year = {2025}, author = {Guo, Y and Guo, W and Li, C and Xu, H and Zhang, X and Zou, X and Sun, Z}, title = {Fe3O4@Au Nanoparticle-Enabled Magnetic Separation Coupled with CRISPR/Cas12a for Ultrasensitive Detection of Foodborne Pathogens.}, journal = {Journal of agricultural and food chemistry}, volume = {73}, number = {22}, pages = {13949-13959}, doi = {10.1021/acs.jafc.5c04580}, pmid = {40411834}, issn = {1520-5118}, mesh = {*Biosensing Techniques/instrumentation/methods ; Gold/chemistry ; CRISPR-Cas Systems ; *Staphylococcus aureus/isolation &amp; purification/genetics ; Animals ; Milk/microbiology ; *Salmonella/isolation &amp; purification/genetics ; Food Contamination/analysis ; Metal Nanoparticles/chemistry ; Food Microbiology ; Electrochemical Techniques/instrumentation/methods ; Limit of Detection ; *Endodeoxyribonucleases/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; }, abstract = {The rapid detection of foodborne pathogens, such as Staphylococcus aureus and Salmonella, is critical for ensuring food safety. Herein, we present a magnetically controlled electrochemical biosensor integrating CRISPR/Cas12a with Fe3O4@Au nanoparticles designed to achieve ultrasensitive and multiplexed detection. By utilization of the magnetic separation of CRISPR-cleaved ssDNA from Fe3O4@Au nanoparticles, the sensor circumvents intricate electrode modifications, enabling direct signal readout. This approach expedites the workflow to 65 min while achieving a detection limit of 2 CFU/mL. Additionally, the sensor exhibits signal stability over 45 days and demonstrates its versatility by enabling the separate detection of both Gram-positive (S. aureus) and Gram-negative (Salmonella) pathogens. With validation in milk samples with high interference resistance, this platform bridges CRISPR programmability with practical deployability, offering a robust solution for on-site monitoring. The innovation lies in its simplified design, enhanced stability, and clinical versatility, setting a new benchmark for rapid, low-cost pathogen detection in resource-limited environments.}, }
@article {pmid40411798, year = {2025}, author = {Shi, K and Zhang, Y and Tao, Y and Wang, Y and Yang, J and Deng, R and Yang, H}, title = {Preamplification-Free Detection of RNA N6-Methyladenosine Modification at Single-Base Resolution Using the CRISPR Tandem Assay.}, journal = {Analytical chemistry}, volume = {97}, number = {22}, pages = {11454-11461}, doi = {10.1021/acs.analchem.4c06782}, pmid = {40411798}, issn = {1520-6882}, mesh = {*Adenosine/analogs &amp; derivatives/analysis/metabolism ; Humans ; *CRISPR-Cas Systems ; RNA, Long Noncoding/genetics ; *RNA ; Lung Neoplasms/diagnosis/genetics ; }, abstract = {N6-Methyladenosine (m[6]A) ranks among the most prevalent modifications in RNA, which serves as a biomarker for diseases, such as lung cancer. Herein, we developed a CRISPR/Cas13a-Csm6 tandem assay (termed CRISPRm[6]A assay) allowing for preamplification-free, sensitive, and rapid detection of RNA m[6]A modifications. The coupling of Cas13a-Csm6 tandem with MazF endoribonuclease enables the assay to identify m[6]A RNA with single-base resolution. Compared to the CRISPRm[6]A assay using Cas13a alone, the tandem CRISPRm[6]A assay yielded an improved sensitivity for RNA detection by ∼22 times, thus enabling preamplification-free detection of RNA m[6]A. Particularly, the proposed assay enabled quantification of m[6]A abundance down to 0.5% at the picomole level in lncRNA MALAT1 and demonstrated a 100% correlation in diagnosing nonsmall cell lung cancer. In summary, the CRISPRm[6]A assay supports two key applications in biological samples: (1) precise determination of m[6]A sites and (2) quantitative measurement of m[6]A fractions. Therefore, the CRISPR tandem method presents a promising tool for RNA epigenetics-based diagnostics.}, }
@article {pmid40411669, year = {2025}, author = {Hassan, HM and Zubair, A and Helal, MH and Almagharbeh, WT and Elmagzoub, RM}, title = {New hope and promise with CRISPR-Cas9 technology for the treatment of HIV.}, journal = {Functional &amp; integrative genomics}, volume = {25}, number = {1}, pages = {108}, pmid = {40411669}, issn = {1438-7948}, mesh = {*CRISPR-Cas Systems ; Humans ; *HIV Infections/therapy/genetics/virology ; *Gene Editing/methods ; *HIV-1/genetics ; Genetic Therapy/methods ; }, abstract = {The commencement of Highly Active Antiretroviral Therapy almost completely stopped viral replication, enabling the immune system to restore its full functionality. The rise in life expectancy has resulted in a decrease in the incidence of classical infections and HIV-associated cancers. HAART has raised concerns, including its exorbitant cost (which hinders its implementation in developing nations), the need for strict adherence, and the potential for both immediate and prolonged ill effects. Lipodystrophy is a significant long-term consequence of HIV that may result in central fat accumulation and severe peripheral fat depletion. Current initiatives to tackle these difficulties include the global expansion of access to HAART, the development of novel drugs that mitigate early side effects, and the introduction of once-daily drug combinations that enhance adherence. The CRISPR-Cas9 system has facilitated the creation of a powerful instrument for precise gene editing. This method has lately established itself as the gold standard for efficient HIV-1 genome editing in HIV therapy, owing to progress in related disciplines. CRISPR may be customized to cleave specific sequences by altering Cas9. This article offers a concise overview of promising CRISPR-Cas9 technology. This technique has the potential to halt the transmission of HIV-1 and alleviate its symptoms. CRISPR-Cas9 technology will be significant in the fight against HIV-1 in the future.}, }
@article {pmid40411467, year = {2025}, author = {Wang, X and Zhong, L and Zhang, W and Wu, P and Wang, M and Li, D and Dong, L and Wang, G}, title = {CRISPR Digital Sensing: From Micronano-Collaborative Chip to Biomolecular Detection.}, journal = {ACS nano}, volume = {19}, number = {22}, pages = {20427-20451}, doi = {10.1021/acsnano.5c03474}, pmid = {40411467}, issn = {1936-086X}, mesh = {Humans ; *COVID-19/diagnosis/genetics/virology ; *CRISPR-Cas Systems/genetics ; SARS-CoV-2/genetics/isolation &amp; purification ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Biosensing Techniques/methods ; *Lab-On-A-Chip Devices ; }, abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) sensing technology proved to be valuable during the COVID-19 pandemic through its sensitivity, specificity, robustness, and versatility. However, issues such as overreliance on amplification, susceptibility to false positives, lack of quantification strategies, and complex operation procedures have hindered its broader application in bioanalysis and clinical diagnostics. The collision between micronano-collaborative chips and CRISPR technology has effectively addressed these bottlenecks, offering innovative solutions for diagnosis and treatment. Unlike conventional micronano chips, micronano digital chips enhance CRISPR's response to trace amounts of target molecules by leveraging highly controllable local environments and compartmentalized microreactors. This advancement improves detection efficiency and revolutionizes traditional in vitro bioanalytical processes. First, the working principles, fabrication techniques, and performance metrics of CRISPR-based digital droplet microfluidics and microarray chips are examined. Then, the applications of CRISPR digital sensing chips in bioassays are reviewed, emphasizing their importance in advancing in vitro detection systems for gene editing. Finally, the prospects of CRISPR digital sensing technology are explored, particularly its potential for body surface biomonitoring and its broader development opportunities in the biomedical field.}, }
@article {pmid40410287, year = {2025}, author = {Wei, J and Shao, Y and Liang, Y and Bu, X and Zhou, W and Liang, Y and Li, Y}, title = {A type III-associated Cas6 functions as a negative regulator of type I-B CRISPR-Cas system in Thermus thermophilus.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {793}, pmid = {40410287}, issn = {2399-3642}, support = {32170096//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {*Thermus thermophilus/genetics/metabolism/enzymology ; *CRISPR-Cas Systems ; *Bacterial Proteins/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; RNA, Bacterial/metabolism/genetics ; Gene Expression Regulation, Bacterial ; }, abstract = {CRISPR-Cas systems are small RNA-guided immune systems in prokaryotes. CRISPR RNA (crRNA) provides sequence specificity and programmability, guiding the effector complex to cleave target nucleic acids. Cas6 family ribonucleases can cleave precursor crRNA to generate functional crRNAs in most type I and type III CRISPR-Cas systems. Most existing studies of Cas6 functions are mainly focused on nuclease activity in vitro and Cas6-processed product characterization in vivo. However, in hosts harboring multiple CRISPR systems, the biological functions of the co-occurrence of various Cas6 proteins and their cross-cleavage activity toward different types of crRNAs remain largely unexplored. In this study, we biochemically characterized the cross-cleavage activity of two Cas6 proteins in Thermus thermophilus HB27 and first found that Cas6 could anchor the mature crRNA and interact with Cas5 subunit of type I-B system, revealing the functions of Cas6 to mediate the assembly of type I Cascade complex. We further demonstrated that the type III-associated Cas6 protein could act as a negative regulator by competing with the I-B Cas6 protein during the assembly of type I-B Cascade complex, significantly suppressing the interference activity of type I-B system. Our findings provide an insight into the functional coupling and regulation mechanisms underlying multiple CRISPR-Cas systems.}, }
@article {pmid40409866, year = {2025}, author = {Wang, L and Wu, Y and Pan, S and Pan, F and Chen, J}, title = {Rapid and sensitive detection of Karlodinium veneficum using RAA and CRISPR-Cas12a technologies.}, journal = {Harmful algae}, volume = {146}, number = {}, pages = {102864}, doi = {10.1016/j.hal.2025.102864}, pmid = {40409866}, issn = {1878-1470}, mesh = {*Dinoflagellida/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Harmful Algal Bloom ; }, abstract = {The harmful algal species Karlodinium veneficum (K. veneficum) poses a significant threat to aquatic ecosystems, economic stability, and human health due to its toxin production and widespread occurrence. Rapid climatic changes and eutrophication have intensified harmful algal blooms (HABs), making the timely detection of K. veneficum critical. To address this need, we developed a rapid and accurate detection method of K. veneficum by combining Recombinase Aided Amplification (RAA) with CRISPR/LbCas12a. This method targets the internal transcribed spacer (ITS) sequence of K. veneficum and utilizes the "collateral activity" of CRISPR/LbCas12a for visualization. Our method can detect plasmid DNA as low as 5.9 × 10[2] copies/µL and genomic DNA as low as 3.6 × 10[-2] ng/µL, achieving a detection limit of 10 cells of K. veneficum through a simplified DNA extraction process. The entire detection process, from DNA crude extract to result visualization, can be completed in as fast as 90 min, making it suitable for field applications requiring a rapid response. In addition, our method was validated against a wide range of non-target microalgae species, confirming its specificity to K. veneficum and eliminating the risk of cross-reactivity. Overall, the RAA-CRISPR/LbCas12a system is simple, accurate, and sensitive, showing great potential for field applications in monitoring K. veneficum.}, }
@article {pmid40409549, year = {2025}, author = {Okada, R and Sakaguchi, R and Komaki, T and Nonaka, R and Polat, OK and Kihara, T and Asanuma, K and Yamamoto, T and Isaka, Y and Mori, Y and Mori, MX}, title = {Delayed inactivation of TRPC6 as a determinative characteristic of FSGS-associated variants.}, journal = {The Journal of biological chemistry}, volume = {301}, number = {6}, pages = {110256}, doi = {10.1016/j.jbc.2025.110256}, pmid = {40409549}, issn = {1083-351X}, abstract = {Transient receptor potential canonical 6 (TRPC6) is a receptor-operated nonspecific cation channel. To date, more than 30 TRPC6 variants have been reported to focal segmental glomerulosclerosis (FSGS), which can present from infancy to adulthood and is characterized by proteinuria and often nephrotic syndrome leading to kidney failure. These variants may exhibit gain-of-function (e.g. K874X) or loss-of-function (e.g. L395A, G757D) phenotypes, making the role of TRPC6 in FSGS controversial. Here, we characterized Ca[2+]-dependent inactivation (CDI) of TRPC6 after the receptor activation and found that >85% of TRPC6 variants exhibit delayed CDI. Thus, prolonged TRPC6 channel opening due to impaired inactivation may be a common feature of FSGS-associated variants. This effect was confirmed in immortalized mouse podocytes (MPC-5) in which the coiled-coil (CC) domain was deleted from the channel (C6ΔCC). Podocytes expressing C6ΔCC exhibited delayed CDI and increased basal Ca[2+] levels as well as disruption of the F-actin cytoskeleton. Moreover, transcriptomic data from C6ΔCC-expressing podocytes showed weak expression of the podocyte markers Synpo and Magi2. These results indicate that CDI of TRPC6 is critical for maintaining proper podocyte function. Notably, we observed a correlation between the magnitude of the prolongation of TRPC6 channel activity and the age diagnosed with FSGS. Our findings thus demonstrate that delayed inactivation due to lack of CDI is a determinative characteristic of FSGS-associated TRPC6 variants, affecting both the structure and the function of glomerular podocytes.}, }
@article {pmid40409480, year = {2025}, author = {Yi, B and Zhou, B and Zhou, D and Yang, L and Xu, H}, title = {CRISPR/Cas-powered nucleic acid amplification and amplification-free biosensors for public safety detection: Principles, advances and prospects.}, journal = {Biotechnology advances}, volume = {83}, number = {}, pages = {108609}, doi = {10.1016/j.biotechadv.2025.108609}, pmid = {40409480}, issn = {1873-1899}, abstract = {Rapid, accurate, cost-effective, and efficient ultrasensitive detection strategies are essential for public health safety (including food safety, disease prevention and environmental governance). The CRISPR/CRISPR-associated (Cas) detection is a cutting-edge technology that has been widely used in the detection of public health safety due to its targeted cleavage properties (signal amplification), attomolar level sensitivity, high specificity (recognizing single-base mismatches), and rapid turnover time. However, the current research about CRISPR/Cas-based biosensors is not clear, such as mechanism problem and application differences of integrating CRISPR/Cas system with other technologies, and how to further innovate and develop in the future. Therefore, further detailed analysis and comparative discussion of CRISPR/Cas-based biosensors is needed. Currently, CRISPR/Cas system powered biosensors can be mainly categorized into two types: CRISPR/Cas system powered nucleic acid amplification biosensors and CRISPR/Cas system powered nucleic acid amplification-free biosensors. The two biosensors have different characteristics and advantages. This paper first provides an in-depth investigation of the enzymatic mechanism of CRISPR/Cas system at the molecular level. Then, this paper summarizes the principles and recent advances of CRISPR/Cas system powered nucleic acid amplification biosensors and CRISPR/Cas system powered nucleic acid amplification-free biosensors and discusses their integration mechanisms in depth. More, the differences and application-oriented between the two biosensors are further discussed. Finally, the application orientation and future perspectives of the two biosensors are discussed, and unique insights into the future development of CRISPR/Cas system are provided.}, }
@article {pmid40409263, year = {2025}, author = {Yang, Z and Yao, Y and Chen, X and Madigan, V and Pu, S and Fan, X and Pu, J and Bei, F}, title = {Cross-species tropism of AAV.CPP.16 in the respiratory tract and its gene therapies against pulmonary fibrosis and viral infection.}, journal = {Cell reports. Medicine}, volume = {6}, number = {6}, pages = {102144}, doi = {10.1016/j.xcrm.2025.102144}, pmid = {40409263}, issn = {2666-3791}, mesh = {*Dependovirus/genetics/physiology ; Animals ; *Genetic Therapy/methods ; Humans ; Mice ; Genetic Vectors/genetics ; *Viral Tropism ; Administration, Intranasal ; Lung/virology/pathology ; SARS-CoV-2/genetics ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; *Respiratory System/virology/metabolism ; Gene Editing ; *Pulmonary Fibrosis/therapy/genetics ; Vascular Endothelial Growth Factor A/metabolism/genetics ; COVID-19/virology ; *Idiopathic Pulmonary Fibrosis/therapy ; *Virus Diseases/therapy ; Disease Models, Animal ; Female ; }, abstract = {Efficient gene delivery vectors are crucial for respiratory and lung disease therapies. We report that AAV.CPP.16, an engineered adeno-associated virus (AAV) variant derived from AAV9, efficiently transduces airway and lung cells in mice and non-human primates via intranasal administration. AAV.CPP.16 outperforms AAV6 and AAV9, two wild-type AAVs with demonstrated tropism for respiratory tissues, and efficiently targets key respiratory cell types. It supports gene supplementation and editing therapies in two clinically relevant mouse models of respiratory and lung diseases. A single intranasal dose of AAV.CPP.16 expressing a dual-target, vascular endothelial growth factor (VEGF)/transforming growth factor (TGF)-β1-neutralizing protein protected lungs from idiopathic pulmonary fibrosis, while a similar application of AAV.CPP.16 carrying an "all-in-one" CRISPR-Cas13d system inhibited transcription of the SARS-CoV-2-derived RNA-dependent RNA polymerase (Rdrp) gene. Our findings highlight AAV.CPP.16 as a promising vector for respiratory and lung gene therapy.}, }
@article {pmid40408898, year = {2025}, author = {Li, M and Tao, C and Tang, Z and Li, K and Wang, Y and Zhao, C and Tao, C and Geng, J and Sun, K}, title = {Ultrasensitive clinical identification of hepatitis B surface antigen (HBsAg) by CRISPR-assisted nanopore sensing.}, journal = {Biosensors &amp; bioelectronics}, volume = {286}, number = {}, pages = {117579}, doi = {10.1016/j.bios.2025.117579}, pmid = {40408898}, issn = {1873-4235}, mesh = {*Biosensing Techniques/methods ; Humans ; *Hepatitis B Surface Antigens/blood/isolation &amp; purification/genetics ; *Nanopores/ultrastructure ; *Hepatitis B virus/isolation &amp; purification/genetics ; *CRISPR-Cas Systems/genetics ; *Hepatitis B/diagnosis/virology/blood ; Limit of Detection ; Aptamers, Nucleotide/chemistry ; }, abstract = {Hepatitis B virus (HBV) infection is a major global health issue. The underdiagnosis of HBV contributes to the increasing mortality from hepatitis B-related complications. Hepatitis B surface antigen is a biomarker guiding the clinical management of chronic hepatitis B, and its disappearance from the blood is a key sign of functional cure. There is a need for highly sensitive detection methods for early intervention and prevention of disease recurrence. We presented a new CRISPR-assisted nanopore sensing method for ultrasensitive detection of hepatitis B surface antigen. It uses the high specificity and turnover efficiency of the CRISPR-Cas12a system. The system is activated by the competitive binding between hepatitis B surface antigen and its aptamer, followed by restriction enzyme digestion. The products are detected by a nanopore for precise quantification at very low concentrations. The result achieves the limit of quantification (LOQ) of 10 fM, outperforming conventional assays. Clinical validation with patient samples confirms its superiority. This integrated technology is a powerful tool for HBV early diagnosis, treatment monitoring, and disease assessment, and paves the way for nanopore technology in clinical diagnostics.}, }
@article {pmid40408895, year = {2025}, author = {Xu, Y and Deng, R and Liu, X and Zhou, Y}, title = {CRISPR/Cas12a-triggered electrochemiluminescence biosensor to ultrasensitive detect herpes simplex virus via self-enhanced near-infrared selenium-based polymer dots.}, journal = {Biosensors &amp; bioelectronics}, volume = {286}, number = {}, pages = {117597}, doi = {10.1016/j.bios.2025.117597}, pmid = {40408895}, issn = {1873-4235}, mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; *Polymers/chemistry ; Humans ; Limit of Detection ; *Simplexvirus/isolation &amp; purification/genetics ; *Selenium/chemistry ; Luminescent Measurements/methods ; *Herpes Simplex/virology/diagnosis ; Electrochemical Techniques/methods ; *Quantum Dots/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Herpes simplex virus (HSV) as one of the most popular herpesviruses exhibits nearly identical clinical symptoms among the family of herpesvirus species and it is urgent to develop high specific biosensors to realize accurate detection of HSV. CRISPR/Cas12a systems with programmability and high specificity could serve an essential role in accurate diagnosis of HSV in complex samples. Herein, a novel near-infrared selenium-based polymer dots with self-enhanced effect were unprecedentedly designed and successfully synthesized in this work, and an ultrasensitive biosensor has been constructed together with the CRISPR/Cas12a system for the specific detection of HSV. Notably, the as-prepared selenium-based polymer dots exhibited near-infrared emission with a peak at 760 nm, which could significantly minimize background noise and achieve high sensitivity. The proposed biosensor demonstrated a wide linear range from 1 fM to 1 nM and the limit of detection as low as 0.1 fM (S/N = 3). Undoubtedly, the CRISPR/Cas12a-triggered ECL biosensor proposed in this work could provide a simple, sensitive, and environmentally friendly approach to detect HSV in clinical applications.}, }
@article {pmid40408893, year = {2025}, author = {Kong, H and Yi, K and Zhu, X and Chen, L and Wang, H and Ju, E and Lv, S and Lao, YH and Shao, D and Xie, X and Cheng, D and Zhang, Y and Tao, Y and Li, M}, title = {Antifouling fusion-mediated diagnostic platform to detect viral DNA-positive extracellular vesicles for in situ blood-based liquid biopsy.}, journal = {Biosensors &amp; bioelectronics}, volume = {286}, number = {}, pages = {117568}, doi = {10.1016/j.bios.2025.117568}, pmid = {40408893}, issn = {1873-4235}, mesh = {Humans ; *Extracellular Vesicles/virology/chemistry/genetics ; Liquid Biopsy ; *DNA, Viral/blood/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; }, abstract = {In liquid biopsy, extracellular vesicles (EVs) have emerged as promising biomarkers due to their ability to carry protected nucleic acids. In particular, DNA enclosed within these vesicles shows great diagnostic potential for monitoring oncovirus-related disease progression. However, current methods still require labor-intensive procedures and bulk analysis. Additionally, in situ detection from blood is hindered by abundant serum proteins, interfering with the accuracy of diagnosis. To address these limitations, we developed an antifouling fusion-mediated CRISPR/Cas detector (AFFECTOR) as a user-friendly and efficient diagnostic platform for directly detecting EV-contained viral DNA in serum samples. Leveraging zwitterionic phosphatidylcholine to resist protein interference, the platform enables stable membrane fusion with intact EVs even in serum-containing environments, allowing highly specific and sensitive detection of internal DNA via the CRISPR/Cas12a sensing system, lasting just 2 h at 37 °C. In clinical samples from oncovirus-infected patients and healthy donors, the platform achieved one-step detection of viral DNA-positive EVs. Notably, viral DNA in circulating EVs was found for the first time to correlate with oncovirus infection stages. Overall, this platform provides a practical tool for diagnostic applications and expands the detection window in liquid biopsy.}, }
@article {pmid40405306, year = {2025}, author = {Marei, HE}, title = {Stem cell therapy: a revolutionary cure or a pandora's box.}, journal = {Stem cell research &amp; therapy}, volume = {16}, number = {1}, pages = {255}, pmid = {40405306}, issn = {1757-6512}, mesh = {Humans ; *Stem Cell Transplantation/ethics ; *Stem Cell Research/ethics/legislation &amp; jurisprudence ; Gene Editing ; *Cell- and Tissue-Based Therapy/ethics ; Embryonic Stem Cells ; Animals ; CRISPR-Cas Systems ; }, abstract = {This review article examines how stem cell therapies can cure various diseases and injuries while also discussing the difficulties and moral conundrums that come with their application. The article focuses on the revolutionary developments in stem cell research, especially the introduction of gene editing tools like CRISPR-Cas9, which can potentially improve the safety and effectiveness of stem cell-based treatments. To guarantee the responsible use of stem cells in clinical applications, it is also argued that standardizing clinical procedures and fortifying ethical and regulatory frameworks are essential first steps. The assessment also highlights the substantial obstacles that still need to be addressed, such as the moral dilemmas raised by the use of embryonic stem cells, the dangers of unlicensed stem cell clinics, and the difficulties in obtaining and paying for care for patients. The study emphasizes how critical it is to address these problems to stop exploitation, guarantee patient safety, and increase the accessibility of stem cell therapy. The review also addresses the significance of thorough clinical trials, public education, and policy development to guarantee that stem cell research may fulfill its full potential. The review concludes by describing stem cell research as a promising but complicated topic that necessitates a thorough evaluation of both the hazards and the benefits. To overcome the ethical, legal, and accessibility obstacles and eventually guarantee that stem cell treatments may be safely and fairly included in conventional healthcare, it urges cooperation between the scientific community, legislators, and the general public.}, }
@article {pmid40404851, year = {2025}, author = {Syed, M and Khan, RS and Nazir, S and Khan, S and Ul Islam, Z and Khan, S and Ikuo, N}, title = {Silencing of disease susceptibility genes: an effective disease resistance strategy against fungal pathogens.}, journal = {Plant cell reports}, volume = {44}, number = {6}, pages = {127}, pmid = {40404851}, issn = {1432-203X}, mesh = {*Plant Diseases/microbiology/genetics/immunology ; *Disease Resistance/genetics ; *Gene Silencing ; *Fungi/pathogenicity/physiology ; RNA Interference ; Host-Pathogen Interactions/genetics ; Genes, Plant ; CRISPR-Cas Systems ; }, abstract = {Silencing of target susceptibility (S) genes in plants exhibits a promising and durable strategy for enhanced resistance to fungal pathogens by causing disruption in the host mechanisms that the pathogens exploit, offering an alternative to the traditional resistance gene-based approaches. Devastating fungal diseases have significantly reduced crop productivity, posing a potential threat to global food security. Producing disease-resistant cultivars is the most effective strategy for protecting crops against these fungal pathogens. Typically, susceptibility (S) genes in host plants facilitate the penetration and proliferation of phytopathogens. Perturbation of these S genes can potentially impede the compatibility between the host and the fungal pathogens, thereby providing broad-spectrum and lasting resistance. Consequently, the identification and targeting of S-genes have gained increasing interest in enhancing disease resistance in plants. In this review, we describe three distinct categories of S genes that function during different stages of the infection process. We focus on various gene silencing technologies, including RNA interference (RNAi), virus-induced gene silencing (VIGS), and CRISPR-Cas9, to improve plant disease resistance against fungal pathogens. The numerous examples discussed here illustrate the potential of S-genes for use in plant disease-resistance breeding.}, }
@article {pmid40403777, year = {2025}, author = {Wu, HJ and Yang, JP and Ma, WJ and Li, ZH and Feng, HY and Yang, ZN and Xu, HJ}, title = {A CRISPR/Cas9-induced point mutation on the GABA receptor subunit RDL confers high resistance to phenylpyrazole insecticides in the rice planthopper Laodelphax striatellus.}, journal = {Insect biochemistry and molecular biology}, volume = {181}, number = {}, pages = {104327}, doi = {10.1016/j.ibmb.2025.104327}, pmid = {40403777}, issn = {1879-0240}, mesh = {Animals ; *Hemiptera/genetics/drug effects/metabolism ; *Insecticide Resistance/genetics ; *Insecticides/pharmacology ; *Pyrazoles/pharmacology ; CRISPR-Cas Systems ; *Receptors, GABA/genetics/metabolism ; *Point Mutation ; *Insect Proteins/genetics/metabolism ; Female ; }, abstract = {The Delphacidae planthopper Laodelphax striatellus (Order: Hemiptera) is one of the most damaging insect pests of rice crops in Asia. The phenylpyrazole insecticide fipronil was introduced in China in the mid-1990s to control these pests, but its widespread use has led to the development of high levels of resistance. Field sampling coupled with in vitro assays indicated that an A2'N-point mutation in the gamma-aminobutyric acid receptor RDL has been linked to fipronil resistance; however, genetic evidence supporting this association has been lacking. Here, we generated an A2'N-point mutation (LsRDL[A/N]) in L. striatellus using CRISPR/Cas9-mediated homologous recombination. Bioassay revealed that the LsRDL[A/N] mutants conferred a 1211-fold higher resistance to fipronil compared to wild-type planthoppers. Furthermore, these mutants also showed significant resistance (>1000-fold) to ethiprole but not to imidacloprid, demonstrating that the A2'N mutation confers resistance specific to phenylpyrazole insecticides. However, the LsRDL[A/N] mutants displayed reduced fecundity, lower egg hatching rates, and prolonged egg and nymph stages, indicating that fipronil resistance carries a substantial fitness cost. These findings enhance our understanding of the genetic mechanisms underlying phenylpyrazole resistance and provide valuable insights for developing effective resistance management strategies for planthoppers.}, }
@article {pmid40403646, year = {2025}, author = {Wu, Y and Kilgore, R and Hetzler, Z and Chu, W and Moore, B and Barbieri, E and Wei, Q and Menegatti, S}, title = {Purification of CRISPR Cas12a from E. coli cell lysates using peptide affinity ligands.}, journal = {Journal of chromatography. A}, volume = {1755}, number = {}, pages = {466065}, doi = {10.1016/j.chroma.2025.466065}, pmid = {40403646}, issn = {1873-3778}, mesh = {*Escherichia coli/chemistry/genetics/enzymology ; *Chromatography, Affinity/methods ; Ligands ; *CRISPR-Associated Proteins/isolation &amp; purification/metabolism/chemistry ; *Peptides/chemistry/metabolism ; CRISPR-Cas Systems ; *Bacterial Proteins/isolation &amp; purification/metabolism/chemistry ; *Endodeoxyribonucleases/isolation &amp; purification/metabolism/chemistry ; Acidaminococcus/enzymology ; }, abstract = {CRISPR Cas nucleases are revolutionizing gene therapy by providing a precise and efficient tool for editing the human genome, and are increasingly applied for engineering microorganisms for bioremediation, drought-resistant crops, and livestock with higher productivity. While Cas9 is currently the most widely utilized member of the CRISPR family, Cas12a stands is gaining prominence for its ability to produce staggered cuts in the target DNA while requiring a shorter guide RNA (crRNA). Current methods of Cas purification such as affinity tag, immunoaffinity, and ion exchange chromatography do not provide either the productivity or the purity needed to meet the demand of clinics and biotechnology industries. Responding to this need, this study presents the first affinity ligands for Cas12a purification via affinity chromatography. The ligands were initially designed in silico as peptide mimetics of anti-CRISPR protein inhibitors AcrVA1 and AcrVA4, and ranked experimentally by Cas12a dynamic binding. Selected ligands P5 and P9 were utilized for purifying Cas12a derived from Acidaminococcus sp. (AsCas12a) and Lachnospiraceae sp. (LbCas12a) from clarified Escherichia coli cell lysates. P5-functionalized resin afforded high yield (up to 80 %), purity (> 93 %), and DNA editing activity (∼72 %) of Cas12a from E. coli lysates featuring different Cas12a and host cell protein titers. The characterization of ligand P5 by surface plasmon resonance (SPR) indicated adsorption kinetics (ka ∼ 1.21·10[5] M[-1]s[-1]) and dissociation constant (KD ∼ 1.76·10[-6] M) that confirmed the ligand design criteria and are characteristic of peptide affinity ligands.}, }
@article {pmid40403622, year = {2025}, author = {Choi, H and Yi, TG and Gho, YS and Kim, JH and Kim, S and Choi, YJ and Lim, S and Eom, SH and Jung, KH and Ha, SH}, title = {Augmenting carotenoid accumulation by multiplex genome editing of the redundant CCD family in rice.}, journal = {Plant physiology and biochemistry : PPB}, volume = {225}, number = {}, pages = {110008}, doi = {10.1016/j.plaphy.2025.110008}, pmid = {40403622}, issn = {1873-2690}, mesh = {*Oryza/genetics/metabolism ; *Gene Editing/methods ; *Carotenoids/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; *Dioxygenases/genetics/metabolism ; Gene Knockout Techniques ; Plants, Genetically Modified ; }, abstract = {The biodegradation of carotenoid is carried out and regulated by a family of carotenoid cleavage dioxygenases (CCDs). In rice, potential redundancy of OsCCD1, OsCCD4a, and OsCCD4b, among multiple CCDs, was predicted through in silico protein-ligand docking simulations, which were based on the interactions of diverse carotene and xanthophyll substrates in the active sites. To elucidate the roles of the three CCDs in planta, we generated single, double, and triple knockout (KO) rice lines using CRISPR-Cas9 technology and confirmed their genetic stabilities. Triple KO (osccd1/osccd4a/osccd4b) lines exhibited enhanced carotenoid contents notably in both leaves after the harvesting stage and under dark-induced senescence, verifying that was caused by a simultaneously successful blocking of OsCCD1/OsCCD4a/OsCCD4b activities in these conditions. Transcriptional profiling of this triple KO line revealed downregulation of key genes involved in carotenoid biosynthesis, suggesting a feedback mechanism to regulate carotenoid levels. In order to explore and increase practical agricultural use of this triple KO line, we performed agronomic assessments that showed no adverse effects on major traits such as photosynthetic rate and seed productivity and then developed a T-DNA-free version of this triple KO rice line with high carotenoid content. Our study highlights the capacity of OsCCD1/OsCCD4a/OsCCD4b as promising targets for genome editing in biofortification strategies aimed at improving the functionality of rice and suggests their applicability to other forage crops.}, }
@article {pmid40403614, year = {2025}, author = {Dong, H and Qin, Y and Zhang, P and Lv, L and Yu, C and Jia, P and Zhao, J and Du, F and Guo, Y and Sun, X}, title = {"Turn-on" aptamer-immune lateral flow assays for the detection of small molecule targets based on CHA-assisted and CRISPR/Cas12a mediated signal transduction and amplification.}, journal = {Biosensors &amp; bioelectronics}, volume = {285}, number = {}, pages = {117593}, doi = {10.1016/j.bios.2025.117593}, pmid = {40403614}, issn = {1873-4235}, mesh = {*Aptamers, Nucleotide/chemistry ; CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Limit of Detection ; Signal Transduction ; }, abstract = {Lateral flow assays (LFAs) have emerged as crucial tools for on-site food safety detection due to their simple operation and intuitive detection results. Nevertheless, LFAs for small molecule targets such as pesticides often present a "Turn-off" signal output, which leads to their low sensitivity and the risk of false positives. In this study, a CRISPR/Cas12a system-mediated strategy was employed to convert aptamer signals into the signals of immune LFAs, achieving a "Turn-on" signal output for highly sensitive detection of small molecule targets. The binding of aptamers to targets released the trigger sequence to initiate the catalytic hairpin assembly (CHA) reaction, generating double-stranded DNA, which subsequently activated the CRISPR/Cas12a system to cleave the FAM-labeled Reporter. Eventually, the "Turn-on" visual output of the signal was realized through an anti-6-FAM immune LFAs. The experiment optimized the sample pool preparation, CHA reaction conditions, CRISPR/Cas12a activation parameters, and the assembly process of the LFAs. The limit of detection for procymidone was as low as 0.015 ng/mL, which was 52.67 times more sensitive than those of conventional aptamer-based LFAs without signal amplification strategies. This method exhibits high specificity for procymidone and a recovery rate ranging from 94.00% to 104.20% in vegetable samples, demonstrating excellent stability and practicability.}, }
@article {pmid40403612, year = {2025}, author = {Lin, L and Xue, Y and Tan, L and Jiang, C and Liu, M and Li, X and Qiu, J and Zhang, H and Zhou, J and Shu, B}, title = {Micro-scale thermofluidics enable autonomous and scalable CRISPR diagnostics for sexually transmitted infections screening.}, journal = {Biosensors &amp; bioelectronics}, volume = {285}, number = {}, pages = {117591}, doi = {10.1016/j.bios.2025.117591}, pmid = {40403612}, issn = {1873-4235}, mesh = {Humans ; *Biosensing Techniques/instrumentation ; *Sexually Transmitted Diseases/diagnosis/microbiology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Nucleic Acid Amplification Techniques/instrumentation ; Equipment Design ; CRISPR-Cas Systems ; Lab-On-A-Chip Devices ; }, abstract = {The development of clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection has recently been a center of interest for next-generation molecular diagnostics. Despite considerable advances, simple and effective strategies to harness the isothermal amplification reaction and CRISPR-based detection for maximal performance and minimal complexity are still desirable. Here, a thermofluidic approach leverages the micro-scale chemical and physical mechanism to perform autonomous and scalable CRISPR-based diagnostics (CRISPR-Dx) in a greatly simplified format, which was called "Thermofluidic CRISPR". Originating from the concept of convective PCR, it utilizes looped microchannel reactors to perform approximatively undisturbed isothermal amplification reaction at balanced temperature by virtue of the restricted molecular diffusion across the microchannel, in which the reagents of two reactions are compartmentalized virtually; then it creates circulatory flow within the loop channel to mix the amplificons and CRISPR reagents via Rayleigh-Bénard thermal convection, by simply warming up one side of the loop channel. Due to the simplicity and scalability, a low-cost, battery-powered, portable diagnostic platform, incorporating with smartphone-enabled real-time fluorescence readout, to perform rapid (<30 min), highly sensitive (2 copies per reaction), quantitative and multiplexed CRISPR-Dx was constructed. Its diagnostic performance in rapid screening of multiple pathogens from 196 clinical samples for syndromic testing of sexually transmitted infections was evaluated, exhibiting 97.4 % sensitivity and 100 % specificity benchmarked against the laboratory-based testing. Leveraging the micro-scale chemical and physical mechanism to simplify workflows for CRISPR-Dx may enhance their versatility and facilitate their broader applicability at the point of care.}, }
@article {pmid40403178, year = {2025}, author = {Lee, Y and Lee, JH and Lee, T and Shin, M and Yoon, J}, title = {Amplification-Free CRISPR/Cas12a-Based Electrochemical Biosensor with Enhanced Sensitivity for Viral Detection.}, journal = {ACS sensors}, volume = {}, number = {}, pages = {}, doi = {10.1021/acssensors.5c00576}, pmid = {40403178}, issn = {2379-3694}, abstract = {To detect contagious viral nucleic acids, traditional biosensors often require target amplification steps or use fluorescence and Raman probes tagged on nucleic acids, which are time-consuming, complex, and expensive. Recently, the CRISPR/Cas12a has received the attraction for development of nucleic acid biosensors, beyond its conventional role-like gene editing, but the enhancement of the sensitivity of CRISPR/Cas-based biosensors is still required to simplify the biosensing steps. Here, we develop a CRISPR/Cas12a-based electrochemical biosensor for the detection of viral nucleic acids in a simple manner. The novel mismatch Ag probe (MAP), as a sensing probe, and the highly conductive gold electrode on indium tin oxide with a nano array (GELITION) are introduced that enable the amplification-free and ultrasensitive detection of nucleic acids using a CRISPR/Cas12a system. The biosensing ability of the developed biosensor is validated using human papillomavirus type 16 and 18 viral DNAs (HPV16 and HPV18), achieving a limit of detection (LOD) of 1 fM without amplification and complex steps. Our developed biosensor is expected to be applicable in detecting various viruses and could contribute to the early detection of future pandemics.}, }
@article {pmid40402294, year = {2025}, author = {Hassane, AMA and Obiedallah, M and Karimi, J and Khattab, SMR and Hussein, HR and Abo-Dahab, Y and Eltoukhy, A and Abo-Dahab, NF and Abouelela, ME}, title = {Unravelling fungal genome editing revolution: pathological and biotechnological application aspects.}, journal = {Archives of microbiology}, volume = {207}, number = {7}, pages = {150}, pmid = {40402294}, issn = {1432-072X}, mesh = {*Gene Editing/methods ; *Genome, Fungal ; CRISPR-Cas Systems ; *Biotechnology/methods ; *Fungi/genetics/pathogenicity ; Humans ; Candida albicans/genetics/pathogenicity ; Aspergillus fumigatus/genetics/pathogenicity ; }, abstract = {Fungi represent a broad and evolutionarily unique group within the eukaryotic domain, characterized by extensive ecological adaptability and metabolic versatility. Their inherent biological intricacy is evident in the diverse and dynamic relationships they establish with various hosts and environmental niches. Notably, fungi are integral to disease processes and a wide array of biotechnological innovations, highlighting their significance in medical, agricultural, and industrial domains. Recent advances in genetic engineering have revolutionized fungal research, with CRISPR/Cas emerging as the most potent and versatile genome editing platform. This technology enables precise manipulation of fungal genomes, from silencing efflux pump genes in Candida albicans (enhancing antifungal susceptibility) to targeting virulence-associated sirtuins in Aspergillus fumigatus (attenuating pathogenicity). Its applications span gene overexpression, multiplexed mutagenesis, and secondary metabolite induction, proving transformative for disease management and biotechnological innovation. CRISPR/Cas9's advantages-unmatched precision, cost-effectiveness, and therapeutic potential-are tempered by challenges like off-target effects, ethical dilemmas, and regulatory gaps. Integrating nanoparticle delivery systems and multi-omics approaches may overcome technical barriers, but responsible innovation requires addressing these limitations. CRISPR-driven fungal genome editing promises to redefine solutions for drug-resistant infections, sustainable bioproduction, and beyond as the field evolves. In conclusion, genome editing technologies have enhanced our capacity to dissect fungal biology and expanded fungi's practical applications across various scientific and industrial domains. Continued innovation in this field promises to unlock the vast potential of fungal systems further, enabling more profound understanding and transformative biotechnological progress.}, }
@article {pmid40401996, year = {2025}, author = {Pandey, N and Misra, CS and Rath, D}, title = {Cas11 augments Cascade functions in type I-E CRISPR system but is redundant for gene silencing and plasmid interference.}, journal = {The Biochemical journal}, volume = {482}, number = {12}, pages = {}, doi = {10.1042/BCJ20253056}, pmid = {40401996}, issn = {1470-8728}, mesh = {*CRISPR-Cas Systems ; *Plasmids/genetics/metabolism ; *Escherichia coli Proteins/genetics/metabolism ; *Gene Silencing ; *Escherichia coli/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; Gene Editing ; *Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {The structural and mechanistic complexity of Escherichia coli's type I Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR-Cas) system, compared with the multidomain, single effector protein-based type II systems, limits its application in genome editing and silencing. Despite the higher prevalence of the type I endogenous systems in bacteria, significant research has focused on improving the type II systems. While the type-I CRISPR system possesses several advantages over others, it may benefit from further studies to simplify the system for ease of use. To enable this, the dispensability of the type-I Cascade components (Cas8, Cas11, Cas7, Cas5 and Cas6) for genome editing and silencing applications was evaluated in vivo. We created deletion variants of each of the Cascade components and investigated their effects on gene silencing and plasmid interference in two genetically distinct E. coli lineages: BW25113, a K-12 strain that bears an endogenous, albeit repressed type I-E CRISPR system; and BL21, a natural mutant lacking the type I-E CRISPR-Cas system. Cas8, Cas7 and Cas5 were found to be indispensable for gene silencing and plasmid interference. Dispensability of Cas6, which is involved in crRNA maturation, was strain-dependent. Notably, Cas11, which has no definitive function assigned to it, was found to be dispensable for gene silencing and plasmid interference.}, }
@article {pmid40401794, year = {2025}, author = {Jiao, L and Zhou, Q and Sun, D}, title = {CRISPR-Based Regulation for High-Throughput Screening.}, journal = {ACS synthetic biology}, volume = {14}, number = {6}, pages = {1890-1904}, doi = {10.1021/acssynbio.5c00076}, pmid = {40401794}, issn = {2161-5063}, abstract = {CRISPR technology has revolutionized genome editing by enabling precise, permanent modifications to genetic material. To circumvent the irreversible alterations associated with traditional CRISPR methods and facilitate research on both essential and nonessential genes, CRISPR interference or inhibition (CRISPRi) and CRISPR activation (CRISPRa) were developed. The gene-silencing approach leverages an inactivated Cas effector protein paired with guide RNA to obstruct transcription initiation or elongation, while the gene-activation approach exploits the programmability of CRISPR to activate gene expression. Recent advances in CRISPRi technology, in combination with other technologies (e.g., biosensing, sequencing), have significantly expanded its applications, allowing for genome-wide high-throughput screening (HTS) to identify genetic determinants of phenotypes. These screening strategies have been applied in biomedicine, industry, and basic research. This review explores the CRISPR regulation mechanisms, offers an overview of the workflow for genome-wide CRISPR-based regulation for screens, and highlights its superior suitability for HTS across biomedical and industrial applications. Finally, we discuss the limitations of current CRISPRi/a HTS screening methods and envision future directions in CRISPR-mediated HTS research, considering its potential for broader application across diverse fields.}, }
@article {pmid40401239, year = {2025}, author = {Mak, JK and Bendandi, A and Salim, JA and Mazoni, I and de Moraes, FR and Borro, L and Störtz, F and Rocchia, W and Neshich, G and Minary, P}, title = {Learning to utilize internal protein 3D nanoenvironment descriptors in predicting CRISPR-Cas9 off-target activity.}, journal = {NAR genomics and bioinformatics}, volume = {7}, number = {2}, pages = {lqaf054}, pmid = {40401239}, issn = {2631-9268}, mesh = {*CRISPR-Cas Systems ; *Machine Learning ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; Gene Editing ; Mutation ; }, abstract = {Despite advances in determining the factors influencing cleavage activity of a CRISPR-Cas9 single guide RNA (sgRNA) at an (off-)target DNA sequence, a comprehensive assessment of pertinent physico-chemical/structural descriptors is missing. In particular, studies have not yet directly exploited the information-rich internal protein 3D nanoenvironment of the sgRNA-(off-)target strand DNA pair, which we obtain by harvesting 634 980 residue-level features for CRISPR-Cas9 complexes. As a proof-of-concept study, we simulated the internal protein 3D nanoenvironment for all experimentally available single-base protospacer-adjacent motif-distal mutations for a given sgRNA-target strand pair. By determining the most relevant residue-level features for CRISPR-Cas9 off-target cleavage activity, we developed STING_CRISPR, a machine learning model delivering accurate predictive performance of off-target cleavage activity for the type of single-base mutations considered in this study. By interpreting STING_CRISPR, we identified four important Cas9 residue spatial hotspots and associated structural/physico-chemical descriptor classes influencing CRISPR-Cas9 (off-)target cleavage activity for the sgRNA-target strand pairs covered in this study.}, }
@article {pmid40400348, year = {2025}, author = {Khatri, M and Shanmugam, NRS and Zhang, X and Patel, RSKR and Yin, Y}, title = {AcrDB update: Predicted 3D structures of anti-CRISPRs in human gut viromes.}, journal = {Protein science : a publication of the Protein Society}, volume = {34}, number = {6}, pages = {e70177}, pmid = {40400348}, issn = {1469-896X}, support = {R01GM140370/NH/NIH HHS/United States ; R21AI171952/NH/NIH HHS/United States ; 58-8042-9-089//U.S. Department of Agriculture (USDA)/ ; }, mesh = {Humans ; *Bacteriophages/chemistry/genetics ; Protein Conformation ; Databases, Protein ; *Viral Proteins/chemistry/genetics/metabolism ; *Gastrointestinal Microbiome ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Models, Molecular ; }, abstract = {Anti-CRISPR (Acr) proteins play a key role in phage-host interactions and hold great promise for advancing genome-editing technologies. However, finding new Acrs has been challenging due to their low sequence similarity. Recent advances in protein structure prediction have opened new pathways for Acr discovery by using 3D structure similarity. This study presents an updated AcrDB, with the following new features not available in other databases: (1) predicted Acrs from human gut virome databases, (2) Acr structures predicted by AlphaFold2, (3) a structural similarity search function to allow users to submit new sequences and structures to search against 3D structures of experimentally known Acrs. The updated AcrDB contains predicted 3D structures of 795 candidate Acrs with structural similarity (TM-score ≥0.7) to known Acrs supported by at least two of the three non-sequence similarity-based tools (TM-Vec, Foldseek, AcrPred). Among these candidate Acrs, 121 are supported by all three tools. AcrDB also includes 3D structures of 122 experimentally characterized Acr proteins. The 121 most confident candidate Acrs were combined with the 122 known Acrs and clustered into 163 sequence similarity-based Acr families. The 163 families were further subject to a structure similarity-based hierarchical clustering, revealing structural similarity between 44 candidate Acr (cAcr) families and 119 known Acr families. The bacterial hosts of these 163 Acr families are mainly from Bacillota, Pseudomonadota, and Bacteroidota, which are all dominant gut bacterial phyla. Many of these 163 Acr families are also co-localized in Acr operons. All the data and visualization are provided on our website: https://pro.unl.edu/AcrDB.}, }
@article {pmid40400168, year = {2025}, author = {Jiang, M and Zhao, X and Zhang, C and Liu, R and Hu, J and Lv, Y}, title = {Thermus thermophilus Argonaute-Mediated Single Particle Counting Platform for Multiplex Cancer-Related Biomarkers Detection.}, journal = {Analytical chemistry}, volume = {97}, number = {22}, pages = {11907-11916}, doi = {10.1021/acs.analchem.5c02118}, pmid = {40400168}, issn = {1520-6882}, mesh = {Humans ; *Thermus thermophilus/metabolism/chemistry ; *Argonaute Proteins/metabolism/chemistry ; *MicroRNAs/analysis/blood/genetics ; *Biomarkers, Tumor/analysis/blood/genetics ; *Bacterial Proteins/metabolism/chemistry ; Mass Spectrometry ; *Colorectal Neoplasms/diagnosis ; }, abstract = {The clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) system has achieved remarkable success in the field of nucleic acid detection, while its Achilles' heel lies in the difficulties encountered in flexibility regarding the multiplex detection. As a sister system of CRISPR-Cas, prokaryotic Argonautes (pAgos) have precise recognition, multiturnover, and more importantly multiple specific cleavage characteristics, which is a potential candidate for the next generation of multiplex detection. Herein, a single particle counting platform was developed for the simultaneous detection of three colorectal cancer-related miRNAs (miR-141, miR-31, and miR-21) by combining single particle inductively coupled plasma mass spectrometry (SP-ICPMS) with the Thermus thermophilus Argonaute protein (TtAgo), with nanoparticles as signal probes for cleavage. The platform demonstrated high sensitivity (aM level) and specificity due to the dual-cycle mechanism of exponential isothermal amplification (EXPAR) and TtAgo cleavage, as well as the combination of TtAgo's specific cleavage capability and the multiplex detection advantages of metal stable isotope tagging. Additionally, the platform showed good robustness in human serum and cell extracts, indicating significant potential in clinical applications.}, }
@article {pmid40400140, year = {2025}, author = {Jordt, LM and Gellert, M and Zelms, F and Bekeschus, S and Lillig, CH}, title = {The thioredoxin-like and one glutaredoxin domain are required to rescue the iron-starvation phenotype of HeLa GLRX3 knock out cells.}, journal = {FEBS letters}, volume = {}, number = {}, pages = {}, doi = {10.1002/1873-3468.70072}, pmid = {40400140}, issn = {1873-3468}, support = {Li984/3-1//Deutsche Forschungsgemeinschaft/ ; Li984/4-1//Deutsche Forschungsgemeinschaft/ ; }, abstract = {Glutaredoxin 3 (Grx3) is a multidomain protein (Trx-GrxA-GrxB) with a Trx-like domain and two Grx domains containing a CGFS motif for binding Fe2S2 clusters. To study the function of these domains, HeLa cells with GLRX3 knockout were generated via CRISPR/Cas. The knockout activated iron-regulatory protein 1, indicating iron starvation due to impaired iron metabolism. Transfection with constructs encoding wild-type or individual domains showed that only the Trx-GrxA construct could rescue the phenotype, matching the effect of full-length Grx3. The specific role of the second Grx domain in human Grx3, absent in simpler eukaryotes such as yeast, remains unclear. While the individual domains are insufficient to rescue the knockout of full-length Grx3, the Trx-GrxA module is functionally critical. Impact statement Glutaredoxin 3 (Grx3) contains a Trx-like domain and two Grx domains. The importance of the domains in higher eukaryotes has not previously been addressed in physiological or cellular contexts. Here, we report GLRX3 knockout results in activation of iron regulatory protein 1, and a Trx-GrxA construct could rescue the phenotype.}, }
@article {pmid40400023, year = {2025}, author = {Kermanshahi, AZ and Ebrahimi, F and Taherpoor, A and Eslami, N and Baghi, HB}, title = {HPV-driven cancers: a looming threat and the potential of CRISPR/Cas9 for targeted therapy.}, journal = {Virology journal}, volume = {22}, number = {1}, pages = {156}, pmid = {40400023}, issn = {1743-422X}, mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Papillomavirus Infections/therapy/virology/complications ; *Neoplasms/therapy/virology ; *Papillomaviridae/genetics ; *Genetic Therapy/methods ; Oncogene Proteins, Viral/genetics ; Female ; *Uterine Cervical Neoplasms/therapy/virology ; }, abstract = {Cervical and other anogenital malignancies are largely caused by E6 and E7 oncogenes of high-risk human papillomaviruses (HPVs), which inhibit important tumor suppressors like p53 and pRb when they are persistently activated. The main goal of traditional treatments is to physically or chemically kill cancer cells, but they frequently only offer temporary relief, have serious side effects, and have a high risk of recurrence. Exploring the efficacy and accuracy of CRISPR-Cas9 gene editing in both inducing death in HPV-infected cancer cells and restoring the activity of tumor suppressors is our main goal. In this study, we propose a novel precision oncology strategy that targets and inhibits the detrimental effects of the E6 and E7 oncogenes using the CRISPR-Cas9 gene editing system. In order to do this, we create unique guide RNAs that target the integrated HPV DNA and reactivate p53 and pRb. Reactivation is meant to halt aberrant cell development and restart the cell's natural dying pathways. This review discusses the potential of CRISPR/Cas9 in targeting HPV oncogenes, with a focus on studies that have demonstrated its promise in cancer treatment. Given the absence of a definitive treatment for papillomavirus infection and its subsequent association with various cancers, future clinical trials and experimental investigations appear essential to establish and evaluate the therapeutic potential of CRISPR-based approaches. This approach provides a less invasive alternative to conventional treatments and opens the door to personalized care that considers the genetic makeup of each patient's tumor.}, }
@article {pmid40399682, year = {2025}, author = {Panagopoulos, A and Stout, M and Kilic, S and Leary, P and Vornberger, J and Pasti, V and Galarreta, A and Lezaja, A and Kirschenbühler, K and Imhof, R and Rehrauer, H and Ziegler, U and Altmeyer, M}, title = {Multigenerational cell tracking of DNA replication and heritable DNA damage.}, journal = {Nature}, volume = {642}, number = {8068}, pages = {785-795}, pmid = {40399682}, issn = {1476-4687}, mesh = {*DNA Replication/genetics ; Humans ; *DNA Damage/genetics ; *Single-Cell Analysis ; *Cell Lineage/genetics ; *Cell Tracking/methods ; DNA Repair ; Gene Editing ; Polyploidy ; CRISPR-Cas Systems/genetics ; Cell Cycle/genetics ; Epigenesis, Genetic ; }, abstract = {Cell heterogeneity is a universal feature of life. Although biological processes affected by cell-to-cell variation are manifold, from developmental plasticity to tumour heterogeneity and differential drug responses, the sources of cell heterogeneity remain largely unclear[1,2]. Mutational and epigenetic signatures from cancer (epi)genomics are powerful for deducing processes that shaped cancer genome evolution[3-5]. However, retrospective analyses face difficulties in resolving how cellular heterogeneity emerges and is propagated to subsequent cell generations. Here, we used multigenerational single-cell tracking based on endogenously labelled proteins and custom-designed computational tools to elucidate how oncogenic perturbations induce sister cell asymmetry and phenotypic heterogeneity. Dual CRISPR-based genome editing enabled simultaneous tracking of DNA replication patterns and heritable endogenous DNA lesions. Cell lineage trees of up to four generations were tracked in asynchronously growing cells, and time-resolved lineage analyses were combined with end-point measurements of cell cycle and DNA damage markers through iterative staining. Besides revealing replication and repair dynamics, damage inheritance and emergence of sister cell heterogeneity across multiple cell generations, through combination with single-cell transcriptomics, we delineate how common oncogenic events trigger multiple routes towards polyploidization with distinct outcomes for genome integrity. Our study provides a framework to dissect phenotypic plasticity at the single-cell level and sheds light onto cellular processes that may resemble early events during cancer development.}, }
@article {pmid40399557, year = {2025}, author = {Bourgade, B and Xie, H and Lindblad, P and Stensjö, K}, title = {Development of a CRISPR activation system for targeted gene upregulation in Synechocystis sp. PCC 6803.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {772}, pmid = {40399557}, issn = {2399-3642}, support = {2021-01669//Svenska Forskningsr&#xe5;det Formas (Swedish Research Council Formas)/ ; }, mesh = {*Synechocystis/genetics/metabolism ; *Up-Regulation ; *Gene Expression Regulation, Bacterial ; Metabolic Engineering/methods ; Butanols/metabolism ; Biofuels ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {The photosynthetic cyanobacterium Synechocystis sp. PCC 6803 offers a promising sustainable solution for simultaneous CO2 fixation and compound bioproduction. While various heterologous products have now been synthesised in Synechocystis, limited genetic tools hinder further strain engineering for efficient production. Here, we present a versatile CRISPR activation (CRISPRa) system for Synechocystis, enabling robust multiplexed activation of both heterologous and endogenous targets. Following tool characterisation, we applied CRISPRa to explore targets influencing biofuel production, specifically isobutanol (IB) and 3-methyl-1-butanol (3M1B), demonstrating a proof-of-concept approach to identify key reactions constraining compound biosynthesis. Notably, individual upregulation of target genes, such as pyk1, resulted in up to 4-fold increase in IB/3M1B formation while synergetic effects from multiplexed targeting further enhanced compound production, highlighting the value of this tool for rapid metabolic mapping. Interestingly, activation efficacy did not consistently predict increases in compound formation, suggesting complex regulatory interactions influencing bioproduction. This work establishes a CRISPRa system for targeted upregulation in cyanobacteria, providing an adaptable platform for high-throughput screening, metabolic pathway optimisation and functional genomics. Our CRISPRa system provides a crucial advance in the genetic toolbox available for Synechocystis and will facilitate innovative applications in both fundamental research and metabolic engineering in cyanobacteria.}, }
@article {pmid40399309, year = {2025}, author = {Schmerer, N and Janga, H and Aillaud, M and Hoffmann, J and Aznaourova, M and Wende, S and Steding, H and Halder, LD and Uhl, M and Boldt, F and Stiewe, T and Nist, A and Jerrentrup, L and Kirschbaum, A and Ruppert, C and Rossbach, O and Ntini, E and Marsico, A and Valasarajan, C and Backofen, R and Linne, U and Pullamsetti, SS and Schmeck, B and Schulte, LN}, title = {A searchable atlas of pathogen-sensitive lncRNA networks in human macrophages.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {4733}, pmid = {40399309}, issn = {2041-1723}, support = {LOEWE/2/13/519/03/06.001(0002)/74//Hessisches Ministerium f&#xfc;r Wissenschaft und Kunst (Hessen State Ministry of Higher Education, Research and the Arts)/ ; LOEWE/5/A004/519/06/00.005(0008)/E31//Hessisches Ministerium f&#xfc;r Wissenschaft und Kunst (Hessen State Ministry of Higher Education, Research and the Arts)/ ; LOEWE/2/13/519/03/06.001(0002)/74//Hessisches Ministerium f&#xfc;r Wissenschaft und Kunst (Hessen State Ministry of Higher Education, Research and the Arts)/ ; 10.21.2.024MN//Fritz Thyssen Stiftung (Fritz Thyssen Foundation)/ ; SFB-TR-84 (subproject C10)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; BA 2168/25-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; TRR 167/2 NeuroMac//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CIBSS - EXC-2189 - Project ID 390939984//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; SFB-1213 (Project A01)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; SFB-TR-84 (subproject C1)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; PerMed-COPD (01EK2203A)//Bundesministerium f&#xfc;r Bildung und Forschung (Federal Ministry of Education and Research)/ ; Deep Legion (031L0288A)//Bundesministerium f&#xfc;r Bildung und Forschung (Federal Ministry of Education and Research)/ ; }, mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Macrophages/metabolism/immunology ; *Gene Regulatory Networks ; GATA2 Transcription Factor/genetics/metabolism ; NF-kappa B/metabolism ; Macrophage Activation/genetics ; Gene Expression Regulation ; CRISPR-Cas Systems ; }, abstract = {Long noncoding RNAs (lncRNA) are crucial yet underexplored regulators of human immunity. Here we develop GRADR, a method integrating gradient profiling with RNA-binding proteome analysis, to map the protein interactomes of all expressed RNAs in a single experiment to study mechanisms of lncRNA-mediated regulation of human primary macrophages. Applying GRADR alongside CRISPR-multiomics, we reveal a network of NFκB-dependent lncRNAs, including LINC01215, AC022816.1 and ROCKI, which modulate distinct aspects of macrophage immunity, particularly through interactions with mRNA-processing factors, such as hnRNP proteins. We further uncover the function of ROCKI in repressing the messenger of the anti-inflammatory GATA2 transcription factor, thus promoting macrophage activation. Lastly, all data are consolidated in the SMyLR web interface, a searchable reference catalog for exploring lncRNA functions and pathway-dependencies in immune cells. Our results thus not only highlight the important functions of lncRNAs in immune regulation, but also provide a rich resource for lncRNA studies.}, }
@article {pmid40399255, year = {2025}, author = {Riesenberg, S and Kanis, P and Karlic, R and Maricic, T}, title = {Robust prediction of synthetic gRNA activity and cryptic DNA repair by disentangling cellular CRISPR cleavage outcomes.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {4717}, pmid = {40399255}, issn = {2041-1723}, mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Humans ; *DNA Repair/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; INDEL Mutation ; CRISPR-Associated Protein 9/metabolism/genetics ; DNA Cleavage ; CRISPR-Associated Proteins/metabolism/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; HEK293 Cells ; Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins/metabolism/genetics ; }, abstract = {The ability to robustly predict guide RNA (gRNA) activity is a long-standing goal for CRISPR applications, as it would reduce the need to pre-screen gRNAs. Quantification of formation of short insertions and deletions (indels) after DNA cleavage by transcribed gRNAs has been typically used to measure and predict gRNA activity. We evaluate the effect of chemically synthesized Cas9 gRNAs on different cellular DNA cleavage outcomes and find that the activity of different gRNAs is largely similar and often underestimated when only indels are scored. We provide a simple linear model that reliably predicts synthetic gRNA activity across cell lines, robustly identifies inefficient gRNAs across different published datasets, and is easily accessible via online genome browser tracks. In addition, we develop a homology-directed repair efficiency prediction tool and show that unintended large-scale repair events are common for Cas9 but not for Cas12a, which may be relevant for safety in gene therapy applications.}, }
@article {pmid40398759, year = {2025}, author = {Zhao, S and Zhang, J and Yu, S and He, D and Li, B and Fan, Y and Liu, G and Tang, Y and Diao, Y}, title = {Integration of RPA and CRISPR-Cas13a collateral activity for one-step detection of DHAV-3: A biological macromolecule-enabled diagnostic platform.}, journal = {International journal of biological macromolecules}, volume = {314}, number = {}, pages = {144400}, doi = {10.1016/j.ijbiomac.2025.144400}, pmid = {40398759}, issn = {1879-0003}, mesh = {*CRISPR-Cas Systems/genetics ; Animals ; *Recombinases/metabolism ; RNA, Viral/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Picornaviridae/genetics/isolation &amp; purification ; Ducks/virology ; }, abstract = {Duck hepatitis A virus type 3 (DHAV-3), which is families of the Picornaviridae, poses severe threats to waterfowl industries due to rapid antigenic evolution and limitations in conventional diagnostics. Herein, we engineered a CRISPR-Cas13a-mediated RNA detection system by leveraging the intrinsic HEPN domain-dependent collateral cleavage activity of Cas13a, synergistically integrated with recombinase polymerase amplification (RPA) to target DHAV-3 RNA. This biological macromolecule-driven platform achieved ultrasensitive detection (1 copies/μL) within 35 min through sequence-specific crRNA guidance and Cas13a-triggered fluorescent/lateral flow signal amplification. Rigorous validation against four avian pathogens (ARV, H9N2 AIV, TMUV, AstV) confirmed 100 % specificity, highlighting the precise macromolecular interactions between Cas13a and target RNA. Clinical evaluation of 30 field samples demonstrated complete concordance with RT-qPCR. By harnessing the programmable functionality of Cas13a and the thermostable enzymes in RPA, this study provides a novel paradigm for RNA-guided biological macromolecule applications in point-of-care diagnostics, bridging molecular mechanisms with agricultural biosecurity needs.}, }
@article {pmid40398569, year = {2025}, author = {Zhou, Y and Duan, Y and Chen, L and Yang, Y and Ma, L and Chen, W and Liao, Z and Cai, J and Li, D}, title = {Engineering dispersed mycelium morphology in Aspergillus niger for enhanced mycoprotein production via CRISPR/Cas9-mediated genome editing.}, journal = {Bioresource technology}, volume = {432}, number = {}, pages = {132652}, doi = {10.1016/j.biortech.2025.132652}, pmid = {40398569}, issn = {1873-2976}, mesh = {*Aspergillus niger/genetics/metabolism/cytology ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Mycelium/genetics/metabolism/cytology ; Fermentation ; *Fungal Proteins/biosynthesis/genetics/metabolism ; Biomass ; }, abstract = {Filamentous fungi are widely utilized in industrial fermentation processes due to their high productivity, with mycelial morphology directly influencing fermentation broth viscosity and target product yield, which is a critical parameter for process optimization. Aspergillus niger, an FDA-approved safe filamentous fungus, typically forms tightly packed mycelial pellets in submerged cultures, which severely restricts its industrial application potential by limiting mass transfer efficiency. To address this challenge, CRISPR/Cas9 mediated genome editing coupled with fermentation optimization enhanced microbial protein production in A. niger. Endogenous α-1,3-glucan synthase genes (agsA, agsB) and galactosaminogalactan (GAG) synthase genes (sph3, uge3) were disrupted using CRISPR/Cas9, achieving complete dispersion of filamentous pellets in liquid media. This morphological engineering strategy resulted in a 77.52 % increase in biomass and 39.98 % enhancement in mycelial protein content compared to the wild-type strain (A. niger Li2). Transcriptomic analysis revealed that the engineered strain (A. niger AnΔABSU) exhibited upregulated transporter proteins (ABC transporters, MFS transporters, sugar transporters), accelerating nutrient uptake and energy metabolism; altered cell wall integrity pathways, including activation of the MAPK signaling cascade and increased sensitivity to cell wall stressors; enhanced amino acid biosynthesis, driven by upregulated gene expression in key metabolic pathways. Furthermore, response surface methodology (RSM) with Box-Behnken design optimized the fermentation medium, yielding 16.67 g/L biomass and 45.91 % protein content, representing 115.37 % and 67.01 % improvements over the unoptimized wild-type control. This study establishes a novel paradigm for constructing high-efficiency microbial protein cell factories via integrated morphological-engineering and fermentation optimization.}, }
@article {pmid40398216, year = {2025}, author = {Huang, Y and Li, H and Qi, L and Wang, Z and Liu, Z and Wu, R and Chen, Q and Zhu, C and Sun, D and Liu, L and Zhang, L and Feng, G}, title = {NanoCRISPR-assisted biomimetic tissue-equivalent patch regenerates the intervertebral disc by inhibiting endothelial-to-mesenchymal transition.}, journal = {Biomaterials}, volume = {322}, number = {}, pages = {123404}, doi = {10.1016/j.biomaterials.2025.123404}, pmid = {40398216}, issn = {1878-5905}, mesh = {Animals ; Humans ; *Intervertebral Disc/physiology ; *Regeneration ; Intervertebral Disc Degeneration/therapy/pathology ; Tissue Scaffolds/chemistry ; Vascular Endothelial Growth Factor A/metabolism ; *Biomimetic Materials/chemistry/pharmacology ; *Nanoparticles/chemistry ; CRISPR-Cas Systems ; Annulus Fibrosus ; Indoles/chemistry ; Polymers/chemistry ; }, abstract = {The integrity of the intervertebral disc (IVD), an immune-privileged organ protected by the blood-disc barrier, is compromised following annulus fibrosus (AF) injury. This breach facilitates angiogenesis, immune cell infiltration, and inflammation, accelerating intervertebral disc degeneration (IDD) and resulting in various clinical disorders. Current treatments fail to adequately address biological repair of AF defects and angiogenesis. Single-cell RNA sequencing analyses reveal that vascular endothelial growth factor (VEGF), secreted by IDD-associated fibrochondrocytes, is crucial in promoting angiogenesis by inducing endothelial-to-mesenchymal transition (EndoMT). This study proposes a nano-clustered regularly interspaced short palindromic repeats (CRISPR)-assisted AF patch with an aligned, polydopamine-modified nano-lamellae nanofibrous scaffold that replicates the hierarchical structure of natural AF, providing a conducive microenvironment for AF repair. A zeolitic imidazolate framework-8-based nanoCRISPR system encapsulates the CRISPR/CRISPR-associated protein 9 complex to target and eliminate VEGF-mediated angiogenic factors. In vitro studies demonstrate that the nanoCRISPR-assisted patch can enhance AF cell adhesion and migration, promote extracellular matrix deposition, knock out VEGF expression, and inhibit EndoMT. In vivo studies show its significant efficacy in promoting AF repair, inhibiting abnormal angiogenesis, and delaying IDD progression. This study presents a promising approach for structural and biological AF regeneration, addressing physical and angiogenic barriers in IVD regeneration.}, }
@article {pmid40397713, year = {2025}, author = {Teng, H and Hang, Q and Zheng, C and Yan, Y and Liu, S and Zhao, Y and Deng, Y and Nie, L and Wu, W and Sheldon, M and Yu, Z and Shi, W and Gao, J and Meng, C and Martinez, C and Zhang, J and Yao, F and Sun, Y and Zhao, D and Gan, B and Meng, T and Ma, L}, title = {In vivo CRISPR activation screen identifies acyl-CoA-binding protein as a driver of bone metastasis.}, journal = {Science translational medicine}, volume = {17}, number = {799}, pages = {eado7225}, doi = {10.1126/scitranslmed.ado7225}, pmid = {40397713}, issn = {1946-6242}, mesh = {Animals ; *Bone Neoplasms/secondary/genetics/metabolism/pathology ; Humans ; Cell Line, Tumor ; Mice ; *Diazepam Binding Inhibitor/metabolism/genetics ; Lipid Metabolism ; Fatty Acids/metabolism ; Reactive Oxygen Species/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Ferroptosis ; Oxidation-Reduction ; Neoplasm Metastasis ; Female ; *CRISPR-Cas Systems/genetics ; }, abstract = {One of the most common sites of cancer metastasis is to the bone. Bone metastasis is associated with substantial morbidity and mortality, and current therapeutic interventions remain largely palliative. Metastasizing tumor cells need to reprogram their metabolic states to adapt to the nutrient environment of distant organs; however, the role and translational relevance of lipid metabolism in bone metastasis remain unclear. Here, we used an in vivo CRISPR activation screening system coupled with positive selection to identify acyl-coenzyme A (CoA) binding protein (ACBP) as a bone metastasis driver. In nonmetastatic and weakly metastatic cancer cells, overexpression of wild-type ACBP, but not the acyl-CoA-binding deficient mutant, stimulated fatty acid oxidation (FAO) and bone metastasis. Conversely, knockout of ACBP in highly bone metastatic cancer cells abrogated metastatic bone colonization. Mechanistically, ACBP-mediated FAO increased ATP and NADPH production, reduced reactive oxygen species, and inhibited lipid peroxidation and ferroptosis. We found that ACBP expression correlated with metabolic signaling, bone metastatic ability, and poor clinical outcomes. In mouse models, pharmacological blockade of FAO or treatment with a ferroptosis inducer inhibited bone metastasis. Together, our findings reveal the role of lipid metabolism in tumor cells adapting and thriving in the bone and identify ACBP as a key regulator of this process. Agents that target FAO or induce ferroptosis represent a promising therapeutic approach for treating bone metastases.}, }
@article {pmid40397663, year = {2025}, author = {Gast, K and Baker, S and Borges, AL and Ward, S and Banfield, JF and Barrangou, R}, title = {Metagenome-Derived CRISPR-Cas12a Mining and Characterization.}, journal = {The CRISPR journal}, volume = {8}, number = {3}, pages = {189-204}, doi = {10.1089/crispr.2024.0099}, pmid = {40397663}, issn = {2573-1602}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Metagenome/genetics ; Escherichia coli/genetics ; Animals ; Cattle ; *CRISPR-Associated Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins/genetics ; }, abstract = {The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies has revolutionized genome editing, with continued interest in expanding the CRISPR-associated proteins (Cas) toolbox with diverse, efficient, and specific effectors. CRISPR-Cas12a is a potent, programmable RNA-guided dual nickase, broadly used for genome editing. Here, we mined dairy cow microbial metagenomes for CRISPR-Cas systems, unraveling novel Cas12a enzymes. Using in silico pipelines, we characterized and predicted key drivers of CRISPR-Cas12a activity, encompassing guides and protospacer adjacent motifs for five systems. We next assessed their functional potential in cell-free transcription-translation assays with GFP-based fluorescence readouts. Lastly, we determined their genome editing potential in vivo in Escherichia coli by generating 1 kb knockouts. Unexpectedly, we observed natural sequence variation in the bridge-helix domain of the best-performing candidate and used mutagenesis to alter the activity of Cas12a orthologs, resulting in increased gene editing capabilities of a relatively inefficient candidate. This study illustrates the potential of underexplored metagenomic sequence diversity for the development and refinement of genome editing effectors.}, }
@article {pmid40396759, year = {2025}, author = {Vergnes, J-B and Roger, B and Iggo, R and Wodrich, H}, title = {Advanced viral genome in vitro Cas9 editing (AdVICE): an overnight method for traceless and limitless manipulation of adenoviral and vector genomes with large transgenes.}, journal = {Journal of virology}, volume = {99}, number = {6}, pages = {e0226524}, pmid = {40396759}, issn = {1098-5514}, support = {ANR-19-CE15-0013//Agence Nationale de la Recherche/ ; ANR-21-CE14-0074//Agence Nationale de la Recherche/ ; 2021-0261/2022-0225//French Cancer Ligue Comite des Landes/ ; 2021-0174//French Cancer Ligue Comite des Pyrenees-Atlantiques/ ; }, mesh = {*Genome, Viral ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Adenoviridae/genetics ; *Genetic Vectors/genetics ; *Transgenes ; Humans ; }, abstract = {The size and complexity of large viral genomes limit the technical possibilities for genome manipulations in fundamental research and medical or technological applications. State-of-the-art recombineering in bacteria has partially overcome this limit but remains a time-consuming and complex procedure requiring specialist expertise. Here, we describe a simplified and highly efficient in vitro protocol for unlimited and traceless manipulation applicable to large viral genomes from DNA viruses using a combination of CRISPR/Cas9 cleavage and in vitro DNA assembly. We successfully used the protocol to manipulate adenovirus genomes, showing that genome rescue from viruses, insertions, deletions, and mutagenesis can be performed in a simple overnight procedure in a standard laboratory setting without the need for advanced knowledge of molecular biology. Finally, we use our approach to demonstrate the de novo, multi-step construction of an adenovirus vector suitable for delivering very large transgenes for gene editing.IMPORTANCEThe 36 kb size of the adenoviral genome has long been a deterrent to the construction of adenoviral mutants by scientists wishing to study the virus itself or to construct adenoviral vectors for cell biology and gene therapy. Most previous techniques, such as recombineering and yeast gap repair, impress more by their elegance than by their ease. In this paper, we use Cas9 ribonucleoprotein particles (RNPs) to target cleavage to specific sites in an adenoviral plasmid, then repair the break by Gibson assembly. Gibson assembly with synthetic DNA fragments has transformed basic cloning. Combining it with Cas9 RNPs, which act like highly specific restriction enzymes, makes adenoviral mutagenesis as easy as traditional plasmid cloning. We have used the approach to modify multiple sites in the adenoviral genome, but it could be applied to any large DNA virus for which the genome can be cloned in a plasmid.}, }
@article {pmid40394594, year = {2025}, author = {Guo, J and Shan, Y and Hu, G and Zhu, X and Zhao, K and Wu, T and Qiao, Q and Chi, Y and Cui, L and Ge, Y}, title = {Rapid visual detection of Monkeypox virus by one-step LAMP-CRISPR/Cas12b assay.}, journal = {Virology journal}, volume = {22}, number = {1}, pages = {151}, pmid = {40394594}, issn = {1743-422X}, support = {2023YFC2605100, 2023YFC2605104,BK20231374, BK20221413//National Key R&amp;D Program of China&#xff1b;Natural science foundation of Jiangsu Province/ ; }, mesh = {*Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Monkeypox virus/isolation &amp; purification/genetics ; Humans ; *Mpox, Monkeypox/diagnosis/virology ; Limit of Detection ; }, abstract = {BACKGROUND: Monkeypox virus (MPXV) infection has garnered significant global attention due to its rising incidence and substantial public health implications. A rapid, sensitive, and accurate diagnostic method is urgently required to enable early intervention and effective management of MPXV outbreaks.<br><br>RESULTS: In this study, we developed a novel one-step assay that integrates loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12b in one-pot for the detection of MPXV. The entire detection process did not require opening the lid of the reaction tube and could be completed within 40&#xa0;min using extracted viral nucleic acids, which is faster than real-time quantitative PCR (qPCR). And the results could be interpreted through either real-time fluorescence or naked-eye visualization. The limit of detection (LOD) of the assay was demonstrated to be 6.5 copies per reaction and no cross-reactivity with other pathogens such as HSV, EBV, CVA16, EV-A71, and MV was found. Furthermore, when evaluated using 113 clinical samples, the assay achieved 100% sensitivity (71/71) and 100% specificity (42/42) compared to the qPCR.<br><br>CONCLUSIONS: In resource-limited settings, our method requires only a portable heat block or water bath and a blue light or ultraviolet flashlight for visual detection of MPXV, making it highly accessible. The integration of LAMP and CRISPR/Cas12b provides a robust, user-friendly platform for point-of-care testing, with promising potential for the rapid molecular diagnosis of infectious diseases.}, }
@article {pmid40394064, year = {2025}, author = {Gu, S and Bodai, Z and Anderson, RA and So, HYA and Cowan, QT and Komor, AC}, title = {Elucidating the genetic mechanisms governing cytosine base editing outcomes through CRISPRi screens.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {4685}, pmid = {40394064}, issn = {2041-1723}, support = {T32 GM146648/GM/NIGMS NIH HHS/United States ; T32GM146648//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; 27975//Research Corporation for Science Advancement (Research Corporation)/ ; T32GM008326//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; MCB-2048207//National Science Foundation (NSF)/ ; T32 GM008326/GM/NIGMS NIH HHS/United States ; }, mesh = {*Cytosine/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; DNA Repair/genetics ; DNA Mismatch Repair/genetics ; HEK293 Cells ; Ubiquitin-Protein Ligases/metabolism/genetics ; }, abstract = {Cytosine base editors enable programmable and efficient genome editing using an intermediate featuring a U•G mismatch across from a DNA nick. This intermediate facilitates two major outcomes, C•G to T•A and C•G to G•C point mutations, and it is not currently well-understood which DNA repair factors are involved. Here, we couple reporters for cytosine base editing activity with knockdown of 2015 DNA processing genes to identify genes involved in these two outcomes. Our data suggest that mismatch repair factors facilitate C•G to T•A outcomes, while C•G to G•C outcomes are mediated by RFWD3, an E3 ubiquitin ligase. We also propose that XPF, a 3'-flap endonuclease, and LIG3, a DNA ligase, are involved in repairing the intermediate back to the original C•G base pair. Our results demonstrate that competition and collaboration among different DNA repair pathways shape cytosine base editing outcomes.}, }
@article {pmid40393960, year = {2025}, author = {Liu, Z and Luan, X and Lu, Q and Qin, S and Zeng, F and Li, Z and He, B and Song, Y}, title = {Reactive oxygen species responsive nanomotors for gene edited metabolic disruption and immunotherapy.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {4708}, pmid = {40393960}, issn = {2041-1723}, mesh = {*Reactive Oxygen Species/metabolism ; *Immunotherapy/methods ; *Gene Editing/methods ; Humans ; Animals ; Mice ; CRISPR-Cas Systems ; Cell Line, Tumor ; Tumor Microenvironment ; Heme Oxygenase-1/metabolism ; *Neoplasms/therapy/immunology/genetics ; CD8-Positive T-Lymphocytes/immunology ; Glycolysis ; Nanoparticles/chemistry ; Female ; }, abstract = {While gene-editing-based tumor therapy holds promise, conventional passive-diffusion vectors face limited penetration in dense solid tumors. Here, we developed a ROS-driven gene editing nanomotor (RDN@PL), which takes hemin as the core and encapsulates CRISPR/Cas9 plasmids targeting LDHA (A glycolysis key enzyme). In tumor microenvironments, RDN@PL consumes extracellular ROS to fuel self-diffusiophoresis, achieving higher intratumoral accumulation than passive particles. Upon internalization, heme oxygenase-1 (HO-1) degrades RDN@PL, releasing CO and plasmids. LDHA knockout suppresses glycolysis while CO elevates mitochondrial ROS, which triggers apoptosis by disrupting metabolism and enhancing immunity. Simultaneously, extracellular ROS depletion by non-internalized nanomotors reverses immunogenic cell death (ICD) inhibition, enhancing CD8+ T cell infiltration in tumor. The Janus nanomotor enables extracellular ROS scavenging and intracellular ROS increment via HO-1-responsive cargo release and gene editing. This multi-level intervention strategy demonstrates 93.9 % tumor growth suppression in solid tumor models, providing a blueprint for engineering intelligent nanovesicles in precision oncology.}, }
@article {pmid40393243, year = {2025}, author = {Li, H and Lian, S and Zhang, Z and Bi, W and Meng, Q and Ding, Q}, title = {The CRISPR/Cas13a-assisted electrochemiluminescence sensing device combined with entropy-driven and hybrid chain reaction nucleic acid amplification techniques for ultra-sensitive analysis of brain natriuretic peptide.}, journal = {Talanta}, volume = {295}, number = {}, pages = {128310}, doi = {10.1016/j.talanta.2025.128310}, pmid = {40393243}, issn = {1873-3573}, abstract = {Brain natriuretic peptide (BNP) is considered a reliable marker of heart failure disease, and its timely detection can provide important pathological information to prevent or treat heart failure. In this article, an electrochemiluminescence (ECL) sensing device based on a boron carbon nitride/gold nanoparticle (BCN/AuNPs) complex is developed to determine BNP. Prominently, the CRISPR/CAS 13a enzyme reverse cleavage mode, the entropy-driven and hammer hybridization chain reaction processes were involved in the entire detection scheme. Ultimately, with multiple reaction methods and amplification reactions of nucleic acids, this ECL sensing device is able to achieve a detection limit as low as of 0.03 pg/mL and linear range from 0.1 pg/mL to 30 ng/mL for BNP. In addition, the ECL sensing device based on BCN/AuNPs complex obtained satisfactory stability and specificity, and can also be extended to the detection of other pathological markers.}, }
@article {pmid40390640, year = {2025}, author = {Zhang, C and Chen, Y and Chen, X and Lin, X and Huang, Z and Zhang, L and Liu, R and Lv, Y}, title = {Single nanoparticle analysis-based CRISPR/Cas12 bioassay for amplification-free HIV detection.}, journal = {Chemical communications (Cambridge, England)}, volume = {61}, number = {48}, pages = {8759-8762}, doi = {10.1039/d5cc01716e}, pmid = {40390640}, issn = {1364-548X}, mesh = {Humans ; *CRISPR-Cas Systems ; Limit of Detection ; *Nanoparticles/chemistry ; *Biological Assay/methods ; *HIV/isolation &amp; purification ; *Biosensing Techniques/methods ; *HIV-1/isolation &amp; purification ; *HIV Infections/diagnosis ; *Metal Nanoparticles/chemistry ; }, abstract = {To reduce the "window period" in HIV detection, most analytical methods require additional enzymes for signal amplification. Exempting challenges like primer interference and false positives in amplification strategies, we developed an amplification-free bioassay that uses CRISPR's potent cleavage activity and the competent sensitivity of single-nanoparticle analysis. An attomolar detection limit was achieved with adequate selectivity. Serum and cell tests confirm the bioassay's accurate and sensitive HIV detection.}, }
@article {pmid40389868, year = {2025}, author = {Amoroso, CG and Andolfo, G}, title = {Hazelnut allergome overview and Cor a gRNAs identification.}, journal = {BMC plant biology}, volume = {25}, number = {1}, pages = {661}, pmid = {40389868}, issn = {1471-2229}, support = {E53D23020840001P//Ministero dell'Istruzione, dell'Universit&#xe0; e della Ricerca/ ; }, mesh = {*Corylus/genetics/immunology ; *Allergens/genetics/immunology ; *Plant Proteins/genetics/immunology ; Genome, Plant ; CRISPR-Cas Systems ; Phylogeny ; *Antigens, Plant/genetics/immunology ; }, abstract = {BACKGROUND: Corylus species (hazelnuts) are a valuable source of nutrients and are widely consumed worldwide. Nevertheless, Corylus avellana (Cor a) contains 13 allergens (Cor a 1, Cor a 2, Cor a 6, Cor a 8, Cor a 9, Cor a 10, Cor a 11, Cor a 12, Cor a 13, Cor a 14, Cor a 15, Cor a 16, and Cor a TLP) that have been deposited into the official database (WHO/IUIS) for allergen nomenclature. The recent availability of several Corylus genomes provided opportunities to explore allergome variability, and thus to develop hypoallergenic varieties using modern biotech approaches. Certainly, the identification of CRISPR-Cas9 guide RNA (gRNA) is a pivotal step in achieving this goal. User-friendly web tools include limited reference genomes to design CRISPR-Cas9 gRNAs, while bioinformatic software for custom analysis require advanced command-line skills.<br><br>RESULTS: This work explored the evolutionary trajectories of allergenic Cor a homologs in C. avellana, C. americana, C. heterophylla, and C. mandshurica genome assemblies. 52 Cor a orthologs were found in the analyzed species, and a recent tandem duplication of Cor a 1 was found in C. americana. Three new gene models were predicted in C. avellana and C. mandshurica for Cor a 16 and Cor a 10. Additionally, we identified 56 Cor a isoallergens, of which ten Cor a isoforms. Furthermore, phylogenetic analysis sheds light on the evolutionary dynamics of three hazelnut allergens revealing the evolutionary complexity of Cor a 1, Cor a 2, and Cor a TLP within the Corylus genus. A list of multiple gRNAs designed for the CRISPR-Cas9 system was provided for the singular and multiple silencing of Cor a homologs in each Corylus genome.<br><br>CONCLUSIONS: This study enhances our knowledge on the evolutionary path of Cor a allergens among Corylus species and provides highly accurate on-target guides targeting hazelnut allergome.}, }
@article {pmid40389577, year = {2025}, author = {Lv, C and Zhang, F and Ren, L and Zhu, P and Cheng, X and Yang, X and Chen, C and Liu, B}, title = {Rapid visual detection assay for Bactrocera dorsalis (Hendel) using recombinase polymerase amplification and CRISPR/Cas12b.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {17328}, pmid = {40389577}, issn = {2045-2322}, support = {no. 62272060 and 62272061//National Natural Science Foundation of China/ ; no. 62272060 and 62272061//National Natural Science Foundation of China/ ; no. 62272060 and 62272061//National Natural Science Foundation of China/ ; no. 62272060 and 62272061//National Natural Science Foundation of China/ ; }, mesh = {Animals ; *Tephritidae/genetics ; *CRISPR-Cas Systems ; *Recombinases/metabolism/genetics ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; }, abstract = {The oriental fruit fly Bactrocera dorsalis (Hendel) is considered as a quarantine pest in many countries and regions. Challenges remain in distinguishing this species with morphological similarities, especially in relevant development stages. In recent years, CRISPR/Cas12b genetic diagnostics has seen rapid advancements and offers an efficient tool for the identification of pathogens, viruses, and other genetic targets. Here we developed a new and rapid visual detection assay of B. dorsalis using recombinase polymerase amplification (RPA) and the CRISPR/Cas12b system. The system can detect different developmental stages of B. dorsalis within 30-35 min at 43 ℃ and the results are easily observed by the naked eye based on the color change in the tube during the reaction. The specificity and high sensitivity of this method was demonstrated, allowing for detection from 3.2 pg µL[- 1] of DNA. With crude DNA, this diagnostic system successfully identified B. dorsalis by holding the reaction tubes in the hand. Our study demonstrates that RPA-CRISPR/Cas12b visualization system is effective to detect B. dorsalis rapidly and accurately. This approach can be applied for monitoring and identification of other pests in border and relevant locations, preventing biological invasions and ensuring pest control.}, }
@article {pmid40389480, year = {2025}, author = {Troester, S and Eder, T and Wukowits, N and Piontek, M and Fernández-Pernas, P and Schmoellerl, J and Haladik, B and Manhart, G and Allram, M and Maurer-Granofszky, M and Scheidegger, N and Nebral, K and Superti-Furga, G and Meisel, R and Bornhauser, B and Valent, P and Dworzak, MN and Zuber, J and Boztug, K and Grebien, F}, title = {Transcriptional and epigenetic rewiring by the NUP98::KDM5A fusion oncoprotein directly activates CDK12.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {4656}, pmid = {40389480}, issn = {2041-1723}, support = {P35628//Austrian Science Fund (Fonds zur F&#xf6;rderung der Wissenschaftlichen Forschung)/ ; P35298//Austrian Science Fund (Fonds zur F&#xf6;rderung der Wissenschaftlichen Forschung)/ ; TAI490//Austrian Science Fund (Fonds zur F&#xf6;rderung der Wissenschaftlichen Forschung)/ ; }, mesh = {Humans ; *Nuclear Pore Complex Proteins/metabolism/genetics ; *Oncogene Proteins, Fusion/metabolism/genetics ; *Leukemia, Myeloid, Acute/genetics/metabolism/pathology ; *Cyclin-Dependent Kinases/metabolism/genetics ; *Epigenesis, Genetic ; Cell Line, Tumor ; Animals ; Transcription, Genetic ; Mice ; Gene Expression Regulation, Leukemic ; DNA Damage ; CRISPR-Cas Systems ; Retinoblastoma-Binding Protein 2 ; }, abstract = {Nucleoporin 98 (NUP98) fusion oncoproteins are strong drivers of pediatric acute myeloid leukemia (AML) with poor prognosis. Here we show that NUP98 fusion-expressing AML harbors an epigenetic signature that is characterized by increased accessibility of hematopoietic stem cell genes and enrichment of activating histone marks. We employ an AML model for ligand-induced degradation of the NUP98::KDM5A fusion oncoprotein to identify epigenetic programs and transcriptional targets that are directly regulated by NUP98::KDM5A through CUT&amp;Tag and nascent RNA-seq. Orthogonal genome-wide CRISPR/Cas9 screening identifies 12 direct NUP98::KDM5A target genes, which are essential for AML cell growth. Among these, we validate cyclin-dependent kinase 12 (CDK12) as a druggable vulnerability in NUP98::KDM5A-expressing AML. In line with its role in the transcription of DNA damage repair genes, small-molecule-mediated CDK12 inactivation causes increased DNA damage, leading to AML cell death. Altogether, we show that NUP98::KDM5A directly regulates a core set of essential target genes and reveal CDK12 as an actionable vulnerability in AML with oncogenic NUP98 fusions.}, }
@article {pmid40389438, year = {2025}, author = {Pandey, AC and Bezney, J and DeAscanis, D and Kirsch, EB and Ahmed, F and Crinklaw, A and Choudhary, KS and Mandala, T and Deason, J and Hamidi, JS and Siddique, A and Ranganathan, S and Brown, K and Armstrong, J and Head, S and Ordoukhanian, P and Steinmetz, LM and Topol, EJ}, title = {A CRISPR/Cas9-based enhancement of high-throughput single-cell transcriptomics.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {4664}, pmid = {40389438}, issn = {2041-1723}, support = {UL1TR002550//U.S. Department of Health &amp; Human Services | NIH | National Center for Advancing Translational Sciences (NCATS)/ ; 20CDA35310187//American Heart Association (American Heart Association, Inc.)/ ; KL2 TR002552/TR/NCATS NIH HHS/United States ; UL1 TR002550/TR/NCATS NIH HHS/United States ; KL2TR002552//U.S. Department of Health &amp; Human Services | NIH | National Center for Advancing Translational Sciences (NCATS)/ ; }, mesh = {*Single-Cell Analysis/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Transcriptome/genetics ; *Gene Expression Profiling/methods ; Animals ; High-Throughput Nucleotide Sequencing/methods ; Muscle, Smooth, Vascular/cytology/metabolism ; RNA-Seq/methods ; Sequence Analysis, RNA/methods ; Mice ; Myocytes, Smooth Muscle/metabolism ; }, abstract = {Single-cell RNA-seq (scRNAseq) struggles to capture the cellular heterogeneity of transcripts within individual cells due to the prevalence of highly abundant and ubiquitous transcripts, which can obscure the detection of biologically distinct transcripts expressed up to several orders of magnitude lower levels. To address this challenge, here we introduce single-cell CRISPRclean (scCLEAN), a molecular method that globally recomposes scRNAseq libraries, providing a benefit that cannot be recapitulated with deeper sequencing. scCLEAN utilizes the programmability of CRISPR/Cas9 to target and remove less than 1% of the transcriptome while redistributing approximately half of reads, shifting the focus toward less abundant transcripts. We experimentally apply scCLEAN to both heterogeneous immune cells and homogenous vascular smooth muscle cells to demonstrate its ability to uncover biological signatures in different biological contexts. We further emphasize scCLEAN's versatility by applying it to a third-generation sequencing method, single-cell MAS-Seq, to increase transcript-level detection and discovery. Here we show the possible utility of scCLEAN across a wide array of human tissues and cell types, indicating which contexts this technology proves beneficial and those in which its application is not advisable.}, }
@article {pmid40389165, year = {2025}, author = {Öktem, M and Nguyen, TH and Bosman, EDC and Fens, MHAM and Caiazzo, M and Mastrobattista, E and Lei, Z and de Jong, OG}, title = {Lipopeptide-mediated delivery of CRISPR/Cas9 ribonucleoprotein complexes for gene editing and correction.}, journal = {Journal of controlled release : official journal of the Controlled Release Society}, volume = {383}, number = {}, pages = {113854}, doi = {10.1016/j.jconrel.2025.113854}, pmid = {40389165}, issn = {1873-4995}, mesh = {*Gene Editing/methods ; Animals ; *CRISPR-Cas Systems ; *Ribonucleoproteins/administration &amp; dosage/genetics/chemistry ; Humans ; *Lipopeptides/chemistry/administration &amp; dosage ; *CRISPR-Associated Protein 9/administration &amp; dosage/genetics ; Mice ; *Cell-Penetrating Peptides/chemistry/administration &amp; dosage ; HEK293 Cells ; Gene Transfer Techniques ; }, abstract = {CRISPR/Cas gene editing is a highly promising technology for the treatment and even potential cure of genetic diseases. One of the major challenges for its therapeutic use is finding safe and effective vehicles for intracellular delivery of the CRISPR/Cas9 ribonucleoprotein (RNP) complex. In this study, we tested and characterized a series of novel fatty acid-modified versions of a previously reported Cas9 RNP carrier, consisting of a complex of the cell-penetrating peptide (CPP) LAH5 with Cas9 RNP and homology-directed DNA repair templates. Comparative experiments demonstrated that RNP/peptide nanocomplexes showed various improvements depending on the type of fatty acid modification. These improvements included enhanced stability in serum, improved membrane disruption capability and increased transfection efficacy. Cas9 RNP/oleic acid LAH5 peptide nanocomplexes showed the overall best performance for both gene editing and correction. Moreover, Cas9 RNP/oleic acid LAH5 nanocomplexes significantly protected the Cas9 protein cargo from enzymatic protease digestion. In addition, in vivo testing demonstrated successful gene editing after intramuscular administration. Despite the inherent barriers of the tightly organized muscle tissues, we achieved approximately 10 % gene editing in the skeletal muscle tissues when targeting the CAG-tdTomato locus in the transgenic Ai9 Cre-LoxP reporter mouse strain and 7 % gene editing when targeting the Ccr5 gene, without any observable short-term toxicity. In conclusion, the oleic acid-modified LAH5 peptide is an effective delivery platform for direct Cas9/RNP delivery, and holds great potential for the development of new CRISPR/Cas9-based therapeutic applications for the treatment of genetic diseases.}, }
@article {pmid40388365, year = {2025}, author = {Wang, Y and Hu, J and Zhang, J}, title = {Evaluation of Abnormal Growth-related Genes of Hematopoietic Stem and Progenitor Cells by Combining CRISPR/Cas9 Technology with Cell Counting.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {219}, pages = {}, doi = {10.3791/67508}, pmid = {40388365}, issn = {1940-087X}, mesh = {Animals ; *Hematopoietic Stem Cells/cytology/physiology/metabolism ; Mice ; *CRISPR-Cas Systems ; Tumor Suppressor Protein p53/genetics ; Cell Proliferation ; Transfection ; }, abstract = {Hematopoietic stem cells possess the ability for long-term self-renewal and the potential to differentiate into various types of mature blood cells. However, the accumulation of cancerous mutations in hematopoietic stem and progenitor cells (HSPCs) can block normal differentiation, induce aberrant proliferation, and ultimately lead to leukemogenesis. To identify and/or evaluate the cancerous mutations, we integrated CRISPR/Cas9 technology with the Cell Counting Kit-8 (CCK-8) assay to investigate a model gene Trp53, that is essential for abnormal proliferative ability of HSPCs. Specifically, bone marrow cells enriched for HSPCs from Cas9 mice were harvested and then subjected to viral transfection with single-guide RNAs (sgRNAs) targeting one or several candidate genes to introduce genetic alterations in HSPCs. Then, a CCK-8 assay was performed to investigate the proliferative capacity of transfected HSPCs. The sgRNA targeting efficiency was confirmed by a Tracking of Indels by Decomposition assay. These identified genes may play crucial roles in leukemogenesis and could serve as potential therapeutic targets.}, }
@article {pmid40387535, year = {2025}, author = {Liu, S and Yu, T and Song, L and Kalantar-Zadeh, K and Liu, G}, title = {CRISPR/Cas on Microfluidic Paper-Based Analytical Devices for Point-of-Care Screening of Cervical Cancer.}, journal = {ACS sensors}, volume = {}, number = {}, pages = {}, doi = {10.1021/acssensors.5c00863}, pmid = {40387535}, issn = {2379-3694}, abstract = {Highly sensitive point-of-care early screening for high-risk human papillomavirus (HPV) infections is urgently needed, particularly in resource-limited settings. Nucleic acid amplification methods, especially CRISPR/Cas-based biosensors, have emerged as promising tools for sensitive HPV detection; however, current approaches typically rely on tedious tube-based formats coupled with lateral flow assays for signal readout in point-of-care testing (POCT). Here, we developed customized microfluidic paper-based analytical devices (μPADs) with valves that seamlessly integrated recombinase polymerase amplification (RPA) with CRISPR/Cas12a biosensing (RPA-CRISPR/Cas12a) on the filter paper substrate. This innovation achieved sensitive and cost-effective high-risk HPV detection in POCT. The RPA-CRISPR/Cas12a system with a linear reporter on μPADs, enabled fluorescence detection of the E7 gene, achieving a sensitivity of 1 pM at approximately 1 h. The sensitivity was further enhanced by introducing a circular reporter into the fluorescence-based RPA-CRISPR/Cas12a system on μPADs, enabling detection of the E7 gene with a detection limit of 1 fM and an assay time of 35 min. The system was validated using 50 cervical swab clinical samples, demonstrating 95% sensitivity and 100% specificity when compared to qPCR. This sample-to-answer detection platform holds significant promise for early screening of high-risk HPV infections in point-of-care scenarios.}, }
@article {pmid40387367, year = {2025}, author = {Li, J and Liu, Y and Jiang, J and Chen, F and Zhang, N and Kang, X and Liu, L and Wang, Y and Xia, Q and Zhu, C and Kuang, D}, title = {Type I-E* CRISPR-Cas of Klebsiella pneumoniae upregulates bacterial virulence by targeting endogenous histidine utilization system.}, journal = {mSphere}, volume = {}, number = {}, pages = {e0021525}, doi = {10.1128/msphere.00215-25}, pmid = {40387367}, issn = {2379-5042}, abstract = {Klebsiella pneumoniae is a globally recognized microbial pathogen with significant clinical impact. The bacterium harbors the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, which provide adaptive immunity against invading foreign nucleic acids. Recent studies suggest that certain CRISPR-Cas systems can regulate endogenous genes, influencing bacterial virulence. However, their role in regulating pathogenicity in K. pneumoniae remains poorly understood. This study investigates the regulatory role of the type I-E* CRISPR-Cas system in a hypervirulent K. pneumoniae strain, focusing on its impact on histidine metabolism and pathogenicity. Transcriptome analyses identified differentially expressed genes (DEGs) between the casABECD-deletion and wild-type strains, including significant upregulation of the histidine utilization (Hut) operon and downregulation of biofilm-related genes. These molecular changes resulted in enhanced histidine metabolic activity, reduced biofilm formation, attenuated virulence in A549 lung epithelial cells, and improved survival of Galleria mellonella, as validated through phenotypic and virulence assays. Our bioinformatic analysis indicated that the CRISPR-Cas system in K. pneumoniae targets the hutT sequence, which is part of the Hut operon. Furthermore, the overexpression of hutT mitigated CRISPR-Cas-mediated repression of the Hut operon, as observed in virulence assays, while simultaneous deletion of hutH and casABECD restored the reduced virulence in the ΔcasABECD strain. Additionally, deletion of casABECD significantly enhances the growth of the strain in medium with histidine as the sole carbon source, highlighting the intricate regulatory role of the CRISPR-Cas system in metabolic adaptation. Collectively, these findings uncover a novel role for the CRISPR-Cas system in regulating metabolic pathways and virulence in hypervirulent K. pneumoniae.IMPORTANCEClustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are primarily recognized for their roles in adaptive immunity against foreign genetic elements in bacteria. However, emerging evidence indicates that these systems can also regulate endogenous genes, thereby influencing bacterial physiology and virulence. In this study, we demonstrate that the type I-E* CRISPR-Cas system in Klebsiella pneumoniae targets the hutT gene, a critical component of the histidine utilization (Hut) pathway. This targeting potentially impacts hutT transcription and alters the expression of other hut genes, ultimately enhancing bacterial virulence. Our findings reveal a previously unrecognized regulatory mechanism through which CRISPR-Cas systems facilitate metabolic adaptation and pathogenicity in K. pneumoniae. This study broadens our understanding of the multifaceted roles of CRISPR-Cas systems in bacterial physiology and pathobiology, with implications for clinically relevant pathogens.}, }
@article {pmid40387084, year = {2025}, author = {Belato, HB and Knight, AL and D'Ordine, AM and Pindi, C and Fan, Z and Luo, J and Palermo, G and Jogl, G and Lisi, GP}, title = {Structural and dynamic impacts of single-atom disruptions to guide RNA interactions within the recognition lobe of Geobacillus stearothermophilus Cas9.}, journal = {eLife}, volume = {13}, number = {}, pages = {}, pmid = {40387084}, issn = {2050-084X}, support = {P30 GM133893/GM/NIGMS NIH HHS/United States ; R01GM141329/NH/NIH HHS/United States ; R01GM136815/NH/NIH HHS/United States ; R01 GM136815/GM/NIGMS NIH HHS/United States ; TC-24-063//Camille and Henry Dreyfus Foundation/ ; CHE2144823//National Science Foundation/ ; R01 GM141329/GM/NIGMS NIH HHS/United States ; MCB2143760//National Science Foundation/ ; FG-2023-20431//Alfred P. Sloan Foundation/ ; }, mesh = {*Geobacillus stearothermophilus/enzymology/genetics ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry ; *CRISPR-Associated Protein 9/metabolism/chemistry/genetics ; *Bacterial Proteins/metabolism/chemistry/genetics ; CRISPR-Cas Systems ; Protein Conformation ; }, abstract = {The intuitive manipulation of specific amino acids to alter the activity or specificity of CRISPR-Cas9 has been a topic of great interest. As a large multi-domain RNA-guided endonuclease, the intricate molecular crosstalk within the Cas9 protein hinges on its conformational dynamics, but a comprehensive understanding of the extent and timescale of the motions that drive its allosteric function and association with nucleic acids remains elusive. Here, we investigated the structure and multi-timescale molecular motions of the recognition (Rec) lobe of GeoCas9, a thermophilic Cas9 from Geobacillus stearothermophilus. Our results provide new atomic details about the GeoRec subdomains (GeoRec1, GeoRec2) and the full-length domain in solution. Two rationally designed mutants, K267E and R332A, enhanced and redistributed micro-millisecond flexibility throughout GeoRec, and NMR studies of the interaction between GeoRec and its guide RNA showed that mutations reduced this affinity and the stability of the ribonucleoprotein complex. Despite measured biophysical differences due to the mutations, DNA cleavage assays reveal no functional differences in on-target activity, and similar specificity. These data suggest that guide RNA interactions can be tuned at the biophysical level in the absence of major functional losses but also raise questions about the underlying mechanism of GeoCas9, since analogous single-point mutations have significantly impacted on- and off-target DNA editing in mesophilic Streptococcus pyogenes Cas9. A K267E/R332A double mutant did also did not enhance GeoCas9 specificity, highlighting the robust tolerance of mutations to the Rec lobe of GeoCas9 and species-dependent complexity of Rec across Cas9 paralogs. Ultimately, this work provides an avenue by which to modulate the structure, motion, and guide RNA interactions at the level of the Rec lobe of GeoCas9, setting the stage for future studies of GeoCas9 variants and their effect on its allosteric mechanism.}, }
@article {pmid40382983, year = {2025}, author = {Pulito, C and Vaccarella, S and Palcau, AC and Ganci, F and Brandi, R and Frascolla, C and Sacconi, A and Canu, V and Benedetti, A and De Pascale, V and Donzelli, S and Fisch, AS and Manciocco, V and Covello, R and Pimpinelli, F and Morrone, A and Fazi, F and Pellini, R and Muti, P and Meens, J and Karamboulas, C and Nichols, AC and Strano, S and Klinghammer, K and Tinhofer, I and Ailles, L and Fontemaggi, G and Blandino, G}, title = {MicroRNA-mediated PTEN downregulation as a novel non-genetic mechanism of acquired resistance to PI3Kα inhibitors of head &amp; neck squamous cell carcinoma.}, journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy}, volume = {81}, number = {}, pages = {101251}, doi = {10.1016/j.drup.2025.101251}, pmid = {40382983}, issn = {1532-2084}, mesh = {Humans ; *PTEN Phosphohydrolase/genetics/metabolism ; *MicroRNAs/genetics ; *Drug Resistance, Neoplasm/genetics/drug effects ; *Squamous Cell Carcinoma of Head and Neck/drug therapy/genetics/pathology ; Down-Regulation ; *Head and Neck Neoplasms/drug therapy/genetics/pathology ; Cell Line, Tumor ; *Class I Phosphatidylinositol 3-Kinases/antagonists &amp; inhibitors/genetics ; Gene Expression Regulation, Neoplastic/drug effects ; Thiazoles/pharmacology/therapeutic use ; Polo-Like Kinase 1 ; Cell Cycle Proteins/antagonists &amp; inhibitors/metabolism ; *Phosphoinositide-3 Kinase Inhibitors/pharmacology ; Protein Serine-Threonine Kinases/antagonists &amp; inhibitors/metabolism ; Proto-Oncogene Proteins/antagonists &amp; inhibitors/metabolism ; Animals ; Signal Transduction/drug effects ; CRISPR-Cas Systems ; Phosphorylation ; RNA, Long Noncoding ; }, abstract = {AIMS: Head and neck squamous cell carcinomas (HNSCCs) frequently harbor alterations in the PI3K signalling axis and, particularly, in the PIK3CA gene. The promising rationale of using PI3K inhibitors for the treatment of HNSCC has, however, clashed with the spontaneous development of resistance over time.<br><br>METHODS: To identify valuable targets for overcoming acquired resistance to PI3K&#x3b1; inhibitors in HNSCC, we performed microRNA profiling on a cohort of HNSCC PDXs that were treated with alpelisib, including both responsive and resistant tumors. Using CRISPR/Cas9, siRNA, and PTEN-/- isogenic and alpelisib-resistant cell models, we examined the role of PTEN in resistance acquisition. Phospho-proteomic analysis identified PTEN-dependent phosphorylation events, while PI3K&#x3b1; inhibitor-resistant organoids were used to assess PLK1 inhibitor efficacy.<br><br>RESULTS: We identified microRNAs altered in resistant PDXs, including members of the miR-17-92 cluster. Mechanistically, we observed that the hyperactive c-Myc was recruited to MIR17HG regulatory regions in alpelisib-resistant cells, sustaining miR-17-5p, miR-19b-3p, and miR-20a-5p expression, which downregulated PTEN. PTEN knockout or depletion conferred alpelisib resistance in HNSCC cells. We identified PTEN-dependent phosphorylation events, such as p-PLK1-T210, involved in resistance. Interestingly, pharmacological inhibition of PLK1 strongly reduced the viability of PI3K&#x3b1;-resistant organoids derived from HNSCC PDXs and cell line models.<br><br>CONCLUSION: Overall, this study unveils a novel, microRNA-driven, non-genetic mechanism contributing to acquired resistance to PI3K&#x3b1; inhibitors in HNSCC. Indeed, linking hyperactive c-Myc to sustain miR-17-92 expression and consequent PTEN downregulation, we also propose that targeting PTEN-dependent downstream effectors, such as PLK1, may offer a powerful therapeutic strategy for resistant HNSCC.}, }
@article {pmid40380978, year = {2025}, author = {Pipes, BL and Nishiguchi, MK}, title = {Generation and validation of a versatile inducible multiplex CRISPRi system to examine bacterial regulation in the Euprymna-Vibrio fischeri symbiosis.}, journal = {Archives of microbiology}, volume = {207}, number = {7}, pages = {147}, pmid = {40380978}, issn = {1432-072X}, support = {EXO 80NSSC18K1053//National Aeronautical and Space Adminstration/ ; EXO 80NSSC18K1053//National Aeronautical and Space Adminstration/ ; DBI-2214028//National Science Foundation/ ; DBI-2214028//National Science Foundation/ ; }, mesh = {*Aliivibrio fischeri/genetics/physiology ; *Symbiosis/genetics ; Animals ; *Gene Expression Regulation, Bacterial ; *CRISPR-Cas Systems ; *Decapodiformes/microbiology ; Genetic Vectors ; Bacterial Proteins/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {The Vibrio fischeri-Euprymna scolopes symbiosis has become a powerful animal-microbe model system to examine the genetic underpinnings of symbiont development and regulation. Although there has been a number of elegant bacterial genetic technologies developed to examine this symbiosis, there is still a need to develop more sophisticated methodologies to better understand complex regulatory pathways that lie within the association. Therefore, we have developed a suite of CRISPR interference (CRISPRi) vectors for inducible repression of specific V. fischeri genes associated with symbiotic competence. The suite utilizes both Tn7-integrating and shuttle vector plasmids that allow for inducible expression of CRISPRi dCas9 protein along with single-guide RNAs (sgRNA) modules. We validated this CRISPRi tool suite by targeting both exogenous (an introduced mRFP reporter) and endogenous genes (luxC in the bioluminescence producing lux operon, and flrA, the major regulatory gene controlling flagella production). The suite includes shuttle vectors expressing both single and multiple sgRNAs complementary to the non-template strand of multiple targeted genetic loci, which were effective in inducible gene repression, with significant reductions in targeted gene expression levels. V. fischeri cells harboring a version of this system targeting the luxC gene and suppressing the production of luminescence were used to experimentally validate the hypothesis that continuous luminescence must be produced by the symbiont in order to maintain the symbiosis at time points longer than the known 24-h limit. This robust new CRISPRi genetic toolset has broad utility and will enhance the study of V. fischeri genes, bypassing the need for gene disruptions by standard techniques of allelic knockout-complementation-exchange and the ability to visualize symbiotic regulation in vivo.}, }
@article {pmid40380066, year = {2025}, author = {Garduño-Tamayo, YQ and Acosta-García, G}, title = {Gene Silencing in Plants Through Exogenous Application of miRNAs.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2900}, number = {}, pages = {249-255}, pmid = {40380066}, issn = {1940-6029}, mesh = {*MicroRNAs/genetics ; *Gene Silencing ; *Plants/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; RNA Interference ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; RNA, Plant/genetics ; }, abstract = {Although omics technologies allow us to identify genes involved in various biological processes, we still rely on the analysis of the function of individual genes. Studies of the function of a specific gene include technologies such as gene silencing by RNAi or genome editing by CRISPR-cas9. However, most of them depend on the availability of transformation methods, so they are not established for all plants. In this chapter, we report a protocol that involves the exogenous application of miRNAs for the specific silencing of genes of interest. The advantage of this protocol is that it does not require a transformation event and can be applied to a certain tissue or developmental stage. This novel technology facilitates the analysis of specific gene functions in crops of economic interest.}, }
@article {pmid40380065, year = {2025}, author = {Rivera-Toro, DM and Alvarez-Venegas, R}, title = {CRISPR-Activation: Boosting Expression of Plant MIRs.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2900}, number = {}, pages = {229-247}, pmid = {40380065}, issn = {1940-6029}, mesh = {*MicroRNAs/genetics ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Plant ; *Gene Editing/methods ; Promoter Regions, Genetic ; *Plants/genetics ; Plants, Genetically Modified/genetics ; Genetic Vectors/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {In the field of plant genome editing, the CRISPR/Cas system stands out as an exceptionally versatile tool. Originally conceived for gene editing, the catalytically inactive Cas9 variant, known as dead Cas9 (dCas9), takes on a new role when coupled with transcriptional effectors, offering a finely tuned mechanism for controlling gene expression. This innovation has paved the way for the evolution of sophisticated systems such as CRISPR-Act 2.0 and CRISPR-Act 3.0, which significantly enhance transcriptional activation in plants. This chapter serves as an introduction to a protocol that harnesses CRISPR-activation (CRISPRa) to modulate the transcription of plant miRNA genes. Recognizing the imperative for meticulous construct design and promoter identification, the protocol outlines steps encompassing vector construction. It underscores the critical importance of comprehending miRNA promoter regions and cis-acting elements, presenting a holistic approach for effectively employing CRISPRa in manipulating miRNA expression to modulate traits in plants.}, }
@article {pmid40380064, year = {2025}, author = {Martínez-Estrada, E and de la Mora-Franco, D and de Folter, S}, title = {A Simple and Efficient Protocol to Transform and Regenerate CRISPR-Cas9-Mediated Genome-Edited Tomato Plants.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2900}, number = {}, pages = {213-228}, pmid = {40380064}, issn = {1940-6029}, mesh = {*Solanum lycopersicum/genetics/growth &amp; development ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genome, Plant ; Plants, Genetically Modified/genetics ; Cotyledon/genetics/growth &amp; development ; Transformation, Genetic ; Agrobacterium/genetics ; }, abstract = {CRISPR-Cas9-mediated genome editing has revolutionized functional genomics and crop improvement. However, to maximize the adoption of the CRISPR-Cas9 technology, an efficient method to transform and regenerate genetically edited plants is necessary. In this protocol, we describe a detailed method to generate a CRISPR-Cas9 construct based on the Golden Gate cloning system and a simple and efficient method to transform and regenerate tomato plants from cotyledons co-cultured with Agrobacterium. Our protocol allows the production of at least ten Cas-positive independent lines from one hundred cotyledons. This protocol is routinely used in our laboratory to obtain tomato mutant lines and has been proven effective across several genotypes.}, }
@article {pmid40379664, year = {2025}, author = {Nam, H and Xie, K and Majumdar, I and Wang, J and Yang, S and Starzyk, J and Lee, D and Shan, R and Li, J and Wu, H}, title = {Engineering tripartite gene editing machinery for highly efficient non-viral targeted genome integration.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {4569}, pmid = {40379664}, issn = {2041-1723}, support = {DP2 GM154019/GM/NIGMS NIH HHS/United States ; }, mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems/genetics ; Transgenes/genetics ; DNA, Single-Stranded/genetics/metabolism ; HEK293 Cells ; Receptors, Chimeric Antigen/genetics/metabolism ; T-Lymphocytes/metabolism ; DNA, Circular/genetics ; Genetic Engineering/methods ; CRISPR-Associated Protein 9/metabolism/genetics ; Homologous Recombination ; Gene Knock-In Techniques/methods ; }, abstract = {Non-viral DNA donor templates are commonly used for targeted genomic integration via homologous recombination (HR), with efficiency improved by CRISPR/Cas9 technology. Circular single-stranded DNA (cssDNA) has been used as a genome engineering catalyst (GATALYST) for efficient and safe gene knock-in. Here, we introduce enGager, an enhanced GATALYST associated genome editor system that increases transgene integration efficiency by tethering cssDNA donors to nuclear-localized Cas9 fused with single-stranded DNA binding peptide motifs. This approach further improves targeted integration and expression of reporter genes at multiple genomic loci in various cell types, showing up to 6-fold higher efficiency compared to unfused Cas9, especially for large transgenes in primary cells. Notably, enGager enables efficient integration of a chimeric antigen receptor (CAR) transgene in 33% of primary human T cells, enhancing anti-tumor functionality. This 'tripartite editor with ssDNA optimized genome engineering (TESOGENASE) offers a safer, more efficient alternative to viral vectors for therapeutic gene modification.}, }
@article {pmid40378977, year = {2025}, author = {Qin, B and Shen, S and Chen, H and Wang, Y and Ding, J and Ding, J}, title = {Inactivation of the key ORFs of HBV for antiviral therapy by non-cleavage base editing.}, journal = {Microbial pathogenesis}, volume = {205}, number = {}, pages = {107689}, doi = {10.1016/j.micpath.2025.107689}, pmid = {40378977}, issn = {1096-1208}, mesh = {*Hepatitis B virus/genetics/drug effects ; Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Open Reading Frames/genetics ; Antiviral Agents/pharmacology/therapeutic use ; Virus Replication/genetics ; DNA, Viral/genetics ; Point Mutation ; Hepatitis B/therapy/virology ; }, abstract = {OBJECTIVES: Hepatitis B virus (HBV) infection is the key cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Currently available anti-HBV drugs are more or less defective owing to the unremovable covalently closed circular DNA (cccDNA). Thus, CRISPR/Cas9 is a promising therapeutic strategy for anti-HBV therapy. Double-strand breaks (DSBs) and uncontrolled genomic rearrangements occur inevitably. In this study, we aimed to use base editors to control HBV infection.<br><br>METHODS: Base editors precisely instal targeted point mutations without requiring DSBs or donor DNA templates, and without relying on homology-directed repair (HDR) or nonhomologous end joining (NHEJ). Adenine base editors (ABEs) and cytosine base editors (CBEs) catalyse A&#x2022; T to G &#x2022;C and C&#x2022; G to T &#x2022;A conversions, respectively. In this study, to control HBV replication by modifying and inactivating key HBV genes, recently developed CRISPR/Cas-mediated SpRY-ABE8e and CBE4-max were utilised to falsify and invalidate the ATG initiation codons of the S, Pre-S1, PreS2, C, Pre-C, X, and P genes.<br><br>RESULTS: The ATG initiation codons of HBV genes were edited by ABE/CBE. The expected point mutations were successfully introduced, resulting in the simultaneous suppression of HBV antigen expression and replication to varying degrees.<br><br>CONCLUSIONS: Our study focused on clearing HBV using base and provided experimental and theoretical evidence for the treatment of chronic HBV infection. Thus, base editing is a potential strategy for curing CHB by permanently inactivating the integrated DNA and cccDNA without using DSBs.}, }
@article {pmid40378975, year = {2025}, author = {Kumar, V and Nagano, T and Takasuka, TE}, title = {Genome integration and expression of β-glucosidase in Priestia megaterium enhanced poly(3-hydroxybutyrate) production from cellobiose and cellulose.}, journal = {Bioresource technology}, volume = {432}, number = {}, pages = {132681}, doi = {10.1016/j.biortech.2025.132681}, pmid = {40378975}, issn = {1873-2976}, mesh = {*Cellobiose/metabolism ; *Cellulose/metabolism ; *Hydroxybutyrates/metabolism ; *beta-Glucosidase/genetics/metabolism ; *Polyesters/metabolism ; Promoter Regions, Genetic/genetics ; Polyhydroxyalkanoates ; *Genome, Bacterial/genetics ; Polyhydroxybutyrates ; }, abstract = {Polyhydroxyalkanoates (PHAs) production using cellulosic biomass is a promising way for sustainable manufacturing of bioplastics. Priestia megaterium is an ideal choice as it can use glucose and xylose for PHA production. To further improve the strain for PHA production from cellobiose, we integrate exogenous β-glucosidase (Bgl) from Bacillus sp. GL1 (Bsbgl) in the PHA depolymerase (phaZ1) deletion background (ΔZ1) using CRISPR-Cas. The deletion of phaZ1 in P. megaterium showed a significant improvement in the PHA accumulation whereas BsBgl expression resulted in robust activity and improved growth using cellobiose as a sole carbon source compared to other Bgl targets. To further improve the strain, four native promoters were examined for intracellular BsBgl expression, and the PHA promoter (PphaR) and citrate synthase promoter (Pcitz) showed 2.0- and 4.5-fold higher activities of BsBgl, compared to the xylose promoter (Pxyl). The rate of cellobiose utilization in engineered strains P2 (PphaRBsbgl_ΔZ1) and P4 (PcitzBsbgl_ΔZ1) was improved to 1.6-fold and 2.6-fold, whereas poly(3-hydroxybutyrate) (P3HB) yield for the respective strains was around 3-fold to the wild-type. The strain P2 turned out to be better for cellobiose to PHA production. Further, the strain P2 in a co-culture with cellulolytic Streptomyces sp. SirexAA-E in a consolidated bioprocessing yielded 76 mg of P3HB/ g of carboxymethylcellulose, which is 4.3-times higher than the co-culture with the wild-type. Thus, the present work improved the cellobiose utilization and P3HB accumulation of P. megaterium. The current study paves the way for designing efficient cell factories for cellulosic biomass into bioplastic in the future.}, }
@article {pmid40378589, year = {2025}, author = {Chen, Q and Wang, H and Xu, H and Peng, Y and Yao, B and Chen, Z and Yang, J and Adeloju, S and Chen, W}, title = {One-pot RPA-CRISPR/Cas12a integrated dual-mode electrochemical lateral flow strip for ultrasensitive and precise detection of Salmonella.}, journal = {Biosensors &amp; bioelectronics}, volume = {285}, number = {}, pages = {117529}, doi = {10.1016/j.bios.2025.117529}, pmid = {40378589}, issn = {1873-4235}, mesh = {*Biosensing Techniques/methods ; *Salmonella/isolation &amp; purification/genetics ; *Electrochemical Techniques/methods ; Limit of Detection ; CRISPR-Cas Systems/genetics ; Food Microbiology ; Equipment Design ; Salmonella Infections/microbiology/diagnosis ; Humans ; }, abstract = {Rapid and accurate screening of pathogenic contamination is essential for timely intervention and infection prevention. In this work, one-pot RPA-CRISPR/Cas12a strategy combined with an innovative electrochemical lateral flow strip (OPRCC-eLFS) was presented for ultrasensitive and precise detection of Salmonella. Highly sensitive dual-mode detection of Salmonella in various samples has been simultaneously achieved with electrochemical detection limit of 3.84 CFU/mL and visual detection limit of 384 CFU/mL, respectively, with improved detection efficiency and prevention of uncapping-related aerosol-contamination. This dual-mode biosensing platform demonstrates exceptional stability, remarkable sensitivity, and robust on-site quantification capability, emphasizing its potential in food safety monitoring and disease prevention.}, }
@article {pmid40377871, year = {2025}, author = {Rahmati, R and Zarimeidani, F and Ghanbari Boroujeni, MR and Sadighbathi, S and Kashaniasl, Z and Saleh, M and Alipourfard, I}, title = {CRISPR-Assisted Probiotic and In Situ Engineering of Gut Microbiota: A Prospect to Modification of Metabolic Disorders.}, journal = {Probiotics and antimicrobial proteins}, volume = {}, number = {}, pages = {}, pmid = {40377871}, issn = {1867-1314}, abstract = {The gut microbiota, a substantial group of microorganisms residing in the human body, profoundly impacts various physiological and pathological mechanisms. Recent studies have elucidated the association between gut dysbiosis and multiple organ diseases. Gut microbiota plays a crucial role in maintaining gastrointestinal stability, regulating the immune system and metabolic processes not only within the gastrointestinal tract but also in other organs such as the brain, lungs, and skin. Dysbiosis of the gut microbiota can disrupt biological functioning and contribute to the development of metabolic disorders. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (Cas) modules are adaptive immune systems in numerous archaea and bacteria. CRISPR/Cas is a versatile gene-editing tool that enables modification of the genome in live cells, including those within the gut microbiota. This technique has revolutionized gene editing due to its simplicity and effectiveness. It finds extensive applications in diverse scientific arenas, facilitating the functional screening of genomes during various biological processes. Additionally, CRISPR has been instrumental in creating model organisms and cell lines for research purposes and holds great potential for developing personalized medical treatments through precise genetic alterations. This review aims to explore and discuss the possibilities of CRISPR/Cas and the current trends in using this technique for editing gut microbiota genes in various metabolic disorders. By uncovering the valuable potential of CRISPR/Cas in modifying metabolic disorders through the human gut microbiota, we shed light on its promising applications.}, }
@article {pmid40377313, year = {2025}, author = {Scarrone, M and Turner, D and Dion, M and Tremblay, D and Moineau, S}, title = {In silico and in vitro comparative analysis of 79 Acinetobacter baumannii clinical isolates.}, journal = {Microbiology spectrum}, volume = {}, number = {}, pages = {e0284924}, doi = {10.1128/spectrum.02849-24}, pmid = {40377313}, issn = {2165-0497}, abstract = {Acinetobacter baumannii is a significant nosocomial bacterial pathogen that poses a substantial infection risk due to its high resistance to antibiotics and ability to survive in hospital environments. In this study, we performed comprehensive in silico and in vitro analyses on 79 A. baumannii clinical isolates from different geographical locations to uncover their genomic and epidemiological characteristics as well as their antibiotic and phage susceptibilities. Our findings revealed considerable genomic diversity among the isolates, as shown by average nucleotide identity (ANI) heat maps, multilocus sequence typing (MLST), and core genome MLST (cgMLST). We identified several international clones known for their high antibiotic resistance and global prevalence. Surprisingly, we also observed that the number of antimicrobial resistance genes (ARGs) was higher in isolates containing CRISPR-Cas systems. Plaque assays with 13 phages indicated that Acinetobacter phages have a narrow host range, with capsule loci (KL) serving as a good indicator of phage-bacteria interactions. The presence of CRISPR-Cas systems and other antiviral defense mechanisms in A. baumannii genomes also appears to play a key role in providing phage resistance, regardless of the phage receptors. We also found that spacers associated with subtypes I-F1 and I-F2 CRISPR-Cas systems predominantly target prophages, suggesting a role in maintaining genomic stability and contributing to phage-bacteria co-evolution. Overall, this study provides a set of highly characterized A. baumannii clinical isolates for future studies on antibiotic-phage-bacteria interactions.IMPORTANCEAcinetobacter baumannii poses a significant challenge to the healthcare system due to its antibiotic resistance and strong survival mechanisms. This study examines a diverse collection of 79 clinical isolates to deepen our understanding of A. baumannii's genetic characteristics and its defense mechanisms against both antibiotics and phages. Genomic analysis revealed globally prevalent, highly resistant clones and uncovered a complex role for CRISPR-Cas systems. Although CRISPR-Cas systems were not widespread among these isolates, they primarily targeted prophages. Additionally, the study emphasizes the importance of capsule types as indicators of phage susceptibility. Together, these findings provide insights into the pathogen's resilience and evolutionary adaptations, potentially guiding future research on infection control strategies and new therapeutic approaches to combat A. baumannii infections.}, }
@article {pmid40377112, year = {2025}, author = {Kellari, LM and Dalakouras, A and Tsiouri, O and Vletsos, P and Katsaouni, A and Uslu, VV and Papadopoulou, KK}, title = {Cross-kingdom RNAi induced by a beneficial endophytic fungus to its host requires transitivity and amplification of silencing signals.}, journal = {Plant biology (Stuttgart, Germany)}, volume = {27}, number = {4}, pages = {504-514}, pmid = {40377112}, issn = {1438-8677}, support = {6236//the Hellenic Foundation for Research and Innovation (HFRI)/ ; 7322//Research Committee of UTH/ ; //PRIMA Programme/ ; }, mesh = {*Nicotiana/microbiology/genetics/metabolism ; *Endophytes/physiology ; *Fusarium/physiology/genetics ; *RNA Interference ; Green Fluorescent Proteins/metabolism/genetics ; }, abstract = {Cross-kingdom transfer of small RNA (sRNA) molecules has been identified as a means of communication between plants and interacting microorganisms, but the mechanistic details of this sRNA-based interaction remain elusive. We have previously shown that the beneficial root-colonizing fungus Fusarium solani strain K (FsK) translocates sRNAs to its host, Nicotiana benthamiana (Nb), leading to systemic silencing of a reporter gene. Here, we investigated the mechanistic details of the endophyte-induced systemic silencing using an RNAi sensor system. We inoculated three Nb GFP expressing lines with conidia of an FsK transformant containing a transgene that targets host GFP (FsK-hpGF). The efficiency of silencing mediated by FsK-hpGF was monitored both phenotypically under ultraviolet light as well as quantitatively by RT-qPCR. sRNA sequencing was performed to evaluate the production of sRNAs targeting host GFP. Finally, bisulfite sequencing was used to assess plant GFP methylation levels. We show that the translocated fungal sRNAs induced production of secondary sRNAs, mainly of 22-24-nt in size, with the conspicuous absence of 21-nt sRNAs. Importantly, systemic silencing could not be induced in an RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) CRISPR/Cas knockout background, nor in an intron-containing target gene. Overall, our data show that endophyte-induced silencing in the host requires RDR6-mediated transitivity and amplification of silencing signals. Despite being based on an artificial RNAi sensor system, our observations may reflect a more generalized and so far unexplored facet of cross-kingdom RNAi, with RDR6-based transitivity influencing the way symbionts and pathogens elicit systemic phenotypes in their host plants.}, }
@article {pmid40376763, year = {2025}, author = {Wang, K and Liu, Z}, title = {Plant synthetic biology-based biofortification, strategies and recent progresses.}, journal = {Journal of integrative plant biology}, volume = {}, number = {}, pages = {}, doi = {10.1111/jipb.13934}, pmid = {40376763}, issn = {1744-7909}, support = {startup fund//Shenzhen University of Advanced Technology/ ; }, abstract = {Hidden hunger, caused by chronic micronutrient deficiencies, affects billions of people worldwide and remains a critical public health issue despite progress in food production. Biofortification offers a promising solution by enhancing nutrient levels within plant tissues through traditional breeding or advanced biotechnologies. Recent advancements in plant synthetic biology have significantly improved biofortification strategies, enabling precise and targeted nutrient enrichment. This mini-review outlines five core strategies in synthetic biology-based biofortification: overexpression of endogenous biosynthetic genes, introduction of heterologous biosynthetic pathways, expression of nutrient-specific transporters, optimization of transcriptional regulation, and protein (directed) evolution. Vitamin B1 biofortification serves as a primary illustrative example due to its historical importance and ongoing relevance. Recent breakthroughs, particularly from Chinese research teams, are also highlighted. Together, these strategies offer transformative potential for addressing global nutritional challenges through precise, sustainable and innovative plant-based approaches.}, }
@article {pmid40376717, year = {2025}, author = {Marzluf, JP and Daniela, K and Klein, J and Zehe, C and Leroux, AC}, title = {Utilizing Stable Gene-Edited Knockout Pools for Genetic Screening and Engineering in Chinese Hamster Ovary Cells.}, journal = {Biotechnology journal}, volume = {20}, number = {5}, pages = {e70033}, pmid = {40376717}, issn = {1860-7314}, mesh = {CHO Cells ; Animals ; Cricetulus ; *Gene Knockout Techniques/methods ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Cricetinae ; *Genetic Testing/methods ; Fibronectins/genetics ; Genetic Engineering/methods ; }, abstract = {Chinese hamster ovary (CHO) cells are the primary host for biopharmaceutical production. To meet increasing demands for productivity, quality, and complex molecule expression, genetic engineering, particularly clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene knockout (KO), is widely used to optimize host cell performance. However, systematic screening of KO targets remains challenging due to the labor-intensive process of generating and evaluating individual clones. In this study, we present a robust, high-throughput CRISPR workflow using stable KO pools in CHO cells. These pools maintain genetic stability for over 6 weeks, including in multiplexed configurations targeting up to seven genes simultaneously. Compared to clonal approaches, KO pools reduce variability caused by clonal heterogeneity and better reflect the host cell population phenotype. We demonstrate the utility of this approach by reproducing the beneficial phenotypic effects of fibronectin 1 (FN1) KO, specifically prolonged culture duration and improved late-stage viability in fed-batch processes. This workflow enables efficient identification and evaluation of promising KO targets without the need to generate and test large numbers of clones. Overall, screening throughput is increased 2.5-fold and timelines are compressed from 9 to 5 weeks. This provides a scalable, efficient alternative to traditional clonal screening, accelerating discovery for CHO cell line engineering for biopharmaceutical development.}, }
@article {pmid40376300, year = {2025}, author = {Feussner, M and Migur, A and Mitrofanov, A and Alkhnbashi, OS and Backofen, R and Beisel, CL and Weinberg, Z}, title = {Disparate mechanisms counteract extraneous CRISPR RNA production in type II-C CRISPR-Cas systems.}, journal = {microLife}, volume = {6}, number = {}, pages = {uqaf007}, pmid = {40376300}, issn = {2633-6693}, abstract = {CRISPR-Cas adaptive immune systems in bacteria and archaea enable precise targeting and elimination of invading genetic elements. An inherent feature of these systems is the 'extraneous' CRISPR RNA (ecrRNA), which is produced via the extra repeat in a CRISPR array lacking a corresponding spacer. As ecrRNAs would interact with the Cas machinery yet not direct acquired immunity, they pose a potential barrier to defence. Type II-A CRISPR-Cas systems resolve this barrier through the leader sequence upstream of a CRISPR array, which forms a hairpin structure with the extra repeat that inhibits ecrRNA production. However, the fate of ecrRNAs in other CRISPR types and subtypes remains to be explored. Here, we report that II-C systems likely employ disparate strategies to resolve the ecrRNA due to their distinct configuration in comparison to II-A. Applying bioinformatics analyses to over 650 II-C systems followed by experimental validation, we identified three strategies applicable to these systems: formation of an upstream Rho-independent terminator, formation of a hairpin that sequesters the ecrRNA guide, and mutations in the repeat expected to disrupt ecrRNA formation. These findings expand the list of mechanisms in CRISPR-Cas systems that could resolve the ecrRNA to optimize immune response.}, }
@article {pmid40376084, year = {2024}, author = {Shi, H and Al-Sayyad, N and Wasko, KM and Trinidad, MI and Doherty, EE and Vohra, K and Boger, RS and Colognori, D and Cofsky, JC and Skopintsev, P and Bryant, Z and Doudna, JA}, title = {Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, doi = {10.1101/2024.10.01.616117}, pmid = {40376084}, issn = {2692-8205}, abstract = {RNA-guided CRISPR-Cas enzymes initiate programmable genome editing by recognizing a 20-base-pair DNA sequence adjacent to a short protospacer-adjacent motif (PAM). To uncover the molecular determinants of high-efficiency editing, we conducted biochemical, biophysical and cell-based assays on S. pyogenes Cas9 (Spy Cas9) variants with wide-ranging genome editing efficiencies that differ in PAM binding specificity. Our results show that reduced PAM specificity causes persistent non-selective DNA binding and recurrent failures to engage the target sequence through stable guide RNA hybridization, leading to reduced genome editing efficiency in cells. These findings reveal a fundamental trade-off between broad PAM recognition and genome editing effectiveness. We propose that high-efficiency RNA-guided genome editing relies on an optimized two-step target capture process, where selective but low-affinity PAM binding precedes rapid DNA unwinding. This model provides a foundation for engineering more effective CRISPR-Cas and related RNA-guided genome editors.}, }
@article {pmid40375877, year = {2025}, author = {Khamwut, A and Nimnual, J and Chomta, N and Nimsamer, P and Mayuramart, O and Kaewsapsak, P and Pasittungkul, S and Poovorawan, Y and Payungporn, S}, title = {Detection of respiratory syncytial virus based on RT-RPA and CRISPR-Cas12a.}, journal = {Experimental biology and medicine (Maywood, N.J.)}, volume = {250}, number = {}, pages = {10387}, pmid = {40375877}, issn = {1535-3699}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Respiratory Syncytial Virus, Human/isolation &amp; purification/genetics ; *Respiratory Syncytial Virus Infections/diagnosis/virology ; Sensitivity and Specificity ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Human respiratory syncytial virus (hRSV) is one of the most prevalent viruses infecting children globally. In this study, we employed the RT-RPA with CRISPR/Cas12a detection methodology to detect and differentiate RSV-A and RSV-B, particularly in resource-limited settings. The detection limit for RSV-A and RSV-B was approximately 10[2] and 10[3] copies/reaction, respectively. The assay revealed 100% specificity in detecting both RSV-A and RSV-B. Diagnostic accuracy was 90.32 and 93.55% for RSV-A and RSV-B, respectively, compared to RT-qPCR. These data indicate a proficient strategy for RSV screening, demonstrating promise for prospective applications in detecting diverse viral infections.}, }
@article {pmid40375406, year = {2025}, author = {Rhode, J and Edwards, S and Tzvetkova, A and Jensen, LR and Hossain, MF and Nowack, B and Hagenau, L and Kuss, AW}, title = {Two iPSC lines with a heterozygous frameshift mutation in the floating-harbour syndrome locus of the SRCAP gene.}, journal = {Stem cell research}, volume = {86}, number = {}, pages = {103730}, doi = {10.1016/j.scr.2025.103730}, pmid = {40375406}, issn = {1876-7753}, mesh = {Humans ; *Frameshift Mutation/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Heterozygote ; Cell Line ; *Intellectual Disability/genetics/pathology ; CRISPR-Cas Systems ; }, abstract = {We present two CRISPR/Cas9-modified human iPSC lines with a heterozygous frameshift mutation (NM_006662.3:c.7300_7301insA) in the FLHS-locus of the SRCAP gene, which is associated with Floating-Harbor syndrome, a congenital neurodevelopmental disorder with symptoms including short stature and intellectual disability. The iPSCs express the pluripotency markers OCT4, SOX2, NANOG and TRA 1-60. They show differentiation into cells from all 3 germ layers, no chromosomal abnormalities and no off-target mutations in the tested regions. The mutation leads to a stop codon previously found in patients. Thus, either cell line can serve as disease-specific model for studying SRCAP in the context of FLHS.}, }
@article {pmid40374732, year = {2025}, author = {Hagman, A and Stenström, O and Carlström, G and Akke, M and Grey, C and Carlquist, M}, title = {Biocatalytic reductive amination with CRISPR-Cas9 engineered yeast.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {16972}, pmid = {40374732}, issn = {2045-2322}, mesh = {*CRISPR-Cas Systems ; Amination ; *Saccharomyces cerevisiae/genetics/metabolism ; *Metabolic Engineering/methods ; Biocatalysis ; Transaminases/genetics/metabolism ; Chromobacterium/enzymology/genetics ; Alanine/metabolism ; Alanine Transaminase/genetics/metabolism ; Bioreactors ; }, abstract = {Metabolically engineered baker's yeast can be used to produce chiral amines through whole-cell bioconversion of prochiral ketones. This study investigates the modulation of the alanine-pyruvate metabolic node to enhance reductive amination, using the stereoselective conversion of benzylacetone to (S)-1-methyl-3-phenylpropylamine (MPPA) as a model reaction. Chromosomal integration of multiple copies of the promiscuous omega transaminase from Chromobacterium violaceum (cv-ATA) resulted in an active yeast catalyst. Physiological characterization in bioreactors under aerobic batch cultivation revealed that amine production occurred only under post-diauxic growth on ethanol. To reduce native alanine utilization, the endogenous alanine aminotransferase (ALT1) was knocked out and replaced with cv-ATA. To rapidly employ this strategy in other strains, a simple CRISPR/cas9 method for universal gene replacement was developed. The replacement of ALT1 with cv-ATA improved the reaction by 2.6-fold compared to the control strain with intact ALT1. NMR measurements of metabolites originating from [15]N L-alanine and [13]C glucose indicated that pyruvate formation during growth on glucose inhibited amine production. Under optimal conditions, the biocatalytic bioconversion of benzylacetone to MPPA reached a yield of 58%.}, }
@article {pmid40373980, year = {2025}, author = {Deng, X and Wang, W and Tao, Y and Yao, L and Li, Y and Huang, X and He, J}, title = {Application of a rapid and sensitive RPA-CRISPR/Cas12a assay for BCSP31-based Brucella detection.}, journal = {Journal of microbiological methods}, volume = {235}, number = {}, pages = {107148}, doi = {10.1016/j.mimet.2025.107148}, pmid = {40373980}, issn = {1872-8359}, mesh = {*Brucella/genetics/isolation &amp; purification ; *Brucellosis/diagnosis/microbiology ; *CRISPR-Cas Systems/genetics ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; Humans ; Recombinases/genetics/metabolism ; DNA, Bacterial/genetics ; *Molecular Diagnostic Techniques/methods ; Animals ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Brucellosis, a zoonotic disease caused by Brucella species, poses significant health risks to humans and animals. Due to the limitations of current diagnostic methods, such as serological testing and PCR, in terms of sensitivity, specificity, and speed, this study explores the potential of integrating recombinase polymerase amplification (RPA) with the CRISPR-Cas12a system for Brucella detection. This combination leverages the strengths of both technologies for rapid, sensitive, and specific molecular diagnostics. RPA primers and CRISPR RNA (crRNA) targeting the Brucella-specific conserved sequence BCSP31 were designed, followed by optimization of the RPA-CRISPR/Cas12a system. Its performance was evaluated using genomic DNA from Brucella and non-Brucella species. The system's capabilities were assessed on clinical blood samples, demonstrating high sensitivity (detection limit of 10 copies per reaction and 16.6 attomoles for Brucella DNA) and excellent specificity. Testing on clinical samples showed strong agreement with qPCR results and an improvement over the RBT. The RPA-CRISPR/Cas12a platform represents a rapid, ultra-sensitive, and accurate method for Brucella detection and holds promise as a valuable tool for brucellosis control.}, }
@article {pmid40373829, year = {2025}, author = {Trujillo, E and Angulo, C}, title = {Perspectives on the use of the CRISPR system in plants to improve recombinant therapeutic protein production.}, journal = {Journal of biotechnology}, volume = {405}, number = {}, pages = {111-123}, doi = {10.1016/j.jbiotec.2025.05.010}, pmid = {40373829}, issn = {1873-4863}, abstract = {The plant-based system is a promising platform for producing biotherapeutics due to its scalability, cost-effectiveness, and lower risk of contamination by human pathogens. However, several challenges remain, including optimizing yield, stability, functionality, and the immunogenic properties of recombinant proteins. In this context, this review explores the application of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology to improve the production of recombinant therapeutic proteins in plants. Traditional tools and strategies for plant-based recombinant protein production are discussed, highlighting their limitations and the potential of CRISPR to overcome these boundaries. It delves into the components of the CRISPR-Cas system and its application in optimizing therapeutic protein function and yield. Major strategies include modifying glycosylation patterns to humanize plant-produced proteins, metabolic pathway engineering to increase protein accumulation, and the precise integration of transgenes into specific genomic loci to enhance expression stability and productivity. These advancements demonstrate how CRISPR system can overcome bottlenecks in plant molecular farming and enable the production of high-quality therapeutic proteins. Lastly, future trends and perspectives are examined, emphasizing ongoing innovations and challenges in the field. The review underscores the potential of CRISPR to reshape plant biotechnology and support the growing demand for recombinant therapeutics, offering new avenues for sustainable and efficient protein production systems. KEY MESSAGE: CRISPR technology has the potential to improve plant-based therapeutic protein production by optimizing yield, stability, and humanization, overcoming bottlenecks, and enabling sustainable, efficient systems for recombinant biotherapeutics.}, }
@article {pmid40373688, year = {2025}, author = {Thakur, RK and Aggarwal, K and Sood, N and Kumar, A and Joshi, S and Jindal, P and Maurya, R and Patel, P and Kurmi, BD}, title = {Harnessing advances in mechanisms, detection, and strategies to combat antimicrobial resistance.}, journal = {The Science of the total environment}, volume = {982}, number = {}, pages = {179641}, doi = {10.1016/j.scitotenv.2025.179641}, pmid = {40373688}, issn = {1879-1026}, mesh = {*Drug Resistance, Microbial ; Humans ; Bacteria/drug effects ; *Anti-Infective Agents ; *Drug Resistance, Bacterial ; Anti-Bacterial Agents ; }, abstract = {Antimicrobial resistance (AMR) is a growing global health crisis, threatening the effectiveness of antibiotics and other antimicrobial agents, leading to increased morbidity, mortality, and economic burdens. This review article provides a comprehensive analysis of AMR, beginning with a timeline of antibiotics discovery and the year of first observed resistance. Main mechanisms of AMR in bacteria, fungi, viruses, and parasites are summarized, and the main mechanisms of bacteria are given in detail. Additionally, we discussed in detail methods for detecting AMR, including phenotypic, genotypic, and advanced methods, which are crucial for identifying and monitoring AMR. In addressing AMR mitigation, we explore innovative interventions such as CRISPR-Cas systems, nanotechnology, antibody therapy, artificial intelligence (AI), and the One Health approach. Moreover, we discussed both finished and ongoing clinical trials for AMR. This review emphasizes the urgent need for global action and highlights promising technologies that could shape the future of AMR surveillance and treatment. By integrating interdisciplinary research and emerging clinical insights, this study aims to guide individuals toward impactful solutions in the battle against AMR.}, }
@article {pmid40373119, year = {2025}, author = {Witte, IP and Lampe, GD and Eitzinger, S and Miller, SM and Berríos, KN and McElroy, AN and King, RT and Stringham, OG and Gelsinger, DR and Vo, PLH and Chen, AT and Tolar, J and Osborn, MJ and Sternberg, SH and Liu, DR}, title = {Programmable gene insertion in human cells with a laboratory-evolved CRISPR-associated transposase.}, journal = {Science (New York, N.Y.)}, volume = {388}, number = {6748}, pages = {eadt5199}, doi = {10.1126/science.adt5199}, pmid = {40373119}, issn = {1095-9203}, mesh = {Humans ; *Transposases/genetics/metabolism ; *CRISPR-Cas Systems ; *Directed Molecular Evolution/methods ; *Gene Editing/methods ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems ; *Mutagenesis, Insertional ; }, abstract = {Programmable gene integration in human cells has the potential to enable mutation-agnostic treatments for loss-of-function genetic diseases and facilitate many applications in the life sciences. CRISPR-associated transposases (CASTs) catalyze RNA-guided DNA integration but thus far demonstrate minimal activity in human cells. Using phage-assisted continuous evolution (PACE), we generated CAST variants with >200-fold average improved integration activity. The evolved CAST system (evoCAST) achieves ~10 to 30% integration efficiencies of kilobase-size DNA cargoes in human cells across 14 tested genomic target sites, including safe harbor loci, sites used for immunotherapy, and genes implicated in loss-of-function diseases, with undetected indels and low levels of off-target integration. Collectively, our findings establish a platform for the laboratory evolution of CASTs and advance a versatile system for programmable gene integration in living systems.}, }
@article {pmid40372912, year = {2025}, author = {Xiang, W and Lin, X and Yang, Y and Huang, L and Chen, Y and Chen, J and Liu, L}, title = {Cas12h is a crRNA-guided DNA nickase that can be utilized for precise gene editing.}, journal = {Cell reports}, volume = {44}, number = {5}, pages = {115718}, doi = {10.1016/j.celrep.2025.115718}, pmid = {40372912}, issn = {2211-1247}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; DNA/metabolism/genetics ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Humans ; *Deoxyribonuclease I/metabolism/chemistry/genetics ; }, abstract = {Type V-H CRISPR-Cas system, an important subtype of type V CRISPR-Cas systems, has remained enigmatic in terms of its structure and function despite being discovered several years ago. Here, we comprehensively characterize the type V-H CRISPR-Cas system and elucidate its role as a DNA nicking system. The unique CRISPR RNA (crRNA) employed by Cas12h effector protein enables specific targeting of double-stranded DNA (dsDNA), while its RuvC domain is responsible for cleaving the non-target strand (NTS) of dsDNA. We present the structure of Cas12h bound to crRNA and target DNA. Our structural analysis reveals that the RuvC domain possesses a narrow active pocket that facilitates recognition of NTS but potentially hinders access to the target strand. Furthermore, we demonstrate that Cas12h confers adaptive immunity against invading mobile genetic elements through transcriptional gene inhibition. We have engineered an adenine base editor by fusing Cas12h with an adenine deaminase, achieving effective A-to-G substitution.}, }
@article {pmid40371045, year = {2025}, author = {Tewari, M and Rana, P and Pande, V}, title = {Nanomaterial-Based Biosensors for the Detection of COVID-19.}, journal = {Indian journal of microbiology}, volume = {65}, number = {1}, pages = {120-136}, pmid = {40371045}, issn = {0046-8991}, abstract = {The COVID-19 outbreak began in December 2019 and has affected people worldwide. It was declared a pandemic in 2020 by the World Health Organization. Developing rapid and reliable diagnostic techniques is crucial for identifying COVID-19 early and preventing the disease from becoming severe. In addition to conventional diagnostic techniques such as RT-PCR, computed tomography, serological assays, and sequencing methods, biosensors have become widely accepted for identifying and screening COVID-19 infection with high accuracy and sensitivity. Their low cost, high sensitivity, specificity, and portability make them ideal for diagnostics. The use of nanomaterials improves the performance of biosensors by increasing their sensitivities and limiting detection by several orders of magnitude. This manuscript briefly reviews the COVID-19 outbreak and its pathogenesis. Furthermore, it comprehensively discusses the currently available biosensors for SARS-CoV-2 detection, with a special emphasis on nanomaterials-based biosensors developed to detect this emerging virus and its variants efficiently.}, }
@article {pmid40370093, year = {2025}, author = {Qiu, S and Chen, L and Zhuang, D and Cao, Y and Wei, R and Cao, X and Chen, Y and Lai, X and Wang, S and Lin, Y and Lin, Z and Zhang, S}, title = {Fluorescence Aptasensor for sST2 Detection Using In Vitro Selected Aptamers.}, journal = {Analytical chemistry}, volume = {97}, number = {20}, pages = {10910-10918}, doi = {10.1021/acs.analchem.5c01855}, pmid = {40370093}, issn = {1520-6882}, mesh = {*Aptamers, Nucleotide/chemistry ; Humans ; *Interleukin-1 Receptor-Like 1 Protein/blood/analysis ; *Biosensing Techniques/methods ; Limit of Detection ; Fluorescence ; CRISPR-Cas Systems ; }, abstract = {Soluble suppression of tumorigenicity 2 (sST2) is a critical biomarker for heart failure (HF) diagnosis and prognosis, yet conventional antibody-based detection methods suffer from time-consuming protocols and high costs and involve complex detection procedures. To address these challenges, we first screened high-affinity aptamers under clinically relevant conditions and then coupled with the CRISPR/Cas12a system to develop a fluorescence aptasensor for rapid and sensitive sST2 detection. A serum matrix was introduced during aptamer selection to enhance specificity and anti-interference performance in real biological environments. Three sST2-specific aptamers (Apt-1, Apt-2, and Apt-3) were identified with dissociation constants (KD) of 8.42, 46.08, and 25.02 nM, respectively. Among these, Apt-1 demonstrated superior performance, which was utilized to construct a fluorescence biosensor combining aptamer recognition with CRISPR/Cas12a trans-cleavage signal amplification. The sensor achieved a broad linear detection range (5-120 ng/mL) and an ultralow limit of detection (LOD, 0.816 ng/mL) when applied in detecting sST2 in both the buffer and human serum. Notably, the platform exhibited exceptional resistance to interference from HF-related proteins and maintained high accuracy in clinical serum samples, showing a strong correlation (R[2] = 0.9794) with enzyme-linked immunosorbent assay (ELISA) results. By integration of serum-matrix screening and CRISPR-based signal enhancement, this work establishes a robust, cost-effective, and rapid diagnostic tool for sST2 detection.}, }
@article {pmid40369543, year = {2025}, author = {Zhang, T and Liu, G and Sun, S and Meng, Z and Qiu, Y and Ding, P}, title = {A novel tri-mode detection platform for ampicillin and drug resistance genes by CRISPR-driven luminescent nanozymes.}, journal = {Journal of nanobiotechnology}, volume = {23}, number = {1}, pages = {346}, pmid = {40369543}, issn = {1477-3155}, support = {82373635//the National Natural Science Foundation of China/ ; }, mesh = {*Ampicillin/analysis/pharmacology ; *Biosensing Techniques/methods ; *Anti-Bacterial Agents/analysis/pharmacology ; *CRISPR-Cas Systems ; Colorimetry ; Aptamers, Nucleotide/chemistry ; Smartphone ; Manganese Compounds/chemistry ; Oxides/chemistry ; Polymers/chemistry ; Ferrosoferric Oxide/chemistry ; Limit of Detection ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Drug Resistance, Bacterial/genetics ; beta-Lactamases/genetics ; Indoles ; }, abstract = {The antibiotic residues pose significant risks for bacterial resistance. To address the practical requirements for rapid, accurate, and on-site detection of antibiotic residues and monitoring the abundance of associated resistance genes, we report a smartphone-integrated multi-mode platform. The platform is aimed to simultaneous, accurate, and visual quantitative detection of ampicillin (AMP) and β-lactam antibiotic resistance genes (blaTEM). Specifically, we developed a magnetically controlled fluorescence, colorimetric, and photothermal biosensor based on a magnetic separation unit (aminated modified complementary DNA chain (NH2-cDNA) loading on the surface of Ferrosoferric Oxide@polydopamine (Fe3O4@PDA, FP), FP@cDNA) and a signal unit (the aptamer nucleic acid chain modified by phosphate group linked to Prussian blue@UiO-66@manganese dioxide (PB@UiO-66@MnO2, PUM) through Zr-O-P bond, PUM@Apt), for the integrated detection of AMP and blaTEM. By utilizing complementary base pairing between FP@cDNA and PUM@Apt, along with precise aptamer recognition the AMP, we achieved the fluorescence quantitative detection of AMP by measuring the signal unit in the supernatant. Subsequently, the difference of signal units in colorimetric process leads to a varying conversion rate of oxidized 3,3',5,5'-Tetramethylbenzidine (oxTMB), enabling the output of colorimetric and photothermal signals. The competitive binding of aptamers permitting the determination of AMP in the range of 0-160 pM with a low detection limit (0.34 pM). Additionally, in the presence of blaTEM, the activated CRISPR/Cas12a indiscriminately cleaves the single-stranded portion of the FP@DNA@PUM complex obtained by magnetic separation. A PUM-based three-signal detection scheme was established for the sensitive determination of blaTEM with the limit of detection (LOD) of 1.03 pM. The integration of smartphone-assisted analysis broadens the potential of the platform for visual detection. Notably, the innovative platform, with its excellent stability, exhibits great potential as a simple yet robust approach for the simultaneously visually monitoring antibiotics and drug resistance genes, and holds promise in the field of kit development.}, }
@article {pmid40369382, year = {2025}, author = {Ye, Y and Li, L and Chen, Y and Li, B and Xu, Z}, title = {Molecular methods for rapid detection and identification of foodborne pathogenic bacteria.}, journal = {World journal of microbiology &amp; biotechnology}, volume = {41}, number = {5}, pages = {175}, pmid = {40369382}, issn = {1573-0972}, mesh = {*Foodborne Diseases/microbiology/diagnosis ; *Bacteria/genetics/isolation &amp; purification/classification/pathogenicity ; *Molecular Diagnostic Techniques/methods ; *Food Microbiology/methods ; Humans ; Nucleic Acid Amplification Techniques/methods ; Polymerase Chain Reaction/methods ; Nucleic Acid Hybridization/methods ; Food Safety ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; }, abstract = {Foodborne pathogenic bacteria are one of the main factors causing food safety issues. The rapid and accurate detection of pathogenic bacteria using molecular techniques is an effective and powerful strategy for preventing and controlling outbreaks of foodborne diseases, thereby ensuring food safety. This article summarizes the rapid and efficient molecular diagnostic techniques for detecting pathogenic bacteria, including polymerase chain reaction and its derivatives, isothermal amplification, DNA hybridization, genomic sequencing, and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated (CRISPR/Cas)-based detection technique. Through a comparative analysis of the technical principles, advantages, and potential limitations of these diagnostic methods, as well as an outlook on the future development directions for molecular biological detection technology, which will provide a valuable reference for developing more accurate, convenient, and sensitive methods for foodborne pathogens detection, and will help better address the challenges posed by foodborne diseases, thereby ensuring public health and safety.}, }
@article {pmid40369306, year = {2025}, author = {Nie, Y and Li, X and Yang, W and Fei, S and Wang, Y and Li, Y and Zhang, K and Kang, J and Cheng, Y and Wang, H and Liu, D}, title = {Concanavalin-A-assisted extraction-free one-pot RPA-CRISPR/Cas12a assay for rapid detection of HPV16.}, journal = {Mikrochimica acta}, volume = {192}, number = {6}, pages = {354}, pmid = {40369306}, issn = {1436-5073}, support = {22QNFC098//Youth Independent Innovation Science Foundation of the General Hospital of the Chinese People's Liberation Army/ ; 22QNFC098//Youth Independent Innovation Science Foundation of the General Hospital of the Chinese People's Liberation Army/ ; 24JSZ12//Military Family Planning Program of China/ ; 24JSZ12//Military Family Planning Program of China/ ; }, mesh = {*Human papillomavirus 16/genetics/isolation &amp; purification ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Concanavalin A/chemistry ; DNA, Viral/genetics/analysis ; Papillomavirus Infections/diagnosis/virology ; Female ; Recombinases/metabolism ; }, abstract = {Human papillomavirus (HPV) infection is a major threat to women's health worldwide. High-risk subtypes, particularly HPV16, require rigorous screening and long-term surveillance to control cervical cancer. However, traditional HPV testing is hampered by the need for nucleic acid extraction, reliance on specialized technicians, and fluorescence detection equipment, limiting its suitability for rapid on-site testing. In this study, we developed a Concanavalin A-assisted extraction-free one-pot recombinase polymerase amplification (RPA) CRISPR/Cas12a assay (ConRCA) for HPV16. Concanavalin A-coated magnetic beads were used for target enrichment and nucleic acid-extraction-free processing. Suboptimal protospacer-adjacent motifs were used to achieve a one-pot RPA-CRISPR/Cas12a assay. The ConRCA assay can be completed in approximately 25 min under isothermal conditions and can detect at least 1.2 copies/μL of HPV16 genomic DNA using a fluorescence reader or test strip, demonstrating comparable sensitivity to qPCR. The feasibility of this detection method was evaluated with 31 unextracted clinical samples. Compared with qPCR, the overall sensitivity was 95% (19/20), and the specificity was 100% (11/11). Our results indicate that the ConRCA assay has great potential utility as a point-of-care testing for the rapid identification of HPV.}, }
@article {pmid40369106, year = {2025}, author = {Guo, T and Yang, J and Zhou, N and Sun, X and Huan, C and Lin, T and Bao, G and Hu, J and Li, G}, title = {Cas3 of type I-Fa CRISPR-Cas system upregulates bacterial biofilm formation and virulence in Acinetobacter baumannii.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {750}, pmid = {40369106}, issn = {2399-3642}, support = {82073611//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {*Biofilms/growth &amp; development ; *Acinetobacter baumannii/pathogenicity/genetics/physiology ; Animals ; Virulence/genetics ; Mice ; *CRISPR-Cas Systems ; *Acinetobacter Infections/microbiology ; *Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Virulence Factors/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; Female ; Mice, Inbred BALB C ; }, abstract = {Acinetobacter baumannii (A. baumannii) is an important pathogen causing various nosocomial infections. CRISPR-Cas system is the adaptive immune system of bacteria, which is also closely related to the drug resistance and virulence of bacteria. However, the effect and mechanism of cas3 (type I-Fa) in A. baumannii is still unclear. In this study, we successfully constructed a cas3 deletion mutant (19606Δcas3) and complemented strain (19606Δcas3/pcas3) to study the regulatory mechanism of type I-Fa cas3 on bacterial virulence. Our results showed that deletion of cas3(type I-Fa) significantly reduced the biofilm formation, virulence and pathogenicity to mice. The organ bacterial load of mice infected with cas3 deletion strain was significantly reduced, the lung inflammation was slightly changed, and the serum cytokine level was also decreased. All results demonstrated that cas3 enhanced the virulence and pathogenicity of A. baumannii. Mechanism analysis showed that deletion of cas3 can lead to the down-regulation of virulence factors such as biofilm formation related factors and outer membrane protein A(ompA). In addition, cas3 was also involved in the regulation of carbon metabolism and oxidative phosphorylation pathway of A. baumannii. Altogether, our study may provide cas3 as a therapeutic target in the future because of the close link to the virulence of A. baumannii.}, }
@article {pmid40368251, year = {2025}, author = {Ramadan, NK and Gaber, N and Ali, NM and Amer, OSO and Soliman, H}, title = {SHERLOCK, a novel CRISPR-Cas13a-based assay for detection of infectious bursal disease virus.}, journal = {Journal of virological methods}, volume = {337}, number = {}, pages = {115185}, doi = {10.1016/j.jviromet.2025.115185}, pmid = {40368251}, issn = {1879-0984}, mesh = {*Infectious bursal disease virus/isolation &amp; purification/genetics ; Animals ; Chickens/virology ; *Birnaviridae Infections/diagnosis/veterinary/virology ; *Poultry Diseases/diagnosis/virology ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; RNA, Viral/genetics ; }, abstract = {Infectious bursal disease (IBD) is an extremely contagious viral infection that primarily affects young chicks, leading to significant economic losses in the poultry industry. The disease is caused by a double-stranded RNA virus of the genus Avibirnavirus, family Birnaviridae, namely, the infectious bursal disease virus (IBDV). Unfortunately, current methods for detecting IBDV lack adequate sensitivity. Accordingly, the advantages of the Specific High Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) assay were employed to develop an ultrasensitive assay (IBD-SHERLOCK assay) for the detection of IBDV in clinical chicken tissues. The assay comprises two steps: isothermal preamplification of the target RNA through reverse transcription recombinase polymerase amplification (RT-RPA) and a subsequent detection step, which is based on the CRISPR-Cas13a system. The integration of lateral flow (LFD) visual detection of the IBD-SHERLOCK products strengthens the feasibility of the assay for use as a point-of-care test in chicken farms. Compared with RT-qPCR, this method exhibited ultra-analytical and clinical sensitivity. The assay has a lower detection limit of 5 aM, which is equivalent to three IBDV-RNA molecules. The assay demonstrated the ability to detect IBDV-RNA in 70 clinical field samples, 15 of which tested negative by RT-qPCR. This evidence highlights its superior sensitivity and potential for early detection of IBDV in chicken tissues. This study effectively established and verified a CRISPR-based diagnostic test for the early detection of IBDV in clinical chicken tissues, demonstrating remarkable specificity and sensitivity. The IBD-SHERLOCK assay can be used as a highly sensitive point-of-care diagnostic tool in chicken farms.}, }
@article {pmid40366179, year = {2025}, author = {Zhang, Y and Li, W and Chen, S and Zhang, Y and Zhu, Y and Lan, F and Du, H and Fan, R and Zhu, J and Pan, W and Situ, B and Zheng, L and Luo, S and Yan, X}, title = {Layered-Responsive Multivalent Tetrahedral DNA Framework-Decorated CRISPR-Cas12a Nanocapsule Enables Precise and Enhanced Tumor Chemotherapy.}, journal = {ACS nano}, volume = {19}, number = {20}, pages = {19274-19286}, doi = {10.1021/acsnano.5c01747}, pmid = {40366179}, issn = {1936-086X}, mesh = {*Doxorubicin/pharmacology/chemistry/administration &amp; dosage ; Humans ; *DNA/chemistry ; *CRISPR-Cas Systems ; *Nanocapsules/chemistry ; Animals ; Cell Line, Tumor ; Mice ; *Antineoplastic Agents/pharmacology/chemistry ; *Antibiotics, Antineoplastic/pharmacology/chemistry ; Neoplasms/drug therapy ; }, abstract = {The lack of selective tumor targeting and the high toxicity of conventional chemotherapy treatments remain major challenges in cancer therapy. Here, we develop a self-controlled DNA nanostructure-CRISPR-12a system, a triple-locked cascade tumor therapy nanocapsule (Tatna), for efficient and targeted tumor treatment. Tatna integrates structural DNA tetrahedrons (DTs) with high drug-loading capacity, Cas12a/crRNA ribonucleoprotein (Cas12a RNP), and doxorubicin (DOX) to enable multisite response for precise drug delivery and augmented tumor treatment. By incorporation of a nucleolin-targeting aptamer, Tatna achieves selective targeting and efficient tumor cell internalization. Encapsulation in pH-responsive poly l-lactic-co-glycolic acid (PLGA) nanocapsule ensures stable circulation and controlled release of both DOX and Cas12a until tumor-specific activation in the acidic microenvironment. The Cas12a RNP, triggered by APE1 mRNA overexpression in tumor cells, induces trans-cleavage of DTs, releasing DOX and Cas12a to transport into the nucleus and induce enhanced cell apoptosis. This self-regulating and multifunctional approach enhances the efficacy of chemotherapy while reducing off-target effects. Tatna's programmable, tumor-specific delivery system represents a powerful strategy for advancing precision medicine and personalized cancer treatment.}, }
@article {pmid40363763, year = {2025}, author = {Wang, L and Liu, Y and Song, H and Zhang, X and Wang, Y}, title = {Conditional Control of CRISPR/Cas9 Function by Chemically Modified Oligonucleotides.}, journal = {Molecules (Basel, Switzerland)}, volume = {30}, number = {9}, pages = {}, pmid = {40363763}, issn = {1420-3049}, support = {22307107//the National Natural Science Foundation of China/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Oligonucleotides/chemistry/genetics ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; Animals ; }, abstract = {The CRISPR (clustered regularly interspaced short palindromic repeats) system has emerged as a revolutionary gene-editing tool with immense potential in gene therapy, functional genomics, and beyond. However, achieving precise spatiotemporal control of gene editing in specific cells and tissues while effectively mitigating potential risks, such as off-target effects, remains a key challenge for its clinical translation. To overcome these limitations, researchers have developed innovative strategies based on chemical modifications of oligonucleotides to enhance the precision, efficiency, and controllability of CRISPR/Cas9-mediated gene editing. By introducing conditional responsive elements, such as photosensitive groups, small-molecule responsive units, and supramolecular structures, they have successfully achieved precise spatiotemporal and dose-dependent regulation of CRISPR/Cas9 function. This review provides a comprehensive overview of recent advancements in gRNA regulation strategies based on chemical modifications of oligonucleotides, discussing their applications in improving the efficiency, specificity, and controllability of CRISPR/Cas9 editing. We also highlight the challenges associated with the conditional control of gRNA and offer insights into future directions for the chemical regulation of gRNA to further advance CRISPR/Cas9 technology.}, }
@article {pmid40362657, year = {2025}, author = {Seijas, A and Cora, D and Novo, M and Al-Soufi, W and Sánchez, L and Arana, &#xc1;J}, title = {CRISPR/Cas9 Delivery Systems to Enhance Gene Editing Efficiency.}, journal = {International journal of molecular sciences}, volume = {26}, number = {9}, pages = {}, pmid = {40362657}, issn = {1422-0067}, support = {ND//Campus Terra, University of Santiago de Compostela/ ; ND//Fundaci&#xf3;n Caixa Rural Galega Tom&#xe1;s Notario Vacas/ ; investment line no.1 of its component number 17//Spain's Recovery and Resilience Plan, Complementary RTDI Plan for Marine Science/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; Animals ; *Gene Transfer Techniques ; Nanoparticles/chemistry ; Lipids/chemistry ; }, abstract = {CRISPR/Cas9 has revolutionized genome editing by enabling precise and efficient genetic modifications across multiple biological systems. Despite its growing therapeutic potential, key challenges remain in mitigating off-target effects, minimizing immunogenicity, and improving the delivery of CRISPR components into target cells. This review provides an integrated analysis of physical, viral, and non-viral delivery systems, highlighting recent advances in the use of lipid nanoparticles, polymeric carriers, and hybrid platforms. We also examine an often overlooked factor: the aggregation behavior of the Cas9 protein, which may interfere with cellular uptake, the encapsulation efficiency, and nuclear localization. By comparing delivery platforms and their reported editing outcomes, we identify critical physicochemical parameters that influence therapeutic success. Finally, we propose standardized methods to assess Cas9 encapsulation and aggregation and discuss translational barriers such as manufacturing scalability and regulatory requirements. These insights aim to guide the development of safer and more effective CRISPR/Cas9-based therapies.}, }
@article {pmid40362595, year = {2025}, author = {Luo, X and Weidinger, E and Burghardt, T and Höhn, M and Wagner, E}, title = {CRISPR/Cas9 Ribonucleoprotein Delivery Enhanced by Lipo-Xenopeptide Carriers and Homology-Directed Repair Modulators: Insights from Reporter Cell Lines.}, journal = {International journal of molecular sciences}, volume = {26}, number = {9}, pages = {}, pmid = {40362595}, issn = {1422-0067}, support = {SFB1032 (project-ID 201269156) sub-project B4//Deutsche Forschungsgemeinschaft/ ; No 825825 UPGRADE//European Union/ ; CNATM, number 03ZU1201AA//Federal Ministry of Education and Research/ ; doctoral fellowship to XL//China Scholarship Council/ ; }, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Recombinational DNA Repair/drug effects ; *Ribonucleoproteins/genetics/metabolism ; HeLa Cells ; DNA End-Joining Repair ; Peptides ; }, abstract = {CRISPR-Cas9 genome editing is a versatile platform for studying and treating various diseases. Homology-directed repair (HDR) with DNA donor templates serves as the primary pathway for gene correction in therapeutic applications, but its efficiency remains a significant challenge. This study investigates strategies to enhance gene correction efficiency using a T-shaped lipo-xenopeptide (XP)-based Cas9 RNP/ssDNA delivery system combined with various HDR enhancers. Nu7441, a known DNA-PKcs inhibitor, was found to be most effective in enhancing HDR-mediated gene correction. An over 10-fold increase in HDR efficiency was achieved by Nu7441 in HeLa-eGFPd2 cells, with a peak HDR efficiency of 53% at a 5 nM RNP concentration and up to 61% efficiency confirmed by Sanger sequencing. Surprisingly, the total gene editing efficiency including non-homologous end joining (NHEJ) was also improved. For example, Nu7441 boosted exon skipping via NHEJ-mediated splice site destruction by 30-fold in a DMD reporter cell model. Nu7441 modulated the cell cycle by reducing cells in the G1 phase and extending the S and G2/M phases without compromising cellular uptake or endosomal escape. The enhancement in genome editing by Nu7441 was widely applicable across several cell lines, several Cas9 RNP/ssDNA carriers (LAF-XPs), and also Cas9 mRNA/sgRNA/ssDNA polyplexes. These findings highlight a novel and counterintuitive role for Nu7441 as an enhancer of both HDR and total gene editing efficiency, presenting a promising strategy for Cas9 RNP-based gene therapy.}, }
@article {pmid40362588, year = {2025}, author = {Arana, &#xc1;J and González-Llera, L and Barreiro-Iglesias, A and Sánchez, L}, title = {Emerging Frontiers in Zebrafish Embryonic and Adult-Derived Cell Lines.}, journal = {International journal of molecular sciences}, volume = {26}, number = {9}, pages = {}, pmid = {40362588}, issn = {1422-0067}, support = {PID2020-115121GB-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; PID2023-147266NB-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; ED431C 2022/33//Xunta de Galicia/ ; }, mesh = {Animals ; *Zebrafish/embryology/genetics ; Cell Line ; *Embryo, Nonmammalian/cytology ; CRISPR-Cas Systems ; Cell Culture Techniques/methods ; Humans ; }, abstract = {Zebrafish (Danio rerio) has become a pivotal vertebrate model in biomedical research, renowned for its genetic similarity to humans, optical transparency, rapid embryonic development, and amenability to experimental manipulation. In recent years, the derivation of cell lines from zebrafish embryos has unlocked new possibilities for in vitro studies across developmental biology, toxicology, disease modeling, and genetic engineering. These embryo-derived cultures offer scalable, reproducible, and ethically favorable alternatives to in vivo approaches, enabling high-throughput screening and mechanistic exploration under defined conditions. This review provides a comprehensive overview of protocols for establishing and maintaining zebrafish embryonic cell lines, emphasizing culture conditions, pluripotency features, transfection strategies, and recent innovations such as genotype-defined mutant lines generated via CRISPR/Cas9 and feeder-free systems. We also highlight emerging applications in oncology, regenerative medicine, and functional genomics, positioning zebrafish cell lines as versatile platforms bridging animal models and next-generation in vitro systems. Its continued optimization holds promise for improved reproducibility, reduced animal use, and expanded translational impact in biomedical research.}, }
@article {pmid40362571, year = {2025}, author = {Herreno-Pachón, AM and Leal, AF and Khan, S and Alméciga-Díaz, CJ and Tomatsu, S}, title = {CRISPR/nCas9-Edited CD34+ Cells Rescue Mucopolysaccharidosis IVA Fibroblasts Phenotype.}, journal = {International journal of molecular sciences}, volume = {26}, number = {9}, pages = {}, pmid = {40362571}, issn = {1422-0067}, support = {1R01HD102545-01A1/GF/NIH HHS/United States ; }, mesh = {Humans ; *CRISPR-Cas Systems ; *Fibroblasts/metabolism/pathology ; *Gene Editing/methods ; *Antigens, CD34/metabolism/genetics ; Genetic Therapy/methods ; *Mucopolysaccharidosis IV/therapy/genetics/metabolism/pathology ; Phenotype ; Animals ; Mice ; Chondroitinsulfatases/genetics/metabolism ; }, abstract = {Mucopolysaccharidosis (MPS) IVA is a bone-affecting lysosomal storage disease (LSD) caused by impaired degradation of the glycosaminoglycans (GAGs) keratan sulfate (KS) and chondroitin 6-sulfate (C6S) due to deficient N-acetylgalactosamine-6-sulfatase (GALNS) enzyme activity. Previously, we successfully developed and validated a CRISPR/nCas9-based gene therapy (GT) to insert an expression cassette at the AAVS1 and ROSA26 loci in human MPS IVA fibroblasts and MPS IVA mice, respectively. In this study, we have extended our approach to evaluate the effectiveness of our CRISPR/nCas9-based GT in editing human CD34+ cells to mediate cross-correction of MPS IVA fibroblasts. CD34+ cells were electroporated with the CRISPR/nCas9 system, targeting the AAVS1 locus. The nCas9-mediated on-target donor template insertion, and the stemness of the CRISPR/nCas-edited CD34+ cells was evaluated. Additionally, MPS IVA fibroblasts were co-cultured with CRISPR/nCas-edited CD34+ cells to assess cross-correction. CRISPR/nCas9-based gene editing did not affect the stemness of CD34+ cells but did lead to supraphysiological levels of the GALNS enzyme. Upon co-culture, MPS IVA fibroblasts displayed a significant increase in the GALNS enzyme activity along with lysosomal mass reduction, pro-oxidant profile amelioration, mitochondrial mass recovery, and pro-apoptotic and pro-inflammatory profile improvement. These results show the potential of our CRISPR/nCas9-based GT to edit CD34+ cells to mediate cross-correction.}, }
@article {pmid40362308, year = {2025}, author = {Haider, S and Mussolino, C}, title = {Fine-Tuning Homology-Directed Repair (HDR) for Precision Genome Editing: Current Strategies and Future Directions.}, journal = {International journal of molecular sciences}, volume = {26}, number = {9}, pages = {}, pmid = {40362308}, issn = {1422-0067}, support = {CA311-41//Deutsche Forschungsgemeinschaft/ ; }, mesh = {*Gene Editing/methods ; Humans ; *Recombinational DNA Repair ; *CRISPR-Cas Systems ; DNA End-Joining Repair ; Animals ; DNA Breaks, Double-Stranded ; }, abstract = {CRISPR-Cas9 is a powerful genome-editing technology that can precisely target and cleave DNA to induce double-strand breaks (DSBs) at almost any genomic locus. While this versatility holds tremendous therapeutic potential, the predominant cellular pathway for DSB repair-non-homologous end-joining (NHEJ)-often introduces small insertions or deletions that disrupt the target site. In contrast, homology-directed repair (HDR) utilizes exogenous donor templates to enable precise gene modifications, including targeted insertions, deletions, and substitutions. However, HDR remains relatively inefficient compared to NHEJ, especially in postmitotic cells where cell cycle constraints further limit HDR. To address this challenge, numerous methodologies have been explored, ranging from inhibiting key NHEJ factors and optimizing donor templates to synchronizing cells in HDR-permissive phases and engineering HDR-enhancing fusion proteins. These strategies collectively aim to boost HDR efficiency and expand the clinical and research utility of CRISPR-Cas9. In this review, we discuss recent advances in manipulating the balance between NHEJ and HDR, examine the trade-offs and practical considerations of these approaches, and highlight promising directions for achieving high-fidelity genome editing in diverse cell types.}, }
@article {pmid40362304, year = {2025}, author = {Shiryaeva, O and Tolochko, C and Alekseeva, T and Dyachuk, V}, title = {Targets and Gene Therapy of ALS (Part 1).}, journal = {International journal of molecular sciences}, volume = {26}, number = {9}, pages = {}, pmid = {40362304}, issn = {1422-0067}, mesh = {Humans ; *Amyotrophic Lateral Sclerosis/therapy/genetics/pathology ; *Genetic Therapy/methods ; Animals ; Superoxide Dismutase-1/genetics ; C9orf72 Protein/genetics ; Mutation ; Gene Editing ; RNA-Binding Protein FUS/genetics ; Oligonucleotides, Antisense/therapeutic use ; CRISPR-Cas Systems ; DNA-Binding Proteins/genetics ; RNA Interference ; MicroRNAs/genetics ; Disease Models, Animal ; RNA, Small Interfering/genetics ; Motor Neurons/metabolism/pathology ; }, abstract = {Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective death of motor neurons, which causes muscle atrophy. Genetic forms of ALS are recorded only in 10% of cases. However, over the past decade, studies in genetics have substantially contributed to our understanding of the molecular mechanisms underlying ALS. The identification of key mutations such as SOD1, C9orf72, FUS, and TARDBP has led to the development of targeted therapy that is gradually being introduced into clinical trials, opening up a broad range of opportunities for correcting these mutations. In this review, we aimed to present an extensive overview of the currently known mechanisms of motor neuron degeneration associated with mutations in these genes and also the gene therapy methods for inhibiting the expression of their mutant proteins. Among these, antisense oligonucleotides, RNA interference (siRNA and miRNA), and gene-editing (CRISPR/Cas9) methods are of particular interest. Each has shown its efficacy in animal models when targeting mutant genes, whereas some of them have proven to be efficient in human clinical trials.}, }
@article {pmid40361099, year = {2025}, author = {Xie, H and Bourgade, B and Stensjö, K and Lindblad, P}, title = {dCas12a-mediated CRISPR interference for multiplex gene repression in cyanobacteria for enhanced isobutanol and 3-methyl-1-butanol production.}, journal = {Microbial cell factories}, volume = {24}, number = {1}, pages = {104}, pmid = {40361099}, issn = {1475-2859}, support = {2021-01669//Svenska Forskningsr&#xe5;det Formas/ ; CTS 20:412//Carl Tryggers Stiftelse f&#xf6;r Vetenskaplig Forskning/ ; 2024-00443//Vinnova/ ; }, mesh = {*Butanols/metabolism ; *Metabolic Engineering/methods ; *CRISPR-Cas Systems ; *Synechocystis/genetics/metabolism ; *Pentanols/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {BACKGROUND: Cyanobacteria of the genera Synechocystis and Synechococcus have emerged as promising platforms for metabolic engineering endeavors aimed at converting carbon dioxide into valuable fuels and chemicals, thus addressing the pressing energy demand and mitigating global climate change. Notably, Synechocystis sp. strain PCC 6803 (Synechocystis) has been engineered to produce isobutanol (IB) and 3-methyl-1-butanol (3M1B) via heterologous expression of α-ketoisovalerate decarboxylase (Kivd). Despite these advances, the achieved IB/3M1B titers remain low. CRISPR interference (CRISPRi), an emerging tool for targeted gene repression, has demonstrated success in various cellular systems to enhance biochemical productivity.<br><br>RESULTS: In this study, we developed a dCas12a-mediated CRISPRi system (CRISPRi-dCas12a) that effectively blocked the transcriptional initiation/elongation of essential gene(s), resulting in up to 60% gene repression in Synechocystis. Subsequently, the CRISPRi-dCas12a system was successfully integrated into an IB/3M1B producer strain, where it exhibited target gene repression under optimal cultivation conditions. To identify gene targets involved in metabolic pathways potentially limiting IB/3M1B biosynthesis, we initially designed a CRISPR RNA (crRNA) library targeting fifteen individual gene(s), where repression of ten genes significantly increased IB/3M1B production per cell. Moreover, a synergetic effect was observed on IB/3M1B production by designing a single crRNA targeting multiple genes for simultaneous repression. A final strain HX106, featuring dual repression of ppc and gltA, both involved in the TCA cycle, resulted in 2.6-fold and 14.8-fold improvement in IB and 3M1B production per cell, respectively.<br><br>CONCLUSIONS: Our findings underscore the effectiveness of the CRISPRi-dCas12a system in Synechocystis for identifying competing pathways and redirecting carbon flux to enhance IB/3M1B production. Furthermore, this study established a solid groundwork for utilizing an expanded CRISPRi-crRNA library to undertake genome-wide exploration of potential competing pathways not only for IB/3M1B biosynthesis but also for other diverse biofuels and biochemical production processes.}, }
@article {pmid40361067, year = {2025}, author = {Avci, FG and Prasun, T and Wendisch, VF}, title = {Metabolic engineering for microbial production of sugar acids.}, journal = {BMC biotechnology}, volume = {25}, number = {1}, pages = {36}, pmid = {40361067}, issn = {1472-6750}, support = {031B1737C//German Federal Ministry of Education and Research (BMBF) as part of the collaborative research project ForceYield2/ ; online publication fund//Deutsche Forschungsgemeinschaft/ ; open access publication fund//Universit&#xe4;t Bielefeld/ ; }, mesh = {*Metabolic Engineering/methods ; *Sugar Acids/metabolism ; Fermentation ; Synthetic Biology ; Oxidation-Reduction ; *Bacteria/metabolism/genetics ; }, abstract = {Carbohydrates including sugar acids are commonly used as carbon sources in microbial biotechnology. These sugar acids are themselves desirable and often overlooked targets for biobased production since they find applications in a broad range of industries, examples include food, construction, medical, textile, and polymer industries. Different stages of oxidation for natural sugar acids can be distinguished. Oxidation of the aldehyde group yields aldonic acids, oxidation of the primary hydroxy group leads to uronic acids, and both oxidations combined yield aldaric acids. While the chemical oxidation of sugars to their acid forms often is a one-pot reaction under harsh conditions, their biosynthesis is much more delicate. Bio-based production can involve enzymatic conversion, whole-cell biotransformation, and fermentation. Generally, the in vivo approaches are preferred because they are less resource-intensive than enzymatic conversion. Metabolic engineering plays a crucial role in optimizing microbial strains for efficient sugar acid production. Strategies include pathway engineering to overexpress key enzymes involved in sugar oxidation, deletion of competing pathways to enhance the precursor availability and eliminate the product consumption, cofactor balancing for efficient redox reactions, and transporter engineering to facilitate precursor import or sugar acid export. Synthetic biology tools, such as CRISPR-Cas and dynamic regulatory circuits, have further improved strain development by enabling precise genetic modifications and adaptive control of metabolic fluxes. The usage of plant biomass hydrolysates for bio-based production further adds to the environmental friendliness of the in vivo approaches. This review highlights the different approaches for the production of C5 and C6 sugar acids, their applications, and their catabolism in microbes.}, }
@article {pmid40360969, year = {2025}, author = {Dong, J and Ma, W and Zhou, S and Li, X and Deng, L and Hou, C and Huo, D}, title = {Tri-Mode CRISPR-Based Biosensor for miRNA Detection: Enhancing Clinical Diagnostics with Cross-Validation.}, journal = {Analytical chemistry}, volume = {97}, number = {20}, pages = {10938-10946}, doi = {10.1021/acs.analchem.5c02348}, pmid = {40360969}, issn = {1520-6882}, mesh = {*MicroRNAs/analysis/genetics ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques ; Limit of Detection ; Electrochemical Techniques ; Metal-Organic Frameworks/chemistry ; Methylene Blue/chemistry ; }, abstract = {In vitro diagnostics require the accurate detection of disease-associated target biomolecules at ultralow concentrations. A multimode sensing strategy is considered as a potential method for in vitro diagnosis because it allows cross-validation of test results through data complementation and self-calibration, and provides double confirmation. Here, we present a CRISPR/Cas12a-powered trimode biosensor (CPTMB) for ultrasensitive and reliable analysis of miRNA. Briefly, the presence of target miRNA initiates rolling circle extension-driven loop-mediated isothermal amplification (R-LAMP), which subsequently activates the trans-cleavage activity of CRISPR/Cas12a. Then, the hairpin probe (HP) biogate on nucleic acid-functionalized MB@Fe-MOF signal probe was degraded by Cas12a, leading to the release of methylene blue (MB) signal molecules encapsulated within Fe-MOF nanocarriers. Due to the capability of MB to generate output responses across three distinct modes: electrochemical (EC), fluorescence (FL), and ultraviolet-visible spectroscopy (UV-vis), a trimodal sensing system is achieved. Benefiting from the efficient signal amplification capabilities of R-LAMP and CRISPR/Cas12a, this strategy enables rapid detection of target miRNA at femtomolar levels within 70 min. Furthermore, the detection results across the three modes cross-validate one another, thereby enhancing the reliability of the analysis. More importantly, the platform has been successfully applied to miRNA analysis in real samples, and the detection results are in good agreement with those of the standard method RT-qPCR, indicating its great potential in the clinical diagnosis of early-stage cancer.}, }
@article {pmid40360740, year = {2025}, author = {Tei, C and Hata, S and Mabuchi, A and Okuda, S and Ito, KK and Genova, M and Fukuyama, M and Yamamoto, S and Chinen, T and Toyoda, A and Kitagawa, D}, title = {Comparative analysis of multiple DNA double-strand break repair pathways in CRISPR-mediated endogenous tagging.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {749}, pmid = {40360740}, issn = {2399-3642}, support = {18K06246, 19H05651, 20K15987, 20K22701, 21H02623, 22H02629, 22K19305, 22K19370, 22K20624, 23K14176//Japan Society for the Promotion of Science London (JSPS London)/ ; JPMJPR21EC//MEXT | JST | Precursory Research for Embryonic Science and Technology (PRESTO)/ ; }, mesh = {*DNA Breaks, Double-Stranded ; *CRISPR-Cas Systems ; Humans ; *DNA End-Joining Repair ; *Gene Editing/methods ; *DNA Repair ; Gene Knock-In Techniques ; Recombinational DNA Repair ; HEK293 Cells ; }, abstract = {CRISPR-mediated endogenous tagging is a powerful tool in biological research. Inhibiting the non-homologous end joining (NHEJ) pathway has been shown to improve the low efficiency of accurate knock-in via homology-directed repair (HDR). However, the influence of alternative double-stranded break (DSB) repair pathways on knock-in remains to be fully explored. In this study, our long-read amplicon sequencing analysis reveals various patterns of imprecise repair in CRISPR-mediated knock-in, even with NHEJ inhibition. Further suppressing either microhomology-mediated end joining (MMEJ) or single-strand annealing (SSA) reduces nucleotide deletions around the cut site, thereby elevating knock-in accuracy. Additionally, imprecise donor integration is reduced by inhibiting SSA, but not MMEJ. Particularly, SSA suppression reduced asymmetric HDR, a specific imprecise integration pattern, which we further confirm using a novel reporter system. These findings demonstrate the complex interplay of multiple DSB repair pathways in CRISPR-mediated knock-in and offer novel strategies, including SSA pathway targeting, to improve precise gene editing efficiency.}, }
@article {pmid40359875, year = {2025}, author = {Morán Torres, JP and Lyu, J and Chen, X and Klaas, AM and Vonk, PJ and Lugones, LG and de Cock, H and Wösten, HAB}, title = {Single and combinatorial gene inactivation in Aspergillus niger using selected as well as genome-wide gRNA library pools.}, journal = {Microbiological research}, volume = {298}, number = {}, pages = {128204}, doi = {10.1016/j.micres.2025.128204}, pmid = {40359875}, issn = {1618-0623}, mesh = {*Aspergillus niger/genetics ; CRISPR-Cas Systems ; Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Plasmids/genetics ; *Gene Silencing ; Gene Library ; Genome, Fungal ; Genetic Vectors ; Phenotype ; Genes, Fungal ; Transformation, Genetic ; }, abstract = {Aspergillus niger is a saprotroph, a pathogen, an endophyte, a food spoiler and an important cell factory. Only a minor fraction of its genes has been experimentally characterized. We here set up a CRISPR/Cas9 mutagenesis screen for functional gene analysis using co-transformation of a pool of gene editing plasmids that are maintained under selection pressure and that each contain a gRNA. First, a pool of gRNA vectors was introduced in A. niger targeting five genes with easy selectable phenotypes. Transformants were obtained with all possible single, double, triple, quadruple and quintuple gene inactivation phenotypes. Their genotypes were confirmed using the gRNA sequences in the transforming vector as barcodes. Next, a gRNA library was introduced in A. niger targeting > 9600 genes. Gene nsdC was identified as a sporulation gene using co-transformation conditions that favored uptake of one or two gRNA construct(s) from the genome-wide vector pool. Together, CRISPR/Cas9 vectors with a (genome-wide) pool of gRNAs can be used for functional analysis of genes in A. niger with phenotypes that are the result of the inactivation of a single or multiple genes.}, }
@article {pmid40359808, year = {2025}, author = {Liu, S and Xiao, G and Li, P and Xu, Y and Fan, X and Tian, L and Zhang, S and Zhang, G}, title = {Plasma-based ultrasensitive detection of Mycobacterium tuberculosis ESAT6/CFP10 fusion antigen using a CRISPR-driven aptamer fluorescence testing (CRAFT).}, journal = {Biosensors &amp; bioelectronics}, volume = {284}, number = {}, pages = {117566}, doi = {10.1016/j.bios.2025.117566}, pmid = {40359808}, issn = {1873-4235}, mesh = {*Mycobacterium tuberculosis/isolation &amp; purification/genetics ; Humans ; *Biosensing Techniques/methods ; *Bacterial Proteins/blood/genetics/isolation &amp; purification ; *Aptamers, Nucleotide/chemistry/genetics ; *Antigens, Bacterial/blood/genetics/isolation &amp; purification ; *Tuberculosis/diagnosis/microbiology/blood ; Limit of Detection ; CRISPR-Cas Systems ; Fluorescence ; SELEX Aptamer Technique ; }, abstract = {Tuberculosis (TB) screening in clinical diagnosis is challenging due to issues such as sputum dependence, time-consuming procedures, and high costs. In this study, we introduce a CRAFT (CRISPR-Driven Aptamer Fluorescence Testing), an aptamer-based CRISPR/Cas12a assay designed for the rapid and sensitive detection of Mycobacterium tuberculosis (Mtb) antigens from peripheral blood. Aptamer 3 (Ap3) and the aptamer-mediated probe (Aptamer-blocker 3-7) were selected through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Ap3 demonstrated a dissociation constant (Kd) of 8.3E-7 M with the ESAT6/CFP10 fusion proteins (EC proteins), which are produced during the replicative phase of Mtb. Upon labeling the EC proteins with Aptamer-blocker 3-7 (Ap-blocker 3-7) probe, single-stranded DNA (ssDNA) blocker 3-7 was released, thereby completing the process for RPA-based CRISPR/Cas12a fluorescence detection. After optimizing multiple parameters, CRAFT achieved a detection limit of 0.1 ag/mL EC proteins (equivalent to 3 protein particles per mL) within 120 min from plasma sample to result. The method was validated with 86 clinical plasma samples confirmed the method's high diagnostic accuracy for Mtb infection (sensitivity: 97.1 %, 95 % confidence interval (CI) [0.849-0.998]); specificity: 98.0 %, 95 % CI [0.897-0.999]), supporting its utility in early therapeutic evaluation of tuberculosis management.}, }
@article {pmid40359574, year = {2025}, author = {Zhou, J and Pang, R and Han, Y and Guo, Y and Wang, Y and Yang, H and Wang, W and Fu, X and Zhang, R and Zheng, X and Zhang, T and Zhang, Y and Wang, Q}, title = {CRISPR-Cas9-mediated knockout of OsKCS11 in rice reveals potential crosstalk between very-long-chain fatty acids and cytokinin.}, journal = {The Plant journal : for cell and molecular biology}, volume = {122}, number = {3}, pages = {e70208}, doi = {10.1111/tpj.70208}, pmid = {40359574}, issn = {1365-313X}, support = {2020B1515120086//Guangdong Basic and Applied Basic Research Foundation/ ; SZKF2103//Open Project Program of Panxi Crops Research and Utilization Key Laboratory of Sichuan Province/ ; 2023ZD04074//the Biological Breeding- National Science and Technology Major Projects/ ; 2021B0707010006//Key-Area Research and Development Program of Guangdong Province/ ; CAAS-CSIAF-202303//Innovation Program of CAAS/ ; 31961143015//National Natural Science Foundation of China/ ; 32070202//National Natural Science Foundation of China/ ; 2021YFYZ0016//Sichuan Science and Technology Program/ ; 2024NSFSC0314//Sichuan Science and Technology Program/ ; }, mesh = {*Oryza/genetics/metabolism ; *Cytokinins/metabolism ; CRISPR-Cas Systems/genetics ; *Fatty Acids/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Knockout Techniques ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; *3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/genetics/metabolism ; }, abstract = {Very-long-chain fatty acids (VLCFAs) play crucial roles in various physiological processes in plants. Through our investigation using a CRISPR-Cas9 knockout mutant library in rice, we identified a semi-dwarf rice mutant named CRISPR-Cas-based dwarf-1 (csd-1). This mutant displayed multiple developmental defects, such as decreased plant height, panicle length, seed size, and seed-setting rate. Whole-genome resequencing analysis revealed that a T-nucleotide insertion in β-ketoacyl-CoA synthase 11 (KCS11), responsible for the initial step in fatty acid elongation, was responsible for the observed defects in csd-1. The identity of csd-1 was confirmed through genetic complementation and CRISPR-Cas9-mediated knockout. Expression analysis indicated that OsKCS11 was present in various tissues, with differential abundance observed through RT-qPCR and promoter GUS staining, and strong localization at the node position by RNA in situ hybridization; furthermore, OsKCS11 protein was confirmed to be in the endoplasmic reticulum. Furthermore, csd-1 exhibited significantly reduced levels of linolenic acid (18:3), C24:0-OH, C28:0-alkanes, C29:0-alkanes, alpha-tocopherol, and C33:0-alkanes, while trans-nonadecenoic acid and behenic acid levels were increased. Cytokinin analysis revealed significant increases in isopentenyladenine (IPA) and cis-zeatin (cZ) levels in csd-1. Molecular investigations indicated upregulation of genes involved in cytokinin biosynthesis or signaling, suggesting a potential link between VLCFAs and cytokinin synthesis through acetyl-CoA. This study not only proposed an alternative gene mapping method based on whole-genome resequencing but also elucidated the mechanism by which VLCFAs influence cytokinin synthesis and signaling.}, }
@article {pmid40358185, year = {2025}, author = {Boff, MO and Xavier, FAC and Diz, FM and Gonçalves, JB and Ferreira, LM and Zambeli, J and Pazzin, DB and Previato, TTR and Erwig, HS and Gonçalves, JIB and Bruzzo, FTK and Marinowic, D and da Costa, JC and Zanirati, G}, title = {mTORopathies in Epilepsy and Neurodevelopmental Disorders: The Future of Therapeutics and the Role of Gene Editing.}, journal = {Cells}, volume = {14}, number = {9}, pages = {}, pmid = {40358185}, issn = {2073-4409}, support = {Not applicable//Pontifical Catholic University of Rio Grande do Sul (PUCRS) - Brain Institute of Rio Grande do Sul (BraIns)/ ; }, mesh = {Humans ; *Gene Editing/methods ; *Neurodevelopmental Disorders/therapy/genetics ; *TOR Serine-Threonine Kinases/metabolism/genetics ; *Epilepsy/therapy/genetics ; Animals ; Genetic Therapy ; CRISPR-Cas Systems ; }, abstract = {mTORopathies represent a group of neurodevelopmental disorders linked to dysregulated mTOR signaling, resulting in conditions such as tuberous sclerosis complex, focal cortical dysplasia, hemimegalencephaly, and Smith-Kingsmore Syndrome. These disorders often manifest with epilepsy, cognitive impairments, and, in some cases, structural brain anomalies. The mTOR pathway, a central regulator of cell growth and metabolism, plays a crucial role in brain development, where its hyperactivation leads to abnormal neuroplasticity, tumor formation, and heightened neuronal excitability. Current treatments primarily rely on mTOR inhibitors, such as rapamycin, which reduce seizure frequency and tumor size but fail to address underlying genetic causes. Advances in gene editing, particularly via CRISPR/Cas9, offer promising avenues for precision therapies targeting the genetic mutations driving mTORopathies. New delivery systems, including viral and non-viral vectors, aim to enhance the specificity and efficacy of these therapies, potentially transforming the management of these disorders. While gene editing holds curative potential, challenges remain concerning delivery, long-term safety, and ethical considerations. Continued research into mTOR mechanisms and innovative gene therapies may pave the way for transformative, personalized treatments for patients affected by these complex neurodevelopmental conditions.}, }
@article {pmid40356437, year = {2025}, author = {Kong, G and Li, R and Huang, W and Yang, Y and Guan, T and Liu, J and Li, W and Hsiang, T and Xi, P and Li, M and Jiang, Z}, title = {A RACK1 family protein regulates pathogenicity of Peronophythora litchii by acting as a scaffold for MAPK signal modules.}, journal = {Virulence}, volume = {16}, number = {1}, pages = {2503429}, pmid = {40356437}, issn = {2150-5608}, mesh = {*Plant Diseases/microbiology ; *Receptors for Activated C Kinase/metabolism/genetics ; *MAP Kinase Signaling System ; *Litchi/microbiology ; Virulence ; *Mitogen-Activated Protein Kinases/metabolism/genetics ; *Fungal Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Fungal ; }, abstract = {Litchi downy blight caused by Peronophythora litchii is the most destructive disease of litchi (Litchi chinensis). RACK1 (Receptor for activated C kinase 1) is a group of scaffold proteins, mainly involved in the regulation of various signaling pathways by interacting with signal transduction proteins and affecting the activity of these proteins. In this study, a RACK1 homologue identified in P. litchii, and named PlRACK1. The protein was found to interact with the mitogen-activated protein kinases, PlMAPK1 and PlMAPK2. CRISPR/Cas9-mediated genome editing technology was used to knock out PlRACK1, and we found that it was involved in mycelial growth, cell wall integrity, ROS metabolism, laccase activity, and pathogenicity of P. litchii. PlMAPK1 interacted with RACK1, and they jointly regulated sporangiophore branching of P. litchii. Transcriptome analysis showed that P. litchii MAPK Phosphatase 1 (PlMKP1) and beta-glucoside (PlBglX) were regulated by PlRACK1, both of which were also required for the pathogenicity of P. litchii. As well, PlMKP1 also interacted with PlMAPK1 and PlMAPK2. These results provide insights into the direct interactions between RACK1, MAPKs, and MKP, and their functions in growth, development, and pathogenesis in a plant pathogenic oomycete.}, }
@article {pmid40356298, year = {2025}, author = {Zubair, A and Sujan, A and Ali, M and Hussain, SM}, title = {Current Challenges With Highly Active Antiretroviral Therapy and New Hope and Horizon With CRISPR-CAS9 Technology for HIV Treatment.}, journal = {Chemical biology &amp; drug design}, volume = {105}, number = {5}, pages = {e70121}, doi = {10.1111/cbdd.70121}, pmid = {40356298}, issn = {1747-0285}, mesh = {Humans ; *CRISPR-Cas Systems ; *HIV Infections/drug therapy/genetics ; Gene Editing ; *Antiretroviral Therapy, Highly Active ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR/Cas system) is now the predominant approach for genome editing. Compared to conventional genetic editing methods, CRISPR/Cas technology offers several advantages that were previously unavailable. Key benefits include the ability to simultaneously modify multiple locations, reduced costs, enhanced efficiency, and a more user-friendly design. By directing Cas-mediated DNA cleavage to specific genomic targets and utilizing intrinsic DNA repair processes, this system can produce site-specific gene modifications. This goal is achieved through an RNA-guided procedure. As the most effective gene editing method currently available, the CRISPR/Cas system has proven to be highly valuable in genomic research across a wide range of species since its discovery as a component of the adaptive immune system in bacteria. Its applicability extends to various organisms, making it increasingly prevalent in the medical field, where it shows great promise in investigating viral infections, cancer, and genetic disorders. Furthermore, it enhances our understanding of fundamental genetics. This article outlines the current antiretroviral therapy and its adverse effects but also CRISPR/Cas technology. This review article also discusses its mechanism of action and potential applications in the treatment of HIV/AIDS.}, }
@article {pmid40356202, year = {2025}, author = {Butt, H and Mandava, M and Jacobsohn, D}, title = {Advances in Gene Therapy for Sickle Cell Disease: From Preclinical Innovations to Clinical Implementation and Access Challenges.}, journal = {The CRISPR journal}, volume = {8}, number = {3}, pages = {174-188}, doi = {10.1089/crispr.2024.0101}, pmid = {40356202}, issn = {2573-1602}, mesh = {*Anemia, Sickle Cell/therapy/genetics ; *Genetic Therapy/methods/trends ; Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; Animals ; beta-Globins/genetics ; Mutation ; }, abstract = {Sickle cell disease (SCD) is a hereditary blood disorder caused by a specific mutation in the β-globin gene, leading to the production of hemoglobin S, which deforms red blood cells, causing occlusion in small blood vessels. This results in pain, anemia, organ damage, infections, and increased stroke risk. Treatment options, including disease-modifying therapies and curative hematopoietic stem cell transplants, have limited accessibility. Recently, autologous gene therapy has emerged as a promising curative option, particularly for SCD. Gene editing techniques such as CRISPR, base editing, and prime editing offer potential to correct this mutation. In this review, we discuss recent preclinical studies and clinical trials of gene and cell therapies, focusing on the progress of FDA-approved treatments like Lyfgenia and Casgevy. We also examine the many challenges, including accessibility, safety, and long-term efficacy, which continue to shape the future of SCD gene therapy.}, }
@article {pmid40355755, year = {2025}, author = {Wolf, G and Leippe, P and Onstein, S and Goldmann, U and Frommelt, F and Teoh, ST and Girardi, E and Wiedmer, T and Superti-Furga, G}, title = {The genetic interaction map of the human solute carrier superfamily.}, journal = {Molecular systems biology}, volume = {21}, number = {6}, pages = {531-559}, pmid = {40355755}, issn = {1744-4292}, support = {777372//Innovative Medicines Initiative 2/ ; }, mesh = {Humans ; CRISPR-Cas Systems ; *Solute Carrier Proteins/genetics/metabolism ; Gene Knockout Techniques ; Mitochondrial Membrane Transport Proteins/genetics/metabolism ; Cation Transport Proteins/genetics/metabolism ; Cell Line, Tumor ; *Epistasis, Genetic ; Gene Regulatory Networks ; Mitochondrial Proteins ; Organic Anion Transporters ; }, abstract = {Solute carriers (SLCs), the largest superfamily of transporter proteins in humans with about 450 members, control the movement of molecules across membranes. A typical human cell expresses over 200 different SLCs, yet their collective influence on cell phenotypes is not well understood due to overlapping substrate specificities and expression patterns. To address this, we performed systematic pairwise gene double knockouts using CRISPR-Cas12a and -Cas9 in human colon carcinoma cells. A total of 1,088,605 guide combinations were used to interrogate 35,421 SLC-SLC and SLC-enzyme double knockout combinations across multiple growth conditions, uncovering 1236 genetic interactions with a growth phenotype. Further exploration of an interaction between the mitochondrial citrate/malate exchanger SLC25A1 and the zinc transporter SLC39A1 revealed an unexpected role for SLC39A1 in metabolic reprogramming and anti-apoptotic signaling. This full-scale genetic interaction map of human SLC transporters is the backbone for understanding the intricate functional network of SLCs in cellular systems and generates hypotheses for pharmacological target exploitation in cancer and other diseases. The results are available at https://re-solute.eu/resources/dashboards/genomics/ .}, }
@article {pmid40355359, year = {2025}, author = {Gao, YM and Chang, LX and Zhu, XF}, title = {[Advancements in CRISPR-Cas9 for Fanconi anemia].}, journal = {Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi}, volume = {46}, number = {3}, pages = {276-280}, pmid = {40355359}, issn = {0253-2727}, mesh = {*Fanconi Anemia/therapy/genetics ; Humans ; *CRISPR-Cas Systems ; *Gene Editing ; *Genetic Therapy ; }, abstract = {Fanconi anemia (FA) is a hereditary bone marrow failure syndrome that is characterized by genomic instability and heightened sensitivity to DNA cross-linking agents. In recent years, the CRISPR-Cas9 technology has exhibited groundbreaking progress in the field of gene therapy for FA. The traditional CRISPR-Cas9 technology has been successfully applied in FA gene editing. Further, single-base editing technology, based on the CRISPR/Cas9 system, performs precise and efficient gene repair for prevalent gene mutations in patients with FA. The prime editing technology provides new possibilities for gene editing; however, its application in FA has not been initiated. Despite significant advancements in FA gene editing technology, several challenges remain, including the collection of sufficient hematopoietic stem cells, the risk of increased tumorigenesis postgene editing, chromosomal instability, and off-target effects. Future research is recommended to focus on optimizing sgRNA and Cas9 nucleases, designing stricter PAM sequences to reduce off-target effects, and devising personalized gene editing strategies. Further, ethical and regulatory issues as well as long-term follow-ups are crucial priorities for future gene editing work. With continuous technological advancements and in-depth clinical trials, we expect more breakthroughs in FA treatment using the CRISPR-Cas9 technology in the future. This article reviews the latest research progress of CRISPR technology in FA treatment and analyzes the advantages and disadvantages of this technology in FA gene therapy.}, }
@article {pmid40354613, year = {2025}, author = {Zhao, S and Zhang, Q and Sun, J and Li, S and Wang, S and Zhou, D and Gong, X}, title = {Simply Engineered crRNA with CRISPR/Cas12a System Enables Wide-Scope Nucleic Acid Biomarker Analysis.}, journal = {Nano letters}, volume = {25}, number = {20}, pages = {8431-8441}, doi = {10.1021/acs.nanolett.5c01939}, pmid = {40354613}, issn = {1530-6992}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *MicroRNAs/genetics/analysis ; Point Mutation ; Animals ; *RNA/genetics/analysis ; Cell Line, Tumor ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {CRISPR/Cas12a systems have emerged as versatile tools for molecular diagnostics, but directly detecting RNA and identifying specific DNA point mutations remain challenging. Herein, we report a simple engineering approach with a split site in the spacer sequence, enabling activation of CRISPR/Cas12a (LbCas12a) for trans-cleavage with similar efficiency to wild-type crRNA. The engineered crRNA facilitated RNA target recognition by replacing the 3'-end with RNA fragments, enhancing point mutation specificity for ssDNA targets. Based on this, we achieved amplification-free detection of microRNAs and DNA point mutations with high sensitivity and specificity. For clinical sample validation, we constructed reverse fluorescence-enhanced lateral flow test strips (rLFTS), which achieved femtomole-level detection. Moreover, the engineered crRNA-based CRISPR/Cas12a system also effectively recognized tumor cells via intracellular and in vivo imaging of miRNA-21. In conclusion, this engineered crRNA platform enhances CRISPR/Cas12a-based nucleic acid detection, promoting its wide application in molecular diagnostics and bioimaging.}, }
@article {pmid40354233, year = {2025}, author = {Zhang, X and Duan, M and Zhao, Y and Zhang, K and Liu, F and Jie, J and Li, C and Chen, D and Li, D and Hua, S and Wang, C and Guan, Q and Wu, J and Liu, B and Song, L}, title = {Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {218}, pages = {}, doi = {10.3791/67542}, pmid = {40354233}, issn = {1940-087X}, mesh = {*Acinetobacter baumannii/isolation &amp; purification/genetics/enzymology ; *Nucleic Acid Amplification Techniques/methods ; *Acinetobacter Infections/diagnosis/microbiology ; *Recombinases/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; *Bacterial Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism/chemistry ; Humans ; DNA, Bacterial/genetics/analysis ; }, abstract = {Acinetobacter baumannii, a gram-negative bacterium, is notorious for causing severe infections with high mortality rates. Rapid and accurate detection of A. baumannii is crucial for prompt treatment, effective infection control, and curbing antibiotic resistance. However, there is no suitable method for rapid and easy on-site detection of A. baumannii. The DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) system offers a rapid, precise, and sensitive approach to A. baumannii detection by integrating the target-specific recognition capabilities of Cas12a with the isothermal amplification efficiency of Recombinase Polymerase Amplification (RPA). This protocol details the detection of A. baumannii using RPA combined with LbaCas12a endonuclease. The following steps are described in this article: extraction of DNA, selection of a specific DNA sequence, design of primer and CRISPR RNA (crRNA), construction of positive recombinant plasmid, setup of Cas12a-RPA assay, optimization of the RPA amplification system, visualization of the RPA-CRISPR/Cas12a assay using a fluorescence detection tool such as a real-time PCR instrument, and evaluation of sensitivity and specificity evaluation.}, }
@article {pmid40354126, year = {2025}, author = {Lv, B and Liang, P and Chang, C and Li, D}, title = {Sensitive aptasensing of tobramycin through a rational design of catalytic hairpin assembly and hybridization chain reaction amplification monomers for CRISPR/Cas12a activation.}, journal = {The Analyst}, volume = {150}, number = {12}, pages = {2580-2590}, doi = {10.1039/d5an00267b}, pmid = {40354126}, issn = {1364-5528}, mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; *Aptamers, Nucleotide/chemistry/genetics ; *Nucleic Acid Amplification Techniques/methods ; Milk/chemistry ; *Tobramycin/analysis ; Nucleic Acid Hybridization ; Animals ; *Anti-Bacterial Agents/analysis ; DNA/chemistry/genetics ; Inverted Repeat Sequences ; *CRISPR-Associated Proteins/metabolism/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {The catalytic hairpin assembly (CHA) and hybridization chain reaction amplification (HCR) are enzyme-free isothermal DNA amplification methods based on the self-assembly of hairpin monomers. Recently, CRISPR/Cas12a-based biosensors in combination with CHA or HCR signal amplification have shown promising performance. Herein, several design strategies for hairpin monomers in CHA and HCR were evaluated in the context of CRISPR/Cas12a-based biosensor construction. The SL-HCR strategy, in which the CRISPR/Cas12a target strand is blocked in the loop of one hairpin monomer DNA and released in the duplex HCR products, demonstrated superior performance in terms of a low background signal, wide linear detection range, and high signal-to-noise ratio. With the assistance of an aptamer-containing probe, a highly sensitive aptasensor was constructed for tobramycin detection, whereby the SL-HCR served the function of signal amplification, whereas the CRISPR/Cas12a system acted to cleave the FQ probes, thereby resulting in the production of a fluorescent signal. After optimization, the aptasensor enables linear detection of tobramycin concentrations ranging from 125 pM to 2500 nM, with a limit of detection (LOD) of 92.87 pM. Moreover, the aptasensor was utilized to detect tobramycin in beef and milk samples, yielding satisfactory results. The assay is concise and cost-effective due to the absence of nanomaterial DNA labeling and magnetic separation procedures. Furthermore, the entire detection workflow operates under isothermal conditions, which makes it suitable for use in food safety control and environmental monitoring. In addition, the results presented here may shed new light on the design of CRISPR/Cas12a-based biosensors in combination with CHA or HCR.}, }
@article {pmid40353913, year = {2025}, author = {Wu, G and Yin, C and Zheng, J and Wang, M and Abdalmegeed, D and Zhang, F and Sun, S and Sun, S and Shao, Y and Xin, Z}, title = {Dynamic regulation of iturin production via reconstructing the quorum-sensing system ComQXPA in Bacillus subtilis.}, journal = {World journal of microbiology &amp; biotechnology}, volume = {41}, number = {5}, pages = {173}, pmid = {40353913}, issn = {1573-0972}, mesh = {*Bacillus subtilis/genetics/metabolism ; *Quorum Sensing/genetics ; Gene Expression Regulation, Bacterial ; *Bacterial Proteins/genetics/metabolism ; Promoter Regions, Genetic ; *Peptides, Cyclic/biosynthesis/genetics/metabolism ; *Lipopeptides/biosynthesis/genetics/metabolism ; CRISPR-Cas Systems ; }, abstract = {Pheromone ComX is a critical element of the quorum-sensing (QS) system in Bacillus subtilis. It activates the surfactin promoter (Psrf) to initiate surfactin production in a cell-density-dependent manner. In this study, the natural promoter Pitu of B. subtilis 1A751 WR-itu, a lipopeptide iturin mono-producing parent strain, was replaced by the constitutive promoter P43, QS Psrf, and the mutated QS PM-srf, generating dynamic regulation systems to improve iturin production. HPLC analysis revealed that the PM-srf promoter-harbouring strain significantly enhanced iturin production to 409.33 ± 16.77 mg·L[- 1], 2.15 times higher than the parent strain. Further identification by UPLC-HRESI-MS/MS and GC-MS indicated that the strain could produce four new C10-C13 iturins. Moreover, the ComX degrading enzymes AprE or NprE were down-regulated by the CRISPR interference (CRISPRi) system, increasing iturin production to 526.46 ± 18.43 mg·L[- 1] and 416.99 ± 17.02 mg·L[- 1], respectively. Interestingly, iturin production was further increased to 579.85 ± 19.83 mg·L[- 1] under simultaneous down-regulation of AprE and NprE, 3.05 times higher than the parent strain. The reconstructed ComQXPA QS system in B. subtilis combines the inhibitory of AprE and NprE to dynamically up and down-regulate the expression of iturin operon, providing an effective pipeline for regulating other bioactive molecules.}, }
@article {pmid40353666, year = {2025}, author = {Lv, L and Luo, H and Yi, J and Zhang, K and Li, Y and Tong, W and Jiang, Y and Zhou, Y and Tong, G and Liu, C}, title = {IFITM proteins are key entry factors for porcine epidemic diarrhea coronavirus.}, journal = {Journal of virology}, volume = {99}, number = {6}, pages = {e0202824}, pmid = {40353666}, issn = {1098-5514}, support = {No. 2022YFD1800800//National Key Research and Development Program of China/ ; No. 21PJ1416300//Shanghai Pujiang Program/ ; No. 2019JB09//Central Public-interest Scientific Institution Basal Research Fund/ ; No. CAAS-ASTIP-2016-SHVRI-2004-1//Agricultural Science and Technology Innovation Program/ ; }, mesh = {Animals ; *Porcine epidemic diarrhea virus/physiology ; Swine ; Humans ; *Virus Internalization ; *Membrane Proteins/metabolism/genetics ; *RNA-Binding Proteins/metabolism/genetics ; *Coronavirus Infections/virology/metabolism/veterinary ; *Antigens, Differentiation/metabolism/genetics ; *Swine Diseases/virology/metabolism ; Cell Line ; Virus Replication ; Host-Pathogen Interactions ; CRISPR-Cas Systems ; }, abstract = {UNLABELLED: Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that poses a substantial threat to the global swine industry. However, our current understanding of the host factors crucial for PEDV infection remains limited. To identify these host factors, we conducted a genome-wide CRISPR/Cas9 gene knockout screen using a PEDV-permissive cell line. Our results indicate that the endogenous expression of human interferon-inducible transmembrane protein 3 (IFITM3) enhances PEDV entry and replication. Silencing or eliminating endogenous IFITM3 in Huh7 cells significantly suppressed PEDV entry, whereas reintroducing IFITM3 partially restored susceptibility to PEDV. Overexpression of human IFITM3 or IFITM2, but not IFITM1, in Huh7.5 cells substantially increased PEDV entry and replication. Importantly, our results suggest that human IFITM3 influences PEDV entry at a later stage. Furthermore, the overexpression of porcine IFITM1 significantly enhanced PEDV infection in LLC-PK1 cells, whereas the overexpression of porcine IFITM2/3 did not produce similar effects. Notably, removing the C-terminal 15 amino acids of porcine IFITM2/3 resulted in increased PEDV entry. Coimmunoprecipitation analyses showed that all IFITMs interacted with the PEDV S1 protein, indicating a direct role in the viral entry process. Additionally, porcine IFITM1 colocalized with the PEDV S protein at the cell nuclear periphery and enhanced PEDV infection in porcine small intestinal organoids. Overall, our results suggest that IFITMs are critical in facilitating PEDV entry into cells. Targeting IFITMs may provide a promising strategy for controlling PEDV transmission and developing interventions to mitigate the virus's impact on the swine industry.<br><br>IMPORTANCE: Understanding the mechanisms underlying porcine epidemic diarrhea virus (PEDV) infection is vital for addressing its significant impact on the swine industry. This study reveals that interferon-inducible transmembrane (IFITM) proteins, particularly human IFITM3 and porcine IFITM1, play crucial roles in facilitating PEDV entry and replication. By elucidating these molecular interactions, the research highlights the potential of IFITMs as therapeutic targets for managing PEDV infections and paves the way for antiviral strategies. Moreover, this research extends beyond PEDV management, underscoring the critical role of host factors in controlling the spread of pathogenic coronaviruses.}, }
@article {pmid40352342, year = {2025}, author = {Kaushik, A and Singh, J and Fatima, Z and Hameed, S}, title = {Establishment and evaluation of a naked-eye diagnostic assay for tuberculosis utilizing reverse isothermal amplification-assisted CRISPR-Cas in resource-limited settings.}, journal = {Drug target insights}, volume = {19}, number = {}, pages = {31-40}, pmid = {40352342}, issn = {1177-3928}, abstract = {INTRODUCTION: The current scenario of tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) has presented an almost insurmountable challenge to hospitals with high patient numbers. Delayed diagnosis of TB is a major hurdle in preventing the employment of efficient therapeutics, leading to the development of drug resistance. Hence, an easily accessible diagnostic method, particularly for resource for resource-limited settings, is pertinent for the rapid identification of MTB-infected patients. In pursuit of developing such an assay, the present study offers a CLAP-TB (CRISPR-Cas coupled RT-LAMP Amplification Protocol for Tuberculosis) assay, which will allow us to diagnose TB rapidly and visually.<br><br>METHODS AND RESULTS: Herein, the visual MTB detection consists of a method utilizing 232 different samples (sputum, urine, serum) from 82 patients for reverse transcription loop-mediated isothermal amplification (RT-LAMP). Additionally, the assay also utilizes the integration of a CRISPR-Cas12-based system using different guide RNAs of IS6110 and an internal control POP7 (human RNase P) genes along with visual detection via lateral flow readout-based dipsticks with the unaided eye (~134 min). Overall, the limit of detection for CLAP-TB assay was up to 1 ag of RNA, while the clinical sensitivity and specificity were 98.27% and 100%, respectively, on the pilot scale.<br><br>CONCLUSION: Together, our CLAP-TB assay offers proof of concept for a rapid, sensitive, and specific method with the minimum technical expertise required for TB diagnosis in developing and resource-limited settings.}, }
@article {pmid40352243, year = {2025}, author = {Prasetia, IG and Kurniati, NF and Riani, C and Mudhakir, D}, title = {Design of lipid nanoparticle (LNP) containing genetic material CRISPR/Cas9 for familial hypercholesterolemia.}, journal = {Narra J}, volume = {5}, number = {1}, pages = {e2217}, pmid = {40352243}, issn = {2807-2618}, mesh = {*Hyperlipoproteinemia Type II/therapy/genetics ; *Nanoparticles/chemistry ; *CRISPR-Cas Systems ; Humans ; Receptors, LDL/genetics ; *Lipids/chemistry ; *Genetic Therapy/methods ; Particle Size ; Liposomes ; }, abstract = {Familial hypercholesterolemia is a genetic disorder caused by mutations in the low- density lipoprotein receptor gene (LDLR) and the current treatment still focuses on symptom management. The aim of this study was to develop a lipid nanoparticle (LNP)- based delivery system for the CRISPR/Cas9 component in correcting LDLR gene mutations. LNPs were prepared using an ultrasonic-solvent emulsification technique by varying the surfactant: oil ratio (SOR), homogenization speed and time, and sonication time. Next, the LNP surface was modified by adding DSPE-PEG2000-NH2 and polyethyleneimine. The next stage is to design the single guide RNA (sgRNA) and Donor DNA wildtype (Donor DNA wt). This genetic material was complexed with LNP and then transfected into Hepa1-6 LDLR mt cells, an in vitro representation of cells suffering from familial hypercholesterolemia. This optimization process produced LNPs with a particle size of 118.6 ± 0.8 nm and a polydispersity index of 0.34 ± 0.03. The LNP surface modification resulted in a zeta potential of +7.5 mV. A transmission electron microscope (TEM) analysis showed spherical morphology with size distribution following a regular pattern. LNP cell viability tests showed good biocompatibility at concentrations <15 mM with a half-maximal inhibitory concentration (IC50) value of 27.7 mM. The dominant cellular uptake mechanism of LNP was through the clathrin-mediated endocytosis (CME) pathway. The Hepa1-6 LDLR mt cell model was successfully produced with the transfecting agent Lipofectamine 3000 by homology-directed repair (HDR) mechanism. The LNP-genetic material complex with a ratio of sgRNA:Cas9:Donor DNA wt (1:1:0.04) showed an increase in LDLR gene expression of 3.3 ± 0.2 times and LDLR protein levels reached 12.95 ± 0.25 ng/mL on day 4 after transfection. The results of this study indicate that the developed LNP-based delivery system has the potential for gene therapy applications in familial hypercholesterolemia.}, }
@article {pmid40351170, year = {2025}, author = {Zang, SS and Zhang, R and Zhang, JR and Zhang, X and Li, J}, title = {Progress, Applications and Prospects of CRISPR-Based Genome Editing Technology in Gene Therapy for Cancer and Sickle Cell Disease.}, journal = {Human gene therapy}, volume = {36}, number = {11-12}, pages = {858-869}, doi = {10.1089/hum.2024.262}, pmid = {40351170}, issn = {1557-7422}, mesh = {Humans ; *Anemia, Sickle Cell/therapy/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; *Neoplasms/therapy/genetics ; Animals ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {The advent of genome-editing technologies, particularly the RNA-guided the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) 9, which originates from prokaryotic CRISPR/Cas adaptive immune mechanisms, has revolutionized molecular biology. Renowned for its simplicity, cost-effectiveness, and capacity for multiplexed gene editing, CRISPR/Cas9 has emerged as the most versatile and widely adopted genome-editing platform. Its applications span fundamental research, biotechnology, medicine, and therapeutics. This review highlights recent advancements in CRISPR-based technologies, focusing on CRISPR/Cas9, CRISPR/Cas12a, and CRISPR/Cas12f. It emphasizes precision editing methods like base editing and prime editing, which enable targeted nucleotide changes without double-strand breaks. The specificity of these tools, including on-target accuracy and off-target risks, is critically evaluated. Additionally, recent preclinical and clinical efforts to treat diseases such as cancer and sickle cell disease using CRISPR are summarized. Finally, the challenges and future directions of CRISPR-mediated gene therapy are discussed, emphasizing its potential to integrate with other molecular approaches to address unmet medical needs.}, }
@article {pmid40350476, year = {2025}, author = {Kumari, G and Gupta, P and Goswami, SG and Jain, R and Anand, S and Biswas, S and Garg, S and Thakur, P and Saravanakumar, V and Arvinden, VR and Goswami, B and Bhowmick, IP and Mohandas, N and Burrows, J and Ramalingam, S and Singh, S}, title = {CRISPR/Cas9-engineering of Kell null erythrocytes to unveil host targeted irresistible antimalarial.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {730}, pmid = {40350476}, issn = {2399-3642}, support = {NER/84/2022-ECD-I//Indian Council of Medical Research (ICMR)/ ; }, mesh = {*Erythrocytes/parasitology/metabolism/drug effects ; *Plasmodium falciparum/drug effects/genetics ; *Antimalarials/pharmacology ; *CRISPR-Cas Systems ; Animals ; Humans ; *Malaria, Falciparum/drug therapy/parasitology ; Mice ; }, abstract = {Malaria elimination faces challenges from drug resistance, stemming from mutations within the parasite's genetic makeup. Genetic adaptations in key erythrocyte proteins offer malaria protection in endemic regions. Emulating nature's approach, and implementing methodologies to render indispensable host proteins inactive, holds the potential to reshape antimalarial therapy. This study delves into the functional implication of the single-span membrane protein Kell ectodomain, which shares consensus sequence with the zinc endopeptidase family, possesses extracellular enzyme activity crucial for parasite invasion into host erythrocytes. Through generating Kell-null erythrocytes from an erythroid progenitor, BEL-A, we demonstrate the indispensable nature of Kell activity in P. falciparum invasion. Additionally, thiorphan, a metallo-endopeptidase inhibitor, which specifically inhibits Kell activity, inhibited Plasmodium infection at nanomolar concentrations. Interestingly, individuals in malaria-endemic regions exhibit low Kell expression and activity, indicating a plausible Plasmodium-induced evolutionary pressure. Both thiorphan and its prodrug racecadotril, demonstrated potent antimalarial activity in vivo, highlighting Kell's protease role in invasion and proposing thiorphan as a promising host-oriented antimalarial therapeutic.}, }
@article {pmid40350377, year = {2025}, author = {Zhang, L and Zhao, D and Wei, Z and Zhu, X and Sha, T and Tang, W and Bi, C and Zhang, X}, title = {The Advancement of Prime Editing Technology.}, journal = {Chembiochem : a European journal of chemical biology}, volume = {26}, number = {12}, pages = {e202500193}, doi = {10.1002/cbic.202500193}, pmid = {40350377}, issn = {1439-7633}, support = {32225031//National Natural Science Foundation of China/ ; 32171449//National Natural Science Foundation of China/ ; 32271483//National Natural Science Foundation of China/ ; TSBICIP-KJGG-017//Tianjin Synthetic Biotechnology Innovation Capacity Improvement/ ; 2022177//the Youth Innovation Promotion Association CAS/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Protein Engineering ; }, abstract = {The advent of CRISPR/Cas genome editing has spurred major breakthroughs across life sciences, offering vast potential across numerous research and application fields. Among the expanding toolkit of CRISPR/Cas-derived methods, prime editing (PE) stands out for its versatility and specificity, enabling precise point mutations and small insertions or deletions without requiring double-stranded DNA breaks. Since its introduction, PE has undergone multiple rounds of optimization to improve performance. In this review, the core components and mechanism of prime editors, followed by four key evolution strategies: protein engineering, pegRNA modifications, accessory protein recruitment, and paired pegRNA approaches are outlined. Further persistent challenges and outline possible refinements are discussed, highlighting how further innovations can expand PE's utility across diverse areas of research, biotechnology, and potential therapeutic interventions.}, }
@article {pmid40349461, year = {2025}, author = {Li, W and Song, H and Cai, X and Wang, Y and Zhang, Y and Sun, C and Han, B and Zhang, J}, title = {Deletion of a novel upstream promoter of p53 impairs cold tolerance capacity in zebrafish.}, journal = {Biochemical and biophysical research communications}, volume = {769}, number = {}, pages = {151969}, doi = {10.1016/j.bbrc.2025.151969}, pmid = {40349461}, issn = {1090-2104}, mesh = {Animals ; *Zebrafish/genetics/physiology ; *Promoter Regions, Genetic/genetics ; *Tumor Suppressor Protein p53/genetics ; *Zebrafish Proteins/genetics/metabolism ; *Cold-Shock Response/genetics ; Oxidative Stress/genetics ; Cold Temperature ; CRISPR-Cas Systems ; Reactive Oxygen Species/metabolism ; *Sequence Deletion ; }, abstract = {P53 is a tumor suppressor that plays a crucial role in stress responses. We previously identified a novel upstream p53 promoter in cold acclimated zebrafish cells, however the functional significance of this upstream promoter under cold stress in zebrafish remains unclear. We generated novel p53 promoter[-/-] zebrafish using CRISPR/Cas9 and characterized their responses to cold stress. While novel p53 promoter[-/-] zebrafish exhibited normal development, growth, and locomotion at 28 °C, they showed enhanced locomotor capacity at 18 °C and reduced cold tolerance capacity at 8 °C. RNA-seq revealed the expression of genes related to oxidative stress was downregulated at 8 °C in the gill of novel p53 promoter[-/-] zebrafish, compared with WT zebrafish. Further experiments confirmed that the ROS levels increased, and the expression of SOD and GSH-PX reduced under cold stress in novel p53 promoter[-/-] zebrafish, compared with WT zebrafish. The novel p53 promoter plays a critical role in maintaining antioxidant defense and low-temperature tolerance in zebrafish. Our findings provide new theoretical insights into the environmental adaptation mechanisms of fish and offer experimental evidence for the regulatory role of the p53 gene in response to low-temperature stress.}, }
@article {pmid40348775, year = {2025}, author = {Jalan, M and Brambati, A and Shah, H and McDermott, N and Patel, J and Zhu, Y and Doymaz, A and Wu, J and Anderson, KS and Gazzo, A and Pareja, F and Yamaguchi, TN and Vougiouklakis, T and Ahmed-Seghir, S and Steinberg, P and Neiman-Golden, A and Azeroglu, B and Gomez-Aguilar, J and da Silva, EM and Hussain, S and Higginson, D and Boutros, PC and Riaz, N and Reis-Filho, JS and Powell, SN and Sfeir, A}, title = {RNA transcripts serve as a template for double-strand break repair in human cells.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {4349}, pmid = {40348775}, issn = {2041-1723}, support = {U01CA231019//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; U2C CA271894/CA/NCI NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; P30 CA016042/CA/NCI NIH HHS/United States ; R01 CA244729/CA/NCI NIH HHS/United States ; P50 CA247749/CA/NCI NIH HHS/United States ; U01 CA231019/CA/NCI NIH HHS/United States ; R01 CA229161/CA/NCI NIH HHS/United States ; }, mesh = {Humans ; *DNA Breaks, Double-Stranded ; *DNA Repair/genetics ; CRISPR-Cas Systems/genetics ; *RNA, Messenger/genetics/metabolism ; *RNA/genetics/metabolism ; DNA-Directed DNA Polymerase/metabolism/genetics ; Templates, Genetic ; Genomic Instability ; }, abstract = {Double-strand breaks (DSBs) are toxic lesions that lead to genome instability. While canonical DSB repair pathways typically operate independently of RNA, growing evidence suggests that RNA:DNA hybrids and nearby transcripts can influence repair outcomes. However, whether transcript RNA can directly serve as a template for DSB repair in human cells remains unclear. In this study, we develop fluorescence and sequencing-based assays to show that RNA-containing oligonucleotides and messenger RNA can serve as templates during DSB repair. We conduct a CRISPR/Cas9-based genetic screen to identify factors that promote RNA-templated DSB repair (RT-DSBR). Of the candidate polymerases, we identify DNA polymerase zeta (Polζ) as a potential reverse transcriptase that facilitates RT-DSBR. Furthermore, analysis of cancer genome sequencing data reveals whole intron deletions - a distinct genomic signature of RT-DSBR that occurs when spliced mRNA guides repair. Altogether, our findings highlight RT-DSBR as an alternative pathway for repairing DSBs in transcribed genes, with potential mutagenic consequences.}, }
@article {pmid40348052, year = {2025}, author = {Koike, A and Brindley, PJ}, title = {CRISPR/Cas genome editing, functional genomics, and diagnostics for parasitic helminths.}, journal = {International journal for parasitology}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.ijpara.2025.05.001}, pmid = {40348052}, issn = {1879-0135}, abstract = {Functional genomics using CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated endonuclease)-based approaches has revolutionized biomedical sciences. Gene editing is also widespread in parasitology generally and its use is increasing in studies on helminths including flatworm and roundworm parasites. Here, we survey the progress, specifically with experimental CRISPR-facilitated functional genomics to investigate helminth biology and pathogenesis, and also with the burgeoning use of CRISPR-based methods to assist in diagnosis of helminth infections. We also provide an historical timeline of the introduction and uses of CRISPR in helminth species to date.}, }
@article {pmid40347811, year = {2025}, author = {Wang, R and He, B and Liang, Z and Liu, Y and Yang, J and Jin, H and Wei, M and Ren, W and Suo, Z and Xu, Y}, title = {A dual-mode sensor for rapid detection of procymidone: "Dark box" qualitative analysis and electrochemical quantification mediated by PdHPCN-222/PEI-rGO and CRISPR/Cas12a.}, journal = {Food chemistry}, volume = {486}, number = {}, pages = {144586}, doi = {10.1016/j.foodchem.2025.144586}, pmid = {40347811}, issn = {1873-7072}, mesh = {*Electrochemical Techniques/methods/instrumentation ; *Biosensing Techniques/methods/instrumentation ; CRISPR-Cas Systems ; Graphite/chemistry ; Limit of Detection ; Polyethyleneimine/chemistry ; Fluorescence ; Food Contamination/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {This study presents an integrated dual-mode sensing strategy, in which qualitative fluorescence screening is followed by quantitative electrochemical detection, improving detection efficiency and enabling rapid PCM analysis. It develops a novel fluorescence electrochemical aptasensor that combines in vitro "Dark-box" applications with CRISPR/Cas12a system electrode surface sensing technology. PCM activates the DNA walker, and the DNAzyme induces cyclic cleavage of DNA strands bearing the Carboxyfluorescein (FMA) group. After magnetic separation, the fluorescence reaction combined with the "Dark box" enables the preliminary qualitative analysis of procymidone (PCM). Following the preliminary qualitative detection, the solution is introduced to the electrochemical aptasensor platform integrated with the CRISPR/Cas12 system. The Cas12a system triggers cyclic cleavage, producing a signal change that enables the electrochemical quantitative detection of PCM. An fluorescence (FL) response occurs when the PCM concentration in spiked samples is at or above 1 pg·mL[-1], allowing for qualitative fluorescence detection. The EC platform's detection limit is 8.51 × 10[-6] ng·mL[-1], with a range of 1 × 10[-2] ng·mL[-1] to 1 × 10[4] ng·mL[-1]. The designed dual-mode sensor provides reliable monitoring of PCM in real samples.}, }
@article {pmid40347696, year = {2025}, author = {Martin, OL and Lynch, CRH and Fleming, R}, title = {Advancing forensic body fluid identification: A comparative analysis of RT-LAMP+CRISPR-Cas12a and established mRNA-based methods.}, journal = {Forensic science international. Genetics}, volume = {78}, number = {}, pages = {103297}, doi = {10.1016/j.fsigen.2025.103297}, pmid = {40347696}, issn = {1878-0326}, mesh = {Humans ; *RNA, Messenger/genetics ; Female ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/genetics ; Sensitivity and Specificity ; Cervix Mucus/chemistry ; Male ; Forensic Genetics/methods ; Semen/chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {In forensic science, the analysis of body fluid evidence determines the cellular origin of a sample, aiding in the reconstruction of a potential crime. Messenger ribonucleic acid (mRNA) based confirmatory tests address limitations of current conventional methods, providing increased specificity and sensitivity, minimal sample consumption, and the detection of a broader range of body fluids. However, they require expensive instrumentation, longer reaction times, and lack portability. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) coupled with clustered regular interspaced short palindromic repeats (CRISPR) with CRISPR-associated protein 12a (Cas12a) has the potential to overcome these challenges. This approach offers reduced testing time and cost, while potentially providing equivalent sensitivity and specificity, as observed in the field of viral diagnostics. Visual detection capabilities enable the development of rapid, portable screening tests suitable for testing at the crime scene. In the context of a sexual assault investigation, RT-LAMP+CRISPR-Cas12a could potentially increase the efficiency and detection rate. This study compares this novel method to two other mRNA-based methods, endpoint reverse transcription polymerase chain reaction (RT-PCR) multiplex assay CellTyper 2, and a real-time reverse transcription quantitative PCR (RT-qPCR) multiplex assay. The tests' sensitivity and specificity were evaluated on single-source and mixed body fluid samples, including rectal mucosa, a fluid which is minimally explored in forensic literature. The RT-qPCR assay demonstrated the highest sensitivity, specificity, and precision in mixed samples. In addition, RT-qPCR offers a greater linear dynamic range, faster processing time and easier methodology compared to CellTyper 2, only limited by its expensive nature. Notably, rectal mucosa samples exhibited non-specific marker expression of CellTyper 2 markers and expression of CYP2B7P (vaginal fluid) for all methods. This emphasises the need for a dedicated rectal mucosa marker. RT-LAMP+CRISPR-Cas12a exhibited a high specificity, displaying off-target expression of CYP2B7P in two fluid types. However, the method lacked sensitivity and precision for most markers except MMP3 (menstrual blood), demonstrating detection down to 1:10,000 with 100 % specificity. RT-LAMP+CRISPR requires further development, but its quick, inexpensive nature and high specificity suggest it has potential as a confirmatory test that could reduce the limitations of existing methods.}, }
@article {pmid40347545, year = {2025}, author = {Wang, S and Guo, X and Yan, H and Zhou, T and Liu, J}, title = {Generation of an anti-CD5 CAR knock-in human induced pluripotent stem cell line using CRISPR/Cas9 technology.}, journal = {Stem cell research}, volume = {86}, number = {}, pages = {103731}, doi = {10.1016/j.scr.2025.103731}, pmid = {40347545}, issn = {1876-7753}, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques ; *CD5 Antigens/immunology/metabolism ; *Receptors, Chimeric Antigen/genetics/metabolism ; Cell Line ; Cell Differentiation ; }, abstract = {Theanti-CD5 chimeric antigen receptor (CAR)isa genetically engineered immune cell therapydevelopedto targetCD5-associatedhematologic malignancies,such asT-cell lymphoma and leukemia.UsingCRISPR/Cas9-mediated gene targeting, wegeneratedananti-CD5 CAR knock-in human induced pluripotent stem cell (iPSC) linethat stably expresses the CAR constructand is detectable via FLAG tag and GFP markers. Thisengineeredcell linemaintainsstem cell morphology,displaysa normal karyotype, andexhibits robustexpression ofpluripotency markers while retaining differentiation potential.}, }
@article {pmid40347261, year = {2025}, author = {Botelho, FBS and Nandy, S and Srivastava, V}, title = {CRISPR/Cas9-based modulation of V-PPase expression in rice improves grain quality and yield under high nighttime temperature.}, journal = {Plant cell reports}, volume = {44}, number = {6}, pages = {119}, pmid = {40347261}, issn = {1432-203X}, support = {1826836//Directorate for Biological Sciences/ ; 2023-11092//National Institute of Food and Agriculture/ ; }, mesh = {*Oryza/genetics/enzymology/growth &amp; development/physiology ; Gene Expression Regulation, Plant ; *Edible Grain/genetics/growth &amp; development ; *CRISPR-Cas Systems/genetics ; *Inorganic Pyrophosphatase/genetics/metabolism ; Plant Proteins/genetics/metabolism ; Hot Temperature ; Plants, Genetically Modified ; Endosperm/genetics ; }, abstract = {Transcriptional modulation of the vacuolar H[+] translocating pyrophosphatase expressed specifically in the endosperm and reproductive tissue of rice improves its spikelet fertility and reduces grain chalkiness under high nighttime temperature.}, }
@article {pmid40346697, year = {2025}, author = {Guo, T and Wang, H and Wu, F and Lu, W and Zhu, M and Ma, S and Zhang, Y and Yan, Y and Zhou, M and Talanaite, D and Liu, S and Qi, M and Lan, F and Liu, X}, title = {Functional analysis of JPH2-knockout cardiomyocytes identifies ECCD as a novel indicator in a human cardiac modelJPH2.}, journal = {Stem cell research &amp; therapy}, volume = {16}, number = {1}, pages = {234}, pmid = {40346697}, issn = {1757-6512}, support = {2021-RC310-12, 2022I2M-2-001 and 2023-I2M-1-003//CAMS Innovation Fund for Medical Sciences/ ; 2021YFC2701703, 2023YFA0915002//National Key Research and Devel opment Program of China/ ; 2022-GSP-GG-7//National High-Level Hospital Clinical Research Funding/ ; 2019PT320026//Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences/ ; ZDSYS20200923172000001//Shenzhen Fundamental Research Program/ ; 2024SKL-TJ001//Open Project Fund of State Key Laboratory of Cardiovascular Diseases&#xff0c;Shanghai East Hospital/ ; 2024T170070//China Postdoctoral Science Foundation/ ; 82400381//National Natural Science Foundation of China/ ; NCRCG-PLAGH-2024008//Open Project Fund of National Clinical Research Center for Geriatric Diseases, Chinese PLA General Hospital/ ; CXPY202304//Chinese PLA General Hospital Sixth medical center Innovation and Culture Grant/ ; }, mesh = {Humans ; *Myocytes, Cardiac/metabolism/cytology ; *Membrane Proteins/genetics/metabolism ; Calcium/metabolism ; Human Embryonic Stem Cells/metabolism/cytology ; *Excitation Contraction Coupling ; Myocardial Contraction ; Animals ; CRISPR-Cas Systems ; Muscle Proteins ; }, abstract = {BACKGROUND: Junctophilin-2 (JPH2) is a vital protein in cardiomyocytes, anchoring T-tubule and sarcoplasmic reticulum membranes to facilitate excitation-contraction coupling, a process essential for cardiac contractile function. Dysfunction of JPH2 is associated with cardiac disorders such as heart failure; however, prior studies using mouse models or primary human cardiomyocytes are limited by interspecies differences or poor cell viability, respectively. This study aimed to investigate JPH2's role in human cardiac function and disease using a novel stem cell-derived model, while introducing a new indicator to evaluate related cardiac impairments.<br><br>METHODS: We generated a JPH2-knockout model using human embryonic stem cell-derived cardiomyocytes (hESC-CMs) with CRISPR/Cas9. Cellular morphology, contractile function, calcium dynamics, and electrophysiological properties were assessed via transmission electron microscopy, the CardioExcyte96 system, calcium imaging with Fluo-4 AM, and multi-electrode array recordings, respectively. Wild-type JPH2 was overexpressed through lentiviral transfection to evaluate rescue effects, and two JPH2 variants-one benign (G505S) and one pathogenic (E85K)-were introduced to study mutation-specific effects.<br><br>RESULTS: JPH2 knockout disrupted excitation-contraction coupling in hESC-CMs by impairing junctional membrane complex structure, leading to heart failure-like phenotypes with reduced contractility, altered calcium dynamics, and electrophysiological irregularities. Overexpression of wild-type JPH2 restored these functions, affirming its critical role in cardiac physiology. We identified excitation-contraction coupling delay (ECCD) as a novel indicator that precisely quantified coupling impairment severity, with its applicability validated across distinct JPH2 variants (G505S and E85K).<br><br>CONCLUSIONS: This study demonstrates JPH2's essential role in sustaining excitation-contraction coupling by stabilizing the junctional membrane complex, with its deficiency driving heart failure-like cardiac dysfunction. ECCD is established as a sensitive, comprehensive indicator for assessing JPH2-related impairment severity. These findings advance our understanding of JPH2 in cardiac pathology and position ECCD as a valuable tool for research and potential clinical evaluation, with JPH2 and calcium regulation emerging as promising therapeutic targets.}, }
@article {pmid40346034, year = {2025}, author = {Villa, S and Jafri, Q and Lazzari-Dean, JR and Sangha, M and Olsson, N and Lefebvre, AEYT and Fitzgerald, ME and Jackson, K and Chen, Z and Feng, BY and Nile, AH and Stokoe, D and Bersuker, K}, title = {BiDAC-dependent degradation of plasma membrane proteins by the endolysosomal system.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {4345}, pmid = {40346034}, issn = {2041-1723}, mesh = {*Lysosomes/metabolism/drug effects ; Humans ; *Proteolysis/drug effects ; Ubiquitination/drug effects ; ErbB Receptors/metabolism ; *Membrane Proteins/metabolism ; *Cell Membrane/metabolism/drug effects ; Proteasome Endopeptidase Complex/metabolism ; Receptor, ErbB-2/metabolism/genetics ; HEK293 Cells ; Endocytosis/drug effects ; CRISPR-Cas Systems ; *Endosomes/metabolism ; Animals ; Protein Transport/drug effects ; }, abstract = {The discovery of bifunctional degradation activating compounds (BiDACs) has led to the development of a new class of drugs that promote the clearance of their protein targets. BiDAC-induced ubiquitination is generally believed to direct cytosolic and nuclear proteins to proteolytic destruction by proteasomes. However, pathways that govern the degradation of other classes of BiDAC targets, such as integral membrane and intraorganellar proteins, have not been investigated in depth. In this study we use morphological profiling and CRISPR/Cas9 genetic screens to investigate the mechanisms by which BiDACs induce the degradation of plasma membrane receptor tyrosine kinases (RTKs) EGFR and Her2. We find that BiDAC-dependent ubiquitination triggers the trafficking of RTKs from the plasma membrane to lysosomes for degradation. Notably, functional proteasomes are required for endocytosis of RTKs upstream of the lysosome. Additionally, our screen uncovers a non-canonical function of the lysosome-associated arginine/lysine transporter PQLC2 in EGFR degradation. Our data show that BiDACs can target proteins to proteolytic machinery other than the proteasome and motivate further investigation of mechanisms that govern the degradation of diverse classes of BiDAC targets.}, }
@article {pmid40345204, year = {2025}, author = {Wu, Y and Zhong, A and Evangelisti, A and Sidharta, M and Danwei, H and Studer, L and Zhou, T}, title = {Leveraging CRISPR activation for rapid assessment of gene editing products in human pluripotent stem cells.}, journal = {Stem cell reports}, volume = {20}, number = {6}, pages = {102499}, pmid = {40345204}, issn = {2213-6711}, mesh = {Humans ; *Gene Editing/methods ; *Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Transcriptional Activation ; DNA Methylation ; Cell Line ; }, abstract = {Verification of genome editing in human pluripotent stem cells (hPSCs), particularly at silent loci, is desirable but challenging, as it often requires complex and time-intensive differentiation to induce their expression. Here, we establish a rapid and effective workflow for verifying genome-edited hPSC lines targeting unexpressed genes using CRISPR-mediated transcriptional activation (CRISPRa). We systematically compared the efficiency of various CRISPRa systems and identified the synergistic activation mediator (SAM) system as the most potent for activating silent genes in hPSCs. Furthermore, combining SAM with TET1, a demethylation module, enhanced the activation of methylated genes. By inducing targeted gene activation in undifferentiated hPSCs using CRISPRa, we successfully verified single- and dual-reporter lines, functionally tested degradation tag (dTAG) knockins, and validated silent gene knockouts within 48 h. This approach bypasses the need to induce target gene expression through differentiation, providing a rapid and effective assay for verifying silent gene editing at the hPSC stage.}, }
@article {pmid40345045, year = {2025}, author = {Li, C and Peng, X and Zhang, Z and Liu, Y and Pedro, GC and Fu, C and Yang, Y and Dong, Q and Duan, Y and Sun, X}, title = {Establishment of Agrobacterium-mediated genetic transformation and CRISPR/Cas9-guided gene editing in Elymus nutans.}, journal = {Journal of plant physiology}, volume = {310}, number = {}, pages = {154513}, doi = {10.1016/j.jplph.2025.154513}, pmid = {40345045}, issn = {1618-1328}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Transformation, Genetic ; *Agrobacterium/genetics ; *Elymus/genetics/physiology ; Plants, Genetically Modified/genetics ; }, abstract = {Elymus nutans, an allohexaploid (2n = 6x = 42) species with a StStHHYY genome, is a native perennial in the alpine grasslands of the Qinghai-Xizang Plateau, and has been widely used for artificial pasture and ecological restoration as a forage grass with highest yield on the plateau. Nevertheless, the lack of a stable transformation system has impeded further efforts to trait improvement of E. nutans. In the present study, we established a reliable Agrobacterium-mediated genetic transformation system for E. nutans, and successfully generated EnTCP4-edited plants using the CRISPR/Cas9 system. The editing efficiency achieved 19.23 % in E. nutans. Knocking out EnTCP4 significantly delayed flowering and enhanced water-deficit stress resistance. This research represents a significant breakthrough in the genetic transformation and gene editing of E. nutans, laying a technological foundation to gain insight into gene functions and molecular breeding in E. nutans.}, }
@article {pmid40344932, year = {2025}, author = {Talanaite, D and Wang, H and Qi, M and Lan, F}, title = {Generation of a human embryonic stem cell line (WAe009-A-3B) carrying homozygous TNNT2 gene knockout by CRISPR/Cas9 editing.}, journal = {Stem cell research}, volume = {86}, number = {}, pages = {103729}, doi = {10.1016/j.scr.2025.103729}, pmid = {40344932}, issn = {1876-7753}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Troponin T/genetics/metabolism ; *Gene Knockout Techniques ; *Gene Editing ; Cell Line ; Homozygote ; }, abstract = {The TNNT2 gene encodes cardiac troponin T (cTnT), a critical protein in cardiac muscle contraction. Mutations in TNNT2 are associated with various cardiomyopathies, including hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM), which contribute to significant morbidity and mortality. In this study, we established a novel TNNT2 knockout human embryonic stem cell (hESC) line, WAe009-A-3B, utilizing the CRISPR/Cas9 genome editing system. This novel hESC line provides an important tool for investigating the molecular mechanisms underlying TNNT2-related cardiomyopathies and may serve as a promising in vitro model for the development of therapeutic strategies targeting TNNT2 mutations in cardiac diseases.}, }
@article {pmid40342201, year = {2025}, author = {Chulei, Y and Yiyuan, Z and Hong, T and Yanhua, G and Limin, W and Ping, Z}, title = {Analysis of Oestrus and Hormonal Changes in the Progeny of 2 Genotypes of FecB Gene-Edited Sheep.}, journal = {Reproduction in domestic animals = Zuchthygiene}, volume = {60}, number = {5}, pages = {e70038}, doi = {10.1111/rda.70038}, pmid = {40342201}, issn = {1439-0531}, support = {2023TSYCTD0021//the foundation Bingtuan Science and Technology Project/ ; NYHXGG2023AA101//Bingtuan Science and Technology Project/ ; NCG202221//Bingtuan Science and Technology Project/ ; }, mesh = {Animals ; Female ; *Estrus/genetics/physiology ; Genotype ; *Gene Editing/veterinary ; Ovarian Follicle ; Luteinizing Hormone/blood ; *Sheep, Domestic/genetics/physiology ; Follicle Stimulating Hormone/blood ; Estradiol/blood ; Sheep/genetics/physiology ; CRISPR-Cas Systems ; }, abstract = {The FecB gene, the first gene identified for multiple births, can improve reproductive performance in sheep. We used the CRISPR/Cas9 editing system to obtain the FecB gene in Kazakh sheep and mated with unedited Kazakh sheep. After the genotypic screening of the progeny, four B+ and ++ Kazakh ewes, each 2 weeks old and healthy, and close to the same body weight, were selected to study oestrus and hormonal changes in the progeny population of FecB gene-edited Kazakh sheep. Our results showed that the FecB gene was moderately polymorphic (0.25 < PIC < 0.5) in the progeny population, was able to be stably inherited, and showed increased lambing numbers. The number of follicles and tiny follicles differed significantly (p < 0.05) between the 2 genotypes of ewes in the progeny population. Physiologically, FecB gene-edited sheep did not differ from wild-type sheep. Throughout the oestrous cycle, FSH, LH, and E2 in serum hormones of both sheep genotypes showed variability (p < 0.05) between 72-360 h, 360-528 h, and 12-72 h, respectively, compared to other periods. The concentration of FSH and PROG in follicular fluid was much higher than that of LH and E2 (p < 0.05). Overall, it is possible to make Kazakh sheep carry the FecB gene by CRISPR/Cas9 editing and stably inherit it into the progeny population. The enhancement of reproductive hormones by the FecB gene is beneficial to follicular development and ovulation and improves the reproductive efficiency of sheep.}, }
@article {pmid40342053, year = {2025}, author = {Allemailem, KS and Rahmani, AH and Almansour, NM and Aldakheel, FM and Albalawi, GM and Albalawi, GM and Khan, AA}, title = {Current updates on the structural and functional aspects of the CRISPR/Cas13 system for RNA targeting and editing: A next‑generation tool for cancer management (Review).}, journal = {International journal of oncology}, volume = {66}, number = {5}, pages = {}, pmid = {40342053}, issn = {1791-2423}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/genetics/therapy ; *Gene Editing/methods ; Animals ; Genetic Therapy/methods ; }, abstract = {For centuries, a competitive evolutionary race between prokaryotes and related phages or other mobile genetic elements has led to the diversification of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR‑associated sequence (Cas) genome‑editing systems. Among the different CRISPR/Cas systems, the CRISPR/Cas9 system has been widely studied for its precise DNA manipulation; however, due to certain limitations of direct DNA targeting, off‑target effects and delivery challenges, researchers are looking to perform transient knockdown of gene expression by targeting RNA. In this context, the more recently discovered type VI CRISPR/Cas13 system, a programmable single‑subunit RNA‑guided endonuclease system that has the capacity to target and edit any RNA sequence of interest, has emerged as a powerful platform to modulate gene expression outcomes. All the Cas13 effectors known so far possess two distinct ribonuclease activities. Pre‑CRISPR RNA processing is performed by one RNase activity, whereas the two higher eukaryotes and prokaryotes nucleotide‑binding domains provide the other RNase activity required for target RNA degradation. Recent innovative applications of the type VI CRISPR/Cas13 system in nucleic acid detection, viral interference, transcriptome engineering and RNA imaging hold great promise for disease management. This genome editing system can also be employed by the Specific High Sensitivity Enzymatic Reporter Unlocking platform to identify any tumor DNA. The discovery of this system has added a new dimension to targeting, tracking and editing circulating microRNA/RNA/DNA/cancer proteins for the management of cancer. However, there is still a lack of thorough understanding of the mechanisms underlying some of their functions. The present review summarizes the recent updates on the type VI CRISPR/Cas system in terms of its structural and mechanistic properties and some novel applications of this genome‑editing tool in cancer management. However, some issues, such as collateral degradation of bystander RNA, impose major limitations on its in vivo application. Furthermore, additional challenges and future prospects for this genome editing system are described in the present review.}, }
@article {pmid40341582, year = {2025}, author = {Dacquay, LC and Antoniou, P and Mentani, A and Selfjord, N and Mårtensson, H and Hsieh, PP and Mustfa, S and Thom, G and Wimberger, S and Firth, M and Akrap, N and Maresca, M and Peterka, M}, title = {Dual inhibition of DNA-PK and Polϴ boosts precision of diverse prime editing systems.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {4290}, pmid = {40341582}, issn = {2041-1723}, mesh = {*Gene Editing/methods ; *DNA-Activated Protein Kinase/antagonists &amp; inhibitors/metabolism/genetics ; Humans ; DNA Polymerase theta ; *DNA-Directed DNA Polymerase/metabolism/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; DNA Repair ; HEK293 Cells ; Animals ; }, abstract = {Prime editing is a genome engineering tool that allows installation of various small edits with high precision. However, prime editing efficiency and purity can vary widely across different edits, genomic targets, and cell types. Prime editing typically offers more precise editing outcomes compared to other genome editing methods such as homology-directed repair. However, it can still result in significant rates of unintended editing outcomes, such as indels or imprecise prime edits. This issue is particularly notable in systems utilizing a second nicking gRNA, such as PE3 and PE5, as well as in dual pegRNA systems and fully active nuclease systems such as PEn, which increase efficiency but compromise precision. In this work, we show that pharmacological inhibition of DNA-PK and Polϴ, two major mediators of mutagenic DNA repair pathways, improves precision of PEn, PE3, PE5, PE7, and dual pegRNA editing systems, including TwinPE, HOPE, and Bi-PE, across multiple genomic loci and edit types. We show that co-inhibition of DNA-PK and Polϴ mitigates both prime editing-unrelated indels and prime editing by-products such as template duplications. Moreover, in the case of PEn, this strategy also substantially improved the off-target editing profile. Together, our data establish small molecule modulation of DNA repair pathways as a general strategy to maximize the precision of diverse prime editing systems.}, }
@article {pmid40341025, year = {2025}, author = {Wang, M and Krueger, JB and Gilkey, AK and Stelljes, EM and Kluesner, MG and Pomeroy, EJ and Skeate, JG and Slipek, NJ and Lahr, WS and Claudio Vázquez, PN and Zhao, Y and Bell, JB and Clement, K and Eaton, EJ and Laoharawee, K and Chang, JW and Webber, BR and Moriarity, BS}, title = {Precision enhancement of CAR-NK cells through non-viral engineering and highly multiplexed base editing.}, journal = {Journal for immunotherapy of cancer}, volume = {13}, number = {5}, pages = {}, pmid = {40341025}, issn = {2051-1426}, support = {R01 AI161017/AI/NIAID NIH HHS/United States ; P30 CA077598/CA/NCI NIH HHS/United States ; R21 CA237789/CA/NCI NIH HHS/United States ; R21 AI163731/AI/NIAID NIH HHS/United States ; T32 HL007062/HL/NHLBI NIH HHS/United States ; U24 OD026641/OD/NIH HHS/United States ; P01 CA254849/CA/NCI NIH HHS/United States ; R01 AI146009/AI/NIAID NIH HHS/United States ; U54 CA268069/CA/NCI NIH HHS/United States ; P50 CA136393/CA/NCI NIH HHS/United States ; }, mesh = {Humans ; *Gene Editing/methods ; *Killer Cells, Natural/immunology/metabolism ; *Receptors, Chimeric Antigen/genetics/metabolism ; Animals ; Mice ; *Immunotherapy, Adoptive/methods ; CRISPR-Cas Systems ; }, abstract = {BACKGROUND: Natural killer (NK) cells' unique ability to kill transformed cells expressing stress ligands or lacking major histocompatibility complexes (MHC) has prompted their development for immunotherapy. However, NK cells have demonstrated only moderate responses against cancer in clinical trials.<br><br>METHODS: Advanced genome engineering may thus be used to unlock their full potential. Multiplex genome editing with CRISPR/Cas9 base editors (BEs) has been used to enhance T cell function and has already entered clinical trials but has not been reported in human NK cells. Here, we report the first application of BE in primary NK cells to achieve both loss-of-function and gain-of-function mutations.<br><br>RESULTS: We observed highly efficient single and multiplex base editing, resulting in significantly enhanced NK cell function in vitro and in vivo. Next, we combined multiplex BE with non-viral TcBuster transposon-based integration to generate interleukin-15 armored CD19 chimeric antigen receptor (CAR)-NK cells with significantly improved functionality in a highly suppressive model of Burkitt's lymphoma both in vitro and in vivo.<br><br>CONCLUSIONS: The use of concomitant non-viral transposon engineering with multiplex base editing thus represents a highly versatile and efficient platform to generate CAR-NK products for cell-based immunotherapy and affords the flexibility to tailor multiple gene edits to maximize the effectiveness of the therapy for the cancer type being treated.}, }
@article {pmid40340328, year = {2025}, author = {Liu, H and Yang, Y and Zhang, N and Hou, Y and Zhang, Z and Yu, X and Huang, Y and Yan, B and Zhou, H and Mu, M and Gao, Y and Guo, K and Yang, M and Sun, X}, title = {Overcoming Photothermal Resistance of Gastric Cancer by Bionic 2D Iron-Based Nanoplatforms with Precise CRISPR/Cas9 Delivery.}, journal = {ACS nano}, volume = {19}, number = {19}, pages = {18188-18202}, doi = {10.1021/acsnano.4c16846}, pmid = {40340328}, issn = {1936-086X}, mesh = {*CRISPR-Cas Systems ; Humans ; Animals ; *Stomach Neoplasms/therapy/pathology/diagnostic imaging ; *Iron/chemistry/pharmacology ; Mice ; *Photothermal Therapy ; Ferroptosis/drug effects ; Cell Line, Tumor ; *Nanoparticles/chemistry ; Mice, Inbred BALB C ; Mice, Nude ; }, abstract = {The development of new CRISPR/Cas9 delivery systems with a synergistic therapeutic mode can provide a new perspective for efficient tumor treatment. In this work, we developed a bionic 2D FeS nanoplatform with high CRISPR/Cas9 loading (FCRM), highlighting the synergy of CRISPR/Cas9 and ferroptosis in regulating heat shock proteins (HSPs) for enhanced tumor photothermal therapy (PTT). Due to the ultrathin structure and pH-response of FeS nanosheets, FCRM can quickly decompose in a tumor microenvironment and effectively release CRISPR/Cas9 and Fe[2+], which can be further enhanced by a photothermal process. CRISPR/Cas9 can accurately downregulate the level of intracellular antiapoptosis protein Survivin. Fe[2+] can induce lipid peroxidation and ferroptosis of tumor cells. Ferroptosis and the regulation of Survivin protein can synergistically downregulate the expression of HSPs, thereby reducing the photothermal resistance of tumor cells in PTT. Additionally, FCRM caused significant tumor magnetic resonance contrast enhancement, which aided in the accurate diagnosis of tumors. Therefore, FCRM has great potential in achieving targeted magnetic resonance imaging and dual regulation of HSPs for ferroptosis-gene enhanced tumor PTT.}, }
@article {pmid40339766, year = {2025}, author = {Gao, L and Yuan, J and Hong, K and Ma, NL and Liu, S and Wu, X}, title = {Technological advancement spurs Komagataella phaffii as a next-generation platform for sustainable biomanufacturing.}, journal = {Biotechnology advances}, volume = {82}, number = {}, pages = {108593}, doi = {10.1016/j.biotechadv.2025.108593}, pmid = {40339766}, issn = {1873-1899}, mesh = {*Saccharomycetales/genetics/metabolism ; *Metabolic Engineering/methods ; Gene Editing ; CRISPR-Cas Systems ; Methanol/metabolism ; *Biotechnology/methods ; }, abstract = {Biomanufacturing stands as a cornerstone of sustainable industrial development, necessitating a shift toward non-food carbon feedstocks to alleviate agricultural resource competition and advance a circular bioeconomy. Methanol, a renewable one‑carbon substrate, has emerged as a pivotal candidate due to its abundance, cost-effectiveness, and high reduction potential, further bolstered by breakthroughs in CO2 hydrogenation-based synthesis. Capitalizing on this momentum, the methylotrophic yeast Komagataella phaffii has undergone transformative technological upgrades, evolving from a conventional protein expression workhorse into an intelligent bioproduction chassis. This paradigm shift is fundamentally driven by converging innovations across CRISPR-empowered advancement in genome editing and AI-powered metabolic pathway design in K. phaffii. The integration of CRISPR systems with droplet microfluidics high-throughput screening has redefined strain engineering efficiency, achieving much higher editing precision than traditional homologous recombination while compressing the "design-build-test-learn" cycle. Concurrently, machine learning-enhanced genome-scale metabolic models facilitate dynamic flux balancing, enabling simultaneous improvements in product titers, carbon yields, and volumetric productivity. Finally, technological advancement promotes the application of K. phaffii, including directing more efficiently metabolic flux toward nutrient products, and strengthening efficient synthesis of excreted proteins. As DNA synthesis automation and robotic experimentation platforms mature, next-generation breakthroughs in genome modification, cofactor engineering, and AI-guided autonomous evolution will further cement K. phaffii as a next-generation platform for decarbonizing global manufacturing paradigms. This technological trajectory positions methanol-based biomanufacturing as a cornerstone of the low-carbon circular economy.}, }
@article {pmid40338137, year = {2025}, author = {Wang, X and Yang, S and Qian, F and Dong, F and Zhou, X and Yin, M and Zhang, Y and Huang, Z and Jiang, Y and Yang, S}, title = {RAGATH-Associated DNA Nuclease Assisted DNA Insertion in Corynebacterium glutamicum.}, journal = {ACS synthetic biology}, volume = {14}, number = {5}, pages = {1861-1867}, doi = {10.1021/acssynbio.5c00022}, pmid = {40338137}, issn = {2161-5063}, mesh = {*Corynebacterium glutamicum/genetics/metabolism ; Plasmids/genetics/metabolism ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Deoxyribonucleases/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; DNA, Bacterial/genetics ; }, abstract = {Corynebacterium glutamicum serves as a key microbial chassis for the industrial production of feed and food ingredients. While long DNA fragment insertion technologies have advanced strain engineering capabilities, previous approaches such as utilizing a chromosome-integrated Cas9-RecET system were constrained by a maximum insertion fragment size of 7.5 kb. Through systematic evaluation of Cas9, gRNA, and recombinase expression driven by five distinct promoters and their implementation on 1 or 2 plasmids with compatible replicons (resulting in a total of 17 combinations), we developed an optimized genome editing vector capable of inserting DNA fragments of up to 8.0 kb in C. glutamicum. Parallel implementation of the Cpf1 system also successfully achieved 8.0 kb of DNA insertions. However, the construction of plasmids carrying insertion sequences larger than 8.0 kb was hindered by the plasmid vector capacity. To address this limitation, we screened six smaller RAGATH-associated DNA nucleases, ultimately identifying two with high cleavage activity in C. glutamicum. These nucleases demonstrated superior editing efficiencies compared to both Cas9 and Cpf1, enabling the integration of DNA fragments up to 11.3 kb─surpassing previously reported size limitations for C. glutamicum. These RAGATH-associated DNA nuclease-based systems effectively overcome the previous size constraints for long fragment insertions, thereby advancing metabolic engineering and fundamental research applications.}, }
@article {pmid40335033, year = {2025}, author = {Bryson, JW}, title = {Array Assembler Provides Greatly Simplified crRNA Array Design for CRISPR Cas12 and Cas13 Variants.}, journal = {ACS synthetic biology}, volume = {14}, number = {5}, pages = {1868-1872}, pmid = {40335033}, issn = {2161-5063}, mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Associated Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {As newer CRISPR variants have emerged and corresponding toolkits have been developed, researchers can now readily target multiple genes simultaneously for knockout, activation, or repression alongside being able to bind or cleave mRNA. However, as larger multitargeting crRNA arrays are required for these experiments, the design process becomes more complicated, taking more time and increasing risks of errors being introduced. The Array Assembler seeks to address the critical bottleneck that emerges during longer crRNA array design by providing a highly user-friendly tool to process input crRNA spacer sequences into the oligos required for efficient assembly of the corresponding crRNA array. By enabling rapid and reliable design of oligos for efficient assembly of crRNA arrays from a user-defined list of crRNA spacer sequences this tool should prove useful for a wide range of laboratories employing genomic perturbations.}, }
@article {pmid40334730, year = {2025}, author = {Amato, P and Mikhalchenko, A and Mitalipov, S}, title = {The case for germline gene correction: state of the science.}, journal = {Fertility and sterility}, volume = {124}, number = {1}, pages = {22-29}, doi = {10.1016/j.fertnstert.2025.04.039}, pmid = {40334730}, issn = {1556-5653}, mesh = {Humans ; *Gene Editing/methods/ethics/trends ; *Germ Cells/metabolism ; *Germ-Line Mutation ; CRISPR-Cas Systems ; *Genetic Therapy/methods/ethics/trends ; Animals ; *Genetic Diseases, Inborn/genetics/therapy/prevention &amp; control ; }, abstract = {Germline gene editing refers to altering the DNA in the reproductive cells (gametes or embryos). Germline gene editing experiments in human embryos have primarily focused on correcting genetic mutations linked to inherited diseases. This technology has the potential to prevent genetic disease before birth and in future generations. Advances in CRISPR-Cas9 and other gene-editing tools have accelerated scientific progress, raising both promise and safety and ethical concerns. A translational pathway for human heritable genome editing will require an approach that integrates scientific validation, ethical oversight, regulatory frameworks, and public engagement.}, }
@article {pmid40334450, year = {2025}, author = {Zhang, X and Wang, X and Zhu, L and Zhu, J and Zheng, Q and Yuan, J and Xu, W and Cao, J}, title = {Target responsive-regulated CRISPR/Cas12a electrochemiluminescence sensing of salmonella typhimurium integrating ultrafine Pt NCs-anchored MXenes-boosted luminol/O2 system.}, journal = {Biosensors &amp; bioelectronics}, volume = {283}, number = {}, pages = {117558}, doi = {10.1016/j.bios.2025.117558}, pmid = {40334450}, issn = {1873-4235}, mesh = {*Salmonella typhimurium/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; Luminol/chemistry ; Luminescent Measurements/methods ; Platinum/chemistry ; Electrochemical Techniques/methods ; Oxygen/chemistry ; Metal Nanoparticles/chemistry ; Limit of Detection ; Humans ; Salmonella Infections/microbiology/diagnosis ; }, abstract = {Salmonella typhimurium (S. typhimurium), as common and highly pathogenic foodborne pathogen, poses a significant risk to public safety worldwide. The development of highly sensitive, rapid and on-site method for S. typhimurium analysis is urgently needed to prevent bacterial infections. Herein, we introduced a CRISPR/Cas12a-driven electrochemiluminescence (ECL) sensor based on luminol/O2 binary systems for S. typhimurium detection, employing ultrafine Pt nanoclusters-anchored 2D delaminated-MXenes (Pt NCs/D-MXenes) as the co-reactant accelerator. The ultrathin D-MXenes support regulates the size and dispersibility of Pt NCs and facilitates the full exposure of active sites, and synergistic interactions between D-MXenes and Pt NCs improves electrocatalytic properties toward the reduction of O2, which promotes the generation of ROS for boosting ECL emission. Using target responsive-regulated CRISPR/Cas12a system, the ECL sensor for S. typhimurium showed a broad concentration range from 10[1] to 10[6] CFU/mL and limit of detection of 6 CFU/mL, with satisfactory recoveries in spiked-actual samples.}, }
@article {pmid40334424, year = {2025}, author = {Baranova, SV and Zhdanova, PV and Pestryakov, PE and Chernonosov, AA and Koval, VV}, title = {Key thermodynamic characteristics of Cas9 and Cas12a endonucleases' cleavage of a DNA substrate containing a nucleotide mismatch in the region complementary to RNA.}, journal = {Biochemical and biophysical research communications}, volume = {768}, number = {}, pages = {151892}, doi = {10.1016/j.bbrc.2025.151892}, pmid = {40334424}, issn = {1090-2104}, mesh = {Thermodynamics ; *Base Pair Mismatch ; *CRISPR-Associated Proteins/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/chemistry ; *DNA/metabolism/chemistry/genetics ; CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/metabolism/chemistry ; Substrate Specificity ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Molecular Dynamics Simulation ; *Bacterial Proteins/metabolism/chemistry ; DNA Cleavage ; Kinetics ; *RNA/metabolism/chemistry/genetics ; }, abstract = {CRISPR-Cas9 and CRISPR-Cas12a are endonuclease systems widely used for genome editing, but their mechanisms of DNA cleavage, particularly in the presence of nucleotide mismatches, remain incompletely understood. This study deals with thermodynamic parameters governing the cleavage of DNA substrates-containing a mismatch in the region complementary to RNA-by Cas9 and Cas12a. Using a series of 55 bp DNA substrates with various mismatches, we investigated the cleavage efficiency, reaction kinetics, and thermodynamic stability of the Cas12a-crRNA complex and compared it with Cas9-sgRNA on the same substrates. Cas12a manifested strict specificity, with a mismatch leading to a significant reduction in cleavage efficiency or to nonspecific trans-cleavage, whereas Cas9 showed higher tolerance to each mismatch, especially in internal and distal regions. Thermodynamic calculations indicated that Cas12a-crRNA complexes are generally stabler with fully complementary DNA but are more destabilized by a mismatch than Cas9-sgRNA complexes are. Molecular dynamics simulations revealed that a mismatch causes greater structural destabilization in Cas12a than in Cas9, correlating with reduced cleavage efficiency. These findings highlight distinct mechanisms of mismatch recognition by Cas9 and Cas12a, provide insights into their enzymatic behavior, and inform the design of more precise genome-editing tools.}, }
@article {pmid40333994, year = {2025}, author = {Chen, H and LaFlamme, CW and Wang, YD and Blan, AW and Koehler, N and Mendonca Moraes, R and Olszewski, AR and Almanza Fuerte, EP and Bonkowski, ES and Bajpai, R and Lavado, A and Pruett-Miller, SM and Mefford, HC}, title = {Patient-derived models of UBA5-associated encephalopathy identify defects in neurodevelopment and highlight potential therapeutic avenues.}, journal = {Science translational medicine}, volume = {17}, number = {797}, pages = {eadn8417}, doi = {10.1126/scitranslmed.adn8417}, pmid = {40333994}, issn = {1946-6242}, mesh = {Humans ; Organoids/metabolism/pathology ; *Ubiquitin-Activating Enzymes/genetics/metabolism ; *Brain Diseases/genetics/pathology/therapy ; Endoplasmic Reticulum/metabolism ; *Models, Biological ; Male ; Female ; Unfolded Protein Response ; CRISPR-Cas Systems/genetics ; Neurons/metabolism/pathology ; }, abstract = {UBA5 encodes for the E1 enzyme of the UFMylation cascade, which plays an essential role in endoplasmic reticulum (ER) homeostasis. The clinical phenotypes of UBA5-associated encephalopathy include developmental delays, epilepsy, and intellectual disability. To date, there is no humanized neuronal model to study the cellular and molecular consequences of UBA5 pathogenic variants. We developed and characterized patient-derived cortical organoid cultures from two patients with compound heterozygous variants in UBA5. Both shared the same missense variant, which encodes a hypomorphic allele (p.A371T), along with a nonsense variant (p.G267* or p.A123fs*4). Single-cell RNA sequencing of 100-day organoids identified defects in GABAergic interneuron development. We demonstrated aberrant neuronal firing and reduction in size of patient-derived organoids. Mechanistically, we showed that ER homeostasis is perturbed along with an exacerbated unfolded protein response pathway in engineered U87-MG cells and patient-derived organoids expressing UBA5 pathogenic variants. We also assessed two potential therapeutic modalities that augmented UBA5 protein abundance to rescue aberrant molecular and cellular phenotypes. We assessed SINEUP, a long noncoding RNA that augments translation efficiency, and CRISPRa, a modified CRISPR-Cas9 approach to augment transcription efficiency to increase UBA5 protein production. Our study provides a humanized model that allows further investigations of UBA5 variants in the brain and highlights promising approaches to alleviate cellular aberrations for this rare, developmental disorder.}, }
@article {pmid40333840, year = {2025}, author = {Chang, J and Yang, X and Zhang, T and Sun, H and Cheng, H and Jia, Z and Li, Y and Ma, S and Sun, T and Cao, J}, title = {High-Throughput Screening to Identify Novel Compounds Affecting the Genome Editing Efficiency of CRISPR System.}, journal = {Molecules (Basel, Switzerland)}, volume = {30}, number = {8}, pages = {}, pmid = {40333840}, issn = {1420-3049}, support = {202203021212372//Applied Basic Research Program of Shanxi Province/ ; 82170523//National Natural Science Foundation of China/ ; }, mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/drug effects ; HEK293 Cells ; *High-Throughput Screening Assays/methods ; }, abstract = {Genome editing is a promising therapeutic strategy for genetic disorders by modifying the genome precisely, especially the CRISPR/Cas9 system. However, a major limitation of CRISPR/Cas9 in gene therapy is the biosafety issues caused by off-target effects. Compounds that can modulate the genome editing efficiency of the CRISPR/Cas9 system, especially those reducing the off-target effects, are potentially useful pharmacological tools for improving the effectiveness and safety of genome editing. Here, we performed high-throughput screening in HEK 293FT cells to discover compounds that decrease or increase the genome editing efficiency of the CRISPR/Cas9 system from 9930 compounds. After two rounds of screening, we identified that CP-724714, a ErbB2 (HER2) tyrosine kinase inhibitor, decreased the CRISPR/Cas9 efficiency and reduced the off-target effects by suppressing the efficiency of CRISPR/Cas9, and was thus named a CRISPR decelerator (or inhibitor), while Clofarabine, a DNA synthesis inhibitor, increased the efficiency of CRISPR/Cas9, and was named a CRISPR accelerator. We further identified four compounds (Tranilast, Cerulenin, Rosolic acid and Resveratrol) that affected the efficiency of single-strand annealing (SSA) repair. Among them, Tranilast, Cerulenin and Rosolic acid are potential SSA decelerators, while Resveratrol is a potential SSA accelerator. These identified compounds may be useful in optimizing mammalian genetic manipulation techniques.}, }
@article {pmid40333207, year = {2025}, author = {Ouyang, Y and Xia, Y and Tang, X and Qin, L and Xia, S}, title = {Trans-Kingdom sRNA Silencing in Sclerotinia sclerotiorum for Crop Fungal Disease Management.}, journal = {Pathogens (Basel, Switzerland)}, volume = {14}, number = {4}, pages = {}, pmid = {40333207}, issn = {2076-0817}, support = {31971836//the National Nature Science Foundation of China/ ; }, mesh = {*Ascomycota/genetics/pathogenicity ; *Plant Diseases/microbiology/prevention &amp; control ; *RNA Interference ; *Crops, Agricultural/microbiology ; Gene Silencing ; Disease Management ; *RNA, Fungal/genetics ; }, abstract = {Sclerotinia sclerotiorum is a globally widespread and vast destructive plant pathogenic fungus that causes significant yield losses in crops. Due to the lack of effective resistant germplasm resources, the control of diseases caused by S. sclerotiorum largely relies on chemical fungicides. However, excessive use of these chemicals not only causes environmental concerns but also leads to the increased development of resistance in S. sclerotiorum. In contrast, trans-kingdom sRNA silencing-based technologies, such as host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS), offer novel, effective, and environmentally friendly methods for the management of S. sclerotiorum infection. This review summarizes recent advances in the identification of S. sclerotiorum pathogenic genes, target gene selection, categories, and application of trans-kingdom RNA interference (RNAi) technologies targeting this pathogen. Although some challenges, including off-target effects and the efficiency of external sRNA uptake, exist, recent findings have proposed solutions for further improvement. Combined with the latest developments in CRISPR/Cas gene editing and other technologies, trans-kingdom RNAi has significant potential to become a crucial tool in the control of sclerotinia stem rot (SSR), mitigating the impact of S. sclerotiorum on crop production.}, }
@article {pmid40333073, year = {2025}, author = {Saenz-Garcia, JL and Souza-Melo, N and Miranda, JS and Borges, B and Pacheco-Lugo, LA and Quintero-Solano, JM and Moretti, N and Wheeler, R and Soares-Medeiros, LC and DaRocha, WD}, title = {Kharon Is Crucial for Trypanosoma cruzi Morphology but Does Not Impair In Vitro Infection.}, journal = {Pathogens (Basel, Switzerland)}, volume = {14}, number = {4}, pages = {}, pmid = {40333073}, issn = {2076-0817}, support = {2020221000012//Funda&#xe7;&#xe3;o Arauc&#xe1;ria/ ; 2018/09948-0//FAPESP/ ; 2020/07870-4//FAPESP/ ; 2022/03075-0//FAPESP/ ; }, mesh = {*Trypanosoma cruzi/genetics/metabolism/growth &amp; development ; *Protozoan Proteins/genetics/metabolism ; *Chagas Disease/parasitology ; Animals ; Cytoskeleton/metabolism ; CRISPR-Cas Systems ; }, abstract = {Chagas disease, caused by Trypanosoma cruzi, is a neglected tropical disease with few options for treatment and no available vaccine. Deletion mutants for live attenuated vaccines, particularly deletions of proteins related to the cytoskeleton, have been widely tested in related parasites but candidates have not been tested in T. cruzi. Kharon is one such protein, identified as being associated with the cytoskeleton in Leishmania and essential for amastigote replication. Here we investigated the T. cruzi Kharon ortholog (TcKharon) to test if it has orthologous function and thus potential in generating a live attenuated vaccine. In silico analysis predicted TcKharon to be an intrinsically disordered protein, consistent with its ortholog feature, and GFP fusion protein revealed that TcKharon is associated with the cytoskeleton of epimastigotes. CRISPR-Cas9-mediated gene disruption impaired epimastigote proliferation and cytokinesis, resulting in altered nucleus-to-kinetoplast ratios and pronounced morphological defects, particularly in the posterior cell region. Despite these abnormalities, TcKharon[-/-] mutants retained the ability to differentiate into metacyclic trypomastigotes and exhibited in vitro infection rates comparable to wild-type parasites. Our data show that TcKharon is crucial for cell morphology. However, in contrast to close related parasites, TcKharon is not essential for in vitro infectivity.}, }
@article {pmid40332816, year = {2025}, author = {Xu, Q and Wang, M and Zeng, J and Sun, H and Wei, X and Jiang, H and Shentu, X and Sun, D}, title = {CRISPR/Cas Technology in Insect Insecticide Resistance.}, journal = {Insects}, volume = {16}, number = {4}, pages = {}, pmid = {40332816}, issn = {2075-4450}, support = {32302354//National Natural Science Foundation of China/ ; LQ24C140002//Zhejiang Provincial Programs for Natural Science/ ; U21A20223//National Natural Science Foundation of China/ ; 2023SNJF034//Zhejiang Provincial Programs for Science and Technology Development/ ; "Pioneer" and "Leading Goose" R&amp;D Program of Zhejiang//"Pioneer" and "Leading Goose" R&amp;D Program of Zhejiang/ ; }, abstract = {Chemicals and biological insecticides play a crucial role as pest management strategies in modern agriculture and forestry. However, their excessive and unreasonable use inevitably leads to varying degrees of resistance among insect populations, which seriously affects the sustainability of insecticide use. One primary reason for this resistance is alterations or mutations in insect gene expression. One class of genes encodes proteins that serve as critical targets for insecticides to exert their toxic effects in insects, while another class of genes encodes proteins involved in the detoxification process of insecticides within insects. Reverse genetics has become a vital research tool for studying the molecular mechanisms underlying changes and mutations in these target genes and their impact on insect resistance. The advent of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and the CRISPR-associated gene Cas as gene-editing technologies has significantly advanced our understanding of how insects adapt to and resist insecticides. This article aims to provide a comprehensive and objective review of the progress made using the CRISPR/Cas system in various arthropods within the field of pest control.}, }
@article {pmid40332635, year = {2025}, author = {Mukhtiar, A and Ullah, S and Yang, B and Jiang, YQ}, title = {Unlocking genetic potential: a review of the role of CRISPR/Cas technologies in rapeseed improvement.}, journal = {Stress biology}, volume = {5}, number = {1}, pages = {31}, pmid = {40332635}, issn = {2731-0450}, abstract = {Rapeseed (Brassica napus L.) is a globally important oil crop, providing edible vegetable oil and other valuable sources for humans. Being an allotetraploid, rapeseed has a complex genome that has undergone whole-genome duplication, making molecular breeding rather difficult. Fortunately, clustered regularly interspacedshort palindromic repeat (CRISPR)/CRISPR-associated (Cas) technologies have emerged as a potent tool in plant breeding, providing unprecedented accuracy as well as effectiveness in genome editing. This review focuses on the application and progresses of CRISPR/Cas technologies in rapeseed. We discussed the principles and mechanisms of CRISPR/Cas systems focusing on their use in rapeseed improvement such as targeted gene knockout, gene editing and transcriptional regulation. Furthermore, we summarized the regulatory frameworks governing CRISPR-edited crops as well as the challenges and opportunities for their commercialization and adoption. The potential advantages of CRISPR-mediated traits in rapeseed such as increased yield, disease and stress resistance and oil quality are discussed along with biosafety and environmental implications. The purpose of this review is to provide insights into the transformative role of CRISPR/Cas technologies in rapeseed breeding and its potential to address global agricultural challenges while ensuring sustainable crop production.}, }
@article {pmid40332465, year = {2025}, author = {Zhang, RX and Zhang, YF and Yang, H and Zhang, XD and Yang, ZG and Li, BB and Sun, WH and Yang, Z and Liu, WT and Chen, KM}, title = {An Optimized Editing Approach for Wheat Genes by Improving sgRNA Design and Transformation Strategies.}, journal = {International journal of molecular sciences}, volume = {26}, number = {8}, pages = {}, pmid = {40332465}, issn = {1422-0067}, support = {32270372//National Natural Science Foundation of China/ ; }, mesh = {*Triticum/genetics ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; *Transformation, Genetic ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Biolistics ; Agrobacterium/genetics ; }, abstract = {Hexaploid wheat has a large genome, making it difficult for transgenes to produce phenotypes due to gene redundancy and tight linkage among genes. Multiple gene copies typically necessitate multiple targeting events during gene editing, followed by several generations of self-crossing to achieve homozygous genotypes. The high cost of transgenesis in wheat is another issue, which hinders the easy availability of gene-edited materials in wheat. In this study, we developed a comprehensive approach to improve wheat gene editing efficiency. First, we established a protoplast-based system to evaluate the relative efficiency of gene editing targets, which enabled the rapid and effective selection of optimal sgRNAs. We then compared two transformation strategies: biolistic bombardment and Agrobacterium-mediated transformation for generating edited wheat lines. Although biolistic bombardment showed higher initial editing efficiency, Agrobacterium-mediated transformation proved more effective for obtaining homozygous mutants. Notably, we discovered that deploying the same sgRNA through different vectors enhanced editing efficiency, whereas overlapping but distinct sgRNAs exhibited interference effects. Finally, we optimized the VITF-edit (virus-induced transgene free editing) technique using BSMV delivery to establish a relatively simple and easily applied wheat gene editing method for general laboratories.}, }
@article {pmid40332425, year = {2025}, author = {Guo, X and Wang, X and Wang, J and Ma, M and Ren, Q}, title = {Current Development of iPSC-Based Modeling in Neurodegenerative Diseases.}, journal = {International journal of molecular sciences}, volume = {26}, number = {8}, pages = {}, pmid = {40332425}, issn = {1422-0067}, support = {81901088//National Natural Science Foundation of China/ ; E2019050019//Hundred Talents Program of Hebei Province/ ; H2022206562//Hebei Provincial Natural Science Foundation Precision Medicine Joint Fund Cultivation Project/ ; NV20230011//Key laboratory of Neural and Vascular Biology, Ministry of Education of China/ ; }, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Neurodegenerative Diseases/pathology/therapy/metabolism/genetics ; Animals ; Gene Editing ; Organoids/metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; *Models, Biological ; Neurons/metabolism ; Huntington Disease/pathology ; }, abstract = {Over the past two decades, significant advancements have been made in the induced pluripotent stem cell (iPSC) technology. These developments have enabled the broader application of iPSCs in neuroscience, improved our understanding of disease pathogenesis, and advanced the investigation of therapeutic targets and methods. Specifically, optimizations in reprogramming protocols, coupled with improved neuronal differentiation and maturation techniques, have greatly facilitated the generation of iPSC-derived neural cells. The integration of the cerebral organoid technology and CRISPR/Cas9 genome editing has further propelled the application of iPSCs in neurodegenerative diseases to a new stage. Patient-derived or CRISPR-edited cerebral neurons and organoids now serve as ideal disease models, contributing to our understanding of disease pathophysiology and identifying novel therapeutic targets and candidates. In this review, we examine the development of iPSC-based models in neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease.}, }
@article {pmid40332149, year = {2025}, author = {Navarro, C and Díaz, MP and Duran, P and Castro, A and Díaz, A and Cano, C and Carbonell-Zabaleta, AK and Solano-Jimenez, DS and Rivera-Porras, D and Contreras-Velásquez, JC and Bermúdez, V}, title = {CRISPR-Cas Systems: A Functional Perspective and Innovations.}, journal = {International journal of molecular sciences}, volume = {26}, number = {8}, pages = {}, pmid = {40332149}, issn = {1422-0067}, mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Bacteria/genetics/virology ; Archaea/genetics ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Adaptation is a fundamental tenet of evolutionary biology and is essential for the survival of all organisms, including prokaryotes. The evolution of clustered regularity exemplifies this principle of interspaced short palindromic repeats (CRISPR) and associated proteins (Cas), an adaptive immune system that confers resistance to viral infections. By integrating short segments of viral genomes into their own, bacteria and archaea develop a molecular memory that enables them to mount a rapid and targeted response upon subsequent viral challenges. The fortuitous discovery of this immune mechanism prompted many studies and introduced researchers to novel tools that could potentially be developed from CRISPR-Cas and become clinically relevant as biotechnology rapidly advances in this area. Thus, a deeper understanding of the underpinnings of CRISPR-Cas and its possible therapeutic applications is required. This review analyses the mechanism of action of the CRISPR-Cas systems in detail and summarises the advances in developing biotechnological tools based on CRISPR, opening the field for further research.}, }
@article {pmid40331919, year = {2025}, author = {Xu, C and Zhang, Y and Zhu, X and Hua, D and Yang, L and Huang, X and Gao, H and Luo, A and Deng, R and Xia, X}, title = {Preamplification-Free Detection of Viable Microorganisms in Fermentation Using Tandem CRISPR Nuclease Probe.}, journal = {Journal of agricultural and food chemistry}, volume = {73}, number = {20}, pages = {12488-12496}, doi = {10.1021/acs.jafc.5c01068}, pmid = {40331919}, issn = {1520-5118}, mesh = {Fermentation ; CRISPR-Cas Systems ; *Fermented Foods/microbiology ; *Lactobacillus/genetics/metabolism/isolation &amp; purification ; *Bacillus/genetics/isolation &amp; purification/metabolism ; Bacterial Proteins/genetics/metabolism ; }, abstract = {Accurate detection of viable bacteria is crucial for evaluating and monitoring the fermentation process. However, the complexity of fermentation samples presents challenges to developing precise and rapid detection tools. Here, we present a Cas13a-Csm6 tandem nuclease probe capable of the one-pot detection of viable microorganisms during fermentation, eliminating the need for nucleic acid preamplification. The RNA-activated CRISPR-Cas13a generates cleavage substrates that serve as activators for the CRISPR/Cas III-A Csm6 system. Leveraging the high specificity and efficient amplification capacity of the CRISPR cascade, this nuclease probe can detect 1% of viable Lactobacillus and Bacillus, facilitating the monitoring of bacterial populations throughout fermentation. This approach completes detection within 30 min and improves sensitivity for bacterial profiling by 16-fold compared with using Cas13 alone. The Cas13a-Csm6 tandem nuclease probe offers a precise and rapid analytical tool for the on-site quality monitoring of fermented foods.}, }
@article {pmid40331901, year = {2025}, author = {Ai, Z and Wang, W and Li, X and Wang, X and Chen, J and Wu, J and Zhou, S}, title = {A CRISPR/Cas12a-coupled multiplexed amplification system for ultrasensitive detection of miRNA-155.}, journal = {Analytical methods : advancing methods and applications}, volume = {17}, number = {19}, pages = {4044-4050}, doi = {10.1039/d5ay00415b}, pmid = {40331901}, issn = {1759-9679}, mesh = {*MicroRNAs/genetics/analysis ; *CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; *Endodeoxyribonucleases/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; }, abstract = {miRNA plays an important role in gene regulation and can be an effective biomarker for disease diagnosis. Herein, a new miRNA detection platform based on the CRISPR/Cas12a-coupled multiplexed amplification system is developed. In this strategy, miRNA-155 acts as an intermediary to trigger the recombinase polymerase amplification (RPA). Due to the introduction of endonuclide recognition sites in the amplification template, the resulting double-stranded DNA (dsDNA) can in turn initiate a strand replacement reaction (SDA), generating a great deal of single-stranded DNA (ssDNA). The ssDNA can directly unlock the trans-cleavage activity of CRSIPR/Cas12a, and the process is independent of PAM sites. Subsequently, the activated Cas12a trans-cleaves nearby signaling molecules, outputting a fluorescence/visualization signal. This method achieves miRNA detection as low as 68.69 fM, with a linear range of 200 fM to 1 nM, and shows good selectivity and repeatability. Meanwhile, the target of 10 pM can be distinguished by the naked eye. Moreover, the proposed method can achieve miRNA-155 detection in complicated cell extracts. The excellent detection sensitivity is mainly due to the integration of two amplification techniques, while the CRISPR/Cas12a system enables fast and accurate visual detection. More importantly, the actual detection results are consistent with standard methods (RT-qPCR), indicating that the CRISPR/Cas12a-coupled multiplexed amplification system is reliable and has potential clinical application value.}, }
@article {pmid40331862, year = {2025}, author = {Anglada, T and Rodriguez-Muñoz, M and Pulido-Artola, N and Genescà, A}, title = {Engineering Chromosome Bridges Through CRISPR/Cas9 to Decipher the Impact of Intercentromeric Distance on Resolution Dynamics.}, journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology}, volume = {39}, number = {9}, pages = {e70599}, pmid = {40331862}, issn = {1530-6860}, support = {//Fundaci&#xf3; la Marat&#xf3; de TV3 (Fundaci&#xf3; la Marat&#xf3;)/ ; //Generalitat de Catalunya (Government of Catalonia)/ ; //Consejo de Seguridad Nuclear (CSN)/ ; }, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Mitosis/genetics ; *Chromatin/genetics/metabolism ; Cell Line ; Kinetochores/metabolism ; *Centromere/genetics ; }, abstract = {Resolution of chromosome bridges during mitosis is a critical yet incompletely understood process with implications for genomic stability and cancer development. In this study, we investigated the impact of the bridging chromatin length on the timing and mechanism of chromosome bridge resolution. Using CRISPR/Cas9 technology, we engineered chromosome bridges with precisely defined intercentromeric distances in human RPE-1 cells. Our study revealed a decline in the frequency of chromosome bridges as cells progressed from early anaphase to late telophase, indicating resolution during mitosis. Moreover, the longer the bridging chromatin length, the higher the frequency of chromosome bridges observed at the mitotic exit, demonstrating that the size of the bridge influences its resolution during mitosis. Additionally, the separation between the bridge kinetochores needed for bridge breakage was strongly dependent on the megabase length of the bridging chromatin, with longer chromosome bridges requiring greater separation for their resolution. Given that chromosome bridge resolution occurs in a concerted manner with spindle elongation and is influenced by the length of the bridging chromatin, we posit that the traction forces generated by microtubules attaching to dicentric chromosomes play a significant role in resolving chromosome bridges during mitosis. Our study underscores the intricate interplay between chromosome bridge geometry and mechanical forces in mitotic chromosome bridge resolution. Our model offers a valuable framework for future investigations into the molecular mechanisms underlying chromosome bridge resolution, with potential implications for cancer biology and genomic stability maintenance.}, }
@article {pmid40330464, year = {2025}, author = {Li, TF and Rothhaar, P and Lang, A and Grünvogel, O and Colasanti, O and Ugarte, SMO and Traut, J and Piras, A and Acosta-Rivero, N and Gonçalves Magalhães, V and Springer, E and Betz, A and Huang, HE and Park, J and Qiu, R and Gnouamozi, GE and Mehnert, AK and Thi, VLD and Urban, S and Muckenthaler, M and Schlesner, M and Wohlleber, D and Binder, M and Bartenschlager, R and Pichlmair, A and Lohmann, V}, title = {RBM39 shapes innate immunity by controlling the expression of key factors of the interferon response.}, journal = {Frontiers in immunology}, volume = {16}, number = {}, pages = {1568056}, pmid = {40330464}, issn = {1664-3224}, mesh = {*RNA-Binding Proteins/genetics/metabolism/immunology ; *Immunity, Innate/genetics ; Humans ; Toll-Like Receptor 3/metabolism/genetics/immunology ; Hepatocytes/immunology/metabolism ; Signal Transduction ; *Interferons/immunology/metabolism ; *Gene Expression Regulation ; CRISPR-Cas Systems ; Cell Line ; }, abstract = {BACKGROUND AND AIMS: The contribution of innate immunity to clearance of viral infections of the liver, in particular sensing via Toll-like receptor 3 (TLR3), is incompletely understood. We aimed to identify the factors contributing to the TLR3 response in hepatocytes via CRISPR/Cas9 screening.<br><br>METHODS: A genome-wide CRISPR/Cas9 screen on the TLR3 pathway was performed in two liver-derived cell lines, followed by siRNA knockdown validation. SiRNA knockdown and indisulam treatment were used to study the role of RNA-binding motif protein 39 (RBM39) in innate immunity upon poly(I:C) or cytokine treatment and viral infections. Transcriptome, proteome, and alternative splicing were studied via RNA sequencing and mass spectrometry upon depletion of RBM39.<br><br>RESULTS: Our CRISPR/Cas9 screen identified RBM39, which is highly expressed in hepatocytes, as an important regulator of the TLR3 pathway. Knockdown of RBM39 or treatment with indisulam, an aryl sulfonamide drug targeting RBM39 for proteasomal degradation, strongly reduced the induction of interferon-stimulated genes (ISGs) in response to double-stranded RNA (dsRNA) or viral infections. RNA sequencing (seq) and mass spectrometry identified that transcription and/or splicing of the key pathway components IRF3, RIG-I, and MDA5 were affected by RBM39 depletion, along with multiple other cellular processes identified previously. RBM39 knockdown further restrained type I and type III IFN pathways by reducing the expression of individual receptor subunits and STAT1/2. The function of RBM39 was furthermore not restricted to hepatocytes.<br><br>CONCLUSION: We identified RBM39 as a regulatory factor of cell intrinsic innate immune signaling. Depletion of RBM39 impaired TLR3, RIG-I/MDA5, and IFN responses by affecting the basal expression of key pathway components.}, }
@article {pmid40329823, year = {2025}, author = {Henkel, E and Li, Z and Uvehag, D and Schmierer, B and Henkel, M and Wermeling, F}, title = {Green Listed v2.0: A Web Application for Streamlined Design of Custom CRISPR Screens.}, journal = {The CRISPR journal}, volume = {8}, number = {3}, pages = {216-223}, doi = {10.1089/crispr.2025.0023}, pmid = {40329823}, issn = {2573-1602}, mesh = {*Software ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Internet ; *Gene Editing/methods ; Gene Library ; User-Computer Interface ; Humans ; }, abstract = {Custom CRISPR screens are powerful tools for rapid, hypothesis-driven discovery, but their design is often complex and time-consuming. Green Listed v2.0 simplifies this process with an intuitive workflow for designing custom CRISPR spacer libraries and supports downstream analysis for all users, irrespective of their computational experience. The web application features a user-friendly graphical interface freely accessible at https://greenlisted.cmm.se. Version 2.0 includes significant upgrades to the original 2016 version that were implemented based on user feedback. This includes a new gene synonym tool, expanded library options, optimized output lists, performance improvements, and linked scripts for the rational design of custom CRISPR screen gene sets.}, }
@article {pmid40329691, year = {2025}, author = {Su, R and Shen, G and Xiao, X and Zheng, Y and Liu, F and Chen, D}, title = {Generation of a Novel Inducible and Dermal Papilla-Specific Wif1-CreER Knock-In Mouse Line for Hair Follicle Research.}, journal = {Experimental dermatology}, volume = {34}, number = {5}, pages = {e70109}, doi = {10.1111/exd.70109}, pmid = {40329691}, issn = {1600-0625}, support = {//National Natural Science Foundation of China/ ; }, mesh = {Animals ; *Hair Follicle/cytology/metabolism ; Mice ; Gene Knock-In Techniques ; Integrases/genetics/metabolism ; *Adaptor Proteins, Signal Transducing/genetics ; Mice, Transgenic ; *Dermis/cytology/metabolism ; CRISPR-Cas Systems ; Tamoxifen/pharmacology ; }, abstract = {Dermal papilla (DP) cells are essential niche cells that regulate hair follicle development, cycling and regeneration. Despite the establishment of several DP cell mouse lines in prior research, these tools are limited by incomplete specificity and spatiotemporal control. The Wnt inhibitory factor 1 (Wif1) has been identified as a DP signature gene. To address the need for precise labelling and manipulation of DP cells, we developed a novel genetic tool-Wif1-CreER knock-in mice. Using CRISPR/Cas9-mediated homologous recombination, the CreERT2 sequences were inserted into the endogenous Wif1 locus, under the control of the native promoter. PCR and sequencing analysis confirmed the accurate insertion of the CreERT2 sequence. Crossing Wif1-CreER mice with a reporter line demonstrated efficient and specific Cre recombinase activity in DP cells during anagen, catagen and telogen upon tamoxifen treatment across hair types. Importantly, DP-restricted labelling was confirmed by immunofluorescence and colocalised with Crabp1 and alkaline phosphatase (AP)-staining activity, exhibiting minimal to negligible expression in other tissues. This innovative mouse model overcomes the limitations of current tools and provides a valuable resource for advancing our understanding of hair biology and developing targeted therapies for hair-related disorders, offering unprecedented precision in the manipulation of dermal papilla cells.}, }
@article {pmid40329173, year = {2025}, author = {Piché, LC and Bories, S and Liato, V and Paquet, VE and Saucier, L and Létourneau-Montminy, MP and Charette, SJ and Dubar, R and Labrie, SJ and Lagüe, P and Vincent, AT}, title = {Evolutionary responses of Escherichia coli to phage pressure: insights into mucoidy and colanic acid overexpression.}, journal = {BMC genomics}, volume = {26}, number = {1}, pages = {448}, pmid = {40329173}, issn = {1471-2164}, support = {IA120638//MAPAQ (Innov'Action)/ ; IA120638//MAPAQ (Innov'Action)/ ; RGPIN-2022-03321//Natural Sciences and Engineering Research Council of Canada/ ; }, mesh = {*Escherichia coli/virology/genetics/metabolism ; *Evolution, Molecular ; *Bacteriophages/physiology ; Escherichia coli Proteins/genetics/metabolism/chemistry ; *Coliphages/physiology ; Mutation ; Polysaccharides ; }, abstract = {BACKGROUND: Antibiotic resistance is a major issue affecting all spheres of human activity, including agriculture. One significant example is the Avian Pathogenic Escherichia coli (APEC), a bacterium that infects poultry and leads to substantial economic losses in the farming industry. As antibiotics lose efficacity, bacteriophages (phages) -viruses that specifically target bacteria-are emerging as a promising alternative to antibiotics for treating and preventing bacterial infections. However, bacteria can develop resistance to phages through various mechanisms. Studying the coevolution between a phage and its host bacterium is important to gain insight into the phage's potential as a therapeutic agent. This study investigates the evolutionary responses of an APEC strain and a laboratory E. coli strain to a commercial phage originally isolated from APEC.<br><br>RESULTS: In most cases, phage resistance resulted in a significant increase in mucoidy. Genomic analysis revealed that this resistance consistently correlated with amino acid changes, particularly in proteins involved in colanic acid production, such as YrfF. Further investigation of a mutation found in the YrfF protein demonstrated that this mutation altered the protein's structure and its interaction with the membrane. Transcriptomic analysis confirmed that the genes involved in colanic acid production were significantly overexpressed. Although the strains possessed a CRISPR-Cas system, it did not contribute to phage resistance.<br><br>CONCLUSIONS: This study suggests that specific amino acid changes in key proteins may be a mechanism employed by E. coli, including APEC, to defend against phage infections.}, }
@article {pmid40328743, year = {2025}, author = {Huang, F and Wang, Y and Zhang, X and Gao, W and Li, J and Yang, Y and Mo, H and Prince, E and Long, Y and Hu, J and Jiang, C and Kang, Y and Chen, Z and Hu, YC and Zeng, C and Yang, L and Chen, CW and Chen, J and Huang, H and Weng, H}, title = {m[6]A/IGF2BP3-driven serine biosynthesis fuels AML stemness and metabolic vulnerability.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {4214}, pmid = {40328743}, issn = {2041-1723}, support = {82270168//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {*Leukemia, Myeloid, Acute/metabolism/genetics/pathology ; Humans ; *Serine/biosynthesis/metabolism ; *RNA-Binding Proteins/metabolism/genetics ; Animals ; *Neoplastic Stem Cells/metabolism/pathology ; *Adenosine/analogs &amp; derivatives/metabolism ; Mice ; Cell Line, Tumor ; Glycine/metabolism ; Methyltransferases/metabolism/genetics ; CRISPR-Cas Systems ; RNA, Messenger/metabolism/genetics ; Gene Expression Regulation, Leukemic ; }, abstract = {Metabolic reprogramming of amino acids represents a vulnerability in cancer cells, yet the mechanisms underlying serine metabolism in acute myeloid leukemia (AML) and leukemia stem/initiating cells (LSCs/LICs) remain unclear. Here, we identify RNA N[6]-methyladenosine (m[6]A) modification as a key regulator of serine biosynthesis in AML. Using a CRISPR/Cas9 screen, we find that depletion of m[6]A regulators IGF2BP3 or METTL14 sensitizes AML cells to serine and glycine (SG) deprivation. IGF2BP3 recognizies m[6]A on mRNAs of key serine synthesis pathway (SSP) genes (e.g., ATF4, PHGDH, PSAT1), stabilizing these transcripts and sustaining serine production to meet the high metabolic demand of AML cells and LSCs/LICs. IGF2BP3 silencing combined with dietary SG restriction potently inhibits AML in vitro and in vivo, while its deletion spares normal hematopoiesis. Our findings reveal the critical role of m[6]A modification in the serine metabolic vulnerability of AML and highlight the IGF2BP3/m[6]A/SSP axis as a promising therapeutic target.}, }
@article {pmid40328729, year = {2025}, author = {Hermann, J and Borteçen, T and Kalis, R and Kowar, A and Pechincha, C and Vogt, V and Schneider, M and Helm, D and Krijgsveld, J and Loayza-Puch, F and Zuber, J and Palm, W}, title = {mTORC1 cooperates with tRNA wobble modification to sustain the protein synthesis machinery.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {4201}, pmid = {40328729}, issn = {2041-1723}, support = {Projektnummer 2024.086.1//Wilhelm Sander-Stiftung (Wilhelm Sander Foundation)/ ; ERC StG No. 759579//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; }, mesh = {*RNA, Transfer/metabolism ; Animals ; Humans ; Mice ; Cell Line, Tumor ; Gene Knockout Techniques ; *Mechanistic Target of Rapamycin Complex 1/antagonists &amp; inhibitors/metabolism ; *Protein Biosynthesis ; CRISPR-Cas Systems ; Codon/metabolism ; Uridine/metabolism ; Ribosomes/metabolism ; }, abstract = {Synthesizing the cellular proteome is a demanding process that is regulated by numerous signaling pathways and RNA modifications. How precisely these mechanisms control the protein synthesis machinery to generate specific proteome subsets remains unclear. Here, through genome-wide CRISPR screens we identify genes that enable mammalian cells to adapt to inactivation of the kinase mechanistic target of rapamycin complex 1 (mTORC1), the central driver of protein synthesis. When mTORC1 is inactive, enzymes that modify tRNAs at wobble uridines (U34-enzymes), Elongator and Ctu1/2, become critically essential for cell growth in vitro and in tumors. By integrating quantitative nascent proteomics, steady-state proteomics and ribosome profiling, we demonstrate that the loss of U34-enzymes particularly impairs the synthesis of ribosomal proteins. However, when mTORC1 is active, this biosynthetic defect only mildly affects steady-state protein abundance. By contrast, simultaneous suppression of mTORC1 and U34-enzymes depletes cells of ribosomal proteins, globally inhibiting translation. Thus, mTORC1 cooperates with tRNA U34-enzymes to sustain the protein synthesis machinery and support the high translational requirements of cell growth.}, }
@article {pmid40328723, year = {2025}, author = {Yang, X and Shi, J and Wang, H and Wang, L and Su, H and Chen, C and Zhao, C}, title = {[Construction of mouse podocyte clone-5 cell lines with Smad3 knockout by CRISPR/Cas9].}, journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology}, volume = {41}, number = {4}, pages = {1658-1670}, doi = {10.13345/j.cjb.240803}, pmid = {40328723}, issn = {1872-2075}, mesh = {Animals ; *Smad3 Protein/genetics ; *CRISPR-Cas Systems/genetics ; Mice ; *Podocytes/cytology/metabolism ; Transforming Growth Factor beta1/pharmacology ; Cell Line ; *Gene Knockout Techniques ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {This study established the mouse podocyte clone-5 (MPC5) with Smad3 knockout and studied the effect of transforming growth factor-beta 1 (TGF-β1) on the dedifferentiation of the MPC5 cells with Smad3 knockout, aiming to provide a cell tool for studying the role of Smad3 in mouse podocytes. The single-guide RNA (sgRNA) sequence targeting Smad3 was designed according to the principles of CRISPR/Cas9 design. The pX458-Smad3 vector was constructed and introduced into competent cells, and then the vector was extracted and used to transfect MPC5 cells. The successfully transfected cells were sorted by a flow cytometer. After single-cell clone expansion, PCR amplification of sequences adjacent to the edition site of Smad3 and sequencing were performed to identify potential cells with gene knockout. Western blotting was employed to verify the knockout efficiency of Smad3. Finally, the effect of Smad3 knockout on TGF-β1-induced dedifferentiation of MPC5 cells was analyzed by reverse transcription-polymerase chain reacting (RT-PCR), Western blotting, and the immunofluorescence method. The sgRNA was designed to target the fifth exon of Smad3. EGFP expression was observed 24 h after transfection of the pX458-Smad3 plasmid into MPC5 cells, with the transfection efficiency of 0.1% as determined by flow cytometry. From the transfected cells, 21 cell clones were obtained through flow cytometric sorting and single-cell clone expansion. PCR amplification and sequencing of the region around the sgRNA target site in Smad3 identified two cell clones with biallelic frameshift mutations. Western blotting results confirmed the absence of Smad3 expression in these clones, indicating successful establishment of the MPC5 cell line with Smad3 knockout. In normal MPC5 cells, TGF-β1 stimulation promoted the expression of fibrosis-related genes fibronectin and Col1a1 (collagen I) and inhibited the expression of the podocyte marker proteins synaptopodin and podocin, which suggested epithelial-mesenchymal transition and podocyte injury. However, in the two MPC5 cell lines with Smad3 knockout, TGF-β1-induced expression of epithelial-mesenchymal transition markers was significantly suppressed. The MPC5 cell lines with Smad3 knockout that were constructed by CRISPR/Cas9 provide a valuable cell model for functional studies of Smad3 protein and highlight the critical role of Smad3 in cell dedifferentiation.}, }
@article {pmid40328722, year = {2025}, author = {Cai, Q and Wang, M and Zhu, J and Wu, J}, title = {[A universal counter-selection strategy based on replacement of sgRNA expression cassettes targeting multi-copy genes].}, journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology}, volume = {41}, number = {4}, pages = {1649-1657}, doi = {10.13345/j.cjb.240505}, pmid = {40328722}, issn = {1872-2075}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Plasmids/genetics ; }, abstract = {Selection markers are essential tools in gene editing, the utility of such systems is inherently constrained by species-specific limitations, governed by divergent host genetic backgrounds and metabolic compatibility. To address this limitation, we leveraged the CRISPR/Cas9 system to develop a universal counter-selection tool. We designed and introduced an sgRNA expression cassettes as counter-selection markers, which directs the Cas9 protein to target and cleave genomic DNA, allowing for the selection of the strains where the sgRNA expression cassette has been replaced. Optimized to target multiple copy sites with sgRNA, this system significantly enhances cell lethality, boosting counter-selection efficiency to over 85.00%. This counter-selection tool is not limited to single strains and is suitable for various scenarios, including multi-copy plasmid assembly and plasmid editing, demonstrating broad application potential.}, }
@article {pmid40327602, year = {2025}, author = {Sharma, I and Hall, K and Moonah, S}, title = {CRISPR genome editing using a combined positive and negative selection system.}, journal = {PloS one}, volume = {20}, number = {5}, pages = {e0321881}, pmid = {40327602}, issn = {1932-6203}, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Polymorphism, Single Nucleotide ; *Selection, Genetic ; beta-Globins/genetics ; }, abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system is a powerful genome editing tool that has revolutionized research. Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation in humans. Only a subset of these SNPs has been shown to be linked to genetic diseases, while the biological relevance of the majority remains unclear. Investigating these variants of unknown significance could provide valuable insights into their roles in biological processes, disease susceptibility, and treatment responses. While CRISPR/Cas has emerged as a transformative technology, its ability to make single nucleotide substitutions remains a significant limitation. Other techniques in single nucleotide editing, such as base editing and prime editing, offer promising possibilities to complement CRISPR/Cas systems, though they also have their own limitations. Hence, alternative approaches are necessary to overcome the limitations of CRISPR. Here, to improve the feasibility of generating single base edits in the genome, we provide a protocol that introduces a multiple expression and dual selection (MEDS) system, which, alongside CRISPR, utilizes the opposing roles of cytosine deaminase/uracil phosphoribosyltransferase (CD/UPRT) for negative selection and neomycin phosphotransferase II (NPT II) for positive selection. As a proof of concept and to demonstrate feasibility of the method, we used MEDS, along with traditional CRISPR-Cas9, to generate sickle hemoglobin by introducing a point mutation (A → T) in the sixth codon of the hemoglobin beta gene.}, }
@article {pmid40327171, year = {2025}, author = {Yang, J and Li, W and Hu, Y and Han, Y and Lei, C and Wang, H}, title = {Establishment of a rapid RAA-CRISPR/Cas12a system targeting the recN gene for on-site detection of Streptococcus suis in livestock and fresh pork meat.}, journal = {Functional &amp; integrative genomics}, volume = {25}, number = {1}, pages = {99}, pmid = {40327171}, issn = {1438-7948}, support = {2025ZNSFSC0209//Natural Science Foundation of Sichuan Province/ ; U21A20257//National Natural Science Foundation of China/ ; 2021ZDZX0010//Sichuan Science and Technology Programs/ ; }, mesh = {*Streptococcus suis/genetics/isolation &amp; purification ; Animals ; *CRISPR-Cas Systems ; Swine ; *Livestock/microbiology ; *Bacterial Proteins/genetics ; *Pork Meat/microbiology ; *Nucleic Acid Amplification Techniques/methods ; }, abstract = {Streptococcus suis is a major bacterial pathogen in the swine industry, causing meningitis, arthritis, and other diseases in infected pigs. It also poses significant public health risks due to its zoonotic potential, particularly in individuals with skin lesions. Current detection methods, including traditional culture-based techniques and PCR assays, are time-consuming, labor-intensive, and lack sufficient accuracy. To address these limitations, this study aimed to develop a rapid and precise detection method for S. suis. By leveraging whole-genome sequencing (WGS) and multiple sequence alignment, the recN gene was identified as a highly specific molecular target. A novel isothermal detection method, integrating recombinase-aided amplification (RAA) with CRISPR/Cas12a, was subsequently established. This RAA-CRISPR/Cas12a-based system demonstrated superior sensitivity compared to conventional PCR (targeting the gdh gene), achieving detection within 30 min without requiring specialized equipment. This method achieves 2.44 × 10[1] copies/µL and 2.1 × 10[1] CFU sensitivity and 100% specificity within 30 min, outperforming conventional PCR in speed and reliability while eliminating dependency on specialized equipment. Designed for field applications, it offers a cost-effective (US$1/test), user-friendly solution for on-site S. suis detection in swine farms and fresh pork meat, enhancing outbreak control and preventive healthcare in the livestock industry.}, }
@article {pmid40326747, year = {2025}, author = {Chen, Y and Yu, K and Jiang, Z and Yang, G}, title = {CRISPR-based genetically modified scaffold-free biomaterials for tissue engineering and regenerative medicine.}, journal = {Biomaterials science}, volume = {13}, number = {12}, pages = {3149-3175}, doi = {10.1039/d5bm00194c}, pmid = {40326747}, issn = {2047-4849}, mesh = {*Tissue Engineering/methods ; Humans ; *Regenerative Medicine/methods ; *Biocompatible Materials/chemistry ; Animals ; Gene Editing ; *CRISPR-Cas Systems ; Tissue Scaffolds ; }, abstract = {CRISPR-based genetically modified scaffold-free biomaterials, including extracellular vehicles, cell sheets, cell aggregates, organoids and organs, have attracted significant attention in the fields of regenerative medicine and tissue engineering in recent years. With a wide range of applications in gene therapy, modeling disease, tissue regeneration, organ xenotransplantation, modeling organogenesis as well as gene and drug screening, they are at a critical juncture from clinical trials to therapeutic applications. Xenografts have already been tested on non-human primates and humans. However, we have to admit that a series of obstacles still need to be addressed, such as immune response, viral infection, off-target effects, difficulty in mass production, and ethical issues. Therefore, future research should pay more attention to improving their safety, accuracy of gene editing, flexibility of production, and ethical rationality. This review summarizes various types of CRISPR-based genetically modified scaffold-free biomaterials, including their preparation procedures, applications, and possible improvements.}, }
@article {pmid40326732, year = {2025}, author = {Li, Z and Li, C and Xiao, S and Liang, H}, title = {Efficient and Precise Integration of Large DNA Sequences Using Precise Interstrand Cross-Linking of Long ssDNA and sgRNA.}, journal = {ACS synthetic biology}, volume = {14}, number = {5}, pages = {1451-1463}, doi = {10.1021/acssynbio.4c00715}, pmid = {40326732}, issn = {2161-5063}, mesh = {Humans ; *DNA, Single-Stranded/genetics/chemistry/metabolism ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; HEK293 Cells ; CRISPR-Cas Systems/genetics ; Cross-Linking Reagents/chemistry ; Recombinational DNA Repair/genetics ; }, abstract = {Homology-directed repair (HDR) allows the precise introduction of functional constructs into the human genome through nonviral gene-editing reagents. However, its application in large DNA sequence gene editing remains limited due to challenges such as low efficiency and the off-target effect. To address these limitations, a new method named AOLP was developed to synthesize chemically modified long single-stranded DNA (lssDNA) as the template donor for Cas9-based gene editing, which has been proven to be more stable than that prepared using the commercial phosphorylation method. We propose a novel strategy involving precise ligation-based interstrand cross-linking between lssDNA and sgRNA using cyanovinylcarbazole nucleoside ([CNV]K), enhancing the upregulation of the HDR pathway for DSB repair induced by Cas9. The light-activated ligation between Cas9/sgRNA and lssDNA improves the knock-in (KI) efficiency, overcomes the challenges of low KI efficiency, and surpasses the low off-target effect accompanied by the lssDNA donor. Moreover, the interstrand cross-linking of lssDNA and sgRNA can subtly control the ligation sites and the degree of cross-linking of lssDNA and sgRNA to enhance the KI accuracy of HDR. Our approach improves the KI efficiency of lssDNA in K562, HEK293T, and HepG2 cells by 4- to 12-fold relative to conventional lssDNA donors prepared using the phosphorylation method. Furthermore, the KI accuracy of HDR pathway in HEK293T cells is enhanced by >4.7-fold relative to previous commercial lssDNA. Leveraging this approach, we achieved an unprecedented KI rate of approximately 36% for a gene-sized 1.4 kilobase lssDNA insertion in HEK293T cells.}, }
@article {pmid40326084, year = {2025}, author = {Barman, K and Goswami, P}, title = {Recent Advances in Diagnostics and Therapeutic Interventions for Drug-Resistant Malaria.}, journal = {ACS infectious diseases}, volume = {11}, number = {6}, pages = {1296-1332}, doi = {10.1021/acsinfecdis.4c00962}, pmid = {40326084}, issn = {2373-8227}, mesh = {Humans ; *Antimalarials/pharmacology/therapeutic use ; *Drug Resistance/genetics ; *Malaria/diagnosis/drug therapy/parasitology ; *Plasmodium/drug effects/genetics ; Animals ; Plasmodium falciparum/drug effects/genetics ; }, abstract = {The emergence of drug-resistant malarial parasites has been a growing challenge to medical science to safeguard public health in the malaria-endemic regions of the globe. With time, the parasite develops newer resistance mechanisms to defunct the drug's action one after another. Genetic mutation is the prime weapon parasites rely upon to initiate the resistance mechanism in a case-specific manner, following various strategies such as structural changes in the target protein, metabolic alterations, and tweaking the drug-transported channels. In order to combat these resistances, different approaches have evolved among these developing inhibitors against critical parasite enzymes and metabolic pathways, combinatorial/hybrid drug therapies, exploring new drug targets and analogues of existing drugs, use of resistance-reversal agents, drug-repurposing, gene blocking/altering using RNA interference and CRISPR/Cas systems are prominent. However, the effectiveness of these approaches needs to be earnestly monitored for better management of the disease, which demands the development of a reliable diagnosis technique. Several methodologies have been investigated in search of a suitable diagnosis technique, such as in vivo, in vitro, ex vivo drug efficacy studies, and molecular techniques. A parallel effort to transform the efficient method into an inexpensive and portable diagnosis tool for rapid screening of drug resistance malaria among masses in the societal landscape is advocated. This review gives an insight into the historical perspectives of drug-resistant malaria and the recent developments in malaria diagnosis and antimalarial drug discovery. Efforts have been made to update recent strategies formulated to combat and diagnose drug-resistant malaria. Finally, a concluding remark with a future perspective on the subject has been forwarded.}, }
@article {pmid40325455, year = {2025}, author = {Gao, Y and Zou, Y and Wu, C and Tao, J and Nie, Z and Yan, J and Wang, P and Huang, X}, title = {Comparative evaluation of immunomodulatory cytokines for oncolytic therapy based on a high-efficient platform for oHSV1 reconstruction.}, journal = {Virology journal}, volume = {22}, number = {1}, pages = {133}, pmid = {40325455}, issn = {1743-422X}, support = {Kunming Medical University graduate Student Innovation Fund(2024S086)//Yufang Zou/ ; National Natural Science Foundation of China (82260486)//Xinwei Huang/ ; }, mesh = {Animals ; *Oncolytic Virotherapy/methods ; *Oncolytic Viruses/genetics/immunology ; *Cytokines/genetics/immunology ; Mice ; *Herpesvirus 1, Human/genetics/immunology ; Humans ; Female ; Cell Line, Tumor ; CRISPR-Cas Systems ; *Triple Negative Breast Neoplasms/therapy/immunology ; Mice, Inbred BALB C ; Tumor Microenvironment ; *Immunologic Factors/genetics ; }, abstract = {BACKGROUND: Triple-negative breast cancer (TNBC) presents significant therapeutic challenges due to its immunosuppressive tumor microenvironment (TME). Oncolytic herpes simplex virus type 1 (oHSV1) offers dual mechanisms of tumor lysis and immune activation, yet the optimal cytokine payloads for TNBC remain undefined.<br><br>METHODS: We developed a CRISPR/Cas9-mediated platform for high-efficiency oHSV1 engineering, replacing the ICP47 locus with murine IFN-&#x3b3;, GM-CSF, or IL-15R&#x3b1;/IL-15 fusion protein (IL15Fu). Constructs were validated for cytokine secretion, MHC modulation, and cytotoxicity in 4T1 TNBC and a panel of human cancer cell lines. Antitumor efficacy and immune remodeling were evaluated in a syngeneic 4T1 model using RNA sequencing and flow cytometry.<br><br>RESULTS: The CRISPR platform achieved 62.5-71.4% homologous recombination efficiency, enabling rapid virus construction. In vitro, OV-IFNG exhibited upregulated MHC I/II expression and potent cytotoxicity, while OV-GMCSF attenuated oncolysis in subsets of breast cancer cell lines. In the 4T1 model, OV-IL15Fu modestly improved tumor control and extended survival without apparent toxicity, while OV-IFNG induced early mortality associated with systemic toxicity. Transcriptomic profiling revealed divergent immune modulation: OV-IL15Fu enriched T cell/NK cytotoxicity pathways, OV-IFNG amplified cytokine/chemokine signaling, and OV-GMCSF paradoxically enhanced myeloid recruitment while inhibiting MHC-II pathways. Flow cytometry confirmed functional differences in immune activation: OV-IL15Fu expanding cytotoxic lymphocytes (CD8&#x207a; T/NK cells), OV-IFNG preferentially promote Th1 polarization and innate immune activation, and OV-GMCSF failed to activate T cells despite myeloid infiltration.<br><br>CONCLUSIONS: Our findings underscore the need for rational cytokine selection in oHSV1-based immunotherapy. While IFN-&#x3b3; increased immunogenic markers, its systemic toxicity and myeloid effects may limit benefit. GM-CSF exacerbated immune suppression in this context, whereas IL15Fu showed favorable immunostimulatory properties without detectable toxicity. These data support IL15Fu as a contextually promising payload for further evaluation in TNBC-targeted oncolytic virotherapy.}, }
@article {pmid40324959, year = {2025}, author = {Namata, MJ and Xu, J and Habyarimana, E and Palakolanu, SR and Wang, L and Li, J}, title = {Genome editing in maize and sorghum: A comprehensive review of CRISPR/Cas9 and emerging technologies.}, journal = {The plant genome}, volume = {18}, number = {2}, pages = {e70038}, pmid = {40324959}, issn = {1940-3372}, support = {32372134//National Natural Science Foundation of China/ ; S202310879188//Student Innovation and Entrepreneurship Project of Anhui Science and Technology University/ ; No.XK-XJGF001//Key Discipline Construction Funds of Anhui Sciences and Technology University/ ; 2023cxcysj179//Chuzhou "Star of Innovation and Entrepreneurship" Industrial Innovation Team, Postgraduate Innovation and Entrepreneurship Practice Project of Anhui Province/ ; }, mesh = {*Sorghum/genetics ; *Zea mays/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Plant Breeding/methods ; *Genome, Plant ; Plants, Genetically Modified ; Crops, Agricultural/genetics ; }, abstract = {The increasing changes in the climate patterns across the globe have deeply affected food systems where unparalleled and unmatched challenges are created. This jeopardizes food security due to an ever-increasing population. The extreme efficiency of C4 crops as compared to C3 crops makes them incredibly significant in securing food safety. C4 crops, maize (Zea mays L.) and sorghum (Sorghum bicolor L. Moench) in particular, have the ability to withstand osmotic stress induced by oxidative stress. Osmotic stress causes a series of physical changes in a plant thus facilitating reduced water uptake and photosynthesis inhibition, such as membrane tension, cell wall stiffness, and turgor changes. There has been a great advancement in plant breeding brought by introduction of clustered regularly interspaced short palindromic repeats (CRISPR) gene editing technology. This technology offers precise alterations to an organism's DNA through targeting specific genes for desired traits in a wide number of crop species. Despite its immense opportunities in plant breeding, it faces limitations such as effective delivery systems, editing efficiency, regulatory concerns, and off-target effects. Future prospects lie in optimizing next-generation techniques, such as prime editing, and developing novel genotype-independent delivery methods. Overall, the transformative role of CRISPR/Cas9 in sorghum and maize breeding underscores the need for responsible and sustainable utilization to address global food security challenges.}, }
@article {pmid40324726, year = {2025}, author = {Kumbhakar, R and Mondal, M and Thakro, V and Tripathi, S and Parida, SK}, title = {Shaping the future: Unravelling regulators modulating plant architecture for next-generation crops.}, journal = {Plant science : an international journal of experimental plant biology}, volume = {358}, number = {}, pages = {112534}, doi = {10.1016/j.plantsci.2025.112534}, pmid = {40324726}, issn = {1873-2259}, mesh = {*Crops, Agricultural/genetics/growth &amp; development/anatomy &amp; histology ; Plant Breeding ; Gene Editing ; }, abstract = {Plant architecture traits in crops are modulated through intricate interactions of various genetic pathways, which helps them to adapt to diverse environmental conditions. Key developmental pathways involved in forming plant architecture include the LAZY-TAC (Tiller Angle Control) module regulating branch and tiller angle, the CLAVATA-WUSCHEL pathway controlling shoot apical meristem fate and the GID1-DELLA pathway governing plant height and tillering in major food crops. These pathways function in concert to shape the overall architecture of plants, which is essential for optimizing light capture, resource allocation, reproductive success and eventual crop yield enhancement. Presently, plant architecture of modern crops has been shaped especially by artificial selection of natural alleles that target yield traits. Recent advances in CRISPR-Cas-based genome editing and genomics-assisted breeding strategies have enabled precise genetic manipulation of natural alleles in the functionally relevant genes regulating plant architecture traits in crops. This will assist researchers to select and introgress superior natural alleles in popular cultivars strategically for restructuring their desirable plant-types suitable for mechanical harvesting as well as enhancing the crop yield potential.}, }
@article {pmid40324585, year = {2025}, author = {Jiang, Y and Zheng, H and Bai, L and Wang, Y and Zhong, J and Zhang, W and Xu, T and Pan, Y and Tang, J and Lu, J and Zhang, B and Wu, Y}, title = {Genomic analysis and mobile genetic elements carriage of Clostridium perfringens type A.}, journal = {Anaerobe}, volume = {93}, number = {}, pages = {102970}, doi = {10.1016/j.anaerobe.2025.102970}, pmid = {40324585}, issn = {1095-8274}, mesh = {*Clostridium perfringens/genetics/classification/isolation &amp; purification/drug effects/pathogenicity ; Humans ; *Clostridium Infections/microbiology/epidemiology ; *Interspersed Repetitive Sequences ; China/epidemiology ; *Genome, Bacterial ; Whole Genome Sequencing ; Genomics ; Animals ; Plasmids/genetics ; Prophages/genetics ; Virulence Factors/genetics ; CRISPR-Cas Systems ; Drug Resistance, Bacterial/genetics ; }, abstract = {OBJECTIVES: To explore a comprehensive genomic analysis of Clostridium perfringens type A strains from diverse regions in China, investigating their virulence genes, antibiotic resistance genes, and mobile genetic elements (MGEs) to inform strategies for infection control and resistance gene surveillance.<br><br>METHODS: We conducted whole-genome sequencing on 168&#xa0;C. perfringens type A strains from nine provinces in China (2016-2021). Previously described alpha-toxin (PLC) sequence typing for C. perfringens was used for comparisons with core genome multilocus sequence typing. Virulence genes, antibiotic resistance genes, and MGEs, including CRISPR/Cas, prophages, and plasmids of C. perfringens type A were investigated by molecular and bioinformatic methods.<br><br>RESULTS: PLC type II contained the largest number of isolates (n&#xa0;=&#xa0;44). The same type strains were largely clustered in the same branches. Tetracycline resistance genes tetA(P) and tetB(P) had high prevalence in type A isolates. 395 prophages were predicted including 265 "incomplete," 55 "questionable," and 75 "intact" prophages. CRISPR/Cas systems were more common in isolates from humans (63&#xa0;%) than in those from animals and food (52&#xa0;% and 46&#xa0;%, respectively). Fifty-seven percent of strains likely had the tcp conjugation locus (tcpC to tcpH), and 12 isolates likely carried the conjugative pCW3 plasmid. Type A strains exhibited fewer plasmid-encoded toxins.<br><br>CONCLUSIONS: cgMLST analysis demonstrated some micro-evolution and regional transmission trends within type A, which exhibited partial correlated with PLC typing. This study highlights the need for enhanced surveillance of antimicrobial resistance and pathogenicity-associated MGEs in C. perfringens type A.}, }
@article {pmid40324075, year = {2025}, author = {Engel, NW and Steinfeld, I and Ryan, D and Anupindi, K and Kim, S and Wellhausen, N and Chen, L and Wilkins, K and Baker, DJ and Rommel, PC and Jarocha, D and Gohil, M and Zhang, Q and Milone, MC and Fraietta, JA and Davis, M and Young, RM and June, CH}, title = {Quadruple adenine base-edited allogeneic CAR T cells outperform CRISPR/Cas9 nuclease-engineered T cells.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {122}, number = {20}, pages = {e2427216122}, pmid = {40324075}, issn = {1091-6490}, support = {U54 CA244711/CA/NCI NIH HHS/United States ; 1-U54 CA244711//US NIH, National Cancer Institute/ ; Thought Leader Award 2020//Agilent Foundation (Agilent Technologies Foundation)/ ; Sponsored Research//Gilead | Kite Pharma/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *T-Lymphocytes/immunology/metabolism ; Animals ; Humans ; Mice ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; *Adenine/metabolism ; Immunotherapy, Adoptive/methods ; Graft vs Host Disease ; }, abstract = {Genome-editing technologies have enabled the clinical development of allogeneic cellular therapies, yet the optimal gene-editing modality for multiplex editing of therapeutic T cell product manufacturing remains elusive. In this study, we conducted a comprehensive comparison of CRISPR/Cas9 nuclease and adenine base editor (ABE) technologies in generating allogeneic chimeric antigen receptor (CAR) T cells, utilizing extensive in vitro and in vivo analyses. Both methods achieved high editing efficiencies across four target genes, critical for mitigating graft-versus-host disease and allograft rejection: TRAC or CD3E, B2M, CIITA, and PVR. Notably, ABE demonstrated higher manufacturing yields and distinct off-target profiles compared to Cas9, with translocations observed exclusively in Cas9-edited products. Functionally, ABE-edited CAR T cells exhibited superior in vitro effector functions under continuous antigen stimulation, including enhanced proliferative capacity and increased surface CAR expression. Transcriptomic analysis revealed that ABE editing resulted in reduced activation of p53 and DNA damage response pathways at baseline, along with sustained activation of metabolic pathways during antigen stress. Consistently, Assay for Transposase-Accessible Chromatin using sequencing data indicated that Cas9-edited, but not ABE-edited, CAR T cells showed enrichment of chromatin accessibility peaks associated with double-strand break repair and DNA damage response pathways. In a preclinical leukemia model, ABE-edited CAR T cells demonstrated improved tumor control and extended overall survival compared to their Cas9-edited counterparts. Collectively, these findings position ABE as superior to Cas9 nucleases for multiplex gene editing of therapeutic T cells.}, }
@article {pmid40323736, year = {2025}, author = {Chen, Y and Li, Y and Li, P and Li, X and Zhao, S and Zuo, Z}, title = {Catching CRISPR-Cas9 in Action.}, journal = {Journal of chemical theory and computation}, volume = {21}, number = {10}, pages = {5023-5036}, doi = {10.1021/acs.jctc.5c00165}, pmid = {40323736}, issn = {1549-9626}, mesh = {Molecular Dynamics Simulation ; *CRISPR-Cas Systems ; DNA/chemistry/metabolism ; *CRISPR-Associated Protein 9/chemistry/metabolism ; Quantum Theory ; }, abstract = {CRISPR-Cas9 has revolutionized genome editing, yet its structural dynamics and functional properties remain incompletely understood, partly due to limited atomic-level characterization of its active conformation with a full R-loop. Capitalizing on recent advances in Cas9 structural determination, we constructed a catalytic-state Cas9 model bound to a bona fide R-loop and performed an integrated computational investigation. Our molecular dynamics simulations reveal substantial conformational heterogeneity in the PAM (protospacer-adjacent motif)-distal nontarget DNA strand and adjacent Cas9 regions, leading to dynamically fluctuating interactions, thereby challenging experimental resolution of the full R-loop complex. Comparative analysis highlights a conformational barrier restricting final activation of the HNH nuclease domain, suggesting that strategic modulation of HNH interactions on its two sides could enhance cleavage efficiency. Furthermore, quantum mechanics/molecular mechanics simulations indicate that with H983 protonated at Nε, the RuvC domain favors a phosphate-mediated over a histidine-mediated pathway for nontarget strand cleavage. Additionally, we identify an alternative HNH-mediated target strand cleavage pathway, involving a water nucleophile aligned at the 5' side of the scissile phosphate. Inspired by the basic residue ladder observed in RuvC, we propose extending a similar ladder in HNH to strengthen DNA binding and catalytic activity. Our study provides critical insights into Cas9 structure, dynamics, and catalysis, laying a foundation for the rational design of next-generation CRISPR-Cas9 systems with optimized specificity-efficiency balance.}, }
@article {pmid40319546, year = {2025}, author = {Tavella, S and di Lillo, A and Conti, A and Iannelli, F and Mancheno-Ferris, A and Matti, V and Di Micco, R and Fagagna, FDD}, title = {Weaponizing CRISPR/Cas9 for selective elimination of cells with an aberrant genome.}, journal = {DNA repair}, volume = {149}, number = {}, pages = {103840}, pmid = {40319546}, issn = {1568-7856}, mesh = {Humans ; *CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; HeLa Cells ; *Gene Editing/methods ; Green Fluorescent Proteins/genetics/metabolism ; DNA Repair ; Cell Survival ; }, abstract = {The CRISPR/Cas9 technology is a powerful and versatile tool to disrupt genes' functions by introducing sequence-specific DNA double-strand breaks (DSBs). Here, we repurpose this technology to eradicate aberrant cells by specifically targeting silent and non-functional genomic sequences present only in target cells to be eliminated. Indeed, an intrinsic challenge of most current therapies against cancer and viral infections is the non-specific toxicity that they can induce in normal tissues because of their impact on important cellular mechanisms shared, to different extents, between unhealthy and healthy cells. The CRISPR/Cas9 technology has potential to overcome this limitation; however, so far effectiveness of these approaches was made dependent on the targeting and inactivation of a functional gene product. Here, we generate proof-of-principle evidence by engineering HeLa and RKO cells with a promoterless Green Fluorescent Protein (GFP) construct. The integration of this construct simulates either a genomic alteration, as in cancer cells, or a silent proviral genome. Cas9-mediated DSBs in the GFP sequence activate the DNA damage response (DDR), reduce cell viability and increase mortality. This is associated with increased cell size, multinucleation, cGAS-positive micronuclei accumulation and the activation of an inflammatory response. Pharmacological inhibition of the DNA repair factor DNA-PK enhances cell death. These results demonstrate the therapeutic potential of the CRISPR/Cas9 system in eliminating cells with an aberrant genome, regardless of the expression or the function of the target DNA sequence.}, }
@article {pmid40319267, year = {2025}, author = {García-Calvo, L and Kummen, C and Rustad, S and Rønning, SB and Fagerlund, A}, title = {A toolkit for facilitating markerless integration of expression cassettes in Komagataella phaffii via CRISPR/Cas9.}, journal = {Microbial cell factories}, volume = {24}, number = {1}, pages = {97}, pmid = {40319267}, issn = {1475-2859}, support = {314743//Norwegian Fund for Research Fees for Agricultural Products (FFL)/ ; 314743//Norwegian Fund for Research Fees for Agricultural Products (FFL)/ ; 314743//Norwegian Fund for Research Fees for Agricultural Products (FFL)/ ; 314743//Norwegian Fund for Research Fees for Agricultural Products (FFL)/ ; 336136//The Research Council of Norway/ ; 336136//The Research Council of Norway/ ; 336136//The Research Council of Norway/ ; 336136//The Research Council of Norway/ ; 336136//The Research Council of Norway/ ; }, mesh = {*CRISPR-Cas Systems ; *Saccharomycetales/genetics/metabolism ; *Gene Editing/methods ; Plasmids/genetics ; Recombinant Proteins/genetics/biosynthesis ; Green Fluorescent Proteins/genetics/metabolism ; }, abstract = {BACKGROUND: The yeast Komagataella phaffii (formerly known as Pichia pastoris) has been widely used for functional expression of recombinant proteins, including plant and animal food proteins. CRISPR/Cas9 genome editing systems can be used for insertion of heterologous genes without the use of selection markers. The study aimed to create a convenient markerless knock-in method for integrating expression cassettes into the chromosome of K. phaffii using CRISPR/Cas9 technology. The approach was based on the hierarchical, modular, Golden Gate assembly employing the GoldenPiCS toolkit. Furthermore, the aim was to evaluate the system's efficiency and suitability for producing secreted recombinant food proteins.<br><br>RESULTS: Three Cas9/sgRNA plasmids were constructed, along with corresponding donor helper plasmids containing homology regions for chromosomal integration via homology-directed repair. The integration efficiency of an enhanced green fluorescent protein (eGFP) expression cassette was assessed at three genomic loci (04576, PFK1, and ROX1). The 04576 locus showed the highest integration efficiency, while ROX1 had the highest transformation efficiency. Whole genome sequencing revealed variable copy numbers of eGFP expression cassettes among clones, corresponding with increasing levels of fluorescence. Furthermore, the system's applicability for producing recombinant food proteins was validated by successfully expressing and secreting chicken ovalbumin. This constitutes the first report of CRISPR/Cas9 applied to produce recombinant chicken ovalbumin.<br><br>CONCLUSIONS: The adapted GoldenPiCS toolkit combined with CRISPR/Cas9 technology enabled efficient and precise genome integration in K. phaffii. This approach holds promise for expanding the production of high-value recombinant proteins. Future research should focus on optimizing integration sites and improving cloning procedures to enhance the system's efficiency and versatility.}, }
@article {pmid40319033, year = {2025}, author = {Cheng, B and Peng, SI and Jia, YY and Tong, E and Atwood, SX and Sun, BK}, title = {Comprehensive secretome profiling and CRISPR screen identifies SFRP1 as a key inhibitor of epidermal progenitor proliferation.}, journal = {Cell death &amp; disease}, volume = {16}, number = {1}, pages = {360}, pmid = {40319033}, issn = {2041-4889}, support = {K08 AR067853/AR/NIAMS NIH HHS/United States ; R03 AR075844/AR/NIAMS NIH HHS/United States ; R03AR075844//U.S. Department of Health &amp; Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)/ ; K08AR067853//U.S. Department of Health &amp; Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)/ ; }, mesh = {Humans ; Cell Proliferation ; *Keratinocytes/metabolism/cytology ; *Membrane Proteins/metabolism/genetics ; *Stem Cells/metabolism/cytology ; *Intercellular Signaling Peptides and Proteins/metabolism/genetics ; *Secretome/metabolism ; *Epidermis/metabolism ; Wnt Signaling Pathway ; CRISPR-Cas Systems/genetics ; *Epidermal Cells/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Secreted proteins are crucial for the structure and functions of the human epidermis, but the full repertoire of the keratinocyte secretome has not been experimentally defined. In this study, we performed mass spectrometry on conditioned media from primary human keratinocytes, identifying 406 proteins with diverse roles in adhesion, migration, proliferation, proteolysis, signal transduction, and innate immunity. To leverage this new dataset, we developed a novel colony formation assay-based CRISPR screen to investigate the functions of uncharacterized secreted proteins on epidermal stem cells. The screen identified six candidate proteins that promoted proliferation of epidermal progenitors and two proteins that inhibited it. Secreted frizzled-related protein-1 (SFRP1) was the most potent inhibitor. We discovered that SFRP1 restrained clonogenic keratinocyte proliferation by inhibiting Wnt signaling as well as blocking ectopic expression of leukemia inhibitory factor (LIF). Collectively, our study expands our knowledge of the keratinocyte secretome, establishes a novel CRISPR screen to assess the function of non-cell autonomous factors, and highlights SFRP1's role in regulating epidermal balance.}, }
@article {pmid40318521, year = {2025}, author = {Chen, H and Liang, Y and Liang, Y and Chen, Y and Li, X and Zhang, R and Duan, C and Li, W and Cui, Z and Gu, J and Ding, C and Sun, X and Chen, J}, title = {Generation of a gene-corrected isogenic human iPS cell line (CSUASOi006-A-2) from a retinitis pigmentosa patient using CRISPR/Cas9 technology.}, journal = {Stem cell research}, volume = {86}, number = {}, pages = {103727}, doi = {10.1016/j.scr.2025.103727}, pmid = {40318521}, issn = {1876-7753}, mesh = {Humans ; *Retinitis Pigmentosa/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Cell Line ; Gene Editing ; }, abstract = {Retinitis pigmentosa (RP) is a heterogeneous group of hereditary eye disorders characterized by a progressive degeneration of the light-sensing photoreceptor cells in the retina. Currently, there are no effective treatments. In a previous study, we generated a human induced pluripotent stem (iPS) cell line (CSUASOi006-A) from an RP patient carrying a PRPF8 (c.C5792T) mutation. In this study, we corrected the c.5792C > T mutation in the PRPF8 gene using CRISPR/Cas9 technology and generated an isogenic control cell line (CSUASOi006-A-2). This provides an important cellular resource for RP research.}, }
@article {pmid40318489, year = {2025}, author = {Sun, J and Bai, J and Huang, Y and Langford, PR and Zhang, Y and Li, G}, title = {A CRISPR/Cas12a-based DNAzyme visualization platform for rapid discrimination of Streptococcus suis serotype 2 versus 1/2 and serotype 1 versus 14.}, journal = {Talanta}, volume = {294}, number = {}, pages = {128241}, doi = {10.1016/j.talanta.2025.128241}, pmid = {40318489}, issn = {1873-3573}, mesh = {*Streptococcus suis/genetics/classification/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; Serogroup ; *DNA, Catalytic/metabolism/chemistry/genetics ; Polymorphism, Single Nucleotide ; Nucleic Acid Amplification Techniques ; Animals ; Swine ; Colorimetry ; G-Quadruplexes ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Streptococcus suis is a major swine pathogen with serotypes 2 and 14 posing zoonotic risks. However, distinguishing serotypes 1/2 from 2 or 1 from 14 remains challenging due to high similarity in their capsule polysaccharide (CPS) loci. Here, we developed a rapid, equipment-free discriminating platform targeting a single nucleotide polymorphism (SNP) at position 483 of the cpsK gene (G in serotypes 2/14 vs. T/C in 1/2/1). The method integrates recombinase polymerase amplification (RPA) with CRISPR/Cas12a and a G-quadruplex-hemin DNAzyme visualization system. RPA enables isothermal amplification, while CRISPR/Cas12a ensures single-nucleotide specificity by cleaving target DNA. Subsequent DNAzyme catalysis converts colorimetric substrates, enabling naked-eye differentiation via distinct color changes (blue for serotypes 1/2/1 vs. colorless for 2/14). This approach achieved a sensitivity of 10[1]-10[2] copies per reaction and demonstrated 100 % specificity across 29 S. suis serotypes and related strains. Compared to PCR-based or sequencing methods, our platform eliminates reliance on thermocyclers or fluorescence detectors, reducing costs and operational complexity. The entire workflow, completed within 70 min, offers a practical solution for point-of-care testing in resource-limited settings. By enabling rapid, accurate discrimination, this tool will become a complementary tool for resolving ambiguous serotypes and enhances outbreak management in swine populations and mitigates zoonotic transmission.}, }
@article {pmid40318042, year = {2025}, author = {Kang, J and Park, C and Lee, G and Koo, J and Oh, H and Kim, EH and Bae, E and Suh, JY}, title = {Structural Investigation of the Anti-CRISPR Protein AcrIE7.}, journal = {Proteins}, volume = {}, number = {}, pages = {}, doi = {10.1002/prot.26832}, pmid = {40318042}, issn = {1097-0134}, support = {NRF-2022R1A2C1009804//National Research Foundation of Korea/ ; NRF-2021R1A2C1003917//National Research Foundation of Korea/ ; RS-2023-00207820//National Research Foundation of Korea/ ; A412550//Korea Basic Science Institute/ ; }, abstract = {The CRISPR-Cas system is an adaptive immune system in prokaryotes that provides protection against bacteriophages. As a countermeasure, bacteriophages have evolved various anti-CRISPR proteins that neutralize CRISPR-Cas immunity. Here, we report the structural and functional investigation of AcrIE7, which inhibits the type I-E CRISPR-Cas system in Pseudomonas aeruginosa. We determined both crystal and solution structures of AcrIE7, which revealed a novel helical fold. In binding assays using various biochemical methods, AcrIE7 did not tightly interact with a single Cas component in the type I-E Cascade complex or the CRISPR adaptation machinery. In contrast, AlphaFold modeling with our experimentally determined AcrIE7 structure predicted that AcrIE7 interacts with Cas3 in the type I-E CRISPR-Cas system in P. aeruginosa. Our findings are consistent with a model where AcrIE7 inhibits Cas3 and also highlight the effectiveness and limitations of AlphaFold modeling.}, }
@article {pmid40317571, year = {2025}, author = {da Silva, GC and Rossi, CC}, title = {The Arms Race Between Actinobacillus pleuropneumoniae and Its Genetic Environment: A Comprehensive Analysis of Its Defensome and Mobile Genetic Elements.}, journal = {Molecular microbiology}, volume = {}, number = {}, pages = {}, doi = {10.1111/mmi.15374}, pmid = {40317571}, issn = {1365-2958}, support = {APQ-03498-22//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de Minas Gerais/ ; APQ-01339-25//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de Minas Gerais/ ; 408564/2023-7//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; }, abstract = {Actinobacillus pleuropneumoniae is the causative agent of pleuropneumonia in swine, a highly contagious and economically significant disease. The genetic variability of A. pleuropneumoniae complicates disease control efforts, as it enables rapid adaptation to various stressors, including antimicrobial treatments. To better understand the molecular mechanisms underlying this adaptability, we investigated the role of the bacterial defensome and its relationship with mobile genetic elements (MGEs), such as prophages, plasmids, and integrative conjugative elements (ICEs). Using bioinformatic tools, we identified a diverse and rich defensome in A. pleuropneumoniae, with an average of 16 different defense systems per strain. We found that CRISPR-Cas systems, along with other defense mechanisms, are actively involved in restricting the entry of foreign genetic material, playing a crucial role in bacterial adaptation. Additionally, we characterized several novel prophages and examined their distribution across different strains, revealing their potential contribution to the bacterium's evolutionary success. Our findings underscore the complex interplay between the bacterium's defense systems and MGEs, shedding light on how A. pleuropneumoniae maintains genetic diversity while also safeguarding itself against external threats. These insights provide a better understanding of the genetic factors that influence the pathogen's adaptability and highlight potential avenues for more effective disease control strategies.}, }
@article {pmid40317300, year = {2025}, author = {Rathore, RS and Jiang, W and Sedeek, K and Mahfouz, M}, title = {Harnessing neo-domestication of wild pigmented rice for enhanced nutrition and sustainable agriculture.}, journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik}, volume = {138}, number = {5}, pages = {108}, pmid = {40317300}, issn = {1432-2242}, mesh = {*Oryza/genetics ; Gene Editing ; *Domestication ; *Plant Breeding ; *Agriculture ; Nutritive Value ; Genetic Variation ; CRISPR-Cas Systems ; Crops, Agricultural/genetics ; }, abstract = {Advances in precision gene editing have enabled the rapid domestication of wild crop relatives, a process known as neo-domestication. During domestication, breeding rice for maximum productivity under optimal growth conditions reduced genetic diversity, eliminating variants for stress tolerance and grain nutrients. Wild rice varieties have rich genetic diversity, including variants for disease resistance, stress tolerance, and grain nutritional quality. For example, the grain of pigmented wild rice has abundant antioxidants (anthocyanins, proanthocyanidins, and flavonoids), but low yield, poor plant architecture, and long life cycle limit its cultivation. In this review, we address the neo-domestication of wild pigmented rice, focusing on recent progress, CRISPR-Cas editing toolboxes, selection of key candidate genes for domestication, identifying species with superior potential via generating genomic and multi-omics resources, efficient crop transformation methods and highlight strategies for the promotion and application pigmented rice. We also address critical outstanding questions and potential solutions to enable efficient neo-domestication of wild pigmented rice and thus enhance food security and nutrition.}, }
@article {pmid40316660, year = {2025}, author = {Chen, Y and Ren, J and Yang, X and Li, Z and Wu, W and Luo, G and Xu, Y and Yang, H and Wang, Q and Zhang, Q}, title = {The rapid detection of human HLA-B*27 gene based on BASIC isothermal detection.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {15427}, pmid = {40316660}, issn = {2045-2322}, support = {82472386//National Natural Science Foundation of China/ ; 23LCYJ038//Sichuan Health Commission/ ; 2023HT073//Industry-University-Research Innovation Fund for Chinese Universities/ ; S21077//Sichuan Medical Association/ ; 23JCYJPT0032, 20SXZRKX0010, 20SXQT0109 and 202001207//Nanchong Science and Technology Fund/ ; 2024CX001, 2023PTZK016 and ZX-51130001-2021-023//Research Fund of Affiliated Hospital of North Sichuan Medical College/ ; 22SXQT0307//Special Fund of the Strategic Science and Technology Cooperation between Nanchong Municipal Science and Technology Bureau and Universities/ ; 2024NSFSC1546//National Natural Science Foundation of Sichuan Province/ ; }, mesh = {Humans ; *HLA-B27 Antigen/genetics ; *Spondylitis, Ankylosing/genetics/diagnosis ; CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Genotype ; }, abstract = {Ankylosing spondylitis (AS) is a chronic, inflammatory arthritis of the spine and peripheral joints which is known to have a strong association with the human leukocyte antigen B27 (HLA-B27). Quantitative real-time PCR and flow cytometry are the predominant methods for HLA-B27 gene and antigen, respectively, which are too time-consuming and labor-intensive to realize rapid analysis. Therefore, a rapid diagnostic tool is highly required. In this study, we developed a rapid HLA-B*27 detection platform (namely BASIC) by combining our previously invented BASIS isothermal amplification method with the widely used CRISPR/Cas12a signal output tool. The BASIS can efficiently amplify all HLA-B*27 genotypes by using a set of universal primers, which target the conserved regions. The amplicons are subsequently applied to CRISPR/Cas12a analysis. The CRISPR/Cas12a recognizes the pathogenic HLA-B*27 amplicons specifically by using a well-designed gRNA, thereby achieving fluorescence signal output. Our results showed that the BASIC can be completed in 1 h with analytical sensitivity up to 100 aM. It could resist interference of homologous genes, hemoglobin, bilirubin, and triglyceride. For clinical sample detection, the BASIC offered completely consistent results with qPCR. Given the advantages of sensitivity, specificity, simplicity and rapidity, the BASIC was demonstrated a promising HLA-B*27 gene rapid detection tool for the early screening and diagnosis of AS.}, }
@article {pmid40316524, year = {2025}, author = {Berman, A and Su, N and Li, Z and Landau, U and Chakraborty, J and Gerbi, N and Liu, J and Qin, Y and Yuan, B and Wei, W and Yanai, O and Mayrose, I and Zhang, Y and Shani, E}, title = {Construction of multi-targeted CRISPR libraries in tomato to overcome functional redundancy at genome-scale level.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {4111}, pmid = {40316524}, issn = {2041-1723}, support = {757683//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 101118769//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Euratom (H2020 Euratom Research and Training Programme 2014-2018)/ ; 101113412//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Euratom (H2020 Euratom Research and Training Programme 2014-2018)/ ; 3419/20//Israel Science Foundation (ISF)/ ; 1462/24//Israel Science Foundation (ISF)/ ; }, mesh = {*Solanum lycopersicum/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Plant ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Library ; Plants, Genetically Modified ; Phenotype ; Plant Breeding ; Clustered Regularly Interspaced Short Palindromic Repeats ; Mutation ; }, abstract = {Genetic variance is vital for breeding programs and mutant screening, yet traditional mutagenesis methods wrestle with genetic redundancy and a lack of specificity in gene targeting. CRISPR-Cas9 offers precise, site-specific gene editing, but its application in crop improvement has been limited by scalability challenges. In this study, we develop genome-wide multi-targeted CRISPR libraries in tomato, enhancing the scalability of CRISPR gene editing in crops and addressing the challenges of redundancy while maintaining its precision. We design 15,804 unique single guide RNAs (sgRNAs), each targeting multiple genes within the same gene families. These sgRNAs are classified into 10 sub-libraries based on gene function. We generate approximately 1300 independent CRISPR lines and successfully identify mutants with distinct phenotypes related to fruit development, fruit flavor, nutrient uptake, and pathogen response. Additionally, we develop CRISPR-GuideMap, a double-barcode tagging system to enable large-scale sgRNA tracking in generated plants. Our results demonstrate that multi-targeted CRISPR libraries are scalable and effective for large-scale gene editing and offer an approach to overcome gene functional redundancy in basic plant research and crop breeding.}, }
@article {pmid40316390, year = {2025}, author = {Shixing, X and Shengjun, B and He, S and Xinyue, Z and Xingdong, Z and Xiaoying, Z and Leng, H and Enyong, D and Wan, J}, title = {A fluorescence biosensor for detecting LncRNA MALAT1 based on isothermal amplification by cyclic extension.}, journal = {Analytica chimica acta}, volume = {1357}, number = {}, pages = {344076}, doi = {10.1016/j.aca.2025.344076}, pmid = {40316390}, issn = {1873-4324}, mesh = {*RNA, Long Noncoding/analysis/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; Fluorescence ; Limit of Detection ; DNA Probes/chemistry/genetics ; }, abstract = {BACKGROUND: Long non-coding RNA (lncRNA) Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1), a crucial regulator of gene expression, has emerged as a highly promising biomarker in the progression of various cancers. The clinical detection of lncRNA MALAT1 primarily relies on Reverse Transcription-Polymerase Chain Reaction (RT-PCR), which requires skilled operators and large, expensive thermal cycling equipment. These limitations have restricted the application of RT-PCR, particularly in resource-constrained settings.<br><br>RESULTS: In this study, we developed a novel signal amplification method, termed Isothermal Amplification by Cyclic Extension (IACE), based on the linear extension of a single-stranded DNA probe. IACE operates through the continuous extension of Probe 1 (a) into long single-stranded DNA with multiple repetitive sequences, facilitated by Probe 2 (a&#x2217;a&#x2217;) and Bst DNA polymerase. We found that the single-stranded DNA product of IACE could directly activate the CRISPR-Cas12a system without requiring a protospacer adjacent motif (PAM). By integrating IACE with a three-way junction structure and a nicking enzyme, we established a one-step signal amplification strategy for the detection of lncRNA MALAT1, achieving a detection limit as low as 37.5&#xa0;fM using the CRISPR-Cas system.<br><br>SIGNIFICANCE: The biosensor developed in the present study simplifies workflows, minimizes contamination risks, and demonstrates exceptional detection performance in tumor patient samples, highlighting its potential to advance clinical tumor diagnostic approaches.}, }
@article {pmid40316384, year = {2025}, author = {Chang, Z and Zhou, J and Li, D and Wang, H and Chen, M and Liu, L and Ding, J and Wang, Y and Gao, Z and Sai, N}, title = {Selection and identification of the ssDNA aptamer against polymyxin B sulfate.}, journal = {Analytica chimica acta}, volume = {1357}, number = {}, pages = {344067}, doi = {10.1016/j.aca.2025.344067}, pmid = {40316384}, issn = {1873-4324}, mesh = {*Aptamers, Nucleotide/chemistry ; *DNA, Single-Stranded/chemistry ; SELEX Aptamer Technique ; *Polymyxin B/analysis ; Biosensing Techniques ; CRISPR-Cas Systems ; *Anti-Bacterial Agents/analysis ; Animals ; }, abstract = {BACKGROUND: Polymyxin B (PMB) is a potent antibiotic, and its sulfate form, Polymyxin B Sulfate (PMBS), is widely used. When PMBS is used as a veterinary drug, excessive use can lead to drug residues in animal bodies, which is a significant food safety issue and raises serious concerns. Therefore, there is an urgent need to strengthen research in this area. In this study, we obtained a ssDNA aptamer against PMBS using the Capture-SELEX method, which has notable advantages in small molecule selection. Then, we integrated the obtained aptamer with a biosensor based on the CRISPR/Cas14a system for a series of validations.<br><br>RESULTS: In this study, specific aptamers against PMBS were discovered via the Capture-SELEX process. The selection process consisted of ten rounds. The affinity of the candidate sequences was determined by Isothermal Titration Calorimetry (ITC), and the PMBS24 aptamer with the highest affinity was ultimately identified, with a dissociation constant (Kd) of 3.89&#xa0;&#xb1;&#xa0;0.46&#xa0;&#x3bc;M. To further obtain high-affinity aptamers, we attempted to truncate the PMBS24 aptamer. However, the results demonstrated that the aptamer with the full-length sequence exhibited superior affinity. Subsequently, it was applied in the CRISPR/Cas14a-based sensor for the detection of PMBS. This method had a LOD of 0.99&#xa0;ng&#xa0;mL[-1], with good specificity, and was successfully used for the detection in milk.<br><br>SIGNIFICANCE: An important recognition element of PMBS has been successfully obtained, filling a part of the gap in the detection of PMBS. Moreover, the aptamer has been applied to the CRISPR/Cas14a-based sensor for amplification-free PMBS detection, yielding favorable detection results. This also opens up the possibility of applying the aptamer to other similar sensors.}, }
@article {pmid40316310, year = {2025}, author = {Kang, H and Fitch, JC and Varghese, RP and Thorne, CA and Cusanovich, DA}, title = {Optimization of a Cas12a-Driven Synthetic Gene Regulatory Network System.}, journal = {ACS synthetic biology}, volume = {14}, number = {5}, pages = {1732-1744}, pmid = {40316310}, issn = {2161-5063}, support = {P30 CA023074/CA/NCI NIH HHS/United States ; R35 GM137896/GM/NIGMS NIH HHS/United States ; R35 GM147128/GM/NIGMS NIH HHS/United States ; }, mesh = {*Gene Regulatory Networks/genetics ; *CRISPR-Cas Systems/genetics ; *Synthetic Biology/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; }, abstract = {Gene regulatory networks, which control gene expression patterns in development and in response to stimuli, use regulatory logic modules to coordinate inputs and outputs. One example of a regulatory logic module is the gene regulatory cascade (GRC), where a series of transcription factor genes turn on in order. Synthetic biologists have derived artificial systems that encode regulatory rules, including GRCs. Furthermore, the development of single-cell approaches has enabled the discovery of gene regulatory modules in a variety of experimental settings. However, the tools available for validating these observations remain limited. Based on a synthetic GRC using DNA cutting-defective Cas9 (dCas9), we designed and implemented an alternative synthetic GRC utilizing DNA cutting-defective Cas12a (dCas12a). Comparing the ability of these two systems to express a fluorescent reporter, the dCas9 system was initially more active, while the dCas12a system was more streamlined. Investigating the influence of individual components of the systems identified nuclear localization as a major driver of differences in activity. Improving nuclear localization for the dCas12a system resulted in 1.5-fold more reporter-positive cells and a 15-fold increase in reporter intensity relative to the dCas9 system. We call this optimized system the "Synthetic Gene Regulatory Network" (SGRN, pronounced "sojourn").}, }
@article {pmid40316115, year = {2025}, author = {Tao, Z and Tian, C and Zhong, C and Ji, B and Li, W and Zhao, Y}, title = {The role of NhaA protein in modulating antibiotic tolerance in Escherichia coli.}, journal = {International journal of biological macromolecules}, volume = {311}, number = {Pt 2}, pages = {143721}, doi = {10.1016/j.ijbiomac.2025.143721}, pmid = {40316115}, issn = {1879-0003}, mesh = {*Escherichia coli/drug effects/genetics/metabolism ; *Anti-Bacterial Agents/pharmacology ; *Escherichia coli Proteins/genetics/metabolism ; *Drug Resistance, Bacterial/genetics ; Adenosine Triphosphate/metabolism ; Proton-Motive Force/drug effects ; Cell Membrane Permeability/drug effects ; Membrane Fluidity/drug effects ; Microbial Sensitivity Tests ; CRISPR-Cas Systems ; }, abstract = {As microbial resistance and recurrent bacterial infections escalate, the growing understanding of the interplay between antibiotic resistance and tolerance has sparked significant interest in the latter. Previous studies have demonstrated that the deletion of cation/proton antiporters (CPAs) induces bacterial phenotypes, such as slow growth and prolonged lag phases, which contribute to the development of tolerance. This study investigates the role of the NhaA protein in antibiotic tolerance in Escherichia coli using CRISPR/Cas9 gene editing to delete the NhaA protein. Our results suggest that the NhaA protein plays a key role in modulating antibiotic tolerance. In response to NhaA deletion, E. coli adapts through multiple mechanisms, including changes in membrane permeability, enhanced efflux activity, increased membrane fluidity, disruption of the proton motive force (PMF), and a reduction in intracellular ATP levels. These adaptive changes collectively promote the development of antibiotic tolerance. Understanding these tolerance mechanisms could uncover new therapeutic targets, help prevent the emergence of tolerance, or sustain bacteria cells in a tolerant state, providing crucial strategies to combat the rise of antibiotic-resistant bacteria.}, }
@article {pmid40315964, year = {2025}, author = {Gupta, MK and Gouda, G and Moazzam-Jazi, M and Vadde, R and Nagaraju, GP and El-Rayes, BF}, title = {CRISPR/Cas9-directed epigenetic editing in colorectal cancer.}, journal = {Biochimica et biophysica acta. Reviews on cancer}, volume = {1880}, number = {3}, pages = {189338}, doi = {10.1016/j.bbcan.2025.189338}, pmid = {40315964}, issn = {1879-2561}, mesh = {Humans ; *Colorectal Neoplasms/genetics/therapy/pathology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Epigenesis, Genetic ; Animals ; DNA Methylation ; Gene Expression Regulation, Neoplastic ; Epigenome Editing ; }, abstract = {Colorectal cancer (CRC) remains a leading cause of cancer-related illness and death worldwide, arising from a complex interplay of genetic predisposition, environmental influences, and epigenetic dysregulation. Among these factors, epigenetic modifications-reversible and heritable changes in gene expression-serve as crucial regulators of CRC progression. Understanding these modifications is essential for identifying potential biomarkers for early diagnosis and developing targeted therapeutic strategies. Epigenetic drugs (epidrugs) such as DNA methyltransferase inhibitors (e.g., decitabine) and bromodomain inhibitors (e.g., JQ1) have shown promise in modulating aberrant epigenetic changes in CRC. However, challenges such as drug specificity, delivery, and safety concerns limit their clinical application. Advances in CRISPR-Cas9-based epigenetic editing offer a more precise approach to modifying specific epigenetic markers, presenting a potential breakthrough in CRC treatment. Despite its promise, CRISPR-based epigenome editing may result in unintended genetic modifications, necessitating stringent regulations and safety assessments. Beyond pharmacological interventions, lifestyle factors-including diet and gut microbiome composition-play a significant role in shaping the epigenetic landscape of CRC. Nutritional and microbiome-based interventions have shown potential in preventing CRC development by maintaining intestinal homeostasis and reducing tumor-promoting epigenetic changes. This review provides a comprehensive overview of epigenetic alterations in CRC, exploring their implications for diagnosis, prevention, and treatment. By integrating multi-omics approaches, single-cell technologies, and model organism studies, future research can enhance the specificity and efficacy of epigenetic-based therapies. Shortly, a combination of advanced gene-editing technologies, targeted epidrugs, and lifestyle interventions may pave the way for more effective and personalized CRC treatment strategies.}, }
@article {pmid40315882, year = {2025}, author = {Lou, E and Choudhry, MS and Starr, TK and Folsom, TD and Bell, J and Rathmann, B and DeFeo, AP and Kim, J and Slipek, N and Jin, Z and Sumstad, D and Klebanoff, CA and Ladner, K and Sarkari, A and McIvor, RS and Murray, TA and Miller, JS and Rao, M and Jensen, E and Ankeny, J and Khalifa, MA and Chauhan, A and Spilseth, B and Dixit, A and Provenzano, PP and Pan, W and Weber, D and Byrne-Steele, M and Henley, T and McKenna, DH and Johnson, MJ and Webber, BR and Moriarity, BS}, title = {Targeting the intracellular immune checkpoint CISH with CRISPR-Cas9-edited T cells in patients with metastatic colorectal cancer: a first-in-human, single-centre, phase 1 trial.}, journal = {The Lancet. Oncology}, volume = {26}, number = {5}, pages = {559-570}, doi = {10.1016/S1470-2045(25)00083-X}, pmid = {40315882}, issn = {1474-5488}, mesh = {Humans ; Middle Aged ; *CRISPR-Cas Systems ; Male ; Female ; *Colorectal Neoplasms/genetics/immunology/pathology/therapy ; Aged ; Adult ; *Lymphocytes, Tumor-Infiltrating/immunology/transplantation ; Gene Editing ; Young Adult ; Adolescent ; *T-Lymphocytes/immunology/transplantation ; }, abstract = {BACKGROUND: Over the past decade, immunotherapeutic strategies-mainly targeting the PD-1-PD-L1 immune checkpoint axis-have altered cancer treatment for many solid tumours, but few patients with gastrointestinal forms of cancer have benefited to date. There remains an urgent need to extend immunotherapy efficacy to more patients while addressing resistance to current immune checkpoint inhibitors. The aim of this study was to determine the safety and anti-tumour activity of knockout of CISH, which encodes cytokine-inducible SH2-containing protein, a novel intracellular immune checkpoint target and a founding member of the SOCS family of E3-ligases, using tumour infiltrating lymphocyte (TILs) genetically edited with CRISPR-Cas9 in patients with metastatic gastrointestinal epithelial cancers.<br><br>METHODS: For this first-in-human, single-centre, phase 1 trial, patients aged 18-70 years with a diagnosis of metastatic gastrointestinal epithelial cancer with progressive disease following at least one first line standard therapy, measurable disease with at least one lesion identified as resectable for TIL generation and at least one other lesion meeting RECIST criteria as measurable to serve as an indicator of disease response, and an ECOG performance status of 0 or 1 were screened and enrolled if meeting these and all other eligibility criteria. TILs procured from tumour biopsies were expanded on the basis of neoantigen reactivity, subjected to CRISPR-Cas9-mediated CISH knockout, and infused intravenously into 12 patients after non-myeloablative lymphocyte depleting chemotherapy (cyclophosphamide 60 mg/kg per dose on study days -6 and -5, and fludarabine 25 mg/m[2] per dose on days -7 to -3) followed by high-dose IL-2 (aldesleukin; 720&#x2009;000 IU/kg per dose). The primary endpoint was safety of administration of neoantigen-reactive TILs with knockout of the CISH gene, and a key secondary endpoint was anti-tumour activity measured as objective radiographic response and progression-free and overall survival. This study is registered with ClinicalTrials.gov, NCT04426669, and is complete.<br><br>FINDINGS: Between May 12, 2020, and Sept 16, 2022, 22 participants were enrolled in the trial (one patient was enrolled twice owing to lack of TIL outgrowth on the first attempt); ten patients were female, and 11 were male (self-defined). One patient was Asian, the remainder were White (self-defined). We successfully manufactured CISH knockout TIL products for 19 (86%) of the patients, of whom 12 (63%) received autologous CISH knockout TIL infusion. The median follow-up time for the study was 129 days (IQR 15-283). All 12 (100%) patients had treatment-related severe adverse events. The most common grade 3-4 adverse events included haematological events (12 patients [100%]) attributable to the preparative lymphodepleting chemotherapy regimen or expected effects of IL-2, fatigue (four patients [33%]), and anorexia (three patients [25%]). Deaths of any cause for patients on study were attributed to the underlying disease under study (metastatic gastrointestinal cancer) and related complications (10 patients) or infection (grade 5 septicaemia in one patient). There were no severe (&#x2265;grade 3) cytokine release or neurotoxicity events. Six (50%) of 12 patients had stable disease by day 28, and four (33%) had stable disease ongoing at 56 days. One young adult patient with microsatellite-instability-high colorectal cancer refractory to anti-PD1/CTLA-4 therapies had a complete and ongoing response (>21 months).<br><br>INTERPRETATION: These results support the safety and potential antitumour activity of inhibiting the immune checkpoint CISH through the administration of neoantigen-reactive CISH-knockout TILs, with implications for patients with advanced metastatic cancers refractory to checkpoint inhibitor immunotherapies, and provide the first evidence that a novel intracellular checkpoint can be targeted with therapeutic effect.<br><br>FUNDING: Intima Bioscience.}, }
@article {pmid40315825, year = {2025}, author = {Brück, M and Randau, L}, title = {TIGR on the loose: A dual-guide RNA system for DNA targeting.}, journal = {Molecular cell}, volume = {85}, number = {9}, pages = {1712-1713}, doi = {10.1016/j.molcel.2025.04.007}, pmid = {40315825}, issn = {1097-4164}, mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *DNA/genetics/metabolism ; *Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; }, abstract = {A recent study[1] unveils tandem interspaced guide RNAs (TIGRs) that simultaneously engage both strands of target DNA and direct Tas protein activity. It offers insights into the evolution of RNA-guided proteins and introduces a promising tool for genome editing.}, }
@article {pmid40313975, year = {2025}, author = {Zeng, S and Ju, Y and Alam, MS and Lu, Z and Hameed, HMA and Li, L and Tian, X and Fang, C and Fang, X and Ding, J and Wang, X and Hu, J and Wang, S and Zhang, T}, title = {A CRISPR-nonhomologous end-joining-based strategy for rapid and efficient gene disruption in Mycobacterium abscessus.}, journal = {mLife}, volume = {4}, number = {2}, pages = {169-180}, pmid = {40313975}, issn = {2770-100X}, abstract = {Mycobacterium abscessus, a fast-growing, non-tuberculous mycobacterium resistant to most antimicrobial drugs, causes a wide range of serious infections in humans, posing a significant public health challenge. The development of effective genetic manipulation tools for M. abscessus is still in progress, limiting both research and therapeutic advancements. However, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) systems have emerged as promising tools for generating highly specific double-strand breaks (DSBs) in its genome. One of the mechanisms that repair these DSBs is the error-prone nonhomologous end-joining (NHEJ) pathway, which facilitates targeted gene editing. In this study, we introduced a novel application of the CRISPR-NHEJ approach in M. abscessus. We demonstrated that NrgA from M. marinum plays a crucial role in repairing DSBs induced by the CRISPR-Cas system in M. abscessus. Contrary to previous findings, our study also revealed that inhibiting or overexpressing components of homologous recombination/single-strand annealing significantly reduces the efficiency of NHEJ repair in M. abscessus. This discovery challenges current perspectives and suggests that NHEJ repair in M. abscessus may involve components from both homologous recombination and single-strand annealing pathways, highlighting the complex interactions among the three DSB repair mechanisms in M. abscessus.}, }
@article {pmid40313468, year = {2025}, author = {Zhang, X and Wu, M and Shi, H and Kim, S and Lu, S and Wang, P and Qin, J}, title = {Amplification-Free Electrochemiluminescent Biosensor for Ultrasensitive Detection of Fusobacterium nucleatum Using Tetrahedral DNA-Based CRISPR/Cas12a.}, journal = {Cyborg and bionic systems (Washington, D.C.)}, volume = {6}, number = {}, pages = {0266}, pmid = {40313468}, issn = {2692-7632}, abstract = {Fusobacterium nucleatum, a bacterium linked to colorectal cancer, possesses a specific gene called fadA that serves as an early diagnostic biomarker. The CRISPR/Cas12a system has demonstrated marked potential for nucleic acid detection due to its satisfactory selectivity and trans-cleavage ability. However, most CRISPR/Cas-based sensors suffer from problems such as probe entanglement or local aggregation, reducing the Cas enzyme efficiency. In this study, an amplification-free biosensing platform for ultrasensitive detection of F. nucleatum was developed by integrating the highly specific CRISPR/AsCas12a with an improved electrochemiluminescence (ECL) biosensor. Different from the conventional 1- or 2-dimensional probes, the platform was constructed by tetrahedral DNA nanostructure (TDN) probes conjugated with quenchers and coralliform gold (CFAu) functionalized with luminescent agents. The TDN serves as an exceptional scaffold to modulate the recognition unit, substantially enhancing the recognition and cleavage efficiency of AsCas12a toward the probes. Furthermore, the high surface area of CFAu provides extensive landing sites for the luminescent agents, thereby improving the detection sensitivity. The prepared ECL biosensor exhibited a wider linear range (10 fM to 100 nM) and was capable of detecting F. nucleatum down to 1 colony-forming unit/ml. Additionally, the high mismatch sensitivity of AsCas12a to protospacer adjacent motifs and nearby areas provides a strategy for distinguishing mutant from wild-type sequences. Finally, by designing CRISPR RNA (crRNA), this diagnostic method can also be easily modified to detect other bacteria or biomarkers for the early diagnosis of various diseases.}, }
@article {pmid40313410, year = {2025}, author = {Liu, J and Zhou, Y and Feng, J and Cai, C and Zhang, S}, title = {Comparative metagenomic analysis reveals the adaptive evolutionary traits of siboglinid tubeworm symbionts.}, journal = {Frontiers in microbiology}, volume = {16}, number = {}, pages = {1533506}, pmid = {40313410}, issn = {1664-302X}, abstract = {Tubeworms flourish in marine cold seeps and hydrothermal vents through the establishment of symbiotic relationships with chemosynthetic bacteria. However, the environmental adaptations and evolutionary relationships of tubeworm symbionts across diverse habitats and hosts remain largely unknown. In this study, we characterized the genomes of 26 siboglinid tubeworm symbionts collected from deep-sea hydrothermal vents, cold seeps, and deep-sea mud, including two sequenced in this study and 24 previously published. Phylogenetic analysis classified the 26 symbiont genomes into five distinct clusters at the genus level. The findings highlight the remarkable diversity in symbiont classification, influenced by the habitat and species of tubeworm, with the symbiont genome characteristics of various genera revealing unique evolutionary strategies. Siboglinid symbionts exhibit functional metabolic diversity, encompassing chemical autotrophic capabilities for carbon, nitrogen, and sulfur metabolism, hydrogen oxidation, and a chemoorganotrophic ability to utilize various amino acids, cofactors, and vitamins. Furthermore, the symbiont's homeostatic mechanisms and CRISPR-Cas system are vital adaptations for survival. Overall, this study highlights the metabolic traits of siboglinid symbionts across different genera and enhances our understanding of how different habitats and hosts influence symbiont evolution, offering valuable insights into the strategies that symbionts use to adapt and thrive in extreme environments.}, }
@article {pmid40312543, year = {2025}, author = {Karan, R and Prasannakumar, MK and Khera, HK and Harish, J and Vamsidharreddy, N and Venkateshbabu, G and Devanna, P and Manjunatha, C and Palanna, KB and Mishra, RK}, title = {Molecular virulotyping and advancing the detection of Magnaporthe oryzae using a CRISPR-Cas12a-based diagnostic tool.}, journal = {World journal of microbiology &amp; biotechnology}, volume = {41}, number = {5}, pages = {159}, pmid = {40312543}, issn = {1573-0972}, mesh = {*Plant Diseases/microbiology ; *Oryza/microbiology ; Phylogeny ; *CRISPR-Cas Systems ; Genetic Variation ; DNA, Fungal/genetics ; Virulence/genetics ; *Ascomycota/genetics/pathogenicity/isolation &amp; purification/classification ; *Magnaporthe/genetics/isolation &amp; purification/pathogenicity/classification ; Plant Leaves/microbiology ; India ; Fungal Proteins/genetics ; }, abstract = {Rice blast, caused by Magnaporthe oryzae, is a major threat to global rice production. The disease affects various growth stages of rice including leaves, nodes, and panicles leading to significant yield loss. Understanding the genetic diversity and early detection of M. oryzae is essential for developing effective intervention and disease management. In current study, 30 isolates of M. oryzae were collected from major rice-growing areas in Karnataka, Tamil Nadu, Andhra Pradesh, and Telangana, revealing considerable morphological and genetic diversity. Molecular characterization using ITS, LSU, and actin primers confirmed all isolates as M. oryzae, showing 100% sequence homology to reference strains. Phylogenetic analysis revealed regional clustering of isolates, with notable diversity observed in populations from Tamil Nadu and Karnataka. Pathogenicity assays identified significant variability in disease severity, with isolates MOK1, MOTN4, and MOK10 being highly virulent. Virulotyping revealed the widespread presence of critical pathogenicity genes, with Avr-Pik, Avr-Pita, MPS1, SLP1 and TYR1 as prominent contributors. Disease severity is highly correlated with the presence of EXO 70. The tyrosinase gene remains well conserved among all the isolates and was used as a target for CRISPR cas12a based detection. A novel CRISPR-Cas12a detection system, coupled with recombinase polymerase amplification (RPA), demonstrated high sensitivity and specificity for M. oryzae DNA, detecting concentrations as low as 10[-1]/µL of DNA copies. Field testing successfully identified the pathogen in leaf, neck, and seed samples with visual confirmation via fluorescence and lateral flow assays. This integrated approach provides valuable insights into M. oryzae diversity, pathogenicity, and a robust diagnostic tool for early pathogen detection, paving the way for targeted management strategies to mitigate rice blast disease.}, }
@article {pmid40312015, year = {2025}, author = {Li, H and Li, C and Pedro, GC and Zhang, Q and Jiang, H and Yang, Y and Duan, Y and Sun, X}, title = {CRISPR/Cas9-mediated gene editing of MsCLE3a confers compact alfalfa architecture.}, journal = {Plant science : an international journal of experimental plant biology}, volume = {357}, number = {}, pages = {112532}, doi = {10.1016/j.plantsci.2025.112532}, pmid = {40312015}, issn = {1873-2259}, mesh = {*Medicago sativa/genetics/growth &amp; development/anatomy &amp; histology ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; *Plant Proteins/genetics/metabolism ; Plant Leaves/genetics/growth &amp; development/anatomy &amp; histology ; Gene Expression Regulation, Plant ; Brassinosteroids/metabolism ; }, abstract = {Alfalfa (Medicago sativa L.) is a globally major forage crop for livestock due to its superior nutritional value and palatability. As a premium forage species, alfalfa breeding has traditionally emphasized high yield. In this study, we designed guide RNA (gRNA) to target the coding sequence of the MsCLE3a gene, and constructed a CRISPR/Cas9 vector to generate knockouts. Five mutant lines were successfully isolated, displaying upright petioles and a compact ternately compound leaf phenotype during the seedling stage. Cryo-scanning electron microscopy (Cryo-SEM) was employed to analyze cell morphology on the abaxial leaf surface. The results indicated that the cells on the abaxial side of the Mscle3a mutants were significantly elongated compared to the wild type (WT). Transcriptomic analysis further uncovered the down-regulation of brassinosteroid (BR) biosynthesis genes in Mscle3a mutants, which might underlie the mechanisms regulating petiole angle. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed a major reduction in the transcript levels of several key genes involved in BR signaling pathways in the Mscle3a mutants relative to WT. This transcriptional down-regulation was strongly associated with the observed alterations in leaf petiole angle (LPA) in the mutant lines. Our findings provide novel insights into the novel regulatory role of MsCLE3a in alfalfa development and pinpoint potential targets for manipulating LPA in crop breeding programs.}, }
@article {pmid40311759, year = {2025}, author = {Soltani, S and Fallah, T and Shafiei, M and Shahraki, AH and Iranbakhsh, A}, title = {Investigating the prevalence of CRISPR-Cas system and their association with antibiotic resistance genes and virulence factors in Enterococcus faecalis and Enterococcus faecium strains isolated from hospitalized patients.}, journal = {Journal of global antimicrobial resistance}, volume = {43}, number = {}, pages = {344-357}, doi = {10.1016/j.jgar.2025.04.022}, pmid = {40311759}, issn = {2213-7173}, abstract = {OBJECTIVES: Enterococcus faecalis and Enterococcus faecium are Gram-positive opportunistic pathogens that rank among the leading causes of nosocomial infections worldwide. This study investigates the prevalence and role of CRISPR-Cas systems in modulating antimicrobial resistance and virulence factors in clinical isolates of E. faecalis and E. faecium collected from patients in Tehran, Iran.<br><br>METHODS: A total of 75 clinical isolates of E. faecalis and E. faecium were collected from various hospitals in Tehran, Iran, between January and April 2023, from adult patients with urinary tract infections (n = 55), blood infections (n = 12), and wound infections (n = 8). Conventional bacteriology tests and PCR were used to isolate and identify Enterococcus species. Phenotypic antibiotic and genotypic resistance were assessed. CRISPR-Cas repeat-spacer array were screened using PCR, and the relationship between CRISPR-Cas systems and antibiotic resistance and virulence genes was statistically analyzed. Phylogenetic, structural, and conservation analyses were performed to assess the degree of conservation in CRISPR1-Cas csn1 and CRISPR3-Cas csn1 genes, identify potential mutations, and evaluate their possible impact on Cas9 protein function.<br><br>RESULTS: 86.6% of the isolates harbored CRISPR-Cas repeat-spacer array, with a significantly higher prevalence in E. faecalis than in E. faecium (100% vs. 66.6%, P = 0.0001). CRISPR1-Cas, CRISPR2, and CRISPR3-Cas loci were identified in 76%, 82.6%, and 64% of isolates, respectively. Notably, the prevalence of CRISPR-Cas systems was significantly reduced in extensively drug-resistant (XDR) isolates (32%) compared to multidrug-resistant (MDR) isolates (68%, P = 0.0001). Conservation analyses of CRISPR1-Cas csn1 and CRISPR3-Cas csn1 genes revealed conserved regions potentially linked to functional activity. Furthermore, CRISPR-Cas repeat-spacer array were correlated with specific antimicrobial resistance phenotypes and genotypes, as well as with virulence factors.<br><br>CONCLUSIONS: These findings suggest that CRISPR-Cas systems may influence the resistance and virulence profiles of clinical Enterococcus isolates, potentially impacting their pathogenicity and adaptability.}, }
@article {pmid40311721, year = {2025}, author = {Liberty, JT and Bromage, S and Peter, E and Ihedioha, OC and Alsalman, FB and Odogwu, TS}, title = {CRISPR revolution: Unleashing precision pathogen detection to safeguard public health and food safety.}, journal = {Methods (San Diego, Calif.)}, volume = {240}, number = {}, pages = {180-194}, doi = {10.1016/j.ymeth.2025.04.018}, pmid = {40311721}, issn = {1095-9130}, mesh = {*Food Safety/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Food Microbiology/methods ; *Foodborne Diseases/microbiology/diagnosis/virology ; Public Health/methods ; Biosensing Techniques/methods ; *Bacteria/genetics/isolation &amp; purification/pathogenicity ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Gene Editing/methods ; }, abstract = {Foodborne pathogens represent a significant challenge to global food safety, causing widespread illnesses and economic losses. The growing complexity of food supply chains and the emergence of antimicrobial resistance necessitate rapid, sensitive, and portable diagnostic tools. CRISPR technology has emerged as a transformative solution, offering unparalleled precision and adaptability in pathogen detection. This review explores CRISPR's role in addressing critical gaps in traditional and modern diagnostic methods, emphasizing its advantages in sensitivity, specificity, and scalability. CRISPR-based diagnostics, such as Cas12 and Cas13 systems, enable rapid detection of bacterial and viral pathogens, as well as toxins and chemical hazards, directly in food matrices. Their integration with isothermal amplification techniques and portable biosensors enhances field applicability, making them ideal for decentralized and real-time testing. Additionally, CRISPR's potential extends beyond food safety, contributing to public health efforts by monitoring antimicrobial resistance and supporting One Health frameworks. Despite these advancements, challenges remain, including issues with performance in complex food matrices, scalability, and regulatory barriers. This review highlights future directions, including AI integration for assay optimization, the development of universal CRISPR platforms, and the adoption of sustainable diagnostic solutions. By tackling these challenges, CRISPR has the potential to redefine global food safety standards and create a more resilient food system. Collaborative research and innovation will be critical to fully unlocking its transformative potential in food safety and public health.}, }
@article {pmid40311478, year = {2025}, author = {Yang, J and Chen, J and Xia, L and Li, G}, title = {Recent progress on biosensors for detection of circulating miRNA biomarkers.}, journal = {Talanta}, volume = {294}, number = {}, pages = {128219}, doi = {10.1016/j.talanta.2025.128219}, pmid = {40311478}, issn = {1873-3573}, mesh = {*Biosensing Techniques/methods ; Humans ; *Circulating MicroRNA/blood/analysis/genetics ; Biomarkers/blood ; *MicroRNAs/blood ; Electrochemical Techniques ; }, abstract = {Circulating miRNAs are a class of non-coding endogenous RNAs found in body fluids which typically consist of 19-24 nucleotides in length. The abnormal expression of miRNAs has been demonstrated to be associated with severe human diseases. Aiming to provide valuable insights for the further development of reliable miRNA detectors for disease early diagnosis and treatment, this work systematically summarizes the latest advancements in signal amplification strategies for miRNA analysis, based on nanomaterials, nucleic acids, enzymes, and CRISPR/Cas system. The emerging techniques for detecting circulating miRNAs in human body fluids over the past decade are highlighted, including electrochemical, optical, and dual-mode biosensors. Furthermore, the challenges of trace miRNA detection in complex samples and the development prospects of miRNA biosensors are also discussed.}, }
@article {pmid40311445, year = {2025}, author = {Yu, Y and Liu, Y and Xu, N and Li, L and Yang, Y and Liu, X and Zhao, L and Bai, X}, title = {A CRISPR/Cas12a mediated click immunoassay catalyzed by in situ formation of clickase for highly sensitive detection of Trichinella spiralis.}, journal = {Biosensors &amp; bioelectronics}, volume = {283}, number = {}, pages = {117521}, doi = {10.1016/j.bios.2025.117521}, pmid = {40311445}, issn = {1873-4235}, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Trichinella spiralis/isolation &amp; purification/genetics ; Immunoassay/methods ; Limit of Detection ; Metal Nanoparticles/chemistry ; Gold/chemistry ; *Trichinellosis/diagnosis/parasitology ; Copper/chemistry ; Click Chemistry ; DNA, Single-Stranded/chemistry ; Meat/parasitology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {The accurate and high-throughput detection of trace protein targets remains an ongoing challenge in the field of food safety testing. This research article presents a highly sensitive CRISPR/Cas12a-mediated click immunoassay for the sensitive detection of Trichinella spiralis (T. spiralis) in meat samples. By simultaneously conjugating activator ssDNA and monoclonal antibodies to gold nanoparticles, the CRISPR/Cas12a system was introduced into the immunoassay. To overcome the challenges associated with the preparation, storage, and transportation of FQ probes in CRISPR/Cas12a systems, the Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction was employed instead. The designed ssDNA was both the substrate of trans-cleavage activity of Cas12a and the synthetic template of the artificial clicking enzyme copper nanoparticles (CuNPs), which could effectively catalyze the CuAAC reaction to generate the desired signal output. The fluorescence intensity showed a linear relationship with T. spiralis crude protein concentration ranging from 3.125 to 100 ng/mL, and the detection limit was 0.35 ng/mL, which is three orders of magnitude lower than that of ELISA (LOD: 309.75 ng/mL). This method can accurately detect a single T. spiralis larva in 100 g of pork. Collectively, the strategy of combining CRISPR/Cas12a system and CuAAC reaction opens up a novel avenue for developing a highly sensitive, simple and convenient fluorescence assay.}, }
@article {pmid40311328, year = {2025}, author = {Wang, S and Feng, Y and Xing, Q and Zhou, T and Liu, J}, title = {Generation of an anti-CD19 CAR knock-in human induced pluripotent stem cell line using CRISPR/Cas9 technology.}, journal = {Stem cell research}, volume = {86}, number = {}, pages = {103721}, doi = {10.1016/j.scr.2025.103721}, pmid = {40311328}, issn = {1876-7753}, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Antigens, CD19/immunology/metabolism ; *Gene Knock-In Techniques ; Cell Line ; Cell Differentiation ; *Receptors, Chimeric Antigen/genetics/metabolism ; }, abstract = {Chimeric antigen receptor T cell (CAR-T) therapy represents a major breakthrough in the field of tumor immunotherapy. CD19-targeted CAR-T cells (CD19 CAR-T) have emerged as an important therapeutic approach for treating B-cell malignancies. We successfully constructed a human induced pluripotent stem cell (iPSC) line with an anti-CD19 CAR knock-in using CRISPR/Cas9-mediated gene targeting technology. This cell line can stably express the CAR gene while maintaining its typical stem cell morphology and normal karyotype. Furthermore, this cell line possesses multilineage differentiation potential and can be directionally differentiated into various chimeric antigen receptor-expressing immune cells for targeting CD19-positive tumors.}, }
@article {pmid40311326, year = {2025}, author = {Bai, R and Fu, W and Hou, X and Wan, J}, title = {Generation of a MYL3 knockout stem cell line (WAe009-A-1H) by episomal vector-based CRISPR/Cas9 system.}, journal = {Stem cell research}, volume = {86}, number = {}, pages = {103723}, doi = {10.1016/j.scr.2025.103723}, pmid = {40311326}, issn = {1876-7753}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Myosin Light Chains/genetics/metabolism ; Cell Line ; Gene Knockout Techniques ; *Human Embryonic Stem Cells/metabolism/cytology ; Genetic Vectors/genetics/metabolism ; *Plasmids/genetics/metabolism ; }, abstract = {The Myosin light chain 3 (MYL3) gene encodes the ventricular essential light chain isoform, which is an important modulator of sarcomeric myosin cross-bridge kinetics. Variants in MYL3 are a cause of hypertrophic cardiomyopathy and dilated cardiomyopathy with cardiac failure and sudden cardiac death (SCD). To further elucidate the involvement of MYL3 in the pathogenesis of cardiomyopathies, we have created a MYL3 knockout human embryonic stem cell line using the CRISPR/Cas9 system. Notably, this MYL3-knockout cell line retains normal morphology, pluripotency, and karyotype. This resource provides a valuable tool for investigating MYL3-related health and disease.}, }
@article {pmid40311322, year = {2025}, author = {He, Z and Cole, KD and He, HJ}, title = {A novel immortalization method for immortalizing human primary CD8[+] T cells by inserting a single copy of human telomerase reverse transcriptase via CRISPR/Cas9.}, journal = {Tissue &amp; cell}, volume = {95}, number = {}, pages = {102908}, doi = {10.1016/j.tice.2025.102908}, pmid = {40311322}, issn = {1532-3072}, mesh = {Humans ; *Telomerase/genetics/metabolism ; *CD8-Positive T-Lymphocytes/cytology/metabolism ; *CRISPR-Cas Systems/genetics ; }, abstract = {BACKGROUND: Existing cell immortalization methods made the cells obtain oncogenesis phenotype and/or caused the cells gain and/or lose chromosomes. Immortalized normal human T cells lines provide critical in vitro models for basic research and therapeutic products development.<br><br>METHODS: We developed a novel method utilizing a CRISPR/Cas9 system to replace the exon 2 of the cell cycle inhibitor gene CDKN2A (encoding p16 and p14 proteins) with a single copy of human telomerase reverse transcriptase (hTERT) to immortalize human primary CD8[+] T cells (hCD8[+]T-TERT).<br><br>RESULTS: By using Cas9 protein and low donor DNA copies/cell, we successfully immortalized hCD8[+]T cells with a single copy of hTERT transgene, which also avoided uncontrolled insertion of Cas9 gene and guide RNA vector. Human primary CD8[+] cells from independent donors were immortalized and expanded more than 2.6&#x202f;&#xd7;&#x202f;10[7] times. Characterization of one of the immortalized CD8[+] T-TERT cell lines revealed that the cells retained most of the cell surface markers and normal karyotype. The CD8[+] T-TERT cells also retained the dependence of IL-2 and CD3/CD28 activator for survival and expansion.<br><br>CONCLUSION: We established a stable immortalized cell lines using the novel immortalization method, and the immortalized CD8[+] T cells had a phenotype consistent with T cells.}, }
@article {pmid40310889, year = {2025}, author = {Andreatta, F and Hendriks, D and Artegiani, B}, title = {Human Organoids as an Emerging Tool for Genome Screenings.}, journal = {Annual review of biomedical engineering}, volume = {27}, number = {1}, pages = {157-183}, doi = {10.1146/annurev-bioeng-103023-122327}, pmid = {40310889}, issn = {1545-4274}, mesh = {Humans ; *Organoids/metabolism/cytology ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Pluripotent Stem Cells/cytology/metabolism ; *Genome, Human ; *Genetic Testing/methods ; }, abstract = {Over the last decade, a plethora of organoid models have been generated to recapitulate aspects of human development, disease, tissue homeostasis, and repair. Organoids representing multiple tissues have emerged and are typically categorized based on their origin. Tissue-derived organoids are established directly from tissue-resident stem/progenitor cells of either adult or fetal origin. Starting from pluripotent stem cells (PSCs), PSC-derived organoids instead recapitulate the developmental trajectory of a given organ. Gene editing technologies, particularly the CRISPR-Cas toolbox, have greatly facilitated gene manipulation experiments with considerable ease and scalability, revolutionizing organoid-based human biology research. Here, we review the recent adaptation of CRISPR-based screenings in organoids. We examine the strategies adopted to perform CRISPR screenings in organoids, discuss different screening scopes and readouts, and highlight organoid-specific challenges. We then discuss individual organoid-based genome screening studies that have uncovered novel genes involved in a variety of biological processes. We close by providing an outlook on how widespread adaptation of CRISPR screenings across the organoid field may be achieved, to ultimately leverage our understanding of human biology.}, }
@article {pmid40310292, year = {2025}, author = {Alejandre-Sixtos, JE and Aguirre-Martínez, K and Cruz-López, J and Mares-Rivera, A and Álvarez-Martínez, SM and Zamorano-Sánchez, D}, title = {Insights on the regulation and function of the CRISPR/Cas transposition system located in the pathogenicity island VpaI-7 from Vibrio parahaemolyticus RIMD2210633.}, journal = {Infection and immunity}, volume = {93}, number = {6}, pages = {e0016925}, pmid = {40310292}, issn = {1098-5522}, support = {project no. IA203323//DGAPA-PAPIIT (UNAM)/ ; }, mesh = {*Vibrio parahaemolyticus/genetics/pathogenicity ; *CRISPR-Cas Systems/genetics ; *Genomic Islands/genetics ; *DNA Transposable Elements/genetics ; *Gene Expression Regulation, Bacterial ; Promoter Regions, Genetic ; Bacterial Proteins/genetics/metabolism ; Gene Transfer, Horizontal ; }, abstract = {CRISPR/Cas-mediated transposition is a recently recognized strategy for horizontal gene transfer in a variety of bacterial species. However, our understanding of the factors that control their function in their natural hosts is still limited. In this work, we report our initial genetic characterization of the elements associated with the CRISPR/Cas-transposition machinery (CASTm) from Vibrio parahaemolyticus (VpaCASTm), which are encoded within the pathogenicity island VpaI-7. Our results revealed that the components of the VpaCASTm and their associated CRISPR arrays (VpaCAST system) are transcriptionally active in their native genetic context. Furthermore, we were able to detect the presence of polycistrons and several internal promoters within the loci that compose the VpaCAST system. Our results also suggest that the activity of the promoter of the atypical CRISPR array is not repressed by the baseline activity of its known regulator VPA1391 in V. parahaemolyticus. In addition, we found that the activity of the promoter of tniQ was modulated by a regulatory cascade involving ToxR, LeuO, and H-NS. Since it was previously reported that the activity of the VpaCAST system was less efficient than that of the VchCAST system at promoting transposition of a miniaturized CRISPR-associated transposon (mini-CAST) in Escherichia coli, we analyzed if the transposition efficiency mediated by the VpaCAST system could be enhanced inside its natural host V. parahaemolyticus. We provide evidence that this might be the case, suggesting that there could be host induction factors in V. parahaemolyticus that could enable more efficient transposition of CASTs.IMPORTANCEMobile genetic elements such as transposons play important roles in the evolutionary trajectories of bacterial genomes. The success of transposon dissemination depends on their ability to carry selectable markers that improve the fitness of the host cell or loci with addictive traits such as the toxin-antitoxin systems. Here we aimed to characterize a transposon from Vibrio parahaemolyticus that carries and could disseminate multiple virulence factors. This transposon belongs to a recently discovered family of transposons whose transposition is guided by crRNA. We showed that the transposition machinery of this transposon is transcribed in V. parahaemolyticus and that there are likely host-associated factors that favor transposition in the natural host V. parahaemolyticus over transposition in Escherichia coli.}, }
@article {pmid40309116, year = {2025}, author = {Kinde, MZ and Kerisew, B and Eshetu, T and Gessese, AT}, title = {Genomic analysis of Listeria monocytogenes strains from dairy products in Ethiopia.}, journal = {Frontiers in bioinformatics}, volume = {5}, number = {}, pages = {1572241}, pmid = {40309116}, issn = {2673-7647}, abstract = {This study explored virulence genes, antibiotic resistance genes, and mobile genetic elements in 14 Listeria monocytogenes strains from milk and dairy products collected from different regions of Ethiopia. The strains were classified into two Multilocus Sequence Typing sequence types (ST2 and ST45) and further grouped into clonal complexes (CC2) and different cgMLST types. Twenty-nine virulence genes were identified across all 14 strains, with lplA1 detected at higher levels in all strains except SAMN28661660. All L. monocytogenes strains also carried four antibiotic resistance genes (fosX, lin, norB, mprF), contributing to their ability to withstand multiple antimicrobial agents. Notably, no plasmids or mobile genetic elements were detected. Stress resistance genes, including stress survival islet 1 (SSI1_lmo0447), lmo 1800, and lmo1799, were identified in all strains. However, genes encoding for disinfectant resistance were not identified from all strains. LGI-2 was found in all the strains and none of the studied strains harbored LGI-1 and LGI-3. Conserved CRISPR-Cas systems were found in some strains. KEGG pathway analysis revealed that inlA and inlB genes facilitate bacterial internalization through host actin polymerization. Overall, the study provided crucial insights into the genomic features of L. monocytogenes in the Ethiopian dairy chain. It is crucial to establish continuous monitoring of L. monocytogenes in dairy products, improve sanitation, enforce stricter antibiotic usage and food safety regulations, and raise public awareness of associated risks.}, }
@article {pmid40307869, year = {2025}, author = {Sitara, A and Hocq, R and Lu, AJ and Pflügl, S}, title = {Hi-TARGET: a fast, efficient and versatile CRISPR type I-B genome editing tool for the thermophilic acetogen Thermoanaerobacter kivui.}, journal = {Biotechnology for biofuels and bioproducts}, volume = {18}, number = {1}, pages = {49}, pmid = {40307869}, issn = {2731-3654}, support = {Y-1513//Austrian Science Fund/ ; }, abstract = {BACKGROUND: Due to its ability to grow fast on CO2, CO and H2 at high temperatures and with high energy efficiency, the thermophilic acetogen Thermoanaerobacter kivui could become an attractive host for industrial biotechnology. In a circular carbon economy, diversification and upgrading of C1 platform feedstocks into value-added products (e. g., ethanol, acetone and isopropanol) could become crucial. To that end, genetic and bioprocess engineering tools are required to facilitate the development of bioproduction scenarios. Currently, the genome editing tools available for T. kivui present some limitations in speed and efficiency, thus restricting the development of a powerful strain chassis for industrial applications.<br><br>RESULTS: In this study, we developed the versatile genome editing tool Hi-TARGET, based on the endogenous CRISPR Type I-B system of T.&#xa0;kivui. Hi-TARGET demonstrated 100% efficiency for gene knock-out (from both purified plasmid and cloning mixture) and knock-in, and 49% efficiency for creating point mutations. Furthermore, we optimized the transformation and plating protocol and increased transformation efficiency by 245-fold to 1.96&#x2009;&#xd7;&#x2009;10[4]&#x2009;&#xb1;&#x2009;8.7&#x2009;&#xd7;&#x2009;10[3]&#xa0;CFU&#xa0;&#x3bc;g[-1]. Subsequently, Hi-TARGET was used to demonstrate gene knock-outs (pyrE, rexA, hrcA), a knock-in (ldh::pFAST), a single nucleotide mutation corresponding to PolC[C629Y], and knock-down of the fluorescent protein pFAST. Analysis of the &#x2206;rexA deletion mutant created with Hi-TARGET revealed that the transcriptional repressor rexA is likely involved in the regulation of the expression of lactate dehydrogenase (ldh). Following genome engineering, an optimized curing procedure for edited strains was devised. In total, the time required from DNA to a clean, edited strain is 12&#xa0;days, rendering Hi-TARGET a fast, robust and complete method for engineering T.&#xa0;kivui.<br><br>CONCLUSIONS: The CRISPR-based genome editing tool Hi-TARGET developed for T. kivui can be used for scarless deletion, insertion, point mutation and gene knock-down, thus fast-tracking the generation of industrially-relevant strains for the production of carbon-negative chemicals and fuels as well as facilitating studies of acetogen metabolism and physiology.}, }
@article {pmid40307455, year = {2025}, author = {Chen, G and Hou, L and Li, Z and Xie, B and Liu, Y}, title = {A new strategy for Cas protein recognition based on graph neural networks and SMILES encoding.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {15236}, pmid = {40307455}, issn = {2045-2322}, mesh = {*Neural Networks, Computer ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; *Computational Biology/methods ; Databases, Protein ; Graph Neural Networks ; }, abstract = {The CRISPR-Cas system, an adaptive immune mechanism found in bacteria and archaea, has evolved into a promising genomic editing tool, with various types of Cas proteins playing a crucial role. In this study, we developed a set of strategies for mining and identifying Cas1 proteins. Firstly, we analyzed the characteristic differences of 14 types of Cas proteins in the protein large language model embedding space in detail; then converted proteins into the Simplified Molecular Input Line Entry System (SMILES) format, thereby constructing graph data representing atom and bond features. Next, based on the characteristic differences of different Cas proteins, we designed and trained an ensemble model composed of two Directed Message Passing Neural Network (DMPNN) models for high-precision identification of Cas1 proteins. This ensemble model performed excellently on both training data and newly designed datasets. The comparison of this method with other methods, such as CRISPRCasFinder, has demonstrated its effectiveness. Finally, the ensemble model was successfully employed to identify potential Cas1 proteins in the Ensemble database, further highlighting its robustness and practicality. The strategies and models from this research may potentially be extended to other types of Cas proteins, though this would require further investigation and validation. Moreover, our work highlights SMILES encoding as a versatile tool for studying biological macromolecules, enabling efficient structural representation and advanced computational applications in protein research and beyond.}, }
@article {pmid40307212, year = {2025}, author = {Wang, YY and Lin, JF and Wu, WW and Fu, Z and Cao, F and Chen, YX and Mo, HY and Sheng, H and Liu, ZX and Zeng, ZL and Guan, XY and Ju, HQ and Liao, K and Xu, RH}, title = {Inhibition of MBTPS1 enhances antitumor immunity and potentiates anti-PD-1 immunotherapy.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {4047}, pmid = {40307212}, issn = {2041-1723}, support = {82303306//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82173128//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {Animals ; Mice ; *Immunotherapy/methods ; Humans ; *Programmed Cell Death 1 Receptor/antagonists &amp; inhibitors/immunology ; Cell Line, Tumor ; *Colorectal Neoplasms/immunology/therapy/genetics ; STAT1 Transcription Factor/metabolism/genetics ; CD8-Positive T-Lymphocytes/immunology ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; Immune Checkpoint Inhibitors/pharmacology ; Female ; }, abstract = {Despite advances in cancer immunotherapy, colorectal cancer patients exhibit limited therapeutic responses. Therefore, the exploration of strategies combining immunotherapy with adjuvant approaches to enhance adaptive immune responses is in demand. Here, we perform a customized in vivo CRISPR-Cas9 screen to target genes encoding membrane and secreted proteins in CRC mouse models with different immune characteristics. We observe that loss of membrane-bound transcription factor site-1 protease (MBTPS1) in tumor cells enhances antitumor immunity and potentiates anti-PD-1 therapy. Mechanistic studies reveal that tumor cell-intrinsic MBTPS1 competes with USP13 for binding to STAT1, thereby disrupting the USP13-dependent deubiquitination-mediated STAT1 stabilization. The upregulated STAT1-transcribed chemokines including CXCL9, CXCL10, and CXCL11, promote CXCR3[+]CD8[+] T cell infiltration. Notably, the regulatory role of MBTPS1 in antitumor immunity operates independently of its classic function in cleaving membrane-bound transcription factors. Collectively, our results provide a theoretical basis for MBTPS1 as a potential immunotherapy target.}, }
@article {pmid40306523, year = {2025}, author = {Sui, B and Liu, C and Sun, Z and Zheng, Y and Zhou, W and Liu, H}, title = {CRISPR/Cas12a-based transition state molecular switch for low-background detection of HPV-16 on a microfluidic platform.}, journal = {International journal of biological macromolecules}, volume = {311}, number = {Pt 1}, pages = {143556}, doi = {10.1016/j.ijbiomac.2025.143556}, pmid = {40306523}, issn = {1879-0003}, mesh = {*Human papillomavirus 16/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; Humans ; Biosensing Techniques/methods ; *Lab-On-A-Chip Devices ; *Microfluidics/methods ; DNA, Viral/genetics ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Human papillomavirus type 16 (HPV-16) is a high-risk oncogenic subtype of HPV, strongly associated with the pathogenesis of multiple cancers. Researchers have developed many detection methods for HPV-16, among which, the detection method based on microfluidic has the characteristics of high efficiency and high sensitivity. However, non-specific adsorption remains a critical challenge, often leading to elevated background signals. Here, we propose an On-Chip assay integrated by transition state molecular switch based on CRISPR-Cas12a (OCTMS-CRISPR) for stable, sensitive, and low-background fluorescence detection of HPV-16. This system leverages a highly integrated molecular switch and Cas12a to perform dual-screening, while the dissociation products of the molecular switch activate trans-cleavage activity. Our data suggest that OCTMS-CRISPR suppresses background signals on microfluidic chip while maintaining the specificity and sensitivity of trans-cleavage. For demonstration, we detected five HPV subtypes and base mismatches at varying positions and quantities. The LOD can reach 7.64 pM (average fluorescence intensity) and 9.91fM (pixel counting), showing great potential in the field of biosensing and DNA chips.}, }
@article {pmid40306516, year = {2025}, author = {Bina, F and Bani, F and Khalilzadeh, B and Gheit, T and Karimi, A}, title = {Advancements in fluorescent nanobiosensors for HPV detection: from integrating nanomaterials to DNA nanotechnology.}, journal = {International journal of biological macromolecules}, volume = {311}, number = {Pt 1}, pages = {143619}, doi = {10.1016/j.ijbiomac.2025.143619}, pmid = {40306516}, issn = {1879-0003}, mesh = {*Biosensing Techniques/methods ; Humans ; *Nanotechnology/methods ; *Nanostructures/chemistry ; *Papillomaviridae/isolation &amp; purification/genetics ; *Papillomavirus Infections/diagnosis/virology ; }, abstract = {Human papillomavirus (HPV) is a leading cause of cervical cancer and other malignancies, necessitating the development of highly sensitive and specific detection tools. This review explores recent advancements in fluorescent nanobiosensors (FNBS) for HPV detection, focusing on the integration of nanomaterials and DNA nanotechnology, highlighting their contributions to improving sensitivity, specificity, and point-of-care (POC) usability. The review critically evaluates a range of nanomaterial-based FNBS, including those employing quantum and carbon dots, nanoclusters, nanosheets, and nanoparticles, discussing their underlying signal amplification mechanisms, target recognition strategies, and limitations related to toxicity, stability, and reproducibility. Furthermore, it examines the application of diverse DNA nanotechnology, such as DNA origami, DNAzyme, catalytic hairpin assembly (CHA), hybridization chain reaction (HCR), and DNA hydrogel in improving FNBS performance. It also addresses the current challenges in clinical translation, emphasizing the necessity for large-scale production methods and thorough clinical validation to ensure biosafety. It also outlines the potential of innovative technologies, such as CRISPR-Cas-based diagnostics and artificial intelligence, to further revolutionize HPV detection and enable accessible, cost-effective screening, particularly in resource-limited settings. This review provides a valuable resource for researchers and clinicians seeking to develop next-generation FNBS for improved HPV diagnostics and cervical cancer prevention.}, }
@article {pmid40305594, year = {2025}, author = {Sun, J and Xiong, X and Lai, W and Wu, Z and Wang, H and Yang, L and Xue, N and Yao, Q and Song, G and Zhao, Y and Li, L and Wang, F and Fan, C and Pei, H}, title = {Implementing complex nucleic acid circuits in living cells.}, journal = {Science advances}, volume = {11}, number = {18}, pages = {eadv6512}, pmid = {40305594}, issn = {2375-2548}, mesh = {Humans ; CRISPR-Cas Systems ; Gene Editing ; *Nucleic Acids/genetics/chemistry ; RNA, Messenger/genetics ; HEK293 Cells ; Synthetic Biology ; }, abstract = {Synthetic nucleic acid-based computing has demonstrated complex computational capabilities in vitro. However, translating these circuits into living cells remains challenging because of instability and cellular interference. We introduce an allosteric strand exchange (ASE) strategy for complex intracellular computing. Leveraging conformational cooperativity to regulate strand exchange, ASE offers a modular platform for designing intracellular circuits with flexible programmability. We engineer a scalable circuit architecture based on ASE that can execute AND and OR logic and scale to an eight-input expression. We demonstrate ASE-based circuits can detect messenger RNAs with high specificity in mammalian cells via AND logic computation. The capacity of ASE-based circuits to accept messenger RNAs as inputs enables integration of endogenous cellular information for efficient multi-input information processing, demonstrated by a multi-input molecular classifier monitoring key cell reprogramming events. Reprogramming ASE-based circuit to interface with CRISPR-Cas9 enables programmable control of Cas9-targeting activity for gene editing, highlighting their potential for advancing intracellular biocomputation.}, }
@article {pmid40305308, year = {2025}, author = {Nayyab, S and Gervasi, MG and Tourzani, DA and Shamailova, Y and Akizawa, H and Taghavi, M and Cui, W and Fissore, R and Salicioni, AM and Georg, GI and Snyder, E and Visconti, PE}, title = {Identification of TSSK1 and TSSK2 as Novel Targets for Male Contraception.}, journal = {Biomolecules}, volume = {15}, number = {4}, pages = {}, pmid = {40305308}, issn = {2218-273X}, support = {1R01HD038082-21A1/NH/NIH HHS/United States ; }, mesh = {Animals ; Male ; Mice ; Female ; Testis/metabolism ; Mice, Knockout ; Spermatogenesis/genetics ; *Protein Serine-Threonine Kinases/genetics/metabolism ; CRISPR-Cas Systems ; Spermatozoa/metabolism ; Sperm Motility ; *Contraception ; Infertility, Male/genetics ; }, abstract = {The testis-specific serine kinases (TSSKs) are post-meiotically expressed in testicular germ cells. Their testis-specific expression, together with their putative role in phosphorylation pathways, suggests that TSSKs have relevant roles in spermiogenesis, sperm function, or both. Independent Tssk3 and Tssk6 knockout mice, as well as the double Tssk1/Tssk2 KO males, are sterile. However, the double KO results are silent regarding the individual roles of TSSK1 and TSSK2. The aim of this study was to develop independent mutant mouse models of Tssk1 and Tssk2, using CRISPR/Cas9, to evaluate their independent roles in reproduction. Male heterozygous pups were used to establish one Tssk1 and two independent Tssk2 mutant lines. Natural mating mutant Tssk1 and Tssk2 homozygous males but not females were found to be sterile. Additionally, homozygous males have lower sperm numbers and decreased motility, and were infertile in vitro. Anti-TSSK2 antibodies were validated against Tssk2 mutants and used in Western blot and immunofluorescence experiments. TSSK2 is localized to the sperm head; importantly, it is present in the testes and sperm from Tssk1 mutant mice, confirming individual mutation. Our results indicate that both TSSK1 and TSSK2 are individually essential for male reproduction and support both kinases as suitable nonhormonal male contraceptive targets.}, }
@article {pmid40304259, year = {2025}, author = {Kim, J and Orozaliev, A and Sahloul, S and Van, AD and Dang, VT and Pham, VS and Oh, Y and Chehade, I and Al-Sayegh, M and Song, YA}, title = {Accelerating Cleavage Activity of CRISPR-Cas13 System on a Microfluidic Chip for Rapid Detection of RNA.}, journal = {Analytical chemistry}, volume = {97}, number = {18}, pages = {9858-9865}, pmid = {40304259}, issn = {1520-6882}, mesh = {*CRISPR-Cas Systems ; Humans ; *Lab-On-A-Chip Devices ; *MicroRNAs/analysis/genetics ; *Microfluidic Analytical Techniques ; SARS-CoV-2/genetics ; }, abstract = {It is extremely advantageous to detect nucleic acid levels in the early phases of disease management; such early detection facilitates timely treatment, and it can prevent altogether certain cancers and infectious diseases. A simple, rapid, and versatile detection platform without enzymatic amplification for both short and long sequences would be highly desirable in this regard. Our study addresses this need by introducing IMACC, an ICP-based Microfluidic Accelerator Combined with CRISPR, for amplification-free nucleic acid detection. It exploits electrokinetically induced ion concentration polarization (ICP) to concentrate target nucleic acids and CRISPR reagents near the depletion zone boundary within a microfluidic channel. This localized accumulation accelerates the CRISPR-guided promiscuous cleavage of reporter molecules while enhancing their fluorescence signals simultaneously. Simultaneous accumulation of RNA and ribonucleoproteins (RNP) in confined spaces was validated experimentally and numerically, showing overlapping regions. IMACC enabled detection of miRNA-21 (22 bp) down to 10 pM within 2 min of ICP. IMACC ensured CRISPR specificity (single mismatch (N = 1) sensitivity) during ICP, as shown by off-target and mismatch sequence experiments. IMACC was applied to long RNA samples (i.e., SARS-CoV-2), but it statistically remained challenging at this point due to nonlinear intensity trends with copy numbers and large deviations. IMACC enabled rapid detection of short RNAs such as microRNAs using only basic CRISPR reagents in a single microfluidic channel, eliminating the need for extra enzymes or buffer sets, streamlining workflow and reducing turnaround time. IMACC has the potential to advance CRISPR diagnostics and holds promise for improved detection and future prescreening applications.}, }
@article {pmid40303110, year = {2024}, author = {Jin, K and Huang, P and Li, B and Cao, Z and Huang, Z and Zhang, Z and Liu, M and Li, H and Niu, L and Zhang, T and Li, Y and Li, X and Wang, H and Zhang, H}, title = {A Single-Copy Sensitive and Field-Deployable One-Pot RT-RPA CRISPR/Cas12a Assay for the Specific Visual Detection of the Nipah Virus.}, journal = {Transboundary and emerging diseases}, volume = {2024}, number = {}, pages = {4118007}, pmid = {40303110}, issn = {1865-1682}, mesh = {*Nipah Virus/isolation &amp; purification/genetics ; Animals ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Henipavirus Infections/virology/diagnosis/veterinary ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Humans ; }, abstract = {Nipah virus (NiV) is an emerging bat-borne zoonotic virus that can be transmitted to humans and other animals through infected bats or contaminated foods. The disease is highly lethal in humans (40%-75%) and has the potential for human-to-human transmission. Currently, there are no approved treatments or vaccines for NiV infection in humans or animals. Consequently, there is a pressing need for a highly sensitive, precise, and visually detectable assay to enable early intervention and mitigate the transmission of NiV infection. Here, we report a single-copy sensitive, field-deployable, one-pot visual reverse transcription-recombinase polymerase amplification (RT-RPA)-clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associate system (Cas)12 for the detection of NiV. The assay works by targeting the N gene of NiV, and the results are directly visible to the naked eye. The assay has demonstrated the ability to detect as few as 5.5 copies/μl of positive plasmids or 5.5 × 10[1] copies/μl of RNA transcripts when reacted at constant temperature for 40 min. It showed high specificity for NiV and had no cross-reaction with other pathogens, including rabies virus (RABV), Japanese encephalitis virus (JEV), herpes simplex virus type 1 (HSV-1), Hendra virus (HeV), and Streptococcus suis (S. suis), that can cause clinical symptoms similar to those of NiV infection. Moreover, this assay had a 100% coincidence rate with the reverse transcription quantitative polymerase chain reaction (RT-qPCR) method recommended by the World Organization for Animal Health (WOAH) for the detection of simulated clinical samples, indicating that it has great potential as an ultrasensitive, simple, and portable novel assay for the onsite diagnosis of NiV infection.}, }
@article {pmid40303064, year = {2024}, author = {Wu, M and Chen, M and Qiu, R and Ge, L and Fan, Z and Hu, B and Wei, H and Li, Y and Wang, F and Song, Y}, title = {Specific Detection of RHDV GI.1 and GI.2 by RT-LAMP-CRISPR/Cas12a Platform.}, journal = {Transboundary and emerging diseases}, volume = {2024}, number = {}, pages = {3881457}, pmid = {40303064}, issn = {1865-1682}, mesh = {Animals ; *Caliciviridae Infections/veterinary/virology/diagnosis/epidemiology ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *Hemorrhagic Disease Virus, Rabbit/isolation &amp; purification/genetics/classification ; Rabbits ; Sensitivity and Specificity ; *Molecular Diagnostic Techniques/veterinary/methods ; CRISPR-Cas Systems ; China/epidemiology ; }, abstract = {Rabbit hemorrhagic disease is a highly contagious and acute fatal disease caused by rabbit hemorrhagic disease virus (RHDV). The first outbreak of RHDV2 in 2020 has posed a serious threat to the rabbit breeding industry in China. An effective and specific detection strategy for RHDV GI.1 (RHDV1) and GI.2 (RHDV2) is urgently needed. In this study, we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP)-CRISPR/Cas12a-based dual readout portable detection platform. The platform showed excellent specificity to identify RHDV1 and RHDV2 strains and no cross-reaction with other prevalent pathogens of rabbit. The detection limit for RHDV1 and RHDV2 by RT-LAMP-CRISPR/Cas12a could reach 10 copies/μl of the VP60 gene per reaction. Furthermore, 74 clinical samples were detected for both RHDV1 and RHDV2. RT-LAMP-CRISPR/Cas12a-based dual readout portable detection platform showed 25.68% (19/74) RHDV1-positive samples, 43.24% (32/74) RHDV2-positive samples, and 8.11% (6/74) RHDV1/RHDV2 double positive samples, respectively. The coincidence rates of detection RHDV1 and RHDV2 between RT-LAMP-CRISPR/Cas12a and quantitative real-time-polymerase chain reaction (qPCR) were both 97.30%. RT-LAMP-CRISPR/Cas12a showed higher sensitivity and detection rate compared with qPCR. Moreover, the results were visible to the naked eye within 1.5 h combined with lateral flow strips (LFSs) and visual fluorescence. The RT-LAMP-CRISPR/Cas12a portable platform has the advantages of high sensitivity, specificity, fast, low equipment requirements, which can be used in clinical practice in rural areas and resource-limited settings.}, }
@article {pmid40303012, year = {2024}, author = {Kan, X and Wu, Y and Zhang, X and Zeng, Z and Lou, C and Wu, L and Huang, X and Wang, X and Huang, B}, title = {Palm Multidiagnostic of Mycoplasma pneumoniae, Chlamydia pneumoniae, Haemophilus influenzae, and Streptococcus pneumoniae Using One-Tube CRISPR/Cas12a.}, journal = {Transboundary and emerging diseases}, volume = {2024}, number = {}, pages = {5002521}, pmid = {40303012}, issn = {1865-1682}, mesh = {*Mycoplasma pneumoniae/isolation &amp; purification/genetics ; *Haemophilus influenzae/isolation &amp; purification/genetics ; *Streptococcus pneumoniae/isolation &amp; purification/genetics ; Humans ; *Chlamydophila pneumoniae/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; *Pneumonia, Mycoplasma/diagnosis/microbiology ; *Haemophilus Infections/diagnosis/microbiology ; Respiratory Tract Infections/diagnosis/microbiology ; }, abstract = {The recent high incidence of Mycoplasma pneumoniae (Mp) infections has raised widespread public health concerns. Therefore, rapid and accurate diagnosis of respiratory pathogenic microbial infections is of paramount importance to provide clinicians with accurate diagnostic insights and guide clinical medication. In response to this urgent need, we developed a one-tube Palm CRISPR/Cas12a Diagnostic (PaCD) method. This method facilitates the rapid detection of Mp infections, as well as three other prevalent respiratory pathogens, Chlamydia pneumoniae (Cp), Haemophilus influenzae (Hi), and Streptococcus pneumoniae (SP). In addition, 3D printing was employed to fabricate a compact detection device that includes a temperature control module set at 39°C and a blue light irradiation module, significantly enhancing the feasibility of point-of-care testing. The PaCD diagnostic process takes only 30 min with a detection limit of 50 copies/test, making it suitable for analysis of sputum and throat swab samples. PaCD demonstrated 100% concordance (72/72) with next-generation sequencing and exhibited high concordance with computed tomography test results. These findings demonstrate the clinical feasibility of PaCD for the rapid and accurate diagnosis of infections caused by four prevalent respiratory pathogens, offering theoretical insights into the versatile application of point-of-care tests for the detection of other respiratory pathogens in various clinical scenarios.}, }
@article {pmid40302337, year = {2025}, author = {Sobah, ML and Liongue, C and Ward, AC}, title = {Socs3a is Dispensable for Zebrafish Hematopoiesis and is Required for Neuromast Formation.}, journal = {Frontiers in bioscience (Landmark edition)}, volume = {30}, number = {4}, pages = {36537}, doi = {10.31083/FBL36537}, pmid = {40302337}, issn = {2768-6698}, mesh = {Animals ; *Zebrafish/genetics/embryology/metabolism ; *Hematopoiesis/genetics ; *Suppressor of Cytokine Signaling 3 Protein/genetics/metabolism ; *Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; }, abstract = {BACKGROUND: Suppressor of cytokine signaling (SOCS)3 is a regulatory protein that participates in an important negative feedback loop downstream of several critical cytokines, especially members of the interleukin-6 (IL-6) family. As a result, SOCS3 has been shown to impact the development and function of blood and immune cells. Zebrafish harbor duplicates of SOCS3, Socs3a and Socs3b, both of which possess conserved functional domains.<br><br>METHODS: This study explored the role of zebrafish Socs3a by creating a whole genome knockout using CRISPR/Cas9, with a focus on hematopoiesis and neuromast formation.<br><br>RESULTS: A zebrafish Socs3a knockout mutant was successfully generated. Characterization of this mutant revealed that normal hematopoiesis was not impacted nor was neutrophils lacking Socs3a displayed normal responses to injury or their production during emergency granulopoiesis. Neuromast formation was severely impacted in Socs3a knockout zebrafish.<br><br>CONCLUSIONS: Zebrafish Socs3a mutants display normal hematopoiesis and myeloid function, but the formation of the lateral line neuromast was affected by the absence of Socs3a.}, }
@article {pmid40302206, year = {2025}, author = {Drebes Dörr, NC and Lemopoulos, A and Blokesch, M}, title = {Exploring Mobile Genetic Elements in Vibrio cholerae.}, journal = {Genome biology and evolution}, volume = {17}, number = {5}, pages = {}, pmid = {40302206}, issn = {1759-6653}, support = {310030_185022/SNSF_/Swiss National Science Foundation/Switzerland ; 724630/ERC_/European Research Council/International ; 55008726/HHMI/Howard Hughes Medical Institute/United States ; }, mesh = {*Vibrio cholerae/genetics ; *Interspersed Repetitive Sequences ; Genome, Bacterial ; Cholera/microbiology ; Bacteriophages/genetics ; Evolution, Molecular ; }, abstract = {Members of the bacterial species Vibrio cholerae are known both as prominent constituents of marine environments and as the causative agents of cholera, a severe diarrheal disease. While strains responsible for cholera have been extensively studied over the past century, less is known about their environmental counterparts, despite their contributions to the species' pangenome. This study analyzed the genome compositions of 46 V. cholerae strains, including pandemic and nonpandemic, toxigenic, and environmental variants, to investigate the diversity of mobile genetic elements (MGEs), embedded bacterial defense systems, and phage-associated signatures. Our findings include both conserved and novel MGEs across strains, pointing to shared evolutionary pathways and ecological niches. The defensome analysis revealed a wide array of antiphage/antiplasmid mechanisms, extending well beyond the traditional CRISPR-Cas and restriction-modification systems. This underscores the dynamic arms race between V. cholerae and MGEs and suggests that nonpandemic strains may act as reservoirs for emerging defense strategies. Moreover, the study showed that MGEs are integrated into genomic hotspots, which may serve as critical platforms for the exchange of defense systems, thereby enhancing V. cholerae's adaptive capabilities against phage attacks and other invading MGEs. Overall, this research offers new insights into V. cholerae's genetic complexity and potential adaptive strategies, offering a better understanding of the differences between environmental strains and their pandemic counterparts, as well as the possible evolutionary pathways that led to the emergence of pandemic strains.}, }
@article {pmid40302137, year = {2025}, author = {Cañizares, E and Giovannini, L and Gumus, BO and Fotopoulos, V and Balestrini, R and González-Guzmán, M and Arbona, V}, title = {Seeds of Change: exploring the transformative effects of seed priming in sustainable agriculture.}, journal = {Physiologia plantarum}, volume = {177}, number = {3}, pages = {e70226}, pmid = {40302137}, issn = {1399-3054}, support = {CNS2023-143759//Agencia Estatal de Investigaci&#xf3;n/ ; PCI2021-121920//Agencia Estatal de Investigaci&#xf3;n/ ; PID2020-118126RB-I00//Agencia Estatal de Investigaci&#xf3;n/ ; PID2023-150080OB-I00//Agencia Estatal de Investigaci&#xf3;n/ ; EXCELLENCE/0421/0462//research and innovation foundation of cyprus/ ; CIGRIS/2021/014//Conselleria de Innovaci&#xf3;n, Universidades, Ciencia y Sociedad Digital, Generalitat Valenciana/ ; DD 16302//Ministero dell'Universit&#xe0; e della Ricerca/ ; }, mesh = {*Seeds/physiology/genetics ; *Agriculture/methods ; Germination/physiology ; Climate Change ; Crops, Agricultural/genetics ; Plants, Genetically Modified ; }, abstract = {The threats posed by climate change on agriculture at a global scale have fostered researchers to explore new and efficient strategies to ensure stable and safe food production. These new strategies must not only be efficient in reducing yield loss but also comply with environmental and consumer safety regulations, which particularly refer to restrictions to pesticide application as well as the implementation of genetically modified organisms, including CRISPR/Cas edited lines. Among other approaches, priming constitutes an easier and relatively cheaper strategy to cope with the effects of abiotic and biotic stresses by boosting plants' endogenous potential. Particularly, pre-sowing seed priming has proven effective in improving germination and seedling establishment as well as tolerance to environmental and biotic factors throughout the plant's life cycle, exhibiting clear long-lasting effects. This tolerance response to a wide range of adverse factors is associated with physiological, metabolic and genetic mechanisms and responses at the seed level and subsequently in the established plant. The genetic and epigenetic mechanisms enabling this tolerance response in plants and their subsequent generation, as a transgenerational effect, will be reviewed. Finally, the potential of the different seed priming approaches contributing to an ecologically and economically more sustainable agriculture will be discussed.}, }
@article {pmid40301471, year = {2025}, author = {Shin, KC and Hasan, W and Ali, G and Abdelrahman, D and Abuarja, T and Stanton, LW and Da'as, SI and Park, Y}, title = {Seizure-like behavior and hyperactivity in napb knockout zebrafish as a model for autism and epilepsy.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {14579}, pmid = {40301471}, issn = {2045-2322}, mesh = {Animals ; Zebrafish/genetics ; *Autistic Disorder/genetics/physiopathology ; Disease Models, Animal ; *Epilepsy/genetics/physiopathology ; *Seizures/genetics/physiopathology ; *Zebrafish Proteins/genetics ; Behavior, Animal ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Humans ; }, abstract = {We identified N-ethylmaleimide-sensitive factor attachment protein beta (NAPB) as a potential risk gene for autism and epilepsy. Notably, Qatari monozygotic triplets with loss of function mutations in NAPB exhibit early onset epileptic encephalopathy and varying degrees of autism. In this study, we generated NAPB zebrafish model using CRISPR-Cas9-sgRNAs technology for gene editing of the two orthologs napba and napbb. We observed that napb crispants (CR) show shorter motor neuron axons length together with altered locomotion behavior, including significant increases in larvae total distance traveled, swimming velocity, and rotation frequency, indicating that these behavioral changes effectively mimic the human epileptic phenotype. We applied microelectrode array (MEA) technology to monitor neural activity and hyperactivity in the zebrafish model. The napb CR shows hyperexcitability in the brain region. By combining behavioral tests with electrophysiological MEA assays, the established NAPB zebrafish model can be employed to study the pathophysiological mechanisms of ASD and epilepsy to screen potential therapeutic drugs.}, }
@article {pmid40301362, year = {2025}, author = {Vlasova, KY and Kerr, A and Pennock, ND and Jozic, A and Sahel, DK and Gautam, M and Murthy, NTV and Roberts, A and Ali, MW and MacDonald, KD and Walker, JM and Luxenhofer, R and Sahay, G}, title = {Synthesis of ionizable lipopolymers using split-Ugi reaction for pulmonary delivery of various size RNAs and gene editing.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {4021}, pmid = {40301362}, issn = {2041-1723}, support = {R01 CA270783/CA/NCI NIH HHS/United States ; R01CA270783-01//U.S. Department of Health &amp; Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; R01HL146736//U.S. Department of Health &amp; Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; SAHAY19XX0//Cystic Fibrosis Foundation (CF Foundation)/ ; R01 HL146736/HL/NHLBI NIH HHS/United States ; }, mesh = {Animals ; *Gene Editing/methods ; Mice ; Humans ; *Lung/metabolism ; Nanoparticles/chemistry ; RNA, Messenger/administration &amp; dosage/genetics/chemistry ; Mice, Knockout ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; *Gene Transfer Techniques ; *Polymers/chemistry/chemical synthesis ; Mice, Inbred C57BL ; Genetic Therapy/methods ; Lipids/chemistry ; CRISPR-Cas Systems ; }, abstract = {We present an efficient method for synthesizing cationic poly(ethylene imine) derivatives using the multicomponent split-Ugi reaction to create a library of functional ionizable lipopolymers. Here we show 155 polymers, formulated into polyplexes, to establish structure-activity relationships essential for endosomal escape and transfection. A lead structure is identified, and lipopolymer-lipid hybrid nanoparticles are developed to deliver mRNA to lung endothelium and immune cells, including T cells, with low in vivo toxicity. These nanoparticles show significant improvements in mRNA delivery to the lung compared to in vivo-JetPEI® and demonstrate effective delivery of therapeutic mRNA(s) of various sizes. IL-12 mRNA-loaded nanoparticles delay Lewis Lung cancer progression, while human CFTR mRNA restores CFTR protein function in CFTR knockout mice. Additionally, we demonstrate in vivo CRISPR-Cas9 mRNA delivery, achieving gene editing in lung tissue and successful PD-1 knockout in T cells in mice. These results highlight the platform's potential for systemic gene therapy delivery.}, }
@article {pmid40301361, year = {2025}, author = {Xing, C and Zhang, C and Xu, Z and Wang, Y and Lu, W and Liu, X and Zhang, Y and Ma, J and Yang, S and Du, Y and Xu, G and Liu, Y}, title = {Genome-wide CRISPR screening identifies LRP1 as an entry factor for SFTSV.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {4036}, pmid = {40301361}, issn = {2041-1723}, support = {81772203//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82302524//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {Animals ; *Low Density Lipoprotein Receptor-Related Protein-1/genetics/metabolism/antagonists &amp; inhibitors ; *Virus Internalization/drug effects ; Mice ; Humans ; Male ; *Phlebovirus/physiology/genetics ; Fibroblasts/virology/metabolism ; Antibodies, Neutralizing/pharmacology ; HEK293 Cells ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease caused by the SFTS virus (SFTSV), which has high mortality rates and poses a significant threat to public health. To identify potential therapeutic targets against SFTSV, we conduct genome-wide knockout screening, which identifies the previously known host factor CCR2, and reveals prolow-density lipoprotein receptor-related protein 1 (LRP1) as an entry factor for SFTSV. Knockdown or knockout of LRP1 significantly attenuate SFTSV infection in mouse embryonic fibroblasts (MEFs). Additionally, inhibition of LRP1 suppresses SFTSV pseudovirus infection in MEFs, suggesting its role in viral entry. The interaction between the SFTSV glycoprotein Gn and LRP1 via the CLI and CLII domains is revealed by co-IP and surface plasmon resonance (SPR). Moreover, LRP1 antagonists and neutralizing antibodies effectively attenuate SFTSV infection in MEFs. Administration of an LRP1-neutralizing antibody in a lethal male mouse model reduces the viral load, mitigates tissue damage, and improves survival. This study identifies LRP1 as a host entry receptor for SFTSV, providing a target for therapeutic strategy development.}, }
@article {pmid40301355, year = {2025}, author = {Liang, X and Gupta, D and Xie, J and Van Wonterghem, E and Van Hoecke, L and Hean, J and Niu, Z and Ghaeidamini, M and Wiklander, OPB and Zheng, W and Wiklander, RJ and He, R and Mamand, DR and Bost, J and Zhou, G and Zhou, H and Roudi, S and Estupiñán, HY and Rädler, J and Zickler, AM and Görgens, A and Hou, VWQ and Slovak, R and Hagey, DW and de Jong, OG and Uy, AG and Zong, Y and Mäger, I and Perez, CM and Roberts, TC and Carter, D and Vader, P and Esbjörner, EK and de Fougerolles, A and Wood, MJA and Vandenbroucke, RE and Nordin, JZ and El Andaloussi, S}, title = {Engineering of extracellular vesicles for efficient intracellular delivery of multimodal therapeutics including genome editors.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {4028}, pmid = {40301355}, issn = {2041-1723}, mesh = {*Extracellular Vesicles/metabolism/genetics ; Animals ; *Gene Editing/methods ; Mice ; Humans ; Integrases/genetics/metabolism ; CRISPR-Cas Systems ; CRISPR-Associated Protein 9/metabolism ; Inflammation/chemically induced/therapy ; HEK293 Cells ; NF-kappa B/metabolism ; *Drug Delivery Systems/methods ; }, abstract = {Intracellular delivery of protein and RNA therapeutics represents a major challenge. Here, we develop highly potent engineered extracellular vesicles (EVs) by incorporating bio-inspired attributes required for effective delivery. These comprise an engineered mini-intein protein with self-cleavage activity for active cargo loading and release, and fusogenic VSV-G protein for endosomal escape. Combining these components allows high efficiency recombination and genome editing in vitro following EV-mediated delivery of Cre recombinase and Cas9/sgRNA RNP cargoes, respectively. In vivo, infusion of a single dose Cre loaded EVs into the lateral ventricle in brain of Cre-LoxP R26-LSL-tdTomato reporter mice results in greater than 40% and 30% recombined cells in hippocampus and cortex respectively. In addition, we demonstrate therapeutic potential of this platform by showing inhibition of LPS-induced systemic inflammation via delivery of a super-repressor of NF-ĸB activity. Our data establish these engineered EVs as a platform for effective delivery of multimodal therapeutic cargoes, including for efficient genome editing.}, }
@article {pmid40300704, year = {2025}, author = {Tarin, M and Saljooghi, AS}, title = {Gasdermin E as a potential target and biomarker for CRISPR-Cas9-based cancer therapy.}, journal = {Biochemical pharmacology}, volume = {237}, number = {}, pages = {116961}, doi = {10.1016/j.bcp.2025.116961}, pmid = {40300704}, issn = {1873-2968}, mesh = {Humans ; *CRISPR-Cas Systems/physiology/genetics ; *Neoplasms/therapy/genetics/metabolism ; *Biomarkers, Tumor/metabolism/genetics/antagonists &amp; inhibitors ; Animals ; Pyroptosis/physiology/drug effects ; Gene Editing/methods ; Gasdermins ; }, abstract = {Gasdermin E (GSDME), a protein pivotal in mediating pyroptosis, has gained significant attention due to its role in cancer pathogenesis and its potential as a therapeutic target. The advent of CRISPR-Cas9, a precise genome editing tool, has revolutionized cancer therapy by enabling the manipulation of GSDME expression and function. This review explores the interplay of GSDME and CRISPR-Cas9 in cancer, emphasizing GSDME's unique mechanism of cleavage-dependent pore formation in the cell membrane and its emerging applications as both a therapeutic target and a diagnostic biomarker. We discuss the potential and challenges of using GSDME-induced pyroptosis as a therapeutic strategy and how can enhance its efficacy and specificity. We conclude by highlighting promising future research directions in this emerging field.}, }
@article {pmid40300480, year = {2025}, author = {Richard, C and Allais-Bonnet, A and André, M and Gelin, V and Deloche, MC and Thépot, D and Canon, E and Laffont, L and Dubois, O and Congar, P and Lasserre, O and Aguirre-Lavin, T and Gomot, G and Moussu, C and Barbey, S and Pailhoux, E and Dardente, H}, title = {CRISPR-Cas9 gene-editing to assess the role of RF-amide-related peptide 3 in ovine seasonal breeding.}, journal = {Animal : an international journal of animal bioscience}, volume = {19}, number = {5}, pages = {101508}, doi = {10.1016/j.animal.2025.101508}, pmid = {40300480}, issn = {1751-732X}, mesh = {Animals ; Seasons ; Female ; *CRISPR-Cas Systems ; *Gene Editing/veterinary ; Sheep/genetics/physiology ; *Neuropeptides/genetics/metabolism ; Male ; Breeding ; Reproduction/genetics ; }, abstract = {Seasonal breeding is an adaptive strategy that ensures the birth of the offspring during the best time of year and allows energy saving in times of food scarcity and adverse environmental conditions. At temperate and polar latitudes, photoperiod is the main synchroniser of seasonal functions, which exerts its action through melatonin. Within the pars tuberalis of the anterior pituitary, melatonin triggers a series of events that lead to enhanced local triiodothyronine (T3) production in the medio-basal hypothalamus specifically under long days and ultimately drive the appropriate GnRH output at the median eminence. How T3 governs GnRH output is mostly unknown but neuronal populations that respectively produce KISS1 and RFRP3 appear to be involved. However, while the role of KISS1 as a major GnRH secretagogue is undisputed, the function of RFRP3 in the control of (seasonal) breeding remains enigmatic, with conflicting results hinting at elusive mechanisms. Therefore, we launched an extensive series of experiments in sheep, aimed at invalidating the NPVF gene, which encodes RFRP3, using CRISPR-Cas9 technology. Here, we report on the generation of six sheep for which the NPVF gene has been successfully edited. Four of these animals bear at least one allele expected to behave as a null and constitute founders for distinct ovine lines. These founder sheep will now enter a standard breeding scheme in order to obtain male and female homozygotes for distinct mutations. These animals are expected to provide a clear delineation of the function of RFRP3 in seasonal breeding.}, }
@article {pmid40300474, year = {2025}, author = {Zhu, T and Jiang, W and Wu, Y and Fang, R and Deng, F and Yang, D}, title = {Advances in CRISPR/Cas13a-based biosensors for non-coding RNA detection.}, journal = {Talanta}, volume = {294}, number = {}, pages = {128223}, doi = {10.1016/j.talanta.2025.128223}, pmid = {40300474}, issn = {1873-3573}, mesh = {*Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *RNA, Untranslated/analysis/genetics ; }, abstract = {Non-coding RNAs play crucial roles in disease initiation and progression, making them promising biomarkers for early diagnosis and treatment monitoring. Conventional nucleic acid diagnostic methods, including polymerase chain reaction (PCR), next-generation sequencing (NGS), and enzyme-linked immunosorbent assay (ELISA), alongside emerging techniques such as single-molecule fluorescence in situ hybridization (smFISH), nanopore sequencing, and single-cell RNA sequencing (scRNA-seq), face inherent limitations in detecting regulatory non-coding RNAs. These challenges include laborious workflows, prolonged processing times, and technical complexities, hindering their broad applicability in rapid and high-throughput RNA analysis. CRISPR/Cas13a-based biosensors, integrated with various signal transduction systems-such as fluorescence, electrochemistry, colorimetry, surface-enhanced Raman spectroscopy (SERS)-show great promise for real-world diagnostic applications. This review provides a comprehensive overview of the CRISPR/Cas13a-mediated RNA detection mechanism, the development of CRISPR/Cas13a-based biosensors, and their integration with innovative signal detection methods. Additionally, we highlight the progress in portable detection devices, including lateral flow assay strips and smartphone-based platforms. Finally, the review discusses the current challenges and future prospects of CRISPR/Cas13a-based biosensors, particularly in the context of clinical diagnostics and personalized medicine.}, }
@article {pmid40300299, year = {2025}, author = {Lu, L and Gan, S and Xiao, S and Mu, X and Zhao, S and Tian, J}, title = {An innovative "double-locked" CRISPR/Cas12a system based on DNAzyme for the precise imaging of microRNAs in living cells.}, journal = {International journal of biological macromolecules}, volume = {310}, number = {Pt 4}, pages = {143605}, doi = {10.1016/j.ijbiomac.2025.143605}, pmid = {40300299}, issn = {1879-0003}, mesh = {*MicroRNAs/genetics/metabolism ; *DNA, Catalytic/metabolism/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; Optical Imaging/methods ; *Molecular Imaging/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {We constructed a "double-locked" CRISPR/Cas12a system based on DNAzyme for miRNA detection and precise imaging. One lock function to close the catalytic activity of DNAzyme, while the other lock serves to inhibit the cleavage function of CRISPR-induced RNA (crRNA). This "double-lock" mechanism ensures that the system is inhibited in the absence of the target molecule miRNA-141, effectively reducing the background signal. When the target miRNA-141 is present, the "lock" of DNAzyme is opened, and DNAzyme further opens the "lock" of crRNA, which activates the trans-cleavage ability of Cas12a on F-Q probe, and the fluorescence signal is restored. The linear range of miRNA-141 was 50 pmol/L ~ 15 nmol/L, and the detection limit was 47 pmol/L (S/N = 3). The system has been successfully applied to detect miRNA-141 expression levels in cell lysates. Meanwhile, this method can be applied for intracellular miRNA-141 fluorescence imaging and fluctuations in intracellular miRNA-141 expression. Overall, this strategy not only offers new prospects for programmable Cas12a detection systems, but also provides new insights for early diagnosis and screening of diseases by combining this in vitro assay with live cell imaging analysis for the detection of cancer markers.}, }
@article {pmid40298457, year = {2025}, author = {Zhang, Y and Liu, J and Zhou, Y and Hu, X and Geng, B and Yang, S}, title = {Establishment of a RecET-Assisted CRISPR-Cas12a System for Large Deoxyribonucleic Acid-Fragment Manipulation in Zymomonas mobilis.}, journal = {ACS synthetic biology}, volume = {14}, number = {5}, pages = {1606-1614}, doi = {10.1021/acssynbio.4c00863}, pmid = {40298457}, issn = {2161-5063}, mesh = {*Zymomonas/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Plasmids/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; Promoter Regions, Genetic ; *DNA/genetics ; }, abstract = {The lack of effective and efficient genome-editing toolkits for large DNA-fragment manipulation impedes the development of robust cell factories to meet the needs of fast-growing biomanufacturing. Zymomonas mobilis is an important nonmodel polyploid industrial strain with excellent industrial characteristics. Although diverse CRISPR-Cas systems have been established in Z. mobilis for gene deletion, replacement, and ssDNA recombination, it is still challenging to achieve large DNA-fragment manipulation due to its low recombination and repair efficiencies for double-strand DNA breaks. In this study, a RecET-assisted CRISPR-Cas12a genome editing system was developed using a chromosome-borne cas12a and recET-encoded recombinase, as well as an all-in-one editing plasmid. Different promoters were used for recET and cas12a to determine optimal expression. The combination of PB-cas12a_Pt-recET had the highest efficiency of 97.92 ± 2.95% for 9-kb DNA-fragment deletion, which also had efficiencies about 100%, 80%, and 5%, respectively, for the deletion of 9-16, 20-25, and 30 kb DNA fragments. The RecET-assisted CRISPR-Cas12a was further applied for deletions of different large gene clusters and had the potential for efficient pathway knock-in. This study highlights the importance of the Cas12a nuclease expression levels and the combination of the RecET system in improving the double-strand DNA repair capability for large DNA-fragment manipulation in Z. mobilis. The RecET-assisted CRISPR-Cas12a system established in this study provides a versatile and powerful tool for large DNA-fragment manipulation in Z. mobilis, which is beneficial for functional genomic research, strain improvement, as well as the development of synthetic microbial chassis.}, }
@article {pmid40298107, year = {2025}, author = {Sun, J and Yu, X and Tang, G and Chen, M and Zheng, Y and Hu, Y and Li, Q and Li, X and Li, N and Li, Z and Li, Y and Lu, N and Tan, W and Yang, Y and Lyu, X and Zhao, G and Wang, H and Dai, L and Zhao, GP and Ai, L and Zhao, W}, title = {A CRISPR-SpCas9M-reporting system for efficient and rapid genome editing in Caulobacter crescentus.}, journal = {Nucleic acids research}, volume = {53}, number = {8}, pages = {}, pmid = {40298107}, issn = {1362-4962}, support = {2024YFA0917100//National Key R&amp;D Program of China/ ; XDB0480000//Chinese Academy of Sciences/ ; 32025029//National Natural Science Foundation of China/ ; 2023A1515030069//Guangdong Basic and Applied Basic Research Foundation/ ; }, mesh = {*Caulobacter crescentus/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Genome, Bacterial ; Bacterial Proteins/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {As members of the α-proteobacteria group, Caulobacter crescentus and its relatives are wildly studied for their unique asymmetric life cycle and versatile applications in industry, agriculture, and biomedicine. However, genetic manipulation in these bacteria remains challenging, typically requiring time-consuming and labor-intensive procedures. Here, we report a practical CRISPR-SpCas9M-reporting system that overcomes the limitations of SpCas9 expression and CRISPR escape, enabling efficient, markerless, and rapid genome editing in C. crescentus. Two genes encoding for a pair of scaffold proteins were knocked out individually or iteratively, demonstrating their direct involvements in cellular signaling asymmetry. Key components, including the Cas protein, Cas inducer, sgRNA, homologous arms, and reporter, were systematically analyzed and optimized in the system, finally achieving the apparent editing efficiency up to 80% in C. crescentus. Furthermore, we applied the CRISPR-SpCas9M-reporting system to two C. crescentus relatives, Agrobacterium fabrum and Sinorhizobium meliloti, establishing it as an efficient and general editing strategy. We anticipate that this system could be applied to other CRISPR-Cas-recalcitrant organisms, accelerating both basic and applied research in α-proteobacteria.}, }
@article {pmid40295218, year = {2025}, author = {Jeong, SO and Kim, HJ and Lee, SJ}, title = {Adaptive Evolution of GatC, a Component of the Galactitol Phosphotransferase System, for Glucose Transport in Escherichia coli.}, journal = {Journal of microbiology and biotechnology}, volume = {35}, number = {}, pages = {e2502021}, pmid = {40295218}, issn = {1738-8872}, mesh = {*Escherichia coli/genetics/metabolism/enzymology ; *Glucose/metabolism ; *Escherichia coli Proteins/genetics/metabolism ; *Phosphoenolpyruvate Sugar Phosphotransferase System/genetics/metabolism ; Directed Molecular Evolution ; Fermentation ; CRISPR-Cas Systems ; Biological Transport ; Mutation, Missense ; Metabolic Engineering ; Mutation ; Succinic Acid/metabolism ; }, abstract = {Microbial adaptive laboratory evolution is a powerful approach for uncovering novel gene functions within metabolic pathways. Building on our previous discovery of ExuT as a glucose transporter in ptsG-deficient Escherichia coli, this study investigates strains lacking recognized glucose transporters (ptsG, manX, and exuT). Successive rounds of experimental evolution revealed key genetic adaptations, including loss-of-function mutations in malI and nagC, which encode repressors of the maltose and N-acetylglucosamine phosphotransferase systems (PTS), respectively. Additionally, a gain-of-function mutation in gatC, a component of the galactitol PTS EIIC, was identified. The functional significance of these mutations was validated through transcript analysis, genetic knockouts, and CRISPR-Cas9-mediated site-specific genome mutagenesis, with a particular focus on the gatC missense mutation (F340C). The resulting modifications were examined for their effects on sugar specificity and metabolic flux. Furthermore, our findings identified succinate as the predominant fermentation product in engineered strains utilizing alternative glucose transport pathways, including the maltose, N-acetylglucosamine, and galactitol PTS. This study advances our understanding of sugar transport mechanisms in E. coli and offers insights into regulatory networks, fermentative metabolism, and substrate specificity, which can be leveraged for evolutionary engineering in biotechnological applications.}, }
@article {pmid40295092, year = {2025}, author = {Dipalo, LL and Mikkelsen, JG and Gijsbers, R and Carlon, MS}, title = {Trojan Horse-Like Vehicles for CRISPR-Cas Delivery: Engineering Extracellular Vesicles and Virus-Like Particles for Precision Gene Editing in Cystic Fibrosis.}, journal = {Human gene therapy}, volume = {}, number = {}, pages = {}, doi = {10.1089/hum.2024.258}, pmid = {40295092}, issn = {1557-7422}, abstract = {The advent of genome editing has kindled the hope to cure previously uncurable, life-threatening genetic diseases. However, whether this promise can be ultimately fulfilled depends on how efficiently gene editing agents can be delivered to therapeutically relevant cells. Over time, viruses have evolved into sophisticated, versatile, and biocompatible nanomachines that can be engineered to shuttle payloads to specific cell types. Despite the advances in safety and selectivity, the long-term expression of gene editing agents sustained by viral vectors remains a cause for concern. Cell-derived vesicles (CDVs) are gaining traction as elegant alternatives. CDVs encompass extracellular vesicles (EVs), a diverse set of intrinsically biocompatible and low-immunogenic membranous nanoparticles, and virus-like particles (VLPs), bioparticles with virus-like scaffold and envelope structures, but devoid of genetic material. Both EVs and VLPs can efficiently deliver ribonucleoprotein cargo to the target cell cytoplasm, ensuring that the editing machinery is only transiently active in the cell and thereby increasing its safety. In this review, we explore the natural diversity of CDVs and their potential as delivery vectors for the clustered regularly interspaced short palindromic repeats (CRISPR) machinery. We illustrate different strategies for the optimization of CDV cargo loading and retargeting, highlighting the versatility and tunability of these vehicles. Nonetheless, the lack of robust and standardized protocols for CDV production, purification, and quality assessment still hinders their widespread adoption to further CRISPR-based therapies as advanced "living drugs." We believe that a collective, multifaceted effort is urgently needed to address these critical issues and unlock the full potential of genome-editing technologies to yield safe, easy-to-manufacture, and pharmacologically well-defined therapies. Finally, we discuss the current clinical landscape of lung-directed gene therapies for cystic fibrosis and explore how CDVs could drive significant breakthroughs in in vivo gene editing for this disease.}, }
@article {pmid40294708, year = {2025}, author = {Gao, X and Zhou, C and Feng, Y and Ye, B and Zhao, Z and Qi, L and Hu, L and Deng, Y and Lin, C and Ding, Q and Liu, G and Wang, C and Song, C and Qian, B and Wu, T and Wang, X and Liu, Z and Lin, Z and Zhang, M}, title = {Research progress of gene editing technology in neurological diseases.}, journal = {Gene}, volume = {962}, number = {}, pages = {149534}, doi = {10.1016/j.gene.2025.149534}, pmid = {40294708}, issn = {1879-0038}, mesh = {*Gene Editing/methods/trends ; Humans ; *Nervous System Diseases/therapy/genetics ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Animals ; }, abstract = {Gene editing (GE) technology is a genetic manipulation technique based on artificial nucleases that enables the precise modification of DNA or RNA. With the development of technology, GE in disease treatment is becoming increasingly widespread, playing an essential role in haematology, cancer, and neurological disorders (ND). This review describes the principles, advantages, and limitations of four GE technologies, focusing on the fourth generation of GE (next-generation GE). The next-generation GE technology breaks the limitations of traditional GE technology, makes GE more precise and stable, and broadens the scope of gene technology applications. Additionally, this review explores the latest gene therapy strategies for ND, focusing on the application of next-generation GE technologies to examine the prospects for the application of GE technologies. This study discusses and analyses the great advantages and potential of GE technology for treating ND and elucidates the shortcomings of GE in this field.}, }
@article {pmid40294287, year = {2025}, author = {Huang, P and Liu, Y and Zhao, C and Wang, C and Wang, L and Luo, M and Wang, W and Shan, W and Liu, X and Li, B and Wang, Z and Deng, H and Chen, X}, title = {Permanent Efferocytosis Prevention by Terminating MerTK Recycle on Tumor-Associated Macrophages for Cancer Immunotherapy.}, journal = {Journal of the American Chemical Society}, volume = {147}, number = {18}, pages = {15901-15914}, doi = {10.1021/jacs.5c05640}, pmid = {40294287}, issn = {1520-5126}, mesh = {*c-Mer Tyrosine Kinase/metabolism/genetics/immunology/antagonists &amp; inhibitors ; Animals ; Proto-Oncogene Mas ; Mice ; *Immunotherapy ; *Tumor-Associated Macrophages/metabolism/immunology ; Humans ; Phagocytosis ; Mice, Inbred C57BL ; Cell Line, Tumor ; Phosphatidylserines/metabolism ; *Neoplasms/therapy/immunology ; CRISPR-Cas Systems ; Efferocytosis ; }, abstract = {Efferocytosis of apoptotic tumor cells by tumor-associated macrophages mediated through the phosphatidylserine (PtdSer)/MER proto-oncogene tyrosine kinase (MerTK) axis can exacerbate tumor immunosuppression, and conversely, prevention of efferocytosis via blocking PtdSer-MerTK association using prevalent antibodies represents a promising strategy for reversing tumor immunosuppression and boosting antitumor immunity. However, it remains unclear whether the antibody blockade can induce durable efferocytosis prevention and achieve sustained tumor growth inhibition. Here, we have shown that utilizing PtdSer and MerTK antibodies induced only a transient rather than a persistent efferocytosis prevention effect, and little enhancement was observed even after improving antibody enrichment in tumor sites. Further mechanistic studies suggested that degradation of anti-MerTK antibody and recycling of the MerTK receptor to the cell membrane would compromise the therapeutic benefits of antibody blockade. Based on these findings, we developed a CRISPR/Cas9 gene editing system deployed using Cas9 mRNA and MerTK sgRNA to permanently knock out MerTK, which achieved durable efferocytosis prevention, elicited persistent in situ vaccination immune responses via enhancing X-ray irradiation-induced immunogenic cell death, and led to sustained tumor suppression effects together with anti-PtdSer antibody and X-ray irradiation treatment in multiple B16 melanoma tumor models. Our findings provide a reliable gene-editing-mediated strategy for long-term modulating MerTK homeostasis and overcoming MerTK-dependent cancer immune evasion, generating adaptive antitumor immune responses for sustained cancer immunotherapy.}, }
@article {pmid40293975, year = {2025}, author = {Deragon, MA and Sharifi, HJ and LaRocca, TJ}, title = {Generation of a RIP1 Knockout U937 Cell Line Using the CRISPR-Cas9 System.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {218}, pages = {}, doi = {10.3791/66077}, pmid = {40293975}, issn = {1940-087X}, mesh = {Humans ; *CRISPR-Cas Systems ; U937 Cells ; *Gene Knockout Techniques/methods ; *Receptor-Interacting Protein Serine-Threonine Kinases/genetics ; *Nuclear Pore Complex Proteins/genetics ; }, abstract = {This protocol outlines a procedure for knocking out the RIP1 gene using CRISPR/Cas9 in the human monocyte U937 cell line. The method utilizes designated guide RNA plasmids and lentiviral packaging plasmids to achieve RIP1 gene knockout. The protocol addresses challenges and improvements to traditional CRISPR methods, enabling its replication for future cell death studies. The resulting mutant cells can be used to investigate mechanistic changes in cell death, where functional RIP1 proteins would otherwise play a role. Viability assays demonstrated a significant reduction in cell death in knockout cells following necroptosis induction. Fluorescence microscopy revealed a marked decrease in mitochondrial reactive oxygen species (ROS) in knockout cells under the same conditions. Together, these functional assays confirm the loss of RIP1 protein. Optimized for use with U937 human monocytes, this procedure may also be adapted to target other key cell death regulators, yielding functional, non-lethal mutants. Potential pitfalls are addressed throughout to provide insights into challenges that may arise during mutant generation.}, }
@article {pmid40293254, year = {2025}, author = {Huang, J and Ding, K and Chen, J and Fan, J and Huang, L and Qiu, S and Wang, L and Du, X and Wang, C and Pan, H and Yuan, Z and Liu, H and Song, H}, title = {Comparison of CRISPR-Cas9, CRISPR-Cas12f1, and CRISPR-Cas3 in eradicating resistance genes KPC-2 and IMP-4.}, journal = {Microbiology spectrum}, volume = {13}, number = {6}, pages = {e0257224}, pmid = {40293254}, issn = {2165-0497}, support = {32141003//National Science Foundation of China/ ; 32300080//National Science Foundation of China/ ; 2021YFC2301102//National Science and Technology Major Project/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics/drug effects ; *Gene Editing/methods ; Plasmids/genetics ; *beta-Lactamases/genetics ; Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests ; *Bacterial Proteins/genetics ; Drug Resistance, Bacterial/genetics ; Carbapenems/pharmacology ; Klebsiella pneumoniae/genetics/drug effects ; }, abstract = {UNLABELLED: Bacterial plasmid encoding antibiotic resistance could be eradicated by various CRISPR systems, such as CRISPR-Cas9, Cas12f1, and Cas3. However, the efficacy of these gene editing tools against bacterial resistance has not been systematically assessed and compared. This study eliminates carbapenem resistance genes KPC-2 and IMP-4 via CRISPR-Cas9, Cas12f1, and Cas3 systems, respectively. The eradication efficiency of the three CRISPR systems was evaluated. First, the target sites for the three CRISPR systems were designed within the regions 542-576 bp of the KPC-2 gene and 213-248 bp of the IMP-4 gene, respectively. The recombinant CRISPR plasmids were transformed into Escherichia coli carrying KPC-2 or IMP-4-encoding plasmid. Colony PCR of transformants showed that KPC-2 and IMP-4 were eradicated by the three different CRISPR systems, and the elimination efficacy was both 100.00%. The drug sensitivity test results showed that the resistant E. coli strain was resensitized to ampicillin. In addition, the three CRISPR plasmids could block the horizontal transfer of drug-resistant plasmids, with a blocking rate as high as 99%. Importantly, a qPCR assay was performed to analyze the copy number changes of drug-resistant plasmids in E. coli cells. The results indicated that CRISPR-Cas3 showed higher eradication efficiency than CRISPR-Cas9 and Cas12f1 systems.<br><br>IMPORTANCE: With the continuous development and application of CRISPR-based resistance removal technologies, CRISPR-Cas9, Cas12f1, and Cas3 have gradually come into focus. However, it remains uncertain which system exhibits more potent efficacy in the removal of bacterial resistance. This study verifies that CRISPR-Cas9, Cas12f1, and Cas3 can eradicate the carbapenem-resistant genes KPC-2 and IMP-4 and restore the sensitivity of drug-resistant model bacteria to antibiotics. Among the three CRISPR systems, the CRISPR-Cas3 system showed the highest eradication efficiency. Although each system has its advantages and characteristics, our results provide guidance on the selection of the CRISPR system from the perspective of resistance gene removal efficiency, contributing to the further application of CRISPR-based bacterial resistance removal technologies.}, }
@article {pmid40292762, year = {2025}, author = {Qin, Y and Xie, JL and Mao, K and Li, YF and Huang, CZ and Zou, HY and Zhen, SJ}, title = {A novel CRISPR-Cas12a-based fluorescence anisotropy method with a high signal-to-background ratio for sensitive biosensing.}, journal = {Chemical communications (Cambridge, England)}, volume = {61}, number = {41}, pages = {7458-7461}, doi = {10.1039/d5cc01565k}, pmid = {40292762}, issn = {1364-548X}, mesh = {*Biosensing Techniques/methods ; *Fluorescence Polarization/methods ; *CRISPR-Cas Systems ; *DNA, Viral/analysis ; *Hepatitis B virus/genetics ; DNA/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Here, a CRISPR-Cas12a system with high trans-cleavage ability integrating a DNA nanochain formed by DNA tetrahedrons with a large molecular mass was employed to enhance the signal-to-background ratio of the fluorescence anisotropy method for achieving sensitive detection of hepatitis B virus DNA.}, }
@article {pmid40292247, year = {2025}, author = {Suthar, MK and Mittal, MK}, title = {Genome-wide identification and characterization of dicer-like genes in Andrographis paniculata (Burm. f.) Wall. ex Nees and their expression response to methyl jasmonate elicitation.}, journal = {3 Biotech}, volume = {15}, number = {5}, pages = {141}, pmid = {40292247}, issn = {2190-572X}, abstract = {Andrographis paniculata, commonly known as the "King of Bitters," is a medicinal plant valued for its bioactive diterpenoids, particularly andrographolides. Dicer-like (DCL) proteins are central to the miRNA pathway, processing precursor miRNAs into mature miRNAs that regulate gene expression. While miRNAs influence plant metabolism, their role in secondary metabolite biosynthesis in A. paniculata remains unexplored. This study identified and characterized five ApDCL genes, mapped to distinct chromosomes in A. paniculata. The structural analysis revealed that ApDCL3 contained the highest number of introns (24), whereas ApDCL2 had the fewest (10). Conserved RNA-processing domains were confirmed, and phylogenetic analysis revealed evolutionary conservation, showing that ApDCLs are closely related to the DCLs of Sesamum indicum and Salvia miltiorrhiza. Expression analysis showed ApDCL1 and ApDCL2 were predominantly expressed in roots, whereas ApDCL3, ApDCL4a, and ApDCL4b were more abundant in leaves. Methyl jasmonate treatment upregulated ApDCL3 (~ 3.5-fold) and ApDCL4a (~ 1.5-fold), but further research is needed to determine whether this response directly influences secondary metabolism or results from MeJ-induced stress. These findings provide a foundation for functional validation through gene knockdown, overexpression, and CRISPR-Cas-based genome editing to elucidate DCL-mediated regulatory mechanisms. Future research leveraging these insights could aid in modulating RNA silencing pathways to enhance the biosynthesis of pharmacologically significant metabolites in A. paniculata through biotechnological interventions.}, }
@article {pmid40288547, year = {2025}, author = {Goméz-Quintero, OS and Morales-Moreno, MD and Valdés-Galindo, EG and Cárdenas-Guerra, RE and Hernandez-Garcia, A}, title = {Enhanced production of functional CRISPR-AsCas12a protein in Escherichia coli.}, journal = {Protein expression and purification}, volume = {232}, number = {}, pages = {106722}, doi = {10.1016/j.pep.2025.106722}, pmid = {40288547}, issn = {1096-0279}, mesh = {*Escherichia coli/genetics/metabolism ; Recombinant Proteins/genetics/biosynthesis/isolation &amp; purification ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics/biosynthesis/isolation &amp; purification ; *Bacterial Proteins/genetics/biosynthesis/isolation &amp; purification ; *Acidaminococcus/genetics/enzymology ; *Endodeoxyribonucleases/genetics/biosynthesis/isolation &amp; purification ; Gene Editing ; }, abstract = {The CRISPR-Cas12a system is a groundbreaking tool widely used for genome editing and diagnostics in biotechnology and biomedicine research laboratories. Despite its growing application, studies optimizing Cas12a protein production at the laboratory scale using straightforward protocols remains scarce. This study aimed to enhance the lab-scale recombinant production of Acidaminococcus sp Cas12a protein (AsCas12a) in E. coli. Through targeted adjustments of simple parameters, AsCas12a production was significantly increased. The optimized conditions included the use of E. coli BL21(DE3), TB medium supplemented with 1 % glucose, induction with 0.3 mM IPTG for at least 6-9 h, and incubation at 30 °C. Notably, these conditions differ from conventional protocols typically used for Cas12a and related proteins, such as Streptococcus pyogenes Cas9. Upon combining all optimized parameters, AsCas12a production increased approximately 3-fold, from 0.95 mg/mL of bacterial lysate under non-optimized conditions to 3.73 mg/mL under optimized ones. After chromatographic purification, the final protein yield rose approximately 4.5-fold, from 5.2 to 23.4 mg/L of culture volume, without compromising functional activity. The purified AsCas12a retained full activity for programmable in vitro DNA cis-cleavage and collateral trans-cleavage, which was successfully applied to detect the N gene of SARS-CoV-2. This optimized method provide an efficient and high-yield approach for producing functional AsCas12a protein using accessible materials and conditions available to many research laboratories worldwide.}, }
@article {pmid40288511, year = {2025}, author = {Sharma, N and Whinn, KS and Ghodke, H and van Oijen, AM and Lewis, JS and Spenkelink, LM}, title = {nCas9-based method for rolling-circle DNA substrate generation.}, journal = {Analytical biochemistry}, volume = {703}, number = {}, pages = {115883}, pmid = {40288511}, issn = {1096-0309}, support = {RM1 GM130450/GM/NIGMS NIH HHS/United States ; }, mesh = {*DNA, Circular/genetics/chemistry/metabolism ; *CRISPR-Associated Protein 9/metabolism ; *DNA Replication ; RNA, Guide, CRISPR-Cas Systems/genetics ; *DNA ; }, abstract = {Rolling-circle DNA replication is a DNA-duplication mechanism whereby circular DNA templates are continuously copied to produce long DNA products. It is widely used in molecular diagnostics, DNA sequencing, nanotechnology, and in vitro DNA replication studies. The efficiency of rolling-circle replication reaction heavily relies on the quality of the rolling-circle DNA template. Existing methods to create rolling-circle DNA substrates often rely on unique restriction sites and have limited control over replication fork topology and position. To address these limitations, we present a straightforward, customizable, and efficient strategy for producing rolling-circle DNA substrates with control over gap size and fork position. Our method relies on the use of nickase Cas9 (nCas9), which can be programmed to target specific DNA sequences using guide RNAs. In a one-pot reaction, we target nCas9 to four sites on an 18-kb plasmid to create 8-11-bp fragments. These fragments are removed and a flap oligo is ligated, to construct a fork with precisely controlled flap length and gap size. We demonstrate the application of this DNA substrate in an in vitro single-molecule rolling-circle DNA-replication assay. With our method, any plasmid DNA can be converted into a rolling-circle template, permitting generation of more physiologically-relevant DNA templates.}, }
@article {pmid40288310, year = {2025}, author = {Qian, J and Zhang, B and Liu, C and Xue, Y and Zhou, H and Huang, L and Zheng, S and Chen, M and Fu, YQ}, title = {Reconfigurable acoustic tweezer for precise tracking and in-situ sensing of trace miRNAs in tumor cells.}, journal = {Biosensors &amp; bioelectronics}, volume = {282}, number = {}, pages = {117505}, doi = {10.1016/j.bios.2025.117505}, pmid = {40288310}, issn = {1873-4235}, mesh = {*MicroRNAs/genetics/isolation &amp; purification/analysis ; Humans ; *Biosensing Techniques/instrumentation ; *Neoplasms/genetics/diagnosis/pathology ; Acoustics/instrumentation ; Cell Line, Tumor ; Equipment Design ; *Cell Tracking/instrumentation ; Biomarkers, Tumor/genetics ; CRISPR-Cas Systems ; }, abstract = {MicroRNAs (miRNAs) have emerged as critical biomarkers for early cancer diagnosis and monitoring. However, their isolation from clinical samples typically yields only trace amounts, significantly limiting the sensitivity and efficiency of cancer detection. To address this challenge, we present a octangular reconfigurable acoustic tweezer (ORAT) as an integrated platform for precise tumor cell tracking and in-situ detection of trace miRNAs. By simultaneously modulating multidirectional acoustic signals and parameters, the ORAT dynamically reshapes the acoustic field, enabling precise control over manipulation areas, particle spacing, array angles, distribution patterns, and node rotation. This device allows selective particle manipulation across entire regions or specific areas through adaptive adjustments of the microchamber boundary. Notably, the ORAT achieves rapid and accurate localization and labeling of rare tumor cells within a large population of normal cells. Furthermore, it enhances the sensitivity of CRISPR/Cas-based miRNA detection in digital microdroplets by three orders of magnitude, if compared to that of the conventional tube-based method. With its versatile capabilities, the ORAT holds remarkable promise for advancing nucleic acid analysis in a wide range of cancers and related diseases.}, }
@article {pmid40287132, year = {2025}, author = {Lancho, VJ and Eisenhut, P and Klanert, G and Ivansson, D and Jonsson, A and Lövgren, A and Borth, N}, title = {Stepwise activation of gene copies results in higher final titers of subclones compared to immediate integration of the full set of active copies.}, journal = {New biotechnology}, volume = {88}, number = {}, pages = {89-99}, doi = {10.1016/j.nbt.2025.04.011}, pmid = {40287132}, issn = {1876-4347}, mesh = {Animals ; Green Fluorescent Proteins/genetics/metabolism ; *Gene Dosage ; CHO Cells ; CRISPR-Cas Systems/genetics ; Cricetulus ; }, abstract = {The increasing demand for production of therapeutic proteins has encouraged both industrial and academic institutions to pursue the development of mammalian expression platforms with high productivities. While protocols for rapid and efficient integration of multiple transgene copies into the genome are available, they require substantial time and resources for screening numerous clones. A contributing factor is the tendency of high producers to disappear from the selected mini-pools due to the stress caused by high productivity without adequate time for adaptation of cellular capacities. Here, we have developed a strategy to stably activate individual copies within an initially repressed multicopy coding cassette harboring 2 GFP-Fc and 2 BFP-Fc genes, each fused to an Fc region for secretion. This toolbox enables gene activation via CRISPR/Cas9-mediated deletion of the repressor elements. Subsequently, producers can be sorted based on increased GFP or BFP fluorescence and assessed by measuring the secreted total Fc protein. We demonstrate that the stepwise activation of initially repressed genes outperforms a control cell line with the same number of genes active from the outset, as evidenced by higher fluorescence signals from GFP and BFP, increased mRNA levels for BFP, GFP, and Fc genes, and enhanced titer of secreted Fc fusion protein. This study demonstrates the ability of cells to adapt to new challenges by modulating both gene expression patterns and channeling of resources to accommodate high production loads.}, }
@article {pmid40287111, year = {2025}, author = {Yang, Q and Zhang, Y and Chen, L and Fang, L and Xiao, S}, title = {Isolation, pathogenicity, and an infectious cDNA clone of the prevalent G2c variant of porcine epidemic diarrhea virus in China.}, journal = {Microbial pathogenesis}, volume = {205}, number = {}, pages = {107637}, doi = {10.1016/j.micpath.2025.107637}, pmid = {40287111}, issn = {1096-1208}, mesh = {Animals ; Swine ; *Porcine epidemic diarrhea virus/genetics/pathogenicity/isolation &amp; purification ; China/epidemiology ; *Swine Diseases/virology/epidemiology ; Chlorocebus aethiops ; Vero Cells ; *Coronavirus Infections/veterinary/virology/epidemiology ; *DNA, Complementary/genetics ; Virulence ; Diarrhea/veterinary/virology/epidemiology ; Spike Glycoprotein, Coronavirus/genetics ; Disease Outbreaks ; Mutation ; Phylogeny ; CRISPR-Cas Systems ; }, abstract = {Porcine epidemic diarrhea virus (PEDV) is a major enteric coronavirus causing diarrhea in piglets, and the recent prevalence of its G2c variant poses a significant threat to the pig industry in China. In this study, we successfully isolated a G2c variant strain (designated EHuB4) from a pig farm experiencing an outbreak of severe diarrhea in China, and constructed its full-length cDNA infectious clone using CRISPR/Cas9 technology. The parental EHuB4 strain and its rescued virus, rEHuB4, exhibited similar biological characteristics in Vero cells, including rapid cytopathic effects and the formation of large syncytia, suggesting strong cell fusion ability and replication efficiency. Pathogenicity experiments in piglets demonstrated that both EHuB4 and rEHuB4 caused severe diarrhea and mortality, further confirming that the prevalence of the G2c variant may be the primary reason for the current high incidence of PED outbreaks. We also analyzed the mutation frequency and variation tendency in the amino acids encoded by the EHuB4 Spike (S) gene and identified multiple high-frequency mutation sites located not only in the S1 subunit but also in the intermediate region between heptad repeat 1 (HR1) and HR2. Furthermore, these mutations have persisted since 2011 and may be closely related to virus prevalence, host adaptation, and pathogenicity. In summary, this study not only characterizes the genetic evolution and pathogenicity of the prevalent G2c strain but also provides a valuable molecular tool for developing novel vaccines through the established full-length cDNA infectious clone.}, }
@article {pmid40286970, year = {2025}, author = {Long, K and Liu, H and Yang, N and Li, J and Hou, C and Yang, M and Huang, Z and Huo, D}, title = {Selenium-enhanced recombinase polymerase amplification-CRISPR/Cas12a: A low-noise single-tube assay for human papillomavirus 16 ultrasensitive detection.}, journal = {International journal of biological macromolecules}, volume = {310}, number = {Pt 4}, pages = {143468}, doi = {10.1016/j.ijbiomac.2025.143468}, pmid = {40286970}, issn = {1879-0003}, mesh = {*Human papillomavirus 16/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; Humans ; *Selenium/chemistry ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism ; DNA, Viral/genetics ; Limit of Detection ; *Endodeoxyribonucleases/genetics/metabolism ; *Papillomavirus Infections/diagnosis/virology ; *CRISPR-Associated Proteins/metabolism ; Bacterial Proteins ; }, abstract = {In vitro amplification represents a critical step in human papillomavirus (HPV) DNA detection. However, DNA polymerases can initiate nonspecific amplification and incorporate erroneous nucleotides due to the lack of cellular repair mechanisms. To address these challenges, we present a novel one-tube selenium-enhanced recombinase polymerase amplification (Se-RPA) coupled with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 12a (Cas12a) (OTSRC) system for the ultrasensitive HPV DNA detection. The Se-RPA incorporates 10 % selenium-modified nucleoside triphosphates (dNTPαSe) into the conventional RPA protocol, effectively suppressing nonspecific amplification while maintaining high-fidelity DNA synthesis. The CRISPR/Cas12a component integrates sequence-specific verification, exponential signal amplification, and fluorescence-based readout capabilities. Optimized in a single-tube format to minimize aerosol contamination, OTSRC exhibits a background signal of 71.77 % compared to the one-tube RPA-CRISPR/Cas12a (OTRC) system. Within a 20-min incubation, the OTSRC demonstrated a detection limit of 169 aM, which is half that of the OTRC without dNTPαSe and comparable to qPCR. Furthermore, the OTSRC system demonstrates the excellent compatibility of dNTPαSe with the RPA-CRISPR/Cas12a system, thereby enhancing HPV detection sensitivity. Overall, OTSRC enables rapid, sensitive, and specific detection of HPV DNA, showing strong potential for clinical point-of-care nucleic acid testing applications.}, }
@article {pmid40286769, year = {2025}, author = {Yang, X and Gao, S}, title = {Competitive rDNA binding by dCas9 induces outside-in disassembly of the nucleolus.}, journal = {Biochemical and biophysical research communications}, volume = {766}, number = {}, pages = {151883}, doi = {10.1016/j.bbrc.2025.151883}, pmid = {40286769}, issn = {1090-2104}, mesh = {*Cell Nucleolus/metabolism/genetics ; Nucleophosmin ; *DNA, Ribosomal/metabolism/genetics ; Humans ; Nuclear Proteins/metabolism/genetics ; HeLa Cells ; Promoter Regions, Genetic ; *CRISPR-Cas Systems ; Transcription, Genetic ; Pol1 Transcription Initiation Complex Proteins/metabolism ; }, abstract = {The inside-out assembly and outside-in disassembly of the nucleolus are well-accepted models, yet direct in-cell evidence remains elusive. Here, we employed a dCas9-based competitive binding system to specifically target the rDNA promoter within the nucleolus, effectively inhibiting rDNA transcription. This transcriptional blockade induced a stepwise, outside-in disassembly of the nucleolus. NPM1 was the first to disappear from the nucleolus, followed by a progressive reduction in the fluorescence intensities of FBL and UBF. Additionally, UBF relocated from the nucleolar core to the periphery. These findings provide the first direct evidence in cells supporting the outside-in disassembly of the nucleolus. Furthermore, our results suggest that the dynamic inside-out assembly and outside-in disassembly of the nucleolus.}, }
@article {pmid40286604, year = {2025}, author = {Härting, N and Ludwik, KA and Jahn, R and Kaiser, FJ and Stachelscheid, H}, title = {Generation of hiPSC lines with fusion tagged thyroid hormone receptor α isoforms to study isoform-specific actions of TRα1 and TRα2.}, journal = {Stem cell research}, volume = {86}, number = {}, pages = {103720}, doi = {10.1016/j.scr.2025.103720}, pmid = {40286604}, issn = {1876-7753}, mesh = {Humans ; *Thyroid Hormone Receptors alpha/metabolism/genetics ; Protein Isoforms/metabolism/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Male ; Cell Line ; CRISPR-Cas Systems ; Gene Editing ; }, abstract = {The role of the nuclear thyroid hormone receptor TRα1 as a mediator of thyroid hormone action on target gene expression is well understood. However, the function of the TRα2 splicing isoform, which does not bind thyroid hormones, remains unexplored. As no reliable antibodies are available to investigate TRα1 and TRα2 specifically, we introduced small fusion tags into the THRA locus of the male healthy donor iPSC lines BIHi001-B and BIHi005-A by CRISPR/Cas9-mediated genome editing. Consequently, the modified lines express C-terminally tagged TRα1-2xHA or TRα2-3xFLAG. These genome-edited lines facilitate the investigation of isoform-specific actions of TRα in different cell types.}, }
@article {pmid40286513, year = {2025}, author = {Tang, Q and Liao, Q and Huang, X and Huang, H and Tang, Q and Zhang, K and Liao, X}, title = {A highly sensitive MiRNA detection sensor powered by CRISPR/Cas13a and entropy-driven amplification.}, journal = {Bioelectrochemistry (Amsterdam, Netherlands)}, volume = {165}, number = {}, pages = {108992}, doi = {10.1016/j.bioelechem.2025.108992}, pmid = {40286513}, issn = {1878-562X}, mesh = {*MicroRNAs/analysis/genetics ; *CRISPR-Cas Systems ; *Entropy ; Humans ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Electrochemical Techniques/methods ; Luminescent Measurements/methods ; }, abstract = {MicroRNAs (miRNAs) are critical regulators of numerous physiological and pathological processes, influencing gene expression and playing essential roles in cellular development, differentiation, and disease progression. Their sensitive and specific detection is vital for advancing biomedical research and clinical diagnostics, particularly for early disease detection and biomarker discovery. However, traditional miRNA detection methods often face significant challenges, such as limited sensitivity, insufficient specificity, and the inability to detect low-abundance miRNAs in complex biological samples. To overcome these limitations, we present a novel miRNA detection electrochemiluminescence (ECL) platform that integrates entropy-driven amplification with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a-mediated RNA cleavage. The entropy-driven amplification strategy exploits the thermodynamic advantages of nucleic acid hybridization, driving spontaneous molecular reorganization to amplify detection signals and achieve ultralow detection limits. CRISPR/Cas13a, an RNA-targeting nuclease, provides exceptional sequence specificity by recognizing and binding to target miRNA sequences, activating a collateral cleavage mechanism. This activity cleaves hairpin (HP) structure, generating a signal that further triggers EDA over DNA tetrahedron (DT) to induce a vigorous ECL response. Based on this strategy, we achieve rapid and precise quantification of miR-17 at femtomolar levels. Experimental results demonstrate high sensitivity, specificity, and the ability to analyze complex biological samples in the laboratory. This innovative approach holds great promise for advancing molecular diagnostics and personalized medicine.}, }
@article {pmid40285901, year = {2025}, author = {Lin, W and Huang, M and Fu, H and Yu, L and Chen, Y and Chen, L and Lin, Y and Wen, T and Luo, X and Cong, Y}, title = {An EXPAR-CRISPR/Cas12a Assay for Rapid Detection of Salmonella.}, journal = {Current microbiology}, volume = {82}, number = {6}, pages = {262}, pmid = {40285901}, issn = {1432-0991}, support = {20231800500552//Dongguan Science and Technology Commissioner Project/ ; 81974299//National Natural Science Foundation of China/ ; 2023A1515010938//Basic and Applied Basic Research Foundation of Guangdong Province/ ; }, mesh = {*Salmonella/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Bacterial Proteins/genetics ; Sensitivity and Specificity ; DNA, Bacterial/genetics ; Salmonella Infections/diagnosis/microbiology ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Salmonella is considered as one of the primary pathogens associated with foodborne diseases globally. The effective treatment of these illnesses depends on the rapid and accurate identification of this organism. Traditional culture methods, however, necessitate extended testing periods, while many alternative techniques often lack precision. This research presents an innovative detection system that employs CRISPR-Cas12a for the detection of Salmonella. The detection system specifically targets the yfiR gene, which is amplified through isothermal exponential amplification (EXPAR). Target DNA hybridizes with the hairpin probe to form the DNA strand. The DNA strand was nicked to generate a nick by nicking endonuclease owing to its recognition sequence toward the hairpin probe. DNA polymerase can extend the 3'-end of the nicked site, which simultaneously displaces the newly synthesized strand. Thus, a large number of single-stranded DNA (ssDNA) were produced in the circle of nicking, polymerization, and strand displacement to achieve exponential amplification. The resultant amplified ssDNA products are subsequently recognized by CRISPR/Cas12a, resulting in the emission of a fluorescence signal. The detection system demonstrates a limit of detection of 10 fM for synthetic DNA and exhibits a strong linear relationship between 10 fM and 100 nM. Furthermore, the EXPAR-CRISPR/Cas12a detection system successfully identifies extracted genomic DNA samples containing Salmonella strains less than one hour, achieving a detection threshold of 1 pg/μL. This assay not only offers rapid results, requiring less than one hour for sample-to-answer outcomes, but is also cost-effective, minimizes aerosol risks, and provides exceptional specificity and sensitivity for the detection of Salmonella.}, }
@article {pmid40285354, year = {2025}, author = {John, T and Czechowicz, A}, title = {Clinical hematopoietic stem cell-based gene therapy.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {33}, number = {6}, pages = {2663-2678}, pmid = {40285354}, issn = {1525-0024}, mesh = {Humans ; *Genetic Therapy/methods ; *Hematopoietic Stem Cells/metabolism/cytology ; Gene Editing/methods ; CRISPR-Cas Systems ; *Hematopoietic Stem Cell Transplantation/methods ; Genetic Vectors/genetics ; Animals ; Gene Transfer Techniques ; Clinical Trials as Topic ; }, abstract = {Hematopoietic stem cell (HSC)-based gene therapies have seen extraordinary progress since their initial conception, now fundamentally transforming the treatment paradigms for various inherited hematologic, immunologic, and metabolic conditions-with additional use cases under exploration. Decades worth of work with advances in viral vector technologies and cell manufacturing have paved the way for HSC gene therapy with marked improvement in the safety and efficiency of gene delivery into HSCs. These have been augmented by the recent rise of innovative genome-editing techniques, particularly using clustered regularly interspaced short palindromic repeats CRISPR-associated proteins (CRISPR-Cas)-based technologies, which have enabled more precise and reproducible genome alterations in HSCs and fostered opportunities for targeted gene modification or gene correction. These breakthroughs have led to the development of many active clinical trials and culminated in the recent federal regulatory-agency approvals of multiple clinical HSC gene therapies for various indications that are now becoming available across different geographies. These treatments aim to offer significant, long-lasting benefits to patients worldwide without the toxicities of alternative treatment approaches. This review explores the history and advancements in HSC gene therapies and provides a comprehensive overview of the latest clinical innovations and cell-therapy products. Further, it concludes with a discussion of the persistent challenges that have limited adoption and potential future opportunities that aspire to enable curative treatment of many different patients through such personalized medicines.}, }
@article {pmid40284926, year = {2025}, author = {Yin, J and Cui, J and Zheng, H and Guo, T and Wei, R and Sha, Z and Gu, S and Ni, B}, title = {Implementation of RT-RAA and CRISPR/Cas13a for an NiV Point-of-Care Test: A Promising Tool for Disease Control.}, journal = {Viruses}, volume = {17}, number = {4}, pages = {}, pmid = {40284926}, issn = {1999-4915}, support = {2021YFD1800301-04//National Key R&amp;D Program for the 14th Five-Year Plan/ ; }, mesh = {Animals ; Swine ; *CRISPR-Cas Systems ; *Swine Diseases/diagnosis/virology ; Sensitivity and Specificity ; *Nipah Virus/genetics/isolation &amp; purification ; *Point-of-Care Testing ; *Nucleic Acid Amplification Techniques/methods ; Point-of-Care Systems ; }, abstract = {Nipah virus (NiV) is a severe zoonotic pathogen that substantially threatens public health. Pigs are the natural hosts of NiV and can potentially transmit this disease to humans. Establishing a rapid, sensitive, and accurate point-of-care detection method is critical in the timely identification of infected pig herds. In this study, we developed an NiV detection method based on reverse transcription-recombinase polymerase amplification (RT-RAA) and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 13a (Cas13a) system for the precise detection of NiV. The highly conserved region of the NiV gene was selected as the detection target. We first designed eleven pairs of RT-RAA primers, and the optimal primer combination and reaction temperature were identified on the basis of RT-RAA efficiency. Additionally, the most efficient crRNA sequence was selected on the basis of the fluorescence signal intensity. The results revealed that the optimal reaction temperature for the developed method was 37 °C. The detection limit was as low as 1.565 copies/μL. Specificity testing revealed no cross-reactivity with nucleic acids from six common swine viruses, including Seneca virus A (SVA), foot-and-mouth disease virus (FMDV), classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), African swine fever virus (ASFV), and pseudorabies virus (PRV). A validation test using simulated clinical samples revealed a 100% concordance rate. The detection results can be visualized via a fluorescence reader or lateral flow strips (LFSs). Compared with conventional detection methods, this RT-RAA-CRISPR/Cas13a-based method is rapid and simple and does not require scientific instruments. Moreover, the reagents can be freeze-dried for storage, eliminating the need for cold-chain transportation. This detection technology provides a convenient and efficient new tool for the point-of-care diagnosis of NiV and for preventing and controlling outbreaks.}, }
@article {pmid40284749, year = {2025}, author = {Hu, M and Chua, SL}, title = {Antibiotic-Resistant Pseudomonas aeruginosa: Current Challenges and Emerging Alternative Therapies.}, journal = {Microorganisms}, volume = {13}, number = {4}, pages = {}, pmid = {40284749}, issn = {2076-2607}, abstract = {Antibiotic-resistant Pseudomonas aeruginosa is a pathogen notorious for its resilience in clinical settings due to biofilm formation, efflux pumps, and the rapid acquisition of resistance genes. With traditional antibiotic therapy rendered ineffective against Pseudomonas aeruginosa infections, we explore alternative therapies that have shown promise, including antimicrobial peptides, nanoparticles and quorum sensing inhibitors. While these approaches offer potential, they each face challenges, such as specificity, stability, and delivery, which require careful consideration and further study. We also delve into emerging alternative strategies, such as bacteriophage therapy and CRISPR-Cas gene editing that could enhance targeted treatment for personalized medicine. As most of them are currently in experimental stages, we highlight the need for clinical trials and additional research to confirm their feasibility. Hence, we offer insights into new therapeutic avenues that could help address the pressing issue of antibiotic-resistant Pseudomonas aeruginosa, with an eye toward practical applications in future healthcare.}, }
@article {pmid40282900, year = {2025}, author = {Ganesh, I and Karthiga, I and Murugan, M and Rangarajalu, K and Ballambattu, VB and Ravikumar, S}, title = {CRISPR/Cas-Based Prenatal Screening for Aneuploidy: Challenges and Opportunities for Early Diagnosis.}, journal = {Medicina (Kaunas, Lithuania)}, volume = {61}, number = {4}, pages = {}, pmid = {40282900}, issn = {1648-9144}, mesh = {Humans ; Female ; Pregnancy ; *Aneuploidy ; *Prenatal Diagnosis/methods/trends ; *CRISPR-Cas Systems/genetics ; Early Diagnosis ; Pregnancy Trimester, First ; Genetic Testing/methods ; }, abstract = {Aneuploidy is increasingly recognized globally as a common cause of miscarriage among expectant mothers. The existing prenatal screening techniques for detecting aneuploidy have several limitations. The ability to diagnose aneuploidy early in a non-invasive manner is not feasible with the current screening methods, as they may produce false positive or false negative results. Recently, the widely used gene editing tool CRISPR/Cas has shown great promise in diagnostics. This review summarizes the prenatal screening tests used in the first trimester to assess aneuploidy conditions. Additionally, we discuss the advantages and disadvantages of molecular diagnostic tests, including the benefits and challenges of CRISPR/Cas-based trisomy detection. Thus, the proposed prenatal screening using CRISPR/Cas could provide significant benefits to expectant mothers by potentially enabling the early diagnosis of trisomy, helping to prevent miscarriage and birth defects. Furthermore, it opens new avenues for research, allowing clinicians and researchers to develop, optimize, and implement CRISPR/Cas-based prenatal screening assays in the future.}, }
@article {pmid40282347, year = {2025}, author = {Zheng, Y and Zou, Q and Li, J and Yang, Y}, title = {CRISPR-MFH: A Lightweight Hybrid Deep Learning Framework with Multi-Feature Encoding for Improved CRISPR-Cas9 Off-Target Prediction.}, journal = {Genes}, volume = {16}, number = {4}, pages = {}, pmid = {40282347}, issn = {2073-4425}, support = {32471860//National Natural Science Foundation of China/ ; Y202352570//scientific research project of the education department of Zhejiang province/ ; }, mesh = {*Deep Learning ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; }, abstract = {BACKGROUND: The CRISPR-Cas9 system has emerged as one of the most promising gene-editing technologies in biology. However, off-target effects remain a significant challenge. While recent advances in deep learning have led to the development of models for off-target prediction, these models often fail to fully leverage sequence pair information. Furthermore, as the models' parameter sizes increase, so do their complexities, limiting their practical applicability.<br><br>METHODS: In this study, we introduce a novel multi-feature independent encoding method, which encodes the gRNA-DNA sequence pair into three distinct feature matrices to minimize information loss. Additionally, we propose a lightweight hybrid deep learning framework, CRISPR-MFH, that integrates multi-scale separable convolutions and hybrid attention mechanisms for efficient and accurate off-target prediction.<br><br>RESULTS: Extensive experiments across multiple benchmark datasets demonstrate that the proposed encoding method effectively captures critical features and that CRISPR-MFH outperforms or matches state-of-the-art models with significantly fewer parameters across multiple evaluation metrics.<br><br>CONCLUSIONS: This study offers a novel perspective for advancing deep learning technology in the realm of CRISPR-Cas9 off-target detection.}, }
@article {pmid40282329, year = {2025}, author = {Rathe, SK and Marko, TA and Edwards, EN and Ridder, PH and Varshney, J and Williams, KB and Johnson, JE and Moriarity, BS and Largaespada, DA}, title = {Techniques for Validating CRISPR Changes Using RNA-Sequencing Data.}, journal = {Genes}, volume = {16}, number = {4}, pages = {}, pmid = {40282329}, issn = {2073-4425}, support = {MSTP grant T32 GM008224/NH/NIH HHS/United States ; R01-CA113636/NH/NIH HHS/United States ; #123939//ACS Research Professorship/ ; NA//Zach Sobiech Osteosarcoma Fund of the Children's Cancer Research Fund, Minneapolis MN/ ; NA//Children's Cancer Research Fund Emerging Scientist Award/ ; NA//Children's Tumor Foundation Young Investigator Award/ ; 5T32CA009138-40//Children's Tumor Foundation/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Sequence Analysis, RNA/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Transcriptome/genetics ; *RNA-Seq/methods ; Gene Editing/methods ; Gene Knockout Techniques/methods ; }, abstract = {UNLABELLED: The use of CRISPR to knockdown or knockout genes is a powerful tool for understanding the specific role of a gene in disease development. However, it can cause many unanticipated changes to the transcriptome that are not detected by DNA amplification and Sanger sequencing of the target site. Various RNA-sequencing techniques can be used to identify these changes and effectively gauge the full impact of the CRISPR knockout, thereby providing a means of selecting appropriate clones for further experimentation.<br><br>BACKGROUND/OBJECTIVES: RNA-seq data from 4 CRISPR knockout experiments were analyzed and techniques developed to both confirm the success of the CRISPR modifications and identify potential issues.<br><br>METHODS: A broad-based analysis of RNA-sequencing data identified many CRISPR-based changes not identified by PCR amplification of DNA around the CRISPR target site. These changes included an inter-chromosomal fusion event, exon skipping, chromosomal truncation, and the unintentional transcriptional modification and amplification of a neighboring gene.<br><br>CONCLUSIONS: The inadvertent modifications identified by the evaluation of 4 CRISPR experiments highlight the value of using RNA-seq to identify transcriptional changes to cells altered by CRISPR, many of which cannot be recognized by evaluating DNA alone. Specific guidelines are presented for designing and analyzing CRISPR experiments using RNA-seq data.}, }
@article {pmid40280947, year = {2025}, author = {Chen, H and Song, F and Wang, B and Huang, H and Luo, Y and Han, X and He, H and Lin, S and Wan, L and Huang, Z and Fu, Z and Ledesma-Amaro, R and Yin, D and Mao, H and He, L and Yang, T and Chen, Z and Ma, Y and Xue, EY and Wan, Y and Mao, C}, title = {Ultrasensitive detection of clinical pathogens through a target-amplification-free collateral-cleavage-enhancing CRISPR-CasΦ tool.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {3929}, pmid = {40280947}, issn = {2041-1723}, support = {42376184//National Science Foundation of China | National Natural Science Foundation of China-Yunnan Joint Fund (NSFC-Yunnan Joint Fund)/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Limit of Detection ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Bacteria/isolation &amp; purification/genetics ; DNA, Bacterial/genetics ; }, abstract = {Clinical pathogen diagnostics detect targets by qPCR (but with low sensitivity) or blood culturing (but time-consuming). Here we leverage a dual-stem-loop DNA amplifier to enhance non-specific collateral enzymatic cleavage of an oligonucleotide linker between a fluophore and its quencher by CRISPR-CasΦ, achieving ultrasensitive target detection. Specifically, the target pathogens are lysed to release DNA, which binds its complementary gRNA in CRISPR-CasΦ to activate the collateral DNA-cleavage capability of CasΦ, enabling CasΦ to cleave the stem-loops in the amplifier. The cleavage product binds its complementary gRNA in another CRISPR-CasΦ to activate more CasΦ. The activated CasΦ collaterally cleaves the linker, releasing the fluophore to recover its fluorescent signal. The cycle of stem-loop-cleavage/CasΦ-activation/fluorescence-recovery amplifies the detection signal. Our target amplification-free collateral-cleavage-enhancing CRISPR-CasΦ method (TCC), with a detection limit of 0.11 copies/μL, demonstrates enhanced sensitivity compared to qPCR. It can detect pathogenic bacteria as low as 1.2 CFU/mL in serum within 40 min.}, }
@article {pmid40280080, year = {2025}, author = {Xiong, Q and Zhu, C and Yin, X and Zhu, L}, title = {CRISPR/Cas and Argonaute-based biosensors for nucleic acid detection.}, journal = {Talanta}, volume = {294}, number = {}, pages = {128210}, doi = {10.1016/j.talanta.2025.128210}, pmid = {40280080}, issn = {1873-3573}, mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Argonaute Proteins/metabolism/genetics ; *Nucleic Acids/analysis/genetics ; Humans ; }, abstract = {Nowadays, nucleic acid detection technology has been applied to disease diagnosis, prevention, food safety, environmental testing and many other aspects. However, traditional methods still have shortcomings. Therefore, there is an urgent need for a simple, rapid, sensitive, and specific new method to supersede traditional nucleic acid detection technology. CRISPR/Cas(Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) system and Argonaute (Ago) system play an important role in microbial immune defense. Their targeting specificity, programmability and special trans-cleavage activity make it possible to develop some new platforms for nucleic acid detection in combination with a variety of biosensors. We introduce the origins of these two systems and the biosensors developed based on CRISPR/Cas system and Ago system, respectively, especially the prospects for the future development of Cascade Amplification biosensors. This review is expected to provide useful guidance for researchers in related fields and provide inspiration for the development of Cascade Amplification biosensors in the future.}, }
@article {pmid40278504, year = {2025}, author = {Lv, J and Jin, J and Ding, L and Xiang, L and Xie, B and Wu, K and Chen, Q}, title = {Directed Evolution of OgeuIscB With Enhanced Activity in Human Cells.}, journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology}, volume = {39}, number = {8}, pages = {e70570}, doi = {10.1096/fj.202500082R}, pmid = {40278504}, issn = {1530-6860}, support = {Y20220148//Science and Technology Project of Wenzhou City/ ; Y2023784//Science and Technology Project of Wenzhou City/ ; ZCLTGD24H1202//MOST | NSFC | NSFC-Zhejiang Joint Fund | | Natural Science Foundation of Zhejiang Province (ZJNSF)/ ; LTGD23H120001//MOST | NSFC | NSFC-Zhejiang Joint Fund | | Natural Science Foundation of Zhejiang Province (ZJNSF)/ ; LTGC23H120001//MOST | NSFC | NSFC-Zhejiang Joint Fund | | Natural Science Foundation of Zhejiang Province (ZJNSF)/ ; 82101169//National Natural Science Foundation of China/ ; }, mesh = {Humans ; *Gene Editing/methods ; *Directed Molecular Evolution/methods ; HEK293 Cells ; Protein Engineering/methods ; Mutation ; CRISPR-Cas Systems ; }, abstract = {The miniature RNA-guided endonuclease IscB, as the evolutionary progenitor of Cas9, is attracting increased attention for genome editing due to its compact size and suitability for in vivo delivery. However, the poor editing efficiency of IscB in eukaryotic cells presents a significant challenge to its widespread application in precise site-specific human genome editing. In this study, we employed structure-guided rational design and protein engineering to optimize OgeuIscB, resulting in the identification of enIscB-F138R, which further enhanced editing activity up to 3.49-fold in mammalian cells compared to the high-activity OgeuIscB variant enIscB. Furthermore, we engineered an enIscB-F138R nickase-based adenine base editor, termed miABE-F138R, exhibiting enhanced base editing efficiency relative to miABE. To illustrate the practical applications of miABE-F138R, we applied it to rectify the prevalent R560C mutation in Pde6β associated with autosomal recessive retinitis pigmentosa, resulting in a significant improvement in activity compared to miABE. In conclusion, enIscB-F138R and miABE-F138R offer adaptable platforms for genome editing with potential significance in future biomedical applications.}, }
@article {pmid40277901, year = {2025}, author = {Plümers, R and Jelinek, S and Lindenkamp, C and Osterhage, MR and Knabbe, C and Hendig, D}, title = {Investigation on ABCC6-Deficient Human Hepatocytes Generated by CRISPR-Cas9 Genome Editing.}, journal = {Cells}, volume = {14}, number = {8}, pages = {}, pmid = {40277901}, issn = {2073-4409}, mesh = {Humans ; *Hepatocytes/metabolism ; *CRISPR-Cas Systems/genetics ; *Multidrug Resistance-Associated Proteins/deficiency/genetics/metabolism ; *Gene Editing/methods ; Pseudoxanthoma Elasticum/genetics/pathology/metabolism ; }, abstract = {Patients affected by the rare disease pseudoxanthoma elasticum (PXE) exhibit the calcification of elastic fibers in ocular, dermal, and vascular tissues. These symptoms are triggered by mutations in the ATP-binding cassette transporter subfamily C member 6 (ABCC6), whose substrate remains unknown. Interestingly, ABCC6 is predominantly expressed in the liver tissue, leading to the hypothesis that PXE is a metabolic disorder. We developed a genome-editing system targeting ABCC6 in human immortalized hepatocytes (HepIms) for further investigations. The HepIms were transfected with an ABCC6-specific clustered regulatory interspaced short palindromic repeat (CRISPR-Cas9) genome-editing plasmid, resulting in the identification of a heterozygous (ht[ABCC6]HepIm) and a compound heterozygous (cht[ABCC6]HepIm) clone. These clones were analyzed for key markers associated with the PXE pathobiochemistry. Hints of impaired lipid trafficking, defects in the extracellular matrix remodeling, the induction of calcification inhibitor expression, and the down regulation of senescence and inflammatory markers in ABCC6-deficienct HepIms were found. Our ABCC6 knock-out model of HepIms provides a valuable tool for studying the metabolic characteristics of PXE in vitro. The initial analysis of the clones mirrors various features of the PXE pathobiochemistry and provides an outlook on future research approaches.}, }
@article {pmid40277547, year = {2025}, author = {Xue, B and Qiao, B and Jia, L and Chi, J and Su, M and Song, Y and Du, J}, title = {A Sensitive and Fast microRNA Detection Platform Based on CRlSPR-Cas12a Coupled with Hybridization Chain Reaction and Photonic Crystal Microarray.}, journal = {Biosensors}, volume = {15}, number = {4}, pages = {}, pmid = {40277547}, issn = {2079-6374}, support = {2021YFA0805100//National Key Research and Development Program of China/ ; 82070407//National Natural Science Foundation of China/ ; 82200459//National Natural Science Foundation of China/ ; }, mesh = {*MicroRNAs/analysis ; Nucleic Acid Hybridization ; CRISPR-Cas Systems ; *Biosensing Techniques ; Oligonucleotide Array Sequence Analysis ; Humans ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Changes in microRNA (miRNA) levels are closely associated with the pathological processes of many diseases. The sensitive and fast detection of miRNAs is critical for diagnosis and prognosis. Here, we report a platform employing CRISPR/Cas12a to recognize and report changes in miRNA levels while avoiding complex multi-thermal cycling procedures. A non-enzyme-dependent hybridization chain reaction (HCR) was used to convert the miRNA signal into double-stranded DNA, which contained a Cas12a activation sequence. The target sequence was amplified simply and isothermally, enabling the test to be executed at a constant temperature of 37 °C. The detection platform had the capacity to measure concentrations down to the picomolar level, and the target miRNA could be distinguished at the nanomolar level. By using photonic crystal microarrays with a stopband-matched emission spectrum of the fluorescent-quencher modified reporter, the fluorescence signal was moderately enhanced to increase the sensitivity. With this enhancement, analyzable fluorescence results were obtained in 15 min. The HCR and Cas12a cleavage processes could be conducted in a single tube by separating the two procedures into the bottom and the cap. We verified the sensitivity and specificity of this one-pot system, and both were comparable to those of the two-step method. Overall, our study produced a fast and sensitive miRNA detection platform based on a CRISPR/Cas12a system and enzyme-free HCR amplification. This platform may serve as a potential solution for miRNA detection in clinical practice.}, }
@article {pmid40277544, year = {2025}, author = {Chao, A and Hu, Q and Yin, K}, title = {A Label-Free CRISPR/Cas12a-G4 Biosensor Integrated with FTA Card for Detection of Foodborne Pathogens.}, journal = {Biosensors}, volume = {15}, number = {4}, pages = {}, pmid = {40277544}, issn = {2079-6374}, support = {ZDYF2022SHFZ321//Hainan Province Science and Technology Special Fund/ ; X2020-02-08-00-12-F01110//Shanghai Agriculture Applied Technology Development Program/ ; 202340069//Project of Shanghai Municipal Health Commission/ ; 22104090//National Natural Science Foundation of China/ ; 22ZR1436200//Natural Science Foundation of Shanghai/ ; NA//Shanghai Oriental Talent Program (Youth Project)/ ; 22474076//National Natural Science Foundation of China/ ; }, mesh = {*Biosensing Techniques ; *Escherichia coli O157/isolation &amp; purification ; *CRISPR-Cas Systems ; *Food Microbiology ; G-Quadruplexes ; Limit of Detection ; }, abstract = {CRISPR/Cas-based diagnostics offer unparalleled specificity, but their reliance on fluorescently labeled probes and complex nucleic acid extraction limits field applicability. To tackle this problem, we have developed a label-free, equipment-free platform integrating FTA card-based extraction, CRISPR/Cas12a, and pre-folded G-quadruplex (G4)-Thioflavin T (ThT) signal reporter. This system eliminates costly fluorescent labeling by leveraging G4-ThT structural binding for visible fluorescence output, while FTA cards streamline nucleic acid isolation without centrifugation. Achieving a limit of detection (LOD) to 10[1] CFU/mL for Escherichia coli O157:H7 in spiked food samples, the platform demonstrated 100% concordance with qPCR and standard fluorescent probe-based CRISPR/Cas12a system. Its simplicity, minimal equipment (portable heating/imaging), and cost-effectiveness make it a revolutionary tool for detecting foodborne pathogens in resource-limited environments.}, }
@article {pmid40277382, year = {2025}, author = {Xu, D and Wu, Q and Yang, F and Zhang, Q and Jiang, Q and Zeng, X and Zhang, Y and Lv, T and Wang, J and Li, F}, title = {Fast-Flu: RT-RPA-CRISPR/Cas12a assisted one-step platform for rapid influenza B virus detection.}, journal = {Microbiology spectrum}, volume = {13}, number = {6}, pages = {e0036525}, pmid = {40277382}, issn = {2165-0497}, mesh = {*Influenza B virus/isolation &amp; purification/genetics ; Humans ; *Influenza, Human/diagnosis/virology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/genetics ; *Molecular Diagnostic Techniques/methods ; Recombinases ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {UNLABELLED: Influenza B virus (Flu B) is a prevalent respiratory pathogen responsible for seasonal influenza epidemics. Despite its clinical significance, there remains a lack of rapid and accurate diagnostic methods for Flu B detection. In this study, we developed a novel Flu B detection system, named Fast-Flu, by integrating reverse transcription recombinase polymerase amplification (RT-RPA) with the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system (CRISPR/Cas). Through optimization of reaction temperature and adjustment of Cas12a concentrations, we successfully balanced RPA amplification and CRISPR/Cas12a trans-cleavage activity, enabling the establishment of a one-step detection system. The one-step Fast-Flu system demonstrated the ability to specifically identify Flu B within 45 min, with a limit of detection of 58 copies per test. It eliminates the need for uncapping operations and minimizes the risk of cross-contamination, without cross-reactivity with other pathogens. When evaluated using 101 clinical throat swab samples, the one-step Fast-Flu system achieved a sensitivity of 56.25% and a specificity of 100% compared to the PCR-based method, with an overall concordance rate of 93.06% (94/101). The development of this one-step RT-RPA-CRISPR/Cas12a system represents a significant advancement in the rapid, convenient, and accurate detection of Flu B, highlighting its potential for clinical diagnosis. Furthermore, with future technical improvements to enhance sensitivity, this one-step RT-RPA-CRISPR assay holds promise as a versatile tool for the rapid nucleic acid detection of other RNA viruses.<br><br>IMPORTANCE: Influenza B virus (Flu B) is a significant global health concern, and rapid, accurate pathogen diagnosis is crucial for effective influenza prevention and control. The integration of isothermal amplification methods with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has achieved high sensitivity and specificity for nucleic acid detection. Although CRISPR/Cas-based systems have been developed for influenza detection, existing platforms require the transfer of amplified products into the CRISPR/Cas12a detection system through uncapping operations, which increases the risk of cross-contamination. In this study, we developed a one-step reverse transcription recombinase polymerase amplification-CRISPR/Cas12a Flu B detection method using a one-pot detection system. By optimizing the reaction temperature and Cas12a concentration, we achieved a streamlined and contamination-free workflow. This innovative approach not only improves Flu B detection but also serves as a valuable reference for constructing CRISPR/Cas systems for the detection of other pathogens and targets, paving the way for broader applications in molecular diagnostics.}, }
@article {pmid40277083, year = {2025}, author = {Dillard, KE and Zhang, H and Dubbs, LZ and Chou, CW and Terrace, C and Javanmardi, K and Kim, W and Forsberg, KJ and Finkelstein, IJ}, title = {Mechanism of Cas9 inhibition by AcrIIA11.}, journal = {Nucleic acids research}, volume = {53}, number = {8}, pages = {}, pmid = {40277083}, issn = {1362-4962}, support = {R01GM124141/GF/NIH HHS/United States ; R01 GM124141/GM/NIGMS NIH HHS/United States ; //Howard Hughes Medical Institute Emerging Pathogens Initiative/ ; F31 GM125201/GM/NIGMS NIH HHS/United States ; //Searle Scholars Award/ ; R01 GM104896/GM/NIGMS NIH HHS/United States ; F-1808//Welch Foundation/ ; //University of Texas Southwestern Medical Center/ ; DP2 AI154402/AI/NIAID NIH HHS/United States ; }, mesh = {*CRISPR-Associated Protein 9/metabolism/antagonists &amp; inhibitors/genetics/chemistry ; Humans ; *CRISPR-Cas Systems/genetics ; Staphylococcus aureus/genetics/enzymology ; Streptococcus pyogenes/genetics/enzymology ; DNA Cleavage ; *Bacterial Proteins/metabolism/genetics/antagonists &amp; inhibitors ; Francisella/genetics/enzymology ; DNA/metabolism ; HEK293 Cells ; }, abstract = {Mobile genetic elements evade CRISPR-Cas adaptive immunity by encoding anti-CRISPR proteins (Acrs). Acrs inactivate CRISPR-Cas systems via diverse mechanisms but generally coevolve with a narrow subset of Cas effectors that share high sequence similarity. Here, we demonstrate that AcrIIA11 inhibits Streptococcus pyogenes (Sp), Staphylococcus aureus (Sa), and Francisella novicida (Fn) Cas9s in vitro and in human cells. Single-molecule imaging reveals that AcrIIA11 hinders SaCas9 target search by reducing its diffusion on nonspecific DNA. DNA cleavage is inhibited because the AcrIIA11:SaCas9 complex binds to protospacer adjacent motif (PAM)-rich off-target sites, preventing SaCas9 from reaching its target. AcrIIA11 also greatly slows down DNA cleavage after SaCas9 reaches its target site. A negative-stain electron microscopy reconstruction of an AcrIIA11:SaCas9 RNP complex reveals that the heterodimer assembles with a 1:1 stoichiometry. Physical AcrIIA11-Cas9 interactions across type IIA and IIB Cas9s correlate with nuclease inhibition and support its broad-spectrum activity. These results add a kinetic inhibition mechanism to the phage-CRISPR arms race.}, }
@article {pmid40275527, year = {2025}, author = {Chen, Y and Li, M and Liu, X and Duan, Q and Xiao, L and Wang, L and Huang, C and Song, H and Cao, Y}, title = {Establishment of CRISPR-STAR System to Realise Simultaneous Transcriptional Activation and Repression in Yarrowia lipolytica.}, journal = {Microbial biotechnology}, volume = {18}, number = {4}, pages = {e70151}, pmid = {40275527}, issn = {1751-7915}, support = {2021YFC2104400//National Key Research and Development Program of China/ ; NSFC22478294//the National Natural Science Foundation of China/ ; NSFC22078240//the National Natural Science Foundation of China/ ; }, mesh = {*Yarrowia/genetics/metabolism ; *Transcriptional Activation ; *Gene Expression Regulation, Fungal ; *CRISPR-Cas Systems ; Promoter Regions, Genetic ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {The ability to regulate gene expression in multiple directions is crucial to maximise the production of microbial cell factories. However, the lack of a regulatory tool that can simultaneously activate and repress multiple genes restricts the manipulation diversity of Yarrowia lipolytica, which is an industrial workhorse for bioproduction. To address this issue, we developed a CRISPR scaffold RNAs (scRNAs)-mediated transcriptional activation and repression (CRISPR-STAR) platform. Firstly, we evaluated different methods for bidirectional regulation using CRISPR on both endogenous and synthetic promoters in Y. lipolytica, and chose the utilisation of orthogonal scRNAs to recruit activation and inhibition domains. Secondly, CRISPR-STAR was optimised by the introduction of alternative dCas proteins, scRNA structures and activators. 2.6-fold and 54.9-fold activation were achieved for synthetic and endogenous promoters, respectively, when the VPR transcriptional activator was recruited via MS2 hairpin. The repression of several genes was successfully achieved, with repression levels ranging from 3% to 32%, when the MXI1 transcriptional repressor was recruited via PP7 hairpin. Finally, CRISPR-STAR was applied to enhance fatty alcohol production by activating the FAR gene (encodes fatty acyl-CoA reductase) and repression of the PEX10 gene (encodes an integral membrane protein required for peroxisome biogenesis and matrix protein import). Compared to the non-targeting control, the bidirectionally regulated strain showed a 55.7% increase in yield to 778.8 mg/L. Our findings demonstrate that the CRISPR-STAR platform enables multi-mode regulation of genes, offering engineering opportunities to improve the productive performance of Y. lipolytica.}, }
@article {pmid40275470, year = {2025}, author = {Kelly, G and Plesser, E and Bdolach, E and Arroyave, M and Belausov, E and Doron-Faigenboim, A and Rozen, A and Zemach, H and Zach, YY and Goldenberg, L and Arad, T and Yaniv, Y and Sade, N and Sherman, A and Eyal, Y and Carmi, N}, title = {In planta genome editing in citrus facilitated by co-expression of CRISPR/Cas and developmental regulators.}, journal = {The Plant journal : for cell and molecular biology}, volume = {122}, number = {2}, pages = {e70155}, pmid = {40275470}, issn = {1365-313X}, mesh = {*Citrus/genetics/growth &amp; development ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Genome, Plant/genetics ; Agrobacterium tumefaciens/genetics ; Plant Shoots/genetics/growth &amp; development ; }, abstract = {Recent advances in the field of genome editing offer a promising avenue for targeted trait improvements in fruit trees. However, the predominant method taken for genome editing in citrus (and other fruit trees) involves the time-consuming tissue culture approach, thereby prolonging the overall citrus breeding process and subjecting it to the drawbacks associated with somaclonal variation. In this study, we introduce an in planta approach for genome editing in soil-grown citrus plants via direct transformation of young seedlings. Our editing system, abbreviated here as IPGEC (in planta genome editing in citrus), is designed to transiently co-express three key gene groups in citrus tissue via Agrobacterium tumefaciens: (i) a genome-editing catalytic group, (ii) a shoot induction and regeneration group, and (iii) a T-DNA enhanced delivery group. This integrated system significantly improves de novo shoot induction and regeneration efficiency of edited tissue. By incorporating single-guides RNA's (sgRNA's) targeting the carotenoid biosynthetic gene PHYTOENE DESATURASE (CsPDS), the IPGEC system effectively produced mutated albino shoots, confirming its ability to generate homozygous/biallelic genome-edited plants. By using high throughput screening, we provide evidence that transgene-free genome-edited plants could be obtained following the IPGEC approach. Our findings further suggest that the efficiency of specific developmental regulators in inducing transformation and regeneration rates may be cultivar-specific and therefore needs to be optimized per cultivar. Finally, targeted breeding for specific trait improvements in already successful cultivars is likely to revolutionize fruit tree breeding and will pave the way for accelerating the development of high-quality citrus cultivars.}, }
@article {pmid40275435, year = {2025}, author = {Yang, Z and Bai, W and Guo, G and Huang, S and Wang, Y and Zhou, Y and Zhang, Y and Sun, J}, title = {The Q-interacted protein QIP3 recruits TaTPL to regulate spike architecture in wheat.}, journal = {The Plant journal : for cell and molecular biology}, volume = {122}, number = {2}, pages = {e70149}, doi = {10.1111/tpj.70149}, pmid = {40275435}, issn = {1365-313X}, support = {32472096//National Natural Science Foundation of China Grant/ ; 2023YFF1000402//National Key Research and Development Program of China/ ; //Innovation Program of the Chinese Academy of Agricultural Sciences/ ; }, mesh = {*Triticum/genetics/metabolism/anatomy &amp; histology/growth &amp; development ; *Plant Proteins/metabolism/genetics ; *Transcription Factors/metabolism/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; }, abstract = {Spike architecture is a critical determinant of grain yield in wheat; yet the regulatory mechanisms remain poorly understood. Here, we demonstrate that the AP2 transcription factor Q directly represses the expression of TaMYB30-6A, a gene associated with spike length in wheat. We further identify QIP3 as a Q-interacting protein harboring an N-terminal EAR motif. Simultaneously, we reveal that QIP3 exhibits transcriptional repression activity, dependent on the EAR motif, and physically interacts with the transcriptional corepressor TaTPL. Importantly, the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-generated qip3-aabbdd mutants exhibit reduced plant height and increased spike length phenotypes. Furthermore, RNA-seq and RT-qPCR assays show that QIP3 negatively regulates the expression of the Q target gene TaMYB30-6A in wheat. Collectively, we propose that the EAR motif-containing QIP3 interacts with Q to regulate spike architecture by recruiting the transcriptional corepressor TaTPL in wheat.}, }
@article {pmid40274952, year = {2025}, author = {Zhang, Z and Abreu, B and Brothwood, JL and Alexander, J and Sims, MJ and Lyons, JF and Munck, JM and Hindley, CJ}, title = {The identification of functional regions of MEK1 using CRISPR tiling screens.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {656}, pmid = {40274952}, issn = {2399-3642}, mesh = {*MAP Kinase Kinase 1/genetics/antagonists &amp; inhibitors/metabolism/chemistry ; Humans ; *CRISPR-Cas Systems ; Protein Kinase Inhibitors/pharmacology ; Cell Line, Tumor ; Drug Resistance, Neoplasm/genetics ; Mutation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Discovery/methods ; Cell Survival/drug effects ; }, abstract = {CRISPR tiling screen is a powerful tool to identify protein regions relevant to its biological function. Understanding the functional relevance of the regions of target protein is of great help for structure-based drug discovery. Studying the drug resistance mechanisms of small-molecule inhibitors is important for the development and clinical application of the compounds. Using MEK1 and MEK inhibitors as example here, we demonstrate the utility of CRISPR tiling to identify regions essential for cancer cell viability and regions where mutations are resistant to MEK inhibitors. We study the drug resistance mechanisms of the regions and discussed the potential, as well as limitations, of applying the technology to drug development. Our findings demonstrate the value and prompt the utilization of CRISPR tiling technology in structure-based drug discovery.}, }
@article {pmid40274871, year = {2025}, author = {Chai, R and Guo, J and Yang, C and Zhu, D and Li, T and Yang, W and Liu, X and Chen, X and Huang, S and Wang, H and Yao, X and Gao, Y and Qiu, L}, title = {Enhanced chemotaxis and degradation of nonylphenol in Pseudoxanthomonas mexicana via CRISPR-mediated receptor modification.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {14296}, pmid = {40274871}, issn = {2045-2322}, support = {25B610014//the Key scientific research projects of colleges and universities in Henan Province/ ; GTTXM202412//the Consortium Construction Research Project of China Biotechnology Vocational Education Teaching Steering Committee/ ; 2403107//the Kaifeng Science and Technology Development Project/ ; 252102320066//the Key Research and Development Special Project of Henan Provincial Science and Technology/ ; }, mesh = {*Chemotaxis/genetics ; *Phenols/metabolism ; Biodegradation, Environmental ; Phylogeny ; Bacterial Proteins/genetics/metabolism ; Wastewater/microbiology ; Sewage/microbiology ; *CRISPR-Cas Systems ; }, abstract = {In this study, a novel nonylphenol (NP)-degrading bacterium, Pseudoxanthomonas mexicana CH, was isolated from wastewater treatment plant effluent. Phylogenetic analysis showed its close relationship to P. mexicana AMX 26BT. The strain displayed chemotaxis toward NP, with Mcp24 as the key chemoreceptor. The Mcp24 deletion mutant (CH- 1) had weaker chemotaxis and NP degradation (over 30% lower in solution and 8% lower in sludge than the wild type). In vitro, Mcp15's C-terminal pentapeptide DWQEF was methylated by CheR. Using CRISPR, this pentapeptide was added to Mcp24 to create CH- 2. CH- 2 showed better NP chemotaxis (17% higher in plate assays and 39% higher in capillary assays) and higher NP degradation rates (23.5% and 24.2% higher in solution and sludge, respectively). These findings demonstrate that NP acts as a bacterial chemoattractant, with Mcp24 as the receptor. Enhancing Mcp24's C-terminal pentapeptide improves chemotaxis and degradation efficiency, representing a significant advancement in bioremediation by strengthening bacterial responses to pollutants.}, }
@article {pmid40274854, year = {2025}, author = {Balakrishnan, A and Hunziker, M and Tiwary, P and Pandey, V and Drew, D and Billker, O}, title = {A CRISPR homing screen finds a chloroquine resistance transporter-like protein of the Plasmodium oocyst essential for mosquito transmission of malaria.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {3895}, pmid = {40274854}, issn = {2041-1723}, support = {LT000131/2020-L//Human Frontier Science Program (HFSP)/ ; LT000131/2020-L//Human Frontier Science Program (HFSP)/ ; 895744//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 Marie Sk&#x142;odowska-Curie Actions (H2020 Excellent Science - Marie Sk&#x142;odowska-Curie Actions)/ ; 788516//EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: "Ideas" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013))/ ; 895744//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; P2SKP3_187635//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; }, mesh = {*Plasmodium berghei/genetics/drug effects/metabolism ; Animals ; *Malaria/transmission/parasitology ; *Oocysts/metabolism/genetics/drug effects/growth &amp; development ; *Protozoan Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing/methods ; Mice ; Mosquito Vectors/parasitology ; Drug Resistance/genetics ; Chloroquine/pharmacology ; Female ; Anopheles/parasitology ; }, abstract = {Genetic screens with barcoded PlasmoGEM vectors have identified thousands of Plasmodium berghei gene functions in haploid blood stages, gametocytes and liver stages. However, the formation of diploid cells by fertilisation has hindered similar research on the parasites' mosquito stages. In this study, we develop a scalable genetic system that uses barcoded gene targeting vectors equipped with a CRISPR-mediated homing mechanism to generate homozygous loss-of-function mutants after one parent introduces a modified allele into the zygote. To achieve this, we use vectors additionally expressing a target gene specific gRNA. When integrated into one of the parental alleles it directs Cas9 to the intact allele after fertilisation, leading to its disruption. This homing strategy is 90% effective at generating homozygous gene editing of a fluorescence-tagged reporter locus in the oocyst. A pilot screen identifies PBANKA_0916000 as a chloroquine resistance transporter-like protein (CRTL) essential for oocyst growth and sporogony, pointing to an unexpected importance for malaria transmission of the poorly understood digestive vacuole of the oocyst that contains hemozoin granules. Homing screens provide a method for the systematic discovery of malaria transmission genes whose first essential functions are after fertilisation in the bloodmeal, enabling their potential as targets for transmission-blocking interventions to be assessed.}, }
@article {pmid40274667, year = {2025}, author = {Mukherjee, A and Samanta, S and Das, S and Haque, MZ and Jana, PS and Samanta, I and Kar, I and Das, S and Nanda, PK and Thomas, P and Dandapat, P}, title = {Leveraging CRISPR-Cas-Enhanced Isothermal Amplification Tools for Quick Identification of Pathogens Causing Livestock Diseases.}, journal = {Current microbiology}, volume = {82}, number = {6}, pages = {260}, pmid = {40274667}, issn = {1432-0991}, support = {STBT-11012(27)/5/2024-ST SEC//Department of Science and Technology, Government of West Bengal/ ; }, mesh = {Animals ; *Nucleic Acid Amplification Techniques/methods ; *Livestock/microbiology/virology ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; *Animal Diseases/diagnosis/microbiology/virology ; *Bacteria/genetics/isolation &amp; purification ; }, abstract = {Prompt and accurate diagnosis of infectious pathogens of livestock origin is of utmost importance for epidemiological surveillance and effective therapeutic strategy formulation. Among various methods, nucleic acid-based detection of pathogens is the most sensitive and specific; but the majority of these assays need expensive equipment and skilled workers. Due to the rapid advancement of clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas)-based nucleic acid detection methods, these are now being widely used for pathogen detection. CRISPR-Cas is a bacterial counterpart of "adaptive immunity", generally used for editing genome. Many CRISPR systems have been modified for nucleic acid detection due to their excellent selectivity in detecting DNA and RNA sequences. The combination of CRISPR with suitable isothermal amplification technologies has made it more sensitive, specific, versatile, and reproducible for the detection of pathogen nucleic acids at the point of care. Amplification of pathogen nucleic acid by isothermal amplification followed by CRISPR-Cas-based detection has several advantages, including short sample-to-answer times and no requirement for laboratory set-up. They are also significantly less expensive than the existing nucleic acid detection methods. This review focuses on the recent trends in the use of this precision diagnostic method for diagnosis of a wide range of animal pathogens with or without zoonotic potential, particularly various isothermal amplification strategies, and visualization methods for sensing bacteria, viruses, and parasites of veterinary and public health importance.}, }
@article {pmid40273916, year = {2025}, author = {Shi, H and Al-Sayyad, N and Wasko, KM and Trinidad, MI and Doherty, EE and Vohra, K and Boger, RS and Colognori, D and Cofsky, JC and Skopintsev, P and Bryant, Z and Doudna, JA}, title = {Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing.}, journal = {Molecular cell}, volume = {85}, number = {9}, pages = {1730-1742.e9}, doi = {10.1016/j.molcel.2025.03.024}, pmid = {40273916}, issn = {1097-4164}, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Streptococcus pyogenes/genetics/enzymology ; Humans ; *CRISPR-Associated Protein 9/genetics/metabolism ; HEK293 Cells ; *Bacterial Proteins/genetics/metabolism ; *DNA/genetics/metabolism ; Nucleotide Motifs ; }, abstract = {RNA-guided CRISPR-Cas enzymes initiate programmable genome editing by recognizing a ∼20-base-pair DNA sequence next to a short protospacer-adjacent motif (PAM). To uncover the molecular determinants of high-efficiency editing, we conducted biochemical, biophysical, and cell-based assays on Streptococcus pyogenes Cas9 (SpyCas9) variants with wide-ranging genome-editing efficiencies that differ in PAM-binding specificity. Our results show that reduced PAM specificity causes persistent non-selective DNA binding and recurrent failures to engage the target sequence through stable guide RNA hybridization, leading to reduced genome-editing efficiency in cells. These findings reveal a fundamental trade-off between broad PAM recognition and genome-editing effectiveness. We propose that high-efficiency RNA-guided genome editing relies on an optimized two-step target capture process, where selective but low-affinity PAM binding precedes rapid DNA unwinding. This model provides a foundation for engineering more effective CRISPR-Cas and related RNA-guided genome editors.}, }
@article {pmid40273800, year = {2025}, author = {Zheng, M and Bao, N and Wang, Z and Song, C and Jin, Y}, title = {Alternative splicing in autism spectrum disorder: Recent insights from mechanisms to therapy.}, journal = {Asian journal of psychiatry}, volume = {108}, number = {}, pages = {104501}, doi = {10.1016/j.ajp.2025.104501}, pmid = {40273800}, issn = {1876-2026}, mesh = {Humans ; *Autism Spectrum Disorder/genetics/therapy ; *Alternative Splicing/genetics ; }, abstract = {Alternative splicing (AS) is a vital and highly dynamic RNA regulatory mechanism that allows a single gene to generate multiple mRNA and protein isoforms. Dysregulation of AS has been identified as a key contributor to the pathogenesis of autism spectrum disorders (ASD). A comprehensive understanding of aberrant splicing mechanisms and their functional consequences in ASD can help uncover the molecular basis of the disorder and facilitate the development of therapeutic strategies. This review focuses on the major aberrant splicing events and key splicing regulators associated with ASD, highlighting their roles in linking defective splicing to ASD pathogenesis. In addition, a discussion of how emerging technologies, such as long-read sequencing, single-cell sequencing, spatial transcriptomics and CRISPR-Cas systems are offering novel insights into the role and mechanisms of AS in ASD is presented. Finally, the RNA splicing-based therapeutic strategies are evaluated, emphasizing their potential to address unmet clinical needs in ASD treatment.}, }
@article {pmid40273687, year = {2025}, author = {Chen, X and Zhang, S and Lin, S and Huang, M and Chen, S and Wang, S}, title = {From lab to field: Innovative RPA‒CRISPR/Cas12a platform for early short-beak and dwarfism syndrome virus nucleic acids detection.}, journal = {Poultry science}, volume = {104}, number = {7}, pages = {105191}, pmid = {40273687}, issn = {1525-3171}, mesh = {Animals ; *Poultry Diseases/diagnosis/virology ; CRISPR-Cas Systems ; *Parvoviridae Infections/veterinary/diagnosis/virology ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *Ducks ; *Parvovirinae/isolation &amp; purification ; Sensitivity and Specificity ; }, abstract = {Short beak and dwarfism syndrome virus (SBDSV) is the causative agent of short beak and dwarfism syndrome (SBDS), which is characterized by beak atrophy and dwarfism. SBDS has caused substantial economic losses for waterfowl husbandry industries. To address the urgent need for rapid and accurate SBDSV diagnostics, the study developed a dual-mode detection platform integrating recombinase-aided amplification (RPA) with CRISPR/Cas12a-mediated fluorescence and lateral flow strip readouts. The optimized assay achieved a detection limit of 10 copies/μL. Notably, the platform demonstrated superior specificity to distinguish SBDSV from genetically related Muscovy duck-origin goose parvovirus (GPV) and classical GPV, a distinction unachievable by qPCR. Clinical validation using 36 field samples confirmed 100% concordance with qPCR and indirect immunofluorescence assays, with no cross-reactivity against other common duck pathogens. This innovative detection system provides a robust toolkit for field-deployable SBDSV surveillance and lays a solid foundation for the development of novel diagnostic methodologies applicable to various waterfowl-related pathogens.}, }
@article {pmid40273641, year = {2025}, author = {Bhoir, K and Prakash, G and Odaneth, A}, title = {Genetic Engineering of Yarrowia lipolytica for 1,8-cineole production: A sustainable approach.}, journal = {Enzyme and microbial technology}, volume = {189}, number = {}, pages = {110659}, doi = {10.1016/j.enzmictec.2025.110659}, pmid = {40273641}, issn = {1879-0909}, mesh = {*Yarrowia/genetics/metabolism ; *Eucalyptol/metabolism ; *Metabolic Engineering/methods ; Streptomyces/enzymology/genetics ; CRISPR-Cas Systems ; Mevalonic Acid/metabolism ; Genetic Engineering ; Intramolecular Lyases/genetics/metabolism ; Squalene/metabolism ; Diterpenes ; Diphosphates ; }, abstract = {1,8-Cineole, a monoterpene with diverse industrial and pharmaceutical applications, has garnered significant interest due to its unique properties. This study aims to achieve sustainable production of 1,8-cineole from Yarrowia lipolytica through metabolic and media engineering strategies. The heterologous 1,8-cineole synthase from Streptomyces clavuligerus was integrated through CRISPR-Cas9, along with overexpression of key genes in the mevalonate pathway and a double mutation in the Erg20p to enhance flux towards geranyl pyrophosphate. The modified strain was further investigated for varying carbon and nitrogen sources with MgSO4 addition. The above approaches achieved a titer of 4.68 mg/L of 1,8-cineole along with 1108.53 mg/L of intracellular squalene when grown on 5 % WCO, marking the first report of genetic engineering of Y. lipolytica for 1,8-cineole production. Further studies are in progress to redirect internal fluxes to 1,8-cineole for improvising yields and productivities. This work shows a sustainable and innovative approach to biotechnology improvements in terpene biosynthesis and waste valorization.}, }
@article {pmid40273314, year = {2025}, author = {Montagud-Martínez, R and Márquez-Costa, R and Ruiz, R and Martínez-Aviñó, A and Ballesteros-Garrido, R and Navarro, D and Campins-Falcó, P and Rodrigo, G}, title = {Virus Detection by CRISPR-Cas9-Mediated Strand Displacement in a Lateral Flow Assay.}, journal = {ACS applied bio materials}, volume = {8}, number = {5}, pages = {4221-4229}, pmid = {40273314}, issn = {2576-6422}, mesh = {*CRISPR-Cas Systems ; Humans ; *SARS-CoV-2/isolation &amp; purification/genetics ; *COVID-19/diagnosis/virology ; Nucleic Acid Amplification Techniques/methods ; Chromatography, Affinity/methods ; }, abstract = {In public health emergencies or in resource-constrained settings, laboratory-based diagnostic methods, such as RT-qPCR, need to be complemented with accurate, rapid, and accessible approaches to increase testing capacity, as this will translate into better outcomes in disease prevention and management. Here, we develop an original nucleic acid detection platform by leveraging CRISPR-Cas9 and lateral flow immunochromatography technologies. In combination with an isothermal amplification that runs with a biotinylated primer, the system exploits the interaction between the CRISPR-Cas9 R-loop formed upon targeting a specific nucleic acid and a fluorescein-labeled probe to generate a visual readout on a lateral flow device. Our method enables rapid, sensitive detection of nucleic acids, achieving a limit of 1-10 copies/μL in 1 h at a low temperature. We validated the efficacy of the method by using clinical samples of patients infected with SARS-CoV-2. Compared with other assays, it operates with more accessible molecular elements and showcases a robust signal-to-noise ratio. Moreover, multiplexed detection was demonstrated using primers labeled with biotin and digoxigenin, achieving the simultaneous identification of target genes on lateral flow devices with two test lines. We successfully detected SARS-CoV-2 and Influenza A (H1N1) in spiked samples, highlighting the potential of the method for multiplexed diagnostics of respiratory viruses. All in all, this represents a versatile and manageable platform for point-of-care testing, thereby supporting better patient outcomes and enhanced pandemic preparedness.}, }
@article {pmid40273258, year = {2025}, author = {Hayes, VM and Zhang, JT and Katz, MA and Li, Y and Kocsis, B and Brinkley, DM and Jia, N and Meeske, AJ}, title = {RNA-mediated CRISPR-Cas13 inhibition through crRNA structural mimicry.}, journal = {Science (New York, N.Y.)}, volume = {388}, number = {6745}, pages = {387-391}, pmid = {40273258}, issn = {1095-9203}, support = {R35 GM142460/GM/NIGMS NIH HHS/United States ; }, mesh = {*Bacteriophages/genetics ; *CRISPR-Associated Proteins/chemistry/genetics ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; *Listeria/virology/genetics ; *Molecular Mimicry ; Nucleic Acid Conformation ; Plasmids/genetics ; *RNA, Viral/chemistry/genetics ; *RNA, Small Untranslated/chemistry/genetics ; }, abstract = {To circumvent CRISPR-Cas immunity, phages express anti-CRISPR factors that inhibit the expression or activities of Cas proteins. Whereas most anti-CRISPRs described to date are proteins, recently described small RNAs called RNA anti-CRISPRs (rAcrs) have sequence homology to CRISPR RNAs (crRNAs) and displace them from cognate Cas nucleases. In this work, we report the discovery of rAcrVIA1-a plasmid-encoded small RNA that inhibits the RNA-targeting CRISPR-Cas13 system in its natural host, Listeria seeligeri. We solved the cryo-electron microscopy structure of the Cas13-rAcr complex, which revealed that rAcrVIA1 adopts a fold nearly identical to crRNA despite sharing negligible sequence similarity. Collectively, our findings expand the diversity of rAcrs and reveal an example of immune antagonism through RNA structural mimicry.}, }
@article {pmid40273189, year = {2025}, author = {Akiyama, C and Sakata, S and Ono, F}, title = {Normal locomotion in zebrafish lacking the sodium channel NaV1.4 suggests that the need for muscle action potentials is not universal.}, journal = {PLoS biology}, volume = {23}, number = {4}, pages = {e3003137}, pmid = {40273189}, issn = {1545-7885}, mesh = {Animals ; *Zebrafish/physiology/genetics ; *NAV1.4 Voltage-Gated Sodium Channel/genetics/metabolism/physiology ; *Action Potentials/physiology ; *Locomotion/physiology/genetics ; Muscle Contraction/physiology ; *Zebrafish Proteins/genetics/metabolism ; Muscle, Skeletal/physiology/metabolism ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Tetrodotoxin/pharmacology ; Calcium/metabolism ; }, abstract = {Extensive studies over decades have firmly established the concept that action potentials (APs) in muscles are indispensable for muscle contraction. To re-examine the significance of APs, we generated zebrafish lacking APs by editing the scn4aa and scn4ab genes, which together encode NaV1.4 (NaVDKO), using the CRISPR-Cas9 system. Surprisingly, the escape response of NaVDKOs to tactile stimuli, both in the embryonic and adult stages, was indistinguishable from that of wild-type (WT) fish. Ca2+ imaging using the calcium indicator protein GCaMP revealed that myofibers isolated from WT fish could be excited by the application of acetylcholine (ACh), even in the presence of tetrodotoxin (TTX) indicating that NaVs are dispensable for skeletal muscle contraction in zebrafish. Mathematical simulations showed that the end-plate potential was able to elicit a change in membrane potential large enough to activate the dihydropyridine receptors of the entire muscle fiber owing to the small fiber size and the disseminated distribution of neuromuscular synapses in both adults and embryos. Our data demonstrate that NaVs are not essential for muscle contraction in zebrafish and that the physiological significance of NaV1.4 in muscle is not uniform across vertebrates.}, }
@article {pmid40272173, year = {2025}, author = {Paz, M and Moratorio, G}, title = {Deep mutational scanning and CRISPR-engineered viruses: tools for evolutionary and functional genomics studies.}, journal = {mSphere}, volume = {10}, number = {5}, pages = {e0050824}, pmid = {40272173}, issn = {2379-5042}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Genomics/methods ; *Genome, Viral ; *Viruses/genetics ; *Evolution, Molecular ; Humans ; Mutation ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Mutational Analysis/methods ; }, abstract = {Recent advancements in synthetic biology and sequencing technologies have revolutionized the ability to manipulate viral genomes with unparalleled precision. This review focuses on two powerful methodologies: deep mutational scanning and CRISPR-based genome editing, that enable comprehensive mutagenesis and detailed functional characterization of viral proteins. These approaches have significantly deepened our understanding of the molecular determinants driving viral evolution and adaptation. Furthermore, we discuss how these advances provide transformative insights for future vaccine development and therapeutic strategies.}, }
@article {pmid40270959, year = {2025}, author = {Caras, I and Ionescu, IE and Pantazica, AM and van Eerde, A and Steen, H and Heldal, I and Haugslien, S and Tucureanu, C and Chelmus, RE and Tofan, VC and Costache, A and Onu, A and Su, H and Branza-Nichita, N and Liu-Clarke, J and Stavaru, C}, title = {Humanized mouse model reveals the immunogenicity of Hepatitis B Virus vaccine candidates produced in CRISPR/Cas9-edited Nicotiana benthamiana.}, journal = {Frontiers in immunology}, volume = {16}, number = {}, pages = {1479689}, pmid = {40270959}, issn = {1664-3224}, mesh = {Animals ; *Nicotiana/genetics ; *Hepatitis B Vaccines/immunology/genetics ; Humans ; Mice ; *Hepatitis B virus/immunology ; *CRISPR-Cas Systems ; Disease Models, Animal ; *Hepatitis B/immunology/prevention &amp; control ; Mice, Inbred NOD ; *Immunogenicity, Vaccine ; Hepatitis B Antibodies/immunology ; Antibodies, Neutralizing/immunology ; Hepatitis B Surface Antigens/immunology/genetics ; Female ; Gene Editing ; Leukocytes, Mononuclear/immunology ; Mice, Inbred BALB C ; }, abstract = {INTRODUCTION: Hepatitis B Virus (HBV) infection is still an ongoing public health issue worldwide. The most efficient tool in preventing HBV infection remains vaccination and significant efforts have been made in the last decade to improve current HBV vaccines. Owing to the strict HBV tropism for the human liver, developing animal models for preclinical screening of vaccine candidates is extremely challenging. To date, there are only a few reports regarding the use of humanized mouse models for the evaluation of the immunogenic properties of viral antigens.<br><br>METHODS: Previously we showed that a Nicotiana benthamiana-produced HBV-S/preS1[16-42] antigen elicited strong HBV-specific immune responses in BALB/c mice. In the current study, we used immunodeficient NOD.Cg-Prkdc[scid] Il2rg[tm1Wjl]/SzJ (NSG) mice as recipients of human peripheral blood mononuclear cells (hPBMCs), to evaluate the immunogenicity of the recently developed chimeric HBV immunogen produced in CRISPR/Cas9-edited N. benthamiana, under more "humanized" conditions.<br><br>RESULTS: Analysis of the immune response in NSG mice immunized with the chimeric antigen demonstrated induction of virus infection-neutralizing antibodies, indicating activation of antigen-specific B cells.<br><br>DISCUSSION: The ability of hPBMCs-engrafted NSG mice to mount specific humoral immune responses after immunization with viral antigens supports this animal model as a promising tool for pre-clinical evaluation of human vaccine antigens.}, }
@article {pmid40270429, year = {2025}, author = {Shi, K and Luo, W and Cheng, Y and Li, H and Peng, L and Luo, X and Hu, Y and Zhang, J and Chen, J}, title = {Phosphorothioate-Modified Hairpin G-Triplex Reporter-Assisted Split CRISPR/Cas12a-Powered Biosensor for "Turn-On" Fluorescent Detection of Nucleic Acid and Non-Nucleic Acid Targets.}, journal = {Analytical chemistry}, volume = {97}, number = {17}, pages = {9361-9366}, doi = {10.1021/acs.analchem.5c00140}, pmid = {40270429}, issn = {1520-6882}, mesh = {*Biosensing Techniques/methods ; G-Quadruplexes ; *CRISPR-Cas Systems ; *MicroRNAs/analysis ; *DNA/analysis/chemistry ; Benzothiazoles/chemistry ; *Phosphorothioate Oligonucleotides/chemistry ; Fluorescent Dyes/chemistry ; Limit of Detection ; Humans ; Spectrometry, Fluorescence ; }, abstract = {CRISPR/Cas12a-powered biosensors with guanine (G)-rich sequence reporters (e.g., G-quadruplex and G-triplex) are widely used in detection applications due to their simplicity and sensitivity. However, when these biosensors are employed for molecular detection in complex samples, they may encounter difficulties such as high background signal and susceptibility to interference because of the "turn-off" signal output. Herein, we explore, for the first time, a set of phosphorothioate (ps)-modified G-quadruplex (G4) and G-triplex (G3) sequences that can bind with thioflavin T (ThT) in an active split CRISPR/Cas12a system (SCas12a) to generate a "turn-on" fluorescent signal. To apply this new phenomenon, we develop a universal SCas12a-powered biosensor for "turn-on" fluorescent detection of nucleic acid (miRNA-21) and non-nucleic acid (kanamycin) targets by using ps-modified hairpin G3 as a reporter (SCas12a/psHG3). Target recognition activates SCas12a's trans-cleavage activity, leading to cleavage at the loop region of the psHG3 reporter. The released prelocked psG3 DNA binds ThT to produce a strong fluorescence signal. Without preamplification, this strategy can detect miRNA-21 with a detection limit of 100 fM. Moreover, the SCas12a/psHG3 system was further utilized for detecting kanamycin by incorporating its aptamers, enabling the detection of kanamycin at concentrations as low as 100 pM. This work is the first to develop a "turn-on" SCas12a/psHG3 system, showcasing its improved performance and wide range of applications in synthetic biology-based sensing technology.}, }
@article {pmid40270035, year = {2025}, author = {Lin, XL and Zhou, YM and Meng, K and Yang, JY and Zhang, H and Lin, JH and Wu, HY and Wang, XY and Zhao, H and Feng, SS and Park, KS and Cai, DQ and Zheng, L and Qi, XF}, title = {CRISPR/Cas-mediated mRNA knockdown in the embryos of Xenopus tropicalis.}, journal = {Cell &amp; bioscience}, volume = {15}, number = {1}, pages = {52}, pmid = {40270035}, issn = {2045-3701}, support = {21623110//Fundamental Research Funds for Central Universities of the Jinan University/ ; ZSYXM202303//Key Laboratory of Regenerative Medicine of Ministry of Education, Jinan University/ ; ZSYXM202402//Key Laboratory of Regenerative Medicine of Ministry of Education, Jinan University/ ; 82370247, 82070257, 81770240//National Natural Science Foundation of China/ ; 2023A1515012147//Basic and Applied Basic Research Foundation of Guangdong Province/ ; }, abstract = {The Xenopus tropicalis (Western clawed frog) is an important amphibian model for genetics, developmental and regenerative biology, due to its diploid genetic background and short generation time. CRISPR-Cas13 and CRISPR interference (CRISPRi) systems have recently been employed to suppress mRNA expression in many organisms such as yeast, plants, and mammalian cells. However, no systematic study of these two systems has been carried out in Xenopus tropicalis. Here, we show that CRISPRi rather than CRISPR-Cas13 is an effective and suitable approach to suppress specific mRNA transcription in Xenopus tropicalis embryos. We demonstrated that CRISPRi composed of dCas9 and KRAB-MeCP2 (dCas9-KM) can efficiently target exogenous and endogenous transcripts in Xenopus tropicalis embryos. Moreover, our data suggest that the new KRAB domain from ZIM3 protein (ZIM3-KRAB, ZIM3K) alone has a comparable transcript targeting capacity in Xenopus tropicalis embryos to the traditional fusion repressor KRAB-MeCP2 in which the KRAB domain from KOX1 protein. In conclusion, our results demonstrate that CRISPRi rather than CRISPR-Cas13 is an efficient knockdown platform to explore specific gene function in Xenopus tropicalis embryos.}, }
@article {pmid40269535, year = {2025}, author = {Abe, T and Inoue, KI and Kiyonari, H}, title = {Efficient CRISPR/Cas9-mediated knockin of reporter genes in rats at ROSA26 by pronuclear microinjection.}, journal = {Development, growth &amp; differentiation}, volume = {67}, number = {4}, pages = {215-225}, pmid = {40269535}, issn = {1440-169X}, support = {//RIKEN Intramural funding/ ; }, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Rats ; Microinjections/methods ; *Genes, Reporter/genetics ; *Gene Knock-In Techniques/methods ; Female ; Zygote/metabolism ; Gene Editing/methods ; Rats, Transgenic ; }, abstract = {The genetic modification of rats is a key technology for advancing biomedical research on human diseases. CRISPR/Cas9-mediated genome editing enables the generation of knockout rats in a single step, without the need for embryonic stem cells, by directly injecting genome editing components into zygotes. This simplifies the process, reduces costs, and accelerates gene function analysis in rats. However, the insertion of a gene cassette into a target site has remained inefficient, limiting the generation of knockin (KI) rats. To overcome this issue, we developed an optimized method that covers the entire process from zygote harvesting with superovulation to timed microinjection, ensuring the consistent generation of KI rats. We successfully generated four different fluorescent reporter lines at the ROSA26 locus in rats. Our study provides detailed, step-by-step protocols for donor vector design, zygote collection, microinjection, founder screening, and cryopreservation in rats.}, }
@article {pmid40269480, year = {2025}, author = {Chen, Z and Pilehvar, E and Sadeghi, H and Pilehvar, Y}, title = {Precision Reimagined: CRISPR and Multiomics Transform Systemic Lupus Erythematosus Diagnosis and Therapy.}, journal = {International journal of rheumatic diseases}, volume = {28}, number = {4}, pages = {e70189}, doi = {10.1111/1756-185X.70189}, pmid = {40269480}, issn = {1756-185X}, mesh = {Humans ; *Lupus Erythematosus, Systemic/genetics/diagnosis/therapy/immunology ; *Precision Medicine/methods ; *Gene Editing/methods ; *Genomics/methods ; Artificial Intelligence ; *CRISPR-Cas Systems ; Predictive Value of Tests ; *Proteomics/methods ; Phenotype ; Genetic Predisposition to Disease ; *Genetic Therapy/methods ; Multiomics ; }, abstract = {Systemic lupus erythematosus (SLE) is a complex autoimmune disorder with diverse clinical manifestations and unpredictable progression, posing significant challenges to accurate diagnosis and effective treatment. Traditional biomarkers and treatments often fail to address the disease's molecular and clinical heterogeneity. Recent advancements in CRISPR gene-editing technology and multiomics approaches offer transformative opportunities for personalized SLE care by unraveling its underlying molecular complexity and enabling precise therapeutic interventions. CRISPR technology allows targeted editing of SLE-associated genetic mutations, addressing disease drivers directly, while multiomics-including genomics, transcriptomics, and proteomics-provides insights into dysregulated immune networks, identifying biomarkers and therapeutic targets. Integrating these approaches can refine patient stratification and enhance the precision of treatments. Artificial intelligence (AI) complements these technologies by synthesizing high-dimensional data, enabling personalized treatment plans, predicting disease trajectories, and optimizing therapeutic strategies. However, the integration of CRISPR and multiomics in clinical settings raises challenges, including technical limitations, ethical concerns, and economic barriers. Emerging clinical trials and case studies demonstrate the potential of these innovations to personalize care and improve outcomes. Nonetheless, the transition from experimental research to routine clinical application requires robust regulatory frameworks and strategies to address these challenges. This review aims to explore the potential of CRISPR and multiomics technologies to revolutionize SLE diagnosis and therapy, emphasizing their integration with AI to advance personalized care. By addressing existing barriers, the review envisions a future where precision medicine transforms SLE management, paving the way for individualized, patient-centered autoimmune therapy.}, }
@article {pmid40268754, year = {2025}, author = {Liu, Y and Yao, F and Zou, J and Guo, D and Jiang, W and Fan, J and Li, R and Yang, Z and Ma, Y and Deng, H and Huang, J and Tan, L}, title = {RPAD locus controls prostrate growth habit in Oryza nivara.}, journal = {The plant genome}, volume = {18}, number = {2}, pages = {e70032}, pmid = {40268754}, issn = {1940-3372}, support = {2023YFF1000401//National Key Research and Development Program of China/ ; 32125029//National Natural Science Foundation of China/ ; PC2023A01003//Pinduoduo-China Agricultural University Research Fund/ ; }, mesh = {*Oryza/genetics/growth &amp; development ; *Quantitative Trait Loci ; Domestication ; CRISPR-Cas Systems ; Chromosomes, Plant/genetics ; Transcription Factors/genetics ; Chromosome Mapping ; Plant Breeding ; Plant Proteins/genetics ; }, abstract = {The development of ideal plant architecture is crucial for optimizing grain yield in crop breeding. The transition from prostrate growth habit in wild rice to erect growth habit in cultivated rice is one of the important events during rice domestication. Here, we identified a yield-related quantitative trait locus (QTL) cluster on the short arm of chromosome 7 using Teqing/W2014 (Oryza nivara) derived BC3F6 population. The introgression line TIL81 containing this QTL cluster exhibited significantly larger tiller angle, increased tiller numbers, and prostrate growth habit compared to the recipient parent Teqing. Using a segregating F2 population derived from a cross between TIL81 and Teqing, this yield-related QTL cluster was mapped to a similar position as the known rice plant architecture domestication (RPAD) locus controlling rice plant architecture domestication. CRISPR/Cas9-mediated genome (where CRISPR is clustered regularly interspaced short palindromic repeats) editing of four zinc finger transcription factors (OnZnF1, OnZnF6, OnZnF8, and OnZnF9) within the RPAD locus demonstrated their collective involvement in regulating plant architecture and yield-related traits. Notably, the knockout lines harboring all four zinc finger gene mutations exhibited plant architecture traits and grain yield per plant comparable to the control Teqing. These findings demonstrated that RPAD locus in O. nivara functions in prostrate growth habit and provided new insights into the molecular mechanism of plant architecture during rice domestication.}, }
@article {pmid40268745, year = {2025}, author = {Zhang, X and Ma, D and Liu, F}, title = {CRISPR Technology and Its Emerging Applications.}, journal = {Genomics, proteomics &amp; bioinformatics}, volume = {}, number = {}, pages = {}, doi = {10.1093/gpbjnl/qzaf034}, pmid = {40268745}, issn = {2210-3244}, abstract = {The discovery and iteration of clustered regularly interspaced short palindromic repeats (CRISPR) systems have revolutionized genome editing due to their remarkable efficiency and easy programmability, enabling precise manipulation of genomic elements. Owing to these unique advantages, CRISPR technology has the transformative potential to elucidate biological mechanisms and clinical treatments. This review provides a comprehensive overview of the development and applications of CRISPR technology. After describing the three primary CRISPR-Cas systems-CRISPR-associated protein 9 (Cas9) and Cas12a targeting DNA, and Cas13 targeting RNA-which serve as the cornerstone for technological advancements, we describe a series of novel CRISPR-Cas systems that offer new avenues for research, and then explore the applications of CRISPR technology in large-scale genetic screening, lineage tracing, genetic diagnosis, and gene therapy. As this technology evolves, it holds significant promise for studying gene functions and treating human diseases in the near future.}, }
@article {pmid40268248, year = {2025}, author = {Biber, J and Gandor, C and Becirovic, E and Michalakis, S}, title = {Retina-directed gene therapy: Achievements and remaining challenges.}, journal = {Pharmacology &amp; therapeutics}, volume = {271}, number = {}, pages = {108862}, doi = {10.1016/j.pharmthera.2025.108862}, pmid = {40268248}, issn = {1879-016X}, mesh = {*Genetic Therapy/methods ; Humans ; Dependovirus/genetics ; Animals ; *Retinal Diseases/therapy/genetics ; Genetic Vectors ; *Retina/metabolism ; Gene Transfer Techniques ; cis-trans-Isomerases/genetics ; }, abstract = {Gene therapy is an innovative medical approach that offers new treatment options for congenital and acquired diseases by transferring, correcting, inactivating or regulating genes to supplement, replace or modify a gene function. The approval of voretigene neparvovec (Luxturna), a gene therapy for RPE65-associated retinopathy, has marked a milestone for the field of retinal gene therapy, but has also helped to accelerate the development of gene therapies for genetic diseases affecting other organs. Voretigene neparvovec is a vector based on adeno-associated virus (AAV) that delivers a functional copy of RPE65 to supplement the missing function of this gene. The AAV-based gene delivery has proven to be versatile and safe for the transfer of genetic material to retinal cells. However, challenges remain in treating additional inherited as well as acquired retinopathies with this technology. Despite the high level of activity in this field, no other AAV gene therapy for retinal diseases has been approved since voretigene neparvovec. Ongoing research focuses on overcoming the current restraints through innovative strategies like AAV capsid engineering, dual-AAV vector systems, or CRISPR/Cas-mediated genome editing. Additionally, AAV gene therapy is being explored for the treatment of complex acquired diseases like age-related macular degeneration (AMD) and diabetic retinopathy (DR) by targeting molecules involved in the pathobiology of the degenerative processes. This review outlines the current state of retinal gene therapy, highlighting ongoing challenges and future directions.}, }
@article {pmid40267574, year = {2025}, author = {Zhang, D and Gao, T and Guo, Q and Ren, Y and Zhu, W and Zhuang, H and Pan, Z and Wang, F and Chen, Y and Guo, M and Liu, T and Wang, C and Ji, L and Qian, W and Li, J and Hou, S and Sun, Z and Wang, X and Xu, J and Guo, H}, title = {Rapid and sensitive Mycoplasma detection in antibody bioprocessing via RPA-CRISPR/Cas12a.}, journal = {Journal of pharmaceutical and biomedical analysis}, volume = {263}, number = {}, pages = {116904}, doi = {10.1016/j.jpba.2025.116904}, pmid = {40267574}, issn = {1873-264X}, mesh = {*Mycoplasma/isolation &amp; purification/genetics ; *CRISPR-Cas Systems/genetics ; *Antibodies, Monoclonal ; *Nucleic Acid Amplification Techniques/methods ; RNA, Ribosomal, 16S/genetics ; Drug Contamination/prevention &amp; control ; Recombinases/metabolism ; Sensitivity and Specificity ; }, abstract = {Mycoplasma species are prevalent microbial contaminants in the production of biological products, such as monoclonal antibodies, posing significant threats to the safety and efficacy of these products. Current regulatory guidelines as well as pharmacopoeias mandate the demonstration of the absence of Mycoplasma in the cell culture and further downstream processing to ensure product safety. Despite recent advancements in sensitive detection techniques for Mycoplasma in eucaryotic expression systems, these methods remain complex and time-consuming. There is a pressing need for a rapid, simple, and sensitive process analytical technology (PAT) for Mycoplasma detection. Here, we report the first development and application of a recombinase polymerase amplification (RPA)-assisted CRISPR-Cas12a (RPA-CRISPR/Cas12a) system spcifically tailored for Mycoplasma detection in biopharmaceutical production. This system combines the high-sensitivity isothermal nucleic acid amplification capabilities of RPA with the trans-cleavage activity of CRISPR-Cas12a reporter probes, enabling the rapid and accurate detection of Mycoplasma, accommodating various experimental requirements and application scenarios. By designing RPA universal primers and crRNA targeting the highly conserved sequences of Mycoplasma 16S rRNA and optimizing reaction conditions, we achieved dual-specific recognition with unprecedented efficiency in bioprocessing samples. All tested Mycoplasma specimens were detectable with limits between 10 and 0.1 copies/μL, with the whole process taking less than 1 hour. We further evaluated the feasibility of this method in detecting Mycoplasma in the cell culture of antibody products and further downstream processing samples. This method reduces the risk of false-positive signals due to non-specific amplification, enhancing detection sensitivity and specificity while significantly reducing analysis, representing the first PAT-compatible method for rapid Mycoplasma monitoring in antibody manufacturing, thereby providing robust assurance for the quality and safety of biological products.}, }
@article {pmid40267240, year = {2025}, author = {Rosengarten, H and D'Amore, A and Kim, HM and Ebrahimi-Fakhari, D}, title = {ap4b1 -/- zebrafish demonstrate morphological and motor abnormalities.}, journal = {Human molecular genetics}, volume = {34}, number = {12}, pages = {1034-1039}, doi = {10.1093/hmg/ddaf056}, pmid = {40267240}, issn = {1460-2083}, support = {K08NS123552-01//NIH/NINDS/ ; //CureAP4 Foundation/ ; }, mesh = {Animals ; *Zebrafish/genetics ; *Zebrafish Proteins/genetics/metabolism ; Disease Models, Animal ; Motor Neurons/metabolism/pathology ; *Spastic Paraplegia, Hereditary/genetics/physiopathology/pathology ; CRISPR-Cas Systems ; Seizures/genetics ; Axons/pathology/metabolism ; Gene Editing ; Humans ; }, abstract = {OBJECTIVE: Hereditary spastic paraplegia type 47 (SPG47) is caused by biallelic loss-of-function variants in the AP4B1 gene, leading to neurodevelopmental and progressive motor impairment. This study aimed to generate and characterize a zebrafish (Danio rerio) model of SPG47 to investigate the role of ap4b1 in neurodevelopment and motor function.<br><br>METHODS: We employed CRISPR/Cas9 gene-editing to generate a stable ap4b1-/- zebrafish line. Behavioral, morphological, and motor function analyses were performed, including survival under stress conditions, spontaneous locomotor activity, light-dark transition assays, and coiling behavior. Axonal length was assessed via immunofluorescence targeting spinal motor neurons. Seizure susceptibility was evaluated using a PTZ paradigm.<br><br>RESULTS: ap4b1-/- zebrafish exhibited significantly reduced axonal length of spinal motor neurons, impaired motor function, and developmental malformations, including brachycephaly, reduced body length, bent spines, and craniofacial defects. Increased tail coiling and reduced spontaneous activity were observed in larvae, alongside absent habituation to light-dark stimuli. Under stress conditions, survival rates were significantly lower in the knockout group compared to controls. Despite early hyperexcitability, no significant increase in PTZ-induced seizures was observed.<br><br>INTERPRETATION: This study characterizes an ap4b1-/- zebrafish model that recapitulates some phenotypes of SPG47, including motor deficits and morphological abnormalities. These findings support the utility of zebrafish for studying AP-4 deficiency and provide a platform for investigating the molecular mechanisms underlying SPG47.}, }
@article {pmid40267200, year = {2025}, author = {Brumage, L and Best, S and Hippe, DS and Grunblatt, E and Chanana, P and Wu, F and Lee, MC and Ying, Z and Ibrahim, A and Chung, JH and Vigil, A and Fatherree, J and Beronja, S and Paddison, P and Sullivan, L and Nabet, B and MacPherson, D}, title = {In vivo functional screens reveal KEAP1 loss as a driver of chemoresistance in small cell lung cancer.}, journal = {Science advances}, volume = {11}, number = {17}, pages = {eadq7084}, pmid = {40267200}, issn = {2375-2548}, mesh = {*Small Cell Lung Carcinoma/drug therapy/genetics ; Humans ; *Drug Resistance, Neoplasm ; *Kelch-Like ECH-Associated Protein 1/genetics ; DNA, Complementary/analysis ; Proto-Oncogene Proteins c-myc/metabolism ; Animals ; Mice ; CRISPR-Cas Systems ; Signal Transduction ; *Lung Neoplasms/drug therapy/genetics ; Male ; Female ; Immunologic Deficiency Syndromes ; HEK293 Cells ; }, abstract = {Exquisitely chemosensitive initially, small cell lung cancer (SCLC) exhibits dismal outcomes owing to rapid transition to chemoresistance. Elucidating the genetic underpinnings has been challenging owing to limitations with cellular models. As SCLC patient-derived xenograft (PDX) models mimic therapeutic responses, we perform genetic screens in chemosensitive PDX models to identify drivers of chemoresistance. cDNA overexpression screens identify MYC, MYCN, and MYCL, while CRISPR deletion screens identify KEAP1 loss as driving chemoresistance. Deletion of KEAP1 switched a chemosensitive SCLC PDX model to become chemoresistant and resulted in sensitivity to inhibition of glutamine metabolism. Data from the IMpower133 clinical trial revealed ~6% of patients with extensive-stage SCLC exhibit KEAP1 genetic alterations, with activation of a KEAP1/NRF2 transcriptional signature associated with reduced survival upon chemotherapy treatment. While roles for KEAP1/NRF2 have been unappreciated in SCLC, our genetic screens revealed KEAP1 loss as a driver of chemoresistance, while patient genomic analyses demonstrate clinical importance.}, }
@article {pmid40266686, year = {2025}, author = {Zeng, Y and Tan, X and Xiao, P and Gao, P and Wang, L and Zhang, A}, title = {Natronobacterium gregoryi Argonaute inhibits class 1 integron integrase-mediated excision and integration.}, journal = {Nucleic acids research}, volume = {53}, number = {8}, pages = {}, pmid = {40266686}, issn = {1362-4962}, support = {2021YFD1800402//National Key Research and Development Program of China/ ; 32172848//National Natural Science Foundation of China/ ; }, mesh = {*Argonaute Proteins/metabolism/genetics/chemistry ; *Integrases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics/chemistry ; Protein Binding ; *Integrons/genetics ; }, abstract = {Argonaute (Ago) proteins, ubiquitous in all domains of life, serve as key components in defense against foreign nucleic acids. While eukaryotic Agos (eAgos) are well characterized for guide RNA-mediated RNA targeting, prokaryotic Agos (pAgos) exhibit diverse functions, particularly in protecting bacteria from invasive DNA. The previous study identified Class 1 integron integrase (IntI-1), a tyrosine site-specific recombinase involved in horizontal transfer of antibiotic resistance genes, as a potential interaction partner of Natronobacterium gregoryi Argonaute (NgAgo), a member of pAgos. Here, we demonstrated that this interaction was direct, depended on the PIWI domain, and was independent of the catalytic activity of NgAgo. Notably, no interaction occurred between NgAgo and Cre (another tyrosine site-specific recombinase), highlighting the specificity of NgAgo-IntI-1 interaction. Furthermore, NgAgo could inhibit binding of IntI-1 to its target DNA, and then impede IntI-1-mediated integration and excision. Consistent with the above finding, few pAgos could be found in prokaryotic genomes containing IntI, whereas IntI showed significant co-occurrence with another bacterial defense system, CRISPR-Cas. In summary, our study elucidated a novel defense mechanism of pAgos through interaction with IntI-1 for inhibiting IntI-1-mediated gene excision/integration process.}, }
@article {pmid40265805, year = {2025}, author = {Han, X and Deng, Z and Liu, H and Ji, X}, title = {Current Advancement and Future Prospects in Simplified Transformation-Based Plant Genome Editing.}, journal = {Plants (Basel, Switzerland)}, volume = {14}, number = {6}, pages = {}, pmid = {40265805}, issn = {2223-7747}, support = {32370432//National Natural Science Foundation of China/ ; }, abstract = {Recent years have witnessed remarkable progress in plant biology, driven largely by the rapid evolution of CRISPR/Cas-based genome editing (GE) technologies. These tools, including versatile CRISPR/Cas systems and their derivatives, such as base editors and prime editors, have significantly enhanced the universality, efficiency, and convenience of plant functional genomics, genetics, and molecular breeding. However, traditional genetic transformation methods are essential for obtaining GE plants. These methods depend on tissue culture procedures, which are time-consuming, labor-intensive, genotype-dependent, and challenging to regenerate. Here, we systematically outline current advancements in simplifying plant GE, focusing on the optimization of tissue culture process through developmental regulators, the development of in planta transformation methods, and the establishment of nanomaterial- and viral vector-based delivery platforms. We also discuss critical challenges and future directions for achieving genotype-independent, tissue culture-free plant GE.}, }
@article {pmid40264801, year = {2025}, author = {Zueva, AS and Shevchenko, AI and Medvedev, SP and Elisaphenko, EA and Sleptcov, AA and Nazarenko, MS and Tmoyan, NA and Zakian, SM and Zakharova, IS}, title = {Isogenic induced pluripotent stem cell line ICGi036-A-1 from a patient with familial hypercholesterolaemia, derived by correcting a pathogenic variant of the gene LDLR c.530C&gt;T.}, journal = {Vavilovskii zhurnal genetiki i selektsii}, volume = {29}, number = {2}, pages = {189-199}, doi = {10.18699/vjgb-25-22}, pmid = {40264801}, issn = {2500-0462}, abstract = {Familial hypercholesterolaemia is a common monogenic disorder characterized by high plasma cholesterol levels leading to chronic cardiovascular disease with high risk and often early manifestation due to atherosclerotic lesions of the blood vessels. The atherosclerotic lesions in familial hypercholesterolaemia are mainly caused by pathogenic variants of the low-density lipoprotein receptor (LDLR) gene, which plays an important role in cholesterol metabolism. Normally, cholesterol-laden low-density lipoproteins bind to the LDLR receptor on the surface of liver cells to be removed from the bloodstream by internalisation with hepatocytes. In familial hypercholesterolaemia, the function of the receptor is impaired and the uptake of low-density lipoproteins is significantly reduced. As a result, cholesterol accumulates in the subendothelial space on the inner wall of blood vessels, triggering atherogenesis, the formation of atherosclerotic plaques. At present, there are no effective and universal approaches to the diagnosis and treatment of familial hypercholesterolaemia. A relevant approach to study the molecular genetic mechanisms of the disease and to obtain systems for screening chemical compounds as potential drugs is the generation of cellular models based on patient-specific induced pluripotent stem cells. The aim of our work was to derive an isogenic genetically modified induced pluripotent stem cell line by correcting the pathogenic allelic variant c.530C of the LDLR gene in the original iPSC previously obtained from a compound heterozygote patient with familial hypercholesterolaemia. The resulting isogenic iPSC line differs from the original by only one corrected nucleotide substitution, allowing us to study the direct effect of this pathogenic genetic variant on physiological changes in relevant differentiated cells. CRISPR/Cas-mediated base editing was used to correct the single nucleotide substitution. The resulting genetically modified iPSC line has pluripotency traits, a normal karyotype, a set of short tandem repeats identical to that in the original line and can be used to obtain differentiated derivatives necessary for the elaboration of relevant cell models.}, }
@article {pmid40264105, year = {2025}, author = {Yahata, T and Toujima, S and Sasaki, I and Iwahashi, N and Fujino, M and Nishioka, K and Noguchi, T and Tanizaki-Horiuchi, Y and Kaisho, T and Ino, K}, title = {Adeno-associated virus-clustered regularly interspaced short palindromic repeats/cas9‑mediated ovarian cancer treatment targeting PD-L1.}, journal = {BMC cancer}, volume = {25}, number = {1}, pages = {749}, pmid = {40264105}, issn = {1471-2407}, mesh = {Female ; Animals ; *B7-H1 Antigen/genetics/antagonists &amp; inhibitors/metabolism ; *Ovarian Neoplasms/therapy/genetics/immunology/pathology ; *Dependovirus/genetics ; Mice ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; Humans ; Cell Line, Tumor ; Genetic Vectors/genetics ; Immunotherapy/methods ; }, abstract = {The response rate of antibody therapy targeting immune checkpoint molecules in ovarian cancer is insufficient. This study aimed to develop a novel gene immunotherapy model targeting programmed death ligand 1 (PD-L1) in vivo in ovarian cancer using adeno-associated virus (AAV)-clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and investigate its efficacy. In vitro, we produced PD-L1-AAV particles to knock out PD-L1. PD-L1-AAV particles were transduced into the murine ovarian cancer cell line ID8. PD-L1 expression at the cellular level was significantly decreased following treatment with PD-L1-AAV particles compared with control-AAV particles. In the peritoneal dissemination model, the survival time was significantly longer in the PD-L1-AAV particles intraperitoneally injected group than that in the control group. Furthermore, intratumoral lymphocyte recruitment was analyzed by immunohistochemistry, and the number of intratumoral CD4[+] and CD8[+] T cells was significantly higher, whereas that of Foxp3[+] Treg cells was significantly lower in the PD-L1-AAV particles injected group than in the control group. No severe adverse events in normal organs, such as the lungs, spleen, liver, and kidney, were observed. These results suggest that PD-L1-targeted therapy by genome editing using AAV-CRISPR/Cas9 is a novel gene-immune therapeutic strategy for ovarian cancer.}, }
@article {pmid40264050, year = {2025}, author = {Pang, KL and Li, P and Yao, XR and Xiao, WT and Ren, X and He, JY}, title = {Deciphering a proliferation-essential gene signature based on CRISPR-Cas9 screening to predict prognosis and characterize the immune microenvironment in HNSCC.}, journal = {BMC cancer}, volume = {25}, number = {1}, pages = {756}, pmid = {40264050}, issn = {1471-2407}, support = {82304083//National Natural Science Foundation of China/ ; 2023JJ40584//Natural Science Foundation of Hunan Province/ ; }, mesh = {Humans ; Prognosis ; *Squamous Cell Carcinoma of Head and Neck/genetics/immunology/pathology/mortality ; *Tumor Microenvironment/immunology/genetics ; *CRISPR-Cas Systems ; *Head and Neck Neoplasms/genetics/immunology/pathology/mortality ; Cell Proliferation/genetics ; *Biomarkers, Tumor/genetics ; Gene Expression Regulation, Neoplastic ; Female ; *Genes, Essential ; Gene Expression Profiling ; Male ; Gene Regulatory Networks ; }, abstract = {BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a highly aggressive malignancy with a poor prognosis. Identifying reliable prognostic biomarkers and therapeutic targets is crucial for improving patient outcomes. This study aimed to systematically identify proliferation-essential genes (PEGs) associated with HNSCC prognosis using CRISPR-Cas9 screening data.<br><br>METHODS: CRISPR-Cas9 screening data from the DepMap database were used to identify PEGs in HNSCC cells. A prognostic PEGs signature was constructed using univariate Cox regression, least absolute shrinkage and selection operator (LASSO) Cox regression, and multivariate Cox regression analyses. The predictive accuracy of the signature was validated in internal and external datasets. Weighted gene co-expression network analysis (WGCNA), gene set enrichment analysis (GSEA), and immune infiltration analysis were used to investigate the underlying mechanism between high and low-risk patients. Random forest analysis and functional experiments were conducted to investigate the role of key proliferation essential genes in HNSCC progression.<br><br>RESULTS: A total of 1511 PEGs were identified. A seven-gene prognostic PEGs signature (MRPL33, NAT10, PSMC1, PSMD11, RPN2, TAF7, and ZNF335) was developed and validated, demonstrating robust prognostic performance in stratifying HNSCC patients by survival risk. WGCNA and GSEA analyses revealed a marked downregulation of immune-related pathways in high-risk patients. Immune infiltration analysis validated those high-risk patients had reduced immune scores, stromal scores, and ESTIMATE scores, as well as decreased infiltration of multiple immune cell types. Among the identified genes, PSMC1 was highlighted as a pivotal regulator of HNSCC proliferation and migration, as confirmed by functional experiments.<br><br>CONCLUSIONS: This study identifies a novel PEGs signature that effectively predicts HNSCC prognosis and stratifies patients by survival risk. PSMC1 was identified as a key gene promoting malignant progression, offering potential as a therapeutic target for HNSCC.}, }
@article {pmid40263956, year = {2025}, author = {Yan, H and Qi, M and Li, H}, title = {Characterization and Full Sequencing of 100 Nt sgRNA and Large RNA Using Site-Directed Cleavage and Liquid Chromatography Tandem Mass Spectrometry.}, journal = {Analytical chemistry}, volume = {97}, number = {17}, pages = {9228-9238}, doi = {10.1021/acs.analchem.4c06606}, pmid = {40263956}, issn = {1520-6882}, mesh = {Tandem Mass Spectrometry/methods ; Ribonuclease H/metabolism ; Chromatography, Liquid ; DNA, Catalytic/metabolism/chemistry ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; *RNA/genetics/chemistry ; *Sequence Analysis, RNA/methods ; CRISPR-Cas Systems ; }, abstract = {CRISPR/Cas9 is widely recognized as the most effective, efficient, and precise genome editing tool, inspiring numerous applications in basic science, medicine, and biotechnology. In the CRISPR/Cas9 system, single guide RNA (sgRNA) and Cas9 enzyme form a ribonucleoprotein complex that specifically and effectively cleaves target DNA. Accurate sequencing of sgRNA, particularly identifying the target sequence within the first 20 nucleotides (nt) at the 5'-end, is crucial for quality assurance and regulatory compliance. In this study, we used site-directed cleavage using ribonuclease H (RNase H) and DNAzyme for the first time to digest 100 nt sgRNA, achieving full sequencing with 100% coverage by analyzing the two cleaved fragments separately via LC MS/MS. We evaluated four different DNA-RNA chimeras as capture probes for the RNase H site-directed cleavage approach, finding that the chimera with four deoxynucleotides provided the most specific cleavage. Compared to RNase H, the DNAzyme demonstrated higher specificity and stability for 100 nt sgRNA digestion, successfully identifying up to 200 nucleotides of large RNA with 100% sequence coverage by fully sequencing the four short cleaved fragments. Due to the high specificity of DNAzyme cleavage, we used this method to study the designed 5'-end N-X truncated impurities of 100 nt sgRNA, demonstrating accurate identification and relative quantification. For 100 nt sgRNA, the limited available cleavage site was set on the scaffold sequence for both site-directed cleavage approaches, and the captured probes designed for RNase H and DNAzyme can be universally applied to sequence all 100 nt sgRNAs because of the conserved scaffold sequence.}, }
@article {pmid40263938, year = {2025}, author = {Thornburg, CD and Pipe, SW and Cantore, A and Unzu, C and Jones, M and Miesbach, WA}, title = {Clinical perspective: Advancing hemophilia treatment through gene therapy approaches.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {33}, number = {6}, pages = {2350-2362}, pmid = {40263938}, issn = {1525-0024}, mesh = {Humans ; *Hemophilia A/therapy/genetics ; *Genetic Therapy/methods ; Gene Editing/methods ; Genetic Vectors/genetics ; Dependovirus/genetics ; Lentivirus/genetics ; Animals ; CRISPR-Cas Systems ; Clinical Trials as Topic ; }, abstract = {Hemophilia, a congenital bleeding disorder, can cause arthropathy, impaired mobility, pain, and life-threatening hemorrhage events, significantly impacting quality of life for patients and caregivers. Current therapies, although effective, necessitate costly lifelong treatment, often in specialized settings. However, as a monogenic disorder caused by loss-of-function genetic variants, hemophilia is amenable to gene therapy. In this article, three primary gene therapy approaches at the forefront of clinical development are reviewed. Adeno-associated virus-based gene therapy, having secured approval in the EU, UK, and US after promising phase 3 trial results, demonstrates clear superiority over standard-of-care treatment. Lentivirus-based approaches capable of transducing dividing and nondividing cells may improve the durability of treatment and have low susceptibility to pre-existing neutralizing antibodies to viral vectors. Finally, gene editing techniques such as zinc finger nucleases and CRISPR aim to correct genetic defects directly, holding promise as novel, effective, and highly durable therapeutic strategies in adults and children with hemophilia. This review provides a comprehensive summary of the current status of these gene therapy approaches, highlighting advantages, limitations, and potential future developments.}, }
@article {pmid40263874, year = {2025}, author = {Huang, X and Li, R and Xu, J and Kang, J and Chen, X and Han, B and Xue, Y}, title = {Integrated multi-omics uncover viruses, active fermenting microbes and their metabolic profiles in the Daqu microbiome.}, journal = {Food research international (Ottawa, Ont.)}, volume = {208}, number = {}, pages = {116061}, doi = {10.1016/j.foodres.2025.116061}, pmid = {40263874}, issn = {1873-7145}, mesh = {*Fermentation ; *Microbiota ; Fungi/metabolism/genetics ; *Bacteria/metabolism/genetics ; *Viruses/genetics/classification ; Metagenomics ; Food Microbiology ; *Fermented Foods/microbiology/virology ; Bacteriophages/genetics ; *Metabolome ; Multiomics ; }, abstract = {The coexistence and coevolution of viruses and fermenting microbes have a significant impact on the structure and function of microbial communities. Although the presence of viruses in Daqu, the fermentation starter for Chinese Baijiu, has been documented, their specific effects on the community composition and metabolic functions of low, medium, and high-temperature Daqu remain unclear. In this study, we employed multi-omics technology to explore the distribution of viruses and active bacteria and fungi in various Daqu and their potential metabolic roles. Viral metagenomic sequencing showed a predominance of Parvoviridae in High-Temperature Daqu (HTQ), while Genomoviridae were dominant in Medium-Temperature Daqu (MTQ) and Low- Temperature Daqu (LTQ). Phages belonging to the Siphoviridae, Podoviridae, Herelleviridae, and Myoviridae families showed significantly different abundances across three Daqu groups. Metatranscriptomic analysis showed that fungal communities were most active in LTQ, whereas bacterial communities were dominant in MTQ and HTQ. By employing the CRISPR-Cas spacer, a higher predicted number of phage-host linkages was identified in LTQ, particularly with hosts including Lactobacillus, Staphylococcus, Acinetobacter, Enterobacter, and Bacillus. Correlation analysis showed that bacteria like Acinetobacter, Lactobacillus, and Streptococcus exhibited the strongest associations with metabolites, particularly amino acids and organic acids. The potential phage-induced metabolic differences in the three Daqu groups were mainly linked to pathways involved in the metabolism of amino acids, sugars, and organic acids. Overall, our study elucidates the impact of viruses on shaping microbial composition and influencing metabolic functions in Daqu. These results improve our comprehension of viruses and microbes in Daqu microbial communities and provide valuable insights for enhancing quality control in Daqu production.}, }
@article {pmid40263707, year = {2025}, author = {Zhu, Z and Li, X and Ding, L and Wu, T}, title = {Exploring the effect of activator topology on CRISPR-Cas12a trans-cleavage activity.}, journal = {Nucleic acids research}, volume = {53}, number = {8}, pages = {}, pmid = {40263707}, issn = {1362-4962}, support = {22474045//National Natural Science Foundation of China/ ; //Fundamental Research Funds for the Central Universities/ ; 2024JYCXJJ010//Huazhong University of Science and Technology/ ; }, mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *Biosensing Techniques/methods ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; *Bacterial Proteins/metabolism/chemistry/genetics ; }, abstract = {The CRISPR-Cas12a system is widely used in nucleic acid detection and biosensing due to its high sensitivity, selectivity, and simple design. However, traditional CRISPR-Cas12a sensors, which rely on linear activators, face challenges such as limited operability and low stability. This study explored the impact of three different activator topologies-linear, planar, and steric-on the trans-cleavage activity of Cas12a. We developed a Cas12a-based switch using a planar activator, which demonstrated superior operability and maintained higher activity compared to linear activators. Using this planar activator, we achieved highly sensitive detection of hypochlorous acid, with a detection limit as low as 88 nM, outperforming chemical probe-based methods. The introduction of topological activators will open new avenues for the development of CRISPR-Cas12a-based biosensors, offering broad potential for diverse applications.}, }
@article {pmid40263616, year = {2025}, author = {Wood, TWP and Henriques, WS and Cullen, HB and Romero, M and Blengini, CS and Sarathy, S and Sorkin, J and Bekele, H and Jin, C and Kim, S and Wang, X and Laureau, R and Chemiakine, A and Khondker, RC and Isola, JVV and Stout, MB and Gennarino, VA and Mogessie, B and Jain, D and Schindler, K and Suh, Y and Wiedenheft, B and Berchowitz, LE}, title = {The retrotransposon-derived capsid genes PNMA1 and PNMA4 maintain reproductive capacity.}, journal = {Nature aging}, volume = {5}, number = {5}, pages = {765-779}, pmid = {40263616}, issn = {2662-8465}, support = {R35 GM134867/GM/NIGMS NIH HHS/United States ; NA//Irma T. Hirschl Trust (Irma T. Hirschl Charitable Trust)/ ; NA//M.J. Murdock Charitable Trust (Murdock Charitable Trust)/ ; R01 AG069750/AG/NIA NIH HHS/United States ; R01 NS109858/NS/NINDS NIH HHS/United States ; R01 AG069742/AG/NIA NIH HHS/United States ; R35 GM136340/GM/NIGMS NIH HHS/United States ; 213470/A/18/Z//Wellcome Trust (Wellcome)/ ; R35 GM146725/GM/NIGMS NIH HHS/United States ; R35 GM124633/GM/NIGMS NIH HHS/United States ; R35 GM147130/GM/NIGMS NIH HHS/United States ; }, mesh = {Animals ; Humans ; *Retroelements/genetics ; Female ; Mice ; Male ; *Reproduction/genetics ; *Capsid Proteins/genetics/metabolism ; Ovary/metabolism ; }, abstract = {Almost half of the human genome consists of retrotransposons-'parasitic' sequences that insert themselves into the host genome via an RNA intermediate. Although most of these sequences are silenced or mutationally deactivated, they can present opportunities for evolutionary innovation: mutation of a deteriorating retrotransposon can result in a gene that provides a selective advantage to the host in a process termed 'domestication'[1-3]. The PNMA family of gag-like capsid genes was domesticated from an ancient vertebrate retrotransposon of the Metaviridae clade at least 100 million years ago[4,5]. PNMA1 and PNMA4 are positively regulated by the master germ cell transcription factors MYBL1 and STRA8, and their transcripts are bound by the translational regulator DAZL during gametogenesis[6]. This developmental regulation of PNMA1 and PNMA4 expression in gonadal tissue suggested to us that they might serve a reproductive function. Through the analysis of donated human ovaries, genome-wide association studies (GWASs) and mouse models, we found that PNMA1 and PNMA4 are necessary for the maintenance of a normal reproductive lifespan. These proteins self-assemble into capsid-like structures that exit human cells, and we observed large PNMA4 particles in mouse male gonadal tissue that contain RNA and are consistent with capsid formation.}, }
@article {pmid40263581, year = {2025}, author = {Weiss, T and Kamalu, M and Shi, H and Li, Z and Amerasekera, J and Zhong, Z and Adler, BA and Song, MM and Vohra, K and Wirnowski, G and Chitkara, S and Ambrose, C and Steinmetz, N and Sridharan, A and Sahagun, D and Banfield, JF and Doudna, JA and Jacobsen, SE}, title = {Viral delivery of an RNA-guided genome editor for transgene-free germline editing in Arabidopsis.}, journal = {Nature plants}, volume = {11}, number = {5}, pages = {967-976}, pmid = {40263581}, issn = {2055-0278}, support = {2334027//National Science Foundation (NSF)/ ; }, mesh = {*Arabidopsis/genetics ; *Gene Editing/methods ; *Plant Viruses/genetics ; CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Transgenes ; Genome, Plant ; }, abstract = {Genome editing is transforming plant biology by enabling precise DNA modifications. However, delivery of editing systems into plants remains challenging, often requiring slow, genotype-specific methods such as tissue culture or transformation[1]. Plant viruses, which naturally infect and spread to most tissues, present a promising delivery system for editing reagents. However, many viruses have limited cargo capacities, restricting their ability to carry large CRISPR-Cas systems. Here we engineered tobacco rattle virus (TRV) to carry the compact RNA-guided TnpB enzyme ISYmu1 and its guide RNA. This innovation allowed transgene-free editing of Arabidopsis thaliana in a single step, with edits inherited in the subsequent generation. By overcoming traditional reagent delivery barriers, this approach offers a novel platform for genome editing, which can greatly accelerate plant biotechnology and basic research.}, }
@article {pmid40263339, year = {2025}, author = {Zhao, C and Cao, Y and Ibrahim, N and Wang, Y and Martemyanov, KA}, title = {Efficient in vivo labeling of endogenous proteins with SMART delineates retina cellular and synaptic organization.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {3768}, pmid = {40263339}, issn = {2041-1723}, support = {EY028033//U.S. Department of Health &amp; Human Services | NIH | National Eye Institute (NEI)/ ; K99 EY030554/EY/NEI NIH HHS/United States ; R01 EY018139/EY/NEI NIH HHS/United States ; EY018139//U.S. Department of Health &amp; Human Services | NIH | National Eye Institute (NEI)/ ; R00 EY030554/EY/NEI NIH HHS/United States ; EY030554//U.S. Department of Health &amp; Human Services | NIH | National Eye Institute (NEI)/ ; R01 EY028033/EY/NEI NIH HHS/United States ; }, mesh = {Animals ; *Retina/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Synapses/metabolism ; Mice ; }, abstract = {A key application of CRISPR/Cas9-based genomic editing is modification of genes to introduce engineered sequences. However, the editing flexibility is severely constrained by the requirement for targeting sites in proximity to the desired modification site, which makes many modifications intractable. Here, we develop a strategy that overcomes this key limitation to allow CRISPR-based editing at any position with high efficiency. It relies on reconstructing the targeted gene using Silently Mutate And Repair Template (SMART) where we mutate the gap sequence in the repair template to prevent its base pairing with the target DNA while maintaining the same amino acid coding. Using vertebrate retina as a neuronal model system we document the application of SMART editing for labeling endogenous proteins in vivo with high efficiency. We show that SMART editing allows us to access numerous cell types in the retina and address fundamental cell biological questions pertaining to its organization. We propose that this approach will facilitate functional genomic studies in a wide range of systems and increase the precision of corrective gene therapies.}, }
@article {pmid40263274, year = {2025}, author = {Rengifo-Gonzalez, M and Mazzuoli, MV and Janssen, AB and Rueff, AS and Burnier, J and Liu, X and Veening, JW}, title = {Make-or-break prime editing for genome engineering in Streptococcus pneumoniae.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {3796}, pmid = {40263274}, issn = {2041-1723}, support = {310030_200792//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; NCCR AntiResist 51NF40_180541//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; TMPFP3_210202//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; 771534-PneumoCaTChER//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; }, mesh = {*Streptococcus pneumoniae/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Bacterial ; RNA, Guide, CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; *Genetic Engineering/methods ; CRISPR-Associated Protein 9/metabolism/genetics ; }, abstract = {CRISPR-Cas9 has revolutionized genome engineering by allowing precise introductions of DNA double-strand breaks (DSBs). However, genome engineering in bacteria is still a complex, multi-step process requiring a donor DNA template for repair of DSBs. Prime editing circumvents this need as the repair template is indirectly provided within the prime editing guide RNA (pegRNA). Here, we developed make-or-break Prime Editing (mbPE) that allows for precise and effective genetic engineering in the opportunistic human pathogen Streptococcus pneumoniae. In contrast to traditional prime editing in which a nicking Cas9 is employed, mbPE harnesses wild type Cas9 in combination with a pegRNA that destroys the seed region or protospacer adjacent motif. Since most bacteria poorly perform template-independent end joining, correctly genome-edited clones are selectively enriched during mbPE. We show that mbPE is RecA-independent and can be used to introduce point mutations, deletions and targeted insertions, including protein tags such as a split luciferase, at selection efficiencies of over 93%. mbPE enables sequential genome editing, is scalable, and can be used to generate pools of mutants in a high-throughput manner. The mbPE system and pegRNA design guidelines described here will ameliorate future bacterial genome editing endeavors.}, }
@article {pmid40262634, year = {2025}, author = {Silverstein, RA and Kim, N and Kroell, AS and Walton, RT and Delano, J and Butcher, RM and Pacesa, M and Smith, BK and Christie, KA and Ha, LL and Meis, RJ and Clark, AB and Spinner, AD and Lazzarotto, CR and Li, Y and Matsubara, A and Urbina, EO and Dahl, GA and Correia, BE and Marks, DS and Tsai, SQ and Pinello, L and De Ravin, SS and Liu, Q and Kleinstiver, BP}, title = {Custom CRISPR-Cas9 PAM variants via scalable engineering and machine learning.}, journal = {Nature}, volume = {}, number = {}, pages = {}, pmid = {40262634}, issn = {1476-4687}, abstract = {Engineering and characterizing proteins can be time-consuming and cumbersome, motivating the development of generalist CRISPR-Cas enzymes[1-4] to enable diverse genome-editing applications. However, such enzymes have caveats such as an increased risk of off-target editing[3,5,6]. Here, to enable scalable reprogramming of Cas9 enzymes, we combined high-throughput protein engineering with machine learning to derive bespoke editors that are more uniquely suited to specific targets. Through structure-function-informed saturation mutagenesis and bacterial selections, we obtained nearly 1,000 engineered SpCas9 enzymes and characterized their protospacer-adjacent motif (PAM)[7] requirements to train a neural network that relates amino acid sequence to PAM specificity. By utilizing the resulting PAM machine learning algorithm (PAMmla) to predict the PAMs of 64 million SpCas9 enzymes, we identified efficacious and specific enzymes that outperform evolution-based and engineered SpCas9 enzymes as nucleases and base editors in human cells while reducing off-targets. An in silico-directed evolution method enables user-directed Cas9 enzyme design, including for allele-selective targeting of the RHO[P23H] allele in human cells and mice. Together, PAMmla integrates machine learning and protein engineering to curate a catalogue of SpCas9 enzymes with distinct PAM requirements, motivating a shift away from generalist enzymes towards safe and efficient bespoke Cas9 variants.}, }
@article {pmid40262340, year = {2025}, author = {Li, X and Li, Y and Wang, C and Chai, J and Liu, H and Jiang, X and Li, Y}, title = {Structure-switchable dsDNA promoter regulates the activity of CRISPR-Cas12a for APE1 detection.}, journal = {Talanta}, volume = {294}, number = {}, pages = {128161}, doi = {10.1016/j.talanta.2025.128161}, pmid = {40262340}, issn = {1873-3573}, mesh = {*DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics/analysis/metabolism/antagonists &amp; inhibitors ; Humans ; *CRISPR-Cas Systems/genetics ; *Promoter Regions, Genetic ; *DNA/genetics/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Apurinic/apyrimidinic endonuclease 1 (APE1) has been considered as a promising biomarker since it is associated with numerous human diseases, involving neurodegenerative diseases and cancer. However, current APE1 detection methods mainly rely on immunology-based methods, which are burdened by time-consuming and procedural complexity. To overcome these shortcomings, we have developed an innovative all-in-one technique that simplifies APE1 detection by integrating enzyme-responsive elements structure-switchable dsDNA promoter with CRISPR/AsCas12a methodology, namely EDC. In this work, the structure-switchable dsDNA promoter has been well-designed to trigger the site-directed incision of APE1 and then release the split activator to illumine the CRISPR/AsCas12a catalyst system by coupling it with another truncated activator. Under optimal circumstances, the proposed strategy enables sensitive detection of the target APE1 with a detection limit of 4.8 × 10[-5] U/mL and a wide linear range from 5.0 × 10[-5] to 1.0 × 10[-1] U/mL. Moreover, this strategy could be gratifyingly applied to screen APE1 inhibitors and monitor APE1 in lysates from cell extractions or clinical serum samples. Overall, this study presents a novel approach that utilizes dsDNA promoter as programmable switching components, effectively enhancing CRISPR/Cas12a-based diagnostic platforms and demonstrating the significant potential for clinical translation.}, }
@article {pmid40262200, year = {2025}, author = {Tian, SC and Song, XH and Feng, KK and Li, CL and Tu, YF and Hu, YS and Shao, JW}, title = {Self-oxygenating nanoplatform integrating CRISPR/Cas9 gene editing and immune activation for highly efficient photodynamic therapy.}, journal = {Journal of colloid and interface science}, volume = {693}, number = {}, pages = {137632}, doi = {10.1016/j.jcis.2025.137632}, pmid = {40262200}, issn = {1095-7103}, mesh = {*CRISPR-Cas Systems ; *Photochemotherapy ; *Gene Editing ; Animals ; Humans ; Mice ; *Manganese Compounds/chemistry/pharmacology ; *Oxides/chemistry/pharmacology ; *Nanoparticles/chemistry ; Reactive Oxygen Species/metabolism ; *Photosensitizing Agents/pharmacology/chemistry ; Tumor Microenvironment/drug effects ; Particle Size ; *Oxygen/chemistry/metabolism ; Phosphoric Monoester Hydrolases/genetics ; Surface Properties ; *Antineoplastic Agents/pharmacology/chemistry ; }, abstract = {Photodynamic therapy (PDT) has arisen as a promising method due to its spatiotemporal precision and minimal invasiveness. It encounters significant obstacles in solid tumors due to hypoxia-induced therapeutic resistance and the self-protective mechanisms of cancer cells facilitated by MutT homolog 1 (MTH1), an enzyme involved in oxidative damage repair. Herein, we fabricate a tumor-microenvironment responsive CRISPR nanoplatform based on hollow mesoporous manganese dioxide (H-MnO2) for PDT. This platform utilizes H-MnO2 to produce oxygen (O2) through the decomposition of hydrogen peroxide (H2O2) in TME, thereby mitigating hypoxia and enhancing reactive oxygen species (ROS) generation. The high concentration of glutathione (GSH) and hyaluronidase (HAase) in TME induces the release of CRISPR/Cas9 ribonucleoproteins (RNP) to target the MTH1 gene, thereby impairs oxidative damage repair pathways and amplifys ROS-mediated cytotoxicity. The released Mn[2+] ions function as immunomodulatory agents, activate innate immune responses via stimulating STING signal pathway. In vitro, IHMRH NPs markedly increased intracellular O2 levels, ROS production, lipid peroxidation and DNA damage, leading to tumor cell death, immune activation, and effective gene editing. In vivo, the nanoplatform suppressed tumor growth, diminished MTH1 gene expression, stimulated dendritic cell (DC) maturation through immunogenic cell death (ICD). This multimodal nanosystem may amplifies oxidative stress, collaborates with innate and adaptive immune activation to surpass the constraints of traditional PDT. The research presents a novel framework for cancer combination therapy by systematically integrating nanotechnology with precision gene editing.}, }
@article {pmid40261967, year = {2025}, author = {Liu, X and Wang, P and Wang, S and Liao, W and Ouyang, M and Lin, S and Lin, R and Sarris, PF and Michalopoulou, V and Feng, X and Zhang, Z and Xu, Z and Chen, G and Zhu, B}, title = {The circular RNA circANK suppresses rice resistance to bacterial blight by inhibiting microRNA398b-mediated defense.}, journal = {The Plant cell}, volume = {37}, number = {4}, pages = {}, pmid = {40261967}, issn = {1532-298X}, support = {32272479//National Natural Science Foundation of China/ ; CSTB2022NSCQ-MSX0524//Chongqing Natural Science Foundation/ ; 2025YFHZ0043//Science &amp; Technology Department of Sichuan Province/ ; }, mesh = {*Oryza/genetics/microbiology/immunology ; *MicroRNAs/genetics/metabolism ; *RNA, Circular/genetics/metabolism ; *Plant Diseases/microbiology/genetics/immunology ; *Disease Resistance/genetics ; *Xanthomonas/pathogenicity/physiology ; Gene Expression Regulation, Plant ; *RNA, Plant/genetics/metabolism ; Reactive Oxygen Species/metabolism ; CRISPR-Cas Systems/genetics ; Plant Proteins/genetics/metabolism ; }, abstract = {Circular RNAs (circRNAs) are prevalent in eukaryotic cells and have been linked to disease progressions. Their unique circular structure and stability make them potential biomarkers and therapeutic targets. Compared with animal models, plant circRNA research is still in its infancy. The lack of effective tools to specifically knock down circRNAs without affecting host gene expression has slowed the progress of plant circRNA research. Here, we have developed a CRISPR-Cas13d tool that can specifically knock down circRNAs in plant systems, successfully achieving the targeted knockdown of circRNAs in rice (Oryza sativa). We further focused on Os-circANK (a circRNA derived from Ankyrin repeat-containing protein), a circRNA differentially expressed in rice upon pathogen infection. Physiological and biochemical experiments revealed that Os-circANK functions as a sponge for miR398b, suppressing the cleavage of Cu/Zn-superoxidase dismutase (CSD)1/CSD2/copper chaperone for superoxide dismutase/superoxidase dismutaseX through competing endogenous RNA, leading to reduced reactive oxygen species levels following Xanthomonas oryzae pv. oryzae (Xoo) infection and a negative regulation of rice resistance to bacterial blight. Our findings indicate Os-circANK inhibits rice resistance to bacterial blight via the microRNA398b(miR398b)/CSD/SOD pathway.}, }
@article {pmid40261966, year = {2025}, author = {Cheng, Y and Li, G and Qi, A and Mandlik, R and Pan, C and Wang, D and Ge, S and Qi, Y}, title = {A comprehensive all-in-one CRISPR toolbox for large-scale screens in plants.}, journal = {The Plant cell}, volume = {37}, number = {4}, pages = {}, pmid = {40261966}, issn = {1532-298X}, support = {IOS-2029889//NSF Plant Genome Research Program/ ; 2021-67013-34554//USDA-NIFA Agricultural Innovation through Gene Editing/ ; 2020-33522-32274//USDA-NIFA Biotechnology Risk Assessment Research/ ; 21010111//Foundation for Food and Agriculture Research/ ; }, mesh = {Oryza/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Solanum lycopersicum/genetics ; Protoplasts/metabolism ; Plants, Genetically Modified ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome, Plant/genetics ; Acetolactate Synthase/genetics ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (Cas) technologies facilitate routine genome engineering of one or a few genes at a time. However, large-scale CRISPR screens with guide RNA libraries remain challenging in plants. Here, we have developed a comprehensive all-in-one CRISPR toolbox for Cas9-based genome editing, cytosine base editing, adenine base editing (ABE), Cas12a-based genome editing and ABE, and CRISPR-Act3.0-based gene activation in both monocot and dicot plants. We evaluated all-in-one T-DNA expression vectors in rice (Oryza sativa, monocot) and tomato (Solanum lycopersicum, dicot) protoplasts, demonstrating their broad and reliable applicability. To showcase the applications of these vectors in CRISPR screens, we constructed guide RNA (gRNA) pools for testing in rice protoplasts, establishing a high-throughput approach to select high-activity gRNAs. Additionally, we demonstrated the efficacy of sgRNA library screening for targeted mutagenesis of ACETOLACTATE SYNTHASE in rice, recovering novel candidate alleles for herbicide resistance. Furthermore, we carried out a CRISPR activation screen in Arabidopsis thaliana, rapidly identifying potent gRNAs for FLOWERING LOCUS T activation that confer an early-flowering phenotype. This toolbox contains 61 versatile all-in-one vectors encompassing nearly all commonly used CRISPR technologies. It will facilitate large-scale genetic screens for loss-of-function or gain-of-function studies, presenting numerous promising applications in plants.}, }
@article {pmid40261268, year = {2025}, author = {Liu, Y and Wu, Y and Liu, Y and Zhang, Q and Yuan, H and Li, S and Li, Z and Wang, B and Chang, Y and Liu, M}, title = {Arrest of CRISPR-Cas12a by Nonspecific Single-Stranded DNA for Biosensing.}, journal = {Analytical chemistry}, volume = {97}, number = {17}, pages = {9310-9315}, doi = {10.1021/acs.analchem.4c07081}, pmid = {40261268}, issn = {1520-6882}, mesh = {*Biosensing Techniques/methods ; *DNA, Single-Stranded/chemistry/metabolism ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism ; Escherichia coli/isolation &amp; purification ; DNA, Catalytic/metabolism/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {CRISPR-Cas technologies have emerged as powerful biosensing tools for the sensitive and specific detection of non-nucleic acid targets. However, existing biosensing strategies suffer from poor compatibility across diverse targets due to the complicated engineering of crRNA and DNA activator required for the CRISPR-Cas activity regulation. Herein, we report a novel and straightforward strategy for designing CRISPR-Cas12a-based biosensors that function by switching structures from single-stranded (ss)DNA/CRISPR-Cas12a assembly to DNA activator/CRISPR-Cas12a complex in the presence of target bacterium. The strategy begins with a ssDNA assembly made of a trans-acting RNA-cleaving DNAzyme (tRCD) and an RNA/DNA chimeric substrate (RCS). The ssDNA assembly has the ability to bind Cas12a nonspecifically, thus indeed blocking the CRISPR-Cas12a activity. By exploiting the specific recognition and cleavage capacities of tRCD for RCS in the presence of a target, the target-bound tRCD and the cleaved RCS are released from Cas12a, thus restoring the CRISPR-Cas12a activity. This method has been successfully applied for the sensitive (detection limit: 10[2] CFU/mL) detection of Escherichia coli (E. coli, EC) and Burkholderia gladioli (B. gladioli, BG). For the blind testing of 30 clinical urine samples, it exhibited 100% sensitivity and 100% specificity in identifying E. coli-associated urinary tract infections (UTIs).}, }
@article {pmid40261207, year = {2025}, author = {Liao, X and Li, Y and Wu, Y and Li, X and Shang, X}, title = {Deep Learning-Based Classification of CRISPR Loci Using Repeat Sequences.}, journal = {ACS synthetic biology}, volume = {14}, number = {5}, pages = {1813-1828}, doi = {10.1021/acssynbio.5c00174}, pmid = {40261207}, issn = {2161-5063}, mesh = {*Deep Learning ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Neural Networks, Computer ; *Repetitive Sequences, Nucleic Acid/genetics ; }, abstract = {With the widespread application of the CRISPR-Cas system in gene editing and related fields, along with the increasing availability of metagenomic data, the demand for detecting and classifying CRISPR-Cas systems in metagenomic data sets has grown significantly. Traditional classification methods for CRISPR-Cas systems primarily rely on identifying cas genes near CRISPR arrays. However, in cases where cas gene information is absent, such as in metagenomes or fragmented genome assemblies, traditional methods may fail. Here, we present a deep learning-based method, CRISPRclassify-CNN-Att, which classifies CRISPR loci solely based on repeat sequences. CRISPRclassify-CNN-Att utilizes convolutional neural networks (CNNs) and self-attention mechanisms to extract features from repeat sequences. It employs a stacking strategy to address the imbalance of samples across different subtypes and uses transfer learning to improve classification accuracy for subtypes with fewer samples. CRISPRclassify-CNN-Att demonstrates outstanding performance in classifying multiple subtypes, particularly those with larger sample sizes. Although CRISPR loci classification traditionally depends on cas genes, CRISPRclassify-CNN-Att offers a novel approach that serves as a significant complement to cas-based methods, enabling the classification of orphan or distant CRISPR loci. The proposed tool is freely accessible via https://github.com/Xingyu-Liao/CRISPRclassify-CNN-Att.}, }
@article {pmid40259370, year = {2025}, author = {Wang, T and Brown, C and Doherty, N and Byrne, NM and Islam, R and Doherty, M and Feng, J and Yin, C and Chambers, S and McQuoid, L and Mohamed-Smith, L and Butterworth, KT and Kerr, EM and Coulter, JA}, title = {Mannose and PMI depletion overcomes radiation resistance in HPV-negative head and neck cancer.}, journal = {Cell communication and signaling : CCS}, volume = {23}, number = {1}, pages = {189}, pmid = {40259370}, issn = {1478-811X}, support = {EP/X525625/1//Engineering and Physical Sciences Research Council/ ; MR/X502881/1/MRC_/Medical Research Council/United Kingdom ; }, mesh = {Humans ; *Radiation Tolerance/drug effects ; *Mannose/metabolism/pharmacology ; *Head and Neck Neoplasms/radiotherapy/pathology/metabolism ; *Mannose-6-Phosphate Isomerase/metabolism/deficiency/genetics ; Animals ; Cell Line, Tumor ; Mice ; Glycolysis ; CRISPR-Cas Systems ; }, abstract = {Radiotherapy is critical component of multidisciplinary cancer care, used as a primary and adjuvant treatment for patients with head and neck squamous cell carcinoma. This study investigates how mannose, a naturally occurring monosaccharide, combined with phosphomannose isomerase (PMI) depletion, enhances the sensitivity of HPV-negative head and neck tumour models to radiation. Isogenic PMI knockout models were generated by CRISPR/Cas9 gene editing, yielding a 20-fold increase in sensitivity to mannose in vitro, and causing significant tumour growth delay in vivo. This effect is driven by metabolic reprogramming, resulting in potent glycolytic suppression coupled with consistent depletion of ATP and glycolytic intermediates in PMI-depleted models. Functionally, these changes impede DNA damage repair following radiation, resulting in a significant increase in radiation sensitivity. Mannose and PMI ablation supressed both oxygen consumption rate and extracellular acidification, pushing cells towards a state of metabolic quiescence, effects contributing to increased radiation sensitivity under both normoxic and hypoxic conditions. In 3D-tumoursphere models, metabolic suppression by mannose and PMI depletion was shown to elevate intra-tumoursphere oxygen levels, contributing to significant in vitro oxygen-mediated radiosensitisation. These findings position PMI as a promising anti-tumour target, highlighting the potential of mannose as a metabolic radiosensitiser enhancing cancer treatment efficacy.}, }
@article {pmid40259121, year = {2025}, author = {Potlapalli, BP and Dassau, F and Fuchs, J and Sushmoy, DR and Houben, A}, title = {CRISPR-CISH: an in situ chromogenic DNA repeat detection system for research and life science education.}, journal = {Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology}, volume = {33}, number = {1}, pages = {7}, pmid = {40259121}, issn = {1573-6849}, support = {HO1779/33-1//Deutsche Forschungsgemeinschaft/ ; }, mesh = {*CRISPR-Cas Systems ; Humans ; *DNA/genetics ; *In Situ Hybridization/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Repetitive Sequences, Nucleic Acid ; *Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {In situ hybridization is a technique to visualize specific DNA sequences within nuclei and chromosomes. Various DNA in situ fluorescent labeling methods have been developed, which typically involve global DNA denaturation prior to the probe hybridization and often require fluorescence microscopes for visualization. Here, we report the development of a CRISPR/dCas9-mediated chromogenic in situ DNA detection (CRISPR-CISH) method that combines chromogenic signal detection with CRISPR imaging. This non-fluorescent approach uses 3' biotin-labeled tracrRNA and target-specific crRNA to form mature gRNA, which activates dCas9 to bind to target sequences. The subsequent application of streptavidin alkaline phosphatase or horseradish peroxidase generates chromogenic, target-specific signals that can be analyzed using conventional bright-field microscopes. Additionally, chromatin counterstains were identified to aid in the interpretation of CRISPR-CISH-generated target signals. This advancement makes in situ DNA detection techniques more accessible to researchers, diagnostic applications, and educational institutions in resource-limited settings.}, }
@article {pmid40259107, year = {2025}, author = {Yang, J and Song, J and Feng, Z and Ma, Y}, title = {Application of CRISPR-Cas9 in microbial cell factories.}, journal = {Biotechnology letters}, volume = {47}, number = {3}, pages = {46}, pmid = {40259107}, issn = {1573-6776}, support = {BY2021KYQD02//Binzhou Medical University/ ; }, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Metabolic Engineering/methods ; *Bacteria/genetics/metabolism ; }, abstract = {Metabolically engineered bacterial strains are rapidly emerging as a pivotal focus in the biosynthesis of an array of bio-based ingredients. Presently, CRISPR (clustered regularly interspaced short palindromic repeats) and its associated RNA-guided endonuclease (Cas9) are regarded as the most promising tool, having ushered in a transformative advancement in genome editing. Because of CRISPR-Cas9's accuracy and adaptability, metabolic engineers are now able to create novel regulatory systems, optimize pathways more effectively, and make extensive genome-scale alterations. Nevertheless, there are still obstacles to overcome in the application of CRISPR-Cas9 in novel microorganisms, particularly those industrial microorganism hosts that are resistant to traditional genetic manipulation techniques. How to further extend CRISPR-Cas9 to these microorganisms is an urgent problem to be solved. This article first introduces the mechanism and application of CRISPR-Cas9, and then discusses how to optimize CRISPR-Cas9 as a genome editing tool, including how to reduce off-target effects and how to improve targeting efficiency by optimizing design. Through offering a comprehensive perspective on the revolutionary effects of CRISPR-Cas9 in microbial cell factories, we hope to stimulate additional research and development in this exciting area.}, }
@article {pmid40259084, year = {2025}, author = {Nadig, A and Replogle, JM and Pogson, AN and Murthy, M and McCarroll, SA and Weissman, JS and Robinson, EB and O'Connor, LJ}, title = {Transcriptome-wide analysis of differential expression in perturbation atlases.}, journal = {Nature genetics}, volume = {57}, number = {5}, pages = {1228-1237}, pmid = {40259084}, issn = {1546-1718}, support = {T32GM007753//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; T32GM007618//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; F31NS115380//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; }, mesh = {*Transcriptome/genetics ; Humans ; *Gene Expression Profiling/methods ; Single-Cell Analysis/methods ; Gene Expression Regulation ; CRISPR-Cas Systems/genetics ; }, abstract = {Single-cell CRISPR screens such as Perturb-seq enable transcriptomic profiling of genetic perturbations at scale. However, the data produced by these screens are noisy, and many effects may go undetected. Here we introduce transcriptome-wide analysis of differential expression (TRADE)-a statistical model for the distribution of true differential expression effects that accounts for estimation error appropriately. TRADE estimates the 'transcriptome-wide impact', which quantifies the total effect of a perturbation across the transcriptome. Analyzing several large Perturb-seq datasets, we show that many transcriptional effects remain undetected in standard analyses but emerge in aggregate using TRADE. A typical gene perturbation affects an estimated 45 genes, whereas a typical essential gene affects over 500. We find moderate consistency of perturbation effects across cell types, identify perturbations where transcriptional responses vary qualitatively across dosage levels and clarify the relationship between genetic and transcriptomic correlations across neuropsychiatric disorders.}, }
@article {pmid40258142, year = {2025}, author = {Filsinger, GT and Mychack, A and Lyerly, E and Henriksen, C and Bartlett, TM and Kuchwara, H and Eitzinger, S and Bernhardt, TG and Walker, S and Church, GM and Wannier, TM}, title = {A diverse single-stranded DNA-annealing protein library enables efficient genome editing across bacterial phyla.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {122}, number = {17}, pages = {e2414342122}, pmid = {40258142}, issn = {1091-6490}, support = {P01 AI083214/AI/NIAID NIH HHS/United States ; U19 AI158028/AI/NIAID NIH HHS/United States ; }, mesh = {*Gene Editing/methods ; *DNA, Single-Stranded/genetics/metabolism ; *Genome, Bacterial ; Bacteriophages/genetics ; CRISPR-Cas Systems ; Gene Library ; *Bacteria/genetics/classification ; *Viral Proteins/genetics/metabolism ; *DNA-Binding Proteins/genetics/metabolism ; Phylogeny ; Homologous Recombination ; Staphylococcus aureus/genetics ; }, abstract = {Genome modification is essential for studying and engineering bacteria, yet making efficient modifications to most species remains challenging. Bacteriophage-encoded single-stranded DNA-annealing proteins (SSAPs) can facilitate efficient genome editing by homologous recombination, but their typically narrow host range limits broad application. Here, we demonstrate that a single library of 227 SSAPs enables efficient genome-editing across six diverse bacteria from three divergent classes: Actinomycetia (Mycobacterium smegmatis and Corynebacterium glutamicum), Alphaproteobacteria (Agrobacterium tumefaciens and Caulobacter crescentus), and Bacilli (Lactococcus lactis and Staphylococcus aureus). Surprisingly, the most effective SSAPs frequently originated from phyla distinct from their bacterial hosts, challenging the assumption that phylogenetic relatedness is necessary for recombination efficiency, and supporting the value of a large unbiased library. Across these hosts, the identified SSAPs enable genome modifications requiring efficient homologous recombination, demonstrated through three examples. First, we use SSAPs with Cas9 in C. crescentus to introduce single amino acid mutations with >70% efficiency. Second, we adapt SSAPs for dsDNA editing in C. glutamicum and S. aureus, enabling one-step gene knockouts using PCR products. Finally, we apply SSAPs for multiplexed editing in S. aureus to precisely map the interaction between a conserved protein and a small-molecule inhibitor. Overall, this library-based SSAP screen expands engineering capabilities across diverse, previously recalcitrant microbes, enabling efficient genetic manipulation for both fundamental research and biotechnological applications.}, }
@article {pmid40256973, year = {2025}, author = {Zhao, J and Wang, X and Zhang, H and Zhang, Q and Bo, D and Zhong, H and Jiao, L and Yuan, H and Lu, G}, title = {Tumor Cell Membrane Biomimetic Mesoporous Silicon Materials in Combination with PD-L1 Knockout Achieved through the CRISPR/Cas9 System for Targeted and Immunotherapeutic Purposes.}, journal = {Bioconjugate chemistry}, volume = {36}, number = {5}, pages = {971-979}, doi = {10.1021/acs.bioconjchem.5c00001}, pmid = {40256973}, issn = {1520-4812}, mesh = {*CRISPR-Cas Systems ; Animals ; *B7-H1 Antigen/genetics ; *Silicon/chemistry ; Mice ; *Cell Membrane/metabolism/chemistry ; Porosity ; *Biomimetic Materials/chemistry ; Cell Line, Tumor ; Immunotherapy/methods ; *Antineoplastic Agents/pharmacology/chemistry ; Gene Knockout Techniques ; Humans ; Mice, Inbred C57BL ; Drug Delivery Systems ; }, abstract = {Nanoparticle-based drug delivery systems, which enable the effective and targeted delivery of chemotherapeutic drugs to tumors, have revolutionized cancer therapy. Mesoporous silicon materials (MSN) have emerged as promising candidates for drug delivery due to their unique properties. The therapeutic efficacy can be significantly enhanced when treatments exhibit both targeting and antiphagocytic properties. In this study, cell membranes extracted from B16-F10 cells were used to encapsulate carboplatin (CBP)-loaded MSN via physical extrusion. Additionally, we intratumorally injected a plasmid containing the CRISPR/Cas9 system to achieve PD-L1 knockout, thereby reactivating the immune system. The cell membrane coating endowed the CBP@MSN with excellent slow-release capability and cytocompatibility. Enhanced tumor cell uptake of CBP@MSN@M was observed due to homologous targeting by cancer cell membranes. Moreover, CBP@MSN@M demonstrated enhanced antitumor efficacy in vivo and promoted the proliferation of immune cells. Finally, the antitumor effect was further improved by the knockout of PD-L1 within the tumor microenvironment. These results suggest that the newly prepared CBP@MSN@M, combined with PD-L1 knockout, holds significant potential as an effective therapeutic approach for treating tumors.}, }
@article {pmid40255449, year = {2025}, author = {Harshini, P and Varghese, R and Pachamuthu, K and Ramamoorthy, S}, title = {Enhanced pigment production from plants and microbes: a genome editing approach.}, journal = {3 Biotech}, volume = {15}, number = {5}, pages = {129}, pmid = {40255449}, issn = {2190-572X}, abstract = {Pigments are known for their vital roles in the growth and development of plants and microbes. In addition, they are also an imperative component of several industries, including textiles, foods, and pharmaceuticals, owing to their immense colours and therapeutic potential. Conventionally, pigments are obtained from plant resources, and the advent of in-vitro propagation techniques boosted the massive production. However, it could not meet the booming demand, leading to the incorporation of new genetic engineering tools. This review focuses on the role of various genetic engineering techniques in enhancing pigment production in plants and microorganisms. It also critically analyzes the efficacy and bottlenecks of these techniques in augmenting pigment biosynthesis. Furthermore, the use of microbes as pigment biofactories and the prospects in the field of genome editing to augment pigment synthesis are discussed. The limitations in the existing techniques underline the need for advanced genome editing strategies to broaden the mass production of pigments to meet the surging needs.}, }
@article {pmid40254669, year = {2025}, author = {Yang, X and Luo, Y and Su, C and Huang, Z and Tang, Y and Zhang, L}, title = {Ultra-sensitive biosensor detection of microRNA based on the CRISPR/Cas12a system and exonuclease-assisted target recycling signal amplification.}, journal = {Analytical sciences : the international journal of the Japan Society for Analytical Chemistry}, volume = {41}, number = {6}, pages = {867-876}, pmid = {40254669}, issn = {1348-2246}, support = {2021GXNSFBA 076001//Natural Science Foundation of Guangxi Zhuang Autonomous Region/ ; 2022KY0491//Middle-aged and Young Teachers' Basic Ability Promotion Project of Guangxi/ ; Z-C20240897//Autonomous region health commission self-funded research project/ ; }, mesh = {*MicroRNAs/analysis/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Exodeoxyribonucleases/metabolism ; *Nucleic Acid Amplification Techniques ; Limit of Detection ; *Exonucleases/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {MicroRNAs (miRNAs) are essential regulators of gene expression and are significantly involved in both preventing and treating a range of diseases. To that end, we developed an ultra-sensitive detection method for miRNA-141 by integrating exonuclease-assisted target recycling signal amplification with the CRISPR/Cas12a system. This method employs a variable hairpin probe (HP) designed to hybridize with miRNA, which, under the action of exonuclease III (ExoIII), cleaves the hairpin probe and triggers target recycling signal amplification. This results in the formation of output DNAs (ODs) containing multiple repeat sequences. The CRISPR/Cas12a system identifies these repeated sequences in ODs through its crRNA component, which in turn triggers the trans-cleavage function of the Cas12a/crRNA complex. It leads to the cleavage of a fluorescently quenched reporter probe. Consequently, this process restores fluorescence, producing a significantly enhanced fluorescent signal that facilitates the detection of miRNA-141, achieving a detection threshold down to 62 fM. This detection approach can specifically differentiate miRNA-141 from other confounding substances and has effectively identified low concentrations of miRNA-141 in actual sample human serum and diverse cancer cell lysates, showcasing its capability for tracing various nucleic acid biomarkers at minimal levels.}, }
@article {pmid40254211, year = {2025}, author = {Wei, J and Chen, F and Lu, X and Fan, J and Li, M and Huang, J and Liu, N and Zhang, J and Chai, Z and Lu, S}, title = {In silico identification and experimental validation of long-range allosteric inhibition of Staphylococcus aureus Cas9 catalytic activity by an anti-CRISPR protein AcrIIA14.}, journal = {International journal of biological macromolecules}, volume = {310}, number = {Pt 2}, pages = {143324}, doi = {10.1016/j.ijbiomac.2025.143324}, pmid = {40254211}, issn = {1879-0003}, mesh = {*Staphylococcus aureus/enzymology/genetics ; Allosteric Regulation ; Molecular Dynamics Simulation ; *CRISPR-Associated Protein 9/antagonists &amp; inhibitors/chemistry/metabolism/genetics ; *Bacterial Proteins/chemistry/genetics/metabolism/antagonists &amp; inhibitors ; *CRISPR-Cas Systems ; Allosteric Site ; Mutagenesis, Site-Directed ; Computer Simulation ; }, abstract = {Effective temporal and spatial regulation of CRISPR-Cas9 catalytic activity remains a key challenge, limiting the clinical application of CRISPR-Cas9 gene-editing. Here, we investigated the long-range allosteric inhibition of Staphylococcus aureus Cas9 (SauCas9) catalytic activity by its anti-CRISPR (Acr) protein, AcrIIA14, aiming to uncover remote allosteric mechanisms in large protein complexes and identify potential allosteric sites for the design of SauCas9 inhibitors. Through a combined computational-experimental framework integrating extensive molecular dynamics simulations, Markov state models, network community modeling, and site-directed mutagenesis, we identified canonical and non-canonical inhibitory states of SauCas9 regulated by AcrIIA14. Key domains, including REC, L1, HNH, L2, and PI, play crucial roles in transmitting the AcrIIA14-meidated inhibitory signal. Introducing point mutations on the routes of allosteric communication and analyzing these mutants using in vitro DNA cleavage assays and surface plasmon resonance analysis revealed that SauCas9 escaped AcrIIA14's inhibition owing to the disruption of AcrIIA14-meidated allosteric communication. Moreover, two cryptic allosteric sites on SauCas9 were identified as mutations of these sites prevented inhibition of SauCas9 by AcrIIA14. Overall, our results provide a dynamic understanding of CRISPR-Cas9 regulation and an avenue to design SauCas9 inhibitors with a broad range of applications in Cas9 enzyme catalysis, biophysics, and gene-editing.}, }
@article {pmid40253348, year = {2025}, author = {Hurtado, JE and Schieferecke, AJ and Halperin, SO and Guan, J and Aidlen, D and Schaffer, DV and Dueber, JE}, title = {Nickase fidelity drives EvolvR-mediated diversification in mammalian cells.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {3723}, pmid = {40253348}, issn = {2041-1723}, support = {T32 GM139780/GM/NIGMS NIH HHS/United States ; }, mesh = {Humans ; Animals ; CRISPR-Cas Systems/genetics ; *DNA-Directed DNA Polymerase/genetics/metabolism ; *Deoxyribonuclease I/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; HEK293 Cells ; *Directed Molecular Evolution/methods ; Mutation ; Mutagenesis ; }, abstract = {In vivo genetic diversifiers have previously enabled efficient searches of genetic variant fitness landscapes for continuous directed evolution. However, existing genomic diversification modalities for mammalian genomic loci exclusively rely on deaminases to generate transition mutations within target loci, forfeiting access to most missense mutations. Here, we engineer CRISPR-guided error-prone DNA polymerases (EvolvR) to diversify all four nucleotides within genomic loci in mammalian cells. We demonstrate that EvolvR generates both transition and transversion mutations throughout a mutation window of at least 40 bp and implement EvolvR to evolve previously unreported drug-resistant MAP2K1 variants via substitutions not achievable with deaminases. Moreover, we discover that the nickase's mismatch tolerance limits EvolvR's mutation window and substitution biases in a gRNA-specific fashion. To compensate for gRNA-to-gRNA variability in mutagenesis, we maximize the number of gRNA target sequences by incorporating a PAM-flexible nickase into EvolvR. Finally, we find a strong correlation between predicted free energy changes underlying R-loop formation and EvolvR's performance using a given gRNA. The EvolvR system diversifies all four nucleotides to enable the evolution of mammalian cells, while nuclease and gRNA-specific properties underlying nickase fidelity can be engineered to further enhance EvolvR's mutation rates.}, }
@article {pmid40253208, year = {2025}, author = {Xin, Y and Guo, T and Qiao, M}, title = {Current application and future prospects of CRISPR-Cas in lactic acid Bacteria: A review.}, journal = {Food research international (Ottawa, Ont.)}, volume = {209}, number = {}, pages = {116315}, doi = {10.1016/j.foodres.2025.116315}, pmid = {40253208}, issn = {1873-7145}, mesh = {*CRISPR-Cas Systems ; *Lactobacillales/genetics/metabolism ; Gene Editing/methods ; *Food Microbiology ; Probiotics ; Fermentation ; Metabolic Engineering ; }, abstract = {Lactic acid bacteria (LABs) have a long history of use in food and beverages fermentation. Recently, several LABs have gained attention as starter or non-starter cultures and probiotics for making functional fermented foods, which have the potential to enhance human health. In addition, certain LABs show great potential as microbial cell factories for producing food-related chemicals. However, enhancing the outcomes of starter and non-starter cultures, exploring the complicated probiotic mechanism of LABs, and engineering strains to enhance the yields of high-value compounds for precision fermentation remains challenging due to the time-consuming and labor-intensive current genome editing tools. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated proteins (Cas) system, originally an adaptive immune system in bacteria, has revolutionized genome editing, metabolic engineering and synthetic biology. Its versatility has resulted in extensive applications across diverse organisms. The widespread distribution of CRISPR-Cas systems and the diversity of CRISPR arrays in LAB genomes highlight their potential for studying the evolution of LABs. This review discusses the current advancement of CRISPR-Cas systems in engineering LABs for food application. Moreover, it outlines future research directions aimed at harnessing CRISPR-Cas systems to advance lactic acid bacterial research and drive innovation in food science.}, }
@article {pmid40252747, year = {2025}, author = {Zou, W and Huo, B and Tu, Y and Zhu, Y and Hu, Y and Li, Q and Yu, X and Liu, B and Tang, W and Tan, S and Xiao, H}, title = {Metabolic reprogramming by chemo-gene co-delivery nanoparticles for chemo-immunotherapy in head and neck squamous cell carcinoma.}, journal = {Acta biomaterialia}, volume = {199}, number = {}, pages = {361-373}, doi = {10.1016/j.actbio.2025.04.031}, pmid = {40252747}, issn = {1878-7568}, mesh = {*Squamous Cell Carcinoma of Head and Neck/therapy/pathology/metabolism/drug therapy/genetics ; Humans ; Animals ; *Immunotherapy ; *Nanoparticles/chemistry/therapeutic use ; Cell Line, Tumor ; *Cisplatin/pharmacology/chemistry/therapeutic use ; *Head and Neck Neoplasms/therapy/pathology/metabolism/drug therapy ; Prodrugs/pharmacology/chemistry ; Mice ; *Gene Transfer Techniques ; CRISPR-Cas Systems ; Tumor Microenvironment/drug effects ; Metabolic Reprogramming ; }, abstract = {The therapeutic effects of platinum-based drugs are closely linked to the dysregulation of tumor metabolic-immune microenvironment, particularly aberrant lactate accumulation. Herein, we engineered multifunctional nanoparticles (PPPt[IV] NPs) through electrostatic self-assembly of poly(β-amino ester) to co-encapsulate a cisplatin prodrug (Pt[IV]) and CRISPR/Cas9-PKM2 plasmids. Mechanistically, PPPt[IV] NPs efficiently entered cells via endocytosis, followed by escape from lysosomal degradation and cargo release. The reduction of Pt[IV] prodrug to active Pt[II] via GSH depletion induced DNA damage and ROS upregulation, thereby triggering apoptosis. Concurrently, CRISPR/Cas9-mediated PKM2 knockdown suppressed the Warburg effect, resulting in reduced lactate production and downregulated expression of HIF-1α and PD-L1. These alterations drove immune microenvironment remodeling through enhanced dendritic cell maturation, polarized M1 macrophages, and altered cytokine profiles (characterized by upregulation of IFN-γ, TNF-α, and IL-12 alongside suppression of IL-10), ultimately activating T cell-mediated antitumor immunity. Compared to conventional cisplatin, PPPt[IV] NPs demonstrated superior efficacy against both primary and recurrent tumors while reducing nephrotoxicity through synergistic chemo-immunotherapeutic effects, offering a valuable strategy for HNSCC treatment. STATEMENT OF SIGNIFICANCE: This study engineered an innovative nanoplatform (PPPt[IV]) that synergistically integrates a Pt[IV] prodrug with a CRISPR/Cas9-PKM2 plasmid for treating head and neck squamous cell carcinoma. By simultaneously enhancing DNA damage and reversing lactate-mediated immunosuppression, PPPt[IV] nanoplatform achieved chemo-immunotherapy that showed greater suppression of primary and recurrent tumors with reduced renal toxicity compared to cisplatin. This nanotechnology-driven strategy provides valuable insights into the combination of platinum-based drugs with immunometabolic interventions.}, }
@article {pmid40252733, year = {2025}, author = {Barreto, JA and Lacôrte E Silva, MVM and Marin, DC and Brienzo, M and Jacobus, AP and Contiero, J and Gross, J}, title = {Engineering adaptive alleles for Escherichia coli growth on sucrose using the EasyGuide CRISPR system.}, journal = {Journal of biotechnology}, volume = {403}, number = {}, pages = {126-139}, doi = {10.1016/j.jbiotec.2025.04.016}, pmid = {40252733}, issn = {1873-4863}, mesh = {*Escherichia coli/genetics/growth &amp; development/metabolism ; *Sucrose/metabolism ; Alleles ; Plasmids/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genetic Engineering/methods ; }, abstract = {Adaptive Laboratory Evolution (ALE) is a powerful approach for mining genetic data to engineer industrial microorganisms. This evolution-informed design requires robust genetic tools to incorporate the discovered alleles into target strains. Here, we introduce the EasyGuide CRISPR, a five-plasmid platform that exploits E. coli's natural recombination system to assemble gRNA plasmids from overlapping PCR fragments. The production of gRNAs and donor DNA is further facilitated by using recombination cassettes generated through PCR with 40-60-mer oligos. With the new CRISPR toolkit, we constructed 22 gene edits in E. coli DH5α, most of which corresponded to alleles mapped in E. coli DH5α and E2348/69 ALE populations selected for sucrose propagation. For DH5α ALE, sucrose consumption was supported by the cscBKA operon expression from a high-copy plasmid. During ALE, plasmid integration into the chromosome, or its copy number reduction due to the pcnB deletion, conferred a 30-35 % fitness gain, as demonstrated by CRISPR-engineered strains. A ∼5 % advantage was also associated with a ∼40.4 kb deletion involving fli operons for flagella assembly. In E2348/69 ALE, inactivation of the hfl system suggested selection pressures for maintaining λ-prophage dormancy (lysogeny). We further enhanced our CRISPR toolkit using yeast for in vivo assembly of donors and expression cassettes, enabling the establishment of polyhydroxybutyrate synthesis from sucrose. Overall, our study highlights the importance of combining ALE with streamlined CRISPR-mediated allele editing to advance microbial production using cost-effective carbon sources.}, }
@article {pmid40252377, year = {2025}, author = {Zhou, T and Shen, G and Zhong, L and Chen, G and Meng, L and He, W and Liu, J and Yang, S and Luo, Y and Wang, X}, title = {crRNA array-mediated CRISPR/Cas12a coupling with dual RPA for highly sensitive detection of Streptomyces aureofaciens Tü117 from hypertension with multi-signal output.}, journal = {Biosensors &amp; bioelectronics}, volume = {282}, number = {}, pages = {117493}, doi = {10.1016/j.bios.2025.117493}, pmid = {40252377}, issn = {1873-4235}, mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Animals ; *Streptomyces/isolation &amp; purification/genetics ; Humans ; Nucleic Acid Amplification Techniques/methods ; Mice ; *Hypertension/microbiology/diagnosis ; Limit of Detection ; }, abstract = {Accurate and sensitive detection of Streptomyces aureofaciens Tü117 is crucial for hypertension classification and early warning. To achieve this, a dual recombinase polymerase amplification coupled with a crRNA array-mediated CRISPR/Cas12a assay (DR-CAMCas) was developed, enabling multi-signal output for precise identification and detection of S. aureofaciens Tü117. The 16S rDNA and LipReg4 genes of S. aureofaciens Tü117 are amplified simultaneously via one-step dual RPA, activating the crRNA array-mediated CRISPR/Cas12a system to cleave exogenous FQ-reporters, releasing fluorescent signals. DR-CAMCas offers high amplification efficiency, multi-site recognition through crRNA array signal superposition, and the programmability of CRISPR/Cas12a, achieving ultrasensitive detection with a linear range of 10 to 10[8] cfu/mL and a limit of detection of approximately 3 cfu/mL. DR-CAMCas successfully detected S. aureofaciens Tü117 in fecal samples from high-salt diet-induced hypertensive mice and hypertensive patients, matching qPCR results and demonstrating high reliability and practicality. Additionally, target-induced cleavage of a DNA linker by DR-CAMCas dispersed AuNPs-DNA probes, enabling colorimetric detection. Integrated onto lateral flow sensors, DR-CAMCas allows point-of-care testing via simple visual strip analysis. Its triple signal output meets diverse detection needs, offering a promising tool for diagnosing salt-sensitive hypertension.}, }
@article {pmid40252041, year = {2025}, author = {Wen, TT and Yang, YM and Zhang, YX and Liu, MQ and Qian, ZY and Zhang, ZY and Dong, CH and Sun, L and Xu, L and Sun, WJ and Cui, FJ}, title = {CRISPR-Cas9/Safe Harbor-Targeted Overexpression of Glucan Synthase Gene CmGls in Edible Mushroom Cordyceps militaris.}, journal = {Journal of agricultural and food chemistry}, volume = {73}, number = {17}, pages = {10456-10469}, doi = {10.1021/acs.jafc.5c01310}, pmid = {40252041}, issn = {1520-5118}, mesh = {*Glucosyltransferases/genetics/metabolism ; *Fungal Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Cordyceps/genetics/enzymology/metabolism/growth &amp; development ; Gene Expression Regulation, Fungal ; Cell Wall/metabolism/genetics ; Fermentation ; }, abstract = {The membrane-integrated β-1,3-glucan synthase is the key enzyme involved in the biosynthesis of the core component β-1,3-glucan of the fungal cell wall. To date, the precise and targeted insertion of the β-1,3-glucan synthase gene into the genomes of edible fungi for safe and predictable overexpression has been extremely difficult due to the large DNA sequences (>5.0 kb) encoding the multitransmembrane domains and large molecular weights. In the present study, a large 5.9 kb DNA sequence of the membrane-bound β-1,3-glucan synthase gene CmGls was successfully and precisely inserted at a genomic safe harbor site CmSh1 of the C. militaris genome for the first time. By comparing mycelial and fermentation performance, overexpression of the β-1,3-glucan synthase gene CmGls resulted in rapid radial growth with a more pronounced yellowish color and increased resistance to cell wall stresses. Overexpression of CmGls significantly improved exopolysaccharide production with higher molecular weights, accompanied by an increase in the transcription levels of genes associated with polysaccharide/glucan synthesis, such as CmPgm, CmPgi, and CmUgp. Our findings provide convincing proof for the elucidation of glucan biosynthetic pathways and a basis for developing safe strains with highly efficient production of polysaccharides/glucans by edible fungi.}, }
@article {pmid40251402, year = {2025}, author = {Gonzalez, E and Anderson, MAE and Ang, JXD and Nevard, K and Shackleford, L and Larrosa-Godall, M and Leftwich, PT and Alphey, L}, title = {Optimization of SgRNA expression with RNA pol III regulatory elements in Anopheles stephensi.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {13408}, pmid = {40251402}, issn = {2045-2322}, support = {INV-008549/GATES/Gates Foundation/United States ; INV-008549/GATES/Gates Foundation/United States ; BBS/E/I/00007033, BBS/E/I/00007038, and BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; }, mesh = {*Anopheles/genetics ; Animals ; *RNA Polymerase III/genetics ; CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic ; Mosquito Vectors/genetics ; }, abstract = {Anopheles stephensi, a major Asian malaria vector, is invading Africa and has been implicated in recent outbreaks of urban malaria. Control of this species is key to eliminating malaria in Africa. Genetic control strategies, and CRISPR/Cas9-based gene drives are emerging as promising species-specific, environmentally friendly, scalable, affordable methods for pest control. To implement these strategies, a key parameter to optimize for high efficiency is the spatiotemporal control of Cas9 and the gRNA. Here, we assessed the ability of four RNA Pol III promoters to bias the inheritance of a gene drive element inserted into the cd gene of An. stephensi. We determined the homing efficiency and examined eye phenotype as a proxy for non-homologous end joining (NHEJ) events in somatic tissue. We found all four promoters to be active, with mean inheritance rates up to 99.8%. We found a strong effect of the Cas9-bearing grandparent (grandparent genotype), likely due to maternally deposited Cas9.}, }
@article {pmid40251060, year = {2025}, author = {Huang, W and Ruyechan, MC and Ralston, KS}, title = {Work with me here: variations in genome content and emerging genetic tools in Entamoeba histolytica.}, journal = {Trends in parasitology}, volume = {41}, number = {5}, pages = {401-415}, doi = {10.1016/j.pt.2025.03.010}, pmid = {40251060}, issn = {1471-5007}, mesh = {*Entamoeba histolytica/genetics ; *Genome, Protozoan/genetics ; CRISPR-Cas Systems ; *Genetic Variation ; Humans ; Entamoebiasis/parasitology ; RNA Interference ; }, abstract = {Entamoeba histolytica is the causative agent of amoebiasis, a significant source of morbidity and mortality in developing nations. Despite this, E. histolytica is understudied, leading to few treatment options and a poor understanding of pathogenesis. Genetic tools have historically been limited. By applying modern approaches, it was recently revealed that the genome is aneuploid. Interestingly, gene expression levels do not correlate with ploidy, potentially highlighting the importance of RNAi in gene regulation. Characterization of the RNAi pathway has led to potent tools for targeted gene knockdown, and the advent of RNAi-based forward genetics. CRISPR/Cas tools for editing the endogenous genome are an exciting possibility on the horizon. We celebrate the gains that have made E. histolytica tractable and anticipate continued advances.}, }
@article {pmid40250956, year = {2025}, author = {Eidelman, M and Eisenberg, E and Levanon, EY}, title = {Global quantification of off-target activity by base editors.}, journal = {Methods in enzymology}, volume = {713}, number = {}, pages = {255-270}, doi = {10.1016/bs.mie.2024.11.036}, pmid = {40250956}, issn = {1557-7988}, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Adenosine Deaminase/genetics/metabolism ; }, abstract = {Base editors are engineered deaminases combined with CRISPR components. These engineered deaminases are designed to target specific sites within DNA or RNA to make a precise change in the molecule. In therapeutics, they hold promise for correcting mutations associated with genetic diseases. However, a key challenge is minimizing unintended edits at off-target sites, which could lead to harmful mutations. Researchers are actively addressing this concern through a variety of optimization efforts that aim to improve the precision of base editors and minimize off-target activity. Here, we examine the various types of off-target activity, and the methods used to evaluate them. Current methods for finding off-target activity focus on identifying similar sequences in the genome or in the transcriptome, assuming the guide RNA misdirects the editor. The main method presented here, that was originally developed to quantify editing levels mediated by the ADAR enzyme, takes a different approach, investigating the inherent activity of base editors themselves, which might lead to off-target edits beyond sequence similarity. The editing index tool quantifies global off-target editing, eliminates the need to detect individual off-target sites, and allows for assessment of the global load of mutations.}, }
@article {pmid40250676, year = {2025}, author = {Gaba, S and Sahu, M and Chauhan, N and Jain, U}, title = {Transforming growth factor alpha: Key insights into physiological role, cancer therapeutics, and biomarker potential (A review).}, journal = {International journal of biological macromolecules}, volume = {310}, number = {Pt 2}, pages = {143212}, doi = {10.1016/j.ijbiomac.2025.143212}, pmid = {40250676}, issn = {1879-0003}, mesh = {Humans ; *Neoplasms/metabolism/therapy/genetics/drug therapy ; *Biomarkers, Tumor/metabolism/genetics ; *Transforming Growth Factor alpha/metabolism/genetics ; Animals ; Signal Transduction ; }, abstract = {Transforming Growth Factor Alpha (TGF-α) is a critical member of the epidermal growth factor (EGF) family and a key regulator of various physiological processes, including cellular proliferation, survival, differentiation, wound repair, and tissue regeneration. Deficiencies or mutations in TGF-α have been associated with impaired tissue development and organ growth, underscoring its critical role in maintaining normal and healthy physiology. Alterations in its levels are frequently implicated in the neoplastic transformation of cells, contributing to cancer development. Several strategies for targeting TGF-α in cancer therapy have been explored, such as the use of antibodies, recombinant proteins, oligonucleotide-mediated interference in ligand synthesis, ligand sequestration via binding proteins, and modulation of the signal transduction pathway. Furthermore, there is growing interest in the potential of TGF-α as a diagnostic or prognostic biomarker for cancer. This review delves into the role of TGF-α in normal physiology and its involvement in carcinogenesis. It highlights therapies targeting TGF-α and explores future directions in targeting TGF-α/EGFR signaling using advancing approaches, including nanoparticle-based drug delivery systems, CRISPR-Cas genome editing tool, PROTAC, and combination therapies. By bringing attention to this molecule, we aim to explore its untapped potential in cancer treatment and inspire further research into its promising applications across related fields. While recent studies highlight the promise of TGF-α as a clinical biomarker, further research is needed to validate its specificity and integration into personalized medicine. By providing a comprehensive overview of TGF-α in both normal and pathological contexts, this review aims to offer new insights into its translational applications in cancer therapeutics and biomarker discovery.}, }
@article {pmid40250571, year = {2025}, author = {Ali, A and Azmat, U and Khatoon, A and Akbar, K and Murtaza, B and Ji, Z and Irshad, U and Su, Z}, title = {From gene editing to tumor eradication: The CRISPR revolution in cancer therapy.}, journal = {Progress in biophysics and molecular biology}, volume = {196}, number = {}, pages = {114-131}, doi = {10.1016/j.pbiomolbio.2025.04.003}, pmid = {40250571}, issn = {1873-1732}, mesh = {*Gene Editing/methods ; Humans ; *Neoplasms/therapy/genetics/immunology ; *CRISPR-Cas Systems/genetics ; Animals ; *Genetic Therapy/methods ; Immunotherapy/methods ; }, abstract = {Cancer continues to be a significant worldwide health concern, characterized by high rates of occurrence and death. Unfortunately, existing treatments frequently fall short of delivering satisfying therapeutic outcomes. Immunotherapy has ushered in a new era in the treatment of solid tumors, yet its effectiveness is still constrained and comes with unwanted side effects. The advancement of cutting-edge technology, propelled by gene analysis and manipulation at the molecular scale, shows potential for enhancing these therapies. The advent of genome editing technologies, including CRISPR-Cas9, can greatly augment the efficacy of cancer immunotherapy. This review explores the mechanism of CRISPR-Cas9-mediated genome editing and its wide range of tools. The study focuses on analyzing the effects of CRISPR-induced double-strand breaks (DSBs) on cancer immunotherapy, specifically by gene knockdown or knockin. In addition, the study emphasizes the utilization of CRISPR-Cas9-based genome-wide screening to identify targets, the potential of spatial CRISPR genomics, and the extensive applications and difficulties of CRISPR-Cas9 in fundamental research, translational medicine, and clinical environments.}, }
@article {pmid40250544, year = {2025}, author = {Bai, X and Xu, W and Zhu, Y and Luo, B and Ye, D}, title = {Transcriptomics and phenotypic analysis of OTOF gene knockdown in zebrafish mediated by CRISPR/Cas9.}, journal = {Gene expression patterns : GEP}, volume = {55}, number = {}, pages = {119394}, doi = {10.1016/j.gep.2025.119394}, pmid = {40250544}, issn = {1872-7298}, mesh = {Animals ; *Zebrafish/genetics ; *Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Membrane Proteins/genetics/metabolism ; *Transcriptome ; Gene Knockdown Techniques ; Phenotype ; *Deafness/genetics ; Gene Editing ; Apoptosis ; Disease Models, Animal ; }, abstract = {Deafness is a common genetic disorder, where mutations,in the OTOF gene can disrupt the normal functionof the Otoferlin protein, leading to impaired neurotransmitter release in the inner ear and subsequent deafness. Despite the complexity of the pathogenic mechanism,it is not fully understood. Zebrafish are an excellent model for studying genetically-induced deafness,but there have been no previous reports on the pathogenesis of OTOF in zebrafish.This study successfully established a zebrafish model with mutated OTOF genes using CRISPR/Cas9 gene editing technology to investigate the molecular basis of OTOF-induced deafness. Compared to AB wild type zebrafish, those with low otof expression showed injury and apoptosis of hair cells in the posterior lateral neuromasts along with significant increase in the number of macrophages and apoptotic cells in this region. Additionally, these mutants exhibited a reduction in body length. To further elucidate differences at 5dpf (days post-fertilization) between mutant and wild type zebrafish embryos, RNA-seq analysis was conducted to examine differentially expressed genes (DEGs).A total of 334 up-regulated DEGs and 111 down-regulated DEGs were identified in mutants compared to wild types.KEGG and GO enrichment analyses were performed on these DEGs to identify key signaling pathways and hub DEGs. The findings revealedan increased expression of several genes involved in the HSP70 oxidative stress system, suggesting that OTOF may protect cochlear hair cell from apoptosis induced by oxidative stress through regulation of MAPK signal and HSP70 expression.In summary, the establishment of a zebrafish model with OTOF knockout provides a valuable tool for investigating the function of Otoferlin and understanding the role of the OTOF gene in deafness. These potential molecular insights offer significant contributions towards understanding the pathogenesis of deafness experimental models and serves as a foundation for comprehending the involvement of the OTOF gene.}, }
@article {pmid40249536, year = {2025}, author = {Wang, F and Chen, Y and Huang, R and Lu, D and Zhang, J and Yang, Y and Dang, H and Liu, M and Chen, Z and Sun, X and Wang, Z}, title = {Identification of SURF4 and RALGAPA1 as promising therapeutic targets in glioblastoma and pan-cancer through integrative multi-omics, CRISPR-Cas9 screening and prognostic meta-analysis.}, journal = {Cancer immunology, immunotherapy : CII}, volume = {74}, number = {6}, pages = {175}, pmid = {40249536}, issn = {1432-0851}, support = {No. 82171309//National Natural Science Foundation of China/ ; No. 82201445//National Natural Science Foundation of China/ ; }, mesh = {Humans ; *Biomarkers, Tumor/genetics ; *Brain Neoplasms/genetics/pathology ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; *Glioblastoma/genetics/pathology/mortality ; *Membrane Proteins/genetics/metabolism ; Multiomics ; Prognosis ; Tumor Microenvironment ; Meta-Analysis as Topic ; }, abstract = {Glioblastoma (GBM) is the most aggressive and malignant type of primary brain tumor, with a median survival time of less than two years and a uniformly poor prognosis, despite multimodal therapeutic approaches, which highlights an urgent need for novel therapeutic targets. In this study, by integrative multi-omics analysis from CPTAC database, DepMap database and seven independent GBM cohorts, four hub genes (CD44, SURF4, IGSF3 and RALGAPA1) were identified as essential genes regulated by cancer driver genes with robust prognostic value. GBM multi-omics data from public and in-house cohorts validated that CD44 and SURF4 might be synthetic lethal partners of loss-of-function tumor suppressor genes. Analysis for immune-related pathway activity revealed complex regulation relationships of the four hub genes in tumor microenvironment (TME). Further investigation on SURF4 in pathway activity, immune therapy response and drug sensitivity proposed that SURF4 emerged as a promising therapeutic target for GBM, even for pan-cancer. Pan-cancer multi-omics exploration suggested that RALGAPA1 may be a tumor suppressor gene. By screening the first-generation and second-generation DepMap database, four genes (CCDC106, GAL3ST1, GDI2 and HSF1) might be considered as synthetic targets after mutation of RALGAPA1 as a tumor suppressor gene.}, }
@article {pmid40247765, year = {2025}, author = {Nie, H and Jin, M and Wang, Z and Zheng, J and Zhang, Y and Li, X}, title = {Heterologous Expression of Candida antarctica Lipase B in Aspergillus niger Using CRISPR/Cas9-mediated Multi-Gene Editing.}, journal = {Biotechnology and bioengineering}, volume = {122}, number = {7}, pages = {1770-1779}, doi = {10.1002/bit.29000}, pmid = {40247765}, issn = {1097-0290}, support = {//This study was supported by Application Development Special Key Project of Chongqing (cstc2021jscx-jbgsX0002) and Science and Technology Project Founded by the Education Department of Jiangxi Province (GJJ202305)./ ; }, mesh = {*Lipase/genetics/biosynthesis/metabolism ; *Aspergillus niger/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Fungal Proteins/genetics/metabolism/biosynthesis ; Recombinant Proteins/genetics/metabolism/biosynthesis ; }, abstract = {Aspergillus niger, a filamentous fungus, is known as a cell factory due to its ability to produce large amounts of organic acids and industrial enzymes. Lipase B from Candida antarctica (CALB) is one of the most widely used lipases in industrial applications, including oil processing, papermaking, food, pharmaceuticals, and personal care products. In this study, the CRISPR/Cas9 technique was employed to knock out the pyrG and kusA genes in A. niger. The CALB gene was integrated into the high-production protein gene loci, such as glaA and amyA, to construct a multi-copy CALB production engineered strain. Additionally, the pepA, aglU, and bglA genes were deleted, which minimized the background level of secreted proteins in A. niger and increased the production of CALB. After two rounds of gene editing, the A. niger with multi-copy CALB was created, and the engineered A. niger CCTCC 206047.09 with high CALB yield was isolated. After 120 h of liquid fermentation, the lipase activity reached 17.84 U/mL and the protein yield reached 10.21 mg/mL. In summary, an engineered A. niger strain with high lipase activity was successfully isolated by employing a CRISPR/Cas9 system to integrate CALB into high-expression loci, while simultaneously knocking out the host's highly expressed protein genes. These results provide an effective strategy for the high expression of both heterologous and homologous enzymes in A. niger.}, }
@article {pmid40247031, year = {2025}, author = {Loughran, AJ and Narina, S and Klein, J and Siwak, JF and Connelly, JP and Pruett-Miller, SM}, title = {Rapid and robust validation of pooled CRISPR knockout screens using CelFi.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {13358}, pmid = {40247031}, issn = {2045-2322}, support = {P30 CA021765/CA/NCI NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems ; Humans ; *Gene Knockout Techniques/methods ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; INDEL Mutation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Pooled CRISPR screens are vital in the unbiased interrogation of gene function and are instrumental in uncovering therapeutic targets and biological processes. However, follow-up hit validation is critical to confirm observed results. Researchers need a simple and robust approach to rapidly verify putative hits and test resulting observations. Thus, we developed a CRISPR-based method for hit validation that tests the effect of a genetic perturbation on cell fitness. By editing target loci and monitoring the indel profiles over time, we have created a Cellular Fitness (CelFi) assay that can elucidate cellular vulnerabilities and verify hits from pooled CRISPR knockout screens. Unlike traditional cellular fitness assays that evaluate viability over time, the CelFi assay correlates changes in the indel profile at the target gene with a selective growth advantage or disadvantage in individual cells over time. Moreover, the CelFi assay can be utilized to evaluate gene dependencies and test new hypotheses, regardless of variations in single guide RNA optimization, ribonucleoprotein concentration, and gene copy number.}, }
@article {pmid40246839, year = {2025}, author = {Nakamura, K and Aoyama-Ishiwatari, S and Nagao, T and Paaran, M and Obara, CJ and Sakurai-Saito, Y and Johnston, J and Du, Y and Suga, S and Tsuboi, M and Nakakido, M and Tsumoto, K and Kishi, Y and Gotoh, Y and Kwak, C and Rhee, HW and Seo, JK and Kosako, H and Potter, C and Carragher, B and Lippincott-Schwartz, J and Polleux, F and Hirabayashi, Y}, title = {Mitochondrial complexity is regulated at ER-mitochondria contact sites via PDZD8-FKBP8 tethering.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {3401}, pmid = {40246839}, issn = {2041-1723}, support = {R35 NS127232/NS/NINDS NIH HHS/United States ; JP19dm0207082//Japan Agency for Medical Research and Development (AMED)/ ; JP20H04898//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; N/A//Secom Science and Technology Foundation (SSTF)/ ; JP22H05532//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP24H01348//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP24H01269//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP21wm0525015//Japan Agency for Medical Research and Development (AMED)/ ; N/A//Uehara Memorial Foundation/ ; }, mesh = {*Mitochondria/metabolism/ultrastructure ; *Endoplasmic Reticulum/metabolism/ultrastructure ; Humans ; *Tacrolimus Binding Proteins/metabolism/genetics ; *Mitochondrial Membranes/metabolism/ultrastructure ; HeLa Cells ; HEK293 Cells ; Proteomics ; Membrane Proteins/metabolism ; Animals ; Cryoelectron Microscopy ; CRISPR-Cas Systems ; }, abstract = {Mitochondria-ER membrane contact sites (MERCS) represent a fundamental ultrastructural feature underlying unique biochemistry and physiology in eukaryotic cells. The ER protein PDZD8 is required for the formation of MERCS in many cell types, however, its tethering partner on the outer mitochondrial membrane (OMM) is currently unknown. Here we identify the OMM protein FKBP8 as the tethering partner of PDZD8 using a combination of unbiased proximity proteomics, CRISPR-Cas9 endogenous protein tagging, Cryo-electron tomography, and correlative light-electron microscopy. Single molecule tracking reveals highly dynamic diffusion properties of PDZD8 along the ER membrane with significant pauses and captures at MERCS. Overexpression of FKBP8 is sufficient to narrow the ER-OMM distance, whereas independent versus combined deletions of these two proteins demonstrate their interdependence for MERCS formation. Furthermore, PDZD8 enhances mitochondrial complexity in a FKBP8-dependent manner. Our results identify a novel ER-mitochondria tethering complex that regulates mitochondrial morphology in mammalian cells.}, }
@article {pmid40245860, year = {2025}, author = {Martyn, GE and Montgomery, MT and Jones, H and Guo, K and Doughty, BR and Linder, J and Bisht, D and Xia, F and Cai, XS and Chen, Z and Cochran, K and Lawrence, KA and Munson, G and Pampari, A and Fulco, CP and Sahni, N and Kelley, DR and Lander, ES and Kundaje, A and Engreitz, JM}, title = {Rewriting regulatory DNA to dissect and reprogram gene expression.}, journal = {Cell}, volume = {188}, number = {12}, pages = {3349-3366.e23}, pmid = {40245860}, issn = {1097-4172}, support = {R00 HG009917/HG/NHGRI NIH HHS/United States ; UM1 HG011972/HG/NHGRI NIH HHS/United States ; U01 HG012069/HG/NHGRI NIH HHS/United States ; U01 HG012041/HG/NHGRI NIH HHS/United States ; K99 HG009917/HG/NHGRI NIH HHS/United States ; }, mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; *DNA/genetics/metabolism ; Transcription Factors/metabolism ; *Regulatory Sequences, Nucleic Acid/genetics ; *Gene Expression Regulation ; Binding Sites ; Animals ; Mice ; HEK293 Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Regulatory DNA provides a platform for transcription factor binding to encode cell-type-specific patterns of gene expression. However, the effects and programmability of regulatory DNA sequences remain difficult to map or predict. Here, we develop variant effects from flow-sorting experiments with CRISPR targeting screens (Variant-EFFECTS) to introduce hundreds of designed edits to endogenous regulatory DNA and quantify their effects on gene expression. We systematically dissect and reprogram 3 regulatory elements for 2 genes in 2 cell types. These data reveal endogenous binding sites with effects specific to genomic context, transcription factor motifs with cell-type-specific activities, and limitations of computational models for predicting the effect sizes of variants. We identify small edits that can tune gene expression over a large dynamic range, suggesting new possibilities for prime-editing-based therapeutics targeting regulatory DNA. Variant-EFFECTS provides a generalizable tool to dissect regulatory DNA and to identify genome editing reagents that tune gene expression in an endogenous context.}, }
@article {pmid40245636, year = {2025}, author = {Pu, Q and Ren, H and Ou, Q and Yang, X and Wei, T and Zhao, L and Han, Y and Lou, Y and Kashyap, S and Liu, S}, title = {SHMT, SHMTML and PRPS1 synergize to regulate blood digestion and nutrient metabolism in Aedes aegypti mosquitoes.}, journal = {International journal of biological macromolecules}, volume = {309}, number = {Pt 4}, pages = {143243}, doi = {10.1016/j.ijbiomac.2025.143243}, pmid = {40245636}, issn = {1879-0003}, mesh = {Animals ; *Aedes/metabolism/genetics ; *Glycine Hydroxymethyltransferase/metabolism/genetics/chemistry ; Female ; *Nutrients/metabolism ; *Insect Proteins/metabolism/genetics ; Digestion ; CRISPR-Cas Systems ; }, abstract = {Mosquitoes rely on vertebrate blood for nutrients vital for ovarian development. The enzyme serine hydroxymethyltransferase (SHMT) is crucial for amino acid and one‑carbon metabolism, playing a significant role in blood digestion and nutrient use in mosquitoes, though its functional mechanism remains further investigation. Using CRISPR/Cas9 to knock out the SHMT gene, we observed impaired blood digestion, delayed ovarian development, and inability to fly in mosquitoes. Multi-omics analysis revealed that SHMT deletion affected genes and metabolites related to amino acid metabolism. Knocking down SHMT-responsive genes mitochondrial-like serine hydroxymethyltransferase (SHMTML) and ribose-phosphate pyrophosphokinase 1 (PRPS1) also hindered blood digestion and ovarian development, mirroring SHMT-deficient mosquitoes. The interaction between SHMT, SHMTML, and PRPS1 was confirmed through various experiments, including Co-IP, GST pull-down, immunofluorescence colocalization, BiFC, molecular docking, and functional studies. Further research reveals that missing any of these proteins in mosquitoes results in ammonia and reactive oxygen species buildup, leading to mitochondrial problems, midgut cell damage, and abnormal enzyme expression. This study highlights a new molecular mechanism of SHMT and emphasizes its crucial interaction with SHMTML and PRPS1 in blood digestion and nutrient metabolism in vector mosquitoes. These findings may offer a strategic foundation for the development of innovative mosquito control measures.}, }
@article {pmid40244677, year = {2025}, author = {Wang, X and Yue, Y and Zhai, Y and Wang, F and Zhuang, X and Wu, S and Yang, Y and Tabashnik, BE and Wu, Y}, title = {Functional redundancy in the toxic pathway of Bt protein Cry1Ab, but not Cry1Fa, against the Asian corn borer.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {122}, number = {16}, pages = {e2503674122}, pmid = {40244677}, issn = {1091-6490}, support = {2022YFD1400901//Ministry of Science and Technology of China, National Key R&amp;D Program/ ; 32372587)//National Natural Science Foundation of China/ ; 2020-67013-31924//USDA National Institute of Food and Agriculture, Agriculture and Food Research Initiative/ ; }, mesh = {Animals ; *Endotoxins/genetics/toxicity/metabolism ; Bacillus thuringiensis Toxins ; *Bacterial Proteins/toxicity/genetics/metabolism/pharmacology ; *Hemolysin Proteins/genetics/toxicity/metabolism/pharmacology ; *Moths/genetics/drug effects ; Multidrug Resistance-Associated Protein 2 ; Insecticide Resistance/genetics ; Zea mays/parasitology/genetics ; Multidrug Resistance-Associated Proteins/genetics/metabolism ; Cadherins/genetics/metabolism ; Larva/drug effects/genetics ; Bacillus thuringiensis/genetics/metabolism ; Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Pest Control, Biological ; Mutation ; }, abstract = {Crops genetically engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have been used extensively to control some major crop pests, but their benefits decrease when pests evolve resistance. Better understanding of the genetic basis of resistance is needed to effectively monitor, manage, and counter pest resistance to Bt crops. Resistance to Bt proteins in at least 11 species of Lepidoptera, including many important crop pests, is associated with naturally occurring mutations that disrupt one or more of three larval midgut proteins: cadherin and ATP-binding cassette proteins ABCC2 and ABCC3. Here, we determined how CRISPR/Cas9-mediated mutations disrupting cadherin, ABCC2, and ABCC3 singly and in pairs affect resistance to Bt proteins Cry1Ab and Cry1Fa in the Asian corn borer (Ostrinia furnacalis), which is the most damaging pest of corn in Asia and is closely related to the European corn borer (Ostrinia nubilalis), a major pest in Europe and North America. The results from bioassays of six knockout strains and their parent susceptible strain support a model in which Cry1Ab can kill larvae via one path requiring ABCC2 or another path requiring cadherin and ABCC3, whereas Cry1Fa uses only the first path. The model's predictions are generally supported by results from genetic linkage analyses and responses to Cry1Ab and Cry1Fa of Sf9 cells and Xenopus oocytes modified to produce cadherin, ABCC2, and ABCC3 singly or in pairs. The functional redundancy identified here for Cry1Ab could sustain its efficacy against O. furnacalis and may exemplify a widespread natural strategy for delaying resistance.}, }
@article {pmid40244417, year = {2025}, author = {Bono, H}, title = {Recent Advances in Genome Editing and Bioinformatics: Addressing Challenges in Genome Editing Implementation and Genome Sequencing.}, journal = {International journal of molecular sciences}, volume = {26}, number = {7}, pages = {}, pmid = {40244417}, issn = {1422-0067}, support = {JPMJPF2010//Japan Science and Technology Agency/ ; }, mesh = {*Gene Editing/methods ; *Computational Biology/methods ; Humans ; CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing/methods ; Animals ; Genome ; }, abstract = {Genome-editing technology has advanced significantly since the 2020 Nobel Prize in Chemistry was awarded for the development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). While CRISPR-Cas9 has become widely used in academic research, its social implementation has lagged due to unresolved patent disputes and slower progress in gene function analysis. To address this, new approaches bypassing direct gene function analysis are needed, with bioinformatics and next-generation sequencing (NGS) playing crucial roles. NGS is essential for sequencing the genome of target species, but challenges such as data quality, genome heterogeneity, ploidy, and small individual sizes persist. Despite these issues, advancements in sequencing technologies, like PacBio high-fidelity (HiFi) long reads and high-throughput chromosome conformation capture (Hi-C), have improved genome sequencing. Bioinformatics contributes to genome editing through off-target prediction and target gene selection, both of which require accurate genome sequence information. In this review, I will give updates on the development of genome editing and bioinformatics technologies with a focus on the rapid progress in genome sequencing.}, }
@article {pmid40244221, year = {2025}, author = {Fan, S and Jia, L and Wu, J and Zhao, Y}, title = {Harnessing the Potential of CRISPR/Cas in Targeted Alfalfa Improvement for Stress Resilience.}, journal = {International journal of molecular sciences}, volume = {26}, number = {7}, pages = {}, pmid = {40244221}, issn = {1422-0067}, support = {42320104006//National Natural Science Foundation of China/ ; }, mesh = {*Medicago sativa/genetics/physiology ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Stress, Physiological/genetics ; Plant Breeding/methods ; Climate Change ; Genome, Plant ; }, abstract = {Alfalfa (Medicago sativa), recognized as the most valuable legume feed crop, faces significant challenges in enhancing both qualitative and quantitative production amidst the pressures of climate change. This review highlights these challenges, including the underutilization of genomic and genetic resources, while proposing potential solutions through genome editing. Our focus is on leveraging CRISPR/Cas technology in conjunction with decades of advancements in conventional breeding to expedite the improvement of alfalfa. By adopting this approach, we aim to overcome the limitations of traditional alfalfa improvement approaches and accelerate the development of improved cultivars capable of thriving in changing climates. Key candidate traits for CRISPR/Cas genome editing, as reviewed in the latest literature, include nutrient use efficiency, freezing tolerance, and resistance to pests and diseases. We dissect literature on potential gene pathways associated with these traits, providing molecular breeders with valuable insights for utilizing CRISPR/Cas genome editing. Furthermore, we propose editing modalities to expedite the development of stress-resilient, genome-edited alfalfa that can effectively cope with climate change.}, }
@article {pmid40244157, year = {2025}, author = {Zanatta, CB and Narendja, F and El Jawhary, H and Abou-Sleymane, G and Subburaj, S and Nodari, RO and Agapito-Tenfen, SZ}, title = {Suitability of Real-Time PCR Methods for New Genomic Technique Detection in the Context of the European Regulations: A Case Study in Arabidopsis.}, journal = {International journal of molecular sciences}, volume = {26}, number = {7}, pages = {}, pmid = {40244157}, issn = {1422-0067}, support = {301911//The Research Council of Norway/ ; }, mesh = {*Arabidopsis/genetics ; *Real-Time Polymerase Chain Reaction/methods ; *Plants, Genetically Modified/genetics ; Europe ; Polymorphism, Single Nucleotide ; *Genomics/methods ; CRISPR-Cas Systems ; Gene Editing/methods ; DNA, Plant/genetics ; Genome, Plant ; }, abstract = {PCR methods are widely applied for the detection of genetically modified organisms (GMOs) in Europe, facilitating compliance with stringent regulatory requirements and enabling the accurate identification and quantification of genetically modified traits in various crops and foodstuffs. This manuscript investigates the suitability of real-time PCR methods for detecting organisms generated through new genomic techniques (NGTs), specifically focusing on a case study using Arabidopsis thaliana as a model gene-edited plant. Given the complexities of European regulations regarding genetically modified organisms (GMOs) and the classification of gene-edited plants, there is a pressing need for robust detection methods. Our study highlights the development and validation of a novel single-plex real-time PCR method targeting a specific single nucleotide polymorphism (SNP) in the grf1-3 gene modified using CRISPR-Cas9 technology. We emphasize the effectiveness of locked nucleic acid (LNA)-modified primers in improving specificity. The results demonstrate that while the grf1-3 LNA method successfully detected and quantified gene-edited Arabidopsis DNA, achieving absolute specificity remains a challenge. This study also addresses the significance of the cross-laboratory method for validation, demonstrating that the method developed for an SNP-modified allele can be performed in accordance with the precision and trueness criteria established by the European Network of GMO Laboratories (ENGL). Furthermore, we call for continued collaboration among regulatory agencies, academia, and industry stakeholders to refine detection strategies. This proactive approach is essential not only for regulatory compliance but also for maintaining public trust in the safe integration of gene-edited organisms into food products.}, }
@article {pmid40244092, year = {2025}, author = {Ciapaite, J and Albersen, M and Savelberg, SMC and Bosma, M and Meijer, NWF and Tessadori, F and Bakkers, JPW and van Haaften, G and Jans, JJ and Verhoeven-Duif, NM}, title = {Broad Vitamin B6-Related Metabolic Disturbances in a Zebrafish Model of Hypophosphatasia (TNSALP-Deficiency).}, journal = {International journal of molecular sciences}, volume = {26}, number = {7}, pages = {}, pmid = {40244092}, issn = {1422-0067}, support = {OZF 2014-2015//Wilhelmina Children's Hospital Research Fund/ ; }, mesh = {Animals ; *Zebrafish/metabolism/genetics ; *Vitamin B 6/metabolism ; *Hypophosphatasia/metabolism/genetics ; Disease Models, Animal ; *Alkaline Phosphatase/genetics/metabolism/deficiency ; Pyridoxal Phosphate/metabolism ; Pyridoxine ; Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; }, abstract = {Hypophosphatasia (HPP) is a rare inborn error of metabolism caused by pathogenic variants in ALPL, coding for tissue non-specific alkaline phosphatase. HPP patients suffer from impaired bone mineralization, and in severe cases from vitamin B6-responsive seizures. To study HPP, we generated alpl[-/-] zebrafish using CRISPR/Cas9 gene-editing technology. At 5 days post fertilization (dpf), no alpl mRNA and 89% lower total alkaline phosphatase activity was detected in alpl[-/-] compared to alpl[+/+] embryos. The survival of alpl[-/-] zebrafish was strongly decreased. Alizarin red staining showed decreased bone mineralization in alpl[-/-] embryos. B6 vitamer analysis revealed depletion of pyridoxal and its degradation product 4-pyridoxic acid in alpl[-/-] embryos. Accumulation of d3-pyridoxal 5'-phosphate (d3-PLP) and reduced formation of d3-pyridoxal in alpl[-/-] embryos incubated with d3-PLP confirmed Alpl involvement in vitamin B6 metabolism. Locomotion analysis showed pyridoxine treatment-responsive spontaneous seizures in alpl[-/-] embryos. Metabolic profiling of alpl[-/-] larvae using direct-infusion high-resolution mass spectrometry showed abnormalities in polyamine and neurotransmitter metabolism, suggesting dysfunction of vitamin B6-dependent enzymes. Accumulation of N-methylethanolaminium phosphate indicated abnormalities in phosphoethanolamine metabolism. Taken together, we generated the first zebrafish model of HPP that shows multiple features of human disease and which is suitable for the study of the pathophysiology of HPP and for the testing of novel treatments.}, }
@article {pmid40243700, year = {2025}, author = {He, J and Liu, J and Yue, Y and Wang, L and Liu, Z and Xi, G and An, L and Tian, J and Wang, Y}, title = {Genome Editing in Mouse Embryo Using the CRISPR/Cas12i3 System.}, journal = {International journal of molecular sciences}, volume = {26}, number = {7}, pages = {}, pmid = {40243700}, issn = {1422-0067}, support = {2022YFD1302202//National Key R&amp;D Program of China/ ; No.2023TC019//Chinese Universities Scientific Fund/ ; 2022TC114//Chinese Universities Scientific Fund/ ; 2022TC123//Chinese Universities Scientific Fund/ ; }, mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Mice ; *Embryo, Mammalian/metabolism ; Nanog Homeobox Protein/genetics ; Female ; Embryonic Development/genetics ; Blastocyst/metabolism ; }, abstract = {The CRISPR/Cas system is a sizable family that is currently a popular and efficient gene editing tool. Cas12i3, as a member of the Type V-I family, has the characteristics of recognizing T-rich PAM sequences and being guided by shorter crRNA and has higher gene editing efficiency than Cas9 in rice. However, as a potential tool in accelerating the breeding process, the application of Cas12i3 in mammalian embryos has not yet been reported. Our study systematically evaluated the feasibility of applying CRISPR/Cas12i3 to gene editing in mouse embryos, with the core pluripotency regulator gene Nanog as the target. We successfully constructed a Nanog loss-of-function mouse embryo model using CRISPR/Cas12i3. At the targeted Nanog locus, its editing efficiency exceeded that of the Cas9 system under matched experimental conditions; no off-target phenomenon was detected. Moreover, the Cas12i3 system exhibited no side effect on mouse embryo development and proliferation of blastocyst cells. Finally, we obtained healthy chimeric gene-edited offspring by optimizing the concentration of the Cas12i3 mixture. These results confirm the feasibility and safety of CRISPR/Cas12i3 for gene editing in mammals, which provides a reliable tool for one-step generation of gene-edited animals for applications in biology, medical research, and large livestock breeding.}, }
@article {pmid40243657, year = {2025}, author = {Daliri, K and Hescheler, J and Newby, GA and Clement, K and Liu, DR and Pfannkuche, K}, title = {Modulating Collagen I Expression in Fibroblasts by CRISPR-Cas9 Base Editing of the Collagen 1A1 Promoter.}, journal = {International journal of molecular sciences}, volume = {26}, number = {7}, pages = {}, pmid = {40243657}, issn = {1422-0067}, support = {4346564//Imhoff foundation/ ; }, mesh = {Humans ; *Fibroblasts/metabolism ; *Promoter Regions, Genetic ; *Gene Editing/methods ; *Collagen Type I/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Collagen Type I, alpha 1 Chain ; MCF-7 Cells ; Fibrosis/genetics ; }, abstract = {Fibrotic diseases, contributing to a significant portion of global mortality, highlight the need for innovative therapies. This study explores a novel approach to disrupt the expression of collagen by using adenine base editing to target Col1a1, a key gene driving both fibrosis and cancer metastasis. Editing Col1a1 in fibroblasts demonstrated 18% editing efficiency. An analysis of a specific clone harboring a CCAAT-to-CCGGA mutation in the Col1a1 promoter revealed reduced collagen production. Notably, when wild-type fibroblasts were cultured on the Col1a1-edited matrix, no compensatory collagen upregulation was detected, suggesting a lack of feedback mechanism in fibroblasts. Furthermore, the matrix derived from edited fibroblasts did not support the growth of MCF-7 cancer cells. These findings suggest that Col1a1 gene editing holds promise as a potential therapeutic strategy for fibrotic diseases. Further investigation is warranted to fully elucidate the implications of these findings for fibrosis and cancer.}, }
@article {pmid40243589, year = {2025}, author = {Kim, T and Park, BS and Heo, S and Jeon, H and Kim, J and Kim, D and Kook Lee, S and Jung, SY and Kong, SY and Lu, T}, title = {Combinatorial CRISPR screen reveals FYN and KDM4 as targets for synergistic drug combination for treating triple negative breast cancer.}, journal = {eLife}, volume = {13}, number = {}, pages = {}, pmid = {40243589}, issn = {2050-084X}, support = {2021R1A2C1093499//National Research Foundation of Korea/ ; 2E32331//Korea Institute of Science and Technology/ ; 2020M3A9A5036362//National Research Foundation of Korea/ ; }, mesh = {*Triple Negative Breast Neoplasms/drug therapy/genetics ; Humans ; Animals ; *Proto-Oncogene Proteins c-fyn/genetics/metabolism/antagonists &amp; inhibitors ; Female ; Cell Line, Tumor ; Mice ; *Jumonji Domain-Containing Histone Demethylases/genetics/metabolism/antagonists &amp; inhibitors ; Drug Synergism ; *Protein Kinase Inhibitors/pharmacology ; CRISPR-Cas Systems ; *Antineoplastic Agents/pharmacology ; Drug Resistance, Neoplasm ; }, abstract = {Tyrosine kinases play a crucial role in cell proliferation and survival and are extensively investigated as targets for cancer treatment. However, the efficacy of most tyrosine kinase inhibitors (TKIs) in cancer therapy is limited due to resistance. In this study, we identify a synergistic combination therapy involving TKIs for the treatment of triple negative breast cancer. By employing pairwise tyrosine kinase knockout CRISPR screens, we identify FYN and KDM4 as critical targets whose inhibition enhances the effectiveness of TKIs, such as NVP-ADW742 (IGF-1R inhibitor), gefitinib (EGFR inhibitor), and imatinib (ABL inhibitor) both in vitro and in vivo. Mechanistically, treatment with TKIs upregulates the transcription of KDM4, which in turn demethylates H3K9me3 at FYN enhancer for FYN transcription. This compensatory activation of FYN and KDM4 contributes to the resistance against TKIs. FYN expression is associated with therapy resistance and persistence by demonstrating its upregulation in various experimental models of drug-tolerant persisters and residual disease following targeted therapy, chemotherapy, and radiotherapy. Collectively, our study provides novel targets and mechanistic insights that can guide the development of effective combinatorial targeted therapies, thus maximizing the therapeutic benefits of TKIs.}, }
@article {pmid40243584, year = {2025}, author = {Zhao, X and Mai, C and Xia, L and Jia, G and Li, X and Lu, Y and Li, Z and Yang, H and Wang, L}, title = {Molecular Insights into the Positive Role of Soybean Nodulation by GmWRKY17.}, journal = {International journal of molecular sciences}, volume = {26}, number = {7}, pages = {}, pmid = {40243584}, issn = {1422-0067}, support = {32241046, 32472158//National Natural Science Foundation of China/ ; 2025xczx03//Shanxi Breeding Innovation Joint research and development projects/ ; 2023ZD040350103//Scientific and Technological Innovation 2030-Major Projects/ ; 202204051001020//Science and Technology Innovation Young Talent Team of Shanxi Province/ ; 2021xG003, 2022xG0014//Scientific research fund for talents of Shanxi Agricultural University/ ; }, mesh = {*Glycine max/genetics/microbiology/metabolism ; *Plant Root Nodulation/genetics ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Root Nodules, Plant/genetics/metabolism/microbiology ; CRISPR-Cas Systems ; Symbiosis ; *Transcription Factors/genetics/metabolism ; Cytokinins/metabolism ; Plants, Genetically Modified ; Abscisic Acid/metabolism ; RNA Interference ; Nitrogen Fixation ; }, abstract = {Soybean is an important economic oilseed crop, being rich in protein and plant oil, it is widely cultivated around the world. Soybeans have been shown to establish a symbiotic nitrogen fixation (SNF) with their compatible rhizobia, resulting in the formation of nodules. Previous studies have demonstrated the critical roles of phytohormones, such as abscisic acid and cytokinin, in the process of legume nodulation. The present study investigated the role of GmWRKY17, a homolog of Rosa hybrida (Rh)WRKY13 in regulating plant immunity through cytokinin content and abscisic acid signaling in soybean nodulation. Utilizing real-time PCR and histochemical staining, we demonstrated that GmWRKY17 is predominantly expressed in soybean root nodules. Subsequently, we analyzed the function of GmWRKY17-overexpression, RNA interference (RNAi), and the CRISPR/Cas9 system. Overexpression of GmWRKY17 significantly increases soybean nodule number, while RNAi or CRISPR/Cas9-mediated knockout of GmWRKY17 resulted in a dramatic repression of nodule formation in soybeans. These results highlight that GmWRKY17 functions as a positive regulator involved in soybean nodulation. Furthermore, manipulation of GmWRKY17 expression impacts the expression of genes associated with the nod factor (NF) signaling pathway, thereby influencing soybean nodulation. This study demonstrated that WRKY-type transcription factors are involved in the regulation of legume nodulation, offering new light on the molecular basis of the symbiotic interaction between legumes and rhizobia.}, }
@article {pmid40243059, year = {2025}, author = {Liu, Q and Jiang, Z and Li, S and Li, Y and Wan, Y and Hu, Z and Ma, S and Zou, Z and Yang, R}, title = {Nonequilibrium hybridization-driven CRISPR/Cas adapter with extended energetic penalty for discrimination of single-nucleotide variants.}, journal = {Nucleic acids research}, volume = {53}, number = {7}, pages = {}, pmid = {40243059}, issn = {1362-4962}, support = {22334005//National Natural Science Foundation of China/ ; 2022JJ20038//Natural Science Foundation of Hunan Province/ ; 23A0059//Scientific Research Fund of Hunan Provincial Education Department/ ; 2022XKQ0205//Hunan Normal University/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Polymorphism, Single Nucleotide ; Humans ; Nucleic Acid Hybridization ; Proto-Oncogene Proteins B-raf/genetics ; ErbB Receptors/genetics ; CRISPR-Associated Proteins/metabolism/genetics ; Mutation ; Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Base Pair Mismatch ; }, abstract = {Accurate identification of single-nucleotide variants (SNVs) is critical in clinical diagnostics but remains challenging due to subtle free energy variations, particularly for hard-to-detect SNVs such as wobble base pairs and those in high guanine-cytosine (GC) regions. Here we report a high-energetic-penalty SNV detection (HEPSD) platform that redesigns the hybridization regions of CRISPR RNA (crRNA) in the CRISPR/Cas12a system. This system employs a binary crRNA architecture design that enables the activation of the cleavage activity of Cas12a while amplifying the energetic penalty for single-nucleotide mismatches through nonequilibrium hybridization-driven regulation. Consequently, the entire targeting region of CRISPR/Cas exhibits a marked preference for mutations in genomic DNA, while preventing false activation induced by sequences containing a single mismatched nucleotide. Moreover, HEPSD exhibits exceptional differentiation performance for hard-to-detect SNVs including wobble mutations at extreme GC contents. As proof of principle, profiling of BRAF V600E and EGFR L858R tumor mutations down to a 0.01% variant allele frequency was achieved, enabling accurate discrimination of 132 clinical sample pairs, which showed high consistency with quantitative polymerase chain reaction-based techniques and next-generation sequencing. The proven effectiveness of this platform showcases its potential for clinical molecular diagnostics and expands the fundamental scope of hybridization-based protocols.}, }
@article {pmid40242952, year = {2025}, author = {Aloliqi, AA and Alnuqaydan, AM and Albutti, A and Alharbi, BF and Rahmani, AH and Khan, AA}, title = {Current updates regarding biogenesis, functions and dysregulation of microRNAs in cancer: Innovative approaches for detection using CRISPR/Cas13‑based platforms (Review).}, journal = {International journal of molecular medicine}, volume = {55}, number = {6}, pages = {}, pmid = {40242952}, issn = {1791-244X}, mesh = {Humans ; *MicroRNAs/genetics/metabolism ; *Neoplasms/genetics/diagnosis ; *CRISPR-Cas Systems/genetics ; *Gene Expression Regulation, Neoplastic ; Animals ; Biomarkers, Tumor/genetics ; }, abstract = {MicroRNAs (miRNAs) are short non‑coding RNAs, which perform a key role in cellular differentiation and development. Most human diseases, particularly cancer, are linked to miRNA functional dysregulation implicated in the expression of tumor‑suppressive or oncogenic targets. Cancer hallmarks such as continued proliferative signaling, dodging growth suppressors, invasion and metastasis, triggering angiogenesis, and avoiding cell death have all been demonstrated to be affected by dysregulated miRNAs. Thus, for the treatment of different cancer types, the detection and quantification of this type of RNA is significant. The classical and current methods of RNA detection, including northern blotting, reverse transcription‑quantitative PCR, rolling circle amplification and next‑generation sequencing, may be effective but differ in efficiency and accuracy. Furthermore, these approaches are expensive, and require special instrumentation and expertise. Thus, researchers are constantly looking for more innovative approaches for miRNA detection, which can be advantageous in all aspects. In this regard, an RNA manipulation tool known as the CRISPR and CRISPR‑associated sequence 13 (CRISPR/Cas13) system has been found to be more advantageous in miRNA detection. The Cas13‑based miRNA detection approach is cost effective and requires no special instrumentation or expertise. However, more research and validation are required to confirm the growing body of CRISPR/Cas13‑based research that has identified miRNAs as possible cancer biomarkers for diagnosis and prognosis, and as targets for treatment. In the present review, current updates regarding miRNA biogenesis, structural and functional aspects, and miRNA dysregulation during cancer are described. In addition, novel approaches using the CRISPR/Cas13 system as a next‑generation tool for miRNA detection are discussed. Furthermore, challenges and prospects of CRISPR/Cas13‑based miRNA detection approaches are described.}, }
@article {pmid40241992, year = {2025}, author = {Haque, US and Yokota, T}, title = {Gene Editing for Duchenne Muscular Dystrophy: From Experimental Models to Emerging Therapies.}, journal = {Degenerative neurological and neuromuscular disease}, volume = {15}, number = {}, pages = {17-40}, pmid = {40241992}, issn = {1179-9900}, abstract = {The CRISPR system has emerged as a ground-breaking gene-editing tool, offering promising therapeutic potential for Duchenne muscular dystrophy (DMD), a severe genetic disorder affecting approximately 1 in 5000 male births globally. DMD is caused by mutations in the dystrophin gene, which encodes a critical membrane-associated protein essential for maintaining muscle structure, function and repair. Patients with DMD experience progressive muscle degeneration, loss of ambulation, respiratory insufficiency, and cardiac failure, with most succumbing to the disease by their third decade of life. Despite the well-characterized genetic basis of DMD, curative treatments- such as exon skipping therapies, micro-dystrophin, and steroids- remain elusive. Recent preclinical studies have demonstrated the promise of CRISPR-based approaches in restoring dystrophin expression across various models, including human cells, murine systems, and large animal models. These advancements highlight the potential of gene editing to fundamentally alter the trajectory of the disease. However, significant challenges persist, including immunogenicity, off-target effects, and limited editing efficiency, which hinder clinical translation. This review provides a comprehensive analysis of the latest developments in CRISPR-based therapeutic strategies for DMD. It emphasizes the need for further innovation in gene-editing technologies, delivery systems, and rigorous safety evaluations to overcome current barriers and harness the full potential of CRISPR/Cas as a durable and effective treatment for DMD.}, }
@article {pmid40240328, year = {2025}, author = {Torelli, F and Butterworth, S and Lockyer, E and Matias, AN and Hildebrandt, F and Song, OR and Pearson-Farr, J and Treeck, M}, title = {GRA12 is a common virulence factor across Toxoplasma gondii strains and mouse subspecies.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {3570}, pmid = {40240328}, issn = {2041-1723}, support = {WGF2024-0027//Wenner-Gren Foundation (Wenner-Gren Foundation for Anthropological Research, Inc.)/ ; TO 1349/1-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CC2132/WT_/Wellcome Trust/United Kingdom ; CR2132//Francis Crick Institute (Francis Crick Institute Limited)/ ; CR2023/030/2132//Francis Crick Institute (Francis Crick Institute Limited)/ ; 2023.06167.CEECIND//Ministry of Education and Science | Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia (Portuguese Science and Technology Foundation)/ ; CC0199/WT_/Wellcome Trust/United Kingdom ; 223192/Z/21/Z//Wellcome Trust (Wellcome)/ ; }, mesh = {Animals ; *Toxoplasma/pathogenicity/genetics/metabolism ; Mice ; *Virulence Factors/genetics/metabolism ; *Protozoan Proteins/genetics/metabolism ; Macrophages/parasitology/immunology ; CRISPR-Cas Systems ; *Toxoplasmosis, Animal/parasitology/immunology ; Female ; Mice, Inbred C57BL ; Toxoplasmosis/parasitology ; Host-Parasite Interactions ; Interferon-gamma ; }, abstract = {Toxoplasma gondii parasites exhibit extraordinary host promiscuity owing to over 250 putative secreted proteins that disrupt host cell functions, enabling parasite persistence. However, most of the known effector proteins are specific to Toxoplasma genotypes or hosts. To identify virulence factors that function across different parasite isolates and mouse strains that differ in susceptibility to infection, we performed systematic pooled in vivo CRISPR-Cas9 screens targeting the Toxoplasma secretome. We identified several proteins required for infection across parasite strains and mouse species, of which the dense granule protein 12 (GRA12) emerged as the most important effector protein during acute infection. GRA12 deletion in IFNγ-activated macrophages results in collapsed parasitophorous vacuoles and increased host cell necrosis, which is partially rescued by inhibiting early parasite egress. GRA12 orthologues from related coccidian parasites, including Neospora caninum and Hammondia hammondi, complement TgΔGRA12 in vitro, suggesting a common mechanism of protection from immune clearance by their hosts.}, }
@article {pmid40239991, year = {2025}, author = {Nam, HJ and Han, JH and Yu, J and Cho, CS and Kim, D and Kim, YE and Kim, MJ and Kim, JH and Jo, DH and Bae, S}, title = {Autophagy induction enhances homologous recombination-associated CRISPR-Cas9 gene editing.}, journal = {Nucleic acids research}, volume = {53}, number = {7}, pages = {}, pmid = {40239991}, issn = {1362-4962}, support = {2020M3A9I4036072//NRF/ ; KK2431-10//Korea Research Institute of Chemical Technology/ ; //National Research Council of Science &amp; Technology/ ; GTL24021-300//MSIT/ ; 18-2023-0010//Seoul National University Hospital/ ; RS-2023-00260351//Genome Editing Research Program/ ; }, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; *Autophagy/genetics ; Mice ; *Homologous Recombination ; Humans ; HEK293 Cells ; CRISPR-Associated Protein 9/metabolism ; }, abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9)-based gene editing via homologous recombination (HR) enables precise gene correction and insertion. However, its low efficiency poses a challenge due to the predominance of nonhomologous end-joining during DNA repair processes. Although numerous efforts have been made to boost HR efficiency, there remains a critical need to devise a novel method that can be universally applied across cell types and in vivo animals, which could ultimately facilitate therapeutic treatments. This study demonstrated that autophagy induction using different protocols, including nutrient deprivation or chemical treatment, significantly improved HR-associated gene editing at diverse genomic loci in mammalian cells. Notably, interacting cofactor proteins that bind to Cas9 under the autophagic condition have been identified, and autophagy induction could also enhance in vivo HR-associated gene editing in mice. These findings pave the way for effective gene correction or insertion for in vivo therapeutic treatments.}, }
@article {pmid40239628, year = {2025}, author = {Chen, J and Tan, J and Wang, N and Li, H and Cheng, W and Li, J and Wang, B and Sedgwick, AC and Chen, Z and Chen, G and Zhang, P and Zheng, W and Liu, C and Chen, J}, title = {Specific macrophage RhoA targeting CRISPR-Cas9 for mitigating osteoclastogenesis-induced joint damage in inflammatory arthritis.}, journal = {Cell reports. Medicine}, volume = {6}, number = {4}, pages = {102046}, pmid = {40239628}, issn = {2666-3791}, mesh = {*rhoA GTP-Binding Protein/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *Macrophages/metabolism/pathology ; *Osteogenesis/genetics ; Animals ; *Osteoclasts/metabolism/pathology ; *Arthritis, Rheumatoid/pathology/genetics/metabolism ; Mice ; Humans ; Gene Editing ; *Joints/pathology ; Inflammation/pathology ; RAW 264.7 Cells ; Mice, Inbred C57BL ; }, abstract = {Rheumatoid arthritis (RA) is the most prevalent inflammatory arthritis with unknown etiology, characterized by synovial inflammation and articular bone erosion. Studies have highlighted that inhibiting macrophage-induced osteoclastogenesis holds promise in mitigating bone destruction. However, specifically halting this pathological cascade remains a challenge for the management of RA. Here, initially, we identify that Ras homolog gene family member A (RhoA) is a pivotal target in inducing osteoclastogenesis of macrophages. Subsequently, we develop a strategy termed specific macrophages RhoA targeting (SMART), in which phosphatidylserine (PS)-enriched macrophage membranes are engineered to deliver macrophage-specific promoter-containing CRISPR-Cas9 plasmids (SMART-Cas9), enabling targeted editing of RhoA in RA joint macrophages. Multiscale imaging techniques confirm the highly specific targeted effect of SMART-Cas9 on the macrophages of inflamed joints. SMART-Cas9 successfully reduces osteoclastogenesis by macrophages, thus mitigating bone erosion by modulating cytoskeletal dynamics and immune balance in inflammatory arthritis, representing a therapeutic avenue for RA and other inflammatory bone diseases.}, }
@article {pmid40239471, year = {2025}, author = {Li, J and Huang, H and Song, Z and Chen, S and Xu, J and Yang, J and Zheng, C and Liu, Y and Zhang, J and Cao, L and Liu, Q and Li, Q and Li, M and Gu, Z and Wang, H}, title = {Palm-sized CRISPR sensing platform for on-site Mycoplasma pneumoniae detection.}, journal = {Biosensors &amp; bioelectronics}, volume = {281}, number = {}, pages = {117458}, doi = {10.1016/j.bios.2025.117458}, pmid = {40239471}, issn = {1873-4235}, mesh = {*Mycoplasma pneumoniae/isolation &amp; purification/genetics/pathogenicity ; *Biosensing Techniques/instrumentation ; Humans ; *Pneumonia, Mycoplasma/diagnosis/microbiology ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Equipment Design ; Nucleic Acid Amplification Techniques ; DNA, Bacterial/genetics ; }, abstract = {Pneumonia remains a prevalent childhood illness and is the foremost cause of mortality due to infectious diseases among children under the age of five. Mycoplasma pneumoniae (M. pneumoniae) causes the most frequent type of atypical pneumonia in this age group, has raised global health concern. Therefore, there is a pressing need for a rapid, low cost, and user-friendly method for the early diagnosis of M. pneumoniae pneumonia. Herein, we develop a CRISPR sensing platform for on-site M. pneumoniae detection, termed CRAFT (Crispr-based rapid assay device for field testing). The CRAFT provides "sample in-result out" functionality. It completed sample processing and nucleic acid extraction within 5 min at room temperature, with efficiency comparable to commercial kits. RPA and CRISPR/Cas12a reagents were isolated in a closed tube using a movable magnetic bead valve, and the RPA product was then mixed with the CRISPR reagent. The limit of detection for M. pneumoniae using CRAFT was 100 copies/μL, and the method exhibited no cross-reactivity with other respiratory pathogens. CRAFT was utilized to validate 50 clinical samples, and the results demonstrated 100 % consistency with those obtained by qPCR. This versatile platform holds significant potential for point-of-care testing of M. pneumoniae, particularly in resource-limited settings.}, }
@article {pmid40238250, year = {2025}, author = {Zhao, J and Yang, W and Cai, H and Cao, G and Li, Z}, title = {Current Progress and Future Trends of Genomics-Based Techniques for Food Adulteration Identification.}, journal = {Foods (Basel, Switzerland)}, volume = {14}, number = {7}, pages = {}, pmid = {40238250}, issn = {2304-8158}, support = {Grant No. ZDYF2024GXJS316//Key Research and Development Projects in Hainan Province/ ; }, abstract = {Addressing the pervasive issue of food adulteration and fraud driven by economic interests has long presented a complex challenge. Such adulteration not only compromises the safety of the food supply chain and destabilizes the market economy but also poses significant risks to public health. Food adulteration encompasses practices such as substitution, process manipulation, mislabeling, the introduction of undeclared ingredients, and the adulteration of genetically modified foods. Given the diverse range of deceptive methods employed, genomics-based identification techniques have increasingly been utilized for detecting food adulteration. Compared to traditional detection methods, technologies such as polymerase chain reaction (PCR), next-generation sequencing (NGS), high-resolution melt (HRM) analysis, DNA barcoding, and the CRISPR-Cas system have demonstrated efficacy in accurately and sensitively detecting even trace amounts of adulterants. This paper provides an overview of genomics-based approaches for identifying food adulteration, summarizes the latest applications in certification procedures, discusses current limitations, and explores potential future trends, thereby offering new insights to enhance the control of food quality and contributing to the development of more robust regulatory frameworks and food safety policies.}, }
@article {pmid40235216, year = {2025}, author = {Zuo, T and Chen, X and Yu, Y and Qin, L and Xu, G and Wei, F and Yang, J and Zhou, C and Fan, L and Hu, Q and Zhao, Z and Tang, BZ and Cen, Y}, title = {BE-CATCH: Bioamplifier-Equipped CRISPR-Cas12a Transduction System Coupled with Commercial Pregnancy Test Strips to Harness Signal-on Point-of-Care Detection.}, journal = {Analytical chemistry}, volume = {97}, number = {16}, pages = {8947-8956}, doi = {10.1021/acs.analchem.5c00342}, pmid = {40235216}, issn = {1520-6882}, mesh = {*CRISPR-Cas Systems ; Humans ; Female ; Pregnancy ; *Point-of-Care Testing ; *Pregnancy Tests/methods ; *Reagent Strips ; Animals ; *Endodeoxyribonucleases/genetics/metabolism ; *Point-of-Care Systems ; Chorionic Gonadotropin ; *CRISPR-Associated Proteins/metabolism/genetics ; Colorimetry ; Bacterial Proteins ; }, abstract = {Repurposing existing commercial diagnostic equipment to enable portable analysis of diverse targets is driving the development of affordable point-of-care testing (POCT). Interestingly, we found that goat antimouse IgG could replace human chorionic gonadotropin (hCG) to make the T line of pregnancy test strips (PTS) appear red color and accordingly synthesized a novel signal output probe, which eliminated the intricate hCG covalent coupling steps, and could meet the multiple needs of expanded POCT. Given this, we introduced a novel separation-free universal POCT strategy termed bioamplifier-equipped CRISPR-Cas12a transduction system coupled with PTS to harness signal-on detection (BE-CATCH). Specifically, target inputs were converted and amplified by the multiplied strand displacement amplification-based bioamplifier, thereby activating Cas12a's trans-cleavage activity. Then, the activated Cas12a would cleave the connector indiscriminately, which ultimately kept the signal output probe in a free state; thus, the inputs could be translated into a colorimetric signal on the PTS. This strategy not only provided boosted sensitivity and specificity but also enhanced user-friendliness by maintaining the signal-on detection mode. We also demonstrated the versatility of the BE-CATCH strategy through selectively detecting miR-155 and flap endonuclease 1. Given its broad adaptability, the BE-CATCH strategy could provide an appealing option to broaden the application of PTS in biomedical diagnostics.}, }
@article {pmid40234780, year = {2025}, author = {Tian, L and Gao, Y and Zi, L and Zhe, R and Yang, J}, title = {Dual Cas12a and multiplex crRNA CRISPR strategy ultrasensitive detection novel circRNA biomarker for the diagnosis of ovarian cancer.}, journal = {BMC cancer}, volume = {25}, number = {1}, pages = {695}, pmid = {40234780}, issn = {1471-2407}, support = {2022JJ12SN049//Dalian Science and Technology Innovation Fund/ ; 2022JJ12SN049//Dalian Science and Technology Innovation Fund/ ; 2022JJ12SN049//Dalian Science and Technology Innovation Fund/ ; 2022JJ12SN049//Dalian Science and Technology Innovation Fund/ ; 2022JJ12SN049//Dalian Science and Technology Innovation Fund/ ; DUT22YG121//Fundamental Research Funds for the Central Universities/ ; DUT22YG121//Fundamental Research Funds for the Central Universities/ ; DUT22YG121//Fundamental Research Funds for the Central Universities/ ; DUT22YG121//Fundamental Research Funds for the Central Universities/ ; DUT22YG121//Fundamental Research Funds for the Central Universities/ ; }, mesh = {Humans ; Female ; *RNA, Circular/genetics ; *Ovarian Neoplasms/diagnosis/genetics/blood ; *Biomarkers, Tumor/genetics ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *CRISPR-Associated Proteins/genetics ; *Endodeoxyribonucleases/genetics ; Limit of Detection ; Bacterial Proteins ; }, abstract = {BACKGROUND: Ovarian cancer (OC), as a malignant tumor, currently lacks effective screening early diagnosis measures. Clinical biomarkers CA-125 and HE4 are limited by false positives and insufficient sensitivity. Therefore, it's of great significance to search for new biomarker and construct sensitive detection method.<br><br>METHODS: We found a novel circRNA biomarker (hsa_circ_0049101) by RNA sequencing, and simultaneously propose a strategy, which integrates reverse transcription rolling circle amplification (RT-RCA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a to amplify and detect novel circRNA biomarker. This strategy use Dual Cas12a protein (FnCas12a and LbCas12a) and Multiplex CrRNA (DCMC-CRISPR) to enhance detection sensitivity. The sensitivity mechanism of CRISPR to detect circRNA was verified in detail.<br><br>RESULTS: The DCMC-CRISPR assay exhibited a broad detection range of 2000&#xa0;pM to 0.5 fM and the limit of detection (LOD) as low as 0.5&#xa0;fM. The DCMC-CRISPR system has 4-11 times higher sensitivity than single-crRNA CRISPR/Cas12a system. Clinical assessment of RNA extracts from patient's peripheral blood of 22 clinical OC patients and 28 controls demonstrates the DCMC-CRISPR strategy outperformed CA-125, HE4, and the ROMA index. The assay demonstrated comparable performance to RT-qPCR, exhibiting favorable sensitivity and specificity in this pilot cohort.<br><br>CONCLUSIONS: The DCMC-CRISPR platform offers a promising solution for circRNA biomarker screening and circRNA diagnostic. It highlights the possibility of expanding its applicability to address other cancer diseases.}, }
@article {pmid40234414, year = {2025}, author = {Kusumawardhani, H and Zoppi, F and Avendaño, R and Schaerli, Y}, title = {Engineering intercellular communication using M13 phagemid and CRISPR-based gene regulation for multicellular computing in Escherichia coli.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {3569}, pmid = {40234414}, issn = {2041-1723}, support = {310030_200532//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; }, mesh = {*Escherichia coli/genetics/physiology ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Cell Communication/genetics ; *Gene Expression Regulation, Bacterial ; Synthetic Biology/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Computers, Molecular ; *Genetic Engineering/methods ; }, abstract = {Engineering multicellular consortia, where information processing is distributed across specialized cell types, offers a promising strategy for implementing sophisticated biocomputing systems. However, a major challenge remains in establishing orthogonal intercellular communication, or "wires," within synthetic bacterial consortia. In this study, we address this bottleneck by integrating phagemid-mediated intercellular communication with CRISPR-based gene regulation for multicellular computing in synthetic E. coli consortia. We achieve intercellular communication with high sensitivity by regulating the transfer of single guide RNAs (sgRNAs) encoded on M13 phagemids from sender to receiver cells. Once inside the receiver cells, the transferred sgRNAs mediate gene regulation via CRISPR interference. Leveraging this approach, we successfully constructed one-, two-, and four-input logic gates. Our work expands the toolkit for intercellular communication and paves the way for complex information processing in synthetic microbial consortia, with diverse potential applications, including biocomputing, biosensing, and biomanufacturing.}, }
@article {pmid40233430, year = {2025}, author = {Li, Z and Wang, N and Wang, H and Yang, L and Li, Y and Gu, L and Fu, C and Yue, P and Yu, H}, title = {Single-cell transcriptomics reveals the mechanisms of lung injury induced by galt gene editing in mouse.}, journal = {Biochemical and biophysical research communications}, volume = {763}, number = {}, pages = {151780}, doi = {10.1016/j.bbrc.2025.151780}, pmid = {40233430}, issn = {1090-2104}, mesh = {Animals ; Mice ; *Gene Editing ; *Single-Cell Analysis ; *Lung Injury/genetics/pathology/metabolism ; *Transcriptome ; Lung/pathology/metabolism ; Mice, Inbred C57BL ; Disease Models, Animal ; CRISPR-Cas Systems ; }, abstract = {Galactosemia, caused by mutations in the GALT gene, leads to multi-organ damage. This study investigates the impact of Galt c.847 + 1G > T mutation on lung tissue using single-cell transcriptomics. We employed CRISPR/Cas9 to generate a Galt gene-edited mouse model with the Galt c. 847 + 1G > T mutation and assessed Galt expression through PCR and Western blotting. Histopathological analysis revealed significant structural lung changes, including alveolar congestion and inflammation. Single-cell RNA sequencing demonstrated a marked reduction in immune cells (NK, T, macrophages, B cells) and an increase in alveolar type II cells, vascular endothelial cells, and myofibroblasts in the GAL mouse. The increased abundance of alveolar type II cells indicated impaired differentiation and repair. Metabolic analysis revealed significant abnormalities linked to Galt c.847 + 1G > T mutation, with disruptions in TGF-β1, FGF, and Mif pathways contributing to cellular dysfunction and exacerbated lung injury. This model provides insights into the molecular mechanisms of lung injury in galactosemia, highlighting significant alterations in lung cell populations and key signaling pathways.}, }
@article {pmid40232991, year = {2025}, author = {Sahin, GN and Seli, E}, title = {Gene editing using CRISPR-Cas9 technology: potential implications in assisted reproduction.}, journal = {Current opinion in obstetrics &amp; gynecology}, volume = {37}, number = {3}, pages = {141-148}, doi = {10.1097/GCO.0000000000001022}, pmid = {40232991}, issn = {1473-656X}, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; *Reproductive Techniques, Assisted/trends ; Animals ; Female ; Genetic Therapy/methods ; }, abstract = {PURPOSE OF REVIEW: This article reviews the mechanisms, advancements, and potential implications of clustered regularly interspaced short palindromic repeats-associated (CRISPR-Cas) gene editing technology, with a specific focus on its applications in reproductive biology and assisted reproduction. It aims to explore the benefits and challenges of integrating this revolutionary technology into clinical and research settings.<br><br>RECENT FINDINGS: CRISPR-Cas9 is a transformative tool for precise genome editing, enabling targeted modifications through mechanisms like nonhomologous end joining (NHEJ) and homology-directed repair (HDR). Innovations such as Cas9 nickase and dCas9 systems have improved specificity and expanded applications, including gene activation, repression, and epigenetic modifications. In reproductive research, CRISPR has facilitated gene function studies, corrected genetic mutations in animal models, and demonstrated potential in addressing human infertility and hereditary disorders. Emerging applications include mitochondrial genome editing, population control of disease vectors via gene drives, and detailed analyses of epigenetic mechanisms.<br><br>SUMMARY: CRISPR-Cas9 technology has revolutionized genetic engineering by enabling precise genome modifications. This article discusses its mechanisms, focusing on the repair pathways (NHEJ and HDR) and methods to mitigate off-target effects. In reproductive biology, CRISPR has advanced our understanding of fertility genes, allowed corrections of hereditary mutations, and opened avenues for novel therapeutic strategies. While its clinical application in human-assisted reproduction faces ethical and safety challenges, ongoing innovations hold promise for broader biomedical applications.}, }
@article {pmid40231967, year = {2025}, author = {Zhang, X and Chen, S and Li, J and Liu, DA and Lai, J and Song, X and Hu, R and Qiu, Y and Chen, K and Xu, Y and Li, X}, title = {One-Step RAA and CRISPR-Cas13a Method for Detecting Influenza B Virus.}, journal = {Microbial biotechnology}, volume = {18}, number = {4}, pages = {e70144}, pmid = {40231967}, issn = {1751-7915}, support = {2024XZ011//Zhejiang Shuren University Basic Scientific Research Special Funds/ ; }, mesh = {*Influenza B virus/isolation &amp; purification/genetics ; Sensitivity and Specificity ; Humans ; *Influenza, Human/diagnosis/virology ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; *Recombinases/metabolism ; CRISPR-Cas Systems ; }, abstract = {We developed a sensitive and specific method based on recombinase-aided amplification (RAA) and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 13a (Cas13a). This method, named CRISPR-based Rapid and Efficient Test (CRISPRET), is designed for the early diagnosis of Influenza B (FluB) with the aim of shortening its transmission chain. We identified conserved regions in the Influenza B Virus (IBV) NS gene and designed forward and reverse primers along with crRNAs. We then established and optimised the reaction system, and Nucleic Acid Positive Reference Materials of IBV were used to evaluate the detection limit (DL) of CRISPRET. Additionally, we collected 257 clinical samples, comprising 127 samples from patients with IBV infection and 130 samples from healthy individuals, and subjected them to dual detection using CRISPRET and qPCR to evaluate the positive predictive value (PPV), negative predictive value (NPV), sensitivity and specificity of CRISPRET. We designed one forward primer, two reverse primers, and two crRNAs to establish and optimise the CRISPR ET. The method demonstrated the DL of 500 copies·μL[-1] when assisted by appropriate equipment. Despite requiring auxiliary equipment and a 30-min reaction, the CRISPR ET method enables the detection of IBV nucleic acid within approximately the first 5 min, achieving high sensitivity (100%), specificity (97.69%), PPV (97.69%) and NPV (100%), with a concordance rate of 98.83% to qPCR. CRISPRET offers a simple, field-applicable, one-step method for the rapid detection of IBV. It has strong potential for field-testing applications and intelligent integration into existing diagnostic systems.}, }
@article {pmid40231554, year = {2025}, author = {Halat, M and Klimek-Chodacka, M and Domagała, A and Zając, G and Oleszkiewicz, T and Kapitán, J and Baranski, R}, title = {Chiral sensing combined with nuclease activity assay to track Cas9 dynamics in solution: ROA and CPL study.}, journal = {Chemical communications (Cambridge, England)}, volume = {61}, number = {38}, pages = {6905-6908}, doi = {10.1039/d5cc00971e}, pmid = {40231554}, issn = {1364-548X}, mesh = {Spectrum Analysis, Raman ; *CRISPR-Associated Protein 9/metabolism/chemistry ; CRISPR-Cas Systems ; Solutions ; Stereoisomerism ; Luminescence ; }, abstract = {Chiroptical studies of the SpyCas9 protein are extremely rare. Nondestructive methods are needed to characterize its active ribonucleoprotein form. Using Raman optical activity (ROA) and circularly polarized luminescence (CPL), we present a new approach to detect key biomolecules involved in CRISPR-Cas technology while preserving their original nucleolytic activity.}, }
@article {pmid40230958, year = {2025}, author = {Rust, S and Randau, L}, title = {Real-time imaging of bacterial colony growth dynamics for cells with Type IV-A1 CRISPR-Cas activity.}, journal = {microLife}, volume = {6}, number = {}, pages = {uqaf006}, pmid = {40230958}, issn = {2633-6693}, abstract = {The Type IV-A1 CRISPR-Cas system of Pseudomonas oleovorans provides defense against mobile genetic elements in the absence of target DNA degradation. In recent studies, Escherichia coli BL21-AI cells with Type IV-A1 CRISPR-Cas activity displayed a heterogeneous colony growth phenotype. Here, we developed a convenient smartphone-mediated automatic remote-controlled time-lapse imaging system (SMARTIS), that enables monitoring of growing bacteria over time. The system's design includes a custom-built imaging box equipped with LED lights, an adjustable heating system and a smartphone that can be remotely controlled using freely available, user-friendly applications. SMARTIS allowed long-term observation of growing colonies and was utilized to analyze different growth behaviors of E. coli cells expressing Type IV-A1 CRISPR ribonucleoproteins. Our findings reveal that heterogeneity in colonies can emerge within hours of initial growth. We further examined the influence of different expression systems on bacterial growth and CRISPR interference activity and demonstrated that the observed heterogeneity of colony-forming units is strongly influenced by plasmid design and backbone identity. This study highlights the importance of careful assessment of heterogenous colony growth dynamics and describes a real-time imaging system with wide applications beyond the study of CRISPR-Cas activity in bacterial hosts.}, }
@article {pmid40229905, year = {2025}, author = {Chen, RD and Yang, Y and Liu, KM and Hu, JZ and Feng, YL and Yang, CY and Jiang, RR and Liu, SC and Wang, Y and Han, PA and Tian, RG and Wang, YL and Xu, SM and Xie, AY}, title = {Post-cleavage target residence determines asymmetry in non-homologous end joining of Cas12a-induced DNA double strand breaks.}, journal = {Genome biology}, volume = {26}, number = {1}, pages = {96}, pmid = {40229905}, issn = {1474-760X}, support = {32071439//National Natural Science Foundation of China/ ; 32371348//National Natural Science Foundation of China/ ; 2023ZD04048//National Science and Technology Major Project of China/ ; 2023ZD0500501//National Major Science and Technology Projects of China/ ; }, mesh = {*DNA End-Joining Repair ; *DNA Breaks, Double-Stranded ; *CRISPR-Associated Proteins/metabolism/genetics ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Bacterial Proteins ; }, abstract = {BACKGROUND: After Cas12a cleaves its DNA target, it generates a DNA double strand break (DSB) with two compatible 5'-staggered ends. The Cas12a-gRNA complex remains at the protospacer adjacent motif (PAM)-proximal end (PPE) while releasing the PAM-distal end (PDE). The effects of this asymmetric retention on DSB repair are currently unknown.<br><br>RESULTS: Post-cleavage retention of LbCas12a at PPEs suppresses the recruitment of classical non-homologous end joining (c-NHEJ) core factors, leading to longer deletions at PPEs compared to PDEs. This asymmetry in c-NHEJ engagement results in approximately tenfold more accurate ligation between two compatible PDEs induced by paired LbCas12a than ligation involving a compatible PPE. Moreover, ligation to a given end of SpCas9-induced DSBs demonstrates more efficient ligation with a PDE from Cas12a-induced DSBs than with a PPE. In LbCas12a-induced NHEJ-mediated targeted integration, only two compatible PDEs from LbCas12a-induced DSBs-one from donor templates and the other from target sites-promote accurate and directional ligation. Based on these findings, we developed a strategy called Cas12a-induced PDE ligation (CIPDEL) for NHEJ-mediated efficient and precise gene correction and insertion.<br><br>CONCLUSIONS: The asymmetric retention of CRISPR-LbCas12a at DSB ends suppresses c-NHEJ at PPEs, not at PDEs. This unique repair mechanism can be utilized in the CIPDEL strategy, offering a potentially better alternative for homology-directed targeted integration.}, }
@article {pmid40228775, year = {2025}, author = {Qiao, J and Sun, W and Yin, W and Hu, L and Wang, X and Liu, Y}, title = {Direct preparation of Cas9 ribonucleoproteins with an extended 'gRNA-shRNA' construct in Escherichia coli for precise genome manipulation.}, journal = {International journal of biological macromolecules}, volume = {309}, number = {Pt 4}, pages = {143121}, doi = {10.1016/j.ijbiomac.2025.143121}, pmid = {40228775}, issn = {1879-0003}, mesh = {*Escherichia coli/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Ribonucleoproteins/genetics/metabolism ; Humans ; *CRISPR-Cas Systems ; *RNA, Small Interfering/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; DNA Ligase ATP/genetics ; }, abstract = {Gene perturbation approaches have emerged as powerful tools for elucidating gene function and treating hereditary disorders. Previously, we developed a method for streamlined production of ready-to-use Cas9 ribonucleoproteins (RNPs) in Escherichia coli BL21(DE3). In this study, we present an improved approach by assembling Cas9 RNPs with an extended 'gRNA-shRNA' construct in the RNase III deficient strain HT115(DE3). Transfection of these engineered Cas9 RNPs into mammalian cells enables multidimensional genome manipulation, including simultaneous knockdown and knockout of target genes. Furthermore, the design of shRNA specifically targeting human DNA ligase IV (LIG4) significantly enhances efficiency in homology-directed repair genome editing. Collectively, our findings establish a user-friendly CRISPR/Cas9 RNP tool with immense potential for precise genome editing, gene function analysis, and gene therapy.}, }
@article {pmid40228657, year = {2025}, author = {Lee, M and Han, SH and Kim, D and Yun, S and Yeom, J and Kyeong, M and Park, SY and Lee, DY}, title = {Systematic identification of genomic hotspots for high-yield protein production in CHO cells.}, journal = {New biotechnology}, volume = {88}, number = {}, pages = {61-72}, doi = {10.1016/j.nbt.2025.04.006}, pmid = {40228657}, issn = {1876-4347}, mesh = {CHO Cells ; Animals ; Cricetulus ; *Genomics ; CRISPR-Cas Systems ; *Recombinant Proteins/biosynthesis/genetics ; *Genome ; }, abstract = {The efficient and stable production of therapeutic proteins in Chinese hamster ovary (CHO) cells hinges on robust cell line development (CLD). Traditional methods relying on random transgene integration often result in clonal variability, requiring extensive and resource-intensive screening. To address this limitation, we established a systematic, multiomics-driven framework that integrates 202 RNA-sequencing datasets and whole-genome sequencing data to identify genomic "hotspot" loci for precise and high-yield transgene integration. From an initial pool of 20 candidate loci, 5 top-performing hotspots were validated using site-specific integration in CHO-DG44 cells via the CRISPR/Cas9 system with Recombinase-mediated cassette exchange (RMCE). These genomic hotspots achieved 2.2- to 15.0-fold higher relative specific productivity compared to previously known controls (Fer1L4 and Locus1 sites), across multiple therapeutic proteins, including a lysosomal storage disorder-related enzyme and an Immunoglobulin G (IgG)-related monoclonal antibody (mAb) expression. This study offers a transformative approach to CLD, achieving significant improvements in productivity, genomic stability, and efficiency, as well as paving the way for enhanced biopharmaceutical manufacturing.}, }
@article {pmid40227971, year = {2025}, author = {Chen, WD and Liu, L and Cheng, L}, title = {Functionally Tunable Star-Shaped Multivalent crRNAs for Photocontrol CRISPR/Cas Editing.}, journal = {Angewandte Chemie (International ed. in English)}, volume = {64}, number = {25}, pages = {e202506527}, doi = {10.1002/anie.202506527}, pmid = {40227971}, issn = {1521-3773}, support = {XDB0960103//Research Program of the Chinese Academy of Sciences/ ; BNLMS-CXTD-202401//Beijing National Laboratory for Molecular Sciences/ ; 22271291//National Natural Science Foundation of China/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Photochemical Processes ; *RNA/genetics/chemistry ; }, abstract = {Clustered regularly interspaced shortpalindromic repeats/CRISPR-associated (CRISPR/Cas)-based genome editing has significantly advanced genetic engineering due to its precision, simplicity, and versatility. However, achieving precise spatial and temporal control remains challenging, restricting therapeutic and research applications. Herein, we introduce a novel class of star-shaped, multivalent crRNAs engineered for precise spatiotemporal control of CRISPR/Cas9 and Cas12a editing systems. These crRNAs are synthesized via single-site chemical modification and can be efficiently purified. By integrating distinct photo-responsive chemical linkages, we achieved selective activation of crRNA activity upon irradiation with specific wavelengths, enabling orthogonal regulation of multiple genetic targets simultaneously. This method demonstrated robust OFF-ON switching capabilities in vitro, characterized by minimal leakage and rapid activation. Importantly, the approach also proved highly effective for temporally controlled gene editing in mammalian cells in vivo, achieving considerable editing efficiency following brief photoactivation. Due to its target sequence-independent, single-site modification design, this strategy may serve as a universal solution for diverse CRISPR/Cas systems, eliminating cumbersome optimization processes. Future advancements incorporating long-wavelength responsive and reversible linkers promise further enhancement of tissue penetration and control, significantly broadening the applicability and impact of this approach in biological research and therapeutic interventions.}, }
@article {pmid40226913, year = {2025}, author = {Lin, CP and Li, H and Brogan, DJ and Wang, T and Akbari, OS and Komives, EA}, title = {CRISPR RNA binding drives structural ordering that primes Cas7-11 for target cleavage.}, journal = {Nucleic acids research}, volume = {53}, number = {7}, pages = {}, pmid = {40226913}, issn = {1362-4962}, support = {R01 AI151004/AI/NIAID NIH HHS/United States ; T32 GM008326/GM/NIGMS NIH HHS/United States ; R01AI151004/AI/NIAID NIH HHS/United States ; T32 GM008326/GF/NIH HHS/United States ; }, mesh = {*CRISPR-Associated Proteins/chemistry/metabolism/genetics ; Protein Binding ; *CRISPR-Cas Systems ; Catalytic Domain ; Models, Molecular ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Hydrogen Deuterium Exchange-Mass Spectrometry ; *RNA/metabolism/chemistry ; RNA Recognition Motif ; }, abstract = {Type III-E CRISPR-Cas effectors, referred to as Cas7-11 or giant Repeat-Associated Mysterious Protein, are single proteins that cleave target RNAs (tgRNAs) without nonspecific collateral cleavage, opening new possibilities for RNA editing. Here, biochemical assays combined with amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments reveal the dynamics of apo Cas7-11. The HDX-MS results suggest a mechanism by which CRISPR RNA (crRNA) stabilizes the folded state of the protein and subsequent tgRNA binding remodels it to the active form. HDX-MS shows that the four Cas7 RNA recognition motif (RRM) folds are well-folded, but insertion sequences, including disordered catalytic loops and β-hairpins of the Cas7.2/Cas7.3 active sites, fold upon binding crRNA leading to stronger interactions at domain-domain interfaces, and folding of the Cas7.1 processing site. TgRNA binding causes conformational changes around the catalytic loops of Cas7.2 and Cas7.3. We show that Cas7-11 cannot independently process the CRISPR array and that binding of partially processed crRNA induces multiple states in Cas7-11 and reduces tgRNA cleavage. The insertion domain interacts most stably with mature crRNA. Finally, we show a crRNA-induced conformational change in one of the tetratricopeptide repeat fused with Cas/HEF1-associated signal transducer (TPR-CHAT) binding sites providing an explanation for why crRNA binding facilitates TPR-CHAT binding.}, }
@article {pmid40225577, year = {2025}, author = {Karapurkar, JK and Rajkumar, S and Jung, JH and Kim, JY and Birappa, G and Gowda, DAA and Colaco, JC and Suresh, B and Choi, JY and Woo, SH and Jo, WJ and Lee, JH and Kim, KS and Hong, SH and Ramakrishna, S}, title = {Targeting USP11 counteracts SFTPC[I73T] -associated interstitial lung disease in hiPSCs-derived alveolar organoids and in vivo models.}, journal = {Theranostics}, volume = {15}, number = {10}, pages = {4526-4549}, pmid = {40225577}, issn = {1838-7640}, mesh = {Animals ; Mice ; Humans ; *Organoids/metabolism ; *Lung Diseases, Interstitial/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism ; Disease Models, Animal ; CRISPR-Cas Systems ; *Pulmonary Surfactant-Associated Protein C/genetics/metabolism ; Bleomycin ; Alveolar Epithelial Cells/metabolism ; Ubiquitination ; Mutation ; Pulmonary Alveoli/metabolism/pathology ; }, abstract = {Background: Interstitial lung disease (ILD) is a pulmonary disorder characterized by a combination of inflammation and fibrosis in the lung parenchyma, which initiates with the dysfunction of alveolar epithelial cells (AECs). The alveolar cells secrete surfactant proteins that lowers the surface tension of fluids in the lungs and maintains the stability of pulmonary tissue. Mutations on surfactant protein C (SFTPC), particularly I73T, are associated with a toxic gain of function that causes misfolding and the accumulation of immature SFTPC proteins, triggering pulmonary fibrosis (PF). Therefore, it is crucial to block the accumulation of the SFTPC [I73T] protein during ILD progression. Methods: We used a loss-of-function-based CRISPR/Cas9 library kit to screen genome-wide for deubiquitinating enzymes that regulate the SFTPC protein. The interaction between USP11 and SFTPC and its ubiquitination status was validated by immunoprecipitation and the TUBEs assay. HDR-directed knock-in of the I73T mutation into the SFTPC locus in human induced pluripotent stem cells (hiPSCs) was performed using the CRISPR/Cas9 system, and then those cells were differentiated into alveolar organoids (AOs) using a forced aggregation protocol. The clinical relevance of the USP11 inhibitor and its effect on preventing PF were investigated in a TGF-β-induced fibrosis in AOs and bleomycin (BLM)-induced mouse model. Results: We identified USP11 as a novel deubiquitinase that interacts with, stabilizes, deubiquitinates, and extends the half-life of SFTPC. Remarkably, USP11 stabilized and prolonged the half-life of the SFTPC [I73T] mutant protein significantly more than the wild type. In vitro functional studies revealed that USP11 exacerbates SFTPC [I73T] -induced fibrosis and enhances the epithelial-to-mesenchymal transition. Furthermore, we present a human in vitro model for investigating SFTPC[I73T] -induced fibrosis: hiPSCs-derived-AOs carrying the pathogenic SFTPC[I73T] variant. Interestingly, USP11 depletion in the organoids mitigated SFTPC [I73T] -induced fibrosis. Finally, pharmacological inhibition of USP11 prevented PF caused by TGF-β in hiPSCs-SFTPC[I73T]-AOs and BLM-induced mouse model, underscoring its therapeutic potential. Conclusions: Altogether, USP11 is a major protein stabilizer of SFTPC, and the clinical inhibition of USP11 during PF could be a novel therapeutic approach for ILD patients.}, }
@article {pmid40223602, year = {2025}, author = {Mukhare, R and Gandhi, KA and Kadam, A and Raja, A and Singh, A and Madhav, M and Chaubal, R and Pandey, S and Gupta, S}, title = {Integration of Organoids With CRISPR Screens: A Narrative Review.}, journal = {Biology of the cell}, volume = {117}, number = {4}, pages = {e70006}, pmid = {40223602}, issn = {1768-322X}, mesh = {*Organoids/metabolism ; Humans ; *CRISPR-Cas Systems/genetics ; Animals ; *Neoplasms/genetics/pathology ; Gene Editing/methods ; }, abstract = {Organoids represent a significant advancement in disease modeling, demonstrated by their capacity to mimic the physiological/pathological structure and functional characteristics of the native tissue. Recently CRISPR/Cas9 technology has emerged as a powerful tool in combination with organoids for the development of novel therapies in preclinical settings. This review explores the current literature on applications of pooled CRISPR screening in organoids and the emerging role of these models in understanding cancer. We highlight the evolution of genome-wide CRISPR gRNA library screens in organoids, noting their increasing adoption in the field over the past decade. Noteworthy studies utilizing these screens to investigate oncogenic vulnerabilities and developmental pathways in various organoid systems are discussed. Despite the promise organoids hold, challenges such as standardization, reproducibility, and the complexity of data interpretation remain. The review also addresses the ideas of assessing tumor organoids (tumoroids) against established cancer hallmarks and the potential of studying intercellular cooperation within these models. Ultimately, we propose that organoids, particularly when personalized for patient-specific applications, could revolutionize drug screening and therapeutic approaches, minimizing the reliance on traditional animal models and enhancing the precision of clinical interventions.}, }
@article {pmid40223236, year = {2025}, author = {Santiago-Rivera, E and Scheibel, T}, title = {Spider Eye Development Editing and Silk Fiber Engineering Using CRISPR-Cas.}, journal = {Angewandte Chemie (International ed. in English)}, volume = {64}, number = {25}, pages = {e202502068}, pmid = {40223236}, issn = {1521-3773}, support = {N62909-20-1-2068//Office of Naval Research Global and Air Force Office for Scientific Research/ ; }, mesh = {Animals ; *Spiders/genetics/growth &amp; development ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; *Silk/genetics/chemistry/metabolism ; *Eye/growth &amp; development/metabolism ; Red Fluorescent Protein ; }, abstract = {CRISPR-Cas9 gene editing represents an effective and precise technology to induce mutations in the genome, and it has been applied to a wide range of organisms for diverse purposes. However, CRISPR-based gene editing in spiders has not been reported to date. In this study, we demonstrate CRISPR-mediated microinjection in parental spiders leading to both knock-out (KO) and knock-in (KI) mutations within the spider's offspring. The KO of the gene sine oculis causes total eye loss, confirming the role of the gene in the development of all spider eyes. The KI of a monomeric red fluorescent protein (mRFP-KI) within a spider silk gene encoding one compound of the major ampullate silk of the spider Parasteatoda tepidariorum yields red fluorescent silk fibers. This finding demonstrates the feasibility of functionalizing silk proteins in spiders using CRISPR-based gene editing without influencing silk assembly. Our study expands the application of CRISPR to spiders and provides insights in the fields of developmental genetics as well as material sciences.}, }
@article {pmid40222963, year = {2025}, author = {Snider, DM and Coffin, ML and Armijo, BJ and Khetan, R and Duchow, MW and Capasso, A and Samanta, D}, title = {Conformationally Locked Peptide-DNA Nanostructures for CRISPR-Amplified Activity-Based Sensing.}, journal = {Angewandte Chemie (International ed. in English)}, volume = {64}, number = {25}, pages = {e202500649}, pmid = {40222963}, issn = {1521-3773}, support = {CHE 2404334//National Science Foundation/ ; 2024-77398//David and Lucile Packard Foundation/ ; RR 160093//Cancer Prevention and Research Institute of Texas/ ; IRG-21-135-01-IRG//American Cancer Society/ ; }, mesh = {Humans ; *Peptides/chemistry/metabolism ; *DNA/chemistry ; *CRISPR-Cas Systems ; *Nanostructures/chemistry ; SARS-CoV-2/enzymology ; *Biosensing Techniques/methods ; Cathepsin B/analysis/metabolism ; Matrix Metalloproteinase 7/analysis/metabolism ; Caspase 3/analysis/metabolism ; Limit of Detection ; }, abstract = {We introduce a new class of chemical probes for activity-based sensing of proteases, termed cleavable, locked initiator probes (CLIPs). CLIPs contain a protease-cleavable peptide linked between two programmable DNA strands-an "initiator" DNA and a shorter "blocking" DNA. These DNA sequences are designed to hybridize, creating a "locked" hairpin-like structure. Upon proteolytic cleavage, the initiator strand is released, triggering the activation of CRISPR-Cas12a enzymes and producing an amplified fluorescence response. CLIPs generate more than 20-fold turn-on signals at room temperature (25 °C), significantly outperforming commercial probes by yielding ∼40-fold lower limits of detection (LOD) at 100-fold lower concentrations. Their versatility enables the detection of various disease-relevant proteases-including the SARS-CoV-2 main protease, caspase-3, matrix metalloproteinase-7, and cathepsin B-simply by altering the peptide sequence. Importantly, CLIPs detect cathepsin B in four different colorectal cancer cell lines, highlighting their clinical potential. Taken together, the sensitivity (LOD: ∼88 pM), selectivity, and rapid assay time (down to 35 min), combined with the ability to operate in complex biological media with minimal sample preparation, position CLIPs as powerful chemical tools for activity-based sensing of functional enzymes.}, }
@article {pmid40222687, year = {2025}, author = {Silver, AJ and Brown, DJ and Olmstead, SD and Watke, JM and Gorska, AE and Tanner, L and Ramsey, HE and Savona, MR}, title = {Interallelic gene conversion of leukemia-associated single nucleotide variants.}, journal = {Gene}, volume = {958}, number = {}, pages = {149493}, doi = {10.1016/j.gene.2025.149493}, pmid = {40222687}, issn = {1879-0038}, mesh = {Humans ; *Leukemia/genetics ; CRISPR-Cas Systems ; Animals ; *Polymorphism, Single Nucleotide ; Mice ; *Gene Conversion ; Alleles ; Cell Line, Tumor ; *Repressor Proteins/genetics ; Gene Editing/methods ; }, abstract = {CRISPR-Cas9 is a useful tool for inserting precise genetic alterations through homology-directed repair (HDR), although current methods largely rely on provision of an exogenous repair template. Here, we tested the possibility of interchanging heterozygous single nucleotide variants (SNVs) using mutation-specific guide RNA, and the cell's own wild-type allele rather than an exogenous template. Using high-fidelity Cas9 to perform allele-specific CRISPR across multiple human leukemia cell lines as well as in primary hematopoietic cells from patients with leukemia, we find high levels of reversion to wild-type in the absence of exogenous template. Moreover, we demonstrate that bulk treatment to revert a truncating mutation in ASXL1 using CRISPR-mediated interallelic gene conversion (IGC) is sufficient to prolong survival in a human cell line-derived xenograft model (median survival 33 days vs 27.5 days; p = 0.0040). These results indicate that IGC is a useful laboratory tool which can be applied to numerous types of leukemia and can meaningfully alter cellular phenotypes at scale. Because our method targets single-base mutations, rather than larger variants targeted by IGC in prior studies, it greatly expands the pool of genetic lesions which could potentially be targeted by IGC. This technique may reduce cost and complexity for experiments modeling phenotypic consequences of SNVs. The principles of SNV-specific IGC demonstrated in this proof-of-concept study could be applied to investigate the phenotypic effects of targeted clonal reduction of leukemogenic SNV mutations.}, }
@article {pmid40222553, year = {2025}, author = {Sadler, RL and Greenman, AC and Methawasin, M and Fan, J and Harris, SP}, title = {The L348P point mutation in cardiac myosin binding protein-C alters transient responses to stretch, slows cardiac relaxation, and is embryonic lethal in homozygous CRISPR gene-edited mice.}, journal = {Journal of molecular and cellular cardiology}, volume = {203}, number = {}, pages = {35-46}, doi = {10.1016/j.yjmcc.2025.04.007}, pmid = {40222553}, issn = {1095-8584}, mesh = {Animals ; *Point Mutation/genetics ; *Carrier Proteins/genetics/metabolism ; Mice ; *Homozygote ; *Gene Editing ; Myocytes, Cardiac/metabolism ; Mice, Transgenic ; Gene Knock-In Techniques ; *Embryo Loss/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems ; Myocardial Contraction/genetics ; Amino Acid Substitution ; Disease Models, Animal ; Female ; }, abstract = {Mutations in cardiac myosin binding protein-C (cMyBP-C) are a common cause of hypertrophic cardiomyopathy (HCM), an inherited autosomal dominant disease affecting 1 in 250-500 people. We previously identified a single amino acid substitution (L348P) in the regulatory motif (M-domain) of cMyBP-C that slowed relaxation and caused diastolic dysfunction in transgenic mice. Here we attempted to increase expression of the mutant protein by creating a CRISPR gene-edited knock-in mouse model (L348P-CR) and breeding mice to homozygosity for the mutant allele. Results showed that L348P-CR homozygous mice died in utero, but that heterozygous knock-in mice developed contractile deficits and diastolic dysfunction comparable to transgenic mice. To overcome the lethal homozygous expression of the L348P mutation, we used our "cut-and-paste" approach to fully replace endogenous wild-type cMyBP-C with recombinant L348P cMyBP-C in permeabilized cardiomyocytes from SpyC3 mice. Results showed that replacement of wild-type cMyBP-C with recombinant L348P recapitulated mechanical effects seen in transgenic and L348P-CR mice, validating the utility of our cut-and-paste method for evaluating functional effects of cMyBP-C. We conclude that L348P-CR knock-in mice are a robust model of diastolic dysfunction due to a single point mutation in cMyBP-C and that the cut-and-paste approach offers a rapid and cost-effective approach for evaluating mutations in cMyBP-C, especially those that are lethal in traditional animal models.}, }
@article {pmid40222509, year = {2025}, author = {Xiao, G and Shi, H and Liu, M and Huang, M and Li, S and Zhou, X and Li, H and Zhang, G}, title = {Trans-cleavage activity of Cas12a effectors can be unleashed by both double-stranded DNA and single-stranded RNA targeting in absence of PAM.}, journal = {International journal of biological macromolecules}, volume = {309}, number = {Pt 4}, pages = {142992}, doi = {10.1016/j.ijbiomac.2025.142992}, pmid = {40222509}, issn = {1879-0003}, mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *DNA/metabolism/genetics/chemistry ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; DNA, Single-Stranded/genetics ; *Bacterial Proteins/metabolism/chemistry/genetics ; Humans ; }, abstract = {CRISPR-Cas12a is a powerful tool in nucleic acid detection, but the relationship between its trans-cleavage activity and protospacer adjacent motif (PAM) sequences remains incompletely understood. In this study, we synthesized diverse PAM-sequence substrates and conducted systematic cis-cleavage and trans-cleavage experiments with three Cas12a orthologs. We found that double-stranded DNA (dsDNA) can activate Cas12a's trans-cleavage activity even without PAM and this activation occurring independently of cis-cleavage. Notably, our results also revealed that single-stranded RNA (ssRNA) can directly initiate the trans-cleavage activity of Cas12a.We also experimentally validated the feasibility of CRISPR-Cas12a in detecting target dsDNA lacking PAM sequences, including identifying mutated sites in clinical samples. Structural prediction using AlphaFold 3 revealed the potential mechanism of Cas12a's PAM-independent trans-cleavage. Our research expands the understanding of Cas12a's trans-cleavage mechanism and demonstrates its potential for nucleic acid detection beyond PAM-dependent targets. This discovery broadens the application scope of Cas12a, providing new opportunities for developing highly sensitive and versatile diagnostic platforms.}, }
@article {pmid40222460, year = {2025}, author = {Gnaim, R and Ledesma-Amaro, R}, title = {Synthetic biology of Fusarium for the sustainable production of valuable bioproducts.}, journal = {Biotechnology advances}, volume = {81}, number = {}, pages = {108579}, doi = {10.1016/j.biotechadv.2025.108579}, pmid = {40222460}, issn = {1873-1899}, mesh = {*Fusarium/genetics/metabolism ; *Synthetic Biology/methods ; *Metabolic Engineering/methods ; Metabolic Networks and Pathways ; Biofuels ; CRISPR-Cas Systems ; }, abstract = {Synthetic biology offers transformative opportunities to optimise Fusarium species as efficient platforms for the sustainable production of diverse bioproducts. Advanced engineering techniques, including CRISPR/Cas9, RNA interference and synthetic promoters, have enhanced the manipulation of metabolic pathways, enabling higher yields of industrially relevant compounds. Recent insights from next-generation sequencing and omics technologies have significantly expanded our understanding of Fusarium's metabolic networks, leading to more precise strain engineering. Despite these advances, challenges such as metabolic bottlenecks, regulatory complexities and strain stability remain significant barriers to industrial-scale applications. The development of efficient genetic tools, together with the expansion of our knowledge of Fusarium physiology and genetics thanks to systems biology approaches, holds promise to unlock Fusarium's full potential as a sustainable cell factory. This review focuses on the genetic and metabolic tools available to enhance Fusarium's capacity to produce biofuels, pharmaceuticals, enzymes and other valuable compounds. It also highlights key innovations and discusses future directions for leveraging Fusarium as an environmentally friendly bioproduction system.}, }
@article {pmid40222082, year = {2025}, author = {Karl-Schöller, F and Breyer, M and Klopocki, E and Üçeyler, N}, title = {Generation of a gene-corrected human isogenic iPSC line from a patient with Fabry disease carrying the GLA variant c.1069C&gt;T using CRISPR/Cas9-mediated homology directed repair.}, journal = {Stem cell research}, volume = {86}, number = {}, pages = {103711}, doi = {10.1016/j.scr.2025.103711}, pmid = {40222082}, issn = {1876-7753}, mesh = {Humans ; *Fabry Disease/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *alpha-Galactosidase/genetics/metabolism ; Cell Line ; Male ; Cell Differentiation ; Gene Editing ; Mutation ; }, abstract = {Fabry disease (FD) is an X-linked genetic disorder caused by mutations in the GLA gene, leading to α-galactosidase A deficiency and intracellular globotriaosylceramide (Gb3) accumulation. To study FD-associated pathomechanisms, we generated an isogenic control induced pluripotent stem cell (iPSC) line (IsoFD-1) from a patient-derived FD-iPSC line (FD-1) carrying the GLA c.1069C>T mutation. Using CRISPR/Cas9 gene correction, we restored the wild-type sequence, confirmed by Sanger sequencing and absence of Gb3 deposits. IsoFD-1 exhibited typical pluripotency markers, normal karyotype, and trilineage differentiation capacity. This line provides a valuable tool for investigating Gb3-related cellular dysfunction in FD.}, }
@article {pmid40221789, year = {2025}, author = {Davydova, S and Liu, J and Liu, Y and Prince, K and Mann, J and Kandul, NP and Braswell, WE and Champer, J and Akbari, OS and Meccariello, A}, title = {A self-limiting sterile insect technique alternative for Ceratitis capitata.}, journal = {BMC biology}, volume = {23}, number = {1}, pages = {97}, pmid = {40221789}, issn = {1741-7007}, support = {AP23PPQS&amp;T00C108//U.S. Department of Agriculture/ ; AP23PPQS&amp;T00C108//U.S. Department of Agriculture/ ; 23-8130-1007-IA//U.S. Department of Agriculture/ ; 101059523//Horizon 2020/ ; }, mesh = {*Ceratitis capitata/genetics/physiology ; Animals ; *CRISPR-Cas Systems ; *Pest Control, Biological/methods ; Female ; Male ; }, abstract = {BACKGROUND: Genetic biocontrol systems have broad applications in population control of insects implicated in both disease spread and food security. Ceratitis capitata (the Mediterranean fruit fly), a major agricultural pest with a global distribution, is one of the appealing targets for such genetic control.<br><br>RESULTS: In this study, we establish and characterise a novel split-CRISPR/Cas9 system we term Sex Conversion Induced by CRISPR (SCIC) in C. capitata. Using the white eye gene for toolkit selection we achieved up to 100% CRISPR/Cas9 efficiency, displaying the feasibility of C. capitata split-CRISPR/Cas9 systems using constitutive promoters. We then induce sex conversion by targeting the transformer gene in a SCIC approach aimed for SIT-mediated releases upon radiation-based sterilisation. Knock-out of transformer induced partial to full female-to-male sex conversion, with the remaining individuals all being intersex and sterile. SCIC population modelling shows a strong potential to outcompete traditional SIT, allowing for faster population elimination with fewer released sterile males.<br><br>CONCLUSION: Overall, we construct an appropriate CRISPR/Cas9 toolkit for the use in C capitata. Our results build the foundation for further genetic pest control methods in the species and related tephritid agricultural pests.}, }
@article {pmid40221460, year = {2025}, author = {Myers, G and Friedman, A and Yu, L and Pourmandi, N and Kerpet, C and Ito, MA and Saba, R and Tang, V and Ozel, AB and Bergin, IL and Johnson, CN and Ku, CJ and Wang, Y and Balbin-Cuesta, G and Lim, KC and Lin, Z and Drysdale, C and McGee, B and Kurita, R and Nakamura, Y and Liu, X and Siemieniak, D and Singh, SA and Lyssiotis, CA and Maillard, I and Weisman, LS and Engel, JD and Khoriaty, R}, title = {A genome-wide screen identifies genes required for erythroid differentiation.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {3488}, pmid = {40221460}, issn = {2041-1723}, support = {R01 AI091627/AI/NIAID NIH HHS/United States ; U2C DK129445/DK/NIDDK NIH HHS/United States ; AI091627//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; F31 HL162544/HL/NHLBI NIH HHS/United States ; K08 DK127013/DK/NIDDK NIH HHS/United States ; P01 HL146372/HL/NHLBI NIH HHS/United States ; R01 HL157062/HL/NHLBI NIH HHS/United States ; U01 HL117658/HL/NHLBI NIH HHS/United States ; R01 HL148333/HL/NHLBI NIH HHS/United States ; T32 GM007315/GM/NIGMS NIH HHS/United States ; TL1 DK136046/DK/NIDDK NIH HHS/United States ; T32-GM007315//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; }, mesh = {Humans ; *Erythropoiesis/genetics ; *Cell Differentiation/genetics ; CRISPR-Cas Systems ; Animals ; Genome-Wide Association Study ; *Erythroid Precursor Cells/cytology/metabolism ; Erythroblasts/metabolism/cytology ; Mice ; Cell Line ; Gene Knockout Techniques ; Erythroid Cells/cytology ; Mutation ; }, abstract = {The complete array of genes required for terminal erythroid differentiation remains unknown. To address this knowledge gap, we perform a genome-scale CRISPR knock-out screen in the human erythroid progenitor cell line HUDEP-2 and validate candidate regulators of erythroid differentiation in a custom secondary screen. Comparison of sgRNA abundance in the CRISPR library, proerythroblasts, and orthochromatic erythroblasts, resulted in the identification of genes that are essential for proerythroblast survival and genes that are required for terminal erythroid differentiation. Among the top genes identified are known regulators of erythropoiesis, underscoring the validity of this screen. Notably, using a Log2 fold change of <-1 and false discovery rate of <0.01, the screen identified 277 genes that are required for terminal erythroid differentiation, including multiple genes not previously nominated through GWAS. NHLRC2, which was previously implicated in hemolytic anemia, was a highly ranked gene. We suggest that anemia due to NHLRC2 mutation results at least in part from a defect in erythroid differentiation. Another highly ranked gene in the screen is VAC14, which we validated for its requirement in erythropoiesis in vitro and in vivo. Thus, data from this CRISPR screen may help classify the underlying mechanisms that contribute to erythroid disorders.}, }
@article {pmid40221070, year = {2025}, author = {Khademi, Z and Mottaghi-Dastjerdi, N and Morad, H and Sahebkar, A}, title = {The role of CRISPR-Cas9 and CRISPR interference technologies in the treatment of autoimmune diseases.}, journal = {Autoimmunity reviews}, volume = {24}, number = {7}, pages = {103816}, doi = {10.1016/j.autrev.2025.103816}, pmid = {40221070}, issn = {1873-0183}, mesh = {Humans ; *Autoimmune Diseases/therapy/genetics/immunology ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Therapy/methods ; Animals ; }, abstract = {Autoimmune disorders can be described as inappropriate immune responses directed against self-antigens, which account for substantial healthcare concerns around the world. Immunosuppression or immune modulation are the main therapeutic modalities for autoimmune disorders. These modalities, however, impair the ability of the immune system to fight against infections, thereby predisposing to opportunistic diseases. This review explores existing therapies for autoimmune disorders, highlighting their limitations and challenges. Additionally, it describes the potential of CRISPR-Cas9 technology as a novel therapeutic approach to address these challenges.}, }
@article {pmid40220102, year = {2025}, author = {Romanowski, AJ and Richardson, RR and Plachez, C and Erzurumlu, RS and Poulopoulos, A}, title = {Gene Knockout in the Developing Brain of Wild-Type Rodents by CRISPR In Utero Electroporation.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2910}, number = {}, pages = {221-238}, pmid = {40220102}, issn = {1940-6029}, mesh = {Animals ; *Electroporation/methods ; *CRISPR-Cas Systems ; Mice ; *Gene Knockout Techniques/methods ; Female ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Brain/metabolism/embryology/growth &amp; development ; Pregnancy ; Hippocampus/metabolism ; Plasmids/genetics ; Neurons/metabolism ; }, abstract = {CRISPR/Cas9 constructs can be delivered by in utero electroporation to knock out a gene of interest in neurons of the developing brain in wild-type rodents. This approach allows for high-throughput genetic screening, circuit-specific gene knockout, and knockout cell phenotyping using sparse labeling within a wild-type in vivo context. Here we outline the methods and steps of designing guide RNAs in silico, cloning guide RNAs into plasmid backbones, and introducing these plasmids into the developing mouse cortex and hippocampus.}, }
@article {pmid40220094, year = {2025}, author = {Robertson, CD and Richardson, RR and Steyert, M and Martin, CA and Flynn, C and Poulopoulos, A}, title = {Prime Editing of Mouse Primary Neurons.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2910}, number = {}, pages = {69-84}, pmid = {40220094}, issn = {1940-6029}, mesh = {Animals ; *Gene Editing/methods ; Mice ; *Neurons/metabolism/cytology ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Plasmids/genetics ; High-Throughput Nucleotide Sequencing ; Cells, Cultured ; }, abstract = {Prime editing is a hybrid genome editing technology that introduces small edits on the genome with high precision. It combines nickase Cas9 with reverse transcriptase to prime and synthesizes edited DNA from RNA, reducing unintended insertions and deletions on the genome. This protocol describes the design of prime editing guide RNAs (pegRNAs), cloning of plasmids, nucleofection of mouse primary neurons, and preparation for next-generation sequencing. Directions are given for pegRNA and PE3b gRNA design and construction using PegAssist, a publicly available webtool and plasmid set. Prime editing in neurons allows genome manipulation while maintaining endogenous gene expression, making it ideal for studying protein structure/function relationships and pathogenic variants in a native neuronal context.}, }
@article {pmid40219967, year = {2025}, author = {Liu, X and Qi, Q and Xiong, W and Zhang, Y and Shen, W and Xu, X and Zhao, Y and Li, M and Zhou, E and Tian, T and Zhou, X}, title = {Tailoring and reversing m6A editing with sequential RNA bioorthogonal chemistry.}, journal = {Nucleic acids research}, volume = {53}, number = {7}, pages = {}, pmid = {40219967}, issn = {1362-4962}, support = {22037004//National Natural Science Foundation of China/ ; 2042023kf0204//Fundamental Research Funds for the Central Universities/ ; }, mesh = {Humans ; *RNA/chemistry/genetics/metabolism ; *Adenosine/analogs &amp; derivatives/chemistry/metabolism ; CRISPR-Cas Systems ; Methylation ; *RNA Editing ; Azides/chemistry ; HEK293 Cells ; }, abstract = {Many existing methods for post-transcriptional RNA modification rely on a single-step approach, limiting the ability to reversibly control m6A methylation at specific sites. Here, we address this challenge by developing a multi-step system that builds on the concept of sequential RNA bioorthogonal chemistry. Our strategy uses an azide-based reagent (NAI-N3) capable of both cleavage and ligation reactions, thereby allowing iterative and reversible modifications of RNA in living cells. By applying this approach in CRISPR (clustered regularly interspaced short palindromic repeats)-based frameworks, we demonstrate tailored editing of m6A marks at targeted RNA sites, overcoming the one-way restriction of conventional bioorthogonal methods. This sequential protocol not only broadens the scope for fine-tuned RNA regulation but also provides a versatile platform for exploring dynamic m6A function in genetic and epigenetic research.}, }
@article {pmid40219064, year = {2025}, author = {Marques, BM and Sulis, DB and Suarez, B and Yang, C and Cofre-Vega, C and Thomas, RD and Whitehill, JGA and Whetten, RW and Barrangou, R and Wang, JP}, title = {A Protoplast System for CRISPR-Cas Ribonucleoprotein Delivery in Pinus taeda and Abies fraseri.}, journal = {Plants (Basel, Switzerland)}, volume = {14}, number = {7}, pages = {}, pmid = {40219064}, issn = {2223-7747}, support = {20-070-4013//North Carolina Specialty Crop Block Grants/ ; NCZ04214//Cooperative State Research Service of the U.S. Department of Agriculture/ ; 2044721//U.S. National Science Foundation Small Business Technology Transfer Program/ ; 190549MA//North Carolina State University Chancellor's Innovation Fund/ ; }, abstract = {Climate change profoundly impacts the health, productivity, and resilience of forest ecosystems and threatens the sustainability of forest products and wood-based industries. Innovations to enhance tree growth, development, and adaptation offer unprecedented opportunities to strengthen ecosystem resilience and mitigate the effects of climate change. Here, we established a method for protoplast isolation, purification, and CRISPR-Cas ribonucleoprotein (RNP) delivery in Pinus taeda and Abies fraseri as a step towards accelerating the genetic improvement of these coniferous tree species. In this system, purified protoplasts could be isolated from somatic embryos with up to 2 × 10[6] protoplasts/g of tissue and transfected with proteins and nucleotides, achieving delivery efficiencies up to 13.5%. The delivery of functional RNPs targeting phenylalanine ammonia lyase in P. taeda and phytoene desaturase in A. fraseri yielded gene editing efficiencies that reached 2.1% and 0.3%, respectively. This demonstration of RNP delivery for DNA-free genome editing in the protoplasts of P. taeda and A. fraseri illustrates the potential of CRISPR-Cas to enhance the traits of value in ecologically and economically important tree species. The editing system provides a foundation for future efforts to regenerate genome-edited forest trees to improve ecosystem health and natural resource sustainability.}, }
@article {pmid40217300, year = {2025}, author = {Khalili-Tanha, G and Radisky, ES and Radisky, DC and Shoari, A}, title = {Matrix metalloproteinase-driven epithelial-mesenchymal transition: implications in health and disease.}, journal = {Journal of translational medicine}, volume = {23}, number = {1}, pages = {436}, pmid = {40217300}, issn = {1479-5876}, support = {R01CA237406/NH/NIH HHS/United States ; R01CA258274/NH/NIH HHS/United States ; R01 CA258274/CA/NCI NIH HHS/United States ; R01GM132100/NH/NIH HHS/United States ; R01HL157424/NH/NIH HHS/United States ; }, mesh = {*Epithelial-Mesenchymal Transition ; Humans ; *Matrix Metalloproteinases/metabolism ; Animals ; *Health ; *Disease ; Extracellular Matrix/metabolism ; Neoplasms/pathology ; }, abstract = {Epithelial-mesenchymal transition (EMT) is a process in which epithelial cells, defined by apical-basal polarity and tight intercellular junctions, acquire migratory and invasive properties characteristic of mesenchymal cells. Under normal conditions, EMT directs essential morphogenetic events in embryogenesis and supports tissue repair. When dysregulated, EMT contributes to pathological processes such as organ fibrosis, chronic inflammation, and cancer progression and metastasis. Matrix metalloproteinases (MMPs)-a family of zinc-dependent proteases that degrade structural components of the extracellular matrix-sit at the nexus of this transition by dismantling basement membranes, activating pro-EMT signaling pathways, and cleaving adhesion molecules. When normally regulated, MMPs promote balanced ECM turnover and support the cyclical remodeling necessary for proper development, wound healing, and tissue homeostasis. When abnormally regulated, MMPs drive excessive ECM turnover, thereby promoting EMT-related pathologies, including tumor progression and fibrotic disease. This review provides an integrated overview of the molecular mechanisms by which MMPs both initiate and sustain EMT under physiological and disease conditions. It discusses how MMPs can potentiate EMT through TGF-β and Wnt/β-catenin signaling, disrupt cell-cell junction proteins, and potentiate the action of hypoxia-inducible factors in the tumor microenvironment. It discusses how these pathologic processes remodel tissues during fibrosis, and fuel cancer cell invasion, metastasis, and resistance to therapy. Finally, the review explores emerging therapeutic strategies that selectively target MMPs and EMT, ranging from CRISPR/Cas-mediated interventions to engineered tissue inhibitors of metalloproteinases (TIMPs), and demonstrates how such approaches may suppress pathological EMT without compromising its indispensable roles in normal biology.}, }
@article {pmid40217120, year = {2025}, author = {Ishikawa, J and Kato, K and Kannan, S and Okazaki, S and Ishiguro, S and Yamashita, K and Yachie, N and Nishizawa, T and Zhang, F and Nishimasu, H}, title = {Structural insights into RNA-guided RNA editing by the Cas13b-ADAR2 complex.}, journal = {Nature structural &amp; molecular biology}, volume = {}, number = {}, pages = {}, pmid = {40217120}, issn = {1545-9985}, abstract = {Cas13 is an RNA-guided RNA endonuclease derived from the type VI CRISPR-Cas system, which has been used in numerous RNA-targeting technologies, such as RNA knockdown, detection and editing. The catalytically inactive Prevotella sp. Cas13b (dPspCas13b) fused to the human adenosine deaminase acting on RNA 2 (ADAR2) deaminase domain can edit adenosine in target transcripts to inosine, in an RNA-editing technology called REPAIR (RNA editing for programmable A-to-I replacement), which has potential for gene therapy. Here we report the cryo-electron microscopy structures of the PspCas13b-guide RNA binary complex, the PspCas13b-guide RNA-target RNA ternary complex and the dPspCas13b-ADAR2-guide RNA-target RNA complex. These structures provide mechanistic insights into RNA cleavage and editing. We applied our structural insights to engineer a compact and efficient dPspCas13b-ADAR2 complex (REPAIR-mini). Overall, our findings advance the understanding of CRISPR-Cas13 effector nucleases and could enable the development of improved RNA-targeting technologies.}, }
@article {pmid40216992, year = {2025}, author = {Sogabe, S and Nakano, H and Ogasahara, Y and Cha, PC and Ando, Y and Taniguchi-Ikeda, M and Matsumoto, R and Kanagawa, M and Kobayashi, K and Murayama, S and Aoi, T and Toda, T and Satake, W}, title = {Regulation of MCCC1 expression by a Parkinson's disease-associated intronic variant: implications for pathogenesis.}, journal = {Journal of human genetics}, volume = {70}, number = {7}, pages = {371-374}, pmid = {40216992}, issn = {1435-232X}, support = {JP23K21409//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP22K19487//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP22H04923//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP21wm0425019//Japan Agency for Medical Research and Development (AMED)/ ; }, mesh = {Humans ; *Parkinson Disease/genetics/pathology ; *Introns/genetics ; Dopaminergic Neurons/metabolism/pathology ; Polymorphism, Single Nucleotide ; Genetic Predisposition to Disease ; Genome-Wide Association Study ; Quantitative Trait Loci ; Induced Pluripotent Stem Cells/metabolism ; Mitochondria/genetics/metabolism ; Gene Expression Regulation ; Brain/metabolism/pathology ; Alleles ; Male ; RNA, Messenger/genetics ; CRISPR-Cas Systems ; Female ; }, abstract = {Parkinson's disease (PD) is a common neurodegenerative disorder characterized by dopaminergic neuron loss and α-synuclein aggregation. While some familial cases result from single-gene mutations, most are sporadic, involving complex genetic and environmental interactions. Among PD risk loci identified through genome-wide association studies, MCCC1 encodes a mitochondrial enzyme essential for leucine catabolism; however, the causal variant remains unclear. Here, we investigated whether the intronic variant rs12637471 regulates MCCC1 mRNA expression and influences PD risk. Postmortem brain analysis revealed significantly elevated MCCC1 mRNA levels in G-allele carriers, consistent with peripheral tissue eQTL data from GTEx. Using CRISPR/Cas9-edited induced pluripotent stem cells, we generated isogenic lines differing only at rs12637471 and observed increased MCCC1 expression in G-allele dopaminergic neurons. Given MCCC1's mitochondrial role, its dysregulation may impact mitochondrial homeostasis, autophagy, or inflammation, potentially contributing to PD pathogenesis.}, }
@article {pmid40216739, year = {2025}, author = {Jeong, TY and Yoon, DE and Kim, SP and Yang, J and Lim, SY and Ok, S and Ju, S and Park, J and Lee, SB and Park, SJ and Kim, S and Lee, H and Lee, D and Kang, SK and Lee, SE and Kim, HS and Seong, JK and Kim, K}, title = {An innovative approach using CRISPR-ribonucleoprotein packaged in virus-like particles to generate genetically engineered mouse models.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {3451}, pmid = {40216739}, issn = {2041-1723}, support = {RS-2023-00261905//National Research Foundation of Korea (NRF)/ ; RS-2023-00220894//National Research Foundation of Korea (NRF)/ ; RS-2023-NR077033//National Research Foundation of Korea (NRF)/ ; RS-2024-00441068//National Research Foundation of Korea (NRF)/ ; NRF-2014M3A9D5A01074636//National Research Foundation of Korea (NRF)/ ; NRF-2014M3A9D5A01075128//National Research Foundation of Korea (NRF)/ ; RS-2023-00220894//National Research Foundation of Korea (NRF)/ ; }, mesh = {Animals ; *Gene Editing/methods ; *Ribonucleoproteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Mice ; Mice, Knockout ; *Genetic Engineering/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; Female ; Male ; Gene Knock-In Techniques ; Mice, Inbred C57BL ; *Virion/genetics ; }, abstract = {Genetically engineered mouse models (GEMMs) are crucial for investigating disease mechanisms, developing therapeutic strategies, and advancing fundamental biological research. While CRISPR gene editing has greatly facilitated the creation of these models, existing techniques still present technical challenges and efficiency limitations. Here, we establish a CRISPR-VLP-induced targeted mutagenesis (CRISPR-VIM) strategy, enabling precise genome editing by co-culturing zygotes with virus-like particle (VLP)-delivered gene editing ribonucleoproteins (RNPs) without requiring physical manipulation or causing cellular damage. We generate Plin1- and Tyr-knockout mice through VLP-based SpCas9 or adenine base editor (ABE)/sgRNA RNPs and characterize their phenotype and germline transmission. Additionally, we demonstrate cytosine base editor (CBE)/sgRNA-based C-to-T substitution or SpCas9/sgRNA-based knock-in using VLPs. This method further simplifies and accelerates GEMM generation without specialized techniques or equipment. Consequently, the CRISPR-VIM method can facilitate mouse modeling and be applied in various research fields.}, }
@article {pmid40216127, year = {2025}, author = {Bacha, SAS and Kiran, S and Cui, FJ and Elboughdiri, N and Ahmad, Z and Sun, WJ}, title = {The potential of advanced crop breeding technologies for sustainable food security.}, journal = {International journal of biological macromolecules}, volume = {309}, number = {Pt 3}, pages = {143025}, doi = {10.1016/j.ijbiomac.2025.143025}, pmid = {40216127}, issn = {1879-0003}, mesh = {*Plant Breeding/methods ; *Food Security ; *Crops, Agricultural/genetics/growth &amp; development ; Gene Editing ; CRISPR-Cas Systems ; Agriculture ; Plants, Genetically Modified/genetics ; Food Supply ; }, abstract = {Considering the increasing demands of a growing global population, shortages of resources, and climate change, exploring the potential of modern plant breeding technology seems to be an important and feasible method for ensuring food security. The current review shed light on the dramatic application of modern plant breeding techniques, which not only increase yields of crops but also lead a way for sustainable agriculture and resilience in dealing with of environmental challenges. Modern plant breeding technologies, such as Clustered regularly interspaced short palindromic repeats-associated protein (CRISPR-Cas) genome editing tools, omics, marker-Assisted Selection (MAS), and RNA Interference (RNAi) for Crop Enhancement exhibited the potential to significantly enhance crop production and diversity. Modern plant breeding technologies offers a method for developing crops that are resistant to the effects of climate change, pests, and diseases, improving crop yield and nutritional quality while decreasing the demand for harmful pesticides. Finally, this review emphasizes the enormous potential of modern plant breeding methods in ensuring global food security, as well as the importance of continued research, collaboration, and strategic application for a resilient and sustainable agricultural future.}, }
@article {pmid40216108, year = {2025}, author = {Yi, JY and Park, S and Kim, M and Jeong, Y and Shin, H and Cho, Y and Jeon, M and Oh, MK and Sung, C}, title = {Emerging wound-healing injectable polydeoxyribonucleotide: potential as a prohibited doping method and its simple detection via CRISPR/Cas12a system.}, journal = {International journal of biological macromolecules}, volume = {309}, number = {Pt 3}, pages = {142999}, doi = {10.1016/j.ijbiomac.2025.142999}, pmid = {40216108}, issn = {1879-0003}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Polydeoxyribonucleotides/administration &amp; dosage/pharmacology/urine/genetics ; *Doping in Sports/prevention &amp; control ; Animals ; *Wound Healing/drug effects ; }, abstract = {Polydeoxyribonucleotide (PDRN), derived from chum salmon (Oncorhynchus keta), is a mixture of hydrolyzed DNA fragments used in various clinical applications. Its therapeutic value stems from its ability to promote wound healing by upregulating growth factors like VEGF, FGF, and HIF-1. However, PDRN's regenerative properties raise concerns about its potential misuse in sports. Studies suggest it may enhance athletic performance by stimulating muscle growth, recovery, and endurance through mechanisms such as satellite cell activation, angiogenesis, and anti-inflammatory effects. These potential performance-enhancing effects could be considered gene or cell doping, prohibited by the World Anti-Doping Agency (WADA). To address this concern, we developed a sensitive and specific detection method for PDRN misuse based on the CRISPR-Cas12a system. This method targets conserved 12S and 16S rDNA sequences unique to salmonids. A direct PCR method was optimized to amplify these target sequences from human plasma and urine without prior DNA extraction. The amplified DNA was then subjected to Cas12a-mediated detection, resulting in a fluorescent signal upon successful target recognition. This method demonstrated high sensitivity, detecting as little as 0.8 pg(0.3 genome copies) of O. keta DNA in 10 μL of biological samples within 90 min, surpassing the detection limits of many current doping agents.}, }
@article {pmid40215828, year = {2025}, author = {Miskel, D and Kurzella, J and Rings, F and Tholen, E and Tesfaye, D and Schellander, K and Salilew-Wondim, D and Held-Hoelker, E and Große-Brinkhaus, C and Hoelker, M}, title = {Functional COPA is indispensable for early embryo development beyond major genome activation in bovines.}, journal = {Theriogenology}, volume = {241}, number = {}, pages = {117415}, doi = {10.1016/j.theriogenology.2025.117415}, pmid = {40215828}, issn = {1879-3231}, mesh = {Animals ; *Embryonic Development/genetics/physiology ; Cattle/embryology/genetics ; CRISPR-Cas Systems ; Gene Expression Regulation, Developmental/physiology ; *Vesicular Transport Proteins/genetics/metabolism ; Blastocyst ; Female ; }, abstract = {Embryonic genome activation is divided into a minor and a major wave of transition to endogenous transcription. In bovines, minor genome activation begins early in the 2-cell stage and is completed by the 8-cell stage when major genome activation becomes dominant. While the activation of genes known to regulate early development have been studied extensively, genes involved in more central cellular functions have not been examined. Taking advantage of the CRISPR Cas9 system, the present study investigated the effect of knocking out the Golgi retrograde protein transporter COPA on early bovine development. After the electroporation of presumptive zygotes with Cas9 ribonucleoproteins targeting COPA exon 6, sequences of 2 (11 %) and 4-cell (16 %) embryos showed knockouts of COPA whereas 8-cell embryos and blastocysts did not, demonstrating that COPA is necessary for development to the 8-cell stage and beyond. Using a repair template containing silent mutations along the target site, COPA loss of wildtype was observed in 5 blastocysts, with successful knock-in of the template on at least one allele. This shows that an edited yet functional copy of COPA can save the developmental capacity of the embryo and demonstrates that Cas9 activity at the target region itself is not responsible for the loss of function. Together, the present study revealed that COPA is necessary for embryonic development, and that the timing of this necessity is before major genome activation onset. More generally, this study further demonstrates the utility of genome editing within reproductive biotechnology for the interrogation of gene function and early embryonic development.}, }
@article {pmid40215680, year = {2025}, author = {Ito, R and Nakano, T and Sugawara, A and Yokoyama, A}, title = {A CRISPR-based high-throughput screening system identifies bromodomain inhibitors as transcriptional suppressors of CYP11B1.}, journal = {Biochemical and biophysical research communications}, volume = {762}, number = {}, pages = {151779}, doi = {10.1016/j.bbrc.2025.151779}, pmid = {40215680}, issn = {1090-2104}, mesh = {*Steroid 11-beta-Hydroxylase/genetics/antagonists &amp; inhibitors/metabolism ; *High-Throughput Screening Assays/methods ; Humans ; *Triazoles/pharmacology ; *Azepines/pharmacology ; *CRISPR-Cas Systems ; *Transcription, Genetic/drug effects ; Cell Line ; Promoter Regions, Genetic/drug effects ; *Enzyme Inhibitors/pharmacology ; Epigenesis, Genetic/drug effects ; }, abstract = {CYP11B1 encodes steroid 11β-hydroxylase, the final rate-limiting enzyme for cortisol biosynthesis in the adrenal cortex. Excessive cortisol production is a hallmark of Cushing's disease (CD). While direct enzymatic inhibitors have been explored, achieving specificity remains a challenge due to the high homology between CYP11B1 and CYP11B2, highlighting transcriptional suppression of CYP11B1 as an alternative therapeutic strategy. To identify transcriptional regulators of CYP11B1, we generated genome-edited H295R adrenal cells carrying a luciferase reporter knocked into the endogenous CYP11B1 locus. Using this reporter cell line, we established a high-throughput screening (HTS) platform and screened a focused chemical library targeting epigenetic-related factors, given the importance of epigenetic mechanisms in gene regulation. Among eight candidate compounds identified, we focused on JQ1, a bromodomain inhibitor. JQ1 significantly suppressed Forskolin-induced CYP11B1 promoter activity and mRNA expression without causing cytotoxicity, suggesting the involvement of epigenetic readers in the transcriptional regulation of steroidogenic genes. Furthermore, the reporter-based HTS platform developed here, when combined with our previously established CYP11B2-luciferase system, may facilitate the identification of compounds that selectively modulate adrenal steroidogenic pathways. These findings provide a foundation for the development of novel transcription-targeted therapies for CD.}, }
@article {pmid40215333, year = {2025}, author = {Macias, LA and Lowther, J and Tillotson, EL and Rohde, E and Madsen, JA}, title = {Ion Mobility Gas-Phase Separation Enhances Top-Down Mass Spectrometry of Heavily Modified Guide RNA.}, journal = {Analytical chemistry}, volume = {97}, number = {17}, pages = {9430-9437}, pmid = {40215333}, issn = {1520-6882}, mesh = {*Mass Spectrometry/methods ; *RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/analysis ; Ion Mobility Spectrometry ; Gases/chemistry ; Phase Separation ; }, abstract = {As gene editing technologies enter the clinic, state-of-the-art characterization methods have been developed in parallel to assess the components of these paradigm-shifting medicines. One such component, the guide RNA (gRNA) element of CRISPR-based drugs, is a large synthetic heavily modified oligonucleotide that programs for the desired gene edit. Conventional oligonucleotide sequencing technologies can inform gRNA composition, but these methods may not completely capture the chemical modifications that are introduced during synthesis. Circumventing these challenges, mass spectrometry has demonstrated use in oligonucleotide analyses and has been combined here with ion mobility to deepen its characterization power. The use of ion mobility enabled us to perform gas-phase separation of the fragment ions produced by top-down mass spectrometry, yielding a significant increase in fragment identifications for a highly modified 100-mer gRNA by uncovering high-confidence assignments for heavily modified regions and for the important spacer region. Furthermore, the high-confidence fragment assignments empowered simultaneous de novo sequencing and chemical modification localization for the 5'-end spacer region as well as for 15 nucleotides on the heavily modified 3'-end. Overall, a total sequence coverage of 95% was achieved for the heavily modified 100-mer, ushering near complete sequence and chemical modification confirmation by top-down mass spectrometry.}, }
@article {pmid40214787, year = {2025}, author = {Liu, M and Yan, L and Lin, Z and Wu, D and Qiu, B and Weng, S}, title = {CHA-based microarray with Cas12a universal readout for multiple microRNA detection.}, journal = {Mikrochimica acta}, volume = {192}, number = {5}, pages = {293}, pmid = {40214787}, issn = {1436-5073}, support = {2022QH1217//Startup Fund for scientific research, Fujian Medical University/ ; }, mesh = {*MicroRNAs/genetics/analysis ; Humans ; CRISPR-Cas Systems ; *Oligonucleotide Array Sequence Analysis/methods ; *Endodeoxyribonucleases/metabolism/genetics ; Hirschsprung Disease/genetics/diagnosis ; *Biosensing Techniques/methods ; *CRISPR-Associated Proteins/metabolism/genetics ; Limit of Detection ; Bacterial Proteins ; }, abstract = {Hirschsprung's disease (HSCR), a congenital condition characterized by the absence of nerve cells in the intestinal wall, often requires early and accurate diagnosis for optimal patient outcomes. In this study, we developed a novel and ultrasensitive biosensing strategy for the detection of HSCR-related microRNAs (miRNAs) by integrating catalytic hairpin assembly (CHA) with CRISPR-Cas12a technology. A two-stage process consists of array recognition, and a universal readout is designed. In the first stage, target miRNAs are recognized and amplified on a solid-phase microarray, while in the second stage, the accumulated conversion chains which are not related to target sequences, activate Cas12a, leading to the cleavage of reporter DNA and the generation of a fluorescence signal spatially separated from the first stage. The proposed method was validated for the comprehensive detection of HSCR-related miRNAs and demonstrated high sensitivity and specificity. This work represents a significant advancement in miRNA diagnostics and holds potential for broader clinical applications.}, }
@article {pmid40214496, year = {2025}, author = {Parikh, SJ and Terron, HM and Burgard, LA and Maranan, DS and Butler, DD and Wiseman, A and LaFerla, FM and Lane, S and Leissring, MA}, title = {Targeted Control of Gene Expression Using CRISPR-Associated Endoribonucleases.}, journal = {Cells}, volume = {14}, number = {7}, pages = {}, pmid = {40214496}, issn = {2073-4409}, support = {R01 AG066928/AG/NIA NIH HHS/United States ; 1R01AG066928-04A1/NH/NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Endoribonucleases/metabolism/genetics ; *Gene Expression Regulation ; *CRISPR-Associated Proteins/metabolism/genetics ; HEK293 Cells ; Gene Editing/methods ; }, abstract = {CRISPR-associated endoribonucleases (Cas RNases) cleave single-stranded RNA in a highly sequence-specific manner by recognizing and binding to short RNA sequences known as direct repeats (DRs). Here, we investigate the potential of exploiting Cas RNases for the regulation of target genes with one or more DRs introduced into the 3' untranslated region, an approach we refer to as DREDGE (direct repeat-enabled downregulation of gene expression). The DNase-dead version of Cas12a (dCas12a) was identified as the most efficient among five different Cas RNases tested and was subsequently evaluated in doxycycline-regulatable systems targeting either stably expressed fluorescent proteins or an endogenous gene. DREDGE performed superbly in stable cell lines, resulting in up to 90% downregulation with rapid onset, notably in a fully reversible and highly selective manner. Successful control of an endogenous gene with DREDGE was demonstrated in two formats, including one wherein both the DR and the transgene driving expression of dCas12a were introduced in one step by CRISPR-Cas. Our results establish DREDGE as an effective method for regulating gene expression in a targeted, highly selective, and fully reversible manner, with several advantages over existing technologies.}, }
@article {pmid40214251, year = {2025}, author = {Mao, S and Yang, X and Wang, Y and Chen, F and Jiang, H and Wang, Y and Kang, Y and Li, S}, title = {Ultrasensitive and highly specific detection of the Brucella genus and B. melitensis by CRISPR/Cas12b-multiple cross displacement amplification technique.}, journal = {Journal of clinical microbiology}, volume = {63}, number = {5}, pages = {e0153224}, pmid = {40214251}, issn = {1098-660X}, support = {82273758//National Natural Science Foundation of China/ ; gzwkj2024-055//Health Commission of Guizhou Province/ ; Guo Jikong Zong Ren Han [2024]122//National Disease Control and Prevention Administration/ ; QJJ [2022]019//Foundation of Key Laboratory of Microbiology and Parasitology of Education Department of Guizhou/ ; }, mesh = {*Brucellosis/diagnosis/microbiology ; Sensitivity and Specificity ; *Brucella/genetics/isolation &amp; purification/classification ; *Brucella melitensis/genetics/isolation &amp; purification ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *CRISPR-Cas Systems ; Animals ; *Molecular Diagnostic Techniques/methods ; DNA Primers/genetics ; }, abstract = {Brucellosis is caused by members of the Brucella spp. and remains one of the world's major zoonotic diseases. Brucella melitensis (B. melitensis) as the most contagious Brucella species cannot be ignored as an essential source of infection for brucellosis, especially in countries/regions dominated by animal husbandry. Thus, the identification of the Brucella genus and B. melitensis is crucial for rapid diagnosis of brucellosis to control disease transmission and clinical treatment. Here, we developed the CRISPR/Cas12b nuclease combined with a multiple cross displacement amplification (MCDA) assay (CRISPR-MCDA) for highly specific and sensitive detection of Brucella genus and B. melitensis in clinical applications. Two sets of specific primers were designed targeting the specific gene of Brucella genus (Bcsp31) and B. melitensis (BMEII0466), respectively. The CRISPR-MCDA assay showed high specificity and sensitivity in 28 non-Brucella isolates and 64 clinical samples. The detection limit of CRISPR-MCDA assay was 2 copies/μL in the plasmid dilution template, and the whole detection process took within 90 minutes with nanoparticle-based lateral flow biosensor (LFB) to validate experimental results. Taken together, the CRISPR-MCDA-LFB assay is a visual, sensitive, and highly specific detection technique that can be used as an attractive potential identification tool for Brucella genus and B. melitensis.IMPORTANCEThe prevention and control of Brucellosis urgently require rapid and accurate diagnostic methods. This work validates a new method for the simultaneous detection of Brucella genus and B. melitensis. The method can effectively reduce the chances of contamination and provides a more rapid, sensitive, and specific on-site detection of Brucella. It also offers a solution for the rapid screening of Brucellosis in resource-limited environments, which is crucial for effective disease prevention and control. This technology can also be widely applied to the rapid detection of other pathogens beyond Brucella.}, }
@article {pmid40213940, year = {2025}, author = {Hou, Z and Yi, Q and Wu, M and Wu, L and Li, F and Wang, T and Bian, P}, title = {Assessment and Mitigation of CRISPR-Cas9-Induced Nontargeted Translocations.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {12}, number = {21}, pages = {e2414415}, pmid = {40213940}, issn = {2198-3844}, support = {12135016//National Natural Science Foundation of China/ ; 12075275//National Natural Science Foundation of China/ ; 12475335//National Natural Science Foundation of China/ ; HYZHXMH02002//Space Medical Experiment Project of China Manned Space Program/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Translocation, Genetic/genetics ; Escherichia coli/genetics ; }, abstract = {The performance of CRISPR-mediated genome editing near inverted repeats (IRs) potentially results in chromosomal translocations and other catastrophic rearrangements. However, the extent of this risk may be significantly underestimated because current reporter systems focus solely on site-specific translocations. Here, trans-acting reporter systems in Escherichia coli are developed to detect nontargeted translocations. Markedly increased frequency of translocations following CRISPR-Cas9 activation is observed, with the magnitude determined primarily by the length of the IRs and the proximity between Cas9 target sites and IRs. These translocations arise through a combination of intramolecular single-strand annealing and alternative end-joining mechanisms. Furthermore, it is discovered that introducing segments homologous to IR loci can substantially mitigate nontargeted translocations without significantly compromising CRISPR-Cas9-mediated editing. The study provides valuable insights into the genetic risks associated with CRISPR technologies and suggests a viable strategy for developing genetically safer CRISPR systems.}, }
@article {pmid40213544, year = {2025}, author = {Sánchez Hernández, S and Bjerg, TW and Nielsen, IH and Laustsen, A and Q Tang, H and Pedersen, LH and Klechevsky, E and Jakobsen, MR and Bak, RO}, title = {Characterization of TLR9 responsiveness in cell subsets derived from in vitro pDC differentiation of hematopoietic stem and progenitor cells.}, journal = {Frontiers in immunology}, volume = {16}, number = {}, pages = {1550397}, pmid = {40213544}, issn = {1664-3224}, mesh = {*Cell Differentiation/immunology ; *Hematopoietic Stem Cells/immunology/cytology/metabolism ; Humans ; *Toll-Like Receptor 9/metabolism/immunology ; *Dendritic Cells/immunology/cytology/metabolism ; Cells, Cultured ; Interferon-alpha/metabolism ; Transcriptome ; }, abstract = {Plasmacytoid dendritic cells (pDCs) are multifunctional immune cells with roles in both the innate and adaptive immune system. Their hallmark function is production of large amounts of type I interferons in response to viral infections, but they are also capable of producing a range of other cytokines, antigen presentation, and cytotoxicity. Their potential as an immunotherapy for cancer and infectious disease is being explored, but broad application of these cells is challenged by low frequency in the blood and low viability during ex vivo culturing. We have previously developed an effective in vitro differentiation protocol for producing pDCs from CD34+ hematopoietic stem and progenitor cells (HSPC-pDCs), which provides an attainable and large source of pDCs. HSPC-pDCs present pDC characteristics and functions, and like naturally occurring pDCs they exhibit large phenotypic and functional heterogeneity. Here, we characterize different cell subsets from in vitro pDC differentiation and identify a distinct population, which is the major producer of IFNα in response to TLR9 stimulation and display a transcriptomic profile similar to what is seen for pDCs circulating in the blood. We also investigate the possibility of rerouting subset specification during HSPCs-to-pDC differentiation by controlling gene expression of key master transcription factors (TFs). We identify TFs associated with the pDC differentiation trajectory that are essential for the development of TLR9-responsive HSPC-pDCs, and we also identify TFs that increase their frequency. In conclusion, we phenotypically and functionally characterize different cell subsets and modulate their relative frequencies by manipulating TF expression during pDC differentiation. These findings provide a deeper understanding of in vitro-differentiated pDC cultures that may spur further developments in their use as an immunomodulatory cell therapy.}, }
@article {pmid40213198, year = {2025}, author = {Khan, A and Barapatre, AR and Babar, N and Doshi, J and Ghaly, M and Patel, KG and Nawaz, S and Hasana, U and Khatri, SP and Pathange, S and Pesaru, AR and Puvvada, CS and Billoo, M and Jamil, U}, title = {Genomic medicine and personalized treatment: a narrative review.}, journal = {Annals of medicine and surgery (2012)}, volume = {87}, number = {3}, pages = {1406-1414}, pmid = {40213198}, issn = {2049-0801}, abstract = {Genomic medicine, which integrates genomics and bioinformatics into clinical care and diagnostics, is transforming healthcare by enabling personalized treatment approaches. Advances in technologies such as DNA sequencing, proteomics, and computational power have laid the foundation for individualized therapies that account for genetic variations influencing disease risk, progression, and treatment response. This review explores the historical milestones leading to current applications of genomic medicine, such as targeted therapies, gene therapies, and precision medicine, in fields including cardiovascular diseases, oncology, and rare genetic disorders. It highlights the use of next-generation sequencing and third-generation sequencing to improve diagnostic accuracy and treatment outcomes, emphasizing the role of genomic data in advancing personalized treatments. Furthermore, emerging therapies such as CRISPR/Cas-based genome editing and adeno-associated viral vectors showcase the potential of gene therapy in addressing complex diseases, including rare genetic disorders. Despite promising advancements, challenges remain in fully integrating genomic medicine into routine clinical practice, including cost barriers, data interpretation complexities, and the need for widespread genomic literacy among healthcare professionals. The future of genomic medicine holds transformative potential for revolutionizing the diagnosis, treatment, and management of both common and rare diseases.}, }
@article {pmid40212051, year = {2025}, author = {Liyanage, W and Kannan, G and Kannan, S and Kannan, RM}, title = {Efficient Intracellular Delivery of CRISPR-Cas9 Ribonucleoproteins Using Dendrimer Nanoparticles for Robust Genomic Editing.}, journal = {Nano today}, volume = {61}, number = {}, pages = {}, pmid = {40212051}, issn = {1748-0132}, support = {P30 EY001765/EY/NEI NIH HHS/United States ; R01 EY025304/EY/NEI NIH HHS/United States ; R01 NS093416/NS/NINDS NIH HHS/United States ; }, abstract = {CRISPR-Cas9, a flexible and efficient genome editing technology, is currently limited by the challenge of delivering the large ribonucleoprotein complex intracellularly and into the nucleus. Existing delivery techniques/vectors are limited by their toxicity, immunogenicity, scalability, and lack of specific cell-targeting ability. This study presents a neutral, non-toxic dendrimer conjugate construct that shows promise in overcoming these limitations. We covalently-conjugated S. pyogenes Cas9-2NLS (Cas9-nuclear localization sequence) endonuclease to a hydroxyl PAMAM dendrimer through a glutathione-sensitive disulfide linker via highly specific inverse Diels-alder click reaction (IEDDA), and a single guide RNA (sgRNA) was complexed to the Cas9-dendrimer conjugate nano-construct (D-Cas9). D-Cas9- RNP produces robust genomic deletion in vitro of GFP in HEK293 cells (~100%) and VEGF in a human pigmental epithelium cell line (ARPE-19) (20%). The uptake of the D-Cas9-RNP constructs on similar timescales as small molecules highlights the robustness of the biophysical mechanisms enabling the dendrimer to deliver payloads as large as Cas9, while retaining payload functionality. This promising conjugation approach enabled better stability to the neutral construct. Combined with recent advances in hydroxyl dendrimer delivery technologies in the clinic, this approach may lead to advances in 'neutral' dendrimer-enabled non-toxic, cell-specific, highly efficient in vitro and in vivo genome editing.}, }
@article {pmid40211002, year = {2025}, author = {Cheng, Y and Hu, M and Yang, B and Jensen, TB and Zhang, Y and Yang, T and Yu, R and Ma, Z and Radda, JSD and Jin, S and Zang, C and Wang, S}, title = {Perturb-tracing enables high-content screening of multi-scale 3D genome regulators.}, journal = {Nature methods}, volume = {22}, number = {5}, pages = {950-961}, pmid = {40211002}, issn = {1548-7105}, support = {UH3 CA268202/CA/NCI NIH HHS/United States ; R35 GM133712/GM/NIGMS NIH HHS/United States ; 2T32GM007499//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01 HG012969/HG/NHGRI NIH HHS/United States ; R01 HG013503/HG/NHGRI NIH HHS/United States ; T32 GM007499/GM/NIGMS NIH HHS/United States ; 5T32GM007205//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; P50 CA196530/CA/NCI NIH HHS/United States ; U01 CA260701/CA/NCI NIH HHS/United States ; DP2GM137414//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01 CA292936/CA/NCI NIH HHS/United States ; DP2 GM137414/GM/NIGMS NIH HHS/United States ; T32 GM007205/GM/NIGMS NIH HHS/United States ; R33 CA251037/CA/NCI NIH HHS/United States ; R01 HG011245/HG/NHGRI NIH HHS/United States ; R01HG013503//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R35GM133712//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; }, mesh = {Humans ; *Chromatin/genetics/chemistry/metabolism ; *Genome, Human ; CRISPR-Cas Systems ; In Situ Hybridization, Fluorescence/methods ; }, abstract = {Three-dimensional (3D) genome organization becomes altered during development, aging and disease, but the factors regulating chromatin topology are incompletely understood and currently no technology can efficiently screen for new regulators of multi-scale chromatin organization. Here, we developed an image-based high-content screening platform (Perturb-tracing) that combines pooled CRISPR screens, a cellular barcode readout method (BARC-FISH) and chromatin tracing. We performed a loss-of-function screen in human cells, and visualized alterations to their 3D chromatin folding conformations, alongside perturbation-paired barcode readout in the same single cells. We discovered tens of new regulators of chromatin folding at different length scales, ranging from chromatin domains and compartments to chromosome territory. A subset of the regulators exhibited 3D genome effects associated with loop extrusion and A-B compartmentalization mechanisms, while others were largely unrelated to these known 3D genome mechanisms. Finally, we identified new regulators of nuclear architectures and found a functional link between chromatin compaction and nuclear shape. Altogether, our method enables scalable, high-content identification of chromatin and nuclear topology regulators that will stimulate new insights into the 3D genome.}, }
@article {pmid40210885, year = {2025}, author = {Tian, M and Wu, D and Gou, X and Li, R and Zhang, X}, title = {Genetic modulation of rare earth nanoparticle biotransformation shapes biological outcomes.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {3429}, pmid = {40210885}, issn = {2041-1723}, mesh = {Animals ; Biotransformation/genetics ; *Metals, Rare Earth/metabolism/chemistry ; Lysosomes/metabolism ; Humans ; *Nanoparticles/metabolism/chemistry ; CRISPR-Cas Systems ; *Metal Nanoparticles/chemistry ; Pyroptosis ; Sea Urchins/metabolism ; Mice ; }, abstract = {The biotransformation of nanoparticles plays a crucial role in determining their biological fate and responses. Although a few engineering strategies (e.g., surface functionalization and shape control) have been employed to regulate the fate of nanoparticles, the genetic control of nanoparticle biotransformation remains an unexplored avenue. Herein, we utilized a CRISPR-based genome-scale knockout approach to identify genes involved in the biotransformation of rare earth oxide (REO) nanoparticles. We found that the biotransformation of REOs in lysosomes could be genetically controlled via SMPD1. Specifically, suppression of SMPD1 inhibited the transformation of La2O3 into sea urchin-shaped structures, thereby protecting against lysosomal damage, proinflammatory cytokine release, pyroptosis and RE-induced pneumoconiosis. Overall, our study provides insight into how to control the biological fate of nanomaterials.}, }
@article {pmid40210680, year = {2025}, author = {Koh, GCC and Nanda, AS and Rinaldi, G and Boushaki, S and Degasperi, A and Badja, C and Pregnall, AM and Zhao, SJ and Chmelova, L and Black, D and Heskin, L and Dias, J and Young, J and Memari, Y and Shooter, S and Czarnecki, J and Brown, MA and Davies, HR and Zou, X and Nik-Zainal, S}, title = {A redefined InDel taxonomy provides insights into mutational signatures.}, journal = {Nature genetics}, volume = {57}, number = {5}, pages = {1132-1141}, pmid = {40210680}, issn = {1546-1718}, support = {C60100/A25274//Cancer Research UK (CRUK)/ ; NIHR301627//DH | National Institute for Health Research (NIHR)/ ; Award 2019//Dr. Josef Steiner Krebsstiftung (Dr. Josef Steiner Cancer Research Foundation)/ ; CGCATF-2021/100013//Cancer Research UK (CRUK)/ ; C60100/A23433//Cancer Research UK (CRUK)/ ; C60100/A27815//Cancer Research UK (CRUK)/ ; /WT_/Wellcome Trust/United Kingdom ; BRC-1215-20014//DH | National Institute for Health Research (NIHR)/ ; }, mesh = {Humans ; *INDEL Mutation/genetics ; DNA Mismatch Repair/genetics ; *Neoplasms/genetics ; DNA Polymerase II/genetics ; DNA Polymerase III/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; Cell Line, Tumor ; }, abstract = {Despite their deleterious effects, small insertions and deletions (InDels) have received far less attention than substitutions. Here we generated isogenic CRISPR-edited human cellular models of postreplicative repair dysfunction (PRRd), including individual and combined gene edits of DNA mismatch repair (MMR) and replicative polymerases (Pol ε and Pol δ). Unique, diverse InDel mutational footprints were revealed. However, the prevailing InDel classification framework was unable to discriminate these InDel signatures from background mutagenesis and from each other. To address this, we developed an alternative InDel classification system that considers flanking sequences and informative motifs (for example, longer homopolymers), enabling unambiguous InDel classification into 89 subtypes. Through focused characterization of seven tumor types from the 100,000 Genomes Project, we uncovered 37 InDel signatures; 27 were new. In addition to unveiling previously hidden biological insights, we also developed PRRDetect-a highly specific classifier of PRRd status in tumors, with potential implications for immunotherapies.}, }
@article {pmid40210053, year = {2025}, author = {Bai, J and Lu, H and Xue, Y and Li, M and Fang, S and Cui, L and He, Y and Wang, Y and Li, H and He, F and Zhao, B and Ai, L and Wang, S}, title = {Deletion of the DEAD-box helicase CshA in Lacticaseibacillus paracasei elicits heightened low-temperature sensitivity and reduced post-acidification capacity.}, journal = {International journal of biological macromolecules}, volume = {309}, number = {Pt 4}, pages = {142970}, doi = {10.1016/j.ijbiomac.2025.142970}, pmid = {40210053}, issn = {1879-0003}, mesh = {*Cold Temperature ; *DEAD-box RNA Helicases/genetics/metabolism ; *Gene Deletion ; *Bacterial Proteins/genetics/metabolism ; Hydrogen-Ion Concentration ; Fermentation ; CRISPR-Cas Systems ; Animals ; }, abstract = {While cold chain logistics remains the primary strategy to mitigate post acidification in fermented dairy products, maintaining reliable refrigeration systems presents substantial challenges, especially in resource-limited settings. Biological approaches targeting strain low-temperature sensitivity modulation for post-acidification control are increasingly recognized as promising alternatives. This study utilized CRISPR/Cas9-mediated knockout of DEAD-box helicase genes in Lacticaseibacillus paracasei, successfully identifying a low-temperature-sensitive strain, ∆AF91_12,710. Under optimal conditions, ∆AF91_12,710 showed comparable growth patterns and pH profiles to the wild-type strain. However, under low-temperature conditions, ∆AF91_12,710 demonstrated significantly impaired growth capacity and distinct pH regulation patterns. Metabolomic analysis of post-acidification revealed 39 unique differential metabolites in ∆AF91_12,710 fermented milk over 21 days, featuring decreased ATP, γ-linolenic acid, and dGMP concentrations, alongside elevated glucose-6-phosphate, cAMP, and D-galactose levels. These metabolic alterations indicate impaired glycolysis, likely resulting from DEAD-box helicase gene deletion-induced RNA instability and reduced metabolic activity in ∆AF91_12,710 under cold stress. The AF91_12,710 deletion significantly enhanced low-temperature sensitivity while reducing post-acidification capability in fermented milk. This research provides valuable insights into post-acidification attenuation in low-temperature-sensitive strains and demonstrates the potential for developing robust starter cultures in dairy applications.}, }
@article {pmid40209969, year = {2025}, author = {Dündar, T and Köksal Çakırlar, F}, title = {Antimicrobial resistance in coagulase negative staphylococci: Genome analysis and role of horizontal gene transfer.}, journal = {Research in microbiology}, volume = {}, number = {}, pages = {104298}, doi = {10.1016/j.resmic.2025.104298}, pmid = {40209969}, issn = {1769-7123}, abstract = {Coagulase-negative staphylococci (CNS) are emerging as significant contributors to antimicrobial resistance, yet their genomic characteristics remain incompletely understood. This study presents a whole-genome analysis of 12 multidrug-resistant CNS strains (Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis) isolated from blood cultures, focusing on antimicrobial resistance genes, mobile genetic elements (MGEs), and horizontal gene transfer (HGT) mechanisms. We identified 22 resistance genes conferring resistance to 11 antimicrobial classes, many of which were plasmid-associated. Notably, we report the first detection of the ISSha1 insertion sequence in S. hominis, along with novel resistance plasmids, including pGO1 and VRSAp in S. haemolyticus and pAMα1 in S. hominis. The identification of bacteriophage-derived sequences in S. haemolyticus and S. hominis suggests a role for phages in genetic exchange. CRISPR sequences and a Cas gene were detected in S. hominis, suggesting a potential but unconfirmed role in restricting gene transfer. Additionally, pGO1 was identified as a conjugative plasmid, while pAMα1 and VRSAp were determined to be mobilizable, reinforcing the role of CNS in resistance dissemination. These results highlight CNS as reservoirs of antimicrobial resistance genes and emphasize the importance of species-specific genomic surveillance. Proactive monitoring of CNS is crucial for controlling antimicrobial resistance in clinical settings.}, }
@article {pmid40209705, year = {2025}, author = {Geilenkeuser, J and Armbrust, N and Steinmaßl, E and Du, SW and Schmidt, S and Binder, EMH and Li, Y and Warsing, NW and Wendel, SV and von der Linde, F and Schiele, EM and Niu, X and Stroppel, L and Berezin, O and Santl, TH and Orschmann, T and Nelson, K and Gruber, C and Palczewska, G and Menezes, CR and Risaliti, E and Engfer, ZJ and Koleci, N and Schmidts, A and Geerlof, A and Palczewski, K and Westmeyer, GG and Truong, DJ}, title = {Engineered nucleocytosolic vehicles for loading of programmable editors.}, journal = {Cell}, volume = {188}, number = {10}, pages = {2637-2655.e31}, doi = {10.1016/j.cell.2025.03.015}, pmid = {40209705}, issn = {1097-4172}, mesh = {*Gene Editing/methods ; Animals ; Mice ; Humans ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Ribonucleoproteins/metabolism ; T-Lymphocytes/metabolism ; HEK293 Cells ; }, abstract = {Advanced gene editing methods have accelerated biomedical discovery and hold great therapeutic promise, but safe and efficient delivery of gene editors remains challenging. In this study, we present a virus-like particle (VLP) system featuring nucleocytosolic shuttling vehicles that retrieve pre-assembled Cas-effectors via aptamer-tagged guide RNAs. This approach ensures preferential loading of fully assembled editor ribonucleoproteins (RNPs) and enhances the efficacy of prime editing, base editing, trans-activators, and nuclease activity coupled to homology-directed repair in multiple immortalized, primary, stem cell, and stem-cell-derived cell types. We also achieve additional protection of inherently unstable prime editing guide RNAs (pegRNAs) by shielding the 3'-exposed end with Csy4/Cas6f, further enhancing editing performance. Furthermore, we identify a minimal set of packaging and budding modules that can serve as a platform for bottom-up engineering of enveloped delivery vehicles. Notably, our system demonstrates superior per-VLP editing efficiency in primary T lymphocytes and two mouse models of inherited retinal disease, highlighting its therapeutic potential.}, }
@article {pmid40209498, year = {2025}, author = {Cen, C and Liu, X and He, W and Tan, X and Li, G and Jintong, N}, title = {Novel approaches in CRISPR/Cas12a-based sensing for HCC diagnosis - A review (2020-2025).}, journal = {Journal of pharmaceutical and biomedical analysis}, volume = {262}, number = {}, pages = {116878}, doi = {10.1016/j.jpba.2025.116878}, pmid = {40209498}, issn = {1873-264X}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Carcinoma, Hepatocellular/diagnosis/genetics ; *Liver Neoplasms/diagnosis/genetics ; *Biosensing Techniques/methods ; Biomarkers, Tumor/genetics ; Early Detection of Cancer/methods ; Sensitivity and Specificity ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Early diagnosis of hepatocellular carcinoma (HCC) is crucial for improving patient survival and treatment outcomes and the early detection of biomarkers for HCC is key to achieving this goal. However, conventional detection methods often lack sufficient specificity and sensitivity. In recent years, CRISPR/Cas12a-based biosensing has gained significant attention due to its ease of use and high sensitivity, demonstrating its potential to address the limitations of conventional detection methods. This paper primarily reviews the research progress of CRISPR/Cas12a-based biosensors for HCC detection, introducing their fluorescence, electrochemical, colorimetric, and other detection principles, as well as practical applications in detail. Additionally, the differences in sensitivity, specificity, and detection speed among different types of CRISPR/Cas12a biosensors are comparatively analyzed. Finally, the potential future directions for the development and application of CRISPR/Cas12a technology in clinical settings are explored.}, }
@article {pmid40209323, year = {2025}, author = {Zhang, Y and Lu, Z and Yang, H and Cheng, L and Zaklyazminskaya, E and Sokolova, O and Tan, H and Zhang, JZ}, title = {Generation of stable Cas9-EGFP expressing human induced pluripotent stem cell lines based on SeLection by Essential-gene Exon Knock-in technology.}, journal = {Stem cell research}, volume = {85}, number = {}, pages = {103710}, doi = {10.1016/j.scr.2025.103710}, pmid = {40209323}, issn = {1876-7753}, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Exons/genetics ; *Gene Knock-In Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Green Fluorescent Proteins/metabolism/genetics ; Cell Line ; Gene Editing ; *Genes, Essential ; Cell Differentiation ; *CRISPR-Associated Protein 9/metabolism/genetics ; }, abstract = {Here, we used SeLection by Essential-gene Exon Knock-in technology to generate the iPSC line with constitutive expression of Cas9-EGFP, while retaining all functions of the essential gene. Cas9-EGFP was inserted into the GAPDH exon9 via the homologous recombination, avoiding Cas9 silencing that often occurs during iPSC differentiation. The edited cell line shows precise knock-in locus with the typical characteristics and pluripotency of iPSCs. Therefore, this iPSC line is valuable for CRISPR screening or related experiments and could be widely used in the CRISPR/Cas9-based gene editing.}, }
@article {pmid40208959, year = {2025}, author = {Baca, CF and Majumder, P and Hickling, JH and Patel, DJ and Marraffini, LA}, title = {Cat1 forms filament networks to degrade NAD[+] during the type III CRISPR-Cas antiviral response.}, journal = {Science (New York, N.Y.)}, volume = {388}, number = {6752}, pages = {eadv9045}, pmid = {40208959}, issn = {1095-9203}, support = {T32 GM136640/GM/NIGMS NIH HHS/United States ; R01 GM129430/GM/NIGMS NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 AI141507/AI/NIAID NIH HHS/United States ; U24 GM129539/GM/NIGMS NIH HHS/United States ; R01 GM149834/GM/NIGMS NIH HHS/United States ; R01 GM145888/GM/NIGMS NIH HHS/United States ; }, mesh = {Catalytic Domain ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; *CRISPR-Cas Systems ; *NAD/metabolism ; Protein Multimerization ; *Chloroflexi/enzymology ; *Bacterial Proteins/chemistry/genetics/metabolism ; }, abstract = {Type III CRISPR-Cas systems defend against viral infection in prokaryotes by using an RNA-guided complex that recognizes foreign transcripts and synthesizes cyclic oligoadenylate (cOA) messengers to activate CRISPR-associated Rossmann-fold (CARF) immune effectors. In this study, we investigated a protein containing a CARF domain-fused Toll/interleukin-1 receptor (TIR) domain, Cat1. We found that Cat1 provides immunity by cleaving and depleting oxidized nicotinamide adenine dinucleotide (NAD[+]) molecules from the infected host, inducing a growth arrest that prevents viral propagation. Cat1 forms dimers that stack upon each other to generate long filaments that are maintained by bound cOA ligands, with stacked TIR domains forming the NAD[+] cleavage catalytic sites. Furthermore, Cat1 filaments assemble into distinct trigonal and pentagonal networks that enhance NAD[+] degradation. Cat1 presents an unprecedented chemistry and higher-order protein assembly for the CRISPR-Cas response.}, }
@article {pmid40207628, year = {2025}, author = {Yuan, K and Xi, X and Han, S and Han, J and Zhao, B and Wei, Q and Zhou, X}, title = {Selict-seq profiles genome-wide off-target effects in adenosine base editing.}, journal = {Nucleic acids research}, volume = {53}, number = {7}, pages = {}, pmid = {40207628}, issn = {1362-4962}, support = {2023YFC3402200//National Key R&amp;D Program of China/ ; 92153303//National Natural Science Foundation of China/ ; }, mesh = {*Gene Editing/methods ; Humans ; *Adenosine/genetics/metabolism ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Inosine/genetics ; Genome, Human ; Anemia, Sickle Cell/genetics ; Mutation ; }, abstract = {Adenosine base editors (ABEs) facilitate A·T to G·C base pair conversion with significant therapeutic potential for correcting pathogenic point mutations in human genetic diseases, such as sickle cell anemia and β-thalassemia. Unlike CRISPR-Cas9 systems that induce double-strand breaks, ABEs operate through precise deamination, avoiding chromosomal instability. However, the off-target editing effects of ABEs remain inadequately characterized. In this study, we present a biochemical method Selict-seq, designed to evaluate genome-wide off-target editing by ABEs. Selict-seq specifically captures deoxyinosine-containing single-stranded DNA and precisely identifies deoxyadenosine-to-deoxyinosine (dA-to-dI) mutation sites, elucidating the off-target effects induced by ABEs. Through investigations involving three single-guide RNAs, we identified numerous unexpected off-target edits both within and outside the protospacer regions. Notably, ABE8e(V106W) exhibited distinct off-target characteristics, including high editing rates (>10%) at previously unreported sites (e.g. RNF2 and EMX1) and out-of-protospacer mutations. These findings significantly advance our understanding of the off-target landscape associated with ABEs. In summary, our approach enables an unbiased analysis of the ABE editome and provides a widely applicable tool for specificity evaluation of various emerging genome editing technologies that produce intermediate products as deoxyinosine.}, }
@article {pmid40207549, year = {2025}, author = {Bu, Y and Wang, R and Liu, Y and Xing, K and Zhang, X and Sun, Y and Zhang, J}, title = {CRISPR/Cas9-mediated myostatin disruption elevates the expression of genes associated with myofiber composition and growth in Exopalaemon carinicauda.}, journal = {The Journal of experimental biology}, volume = {228}, number = {10}, pages = {}, doi = {10.1242/jeb.250148}, pmid = {40207549}, issn = {1477-9145}, support = {32373121//Innovative Research Group Project of the National Natural Science Foundation of China/ ; 22323201D//Hebei Provincial Key Research Projects/ ; HBU2023BS027//Hebei Province Graduate Innovation Funding Project/ ; D2023201002//Steel and Iron Foundation of Hebei Province/ ; 32373121//National Natural Science Foundation of China/ ; 32172954//National Natural Science Foundation of China/ ; 22323201D//Key Research and Development Project of Hebei Province/ ; HBU2023BS027//Post-graduate Innovation Fund Project of Hebei University/ ; D2023201002//Natural Science Foundation of Hebei Province/ ; }, mesh = {Animals ; *Myostatin/genetics/metabolism ; *CRISPR-Cas Systems ; *Palaemonidae/genetics/growth &amp; development/metabolism ; *Arthropod Proteins/genetics/metabolism ; Gene Editing ; }, abstract = {Myostatin (MSTN) is a negative regulator of skeletal muscle development and growth in vertebrates, but its role in crustaceans remains debated. To explore the functional role of MSTN in Exopalaemon carinicauda (EcMSTN) and to facilitate the development of new strains with enhanced growth rates, we investigated the molecular characteristics, expression patterns and functional implications of EcMSTN. We employed CRISPR/Cas9-mediated gene editing technology to generate EcMSTN knockout (EcMSTN-KO) prawns and subsequently monitored their hatching rate, survival rate and growth performance. The findings revealed that the hatching rate in the EcMSTN-KO group was only 11%, significantly lower than the 50% in the control group (P<0.05). In comparison to their wild-type (WT) siblings (1.212±0.114 cm), the EcMSTN-KO prawns (1.481±0.192) demonstrated a markedly enhanced body length (P<0.001). The expression of genes associated with myofiber composition and growth, including myosin heavy chain 2 (EcMHC2) and myosin light chain 1 (EcMLC1), exhibited a highly significant increase (P<0.001) in EcMSTN-KO prawns. Additionally, the expression of ecdysone receptor (EcEcR), a molt-related gene, was significantly elevated (P<0.001), while the expression of retinoid X receptor (EcRXR) showed no significant difference (P>0.05). The above studies indicate that EcMSTN functions as a negative regulator of muscle growth in E. carinicauda. Moreover, EcMSTN may play a role in molting. These results underscore the significant potential of MSTN as a genetic target for improving crustacean aquaculture, particularly through gene editing technologies aimed at enhancing growth traits.}, }
@article {pmid40207041, year = {2025}, author = {Hoffmann, S and Seeger, T}, title = {Advances in human induced pluripotent stem cell (hiPSC)-based disease modelling in cardiogenetics.}, journal = {Medizinische Genetik : Mitteilungsblatt des Berufsverbandes Medizinische Genetik e.V}, volume = {37}, number = {2}, pages = {137-146}, pmid = {40207041}, issn = {1863-5490}, abstract = {Human induced pluripotent stem cell (hiPSC)-based disease modelling has significantly advanced the field of cardiogenetics, providing a precise, patient-specific platform for studying genetic causes of heart diseases. Coupled with genome editing technologies such as CRISPR/Cas, hiPSC-based models not only allow the creation of isogenic lines to study mutation-specific cardiac phenotypes, but also enable the targeted modulation of gene expression to explore the effects of genetic and epigenetic deficits at the cellular and molecular level. hiPSC-based models of heart disease range from two-dimensional cultures of hiPSC-derived cardiovascular cell types, such as various cardiomyocyte subtypes, endothelial cells, pericytes, vascular smooth muscle cells, cardiac fibroblasts, immune cells, etc., to cardiac tissue cultures including organoids, microtissues, engineered heart tissues, and microphysiological systems. These models are further enhanced by multi-omics approaches, integrating genomic, transcriptomic, epigenomic, proteomic, and metabolomic data to provide a comprehensive view of disease mechanisms. In particular, advances in cardiovascular tissue engineering enable the development of more physiologically relevant systems that recapitulate native heart architecture and function, allowing for more accurate modelling of cardiac disease, drug screening, and toxicity testing, with the overall goal of personalised medical approaches, where therapies can be tailored to individual genetic profiles. Despite significant progress, challenges remain in the maturation of hiPSC-derived cardiomyocytes and the complexity of reproducing adult heart conditions. Here, we provide a concise update on the most advanced methods of hiPSC-based disease modelling in cardiogenetics, with a focus on genome editing and cardiac tissue engineering.}, }
@article {pmid40205037, year = {2025}, author = {Fielden, J and Siegner, SM and Gallagher, DN and Schröder, MS and Dello Stritto, MR and Lam, S and Kobel, L and Schlapansky, MF and Jackson, SP and Cejka, P and Jost, M and Corn, JE}, title = {Comprehensive interrogation of synthetic lethality in the DNA damage response.}, journal = {Nature}, volume = {640}, number = {8060}, pages = {1093-1102}, pmid = {40205037}, issn = {1476-4687}, support = {DP2 GM154152/GM/NIGMS NIH HHS/United States ; R00 GM130964/GM/NIGMS NIH HHS/United States ; }, mesh = {Humans ; Chromosome Breakage ; CRISPR-Cas Systems ; DNA Damage ; *DNA Repair ; DNA-Binding Proteins/metabolism ; Endonucleases/metabolism ; Flap Endonucleases/metabolism/deficiency/genetics ; Genomic Instability/genetics ; Homeostasis/genetics ; Proliferating Cell Nuclear Antigen/metabolism ; Proteolysis ; *Synthetic Lethal Mutations ; Ubiquitination ; HeLa Cells ; K562 Cells ; Intracellular Signaling Peptides and Proteins/metabolism ; Ubiquitin-Specific Proteases/metabolism ; DNA Replication ; DNA Helicases/metabolism ; }, abstract = {The DNA damage response (DDR) is a multifaceted network of pathways that preserves genome stability[1,2]. Unravelling the complementary interplay between these pathways remains a challenge[3,4]. Here we used CRISPR interference (CRISPRi) screening to comprehensively map the genetic interactions required for survival during normal human cell homeostasis across all core DDR genes. We captured known interactions and discovered myriad new connections that are available online. We defined the molecular mechanism of two of the strongest interactions. First, we found that WDR48 works with USP1 to restrain PCNA degradation in FEN1/LIG1-deficient cells. Second, we found that SMARCAL1 and FANCM directly unwind TA-rich DNA cruciforms, preventing catastrophic chromosome breakage by the ERCC1-ERCC4 complex. Our data yield fundamental insights into genome maintenance, provide a springboard for mechanistic investigations into new connections between DDR factors and pinpoint synthetic vulnerabilities that could be exploited in cancer therapy.}, }
@article {pmid40205022, year = {2025}, author = {Gracia-Rodriguez, C and Martínez-Medina, AE and Torres-Cosio, L and Lopez-Ortiz, C and Nimmakayala, P and Luévanos-Escareño, MP and Hernández-Almanza, AY and Castro-Alonso, MJ and Sosa-Martínez, JD and Reddy, UK and Balagurusamy, N}, title = {Can the molecular and transgenic breeding of crops be an alternative and sustainable technology to meet food demand?.}, journal = {Functional &amp; integrative genomics}, volume = {25}, number = {1}, pages = {83}, pmid = {40205022}, issn = {1438-7948}, mesh = {*Crops, Agricultural/genetics/growth &amp; development ; *Plant Breeding/methods ; *Plants, Genetically Modified/genetics ; Gene Editing ; CRISPR-Cas Systems ; }, abstract = {The gradual increase in the worldwide population represents various challenges, and one of the most alarming being the food demand. Historically technological advances led to the development of crops that meets the requirements and demands. Currently, molecular breeding unlocks the genetic potential of crops for their improvement, positioning it as a key technology for the development of new crops. The implementation of OMICs sciences, such spatial and single cell transcriptomics is providing a large and precise information, which can be exploited for crop improvement related to increasing yield, improving the nutritional value; designing new strategies for diseases resistance and management and for conserving biodiversity. Furthermore, the use of new technologies such CRISPR/CAS9 brought us the ability to modify the selected regions of the genome to select the superior's genotypes at a short time and the use of artificial intelligence aid in the analysis of big data generated by OMICS sciences. On the other hand, the application of molecular improvement technologies open up discussion on global regulatory measures, the socio-economic and socio-ethics, as the frameworks on its global regulation and its impact on the society create the public perception on its acceptance. In this review, the use and impact of OMICs sciences and genetic engineering in crops development, the regulatory measures, the socio-economic impact and as well as the mediatic information on genetically modified crops worldwide is discussed along with comprehensive insights on the potential of molecular plant breeding as an alternative and sustainable technology to meet global food demand.}, }
@article {pmid40205014, year = {2025}, author = {Tan, JYP and Thevendran, R and Quek, YS and Maheswaran, S}, title = {Studying the cellular efficacy and tolerability of using CRISPR-gRNA ribonucleoprotein (RNP) complex for in-vitro knockdown of TRIB2 in acute myeloid leukaemia (AML) cells as preliminary clinical evaluations.}, journal = {Molecular biology reports}, volume = {52}, number = {1}, pages = {379}, pmid = {40205014}, issn = {1573-4978}, mesh = {Humans ; *Leukemia, Myeloid, Acute/genetics/metabolism ; *Ribonucleoproteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Knockdown Techniques/methods ; Cell Line, Tumor ; *Calcium-Calmodulin-Dependent Protein Kinases/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Intracellular Signaling Peptides and Proteins/genetics ; }, abstract = {BACKGROUND: Acute Myeloid Leukemia (AML) is a complex, genetically driven cancer disease with several mutations that complicate therapy regimes. The Tribbles gene family, specifically the TRIB2 gene, has garnered substantial interest as a crucial oncogenic factor of AML progression. Studying cancer through gene expression studies and focusing on the primary oncogenes provide accurate information for future therapies. Here, CRISPR stands as the most exceptional tool used to explore both oncogene functionalities and therapeutic utilities. The ribonucleoprotein (RNP) mode of CRISPR formation and usage are preferred compared to plasmid-mediated CRISPR systems, however, it can lead to complications post-transfection to sensitive cellular entities such as human cancer cells in-vitro, transcribing to similar outcomes in-vivo as well. Therefore, this study describes the use of in-house designed CRISPR-RNP systems targeting the TRIB2 oncogene and evaluates their post-transfection cellular safety and efficacy aspects for future clinical applications.<br><br>METHODS: This study uses a designed guide RNA targeting the TRIB2 gene assembled via In-vitro synthesis. The gRNA with Cas9 protein leads to the formation of CRISPR-RNP structures, which target and cleave the TRIB2 gene. The assembled CRISPR-RNP system is transfected into target AML cell and control cell lines (i.e. HEK cells), and the subsequent gene cleavage and resulting changes to the AML cells in terms of cellular safety/tolerability and gene knockdown efficacy were studied via RT-qPCR, flow cytometry, and cell viability analysis.<br><br>RESULTS: The outcome demonstrates the well-tolerated transfection of the in-vitro assembled CRISPR RNP system with no signs of cellular toxicity and disruptions towards the AML cell's metabolic activities, promoting the safety aspects of CRISPR RNP post-transfection to human cells. The study further highlights the in-vitro efficacy of the CRISPR RNP in targeting the TRIB2 oncogene, where a statistically significant gene knockdown of more than 80% was detected via qPCR analysis of TRIB2 gene expression with minimal to no background effects from individual RNP components, equating to their targeted gene cleavage effects. In addition, the CRISPR TRIB2 gene knockdown also indicated the possibilities of induced AML cell death measured via flow cytometry markers, translating to favourable outcomes in eliminating cancerous growths.<br><br>CONCLUSIONS: This study research contributes to the refinement of the CRISPR-RNP strategy and preliminary evaluation for future clinical uses.}, }
@article {pmid40204947, year = {2025}, author = {Andel, D and Nouwens, AJ and Klaassen, S and Laoukili, J and Viergever, B and Verheem, A and Intven, MPW and Zandvliet, M and Hagendoorn, J and Borel Rinkes, IHM and Kranenburg, O}, title = {Rational design of alternative treatment options for radioresistant rectal cancer using patient-derived organoids.}, journal = {British journal of cancer}, volume = {132}, number = {10}, pages = {973-981}, pmid = {40204947}, issn = {1532-1827}, mesh = {Humans ; *Organoids/radiation effects/drug effects/pathology/metabolism ; *Rectal Neoplasms/radiotherapy/genetics/pathology/drug therapy ; *Radiation Tolerance/drug effects/genetics ; DNA Repair/drug effects ; }, abstract = {BACKGROUND: Resistance to radiation therapy is a common challenge in the field of oncology. Cancer cells with an increased ability to effectively repair DNA or cells with higher levels of antioxidants are more resistant to radiation. As cancer cells rely on these traits for survival, they may offer vulnerabilities that could be exploited.<br><br>METHODS: In the current study, rectal cancer organoids that showed different responses to radiation treatment were identified. RNA sequencing was used to compare radioresistant and radiosensitive organoids. In vitro combination drug screens were performed. The selection of drugs was guided by the RNA sequencing results.<br><br>RESULTS: Radioresistant organoids exhibited superior transcriptional adaptability and activated more DNA repair pathways when irradiated. Additionally, radioresistant organoids displayed enhanced antioxidant metabolism, including pathways related to the detoxification of reactive oxygen species and the synthesis of glutathione. Combinatorial drug screens identified the combination of RRx-001 (an inducer of oxidative stress) with GCLC inhibitor BSO as a highly effective and synergistic drug combination in killing radioresistant organoids. CRISPR-CAS-mediated knockout of GCLC sensitised organoids to RRx-001.<br><br>CONCLUSION: Combining RRx-001 with the inhibition of GCLC may be a promising alternative treatment strategy in radioresistant rectal cancer.}, }
@article {pmid40204795, year = {2025}, author = {Wang, Y and Cheng, Y and Sun, H and Wang, Z and Chen, N and Shi, C and Liu, H and Yang, J and Xu, Y}, title = {Genome editing in spinocerebellar ataxia type 3 cells improves Golgi apparatus structure.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {12106}, pmid = {40204795}, issn = {2045-2322}, support = {82001973//the Youth Fund of National Natural Science Foundation of China grant/ ; SBGJ2020003017//the Youth Project Co-established by Henan Province and the Ministry grant/ ; YXKC2020057//the Health Science and Technology Innovation Excellent Youth Training Project in Henan province grant/ ; 2017YFA0105000//National Key R&amp;D Program of China grant/ ; U1904207//the National Natural Science Foundation of China grants/ ; }, mesh = {*Machado-Joseph Disease/genetics/therapy/pathology ; *Gene Editing/methods ; *Golgi Apparatus/metabolism/genetics/ultrastructure ; Humans ; *Ataxin-3/genetics/metabolism ; Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems ; Mutation ; Repressor Proteins ; }, abstract = {Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disease caused by repeat expansion of the CAG trinucleotide within exon 10 of the ATXN3 gene. This mutation results in the production of an abnormal ataxin-3 protein containing an extended polyglutamine tract, referred to as mutant ataxin-3. In this study, we investigated the therapeutic potential of CRISPR/Cas9-mediated genome editing for SCA3. First, we designed a specific single-guide RNA targeting the ATXN3 gene and constructed the corresponding targeting vector. Induced pluripotent stem cells (iPSCs) derived from a SCA3 patient were then electroporated with the CRISPR/Cas9 components. Positive clones were screened and validated by PCR and Sanger sequencing to obtain genome-editing iPSCs (GE-iPSCs). Subsequently, the pluripotency of GE-iPSCs was confirmed, and the effects of genome editing on mutant ataxin-3 protein expression and Golgi apparatus morphology were assessed using Western blotting and immunofluorescence analyses. Our results demonstrated that targeted insertion of polyadenylation signals (PAS) upstream of the abnormal CAG repeats effectively suppressed the production of mutant ataxin-3. This intervention also reduced the formation of neuronal nuclear inclusions in differentiated neurons, restored the structural integrity of the Golgi apparatus (which exhibited a loose and enlarged morphology in SCA3 cells), and increased the expression levels of Golgi structural proteins (GM130 and GORASP2). In conclusion, our findings indicate that the targeted insertion of PAS upstream of the abnormal CAG repeats in the ATXN3 gene represents a promising therapeutic strategy for SCA3 through genome editing.}, }
@article {pmid40204736, year = {2025}, author = {Antoniou, P and Dacquay, L and Mårtensson, H and Madeyski-Bengtson, K and Loyd, AL and Shiriaeva, A and Gordon, E and Mustfa, S and Thom, G and Hsieh, PP and Šviković, S and Firth, M and Akrap, N and Maresca, M and Peterka, M}, title = {Modified pegRNAs mitigate scaffold-derived prime editing by-products.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {3374}, pmid = {40204736}, issn = {2041-1723}, mesh = {*Gene Editing/methods ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *RNA-Directed DNA Polymerase/metabolism/genetics ; CRISPR-Cas Systems ; HEK293 Cells ; }, abstract = {Prime editors (PEs) employ reverse transcriptase (RT) to install genomic edits using a template within the prime editing guide RNA (pegRNA). RT creates a 3' genomic flap containing the intended edit. However, reverse transcription can continue beyond the template, incorporating the pegRNA scaffold sequence into the 3' flap. These scaffold-derived by-products can be installed alongside the intended edit, reducing prime editing precision. Here, we develop a method that prevents RT from accessing the scaffold, thereby mitigating such by-products. We demonstrate that an internal abasic spacer or 2'-O-methylation within the pegRNAs terminates RT at the end of the template. This prevents scaffold-derived sequences from being incorporated into the target locus. We benchmark these pegRNAs in different cell types and demonstrate that they can be used with processive PEs such as PE6d or PE**. Our findings provide a simple approach to mitigate a common prime editing by-product and improve prime editing precision.}, }
@article {pmid40203649, year = {2025}, author = {Moyo, B and Brown, LBC and Khondaker, II and Bao, G}, title = {Engineering adeno-associated viral vectors for CRISPR/Cas based in vivo therapeutic genome editing.}, journal = {Biomaterials}, volume = {321}, number = {}, pages = {123314}, pmid = {40203649}, issn = {1878-5905}, support = {R01 HL169761/HL/NHLBI NIH HHS/United States ; }, mesh = {*Dependovirus/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; *Genetic Vectors/genetics ; Animals ; *Genetic Therapy/methods ; Anemia, Sickle Cell/therapy/genetics ; }, abstract = {The recent approval of the first gene editing therapy for sickle cell disease and transfusion-dependent beta-thalassemia by the U.S. Food and Drug Administration (FDA) demonstrates the immense potential of CRISPR (clustered regularly interspaced short palindromic repeats) technologies to treat patients with genetic disorders that were previously considered incurable. While significant advancements have been made with ex vivo gene editing approaches, the development of in vivo CRISPR/Cas gene editing therapies has not progressed as rapidly due to significant challenges in achieving highly efficient and specific in vivo delivery. Adeno-associated viral (AAV) vectors have shown great promise in clinical trials as vehicles for delivering therapeutic transgenes and other cargos but currently face multiple limitations for effective delivery of gene editing machineries. This review elucidates these challenges and highlights the latest engineering strategies aimed at improving the efficiency, specificity, and safety profiles of AAV-packaged CRISPR/Cas systems (AAV-CRISPR) to enhance their clinical utility.}, }
@article {pmid40203601, year = {2025}, author = {Li, X and Zhang, Y and He, M and Sun, J and Xiong, B and Wang, G}, title = {An ultrasensitive and specific fluorescence split-aptasensor for D-VP detection based on target-induced self-propelled 3D DNA walkers coupled with CRISPR-Cas12a.}, journal = {Talanta}, volume = {293}, number = {}, pages = {128102}, doi = {10.1016/j.talanta.2025.128102}, pmid = {40203601}, issn = {1873-3573}, mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; *Aptamers, Nucleotide/chemistry/genetics/metabolism ; Humans ; *DNA/chemistry/genetics ; Fluorescence ; *Endodeoxyribonucleases/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism ; *Bacterial Proteins/metabolism ; DNA, Catalytic/chemistry/metabolism ; Spectrometry, Fluorescence ; }, abstract = {In this work, we present an ultrasensitive, specific, and high-signal-to-background ratio fluorescence split-aptasensor for D-vasopressin (D-VP) detection. This sensor is based on target-induced self-propelled 3D DNA walkers in conjunction with CRISPR-Cas12a technology. Two split probes (SDA 1 and SDA 2) were designed to undergo structural recombination and function as a walking chain (SDA) under the induction of D-VP. Simultaneously, an intact Mg[2+]-dependent DNAzyme domain was formed at the tail of SDA and subsequently activated. The activated Mg[2+]-dependent DNAzyme continuously propelled the 3D DNA walker, enabling the generation of signal strand DNA (activator DNA). The activator DNA can subsequently trigger the activation of the Cas12a protein, enabling it to cleave the FAM-ssDNA-BHQ1 substrate. This process leads to signal amplification and the specific detection of D-VP. Under optimal conditions, the designed split-aptasensor exhibits excellent linearity across a concentration range of 5 ng/mL to 1215 ng/mL, with a detection limit (LOD) as low as 0.22 ng/mL. This split-aptasensor were employed to identify D-VP in human serum and urine samples, yielding highly satisfactory results. This unique design acts as a proof of concept and illustrates considerable promise for the detection of a wide range of analytes.}, }
@article {pmid40202350, year = {2025}, author = {Dudley, EG}, title = {The E. coli CRISPR-Cas conundrum: are they functional immune systems or genomic singularities?.}, journal = {EcoSal Plus}, volume = {}, number = {}, pages = {eesp00402020}, doi = {10.1128/ecosalplus.esp-0040-2020}, pmid = {40202350}, issn = {2324-6200}, abstract = {The discovery and subsequent characterization and applications of CRISPR-Cas is one of the most fascinating scientific stories from the past two decades. While first identified in Escherichia coli, this microbial workhorse often took a back seat to other bacteria during the early race to detail CRISPR-Cas function as an adaptive immune system. This was not a deliberate slight, but the result of early observations that the CRISPR-Cas systems found in E. coli were not robust phage defense systems as first described in Streptococcus thermophilus. This apparent lack of activity was discovered to result from transcriptional repression by the nucleoid protein H-NS. Despite extensive evidence arguing against such roles, some studies still present E. coli CRISPR-Cas systems in the context of anti-phage and/or anti-plasmid activities. Here, the studies that led to our understanding of its cryptic nature are highlighted, along with ongoing research to uncover potential alternative functions in E. coli.}, }
@article {pmid40202271, year = {2025}, author = {Sun, T and He, W and Chen, X and Shu, X and Liu, W and Ouyang, G}, title = {Nanomaterials-Integrated CRISPR/Cas Systems: Expanding the Toolbox for Optical Detection.}, journal = {ACS sensors}, volume = {10}, number = {4}, pages = {2453-2473}, doi = {10.1021/acssensors.5c00020}, pmid = {40202271}, issn = {2379-3694}, mesh = {*CRISPR-Cas Systems ; *Nanostructures/chemistry ; *Biosensing Techniques/methods ; Humans ; Spectrum Analysis, Raman ; }, abstract = {Nanomaterials-integrated CRISPR/Cas systems have rapidly emerged as powerful next-generation platforms for optical biosensing. These integrated platforms harness the precision of CRISPR/Cas-mediated nucleic acid detection while leveraging the unique properties of nanomaterials to achieve enhanced sensitivity and expanded analytical capabilities, thereby broadening their diagnostic potential. By incorporating a diverse range of nanomaterials, these systems effectively expand the analytical toolbox for optical detection, offering adaptable solutions tailored to various diagnostic challenges. This review provides a comprehensive overview of the nanomaterials successfully integrated into CRISPR/Cas-based optical sensing systems. It examines multiple optical detection modalities, including fluorescence, electrochemiluminescence, colorimetry, and surface-enhanced Raman spectroscopy, highlighting how nanomaterials facilitate signal amplification, enable multiplexing, and support the development of point-of-care applications. Additionally, practical applications of these integrated systems in critical fields such as healthcare diagnostics and environmental monitoring are showcased. While these platforms offer considerable advantages, several real-world challenges such as the complexity of assay workflows, environmental impact of nanomaterials, cost, and regulatory hurdles must be addressed before widespread implementation can be achieved. By identifying these critical obstacles and proposing strategic solutions, we aim to pave the way for the continued advancement and adoption of nanomaterial-integrated CRISPR/Cas optical biosensing technologies.}, }
@article {pmid40199340, year = {2025}, author = {Miao, X and Cao, F and Yu, XF and Li, TP and Su, HY and Guo, J and Hu, GL and Chen, BW and You, MS and Liu, YY and Lei, GK and You, S}, title = {A zinc finger protein shapes the temperature adaptability of a cosmopolitan pest.}, journal = {Open biology}, volume = {15}, number = {4}, pages = {240346}, pmid = {40199340}, issn = {2046-2441}, support = {//The Youth Teachers Visit and Study Program at the Haixia Institute of Science and Technology/ ; //Fujian Provincial Science and Technology Department/ ; //The Natural Science Foundation for Distinguished Young Scholars of Fujian Province/ ; }, mesh = {Animals ; *Zinc Fingers ; *Insect Proteins/genetics/metabolism/chemistry ; *Moths/genetics/physiology ; *Thermotolerance/genetics ; Female ; Temperature ; Male ; *Adaptation, Physiological ; CRISPR-Cas Systems ; }, abstract = {Global climate change is characterized by increased extreme temperatures affecting insects at all trophic levels. Zinc finger proteins (ZFPs) are key regulators of gene expression and cell differentiation in eukaryotes, essential for stress resistance in both animals and plants. Using CRISPR/Cas9 for gene deletion, this study predicted and examined the structure of ZFP320 in the diamondback moth (Plutella xylostella) and investigated its function in temperature stress response through a comprehensive age-stage, two-sex life table analysis. We found ZFP320 encodes a 387 amino acid protein (43 kDa) with no transmembrane domains, featuring a ZnF-C2H2 domain. Quantitative fluorescence analysis showed that ZFP320 expression increased under high temperatures. ZFP320 knockout altered antioxidant gene expression, resulting in higher levels of superoxide dismutase and catalase in mutant strains compared with wild-type strain. Life table analysis revealed that the mutant strains had shorter fecundity and oviposition periods under both normal and high temperatures. Additionally, mutant strains exhibited lower parameters (r, λ, R0), as well as reduced survival rates and critical thermal maxima. Notably, PxZFP320 plays a crucial role in temperature adaptation, paving the way for future investigations on the significance of ZFPs in P. xylostella's temperature tolerance.}, }
@article {pmid40199100, year = {2025}, author = {Su, M and Zhang, HS and Liu, H and Yang, K and Ying, ZM}, title = {Allosteric ribozyme-driven crRNA switch for the amplification-free detection of biomolecules.}, journal = {Biosensors &amp; bioelectronics}, volume = {280}, number = {}, pages = {117450}, doi = {10.1016/j.bios.2025.117450}, pmid = {40199100}, issn = {1873-4235}, mesh = {*Biosensing Techniques/methods ; *RNA, Catalytic/chemistry/genetics ; Humans ; *MicroRNAs/blood/genetics/isolation &amp; purification/analysis ; *Adenosine Triphosphate/isolation &amp; purification/blood/analysis ; Allosteric Regulation ; CRISPR-Cas Systems/genetics ; Limit of Detection ; CRISPR-Associated Proteins/genetics/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {Currently, CRISPR-mediated biosensors are concentrating on the design of the crRNA or the activator strand to regulate the trans-cleavage activity of Cas12a. Herein, we report an allosteric ribozyme-driven crRNA switch-regulated CRISPR/Cas12a sensor for amplification-free detection of biomolecules. An allosteric ribozyme is meticulously engineered to connect the target recognition sequence with the 5' binding arm of the hammerhead ribozyme, resulting in the formation of a hairpin structure through complementary hybridization. The presence of target induces the conformational change in the allosteric module and disrupts the hairpin structure, restoring multiple-turnover cleavage RNA activity of ribozyme. Then, the activated ribozyme specifically cuts the cleavage site of the substrate-locked crRNA and releases the native crRNA to initiate CRISPR/Cas12a functions for signal reporting. The reported biosensor exhibited high sensitivity and excellent specificity for miR-155 and adenosine triphosphate (ATP) detection, giving the detection limits of 256 fM and 160 nM, respectively. For clinical validation, our proposed strategy can quantify miR-155 expression levels in cells and serum of cancer patients. Furthermore, we also demonstrate that the allosteric ribozyme-driven crRNA switch can be easily compatible with lateral flow assays, realizing visualization and the portable monitoring of target. Hence, the biosensor not only has outstanding potential in point-of-care testing, but also enables the detection of various biomolecules by flexibly substituting target recognition sequences for molecular diagnosis in the clinic.}, }
@article {pmid40199056, year = {2025}, author = {Li, M and Pan, L and Ma, C and Wu, H and Xiang, G and Li, LF and Wang, T and Luo, R and Li, Y and Liu, D and Zhai, H and Assad, M and Song, X and Wang, Y and Gallardo, F and Qiu, HJ and Sun, Y}, title = {Tracking of single virus: Dual fluorescent labeling of pseudorabies virus for observing entry and replication in the N2a cells.}, journal = {Veterinary microbiology}, volume = {304}, number = {}, pages = {110503}, doi = {10.1016/j.vetmic.2025.110503}, pmid = {40199056}, issn = {1873-2542}, mesh = {*Herpesvirus 1, Suid/physiology/genetics ; Animals ; *Virus Replication ; Mice ; *Virus Internalization ; Genome, Viral ; Staining and Labeling/methods ; Cell Line ; Pseudorabies/virology ; Cell Line, Tumor ; Luminescent Proteins/genetics ; CRISPR-Cas Systems ; Red Fluorescent Protein ; }, abstract = {Pseudorabies virus (PRV) is a neurotropic herpesvirus. It is not easy to be track the whole replication progress of PRV, especially the nascent viral genome in the host cells. In this study, we developed a dual-fluorescence-labeled PRV (rPRV-Anchor3-mCherry) with the viral genome and the envelope protein gM labeled by ANCHOR DNA labeling system and mCherry, respectively. Through single-virus tracking of rPRV-Anchor3-mCherry, we observed that PRV invaded mouse neuroblastoma Neuro-2a cells via both endocytosis and plasma membrane fusion pathway. During the replication stage, parental and progeny viral genome of rPRV-Anchor3-mCherry in the cell nuclei could be visible, and viral nucleocapsid appeared more specifically than traditional capsid protein labeled PRV particles (rPRV-VP26-EGFP). We found that numerous progeny viral particles were produced in the nuclear, causing the nucleus membrane to break using three-dimensional (3D) live-cell imaging and electron microscopy. Moreover, our findings confirmed that simultaneously targeting of the UL9 and UL54 genes using a CRISPR-Cas9 system led to the complete inhibition PRV replication. rPRV-Anchor3-mCherry can be used to research multiple steps of the viral cycle.}, }
@article {pmid40198713, year = {2025}, author = {Ashokkumar, M and Hafer, TL and Felton, A and Archin, NM and Margolis, DM and Emerman, M and Browne, EP}, title = {A targeted CRISPR screen identifies ETS1 as a regulator of HIV-1 latency.}, journal = {PLoS pathogens}, volume = {21}, number = {4}, pages = {e1012467}, pmid = {40198713}, issn = {1553-7374}, support = {DP1 DA051110/DA/NIDA NIH HHS/United States ; R01 AI143381/AI/NIAID NIH HHS/United States ; UM1 AI164567/AI/NIAID NIH HHS/United States ; }, mesh = {*Proto-Oncogene Protein c-ets-1/genetics/metabolism ; *HIV-1/physiology/genetics ; Humans ; *Virus Latency/physiology/genetics ; *CD4-Positive T-Lymphocytes/virology/metabolism ; *HIV Infections/virology/genetics/metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Viral ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Human Immunodeficiency virus (HIV) infection is regulated by a wide array of host cell factors that combine to influence viral transcription and latency. To understand the complex relationship between the host cell and HIV-1 latency, we performed a lentiviral CRISPR screen that targeted a set of host cell genes whose expression or activity correlates with HIV-1 expression. We further investigated one of the identified factors - the transcription factor ETS1, and found that it is required for maintenance of HIV-1 latency in both latently infected cell lines and in a primary CD4 T cell latency model. Interestingly, ETS1 played divergent roles in actively infected and latently infected CD4 T cells, with knockout of ETS1 leading to reduced HIV-1 expression in actively infected cells, but increased HIV-1 expression in latently infected cells, indicating that ETS1 can play both a positive and negative role in HIV-1 expression. CRISPR/Cas9 knockout of ETS1 in CD4 T cells from ART-suppressed people with HIV-1 (PWH) confirmed that ETS1 maintains transcriptional repression of the clinical HIV-1 reservoir. Transcriptomic profiling of ETS1-depleted cells from PWH identified a set of host cell pathways involved in viral transcription that are controlled by ETS1 in resting CD4 T cells. In particular, we observed that ETS1 knockout increased expression of the long non-coding RNA MALAT1 that has been previously identified as a positive regulator of HIV-1 expression. Furthermore, the impact of ETS1 depletion on HIV-1 expression in latently infected cells was partially dependent on MALAT1. Additionally, we demonstrate that ETS1 knockout resulted in enhanced abundance of activating modifications (H3K9Ac, H3K27Ac, H3K4me3) on histones located at the HIV-1 long terminal repeat (LTR), indicating that ETS1 regulates the activity of chromatin-targeting complexes at the HIV-1 LTR. Overall, these data demonstrate that ETS1 is an important regulator of HIV-1 latency that impacts HIV-1 expression through repressing MALAT1 expression and by regulating modification of proviral histones.}, }
@article {pmid40198566, year = {2025}, author = {Kaur, K and Biswal, AK and Kaur, R and Singh, M and Dhugga, KS and Singh, J}, title = {A Protocol for High-efficiency Transformation and Genome Editing in Elite Wheat Cultivars.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2898}, number = {}, pages = {307-320}, pmid = {40198566}, issn = {1940-6029}, mesh = {*Triticum/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Transformation, Genetic ; Plants, Genetically Modified/genetics ; *Genome, Plant ; Genetic Vectors/genetics ; }, abstract = {CRISPR-Cas9-based editing offers a simple yet efficient method for introducing targeted mutations in the genes of interest. Low efficiency of transformation and a lack of reproducibility in editing genes in elite wheat cultivars are the bottlenecks in improving simply inherited traits. We describe a high-efficiency transformation protocol for editing elite wheat cultivars using JD633-GRF4-GIF1 CRISPR vector. This vector reduces the regeneration time of transformants to less than 90 days and enables transformation of elite cultivars.}, }
@article {pmid40197046, year = {2025}, author = {Irfan, M and Solbiati, J and Duran-Pinedo, A and Rocha, FG and Gibson, FC and Frias-Lopez, J}, title = {Erratum for Irfan et al., "A Porphyromonas gingivalis hypothetical protein controlled by the type I-B CRISPR-Cas system is a novel adhesin important in virulence".}, journal = {mSystems}, volume = {10}, number = {5}, pages = {e0029525}, doi = {10.1128/msystems.00295-25}, pmid = {40197046}, issn = {2379-5077}, }
@article {pmid40196691, year = {2025}, author = {Tang, S and Žedaveinytė, R and Burman, N and Pandey, S and Ramirez, JL and Kulber, LM and Wiegand, T and Wilkinson, RA and Ma, Y and Zhang, DJ and Lampe, GD and Berisa, M and Jovanovic, M and Wiedenheft, B and Sternberg, SH}, title = {Protein-primed DNA homopolymer synthesis by an antiviral reverse transcriptase.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, pmid = {40196691}, issn = {2692-8205}, support = {F30 AI183830/AI/NIAID NIH HHS/United States ; R35 GM134867/GM/NIGMS NIH HHS/United States ; P30 GM140963/GM/NIGMS NIH HHS/United States ; T32 GM145440/GM/NIGMS NIH HHS/United States ; F31 GM153146/GM/NIGMS NIH HHS/United States ; R35 GM152258/GM/NIGMS NIH HHS/United States ; P20 GM103474/GM/NIGMS NIH HHS/United States ; }, abstract = {Bacteria defend themselves from viral predation using diverse immune systems, many of which sense and target foreign DNA for degradation[1]. Defense-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this strategy by leveraging DNA synthesis instead[2,3]. We and others recently showed that DRT2 systems use an RNA template to assemble a de novo gene, leading to expression of an antiviral effector protein, Neo[4,5]. It remains unknown whether similar mechanisms of defense are employed by other DRT families. Focusing on DRT9, here we uncover an unprecedented mechanism of DNA homopolymer synthesis, in which viral infection triggers polydeoxyadenylate (poly-dA) accumulation in the cell to drive abortive infection and population-level immunity. Cryo-EM structures reveal how a conserved noncoding RNA serves as both a structural scaffold and reverse transcription template to direct hexameric complex assembly and RNA-templated poly-dA synthesis. Remarkably, biochemical and functional experiments identify conserved tyrosine residues within the reverse transcriptase itself that prime DNA synthesis, leading to the formation of high-molecular weight protein-DNA covalent adducts. Synthesis of poly-dA in vivo is regulated by the competing activities of phage-encoded triggers and host-encoded silencers of DRT9. Collectively, our work unveils a novel nucleic acid-driven defense system that expands the paradigm of bacterial immunity and broadens the known functions of reverse transcriptases.}, }
@article {pmid40196388, year = {2025}, author = {Luo, L and Miao, L and Ma, X and Hu, J and Li, S and Yang, W and Ma, S and Chen, R and Liu, X}, title = {Establishing an Immune System Conferring DNA and RNA Virus Resistance in Plants Using CRISPR/Cas12a Multiplex Gene Editing.}, journal = {Plant direct}, volume = {9}, number = {4}, pages = {e70070}, pmid = {40196388}, issn = {2475-4455}, abstract = {Two types of CRISPR/Cas systems (Cas9 and Cas13) have been used to combat eukaryotic viruses successfully. In this study, we established resistance to the DNA virus BSCTV and RNA virus TMV in Nicotiana benthamiana using the CRISPR-Cas12a multiplex gene editing system. We employed two effector proteins LbCas12a and FnCas12a coupled with six guide RNAs targeting virus genome and a novel mRNA-gRNA nucleic acid complex to transport gRNA efficiently. Compared with the BSCTV accumulation in the wild-type N. benthamiana, it was reduced by more than 90% by most transgenic events derived at 7 days post-inoculation. Additionally, the shoot-tip leaves were normal in the transgenic plants, whereas they appeared severely curled and stunted in wild-type N. benthamiana at 15 days post-infection. Target sites evaluation revealed that the editing system can directly destroy the structure of BSCTV viral genomes via large fragment deletions. We quantified TMV virus accumulation in the transgenic N. benthamiana lines by monitoring dynamic changes in GFP fluorescence and quantitative analysis by qPCR showed that the CRISPR-Cas12a system can introduce TMV virus resistance to N. benthamiana by preventing its systemic spread. Our study provides an innovative strategy-an mRNA-gRNA nucleic acid complex-which has proven to be highly effective in the gene-editing system and offers an efficient antiviral approach for generating virus-resistant plants.}, }
@article {pmid40195937, year = {2025}, author = {Wu, Q and Li, S and Long, X and Liu, L and Zhao, Q and Cui, Y and Zhang, Y and He, Y}, title = {β-Galactosidase-Mediated, Mn[2+]-Activated CRISPR/Cas12a Cascade Reaction for Immunosorbent Assay of Carbendazim.}, journal = {Analytical chemistry}, volume = {97}, number = {15}, pages = {8402-8410}, doi = {10.1021/acs.analchem.4c07110}, pmid = {40195937}, issn = {1520-6882}, mesh = {*Benzimidazoles/analysis/metabolism ; *CRISPR-Cas Systems ; *beta-Galactosidase/metabolism/chemistry ; *Carbamates/analysis ; Enzyme-Linked Immunosorbent Assay/methods ; *Manganese/chemistry/metabolism ; Manganese Compounds/chemistry ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {The CRISPR/Cas12a system is an emerging enzymatic tool for the development of enzyme-linked immunosorbent assay (ELISA) methods, owing to its robust signal amplification capability. Currently, most CRISPR/Cas12a-based ELISA approaches rely on strategies that convert target detection into nucleic acid analysis. This report presents a novel enzymatic cascade reaction for signal transduction and amplification in the development of a CRISPR/Cas12a-based ELISA method, utilizing β-galactosidase (β-gal)-mediated activation of the CRISPR/Cas12a system. Carbendazim (CBD), a widely used and versatile broad-spectrum benzimidazole fungicide, was chosen as the model analyte. In the absence of CBD, streptavidin-labeled β-gal is captured by a biotinylated secondary antibody immobilized on the microplate. The captured β-gal catalyzes the hydrolysis of p-aminophenyl β-D-galactopyranoside to generate p-aminophenol. This compound subsequently facilitates the decomposition of MnO2 nanosheets, leading to the generation of Mn[2+] ions. The Mn[2+] ions modulate the activity of the CRISPR/Cas12a system, thus producing high fluorescence in the detection solution. In the presence of CBD, the amount of β-gal captured on the microplate is reduced, thereby preventing effective cleavage of the reporter molecule by Cas12a, which results in a low fluorescence signal. After systematically optimizing experimental conditions, the developed method successfully detected CBD, demonstrating high sensitivity, selectivity, and applicability in complex food matrices. In comparison to the traditional nucleic acid-activated CRISPR/Cas12a-based ELISA method, our approach, which integrates β-gal-mediated, Mn[2+]-activated CRISPR/Cas12a cascade reactions into ELISA, exhibits superior analytical performance, thereby broadening the applicability of CRISPR/Cas12a for sensitive and convenient small-molecule analysis.}, }
@article {pmid40195838, year = {2025}, author = {Tran, QG and Le, TT and Choi, DY and Cho, DH and Yun, JH and Choi, HI and Kim, HS and Lee, YJ}, title = {Progress and challenges in CRISPR/Cas applications in microalgae.}, journal = {Journal of microbiology (Seoul, Korea)}, volume = {63}, number = {3}, pages = {e2501028}, doi = {10.71150/jm.2501028}, pmid = {40195838}, issn = {1976-3794}, support = {RS-2024-00459155//National Research Foundation of Korea/ ; //Ministry of Science and ICT/ ; }, mesh = {*Microalgae/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Biotechnology/methods ; Metabolic Engineering/methods ; }, abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technologies have emerged as powerful tools for precise genome editing, leading to a revolution in genetic research and biotechnology across diverse organisms including microalgae. Since the 1950s, microalgal production has evolved from initial cultivation under controlled conditions to advanced metabolic engineering to meet industrial demands. However, effective genetic modification in microalgae has faced significant challenges, including issues with transformation efficiency, limited target selection, and genetic differences between species, as interspecies genetic variation limits the use of genetic tools from one species to another. This review summarized recent advancements in CRISPR systems applied to microalgae, with a focus on improving gene editing precision and efficiency, while addressing organism-specific challenges. We also discuss notable successes in utilizing the class 2 CRISPR-associated (Cas) proteins, including Cas9 and Cas12a, as well as emerging CRISPR-based approaches tailored to overcome microalgal cellular barriers. Additionally, we propose future perspectives for utilizing CRISPR/Cas strategies in microalgal biotechnology.}, }
@article {pmid40195420, year = {2025}, author = {Gabaev, I and Rowland, A and Jovanovic, E and Gawden-Bone, CM and Crozier, TWM and Teixeira-Silva, A and Greenwood, EJD and Gerber, PP and Wit, N and Nathan, JA and Matheson, NJ and Lehner, PJ}, title = {CRISPR-Cas9 genetic screens reveal regulation of TMPRSS2 by the Elongin BC-VHL complex.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {11907}, pmid = {40195420}, issn = {2045-2322}, support = {/WT_/Wellcome Trust/United Kingdom ; MR/V011561/1/MRC_/Medical Research Council/United Kingdom ; }, mesh = {Humans ; *CRISPR-Cas Systems ; *Serine Endopeptidases/genetics/metabolism ; SARS-CoV-2 ; COVID-19/virology/genetics/metabolism ; }, abstract = {The TMPRSS2 cell surface protease is used by a broad range of respiratory viruses to facilitate entry into target cells. Together with ACE2, TMPRSS2 represents a key factor for SARS-CoV-2 infection, as TMPRSS2 mediates cleavage of viral spike protein, enabling direct fusion of the viral envelope with the host cell membrane. Since the start of the COVID-19 pandemic, TMPRSS2 has gained attention as a therapeutic target for protease inhibitors which would inhibit SARS-CoV-2 infection, but little is known about TMPRSS2 regulation, particularly in cell types physiologically relevant for SARS-CoV-2 infection. Here, we performed an unbiased genome-wide CRISPR-Cas9 library screen, together with a library targeted at epigenetic modifiers and transcriptional regulators, to identify cellular factors that modulate cell surface expression of TMPRSS2 in human colon epithelial cells. We find that endogenous TMPRSS2 is regulated by the Elongin BC-VHL complex and HIF transcription factors. Depletion of Elongin B or treatment of cells with PHD inhibitors resulted in downregulation of TMPRSS2 and inhibition of SARS-CoV-2 infection. We show that TMPRSS2 is still utilised by SARS-CoV-2 Omicron variants for entry into colonic epithelial cells. Our study enhances our understanding of the regulation of endogenous surface TMPRSS2 in cells physiologically relevant to SARS-CoV-2 infection.}, }
@article {pmid40195317, year = {2025}, author = {Allen, TP and Roennfeldt, AE and Reckdharajkumar, M and Sullivan, AE and Liu, M and Quinn, RJ and Russell, DL and Peet, DJ and Whitelaw, ML and Bersten, DC}, title = {dFLASH; dual FLuorescent transcription factor activity sensor for histone integrated live-cell reporting and high-content screening.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {3298}, pmid = {40195317}, issn = {2041-1723}, support = {INV-001616/GATES/Gates Foundation/United States ; OPP1171844, INV-001616//Bill and Melinda Gates Foundation (Bill &amp; Melinda Gates Foundation)/ ; }, mesh = {Humans ; *Histones/metabolism ; *Transcription Factors/metabolism/genetics ; Receptors, Progesterone/metabolism/genetics ; HEK293 Cells ; High-Throughput Screening Assays/methods ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; Drug Discovery/methods ; }, abstract = {Live-cell transcription factor (TF) activity reporting is crucial for synthetic biology, drug discovery and functional genomics. Here we present dFLASH (dual FLuorescent transcription factor Activity Sensor for Histone-integrated live-cell reporting), a modular, genome-integrated TF sensor. dFLASH homogeneously and specifically detects endogenous Hypoxia Inducible Factor (HIF) and Progesterone Receptor (PGR) activities, as well as coactivator recruitment to synthetic TFs. The dFLASH system produces dual-color nuclear fluorescence, enabling normalized, dynamic, live-cell TF activity sensing with strong signal-to-noise ratios and robust screening performance (Z' = 0.61-0.74). We validate dFLASH for functional genomics and drug screening, demonstrating HIF regulation via CRISPRoff and application to whole-genome CRISPR KO screening. Additionally, we apply dFLASH for drug discovery, identifying HIF pathway modulators from a 1600-compound natural product library using high-content imaging. Together, this versatile platform provides a powerful tool for studying TF activity across diverse applications.}, }
@article {pmid40195165, year = {2025}, author = {Rad, MB and Mohebbi, SR and Yadegar, A and Ghourchian, H}, title = {Porous GNPs assisted LAMP-CRISPR/Cas12a amperometric biosensor as a potential point of care testing system for SARS-CoV-2.}, journal = {Mikrochimica acta}, volume = {192}, number = {5}, pages = {280}, pmid = {40195165}, issn = {1436-5073}, mesh = {*Biosensing Techniques/methods ; *SARS-CoV-2/isolation &amp; purification/genetics ; Gold/chemistry ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *Metal Nanoparticles/chemistry ; *Point-of-Care Testing ; *COVID-19/diagnosis ; CRISPR-Cas Systems ; Electrochemical Techniques/methods ; Porosity ; Limit of Detection ; CRISPR-Associated Proteins/metabolism ; Molecular Diagnostic Techniques/methods ; Electrodes ; Endodeoxyribonucleases ; Bacterial Proteins ; }, abstract = {A simple and ultrasensitive amperometric biosensor is introduced which has the potential to be applied as a point of care test for SARS-CoV-2 monitoring. It was prepared by integrating the reverse transcription loop-mediated isothermal amplification (RT‑LAMP) and CRISPR/Cas12a nuclease activity on a modified gold screen-printed electrode (GSPE). The GSPE is modified with double-end thiolated oligonucleotide reporters conjugated to porous gold nanoparticles (PGNPs) and inserted into a homemade poly-methyl methacrylate cartridge. This biosensor was integrated with a low-cost electronic kit to make a platform with the potential to be applied as a point-of-care testing system. The PGNPs on the reporters create a dense, negatively charged barrier that repels the redox couple of [Fe(CN)6][3-/4-] from the GSPE surface. Upon the addition of a real sample, followed by LAMP amplification and Cas12a nuclease activity on disposable GSPE, in the presence of SARS-CoV-2, the single-guide RNA binds to the target sequence and activates Cas12a. The activated Cas12a then cleaves the reporters, releasing the PGNPs. This removal of electrostatic hindrance allows the redox couple of [Fe(CN)6][3-/4-] to approach the positively charged GSPE, enhancing the amperometric signal. This biosensor offers an outstanding detection limit of 143 zM (~ 86 copies/mL) and a linear response from 4.7 to 7062 aM for SARS-CoV-2 real samples. By using double-end thiolated reporters and porous GNPs, this novel testing system makes it possible to minimize the required sample volume and reagent costs.}, }
@article {pmid40194617, year = {2025}, author = {Wu, Z and Xu, Y and Zhou, W and Shi, L and Shi, W and Pu, L and Jiang, J}, title = {Rapid detection of Klebsiella pneumoniae based on one-tube RPA-CRISPR/Cas12a system.}, journal = {Clinica chimica acta; international journal of clinical chemistry}, volume = {573}, number = {}, pages = {120281}, doi = {10.1016/j.cca.2025.120281}, pmid = {40194617}, issn = {1873-3492}, mesh = {*Klebsiella pneumoniae/isolation &amp; purification/genetics ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *Recombinases/metabolism ; Limit of Detection ; Time Factors ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Klebsiella pneumoniae (KP) is a prevalent pathogen implicated in both community-acquired and nosocomial infections, often leading to severe clinical outcomes. The conventional methods for KP identification are characterized by intricacy and suboptimal efficiency. In this research, we have engineered a novel One-Tube RPA- CRISPR/Cas12a system, integrating recombinase polymerase amplification (RPA) method with the CRISPR/Cas12a diagnostic platform, to facilitate the detection of K. pneumoniae. To minimize the likelihood of aerosol-based contamination, the RPA components are positioned at the base of the tube, while the CRISPR/Cas12a components are placed at the tube's cap. The systems are combined post-RPA amplification through a brief centrifugation step, ensuring that RPA reactions are conducted independently to produce an adequate amount of target DNA before interaction with the CRISPR/Cas12a system. This method was validated using both fluorescent and lateral flow strip assays, achieving a limit of detection (LOD) of 10[0] copies/μL and 10[1] copies/μL respectively. The specificity for KP detection was found to be 100 %. Furthermore, the system demonstrated a positivity rate of 78 % (18/23) when directly extracting DNA from sputum samples, corroborated by culture and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). The simplicity and rapidity of the assay are augmented by a straightforward sample processing without extraction. The complete assay duration from specimen receipt to result is approximately 40 min, significantly reducing the turnaround time (TAT). Collectively, this system presents a streamlined, expeditious, and highly specific diagnostic approach for the detection of Klebsiella pneumoniae strains.}, }
@article {pmid40194458, year = {2025}, author = {Liu, S and Hu, J and Zhang, R and Tian, H and Wang, F and Chou, SH and He, J and Ma, L and Yin, W}, title = {Catalytic hairpin assembly assists CRISPR/Cas12a-mediated high-sensitivity detection of aflatoxin B1.}, journal = {Talanta}, volume = {293}, number = {}, pages = {128043}, doi = {10.1016/j.talanta.2025.128043}, pmid = {40194458}, issn = {1873-3573}, mesh = {*Aflatoxin B1/analysis ; *CRISPR-Cas Systems ; *Food Contamination/analysis ; Limit of Detection ; Aptamers, Nucleotide/chemistry ; *Biosensing Techniques/methods ; *Endodeoxyribonucleases/metabolism/genetics ; Biocatalysis ; *Inverted Repeat Sequences ; Bacterial Proteins ; CRISPR-Associated Proteins ; }, abstract = {Aflatoxin B1 (AFB1) is recognized the most toxic and carcinogenic mycotoxin and is widely present in cereals and various foods. Therefore, its precise detection is crucial to safeguard food quality and human health. In this study, we proposed a highly sensitive detection system for AFB1 by combining the catalytic hairpin assembly (CHA) and CRISPR/Cas12a techniques. The Aptamer of Aptamer-Initiator interacts with AFB1 to release the blocked Antisense. As a result, the Initiator of the Aptamer-Initiator becomes free and can act as a toehold to bind with H1, which can initiate the CHA to generate a large amount of double-stranded DNA, which hybridized with the Cas12a-crRNA duplex to form the Cas12a-crRNA-DNA ternary complex, wherein Cas12a subsequently cleaves the FAM-ssDNA-BHQ1 probe in trans to generate fluorescence signals. After optimization, we observed a linear relationship between fluorescence intensity and the AFB1 concentration in the range of 50 pM to 1 nM, with a limit of detection (LOD) of 10 pM. Also, the system was robust and could operate with excellent reliability and accuracy even in complex samples. The recovery values in food samples ranged from 92.23 % to 111.72 %, with relative standard deviation (RSD) below 5.68 %. The system exhibited remarkable advantages, including high sensitivity, strong specificity, and rapid response, thereby showed great potential in the efficient detection of AFB1 contaminants in food.}, }
@article {pmid40194350, year = {2025}, author = {Hu, H and Xue, H and Dong, K and Li, Y and Liu, P and Wang, H and Li, L and Xiao, X and Chen, H}, title = {Strand displacement-enhanced CRISPR-Cas13a system for ultra-specific detection of RNA single nucleotide variation.}, journal = {Biosensors &amp; bioelectronics}, volume = {280}, number = {}, pages = {117445}, doi = {10.1016/j.bios.2025.117445}, pmid = {40194350}, issn = {1873-4235}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *SARS-CoV-2/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; *Polymorphism, Single Nucleotide/genetics ; *COVID-19/virology/diagnosis ; *MicroRNAs/genetics/analysis ; *RNA, Viral/genetics/analysis ; Limit of Detection ; }, abstract = {RNA plays a critical role in biological systems, mediating genetic information transfer and regulating gene expression. However, RNA is susceptible to variations from endogenous and exogenous sources, with potentially profound biological consequences. The CRISPR-Cas13a system has emerged as a promising tool for RNA variation detection due to its cost-effectiveness, sensitivity, and user-friendly nature. Despite this, designing a simple, universal system with high discrimination factor (DF) for single-nucleotide variations remains a challenge. Here, we present the strand displacement-enhanced Cas13a single-nucleotide variation detection assay (SECND), a sensitive, universal, and easy-to-implement method with a high DF for RNA variations. Using SECND, we detected 5 types of single-nucleotide variations, achieving a maximum DF of 1083.2. We validated the assay's effectiveness on miRNA and SARS-CoV-2 genomic RNA simulants, incorporating a 4-way strand displacement mechanism to enhance detection limits to 10 pmol/L and 50 pmol/L, and to identify variations at frequencies as low as 0.01 % and 0.1 %. Additionally, we demonstrated SECND's utility in quantifying single-nucleotide variants of miR-200b and miR-200c in ovarian cancer and retinal glioma cells. This versatile tool not only advances RNA variation detection but also has significant implications for disease research, diagnostics, and viral classification, enhancing our understanding of the CRISPR-Cas13a system and its potential applications.}, }
@article {pmid40193707, year = {2025}, author = {Cueny, RR and Voter, AF and McKenzie, AM and Morgenstern, M and Myers, KS and Place, MM and Peters, JM and Coon, JJ and Keck, JL}, title = {Altering translation allows E. coli to overcome G-quadruplex stabilizers.}, journal = {Nucleic acids research}, volume = {53}, number = {6}, pages = {}, pmid = {40193707}, issn = {1362-4962}, support = {R35 GM118110/GM/NIGMS NIH HHS/United States ; R35 GM127088/GM/NIGMS NIH HHS/United States ; R01GM098885/GM/NIGMS NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems ; *DNA Transposable Elements ; *DNA, Bacterial/chemistry/genetics ; *Escherichia coli/chemistry/genetics ; Escherichia coli Proteins/genetics/metabolism ; *G-Quadruplexes ; Gene Deletion ; Peptide Elongation Factor Tu/genetics/metabolism ; Protein Biosynthesis ; Proteomics ; Ribosomes/metabolism ; Gene Expression Regulation, Bacterial ; }, abstract = {G-quadruplex (G4) structures can form in guanine-rich DNA or RNA and have been found to modulate cellular processes, including replication, transcription, and translation. Many studies on the cellular roles of G4s have focused on eukaryotic systems, with far fewer probing bacterial G4s. Using a chemical-genetic approach, we identified genes in Escherichia coli that are important for growth in G4-stabilizing conditions. Reducing levels of translation elongation factor Tu or slowing translation initiation or elongation with kasugamycin, chloramphenicol, or spectinomycin suppress the effects of G4-stabilizing compounds. In contrast, reducing the expression of specific translation termination or ribosome recycling proteins is detrimental to growth in G4-stabilizing conditions. Proteomic and transcriptomic analyses reveal decreased protein and transcript levels, respectively, for ribosome assembly factors and proteins associated with translation in the presence of G4 stabilizer. Our results support a model in which reducing the rate of translation by altering translation initiation, translation elongation, or ribosome assembly can compensate for G4-related stress in E. coli.}, }
@article {pmid40193385, year = {2025}, author = {Breunig, M and Hohwieler, M and Haderspeck, J and von Zweydorf, F and Hauff, N and Pasquini, LP and Wiegreffe, C and Zimmer, E and Mulaw, MA and Julier, C and Simon, E and Gloeckner, CJ and Liebau, S and Kleger, A}, title = {PPDPF is not a key regulator of human pancreas development.}, journal = {PLoS genetics}, volume = {21}, number = {4}, pages = {e1011657}, pmid = {40193385}, issn = {1553-7404}, mesh = {Humans ; *Pancreas/metabolism/growth &amp; development/cytology ; Animals ; Cell Differentiation/genetics ; Zebrafish/genetics ; CRISPR-Cas Systems ; Human Embryonic Stem Cells/metabolism/cytology ; Gene Expression Regulation, Developmental ; Pluripotent Stem Cells/metabolism/cytology ; Gene Knockout Techniques ; Cell Lineage/genetics ; }, abstract = {Given their capability to differentiate into each cell type of the human body, human pluripotent stem cells (hPSCs) provide a unique platform for developmental studies. In the current study, we employed this cell system to understand the role of pancreatic progenitor differentiation and proliferation factor (PPDPF), a protein that has been little explored so far. While the zebrafish orthologue exdpf is essential for exocrine pancreas specification, its importance for mammalian and human development has not been studied yet. We implemented a four times CRISPR/Cas9 nicking approach to knockout PPDPF in human embryonic stem cells (hESCs) and differentiated PPDPFKO/KO and PPDPFWT/WT cells towards the pancreatic lineage. In contrast to data obtained from zebrafish, a very modest effect of the knockout was observed in the development of pancreatic progenitors in vitro, not affecting lineage specification upon orthotopic transplantation in vivo. The modest effect is in line with the finding that genetic variants near PPDPF are associated with random glucose levels in humans, but not with type 2 diabetes risk, supporting that dysregulation of this gene may only result in minor alterations of glycaemic balance in humans. In addition, PPDPF is less organ- and cell type specifically expressed in higher vertebrates and its so far reported functions appear highly context-dependent.}, }
@article {pmid40193303, year = {2025}, author = {Coutinho-Abreu, IV and Chen, F and Li, HH and Rose, NH and Akbari, OS}, title = {Genome Editing in the Yellow Fever Mosquito Aedes aegypti using CRISPR-Cas9.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {217}, pages = {}, doi = {10.3791/67732}, pmid = {40193303}, issn = {1940-087X}, mesh = {Animals ; *Aedes/genetics/embryology ; *CRISPR-Cas Systems ; Microinjections/methods ; *Gene Editing/methods ; Yellow Fever/transmission ; Female ; Genome, Insect ; }, abstract = {The emergence of the clustered, regularly interspersed, short palindromic repeats (CRISPR)-Cas9 technology has revolutionized the genetic engineering field and opened the doors for precise genome editing in multiple species, including non-model organisms. In the mosquito Aedes aegypti, loss-of-function mutations and DNA insertions have been accomplished with this technology. Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology, focusing on both the generation of gene knockout and knockin lines. In this protocol, quartz needles are filled with a mixture of guide RNA, recombinant Cas9, and a plasmid containing a DNA cassette encoding a gene for a fluorescent marker, if gene knockin is desired. Embryos at the preblastoderm stage are lined up onto a strip of double-sided sticky tape placed onto a coverslip, which is subsequently mounted onto a glass slide. With the help of a microinjector, the needles are inserted gently into the posterior end of the embryos and a small volume of the CRISPR mixture is dispensed. When the embryos are hatched, the larvae are checked under the fluorescent scope, and the pupae are sex-sorted and separated in different cages. Once the adults emerge, these are reciprocally crossed with wild-type individuals, blood-fed, and placed for egg laying. Once these eggs are hatched, the fluorescent larvae collected represent individuals with stable insertion of the DNA cassette into their genome. These larvae are then grown to the adult stage, outcrossed to wild-type individuals, and then further assessed through molecular techniques to confirm that the exact sequence of the DNA cassette is present at the desired site of the mosquito genome. Homozygous lines can also be obtained by following the provided pipeline of crossing schema and molecular screening of the mutations.}, }
@article {pmid40192002, year = {2025}, author = {Wang, W and Shan, Y and Liu, R and Li, D and Zhou, J and Lu, Q and Zhao, H}, title = {Coordination of IFT20 With Other IFT Components Is Required for Ciliogenesis.}, journal = {Journal of clinical laboratory analysis}, volume = {39}, number = {9}, pages = {e70000}, pmid = {40192002}, issn = {1098-2825}, support = {2022HWYQ-075;ZR2024MC040//Natural Science Foundation of Shandong Province/ ; 32170695//National Natural Science Foundation of China/ ; }, mesh = {*Cilia/metabolism/physiology ; Humans ; *Carrier Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Gene Knockout Techniques ; }, abstract = {BACKGROUND: Primary cilia are organelles formed on the cell surface. They can act as cellular antennae to sense signals and play important roles in various biological processes. Abnormalities in primary cilia lead to a variety of diseases collectively known as ciliopathies. Intraflagellar transport protein 20 (IFT20) has been implicated in ciliogenesis.<br><br>METHODS: IFT20 knockout cell lines were established using the CRISPR-Cas9 gene editing technology. The GFP-IFT20 plasmid was constructed with the Gateway cloning system. Protein levels were detected via immunoblotting, and the localization of IFT20, acetylated &#x3b1;-tubulin, ARL13B, CP110, MKS3, IFT88, and IFT140 in wild-type and IFT20 knockout cells was examined by immunofluorescence microscopy. The fluorescence intensities were analyzed using ImageJ. Data quantifications and mass spectrometry results were analyzed using GraphPad Prism and Metascape.<br><br>RESULTS: The IFT20 deficiency impaired ciliogenesis and reduced cilium length. IFT20 depletion did not affect the removal of centriolar coiled-coil protein 110 (CP110) from the mother centriole or the recruitment of Meckel-Gruber syndrome type 3 (MKS3) to the transition zone. Mass spectrometry analysis revealed that proteins interacting with IFT20 were mainly IFT components. IFT20 knockout decreased the levels of both IFT88 and IFT140, and abrogated IFT88 localization at the basal body and ciliary axoneme. IFT20 knockout also impaired IFT140 localization at the ciliary axoneme but did not affect its localization at the basal body.<br><br>CONCLUSIONS: IFT20 is involved in ciliogenesis by regulating the level and localization of other IFT proteins and may have important implications in ciliopathies and related diseases.}, }
@article {pmid40191992, year = {2025}, author = {Qu, X and Yue, Y and Tang, Y and Jiao, J and Miao, P}, title = {Polydopamine Powered Droplet Electricity Generator for Protein Assay with CRISPR/Cas Enabled Amplification.}, journal = {Nano letters}, volume = {25}, number = {15}, pages = {6253-6259}, pmid = {40191992}, issn = {1530-6992}, mesh = {*CRISPR-Cas Systems ; *Polymers/chemistry ; *Indoles/chemistry ; Electricity ; DNA/chemistry ; *Flap Endonucleases/analysis/metabolism/genetics ; *Biosensing Techniques/methods ; Humans ; }, abstract = {Protein function investigation and clinical assay are fundamental to modern biology and medical diagnostics. Flap endonuclease 1 (FEN1), a key enzyme in DNA replication and repair, plays a critical role in the progression of many diseases. Taking FEN1 as an example, we present a novel protein detection platform combining triboelectric nanogenerator (TENG) and CRISPR/Cas technologies. As a specific form of TENG, the transistor-droplet electricity generator (TDEG) is explored, which offers a low-cost, simple fabrication approach with real-time detection capability. Meanwhile, the FEN1 activated CRISPR/Cas system catalyzes the reactions on the three-dimensional DNA tetrahedron interface, promising the high sensitivity. This work not only demonstrates a powerful method for rapid protein detection but also pioneers the integration of CRISPR/Cas with TENG. It has a great prospect for future development of TENG sensors.}, }
@article {pmid40191453, year = {2025}, author = {Patnaik, A and Rai, SK and Dhaked, RK}, title = {Analytical techniques and molecular platforms for detection and surveillance of antimicrobial resistance: advancements of the past decade.}, journal = {3 Biotech}, volume = {15}, number = {5}, pages = {108}, pmid = {40191453}, issn = {2190-572X}, abstract = {Developing countries have been able to control and minimise the mortality rates caused by pathogenic infections by ensuring affordable access to antibiotics. However, a large number of bacterial ailments are treated with wrong antibiotic prescription due to improper disease diagnosis. Apart from healthcare, antibiotics are also imprudently utilised in crop processing and animal husbandry. This unsupervised usage of antibiotics has propelled the generation of multidrug-resistant species of bacteria. Presently, several traditional antimicrobial susceptibility/resistance tests (AST/ART) are available; however, the accuracy and reproducibility of these tests are often debatable. Rigorous efforts are essential to develop techniques and methods which substantially decrease turnaround time for resistance screening. The present review has comprehensively incorporated the improvements in instrumentation and molecular methods for antimicrobial resistance studies. We have enlisted some innovative takes on conventional techniques such as isothermal calorimetry, Raman spectroscopy, mass spectrometry and microscopy. The contributions of modern molecular tools such as CRISPR-Cas, aptamers and Oxford-MinION sequencers have also been discussed. Persistent evolution has been observed towards adding innovation in diagnostic platforms for drug resistome screening, with the major attraction being the involvement of non-conventional analytical methods and technological improvements in existing setups. This review highlights these updates and provides a detailed account of principal developments in molecular methods for the testing of drug resistance in bacteria.}, }
@article {pmid40191137, year = {2025}, author = {Liu, H and Xu, L and Xiu, Y and Ta, N and Xu, Q and Fan, Y and Li, K and Zhao, H and Piao, D and Ren, F and Jiang, H}, title = {A CRISPR/cas13a-assisted precise and portable test for Brucella nucleic acid detection.}, journal = {Frontiers in cellular and infection microbiology}, volume = {15}, number = {}, pages = {1545953}, pmid = {40191137}, issn = {2235-2988}, mesh = {*Brucellosis/diagnosis/microbiology/veterinary ; Animals ; *Brucella/genetics/isolation &amp; purification ; Humans ; *CRISPR-Cas Systems ; Sheep ; Sensitivity and Specificity ; Milk/microbiology ; Female ; *Molecular Diagnostic Techniques/methods ; DNA, Bacterial/genetics ; Nucleic Acid Amplification Techniques/methods ; Vagina/microbiology ; Bacterial Proteins/genetics ; }, abstract = {INTRODUCTION: Brucella infection in humans or animals can lead to brucellosis, which has the potential to significantly impact both the economy and public health. Currently, molecular biological methods for diagnosing brucellosis are either complex or have low sensitivity, and it is difficult to apply them in real-life settings in the field. Therefore, this study aims to establish a rapid and convenient nucleic acid-based molecular biology method for on-site rapid detection of Brucella and early clinical screening of brucellosis.<br><br>METHODS: Based on the conserved sequence of the Brucella Bcsp31 gene, we designed CRISPR RNA (crRNA) and RAA primers. We developed a fluorescence detection method and a paper strip detection method by integrating RAA amplification with CRISPR/Cas13a detection. We applied these methods to analyze 100 samples of suspected brucellosis-infected milk, 123 samples of human whole blood, and 100 samples of sheep vaginal swabs in order to validate their practical utility.<br><br>RESULTS: The RAA-CRISPR/Cas13a Brucella fluorescence detection method and the strip test method had detection limits of 100 copies/&#x3bc;L and 101 copies/&#x3bc;L, respectively, and both methods had a specificity of 100%. The positivity rate of the RAA-CRISPR/Cas13a fluorescence detection method for the milk, human whole blood, and sheep vaginal swab samples was 93% (93/100), 82.12% (101/123), and 91% (91/100), respectively; the strip test method, 87% (87/100), 64.23% (79/123), and 76% (76/100), respectively.<br><br>CONCLUSION: In this study, we have developed a RAA-CRISPR detection method based on the Brucella BCSP31 gene, with potential applications in the identification of Brucella nucleic acid and implications for clinical diagnosis of brucellosis.}, }
@article {pmid40191042, year = {2025}, author = {Kim, HK and Cheong, H and Kim, MY and Jin, HE}, title = {Therapeutic Targeting in Ovarian Cancer: Nano-Enhanced CRISPR/Cas9 Gene Editing and Drug Combination Therapy.}, journal = {International journal of nanomedicine}, volume = {20}, number = {}, pages = {3907-3931}, pmid = {40191042}, issn = {1178-2013}, mesh = {Humans ; *Ovarian Neoplasms/genetics/therapy/drug therapy ; Female ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Nanoparticles/chemistry ; *Genetic Therapy/methods ; Animals ; Drug Delivery Systems/methods ; Drug Resistance, Neoplasm/genetics ; Molecular Targeted Therapy/methods ; Antineoplastic Agents/administration &amp; dosage/therapeutic use ; }, abstract = {Ovarian cancer is the third most common gynecological cancer worldwide. Due to the high recurrence rate of advanced-stage ovarian cancer, often resulting from drug-resistant and refractory disease, various treatment strategies are under investigation. Genome editing of therapeutic target genes holds promise in enhancing cancer treatment efficacy by elucidating gene functions and mechanisms involved in cancer progression. The CRISPR/Cas9 system, in particular, shows great potential in ovarian cancer gene therapy and drug development. Targeting therapeutic genes such as BRCA1/2, P53, Snai1 etc, could improve the therapeutic strategy in ovarian cancer. CRISPR/Cas9 is a powerful gene-editing tool that there are many on-going clinical trials to treat various diseases including cancer. Nano-based delivery systems for CRISPR/Cas9 offer further therapeutic benefits, leveraging the unique properties of nanoparticles to improve delivery efficiency. Nano-based delivery systems could enhance the stability of CRISPR/Cas9 delivery formats (such as plasmid, mRNA, etc) and improve the delivery precision of delivery to target tumors. Additionally, combining CRISPR/Cas9 with targeted drug treatments, especially those aimed at genes associated with drug resistance, may significantly improve therapeutic outcomes in ovarian cancer. In this review, we discuss therapeutic target genes and their mechanisms in ovarian cancer, advances in nano-based CRISPR/Cas9 delivery, and the therapeutic potential of combining CRISPR/Cas9 with drug treatments for ovarian cancer.}, }
@article {pmid40189908, year = {2025}, author = {Davidson, AE and Straquadine, NRW and Cook, SA and Liu, CG and Nie, C and Spaulding, MC and Ganz, J}, title = {A Rapid F0 CRISPR Screen in Zebrafish to Identify Regulator Genes of Neuronal Development in the Enteric Nervous System.}, journal = {Neurogastroenterology and motility}, volume = {37}, number = {5}, pages = {e70009}, pmid = {40189908}, issn = {1365-2982}, support = {R21 NS123629/NS/NINDS NIH HHS/United States ; //American Gastroenterological Association/ ; /NH/NIH HHS/United States ; //National Science Foundation/ ; //Simons Foundation/ ; }, mesh = {Animals ; *Enteric Nervous System/growth &amp; development/metabolism ; Zebrafish/genetics ; *Neurogenesis/genetics ; *CRISPR-Cas Systems ; Zebrafish Proteins/genetics ; *Neurons/physiology ; Transcription Factors/genetics ; Gene Editing/methods ; }, abstract = {BACKGROUND: The neural crest-derived enteric nervous system (ENS) provides the intrinsic innervation of the gut with diverse neuronal subtypes and glial cells. The ENS regulates all essential gut functions, such as motility, nutrient uptake, immune response, and microbiota colonization. Deficits in ENS neuron numbers and composition cause debilitating gut dysfunction. Yet, few studies have identified genes that control neuronal differentiation and the generation of the diverse neuronal subtypes in the ENS.<br><br>METHODS: Utilizing existing CRISPR/Cas9 genome editing technology in zebrafish, we have developed a rapid and scalable screening approach for identifying genes that regulate ENS neurogenesis.<br><br>KEY RESULTS: As a proof-of-concept, F0 guide RNA-injected larvae (F0 crispants) targeting the known ENS regulator genes sox10, ret, or phox2bb phenocopied known ENS phenotypes with high efficiency. We evaluated 10 transcription factor candidate genes as regulators of ENS neurogenesis and function. F0 crispants for five of the tested genes have fewer ENS neurons. Secondary assays in F0 crispants for a subset of the genes that had fewer neurons reveal no effect on enteric progenitor cell migration but differential changes in gut motility.<br><br>CONCLUSIONS: Our multistep, yet straightforward CRISPR screening approach in zebrafish tests the genetic basis of ENS developmental and disease gene functions that will facilitate the high-throughput evaluation of candidate genes from transcriptomic, genome-wide association, or other ENS-omics studies. Such in&#xa0;vivo ENS F0 crispant screens will contribute to a better understanding of ENS neuronal development regulation in vertebrates and what goes awry in ENS disorders.}, }
@article {pmid40188791, year = {2025}, author = {Huang, X and Tan, Z and Wei, J and Bai, X}, title = {Super-robust synthetic microorganism can get chlorine resistance in advance and transfer their inserted DNA sequence in genome to indigenous bacteria in water.}, journal = {Water research}, volume = {281}, number = {}, pages = {123594}, doi = {10.1016/j.watres.2025.123594}, pmid = {40188791}, issn = {1879-2448}, mesh = {*Chlorine/pharmacology ; *Bacteria/genetics ; Water Microbiology ; CRISPR-Cas Systems ; Gene Editing ; }, abstract = {CRISPR-Cas gene editing tools have brought us to an era of synthetic biology that will change the world. Synthetic microorganisms (SMs) have brought enormous economic benefits and will contribute more in the future. Among them, super-robust SMs can overcome the stresses in bioproduction and further increase yield. However, when they are released into the environments, little is known about their fates and risks to human health. In this study, it was found that the gene editing super-robust SM could transfer its inserted DNA sequence in genome to the indigenous bacteria in surface water and showed stronger resistance to chlorine compared with wild-type bacteria. Chlorine disinfection did slight damage on cell membrane of super-robust SM, which decreased ATP leakage and DNA damage, and thereby promoted bacterial survival. Chlorine-injured super-robust SM retained high respiratory activity, and could resuscitate and regenerate. Less damage on super-robust SM cell membrane could prevent chlorine from entering the cells and resulted in lower ROS generation. Its DNA repair system and antioxidant system could still function under high concentrations of chlorine exposure. These findings provided new insights into the fates and environmental risks of SMs as an emerging biological pollutant in water supply system.}, }
@article {pmid40187879, year = {2025}, author = {Ding, F and Hang, X and Tian, S and Cao, W and Wu, J and Wang, L}, title = {Nicking endonuclease-mediated primer exchange reaction for rapid and sensitive miRNA detection.}, journal = {Analytica chimica acta}, volume = {1351}, number = {}, pages = {343902}, doi = {10.1016/j.aca.2025.343902}, pmid = {40187879}, issn = {1873-4324}, mesh = {*MicroRNAs/blood/analysis/genetics ; Humans ; Limit of Detection ; CRISPR-Cas Systems ; *Endonucleases/metabolism ; DNA/chemistry/metabolism ; *Biosensing Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Nucleic Acid Hybridization ; *DNA Primers/chemistry/metabolism ; }, abstract = {Primer exchange reaction (PER) is a novel and simple nucleic acid-templated extension technique that has recently attracted much attention in the field of biosensing. However, current PER reactions have shown relatively slow rates and low amplification performances, resulting in long assay times and limited detection sensitivities. Here we report a nicking endonuclease-mediated PER reaction (named NEPER) that rapidly releases amplified DNA products by adding a nicking endonuclease to hydrolyze the hybridized double-stranded DNA (dsDNA), and consequently has a maximum speed that is thirty orders of magnitude greater than the maximum for conventional PER. We further combined a CRISPR/Cas12a signal readout technique and developed a cascade NEPER-CRISPR/Cas12a method that can detect miRNA-155 with a limit of detection (LOD) down to 3.1 fM. We also show that the NEPER-CRISPR/Cas12a can be used to detect targets in serum samples.}, }
@article {pmid40187665, year = {2025}, author = {Pablo-Marcos, D and Fernández-Diego, L and Rodríguez-Grande, J and Fraile-Valcárcel, N and Ortiz-Cartagena, C and Pacios, O and García-García, S and García-Fernández, S and Blasco, L and Ocampo-Sosa, A and Calvo-Montes, J and Tomás, M}, title = {An accurate amplification-free CRISPR/Cas12a-based assay for GES β-lactamase detection.}, journal = {International journal of antimicrobial agents}, volume = {66}, number = {1}, pages = {107506}, doi = {10.1016/j.ijantimicag.2025.107506}, pmid = {40187665}, issn = {1872-7913}, mesh = {*beta-Lactamases/genetics/analysis ; *Pseudomonas aeruginosa/genetics/enzymology/isolation &amp; purification ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; Humans ; *Molecular Diagnostic Techniques/methods ; *Enterobacteriaceae/genetics/enzymology/isolation &amp; purification ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {OBJECTIVE: Guiana-Extended-Spectrum (GES) β-lactamases belong to the minor class A β-lactamases and are probably underdiagnosed due to a lack of specific diagnostic tests. There is therefore an urgent need to develop new molecular diagnostic tools that will be able to fill the gap in the detection of rare β-lactamases. Here, we propose an optimized, amplification-free CRISPR/Cas12a-based assay for the accurate detection of GES β-lactamases and we validate its application with clinical isolates (Graphic abstract). Based on the results of examination of 79 standard collection, the proposed assay exhibited 100% sensitivity and specificity, as well as 100% positive and negative predictive values in less than 1.5 hours.<br><br>METHODS: We optimized the CRISPR/Cas12a method by harnessing a multiplex crRNA strategy, a highly efficient DNA reporter (TTATT-5C) and the Murine RNase Inhibitor to prevent crRNA degradation.<br><br>RESULTS: Our yielded limits of detection of 1 ng/&#xb5;L and 3 ng/&#xb5;L in Enterobacterales and Pseudomonas aeruginosa, respectively. The observed difference is due to the location of the blaGES gene. The gene occurs in a chromosomal integron present only in one to three copies in P. aeruginosa, whereas it occurs in plasmids present in multiple copies in Enterobacterales.<br><br>CONCLUSIONS: The proposed method could be established as a routine diagnostic tool in clinical microbiology laboratories to fill the gap in availability of commercial diagnostic tests for GES &#x3b2;-lactamases.}, }
@article {pmid40187387, year = {2025}, author = {Tajer, BJ and Kalu, G and Jay, S and Wynn, E and Decaux, A and Gilbert, P and Singer, HD and Kidd, MD and Nelson, JA and Harake, N and Lopez, NJ and Souchet, NR and Luong, AG and Savage, AM and Min, S and Karabacak, A and Böhm, S and Kim, RT and Froitzheim, T and Sousounis, K and Courtemanche, K and Han, J and Payzin-Dogru, D and Blair, SJ and Roy, S and Fei, JF and Tanaka, EM and Whited, JL}, title = {Optimized toolkit for the manipulation of immortalized axolotl fibroblasts.}, journal = {Methods (San Diego, Calif.)}, volume = {240}, number = {}, pages = {21-34}, doi = {10.1016/j.ymeth.2025.03.019}, pmid = {40187387}, issn = {1095-9130}, mesh = {Animals ; *Fibroblasts/transplantation/cytology/metabolism ; *Ambystoma mexicanum/genetics ; Transfection/methods ; CRISPR-Cas Systems/genetics ; *Cell Culture Techniques/methods ; Cell Line ; Regeneration ; }, abstract = {The axolotl salamander model has broad utility for regeneration studies, but this model is limited by a lack of efficient cell-culture-based tools. The Axolotl Limb-1 (AL-1) fibroblast line, the only available immortalized axolotl cell line, was first published over 20 years ago, but many established molecular biology techniques, such as lipofectamine transfection, CRISPR-Cas9 mutagenesis, and antibiotic selection, work poorly or remain untested in AL-1 cells. Innovating technologies to manipulate AL-1 cells in culture and study their behavior following transplantation into the axolotl will complement in-vivo studies, decrease the number of animals used, and enable the faster, more streamlined investigation of regenerative biology questions. Here, we establish transfection, mutagenesis, antibiotic selection, and in-vivo transplantation techniques in axolotl AL-1 cells. These techniques will enable efficient culture with AL-1 cells and guide future tool development for the culture and manipulation of other salamander cell lines.}, }
@article {pmid40187291, year = {2025}, author = {Meng, T and Kang, Q and Xu, J and Zhao, S and Liu, T and Zhou, D and Gong, X and Zhang, J}, title = {A hairpin reporter-driven feedback CRISPR/Cas signal amplification loop for terminal deoxynucleotidyl transferase activity detection.}, journal = {Talanta}, volume = {293}, number = {}, pages = {128061}, doi = {10.1016/j.talanta.2025.128061}, pmid = {40187291}, issn = {1873-3573}, mesh = {*CRISPR-Cas Systems/genetics ; *DNA Nucleotidylexotransferase/metabolism/blood ; Humans ; *Biosensing Techniques/methods ; Nucleic Acid Amplification Techniques ; CRISPR-Associated Proteins/metabolism ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {The CRISPR/Cas12a system has become a powerful tool in biosensing because of its specific target recognition ability and highly efficient trans-cleavage activity. However, a problem faced by the CRISPR/Cas12a system when directly used for trace detection is the linear amplification efficiency of single-cycle digestion. Here, we present a novel hairpin reporter-driven CRISPR/Cas12a (HR-CRISPR) amplification system that establishes a positive feedback loop within the CRISPR/Cas12a platform to finish an exponential and sensitive signal amplification in a one-step reaction. As proof of concept, we applied this strategy to the terminal deoxynucleotidyl transferase (TdT) activity assay without pre-amplification procedure. The polyT strand extended by TdT hybridizes with crRNA, activating Cas12a, which then cleaves the FQ-hairpin reporter. The cleavage products are further elongated by reverse transcriptase using crRNA as a template, reactivating Cas12a and producing exponentially amplified fluorescence signals. This assay offers a simple yet highly sensitive approach for quantifying TdT activity, achieving a low detection limit of 4.55 × 10[-6] U. Moreover, it is applicable for inhibitor screening and monitoring TdT activity in human serum samples.}, }
@article {pmid40187288, year = {2025}, author = {Li, Z and Wang, J and Shen, K and Zhao, X and Lin, Z and Yi, H}, title = {Signal-on electrochemiluminescence resonance energy transfer biosensor for miRNA-543 based on CRISPR/Cas13a and magnetic separation.}, journal = {Talanta}, volume = {293}, number = {}, pages = {128085}, doi = {10.1016/j.talanta.2025.128085}, pmid = {40187288}, issn = {1873-3573}, mesh = {*MicroRNAs/analysis/genetics/blood ; *Biosensing Techniques/methods ; Humans ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; Silicon Dioxide/chemistry ; *Luminescent Measurements/methods ; Gold/chemistry ; Energy Transfer ; Female ; Limit of Detection ; Metal Nanoparticles/chemistry ; DNA, Single-Stranded/chemistry ; }, abstract = {In this study, an electrochemiluminescence resonance energy transfer (ECL-RET) biosensor with high sensitivity and strong resistance to interference was constructed based on the CRISPR/Cas13a system and magnetic separation for ovarian cancer biomarker miR-543 detection. Mesoporous silica nanoparticles embedded with Ru(bpy)3[2+] (Ru@SiO2) have high electrochemiluminescence (ECL) response was chosen as energy donor. Single-stranded DNA S1 containing "rUrU" motif was immobilized on AuNRs (AuNRs-S1), which hybridized with single-stranded DNA S2 modified SAMBs (SAMBs-S2) to form AuNRs-S1/S2-SAMBs complex, this has been used as energy acceptor. In the absence of the target, Cas13a remained inactive, preventing the cleavage of S1, thereby maintaining the association of AuNRs with SAMBs. Then they were added in Ru@SiO2 solution after magnetic separation. The electrostatic adsorption between the negatively charged AuNRs and the positively charged Ru@SiO2 cause the occurrence of ECL-RET and low ECL signal had been detected. When the target was added, Cas13a was activated and resulted in the non-specifically cleaving of S1, so AuNRs detached from SAMBs. After magnetic separation, fewer AuNRs participated in ECL-RET, leading to an enhanced ECL signal detected. The change in ECL intensity (ΔECL) exhibited a linear correlation with the logarithm of miR-543 concentration within the range of 10 fM to 10 nM, with a detection limit of 6.91 fM. The biosensor had been applied to detect miR-543 in clinical samples with high accuracy.}, }
@article {pmid40186758, year = {2025}, author = {Guo, X and Sun, K and Wu, Z and Xiao, D and Song, Y and Li, S and Wei, G and Li, W and Hao, Y and Xu, B and Zhang, K and Liao, N and Hu, D and Liu, YG and Zong, W and Guo, J}, title = {Improving yield-related traits by editing the promoter and distal regulatory region of heading date genes Ghd7 and PRR37 in elite rice variety Mei Xiang Zhan 2.}, journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik}, volume = {138}, number = {4}, pages = {92}, pmid = {40186758}, issn = {1432-2242}, support = {2023ZD04073//National Key R&amp;D Program of China/ ; 2019B030302006//Major Program of Guangdong Basic and Applied Research/ ; 2022SDZG05//open competition program of top ten critical priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province/ ; }, mesh = {*Oryza/genetics/growth &amp; development ; *Promoter Regions, Genetic ; *Gene Editing ; Plant Breeding ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Phenotype ; *Genes, Plant ; }, abstract = {We revealed that editing the promoter and distal regulatory region of the pleiotropic genes Ghd7 and PRR37 reduces their ability to delay heading date while improving their capacity to boost crop yield, offering valuable resources for rice breeding. Heading date is a crucial agronomic characteristic in rice that governs the adaptability to different latitudes and the yield of various varieties. Optimizing the heading date of superior cultivars in breeding practice can significantly broaden their potential planting areas. Ghd7 and PRR37 are pivotal genes that control heading date and enhance agronomic traits. In the elite indica rice variety Mei Xiang Zhan 2 (MXZ2), we used CRISPR/Cas9 technology to effectively generate homozygous mutant lines with a gradient change in heading date by multi-target editing the promoter and distal regulatory region of Ghd7 and PRR37. Various degrees of down-regulation of Ghd7 or PRR37 expression, impaired gene functions, and advancement of the heading date were observed in the mutant lines. Certain mutant lines exhibited an early heading date and increased yield while preserving the exceptional quality of MXZ2. Our study revealed that editing the promoter and distal regulatory region of the pleiotropic genes Ghd7 and PRR37 reduces their ability to delay heading date while improving their capacity to boost crop yield, offering valuable resources for rice breeding.}, }
@article {pmid40186543, year = {2025}, author = {Nievergelt, AP}, title = {Genome editing in the green alga Chlamydomonas: past, present practice and future prospects.}, journal = {The Plant journal : for cell and molecular biology}, volume = {122}, number = {1}, pages = {e70140}, pmid = {40186543}, issn = {1365-313X}, support = {ALTF 891-2018//European Molecular Biology Organization/ ; LT000515/2019//Human Frontier Science Program/ ; }, mesh = {*Gene Editing/methods/trends ; *Chlamydomonas/genetics ; CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; Genetic Engineering/methods ; }, abstract = {The green alga Chlamydomonas is an important and versatile model organism for research topics ranging from photosynthesis and metabolism, cilia, and basal bodies to cellular communication and the cellular cycle and is of significant interest for green bioengineering processes. The genome in this unicellular green alga is contained in 17 haploid chromosomes and codes for 16 883 protein coding genes. Functional genomics, as well as biotechnological applications, rely on the ability to remove, add, and change these genes in a controlled and efficient manner. In this review, the history of gene editing in Chlamydomonas is put in the context of the wider developments in genetics to demonstrate how many of the key developments to engineer these algae follow the global trends and the availability of technology. Building on this background, an overview of the state of the art in Chlamydomonas engineering is given, focusing primarily on the practical aspects while giving examples of recent applications. Commonly encountered Chlamydomonas-specific challenges, recent developments, and community resources are presented, and finally, a comprehensive discussion on the emergence and evolution of CRISPR/Cas-based precision gene editing is given. An outline of possible future paths for gene editing based on current global trends in genetic engineering and tools for gene editing is presented.}, }
@article {pmid40186294, year = {2025}, author = {Jia, J and Hao, Y and Zhang, L and Cao, X and An, L and Wang, H and Ma, Q and Jin, X and Ma, X}, title = {Development and validation of optimized lentivirus-like particles for gene editing tool delivery with Gag-Only strategy.}, journal = {European journal of medical research}, volume = {30}, number = {1}, pages = {242}, pmid = {40186294}, issn = {2047-783X}, support = {2016YFC1000307//This work was funded by grants from the National Key Research and Development Program of China/ ; }, mesh = {Humans ; *Lentivirus/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; Genetic Vectors/genetics ; HEK293 Cells ; *Gene Transfer Techniques ; *gag Gene Products, Human Immunodeficiency Virus/genetics ; }, abstract = {BACKGROUND: The development of gene editing tools such as CRISPR-Cas9 and base editors (BE) is critical for genetic diseases and cancer. Lentivirus-like particles (LVLPs) grows into an auspicious platform for delivering mRNA or ribonucleic proteins (RNPs) due to it integrates the advantage of viral and non-viral vectors. Current LVLP systems predominantly utilize HIV-Gag and Pol proteins. However, the reverse transcriptase and integrase of Pol, pose risks of genomic integration and potential tumorigenesis. Enhancing the safety of VLP system is essential. This study focuses on improving the LVLP to minimize these risks.<br><br>METHODS: We implemented a Gag-Only strategy, constructing LVLPs with HIV-Gag protein, thereby eliminating the integration risks linked to Pol. By leveraging the interactions between MS2-MCP (MS2 coat protein), PP7 and PP7 BP (PP7 binding protein), and the psi (HIV packaging signal) with HIV-Gag, we encapsulated PAMless andesine base editor (CE-8e-SpRY) mRNA and sgRNA targeting the PD1 start codon (ATG) into the LVLP. Using recombinant lentiviral vector technology, we developed a stable PD1-expressing 293T cell line (PD1-293T) to assess the editing efficiency of LVLP.<br><br>RESULTS: The psi-LVLP demonstrated effective packaging capabilities, achieving 15% base editing efficiency in 293T cells. By optimizing plasmid ratios, we observed increased CE-8e-SpRY mRNA copy numbers, with 30% base editing efficiency. Additionally, the integration of HDVrz (hepatitis delta virus ribozyme) and psi into sgRNA (HDVrz-psi-LVLP) substantially enhanced sgRNA copy numbers, resulting in approximately 50% base editing efficiency in 293T cells and 20% base editing efficiency in Jurkat cells. Mendelian randomization analyses revealed significant genetic correlations between PD1, B2M, CIITA, and TIGIT genes with various cancer risks. Furthermore, HDVrz-psi-LVLPs targeting the start codons of B2M, CIITA, and TIGIT exhibited high base editing activity in both Jurkat and 293T cells.<br><br>CONCLUSION: In conclusion, this optimized platform effectively encapsulates CE-8e-SpRY mRNA and sgRNA, achieving high editing efficiencies across multiple genes and cell types. We introduce a safer and more efficient gene editing tool delivery system by constructing LVLPs based on the Gag-Only strategy. Our study presents a promising implication for cancer immunotherapy.}, }
@article {pmid40186064, year = {2025}, author = {Zainab, R and Mukhtar, A and Saleem, Z and Kaul, H and Ahmad, A and Majeed, M}, title = {Editing gliA, gliP and gliZ of Aspergillus fumigatus Using CRISPR/Cas System Renders Fungus Incapable to Produce Gliotoxin.}, journal = {Molecular biotechnology}, volume = {}, number = {}, pages = {}, pmid = {40186064}, issn = {1559-0305}, support = {13224//Higher Education Commision, Pakistan/ ; }, abstract = {Aspergillus fumigatus is a saprophytic fungus that causes respiratory infections in human, animals, and birds. This fungus produces gliotoxin which is a secondary metabolite that triggers pathogenicity. Gliotoxin is encoded by a 13-gene cluster including gliA, gliP and gliZ. The purpose of this study was to determine whether the fungus produces gliotoxin after these genes are edited using CRISPR/Cas system. For this, crRNAs for gliA, gliP and gliZ were designed using EuPaGDT, while tracrRNA and Cas9 protein were purchased ready-made. These crRNAs were individually annealed with the tracrRNA to make three gRNAs which were then individually combined with the Cas9 to make three ribonucleoprotein (RNP) complexes. A. fumigatus protoplasts were enzymatically generated and transfected with each of the RNP complexes (group 1) in PEGylated conditions. Non-treated protoplasts were simultaneously run as control (group 2). Transfected protoplasts showed reduced growth on SDA plates as compared to their control. Gliotoxin extraction through thin-layer chromatography was carried out for both the groups which showed the absence of gliotoxin in group 1. Sequencing results confirmed the indels in target genes which shows that the CRISPR/Cas9 system effectively targeted A. fumigatus' gliotoxin-related genes that rendered fungus incapable to produce gliotoxin. This work may pave the way to develop effective strategies to control the infections caused by A. fumigatus.}, }
@article {pmid40185749, year = {2025}, author = {Manjunath, L and Santiago, G and Ortega, P and Sanchez, A and Oh, S and Garcia, A and Li, J and Duong, D and Bournique, E and Bouin, A and Semler, BL and Setiaputra, D and Buisson, R}, title = {Cooperative role of PACT and ADAR1 in preventing aberrant PKR activation by self-derived double-stranded RNA.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {3246}, pmid = {40185749}, issn = {2041-1723}, support = {R21 AI185033/AI/NIAID NIH HHS/United States ; RSG-24-1249960-01-DMC//American Cancer Society (American Cancer Society, Inc.)/ ; R01 AI155962/AI/NIAID NIH HHS/United States ; R21 ES036190/ES/NIEHS NIH HHS/United States ; R37 CA252081/CA/NCI NIH HHS/United States ; }, mesh = {*Adenosine Deaminase/metabolism/genetics ; *eIF-2 Kinase/metabolism/genetics ; *RNA, Double-Stranded/metabolism/genetics ; *RNA-Binding Proteins/metabolism/genetics ; Humans ; Animals ; CRISPR-Cas Systems ; Enzyme Activation ; HEK293 Cells ; Mice ; }, abstract = {Double-stranded RNAs (dsRNAs) produced during viral infections are recognized by the innate immune sensor protein kinase R (PKR), triggering a host translation shutoff that inhibits viral replication and propagation. Given the harmful effects of uncontrolled PKR activation, cells must tightly regulate PKR to ensure that its activation occurs only in response to viral infections, not endogenous dsRNAs. Here, we use CRISPR-Translate, a FACS-based genome-wide CRISPR-Cas9 knockout screening method that exploits translation levels as a readout and identifies PACT as a key inhibitor of PKR during viral infection. We find that PACT-deficient cells hyperactivate PKR in response to different RNA viruses, raising the question of why cells need to limit PKR activity. Our results demonstrate that PACT cooperates with ADAR1 to suppress PKR activation from self-dsRNAs in uninfected cells. The simultaneous deletion of PACT and ADAR1 results in synthetic lethality, which can be fully rescued in PKR-deficient cells. We propose that both PACT and ADAR1 act as essential barriers against PKR, creating a threshold of tolerable levels to endogenous dsRNA in cells without activating PKR-mediated translation shutdown and cell death.}, }
@article {pmid40185099, year = {2025}, author = {Lee, JS and Dan, T and Zhang, H and Cheng, Y and Rehfeld, F and Brugarolas, J and Mendell, JT}, title = {An ultraconserved snoRNA-like element in long noncoding RNA CRNDE promotes ribosome biogenesis and cell proliferation.}, journal = {Molecular cell}, volume = {85}, number = {8}, pages = {1543-1560.e10}, pmid = {40185099}, issn = {1097-4164}, support = {P50 CA196516/CA/NCI NIH HHS/United States ; R01 CA282036/CA/NCI NIH HHS/United States ; }, mesh = {*RNA, Long Noncoding/genetics/metabolism ; Humans ; *Cell Proliferation/genetics ; *RNA, Small Nucleolar/genetics/metabolism ; *Ribosomes/metabolism/genetics ; *Carcinoma, Renal Cell/genetics/pathology/metabolism ; *Kidney Neoplasms/genetics/pathology/metabolism ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Cell Nucleolus/metabolism/genetics ; Alternative Splicing ; HEK293 Cells ; RNA, Ribosomal/genetics/metabolism ; Conserved Sequence ; CRISPR-Cas Systems ; }, abstract = {Cancer cells frequently upregulate ribosome production to support tumorigenesis. While small nucleolar RNAs (snoRNAs) are critical for ribosome biogenesis, the roles of other classes of noncoding RNAs in this process remain largely unknown. Here, we performed CRISPR interference (CRISPRi) screens to identify essential long noncoding RNAs (lncRNAs) in renal cell carcinoma (RCC) cells. This revealed that an alternatively spliced isoform of lncRNA colorectal neoplasia differentially expressed (CRNDE) containing an ultraconserved element (UCE), referred to as CRNDE[UCE], is required for RCC cell proliferation. CRNDE[UCE] localizes to the nucleolus and promotes 60S ribosomal subunit biogenesis. The UCE of CRNDE functions as an unprocessed C/D box snoRNA that directly interacts with ribosomal RNA precursors. This facilitates delivery of eukaryotic initiation factor 6 (eIF6), a key 60S biogenesis factor, which binds to CRNDE[UCE] through a sequence element adjacent to the UCE. These findings highlight the functional versatility of snoRNA sequences and expand the known mechanisms through which noncoding RNAs orchestrate ribosome biogenesis.}, }
@article {pmid40185080, year = {2025}, author = {Knight, AL and Lisi, GP}, title = {Spy-ing on nucleic acids: Atomic resolution of the S. pyogenes CRISPR-Cas9 surveillance state.}, journal = {Structure (London, England : 1993)}, volume = {33}, number = {4}, pages = {636-638}, doi = {10.1016/j.str.2025.03.001}, pmid = {40185080}, issn = {1878-4186}, mesh = {*CRISPR-Cas Systems ; *Streptococcus pyogenes/enzymology/genetics ; *DNA/metabolism/chemistry ; *Bacterial Proteins/chemistry/metabolism ; *Endonucleases/chemistry/metabolism ; }, abstract = {In a recent issue of Cell Chemical Biology, De Paula et al.[1] report an extensive methyl-TROSY solution NMR study of the CRISPR-Cas9 holoenzyme. Studying millisecond-to-second protein dynamics using individual domain constructs of Cas9 coupled to structural interrogations of the full-length enzyme, the authors describe the Cas9 "surveillance state," a molecular mechanism driving the discrimination between on- and off-target DNA.}, }
@article {pmid40184725, year = {2025}, author = {Rahimian, E and Koochak, M and Traikov, S and Schroeder, M and Brilloff, S and Schäfer, S and Kufrin, V and Küchler, S and Krüger, A and Mirtschink, P and Baretton, G and Schröck, E and Schewe, DM and Ball, CR and Bornhäuser, M and Glimm, H and Bill, M and Wurm, AA}, title = {A quiescence-like/TGF-β1-specific CRISPRi screen reveals drug uptake transporters as secondary targets of kinase inhibitors in AML.}, journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy}, volume = {81}, number = {}, pages = {101242}, doi = {10.1016/j.drup.2025.101242}, pmid = {40184725}, issn = {1532-2084}, mesh = {Humans ; *Leukemia, Myeloid, Acute/drug therapy/genetics/pathology/mortality ; Cytarabine/pharmacology/therapeutic use ; *Transforming Growth Factor beta1/metabolism/genetics ; *Protein Kinase Inhibitors/pharmacology/therapeutic use ; Drug Resistance, Neoplasm/drug effects/genetics ; Receptor, Transforming Growth Factor-beta Type I/antagonists &amp; inhibitors/genetics ; *Equilibrative Nucleoside Transporter 1/antagonists &amp; inhibitors/genetics/metabolism ; Signal Transduction/drug effects ; Hematopoietic Stem Cells/drug effects/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Prognosis ; }, abstract = {Relapse in acute myeloid leukemia (AML) is driven by resistant subclones that survive chemotherapy. It is assumed that these resilient leukemic cells can modify their proliferative behavior by entering a quiescent-like state, similar to healthy hematopoietic stem cells (HSCs). These dormant cells can evade the effects of cytostatic drugs that primarily target actively dividing cells. Although quiescence has been extensively studied in healthy hematopoiesis and various solid cancers, its role in AML has remained unexplored. In this study, we applied an HSC-derived quiescence-associated gene signature to an AML patient cohort and found it to be strongly correlated with poor prognosis and active TGF-β signaling. In vitro treatment with TGF-β1 induces a quiescence-like phenotype, resulting in a G0 shift and reduced sensitivity to cytarabine. To find potential therapeutic targets that prevent AML-associated quiescence and improve response to cytarabine, we conducted a comprehensive CRISPR interference (CRISPRi) screen combined with TGF-β1 stimulation. This approach identified TGFBR1 inhibitors, like vactosertib, as effective agents for preventing the G0 shift in AML cell models. However, pretreatment with vactosertib unexpectedly induced complete resistance to cytarabine. To elucidate the underlying mechanism, we performed a multi-faceted approach combining a second CRISPRi screen, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and in silico analysis. Our findings revealed that TGFBR1 inhibitors unintentionally target the nucleoside transporter SLC29A1 (ENT1), leading to reduced intracellular cytarabine levels. Importantly, we found that this drug interaction is not unique to TGFBR1 inhibitors, but extends to other clinically significant kinase inhibitors, such as the FLT3 inhibitor midostaurin. These findings may have important implications for optimizing combination therapies in AML treatment.}, }
@article {pmid40184611, year = {2025}, author = {Wu, S and Liu, Y and Zeng, T and Zhou, T and Sun, Y and Deng, Y and Zhang, J and Li, G and Yin, Y}, title = {Enhanced the Trans-Cleavage Activity of CRISPR-Cas12a Using Metal-Organic Frameworks as Stimulants for Efficient Electrochemical Sensing of Circulating Tumor DNA.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {12}, number = {22}, pages = {e2417206}, pmid = {40184611}, issn = {2198-3844}, support = {82202294//National Natural Science Foundation of China/ ; 82272667//National Natural Science Foundation of China/ ; 82372091//National Natural Science Foundation of China/ ; JX21817902/008//Collaborative Innovation Center for Tumor Individualization Program/ ; JSSCBS20211476//Doctoral Program for Entrepreneurship and Innovation of Jiangsu Province/ ; PY2021035//Young Scholars Fostering Fund of the First Affiliated Hospital of Nanjing Medical University/ ; }, mesh = {*Metal-Organic Frameworks/chemistry ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Electrochemical Techniques/methods ; *Circulating Tumor DNA/analysis/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; Limit of Detection ; Bacterial Proteins ; CRISPR-Associated Proteins ; }, abstract = {Continued development of clustered regularly interspaced short palindromic repeats (CRISPR)-powered biosensing system on the electrochemical interface is vital for accurate and timely diagnosis in clinical practice. Herein, an electrochemical biosensor based on manganese metal-organic frameworks (MOFs)-enhanced CRISPR (MME-CRISPR) is proposed that enables the efficient detection of circulating tumor DNA (ctDNA). In this design, customized enzyme stimulants (Mn[2+]) are co-assembled with Cas12a/crRNA to form enzyme-MOF composites, which can be released quickly under mild conditions. The MOFs-induced proximity effect can continuously provide adequate Mn[2+] to sufficiently interact with Cas12a/crRNA during the release process, enhancing the trans-cleavage activity of complex available for biosensor construction. The MOFs-based enzyme biocomposites also afford efficient protection against various external stimulus. It is demonstrated that the developed biosensor can achieve ultrasensitive detection of epidermal growth factor receptor L858R mutation in ctDNA with a low detection limit of 0.28 fm without pre-amplification. Furthermore, the engineered mismatch crRNA enables the biosensor based on MME-CRISPR to detect single nucleotide variant with a high signal-to-noise ratio. More importantly, it has been successfully used to detect the targets in clinical practice, requiring low-dose samples and a short time. This strategy is believed to shed new light on the applications of cancer diagnosis, treatment, and surveillance.}, }
@article {pmid40183844, year = {2025}, author = {Lai, S and Guo, J and Li, X and Yu, X and Lai, G}, title = {Dual DNA recycling amplification-assisted CRISPR/Cas12a cleavage for dual-channel ratiometric fluorescence biosensing of kanamycin antibiotic.}, journal = {Analytical and bioanalytical chemistry}, volume = {417}, number = {14}, pages = {3191-3200}, pmid = {40183844}, issn = {1618-2650}, support = {22076043//National Natural Science Foundation of China/ ; 2023AFD003//Natural Science Foundation of Hubei Province/ ; }, mesh = {*Kanamycin/analysis ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Anti-Bacterial Agents/analysis ; Limit of Detection ; *DNA/chemistry/genetics ; Milk/chemistry ; Fluorescence ; *CRISPR-Associated Proteins/metabolism ; Spectrometry, Fluorescence/methods ; Nucleic Acid Amplification Techniques/methods ; Aptamers, Nucleotide/chemistry ; Animals ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {Fluorescence biosensors hold significant importance for testing antibiotic residues which seriously endanger public health. However, how to adopt appropriate strategies to address the false result disadvantage involved in traditional single-channel biosensors is still a great challenge. Meanwhile, too much attention focused on designing signal amplification strategies of biosensors unavoidably decreases their detection efficiency. Herein, we combined the designed dual DNA recycling amplification strategy with CRISPR/Cas12a-mediated dual-channel signal output mode to successfully develop a novel ratiometric fluorescence biosensor for testing kanamycin (Kana) residues in complex sample matrices. The first recycling was formed from an exonuclease-assisted aptamer recognition reaction, which also triggered another cascade DNA recycling to amplify the release of the Cas12a activator. With the non-discrimination cleavage of Cas12a to cause reverse fluorescence changes of copper nanoclusters and an AMAC-labeled signal DNA, the ratiometric signal transduction strategy was constructed. Under optimal conditions, this biosensor could be applied for ultrasensitive testing of Kana antibiotics in a five-order of magnitude wide linear range with a low detection limit of 17.2 fg mL[-1]. Benefiting from the self-correction function of the ratiometric signal transduction mode, it showed promising practicality in lake water and milk samples with the relative error less than 4.9% to the standard ELISA results. Besides CRISPR/Cas12a-based fluorescence output efficiency improvement, this biosensor also excluded the complicated manipulations and expensive instruments required in traditional methods. Therefore, it provides a good choice for expanding the application of fluorescence biosensing technology for practical analysis application.}, }
@article {pmid40183586, year = {2025}, author = {Arshad, F and Abdillah, AN and Shivanand, P and Ahmed, MU}, title = {Dual-Mode RPA/CRISPR-Cas12a Biosensor Based on Silica and Magnetic Hybrid Nanobeads for Rapid Detection of Campylobacter jejuni.}, journal = {ACS applied bio materials}, volume = {8}, number = {4}, pages = {2977-2984}, doi = {10.1021/acsabm.4c01810}, pmid = {40183586}, issn = {2576-6422}, mesh = {*Biosensing Techniques/methods ; *Silicon Dioxide/chemistry ; CRISPR-Cas Systems ; *Campylobacter jejuni/isolation &amp; purification/genetics ; Nucleic Acid Amplification Techniques ; Particle Size ; *Magnetite Nanoparticles/chemistry ; Colorimetry ; Materials Testing ; Recombinases/metabolism ; Surface Properties ; }, abstract = {In this study, we developed a biosensor that makes use of recombinase polymerase amplification (RPA) along with a CRISPR/Cas12a system integrated with silica nanobeads and a magnetic nanoparticle nanohybrid complex that displayed peroxidase-mimicking properties. This nanohybrid nanozyme (NZ) integration with the CRISPR/Cas system allowed dual-mode fluorometric and colorimetric responses . The nanohybrid NZ was a conjugated ssDNA quencher probe sequence with inherent fluorometric properties. In the presence of target RPA amplicons, the CRISPR/Cas12a system gets activated, cleaving the probe sequence attached to the NZ complex and leading to fluorescence signal generation. Post-CRISPR/Cas12a assay, the presence of the NZ in the reaction mixture, after being cleaved away from the probe sequence, gave a colourimetric response directly proportional to the target DNA concentration, as the ssDNA probe sequence no longer hindered its catalytic activity. Therefore, the dual-mode detection using the CRISPR/Cas12a-based fluorometric response and NZ-based colorimetric detection conferred high sensitivity and selectivity toward Campylobacter detection. The developed sensor could detect the pathogenic DNA at concentrations as low as 0.98 pg/μL and 0.96 pg/μL via fluorescence and absorbance spectroscopy, respectively. In addition, our method was also tested in raw food analysis and showed good recovery.}, }
@article {pmid40183470, year = {2025}, author = {Haley, RM and Padilla, MS and El-Mayta, RD and Joseph, RA and Weber, JA and Figueroa-Espada, CG and Mukalel, AJ and Ricciardi, AS and Palanki, R and Geisler, HC and Jester, MT and Davidson, BL and Mitchell, MJ}, title = {Lipid Nanoparticles for In Vivo Lung Delivery of CRISPR-Cas9 Ribonucleoproteins Allow Gene Editing of Clinical Targets.}, journal = {ACS nano}, volume = {19}, number = {14}, pages = {13790-13804}, doi = {10.1021/acsnano.4c16617}, pmid = {40183470}, issn = {1936-086X}, support = {T90 DE030854/DE/NIDCR NIH HHS/United States ; F99 CA284294/CA/NCI NIH HHS/United States ; T32 GM007170/GM/NIGMS NIH HHS/United States ; F30 HL162465/HL/NHLBI NIH HHS/United States ; DP2 TR002776/TR/NCATS NIH HHS/United States ; P30 CA016520/CA/NCI NIH HHS/United States ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/chemistry/administration &amp; dosage/metabolism ; *Lung/metabolism ; Animals ; *Nanoparticles/chemistry ; Humans ; *Lipids/chemistry ; Mice ; *CRISPR-Associated Protein 9 ; Liposomes ; }, abstract = {In the past 10 years, CRISPR-Cas9 has revolutionized the gene-editing field due to its modularity, simplicity, and efficacy. It has been applied for the creation of in vivo models, to further understand human biology, and toward the curing of genetic diseases. However, there remain significant delivery barriers for CRISPR-Cas9 application in the clinic, especially for in vivo and extrahepatic applications. In this work, high-throughput molecular barcoding techniques were used alongside traditional screening methodologies to simultaneously evaluate LNP formulations encapsulating ribonucleoproteins (RNPs) for in vitro gene-editing efficiency and in vivo biodistribution. This resulted in the identification of a lung-tropic LNP formulation, which shows efficient gene editing in endothelial and epithelial cells within the lung, targeting both model reporter and clinically relevant genomic targets. Further, this LNP shows no off-target indel formation in the liver, making it a highly specific extrahepatic delivery system for lung-editing applications.}, }
@article {pmid40180891, year = {2025}, author = {Cakiroglu, E and Eris, S and Oz, O and Karakülah, G and Senturk, S}, title = {Genome-wide CRISPR screen identifies BUB1 kinase as a druggable vulnerability in malignant pleural mesothelioma.}, journal = {Cell death &amp; disease}, volume = {16}, number = {1}, pages = {241}, pmid = {40180891}, issn = {2041-4889}, support = {117Z227//T&#xfc;rkiye Bilimsel ve Teknolojik Ara&#x15f;tirma Kurumu (Scientific and Technological Research Council of Turkey)/ ; TSA-2024-3533//Dokuz Eyl&#xfc;l &#xdc;niversitesi (Dokuz Eylul University)/ ; }, mesh = {Humans ; *Protein Serine-Threonine Kinases/genetics/metabolism/antagonists &amp; inhibitors ; Cell Line, Tumor ; *Mesothelioma, Malignant/genetics/pathology/drug therapy ; Apoptosis/genetics/drug effects ; Gene Expression Regulation, Neoplastic ; *CRISPR-Cas Systems/genetics ; *Pleural Neoplasms/genetics/pathology/drug therapy ; Cell Proliferation ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Malignant pleural mesothelioma (MPM) is a rare yet highly aggressive malignancy with a severe prognosis. Compounded by the lack of effective treatment modalities, MPM remains a formidable health challenge. Therefore, the identification of actionable liabilities is critical for advancing precision medicine to combat this lethal disease. Here, we exploit an unbiased genome-wide CRISPR screen, integrating and cross-comparing three MPM cell lines with nonmalignant mesothelial cells, to selectively map the gene targets whose depletion indicates a common dependency in MPM cells. This systematic approach unveils a cohort of verifiable genes, among which BUB1, a mitotic checkpoint serine/threonine kinase, emerges as a high-confidence hit in cancer cells. Cellular and molecular studies demonstrate that genetic depletion or pharmacological inhibition of BUB1 profoundly impairs MPM cell survival and growth while inducing G2/M cell cycle arrest, cellular senescence, and apoptosis, and attenuating functional hallmarks of aggressive cancer cells. Transcriptomic profiling of BUB1-depleted cells discloses differential gene expression signatures congruent with cell fate phenotypes, including the reprogramming of mitotic network genes. Mechanistically, BUB1 is indispensable for the proper localization of essential mitotic regulators MAD1, MAD2, and Shugoshin (SGO1), thereby ensuring the functionality of the spindle assembly checkpoint (SAC). Furthermore, BUB1 ablation leads to cytokinesis failure and multinucleation, a phenotype characterized by the downregulation of CDC20, Cyclin A, and Cyclin B, and a reciprocal upregulation of the cyclin-dependent kinase inhibitor p21. Clinically, MPM tumors exhibit elevated levels of BUB1, and high BUB1 expression is associated with shorter patient survival. Our novel findings accentuate comparative CRISPR screens as a powerful platform to explore tumor cell-selective gene essentiality and propose BUB1 kinase as a potential marker and druggable vulnerability with therapeutic implications for MPM.}, }
@article {pmid40180619, year = {2025}, author = {Zhang, D and Wang, W and Tang, M and Qu, C and Jiang, Z and Li, X and Luan, Y}, title = {In Situ Gene Engineering Approach to Overcome Tumor Resistance and Enhance T Cell-Mediated Cancer Immunotherapy.}, journal = {Nano letters}, volume = {25}, number = {15}, pages = {6200-6208}, doi = {10.1021/acs.nanolett.5c00488}, pmid = {40180619}, issn = {1530-6992}, mesh = {Animals ; Interleukin-12/genetics/immunology ; CRISPR-Cas Systems ; *T-Lymphocytes, Cytotoxic/immunology ; *Immunotherapy/methods ; Mice ; Humans ; *Gene Editing ; Cell Line, Tumor ; *Neoplasms/therapy/immunology/genetics/pathology ; Tumor Microenvironment/immunology ; Dendrimers/chemistry ; Endosomal Sorting Complexes Required for Transport/genetics ; Genetic Engineering ; }, abstract = {T cell-mediated cancer immunotherapy harnesses the power of cytotoxic T lymphocytes (CTLs) to target and eradicate tumor cells. However, tumor cells often evade immune attack through membrane repair mechanisms involving endosomal sorting complexes required for transport (ESCRT) and immune suppression within the tumor microenvironment. Here, we developed a robust TMV@PpCHIL nanomedicine to address these issues by reprogramming tumor cells via in situ gene editing. Using CRISPR/Cas9, we disrupted the Chmp4b gene, a key component of the ESCRT machinery, preventing tumor cells from repairing CTL-induced membrane damage. Simultaneously, we genetically engineered tumor cells to produce interleukin-12 (IL-12), a cytokine that enhances CTL activation. The TMV@PpCHIL nanomedicine, designed by coating tumor membrane vesicles (TMVs) onto polyamidoamine (PAMAM) dendrimer-condensed plasmid complexes, ensured efficient CRISPR/Cas9-based gene editing and sustained IL-12 production. This approach significantly enhanced CTL-mediated tumor cell cytotoxicity, suppressed tumor growth, reduced metastasis, and prolonged survival, providing a promising strategy for durable cancer treatment.}, }
@article {pmid40180235, year = {2025}, author = {Zheng, HH and Wang, LQ and Hou, CY and Song, YP and Liu, S and Zheng, LL and Ma, SJ and Chen, HY}, title = {Construction and characterization of a gE/gI/TK-gene-deleted recombinant pseudorabies virus variant expressing the GP5 of the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and NADC30-like PRRSV.}, journal = {Microbial pathogenesis}, volume = {203}, number = {}, pages = {107522}, doi = {10.1016/j.micpath.2025.107522}, pmid = {40180235}, issn = {1096-1208}, mesh = {*Porcine respiratory and reproductive syndrome virus/genetics/immunology/pathogenicity ; Animals ; Swine ; *Viral Envelope Proteins/genetics/immunology ; *Herpesvirus 1, Suid/genetics/immunology ; Antibodies, Viral/blood ; Porcine Reproductive and Respiratory Syndrome/prevention &amp; control/virology/immunology ; *Viral Vaccines/immunology/genetics/administration &amp; dosage ; Antibodies, Neutralizing/blood ; Gene Deletion ; China ; Vaccines, Synthetic/immunology/genetics ; Pseudorabies/prevention &amp; control/virology ; Cell Line ; CRISPR-Cas Systems ; Gene Editing ; }, abstract = {Porcine reproductive and respiratory syndrome (PRRS) and pseudorabies (PR) are still the major problems of the worldwide pork industry. Previous studies showed that PR virus (PRV) and PRRS virus (PRRSV) commercial vaccines available in China could not provide complete protection against PRV variants and currently prevalent PRRSV strains. In the present study, a recombinant pseudorabies virus rPRV-GP5/HP-GP5/NA expressing the GP5 of the highly pathogenic PRRSV (HP-PRRSV) and NADC30-like PRRSV was constructed by transfecting the transfer plasmid pG-GP5/HP-GP5/NA-EGFP into ST cells inoculated with gE/gI/TK-gene-deleted rPRV NY-gE[-]/gI[-]/TK[-] using homologous recombination and CRISPR/Cas9 gene editing technique. The recombinant virus rPRV-GP5/HP was also constructed. The expression of the GP5 protein was confirmed by Western blot and indirect immunofluorescence assay. These two viruses were similar to the parental virus rPRV-gE[-]/gI[-]/TK[-] in terms of growth curve, morphogenesis and virus plaque sizes, and proliferated in different cell types. The animal test results showed that ELISA antibodies against PRRSV could be detected in piglets immunized with these two recombinant viruses, and the antibody levels were slightly lower than those of commercial vaccines, but these two recombinant viruses elicited high levels of PRV ELISA antibody and neutralizing antibody, as is the case with commercial vaccine. These two recombinant viruses could provide some protection against virulent PRRSV and PRV, and could effectively inhibit virus proliferation in tissues. These findings provide insights that these two viruses need to be optimally engineered as promising bivalent vaccine candidates against PRV and PRRSV for the control and eradication of the variant PRV and currently prevalent PRRSV. IMPORTANT: Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine pseudorabies virus (PRV) can infect pigs of all ages with high mortality. Due to the appearance of the PRRSV variant (HP-PRRSV and NADC30-Like-PRRSV) and the outbreak of PRV variant in China, the current commercial vaccines available cannot provide complete protection against PRV variants and prevalent PRRSV strains, which causes a major economic loss in pig industry worldwide. Therefore, safe and effective new vaccines are urgently developed to simultaneously control and even eradicate the two viruses. This study intends to use the modified attenuated PRV strain as the carrier and the main antigen gene ORF5 of PRRSV as the exogenous gene to construct the recombinant virus strain, and further validate the protective effect of recombinant strains in vitro and in vivo against the challenge of PRRSV and PRV, which is expected to become a candidate vaccine strain.}, }
@article {pmid40179700, year = {2025}, author = {Shen, D and Guo, H and Zhang, F and Chen, X and Tong, X and Li, H and Wu, W and Mei, S}, title = {Highly-sensitive and logic platform based on spatially-constrained T7 transcription enhanced Cas13a for DNA repair enzyme detection and intracellular imaging.}, journal = {Biosensors &amp; bioelectronics}, volume = {280}, number = {}, pages = {117406}, doi = {10.1016/j.bios.2025.117406}, pmid = {40179700}, issn = {1873-4235}, mesh = {*DNA-(Apurinic or Apyrimidinic Site) Lyase/analysis/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; Humans ; *Flap Endonucleases/analysis/isolation &amp; purification/genetics ; *CRISPR-Cas Systems/genetics ; *Bacteriophage T7/genetics ; DNA Repair ; Limit of Detection ; Transcription, Genetic ; }, abstract = {The activity of DNA repair enzymes, particularly Flap endonuclease 1 (FEN1) and apurinic/apyrimidinic endonuclease 1 (APE1), plays a critical role in disease prevention, diagnosis, and prognosis. Accurate detection of these enzymes is therefore essential. Recent advancements in CRISPR-Cas technology, particularly its programmable and trans-cleavage activity, have paved the way for the development of innovative detection methods. However, there is a need for a simple, low-background, highly sensitive detection platform with logical capabilities for FEN1 and APE1. In this study, we present a novel detection platform that integrates spatially constrained T7 transcription with the CRISPR-Cas13a system. This biosensor minimizes background interference and achieves high sensitivity, with limits of detection as low as 5 × 10[-7] U/μL for FEN1 and 2 × 10[-8] U/μL for APE1, making it one of the most sensitive methods available for detecting these enzymes. The platform supports both OR and logic detection, offering enhanced versatility. It demonstrates robustness by detecting FEN1 activity at concentrations as low as 1 cell/μL and screening enzyme inhibitors. Additionally, the system was successfully used for intracellular imaging of FEN1 activity in cells and reliably measured APE1 activity in ovarian tissue samples, confirming its clinical applicability. This biosensor represents a promising tool for detecting FEN1 and APE1, further expanding the potential of CRISPR-Cas13a in diagnostic applications.}, }
@article {pmid40179699, year = {2025}, author = {Hu, F and Zhang, Y and Yang, Y and Peng, L and Cui, S and Ma, Q and Wang, F and Wang, X}, title = {A rapid and ultrasensitive RPA-assisted CRISPR-Cas12a/Cas13a nucleic acid diagnostic platform with a smartphone-based portable device.}, journal = {Biosensors &amp; bioelectronics}, volume = {280}, number = {}, pages = {117428}, doi = {10.1016/j.bios.2025.117428}, pmid = {40179699}, issn = {1873-4235}, mesh = {Humans ; *Smartphone/instrumentation ; *SARS-CoV-2/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *COVID-19/diagnosis/virology ; *Nucleic Acid Amplification Techniques/instrumentation ; *Biosensing Techniques/instrumentation ; *COVID-19 Nucleic Acid Testing/instrumentation ; Equipment Design ; Sensitivity and Specificity ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {The spread of infectious diseases can be controlled by early identification of the source of infection and timely diagnosis to stop transmission. Real-time fluorescence quantitative polymerase chain reaction (PCR) is the current gold standard for pathogen diagnosis, with high detection sensitivity and accuracy. However, due to the need for specialized equipment, laboratories, and personnel, it is difficult to achieve rapid and immediate diagnosis during large-scale infectious disease outbreaks. Herein, an optimized CRISPR-based nucleic acid detection method was developed that reduces the CRISPR detection time to 15 min while maintaining high sensitivity. By using nucleic acid extraction-free and lyophilization techniques, the 'sample-in-result-out' detection of the two target genes of SARS-CoV-2, the human internal reference gene, and the negative quality control sample can be completed in 20 min, with a sensitivity of 0.5 copies/μL. Additionally, to facilitate the application, a smartphone-based reverse transcription-recombinase polymerase amplification (RT-RPA)-assisted CRISPR-rapid, portable nucleic acid detection device was developed, integrating functions such as heating, centrifugation, mixing, optical detection and result output. Process control, output, and uploading of detection results were conducted through smartphones. The device is not dependent on a power supply and can perform on-site rapid virus detection in resource-limited settings. Real-time uploading of results helps to rapidly implement epidemic prevention and control measures, providing an innovative means of detection, control, and prevention of virus-based infectious diseases. This important work provides a new and effective tool to manage potential future outbreaks of infectious diseases.}, }
@article {pmid40179192, year = {2025}, author = {Martin-Rufino, JD and Caulier, A and Lee, S and Castano, N and King, E and Joubran, S and Jones, M and Goldman, SR and Arora, UP and Wahlster, L and Lander, ES and Sankaran, VG}, title = {Transcription factor networks disproportionately enrich for heritability of blood cell phenotypes.}, journal = {Science (New York, N.Y.)}, volume = {388}, number = {6742}, pages = {52-59}, pmid = {40179192}, issn = {1095-9203}, support = {R01 CA265726/CA/NCI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R33 HL120791/HL/NHLBI NIH HHS/United States ; R01 DK103794/DK/NIDDK NIH HHS/United States ; R01 HL146500/HL/NHLBI NIH HHS/United States ; R33 CA278393/CA/NCI NIH HHS/United States ; R01 CA292941/CA/NCI NIH HHS/United States ; }, mesh = {Humans ; *Transcription Factors/genetics/metabolism ; *Gene Regulatory Networks ; *Chromatin/metabolism/genetics ; Phenotype ; Single-Cell Analysis ; CRISPR-Cas Systems ; Genome, Human ; Genetic Variation ; *Blood Cells ; Cell Differentiation/genetics ; *Erythropoiesis/genetics ; Erythroid Cells/cytology ; }, abstract = {Most phenotype-associated genetic variants map to noncoding regulatory regions of the human genome, but their mechanisms remain elusive in most cases. We developed a highly efficient strategy, Perturb-multiome, to simultaneously profile chromatin accessibility and gene expression in single cells with CRISPR-mediated perturbation of master transcription factors (TFs). We examined the connection between TFs, accessible regions, and gene expression across the genome throughout hematopoietic differentiation. We discovered that variants within TF-sensitive accessible chromatin regions in erythroid differentiation, although representing <0.3% of the genome, show a ~100-fold enrichment for blood cell phenotype heritability, which is substantially higher than that for other accessible chromatin regions. Our approach facilitates large-scale mechanistic understanding of phenotype-associated genetic variants by connecting key cis-regulatory elements and their target genes within gene regulatory networks.}, }
@article {pmid40178896, year = {2025}, author = {Kim, DY and Lee, SY and Ha, HJ and Park, HH}, title = {AcrIE7 inhibits the CRISPR-Cas system by directly binding to the R-loop single-stranded DNA.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {122}, number = {14}, pages = {e2423205122}, pmid = {40178896}, issn = {1091-6490}, support = {2021R1A2C3003331//National Research Foundation of Korea (NRF)/ ; }, mesh = {*DNA, Single-Stranded/metabolism/chemistry/genetics ; *CRISPR-Cas Systems/genetics ; Protein Binding ; *Bacterial Proteins/metabolism/chemistry/genetics ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; }, abstract = {The CRISPR-Cas system is a well-known adaptive immune system in bacteria, and a prominent mechanism for evading this immunity involves anti-CRISPR (Acr) proteins, which employ various methods to neutralize the CRISPR-Cas system. In this study, using structural and biochemical analyses, we revealed that AcrIE7 binds to the single-stranded DNA in the R-loop formed when Cascade encounters the target DNA, thereby preventing Cas3 from cleaving the DNA. This represents a different inhibition strategy distinct from previously reported Acr mechanisms and offers insights into CRISPR-Cas inhibition.}, }
@article {pmid40177811, year = {2025}, author = {Emran, F and Kays, I and Lo, CA and Li, Y and Chen, BE}, title = {A drug screening platform for protein expression levels in neurological disorders.}, journal = {BioTechniques}, volume = {77}, number = {3}, pages = {113-124}, doi = {10.1080/07366205.2025.2484094}, pmid = {40177811}, issn = {1940-9818}, mesh = {Humans ; *Nervous System Diseases/metabolism/genetics/drug therapy ; Drug Evaluation, Preclinical/methods ; *High-Throughput Screening Assays/methods ; CRISPR-Cas Systems ; Gene Editing ; HEK293 Cells ; *Proteins/genetics/metabolism/analysis ; }, abstract = {Neurological and psychiatric diseases and disorders affect more than half of the population. Many of these diseases are caused by the malfunctioning of protein synthesis, where too little or too much production of a protein harms a cell and its functions within the brain. We developed a drug screening platform to identify compounds that target the primary cause of these diseases, namely protein expression amounts. This cellular assay monitors protein expression of a target disease gene along with the protein expression of a control gene using the Protein Quantitation Ratioing (PQR) technique. PQR tracks protein concentration using fluorescence. We used human cells and CRISPR-Cas9 genome editing to insert the Protein Quantitation Reporter into target genes. These cells are used in high-throughput drug screening measuring the fluorescence as the assay. Drug hits can be validated using the same PQR technique or animal models of the disease.}, }
@article {pmid40177728, year = {2025}, author = {Liu, S and Lun, J and Zhan, Y and Li, Z and Tian, J and Zhang, C and Pan, L}, title = {CRISPR/Cas12a Combined with RAA for On-Site Detection of ALS W574L Mutation in Three Alopecurus Species: A Visual Approach for Herbicide Resistance Monitoring.}, journal = {Journal of agricultural and food chemistry}, volume = {73}, number = {15}, pages = {8907-8914}, doi = {10.1021/acs.jafc.5c02188}, pmid = {40177728}, issn = {1520-5118}, mesh = {*Herbicide Resistance ; CRISPR-Cas Systems ; *Acetolactate Synthase/genetics/metabolism ; *Herbicides/pharmacology ; *Plant Proteins/genetics/metabolism ; Mutation ; *Poaceae/genetics/drug effects/enzymology ; Plant Weeds/genetics/drug effects/enzymology ; }, abstract = {The genus Alopecurus encompasses several weed species, including Alopecurus japonicus, Alopecurus aequalis, and Alopecurus myosuroides, which represent significant threats to agricultural productivity, particularly in wheat and oilseed rape fields. ALS-inhibiting herbicides have been extensively used for controlling Alopecurus weeds. However, the widespread use of these herbicides has led to the rapid emergence of resistance in Alopecurus populations with the Trp-574-Leu (W574L) mutation in the ALS gene being one of the most common resistance mechanisms. This study aims to develop a novel molecular detection method combining recombinase-aided amplification (RAA) with CRISPR/Cas12a technology to detect the W574L mutation in Alopecurus species. The method was optimized for key parameters, balancing efficiency with experimental costs, and was evaluated for specificity, sensitivity, and field applicability. This approach offers a rapid, accurate, and visual tool for identifying W574L target-site resistance in A. japonicus, A. aequalis, and A. myosuroides, with significant potential for monitoring resistance and enhancing weed management strategies.}, }
@article {pmid40176603, year = {2025}, author = {Ham, S and Lee, M and Jeong, D and Son, J and Kim, Y and Lee, T and Ko, K and Moh, SH and Ko, K}, title = {Potential use of human pluripotency-related gene expression reporter cell line for screening small molecules to enhance induction of pluripotency.}, journal = {BMB reports}, volume = {58}, number = {4}, pages = {183-189}, pmid = {40176603}, issn = {1976-670X}, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology/drug effects ; Genes, Reporter/genetics ; Cellular Reprogramming/drug effects/genetics ; Cell Line ; *Small Molecule Libraries/pharmacology ; Octamer Transcription Factor-3/genetics/metabolism ; High-Throughput Screening Assays/methods ; CRISPR-Cas Systems/genetics ; Nanog Homeobox Protein/genetics/metabolism ; Green Fluorescent Proteins/metabolism/genetics ; Fibroblasts/metabolism/cytology ; }, abstract = {The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is a crucial development in regenerative medicine, providing patient-specific cells for therapeutic uses. Traditional methods often utilize viral vectors and transcription factors that pose tumorigenic risks, rendering them unsuitable for clinical applications. This study explored the use of chemicals as a non-tumorigenic alternative for cell reprogramming. Utilizing CRISPR/Cas9 technology, we previously created iPSCs expressing OCT4-EGFP and NANOG-tdTomato, and derived OCT4-EGFP and NANOG-tdTomato fibroblastic cells (ON-FCs). These cells were reprogrammed using episomal vectors, and their pluripotency was validated by fluorescence and FACS analyses. High-content screening was employed to assess small molecules that improve reprogramming efficiency, confirming the usefulness of ON-FCs as a dual reporter cell line for identifying small molecules effective in generating human iPSCs. This study underscores the utility of a dual reporter system and high-content screening in identifying effective reprogramming chemicals, establishing a scalable platform for high-throughput screening. Discovering new chemicals that can reprogram iPSCs would provide a non-tumorigenic method to advance the field of regenerative medicine. [BMB Reports 2025; 58(4): 183-189].}, }
@article {pmid40176181, year = {2025}, author = {Yamamoto, A and Tanaka, Y and Ishibashi, S and Ikeda, M and Sugita, K and Ono, M and Nishi, H and Kurata, M}, title = {Modified screening of MYC promotor region elements using the CRISPR library in ovarian cancer.}, journal = {Journal of ovarian research}, volume = {18}, number = {1}, pages = {68}, pmid = {40176181}, issn = {1757-2215}, support = {21K16800//KAKENHI/ ; }, mesh = {Humans ; Female ; *Ovarian Neoplasms/genetics/pathology ; *Promoter Regions, Genetic ; Proto-Oncogene Mas ; Cell Line, Tumor ; *Proto-Oncogene Proteins c-myc/genetics ; Gene Expression Regulation, Neoplastic ; *Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Gene Library ; }, abstract = {Ovarian cancer remains one of the most lethal gynecological malignancies owing to its high recurrence rate and chemotherapeutic resistance. MYC is a well-known proto-oncogene that is frequently amplified in ovarian cancer and has been implicated in drug resistance. Previously, we established a new promoter-reporter system combined with a CRISPR activation library to identify unknown MYC regulators, and M1AP was identified as a novel MYC regulator. However, considering the insufficient explanation for the absence of guide RNA (gRNA) of MYC, this present study explored methods to prevent the gRNA of MYC itself from binding. This study first modified the promoter-reporter vector to improve its quality, then conducted CRISPR screening and analyzed candidate genes as MYC promoter regulators using next-generation sequencing in OVSAHO ovarian cancer cells. Eighty-six genes had ≥ 1000 reads, and Pearson's correlation coefficient analysis was performed on the cBioPortal of the Cancer Genomics database. Fourteen genes were identified as candidate MYC regulators with positive and significant correlations with MYC. Seven genes, including CYP4v2, ASPH, ANP32D, PCED1A, ABI1, FUZ, and HOOK2, demonstrated significantly higher luciferase activity than the control genes. Four genes, including ABI1, PCED1A, HOOK2, and CYP4v2, activated the MYC promoter, which showed over twofold higher activity than the control when overexpressed using a vector. In conclusion, four genes that activate MYC promoters were identified in an ovarian cancer cell line using the CRISPR library system with a modified promoter-reporter tool. These results will prove helpful in the development of novel treatment strategies for ovarian cancer.}, }
@article {pmid40176174, year = {2025}, author = {Okesanya, OJ and Ahmed, MM and Ogaya, JB and Amisu, BO and Ukoaka, BM and Adigun, OA and Manirambona, E and Adebusuyi, O and Othman, ZK and Oluwakemi, OG and Ayando, OD and Tan, MIRS and Idris, NB and Kayode, HH and Oso, TA and Ahmed, M and Kouwenhoven, MBN and Ibrahim, AM and Lucero-Prisno, DE}, title = {Reinvigorating AMR resilience: leveraging CRISPR-Cas technology potentials to combat the 2024 WHO bacterial priority pathogens for enhanced global health security-a systematic review.}, journal = {Tropical medicine and health}, volume = {53}, number = {1}, pages = {43}, pmid = {40176174}, issn = {1348-8945}, abstract = {BACKGROUND: Antimicrobial resistance (AMR) poses a global health threat, particularly in low- and middle-income countries (LMICs). Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system technology offers a promising tool to combat AMR by targeting and disabling resistance genes in WHO bacterial priority pathogens. Thus, we systematically reviewed the potential of CRISPR-Cas technology to address AMR.<br><br>METHODS: This systematic review adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A comprehensive literature search was conducted using the Scopus and PubMed databases, focusing on publications from 2014 to June 2024. Keywords included "CRISPR/Cas," "antimicrobial resistance," and "pathogen." The eligibility criteria required original studies involving CRISPR/Cas systems that targeted AMR. Data were extracted from eligible studies, qualitatively synthesized, and assessed for bias using the Joanna Briggs Institute (JBI)-standardized tool.<br><br>RESULTS: Data from 48 eligible studies revealed diverse CRISPR-Cas systems, including CRISPR-Cas9, CRISPR-Cas12a, and CRISPR-Cas3, targeting various AMR genes, such as blaOXA-232, blaNDM, blaCTX-M, ermB, vanA, mecA, fosA3, blaKPC, and mcr-1, which are responsible for carbapenem, cephalosporin, methicillin, macrolide, vancomycin, colistin, and fosfomycin resistance. Some studies have explored the role of CRISPR in virulence gene suppression, including enterotoxin genes, tsst1, and iutA in Staphylococcus aureus and Klebsiella pneumoniae. Delivery mechanisms include bacteriophages, nanoparticles, electro-transformation, and conjugative plasmids, which demonstrate high efficiency in vitro and in vivo. CRISPR-based diagnostic applications have demonstrated high sensitivity and specificity, with detection limits as low as 2.7&#x2009;&#xd7;&#x2009;10[2]&#xa0;CFU/mL, significantly outperforming conventional methods. Experimental studies have reported significant reductions in resistant bacterial populations and complete suppression of the targeted strains. Engineered phagemid particles and plasmid-curing systems have been shown to eliminate IncF plasmids, cured plasmids carrying vanA, mcr-1, and blaNDM with 94% efficiency, and restore antibiotic susceptibility. Gene re-sensitization strategies have been used to restore fosfomycin susceptibility in E. coli and eliminate blaKPC-2-mediated carbapenem resistance in MDR bacteria. Whole-genome sequencing and bioinformatics tools have provided deeper insights into CRISPR-mediated defense mechanisms. Optimization strategies have significantly enhanced gene-editing efficiencies, offering a promising approach for tackling AMR in high-priority WHO pathogens.<br><br>CONCLUSIONS: CRISPR-Cas technology has the potential to address AMR across priority WHO pathogens. While promising, challenges in optimizing in vivo delivery, mitigating potential resistance, and navigating ethical-regulatory barriers must be addressed to facilitate clinical translation.}, }
@article {pmid40175930, year = {2025}, author = {Xiao, G and Liu, H and Xu, H and Shi, H and Liu, D and Ou, M and Liu, P and Zhang, G}, title = {Direct detection from sputum for drug-resistant Mycobacterium tuberculosis using a CRISPR-Cas14a-based approach.}, journal = {BMC microbiology}, volume = {25}, number = {1}, pages = {188}, pmid = {40175930}, issn = {1471-2180}, support = {82300128//National Natural Science Foundation of China/ ; 82170009//National Natural Science Foundation of China/ ; 2020YFA0907200//National Key Research and Development Program of China/ ; KCXFZ20211020163545004//Shenzhen Scientific and Technological Foundation/ ; JCYJ20210324130009024//Shenzhen Scientific and Technological Foundation/ ; JCYJ20220530163216036//Shenzhen Scientific and Technological Foundation/ ; RCJC20221008092726022//Shenzhen Scientific and Technological Foundation/ ; 0620220214//Science Fund for Distinguished Young Scholars of Guangdong Province/ ; 2020B1111170014//Science and Technology Planning Project of Guangdong Province/ ; SZZYSM202311009//Sanming Project of Medicine in Shenzen Municipality/ ; }, mesh = {*Sputum/microbiology ; *Mycobacterium tuberculosis/genetics/drug effects/isolation &amp; purification ; Humans ; *CRISPR-Cas Systems ; *Tuberculosis, Multidrug-Resistant/microbiology/diagnosis ; Rifampin/pharmacology ; Microbial Sensitivity Tests ; Sensitivity and Specificity ; Antitubercular Agents/pharmacology ; Isoniazid/pharmacology ; Drug Resistance, Multiple, Bacterial/genetics ; Mutation ; }, abstract = {The increasing prevalence of multidrug-resistant tuberculosis (MDR-TB) highlights the urgent need for an efficient approach to identify Mycobacterium tuberculosis complex (MTBC) strains resistant to rifampicin (RIF) and isoniazid (INH). In response, we developed a CRISPR-Cas14a MTB RIF/INH platform that can detect the most common mutations associated with RIF and INH resistance. To evaluate the sensitivity and specificity of our CRISPR-Cas14a MTB RIF/INH platform, we carried out a comprehensive assessment using clinical isolates of M. tuberculosis and sputum samples from TB patients, making direct comparisons with phenotypic drug susceptibility testing (pDST). A total of 60 clinical isolates from TB patients were utilized, consisting of 18 RIF mono-resistant, 15 INH mono-resistant, 24 MDR isolates, and 3 fully susceptible isolates. Among the 42 RIF-resistant isolates, our platform accurately identified 39, achieving a sensitivity of 93.3% (95% CI, 80.0-98.5) and a specificity of 100% (95% CI, 81.6-100). Similarly, out of the 39 INH-resistant isolates, the platform successfully identified 38, demonstrating a sensitivity of 97.5% (95% CI, 86.5-99.9) and a specificity of 100% (95% CI, 83.8-100) when compared with pDST. Moreover, in the analysis of 55 sputum samples, our platform accurately identified RIF resistance in 10 out of 12 samples (85.7%) and INH resistance in all 11 samples (100%). Notably, excluding the nucleic acid extraction step, the entire testing procedure can be completed in approximately 1.5 h. These results suggest that the CRISPR-Cas14a MTB RIF/INH platform is a reliable and promising novel tool for detecting RIF and INH resistance in isolates or directly from sputum samples.}, }
@article {pmid40175837, year = {2025}, author = {Moreno, DS and Cunha, J and de Melo, LDR and Tanaka, K and Bamba, T and Hasunuma, T and Azeredo, J and Domingues, L}, title = {CRISPR-Cas9 engineered Saccharomyces cerevisiae for endolysin delivery to combat Listeria monocytogenes.}, journal = {Applied microbiology and biotechnology}, volume = {109}, number = {1}, pages = {81}, pmid = {40175837}, issn = {1432-0614}, support = {UIDB/04469/2020//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia/ ; UI/BD/151411/2021//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia/ ; }, mesh = {*Listeria monocytogenes/drug effects ; *Saccharomyces cerevisiae/genetics/metabolism ; *Endopeptidases/genetics/metabolism/pharmacology ; *CRISPR-Cas Systems ; *Anti-Bacterial Agents/pharmacology/metabolism ; Bacteriophages/enzymology/genetics ; Listeriosis/prevention &amp; control/microbiology ; }, abstract = {Listeriosis is an infection caused by the consumption of food contaminated with Listeria monocytogenes. It leads to febrile gastroenteritis, central nervous system infections, and even death in risk populations. Bacteriophage endolysins selectively kill bacteria hydrolyzing their cell walls and have emerged as a potential tool for listeriosis control. Ply511 is an anti-Listeria endolysin that has activity against all serovars of L. monocytogenes. The yeast Saccharomyces cerevisiae has been used to produce endolysins for biocontrol, but prior efforts relied on plasmids, which can lead to gene loss and include selection markers unsuitable for therapeutic use. Integration of endolysins in its genome has also been previously demonstrated, relying however, on selection markers for selection and maintenance of the modifications. This study explores S. cerevisiae as a generally regarded as safe (GRAS) platform for producing and displaying Ply511 through CRISPR-Cas9 integration, offering a marker-free and stable solution for Listeria biocontrol. Our results demonstrate that the surface display of Ply511 does not lead to bacterial reduction. In contrast, we show that yeast secreting endolysin significantly reduces L. monocytogenes in cells, supernatants, and cell extracts. The strongest effect was observed with concentrated spent supernatant and cell extract, which reduced L. monocytogenes below the lower limit of quantification. Additionally, the spent supernatant exhibited active anti-Listeria activity in milk. This study highlights yeast-secreted endolysins as a promising platform for listeriosis control and demonstrates the yeast secretion of endolysins can be used for the biocontrol of pathogenic bacteria. KEY POINTS: • S. cerevisiae was edited using CRISPR-Cas9 to display or secrete endolysin Ply511. • Cells, supernatants, and extracts of yeast secreting Ply511 act against L. monocytogenes. • Demonstrates the yeast-based delivery of endolysins to control L. monocytogenes.}, }
@article {pmid40175650, year = {2025}, author = {Guo, P and Song, S and Niu, Y and Kuang, X and Zhou, D and Zhou, Z and Zhang, Y and Ma, X}, title = {Alternative splicing of bunched confers a dual role in hippo pathway-dependent growth and tumorigenesis.}, journal = {Oncogene}, volume = {44}, number = {24}, pages = {1949-1960}, pmid = {40175650}, issn = {1476-5594}, support = {32322027//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32170824//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32370710//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {Animals ; *Alternative Splicing/genetics ; *Drosophila Proteins/genetics/metabolism ; *Protein Serine-Threonine Kinases/metabolism/genetics ; *Carcinogenesis/genetics ; Signal Transduction/genetics ; Protein Isoforms/genetics ; *Intracellular Signaling Peptides and Proteins/metabolism/genetics ; Drosophila melanogaster/genetics ; YAP-Signaling Proteins ; Hippo Signaling Pathway ; Nuclear Proteins/genetics/metabolism ; Trans-Activators/genetics/metabolism ; Transcription Factors/genetics/metabolism ; Cell Proliferation/genetics ; CRISPR-Cas Systems ; }, abstract = {Alternative splicing is a fundamental mechanism that generates functionally distinct proteins from individual genes, contributing to gene regulation and proteomic diversity. In Drosophila, the bunched (bun) gene, a member of the TSC-22 domain gene family, undergoes alternative splicing, yielding diverse protein isoforms involved in crucial biological processes. Nevertheless, the specific roles and regulatory mechanisms of each isoform remain elusive. Here, we employed CRISPR/Cas9 technology to introduce targeted deletions within the endogenous locus of the bun gene, resulting in the removal of either long or short isoforms. We discovered that the short isoforms demonstrated a growth-suppressive role, whereas the long isoforms exhibited a growth-promoting effect. Surprisingly, the long isoforms exhibited a remarkable dual functionality, as both deletion and amplification of long isoform expression impede the excess growth induced by Hippo pathway inactivation. Mechanistically, ectopically expressed Bun long isoforms act as the transcriptional suppressor by competitively binding to targets' promoter regions in conjunction with Yorkie/Scalloped (Yki/Sd), thereby inhibiting its transcriptional outputs and ultimately leading to the growth suppression. These findings unveil the intricate interaction between distinct spliced isoforms of Bun and oncogenic outcomes, highlighting Bun long isoforms as the critical transcription suppressor regulating Hippo pathway inactivation-mediated growth and tumorigenesis in Drosophila.}, }
@article {pmid40175136, year = {2025}, author = {Adachi, Y}, title = {[Identification of genes regulating human CAR-T cell proliferation by genome-wide CRISPR screening].}, journal = {[Rinsho ketsueki] The Japanese journal of clinical hematology}, volume = {66}, number = {3}, pages = {145-152}, doi = {10.11406/rinketsu.66.145}, pmid = {40175136}, issn = {0485-1439}, mesh = {Humans ; Cell Proliferation ; *T-Lymphocytes/immunology/cytology ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Receptors, Chimeric Antigen/immunology ; Immunotherapy, Adoptive ; }, abstract = {In vivo expansion and long-term maintenance of CAR-T cells are considered to be the hallmark of treatment success after CD19 CAR-T cell therapy. Genome-wide CRISPR screening has emerged as a powerful tool for large-scale gene screens. Genome-wide CRISPR screening revealed that CUL5 gene knockout (KO) improved the proliferation of CD19 CAR-T cells. CUL5KO improved not only the proliferation but also the effector function of CAR-T cells. The JAK-STAT pathway was upregulated in CUL5KO CAR-T cells, and CUL5 was associated with the degradation of JAK3 upon activation through IL-2 signaling. CUL5KO CD19 CAR-T cells efficiently suppressed in vivo tumor progression as compared to control CD19 CAR-T cells.}, }
@article {pmid40174865, year = {2025}, author = {Bardani, E and Katsarou, K and Mitta, E and Andronis, C and Štefková, M and Wassenegger, M and Kalantidis, K}, title = {Broadening the Nicotiana benthamiana research toolbox through the generation of dicer-like mutants using CRISPR/Cas9 approaches.}, journal = {Plant science : an international journal of experimental plant biology}, volume = {356}, number = {}, pages = {112490}, doi = {10.1016/j.plantsci.2025.112490}, pmid = {40174865}, issn = {1873-2259}, mesh = {*Nicotiana/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/physiology ; *Caspase 9/genetics/metabolism ; Streptococcus pyogenes/genetics/metabolism ; *RNA Interference ; Stress, Physiological ; Epigenomics ; Cell Cycle Proteins/genetics ; *CRISPR-Cas Systems/genetics ; Genes, Plant/physiology ; Plant Proteins/genetics/metabolism ; *RNA, Plant/genetics ; }, abstract = {RNA silencing in plants plays a pivotal role in various biological processes, including development, epigenetic modifications and stress response. Key components of this network are Dicer-like (DCL) proteins. Nicotiana benthamiana encodes four DCLs, each responsible for the generation of distinct small RNA (sRNA) populations, which regulate different functions. However, elucidating the precise role of each DCL has been proven challenging, as overlapping functions exist within DCLs. In our present study, we have successfully generated dcl2, dcl3 and dcl4 homozygous mutants, employing two different CRISPR/Cas9 approaches. The first approach is based on a transgene-mediated delivery of the single-guide RNA (sgRNA), while the second approach employs a viral vector for sgRNA delivery. By utilizing a suite of screening techniques, including polymerase chain reaction (PCR), T7 endonuclease I (T7E1) assay, high-resolution melt analysis (HRMA) and DNA sequencing, we successfully generated dcl2, dcl3 and dcl4 homozygous mutants harboring identical mutations in every allele. To evaluate these dcl mutants, we examined their sRNA profiles and phenotypes. We further have indications that homozygous mutations of a gene do not always lead to the desired loss-of-function, highlighting the importance of mutant evaluation. dcl mutants represent invaluable tools to explore how overlapping silencing pathways are connected to essential plant functions, including development, stress responses and pathogen defense. Additionally, they hold potential for biotechnological applications, such as crop improvement and gene silencing tools. We anticipate that our study will make significant contributions to enhance understanding of the role of DCLs in plants.}, }
@article {pmid40174713, year = {2025}, author = {Inada, K and Ohde, T and Daimon, T}, title = {Efficient transgenic system for the firebrat Thermobia domestica utilizing hyPBase and G0 founder prescreening.}, journal = {Gene}, volume = {955}, number = {}, pages = {149449}, doi = {10.1016/j.gene.2025.149449}, pmid = {40174713}, issn = {1879-0038}, mesh = {Animals ; *Animals, Genetically Modified/genetics ; Gene Editing/methods ; CRISPR-Cas Systems ; *Transposases/genetics ; Transgenes ; Green Fluorescent Proteins/genetics ; *Gene Transfer Techniques ; *Diptera/genetics ; Female ; }, abstract = {The firebrat, Thermobia domestica, is an apterygote model insect with favorable traits, including rapid generation turnover, high fecundity, and ease of laboratory rearing. We previously developed a method for embryo injection and CRISPR/Cas9-based genome editing in Thermobia. In the present study, we established a highly efficient transgenesis system using the hyperactive piggyBac transposase (hyPBase) to expand genetic manipulation techniques in Thermobia. By injecting embryos with a mixture of hyPBase mRNA and a donor plasmid expressing GFP under the control of an eye enhancer, we achieved the first successful transgenesis in Thermobia. Eye-specific GFP expression was observed in 5.7 % of G0 individuals hatched from injected eggs. Notably, these GFP-positive G0 founders exhibited significantly elevated germline transmission rates (53.3 %) compared with GFP-negative G0 founders (19.0 %). Additionally, a significant difference in the proportion of G1 transgenic progeny emerged between the GFP-positive and GFP-negative G0 groups (20.0 % vs. 2.7 %), highlighting the utility of GFP expression as a predictor of transgenic G1 offspring from injected G0 founders. Furthermore, multiple transgene insertions mediated by hyPBase contributed to the increased transformation efficiency observed in G0 founders with high transmission rates. Our findings offer valuable genetic toolkits for Thermobia that will facilitate advanced research on fundamental biological processes, such as the evolution of wings and metamorphosis.}, }
@article {pmid40174266, year = {2025}, author = {Khan, TG and Bragazzi Cunha, J and Raut, C and Burroughs, M and Vyas, HS and Leix, K and Goonewardena, SN and Smrcka, AV and Speliotes, EK and Emmer, BT}, title = {Functional interrogation of cellular Lp(a) uptake by genome-scale CRISPR screening.}, journal = {Atherosclerosis}, volume = {403}, number = {}, pages = {119174}, pmid = {40174266}, issn = {1879-1484}, support = {R01 DK131787/DK/NIDDK NIH HHS/United States ; R01 HL167733/HL/NHLBI NIH HHS/United States ; R35 GM127303/GM/NIGMS NIH HHS/United States ; K08 HL148552/HL/NHLBI NIH HHS/United States ; R01 HL171013/HL/NHLBI NIH HHS/United States ; R01 DK128871/DK/NIDDK NIH HHS/United States ; }, mesh = {Humans ; *Lipoprotein(a)/metabolism/blood/genetics ; *Receptors, LDL/genetics/metabolism ; Ubiquitin-Protein Ligases/genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; United Kingdom ; *Hepatocytes/metabolism ; }, abstract = {BACKGROUND AND AIMS: An elevated level of lipoprotein(a), or Lp(a), in the bloodstream has been causally linked to the development of atherosclerotic cardiovascular disease and calcific aortic valve stenosis. Steady state levels of circulating lipoproteins are modulated by their rate of clearance, but the identity of the Lp(a) uptake receptor(s) has been controversial.<br><br>METHODS: We performed a genome-scale CRISPR screen to functionally interrogate all potential Lp(a) uptake regulators in HuH7 cells. Screen validation was performed by single gene disruption and overexpression. Direct binding between purified lipoproteins and recombinant protein was tested using biolayer interferometry. An association between human genetic variants and circulating Lp(a) levels was analyzed in the UK Biobank cohort.<br><br>RESULTS: The top positive and negative regulators of Lp(a) uptake in our screen were LDLR and MYLIP, encoding the LDL receptor and its ubiquitin ligase IDOL, respectively. We also found a significant correlation for other genes with established roles in LDLR regulation. No other gene products, including those previously proposed as Lp(a) receptors, exhibited a significant effect on Lp(a) uptake in our screen. We validated the functional influence of LDLR expression on HuH7 Lp(a) uptake, confirmed in vitro binding between the LDLR extracellular domain and purified Lp(a), and detected an association between loss-of-function LDLR variants and increased circulating Lp(a) levels in the UK Biobank cohort.<br><br>CONCLUSIONS: Our findings support a central role for the LDL receptor in mediating Lp(a) uptake by hepatocytes.}, }
@article {pmid40173419, year = {2025}, author = {Towell, SE and Jareczek, MJ and Cooke, LS and Godfrey, DR and Zhukhovitskiy, AV}, title = {Skeletal Editing of Polymer Backbones and Its Impact Across the Polymer Lifecycle.}, journal = {Accounts of chemical research}, volume = {58}, number = {8}, pages = {1275-1283}, doi = {10.1021/acs.accounts.5c00054}, pmid = {40173419}, issn = {1520-4898}, mesh = {*Polymers/chemistry ; CRISPR-Cas Systems ; }, abstract = {ConspectusIn the last five years, interest in the precise modification of molecular cores─termed skeletal editing─has rapidly expanded in the Chemistry community. Beyond the intrinsic value of these transformations, skeletal editing also has value in the attention it brings to under-explored chemical challenges, whose solutions could transform the practice of Chemistry at large. In few contexts does this perspective ring as true as in the realm of polymers. Inspired by the revolutionary power of biologically derived machinery called CRISPR-Cas9 to edit nucleic acid polymers and, consequently, the genetic meaning encoded in them, we envisioned that skeletal editing of synthetic polymer backbones may also enable control over the structure and "meaning"─i.e., properties and function─of plastics. However, the idea of editing polymer backbones brings about numerous fundamental chemical questions that must be answered to make the vision a reality: for instance, how to constructively activate carbon-carbon and carbon-heteroatom bonds that make up typical polymer backbones and how to do so in a site-selective manner? While many fundamental questions have begun to be answered by the small molecule community, they are yet to be applied to the realm of polymers, and such adaptation often begets new scientific challenges. Moreover, as we begin to tackle these questions, we must always consider how advances in skeletal editing of polymer backbones impact the broader contexts of applications and sustainability of plastics.In this Account, we summarize our efforts to advance the skeletal editing of polymer backbones, focusing on how such methods can affect each stage of the polymer lifecycle: (1) provide an entry to previously challenging-to-access functional polymers or to existing ones but from new feedstocks, (2) evolve one type of polymer into another with associated changes in material properties, and (3) enable the breakdown of otherwise intractable polymer backbones. Along the way, we describe our rationale behind the selection and development of reactions utilized for skeletal editing. We explain how small molecule reactions often need to be adapted to suit polymeric substrates and the methodology optimizations we needed to do to accomplish our edits. We also discuss the considerations involved in the selection or design of polymeric substrates for editing with an eye toward what edits can add to polymer function and how to advance the field. We conclude with an outlook on outstanding challenges that we aim to address in future work establishing areas for future exploration within each of our topic areas.}, }
@article {pmid40173337, year = {2025}, author = {Kandlbinder, A and Peter-Spiess, MH and Leeners, B and Mollaysa, A and Cavazza, T and Meier, A and Braunschweig, M and Ioannidi, E and Schwank, G and Krauthammer, M}, title = {Strategies for Interdisciplinary Human Gene Editing Research: Insights from a Swiss Project.}, journal = {The CRISPR journal}, volume = {8}, number = {2}, pages = {79-88}, pmid = {40173337}, issn = {2573-1602}, support = {214936/SNSF_/Swiss National Science Foundation/Switzerland ; }, mesh = {*Gene Editing/methods/ethics ; Humans ; CRISPR-Cas Systems ; Genetic Therapy/methods ; *Interdisciplinary Research ; Switzerland ; }, abstract = {CRISPR gene editing is a cutting-edge technology that has advanced tremendously in recent years. The first clinical CRISPR applications have been approved, and more gene editing therapies are to be expected in human medicine. Consequently, continuous basic research is needed to assess possibilities and prime future clinical applications. Because this technology not only offers new possibilities for treating diseases but also raises important ethical and societal questions, collaboration between human, life, biomedical, and medical sciences is needed. In this article, we discuss the practical challenges of such interdisciplinary projects and present strategies for addressing them based on our experience of conducting an interdisciplinary project on CRISPR. This work aims to help and encourage interdisciplinary collaborations and discussions on modern scientific endeavors that, such as gene editing, tend to blur the lines between traditional disciplines. The strategies suggested include realistic expectations, shared goals, space setting, and expert and lay dialogue.}, }
@article {pmid40173086, year = {2025}, author = {Liu, X and Zhou, E and Qi, Q and Xiong, W and Tian, T and Zhou, X}, title = {Innovative Chemical Strategies for Advanced CRISPR Modulation.}, journal = {Accounts of chemical research}, volume = {58}, number = {8}, pages = {1262-1274}, doi = {10.1021/acs.accounts.5c00052}, pmid = {40173086}, issn = {1520-4898}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism ; Metal-Organic Frameworks/chemistry ; Humans ; }, abstract = {ConspectusOver the past decade, RNA-guided gene editing technologies, particularly those derived from CRISPR systems, have revolutionized life sciences and opened unprecedented opportunities for therapeutic innovation. Despite their transformative potential, achieving precise control over the activity and specificity of these molecular tools remains a formidable challenge, requiring advanced and innovative regulatory strategies. We and others have developed new approaches that integrate chemical ingenuity with bioorthogonal techniques to achieve remarkable precision in CRISPR regulation. One key innovation lies in the chemical modulation of guide RNA (gRNA), significantly expanding the CRISPR toolkit. Strategies such as CRISPR-ON and CRISPR-OFF switches rely on selective chemical masking and demasking of gRNA. These approaches use either bulky chemical groups to preemptively mask RNA or minor, less obstructive groups to fine-tune its function, followed by bioorthogonal reactions to restore or suppress activity. These methodologies have proven to be pivotal for controlled gene editing and expression, addressing the challenges of precision, reversibility, and dynamic regulation.Parallel to these advances, the development of mesoporous metal-organic frameworks (MOFs) has emerged as a promising solution for RNA deprotection and activation. By serving as catalytic tools, MOFs enhance the versatility and efficiency of CRISPR systems, pushing their applications beyond the conventional boundaries. In addition, the synthesis of novel small molecules for regulating CRISPR-Cas9 activity marks a critical milestone in the evolution of gene therapy protocols. Innovative RNA structural control strategies have also emerged, particularly through the engineering of G-quadruplex (G4) motifs and G-G mismatches. These methods exploit the structural propensities of engineered gRNAs, employing small-molecule ligands to induce specific conformational changes that modulate the CRISPR activity. Whether stabilizing G4 formation or promoting G-G mismatches, these strategies demonstrate the precision and sophistication required for the molecular-level control of gene editing.Further enhancing these innovations, techniques like host-guest chemistry and conditional diacylation cross-linking have been developed to directly alter gRNA structure and function. These approaches provide nuanced, reversible, and safe control over CRISPR systems, advancing both the precision and reliability of gene editing technologies. In conclusion, this body of work highlights the convergence of chemistry, materials science, and molecular biology to create integrative solutions for gene editing. By combination of bioorthogonal chemistry, RNA engineering, and advanced materials, these advancements offer unprecedented accuracy and control for both fundamental research and therapeutic applications. These innovations not only advance genetic research but also contribute to developing safer and more effective gene editing strategies, moving us closer to realizing the full potential of these technologies.}, }
@article {pmid40172964, year = {2025}, author = {Patel, AC and Sinha, S and Arantes, PR and Palermo, G}, title = {Unveiling Cas8 dynamics and regulation within a transposon-encoded Cascade-TniQ complex.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {122}, number = {14}, pages = {e2422895122}, pmid = {40172964}, issn = {1091-6490}, support = {R01 GM141329/GM/NIGMS NIH HHS/United States ; R01GM141329//HHS | National Institutes of Health (NIH)/ ; TC-24-063//Camille and Henry Dreyfus Foundation (Dreyfus Foundation)/ ; FG-2023-20431//Alfred P. Sloan Foundation (APSF)/ ; CHE-2144823//National Science Foundation (NSF)/ ; }, mesh = {*Vibrio cholerae/genetics/metabolism ; *DNA Transposable Elements/genetics ; *Bacterial Proteins/metabolism/chemistry/genetics ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; Protein Binding ; CRISPR-Cas Systems ; Protein Conformation ; }, abstract = {The Vibrio cholerae Cascade-TniQ complex unveiled a new paradigm in biology, demonstrating that CRISPR-associated proteins can direct DNA transposition. Despite the tremendous potential of "knocking-in" genes at desired sites, the mechanisms underlying DNA binding and transposition remain elusive. In this system, a conformational change of the Cas8 protein is essential for DNA binding, yet how it occurs is unclear. Here, structural modeling and free energy simulations reconstruct the Cas8 helical bundle and reveal an open-closed conformational change that is key for the complex's function. We show that when Cascade-TniQ binds RNA, the Cas8 bundle changes conformation mediated by the interaction with the Cas7.1 protein. This interaction promotes the bundle's transition toward the open state, priming the complex for DNA binding. As the target DNA binds the guide RNA, the opening of the Cas8 bundle becomes more favorable, exposing positively charged residues and facilitating their interaction with DNA, which ultimately leads the DNA-binding process to completion. These outcomes provide a dynamic representation of a critical conformational change in one of the largest CRISPR systems and illustrate its role at critical steps of the Cascade-TniQ biophysical function, advancing our understanding of nucleic acid binding and transposition mechanisms.}, }
@article {pmid40172925, year = {2025}, author = {Grubben, J and Bijsterbosch, G and Aktürk, B and Visser, RGF and Schouten, HJ}, title = {CRISPR/Cas9-induced breaks are insufficient to break linkage drag surrounding the ToMV locus of Solanum lycopersicum.}, journal = {G3 (Bethesda, Md.)}, volume = {15}, number = {6}, pages = {}, pmid = {40172925}, issn = {2160-1836}, support = {GSGT.2019.016//NWO Graduate School Groene Topsectoren/ ; TU18048//Dutch Topsector Horticulture &amp; Starting Materials/ ; //BASF Nunhems/ ; //Bejo Zaden/ ; //HZPC/ ; //KWS/ ; //SESVanderHave/ ; //Syngenta/ ; //Wageningen University &amp; Research, Plant Breeding/ ; }, mesh = {*CRISPR-Cas Systems ; *Solanum lycopersicum/genetics/virology ; Gene Editing ; *Genetic Linkage ; Polymorphism, Single Nucleotide ; DNA Breaks, Double-Stranded ; *Genetic Loci ; Recombination, Genetic ; Alleles ; }, abstract = {Despite the success of CRISPR/Cas9 in inducing DNA double-strand breaks for genome editing, achieving targeted recombination in somatic cells remains challenging, particularly at recombination cold spots like the tomato mosaic virus (ToMV) resistance locus in Solanum lycopersicum. We investigated the potential of CRISPR/Cas9-induced targeted recombination in somatic cells to overcome linkage drag surrounding the ToMV locus. We employed two strategies: first, inducing double-strand breaks in both alleles of F1 tomato seedlings to promote nonhomologous end joining and homology-directed repair; second, targeting a single allele in a heterozygous background to induce homology-directed repair in seedlings. CRISPR/Cas9 activity was confirmed in F1 seedlings by detecting nonhomologous end joining-mediated mutations at the target sites in ToMV. We developed a bioinformatics pipeline to identify targeted recombinants by analyzing SNPs between parental haplotypes, allowing precise tracking of SNP variations. A two-dimensional pooling strategy was employed to distinguish genuine recombination events from PCR artifacts. Despite these advances and the active CRISPR/Cas9 system in F1 progeny, no reliable targeted recombinations were found. We extended our research to protoplasts to assess whether CRISPR/Cas9 could induce targeted recombination under different cellular conditions at the same locus. Consistent with our findings in F1 plants, we observed no increase in recombinant patterns compared to wild-type controls in protoplasts. Our findings suggest that CRISPR/Cas9-induced DSBs were insufficient to break the genetic linkage at the ToMV locus on chromosome 9 at a detectable level.}, }
@article {pmid40172222, year = {2025}, author = {Rimple, PA and Olafsson, EB and Markus, BM and Wang, F and Augusto, L and Lourido, S and Carruthers, VB}, title = {Metabolic adaptability and nutrient scavenging in Toxoplasma gondii: insights from ingestion pathway-deficient mutants.}, journal = {mSphere}, volume = {10}, number = {4}, pages = {e0101124}, pmid = {40172222}, issn = {2379-5042}, support = {R01 AI144369/AI/NIAID NIH HHS/United States ; R01AI144369//National Institute of Allergy and Infectious Diseases/ ; //Boehringer Ingelheim Fonds/ ; R01AI120607//National Institute of Allergy and Infectious Diseases/ ; P20GM130447/GM/NIGMS NIH HHS/United States ; P20 GM130447/GM/NIGMS NIH HHS/United States ; R01 AI120607/AI/NIAID NIH HHS/United States ; }, mesh = {*Toxoplasma/genetics/metabolism ; Protozoan Proteins/genetics/metabolism ; Mutation ; Vacuoles/parasitology ; *Nutrients/metabolism ; Endocytosis ; CRISPR-Cas Systems ; }, abstract = {UNLABELLED: The obligate intracellular parasite Toxoplasma gondii replicates within a specialized compartment called the parasitophorous vacuole (PV). Recent work showed that despite living within a PV, Toxoplasma endocytoses proteins from the cytosol of infected host cells via a so-called ingestion pathway. The ingestion pathway is initiated by dense granule protein GRA14, which binds host endosomal sorting complex required for transport (ESCRT) machinery to bud vesicles into the lumen of the PV. The protein-containing vesicles are internalized by the parasite and trafficked to the plant vacuole-like compartment (PLVAC), where cathepsin protease L (CPL) degrades the cargo, and the chloroquine resistance transporter (CRT) exports the resulting peptides and amino acids to the parasite cytosol. However, although the ingestion pathway was proposed to be a conduit for nutrients, there is limited evidence for this hypothesis. We reasoned that if Toxoplasma uses the ingestion pathway to acquire nutrients, then parasites lacking GRA14, CPL, or CRT should rely more on biosynthetic pathways or alternative scavenging pathways. To explore this, we conducted a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screen in wild-type (WT) parasites and Δgra14, Δcpl, and Δcrt mutants to identify genes that become more fitness conferring in ingestion-deficient parasites. Our screen revealed a significant overlap of genes that potentially become more fitness conferring in the ingestion mutants compared to WT. Pathway analysis indicated that Δcpl and Δcrt mutants relied more on pyrimidine biosynthesis, fatty acid biosynthesis, tricarboxylic acid (TCA) cycle, and lysine degradation. Bulk metabolomic analysis showed reduced levels of glycolytic intermediates and amino acids in the ingestion mutants compared to WT, highlighting the pathway's potential role in host resource scavenging. Interestingly, Δcpl and Δcrt showed an exacerbated growth defect when cultured in amino acid-depleted media, suggesting that disrupting proteolysis or the export of proteolytic products from the PLVAC affects parasite survival during nutrient scarcity.<br><br>IMPORTANCE: Toxoplasma gondii is an obligate intracellular pathogen that infects virtually any nucleated cell in most warm-blooded animals. Infections are asymptomatic in most cases, but people with weakened immunity can experience severe disease. For the parasite to replicate within the host, it must efficiently acquire essential nutrients, especially as it is unable to make several key metabolites. Understanding the mechanisms by which Toxoplasma scavenges nutrients from the host is crucial for identifying potential therapeutic targets. Our study suggests that the ingestion pathway contributes to sustaining parasite metabolites and parasite replication under amino acid-limiting conditions. This work advances our understanding of the metabolic adaptability of Toxoplasma.}, }
@article {pmid40172186, year = {2025}, author = {Watson, RG and Hole, CR}, title = {Simple growth conditions improve targeted gene deletion in Cryptococcus neoformans.}, journal = {mSphere}, volume = {10}, number = {4}, pages = {e0107024}, pmid = {40172186}, issn = {2379-5042}, support = {K22 AI148724/AI/NIAID NIH HHS/United States ; 1K22 AI148724//National Institute of Allergy and Infectious Diseases/ ; }, mesh = {*Cryptococcus neoformans/genetics/growth &amp; development ; *Gene Deletion ; CRISPR-Cas Systems ; *Culture Media/chemistry ; Fungal Proteins/genetics ; Transformation, Genetic ; Electroporation ; }, abstract = {Cryptococcus neoformans infections are a significant cause of morbidity and mortality among AIDS patients and the third most common invasive fungal infection in organ transplant recipients. The cryptococcal cell wall is very dynamic and can be modulated depending on growth conditions. It was reported that when C. neoformans is grown in unbuffered yeast nitrogen base (YNB) for 48 hours, the pH of the media drastically drops, and the cells start to shed their cell walls. With this observation, we sought to determine if YNB-grown cells could be used directly for genetic transformation. To test this, we targeted ADE2 using TRACE (transient CRISPR-Cas9 coupled with electroporation) in YNB-grown or competent cells. Deletion of the ADE2 gene results in red-pigmented colonies, allowing visual confirmation of disruption. We were able to successfully delete ADE2 in YNB-grown cells with better efficiency compared to competent cells. Recent studies have shown that gene deletion can be accomplished using short (50 bp) homology arms in place of the normal long arms (~1 kb). However, it was inefficient, leading to more insertions and gene disruption than gene deletions. We tested short homology with YNB-grown cells vs. competent cells and found that gene deletion was significantly improved in YNB-grown cells, at around 60% compared to 6% in competent cells. This was also observed when we deleted LAC1 with the short arms. Altogether, using simple growth conditions, we have greatly improved the speed and efficiency of cryptococcal genetic transformations.IMPORTANCEThe World Health Organization recently ranked C. neoformans as the highest-priority fungal pathogen based on unmet research and development needs and its public health importance. Understanding cryptococcal pathogenicity is key for developing treatments. We found that using simple growth conditions can greatly improve the speed and efficiency of cryptococcal genetic transformations. This finding will advance the field by expanding the ease of cryptococcal genetic manipulations.}, }
@article {pmid40172074, year = {2025}, author = {Boccacci, Y and Dumont, N and Doyon, Y and Laganière, J}, title = {CRISPR-Cas9-driven antigen conversion of clinically relevant blood group systems.}, journal = {Human molecular genetics}, volume = {34}, number = {12}, pages = {1001-1008}, pmid = {40172074}, issn = {1460-2083}, support = {//Centre de recherche du CHU de Qu&#xe9;bec-Universit&#xe9; Laval/ ; //Facult&#xe9; de m&#xe9;decine de l'Universit&#xe9; Laval, Mitacs and H&#xe9;ma-Qu&#xe9;bec/ ; }, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Erythrocytes/immunology/metabolism ; *Blood Group Antigens/genetics/immunology ; *ABO Blood-Group System/genetics/immunology ; Rh-Hr Blood-Group System/genetics/immunology ; Hematopoietic Stem Cells/metabolism/immunology ; Blood Transfusion ; }, abstract = {The common practice of blood transfusion entirely relies on blood donations from the population. Ensuring blood group compatibility between a donor and a recipient is paramount to prevent critical adverse reactions. Finding compatible blood can be challenging given the high diversity of blood group antigens, especially for chronically transfused patients at higher risk of alloimmunization owing to repeated exposures to foreign RBCs. In addition, due to the immunogenicity of the ABO blood group and the highly polymorphic nature of the Rhesus (Rh) system, they both remain of prime importance in transfusion medicine. Cultured red blood cells (cRBCs) may eventually provide an alternative for blood donations-at least in some circumstances. Combining cRBCs with blood group gene editing could broaden transfusion accessibility by making antigen expression compatible with rare phenotypes, thus meeting the needs of more patients. Starting from mobilized, erythroid-primed hematopoietic stem and progenitor cells (HSPCs), we used virus- and selection-free, CRISPR-Cas9-mediated knockouts to produce erythroid cells devoid of AB and Rh antigen. The approach yielded almost complete conversion to O- and RhNull phenotypes, as determined by standard hemagglutination and flow cytometry analyses. Combined with robust cRBC protocols, these clinically relevant phenotypic changes could eventually expand the accessibility of blood transfusion for specific and unmet clinical needs.}, }
@article {pmid40172020, year = {2025}, author = {Cheng, HJ and Liu, Y and Li, HD and Yin, BC and Ma, PQ and Ye, BC}, title = {Catalytic hairpin assembly-coupled CRISPR/Cas12a biosensor for sensitive detection of melamine in dairy products.}, journal = {Chemical communications (Cambridge, England)}, volume = {61}, number = {34}, pages = {6300-6303}, doi = {10.1039/d5cc00114e}, pmid = {40172020}, issn = {1364-548X}, mesh = {*Triazines/analysis ; *Biosensing Techniques/methods ; *Dairy Products/analysis ; *CRISPR-Cas Systems ; Catalysis ; *Food Contamination/analysis ; Limit of Detection ; }, abstract = {We combined catalytic hairpin assembly (CHA) with the Cas12a system for detecting melamine adulteration. This system involved two-step signal conversion and two-level amplification, boosting the sensor's versatility and sensitivity. The sensor showed excellent specificity and applicability for melamine detection in dairy products, and was broadened to viral nucleic acid detection.}, }
@article {pmid40171918, year = {2025}, author = {Wu, W and Miao, R and Li, Z and Fang, Z}, title = {CRISPR/Cas9-mediated editing of BADH2 and Wx genes for the development of novel aromatic and soft-textured black and red rice.}, journal = {Physiologia plantarum}, volume = {177}, number = {2}, pages = {e70194}, doi = {10.1111/ppl.70194}, pmid = {40171918}, issn = {1399-3054}, support = {Qiankehezhongyindi (2023) 008//the Key Laboratory of Molecular Breeding for Grain and Oil Crops in Guizhou Province/ ; qiankehechengguo (2024) General 116//the Guizhou Provincial Science and Technology Projects/ ; qiankehejichu-ZK (2021) General 128//the Guizhou Provincial Science and Technology Projects/ ; qiankehejichu-ZK (2022) Key 008//the Guizhou Provincial Science and Technology Projects/ ; qiankehepingtai-YWZ (2024) 004//the Guizhou Provincial Science and Technology Projects/ ; qiankehepingtairencai-BQW (2024) 001//the Guizhou Provincial Science and Technology Projects/ ; Qiandongnan Kehe Support (2023)06//the Qiandongnan Science and Technology Support Project/ ; Qianjiaoji (2023)007//the Key Laboratory of Functional Agriculture of Guizhou Provincial Department of Education/ ; qiankehepingtairencai-YQK (2023) 002//the Guizhou Provincial Excellent Young Talents Project of Science and Technology/ ; 32260498/32060064//the National Natural Science Foundation of China/ ; }, mesh = {*Oryza/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Plant Proteins/genetics/metabolism ; Amylose/metabolism ; Plants, Genetically Modified ; Gas Chromatography-Mass Spectrometry ; Genes, Plant ; Odorants ; Pyrroles ; }, abstract = {Black and red rice are known for their rich nutritional content, yet most varieties suffer from a firm texture and insufficient fragrance. In this study, we aimed to develop a fragrant and soft-textured black and red rice variety using the CRISPR/Cas9 technology to knock out the OsWx gene, which is associated with amylose content (AC), and the OsBADH2 gene, responsible for rice aroma. Our results showed that, compared to wild-type, CRISPR lines of XHZ, HM, NWZ, and PGZ targeting OsWx and OsBADH2 exhibited a reduction in AC content, altered gel consistency, and a more than 50% increase in gel consistency. Headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) analysis revealed that the 2-acetyl-1-pyrroline (2-AP) content in the grains of xhz-c[BADH2 Wx] and hm-c[BADH2 Wx] reached 189.04 μg kg[-1] and 309.03 μg kg[-1], respectively. Furthermore, we observed a slight increase in anthocyanins and proanthocyanidins in these co-edited lines, without significant effects on their agronomic traits. Furthermore, to investigate the genes involved in the quality formation of black and red rice for the knockout of OsBADH2 and OsWx, we conducted RNA-seq analysis. The results indicated that knockout of OsBADH2 and OsWx affected the expression of genes involved in carotenoid biosynthesis, multiple amino acid metabolism genes, and endosperm starch and sucrose metabolic pathways. These findings suggest that the CRISPR/Cas9 technology can effectively target OsBADH2 and OsWx to develop high-quality black and red rice varieties with enhanced aroma and softer texture, providing a new strategy for the improvement of colored rice.}, }
@article {pmid40171792, year = {2025}, author = {Diril, MK and Esmen, K and Sehitogullari, T and Őztürk, G}, title = {Generation of albino C57BL/6J mice by CRISPR embryo editing of the mouse tyrosinase locus.}, journal = {Journal of genetics}, volume = {104}, number = {}, pages = {}, pmid = {40171792}, issn = {0973-7731}, mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Mice ; *Monophenol Monooxygenase/genetics ; Mice, Inbred C57BL ; Electroporation ; Female ; Zygote ; Embryo, Mammalian/metabolism ; *Genetic Loci ; INDEL Mutation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Knock-In Techniques ; }, abstract = {After the arrival of the CRISPR/Cas9 genome editing technology, genetic engineering of model organisms has become much faster and more efficient. The development of genetically modified mouse models is also facilitated by the application of various CRISPR methodologies. Although the very first studies utilized pronuclear injection (PNI) of Cas9 mRNA and sgRNAs into the zygote stage embryos to create knockout and knockin mutations, the repertoire of techniques and collection of reagents for CRISPR editing has rapidly expanded. This presents researchers in the field with a versatility of choices for genetic engineering. However, there are not many comparative studies that analysed the efficacy of gene editing when Cas9 and sgRNA/ssDNA oligos were transferred to the embryos by different methodologies. Here, we aimed to compare two different methods, electroporation and PNI. One of the recent developments gaining wide use in mouse model research is the application of electroporation for the introduction of Cas9/sgRNA ribonucleoprotein complexes into zygote stage embryos. Here, we have used this technique to generate albino coat-coloured C57BL/6J mice by targeted inactivation of the mouse tyrosinase gene through indel or knockin mutations. We have also applied the PNI protocol with the same set of reagents, to compare the efficiency of the two techniques in generation of indel and knockin mutations. Although PNI results in signifi- cantly higher efficiency for knockin mutations, it requires specialized equipment setup and advanced training in embryo micromanipulation and microinjection. Therefore, for the generation of simple gene knockouts by indel mutations, electroporation can be used.}, }
@article {pmid40171546, year = {2025}, author = {Kaur, N and Qadir, M and Francis, DV and Alok, A and Tiwari, S and Ahmed, ZFR}, title = {CRISPR/Cas9: a sustainable technology to enhance climate resilience in major Staple Crops.}, journal = {Frontiers in genome editing}, volume = {7}, number = {}, pages = {1533197}, pmid = {40171546}, issn = {2673-3439}, abstract = {Climate change is a global concern for agriculture, food security, and human health. It affects several crops and causes drastic losses in yield, leading to severe disturbances in the global economy, environment, and community. The consequences on important staple crops, such as rice, maize, and wheat, will worsen and create food insecurity across the globe. Although various methods of trait improvements in crops are available and are being used, clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) mediated genome manipulation have opened a new avenue for functional genomics and crop improvement. This review will discuss the progression in crop improvement from conventional breeding methods to advanced genome editing techniques and how the CRISPR/Cas9 technology can be applied to enhance the tolerance of the main cereal crops (wheat, rice, and maize) against any harsh climates. CRISPR/Cas endonucleases and their derived genetic engineering tools possess high accuracy, versatile, more specific, and easy to design, leading to climate-smart or resilient crops to combat food insecurity and survive harsh environments. The CRISPR/Cas9-mediated genome editing approach has been applied to various crops to make them climate resilient. This review, supported by a bibliometric analysis of recent literature, highlights the potential target genes/traits and addresses the significance of gene editing technologies in tackling the vulnerable effects of climate change on major staple crops staple such as wheat, rice, and maize.}, }
@article {pmid40170307, year = {2025}, author = {Mao, Y and Chu, G and Liang, Q and Liu, Y and Yang, Y and Liao, X and Wang, M}, title = {[Artificial intelligence-assisted design, mining, and modification of CRISPR-Cas systems].}, journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology}, volume = {41}, number = {3}, pages = {949-967}, doi = {10.13345/j.cjb.240865}, pmid = {40170307}, issn = {1872-2075}, mesh = {*CRISPR-Cas Systems/genetics ; *Artificial Intelligence ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Machine Learning ; Humans ; Genetic Engineering/methods ; Synthetic Biology ; }, abstract = {With the rapid advancement of synthetic biology, CRISPR-Cas systems have emerged as a powerful tool for gene editing, demonstrating significant potential in various fields, including medicine, agriculture, and industrial biotechnology. This review comprehensively summarizes the significant progress in applying artificial intelligence (AI) technologies to the design, mining, and modification of CRISPR-Cas systems. AI technologies, especially machine learning, have revolutionized sgRNA design by analyzing high-throughput sequencing data, thereby improving the editing efficiency and predicting off-target effects with high accuracy. Furthermore, this paper explores the role of AI in sgRNA design and evaluation, highlighting its contributions to the annotation and mining of CRISPR arrays and Cas proteins, as well as its potential for modifying key proteins involved in gene editing. These advancements have not only improved the efficiency and precision of gene editing but also expanded the horizons of genome engineering, paving the way for intelligent and precise genome editing.}, }
@article {pmid40170086, year = {2025}, author = {Wang, H and Qi, Z and Wang, J and He, Z and Lu, L and Chen, Z and Shao, Y and Wang, G and Wang, Z and Tu, J and Song, X}, title = {Rapid and visual detection of transmissible gastroenteritis virus using a CRISPR/Cas12a system combined with loop-mediated isothermal amplification.}, journal = {BMC veterinary research}, volume = {21}, number = {1}, pages = {234}, pmid = {40170086}, issn = {1746-6148}, support = {GXXT-2022-057//University Synergy Innovation Program of Anhui Province/ ; YQYB2023001//Anhui Provincial University Excellent Young Talents Support Program/ ; 2023SJY01//Research Funds of Joint Research Center for Food Nutrition and Health of IHM/ ; }, mesh = {*CRISPR-Cas Systems ; *Transmissible gastroenteritis virus/isolation &amp; purification/genetics ; Animals ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Swine ; *Gastroenteritis, Transmissible, of Swine/diagnosis/virology ; *Molecular Diagnostic Techniques/veterinary/methods ; Sensitivity and Specificity ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {BACKGROUND: Transmissible gastroenteritis (TGE) is a highly contagious intestinal disease caused by transmissible gastroenteritis virus (TGEV). The primary techniques for identifying TGEV involve enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and fluorescent quantitative PCR (qPCR). However, these approaches are complex, demanding specialized tools and significant time. Therefore, a precise, swift, and effective differential diagnosis method is crucial for TGEV prevention. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR) and Cas-associated proteins have become popular for their high specificity, unique cleavage activity, and ease of detection. CRISPR-Cas12a, a novel RNA-guided nucleic acid endonuclease, is emerging as a powerful molecular scissor.<br><br>RESULTS: In this study, we designed three pairs of crRNA targeting the N gene of TGEV. Following the selection of the most appropriate crRNA, we established the loop-mediated isothermal (LAMP) amplification method with a sensitivity of 10[2] copies/&#xb5;L. And based on this, we established the CRISPR-Cas12a fluorescence assay with a sensitivity of 10[0] copies/&#xb5;L. Furthermore, we established a CRISPR/Cas12a lateral-flow dipstick assay with a sensitivity of 10[2] copies/&#xb5;L. Importantly, none of these methods exhibited cross-reactivity with other related viruses, enabling quicker and more straightforward observation of experimental results.<br><br>CONCLUSIONS: We have successfully developed a CRISPR-Cas12a fluorescence assay and a CRISPR/Cas12a lateral-flow dipstick assay for clinical TGEV detection. Overall, we created a portable, quick, and sensitive TGEV assay with strong specificity utilizing the CRISPR-Cas12a system.}, }
@article {pmid40169710, year = {2025}, author = {Palomino, SM and Gabriel, KA and Mwirigi, JM and Cervantes, A and Horton, P and Funk, G and Moutal, A and Martin, LF and Khanna, R and Price, TJ and Patwardhan, A}, title = {Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {11116}, pmid = {40169710}, issn = {2045-2322}, support = {R01 NS116694/NS/NINDS NIH HHS/United States ; NS116694/NS/NINDS NIH HHS/United States ; }, mesh = {Humans ; *Ganglia, Spinal/cytology/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Neurons/metabolism ; TRPV Cation Channels/genetics/metabolism ; Cells, Cultured ; Transfection ; }, abstract = {CRISPR-Cas9 is now the leading method for genome editing and is advancing for the treatment of human disease. CRIPSR has promise in treating neurological diseases, but traditional viral-vector-delivery approaches have neurotoxicity limiting their use. Here we describe a simple method for non-viral transfection of primary human DRG (hDRG) neurons for CRISPR-Cas9 editing. We edited TRPV1, NTSR2, and CACNA1E using a lipofection method with CRISPR-Cas9 plasmids containing reporter tags (GFP or mCherry). Transfection was successfully demonstrated by the expression of the reporters two days post-administration. CRISPR-Cas9 editing was confirmed at the genome level with a T7-endonuclease-I assay; protein level with immunocytochemistry and Western blot; and functional level through capsaicin-induced Ca[2+] accumulation in a high-throughput compatible fluorescent imaging plate reader (FLIPR) system. This work establishes a reliable, target specific, non-viral CRISPR-Cas9-mediated genetic editing in primary human neurons with potential for future clinical application for sensory diseases.}, }
@article {pmid40169039, year = {2025}, author = {Dam, MI and Ding, BJ and Brauburger, K and Wang, HL and Powell, D and Groot, AT and Heckel, DG and Löfstedt, C}, title = {Sex pheromone biosynthesis in the Oriental fruit moth Grapholita molesta involves Δ8 desaturation.}, journal = {Insect biochemistry and molecular biology}, volume = {180}, number = {}, pages = {104307}, doi = {10.1016/j.ibmb.2025.104307}, pmid = {40169039}, issn = {1879-0240}, mesh = {Animals ; *Moths/metabolism/genetics/enzymology ; *Sex Attractants/biosynthesis ; *Fatty Acid Desaturases/metabolism/genetics ; *Insect Proteins/metabolism/genetics ; Female ; Male ; CRISPR-Cas Systems ; }, abstract = {The Oriental fruit moth Grapholita molesta is distributed throughout temperate regions and considered to be a pest in peach production and other high-value fruit crops in the rose family. Insecticide treatment has led to resistance development, but the use of sex pheromones in pest management has shown great promise. We investigated the pheromone biosynthesis pathway in G. molesta with the aim of elucidating pheromone evolution in the Olethreutinae subfamily of moths and harnessing pathway genes in biotechnological production of sex pheromone for use in pest management. In vivo labelling experiments suggested that an uncommon Δ8 fatty acyl desaturase is involved in sex pheromone biosynthesis. CRISPR/Cas9 knock-out of the highly expressed candidate desaturase gene Gmol_CPRQ almost completely blocked the production of Δ8 pheromone components in vivo. Heterologous expression of Gmol_CPRQ protein in yeast- or Sf9 insect cells, however, failed to demonstrate the expected Δ8 desaturase activity. Instead, Δ9 desaturase activity was observed. Co-expression in the yeast system of the electron donor, cytochrome b5, from G. molesta still produced only Δ9 desaturase activity. We suggest that Gmol_CPRQ is intimately involved in pheromone production in vivo, via an unknown reaction mechanism that may possibly involve another co-factor that is absent in the yeast and Sf9 expression systems, or depend on its subcellular site of activity. Solving this puzzle will shed further light on pheromone biosynthesis in the family Tortricidae and will be required for successful biotechnological production of fatty acids and pheromones requiring Δ8 desaturation.}, }
@article {pmid40167233, year = {2025}, author = {Kim, EJ and Hong, WJ and Kim, YJ and Kim, EY and Yun, SD and Moon, S and Lee, SK and Park, SK and Jung, KH}, title = {Global identification of key genes for pollen germination in rice through high-throughput screening and gene editing.}, journal = {Journal of integrative plant biology}, volume = {67}, number = {6}, pages = {1665-1684}, pmid = {40167233}, issn = {1744-7909}, support = {RS-2024-00322278//Rural Development Administration/ ; RS-2021-NR060084//National Research Foundation of Korea/ ; RS-2021-NR062086//National Research Foundation of Korea/ ; NRF-2021R1A2C2010124//National Research Foundation of Korea/ ; RS-2023-00217064//National Research Foundation of Korea/ ; RS-2024-00344197//National Research Foundation of Korea/ ; }, mesh = {*Oryza/genetics ; *Pollen/genetics/growth &amp; development ; *Germination/genetics ; *Gene Editing/methods ; *Genes, Plant/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems/genetics ; *High-Throughput Screening Assays ; Plant Infertility/genetics ; Mutation/genetics ; DNA, Bacterial ; }, abstract = {Successful reproduction depends on the stable germination and growth of the pollen tubes (PT). However, the molecular mechanisms involved in rice PT growth and development remain largely unknown. In a previous study, microarray transcriptome analysis identified 627 genes preferentially expressed in the tricellular and germinating pollen of rice (i.e., Oryza sativa ssp. japonica). To elucidate key genes involved in the gene transfer process facilitated by male gametophytes, we systematically screened T-DNA lines containing disrupted sequences that corresponded to these 627 genes and analyzed the genotypes of heterozygote progeny from 107 T-DNA-indexed lines covering 105 genes. We found that 42 lines exhibited a distorted segregation ratio among the wild-type (WT), heterozygote (HT), and homozygote (HM) genotypes, which deviated from the expected Mendelian ratio of 1:2:1 (WT:HT:HM). Further characterization using CRISPR/Cas9 mutants revealed that knockout mutants of certain genes that exhibited segregation distortion in the T-DNA insertion region were completely sterile. Moreover, even when T-DNA insertion lines followed Mendelian segregation patterns, sterility could be induced by simultaneously mutating functionally redundant genes, thereby overcoming genetic compensation. Interestingly, although some T-DNA insertion lines exhibited segregation ratios approximating 1:1:0, the corresponding CRISPR/Cas9 mutants produced homozygous seeds and showed partial sterility. Partial sterility suggests that despite mutant pollen grains being less competitive than WT pollen, they retain their fertilization potential under relaxed competition from WT pollen. Beyond mutant-based analysis, transcriptomic profiling of sterile mutant lines provided additional insight into the regulatory relationship between key germination regulators and the 105 target genes studied here. Overall, this study demonstrates the effectiveness of a multi-pronged strategy to accelerate the identification of defective phenotypes using mutant studies and provides valuable genetic resources for inducing novel male sterility in rice.}, }
@article {pmid40166066, year = {2025}, author = {Saeed, U and Piracha, ZZ and Khan, M and Tariq, MN and Gilani, SS and Raza, M and Munusamy, R and Bose, N and Ozsahin, DU and Özşahin, &#x130; and Nauli, SM}, title = {Cracking the code of HBV persistence: cutting-edge approaches to targeting cccDNA in chronic hepatitis B with or without pyogenic liver Abscesses.}, journal = {Frontiers in medicine}, volume = {12}, number = {}, pages = {1504736}, pmid = {40166066}, issn = {2296-858X}, abstract = {Chronic Hepatitis B Virus (HBV) infection remains a formidable global health challenge, driving severe liver complications such as hepatocellular carcinoma (HCC) and pyogenic liver abscesses (PLA). At the core of HBV persistence lies covalently closed circular DNA (cccDNA), a viral reservoir that fuels ongoing infection despite antiviral treatments. This review highlights molecular mechanisms governing cccDNA formation, maintenance, and clearance, spotlighting innovative therapeutic strategies to disrupt this key viral element. We explore cutting-edge approaches, including epigenetic modulation to silence cccDNA, RNA interference (RNAi) for viral RNA degradation, and CRISPR/Cas genome editing to excise cccDNA directly. Additionally, emerging antiviral therapies and immunotherapies, such as therapeutic vaccines and immune checkpoint inhibitors, offer new avenues for enhanced treatment efficacy. Special attention is given to the clinical complexities of managing HBV in patients with co-morbid conditions like HCC and PLA, emphasizing the necessity of a multidisciplinary approach. The interplay between antibacterial and antiviral therapies in PLA-associated HBV cases is critically examined to prevent treatment antagonism, ensuring optimal patient outcomes. Advanced therapeutic strategies, including nucleos(t)ide analogs, interferon therapy, and novel genomic interventions, are explored in both isolated HBV infection and PLA co-infections. Personalized regimens remain pivotal in enhancing therapeutic efficacy and long-term disease control. Current review advocates for a shift toward precision medicine, highlighting the critical need for interdisciplinary collaboration to bridge molecular discoveries with clinical innovations. Ultimately, these advancements promise to revolutionize the management of chronic HBV, paving the way for potential cures and improved patient outcomes.}, }
@article {pmid40165637, year = {2025}, author = {Guo, Q and Shen, Q and Hao, Q and Jiang, XL and Zou, LP and Xue, YP and Zheng, YG}, title = {Resolving the Trade-Off Between Toxicity and Efficiency of CRISPR-Cas9 System for Genome Editing Within Escherichia coli.}, journal = {Biotechnology journal}, volume = {20}, number = {4}, pages = {e70010}, doi = {10.1002/biot.70010}, pmid = {40165637}, issn = {1860-7314}, support = {2021YFC2102100//National Key Research and Development Program/ ; 2024C03011//Zhejiang Provincial Science and Technology Plan Project/ ; }, mesh = {*Escherichia coli/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Plasmids/genetics ; Genome, Bacterial ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {Efficient gene editing of Escherichia coli BL21 (DE3) holds significant practical value as a host for heterologous protein expression. Recently reported CRISPR-Cas9 editing systems for this strain exhibit a trade-off between efficiency and toxicity. In this study, we addressed this trade-off by employing the strategy to transiently induce Cas9 expression in the high-copy plasmid during the editing stage. Furthermore, we demonstrated that eliminating the sgRNA-expressing plasmid using a temperature-sensitive replicon, combined with SacB for removing the Cas9-expressing plasmid, exhibited higher efficiency compared to previously reported strategies for editing system removal. We assigned this optimized CRISPR-Cas9 genome editing system as the pEBcas9/pEBsgRNA system, which has successfully achieved efficient five rounds of genome editing and simultaneous editing of multiple loci in E. coli BL21 (DE3). Using this system, we identified several loci suitable for multi-copy integrated expression of exogenous genes. Overall, the pEBcas9/pEBsgRNA system may facilitate the application of E. coli in both industrial and academic fields.}, }
@article {pmid40165508, year = {2025}, author = {Zhang, T and Zhao, Y and Zhu, C and Zhu, X and Zhu, X and Qiu, Y and Nie, Z and Lei, C}, title = {CRISPR/Cas12a Protein Switch Powered Label-Free Electrochemical Biosensor for Sensitive Viral Protease Detection.}, journal = {Analytical chemistry}, volume = {97}, number = {14}, pages = {8039-8047}, doi = {10.1021/acs.analchem.5c00547}, pmid = {40165508}, issn = {1520-6882}, mesh = {*Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; Humans ; *CRISPR-Associated Proteins/metabolism ; *Viral Nonstructural Proteins/analysis/metabolism ; *Endodeoxyribonucleases/metabolism ; Hemin/chemistry ; *Viral Proteins/analysis/metabolism ; Aptamers, Nucleotide/chemistry ; *Bacterial Proteins/metabolism/chemistry ; Enterovirus A, Human/enzymology ; Viral Proteases ; Serine Endopeptidases ; Nucleoside-Triphosphatase ; DEAD-box RNA Helicases ; }, abstract = {Viral proteases are critical molecular targets in viral pathogenesis, representing pivotal biomarkers for understanding viral infection mechanisms and developing antiviral therapeutics. This study introduces a label-free electrochemical biosensor that enables sensitive viral protease detection by integrating protease-responsive CRISPR/Cas protein switches (CasPSs) with a hemin aptamer-functionalized electrochemical interface. The biosensor's mechanism relies on viral protease-mediated proteolysis, which leads to the release of active Cas12a proteins from CasPSs and generates amplified electrochemical responses through continuous cleavage of immobilized redox-active hemin/aptamer complexes. This biosensor achieved specific hepatitis C virus NS3/4A protease sensing with femtomolar sensitivity and could be readily expanded to other viral proteases by replacing the CasPS module. The feasibility of this biosensor was demonstrated by monitoring enterovirus 71 3C protease activities in virus-infected cell samples with different viral loads and postinfection times. This study provides a promising strategy for integrating CRISPR biosensing with electrochemical platforms, offering a helpful analytical tool for viral infection monitoring and antiviral drug screening.}, }
@article {pmid40165374, year = {2025}, author = {Butt, H and Sathish, S and London, E and Lee Johnson, T and Essawi, K and Leonard, A and Tisdale, JF and Demirci, S}, title = {Genome editing strategies for targeted correction of β-globin mutation in sickle cell disease: From bench to bedside.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {33}, number = {5}, pages = {2154-2171}, pmid = {40165374}, issn = {1525-0024}, mesh = {*Anemia, Sickle Cell/genetics/therapy ; Humans ; *Gene Editing/methods ; *beta-Globins/genetics ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; *Mutation ; Animals ; }, abstract = {Sickle cell disease (SCD) includes a range of genotypes that result in a clinical syndrome, where abnormal red blood cell (RBC) physiology leads to widespread complications affecting nearly every organ system. Treatment strategies for SCD can be broadly categorized into disease-modifying therapies and those aimed toward a cure. Although several disease-modifying drugs have been approved, they do not fully address the complexity and severity of SCD. Recent advances in allogeneic transplantation and autologous gene therapy show promising outcomes in terms of efficacy and safety. While these approaches have improved the lives of many patients, achieving a durable and comprehensive cure for all remains challenging. To address this, gene-editing technologies, including zinc-finger nucleases, TALENs, CRISPR-Cas, base editing, and prime editing, have been explored both ex vivo and in vivo for targeted correction of the β-globin gene (HBB) in SCD. However, direct correction of HBB and its translation from the laboratory to the clinic presents ongoing limitations, with challenges involved in achieving robust mutation-correction efficiency, off-target effects, and high costs of therapies. The optimal strategy for curing SCD remains uncertain, but several promising approaches are emerging. This review touches on past, present, and future developments in HBB correction.}, }
@article {pmid40165206, year = {2025}, author = {Lipert, BA and Siemens, KN and Khan, A and Airey, R and Dam, GH and Lu, M and Flinterman, M and Yong, Q and Lee, TW and Hunter, FW and Jamieson, SMF}, title = {CRISPR screens with trastuzumab emtansine in HER2-positive breast cancer cell lines reveal new insights into drug resistance.}, journal = {Breast cancer research : BCR}, volume = {27}, number = {1}, pages = {48}, pmid = {40165206}, issn = {1465-542X}, support = {22/345//Health Research Council of New Zealand/ ; 1921 PG//Cancer Research Trust New Zealand/ ; }, mesh = {Humans ; *Drug Resistance, Neoplasm/genetics ; *Breast Neoplasms/genetics/drug therapy/pathology/metabolism ; *Receptor, ErbB-2/metabolism/genetics ; Female ; *CRISPR-Cas Systems ; *Ado-Trastuzumab Emtansine/pharmacology ; Cell Line, Tumor ; *Antineoplastic Agents, Immunological/pharmacology ; Trastuzumab/pharmacology ; *Maytansine/analogs &amp; derivatives/pharmacology ; Gene Expression Regulation, Neoplastic/drug effects ; }, abstract = {BACKGROUND: Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate that is an effective therapy for HER2-positive breast cancer; however, its efficacy is limited by drug resistance. While multiple mechanisms of resistance have been proposed, these are not yet well understood. Greater understanding of T-DM1 sensitivity and resistance could provide new combination strategies to overcome resistance or predictive biomarkers to guide therapy.<br><br>METHODS: We have conducted CRISPR/Cas9 functional genomics modifier screens in HER2-positive breast cancer cell lines to allow for unbiased discovery of T-DM1 sensitivity and resistance genes. Whole-genome knockout screens were carried out in MDA-MB-361 and MDA-MB-453 cells treated with T-DM1 and its payload cytotoxin DM1. Hits were validated in secondary T-DM1 screens using a focused single-guide RNA (sgRNA) library and subsequently by individual gene knockout.<br><br>RESULTS: The whole-genome CRISPR screens with T-DM1 and DM1 identified 599 genes as potential modifiers of T-DM1 sensitivity and resistance. Of these, 17 genes were significantly enriched and 3 genes depleted at P&#x2009;<&#x2009;0.001 in either or both MDA-MB-361 and MDA-MB-453 libraries in the secondary screens. Among the top hits, were known T-DM1 sensitivity genes ERBB2 and SLC46A3, in addition to negative regulators of mTOR complex 1: TSC1 and TSC2. MDA-MB-453 clones with knockout of TSC1 or partial knockout of TSC2 were more resistant to T-DM1 than wild type cells in competition growth assays and to T-DM1 and other HER2 targeting therapies (T-DXd, lapatinib and neratinib) in growth inhibition assays, and had increased internalisation of T-DM1 at 6&#xa0;h. T-DM1 and the mTOR inhibitor everolimus demonstrated synergistic activity at inhibiting cell proliferation at multiple T-DM1 concentrations across four HER2-positive breast cancer cell lines.<br><br>CONCLUSIONS: Our CRISPR screening approach with T-DM1 in HER2-positive breast cancer cell lines identified genes not previously implicated in T-DM1 sensitivity or resistance, including TSC1 and TSC2. These genes may inform new strategies to enhance T-DM1 therapy in the clinic.}, }
@article {pmid40164595, year = {2025}, author = {Feist, WN and Luna, SE and Ben-Efraim, K and Filsinger Interrante, MV and Amorin, A and Johnston, NM and Bruun, TUJ and Utz, A and Ghanim, HY and Lesch, BJ and McLaughlin, TM and Dudek, AM and Porteus, MH}, title = {Multilayered HIV-1 resistance in HSPCs through CCR5 Knockout and B cell secretion of HIV-inhibiting antibodies.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {3103}, pmid = {40164595}, issn = {2041-1723}, support = {T32 GM007365/GM/NIGMS NIH HHS/United States ; F32 HL154667/HL/NHLBI NIH HHS/United States ; T32 GM119995/GM/NIGMS NIH HHS/United States ; U54 AI170792/AI/NIAID NIH HHS/United States ; F30 AI152943/AI/NIAID NIH HHS/United States ; K99 HL172253/HL/NHLBI NIH HHS/United States ; }, mesh = {*Receptors, CCR5/genetics/immunology/metabolism ; *HIV-1/immunology ; Humans ; *HIV Infections/immunology/therapy/virology ; *HIV Antibodies/immunology/metabolism ; *B-Lymphocytes/immunology/metabolism ; Animals ; *Hematopoietic Stem Cells/immunology/metabolism/virology ; Antibodies, Neutralizing/immunology/metabolism ; Mice ; Hematopoietic Stem Cell Transplantation/methods ; Gene Knockout Techniques ; CRISPR-Cas Systems ; }, abstract = {Allogeneic transplantation of CCR5 null hematopoietic stem and progenitor cells (HSPCs) is the only known cure for HIV-1 infection. However, this treatment is limited because of the rarity of CCR5-null matched donors, the morbidities associated with allogeneic transplantation, and the prevalence of HIV-1 strains resistant to CCR5 knockout (KO) alone. Here, we propose a one-time therapy through autologous transplantation of HSPCs genetically engineered ex vivo to produce both CCR5 KO cells and long-term secretion of potent HIV-1 inhibiting antibodies from B cell progeny. CRISPR-Cas9-engineered HSPCs engraft and reconstitute multiple hematopoietic lineages in vivo and can be engineered to express multiple antibodies simultaneously (in pre-clinical models). Human B cells engineered to express each antibody secrete neutralizing concentrations capable of inhibiting HIV-1 pseudovirus infection in vitro. This work lays the foundation for a potential one-time functional cure for HIV-1 through combining the long-term delivery of therapeutic antibodies against HIV-1 and the known efficacy of CCR5 KO HSPC transplantation.}, }
@article {pmid40164474, year = {2025}, author = {Guia, S and Fenis, A and Baudesson De Chanville, C and Galluso, J and Medjouel, H and Escaliere, B and Modelska, A and Vienne, M and Lopes, N and Pouchin, A and Rossi, B and Gauthier, L and Roulland, S and Vivier, E and Narni-Mancinelli, E}, title = {Genome-wide CRISPR/Cas9 screen reveals factors that influence the susceptibility of tumor cells to NK cell-mediated killing.}, journal = {Journal for immunotherapy of cancer}, volume = {13}, number = {3}, pages = {}, pmid = {40164474}, issn = {2051-1426}, mesh = {Humans ; *Killer Cells, Natural/immunology/metabolism ; *CRISPR-Cas Systems ; Animals ; Mice ; *Cytotoxicity, Immunologic ; HCT116 Cells ; Cell Line, Tumor ; }, abstract = {BACKGROUND: Natural killer (NK) cells exhibit potent cytotoxic activity against various cancer cell types. Over the past five decades, numerous methodologies have been employed to elucidate the intricate molecular mechanisms underlying NK cell-mediated tumor control. While significant progress has been made in elucidating the interactions between NK cells and tumor cells, the regulatory factors governing NK cell-mediated tumor cell destruction are not yet fully understood. This includes the diverse array of tumor ligands recognized by NK cells and the mechanisms that NK cells employ to eliminate tumor cells.<br><br>METHODS: In this study, we employed a genome-wide CRISPR/Cas9 screening approach in conjunction with functional cytotoxicity assays to delineate the pathways modulating the susceptibility of colon adenocarcinoma HCT-116 cells to NK cell-mediated cytotoxicity.<br><br>RESULTS: Analysis of guide RNA distribution in HCT-116 cells that survived co-incubation with NK cells identified ICAM-1 as a pivotal player in the NKp44-mediated immune synapse, with NKp44 serving as an activating receptor crucial for the elimination of HCT-116 tumor cells by NK cells. Furthermore, disruption of genes involved in the apoptosis or interferon (IFN)-&#x3b3; signaling pathways conferred resistance to NK cell attack. We further dissected that NK cell-derived IFN-&#x3b3; promotes mitochondrial apoptosis in vitro and exerts control over B16-F10 lung metastases in vivo.<br><br>CONCLUSION: Monitoring ICAM-1 levels on the surface of tumor cells or modulating its expression should be considered in the context of NK cell-based therapy. Furthermore, promoting FasL expression on the NK cell surface is reaffirmed as an important strategy to enhance NK cell-mediated tumor killing, offering an additional avenue for therapeutic optimization. Additionally, considering the diffusion properties of IFN-&#x3b3;, our findings highlight the potential of leveraging NK cell-derived IFN-&#x3b3; to enhance direct tumor cell killing and facilitate bystander effects via cytokine diffusion, warranting further investigation.}, }
@article {pmid40163950, year = {2025}, author = {Fang, L and Yang, X and Li, Y and Xue, C and Li, Z and Jiang, H and Li, X and Lu, S and Wang, D and He, H and Huang, Z and Guo, X and Luo, G}, title = {SPECIAL: Phosphorothioate dNTP assisted RPA equipped with CRISPR/Cas12a amplifier enables high-specific nucleic acid testing.}, journal = {Biosensors &amp; bioelectronics}, volume = {279}, number = {}, pages = {117421}, doi = {10.1016/j.bios.2025.117421}, pmid = {40163950}, issn = {1873-4235}, mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; Humans ; *Recombinases/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Recombinase polymerase amplification (RPA) is one of the most widely used isothermal amplification methods and considered to be a promising tool for point-of-care testing (POCT) molecular diagnosis. However, RPA is prone to have nonspecific amplification occur, due to the poor recognition accuracy of polymerase and recombinase, which severely hindered its clinical application. It is important to improve the specificity of RPA further. Herein, we developed a novel nucleic acid testing method termed phosphorothioate dNTP (dNTPαS) assisted RPA (S-RPA) that employs dNTPαS as substrates to suppress nonspecific amplification effectively. We found that dNTPαS could improve the recognition accuracy of Bsu polymerase and recombinase, thereby enhancing their amplification specificity. Our S-RPA provided much higher specificity (approximately 40 % improvement compared to classical RPA), realizing detection target with single nucleotide mutation. Based on its outstanding performance, we further combined the S-RPA with CRISPR/Cas12a to achieve highly specific and sensitive fluorescence detection, namely S-RPA equipped with CRISPR/Cas12a amplifier (SPECIAL). Our SPECIAL was more sensitive (10-fold higher) than the classical RPA-CRISPR/Cas12a assay, offering 100 % agreement with the qPCR during clinical validation. In summary, a strategy based on dNTPαS was established to enhance the specificity of RPA, thereby improving its practicability and providing a potential POCT tool for molecular diagnosis.}, }
@article {pmid40163416, year = {2025}, author = {Tordoff, J and Alfonse, LE and Makarova, KS and Ornstein, A and Garrity, AJ and Yan, WX and Scott, DA and Koonin, EV and Cheng, DR}, title = {Initial Characterization of 12 New Subtypes and Variants of Type V CRISPR Systems.}, journal = {The CRISPR journal}, volume = {8}, number = {2}, pages = {149-154}, pmid = {40163416}, issn = {2573-1602}, mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Bacteriophages/genetics ; *CRISPR-Associated Proteins/genetics ; Gene Editing ; Prophages/genetics ; Genetic Variation ; }, abstract = {Type V CRISPR systems are highly diverse in sequence, mechanism, and function. Although recent efforts have greatly expanded our understanding of their evolution, the diversity of type V systems remains to be completely explored, and many clades have not been experimentally characterized. In this work, we mined metagenomic databases to identify three new subtypes and nine new variants of Cas12, the effector of Type V systems, and provide experimental and computational characterization of their Protospacer-Adjacent Motif (PAM), interference activity, loci architecture, and tracrRNA dependence. Half of the new Cas12s are found in phages or prophages. New subtypes Cas12o and Cas12p lack the canonical RuvC catalytic residues, suggesting they interfere with the target without cleavage, possibly by blocking transcription or replication. One variant, Cas12f10, displays substantial activity on PAM-less targets. Our work expands the diversity of the functionally characterized Cas12 effectors and provides some promising candidates for genome engineering tools.}, }
@article {pmid40163415, year = {2025}, author = {Noel, EA and Sahu, SU and Wyman, SK and Krishnappa, N and Jeans, C and Wilson, RC}, title = {Hairpin Internal Nuclear Localization Signals in CRISPR-Cas9 Enhance Editing in Primary Human Lymphocytes.}, journal = {The CRISPR journal}, volume = {8}, number = {2}, pages = {105-119}, doi = {10.1089/crispr.2024.0080}, pmid = {40163415}, issn = {2573-1602}, mesh = {Humans ; *Gene Editing/methods ; *Nuclear Localization Signals/genetics ; *CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; *Lymphocytes/metabolism ; T-Lymphocytes/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Nucleus ; }, abstract = {The incorporation of nuclear localization signal (NLS) sequences at one or both termini of CRISPR enzymes is a widely adopted strategy to facilitate genome editing. Engineered variants of CRISPR enzymes with diverse NLS sequences have demonstrated superior performance, promoting nuclear localization and efficient DNA editing. However, limiting NLS fusion to the CRISPR protein's termini can negatively impact protein yield via recombinant expression. Here we present a distinct strategy involving the installation of hairpin internal NLS sequences (hiNLS) at rationally selected sites within the backbone of CRISPR-Cas9. We evaluated the performance of these hiNLS Cas9 variants by editing genes in human primary T cells following the delivery of ribonucleoprotein enzymes via either electroporation or co-incubation with amphiphilic peptides. We show that hiNLS Cas9 variants can improve editing efficiency in T cells compared with constructs with terminally fused NLS sequences. Furthermore, many hiNLS Cas9 constructs can be produced with high purity and yield, even when these constructs contain as many as nine NLS. These hiNLS Cas9 constructs represent a key advance in optimizing CRISPR effector design and may contribute to improved editing outcomes in research and therapeutic applications.}, }
@article {pmid40163310, year = {2025}, author = {Rathje, F and Sykora, MM and Aberger, F and Krenn, PW}, title = {High Efficiency Lentiviral Transduction of Colon Organoids Using Reversible 2D/3D Culture Techniques.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2905}, number = {}, pages = {245-254}, pmid = {40163310}, issn = {1940-6029}, mesh = {*Organoids/cytology/metabolism ; Animals ; *Lentivirus/genetics ; Mice ; *Colon/cytology/metabolism ; *Transduction, Genetic/methods ; *Cell Culture Techniques/methods ; Genetic Vectors/genetics ; CRISPR-Cas Systems ; Humans ; Stem Cells/cytology/metabolism ; }, abstract = {Organoids are a promising research tool for studying tissue development and disease in vitro. While organoids are frequently considered a replacement or complementary model for in vivo mouse experiments, exploiting their full potential often requires genetically engineered mice as a source of transgenic stem cells, also because genetic manipulation of organoids is rather inefficient and cumbersome. Here, we describe an alternative and optimized murine colon organoid manipulation protocol that reversibly and temporarily interrupts the 3D organoid structure for short-term 2D monolayer culture. This approach allows highly efficient viral transduction and genetic manipulation of stem cells in a 2D setting, followed by 3D stem cell embedding and restoration of the original organoid architecture. This method greatly improves the efficiency of lentiviral-mediated genetic manipulation of organoids and increases their potential applications in CRISPR/Cas9 and compound screens, immune-competent co-cultures, and disease modeling.}, }
@article {pmid40163287, year = {2025}, author = {Oh, Y and Nagalakshmi, U and Dahlbeck, D and Koehler, N and Cho, MJ and Dinesh-Kumar, SP and Staskawicz, BJ}, title = {Heritable virus-induced germline editing in tomato.}, journal = {The Plant journal : for cell and molecular biology}, volume = {122}, number = {1}, pages = {e70115}, pmid = {40163287}, issn = {1365-313X}, support = {ios-2303522//National Science Foundation/ ; ios-2303523//National Science Foundation/ ; //Innovative Genomics Institute/ ; }, mesh = {*Solanum lycopersicum/genetics/virology ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; *Plant Viruses/genetics ; CRISPR-Cas Systems ; Plant Proteins/genetics/metabolism ; CRISPR-Associated Protein 9/genetics ; Caulimovirus/genetics ; Oxidoreductases ; }, abstract = {Here, we report the successful implementation of heritable virus-induced genome editing (VIGE) in tomato (Solanum lycopersicum). We generated three transgenic tomato lines expressing Streptococcus pyogenes Cas9 (SpCas9) under the control of Cauliflower mosaic virus 35S (35S), S. lycopersicum ribosomal protein S5A (SlRPS5A), or S. lycopersicum YAO promoters (SlYAO). These three lines were tested for somatic and heritable editing using the tobacco rattle virus (TRV)-based system carrying guide RNAs (gRNAs) fused with mobile RNA sequences. TRV with gRNA targeted to Phytoene desaturase (SlPDS) and Downy mildew resistance 6 (SlDMR6) genes fused to mobile RNA sequences showed significant somatic editing efficiency in all three tomato lines expressing SpCas9. However, the progenies from the SlYAO promoter-driven SpCas9 tomato infected with TRV with gRNA targeted to SlDMR6 fused to the mobile RNA sequence resulted in monoallelic mutations with a frequency of 3%. Optimization of environmental conditions, such as reduced light intensity, significantly increased heritable editing frequencies, from 0% to 86% at the SlPDS and from 3% to 100% at the SlDMR6, including biallelic mutations. These findings underscore the use of appropriate promoters to express Cas nucleases and optimized environmental conditions to enhance heritable genome editing efficiency in tomato using VIGE. Furthermore, our method enables the generation of mutants without additional tissue culture or transformation once a SpCas9-expressing tomato line is established.}, }
@article {pmid40162959, year = {2025}, author = {Shen, H and Li, Y and Tang, K and Liang, H and Xu, ZL and Liu, Y and Liu, W}, title = {Programmable AIESTA: All-in-One Isothermal Enzymatic Signal Transduction Amplifier for Portable Profiling.}, journal = {Analytical chemistry}, volume = {97}, number = {14}, pages = {8088-8097}, doi = {10.1021/acs.analchem.5c00934}, pmid = {40162959}, issn = {1520-6882}, mesh = {*Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; *Temperature ; *Argonaute Proteins/metabolism/chemistry ; Signal Transduction ; }, abstract = {The Argonaute (Ago) protein exhibits high specificity in nucleic acid recognition and cleavage, making it highly promising for biosensing applications. Its potential is further enhanced by its independence from protospacer adjacent motif (PAM) requirements and the cost-effectiveness of using short DNA guides. Both Ago and CRISPR/Cas systems face challenges in signal amplification, which limit their ability to detect targets at ultralow concentrations. To overcome this limitation, a thermostable quadratic amplification system (T-QAS) was constructed by integrating a thermostable nicking-enzyme-mediated amplification (NEMA) strategy with TtAgo. The system leverages the high stability of T-QAS at elevated temperatures to enhance guide-target interactions and decrease false positives caused by nonspecific amplification. Additionally, nanozyme is integrated with T-QAS to construct the AIESTA platform (all-in-one isothermal enzymatic signal transduction amplifier), which is a single-tube visual sensing platform. Within the AIESTA system, T-QAS improves specificity through high operational temperatures and offers programmable functions, enabling the sensitive detection of miRNA and foodborne toxins. The combination of T-QAS and nanozyme makes AIESTA a candidate of point-of-care testing (POCT) field, showcasing the potential for biosensing in resource-limited and complex environments.}, }
@article {pmid40162949, year = {2025}, author = {Jurgens, JA and Matos Ruiz, PM and King, J and Foster, EE and Berube, L and Chan, WM and Barry, BJ and Jeong, R and Rothman, E and Whitman, MC and MacKinnon, S and Rivera-Quiles, C and Pratt, BM and Easterbrooks, T and Mensching, FM and Di Gioia, SA and Pais, L and England, EM and de Berardinis, T and Magli, A and Koc, F and Asakawa, K and Kawakami, K and O'Donnell-Luria, A and Hunter, DG and Robson, CD and Bulyk, ML and Engle, EC}, title = {Gene Identification for Ocular Congenital Cranial Motor Neuron Disorders Using Human Sequencing, Zebrafish Screening, and Protein Binding Microarrays.}, journal = {Investigative ophthalmology &amp; visual science}, volume = {66}, number = {3}, pages = {62}, pmid = {40162949}, issn = {1552-5783}, support = {R03 HD099358/HD/NICHD NIH HHS/United States ; UM1 HG008900/HG/NHGRI NIH HHS/United States ; UL1 TR002541/TR/NCATS NIH HHS/United States ; R01 HG009141/HG/NHGRI NIH HHS/United States ; T32 NS007473/NS/NINDS NIH HHS/United States ; T32 GM007748/GM/NIGMS NIH HHS/United States ; R01 EY032539/EY/NEI NIH HHS/United States ; T32 EY007145/EY/NEI NIH HHS/United States ; R01 HG010501/HG/NHGRI NIH HHS/United States ; R01 MH115957/MH/NIMH NIH HHS/United States ; R01 EY027421/EY/NEI NIH HHS/United States ; K08 EY027850/EY/NEI NIH HHS/United States ; U01 HG011755/HG/NHGRI NIH HHS/United States ; R56 MH115957/MH/NIMH NIH HHS/United States ; }, mesh = {Animals ; Humans ; CRISPR-Cas Systems ; Disease Models, Animal ; Exome Sequencing ; Genetic Testing/methods ; Protein Array Analysis/methods ; Transcription Factors/genetics ; Zebrafish ; Zebrafish Proteins/genetics ; *Congenital Cranial Dysinnervation Disorders/genetics ; }, abstract = {PURPOSE: To functionally evaluate novel human sequence-derived candidate genes and variants for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs).<br><br>METHODS: Through exome and genome sequencing of a genetically unsolved human oCCDD cohort, we previously reported the identification of variants in many candidate genes. Here, we describe a parallel study that prioritized a subset of these genes (43 human genes, 57 zebrafish genes) using a G0 CRISPR/Cas9-based knockout assay in zebrafish and generated F2 germline mutants for 17. We tested the functionality of variants of uncertain significance in known and novel candidate transcription factor-encoding genes through protein binding microarrays.<br><br>RESULTS: We first demonstrated the feasibility of the G0 screen by targeting known oCCDD genes phox2a and mafba. Approximately 70% to 90% of gene-targeted G0 zebrafish embryos recapitulated germline homozygous null-equivalent phenotypes. Using this approach, we then identified three novel candidate oCCDD genes (SEMA3F, OLIG2, and FRMD4B) with putative contributions to human and zebrafish cranial motor development. In addition, protein binding microarrays demonstrated reduced or abolished DNA binding of human variants of uncertain significance in known and novel sequence-derived transcription factors PHOX2A (p.(Trp137Cys)), MAFB (p.(Glu223Lys)), and OLIG2 (p.(Arg156Leu)).<br><br>CONCLUSIONS: This study nominates three strong novel candidate oCCDD genes (SEMA3F, OLIG2, and FRMD4B) and supports the functionality and putative pathogenicity of transcription factor candidate variants PHOX2A p.(Trp137Cys), MAFB p.(Glu223Lys), and OLIG2 p.(Arg156Leu). Our findings support that G0 loss-of-function screening in zebrafish can be coupled with human sequence analysis and protein binding microarrays to aid in prioritizing oCCDD candidate genes/variants.}, }
@article {pmid40162522, year = {2025}, author = {Pan, Y and Wang, D and Wei, R and Wang, S and Li, Y and Pan, W and Zhou, P and Li, N and Tang, B}, title = {Lateral Flow Platform for Lung Cancer Diagnosis through Simultaneous Detection of ctDNA and MicroRNA.}, journal = {Analytical chemistry}, volume = {97}, number = {13}, pages = {7063-7070}, doi = {10.1021/acs.analchem.4c05502}, pmid = {40162522}, issn = {1520-6882}, mesh = {*Lung Neoplasms/diagnosis/genetics ; Humans ; *MicroRNAs/analysis/genetics ; *Circulating Tumor DNA/analysis/genetics ; Gold/chemistry ; Biomarkers, Tumor/genetics ; Metal Nanoparticles/chemistry ; Nucleic Acid Amplification Techniques ; Proto-Oncogene Proteins p21(ras)/genetics ; CRISPR-Cas Systems ; }, abstract = {Early cancer screening is essential for reducing cancer-related mortality and improving survival rates. Simultaneous detection of multiple tumor markers can enhance the accuracy and specificity of cancer diagnosis, helping us to mitigate false-positive results associated with single-marker analysis. Here, we have developed a lateral flow detection platform that combines recombinase polymerase amplification (RPA), CRISPR Cas9, and catalyzed hairpin assembly (CHA) for the simultaneous detection of KRAS ctDNA and miRNA-223 in lung cancer. The CRISPR Cas9 system acts as a linking element, enabling specific recognition and binding to RPA amplicons of KRAS ctDNA while facilitating the capture of Au-DNA-Bio nanoparticles (NPs), thereby producing a stronger detection signal through Au NPs aggregation. The CHA system enhances this platform by providing sensitive detection of miRNA-223. Our platform was tested on a limited number of clinical saliva samples, demonstrating feasibility but requiring further validation with larger cohorts.}, }
@article {pmid40161755, year = {2025}, author = {Ratna, TA and Sharon, BM and Velin, CAB and Buttaro, BA and Palmer, KL}, title = {Factors affecting CRISPR-Cas defense against antibiotic resistance plasmids harbored by Enterococcus faecalis laboratory model strains and clinical isolates.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, pmid = {40161755}, issn = {2692-8205}, support = {R01 AI116610/AI/NIAID NIH HHS/United States ; }, abstract = {Enterococcus faecalis is a Gram-positive bacterium and opportunistic pathogen that acquires resistance to a wide range of antibiotics by horizontal gene transfer (HGT). The rapid increase of multidrug-resistant (MDR) bacteria including MDR E. faecalis necessitates the development of alternative therapies and a deeper understanding of the factors that impact HGT. CRISPR-Cas systems provide sequence-specific defense against HGT. From previous studies, we know that E. faecalis CRISPR-Cas provides sequence-specific anti-plasmid defense during agar plate biofilm mating and in the murine intestine. Those studies were mainly conducted using laboratory model strains with a single, CRISPR-targeted plasmid in the donor. MDR E. faecalis typically possess multiple plasmids that are diverse in sequence and may interact with each other to impact plasmid transfer and CRISPR-Cas efficacy. Here, we altered multiple parameters of our standard in vitro conjugation assays to assess CRISPR-Cas efficacy, including the number and genotype of plasmids in the donor; laboratory model strains as donor versus recent human isolates as donor; and the biofilm substrate utilized during conjugation. We found that the plasmids pTEF2 and pCF10, which are not targeted by CRISPR-Cas in our recipient, enhance the conjugative transfer of the CRISPR-targeted plasmid pTEF1 into both wild-type and CRISPR-Cas-deficient (via deletion of cas9) recipient cells. However, the effect of pTEF2 on pTEF1 transfer is much more pronounced, with a striking 6-log increase in pTEF1 conjugation frequency when pTEF2 is also present in the donor and recipients are deficient for CRISPR-Cas (compared to 4-log for pCF10). We also identified that E. faecalis Δcas9 has altered biofilm structure and thickness relative to the wild-type strain when cultured on a plastic substrate, but equivalent growth in the agar plate biofilms widely used for conjugation studies. Overall, this study provides insight about the interplay between plasmids and CRISPR-Cas defense, opening avenues for developing novel therapeutic strategies to curb HGT among bacterial pathogens, and highlighting pTEF2 as a plasmid for additional mechanistic study.}, }
@article {pmid40160815, year = {2025}, author = {Schönberg, PY and Muñoz-Ovalle, &#xc1; and Paszkowski-Rogacz, M and Crespo, E and Sürün, D and Feldmann, A and Buchholz, F}, title = {A pooled CRISPR screen identifies the Tα2 enhancer element as a driver of TRA expression in a subset of mature human T lymphocytes.}, journal = {Frontiers in immunology}, volume = {16}, number = {}, pages = {1536003}, pmid = {40160815}, issn = {1664-3224}, mesh = {Humans ; *Enhancer Elements, Genetic ; Jurkat Cells ; *T-Lymphocytes/immunology/metabolism ; *Receptors, Antigen, T-Cell, alpha-beta/genetics ; *CRISPR-Cas Systems ; *Gene Expression Regulation ; *T-Lymphocyte Subsets/immunology/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {The T cell receptor (TCR) is crucial for immune responses and represents a pivotal therapeutic target for CAR T cell therapies. However, which enhancer elements drive the constitutive expression of the TCRα chain in mature, peripheral T cells has not been well defined. Earlier work has suggested that enhancer alpha is inactive in mature peripheral T cells and that an alternative enhancer element in the 5' J region was driving TRA expression, while more recent findings indicated the opposite. Here, we applied a pooled CRISPR screen to probe a large genomic region proximal to the human TRA gene for the presence of regulatory elements. Interestingly, no sgRNA targeting the 5' J region was identified that influenced TRA expression. In contrast, several sgRNAs targeting enhancer alpha element Tα2, were identified that compromised the expression of the TCRα chain in Jurkat E6.1, as well as in a subset of human primary T cells. Our results provide new insights into the regulation of TRA in human peripheral T cells, advancing our understanding of how constitutive TRA expression is driven and regulated.}, }
@article {pmid40160100, year = {2025}, author = {Yang, S and Ren, L and Fan, N and Wang, S and Shen, B and Liu, Z and Li, X and Ding, S}, title = {CRISPR-Cas12a with split crRNA for the direct and sensitive detection of microRNA.}, journal = {The Analyst}, volume = {150}, number = {9}, pages = {1884-1890}, doi = {10.1039/d5an00142k}, pmid = {40160100}, issn = {1364-5528}, mesh = {*MicroRNAs/genetics/blood/analysis ; *CRISPR-Cas Systems/genetics ; Humans ; Limit of Detection ; *Endodeoxyribonucleases/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; DNA/genetics/chemistry ; }, abstract = {microRNAs (miRNAs) have been identified as potential biomarkers. Despite the prevalence of quantitative PCR in the field of miRNA detection, this technology is encumbered by the complexity of its methodology. This study presents a novel CRISPR/Cas12a-based method for the direct and sensitive detection of miRNA-21 using split crRNA. The system comprises Cas12a protein, crRNA-handle, and activator DNA complementary to the target miRNA. In the presence of the target miRNA, it binds to the activator DNA, forming a duplex. The formed duplex, in conjunction with the crRNA-handle, activates Cas12a's trans-cleavage activity. This leads to cleavage of a fluorescent reporter, generating an enhanced signal. The method enables direct RNA detection without reverse transcription or sample amplification, offering simplicity and efficiency. This method demonstrates high sensitivity with a minimum detectable limit of 5 pM. Furthermore, the method's specificity is substantiated by its capacity to discern point mutations in miRNA. This system has been shown to quantitatively analyse miRNA-21 levels present within serum, as evidenced by the recovery experiment. Therefore, the method's simplicity, stability, and cost-effectiveness render it a powerful tool for nucleic acid detection, with potential for clinical applications.}, }
@article {pmid40160040, year = {2025}, author = {Cetin, B and Erendor, F and Eksi, YE and Sanlioglu, AD and Sanlioglu, S}, title = {Advancing CRISPR genome editing into gene therapy clinical trials: progress and future prospects.}, journal = {Expert reviews in molecular medicine}, volume = {27}, number = {}, pages = {e16}, pmid = {40160040}, issn = {1462-3994}, mesh = {Humans ; *Gene Editing/methods ; *Genetic Therapy/methods/trends ; *CRISPR-Cas Systems ; Clinical Trials as Topic ; Anemia, Sickle Cell/therapy/genetics ; Animals ; beta-Thalassemia/therapy/genetics ; }, abstract = {Genome editing has recently evolved from a theoretical concept to a powerful and versatile set of tools. The discovery and implementation of CRISPR-Cas9 technology have propelled the field further into a new era. This RNA-guided system allows for specific modification of target genes, offering high accuracy and efficiency. Encouraging results are being announced in clinical trials employed in conditions like sickle cell disease (SCD) and transfusion-dependent beta-thalassaemia (TDT). The path finally led the way to the recent FDA approval of the first gene therapy drug utilising the CRISPR/Cas9 system to edit autologous CD34+ haematopoietic stem cells in SCD patients (Casgevy). Ongoing research explores the potential of CRISPR technology for cancer therapies, HIV treatment and other complex diseases. Despite its remarkable potential, CRISPR technology faces challenges such as off-target effects, suboptimal delivery systems, long-term safety concerns, scalability, ethical dilemmas and potential repercussions of genetic alterations, particularly in the case of germline editing. Here, we examine the transformative role of CRISPR technologies, including base editing and prime editing approaches, in modifying the genetic and epigenetic codes in the human genome and provide a comprehensive focus, particularly on relevant clinical applications, to unlock the full potential and challenges of gene editing.}, }
@article {pmid40158793, year = {2025}, author = {Sheng, T and Meng, XZ and Yu, Q and Lv, W and Chen, Y and Cong, Q and Li, W and Gui, L and Li, J and Xu, X}, title = {Transcriptome analysis and CRISPR-Cas9-mediated mutagenesis identify gpr116 as a candidate gene for growth promotion in grass carp (Ctenopharyngodon idella).}, journal = {Comparative biochemistry and physiology. Part A, Molecular &amp; integrative physiology}, volume = {305}, number = {}, pages = {111850}, doi = {10.1016/j.cbpa.2025.111850}, pmid = {40158793}, issn = {1531-4332}, mesh = {Animals ; *Carps/genetics/growth &amp; development ; *CRISPR-Cas Systems ; Zebrafish/genetics/growth &amp; development ; *Receptors, G-Protein-Coupled/genetics/metabolism ; Gene Expression Profiling ; *Fish Proteins/genetics/metabolism ; *Transcriptome ; Mutagenesis ; Brain/metabolism ; }, abstract = {Grass carp (Ctenopharyngodon idella) is an economically important aquaculture species known for its considerable variability in growth performance. In this study, we investigated the growth phenotype by comparing fast-growing and slow-growing groups. Microstructural analyses revealed that slow-growing fish exhibited significantly larger myofibrillar gaps and lower muscle fiber density. To elucidate the underlying molecular basis, we performed transcriptome (RNA-Seq) analysis of brain and dorsal muscle tissues. 328 differentially expressed genes (DEGs) were identified in dorsal muscle tissue (33 up and 295 down-regulated) and 228 in brain tissue (17 up and 211 down-regulated). Gene Ontology and KEGG enrichment analyses indicated that the DEGs were closely associated with apoptosis and angiogenesis pathways. Among the candidate genes, gpr116 was significantly up-regulated in the brain and dorsal muscle tissue of the fast-growing group. Finally, CRISPR-Cas9-mediated knockout in a zebrafish model confirmed that gpr116 deletion significantly restricted growth, underscoring its pivotal role in the growth regulation of grass carp. These discoveries lay significant groundwork for deeper exploration of growth regulation mechanisms in grass carp and offer important clues for selective breeding of key growth marker genes in this species.}, }
@article {pmid40158491, year = {2025}, author = {Khosla, NK and Lesinski, JM and Haywood-Alexander, M and deMello, AJ and Richards, DA}, title = {Machine learning and statistical classification in CRISPR-Cas12a diagnostic assays.}, journal = {Biosensors &amp; bioelectronics}, volume = {279}, number = {}, pages = {117402}, doi = {10.1016/j.bios.2025.117402}, pmid = {40158491}, issn = {1873-4235}, mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Machine Learning ; Humans ; Neural Networks, Computer ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {CRISPR-based diagnostics have gained increasing attention as biosensing tools able to address limitations in contemporary molecular diagnostic tests. To maximize the performance of CRISPR-based assays, much effort has focused on optimizing the chemistry and biology of the biosensing reaction. However, less attention has been paid to improving the techniques used to analyze CRISPR-based diagnostic data. To date, diagnostic decisions typically involve various forms of slope-based classification. Such methods are superior to traditional methods based on assessing absolute signals, but still have limitations. Herein, we establish performance benchmarks (total accuracy, sensitivity, and specificity) using common slope-based methods. We compare the performance of these benchmark methods with three different quadratic empirical distribution function statistical tests, finding significant improvements in diagnostic speed and accuracy when applied to a clinical data set. Two of the three statistical techniques, the Kolmogorov-Smirnov and Anderson-Darling tests, report the lowest time-to-result and highest total test accuracy. Furthermore, we developed a long short-term memory recurrent neural network to classify CRISPR-biosensing data, achieving 100 % specificity on our model data set. Finally, we provide guidelines on choosing the classification method and classification method parameters that best suit a diagnostic assay's needs.}, }
@article {pmid40157940, year = {2025}, author = {Fang, L and Hao, X and Fan, J and Liu, X and Chen, Y and Wang, L and Huang, X and Song, H and Cao, Y}, title = {Genome-scale CRISPRi screen identifies pcnB repression conferring improved physiology for overproduction of free fatty acids in Escherichia coli.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {3060}, pmid = {40157940}, issn = {2041-1723}, support = {22478294//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22308256//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2024T170643//China Postdoctoral Science Foundation/ ; }, mesh = {*Escherichia coli/genetics/metabolism/physiology ; *Escherichia coli Proteins/genetics/metabolism ; *Fatty Acids, Nonesterified/biosynthesis/metabolism ; Gene Expression Regulation, Bacterial ; Genome, Bacterial ; Fermentation ; Metabolic Engineering/methods ; CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing ; }, abstract = {Microbial physiology plays a pivotal role in construction of superior microbial cell factories for efficient biosynthesis of desired products. Here we identify that pcnB repression confers improved physiology for overproduction of free fatty acids (FFAs) in Escherichia coli through genome-scale CRISPRi modulation combining fluorescence-activated cell sorting (FACS) and next-generation sequencing (NGS). The repression of pcnB can enhance the stability and abundance of the transcripts of genes involved in the proton-consuming system, thereby supporting global improvements in membrane properties, redox state, and energy level. Based on pcnB repression, further repression of acrD increases FFAs biosynthesis by enhancing FFAs efflux. The engineered strain pcnB[i]-acrD[i]-fadR[+] achieves 35.1 g L[-1] FFAs production in fed-batch fermentation, which is the maximum titer reported to date in E. coli. This study highlights the significance of uncovering hidden genetic determinants that confer improved microbial physiology for enhancing the biosynthesis of desired products.}, }
@article {pmid40157676, year = {2025}, author = {Li, Y and Sun, C and Yao, D and Gao, X and Wei, X and Qi, Y and Liang, Y and Ye, J}, title = {A review of MicroRNAs and flavonoids: New insights into plant secondary metabolism.}, journal = {International journal of biological macromolecules}, volume = {309}, number = {Pt 1}, pages = {142518}, doi = {10.1016/j.ijbiomac.2025.142518}, pmid = {40157676}, issn = {1879-0003}, mesh = {*Flavonoids/biosynthesis/metabolism/genetics ; *MicroRNAs/genetics/metabolism ; *Plants/metabolism/genetics ; *Secondary Metabolism/genetics ; Gene Expression Regulation, Plant ; }, abstract = {Flavonoids, essential plant secondary metabolites, play crucial roles in growth regulation, stress responses, and applications in medicine, agriculture, and industry. However, the complexity of their biosynthetic pathways and regulatory networks poses challenges for industrial-scale production. MicroRNAs (miRNAs), as pivotal post-transcriptional regulators, play significant roles in fine-tuning flavonoid metabolism by targeting key enzyme genes and transcription factors. This review provides a comprehensive analysis of miRNA biogenesis and their molecular mechanisms, emphasizing miRNA-mediated regulation of flavonoid biosynthesis. We introduce the concept of "miRNA-multifactorial synergistic networks", which elucidates the collaborative interactions between miRNAs, non-coding RNAs, transcription factors, and epigenetic regulators. The review explores emerging strategies, including artificial miRNA design and CRISPR/Cas technologies, to precisely manipulate miRNA activity for enhancing flavonoid production. Additionally, integrating CRISPR/Cas13, synthetic biology, and multi-omics technologies offers new opportunities to construct efficient flavonoid metabolic systems. Artificial intelligence (AI) is proposed as a powerful tool to analyze omics data, identify regulatory nodes, and simulate environmental impacts on miRNA networks, thereby optimizing metabolic pathways. By integrating these multidisciplinary approaches, this review provides a novel theoretical framework and technical roadmap for understanding and improving flavonoid metabolism. The insights presented here aim to facilitate breakthroughs in metabolic engineering, offering significant potential for practical applications in plant breeding, functional food production, and pharmaceutical development.}, }
@article {pmid40157661, year = {2025}, author = {Mukherjee, A and Jodder, J and Chowdhury, S and Das, H and Kundu, P}, title = {A novel stress-inducible dCas9 system for solanaceous plants.}, journal = {International journal of biological macromolecules}, volume = {308}, number = {Pt 3}, pages = {142462}, doi = {10.1016/j.ijbiomac.2025.142462}, pmid = {40157661}, issn = {1879-0003}, mesh = {*Solanum lycopersicum/genetics ; Gene Expression Regulation, Plant ; *CRISPR-Cas Systems/genetics ; *Stress, Physiological/genetics ; Plant Proteins/genetics ; Transcription Factors/genetics ; Plants, Genetically Modified/genetics ; Heat-Shock Response/genetics ; }, abstract = {Conditional manipulation of gene expression is essential in plant biology, yet a simple stimuli-based inducible system for regulating any plant gene is lacking. Here, we present an innovative stress-inducible CRISPR/dCas9-based gene-regulatory toolkit tailored for intentional gene regulation in solanaceous plants. We have translationally fused the transmembrane domain of a tomato membrane-bound NAC transcription factor with dCas9 to utilize the reversible-tethering-based activation mechanism. This system sequesters dCas9 to the plasma membrane under normal conditions and allows membrane detachment in response to heat induction and NLS-mediated nuclear transfer, enabling stress-inducible gene regulation. Transient assays with tomato codon-optimized dCas9-assisted inducible CRISPR activation and interference systems confirmed their superior ability on transcriptional control, rapid induction, and reversibility after stimulus withdrawal in solanaceous plants. The transformative potential of the toolkit was exemplified by enhancing tomato immunity against bacterial speck disease under elevated temperatures by precisely regulating crucial salicylic acid signalling components, SlCBP60g and SlSARD1. Additionally, it was instrumental in engineering heat-stress tolerance in tomato plants through multiplex activation of heat-responsive transcription factors, SlNAC2 and SlHSFA6b. These findings demonstrate the unprecedented temporal control offered by this novel stress-inducible toolkit over gene-expression dynamics, paving the way for favourable manipulation of complex traits in environmentally-challenged crops.}, }
@article {pmid40157335, year = {2025}, author = {Paranthaman, S and Uthaiah, CA and Md, S and Alkreathy, HM}, title = {Comprehensive strategies for constructing efficient CRISPR/Cas based cancer therapy: Target gene selection, sgRNA optimization, delivery methods and evaluation.}, journal = {Advances in colloid and interface science}, volume = {341}, number = {}, pages = {103497}, doi = {10.1016/j.cis.2025.103497}, pmid = {40157335}, issn = {1873-3727}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/therapy/genetics ; *Gene Editing/methods ; *Genetic Therapy/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; }, abstract = {Cancer is a complicated disease that results from the interplay between specific changes in cellular genetics and diverse microenvironments. The application of high-performance and customizable clustered regularly interspaced palindromic repeats/associated protein (CRISPR/Cas) nuclease systems has significantly enhanced genome editing for accurate cancer modeling and facilitated simultaneous genetic modification for cancer therapy and mutation identification. Achieving an effective CRISPR/Cas platform for cancer treatment depends on the identification, selection, and optimization of specific mutated genes in targeted cancer tissues. However, overcoming the off-target effects, specificity, and immunogenicity are additional challenges that must be addressed while developing a gene editing system for cancer therapy. From this perspective, we briefly covered the pipeline of CRISPR/Cas cancer therapy, identified target genes to optimize gRNAs and sgRNAs, and explored alternative delivery modalities, including viral, non-viral, and extracellular vesicles. In addition, the list of patents and current clinical trials related to this unique cancer therapy method is discussed. In summary, we have discussed comprehensive start-to-end pipeline strategies for CRISPR/Cas development to advance the precision, effectiveness, and safety of clinical applications for cancer therapy.}, }
@article {pmid40157320, year = {2025}, author = {Li, J and Yin, L and Wang, C and Xu, Y}, title = {Generation of a homozygous ABCA7 knockout cell line (AHMUCNi002-A) in human iPSCs using CRISPR/Cas9.}, journal = {Stem cell research}, volume = {85}, number = {}, pages = {103700}, doi = {10.1016/j.scr.2025.103700}, pmid = {40157320}, issn = {1876-7753}, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *ATP-Binding Cassette Transporters/genetics/metabolism/deficiency ; Homozygote ; Cell Line ; *Gene Knockout Techniques ; Gene Editing ; }, abstract = {ABCA7, located on chromosome 19, encodes an ATP-binding cassette transporter. Loss-of-function variants of ABCA7 are associated with an increased risk of Alzheimer's disease. To explore the role of ABCA7 deficiency in the pathogenesis of Alzheimer's disease, CRISPR/Cas9 genome-editing technology was utilized to generate a homozygous ABCA7 knockout in human induced pluripotent stem cells (hiPSCs). The resulting ABCA7 knockout cell line exhibited normal pluripotency, a stable karyotype, and the ability to differentiate into all three germ layers.}, }
@article {pmid40157150, year = {2025}, author = {Song, Y and Long, J and Wang, H and Tang, W and Yang, W and Zheng, Y and Yuan, R and Zhang, D and Gu, B and Nian, W}, title = {High-efficiency detection of APE1 using a defective PAM-driven CRISPR-Cas12a self-catalytic biosensor.}, journal = {Biosensors &amp; bioelectronics}, volume = {279}, number = {}, pages = {117410}, doi = {10.1016/j.bios.2025.117410}, pmid = {40157150}, issn = {1873-4235}, mesh = {*Biosensing Techniques/methods ; *DNA-(Apurinic or Apyrimidinic Site) Lyase/blood/isolation &amp; purification/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; Limit of Detection ; *Endodeoxyribonucleases/chemistry/genetics ; DNA Probes/chemistry/genetics ; *CRISPR-Associated Proteins/chemistry/genetics ; Bacterial Proteins ; }, abstract = {The trans-cleavage activity of the CRISPR-Cas system offers tremendous potential for developing highly sensitive and selective molecular diagnostic tools. However, conventional methods often face challenges such as limited catalytic efficiency of single Cas proteins and the necessity of complex multi-enzyme preamplification steps. To address these limitations, we present a novel defective PAM-mediated CRISPR-Cas12a self-catalytic signal amplification strategy, termed DEP-Cas-APE, for the rapid, sensitive, and specific detection of apurinic/apyrimidinic endonuclease 1 (APE1) activity. This approach integrates defective PAM-modified DNA probes to synergize Cas12a trans-cleavage with self-catalytic circuit, achieving efficient signal transformation and amplification under isothermal, one-step conditions. We systematically investigated the influence of defective PAM sequences containing apurinic/apyrimidinic (AP) sites on Cas12a activation and validated the feasibility of the DEP-Cas-APE strategy in detecting APE1. Under optimized conditions, DEP-Cas-APE achieved a detection limit as low as 7.66 × 10[-8] U μL[-1] within 30 min using a simple isothermal reaction. Additionally, we developed a point-of-care testing (POCT) platform by integrating DEP-Cas-APE with a colorimetric assay based on gold nanoparticles (AuNPs), enabling portable, equipment-free detection. This sensitive and selective strategy successfully detected APE1 in complex biological samples, including serum from lung cancer patients, and demonstrated the ability to distinguish cancerous from normal samples. DEP-Cas-APE represents a robust and versatile platform for advancing CRISPR-Cas12a biosensing technologies, offering new opportunities for molecular diagnostics and clinical research.}, }
@article {pmid40156991, year = {2025}, author = {Tang, Q and Zhang, J and Pang, J and Huang, Y and Guan, Y and Gong, Y and Tang, Q and Zhang, K and Liao, X}, title = {Hybridization chain reaction and CRISPR/Cas12a-integrated biosensor for precise Ago2 detection.}, journal = {Bioelectrochemistry (Amsterdam, Netherlands)}, volume = {165}, number = {}, pages = {108975}, doi = {10.1016/j.bioelechem.2025.108975}, pmid = {40156991}, issn = {1878-562X}, mesh = {*Argonaute Proteins/analysis/genetics/metabolism ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Humans ; *Nucleic Acid Hybridization ; Luminescent Measurements/methods ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {This study introduces an innovative electrochemiluminescence (ECL) biosensor for the highly sensitive and specific detection of Argonaute 2 (Ago2) activity. Ago2, a key enzyme in the RNA interference (RNAi) pathway, plays a crucial role in gene regulation, and its dysregulation is associated with diseases such as cancer and viral infections. The biosensor integrates hybridization chain reaction (HCR) amplification and the CRISPR/Cas12a system, leveraging a multi-stage signal amplification strategy. The detection mechanism begins with Ago2-mediated cleavage of a designed hairpin RNA (HP-RNA), releasing single-stranded RNA (ssRNA) that triggers HCR. This amplification step generates long DNA polymers, which serve as activators for the CRISPR/Cas12a system. Cas12a's collateral cleavage activity amplifies the signal further by cleaving a DNA reporter labeled with a ruthenium-based luminophore, enhancing the ECL output. This dual amplification strategy achieves exceptional sensitivity, with a detection limit of 0.126 aM. The biosensor demonstrates excellent specificity, distinguishing Ago2 from other Argonaute proteins, and maintains high reproducibility and stability, retaining 94 % of its signal after two weeks of storage. Real-world applicability was confirmed by accurately detecting Ago2 in spiked cell lysates, with recovery rates exceeding 100 %. The combination of HCR, CRISPR/Cas12a, and ECL establishes a robust platform for biomarker detection, offering superior sensitivity and adaptability for clinical diagnostics, disease monitoring, and therapeutic evaluation. This biosensor represents a significant advancement in the development of next-generation diagnostic tools.}, }
@article {pmid40156858, year = {2025}, author = {Schmidt, T and Wiesbeck, M and Egert, L and Truong, TT and Danese, A and Voshagen, L and Imhof, S and Iraci Borgia, M and Deeksha,  and Neuner, AM and Köferle, A and Geerlof, A and Santos Dias Mourão, A and Stricker, SH}, title = {Efficient DNA- and virus-free engineering of cellular transcriptomic states using dCas9 ribonucleoprotein (dRNP) complexes.}, journal = {Nucleic acids research}, volume = {53}, number = {6}, pages = {}, pmid = {40156858}, issn = {1362-4962}, support = {//EpiCrossBorders/ ; //International Helmholtz-Edinburgh Research School for Epigenetics/ ; //Helmholtz Zentrum M&#xfc;nchen/ ; STR 1385/5-1//DFG/ ; INST 86/2110-1//SFB/ ; //REGENERAR/ ; /CAPMC/CIHR/Canada ; //Natural Sciences and Engineering Research Council of Canada/ ; //Humanities Research Council of Canada/ ; }, mesh = {Humans ; *Ribonucleoproteins/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Transcriptome/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; DNA/genetics ; Promoter Regions, Genetic ; HEK293 Cells ; Transcriptional Activation ; }, abstract = {For genome editing, the use of CRISPR ribonucleoprotein (RNP) complexes is well established and often the superior choice over plasmid-based or viral strategies. RNPs containing dCas9 fusion proteins, which enable the targeted manipulation of transcriptomes and epigenomes, remain significantly less accessible. Here, we describe the production, delivery, and optimization of second generation CRISPRa RNPs (dRNPs). We characterize the transcriptional and cellular consequences of dRNP treatments in a variety of human target cells and show that the uptake is very efficient. The targeted activation of genes demonstrates remarkable potency, even for genes that are strongly silenced, such as developmental master transcription factors. In contrast to DNA-based CRISPRa strategies, gene activation is immediate and characterized by a sharp temporal precision. We also show that dRNPs allow very high-target multiplexing, enabling undiminished gene activation of multiple genes simultaneously. Applying these insights, we find that intensive target multiplexing at single promoters synergistically elevates gene transcription. Finally, we demonstrate in human stem and differentiated cells that the preferable features of dRNPs allow to instruct and convert cell fates efficiently without the need for DNA delivery or viral vectors.}, }
@article {pmid40156857, year = {2025}, author = {Vasquez, CA and Osgood, NRB and Zepeda, MU and Sandel, DK and Cowan, QT and Peiris, MN and Donoghue, DJ and Komor, AC}, title = {Precision genome editing and in-cell measurements of oxidative DNA damage repair enable functional and mechanistic characterization of cancer-associated MUTYH variants.}, journal = {Nucleic acids research}, volume = {53}, number = {6}, pages = {}, pmid = {40156857}, issn = {1362-4962}, support = {27502//Research Corporation for Science Advancement/ ; //Cancer Cell Signaling and Communication Training Program/ ; //National Academies of Sciences, Engineering, and Medicine/ ; //University of California, San Diego/ ; /HHMI/Howard Hughes Medical Institute/United States ; //Molecular Biophysics Training/ ; T32 CA009523/GF/NIH HHS/United States ; T32 GM008326/GM/NIGMS NIH HHS/United States ; }, mesh = {*DNA Glycosylases/genetics/metabolism ; Humans ; *DNA Repair/genetics ; *Gene Editing/methods ; *DNA Damage ; Mutation ; Oxidative Stress ; CRISPR-Cas Systems ; Guanine/analogs &amp; derivatives/metabolism ; Cell Line, Tumor ; *Neoplasms/genetics ; Colorectal Neoplasms/genetics ; Genes, Reporter ; }, abstract = {Functional characterization of genetic variants has the potential to advance the field of precision medicine by enhancing the efficacy of current therapies and accelerating the development of new approaches to combat genetic diseases. MUTYH is a DNA repair enzyme that recognizes and repairs oxidatively damaged guanines [8-oxoguanine (8-oxoG)] mispaired with adenines (8-oxoG·A). While some mutations in the MUTYH gene are associated with colorectal cancer, most MUTYH variants identified in sequencing databases are classified as variants of uncertain significance. Convoluting clinical classification is the absence of data directly comparing homozygous versus heterozygous MUTYH mutations. In this study, we present the first effort to functionally characterize MUTYH variants using precision genome editing to generate heterozygous and homozygous isogenic cell lines. Using a MUTYH-specific lesion reporter in which we site-specifically incorporate an 8-oxoG·A lesion in a fluorescent protein gene, we measure endogenous MUTYH enzymatic activity and classify them as pathogenic or benign. Further, we modify this reporter to incorporate the MUTYH repair intermediate (8-oxoG across from an abasic site) and validate it with co-immunoprecipitation experiments to demonstrate its ability to characterize the mechanism by which MUTYH mutants are defective at DNA repair.}, }
@article {pmid40156855, year = {2025}, author = {Miceli, F and Bracaglia, S and Sorrentino, D and Porchetta, A and Ranallo, S and Ricci, F}, title = {MAIGRET: a CRISPR-based immunoassay that employs antibody-induced cell-free transcription of CRISPR guide RNA strands.}, journal = {Nucleic acids research}, volume = {53}, number = {6}, pages = {}, pmid = {40156855}, issn = {1362-4962}, support = {819160/ERC_/European Research Council/International ; 21965//Associazione Italiana per la Ricerca sul Cancro/ ; 2022ANCEK//Italian Ministry of University and Research/ ; 101165168/ERC_/European Research Council/International ; //Associazione Italiana per la Ricerca sul Cancro/ ; 2022FPYZ2N//Italian Ministry of University and Research/ ; }, mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; *Antibodies/immunology/analysis ; Immunoassay/methods ; CRISPR-Associated Proteins/metabolism ; *Transcription, Genetic ; Cell-Free System ; Humans ; Endodeoxyribonucleases/metabolism ; Bacterial Proteins/metabolism ; DNA/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Here we report on the development of a CRISPR-based assay for the sensitive and specific detection of antibodies and antigens directly in complex sample matrices. The assay, called Molecular Assay based on antibody-Induced Guide-RNA Enzymatic Transcription (MAIGRET), is based on the use of a responsive synthetic DNA template that triggers the cell-free in vitro transcription of a guide RNA strand upon recognition of a specific target antibody. Such transcribed guide RNA activates the DNA collateral activity of the Cas12a enzyme, leading to the downstream cleavage of a fluorophore/quencher-labeled reporter and thus resulting in an increase in the measured fluorescence signal. We have used MAIGRET for the detection of six different antibodies with high sensitivity (detection limit in the picomolar range) and specificity (no signal in the presence of non-target antibodies). MAIGRET can also be adapted to a competitive approach for the detection of specific antigens. With MAIGRET, we significantly expand the scope and applicability of CRISPR-based sensing approaches to potentially enable the measurement of any molecular target for which an antibody is available.}, }
@article {pmid40156749, year = {2025}, author = {Reķēna, A and Pals, K and Gavrilović, S and Lahtvee, PJ}, title = {The role of ATP citrate lyase, phosphoketolase, and malic enzyme in oleaginous Rhodotorula toruloides.}, journal = {Applied microbiology and biotechnology}, volume = {109}, number = {1}, pages = {77}, pmid = {40156749}, issn = {1432-0614}, mesh = {*ATP Citrate (pro-S)-Lyase/metabolism/genetics ; *Rhodotorula/enzymology/genetics/growth &amp; development/metabolism ; *Malate Dehydrogenase/metabolism/genetics ; Gene Knockout Techniques ; *Aldehyde-Lyases/metabolism/genetics ; CRISPR-Cas Systems ; Glucose/metabolism ; Culture Media/chemistry ; Lipid Metabolism ; *Oxo-Acid-Lyases/metabolism/genetics ; Xylose/metabolism ; Acetates/metabolism ; Lipids/biosynthesis ; }, abstract = {Rhodotorula toruloides is an oleaginous yeast recognized for its robustness and the production of high content of neutral lipids. Early biochemical studies have linked ATP citrate lyase (ACL), phosphoketolase (PK), and cytosolic malic enzyme (cMAE) with de novo lipid synthesis. In this study, we discovered that upon a CRISPR/Cas9-mediated knockout of the ACL gene, lipid content in R. toruloides IFO0880 decreased from 50 to 9% of its dry cell weight (DCW) in glucose medium and caused severe growth defects (reduced specific growth rate, changes in cell morphology). In xylose medium, the lipid content decreased from 43 to 38% of DCW. However, when grown on acetate as the sole carbon source, the lipid content decreased from 45 to 20% of DCW. Significant growth defects as a result of ACL knockout were observed on all substrates. In contrast, PK knockout resulted in no change in growth or lipid synthesis. Knocking out cMAE gene resulted in lipid increase of 2.9% of DCW and 23% increase in specific growth rate on glucose. In xylose or acetate medium, no change in lipid production as a result of cMAE gene knockout was observed. These results demonstrated that ACL plays a crucial role in lipid synthesis in R. toruloides IFO0880, as opposed to PK pathway or cMAE, whose presence in some conditions even disfavors lipid production. These results provided valuable information for future metabolic engineering of R. toruloides. KEY POINTS: • ACL is crucial for the fatty acid synthesis and growth in R. toruloides IFO0880. • Lipid production and cell growth is are unchanged as a result of PK knockout. • Cytosolic malic enzyme does not play a significant role in lipogenesis.}, }
@article {pmid40156192, year = {2025}, author = {Georgiadis, C and Preece, R and Qasim, W}, title = {Clinical development of allogeneic chimeric antigen receptor αβ-T cells.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {33}, number = {6}, pages = {2426-2440}, pmid = {40156192}, issn = {1525-0024}, mesh = {Humans ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; *Receptors, Antigen, T-Cell, alpha-beta/genetics/immunology/metabolism ; *Immunotherapy, Adoptive/methods ; Gene Editing ; *T-Lymphocytes/immunology/metabolism ; Graft vs Host Disease/immunology/prevention &amp; control ; Animals ; Transplantation, Homologous ; }, abstract = {Ready-made banks of allogeneic chimeric antigen receptor (CAR) T cells, produced to be available at the time of need, offer the prospect of accessible and cost-effective cellular therapies. Various strategies have been developed to overcome allogeneic barriers, drawing on cell engineering platforms including RNA interference, protein-based restriction, and genome editing, including RNA-guided CRISPR-Cas and base editing tools. Alloreactivity and the risk of graft-versus-host disease from non-matched donor cells have been mitigated by disruption of αβ-T cell receptor expression on the surface of T cells and stringent removal of any residual αβ-T cell populations. In addition, host-mediated rejection has been tackled through a combination of augmented lymphodepletion and cell engineering strategies that have allowed infused cells to evade immune recognition or conferred resistance to lymphodepleting agents to promote persistence and expansion of effector populations. Early-phase studies using off-the-shelf universal donor CAR T cells have been undertaken mainly in the context of blood malignancies, where emerging data of clinical responses have supported wider adoption and further applications. These developments offer the prospect of alternatives to current autologous approaches through the emerging application of genome engineering solutions to improve safety, persistence, and function of universal donor products.}, }
@article {pmid40156040, year = {2025}, author = {Zhang, Y and Zou, W and Zhou, Y and Chen, J and Hu, Y and Wu, F}, title = {Pamoic acid and carbenoxolone specifically inhibit CRISPR/Cas9 in bacteria, mammalian cells, and mice in a DNA topology-specific manner.}, journal = {Genome biology}, volume = {26}, number = {1}, pages = {75}, pmid = {40156040}, issn = {1474-760X}, support = {YG2025QNB55//Research Fund of Medicine and Engineering of Shanghai Jiao Tong University/ ; 32271304//National Natural Science Foundation of China/ ; }, mesh = {Animals ; *CRISPR-Cas Systems/drug effects ; Mice ; *CRISPR-Associated Protein 9/antagonists &amp; inhibitors/metabolism ; *Carbenoxolone/pharmacology ; *DNA/chemistry/metabolism ; Humans ; Staphylococcus aureus/genetics/enzymology ; Gene Editing ; Streptococcus pyogenes/enzymology/genetics ; }, abstract = {BACKGROUND: Regulation of the target DNA cleavage activity of CRISPR/Cas has naturally evolved in a few bacteria or bacteriophages but is lacking in higher species. Thus, identification of bioactive agents and mechanisms that can suppress the activity of Cas9 is urgently needed to rebalance this new genetic pressure.<br><br>RESULTS: Here, we identify four specific inhibitors of Cas9 by screening 4607 compounds that could inhibit the endonuclease activity of Cas9 via three distinct mechanisms: substrate-competitive and protospacer adjacent motif (PAM)-binding site-occupation; substrate-targeting; and sgRNA-targeting mechanisms. These inhibitors inhibit, in a dose-dependent manner, the activity of Streptococcus pyogenes Cas9 (SpyCas9), Staphylococcus aureus Cas9 (SauCas9), and SpyCas9 nickase-based BE4 base editors in in vitro purified enzyme assays, bacteria, mammalian cells, and mice. Importantly, pamoic acid and carbenoxolone show DNA-topology selectivity and preferentially inhibit the cleavage of linear DNA compared with a supercoiled plasmid.<br><br>CONCLUSIONS: These pharmacologically selective inhibitors and new mechanisms offer new tools for controlling the DNA-topology selective activity of Cas9.}, }
@article {pmid40155371, year = {2025}, author = {Chen, P and Wu, Y and Wang, H and Liu, H and Zhou, J and Chen, J and Lei, J and Sun, Z and Paek, C and Yin, L}, title = {Highly parallel profiling of the activities and specificities of Cas12a variants in human cells.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {3022}, pmid = {40155371}, issn = {2041-1723}, support = {32371271//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32171210//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32101196//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2021TQ0253//China Postdoctoral Science Foundation/ ; 2022M712468//China Postdoctoral Science Foundation/ ; 2022M722473//China Postdoctoral Science Foundation/ ; }, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; HEK293 Cells ; Deep Learning ; }, abstract = {Several Cas12a variants have been developed to broaden its targeting range, improve the gene editing specificity or the efficiency. However, selecting the appropriate Cas12a among the many orthologs for a given target sequence remains difficult. Here, we perform high-throughput analyses to evaluate the activity and compatibility with specific PAMs of 24 Cas12a variants and develop deep learning models for these Cas12a variants to predict gene editing activities at target sequences of interest. Furthermore, we reveal and enhance the truncation in the integrated tag sequence that may hinder off-targeting detection for Cas12a by GUIDE-seq. This enhanced system, which we term enGUIDE-seq, is used to evaluate and compare the off-targeting and translocations of these Cas12a variants.}, }
@article {pmid40154762, year = {2025}, author = {Awan, MJA and Farooq, MA and Buzdar, MI and Zia, A and Ehsan, A and Waqas, MAB and Hensel, G and Amin, I and Mansoor, S}, title = {Advances in gene editing-led route for hybrid breeding in crops.}, journal = {Biotechnology advances}, volume = {81}, number = {}, pages = {108569}, doi = {10.1016/j.biotechadv.2025.108569}, pmid = {40154762}, issn = {1873-1899}, mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics ; *Plant Breeding/methods ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; }, abstract = {With the global demand for sustainable agriculture on the rise, RNA-guided nuclease technology offers transformative applications in crop breeding. Traditional hybrid breeding methods, like three-line and two-line systems, are often labor-intensive, transgenic, and economically burdensome. While chemical mutagens facilitate these systems, they not only generate weak alleles but also produce strong alleles that induce permanent sterility through random mutagenesis. In contrast, RNA-guided nuclease system, such as clustered regularly interspaced short palindromic repeats (CRISPR)- associated protein (Cas) system, facilitates more efficient hybrid production by inducing male sterility through targeted genome modifications in male sterility genes, such as MS8, MS10, MS26, and MS45 which allows precise manipulation of pollen development or pollen abortion in various crops. Moreover, this approach allows haploid induction for the rapid generation of recombinant and homozygous lines from hybrid parents by editing essential genes, like CENH3, MTL/NLD/PLA, and DMP, resulting in high-yield, transgene-free hybrids. Additionally, this system supports synthetic apomixis induction by employing the MiMe (Mitosis instead of Meiosis) strategy, coupled with parthenogenesis in hybrid plants, to create heterozygous lines and retain hybrid vigor in subsequent generations. RNA-guided nuclease-induced synthetic apomixis also enables genome stacking for autopolyploid progressive heterosis via clonal gamete production for trait maintenance to enhance crop adaptability without compromising yield. Additionally, CRISPR-Cas-mediated de novo domestication of wild relatives, along with recent advances to circumvent tissue culture- recalcitrance and -dependency through heterologous expression of morphogenic regulators, holds great promise for incorporating diversity-enriched germplasm into the breeding programs. These approaches aim to generate elite hybrids adapted to dynamic environments and address the anticipated challenges of food insecurity.}, }
@article {pmid40154215, year = {2025}, author = {Zhang, Z and Ji, Q and Zhang, Z and Lyu, B and Li, P and Zhang, L and Chen, K and Fang, M and Song, J}, title = {Ultra-sensitive detection of melanoma NRAS mutant ctDNA based on programmable endonucleases.}, journal = {Cancer genetics}, volume = {294-295}, number = {}, pages = {47-56}, doi = {10.1016/j.cancergen.2025.02.008}, pmid = {40154215}, issn = {2210-7762}, mesh = {Humans ; *Melanoma/genetics/blood/diagnosis ; *Membrane Proteins/genetics ; *GTP Phosphohydrolases/genetics ; *Circulating Tumor DNA/genetics/blood ; *Mutation ; CRISPR-Cas Systems ; DNA Mutational Analysis/methods ; Biomarkers, Tumor/genetics ; Liquid Biopsy ; }, abstract = {BACKGROUND: Melanoma is a complex and often fatal disease, with NRAS being one of the most frequently mutated genes in this type of cancer. Liquid biopsies, specifically tests for circulating tumor DNA (ctDNA), represent a promising and less invasive approach to diagnosis. This study aims to develop an ultra-sensitive assay for detecting melanoma NRAS mutant ctDNA.<br><br>METHODS: To detect rare NRAS mutant ctDNA, we developed the NRAS PASEA assay by screening CRISPR-Cas proteins that recognize the PAM sequence 5'-TTN-3'. This method employs CRISPR-Cas proteins to continuously shear wild-type alleles during isothermal amplification, resulting in exponential amplification of mutant alleles to a detectable level by Sanger sequencing.<br><br>RESULTS: The developed NRAS Q61R/L/K mutation detection method can detect simulated ctDNA samples with mutant allele fractions (MAF) as low as 0.01&#x202f;% with 30&#x202f;mins of PASEA treatment. Notably, the NRAS Q61&#x202f;K mutation was accurately identified by FnCas12a-based NRAS PASEA, even with the nucleotide at the "N" position in the PAM site "TTN." The method successfully detected ctDNA in patients with malignant melanoma. All patients (5/5) from 15 melanoma blood samples with NRAS Q61R (4/4) and NRAS Q61&#x202f;K (1/1) mutations were accurately identified, with no false positives among patients with wildtype NRAS Q61.<br><br>CONCLUSION: Detecting ctDNA from peripheral blood samples is highly significant for melanomas in areas where imaging evaluation is challenging. Our assay demonstrated 100&#x202f;% consistency with tumor tissue NGS, providing a new analytical strategy for companion diagnosis and dynamic assessment of therapeutic efficacy and disease progression in melanoma.}, }
@article {pmid40153308, year = {2025}, author = {Hayhurst, M and Vink, JNA and Remerand, M and Gerth, ML}, title = {Transient expression of fluorescent proteins and Cas nucleases in Phytophthora agathidicida via PEG-mediated protoplast transformation.}, journal = {Microbiology (Reading, England)}, volume = {171}, number = {3}, pages = {}, pmid = {40153308}, issn = {1465-2080}, mesh = {*Phytophthora/genetics/metabolism ; *Protoplasts/metabolism ; *Transformation, Genetic ; Polyethylene Glycols/chemistry ; Plasmids/genetics ; Chitinases/metabolism/genetics ; Green Fluorescent Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Calcium Chloride/chemistry ; *Luminescent Proteins/genetics/metabolism ; Plant Diseases/microbiology ; }, abstract = {Phytophthora species are eukaryotic plant pathogens that cause root rot and dieback diseases in thousands of plant species worldwide. Despite their significant economic and ecological impacts, fundamental molecular tools such as DNA transformation methods are not yet established for many Phytophthora species. In this study, we have established a PEG/calcium chloride (CaCl2)-mediated protoplast transformation method for Phytophthora agathidicida, the causal agent of kauri dieback disease. Adapting a protocol from Phytophthora sojae, we systematically optimized the protoplast digesting enzymes, recovery media composition and pH. Our findings reveal that chitinases are essential for P. agathidicida protoplast formation, and the optimum pH of the recovery medium is 5. The media type did not significantly impact protoplast regeneration. Using this protocol, we generated transformants using three plasmids (i.e. pTdTomatoN, pYF2-PsNLS-Cas9-GFP and pYF2-PsNLS-Cas12a-GFP), which expressed fluorescent proteins and/or Cas nucleases. The transformants were unstable unless maintained under antibiotic selective pressure; however, under selection, fluorescence was maintained across multiple generations and life cycle stages, including the production of fluorescent zoospores from transformed mycelia. Notably, we observed the expression of GFP-tagged Cas nucleases, which is promising for future CRISPR-Cas genome editing applications. This study demonstrates that P. agathidicida is amenable to PEG/CaCl2-mediated protoplast transformation. Although the resulting transformants require antibiotic selective pressure to remain stable, this transient expression system can be valuable for applications such as cell tracking, chemotaxis studies and CRISPR-Cas genome editing. The protocol also provides a foundation for further optimization of transformation methods. It serves as a valuable tool for exploring the molecular biology of P. agathidicida and potentially other closely related Phytophthora species.}, }
@article {pmid40153053, year = {2025}, author = {Kiraz, D and Özcan, A}, title = {Comparative genome analysis of 15 Streptococcus thermophilus strains isolated from Turkish traditional yogurt.}, journal = {Antonie van Leeuwenhoek}, volume = {118}, number = {4}, pages = {64}, pmid = {40153053}, issn = {1572-9699}, mesh = {*Streptococcus thermophilus/genetics/isolation &amp; purification/classification ; *Yogurt/microbiology ; *Genome, Bacterial ; Turkey ; Phylogeny ; Fermentation ; Whole Genome Sequencing ; Food Microbiology ; }, abstract = {Streptococcus thermophilus plays a pivotal role in yogurt fermentation, yet strains from traditional fermented products remain largely unexplored compared to their industrial counterparts. This study aimed to characterize the genomic diversity and functional potential of 15 S. thermophilus strains isolated from Turkish traditional yogurts, and to compare them with industrial strains. Through whole-genome sequencing and advanced bioinformatics analyses, we revealed distinct phylogenetic patterns and genetic features that differentiate these traditional strains from industrial isolates. The genomes (1.68-1.86 Mb) exhibited high genetic homogeneity (ANI > 98.69%) while maintaining significant functional diversity. Pan-genome analysis identified 1160 core genes and 5694 accessory genes, highlighting substantial genomic plasticity that enables niche adaptation. Our analysis uncovered several distinctive features: (1) unique phylogenetic clustering patterns based on both housekeeping genes and whole-genome SNPs, suggesting geographical isolation effects; (2) an extensive repertoire of carbohydrate-active enzymes (CAZymes), comprising 111 Glycoside Hydrolases, 227 Glycosyl Transferases, and 44 Carbohydrate Esterases and 13 Carbohydrate-Binding Modules, demonstrating sophisticated carbohydrate metabolism adaptation significantly enriched compared to industrial strains; (3) widespread GABA biosynthesis pathways in 8 strains, including complete gadB gene, indicating potential health-promoting properties; (4) multiple genomic islands containing genes for galactose utilization and stress response, suggesting specific adaptation to traditional fermentation environments; (5) diverse exopolysaccharide biosynthesis and bacteriocin gene clusters; and (6) widespread CRISPR-Cas systems with variable spacer content. Notably, we identified vanY glycopeptide resistance genes across all strains, with two strains additionally harboring vanT. These results reveal the genetic mechanisms behind S. thermophilus adaptation to traditional yogurt environments, offering valuable insights for developing starter cultures and preserving the unique qualities and potential health benefits of traditional dairy products.}, }
@article {pmid40152981, year = {2025}, author = {Feng, B and Wang, Y and Zhang, X and Mu, T and Zhang, B and Li, Y and Zhang, H and Hua, W and Yuan, W and Li, H}, title = {Targeted mutagenesis and functional marker development of two Bna.TAC1s conferring novel rapeseed germplasm with compact architecture.}, journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik}, volume = {138}, number = {4}, pages = {86}, pmid = {40152981}, issn = {1432-2242}, support = {32172028//National Natural Science Foundation of China/ ; U22A20477//National Natural Science Foundation of China/ ; }, mesh = {Genetic Markers ; *Brassica napus/genetics/growth &amp; development/anatomy &amp; histology ; Phenotype ; *Plant Proteins/genetics/metabolism ; Plant Breeding ; Mutagenesis ; CRISPR-Cas Systems ; *Brassica rapa/genetics/growth &amp; development ; Genes, Plant ; Gene Expression Regulation, Plant ; }, abstract = {Simultaneous disruption of two Bna.TAC1s, redundantly controlling the branch angle, generates a compact architecture in rapeseed, and two functional markers are developed to facilitate breeding rapeseed cultivars with compact architecture. Shoot branch angle is a key factor in determining the aerial plant architecture. A narrow branch angle can increase yields by facilitating mechanized harvest and high-density planting in rapeseed, a globally important oil crop. However, the available rapeseed varieties with narrow branch angle are very limited. In this study, two Bna.TAC1 members named BnaA5.TAC1 and BnaC4.TAC1 were found to have the four canonical domains of TAC1-like members, including domains I, II, III and IV in rapeseed. Each Bna.TAC1 exhibits dominant expression in the lateral branch with gradual dynamic response to light and encodes a protein localized in the plasma membrane. CRISPR/Cas9-mediated editing system was used to simultaneously knock out the two Bna.TAC1s to obtain two different Bna.tac1 double mutants, designed as CR-Bna.tac1-1 and CR-Bna.tac1-2. These two mutants displayed different degrees of compact architecture without affecting plant height and yield-related traits. The two Bna.TAC1s were also shown to play a redundant role in controlling branch angle by regulating the gravitropic response. In addition, we developed two specific gel-based functional markers in each Bna.TAC1 for the transgene-free mutant CR-Bna.tac1-1, which co-segregate with narrower branch angle and could help to identify the mutant alleles in a segregating population. We also found that the genomic variation of the two Bna.TAC1s is not associated with branch angle variation in the natural rapeseed population. Overall, these results reveal the key roles of Bna.TAC1s in regulation of rapeseed branch angle and provide a novel germplasm and functional markers for breeding superior varieties with compact architecture in rapeseed.}, }
@article {pmid40152921, year = {2025}, author = {Prithiviraj, S and Garcia Garcia, A and Linderfalk, K and Yiguang, B and Ferveur, S and Falck, LN and Subramaniam, A and Mohlin, S and Hidalgo Gil, D and Dupard, SJ and Zacharaki, D and Raina, DB and Bourgine, PE}, title = {Compositional editing of extracellular matrices by CRISPR/Cas9 engineering of human mesenchymal stem cell lines.}, journal = {eLife}, volume = {13}, number = {}, pages = {}, pmid = {40152921}, issn = {2050-084X}, support = {2019-01864_3//Vetenskapsr&#xe5;det/ ; 948588/ERC_/European Research Council/International ; }, mesh = {Humans ; *CRISPR-Cas Systems ; *Mesenchymal Stem Cells/metabolism/physiology ; *Extracellular Matrix/metabolism/genetics ; Animals ; *Gene Editing/methods ; *Tissue Engineering/methods ; Rats ; Cell Line ; Vascular Endothelial Growth Factor A/genetics ; Core Binding Factor Alpha 1 Subunit/genetics ; }, abstract = {Tissue engineering strategies predominantly rely on the production of living substitutes, whereby implanted cells actively participate in the regenerative process. Beyond cost and delayed graft availability, the patient-specific performance of engineered tissues poses serious concerns on their clinical translation ability. A more exciting paradigm consists in exploiting cell-laid, engineered extracellular matrices (eECMs), which can be used as off-the-shelf materials. Here, the regenerative capacity solely relies on the preservation of the eECM structure and embedded signals to instruct an endogenous repair. We recently described the possibility to exploit custom human stem cell lines for eECM manufacturing. In addition to the conferred standardization, the availability of such cell lines opened avenues for the design of tailored eECMs by applying dedicated genetic tools. In this study, we demonstrated the exploitation of CRISPR/Cas9 as a high precision system for editing the composition and function of eECMs. Human mesenchymal stromal/stem cell (hMSC) lines were modified to knock out vascular endothelial growth factor (VEGF) and Runt-related transcription factor 2 (RUNX2) and assessed for their capacity to generate osteoinductive cartilage matrices. We report the successful editing of hMSCs, subsequently leading to targeted VEGF and RUNX2-knockout cartilage eECMs. Despite the absence of VEGF, eECMs retained full capacity to instruct ectopic endochondral ossification. Conversely, RUNX2-edited eECMs exhibited impaired hypertrophy, reduced ectopic ossification, and superior cartilage repair in a rat osteochondral defect. In summary, our approach can be harnessed to identify the necessary eECM factors driving endogenous repair. Our work paves the road toward the compositional eECMs editing and their exploitation in broad regenerative contexts.}, }
@article {pmid40152782, year = {2025}, author = {Guo, C and Yang, L and Zhang, T and Xie, L and Long, X and Ma, L and Zhao, Q and Cui, Y and Zhang, Y and He, Y}, title = {Target-Induced Enzymatic Cascade Reaction Method: Integrating Strand Displacement Amplification and CRISPR/Cas12a for Homogeneous Assays of Biotin.}, journal = {Journal of agricultural and food chemistry}, volume = {73}, number = {14}, pages = {8651-8659}, doi = {10.1021/acs.jafc.5c02441}, pmid = {40152782}, issn = {1520-5118}, mesh = {*Biotin/analysis/metabolism ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Streptavidin/chemistry ; Food Contamination/analysis ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; *Endodeoxyribonucleases/genetics/metabolism/chemistry ; Bacterial Proteins/genetics/metabolism/chemistry ; DNA, Single-Stranded/chemistry/genetics ; Limit of Detection ; }, abstract = {The important biological role of biotin emphasizes the need for a sensitive method to detect it in foodstuffs. This article introduces a homogeneous and sensitive biotin analysis method that leverages a target-induced enzymatic cascade reaction, incorporating strand-displacement amplification (SDA) and the CRISPR/Cas12a system. Without target biotin, streptavidin (SA) specifically binds to the biotinylated probe DNA, hindering Klenow polymerase from extending the primer single-stranded DNA (ssDNA) due to the steric hindrance created by the SA-biotin complex, resulting in low fluorescence. Conversely, competition between the target biotin and the biotin label for binding to SA reduces the amount of SA captured on the primer ssDNA. The SDA process, which involves Klenow polymerase and the Nb.BbvCI enzyme, proceeds smoothly, thereby activating the CRISPR/Cas12a system and producing an intense fluorescence signal. Utilizing this principle, precise and sensitive biotin detection in food matrices was achieved, with a limit of detection of 0.01 nM.}, }
@article {pmid40152384, year = {2025}, author = {Fragale, N and Divvela, SSK and Williams-Ward, VC and Brand-Saberi, B}, title = {Loss of Atoh8 Affects Neurocranial and Axial Skeleton Development in Zebrafish.}, journal = {Frontiers in bioscience (Landmark edition)}, volume = {30}, number = {3}, pages = {26806}, doi = {10.31083/FBL26806}, pmid = {40152384}, issn = {2768-6698}, support = {G1001029//MRC/ ; MR/N021231/1//MRC/ ; }, mesh = {Animals ; *Zebrafish/genetics/embryology/growth &amp; development ; *Zebrafish Proteins/genetics/metabolism ; *Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism ; Gene Expression Regulation, Developmental ; CRISPR-Cas Systems ; Gene Knockout Techniques ; *Bone Development/genetics ; *Skull/embryology/metabolism ; *Bone and Bones/embryology/metabolism ; Phenotype ; }, abstract = {BACKGROUND: The basic helix-loop-helix (bHLH) transcription factor atonal homologue 8 (Atoh8) has been implicated in various developmental and physiological processes by means of transient knockdown and conditional knockout approaches in zebrafish, chick and mouse. Despite its demonstrated involvement in multiple tissues, the role of Atoh8 remains elusive in zebrafish. A recent permanent knockout study in zebrafish investigated the role of Atoh8 on the background of previous morpholino studies which demonstrated various developmental defects but could not find any of the morpholino-based effects in the mutant. In mice, a knockout study demonstrated involvement of the transcription factor in skeletal development, showing that disruption of the atoh8 gene results in reduction of skeletal size. We investigated a mutant fish line generated using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9)-technology for possible phenotypic effects on zebrafish skeletogenesis.<br><br>METHODS: Here, we present a CRISPR/Cas9-generated atoh8 permanent zebrafish mutant and investigate the phenotypic effects of the knockout on the developing zebrafish craniofacial and axial skeleton. We investigated the expression pattern of the gene in wildtype and conducted detailed morphometric analysis for a variety of bone and cartilage elements of the developing skeleton at 12 days post fertilisation (dpf) in zebrafish siblings from a heterozygous mating using detailed morphometric measurements and statistical analysis of the results.<br><br>RESULTS: Homozygous mutants are viable into late adulthood and show no overt morphological phenotype. Despite the prominent appearance of atoh8 signal in various embryonic and larval craniofacial and axial skeletal structures, detailed morphometric analysis revealed only subtle phenotypic effects of the mutation on skeletal development in zebrafish. We found the formation of the orbital cartilages of the developing neurocranium and the progress of chordacentra mineralisation to be negatively affected by loss of the transcription factor.<br><br>CONCLUSIONS: Despite the very subtle phenotypic effect of our mutation, we were able to show involvement of atoh8 in the skeletal development of zebrafish. We attribute the mild phenotype to a compensatory mechanism induced by nonsense-mediated degradation of messenger ribonucleic acid (mRNA) as suggested in the recent literature. The effect of atoh8-disruption on zebrafish skeletal development suggests that the loss of atoh8 cannot be compensated for at interfaces where more than one embryonic cell lineage contributes to bone and cartilage formation.}, }
@article {pmid40151969, year = {2025}, author = {Tsubota, T and Takasu, Y and Yonemura, N and Sezutsu, H}, title = {Enhancements of the CRISPR-Cas System in the Silkworm Bombyx mori.}, journal = {The CRISPR journal}, volume = {8}, number = {2}, pages = {155-164}, doi = {10.1089/crispr.2024.0089}, pmid = {40151969}, issn = {2573-1602}, mesh = {Animals ; *Bombyx/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; }, abstract = {The silkworm (Bombyx mori) is a lepidopteran model insect that has been utilized for basic research and industrial applications. In this species, transcription activator-like effector nucleases (TALENs) have been found to function efficiently, and we previously developed a TALEN-mediated genome editing system for knockout and knock-in experiments using plasmids and single-stranded oligodeoxynucleotides (ssODNs) as donors. By contrast, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated genome editing, especially for gene integration, remains limited. In this study, we attempted to improve CRISPR-Cas systems to expand the utility of genome editing in the silkworm. Codon optimization of Cas9 improved genome editing efficiency, and single-guide RNA utilization also resulted in a higher genome editing efficiency than crRNA/tracrRNA when Cas9 messenger RNA (mRNA) was used. CRISPR-Cas12a-mediated genome editing and targeted sequence integration using ssODNs were both successfully performed. Overall, our study provides a robust technical platform that can facilitate basic and applied silkworm studies.}, }
@article {pmid40151952, year = {2025}, author = {Yuan, H and Song, C and Xu, H and Sun, Y and Anthon, C and Bolund, L and Lin, L and Benabdellah, K and Lee, C and Hou, Y and Gorodkin, J and Luo, Y}, title = {An Overview and Comparative Analysis of CRISPR-SpCas9 gRNA Activity Prediction Tools.}, journal = {The CRISPR journal}, volume = {8}, number = {2}, pages = {89-104}, doi = {10.1089/crispr.2024.0058}, pmid = {40151952}, issn = {2573-1602}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Machine Learning ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; Deep Learning ; Computational Biology/methods ; Computer Simulation ; Software ; Animals ; }, abstract = {Design of guide RNA (gRNA) with high efficiency and specificity is vital for successful application of the CRISPR gene editing technology. Although many machine learning (ML) and deep learning (DL)-based tools have been developed to predict gRNA activities, a systematic and unbiased evaluation of their predictive performance is still needed. Here, we provide a brief overview of in silico tools for CRISPR design and assess the CRISPR datasets and statistical metrics used for evaluating model performance. We benchmark seven ML and DL-based CRISPR-Cas9 editing efficiency prediction tools across nine CRISPR datasets covering six cell types and three species. The DL models CRISPRon and DeepHF outperform the other models exhibiting greater accuracy and higher Spearman correlation coefficient across multiple datasets. We compile all CRISPR datasets and in silico prediction tools into a GuideNet resource web portal, aiming to facilitate and streamline the sharing of CRISPR datasets. Furthermore, we summarize features affecting CRISPR gene editing activity, providing important insights into model performance and the further development of more accurate CRISPR prediction models.}, }
@article {pmid40151930, year = {2025}, author = {Gao, M and Yang, C and Si, W and Xi, X and Chen, L and Zeng, Z and Rong, Y and Yang, Y and Wang, F and Yuan, C}, title = {Combining CRISPR-Cas12a with Microsphere Array-Enhanced Fluorescence for Portable Pathogen Nucleic Acid Detection.}, journal = {ACS applied materials &amp; interfaces}, volume = {17}, number = {14}, pages = {20932-20942}, doi = {10.1021/acsami.5c00655}, pmid = {40151930}, issn = {1944-8252}, mesh = {*Microspheres ; *CRISPR-Cas Systems/genetics ; Humans ; *SARS-CoV-2/genetics/isolation &amp; purification ; Fluorescence ; *COVID-19/diagnosis/virology ; Nucleic Acid Amplification Techniques ; *CRISPR-Associated Proteins/genetics ; Lab-On-A-Chip Devices ; Food Contamination/analysis ; Limit of Detection ; RNA, Viral/genetics/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {The detection of food contamination in a swift and sensitive manner is essential for safeguarding public health. Clustered regularly interspaced short palindromic repeats (CRISPR)-based assays for nucleic acid detection are renowned for their high specificity and convenient, related studies have focused on refining the Cas protein and optimizing the CRISPR (cr)RNAs design within CRISPR-based assays for enhancing the sensitivity of nucleic acid detection. Our research offers innovative insights into enhancing the fluorescence signal output intensity from a physical standpoint, thereby presenting a practical and cost-effective strategy to lower the detection thresholds in CRISPR-based assays. By a layer of microsphere arrays was spread onto the bottom of the microfluidic chip to enhance the fluorescence signal of the sample via self-assembly of the microspheres. Recombinase polymerase amplification (RPA) was used to amplify target sequences, followed by crRNA binding to activate Cas enzyme, cleaving fluorescein amidite (FAM)-labeled reporters and emitting a fluorescent signal. The method successfully identified SARS-CoV-2 positive samples (10 clinical samples and 8 environmental contamination samples) and distinguished them from negative samples. Meanwhile, it successfully detected 4 food contamination Shigella samples and 5 clinical Shigella samples. In this study, the developed method exhibited a detection limit (LoD) of 75 fM for SARS-CoV-2 (POCT with USB camera: 50 fM) and 100 fM for Shigella (POCT with USB camera: 75 fM). It also demonstrated promising sensitivity (100%) and specificity (100%) in a small-sample validation. Combined portable and automated detection was achieved using a smartphone to receive and process the fluorescent signals obtained from the samples. The detection platform developed in this study is not only applicable for the detection of pathogens in cold-chain food products, but also extends to pathogen detection in community hospitals and resource-limited areas, providing an efficient solution for rapid pathogen screening in different settings. Moreover, different nucleic acid samples can be detected by changing the RPA primer and CRISPR crRNA. This method provides a paradigm for studying enhanced fluorescence signaling and holds significant potential to advance the commercialization and practical use of CRISPR fluorescence sensors.}, }
@article {pmid40151342, year = {2025}, author = {Singh, A and Yasheshwar,  and Kaushik, NK and Kala, D and Nagraik, R and Gupta, S and Kaushal, A and Walia, Y and Dhir, S and Noorani, MS}, title = {Conventional and cutting-edge advances in plant virus detection: emerging trends and techniques.}, journal = {3 Biotech}, volume = {15}, number = {4}, pages = {100}, pmid = {40151342}, issn = {2190-572X}, abstract = {Plant viruses pose a significant threat to global agriculture. For a long time, conventional methods including detection based on visual symptoms, host range investigations, electron microscopy, serological assays (e.g., ELISA, Western blotting), and nucleic acid-based techniques (PCR, RT-PCR) have been used for virus identification. With increased sensitivity, speed, and specificity, new technologies like loop-mediated isothermal amplification (LAMP), high-throughput sequencing (HTS), nanotechnology-based biosensors, and CRISPR diagnostics have completely changed the way plant viruses are detected. Recent advances in detection techniques integrate artificial intelligence (AI), machine learning (ML), and the Internet of Things (IoT) for real-time monitoring. Innovations like hyperspectral imaging, deep learning, and cloud-based IoT platforms further support disease identification and surveillance. Nanotechnology-based lateral flow assays and CRISPR-Cas systems provide rapid, field-deployable solutions. Despite these advancements, challenges such as sequence limitations, multiplexing constraints, and environmental concerns remain. Future research should focus on refining portable on-site diagnostic kits, optimizing nanotechnology applications, and enhancing global surveillance systems. Interdisciplinary collaboration across molecular biology, bioinformatics, and engineering is essential to developing scalable, cost-effective solutions for plant virus detection, ensuring agricultural sustainability and ecosystem protection.}, }
@article {pmid40151107, year = {2025}, author = {Zou, G and Si, P and Wang, J and Yang, M and Chen, J and Liu, C and Luo, Z}, title = {Chemical Modification Coupled with Isothermal CRISPR-Based Assay for Sensitive Detection of DNA Hydroxymethylation.}, journal = {ACS sensors}, volume = {10}, number = {3}, pages = {2073-2079}, doi = {10.1021/acssensors.4c03312}, pmid = {40151107}, issn = {2379-3694}, mesh = {Humans ; *5-Methylcytosine/analogs &amp; derivatives/analysis ; *DNA Methylation ; *DNA/chemistry/genetics/analysis ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; *CRISPR-Cas Systems ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {5-Hydroxymethylcytosine (5hmC) plays a key role in the DNA demethylation process and serves as a stable epigenetic marker in the human genome which is closely associated with disease progression, particularly in diabetes, colorectal cancer, and liver cancer. However, convenient and sensitive methods for detecting and quantifying 5hmC in the genome are scarce, especially in complex biological environments. Herein, a novel attempt at hypersensitive quantitative detection of 5hmC was presented. A multifunctional photosensitive probe was therefore introduced for specific labeling, enrichment, and elution of 5hmC-DNA. Combining with isothermal assay leveraging rolling circle amplification and Cas12a for accurate recognition, we achieved quantitative detection of 5hmC DNA in trace amounts at a level of 11 fM. Global 5hmC was measured in various biological samples using as little as 10 ng of input DNA by a real-time PCR instrument. The reported approach imposed no sequence restrictions, demonstrating promising potential for detecting modified bases in trace amounts of nucleic acids within complex environments, such as blood, urine, and saliva samples.}, }
@article {pmid40149851, year = {2025}, author = {Pan, D and Mijit, M and Wang, H and Sun, C and Pingcuo, B and Yu, Z and Xiong, B and Tang, X}, title = {Rapid Genotyping of FecB Mutation in Sheep Using CRISPR-Cas12a Integrated with DNA Nanotree Biosensing Platform.}, journal = {Biomolecules}, volume = {15}, number = {3}, pages = {}, pmid = {40149851}, issn = {2218-273X}, support = {2022YFD1301102//Xiangfang Tang/ ; 2022BBF02020//Xiangfang Tang&#xff0c;Benhai Xiong/ ; Guike AA22068099//Xiangfang Tang/ ; 2004DA125184G2405//Hui Wang/ ; jc-cxgc-ias-09-1//Xiangfang Tang/ ; 2024-YWF-ZYSQ-10//Hui Wang/ ; CAAS-CSSAE-202402//Hui Wang/ ; }, mesh = {Animals ; Sheep/genetics ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Mutation ; *DNA/genetics/chemistry ; *Genotyping Techniques/methods ; *Bone Morphogenetic Protein Receptors, Type I/genetics ; Genotype ; Gold/chemistry ; }, abstract = {The A-to-G mutation (FecB) in the BMPR1B gene is strongly linked to fertility in sheep, significantly increasing ovulation rates and litter sizes compared to wild-type populations. The rapid and reliable screening of the FecB gene is therefore critical for advancing sheep breeding programs. This study aimed to develop a fast and accurate method for detecting the FecB mutation and genotyping the gene to enhance sheep reproduction and productivity. To achieve this, we integrated the CRISPR-Cas12a system with an optimized amplification refractory mutation system (ARMS). A similar DNA origami technique-based fluorescence reporter nanotree structure was synthesized using gold nanomagnetic beads as carriers to amplify the fluorescence signal further. The resulting biosensing platform, termed CRISPR-ARMS, demonstrated excellent sensitivity for detecting FecB mutations, with a detection limit as low as 0.02 pmol. Therefore, this innovative approach shows great promise for single-base mutation detection and represents a pioneering tool for high-yield genetic screening.}, }
@article {pmid40148961, year = {2025}, author = {Zheng, Y and Mo, Y and Yuan, Y and Su, T and Qi, Q}, title = {A rapid and efficient strategy for combinatorial repression of multiple genes in Escherichia coli.}, journal = {Microbial cell factories}, volume = {24}, number = {1}, pages = {74}, pmid = {40148961}, issn = {1475-2859}, support = {ZR2021QC021//Natural Science Foundation of Shandong Province/ ; tsqn202312029//Young Taishan Scholars Program of Shandong Province/ ; No. 32200081//National Natural Science Foundation of China/ ; }, mesh = {*Escherichia coli/genetics/metabolism ; *Gene Expression Regulation, Bacterial ; *Metabolic Engineering/methods ; Promoter Regions, Genetic ; Plasmids/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Escherichia coli Proteins/genetics/metabolism ; CRISPR-Cas Systems ; }, abstract = {BACKGROUND: The regulation of multiple gene expression is pivotal for metabolic engineering. Although CRISPR interference (CRISPRi) has been extensively utilized for multi-gene regulation, the construction of numerous single-guide RNA (sgRNA) expression plasmids for combinatorial regulation remains a significant challenge.<br><br>RESULTS: In this study, we developed a combinatorial repression system for multiple genes by optimizing the expression of multi-sgRNA with various inducible promoters in Escherichia coli. We designed a modified Golden Gate Assembly method to rapidly construct the sgRNA expression plasmid p3gRNA-LTA. By optimizing both the promoter and the sgRNA handle sequence, we substantially mitigated undesired repression caused by the leaky expression of sgRNA. This method facilitates the rapid assessment of the effects of various inhibitory combinations on three genes by simply adding different inducers. Using the biosynthesis of N-acetylneuraminic acid (NeuAc) as an example, we found that the optimal combinatorial inhibition of the pta, ptsI, and pykA genes resulted in a 2.4-fold increase in NeuAc yield compared to the control.<br><br>CONCLUSION: We anticipate that our combinatorial repression system will greatly simplify the regulation of multiple genes and facilitate the fine-tuning of metabolic flow in the engineered strains.}, }
@article {pmid40148327, year = {2025}, author = {Zhang, X and Li, M and Chen, K and Liu, Y and Liu, J and Wang, J and Huang, H and Zhang, Y and Huang, T and Ma, S and Liao, K and Zhou, J and Wang, M and Lin, Y and Rong, Z}, title = {Engineered circular guide RNAs enhance miniature CRISPR/Cas12f-based gene activation and adenine base editing.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {3016}, pmid = {40148327}, issn = {2041-1723}, mesh = {*Gene Editing/methods ; Animals ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Adenine/metabolism ; Mice ; *RNA, Circular/genetics ; Humans ; Liver/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; HEK293 Cells ; }, abstract = {CRISPR system has been widely used due to its precision and versatility in gene editing. Un1Cas12f1 from uncultured archaeon (hereafter referred to as Cas12f), known for its compact size (529 aa), exhibits obvious delivery advantage for gene editing in vitro and in vivo. However, its activity remains suboptimal. In this study, we engineer circular guide RNA (cgRNA) for Cas12f and significantly improve the efficiency of gene activation about 1.9-19.2-fold. When combined with a phase separation system, the activation efficiency is further increased about 2.3-3.9-fold. In addition, cgRNA enhances the editing efficiency and narrows the editing window of adenine base editing about 1.2-2.5-fold. Importantly, this optimization strategy also boosts the Cas12f-induced gene activation efficiency in mouse liver. Therefore, we demonstrate that cgRNA is able to enhance Cas12f-based gene activation and adenine base editing, which holds great potential for gene therapy.}, }
@article {pmid40147870, year = {2025}, author = {Dunipace, L and McGehee, JM and Irizarry, J and Stathopoulos, A}, title = {The proximal enhancer of the snail gene mediates negative autoregulatory feedback in Drosophila melanogaster.}, journal = {Genetics}, volume = {230}, number = {2}, pages = {}, pmid = {40147870}, issn = {1943-2631}, support = {P40 OD018537/OD/NIH HHS/United States ; R35 GM118146/GM/NIGMS NIH HHS/United States ; R35GM118146//National Institute of Health/ ; }, mesh = {Animals ; *Drosophila melanogaster/genetics/embryology ; *Enhancer Elements, Genetic ; *Drosophila Proteins/genetics/metabolism ; *Snail Family Transcription Factors/genetics/metabolism ; Gene Expression Regulation, Developmental ; *Transcription Factors/genetics/metabolism ; Homeostasis ; Feedback, Physiological ; Embryonic Development/genetics ; CRISPR-Cas Systems ; Nuclear Proteins ; Phosphoproteins ; }, abstract = {Autoregulatory feedback is a mechanism in which a gene product regulates its own expression, stabilizing gene activity amid noise and environmental changes. In Drosophila melanogaster, the gene snail encodes a key transcriptional repressor that regulates the expression of many genes during early embryogenesis, including its own expression. This study focuses on Snail occupancy at both distal and proximal enhancers of the snail gene to understand the cis-regulatory mechanisms involved in autoregulatory control. The coordinated action of these enhancers results in precisely constrained levels of snail expression during early embryogenesis. Using genome editing by CRISPR/Cas9, we found that deletion of each enhancer individually is compatible with embryonic viability under normal conditions. However, the double mutant is lethal, suggesting a functional interplay between the 2 enhancers. To gain further insight, we assayed snail gene expression levels in fixed embryos. Our results revealed that negative autoregulation of snail relies on the proximal enhancer. Moreover, increasing the affinity of binding sites for Dorsal, a transcriptional activator, in the proximal enhancer impaired this autoregulation, suggesting that Snail acts locally to counterbalance Dorsal's input. A mathematical model of snail autoregulatory control further supports our findings, reinforcing the view that the proximal enhancer mediates negative autoregulatory feedback, and implicating the distal enhancer in positive autoregulatory feedback. In summary, Snail's role at the proximal enhancer is pivotal for negative autoregulatory control and essential for balancing the activation mediated by the distal enhancer.}, }
@article {pmid40147800, year = {2025}, author = {Yu, WB and Ye, ZH and Shi, JJ and Deng, WQ and Chen, J and Lu, JJ}, title = {Dual blockade of GSTK1 and CD47 improves macrophage-mediated phagocytosis on cancer cells.}, journal = {Biochemical pharmacology}, volume = {236}, number = {}, pages = {116898}, doi = {10.1016/j.bcp.2025.116898}, pmid = {40147800}, issn = {1873-2968}, mesh = {*CD47 Antigen/antagonists &amp; inhibitors/metabolism/immunology/genetics ; *Phagocytosis/physiology/drug effects ; Humans ; *Macrophages/metabolism/drug effects/immunology ; *Glutathione Transferase/antagonists &amp; inhibitors/metabolism/genetics ; Cell Line, Tumor ; Animals ; Mice ; RAW 264.7 Cells ; CRISPR-Cas Systems ; *Neoplasms/metabolism ; }, abstract = {CD47 is a crucial anti-phagocytic signal in regulating macrophage responses and its manipulation offers the therapeutic potential in cancer treatment. However, in many cases, blockade of CD47 by itself is insufficient to activate macrophage effectively, indicating other unidentified phagocytosis-regulating factors to resist the macrophage activity. In this study, a genome-wide human CRISPR-Cas9 library was developed for comprehensive screening of phagocytosis-regulating factors in the context of CD47 blockade. The screening results identified GSTK1 as a potential anti-phagocytic signal counteracting the efficacy of CD47-based phagocytosis. The disruption of GSTK1 significantly increased the phagocytosis rate of cancer cells by macrophages in combination with anti-CD47 antibody. Further mechanism investigation unveiled that GSTK1 blockade increased the membrane exposure of calreticulin in different cancer cells, which might be the primary mechanism driving enhanced macrophage-mediated phagocytosis. To this end, siGSTK1-loaded nanoparticles (siGSTK1-LNPs) were designed to suppress the GSTK1 expression efficiently. The comparable phagocytosis efficacy was also observed when combining siGSTK1-LNPs with anti-CD47 antibody. Above all, GSTK1 blockade was identified as a promising and feasible stimulus for enhancing the effectiveness of anti-CD47 antibody, introducing a novel and effective combination approach in cancer immunotherapy.}, }
@article {pmid40147495, year = {2025}, author = {Panting, M and Holme, IB and Dionisio, G and Brinch-Pedersen, H}, title = {Simplex and multiplex CRISPR/Cas9-mediated knockout of grain protease inhibitors in model and commercial barley improves hydrolysis of barley and soy storage proteins.}, journal = {Plant biotechnology journal}, volume = {23}, number = {6}, pages = {2418-2428}, pmid = {40147495}, issn = {1467-7652}, support = {8055-00038B//Innovationsfonden/ ; NNF19OC005658//Novo Nordisk Fonden/ ; 101060057//Horizon Europe/ ; }, mesh = {*Hordeum/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Protease Inhibitors/metabolism ; *Soybean Proteins/metabolism ; *Seed Storage Proteins/metabolism ; *Plant Proteins/metabolism/genetics ; Hydrolysis ; Edible Grain/genetics/metabolism ; Seeds/metabolism/genetics ; Gene Knockout Techniques ; Glycine max/metabolism ; Proteolysis ; }, abstract = {Anti-nutritional factors in plant seeds diminish the utilization of nutrients in feed and food. Among these, protease inhibitors inhibit protein degradation by exogenous proteases during digestion. Through conventional and selection-gene-free genome editing using ovules as explants, we used simplex and multiplex CRISPR/Cas9 for studying the impact of chymotrypsin inhibitor CI-1A, CI-1B and CI-2, Bowman-Birk trypsin inhibitor, Serpin-Z4, and barley ɑ-amylase/subtilisin inhibitor on barley and soybean storage protein degradation. Mutants were generated in the commercial cultivar Stairway, having a high level of protease inhibition, and the barley model cultivar Golden Promise, having a lower inhibition level. In Golden Promise, all individual knockouts decreased the inhibition of the three proteases α-chymotrypsin, trypsin and the commercial feed protease Ronozyme ProAct significantly. The triple knockout of all chymotrypsin inhibitors further decreased the inhibition of α-chymotrypsin and Ronozyme ProAct proteases. Degradations of recombinant barley storage proteins B- and C-hordeins were significantly improved following mutagenesis. In Stairway, a single knockout of CI-1A almost compares to the effect on the proteases achieved for the triple knockout in Golden promise, uncovering CI-1A as the major protease inhibitor in that cultivar. The Stairway mutant demonstrated significantly improved degradation of recombinant barley hordeins and in the soybean storage proteins glycinin and β-conglycinin. The results of this study provide insights into cereal protease inhibitor genes and their negative effects on the degradation of barley storage protein and the most important plant protein from soybeans. The study suggests a future focus on plant protease inhibitors as a major target for improving feed and food protein digestibility.}, }
@article {pmid40147084, year = {2025}, author = {Chen, X and Zhao, D and Yu, C and Wei, J and Zhou, G}, title = {A novel photosensitive nanoprobe combined with CRISPR/Cas12a for dual signal amplification detection of ANGPTL2.}, journal = {Talanta}, volume = {292}, number = {}, pages = {128010}, doi = {10.1016/j.talanta.2025.128010}, pmid = {40147084}, issn = {1873-3573}, mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Humans ; *Angiopoietin-Like Protein 2/blood ; *Metal Nanoparticles/chemistry ; Nucleic Acid Amplification Techniques ; Colorectal Neoplasms/diagnosis/blood ; *Angiopoietin-like Proteins/blood ; *Photosensitizing Agents/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {The detection of specific protein biomarkers holds significant potential for the early diagnosis of colorectal cancer (CRC). However, the accurate quantification of low-abundance proteins in serum presents a major challenge due to factors such as limited sensitivity and the complexity of the required methodologies. In this work, we established a universal CRISPR/Cas biosensing platform by integrating novel photosensitive nanoprobes (DA/PL@Cu NPs) and CRISPR/Cas12 system (DPC-Cas) for the highly sensitive, specific and user-friendly detection of angiopoietin-like protein 2 (ANGPTL2). The DA/PL@Cu NPs serve as a critical component in the transduction of protein recognition information into nucleic acid amplification events to produce Cas12a activators. The DPC-Cas biosensor integrates DA/PL@Cu NPs-assisted amplification with Cas12a self-amplification, enabling ultrasensitive detection of ANGPTL2 at concentrations as low as 20.00 pg/mL. The proposed DPC-Cas biosensor successfully detected ANGPTL2 in serum, demonstrating significant potential for the early diagnosis of CRC.}, }
@article {pmid40147061, year = {2025}, author = {Zhou, H and Liu, Y and Chen, Q and Huang, K and Ren, PG}, title = {Generation of TP53 knock out induced pluripotent stem cell using CRISPR/Cas9.}, journal = {Stem cell research}, volume = {85}, number = {}, pages = {103699}, doi = {10.1016/j.scr.2025.103699}, pmid = {40147061}, issn = {1876-7753}, mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Tumor Suppressor Protein p53/genetics/deficiency/metabolism ; Animals ; Mice ; Humans ; *Gene Knockout Techniques ; Cell Line ; Teratoma/pathology ; }, abstract = {The TP53 gene is an important tumor suppressor gene. Through CRISPR/Cas9 technology, we have established a TP53 gene knockout cell line in iPSCs (SIIBRi001-A). This cell line maintains normal stem cell-like morphology, karyotype, expresses markers of pluripotency, and is capable of generating teratomas in immunodeficient mice. Quantitative analysis of pluripotency gene expression remains normal. This cell line can be utilized for studying the mechanisms underlying tumorigenesis.}, }
@article {pmid40146774, year = {2025}, author = {Fidelito, G and Todorovski, I and Cluse, L and Vervoort, SJ and Taylor, RA and Watt, MJ}, title = {Lipid-metabolism-focused CRISPR screens identify enzymes of the mevalonate pathway as essential for prostate cancer growth.}, journal = {Cell reports}, volume = {44}, number = {4}, pages = {115470}, doi = {10.1016/j.celrep.2025.115470}, pmid = {40146774}, issn = {2211-1247}, mesh = {Male ; *Mevalonic Acid/metabolism ; *Prostatic Neoplasms/pathology/genetics/metabolism/enzymology ; Humans ; Cell Line, Tumor ; *Lipid Metabolism/genetics ; Animals ; *CRISPR-Cas Systems/genetics ; Cell Proliferation ; Mice ; *Dimethylallyltranstransferase/metabolism/genetics ; Endoplasmic Reticulum Stress ; }, abstract = {Dysregulated lipid metabolism plays an important role in prostate cancer, although the understanding of the essential regulatory processes in tumorigenesis is incomplete. We employ a CRISPR-Cas9 screen using a custom human lipid metabolism knockout library to identify essential genes for prostate cancer survival. Screening in three prostate cancer cell lines reveals 63 shared dependencies, with enrichment in terpenoid backbone synthesis and N-glycan biosynthesis. Independent knockout of key genes of the mevalonate pathway reduces cell proliferation. Further investigation focuses on NUS1, a subunit of cis-prenyltransferase required for dolichol synthesis. NUS1 knockout decreases tumor growth in vivo and viability in patient-derived xenograft (PDX)-derived organoids. Mechanistic studies reveal that loss of NUS1 promotes oxidative stress, lipid peroxidation and ferroptosis sensitivity, endoplasmic reticulum (ER) stress, and G1 cell-cycle arrest, and it dampens androgen receptor (AR) signaling, collectively leading to growth arrest. This study highlights the critical role of the mevalonate-dolichol-N-glycan biosynthesis pathway, particularly NUS1, in prostate cancer survival and growth.}, }
@article {pmid40146761, year = {2025}, author = {Winiarczyk, D and Khodadadi, H and Leszczyński, P and Taniguchi, H}, title = {A simple validation and screening method for CRISPR/Cas9-mediated gene editing in mouse embryos to facilitate genetically modified mice production.}, journal = {PloS one}, volume = {20}, number = {3}, pages = {e0312722}, pmid = {40146761}, issn = {1932-6203}, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mice ; Female ; *Embryo, Mammalian/metabolism ; Mice, Transgenic ; Blastocyst/metabolism ; }, abstract = {Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) is a genome engineering method for generating site-specific editing in target genes in a variety of species. It is a common tool for generating mouse models of different diseases. However, detecting target modifications in mouse embryos can be time-consuming and expensive. Accordingly, developing a screening method to confirm gene modification may be useful. We propose herein an evaluation method (cleavage assay - CA) for CRISPR/Cas9-mediated gene editing in preimplantation mouse embryos that allows us to detect mutants efficiently and later on initiate in vivo production without the extensive number of samples needing to be sent for Sanger sequencing and animal usage. Our method is based on the inability of the RNP complex to recognize the target sequence after CRISPR-mediated genome editing due to modification of the target locus. It allows us to establish gene edited mice in a user-friendly fashion with a limited number of mice usage by confirming each step of CRISPR-mediated gene editing of mouse embryos and, therefore, can be considered as a supportive tool to existing procedures for verification of successful CRISPR/Cas9-mediated gene alterations in mouse embryos and further mutant production.}, }
@article {pmid40146517, year = {2025}, author = {Tripathi, JN and Tripathi, L}, title = {Agrobacterium tumefaciens-Mediated Genome Editing in Banana.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2911}, number = {}, pages = {143-153}, pmid = {40146517}, issn = {1940-6029}, mesh = {*Musa/genetics/growth &amp; development ; *Gene Editing/methods ; *Agrobacterium tumefaciens/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; *Genome, Plant ; Transformation, Genetic ; }, abstract = {This protocol outlines the Agrobacterium tumefaciens-mediated transformation process for genome editing in banana (Musa spp.). As a crop of significant agricultural and economic importance globally, improving banana cultivars is crucial for addressing challenges such as disease resistance, climate resilience and yield enhancement. The procedure presented here involves the establishment of embryogenic cell suspensions (ECSs) from immature male flowers, followed by co-cultivation with Agrobacterium containing the desired gene construct. Key steps include callus induction, ECS generation and maintenance, transformation using plasmid construct containing genome editing reagents such as CRISPR/Cas9, and regeneration. The method ensures the editing of target genes in the banana genome, facilitating genetic improvements.}, }
@article {pmid40146515, year = {2025}, author = {Arruabarrena, A and Vidal, S}, title = {Agrobacterium-Mediated Transformation for Gene Editing Tomato Elite Breeding Lines.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2911}, number = {}, pages = {121-131}, pmid = {40146515}, issn = {1940-6029}, mesh = {*Solanum lycopersicum/genetics ; *Gene Editing/methods ; *Plant Breeding/methods ; CRISPR-Cas Systems ; *Transformation, Genetic ; Plants, Genetically Modified/genetics ; *Agrobacterium/genetics ; }, abstract = {Genome editing success in crop species is largely dependent on the availability of highly efficient plant transformation protocols. Tomato (Solanum lycopersicum) was the first dicotyledonous crop to be successfully mutagenized using CRISPR-Cas9. Despite many efforts, no standardized, simple protocol is available for non-model tomato genotypes. With the increasing availability of gene editing tools, the transformation of elite tomato breeding lines has gained importance because mutant variants can be easily incorporated into breeding programs. This chapter describes a protocol for transforming and gene editing in elite tomato breeding lines, reaching 3.6% transformation efficiency.}, }
@article {pmid40146510, year = {2025}, author = {Castillo Castro, FM and Payacán Ortiz, C}, title = {Agrobacterium-Mediated Transformation for Commercial Wheat Varieties.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2911}, number = {}, pages = {61-70}, pmid = {40146510}, issn = {1940-6029}, mesh = {*Triticum/genetics/growth &amp; development ; *Transformation, Genetic ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; *Agrobacterium tumefaciens/genetics ; CRISPR-Cas Systems ; Plant Breeding/methods ; }, abstract = {Wheat is a crucial crop for global food security, and new breeding techniques face limitations due to low regeneration rates and a lack of transformable genotypes. We follow and adapt a robust Agrobacterium tumefaciens-mediated transformation system in spring commercial wheat varieties from Chile and Argentina grown in speed breeding conditions. By incorporating the developmental gene fusion GRF4-GIF1, we achieved successful regeneration of plantlets in different wheat varieties through CRISPR/Cas9-based gene editing. This advancement reduces genotype dependency, allowing broader use of genome-editing tools in commercial wheat varieties. In addition to delving into technical complexities, this contribution aims to advance fundamental understanding and practical applications in wheat genetics, serving as a valuable resource for researchers expanding their expertise.}, }
@article {pmid40146509, year = {2025}, author = {Faion-Molina, M and Molina-Risco, MD and Bellinatti-Della Gracia, MD and Ibarra, O and Kim, B and Septiningsih, EM and Thomson, MJ}, title = {Agrobacterium tumefaciens-Mediated Plant Transformation and Gene Editing in Rice.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2911}, number = {}, pages = {45-59}, pmid = {40146509}, issn = {1940-6029}, mesh = {*Oryza/genetics/growth &amp; development ; *Gene Editing/methods ; *Agrobacterium tumefaciens/genetics ; *Transformation, Genetic ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; Oxidoreductases/genetics ; }, abstract = {Bottlenecks in plant transformation and regeneration have slowed progress in applying CRISPR/Cas9-based genome editing for crop improvement. Rice (Oryza sativa L.) has highly efficient temperate japonica transformation protocols, along with reasonably efficient indica protocols using immature embryos. However, rapid and efficient protocols are not available for transformation and regeneration in tropical japonica varieties, even though they represent most of the rice production in the USA and South America, along with some regions in Asia. This chapter describes a protocol for CRISPR/Cas9 gene editing using Agrobacterium-mediated transformation for the tropical japonica rice cultivar Presidio leading to knock-out mutations in the phytoene desaturase (PDS) gene.}, }
@article {pmid40146284, year = {2025}, author = {Kaniganti, S and Palakolanu, SR and Thiombiano, B and Damarasingh, J and Bommineni, PR and Che, P and Sharma, KK and Jones, T and Bouwmeester, H and Bhatnagar-Mathur, P}, title = {Developing Striga resistance in sorghum by modulating host cues through CRISPR/Cas9 gene editing.}, journal = {Plant cell reports}, volume = {44}, number = {4}, pages = {90}, pmid = {40146284}, issn = {1432-203X}, support = {DBT/2017/ICRISAT/973//Department of Biotechnology, Minstry of Science and Technology, India (IN)/ ; }, mesh = {*Sorghum/genetics/parasitology ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Striga/physiology ; *Disease Resistance/genetics ; Plants, Genetically Modified ; *Plant Diseases/parasitology/genetics ; Plant Proteins/genetics/metabolism ; Lactones/metabolism ; Gene Expression Regulation, Plant ; Mutation ; }, abstract = {High transformation and gene editing efficiencies in sorghum-produced, transgene-free SDN1-edited plants exhibit precise mutations, reduced germination stimulants, and enhanced resistance to Striga infection. Sorghum (Sorghum bicolor L.) is a primary food staple grain for millions in Sub-Saharan Africa (SSA). It is mainly constrained by the parasitic weed Striga, which causes up to 100% yield losses and affects over 60% of cultivable farmlands and livelihoods. In this study, CRISPR/Cas9 technology is utilized to induce mutations in core strigolactone (SL) biosynthetic genes, i.e., CCD7, CCD8, MAX1, in addition to an uncharacterized gene (DUF) in the fine-mapped 400 kb lgs1 region in sorghum to develop durable Striga resistance. Two sorghum cultivars were delivered with the expression cassettes through immature embryo-based Agrobacterium-mediated transformation. Our study demonstrated transformation and gene editing efficiencies of ~ 70 and up to 17.5% (calculated based on the numuber of established plants), respectively, in two sorghum genotypes. Subsequent analysis of homozygous E0 lines in the E1 generation confirmed stable integration of mutations for all targeted genes. Loss-of-function mutations in the CCD7, CCD8, MAX1, and DUF genes led to a significant downregulation of the expression of associated genes in the SL biosynthetic pathway. The phenotypic analysis of edited lines revealed changes in phenotypic patterns compared to wild-type plants. Analysis of root exudates showed significant reductions in SL production in edited lines compared to wild-type plants. Striga infection experiments demonstrated delayed or reduced emergence rates of Striga in edited lines with lower SL production, highlighting the potential for genetically altering SL production to control Striga infestations. This study provides insights into the functional roles of CCD7, CCD8, MAX1, and DUF genes in sorghum towards reduced and/or altered SL production and improved resistance to Striga infestations.}, }
@article {pmid40145799, year = {2025}, author = {Thanh, LM and Lan, VTH and Cuong, CQ and Lam, HT and Han, LK and Trang, NTH and Nghia, NH}, title = {Development of CRISPR/Cas9-Mediated Saccharomyces cerevisiae Strains for the Cell-Surface Display of a Novel Fusion Acid-Alkaline Phytase.}, journal = {Journal of agricultural and food chemistry}, volume = {73}, number = {14}, pages = {8458-8468}, doi = {10.1021/acs.jafc.5c00550}, pmid = {40145799}, issn = {1520-5118}, mesh = {*6-Phytase/genetics/metabolism/chemistry ; *Saccharomyces cerevisiae/genetics/enzymology/metabolism/chemistry ; CRISPR-Cas Systems ; Phytic Acid/metabolism ; Animal Feed/analysis ; *Saccharomyces cerevisiae Proteins/genetics/metabolism/chemistry ; Kinetics ; Hydrogen-Ion Concentration ; Glycine max/metabolism/chemistry ; Zea mays/metabolism ; Oryza/metabolism ; }, abstract = {Phytases enhance phosphorus bioavailability in animal feed, but their limited reusability hinders their application. To overcome this, Saccharomyces cerevisiae was engineered to display a fusion phytase combining acid and alkaline phytases on its cell surface by using CRISPR/Cas9. The enzyme was anchored via the α-agglutinin-GPI system in two marker-free strains, BY4743::GAP-mGEB and BY4743::GAP-aGEB, employing MFα and Aga2p signal peptides, respectively. Both strains exhibited robust surface activity across a broad pH range, retaining >50% relative activity between pH 1.0 and 7.0, with dual optima at pH 2.0 and 5.0-6.0. Kinetic analysis revealed a Km of 0.377-0.989 mM and a kcat of 0.014-0.019 μmol/min/mg wet-cell weight, with the Aga2p strain showing the highest efficiency. The fusion phytase exhibited ∼ 3.5-4 times higher activity than the single acid phytase. These strains effectively degraded phytate in soybean, corn flour, and rice bran, demonstrating a sustainable approach for improving phosphorus utilization in animal feed.}, }
@article {pmid40144376, year = {2025}, author = {Smirnov, AV and Korablev, AN and Serova, IA and Yunusova, AM and Muravyova, AA and Valeev, ES and Battulin, NR}, title = {Studying concatenation of the Cas9-cleaved transgenes using barcodes.}, journal = {Vavilovskii zhurnal genetiki i selektsii}, volume = {29}, number = {1}, pages = {26-34}, doi = {10.18699/vjgb-25-04}, pmid = {40144376}, issn = {2500-0462}, abstract = {In pronuclear microinjection, the Cas9 endonuclease is employed to introduce in vivo DNA double-strand breaks at the genomic target locus or within the donor vector, thereby enhancing transgene integration. The manner by which Cas9 interacts with DNA repair factors during transgene end processing and integration is a topic of considerable interest and debate. In a previous study, we developed a barcode-based genetic system for the analysis of transgene recombination following pronuclear microinjection in mice. In this approach, the plasmid library is linearized with a restriction enzyme or a Cas9 RNP complex at the site between a pair of barcodes. A pool of barcoded molecules is injected into the pronucleus, resulting in the generation of multicopy concatemers. In the present report, we compared the effects of in vivo Cas9 cleavage (RNP+ experiment) and in vitro production of Cas9- linearized transgenes (RNP- experiment) on concatenation. In the RNP+ experiment, two transgenic single-copy embryos were identified. In the RNP- experiment, six positive embryos were identified, four of which exhibited lowcopy concatemers. Next-generation sequencing (NGS) analysis of the barcodes revealed that 53 % of the barcoded ends had switched their initial library pairs, indicating the involvement of the homologous recombination pathway. Out of the 20 transgene-transgene junctions examined, 11 exhibited no mutations and were presumably generated through re-ligation of Cas9-induced blunt ends. The majority of mutated junctions harbored asymmetrical deletions of 2-4 nucleotides, which were attributed to Cas9 end trimming. These findings suggest that Cas9-bound DNA may present obstacles to concatenation. Conversely, clean DNA ends were observed to be joined in a manner similar to restriction-digested ends, albeit with distinctive asymmetry. Future experiments utilizing in vivo CRISPR/ Cas cleavage will facilitate a deeper understanding of how CRISPR-endonucleases influence DNA repair processes.}, }
@article {pmid40143364, year = {2025}, author = {Chakraborty, M and Nielsen, L and Nash, D and Nissimov, JI and Charles, TC and Aucoin, MG}, title = {Adapting Next-Generation Sequencing to in Process CRISPR-Cas9 Genome Editing of Recombinant AcMNPV Vectors: From Shotgun to Tiled-Amplicon Sequencing.}, journal = {Viruses}, volume = {17}, number = {3}, pages = {}, pmid = {40143364}, issn = {1999-4915}, support = {RGPIN 355513-2017//Natural Sciences and Engineering Research Council/ ; RGPIN-2023-03666//Natural Sciences and Engineering Research Council/ ; }, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Nucleopolyhedroviruses/genetics ; Animals ; *Genetic Vectors/genetics ; *High-Throughput Nucleotide Sequencing/methods ; Sf9 Cells ; *Genome, Viral ; Spodoptera ; Cell Line ; }, abstract = {The alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most commonly used virus in the Baculovirus Expression Vector System (BEVS) and has been utilized for the production of many human and veterinary biologics. AcMNPV has a large dsDNA genome that remains understudied, and relatively unmodified from the wild-type, especially considering how extensively utilized it is as an expression vector. Previously, our group utilized CRISPR-Cas9 genome engineering that revealed phenotypic changes when baculovirus genes are targeted using either co-expressed sgRNA or transfected sgRNA into a stable insect cell line that produced the Cas9 protein. Here, we describe a pipeline to sequence the recombinant AcMNPV expression vectors using shotgun sequencing, provide a set of primers for tiled-amplicon sequencing, show that untargeted baculovirus vector genomes remain relatively unchanged when amplified in Sf9-Cas9 cells, and confirm that AcMNPV gp64 gene disruption can minimize baculovirus contamination in cell cultures. Our findings provide a robust baseline for analyzing in process genome editing of baculoviruses.}, }
@article {pmid40143350, year = {2025}, author = {Wu, H and Wang, Z and Zhang, Y and Hu, L and Yang, J and Zhang, C and Lou, M and Pi, N and Wang, Q and Fan, S and Huang, Z}, title = {A New Human SCARB2 Knock-In Mouse Model for Studying Coxsackievirus A16 and Its Neurotoxicity.}, journal = {Viruses}, volume = {17}, number = {3}, pages = {}, pmid = {40143350}, issn = {1999-4915}, support = {NO.202105AD160018//Technology Innovation Talents Project of Yunnan Province/ ; NO. 2021-I2M-1-043//CAMS Innovation Fund for Medical Sciences (CIFMS)/ ; U2202214//Joint Funds of the National Natural Science Foundation of China/ ; 2023ZD040630//Scientific and Technological Innovation 2030/ ; }, mesh = {Animals ; *Disease Models, Animal ; Mice ; Humans ; *Lysosomal Membrane Proteins/genetics/metabolism ; Gene Knock-In Techniques ; CRISPR-Cas Systems ; *Receptors, Scavenger/genetics ; *Enterovirus/pathogenicity ; Gene Editing ; *Coxsackievirus Infections/virology/pathology ; Hand, Foot and Mouth Disease/virology ; Enterovirus A, Human ; Mice, Transgenic ; }, abstract = {Hand, Foot, and Mouth Disease (HFMD) is a viral illness caused by enterovirus infections. While the introduction of the enterovirus 71 (EV71) vaccine has significantly reduced the number of EV71-related cases, the continued spread of Coxsackievirus A16 (CVA16) remains a major public health threat. Previous studies have shown that human SCARB2 (hSCARB2) knock-in (KI) mice, generated using embryonic stem cell (ESC) technology, are susceptible to CVA16. However, these models have failed to reproduce the clinical pathology and neurotoxicity after CVA16 infection. Therefore, there is an urgent need for a more reliable and effective animal model to study CVA16. In this study, we successfully created a hSCARB2 KI mouse model targeting the ROSA26 locus using CRISPR/Cas9 gene editing technology. The application of CRISPR/Cas9 enabled stable and widespread expression of hSCARB2 in the model. After infection, the KI mice exhibited a clinical pathology that closely mimics human infection, with prominent limb weakness and paralysis. The virus was detectable in multiple major organs of the mice, with peak viral load observed on day 7 post-infection, gradually clearing thereafter. Further analysis revealed widespread neuronal necrosis and infiltration of inflammatory cells in the brain and spinal cord of the KI mice. Additionally, significant activation of astrocytes (GFAP-positive) and microglia (IBA1-positive) was observed in the brain, suggesting that CVA16 infection may induce limb paralysis by attacking neuronal cells. Overall, this model effectively replicates the neuropathological changes induced by CVA16 infection and provides a potential experimental platform for studying CVA16-associated pathogenesis and neurotoxicity.}, }
@article {pmid40143178, year = {2025}, author = {Elshobary, ME and Badawy, NK and Ashraf, Y and Zatioun, AA and Masriya, HH and Ammar, MM and Mohamed, NA and Mourad, S and Assy, AM}, title = {Combating Antibiotic Resistance: Mechanisms, Multidrug-Resistant Pathogens, and Novel Therapeutic Approaches: An Updated Review.}, journal = {Pharmaceuticals (Basel, Switzerland)}, volume = {18}, number = {3}, pages = {}, pmid = {40143178}, issn = {1424-8247}, abstract = {The escalating global health crisis of antibiotic resistance, driven by the rapid emergence of multidrug-resistant (MDR) bacterial pathogens, necessitates urgent and innovative countermeasures. This review comprehensively examines the diverse mechanisms employed by bacteria to evade antibiotic action, including alterations in cell membrane permeability, efflux pump overexpression, biofilm formation, target site modifications, and the enzymatic degradation of antibiotics. Specific focus is given to membrane transport systems such as ATP-binding cassette (ABC) transporters, resistance-nodulation-division (RND) efflux pumps, major facilitator superfamily (MFS) transporters, multidrug and toxic compound extrusion (MATE) systems, small multidrug resistance (SMR) families, and proteobacterial antimicrobial compound efflux (PACE) families. Additionally, the review explores the global burden of MDR pathogens and evaluates emerging therapeutic strategies, including quorum quenching (QQ), probiotics, postbiotics, synbiotics, antimicrobial peptides (AMPs), stem cell applications, immunotherapy, antibacterial photodynamic therapy (aPDT), and bacteriophage. Furthermore, this review discusses novel antimicrobial agents, such as animal-venom-derived compounds and nanobiotics, as promising alternatives to conventional antibiotics. The interplay between clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) in bacterial adaptive immunity is analyzed, revealing opportunities for targeted genetic interventions. By synthesizing current advancements and emerging strategies, this review underscores the necessity of interdisciplinary collaboration among biomedical scientists, researchers, and the pharmaceutical industry to drive the development of novel antibacterial agents. Ultimately, this comprehensive analysis provides a roadmap for future research, emphasizing the urgent need for sustainable and cooperative approaches to combat antibiotic resistance and safeguard global health.}, }
@article {pmid40142492, year = {2025}, author = {Sadanov, AK and Baimakhanova, BB and Orasymbet, SE and Ratnikova, IA and Turlybaeva, ZZ and Baimakhanova, GB and Amitova, AA and Omirbekova, AA and Aitkaliyeva, GS and Kossalbayev, BD and Belkozhayev, AM}, title = {Engineering Useful Microbial Species for Pharmaceutical Applications.}, journal = {Microorganisms}, volume = {13}, number = {3}, pages = {}, pmid = {40142492}, issn = {2076-2607}, support = {BR21882248//Program-targeted funding of the Committee of Science of the Ministry of Science and Higher Education of the Republic of Kazakh-stan/ ; }, abstract = {Microbial engineering has made a significant breakthrough in pharmaceutical biotechnology, greatly expanding the production of biologically active compounds, therapeutic proteins, and novel drug candidates. Recent advancements in genetic engineering, synthetic biology, and adaptive evolution have contributed to the optimization of microbial strains for pharmaceutical applications, playing a crucial role in enhancing their productivity and stability. The CRISPR-Cas system is widely utilized as a precise genome modification tool, enabling the enhancement of metabolite biosynthesis and the activation of synthetic biological pathways. Additionally, synthetic biology approaches allow for the targeted design of microorganisms with improved metabolic efficiency and therapeutic potential, thereby accelerating the development of new pharmaceutical products. The integration of artificial intelligence (AI) and machine learning (ML) plays a vital role in further advancing microbial engineering by predicting metabolic network interactions, optimizing bioprocesses, and accelerating the drug discovery process. However, challenges such as the efficient optimization of metabolic pathways, ensuring sustainable industrial-scale production, and meeting international regulatory requirements remain critical barriers in the field. Furthermore, to mitigate potential risks, it is essential to develop stringent biocontainment strategies and implement appropriate regulatory oversight. This review comprehensively examines recent innovations in microbial engineering, analyzing key technological advancements, regulatory challenges, and future development perspectives.}, }
@article {pmid40141287, year = {2025}, author = {Li, J and Zhang, L and Li, C and Chen, W and Wang, T and Tan, L and Qiu, Y and Song, S and Li, B and Li, L}, title = {The Pentatricopeptide Repeat Protein OsPPR674 Regulates Rice Growth and Drought Sensitivity by Modulating RNA Editing of the Mitochondrial Transcript ccmC.}, journal = {International journal of molecular sciences}, volume = {26}, number = {6}, pages = {}, pmid = {40141287}, issn = {1422-0067}, support = {U24A20396//National Natural Science Foundation of China/ ; 2024CX119//Hunan agricultural science and technology innovation fund project/ ; }, mesh = {*Oryza/genetics/growth &amp; development/metabolism ; *RNA Editing ; *Plant Proteins/genetics/metabolism ; *Mitochondria/genetics/metabolism ; Gene Expression Regulation, Plant ; Droughts ; *Mitochondrial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Mutation ; RNA, Mitochondrial ; }, abstract = {The P-type pentatricopeptide repeat (PPR) proteins are crucial for RNA editing and post-transcriptional regulation in plant organelles, particularly mitochondria. This study investigates the role of OsPPR674 in rice, focusing on its function in mitochondrial RNA editing. Using CRISPR/Cas9 technology, we generated ppr674 mutant and examined its phenotypic and molecular characteristics. The results indicate that ppr674 exhibits reduced plant height, decreased seed-setting rate, and poor drought tolerance. Further analysis revealed that in the ppr674 mutant, RNA editing at the 299th nucleotide position of the mitochondrial ccmC gene (C-to-U conversion) was abolished. REMSAs showed that GST-PPR674 specifically binds to RNA probes targeting this ccmC-299 site, confirming its role in this editing process. In summary, these results suggest that OsPPR674 plays a pivotal role in mitochondrial RNA editing, emphasizing the significance of PPR proteins in organelle function and plant development.}, }
@article {pmid40141236, year = {2025}, author = {Livneh, Y and Agmon, D and Leor-Librach, E and Vainstein, A}, title = {Viral-Based Gene Editing System for Nutritional Improvement of Fructan Content in Lettuce.}, journal = {International journal of molecular sciences}, volume = {26}, number = {6}, pages = {}, pmid = {40141236}, issn = {1422-0067}, support = {12-01-0037//Office of the Chief Scientist/ ; 20-01-0209//The National Center for Genome Editing in Agriculture, Israel/ ; 7500158//Ministry of Science and Technology, Israel/ ; }, mesh = {*Gene Editing/methods ; *Lactuca/genetics/metabolism ; *Fructans/metabolism/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Plant Viruses/genetics ; Plants, Genetically Modified/genetics ; }, abstract = {Lettuce is a globally cultivated and consumed leafy crop. Here we developed an efficient tobacco rattle virus (TRV)-based guide RNA (gRNA) delivery system for CRISPR/Cas editing in the commercial lettuce cultivar 'Noga'. Plants stably expressing Cas9 were inoculated with TRV vectors carrying gRNAs targeting five nutrient-associated genes. The system achieved an average editing efficiency of 48.7%, with up to 78.9% of regenerated plantlets showing independent mutations. This approach eliminates the need for antibiotic selection, simplifying tissue culture processes. The system supports diverse applications, including Cas12a editing and large-fragment deletions using dual gRNA sets. Targeting the fructan 1-exohydrolase 2 (1-FEH2) gene produced knockout lines with significant increases in prebiotic dietary fibre fructan content, up to 5.2-fold, and an average rise in the degree of polymerisation by 2.15 units compared with controls. Combining 1-FEH1 and 1-FEH2 knockouts did not further increase fructan levels, revealing 1-FEH2 as the predominant isozyme in lettuce. RT-qPCR analysis showed reduced expression of the upstream biosynthetic enzyme sucrose:sucrose 1-fructosyl transferase (1-SST), suggesting potential feedback inhibition in fructan metabolism. This TRV-based gene editing approach, utilised here to increase fructan content, could be applied to improve other valuable traits in lettuce, and may inspire similar systems to enhance nutritional content of crops.}, }
@article {pmid40141178, year = {2025}, author = {Tan, Q and Wang, J and Hao, Y and Yang, S and Cao, B and Pan, W and Cao, M}, title = {Elf1 Deficiency Impairs Macrophage Development in Zebrafish Model Organism.}, journal = {International journal of molecular sciences}, volume = {26}, number = {6}, pages = {}, pmid = {40141178}, issn = {1422-0067}, support = {2023YFA1802000//National Key R&amp;D Program of China/ ; ZDBS-LY-SM010//Key Research Program of Frontier Sciences, Chinese Academy of Sciences/ ; YSBR-077//CAS Project for Young Scientists in Basic Research/ ; 21JC1406300//Shanghai Science and Technology In-novation Action Plan for Basic Research Program/ ; }, mesh = {Animals ; *Zebrafish/genetics/metabolism ; *Macrophages/metabolism/cytology ; *Zebrafish Proteins/genetics/metabolism ; *Transcription Factors/genetics/metabolism/deficiency ; Cell Differentiation ; CRISPR-Cas Systems ; Cell Movement ; Receptors, CXCR4/metabolism/genetics ; }, abstract = {The Ets (E-twenty-six) family of transcription factors plays a critical role in hematopoiesis and myeloid differentiation. However, the specific functions of many family members in these processes remain largely underexplored and poorly understood. Here, we identify Elf1 (E74-like factor 1), an Ets family member, as a critical regulator of macrophage development in the zebrafish model organism, with minimal impact on neutrophil differentiation. Through morpholino knockdown screening and CRISPR/Cas9-mediated gene editing, we demonstrate that Elf1 is critical for macrophage development and tissue injury responses. Specific overexpression of dominant-negative Elf1 (DN-Elf1) in macrophages demonstrated a cell-autonomous effect on macrophage infiltration. Furthermore, the overexpression of cxcr4b, a gene downstream of Elf1 regulation and essential for cell migration and injury response, significantly rescued this defect, indicating Elf1 as a key regulator of macrophage function. Our findings shed light on the roles of Elf1 in macrophage development and injury response and also highlight zebrafish as a powerful model for immunity research.}, }
@article {pmid40141141, year = {2025}, author = {Lin, K and Lu, LX and Pan, BZ and Chai, X and Fu, QT and Geng, XC and Mo, Y and Fei, YC and Xu, JJ and Li, M and Ni, J and Xu, ZF}, title = {Agrobacterium rhizogenes-Mediated Hairy Root Genetic Transformation Using Agrobacterium Gel Inoculation and RUBY Reporter Enables Efficient Gene Function Analysis in Sacha Inchi (Plukenetia volubilis).}, journal = {International journal of molecular sciences}, volume = {26}, number = {6}, pages = {}, pmid = {40141141}, issn = {1422-0067}, support = {AD23026337//Guangxi Specific Project for Science and Technology Bases and Talents/ ; XDA24030502//Strategic Priority Research Program of the Chinese Academy of Sciences (Precision Seed Design and Breeding)/ ; YNWR-QNBJ-2020-172//West Light Foundation of the Chinese Academy of Sciences, the Youth Talent Support Program of Yunnana Province/ ; }, mesh = {*Plant Roots/genetics/microbiology/growth &amp; development ; *Agrobacterium/genetics ; *Transformation, Genetic ; Plants, Genetically Modified/genetics ; Genes, Reporter ; CRISPR-Cas Systems ; }, abstract = {Plukenetia volubilis L., a woody oilseed plant rich in α-linolenic acid, represents a promising source of polyunsaturated fatty acids. However, the lack of an efficient genetic transformation system has significantly hindered gene function research and molecular breeding in P. volubilis. In this study, we developed a highly efficient Agrobacterium rhizogenes-mediated hairy root transformation system for P. volubilis via the use of Agrobacterium gel in combination with the visually detectable RUBY reporter for gene function analysis in roots. The results indicate that the optimal transformation method involves infecting P. volubilis seedlings with Agrobacterium gel containing acetosyringone and inducing hairy root formation in perlite. This approach resulted in more than 18.97% of the seedlings producing positive hairy roots overexpressing the RUBY gene. Using this genetic transformation system, we successfully overexpressed the antimicrobial peptide-encoding gene CEMA in hairy roots, which enhanced the resistance of P. volubilis to Fusarium oxysporum. Furthermore, by combining this transformation system with the CRISPR-Cas9 tool, we validated the regulatory role of PvoSHR in the development of root epidermal cells in P. volubilis. Unexpectedly, a 123-bp DNA fragment from the T-DNA region of the A. rhizogenes Ri plasmid was found to be knocked in to the P. volubilis genome, replacing a 110-bp fragment of PvoSHR at CRISPR-Cas9 induced double-strand DNA breaks. Conclusively, this system provides a powerful tool for gene function research in P. volubilis and provides novel insights into the development of transformation and gene editing systems for other woody plants.}, }
@article {pmid40140585, year = {2025}, author = {Chen, KY and Kibayashi, T and Giguelay, A and Hata, M and Nakajima, S and Mikami, N and Takeshima, Y and Ichiyama, K and Omiya, R and Ludwig, LS and Hattori, K and Sakaguchi, S}, title = {Genome-wide CRISPR screen in human T cells reveals regulators of FOXP3.}, journal = {Nature}, volume = {642}, number = {8066}, pages = {191-200}, pmid = {40140585}, issn = {1476-4687}, mesh = {Animals ; Female ; Humans ; Male ; Mice ; Acetylation ; Cell Differentiation/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; *Forkhead Transcription Factors/genetics/metabolism ; Gene Expression Regulation ; *Genome, Human/genetics ; Graft vs Host Disease/immunology/prevention &amp; control ; Histone Deacetylases/metabolism ; Histones/metabolism ; Signal Transduction ; Single-Cell Analysis ; *T-Lymphocytes, Regulatory/metabolism/cytology/immunology ; *Genetic Techniques ; }, abstract = {Regulatory T (Treg) cells, which specifically express the master transcription factor FOXP3, have a pivotal role in maintaining immunological tolerance and homeostasis and have the potential to revolutionize cell therapies for autoimmune diseases[1-3]. Although stimulation of naive CD4[+] T cells in the presence of TGFβ and IL-2 can induce FOXP3[+] Treg cells in vitro (iTreg cells), the resulting cells are often unstable and have thus far hampered translational efforts[4-6]. A systematic approach towards understanding the regulatory networks that dictate Treg differentiation could lead to more effective iTreg cell-based therapies. Here we performed a genome-wide CRISPR loss-of-function screen to catalogue gene regulatory determinants of FOXP3 induction in primary human T cells and characterized their effects at single-cell resolution using Perturb-icCITE-seq. We identify the RBPJ-NCOR repressor complex as a novel, context-specific negative regulator of FOXP3 expression. RBPJ-targeted knockout enhanced iTreg differentiation and function, independent of canonical Notch signalling. Repeated cytokine and T cell receptor signalling stimulation in vitro revealed that RBPJ-deficient iTreg cells exhibit increased phenotypic stability compared with control cells through DNA demethylation of the FOXP3 enhancer CNS2, reinforcing FOXP3 expression. Conversely, overexpression of RBPJ potently suppressed FOXP3 induction through direct modulation of FOXP3 histone acetylation by HDAC3. Finally, RBPJ-ablated human iTreg cells more effectively suppressed xenogeneic graft-versus-host disease than control iTreg cells in a humanized mouse model. Together, our findings reveal novel regulators of FOXP3 and point towards new avenues to improve the efficacy of adoptive cell therapy for autoimmune disease.}, }
@article {pmid40140553, year = {2025}, author = {Xi, JF and Liu, BD and Tang, GR and Ren, ZH and Chen, HX and Lan, YL and Yin, F and Li, Z and Cheng, WS and Wang, J and Chen, L and Yuan, SC and Zhang, Z and Luo, GZ}, title = {m[6]A modification regulates cell proliferation via reprogramming the balance between glycolysis and pentose phosphate pathway.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {496}, pmid = {40140553}, issn = {2399-3642}, mesh = {*Pentose Phosphate Pathway/genetics ; *Cell Proliferation/genetics ; *Glycolysis/genetics ; Humans ; *Adenosine/analogs &amp; derivatives/metabolism/genetics ; Cell Line, Tumor ; Glucosephosphate Dehydrogenase/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; Neoplasms/genetics/metabolism/pathology ; CRISPR-Cas Systems ; }, abstract = {N6-methyladenosine (m[6]A) stands as the predominant modification in eukaryotic mRNA and is involved in various biological functions. Aberrant m[6]A has been implicated in abnormal cellular phenotypes, including defects in stem cell differentiation and tumorigenesis. However, the precise effects of m[6]A on cell proliferation and the underlining mechanism of metabolic gene regulation remain incompletely understood. Here, we established a cellular environment with low-m[6]A levels and observed a severe impairment of cell proliferation. Mechanistic studies revealed that the depletion of m[6]A on TIGAR mRNA led to increased expression, subsequently inhibiting glycolysis while promoting the pentose phosphate pathway (PPP). A genome-wide CRISPR-Cas9 screen identified numerous genes involved in cell proliferation that are sensitive to m[6]A modification, with G6PD emerging as a key regulator. Integration of gene expression and survival data from cancer patients suggested that patients with elevated G6PD expression may exhibit enhanced responsiveness to tumor growth inhibition through m[6]A suppression. Our findings elucidate the critical role of m[6]A in cell proliferation, highlighting the therapeutic potential of targeting m[6]A-mediated metabolic pathways in cancer.}, }
@article {pmid40140468, year = {2025}, author = {Rodríguez-Sánchez, A and Quijada-Álamo, M and Pérez-Carretero, C and Herrero, AB and Arroyo-Barea, A and Dávila-Valls, J and Rubio, A and García de Coca, A and Benito-Sánchez, R and Rodríguez-Vicente, AE and Hernández-Rivas, JM and Hernández-Sánchez, M}, title = {SAMHD1 dysfunction impairs DNA damage response and increases sensitivity to PARP inhibition in chronic lymphocytic leukemia.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {10446}, pmid = {40140468}, issn = {2045-2322}, support = {JCYL-EDU/1868/2022//Predoctoral research fellowship 2022 from Junta de Castilla y Le&#xf3;n/ ; }, mesh = {Humans ; *Leukemia, Lymphocytic, Chronic, B-Cell/genetics/drug therapy/metabolism/pathology ; *SAM Domain and HD Domain-Containing Protein 1/genetics/metabolism ; *DNA Damage ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use ; Mutation ; Female ; Male ; BRCA1 Protein/metabolism/genetics ; Middle Aged ; Aged ; CRISPR-Cas Systems ; Cell Line, Tumor ; }, abstract = {Chronic lymphocytic leukemia (CLL) is a clinically and genetically heterogenous disease. Recent next-generation sequencing (NGS) studies have uncovered numerous low-frequency mutated genes in CLL patients, with SAMHD1 emerging as a candidate driver gene. However, the biological and clinical implications of SAMHD1 mutations remain unclear. Using CRISPR/Cas9, we generated CLL models to investigate the impact of SAMHD1 deficiency on pathogenesis and explore therapeutic strategies. Moreover, we performed NGS in treatment-naïve CLL patients to characterize SAMHD1 mutations and employed RNA-sequencing to evaluate their clinical significance. Our study shows that SAMHD1 inactivation impairs the DNA damage response by reducing homologous recombination efficiency through BRCA1 and RAD51 dysregulation. Importantly, SAMHD1 colocalizes with BRCA1 at DNA damage sites in CLL cells. This research also identifies that SAMHD1-mutated cells are more sensitive to PARP inhibition. Clinically, SAMHD1 dysfunction negatively impacts clinical outcome of CLL cases: SAMHD1 mutations reduce failure-free survival (median 46 vs 57 months, p = 0.033), while low SAMHD1 expression associates with shorter time to first treatment (median 47 vs 77 months; p = 0.00073). Overall, this study elucidates that SAMHD1 dysfunction compromises DNA damage response mechanisms, potentially contributing to unfavorable clinical outcomes in CLL, and proposes PARP-inhibitors as a potential therapeutic approach for SAMHD1-mutated CLL cells.}, }
@article {pmid40140116, year = {2025}, author = {Anyaegbunam, NJ and Okpe, KE and Bello, AB and Ajanaobionye, TI and Mgboji, CC and Olonade, A and Anyaegbunam, ZKG and Mba, IE}, title = {Leveraging innovative diagnostics as a tool to contain superbugs.}, journal = {Antonie van Leeuwenhoek}, volume = {118}, number = {4}, pages = {63}, pmid = {40140116}, issn = {1572-9699}, mesh = {Humans ; *Bacterial Infections/diagnosis/microbiology ; *Bacteria/genetics/isolation &amp; purification/drug effects ; High-Throughput Nucleotide Sequencing ; CRISPR-Cas Systems ; Drug Resistance, Multiple, Bacterial ; }, abstract = {The evolutionary adaptation of pathogens to biological materials has led to an upsurge in drug-resistant superbugs that significantly threaten public health. Treating most infections is an uphill task, especially those associated with multi-drug-resistant pathogens, biofilm formation, persister cells, and pathogens that have acquired robust colonization and immune evasion mechanisms. Innovative diagnostic solutions are crucial for identifying and understanding these pathogens, initiating efficient treatment regimens, and curtailing their spread. While next-generation sequencing has proven invaluable in diagnosis over the years, the most glaring drawbacks must be addressed quickly. Many promising pathogen-associated and host biomarkers hold promise, but their sensitivity and specificity remain questionable. The integration of CRISPR-Cas9 enrichment with nanopore sequencing shows promise in rapid bacterial diagnosis from blood samples. Moreover, machine learning and artificial intelligence are proving indispensable in diagnosing pathogens. However, despite renewed efforts from all quarters to improve diagnosis, accelerated bacterial diagnosis, especially in Africa, remains a mystery to this day. In this review, we discuss current and emerging diagnostic approaches, pinpointing the limitations and challenges associated with each technique and their potential to help address drug-resistant bacterial threats. We further critically delve into the need for accelerated diagnosis in low- and middle-income countries, which harbor more infectious disease threats. Overall, this review provides an up-to-date overview of the diagnostic approaches needed for a prompt response to imminent or possible bacterial infectious disease outbreaks.}, }
@article {pmid40139718, year = {2025}, author = {Tian, M and Luo, L and Jin, B and Liu, J and Chen, T and Tang, J and Shen, Y and Zhang, H and Guo, J and Zhang, H and Cui, G and Huang, L}, title = {Highly efficient Agrobacterium rhizogenes-mediated gene editing system in Salvia miltiorrhiza inbred line bh2-7.}, journal = {Plant biotechnology journal}, volume = {23}, number = {6}, pages = {2406-2417}, pmid = {40139718}, issn = {1467-7652}, support = {CI2021A04109//CACMS Innovation Fund/ ; ZZ14-YQ-049//The Fundamental Research Funds for the Central public welfare research institutes/ ; 2023YFC3503900//National Key Research and Development Program of China/ ; 82274054//National Natural Science Foundation of China/ ; 2060302//Key project at central government level: the ability to establish sustainable use of valuable Chinese Medicine Resources/ ; TSQN202103160//Supported by the Taishan Scholars Program/ ; }, mesh = {*Agrobacterium/genetics ; *Gene Editing/methods ; *Salvia miltiorrhiza/genetics ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Genome, Plant/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {The CRISPR/Cas9 system is a powerful tool for genomic editing with significant potential for gene function validation and molecular breeding in medicinal plants. Salvia miltiorrhiza, a model medicinal plant, was among the pioneers to utilize CRISPR/Cas9 technology, though achieving high-efficiency homozygous knockout mutants has been challenging. In this study, the analysis of variations at 241 single-guide RNA (sgRNA) across different reference genomes and experimental materials was conducted first, leading to the identification of the six-generation inbred line bh2-7 as the most suitable reference genome and experimental material for gene editing research in S. miltiorrhiza. Next, five Agrobacterium rhizogenes strains were evaluated for hairy root induction, editing efficiency, and mutation patterns, with C58C1 and K599 emerging as the most effective delivery systems. Using the CRISPR/Cas9 vector pZKD672, 53 target sites were successfully edited, with K599 achieving 71.07% editing efficiency and 36.74% homozygous or biallelic (HOM) efficiency and C58C1 showing 62.27% editing efficiency and 23.61% HOM efficiency. We thus constructed a large-scale mutant library targeting 121 genes with 170 sgRNAs, yielding 1664 homozygous or biallelic mutants. Analysis of 65 low-efficiency target sites revealed that sgRNA mismatches and secondary structures were key factors reducing HOM efficiency, offering insights for future target design. This study establishes an efficient CRISPR/Cas9 system, advancing precision breeding and metabolic engineering research in medicinal plants.}, }
@article {pmid40139379, year = {2025}, author = {Shuo, Y and Feng, X and Zhao, C and Zhang, J and Wang, Y and Wang, J and Ding, Y and Shi, X and Li, J and Song, X and Wang, J}, title = {A dual enhancement strategy for ultrasensitive detection of norovirus based on catalytic hairpin assembly-assisted CRISPR/Cas12a sensing system.}, journal = {Journal of dairy science}, volume = {108}, number = {5}, pages = {4726-4733}, doi = {10.3168/jds.2024-26150}, pmid = {40139379}, issn = {1525-3198}, mesh = {*Norovirus/isolation &amp; purification ; Animals ; Milk/virology ; CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Noroviruses are a leading cause of food-borne illnesses, responsible for over 50% of global gastroenteritis outbreaks. Whereas reverse transcriptase PCR (RT-PCR) and other nucleic acid amplification methods are crucial for norovirus detection, their reliance on specialized equipment highlights the urgent need for more accessible detection methods. Herein, we propose an isothermal cascade signal amplification assay that integrates catalytic hairpin assembly (CHA) and cluster regularly interspaced short palindromic repeats (CRISPR)/Cas12a for rapid and accurate detection of norovirus in food samples. By incorporating the advantages of CHA and CRISPR/Cas12a, the dual signal enhancement sensing strategy can achieve high sensitivity low to 14 fM within 70 min, and good specificity in adenovirus, human enterovirus, rotavirus, and other interfering agents. The proposed dual enhancement strategy for norovirus detection has satisfactory accuracy and acceptable recoveries in milk samples compared with RT-PCR assay, and holds promise for improving food safety monitoring, particularly in dairy products.}, }
@article {pmid40138716, year = {2025}, author = {Whitford, CM and Gockel, P and Faurdal, D and Gren, T and Sigrist, R and Weber, T}, title = {CASCADE-Cas3 enables highly efficient genome engineering in Streptomyces species.}, journal = {Nucleic acids research}, volume = {53}, number = {6}, pages = {}, pmid = {40138716}, issn = {1362-4962}, support = {NNF20CC0035580//Novo Nordisk Foundation/ ; //DTU Biosustain/ ; }, mesh = {*Streptomyces/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genome, Bacterial ; Plasmids/genetics ; *Genetic Engineering/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; }, abstract = {Type I clustered regularly interspaced short palindromic repeat (CRISPR) systems are widespread in bacteria and archaea. Compared to more widely applied type II systems, type I systems differ in the multi-effector CRISPR-associated complex for antiviral defense needed for crRNA processing and target recognition, as well as the processive nature of the hallmark nuclease Cas3. Given the widespread nature of type I systems, the processive nature of Cas3 and the recombinogenic overhangs created by Cas3, we hypothesized that CASCADE-Cas3 would be uniquely positioned to enable efficient genome engineering in streptomycetes. Here, we report a new type I based CRISPR genome engineering tool for streptomycetes. The plasmid system, called pCRISPR-Cas3, utilizes a compact type I-C CRISPR system and enables highly efficient genome engineering. pCRISPR-Cas3 outperforms pCRISPR-Cas9 and facilitates targeted and random sized deletions. Furthermore, we demonstrate its ability to effectively perform substitutions of large genomic regions such as biosynthetic gene clusters. Without additional modifications, pCRISPR-Cas3 enabled genome engineering in several Streptomyces species at high efficiencies.}, }
@article {pmid40138431, year = {2025}, author = {Fuchs, W and Assad-Garcia, N and Abkallo, HM and Xue, Y and Oldfield, LM and Fedorova, N and Hübner, A and Kabuuka, T and Pannhorst, K and Höper, D and Nene, V and Gonzalez-Juarbe, N and Steinaa, L and Vashee, S}, title = {A synthetic genomics-based African swine fever virus engineering platform.}, journal = {Science advances}, volume = {11}, number = {13}, pages = {eadu7670}, pmid = {40138431}, issn = {2375-2548}, mesh = {*African Swine Fever Virus/genetics ; Animals ; *African Swine Fever/virology/prevention &amp; control ; *Genomics/methods ; Swine ; *Genome, Viral ; CRISPR-Cas Systems ; *Genetic Engineering/methods ; Viral Vaccines ; }, abstract = {African swine fever (ASF) is a deadly viral disease in domestic pigs that has a large global economic impact for the swine industry. It is present in Africa, Europe, Asia, and in the Caribbean island of Hispaniola. There are no effective treatments or broadly licensed vaccines to prevent disease. Efforts to counteract ASF have been hampered because of the lack of convenient tools to engineer its etiological agent, ASF virus (ASFV), largely due to its large noninfectious genome. Here, we report the use of synthetic genomics methodology to develop a reverse genetics system for ASFV using a CRISPR-Cas9-inhibited self-helper virus to reconstitute live recombinant ASFV from synthetic genomes to rapidly generate a variety of combinatorial mutants of ASFV. The method will substantially facilitate the development of therapeutics or subunit and live-attenuated vaccines for ASF. This synthetic genomics-based approach has wide-ranging impact because it can be applied to rapidly develop reverse genetics tools for emerging viruses with noninfectious genomes.}, }
@article {pmid40138428, year = {2025}, author = {Tian, R and Tian, X and Yang, M and Song, Y and Zhao, T and Zhong, C and Zhu, W and Zhou, P and Han, Z and Hu, Z}, title = {Systematic high-throughput evaluation reveals FrCas9's superior specificity and efficiency for therapeutic genome editing.}, journal = {Science advances}, volume = {11}, number = {13}, pages = {eadu7334}, pmid = {40138428}, issn = {2375-2548}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; *Genetic Therapy/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; High-Throughput Nucleotide Sequencing ; Animals ; }, abstract = {CRISPR-Cas9 systems have revolutionized genome editing, but the off-target effects of Cas9 limit its use in clinical applications. Here, we systematically evaluate FrCas9, a variant from Faecalibaculum rodentium, for cell and gene therapy (CGT) applications and compare its performance to SpCas9 and OpenCRISPR-1. OpenCRISPR-1 is a CRISPR system synthesized de novo using large language models (LLMs) but has not yet undergone systematic characterization. Using AID-seq, Amplicon sequencing, and GUIDE-seq, we assessed the on-target activity and off-target profiles of these systems across multiple genomic loci. FrCas9 demonstrated higher on-target efficiency and substantially fewer off-target effects than SpCas9 and OpenCRISPR-1. Furthermore, TREX2 fusion with FrCas9 reduced large deletions and translocations, enhancing genomic stability. Through screening of 1903 sgRNAs targeting 21 CGT-relevant genes using sequential AID-seq, Amplicon sequencing, and GUIDE-seq analysis, we identified optimal sgRNAs for each gene. Our high-throughput screening platform highlights FrCas9, particularly in its TREX2-fused form, as a highly specific and efficient tool for precise therapeutic genome editing.}, }
@article {pmid40138366, year = {2025}, author = {Rivera-Toro, DM and de Folter, S and Alvarez-Venegas, R}, title = {CRISPR/dCas12a-mediated activation of SlPAL2 enhances tomato resistance against bacterial canker disease.}, journal = {PloS one}, volume = {20}, number = {3}, pages = {e0320436}, pmid = {40138366}, issn = {1932-6203}, mesh = {*Solanum lycopersicum/genetics/microbiology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics ; *CRISPR-Cas Systems ; *Phenylalanine Ammonia-Lyase/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified/genetics ; Gene Editing/methods ; Gene Expression Regulation, Plant ; Lignin/metabolism ; Clavibacter/pathogenicity ; }, abstract = {Crop protection is essential for maintaining and improving agricultural productivity. While pesticides are commonly used to control pests, they pose several challenges, including environmental harm and health risks. Alternative strategies to pesticides include breeding resistant crop varieties, biological control, and utilizing genome-editing tools like CRISPR/Cas. However, the application of epigenome editing, particularly CRISPR activation (CRISPRa), in plants remains underexplored. Phenylalanine ammonia-lyase (PAL), a key enzyme in the phenylpropanoid pathway, plays a pivotal role in plant defense by producing lignin and other secondary metabolites essential for pathogen resistance. In this study, we engineered tomato plants by fusing the SET-domain of the SlATX1 coding gene, a histone H3 lysine 4 tri-methyltransferase, to dCas12a, targeting the SlPAL2 promoter with the aim to increase PAL2 gene expression. CRISPRa-edited plants demonstrated increased deposition of the H3K4me3 epigenetic mark and significantly upregulated SlPAL2 expression. This enhanced lignin accumulation and conferred increased resistance to Clavibacter michiganensis subsp. michiganensis (Cmm) without significant reduction in plant height or fruit yield. Disease resistance was also associated with reduced pathogen load and lesion size, and higher lignin levels persisted even after SlPAL2 expression declined post-infection. These findings highlight the potential of CRISPRa for reprogramming plant defense responses through targeted histone modifications, offering a sustainable approach for crop improvement. Furthermore, CRISPRa could also be applied to enhance crop resilience in other contexts, such as addressing food security challenges by enhancing productivity.}, }
@article {pmid40138089, year = {2025}, author = {Chu, Y and Wei, M and Cao, Z and Chen, L and Tan, J and Bao, W and Yang, F and Zhang, Y and Lin, Y and Zhang, Y and Li, S and Lv, C and Zhou, W and Du, H and Shen, L and Huai, C and Wang, Z and Qin, S}, title = {Integrative analysis based on CRISPR screen identifies apilimod as a potential therapeutic agent for cisplatin-induced acute kidney injury treatment.}, journal = {Science China. Life sciences}, volume = {68}, number = {6}, pages = {1770-1785}, pmid = {40138089}, issn = {1869-1889}, mesh = {*Cisplatin/adverse effects ; *Acute Kidney Injury/chemically induced/drug therapy/genetics ; Animals ; Humans ; Mice ; Apoptosis/drug effects ; *CRISPR-Cas Systems ; *Morpholines/pharmacology/therapeutic use ; Reactive Oxygen Species/metabolism ; Lipid Metabolism/drug effects ; Male ; Antineoplastic Agents/adverse effects ; Signal Transduction/drug effects ; Cell Line ; }, abstract = {Acute kidney injury (AKI), a life-threatening side effect of cisplatin therapy, significantly limits the drug's therapeutic potential. In this study, we conducted a genome-wide CRISPR/Cas9 knockout screen in human renal tubular epithelial cells, integrating the results with transcriptome analyses and the Connectivity Map (CMap) database. Apilimod and elacridar emerged as the top two candidates of mitigating cisplatin-induced nephrotoxicity, with apilimod demonstrating superior efficacy in drug matrix experiments. Apilimod reduced cisplatin-induced apoptosis, inflammation and reactive oxygen species (ROS) generation. Transcriptome analyses suggested that apilimod may protect against cisplatin-induced nephrotoxicity via modulating lipid metabolism. In vitro experiments revealed that apilimod significantly ameliorated cisplatin-induced lipotoxicity by enhancing lipid clearance and upregulating PGC1α-mediated fatty acid oxidation. Mechanism experiments showed that apilimod induces the nuclear translocation of TFEB through the inhibition of its target, PIKfyve, thereby enhancing PGC1α expression and ameliorating lipotoxicity. These protective effects of apilimod were simulated by siRNA-mediated PIKfyve knockdown and diminished by the PGC1α inhibitor SR-18292 and siRNA targeting TFEB, confirming the role of the PIKfyve/TFEB/PGC1α signaling axis in apilimod's renoprotective effects. In vivo, apilimod alleviated apoptosis, inflammation, and lipid accumulation in a cisplatin-induced AKI mouse model. Additionally, apilimod treatment did not compromise the antitumor effect of cisplatin in cancer cells or tumor-bearing mice. Overall, our study suggests that apilimod could be a promising therapeutic agent for the treatment of cisplatin-induced AKI and revealed its underlying molecular mechanism.}, }
@article {pmid40136952, year = {2025}, author = {Xin, X and Su, J and Cui, H and Wang, L and Song, S}, title = {Recent Advances in Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated Proteins System-Based Biosensors.}, journal = {Biosensors}, volume = {15}, number = {3}, pages = {}, pmid = {40136952}, issn = {2079-6374}, support = {2023YFB3208200//National Key Research and Development Program of China/ ; 21974147//National Natural Science Foundation of China/ ; }, mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; }, abstract = {High-sensitivity and high-specificity biodetection is critical for advancing applications in life sciences, biosafety, food safety, and environmental monitoring. CRISPR/Cas systems have emerged as transformative tools in biosensing due to their unparalleled specificity, programmability, and unique enzymatic activities. They exhibit two key cleavage behaviors: precise ON-target cleavage guided by specific protospacers, which ensures accurate target recognition, and bystander cleavage activity triggered upon target binding, which enables robust signal amplification. These properties make CRISPR/Cas systems highly versatile for designing biosensors for ultra-sensitive detection. This review comprehensively explores recent advancements in CRISPR/Cas system-based biosensors, highlighting their impact on improving biosensing performance. We discuss the integration of CRISPR/Cas systems with diverse signal readout mechanisms, including electrochemical, fluorescent, colorimetric, surface-enhanced Raman scattering (SERS), and so on. Additionally, we examine the development of integrated biosensing systems, such as microfluidic devices and portable biosensors, which leverage CRISPR/Cas technology for point-of-care testing (POCT) and high-throughput analysis. Furthermore, we identify unresolved challenges, aiming to inspire innovative solutions and accelerate the translation of these technologies into practical applications for diagnostics, food, and environment safety.}, }
@article {pmid40135905, year = {2025}, author = {Zhang, J-Z and Li, Y-Z and Xi, Z-N and Zhang, Y and Liu, Z-Y and Ma, X-Q and Li, F-L}, title = {Inducible promoters of bacterial microcompartments improve the CRISPR/Cas9 tools for efficient metabolic engineering of Clostridium ljungdahlii.}, journal = {Applied and environmental microbiology}, volume = {91}, number = {4}, pages = {e0218324}, pmid = {40135905}, issn = {1098-5336}, support = {XDC0110302//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; U22A20425//National Natural Science Foundation of China-Shandong Joint Fund/ ; 32370039//National Natural Science Foundation of China/ ; SEI S202104//CAS | Shandong Energy Institute, Chinese Academy of Sciences (SEI)/ ; ZR2023MC021//Shandong Provincial Natural Science Foundation/ ; }, mesh = {*Metabolic Engineering/methods ; *CRISPR-Cas Systems ; *Clostridium/genetics/metabolism ; *Promoter Regions, Genetic ; Gene Editing ; Choline/metabolism ; Bacterial Proteins/genetics/metabolism ; Propylene Glycol/metabolism ; }, abstract = {UNLABELLED: Clostridium ljungdahlii, as a model acetogen strain, represents a novel platform for biotechnological production for CO2 fixation. The genome of C. ljungdahlii harbors two gene loci associated with glycyl radical enzyme-associated microcompartments (GRMs), which are predicted to play essential roles in choline and 1,2-propanediol (1,2-PD) metabolism. This study validated the functions of these GRM loci and identified two inducible promoters, of which Pcholine1 was induced by choline, while P1,2-PD was induced by 1,2-PD. Subsequently, the highly expressed P1,2-PD and tightly controlled Pcholine1 were applied to improve CRISPR/Cas9 gene editing tools. Specifically, P1,2-PD was used to develop a highly efficient gene knockout tool based on an all-in-one plasmid, achieving 100% deletion efficiency for multiple genes, including pyrE, pduS, aor2, and eutT. On the other hand, the cas9 gene was integrated downstream of Pcholine1 into the genome. The integrated cas9 efficiently mediated gene editing in C. ljungdahlii by introducing plasmids containing a gRNA cassette along with the relevant homology arms. This was exemplified by the construction of the Δbdh::pdc strain, where the 2,3-butanediol dehydrogenase gene was replaced with a pyruvate decarboxylase gene from Zymomonas mobilis and the 3-HB Syn KI strain, in which an artificial 3-hydroxybutyric acid synthesis pathway was inserted into the genome. This study highlights the effectiveness and convenience of the inducible CRISPR/Cas9 gene editing systems, thereby enriching the CRISPR/Cas toolkit in acetogens.<br><br>IMPORTANCE: A CRISPR/Cas9 genetic tool controlled by a constitutive promoter has been developed for precise gene deletion in Clostridium ljungdahlii. However, its efficiency was hindered by the toxicity resulting from the constitutive expression of cas9 and the large plasmids, leading to a low overall success rate. Inducible promoters, which allow for the transcription of target genes to be switched on and off in the presence or absence of inducers, have a broad range of applications. In this study, we identify two inducible promoters and apply them to enhance the CRISPR/Cas9 tools. The improved CRISPR/Cas9 tools facilitate gene editing with high efficiency, potentially playing significant roles in advancing genetic research and metabolic engineering of C. ljungdahlii.}, }
@article {pmid40135711, year = {2025}, author = {He, S and Lin, W and Liu, X and Li, F and Liang, H and Xu, H and Lu, C and Xing, C}, title = {A DNA concatemer-encoded CRISPR/Cas12a fluorescence sensor for sensitive detection of Pb[2+] based on DNAzymes.}, journal = {The Analyst}, volume = {150}, number = {9}, pages = {1778-1784}, doi = {10.1039/d5an00189g}, pmid = {40135711}, issn = {1364-5528}, mesh = {*Lead/analysis ; *DNA, Catalytic/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; *DNA/chemistry/genetics ; Spectrometry, Fluorescence/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/metabolism/genetics ; *Water Pollutants, Chemical/analysis ; Fluorescence ; Animals ; Milk/chemistry ; Bacterial Proteins ; }, abstract = {Lead pollution presents a significant threat to ecological systems and human health, underscoring the urgent need for highly sensitive detection methods. Herein, we introduce a novel DNA concatemer-encoded CRISPR/Cas12a fluorescence sensor (MDD-Cas12a) for sensitive detection of Pb[2+] based on DNAzymes. To accomplish this, we designed a substrate strand containing a long DNA concatemer encoding multiple protospacer adjacent motifs (PAMs) and protospacer sequences for activation of the CRISPR/Cas12a system. The DNA concatemer was subsequently anchored to the surface of magnetic beads (MBs) to fabricate a MBs-DNA concatemer nanoprobe. In the presence of Pb[2+], the DNAzyme structure catalyzes the cleavage of the substrate strand, leading to the release of DNA concatemers. Following magnetic separation, the released DNA concatemers significantly activate the non-specific trans-cleavage activity of the Cas12a/crRNA complex. The fluorescence reporter DNA is then completely cleaved by the activated Cas12a/crRNA complex, and the Pb[2+] concentration in the sample can be quantified by measuring the fluorescence signal. By harnessing the specific recognition capability of DNAzymes for Pb[2+], the programmability of DNA concatemers, and the self-amplification features of the CRISPR/Cas12a system, the MDD-Cas12a platform demonstrates high sensitivity and specificity for detecting Pb[2+] in milk and lake water samples.}, }
@article {pmid40135601, year = {2025}, author = {Yee, PS and Chai, AWY and Yee, SM and Ooi, S and Tan, YH and Garnett, MJ and Ng, SK and Cheong, SC}, title = {Interferon-Inducible ADAR1 p150 Is Essential for the Survival of Oral Squamous Cell Carcinoma.}, journal = {Molecular carcinogenesis}, volume = {64}, number = {6}, pages = {1066-1077}, doi = {10.1002/mc.23910}, pmid = {40135601}, issn = {1098-2744}, support = {//This study was funded by the Dr. Siti Hasmah Mohd Ali Professorial Chair appointment (PV061-2019) and Fundamental Research Grant Scheme (FRGS/1/2022/SKK10/USM/02/14)./ ; }, mesh = {Humans ; *Adenosine Deaminase/genetics/metabolism ; *RNA-Binding Proteins/genetics/metabolism ; *Mouth Neoplasms/genetics/pathology/metabolism ; Cell Line, Tumor ; *Carcinoma, Squamous Cell/genetics/pathology/metabolism ; Gene Expression Regulation, Neoplastic/drug effects ; Apoptosis ; Cell Survival/genetics ; CRISPR-Cas Systems ; eIF-2 Kinase/metabolism/genetics ; Interferon-Induced Helicase, IFIH1/metabolism/genetics ; *Squamous Cell Carcinoma of Head and Neck/genetics/pathology ; }, abstract = {We identified ADAR1 as one of the top essential genes for oral squamous cell carcinoma (OSCC) survival from our genome-wide CRISPR/Cas9 screen in OSCC cell lines. In this study, we confirm that ADAR1-knockout (KO) inhibits cell viability and colony forming ability, and induces apoptosis. We report that IFN-β treatment sensitizes less-dependent cell lines to ADAR1 KO-induced cell lethality. Overexpression of ADAR1-p150, but not ADAR1-p110, rescued cell lethality upon ADAR1 KO, confirming that the IFN-inducible p150 is responsible for OSCC survival. Using a deaminase inactive mutant, we demonstrate that the editing function of ADAR1 is important for OSCC survival. Furthermore, we show that ADAR1 KO-induced cell death is mediated by both PKR and MDA5. We compute gene signatures of ADAR1 dependency in OSCC tumors, and found that those with high ADAR1 dependency score are associated with well or moderate differentiation, likely due to high PKR expression or activation. While a majority of ADAR1-dependent tumors exhibit a low T cell-inflamed gene expression profile, ADAR1 KO upregulates PD-L1, a marker of anti-PD1 response, indicating that ADAR1 inhibition may enhance immunotherapy response in OSCC. Collectively, these findings suggest that targeting ADAR1-p150 not only induces OSCC cell death but could induce a favorable response to anti-PD1.}, }
@article {pmid40135490, year = {2025}, author = {Gou, S and Liu, Y and Li, Q and Qiu, L and Liu, Z and Zhao, Y}, title = {CRISPR/Cas12-mediated detection of GI and GII Norovirus in different food samples.}, journal = {Journal of food science}, volume = {90}, number = {3}, pages = {e70160}, doi = {10.1111/1750-3841.70160}, pmid = {40135490}, issn = {1750-3841}, support = {22N31900200//Shanghai Science and Technology Innovation action plan in the agricultural field of 2022/ ; }, mesh = {*Norovirus/genetics/isolation &amp; purification/classification ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Food Contamination/analysis ; Sensitivity and Specificity ; Food Microbiology ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Molecular Diagnostic Techniques ; }, abstract = {Norovirus is one of the leading causes of infectious diarrhea, occurring in about 18% of diarrhea cases worldwide. Norovirus is characterized by a low infectious dose, rapid onset, and strong transmission capacity. Given the lack of specific drugs and vaccines, developing efficient and accurate detection technologies is of great significance to prevent and control the spread of diseases. This study combined the reverse transcription loop-mediated isothermal amplification (RT-LAMP) technology with the clustered regularly interspaced short palindromic repeats (CRISPR) technology to develop a sensitive and rapid detection method, which can reduce the reliance on temperature control and expensive real-time fluorescent polymerase chain reaction (PCR) devices. The RT-LAMP/CRISPR Cas12a method demonstrated good specificity and sensitivity, testing food samples of three different substrates with 100% positive qualitative accuracy. The detection sensitivity is 32.8 copies/reaction for Norovirus GI and 22.8 copies/reaction for Norovirus GII. This method helps to effectively identify food products contaminated with Norovirus, thereby preventing human infections and economic losses due to disease outbreaks.}, }
@article {pmid40133712, year = {2025}, author = {Wang, C and Jiang, X and Li, HY and Hu, J and Ji, Q and Wang, Q and Liu, X and Huang, D and Yan, K and Zhao, L and Fan, Y and Wang, S and Ma, S and Belmonte, JCI and Qu, J and Liu, GH and Zhang, W}, title = {RIG-I-driven CDKN1A stabilization reinforces cellular senescence.}, journal = {Science China. Life sciences}, volume = {68}, number = {6}, pages = {1646-1661}, pmid = {40133712}, issn = {1869-1889}, mesh = {*Cellular Senescence/genetics ; Humans ; *Cyclin-Dependent Kinase Inhibitor p21/metabolism/genetics ; *DEAD Box Protein 58/metabolism/genetics ; Mesenchymal Stem Cells/metabolism/cytology ; Receptors, Immunologic/metabolism/genetics ; RNA, Messenger/metabolism/genetics ; CRISPR-Cas Systems ; Signal Transduction ; Immunity, Innate ; }, abstract = {The innate immune signaling network follows a canonical format for signal transmission. The innate immune pathway is crucial for defense against pathogens, yet its mechanistic crosstalk with aging processes remains largely unexplored. Retinoic acid-inducible gene-I (RIG-I), a key mediator of antiviral immunity within this pathway, has an enigmatic role in stem cell senescence. Our study reveals that RIG-I levels increase in human genetic and physiological cellular aging models, and its accumulation drives cellular senescence. Conversely, CRISPR/Cas9-mediated RIG-I deletion or pharmacological inhibition in human mesenchymal stem cells (hMSCs) confers resistance to senescence. Mechanistically, RIG-I binds to endogenous mRNAs, with CDKN1A mRNA being a prominent target. Specifically, RIG-I stabilizes CDKN1A mRNA, resulting in elevated CDKN1A transcript levels and increased p21[Cip1] protein expression, which precipitates senescence. Collectively, our findings establish RIG-I as a post-transcriptional regulator of senescence and suggest potential targets for the mitigation of aging-related diseases.}, }
@article {pmid40133266, year = {2025}, author = {Aris, KDP and Cofsky, JC and Shi, H and Al-Sayyad, N and Ivanov, IE and Balaji, A and Doudna, JA and Bryant, Z}, title = {Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {2939}, pmid = {40133266}, issn = {2041-1723}, support = {F31 GM145173/GM/NIGMS NIH HHS/United States ; R01 GM106159/GM/NIGMS NIH HHS/United States ; U01 AI142817/AI/NIAID NIH HHS/United States ; R01GM106159//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; }, mesh = {CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *DNA, Superhelical/metabolism/chemistry/genetics ; *R-Loop Structures ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Kinetics ; *DNA/metabolism/chemistry/genetics ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; *Bacterial Proteins/metabolism/genetics/chemistry ; }, abstract = {The sequence specificity and programmability of DNA binding and cleavage have enabled widespread applications of CRISPR-Cas12a in genetic engineering. As an RNA-guided CRISPR endonuclease, Cas12a engages a 20-base pair (bp) DNA segment by forming a three-stranded R-loop structure in which the guide RNA hybridizes to the DNA target. Here we use single-molecule torque spectroscopy to investigate the dynamics and mechanics of R-loop formation of two widely used Cas12a orthologs at base-pair resolution. We directly observe kinetic intermediates corresponding to a ~5 bp initial RNA-DNA hybridization and a ~17 bp intermediate preceding R-loop completion, followed by transient DNA unwinding that extends beyond the 20 bp R-loop. The complex multistate landscape of R-loop formation is ortholog-dependent and shaped by target sequence, mismatches, and DNA supercoiling. A four-state kinetic model captures essential features of Cas12a R-loop dynamics and provides a biophysical framework for understanding Cas12a activity and specificity.}, }
@article {pmid40133194, year = {2025}, author = {Wang, KC and Zheng, T and Hubbard, BP}, title = {CRISPR/Cas technologies for cancer drug discovery and treatment.}, journal = {Trends in pharmacological sciences}, volume = {46}, number = {5}, pages = {437-452}, doi = {10.1016/j.tips.2025.02.009}, pmid = {40133194}, issn = {1873-3735}, mesh = {Humans ; *Neoplasms/genetics/drug therapy/therapy ; *CRISPR-Cas Systems ; Animals ; *Drug Discovery/methods ; Gene Editing/methods ; *Antineoplastic Agents/therapeutic use/pharmacology ; Genetic Therapy/methods ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) tools are revolutionizing the establishment of genotype-phenotype relationships and are transforming cell- and gene-based therapies. In the field of oncology, CRISPR/CRISPR-associated protein 9 (Cas9), Cas12, and Cas13 have advanced the generation of cancer models, the study of tumor evolution, the identification of target genes involved in cancer growth, and the discovery of genes involved in chemosensitivity and resistance. Moreover, preclinical therapeutic strategies employing CRISPR/Cas have emerged. These include the generation of chimeric antigen receptor T (CAR-T) cells and engineered immune cells, and the use of precision anticancer gene-editing agents to inactivate driver oncogenes, suppress tumor support genes, and cull cancer cells in response to genetic circuit output. This review summarizes the collective impact that CRISPR technology has had on basic and applied cancer research, and highlights the promises and challenges facing its clinical translation.}, }
@article {pmid40132699, year = {2025}, author = {Chen, F and Chen, L}, title = {CRISPR/Cas-mediated macromolecular DNA methylation editing: Precision targeting of DNA methyltransferases in cancer therapy.}, journal = {International journal of biological macromolecules}, volume = {308}, number = {Pt 2}, pages = {142401}, doi = {10.1016/j.ijbiomac.2025.142401}, pmid = {40132699}, issn = {1879-0003}, mesh = {Humans ; *DNA Methylation/genetics ; *Neoplasms/genetics/therapy ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Epigenesis, Genetic ; Animals ; }, abstract = {Epigenetic modifications, particularly DNA methylation, play a pivotal role in gene regulation, influencing tumor suppressor silencing and oncogene activation in cancer. DNA methyltransferases (DNMTs), Ten-eleven translocation (TET) enzymes, and associated chromatin regulators are key biological macromolecules that mediate these epigenetic processes. Targeting aberrant DNA methylation holds great promise for cancer therapy, but traditional approaches lack precision and specificity. CRISPR/Cas-based epigenetic editing has emerged as a transformative tool for macromolecular DNA methylation reprogramming, offering targeted modifications without altering the genetic sequence. This review explores the role of DNMTs, TET enzymes, and chromatin-associated proteins in cancer epigenetics and discusses how CRISPR/dCas9 fused with DNMT3A or TET1 enables locus-specific DNA methylation editing. We highlight recent advances, including dCas9-DNMT3A for precise hypermethylation and dCas9-TET1 for targeted demethylation, and discuss their applications in reactivating tumor suppressor genes or silencing oncogenic pathways. Novel epigenetic editing systems, such as SunTag-based amplification, KRAB-MeCP2 repression, further enhance targeting efficiency and therapeutic potential. CRISPR/Cas-mediated macromolecular epigenetic editing represents a paradigm shift in cancer therapy, providing unprecedented control over DNA methylation and chromatin regulation. Despite challenges such as tumor heterogeneity and off-target effects, integrating CRISPR-based methylation reprogramming with precision oncology holds immense promise for future clinical applications.}, }
@article {pmid40132674, year = {2025}, author = {Li, Z and Yoon, JS and Zhong, Z and Ruan, Y and Yang, C and Zhou, X and Zhang, Y and Pan, H}, title = {HvStaufenC contributes to the high RNAi efficiency in the 28-spotted ladybeetle, Henosepilachna vigintioctopunctata.}, journal = {Insect biochemistry and molecular biology}, volume = {180}, number = {}, pages = {104304}, doi = {10.1016/j.ibmb.2025.104304}, pmid = {40132674}, issn = {1879-0240}, mesh = {Animals ; *Coleoptera/genetics/growth &amp; development/metabolism ; *RNA Interference ; Larva/genetics/growth &amp; development/metabolism ; *Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Female ; }, abstract = {RNA interference (RNAi) has been shown to be relatively effective in coleopteran insects, with limited exploration into the molecular mechanisms that underlie this effectiveness. This study specifically examines the 28-spotted ladybeetle, Henosepilachna vigintioctopunctata (Hvig), known for its high RNAi efficiency. Here, we utilized RNAi and CRISPR/Cas9 techniques to identify and validate the genes involved in the RNAi pathway that enhance RNAi efficacy in Hvig. We identified a total of 15 potential genes within the RNAi pathway that may impact RNAi efficiency. The bioassay results showed that only knockdown of HvStaufenC in the 3rd instar larvae could block the abnormal body color phenotype and lethality induced by the subsequent silencing of the two marker genes, HvTH (tyrosine hydroxylase) and HvABCH1 (ATP-binding cassette H transporter gene), respectively. Additionally, successful CRISPR/Cas9-mediated knockout of HvStaufenC led to the generation of stable, heritable mutants that exhibited insensitivity to RNAi, displaying no response to RNAi targeting HvTH and HvABCH1. Compared to the wild-type strain, the HvStaufenC knockout (HvStaufenCKO) mutant females demonstrated a 42 % decrease in oviposition rate and a 41.3 % reduction in egg hatchability. This study demonstrates that HvStaufenC gene is crucial for the RNAi efficiency of Hvig and offers new evidence into the RNAi mechanisms in coleopteran species.}, }
@article {pmid40132543, year = {2025}, author = {Keeley, S and Fernández-Lajarín, M and Bergemann, D and John, N and Parrott, L and Andrea, BE and González-Rosa, JM}, title = {Rapid and robust generation of cardiomyocyte-specific crispants in zebrafish using the cardiodeleter system.}, journal = {Cell reports methods}, volume = {5}, number = {3}, pages = {101003}, pmid = {40132543}, issn = {2667-2375}, mesh = {Animals ; *Zebrafish/genetics ; *Myocytes, Cardiac/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Mutation/genetics ; *Gene Editing/methods ; }, abstract = {CRISPR-Cas9 has accelerated loss-of-function studies in zebrafish, but creating tissue-specific mutant lines is still labor intensive. While some tissue-specific Cas9 zebrafish lines exist, standardized methods for gene targeting, including guide RNA (gRNA) delivery, are lacking, limiting broader use in the community. To tackle these limitations, we develop a cardiomyocyte-specific Cas9 line, the cardiodeleter, that efficiently generates biallelic mutations in combination with gene-specific gRNAs. We create transposon-based guide shuttles that deliver gRNAs targeting a gene of interest while permanently labeling cells susceptible to becoming mutant. We validate this modular approach by deleting five genes (ect2, tnnt2a, cmlc2, amhc, and erbb2), resulting in the loss of the corresponding protein or phenocopy of established mutants. We provide detailed protocols for generating guide shuttles, facilitating the adoption of these techniques in the zebrafish community. Our approach enables rapid generation of tissue-specific crispants and analysis of mosaic phenotypes, making it a valuable tool for cell-autonomous studies and genetic screening.}, }
@article {pmid40131645, year = {2025}, author = {Wang, W and He, H and Liu, H and Gao, Y and Dang, F and Zhao, X and Chen, S and Li, L and Lu, Y}, title = {Developing a robust genome editing tool based on an endogenous type I-B CRISPR-Cas system in Saccharopolyspora spinosa.}, journal = {Science China. Life sciences}, volume = {68}, number = {5}, pages = {1324-1336}, pmid = {40131645}, issn = {1869-1889}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Saccharopolyspora/genetics ; Plasmids/genetics ; *Genome, Bacterial ; Gene Deletion ; }, abstract = {Saccharopolyspora spinosa is an industrial rare actinomycete capable of producing important environmental-friendly biopesticides, spinosyns. However, exploitation of S. spinosa has been limited due to its genetic inaccessibility and lack of effective genome engineering tools. In this work, we characterized the activity of an endogenous type I-B CRISPR-Cas system as well as its recognized protospacer adjacent motifs (PAMs) based on bioinformatics analysis combined with a plasmid interference assay in S. spinosa. By delivering editing plasmids containing a designed miniCRISPR array (repeat+self-targeting spacer+repeat) and repair templates, we achieved 100% editing efficiency for gene deletion. Using this tool, the genetic barrier composed of the restriction-modification (RM) systems was systematically disarmed. We showed that by disarming one type I RM system (encoded by A8926_1903/1904/1905) and two type II RM systems (encoded by A8926_1725/1726 and A8926_2652/2653) simultaneously, the transformation efficiency of the replicative and integrative plasmids (pSP01 and pSI01) was increased by approximately 3.9-fold and 4.2-fold, respectively. Using the engineered strain with simultaneous knock-out of these three RM genes as the starting strain, we achieved the deletion of 75-kb spinosyns biosynthetic gene cluster (BGC) as well as gene insertion at high efficiency. Collectively, we developed a reliable and highly efficient genome editing tool based on the endogenous type I CRISPR-Cas system combined with the disarmament of the RM systems in S. spinosa. This is the first time to establish an endogenous CRISPR-Cas-based genome editing tool in the non-model industrial actinomycetes.}, }
@article {pmid40131558, year = {2025}, author = {Yamanaka, T and Sogo, A and Maegawa, S and Kinoshita, M}, title = {Low-temperature embryo incubation suppresses off-target mutagenesis during CRISPR-Cas9 genome editing in medaka (Oryzias latipes) and zebrafish (Danio rerio).}, journal = {Transgenic research}, volume = {34}, number = {1}, pages = {15}, pmid = {40131558}, issn = {1573-9368}, support = {JPJ008723//Ministry of Agriculture, Forestry and Fisheries/ ; }, mesh = {Animals ; *Oryzias/genetics/growth &amp; development ; *Zebrafish/genetics/growth &amp; development ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Mutagenesis/genetics ; Embryo, Nonmammalian ; Cold Temperature ; Animals, Genetically Modified/genetics ; Mutation ; }, abstract = {Gene knockout using CRISPR-Cas9 is often employed in research aimed at elucidating gene functions in fish. However, CRISPR-Cas9 sometimes introduces unintended alterations, known as off-target mutations. These mutations can reduce the robustness of data during phenotypic analysis. In this study, we focused on the culture temperature, which is known to significantly influence mutagenesis, and examined whether low-temperature culture after introducing CRISPR-Cas9 into early embryos of medaka and zebrafish suppresses off-target mutations. Continuous incubation of medaka at 16 °C significantly reduced off-target mutation rates compared to those at 28 °C; the drawback is that it decreased the survival rate of medaka embryos. Therefore, low-temperature incubation was limited to early development in both zebrafish and medaka, and then the temperature was increased to 28 °C. Under these conditions, the mutation rates of the three off-target regions in medaka (Off-D, Off-P, and Off-A) significantly decreased, whereas those of the three target regions (DJ-1, p4hb, and avt) were unaffected. Similarly, the mutation rate of the zebrafish target region (ywhaqa) remained high, whereas the off-target (Off-Y1) mutation rate significantly reduced. Furthermore, this method effectively suppressed the germ line transmission of off-target mutations in medaka. This approach is effective to obtain more reliable data from the G0 generation of medaka and zebrafish and may reduce the screening effort required to remove individuals with off-target mutations in the F1 generation.}, }
@article {pmid40131505, year = {2025}, author = {Darzi, F and Khamesipour, A and Tasbihi, M and Bahraminasab, M and Nateghi-Rostami, M}, title = {CRISPR/Cas9-mediated deletion of a kinetoplast-associated gene attenuates virulence in Leishmania major parasites.}, journal = {Medical microbiology and immunology}, volume = {214}, number = {1}, pages = {16}, pmid = {40131505}, issn = {1432-1831}, mesh = {Animals ; *Leishmania major/genetics/pathogenicity/growth &amp; development/immunology ; *CRISPR-Cas Systems ; Mice ; Virulence ; Mice, Inbred BALB C ; Macrophages/parasitology/immunology ; *Leishmaniasis, Cutaneous/parasitology/immunology/prevention &amp; control ; *Gene Deletion ; *Protozoan Proteins/genetics ; Female ; Cytokines/metabolism ; *DNA, Kinetoplast/genetics ; Disease Models, Animal ; Gene Editing ; }, abstract = {We employed a CRISPR/Cas9 technique in Leishmania major to evaluate its efficiency in editing a kDNA-associated gene, the universal minicircle sequence binding protein (UMSBP), which is involved in mitochondrial respiration and kinetoplast division. Using this toolkit, we generated UMSBP mNG-tagged and single knockout L. major (LmUMSBP[+/-]) parasites, which were confirmed by PCR, confocal microscopy and Western blot analyses. The growth rate of promastigotes in culture and their infectivity in macrophages were analysed in vitro. Mice were immunized with the LmUMSBP[+/-] mutant strain, and lesion size and parasite burden were measured upon challenge with wild-type (WT) L. major. Cytokines were quantified in supernatants of lymph node cell cultures. The results suggested successful expression and localization of the UMSBP mNG-tagged protein within the kinetoplast in both promastigote and intracellular amastigote forms, confirming the consistency of fluorescence tagging throughout various stages of the Leishmania life cycle. Attenuated LmUMSBP[+/-] parasites showed significantly reduced growth in culture (P < 0.05), increased apoptosis (P < 0.05) and downregulation of tryparedoxin peroxidase (TXNPx) and trypanothione synthetase (TryS) gene expression compared to WT L. major. LmUMSBP[+/-] mutant strains did not cause lesions in a susceptible BALB/c mouse model. Furthermore, immunization with LmUMSBP[+/-] parasites elicited a Th1 immune response, characterized by significantly higher IFN-γ and lower IL-4 production in cell culture (P < 0.001), which was associated with partial protection against WT L. major challenge, as evidenced by reduced parasite burden and lesion development in BALB/c mice. In this study, we successfully validated a practical CRISPR/Cas9 toolkit in L. major, targeting the kinetoplast-associated gene UMSBP. Our findings suggest that the UMSBP single-allele knockout mutant holds promise as a valuable tool for studying the role of the kinetoplast in Leishmania biology and as a potential candidate for further investigation as a live-attenuated vaccine against Leishmania infection.}, }
@article {pmid40130324, year = {2025}, author = {Wu, J and Meng, M and Guo, Z and Hao, K and Liang, Y and Meng, H and Fang, G and Shi, Z and Guo, X and Li, H and Feng, Y and Lin, L and Chen, J and Zhang, Y and Tian, H and Chen, X}, title = {Nuclear-Targeted Material Enabled Intranuclear MicroRNA Imaging for Tracking Gene Editing Process.}, journal = {Angewandte Chemie (International ed. in English)}, volume = {64}, number = {22}, pages = {e202500052}, doi = {10.1002/anie.202500052}, pmid = {40130324}, issn = {1521-3773}, support = {51925305//National Natural Science Foundation of China/ ; 52433006//National Natural Science Foundation of China/ ; 52495010//National Natural Science Foundation of China/ ; 52495015//National Natural Science Foundation of China/ ; 52373161//National Natural Science Foundation of China/ ; 2021YFB3800900//National Key Research and Development Program of China/ ; 3502Z202371003//Natural Science Foundation of Xiamen, China/ ; 20720230008//Fundamental Research Funds for the Central Universities/ ; HRTP-[2022] 52//the talent cultivation project Funds for the Innovation Laboratory for Sciences and Technologies of Energy Materials of Fujian Province/ ; }, mesh = {*MicroRNAs/genetics/metabolism ; Humans ; *Gene Editing ; Animals ; Boronic Acids/chemistry ; CRISPR-Cas Systems ; Fluorescent Dyes/chemistry ; *Cell Nucleus/metabolism ; Polyethyleneimine/chemistry ; Mice ; }, abstract = {Gene editing technology based on clustered regularly interspaced short palindromic repeats/associated protein (CRISPR/Cas) systems serves as an efficient tool in cancer therapy. Tracking the gene editing process can help identify the progress of cancer treatment. However, existing techniques for monitoring the gene editing process rely on lysed cells, which can not reflect the dynamic changes of nucleic acid in living cells. It urgently needs in situ and real-time imaging technologies to track the gene editing process at a living single-cell level more effectively and precisely. Here, we reported a highly efficient nuclear-targeted material, phenylboronic acid modified linear PEI (LPBA), for loading gene editing plasmids and fluorescent probes to track gene editing processes of microRNA. Based on LPBA, we achieved efficient intranuclear microRNA imaging at the living cell level, reaching 32.4-fold higher than the linear PEI (LPEI) delivery system, which facilitated further sensitive monitoring of the gene editing process both in living cells and in vivo. Meanwhile, this efficient gene-editing and real-time detection technique could be extended to screening effective gene-editing plasmids. Such LPBA-based imaging technology extended the imaging area of microRNA and offered new insight in the field of gene editing and nucleic acid detection.}, }
@article {pmid40130092, year = {2025}, author = {Moradi, A and Khoshniyat, S and Nzeako, T and Khazeei Tabari, MA and Olanisa, OO and Tabbaa, K and Alkowati, H and Askarianfard, M and Daoud, D and Oyesanmi, O and Rodriguez, A and Lin, Y}, title = {The Future of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 Gene Therapy in Cardiomyopathies: A Review of Its Therapeutic Potential and Emerging Applications.}, journal = {Cureus}, volume = {17}, number = {2}, pages = {e79372}, pmid = {40130092}, issn = {2168-8184}, abstract = {Cardiomyopathies, among the leading causes of heart failure and sudden cardiac death, are often driven by genetic mutations affecting the heart's structural proteins. Despite significant advancements in understanding the genetic basis of hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy (ARVC), effective long-term therapies remain limited. The advent of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) gene editing offers a promising therapeutic strategy to address these genetic disorders at their root. CRISPR-Cas9 enables precise modification of pathogenic variants (PVs) in genes encoding sarcomeric and desmosomal proteins, which are frequently implicated in cardiomyopathies. By inducing site-specific double-stranded breaks in DNA, followed by repair through nonhomologous end joining (NHEJ) or homology-directed repair (HDR), this system allows for targeted correction of mutations. In preclinical models, CRISPR-Cas9 has shown promise in correcting HCM-associated mutations in β-myosin heavy chain 7 (MYH7), preventing disease phenotypes such as ventricular hypertrophy and myocardial fibrosis. Similarly, gene editing has successfully rectified DCM-linked mutations in Titin (TTN) and LMNA, resulting in improved heart function and reduced pathological remodeling. For ARVC, CRISPR-Cas9 has demonstrated the ability to repair mutations in desmosomal genes such as plakophilin 2 (PKP2), thereby restoring normal cardiac function and cellular adhesion. Despite these successes, challenges remain, including mosaicism, delivery efficiency, and off-target effects. Nevertheless, CRISPR-Cas9 represents a transformative approach to treating genetic cardiomyopathies, potentially offering long-lasting cures by directly addressing their underlying genetic causes.}, }
@article {pmid40129982, year = {2025}, author = {Guo, Y and Zhu, M and Yu, Z and Li, Q and Chen, Y and Ci, L and Sun, R and Shen, R}, title = {Generation and characterization of a tamoxifen-inducible lineage tracing tool Cd2-P2A-CreERT2 knock-in mice.}, journal = {Frontiers in immunology}, volume = {16}, number = {}, pages = {1482070}, pmid = {40129982}, issn = {1664-3224}, mesh = {Animals ; *Tamoxifen/pharmacology ; Mice ; *Gene Knock-In Techniques ; *CD2 Antigens/genetics ; Mice, Transgenic ; *Integrases/genetics ; *Cell Lineage ; CRISPR-Cas Systems ; Genes, Reporter ; Transgenes ; }, abstract = {INTRODUCTION: The new targeted gene editing technologies, such as the CRISPR/Cas system, enable researchers to insert or delete genes at targeted loci efficiently. The Cre-loxp recombination system is widely used to activate or inactivate genes with high spatial and temporal specificity.<br><br>METHODS: Using the CRISPR/Cas9 system, we inserted the CreERT2 transgene expression cassette into the Cd2 gene locus to generate conditional Cre-driver line Cd2-CreERT2 knock-in mice, which drove the expression of CreERT2 by the endogenous Cd2 promoter. By mating the Cd2-CreERT2 strain with a Rosa26-LSL-tdTomato reporter mouse strain which contains a tdTomato expression fragment blocked with a loxP-flanked STOP cassette (LSL) driven by a CAG promoter, a Cd2-CreERT2;Rosa26-LSL-tdTomato reporter strain was obtained to evaluate the expression pattern of CD2 in different cell types.<br><br>RESULTS: After treatment with tamoxifen, the Cd2-CreERT2 knock-in mice were induced to perform efficient recombination at the loxP site following CreERT2 activation and cause the expression of tdTomato fluorescence. The tdTomato and CD2 were expressed in the T cells of peripheral blood, spleen and mesenteric lymph nodes, whereas detected in a low proportion in the B cells. While about 20% of cells labeled with tamoxifen-induced tdTomato were CD2[+] monocytes in peripheral blood, 10% of dendritic cells were tdTomato[+]/CD2[+] cells. Tamoxifen-independent expression of tdTomato occurred in approximately 3% of CD2[+] macrophages, but in negligible (~0.5%) in CD2[+] granulocytes.<br><br>DISCUSSION: This work supplied a new transgenic mouse as a valuable tool for lineage tracing in CD2-expressing cells, for conditional mutant studies of immune modulatory effects in a time-dependent manner, and analysis of the potential therapeutic effect of CD2-targeting biologics.}, }
@article {pmid40129518, year = {2025}, author = {Chavhan, RL and Jaybhaye, SG and Hinge, VR and Deshmukh, AS and Shaikh, US and Jadhav, PK and Kadam, US and Hong, JC}, title = {Emerging applications of gene editing technologies for the development of climate-resilient crops.}, journal = {Frontiers in genome editing}, volume = {7}, number = {}, pages = {1524767}, pmid = {40129518}, issn = {2673-3439}, abstract = {Climate change threatens global crop yield and food security due to rising temperatures, erratic rainfall, and increased abiotic stresses like drought, heat, and salinity. Gene editing technologies, including CRISPR/Cas9, base editors, and prime editors, offer precise tools for enhancing crop resilience. This review explores the mechanisms of these technologies and their applications in developing climate-resilient crops to address future challenges. While CRISPR/enables targeted modifications of plant DNA, the base editors allow for direct base conversion without inducing double-stranded breaks, and the prime editors enable precise insertions, deletions, and substitutions. By understanding and manipulating key regulator genes involved in stress responses, such as DREB, HSP, SOS, ERECTA, HsfA1, and NHX; crop tolerance can be enhanced against drought, heat, and salt stress. Gene editing can improve traits related to root development, water use efficiency, stress response pathways, heat shock response, photosynthesis, membrane stability, ion homeostasis, osmotic adjustment, and oxidative stress response. Advancements in gene editing technologies, integration with genomics, phenomics, artificial intelligence (AI)/machine learning (ML) hold great promise. However, challenges such as off-target effects, delivery methods, and regulatory barriers must be addressed. This review highlights the potential of gene editing to develop climate-resilient crops, contributing to food security and sustainable agriculture.}, }
@article {pmid40128655, year = {2025}, author = {Zhang, Y and Xiang, Y and Hou, D and Fang, L and Cai, S and Zhang, J and Wang, Y and Jiang, Y and Liu, B and Bai, J and Ding, Y and Fang, J and Chen, S and Liu, X and Ren, X}, title = {A one-pot method for universal Dengue virus detection by combining RT-RPA amplification and CRISPR/Cas12a assay.}, journal = {BMC microbiology}, volume = {25}, number = {1}, pages = {163}, pmid = {40128655}, issn = {1471-2180}, support = {32400727//National Natural Science Foundation of China/ ; 24ZR1481100//Natural Science Foundation of Shanghai/ ; 2023YFC2307302//National Key Research and Development Program of China/ ; GWVI-11.2-XD29//program of Shanghai outstanding academic leader in public health subject/ ; 23141902300//experimental animal program sponsored by the Science and Technology Commission of Shanghai Municipality/ ; 22AH0901//Talent Project Foundation of PLA/ ; }, mesh = {*Dengue Virus/genetics/isolation &amp; purification/classification ; *CRISPR-Cas Systems ; *Dengue/diagnosis/virology ; Sensitivity and Specificity ; Humans ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/genetics ; Limit of Detection ; RNA, Viral/genetics ; Recombinases/metabolism/genetics ; *Molecular Diagnostic Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {Dengue Virus (DENV) is a life-threatening pathogen leading to dengue fever, which brings about huge public health challenges globally. However, traditional detection methods currently fail to meet the increasing demands of clinic practice in terms of speed, simplicity, and accuracy. To address these limitations, we developed a novel, rapid, and highly sensitive diagnostic method for universal DENV detection by integrating recombinase polymerase amplification (RPA) assay and the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and associated (Cas) protein 12a (CRISPR/Cas12a) system into one-pot. This approach achieves exceptional sensitivity and specificity for DENV detection, with the entire process completed within 40 min, without the need for sophisticated equipment. The limit of detection (LOD) was determined to be 91.7 copies/test. Using this one-pot RT-RPA CRISPR/Cas12a detection system, all four serotypes of DENV (1 to 4) were successfully identified. In terms of specificity, the assay accurately detected DENV-infected positive samples without cross-reactivity with four other interfering viruses-infected samples (VSV, SeV, HSV-1 and IAV). Furthermore, we established a universal DENV RT-RPA-CRISPR/Cas12a-lateral flow dipstick (LFD) platform, which successfully identified all four serotypes of DENV with a sensitivity of approximately 250 copies/test. Collectively, our method not only provides a robust alternative for universal DENV detection but also offers valuable insights for the identification of other viruses.}, }
@article {pmid40128456, year = {2025}, author = {Liesenfelder, S and Elsafi Mabrouk, MH and Iliescu, J and Baranda, MV and Mizi, A and Perez-Correa, JF and Wessiepe, M and Papantonis, A and Wagner, W}, title = {Epigenetic editing at individual age-associated CpGs affects the genome-wide epigenetic aging landscape.}, journal = {Nature aging}, volume = {5}, number = {6}, pages = {997-1009}, pmid = {40128456}, issn = {2662-8465}, support = {WA 1706/12-2 within CRU344/417911533; WA1706/14-1; SFB 1506/1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; ForTra (PrickMe)//Else Kr&#xf6;ner-Fresenius-Stiftung (Else Kroner-Fresenius Foundation)/ ; VIP+: 03VP11580//Bundesministerium f&#xfc;r Bildung und Forschung (Federal Ministry of Education and Research)/ ; thesis stipend//Jos&#xe9; Carreras Leuk&#xe4;mie-Stiftung (Deutsche Jos&#xe9; Carreras Leuk&#xe4;mie-Stiftung)/ ; }, mesh = {Humans ; *Aging/genetics ; *Epigenesis, Genetic/genetics ; *DNA Methylation/genetics ; *CpG Islands/genetics ; *Gene Editing ; Mesenchymal Stem Cells/metabolism ; Aged ; Adult ; Middle Aged ; Genome, Human ; T-Lymphocytes/metabolism ; CRISPR-Cas Systems ; Male ; Epigenome Editing ; }, abstract = {Aging is reflected by genome-wide DNA methylation changes, which form the basis of epigenetic clocks, but it is largely unclear how these epigenetic modifications are regulated and whether they directly affect the aging process. In this study, we performed epigenetic editing at age-associated CpG sites to explore the consequences of interfering with epigenetic clocks. CRISPR-guided editing targeted at individual age-related CpGs evoked genome-wide bystander effects, which were highly reproducible and enriched at other age-associated regions. 4C-sequencing at age-associated sites revealed increased interactions with bystander modifications and other age-related CpGs. Subsequently, we multiplexed epigenetic editing in human T cells and mesenchymal stromal cells at five genomic regions that become either hypermethylated or hypomethylated upon aging. While targeted methylation seemed more stable at age-hypermethylated sites, both approaches induced bystander modifications at CpGs with the highest correlations with chronological age. Notably, these effects were simultaneously observed at CpGs that gain and lose methylation with age. Our results demonstrate that epigenetic editing can extensively modulate the epigenetic aging network and interfere with epigenetic clocks.}, }
@article {pmid40128351, year = {2025}, author = {Ho Ching Chan, B and Hardy, H and Requena, T and Findlay, A and Ioannidis, J and Meunier, D and Toms, M and Moosajee, M and Raper, A and McGrew, MJ and Rainger, J}, title = {A stable NTN1 fluorescent reporter chicken reveals cell specific molecular signatures during optic fissure closure.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {10096}, pmid = {40128351}, issn = {2045-2322}, support = {BBS/E/D/10002071/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; MR/S033165/1//UKRI Future Leaders Fellowship/ ; }, mesh = {Animals ; Chick Embryo ; Green Fluorescent Proteins/genetics/metabolism ; Chickens/genetics ; *Netrin-1/genetics/metabolism ; Genes, Reporter ; Gene Expression Regulation, Developmental ; Coloboma/genetics ; *Eye/embryology/metabolism ; CRISPR-Cas Systems ; }, abstract = {NTN1 is expressed in a wide range of developmental tissues and is essential for normal development. Here we describe the generation of a Netrin-1 reporter chicken line (NTN1-T2A-eGFP) by targeting green fluorescent protein into the NTN1 locus using CRISPR/Cas9 methodology. Our strategy gave 100% transmission of heterozygous (NTN1[T2A - eGFP/+]) embryos in which GFP localisation faithfully replicated endogenous NTN1 expression in the optic fissure and neural tube floorplate. Furthermore, all NTN1[T2A - eGFP/+] embryos and hatched birds appeared phenotypically normal. We applied this resource to a pertinent developmental context - coloboma is a structural eye malformation characterised by failure of epithelial fusion during optic fissure closure (OFC) and NTN1 is specifically expressed in fusion pioneer cells at the edges of the optic fissure. We therefore optimised the isolation of GFP expressing cells from embryonic NTN1[T2A - eGFP/+] eyes using spectral fluorescence cell-sorting and applied transcriptomic profiling of pioneer cells, which revealed multiple new OFC markers and novel pathways for developmental tissue fusion and coloboma. This work provides a novel fluorescent NTN1 chicken reporter line with broad experimental utility and is the first to directly molecularly characterise pioneer cells during OFC.}, }
@article {pmid40127793, year = {2025}, author = {Yan, C and Zhang, Z and Xu, J and Meng, Y and Yan, S and Wei, L and Zou, Q and Zhang, Q and Cui, F}, title = {CasPro-ESM2: Accurate identification of Cas proteins integrating pre-trained protein language model and multi-scale convolutional neural network.}, journal = {International journal of biological macromolecules}, volume = {308}, number = {Pt 1}, pages = {142309}, doi = {10.1016/j.ijbiomac.2025.142309}, pmid = {40127793}, issn = {1879-0003}, mesh = {*Neural Networks, Computer ; *Software ; *CRISPR-Associated Proteins/chemistry/genetics ; *Computational Biology/methods ; *CRISPR-Cas Systems ; Convolutional Neural Networks ; }, abstract = {Cas proteins (CRISPR-associated protein) are the core components of the CRISPR-Cas system, playing critical roles in defending against foreign DNA and RNA invasions. Identifying Cas proteins can provide deeper insights into the immune mechanisms of the CRISPR-Cas system and help uncover the functional mechanisms of Cas proteins. In this study, we developed a computational tool named CasPro-ESM2, which combines the Pre-trained Protein Language Model ESM-2, multi-scale convolutional neural networks, and evolutionary information from protein sequences to identify Cas proteins. Experimental results demonstrate that CasPro-ESM2 outperforms existing models in Cas protein identification, achieving the highest values in metrics such as ACC, SP, SN, and MCC on two different datasets. Furthermore, we deployed this tool on a web server to enable direct access for users (http://www.bioai-lab.com/CasProESM-2).}, }
@article {pmid40127118, year = {2025}, author = {Dhinoja, S and Qaryoute, AA and Deebani, A and De Maria, A and Jagadeeswaran, P}, title = {CRISPR/Cas9 mediated generation of zebrafish f9a mutant as a model for hemophilia B.}, journal = {Blood coagulation &amp; fibrinolysis : an international journal in haemostasis and thrombosis}, volume = {36}, number = {3}, pages = {90-98}, pmid = {40127118}, issn = {1473-5733}, support = {R01 HL159399/HL/NHLBI NIH HHS/United States ; R15 DK117384/DK/NIDDK NIH HHS/United States ; }, mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; *Hemophilia B/genetics ; *Factor IX/genetics/metabolism ; Disease Models, Animal ; Mutation ; Gene Knockout Techniques ; Humans ; }, abstract = {AIM: This study aimed to develop a zebrafish model for hemophilia B by creating a f9a knockout, as f9a has previously demonstrated functional similarity to human Factor IX.<br><br>METHODS: Using CRISPR/Cas9 technology, two gRNAs targeting exon 8 of the f9a gene, were injected along with Cas9 protein into single-cell zebrafish wild-type embryos. DNA was harvested from the tail tips of the resulting adult zebrafish and screened for mutations using PCR. The founder mutant was crossed with wild-type fish to confirm heritability and subsequently reared to homozygosity. Homozygous mutants were analyzed through quantitative RT-PCR and Western blot to assess f9a RNA and F9a protein levels, respectively. Functional assays like kinetic partial thromboplastin time (kPTT), bleeding assay in adult mutants, and venous laser injury on mutant larvae were performed to assess the hemostatic role.<br><br>RESULTS: Around 61 adults from the CRISPR/Cas9 knockouts were screened, which resulted in a mutant line with a 72&#x200a;bp deletion in the exon 8 encoding catalytic domain. Quantitative RT-PCR and Western Blot analysis showed reduced levels of f9a RNA and F9a protein in the homozygous mutants compared to wild-type siblings. At five dpf, f9a homozygous mutant larvae demonstrated prolonged venous occlusion times in a laser injury assay. Additionally, plasma from the mutants displayed delayed fibrin formation in kPTT assays and exhibited increased bleeding after mechanical injury.<br><br>CONCLUSION: This study created a zebrafish f9a knockout model that mimics the bleeding phenotype observed in hemophilia B patients, which will be valuable for evaluating novel therapeutic approaches for hemophilia B.}, }
@article {pmid40126530, year = {2025}, author = {Yu, Y and Gao, X and Zhao, H and Sun, J and Wang, M and Xiong, X and Li, J and Huang, C and Zhang, H and Jiang, G and Xiao, X}, title = {A Genome-Wide Synthetic Lethal Screen Identifies Spermidine Synthase as a Target to Enhance Erdafitinib Efficacy in FGFR-Mutant Bladder Cancer.}, journal = {Cancer research}, volume = {85}, number = {12}, pages = {2288-2301}, pmid = {40126530}, issn = {1538-7445}, support = {82072840//National Natural Science Foundation of China (NSFC)/ ; 82102734//National Natural Science Foundation of China (NSFC)/ ; 82473301//National Natural Science Foundation of China (NSFC)/ ; 82373053//National Natural Science Foundation of China (NSFC)/ ; 82473179//National Natural Science Foundation of China (NSFC)/ ; }, mesh = {*Urinary Bladder Neoplasms/genetics/drug therapy/pathology/metabolism ; Humans ; Animals ; Mice ; Xenograft Model Antitumor Assays ; *Pyrazoles/pharmacology ; *Spermidine Synthase/antagonists &amp; inhibitors/genetics/metabolism ; Cell Line, Tumor ; Drug Resistance, Neoplasm/genetics/drug effects ; *Receptors, Fibroblast Growth Factor/genetics/antagonists &amp; inhibitors ; Mutation ; *Synthetic Lethal Mutations ; CRISPR-Cas Systems ; Mice, Nude ; ErbB Receptors/genetics/metabolism ; Eukaryotic Translation Initiation Factor 5A ; Peptide Initiation Factors/metabolism ; Quinoxalines ; }, abstract = {UNLABELLED: Mutations of the FGFR family members are frequently observed in metastatic bladder cancer. The development of erdafitinib, a pan-FGFR inhibitor, provided a significant therapeutic advance in bladder cancer, but resistance still limits its efficacy. In this study, we performed an unbiased whole-genome CRISPR-Cas9 synthetic lethal screen on FGFR-mutant bladder cancer cell lines treated with erdafitinib and identified spermidine synthase (SRM) as a critical contributor to erdafitinib resistance. Moreover, hypusinated eIF5A, catalyzed by SRM-mediated spermidine production, facilitated the efficient translation of HMGA2, which in turn promoted the expression of EGFR. Notably, pharmacologic inhibition of SRM enhanced the efficacy of erdafitinib both in vitro and in vivo. Together, these results offer evidence that targeting SRM could attenuate the translation of HMGA2 and subsequently reduce EGFR transcription, thus enhancing the sensitivity of FGFR-mutant bladder cancer cells to erdafitinib treatment.<br><br>SIGNIFICANCE: Combined inhibition of polyamine metabolism and FGFR is a promising therapeutic strategy to overcome erdafitinib resistance and improve treatment for patients with FGFR-mutant bladder cancer.}, }
@article {pmid40126363, year = {2025}, author = {Griffin, C and Coppenrath, K and Khan, D and Lin, Z and Horb, M and Saint-Jeannet, JP}, title = {Deletion of sf3b4 causes splicing defects and gene dysregulation that disrupt craniofacial development and survival.}, journal = {Disease models &amp; mechanisms}, volume = {18}, number = {3}, pages = {}, pmid = {40126363}, issn = {1754-8411}, support = {F32 DE030699/DE/NIDCR NIH HHS/United States ; K99 DE034476/DE/NIDCR NIH HHS/United States ; R24 OD030008/OD/NIH HHS/United States ; R01-DE025468/DE/NIDCR NIH HHS/United States ; //New York University/ ; R01 DE025468/DE/NIDCR NIH HHS/United States ; R24-OD030008//National Institutes of Health office of the Director/ ; P40 OD010997/OD/NIH HHS/United States ; }, mesh = {Animals ; *RNA Splicing Factors/genetics/metabolism ; Neural Crest/pathology/embryology/metabolism ; *RNA Splicing/genetics ; Gene Expression Regulation, Developmental ; Xenopus/genetics/embryology ; *Gene Deletion ; *Xenopus Proteins/genetics/metabolism ; *Skull/embryology/pathology ; Exons/genetics ; Cell Survival/genetics ; Cell Movement/genetics ; Embryo, Nonmammalian/pathology/metabolism ; CRISPR-Cas Systems/genetics ; Cartilage/pathology/embryology ; Mandibulofacial Dysostosis/genetics ; *Craniofacial Abnormalities/genetics ; }, abstract = {Nager and Rodriguez syndromes are rare craniofacial and limb disorders characterized by midface retrusion, micrognathia, absent thumbs and radial hypoplasia. These disorders result from haploinsufficiency of SF3B4 (splicing factor 3b, subunit 4), a component of the pre-mRNA spliceosomal machinery. Although the spliceosome is present and functions in all cells of the body, most spliceosomopathies - including Nager and Rodriguez syndromes - are cell- or tissue-specific in their pathology. To understand the pathomechanism underlying these conditions, we generated a Xenopus tropicalis sf3b4 mutant line using CRISPR/Cas9 gene-editing technology. Homozygous deletion of sf3b4 is detrimental to the development of cranial neural crest (NC)-derived cartilage progenitors. Temporal RNA-sequencing analyses of mutant embryos identified an increase in exon-skipping events, followed by important transcriptional changes associated with an enrichment for terms consistent with defects in NC cell migration and survival. We propose that disruption of these processes may underly the pathogenesis of Nager and Rodriguez syndromes.}, }
@article {pmid40126138, year = {2025}, author = {Pirali, A and Jafarpour, F and Hajian, M and Hosseini Moghaddam, SH and Moradi, R and Tanhaie-Vash, N and Rahimi Andani, M and Izadi, T and Shiralian-Esfahani, H and Safaeinejad, Z and Kues, W and Nasr-Esfahani, MH and Eghbalsaied, S}, title = {Editing the CYP19 Gene in Goat Embryos Using CRISPR/Cas9 and Somatic Cell Nuclear Transfer Techniques.}, journal = {Cellular reprogramming}, volume = {27}, number = {2}, pages = {86-93}, doi = {10.1089/cell.2024.0109}, pmid = {40126138}, issn = {2152-4998}, mesh = {Animals ; *Nuclear Transfer Techniques/veterinary ; *CRISPR-Cas Systems ; *Aromatase/genetics ; *Gene Editing/methods ; *Goats/genetics/embryology ; Female ; Blastocyst/metabolism/cytology ; *Embryo, Mammalian/metabolism ; Embryonic Development/genetics ; Gene Knockout Techniques ; Male ; Cloning, Organism ; }, abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) system is revolutionizing genome engineering and is expected to bring significant advancements in livestock traits, including the treatment of genetic diseases. This study focuses on CRISPR/Cas9-mediated modifications of the CYP19 gene, which encodes aromatase, an enzyme crucial for converting testosterone to estrogen and essential for steroid metabolism. Guide RNAs (gRNAs) were designed to target the CYP19 gene and cloned into the pX459 vector. The recombinant plasmid was then electrotransfected into fibroblast cells from a Lori-Bakhtiari buck, and these transfected cells were used for embryo production via somatic cell nuclear transfer (SCNT). The cloned embryos were evaluated for their progression through embryonic stages, showing no significant difference in blastocyst development between knock-out and unedited groups. The knockout efficiency was 78.4% in cells and 68.9% in goat blastocysts, demonstrating the successful depletion of CYP19. We successfully achieved a high rate of CYP19 gene-edited embryos through the combined application of cell electrotransfection and SCNT technologies, while maintaining the normal developmental rate of the embryos. These embryos can be used for transfer to generate knock-out goats, providing a foundation for further studies on CYP19's role in male fertility and production traits.}, }
@article {pmid40125908, year = {2025}, author = {Niu, H and Zou, L and Liu, Y and Li, Z and Ren, H and Liao, H and Zhang, X and An, S and Ren, F and Ge, X and Cheng, L and Yang, F and Pan, H and Rong, S and Chang, D and Ma, H}, title = {CRISPR/Cas System-Based Fluorescent Sensor for Analysis and Detection.}, journal = {Critical reviews in analytical chemistry}, volume = {}, number = {}, pages = {1-16}, doi = {10.1080/10408347.2025.2481409}, pmid = {40125908}, issn = {1547-6510}, abstract = {Fluorescent sensor is an important tool to reliaze qualitative or quantitative detection of target analyte based on the fluorescence principle. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) has been utilized to develop as a precise, efficient, and highly sensitive molecular diagnostic tool due to its efficient targeting and gene editing ability. At present, CRISPR/Cas system-based fluorescent sensors have shown excellent performance in the field of analysis and detection, and have received widespread attention. Therefore, this paper reviews the mechanism of the CRISPR/Cas system, the characteristics of different Cas proteins, and the principle and characteristics of the fluorescent sensor, with a focus on summarizing the application of the CRISPR/Cas system-based fluorescent sensor for analysis and detection.}, }
@article {pmid40125088, year = {2025}, author = {Cao, ML and Han, RY and Chen, SD and Zhao, DY and Shi, MY and Zou, JH and Li, L and Jiang, HK}, title = {Gene Editing: An Effective Tool for the Future Treatment of Kidney Disease.}, journal = {Journal of inflammation research}, volume = {18}, number = {}, pages = {4001-4018}, pmid = {40125088}, issn = {1178-7031}, abstract = {Gene editing technology involves modifying target genes to alter genetic traits and generate new phenotypes. Beginning with zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), the field has evolved through the advent of clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR-Cas) systems, and more recently to base editors (BE) and prime editors (PE). These innovations have provided deep insights into the molecular mechanisms of complex biological processes and have paved the way for novel therapeutic strategies for a range of diseases. Gene editing is now being applied in the treatment of both genetic and acquired kidney diseases, as well as in kidney transplantation and the correction of genetic mutations. This review explores the current applications of mainstream gene editing technologies in biology, with a particular emphasis on their roles in kidney disease research and treatment of. It also addresses the limitations and challenges associated with these technologies, while offering perspectives on their future potential in this field.}, }
@article {pmid40123515, year = {2025}, author = {Hu, C and Shi, X and Guo, B and Yang, Z and Zhou, J and Wang, F}, title = {Toehold-Based CRISPR-dCas9 Transcriptional Activation Platform for Spatiotemporally Controllable Gene Therapy in Tumor and Diabetic Mouse Models.}, journal = {ACS nano}, volume = {19}, number = {12}, pages = {12277-12287}, doi = {10.1021/acsnano.5c01078}, pmid = {40123515}, issn = {1936-086X}, mesh = {Animals ; Mice ; MicroRNAs/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; Humans ; *Transcriptional Activation/genetics ; *Diabetes Mellitus, Experimental/therapy/genetics ; Gene Editing ; Disease Models, Animal ; Cell Line, Tumor ; *Neoplasms/therapy/genetics ; }, abstract = {The CRISPR-Cas system has been extensively employed as a genome editing tool with the dCas9-based transcriptional activation system emerging as a particularly promising approach for gene editing in the treatment of diseases at the gene level. Nevertheless, the challenge of achieving effective spatiotemporal control of the transcriptional activation system of dCas9 has thus far restricted its broader application. In this study, we present an miRNA-responsive CRISPR-dCas9 transcriptional activation (mCTA) system. This system is capable of responding specifically to exogenous and endogenous miRNAs in mammalian cells and enables the specific imaging of miRNAs during neural development or in the deep tissues of mice. Furthermore, the replacement of downstream functional genes with DTA has been demonstrated to result in the effective apoptosis of tumor cells and inhibition of xenografted tumor growth in mice. Finally, in a diabetic mouse model, the m122CTA system was shown to reduce the blood glucose in diabetic mice via the activation of PDX-1 gene. Our work provides an effective platform for miRNA imaging and gene therapy via spatiotemporal control of gene regulation.}, }
@article {pmid40123001, year = {2025}, author = {Zhang, T and Zhang, Y and Wang, X and Hu, H and Lin, CG and Xu, Y and Zheng, H}, title = {Genome-wide CRISPR activation screen identifies ARL11 as a sensitivity determinant of PARP inhibitor therapy.}, journal = {Cancer gene therapy}, volume = {32}, number = {5}, pages = {521-537}, pmid = {40123001}, issn = {1476-5500}, support = {81972462//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {Humans ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use ; Female ; CRISPR-Cas Systems ; Cell Line, Tumor ; *Breast Neoplasms/genetics/drug therapy/pathology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Resistance, Neoplasm/genetics ; }, abstract = {Resistance to poly-(ADP)-ribose polymerase inhibitors (PARPi) remains a significant challenge in clinical practice, leading to treatment failure in many patients. It is crucial to better understand the molecular mechanisms that underlie PARPi resistance. In this study, utilizing a genome-wide CRISPR activation screen with olaparib, we identified ARL11 as a potential modulator of PARPi treatment response in BRCA-wild-type MDA-MB-231 cells. Mechanistically, ARL11 interacts with STING to enhance innate immunity and forms positive feedback with type I interferon (IFN) induction, which induces ARL11 up-regulation and contributes to resistance to PARPi therapy. Additionally, we observed that ARL11 interacts with the RUVBL1 and RUVBL2 (RUVBL1/2) complex, the key DNA double-strand repair proteins, facilitating DNA homologous recombination (HR) repair and significantly reducing PARPi-induced DNA double-strand damages. Clinical sample analysis reveals that the expression levels of ARL11 and RUVBL1/2 are significantly elevated in breast cancer patients compared to healthy controls. Collectively, our findings suggested that ARL11 and RUVBL1/2 may be promising therapeutic targets to sensitize breast cancer cells to PARPi therapy.}, }
@article {pmid40122420, year = {2025}, author = {Chen, Y and Lv, B and Wang, W and Wu, Y and Li, D}, title = {Structure-switching G-quadruplex: An efficient CRISPR/Cas12a signal reporter for label-free colorimetric biosensing.}, journal = {International journal of biological macromolecules}, volume = {307}, number = {Pt 4}, pages = {142410}, doi = {10.1016/j.ijbiomac.2025.142410}, pmid = {40122420}, issn = {1879-0003}, mesh = {*G-Quadruplexes ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Colorimetry/methods ; MicroRNAs/analysis/genetics ; DNA, Catalytic/chemistry ; Adenosine Triphosphate/analysis ; Humans ; Hemin/chemistry ; DNA/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {G-quadruplex is widely used as a signal reporter for colorimetric biosensor construction. However, the effectiveness of CRISPR/Cas12a in trans-cleaving G-quadruplexes is significantly influenced by their resistance to nuclease, resulting in a weak colorimetric signal response. Herein, a structure-switching G-quadruplex regulated by transducer DNA is used as a signal reporter to construct CRISPR/Cas12a-based biosensors. The transducer DNA lacks a stable secondary structure, enabling efficient cleavage by CRISPR/Cas12a, which subsequently affects the catalytic activity of the G-quadruplex/hemin DNAzyme. We used microRNAs (miRNAs) and ATP as model targets to develop a label-free colorimetric detection platform. By optimizing the DNA sequences and reaction conditions, the biosensors exhibit excellent detection selectivity and sensitivity. The reliability of the proposed method was validated by its consistency with RT-qPCR for miRNAs detection and a commercial chemiluminescence kit for ATP assay, demonstrating its potential in clinical diagnosis and bioanalytical studies. The assay is concise and cost-effective because it does not require DNA labeling, magnetic separation, or enzymatic DNA amplification. Our design strategy avoids the use of G-quadruplex as a cleavage substrate for CRISPR/Cas12a while ensuring an efficient response of the G-quadruplex/hemin DNAzyme to CRISPR/Cas12a system, addressing the issue of G-quadruplex resistance to CRISPR/Cas12a nuclease activity.}, }
@article {pmid40122371, year = {2025}, author = {Ismail, AH and Khormi, MA and Mawkili, W and Albaqami, A and Areshi, S and Aborasain, AM and Hegazy, MM and Amin, AH and Abo-Zaid, MA}, title = {Harnessing the potential of gene-editing technology to overcome the current bottlenecks of CAR-T cell therapy in T-cell malignancies.}, journal = {Experimental hematology}, volume = {146}, number = {}, pages = {104762}, doi = {10.1016/j.exphem.2025.104762}, pmid = {40122371}, issn = {1873-2399}, mesh = {Humans ; *Gene Editing/methods ; *Immunotherapy, Adoptive/methods/adverse effects ; *Receptors, Chimeric Antigen/genetics/immunology ; CRISPR-Cas Systems ; *Hematologic Neoplasms/therapy/genetics/immunology ; *T-Lymphocytes/immunology ; Animals ; }, abstract = {T-cell malignancies (TCMs) include a diverse spectrum of hematologic cancers marked by complex biology and aggressive nature. Treating TCMs remains a critical unmet need in oncology with poor response to standard therapies. Chimeric antigen receptor (CAR)-T cell therapy is one of the most successful types of immunotherapy that has revolutionized cancer treatment, as evidenced by various approved products for CD19 B-cell malignancies and multiple myeloma. Nonetheless, due to some unique hurdles, such as the risk of CAR-T cell fratricide, product contamination with malignant cells, and severe T-cell aplasia, the translation of this treatment approach to TCMs has not been particularly successful. Moreover, irrespective of the type of treated cancer, CAR-T cell therapy can also present some complexities and potential side effects, such as cumbersome and costly manufacturing processes, impaired in vivo function, cytokine release syndrome (CRS), neurotoxicity, and leukemic transformation of CAR-T cells. Recent groundbreaking advances in gene-editing technology and the evolution of precise gene-editing tools such as the CRISPR/Cas9 system and its derivatives have opened a new way to overcoming the mentioned bottlenecks and paving the way for CAR-T cell therapy in TCMs. This review sheds light on how gene editing is being incorporated into CAR-T cell therapy to address current hurdles, enhance therapeutic efficacy, and improve the safety profile of CAR-T cell therapy in TCMs. Ongoing/conducted clinical trials are also discussed to provide a comprehensive view of the evolving landscape of genome-edited CAR-T cell therapy for TCMs.}, }
@article {pmid40121659, year = {2025}, author = {Xu, P and Huang, J and Chen, X and Wang, Q and Yin, B and Xian, Q and Zhuang, C and Hu, Y}, title = {Efficient targeted T-DNA integration for gene activation and male germline-specific gene tagging in Arabidopsis.}, journal = {The Plant journal : for cell and molecular biology}, volume = {121}, number = {6}, pages = {e70104}, doi = {10.1111/tpj.70104}, pmid = {40121659}, issn = {1365-313X}, support = {2023A1515010817//Basic and Applied Basic Research Foundation of Guangdong Province/ ; 31771342//National Natural Science Foundation of China/ ; }, mesh = {*Arabidopsis/genetics ; *DNA, Bacterial/genetics ; Arabidopsis Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; Gene Expression Regulation, Plant ; Transcription Factors/genetics/metabolism ; Genetic Engineering/methods ; }, abstract = {Site-specific DNA integration is an important tool in plant genetic engineering. Traditionally, this process relies on homologous recombination (HR), which is known for its low efficiency in plant cells. In contrast, Agrobacterium-mediated T-DNA integration is highly efficient for plant transformation. However, T-DNA is typically inserted randomly into double-strand breaks within the plant genome via the non-homologous end-joining (NHEJ) DNA repair pathway. In this study, we developed an approach of CRISPR/Cas9-mediated targeted T-DNA integration in Arabidopsis, which was more rapid and efficient than the HR-mediated method. This targeted T-DNA integration aided in gene activation and male germline-specific gene tagging. Gene activation was accomplished by positioning the CaMV35S promoter at the left border of T-DNA, thereby activating specific downstream genes. The activation of FT and MYB26 significantly increased their transcriptional expression, which resulted in early flowering and an altered pattern of cell wall thickening in the anther endothelium, respectively. Male germline-specific gene tagging incorporates two reporters, namely, NeoR and MGH3::mCherry, within the T-DNA. This design facilitates the creation of insertional mutants, simplifies the genetic analysis of mutated alleles, and allows for cellular tracking of male germline cells during fertilization. We successfully applied this system to target the male germline-specific gene GEX2. In conclusion, our results demonstrated that site-specific integration of DNA fragments in the plant genome can be rapidly and efficiently achieved through the NHEJ pathway, making this approach broadly applicable in various contexts.}, }
@article {pmid40121601, year = {2025}, author = {Vollen, K and Alonso, JM and Stepanova, AN}, title = {Beyond a few bases: methods for large DNA insertion and gene targeting in plants.}, journal = {The Plant journal : for cell and molecular biology}, volume = {121}, number = {6}, pages = {e70099}, pmid = {40121601}, issn = {1365-313X}, support = {1444561//National Science Foundation/ ; 1750006//National Science Foundation/ ; 1940829//National Science Foundation/ ; 2023356574//National Science Foundation/ ; 2327912//National Science Foundation/ ; }, mesh = {CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Gene Targeting/methods ; Crops, Agricultural/genetics ; *Mutagenesis, Insertional/methods ; *Plants/genetics ; *DNA, Plant/genetics ; Genome, Plant/genetics ; Plants, Genetically Modified/genetics ; Plant Breeding/methods ; }, abstract = {Genome editing technologies like CRISPR/Cas have greatly accelerated the pace of both fundamental research and translational applications in agriculture. However, many plant biologists are functionally limited to creating small, targeted DNA changes or large, random DNA insertions. The ability to efficiently generate large, yet precise, DNA changes will massively accelerate crop breeding cycles, enabling researchers to more efficiently engineer crops amidst a rapidly changing agricultural landscape. This review provides an overview of existing technologies that allow plant biologists to integrate large DNA sequences within a plant host and some associated technical bottlenecks. Additionally, this review explores a selection of emerging techniques in other host systems to inspire tool development in plants.}, }
@article {pmid40121088, year = {2025}, author = {Williams, AA and Redman, O and Domgaard, H and Armbrust, MJ and Jackson, RN}, title = {Determining the biochemical function of type IV CRISPR ribonucleoprotein complexes and accessory proteins.}, journal = {Methods in enzymology}, volume = {712}, number = {}, pages = {79-114}, doi = {10.1016/bs.mie.2025.01.039}, pmid = {40121088}, issn = {1557-7988}, mesh = {*Ribonucleoproteins/metabolism/genetics/isolation &amp; purification ; *Pseudomonas aeruginosa/genetics/metabolism ; *CRISPR-Cas Systems ; *Bacterial Proteins/metabolism/genetics/isolation &amp; purification ; DNA Helicases/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; }, abstract = {Type IV CRISPR systems are phylogenetically diverse and poorly understood. However, recently, major strides have been made toward understanding type IV-A systems. In type IV-A systems, a multi-subunit ribonucleoprotein complex, called the Csf complex, uses a CRISPR-derived guide to bind double-stranded DNA, forming an R-loop to which a helicase called CRISPR-associated DinG (CasDinG) is recruited. It is proposed that the ATP-dependent helicase activity of CasDinG then unwinds duplex DNA near the targeting site, impairing RNA transcription, and gene expression. Here we describe methods used to investigate the type IV-A system from Pseudomonas aeruginosa strain 83 including a plasmid clearance assay, expression and purification of type IV ribonucleoprotein complexes and proteins, nucleic acid binding assays, and CasDinG helicase assays. These methods provide a foundation for future work aimed at understanding these enigmatic systems.}, }
@article {pmid40121087, year = {2025}, author = {Wang, S and Chang, L}, title = {Biochemical reconstitution of a type I-B CRISPR-associated transposon.}, journal = {Methods in enzymology}, volume = {712}, number = {}, pages = {55-79}, doi = {10.1016/bs.mie.2025.01.042}, pmid = {40121087}, issn = {1557-7988}, mesh = {*DNA Transposable Elements/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {CRISPR-associated transposons (CASTs) are potential gene editing tools because of their RNA-guided DNA insertion activity. It is essential to understand the mechanisms underlying the transposition for the application of CASTs. Here, we provide protocols for the biochemical reconstitution of a type I-B CAST for RNA-guided transposition. The procedures may be applicable to other types of CASTs and facilitate the mechanism studies of various CASTs.}, }
@article {pmid40121086, year = {2025}, author = {Pattali, RK and Ornelas, IJ and Nguyen, CD and Xu, D and Divekar, NS and Nuñez, NK}, title = {CRISPRoff epigenome editing for programmable gene silencing in human cell lines and primary T cells.}, journal = {Methods in enzymology}, volume = {712}, number = {}, pages = {517-551}, pmid = {40121086}, issn = {1557-7988}, support = {R35 GM155044/GM/NIGMS NIH HHS/United States ; }, mesh = {Humans ; *Gene Editing/methods ; *Gene Silencing ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; *T-Lymphocytes/metabolism ; *Epigenome ; DNA Methylation ; Epigenesis, Genetic ; Transfection/methods ; Cell Line ; Epigenome Editing ; }, abstract = {The advent of CRISPR-based technologies has enabled the rapid advancement of programmable gene manipulation in cells, tissues, and whole organisms. An emerging platform for targeted gene perturbation is epigenetic editing, the direct editing of chemical modifications on DNA and histones that ultimately results in repression or activation of the targeted gene. In contrast to CRISPR nucleases, epigenetic editors modulate gene expression without inducing DNA breaks or altering the genomic sequence of host cells. Recently, we developed the CRISPRoff epigenetic editing technology that simultaneously establishes DNA methylation and repressive histone modifications at targeted gene promoters. Transient expression of CRISPRoff and the accompanying single guide RNAs in mammalian cells results in transcriptional repression of targeted genes that is memorized heritably by cells through cell division and differentiation. Here, we describe our protocol for the delivery of CRISPRoff through plasmid DNA transfection, as well as the delivery of CRISPRoff mRNA, into transformed human cell lines and primary immune cells. We also provide guidance on evaluating target gene silencing and highlight key considerations when utilizing CRISPRoff for gene perturbations. Our protocols are broadly applicable to other CRISPR-based epigenetic editing technologies, as programmable genome manipulation tools continue to evolve rapidly.}, }
@article {pmid40121085, year = {2025}, author = {Lu, C and Li, Y and Cummings, JR and Banskota, S}, title = {Delivery of genome editors with engineered virus-like particles.}, journal = {Methods in enzymology}, volume = {712}, number = {}, pages = {475-516}, doi = {10.1016/bs.mie.2025.01.007}, pmid = {40121085}, issn = {1557-7988}, mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; *Virion/genetics ; Animals ; *Gene Transfer Techniques ; }, abstract = {Genome editing technologies have revolutionized biomedical sciences and biotechnology. However, their delivery in vivo remains one of the major obstacles for clinical translation. Here, we introduce various emerging genome editing systems and review different delivery systems have been developed to realize the promise of in vivo gene editing therapies. In particular, we focus on virus-like particles (VLPs), an emerging delivery platform and provide in depth analysis on recent advancements to improve VLPs delivery potential and highlight opportunities for future improvements. To this end, we also provide detail workflows for engineered VLP (eVLP) selection, production, and purification, along with methods for characterization and validation.}, }
@article {pmid40121084, year = {2025}, author = {Zhao, A and Chan, MM}, title = {Cloning and validating systems for high throughput molecular recording.}, journal = {Methods in enzymology}, volume = {712}, number = {}, pages = {453-473}, doi = {10.1016/bs.mie.2025.01.015}, pmid = {40121084}, issn = {1557-7988}, mesh = {*Cloning, Molecular/methods ; CRISPR-Cas Systems ; Humans ; Animals ; Gene Editing/methods ; Cell Lineage/genetics ; Single-Cell Analysis/methods ; }, abstract = {Molecular recording technologies record and store information about cellular history. Lineage tracing is one form of molecular recording and produces information describing cellular trajectories during mammalian development, differentiation and maintenance of adult stem cell niches, and tumor evolution. Our molecular recorder technology utilizes CRISPR-Cas9 barcode editing to generate mutations in genomically integrated, engineered DNA cassettes, which are read out by single-cell RNA sequencing and used to produce high-resolution lineage trees. Here, we describe optimized cloning and validation procedures to construct the molecular recorder lineage tracing system. We include information on considerations of technology design, cloning procedures, the generation of lineage tracing cell lines, and time course experiments to assess their performance.}, }
@article {pmid40121083, year = {2025}, author = {Gould, SI and Sánchez-Rivera, FJ}, title = {Using Prime Editing Guide Generator (PEGG) for high-throughput generation of prime editing sensor libraries.}, journal = {Methods in enzymology}, volume = {712}, number = {}, pages = {437-451}, doi = {10.1016/bs.mie.2025.01.006}, pmid = {40121083}, issn = {1557-7988}, mesh = {*Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Software ; Gene Library ; CRISPR-Cas Systems ; High-Throughput Screening Assays/methods ; Humans ; }, abstract = {Prime editing enables the generation of nearly any small genetic variant. However, the process of prime editing guide RNA (pegRNA) design is challenging and requires automated computational design tools. We developed Prime Editing Guide Generator (PEGG), a fast, flexible, and user-friendly Python package that enables the rapid generation of pegRNA and pegRNA-sensor libraries. Here, we describe the installation and use of PEGG (https://pegg.readthedocs.io) to rapidly generate custom pegRNA-sensor libraries for use in high-throughput prime editing screens.}, }
@article {pmid40121082, year = {2025}, author = {Liu, C and Cheng, S and Zhu, J and Zhou, L and Chen, J}, title = {A quick guide to evaluating prime editing efficiency in mammalian cells.}, journal = {Methods in enzymology}, volume = {712}, number = {}, pages = {419-436}, doi = {10.1016/bs.mie.2025.01.016}, pmid = {40121082}, issn = {1557-7988}, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; Animals ; Core Binding Factor Alpha 2 Subunit/genetics ; High-Throughput Nucleotide Sequencing ; Plasmids/genetics ; Transfection/methods ; INDEL Mutation ; }, abstract = {According to the Clinvar database, modeling the diseases associated with pathogenic mutations requires the installation of base substitutions, small insertions or deletions. Prime editor (PE) was recently developed to precisely install any base substitutions and/or small insertions/deletions (indels) in mammalian cells and animals without requiring DSBs or donor DNA templates. PE also offers greater editing and targeting flexibility compared to other precision CRISPR editing methods because the versatile editing information is encoded in the reverse-transcription template of its prime editing guide RNA. However, optimal PE system selection and experimental design can be complex, and there are various factors that can affect PE efficiency. This chapter serves as a rapid entry-level guideline for the application of PE, providing an experimental framework for using PE at a specific genomic locus. RUNX1 was selected as a representative target site to illustrate the detailed methodology for constructing PE plasmids and the process of transfecting these plasmids into 293FT cells. We further examined the efficiency of PE-mediated genome editing in mammalian cells by using next-generation sequencing.}, }
@article {pmid40121081, year = {2025}, author = {Hibshman, GN and Taylor, DW}, title = {Visualizing the conformational landscape of CRISPR-Cas9 through kinetics-informed structural studies.}, journal = {Methods in enzymology}, volume = {712}, number = {}, pages = {41-53}, doi = {10.1016/bs.mie.2025.01.004}, pmid = {40121081}, issn = {1557-7988}, mesh = {*Cryoelectron Microscopy/methods ; Kinetics ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/chemistry/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics ; Protein Conformation ; Gene Editing/methods ; DNA/chemistry/metabolism ; Models, Molecular ; DNA Cleavage ; }, abstract = {CRISPR-Cas9 has transformed genome editing through its programmability and versatility. Its DNA cleavage activity involves dynamic conformational changes during gRNA binding, DNA recognition, R-loop formation, and endonuclease activation. Understanding these molecular transitions is critical for improving the specificity and efficiency of Cas9, but this remains challenging precisely due to these rapid structural rearrangements. Early structural studies provided foundational insights but were limited to static states under catalytically inactive conditions. Cryo-EM has since enabled visualization of the dynamic nature of active Cas9, by enriching for specific conformations. This chapter introduces a kinetics-informed cryo-EM approach to capture the stepwise activation of Cas9 in real time. With thorough kinetic analyses, such as stopped-flow measurements of R-loop formation, we describe how to identify optimal timepoints to visualize key conformational states with cryo-EM. Integration of kinetic and structural data enables precise mapping of the conformational landscape of Cas9 and other dynamic enzymes, advancing our understanding of their molecular mechanisms and providing a framework for engineering enhanced variants.}, }
@article {pmid40121080, year = {2025}, author = {Zhang, H and Ji, Q}, title = {Prime editing in bacteria with BacPE.}, journal = {Methods in enzymology}, volume = {712}, number = {}, pages = {405-418}, doi = {10.1016/bs.mie.2025.01.026}, pmid = {40121080}, issn = {1557-7988}, mesh = {*Gene Editing/methods ; *Escherichia coli/genetics ; *CRISPR-Cas Systems ; Genome, Bacterial ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {Programmable genome editing technologies have revolutionized the ability of researchers to alter the genomes of microorganisms in a straightforward and efficient manner, significantly advancing the field of microbiology. To date, several CRISPR-Cas-based genome-editing systems have been developed for use in E. coli, including CRISPR/Cas9, base editing, and prime editing technologies. In this chapter, we describe the design and experimental application of BacPE, a variant of prime editing technology optimized for E. coli. BacPE facilitates the introduction of point mutations, insertions, and deletions without the need for double-strand DNA breaks. We demonstrate that BacPE is a powerful tool for genome editing in E. coli and highlight its potential applicability to other bacterial species.}, }
@article {pmid40121079, year = {2025}, author = {Osgood, NRB and Zawalick, NM and Sawyer, CB and Cowan, QT and Gu, S and Mawson, SJ and Ranzau, BL and Li, L and Gymrek, M and Goren, A and Komor, AC}, title = {Genome editing with programmable base editors in human cells.}, journal = {Methods in enzymology}, volume = {712}, number = {}, pages = {351-404}, pmid = {40121079}, issn = {1557-7988}, support = {R56 HG013535/HG/NHGRI NIH HHS/United States ; R35 GM138317/GM/NIGMS NIH HHS/United States ; T32 GM112584/GM/NIGMS NIH HHS/United States ; T32 CA009523/CA/NCI NIH HHS/United States ; T32 GM008326/GM/NIGMS NIH HHS/United States ; }, mesh = {Humans ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; Transfection/methods ; Plasmids/genetics ; HEK293 Cells ; Genome, Human ; }, abstract = {Genome editing has garnered significant attention over the last decade, resulting in a massive expansion of the genome engineering toolbox. Base editors encompass a class of tools that enable installing single-nucleotide changes in genomic DNA without the use of double-strand breaks. With the ever-increasing development of new and/or improved base editor systems, it is easy to be overwhelmed by the abundance of options. Here, we provide clear guidance to facilitate the selection of a base editor and to design guide RNAs (gRNAs) to suit various needs. Additionally, we describe in detail how to generate gRNA plasmids, transfect various mammalian cell types, and evaluate editing efficiencies. Finally, we give alternative methods and troubleshooting tips for some common pitfalls encountered during base editing.}, }
@article {pmid40121078, year = {2025}, author = {Mach, RQ and Miller, SM}, title = {Bacterial directed evolution of CRISPR base editors.}, journal = {Methods in enzymology}, volume = {712}, number = {}, pages = {317-350}, doi = {10.1016/bs.mie.2025.01.003}, pmid = {40121078}, issn = {1557-7988}, mesh = {*Gene Editing/methods ; *Directed Molecular Evolution/methods ; *CRISPR-Cas Systems ; *Escherichia coli/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Base editing and other precision editing agents have transformed the utility and therapeutic potential of CRISPR-based genome editing. While some native enzymes edit efficiently with their nature-derived function, many enzymes require rational engineering or directed evolution to enhance the compatibility with mammalian cell genome editing. While many methods of engineering and directed evolution exist, plate-based discrete evolution offers an ideal balance between ease of use and engineering power. Here, we describe a detailed method for the bacterial directed evolution of CRISPR base editors that compounds technical ease with flexibility of application.}, }
@article {pmid40121077, year = {2025}, author = {McAndrew, MJ and King, MB and Lapinaite, A}, title = {Preparation of high-purity RNPs of CRISPR-based DNA base editors.}, journal = {Methods in enzymology}, volume = {712}, number = {}, pages = {277-315}, doi = {10.1016/bs.mie.2025.01.019}, pmid = {40121077}, issn = {1557-7988}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics/isolation &amp; purification/metabolism ; Humans ; DNA/genetics ; }, abstract = {Since their introduction, CRISPR-based DNA base editors (BEs) have become essential in the field of precision genome editing, revolutionizing the correction of pathogenic SNPs for both basic research and therapeutic applications. As this technology advances, more laboratories are implementing these tools into their workflow. The delivery of BEs as BE-guide RNA complexes (RNPs), rather than as mRNA or plasmids, has been shown to exhibit lower off-target effects, establishing it as the preferred method of delivery. However, there are no protocols describing in detail how to obtain high-purity and highly active BE RNPs. Here, we offer a comprehensive guide for the expression, purification, RNP reconstitution, and in vitro activity assessment of TadA-based BEs. The protocol includes guidance on performing activity assays using commercial denaturing gels, which is convenient and uses standard molecular biology equipment. This allows for rapid quality control testing of reconstituted BE RNPs prior to more expensive and time-consuming in vivo genome editing experiments. Overall, this protocol aims to empower more laboratories to generate tailored BE RNPs for diverse in vitro and in vivo applications.}, }
@article {pmid40121076, year = {2025}, author = {Grimm, MS and Myhrvold, C}, title = {Using CRISPR for viral nucleic acid detection.}, journal = {Methods in enzymology}, volume = {712}, number = {}, pages = {245-275}, doi = {10.1016/bs.mie.2025.01.031}, pmid = {40121076}, issn = {1557-7988}, mesh = {*CRISPR-Cas Systems ; Humans ; *RNA, Viral/genetics/analysis/isolation &amp; purification ; *Viruses/genetics/isolation &amp; purification ; }, abstract = {Pathogenic microorganisms, such as viruses, have threatened human health and will continue to contribute to future epidemics and pandemics, highlighting the importance of developing effective diagnostics. To contain viral outbreaks within populations, fast and early diagnosis of infected individuals is essential. Although current standard methods are highly sensitive and specific, like RT-qPCR, some can have slow turnaround times, which can hinder the prevention of viral transmission. The discovery of CRISPR-Cas systems in bacteria and archaea initially revolutionized the world of genome editing. Intriguingly, CRISPR-Cas enzymes also have the ability to detect nucleic acids with high sensitivity and specificity, which sparked the interest of researchers to also explore their potential in diagnosis of viral pathogens. In particular, the CRISPR-Cas13 system has been used as a tool for detecting viral nucleic acids. Cas13's capability to detect both target RNA and non-specific RNAs has led to the development of detection methods that leverage these characteristics through designing specific detection read-outs. Optimization of viral sample collection, amplification steps and the detection process within the Cas13 detection workflow has resulted in assays with high sensitivity, rapid turnaround times and the capacity for large-scale implementation. This review focuses on the significant innovations of various CRISPR-Cas13-based viral nucleic acid detection methods, comparing their strengths and weaknesses while highlighting Cas13's great potential as a tool for viral diagnostics.}, }
@article {pmid40121075, year = {2025}, author = {Omura, SN and Nureki, O}, title = {General and robust sample preparation strategies for cryo-EM studies of CRISPR-Cas9 and Cas12 enzymes.}, journal = {Methods in enzymology}, volume = {712}, number = {}, pages = {23-39}, doi = {10.1016/bs.mie.2025.01.052}, pmid = {40121075}, issn = {1557-7988}, mesh = {*Cryoelectron Microscopy/methods ; *CRISPR-Cas Systems ; Gene Editing ; *CRISPR-Associated Protein 9/chemistry/ultrastructure/genetics/metabolism ; *CRISPR-Associated Proteins/chemistry/ultrastructure/genetics ; *Endodeoxyribonucleases/chemistry/ultrastructure/genetics ; RNA, Guide, CRISPR-Cas Systems ; }, abstract = {Cas9 and Cas12 are RNA-guided DNA endonucleases derived from prokaryotic CRISPR-Cas adaptive immune systems that have been repurposed as versatile genome-engineering tools. Computational mining of genomes and metagenomes has expanded the diversity of Cas9 and Cas12 enzymes that can be used to develop versatile, orthogonal molecular toolboxes. Structural information is pivotal to uncovering the precise molecular mechanisms of newly discovered Cas enzymes and providing a foundation for their application in genome editing. In this chapter, we describe detailed protocols for the preparation of Cas9 and Cas12 enzymes for cryo-electron microscopy. These methods will enable fast and robust structural determination of newly discovered Cas9 and Cas12 enzymes, which will enhance the understanding of diverse CRISPR-Cas effectors and provide a molecular framework for expanding CRISPR-based genome-editing technologies.}, }
@article {pmid40121074, year = {2025}, author = {Chai, HX and Bamert, RS and Knott, GJ}, title = {Methods for Cas13a expression and purification for use in CRISPR diagnostics.}, journal = {Methods in enzymology}, volume = {712}, number = {}, pages = {225-244}, doi = {10.1016/bs.mie.2025.01.030}, pmid = {40121074}, issn = {1557-7988}, mesh = {*SARS-CoV-2/genetics/isolation &amp; purification ; Humans ; *CRISPR-Cas Systems ; *COVID-19/diagnosis/virology ; *Leptotrichia/enzymology/genetics ; RNA, Viral/genetics/analysis ; *CRISPR-Associated Proteins/genetics/isolation &amp; purification ; *Bacterial Proteins/genetics/isolation &amp; purification ; Clustered Regularly Interspaced Short Palindromic Repeats ; *COVID-19 Nucleic Acid Testing/methods ; }, abstract = {The threat of emerging infectious diseases (e.g., SARS-CoV-2 the RNA virus responsible for the COVID-19 pandemic) has highlighted the importance of accurate and rapid testing for screening, patient diagnosis, and effective treatment of infectious disease. Nucleic acid diagnostic tools such as qPCR are considered the gold standard, providing a sensitive, accurate, and robust method of detection. However, these conventional diagnostic platforms are resource intensive, limited in some applications, and are almost always confined to laboratory settings. With the increasing demand for low-cost, rapid, and accurate point-of-care diagnostics, CRISPR-based systems have emerged as powerful tools to augment detection capabilities. Of note is the potent RNA detection enzyme, Leptotrichia buccalis (Lbu) Cas13a, which is capable of rapid RNA detection in complex mixtures with or without pre-amplification. To support its wide-spread use, we describe a detailed method for the expression, purification, and validation of LbuCas13a for use in molecular diagnostics.}, }
@article {pmid40121072, year = {2025}, author = {Druteika, G and Karvelis, T and Šikšnys, V}, title = {Experimental strategy for characterization of novel TnpB orthologs.}, journal = {Methods in enzymology}, volume = {712}, number = {}, pages = {183-195}, doi = {10.1016/bs.mie.2025.01.056}, pmid = {40121072}, issn = {1557-7988}, mesh = {Humans ; *CRISPR-Cas Systems ; *DNA Transposable Elements ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {TnpB proteins encoded in IS200/IS605 and IS607 mobile genetic elements are among the most widespread proteins in the microbial world. They function as RNA-guided DNA nucleases that play a critical role in transposon proliferation and are the predecessors of CRISPR-Cas12 effector proteins of the type V CRISPR-Cas family. Small size of TnpB nucleases makes them an attractive alternative for larger Cas9 and Cas12 proteins in genome editing applications. However, only a small fraction of TnpB nucleases characterized to date are active in human cells, highlighting the need to identify new TnpB variants that can function as genome editors. Here, we present an experimental pipeline for the characterization of TnpB proteins by combining in silico analysis with in vitro assays. To validate it we determined guide RNA and identified TAM for a set of TnpB orthologs. The proposed workflow can be employed for rapid screening and characterization of the huge TnpB protein family to identify novel TnpB variants that might expand the genome editing toolbox.}, }
@article {pmid40121071, year = {2025}, author = {Schut, FT and Hallmark, T and Dmytrenko, O and Jackson, RN and Beisel, CL}, title = {Purification and in vivo, cell-free, and in vitro characterization of CRISPR-Cas12a2.}, journal = {Methods in enzymology}, volume = {712}, number = {}, pages = {143-181}, doi = {10.1016/bs.mie.2025.01.032}, pmid = {40121071}, issn = {1557-7988}, mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/isolation &amp; purification/metabolism/genetics/chemistry ; Escherichia coli/genetics ; Cell-Free System ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Bacterial Proteins/isolation &amp; purification/metabolism/genetics ; *Endodeoxyribonucleases/isolation &amp; purification/metabolism/genetics ; }, abstract = {The CRISPR-associated (Cas) nuclease Cas12a2 from Sulfuricurvum sp. PC08-66 (SuCas12a2) binds RNA targets with a complementary guide (g)RNA. Target RNA binding causes a major conformational rearrangement in Cas12a2 that activates a RuvC nuclease domain to collaterally cleave RNA, ssDNA and dsDNA, arresting growth and providing population-level immunity. Here, we report in vivo, cell-free, and in vitro methods to characterize the collateral cleavage activity of SuCas12a2 as well as a procedure for gRNA design. As part of the in vivo methods, we describe how to capture growth arrest through plasmid interference and induction of an SOS DNA damage response in the bacterium Escherichia coli. We further apply cell-free transcription-translation to affirm collateral cleavage activity triggered by an expressed RNA target. Finally, as part of the in vitro methods, we describe how to purify active nuclease and subsequently conduct biochemical cleavage assays. In total, the outlined methods should accelerate the exploration of SuCas12a2 and other related Cas nucleases, revealing new features of CRISPR biology and helping develop new CRISPR technologies for molecular diagnostics and other applications.}, }
@article {pmid40121070, year = {2025}, author = {Nguyen, GT and Raju, A and Sashital, DG}, title = {Analysis of metal-dependent DNA nicking activities by Cas endonucleases.}, journal = {Methods in enzymology}, volume = {712}, number = {}, pages = {117-142}, doi = {10.1016/bs.mie.2025.01.034}, pmid = {40121070}, issn = {1557-7988}, mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics ; *DNA/metabolism/chemistry ; DNA Cleavage ; *Bacterial Proteins/metabolism/chemistry/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Gene Editing/methods ; *Metals/metabolism/chemistry ; *Enzyme Assays/methods ; }, abstract = {CRISPR-Cas systems use RNA-guided CRISPR-associated (Cas) effectors to neutralize infections in bacteria and archaea. In class 2 CRISPR-Cas systems, Cas9 and Cas12 are single-protein Cas effectors that target double-stranded DNA based on complementarity to the guide RNA before cleaving the target DNA using metal-dependent endonuclease domains. Cas9 and Cas12 proteins can be readily programmed to target any DNA of interest by changing the guiding RNA sequence and have been co-opted for genome editing and other biotechnology purposes. The effect of metal ion concentration is an essential consideration in the physiological role of Cas immunity effectors as well as the biotechnological applications of Cas endonucleases. In this chapter, we describe methods for studying the effect of variable divalent metal ion conditions on the DNA binding and cleavage activities of well-studied Cas9 and Cas12a proteins.}, }
@article {pmid40121069, year = {2025}, author = {Marin-Gonzalez, A and Rybczynski, AT and Zou, RS and Ha, T}, title = {Measuring double-strand break repair events in mammalian cells with multi-target CRISPR.}, journal = {Methods in enzymology}, volume = {712}, number = {}, pages = {1-22}, doi = {10.1016/bs.mie.2025.01.011}, pmid = {40121069}, issn = {1557-7988}, mesh = {*DNA Breaks, Double-Stranded ; *CRISPR-Cas Systems ; Humans ; *DNA Repair ; High-Throughput Nucleotide Sequencing/methods ; Animals ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {A mechanistic understanding of the different pathways involved in the repair of DSBs is a timely, yet challenging task. CRISPR-Cas9 is a powerful tool to induce DNA double-strand breaks (DSB) at defined genomic locations to study the ensuing repair response, but Cas9 studies are typically limited by i) low-throughput induction of DSB, by targeting only one or a few genomic sites, or ii) the use of genetically integrated reporter systems, which do not always reflect endogenous phenotypes. To address these limitations, we developed multi-target CRISPR, a Cas9-based tool to controllably induce DSBs in high-throughput at endogenous sites, by leveraging repetitive genomic regions. In this Chapter, we describe how to design and execute a multi-target CRISPR experiment. We also detail how to analyze next-generation sequencing data for characterization of DSB repair events at multiple cut sites. We envision that multi-target CRISPR will become a valuable tool for the study of mammalian DSB repair mechanisms.}, }
@article {pmid40120675, year = {2025}, author = {Wan, TR and Wei, L and Cheng, LH and Chin, WC and Shen, J and Chan, FF and Kuang, Z and Wang, C and Wong, CC and Wong, CM}, title = {Genome-wide CRISPR Screening Identifies NFκB and c-MET as Druggable Targets to Sensitize Lenvatinib Treatment in Hepatocellular Carcinoma.}, journal = {Cellular and molecular gastroenterology and hepatology}, volume = {19}, number = {7}, pages = {101502}, pmid = {40120675}, issn = {2352-345X}, mesh = {*Carcinoma, Hepatocellular/drug therapy/genetics/pathology ; *Quinolines/pharmacology/therapeutic use ; *Liver Neoplasms/drug therapy/genetics/pathology ; Humans ; *Phenylurea Compounds/pharmacology/therapeutic use ; Animals ; *Proto-Oncogene Proteins c-met/genetics/antagonists &amp; inhibitors/metabolism ; Cell Line, Tumor ; CRISPR-Cas Systems ; Mice ; *NF-kappa B/antagonists &amp; inhibitors/genetics/metabolism ; Drug Resistance, Neoplasm/genetics/drug effects ; Xenograft Model Antitumor Assays ; Antineoplastic Agents/pharmacology ; Apoptosis/drug effects ; Protein Kinase Inhibitors/pharmacology ; }, abstract = {BACKGROUND &amp; AIMS: Hepatocellular carcinoma (HCC), the dominant form of liver cancer, is a leading cause of cancer death worldwide. Sorafenib and lenvatinib have long been the 2 limited options of first-line treatments for patients with unresectable advanced HCC. However, the single-drug treatment strategy only shows modest survival benefit, mostly because of the survival ability of cancer cells to activate alternative pathways for compensation. In this study, we aim to identify druggable targets contributing to lenvatinib resistance and evaluate the efficacy of combining respective inhibitors and lenvatinib on HCC.<br><br>METHODS: Genome-scale clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 knockout library screening was applied on the vehicle group and lenvatinib treatment group. Identified druggable candidates were validated individually on HCC cell models. Therapeutic effects of the combined treatment of inhibitors of candidate genes and lenvatinib were evaluated in vitro and in vivo.<br><br>RESULTS: We successfully identified NFKB1 and MET as critical drivers for the development of lenvatinib resistance in HCC cells. By perturbing the 2 genes with either CRISPR knockout or RNA interference approaches, lenvatinib treatments were significantly sensitized. Moreover, using small molecules QNZ and cabozantinib to target NFKB1 and MET, respectively, this together with lenvatinib could synergistically induce apoptosis and suppress HCC growth in vitro and in vivo.<br><br>CONCLUSION: Our results demonstrated that genome-wide CRISPR/Cas9 screening is a powerful tool for the design of rational combinational cancer therapy and provided candidate genes possible for combined treatments with lenvatinib to improve therapy efficacy.}, }
@article {pmid40120586, year = {2025}, author = {Herger, M and Kajba, CM and Buckley, M and Cunha, A and Strom, M and Findlay, GM}, title = {High-throughput screening of human genetic variants by pooled prime editing.}, journal = {Cell genomics}, volume = {5}, number = {4}, pages = {100814}, pmid = {40120586}, issn = {2666-979X}, mesh = {Humans ; *Gene Editing/methods ; *High-Throughput Screening Assays/methods ; *Genetic Variation/genetics ; MutL Protein Homolog 1/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems/genetics ; Loss of Function Mutation ; }, abstract = {Multiplexed assays of variant effect (MAVEs) enable scalable functional assessment of human genetic variants. However, established MAVEs are limited by exogenous expression of variants or constraints of genome editing. Here, we introduce a pooled prime editing (PE) platform to scalably assay variants in their endogenous context. We first improve efficiency of PE in HAP1 cells, defining optimal prime editing guide RNA (pegRNA) designs and establishing enrichment of edited cells via co-selection. We next demonstrate negative selection screening by testing over 7,500 pegRNAs targeting SMARCB1 and observing depletion of efficiently installed loss-of-function (LoF) variants. We then screen for LoF variants in MLH1 via 6-thioguanine selection, testing 65.3% of all possible SNVs in a 200-bp region including exon 10 and 362 non-coding variants from ClinVar spanning a 60-kb region. The platform's overall accuracy for discriminating pathogenic variants indicates that it will be highly valuable for identifying new variants underlying diverse human phenotypes across large genomic regions.}, }
@article {pmid40120557, year = {2025}, author = {Ludwik, KA and Opitz, R and Jyrch, S and Megges, M and Kühnen, P and Stachelscheid, H}, title = {Correction of the Allan-Herndon-Dudley syndrome-causing SLC16A2 mutation G401R in a patient derived hiPSC line.}, journal = {Stem cell research}, volume = {85}, number = {}, pages = {103698}, doi = {10.1016/j.scr.2025.103698}, pmid = {40120557}, issn = {1876-7753}, mesh = {Humans ; *X-Linked Intellectual Disability/genetics/pathology/therapy ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Monocarboxylic Acid Transporters/genetics/metabolism ; *Muscle Hypotonia/genetics/pathology/therapy ; *Muscular Atrophy/genetics/pathology/therapy ; Cell Line ; *Mutation ; Symporters/genetics ; Male ; CRISPR-Cas Systems ; Gene Editing ; Mutation, Missense ; }, abstract = {The X-linked Allan-Herndon-Dudley syndrome (AHDS) is a genetic disorder characterized by severe psychomotor impairment, resulting from mutations in the SLC16A2 gene, which encodes the thyroid hormone transporter MCT8 (monocarboxylate transporter 8). Previously, we established a hiPSC line from a patient carrying the SLC16A2:R401G mutation (BIHi045-A). Using CRISPR/Cas9-mediated gene editing, we targeted exon 3 of SLC16A2 and used single-stranded oligodeoxynucleotides as homology-directed repair templates to correct the R401G missense mutation, generating an isogenic control cell line.}, }
@article {pmid40120517, year = {2025}, author = {Wang, Q and Chen, X and Li, Y and Yang, S and Fan, S and Xia, J and Wu, H}, title = {Protein-inorganic hybrid flowers with a two-stage accelerated strategy for stimulated activation of CRISPR/Cas12a enhance polynucleotide kinase biosensing.}, journal = {Talanta}, volume = {292}, number = {}, pages = {127981}, doi = {10.1016/j.talanta.2025.127981}, pmid = {40120517}, issn = {1873-3573}, mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Polynucleotide 5'-Hydroxyl-Kinase/metabolism/analysis ; Humans ; DNA Nucleotidylexotransferase/metabolism/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Polynucleotide kinases (PNK) play a crucial role in DNA damage repair and are closely associated with specific diseases, making them promising targets for therapeutic intervention. In this study, we propose a two-stage accelerated strategy that utilizes protein-inorganic hybrid flowers (PHFs) to enhance the performance of the terminal deoxynucleotidyl transferase (TdT)-combined CRISPR/Cas12a system (TCS) for efficient detection of PNK activity. In TCS, the participation of PHFs confines the substrate probes (SPs) to a limited space, thereby significantly enhancing the local concentration of phosphorylated 3' termini of SPs and effectively promoting the enzymatic reaction kinetics as the first step in the accelerated strategy. Upon encountering the target PNK, the phosphorylated 3' termini were promptly recognized and dephosphorylated to 3'-OH termini. Subsequently, TdT catalyzed the assembly of deoxyadenosine triphosphates (dATPs) without a template, rapidly activating the CRISPR/Cas12a system by forming multiple polyadenine (poly-A) chains. PHF-fixed poly-A chains then substantially boosted the localized concentration of CRISPR/Cas12a systems and vastly enhanced their efficacy in cleaving reporter nucleic acids. Our findings indicated that the spatial confinement effect facilitated by PHFs promoted frequent molecular collisions and accelerated multiple enzymatic reactions. The developed sensing strategy allows for the detection of PNK activity within a linear range of 0.001-1 U/mL, with a detection limit of 1.82 × 10[-4] U/mL. Additionally, this strategy has been successfully applied to detect PNK activity in cell extracts and to screen for PNK inhibitors. Owing to these advantages, PNK can be rapidly and accurately detected with a high sensitivity, specificity, and biostability.}, }
@article {pmid40119750, year = {2025}, author = {Li, L and Mueller, YM and Hioki, K and den Dekker, RJH and Brouwers-Haspels, I and Mezzanotte, L and Maas, A and Erkeland, S and Katsikis, PD}, title = {A novel TOX-nanoluciferase reporter mouse for exploring modulators of T cell exhaustion.}, journal = {Journal of immunology (Baltimore, Md. : 1950)}, volume = {}, number = {}, pages = {}, doi = {10.1093/jimmun/vkaf009}, pmid = {40119750}, issn = {1550-6606}, support = {128371//department of Immunology/ ; 201906210055//China Scholarship Council/ ; }, abstract = {Cytotoxic T cell (CTL) exhaustion is driven by chronic T cell receptor (TCR) stimulation, leading to a dysfunctional state of cells. Exhausted CTLs exhibit diminished effector function against chronic infections and cancers. Therefore, reducing CTL exhaustion may re-establish effective adaptive immune responses. One feature of exhausted CTLs is the sustained and stable expression of transcription factor thymocyte selection-associated high mobility group box (TOX). Downregulating TOX expression in CD8+ T cells enhances their antitumor activities and improves immune checkpoint blockade (ICB) efficiency. We generated a reporter transgenic mouse to rapidly detect the expression of TOX by measuring luciferase activity. We knocked in a reporter cassette containing NanoLuc bioluminescent luciferase (Nluc) into the Tox gene locus by CRISPR/Cas9 (Tox-NLuc mice). We further generated Tox-NLuc-OT-I mice by crossing Tox-NLuc mice with OT-I mice, which allows the induction of CTL exhaustion in vitro by repeated stimulation of CD8+ T cells with OVA (257-264) peptide. Luciferase assays showed that higher luminescent signals were detected in exhausted CTLs compared to non-exhausted CTLs, which can be visualized by bioluminescence imaging. Bioluminescence changes were confirmed by measuring TOX expression by flow cytometry. The luminescence in exhausted CTLs decreased significantly when cells treated with ibrutinib and bryostatin-1, drugs that were found to directly modulate T cell exhaustion and decrease TOX expression. In summary, we have developed a novel TOX-nanoluciferase-based reporter system that can be used to monitor TOX expression and may facilitate the screening of molecules that modulate CTL exhaustion.}, }
@article {pmid40119729, year = {2025}, author = {Mao, S and Wu, R and Luo, W and Qin, J and Chen, AK}, title = {Spuriously transcribed RNAs from CRISPR-sgRNA expression plasmids scaffold biomolecular condensate formation and hamper accurate genomic imaging.}, journal = {Nucleic acids research}, volume = {53}, number = {6}, pages = {}, pmid = {40119729}, issn = {1362-4962}, support = {2021YFA1100201//National Key R&amp;D Program of China/ ; 22177001//National Natural Science Foundation of China/ ; }, mesh = {*Plasmids/genetics/metabolism ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems ; RNA-Binding Proteins/metabolism/genetics ; Cell Nucleus/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Transcription, Genetic ; *Genomics/methods ; HEK293 Cells ; HeLa Cells ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based imaging tools that utilize fluorescently tagged single-guide RNAs (sgRNAs) have enabled versatile analysis of the dynamics of single genomic loci, but the accuracy may be hindered by nonspecific subnuclear probe accumulation, generating false-positive foci in cell nuclei. By examining the subcellular localizations of sgRNA expression plasmids, their RNA transcripts, and several RNA-binding proteins, we found that spuriously transcribed (cryptic) transcripts, produced by sgRNA expression plasmids, are the major contributors of false-positive signals, independent of sgRNA scaffold design or effector probe (i.e. RNA aptamer- or oligonucleotide-based probes) used. These transcripts interact with the paraspeckle core proteins, but not with the sgRNA expression plasmids or the paraspeckle RNA scaffold NEAT1_2, to form nuclear bodies that display liquid-like properties including sphericality, fusion competence, and sensitivity to 1,6-hexanediol. Transfecting sgRNA transcription units (i.e. sgRNA expression cassettes), lacking the plasmid backbones, reduces false-positive signals and enhances genomic imaging accuracy. Overall, this study unveils previously undescribed activities of cryptic plasmid transcripts and presents an easy-to-adapt strategy that can potentially improve the precision of CRISPR-based imaging systems that implement fluorescently tagged sgRNAs.}, }
@article {pmid40119516, year = {2025}, author = {Liu, D and Cao, D and Han, R}, title = {Recent advances in therapeutic gene-editing technologies.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {33}, number = {6}, pages = {2619-2644}, pmid = {40119516}, issn = {1525-0024}, mesh = {*Gene Editing/methods/trends ; Humans ; *CRISPR-Cas Systems ; *Genetic Therapy/methods/trends ; Animals ; Epigenesis, Genetic ; }, abstract = {The advent of gene-editing technologies, particularly CRISPR-based systems, has revolutionized the landscape of biomedical research and gene therapy. Ongoing research in gene editing has led to the rapid iteration of CRISPR technologies, such as base and prime editors, enabling precise nucleotide changes without the need for generating harmful double-strand breaks (DSBs). Furthermore, innovations such as CRISPR fusion systems with DNA recombinases, DNA polymerases, and DNA ligases have expanded the size limitations for edited sequences, opening new avenues for therapeutic development. Beyond the CRISPR system, mobile genetic elements (MGEs) and epigenetic editors are emerging as efficient alternatives for precise large insertions or stable gene manipulation in mammalian cells. These advances collectively set the stage for next-generation gene therapy development. This review highlights recent developments of genetic and epigenetic editing tools and explores preclinical innovations poised to advance the field.}, }
@article {pmid40119192, year = {2025}, author = {Rea, A and Santana-Hernández, S and Villanueva, J and Sanvicente-García, M and Cabo, M and Suarez-Olmos, J and Quimis, F and Qin, M and Llorens, E and Blasco-Benito, S and Torralba-Raga, L and Perez, L and Bhattarai, B and Alari-Pahissa, E and Georgoudaki, AM and Balaguer, F and Juan, M and Pardo, J and Celià-Terrassa, T and Rovira, A and Möker, N and Zhang, C and Colonna, M and Spanholtz, J and Malmberg, KJ and Montagut, C and Albanell, J and Güell, M and López-Botet, M and Muntasell, A}, title = {Enhancing human NK cell antitumor function by knocking out SMAD4 to counteract TGFβ and activin A suppression.}, journal = {Nature immunology}, volume = {26}, number = {4}, pages = {582-594}, pmid = {40119192}, issn = {1529-2916}, support = {765104//EC | EC Seventh Framework Programm | FP7 People: Marie-Curie Actions (FP7-PEOPLE - Specific Programme "People" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013))/ ; ICI24/00041//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; SGR863//Generalitat de Catalunya (Government of Catalonia)/ ; 765104//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 Marie Sklodowska-Curie Actions (H2020 Excellent Science - Marie Sklodowska-Curie Actions)/ ; PI21/00002//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; PI22/00040//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; 2024PROD00089//Departament d'Innovaci&#xf3;, Universitats i Empresa, Generalitat de Catalunya (Department of Innovation, Education and Enterprise, Government of Catalonia)/ ; FI23/00075//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; P01 CA111412/CA/NCI NIH HHS/United States ; }, mesh = {*Smad4 Protein/genetics/metabolism ; *Killer Cells, Natural/immunology/metabolism ; Humans ; *Transforming Growth Factor beta/metabolism/antagonists &amp; inhibitors/immunology ; *Activins/metabolism/antagonists &amp; inhibitors/immunology ; Animals ; Mice ; Cytotoxicity, Immunologic ; Cell Line, Tumor ; Signal Transduction ; Gene Knockout Techniques ; Immunotherapy, Adoptive/methods ; CRISPR-Cas Systems ; *Neoplasms/immunology/therapy ; }, abstract = {Transforming growth factor beta (TGFβ) and activin A suppress natural killer (NK) cell function and proliferation, limiting the efficacy of adoptive NK cell therapies. Inspired by the partial resistance to TGFβ of NK cells with SMAD4 haploinsufficiency, we used CRISPR-Cas9 for knockout of SMAD4 in human NK cells. Here we show that SMAD4[KO] NK cells were resistant to TGFβ and activin A inhibition, retaining their cytotoxicity, cytokine secretion and interleukin-2/interleukin-15-driven proliferation. They showed enhanced tumor penetration and tumor growth control, both as monotherapy and in combination with tumor-targeted therapeutic antibodies. Notably, SMAD4[KO] NK cells outperformed control NK cells treated with a TGFβ inhibitor, underscoring the benefit of maintaining SMAD4-independent TGFβ signaling. SMAD4[KO] conferred TGFβ resistance across diverse NK cell platforms, including CD19-CAR NK cells, stem cell-derived NK cells and ADAPT-NK cells. These findings position SMAD4 knockout as a versatile and compelling strategy to enhance NK cell antitumor activity, providing a new avenue for improving NK cell-based cancer immunotherapies.}, }
@article {pmid40119107, year = {2025}, author = {Kim, S and Matsushita, Y and Katagiri, T and Maseda, H}, title = {Efficiency of genome editing using modified single-stranded oligodeoxyribonucleotides in human cells.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {9764}, pmid = {40119107}, issn = {2045-2322}, support = {JP20ck0106410//Japan Agency for Medical Research and Development/ ; JP20K20640//Japan Society for the Promotion of Science/ ; }, mesh = {Humans ; *Gene Editing/methods ; HEK293 Cells ; *Oligodeoxyribonucleotides/genetics ; Oligonucleotides/genetics ; *DNA, Single-Stranded/genetics ; CRISPR-Cas Systems ; *Genome, Human ; }, abstract = {Single-stranded oligodeoxyribonucleotide (ssODN) gene editing has emerged as a promising therapeutic strategy. However, further improvements in efficiency are desired for practical application. The effects of strand length and locked nucleic acid (LNA) modification on ssODN genome editing were investigated by introducing an assay cassette into the genome of HEK293T cells and measuring precise base deletions of eight bases. The introduction of LNAs into ssODNs, five pairs of LNAs at 25-35 nt from the centre and one pair at 20-25 nt, showed approximately 18-fold higher efficiency than unmodified ssODNs of the same length in the study using 70 nt ssODNs. In addition, genome editing efficiency was further improved when LNAs were introduced at the same positions as the 70 nt ssODN, which showed the highest efficiency for the 90 nt ssODN. However, in some cases, the same number of LNA modifications could conversely reduce the efficiency, and the modification positions in the ssODN method were successfully optimised in the present study. Furthermore, the oligo DNA was shown to be effective not only for deletions but also for base substitutions, with an editing efficiency of 0.63% per cell.}, }
@article {pmid40118423, year = {2025}, author = {Lee, MH and Thomas, JL and Lin, YL and Lin, HY}, title = {In vitro activation of anti-cancer gene expression by delivery of CRISPR/dCas9 ribonucleoproteins to suppress glioblastoma.}, journal = {International journal of biological macromolecules}, volume = {308}, number = {Pt 1}, pages = {142289}, doi = {10.1016/j.ijbiomac.2025.142289}, pmid = {40118423}, issn = {1879-0003}, mesh = {Humans ; *Glioblastoma/genetics/therapy/pathology ; *Ribonucleoproteins/genetics ; Cell Line, Tumor ; *CRISPR-Cas Systems/genetics ; *Gene Expression Regulation, Neoplastic ; RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics ; Astrocytes/metabolism ; Apoptosis/genetics ; Tumor Suppressor Protein p53/genetics ; }, abstract = {Cancer has been a leading cause of death for decades. While many anti-cancer drugs exist, precisely targeting malignant cells is crucial for successful tumor treatment. This targeting can be achieved by activating anti-cancer genes, which specifically destroy malignant cells. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) therapeutics provide a promising approach for gene activation. The technology involves utilizing the denatured Cas9 (CRISPR-associated) protein conjugated with a protein activator to deliver a ribonucleoprotein (RNP) complex including guide RNA into cells for the overexpression of specific proteins. In this study, several guide RNAs targeting cancer suppressor genes were employed. These genes included tumor protein p53 (TP53), human alpha-lactalbumin made lethal to tumor cells (HAMLET), melanoma differentiation-associated gene-7 (MDA7, IL24), phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1, NOXA), pro-apoptotic WT1 regulator (PAWR, PAR4), and TNF superfamily member 10 (TNFSF10, TRAIL). The dCas9/guide RNA complexes were then adsorbed onto magnetic epitope-imprinted nanoparticles. Uppsala 87 malignant glioma (U87MG) cells and induced astrocytes (noncancerous cells) were then treated with the RNP / nanoparticles. The overexpression of MDA7 and NOXA was monitored for at least 30 days using enzyme-linked immunosorbent assay (ELISA) kits. Finally, the induced astrocytes, first activated with these anti-cancer genes, were co-cultured with U87MG cells. This resulted in a "bystander" effect: the malignant U87MG cells underwent apoptosis, while the astrocytes survived.}, }
@article {pmid40118400, year = {2025}, author = {Kong, F and Peng, S and Zhang, Y and Zhang, H and Wang, J and Wang, D}, title = {Spatial double-layer hydrogels enabled visual detection of Cladobotryum mycophilum based on recombinase-aided amplification - CRISPR/Cas12a.}, journal = {International journal of biological macromolecules}, volume = {308}, number = {Pt 1}, pages = {142304}, doi = {10.1016/j.ijbiomac.2025.142304}, pmid = {40118400}, issn = {1879-0003}, mesh = {*CRISPR-Cas Systems ; *Hydrogels/chemistry ; *Ascomycota/isolation &amp; purification/genetics ; *Recombinases/metabolism ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Cladobotryum mycophilum, a pathogen responsible for cobweb disease, caused significant and irreversible losses in the mushroom industry. Effective monitoring and early prevention rely on the development of advanced diagnosis methods. This study introduced a novel hydrogel-based C. mycophilum detection method that integrates recombinase-aided amplification (RAA) with the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system (CRISPR/Cas12a), referred as RCCH. The RAA reaction occurs within cross-linked PEG hydrogel, which is subsequently overlaid with a CRISPR/Cas12a-functionalized hydrogel. The porous network of the PEG hydrogel traps essential enzymes, facilitating spatial co-localization of target DNA and the CRISPR/Cas12a-crRNA complex. Upon activation of Cas12a's trans-cleavage activity, clear and countable fluorescent spots are generated for visual detection. RCCH demonstrates a limit of detection as low as 1 fg/μL, and exceptional selectivity against common fungi Trichoderma viride and T. harzianum and the host mushroom Lentinula edodes. The entire process is completed in under 40 min, indicating RCCH's potential as a rapid, accurate, and practical detection method for monitoring mushroom diseases. This innovative approach offers significant support for enhancing safety in the mushroom industry.}, }
@article {pmid40118290, year = {2025}, author = {Cros-Perrial, E and Beaumel, S and Gimbert, M and Camus, N and Vicente, C and Sekiou, I and Figuet, L and Duruisseaux, M and Dumontet, C and Jordheim, LP}, title = {SLX4 and XPF are involved in cell migration and EMT in a cell-specific manner.}, journal = {Biochemical pharmacology}, volume = {236}, number = {}, pages = {116885}, doi = {10.1016/j.bcp.2025.116885}, pmid = {40118290}, issn = {1873-2968}, mesh = {Humans ; *Epithelial-Mesenchymal Transition/physiology/drug effects ; *Cell Movement/physiology/drug effects ; Animals ; *DNA-Binding Proteins/genetics/metabolism/deficiency ; Mice ; HEK293 Cells ; Cell Line, Tumor ; A549 Cells ; HT29 Cells ; CRISPR-Cas Systems ; Cell Proliferation ; }, abstract = {SLX4 and XPF are two proteins involved in DNA repair, but very little is known about their potential roles in other processes of cancer cell biology. We developed original cell models with CRISPR-Cas9-mediated knock-out of SLX4 and/or XPF using five different cell lines (A549, NCI-H1703, COLO-357, HT-29 and HEK-293 T), and performed characterization with cell biology experiments including migration assays, drug sensitivity testing, cell proliferation assessment and Western blots for relevant proteins. Results showed decreased migration of all models in HT-29 cells, of XPF-deficient COLO-357 cells and of SLX4-deficient HEK-293 T cells. Modified cell models had overall increased sensitivity to cisplatin and mitomycine C, and some models showed an increased frequency of double-stranded DNA damages. One NCI-H1703 cell model showed major karyotypic modifications, and epithelial to mesenchymal transition (EMT)-related proteins were modified in several models. Finally, knocking out one or both proteins in A549 cells had not the same impact on in vivo growth in mice. These original cell models allowed us to identify new and DNA repair-unrelated cellular roles of SLX4 and XPF in cancer cell biology. Our results should be considered within work on Nucleotide Excision Repair (NER) inhibition targeting SLX, XPF or other related proteins.}, }
@article {pmid40118112, year = {2025}, author = {Ma, X and Miao, L and Liu, X}, title = {Teach plants to fish based on CRISPR-Cas system self-evolution.}, journal = {Plant biotechnology journal}, volume = {23}, number = {6}, pages = {2361-2363}, pmid = {40118112}, issn = {1467-7652}, support = {32171453//National Natural Science Foundation of China/ ; 1610392020001//Central Public-interest Scientific Institution Basal Research Fund/ ; 251804006//High-level Talent Research Start-up Project Funding of Henan Academy of Sciences/ ; }, }
@article {pmid40118067, year = {2025}, author = {Takesue, H and Okada, S and Doi, G and Sugiyama, Y and Kusumoto, E and Ito, T}, title = {Strategic targeting of Cas9 nickase expands tandem gene arrays.}, journal = {Cell genomics}, volume = {5}, number = {4}, pages = {100811}, pmid = {40118067}, issn = {2666-979X}, mesh = {Humans ; *Tandem Repeat Sequences/genetics ; *CRISPR-Cas Systems/genetics ; *Deoxyribonuclease I/metabolism/genetics ; DNA Replication/genetics ; *CRISPR-Associated Protein 9/metabolism ; Saccharomyces cerevisiae/genetics ; Animals ; }, abstract = {Expanding tandem gene arrays facilitates adaptation through dosage effects and gene family formation via sequence diversification. However, experimental induction of such expansions remains challenging. Here, we introduce a method termed break-induced replication (BIR)-mediated tandem repeat expansion (BITREx) to address this challenge. BITREx places Cas9 nickase adjacent to a tandem gene array to break the replication fork that has just replicated the array, forming a single-ended double-strand break. This break is subsequently end-resected to become single stranded. Since there is no repeat unit downstream of the break, the single-stranded DNA often invades an upstream unit to initiate ectopic BIR, resulting in array expansion. BITREx has successfully expanded gene arrays in budding yeast, with the CUP1 array reaching ∼1 Mb. Furthermore, appropriate splint DNAs allow BITREx to generate tandem arrays de novo from single-copy genes. We have also demonstrated BITREx in mammalian cells. Therefore, BITREx will find various unique applications in genome engineering.}, }
@article {pmid40118066, year = {2025}, author = {Lodewijk, GA and Kozuki, S and Han, CJ and Topacio, BR and Lee, S and Nixon, L and Zargari, A and Knight, G and Ashton, R and Qi, LS and Shariati, SA}, title = {Self-organization of mouse embryonic stem cells into reproducible pre-gastrulation embryo models via CRISPRa programming.}, journal = {Cell stem cell}, volume = {32}, number = {6}, pages = {895-913.e8}, doi = {10.1016/j.stem.2025.02.015}, pmid = {40118066}, issn = {1875-9777}, mesh = {Animals ; Mice ; *Mouse Embryonic Stem Cells/cytology/metabolism ; Cell Differentiation/genetics ; CDX2 Transcription Factor/genetics/metabolism ; *Embryo, Mammalian/cytology/metabolism ; GATA6 Transcription Factor/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Models, Biological ; Cell Lineage ; }, abstract = {Embryonic stem cells (ESCs) can self-organize into structures with spatial and molecular similarities to natural embryos. During development, embryonic and extraembryonic cells differentiate through activation of endogenous regulatory elements while co-developing via cell-cell interactions. However, engineering regulatory elements to self-organize ESCs into embryo models remains underexplored. Here, we demonstrate that CRISPR activation (CRISPRa) of two regulatory elements near Gata6 and Cdx2 generates embryonic patterns resembling pre-gastrulation mouse embryos. Live single-cell imaging revealed that self-patterning occurs through orchestrated collective movement driven by cell-intrinsic fate induction. In 3D, CRISPRa-programmed embryo models (CPEMs) exhibit morphological and transcriptomic similarity to pre-gastrulation mouse embryos. CPEMs allow versatile perturbations, including dual Cdx2-Elf5 activation to enhance trophoblast differentiation and lineage-specific activation of laminin and matrix metalloproteinases, uncovering their roles in basement membrane remodeling and embryo model morphology. Our findings demonstrate that minimal intrinsic epigenome editing can self-organize ESCs into programmable pre-gastrulation embryo models with robust lineage-specific perturbation capabilities.}, }
@article {pmid40117303, year = {2025}, author = {Hren, A and Lollini, N and Carper, DL and Abraham, PE and Cameron, JC and Fox, JM and Eckert, CA}, title = {High-density CRISPRi screens reveal diverse routes to improved acclimation in cyanobacteria.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {122}, number = {12}, pages = {e2412625122}, pmid = {40117303}, issn = {1091-6490}, support = {DE-SC0018368//U.S. Department of Energy (DOE)/ ; W911NF-18-1-0159//DOD | USA | AFC | CCDC | Army Research Office (ARO)/ ; }, mesh = {*Acclimatization/genetics ; *Synechococcus/genetics/growth &amp; development/physiology ; Gene Expression Regulation, Bacterial ; Photosynthesis/genetics ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics/metabolism ; *Cyanobacteria/genetics ; }, abstract = {Cyanobacteria are the oldest form of photosynthetic life on Earth and contribute to primary production in nearly every habitat, from permafrost to hot springs. Despite longstanding interest in the acclimation of these microbes, it remains poorly understood and challenging to rewire. This study uses a high-density, genome-wide CRISPR interference screen to examine the influence of gene-specific transcriptional variation on the growth of Synechococcus sp. PCC 7002 under environmental extremes. Surprisingly, many partial knockdowns enhanced fitness under cold monochromatic conditions. Transcriptional repression of genes for core subunits of the NDH-1 complex, which are important for photosynthesis and carbon uptake, improved growth rates under both red and blue light but at distinct, color-specific optima. Most genes with fitness-improving knockdowns were distinct to each light color, and dual-target transcriptional repression produced nonadditive effects. Findings reveal diverse routes to improved acclimation in cyanobacteria (e.g., attenuation of genes involved in CO2 uptake, light harvesting, translation, and purine metabolism) and provide an approach for using gradients in sgRNA activity to pinpoint biochemically influential transcriptional changes in cells.}, }
@article {pmid40116487, year = {2025}, author = {Firestone, K and Gopalakrishna, KP and Rogers, LM and Peters, A and Gaddy, JA and Nichols, CM and Hall, MH and Varela, HN and Carlin, SM and Hillebrand, GH and Giacobe, EJ and Aronoff, DM and Hooven, TA}, title = {A CRISPRi library screen in group B Streptococcus identifies surface immunogenic protein (Sip) as a mediator of multiple host interactions.}, journal = {Infection and immunity}, volume = {93}, number = {4}, pages = {e0057324}, pmid = {40116487}, issn = {1098-5522}, support = {R01 HD090061/HD/NICHD NIH HHS/United States ; R01 AI182835/AI/NIAID NIH HHS/United States ; R01 AI134036/AI/NIAID NIH HHS/United States ; 6-FY24-0009//March of Dimes Foundation/ ; T32 GM152284/GM/NIGMS NIH HHS/United States ; R01HD090061,R01HD113675/NH/NIH HHS/United States ; R01AI134036/NH/NIH HHS/United States ; 1275387//Burroughs Wellcome Fund/ ; T32GM007347,T32GM152284/NH/NIH HHS/United States ; I01 BX007005/BX/BLRD VA/United States ; T32 GM007347/GM/NIGMS NIH HHS/United States ; I01 BX005352/BX/BLRD VA/United States ; R01AI182835,R01AI177991/NH/NIH HHS/United States ; R01 HD113675/HD/NICHD NIH HHS/United States ; R01 AI177991/AI/NIAID NIH HHS/United States ; R21AI178067/NH/NIH HHS/United States ; I01BX005352,I01BX007005//U.S. Department of Veterans Affairs/ ; R21 AI178067/AI/NIAID NIH HHS/United States ; }, mesh = {*Streptococcus agalactiae/genetics/immunology ; Animals ; Humans ; *Streptococcal Infections/microbiology/immunology ; Mice ; *Bacterial Proteins/genetics/metabolism/immunology ; Female ; *Host-Pathogen Interactions/immunology ; Macrophages/immunology/microbiology ; Biofilms/growth &amp; development ; Interleukin-1beta/metabolism ; *Membrane Proteins/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Cytokines/metabolism ; }, abstract = {Group B Streptococcus (GBS; Streptococcus agalactiae) is an important pathobiont capable of colonizing various host environments, contributing to severe perinatal infections. Surface proteins play critical roles in GBS-host interactions; however, comprehensive studies of these proteins' functions have been limited by genetic manipulation challenges. This study leveraged a CRISPR interference (CRISPRi) library to target genes encoding surface-trafficked proteins in GBS, identifying their roles in modulating macrophage cytokine responses. Bioinformatic analysis of 654 GBS genomes revealed 66 conserved surface protein genes. Using a GBS strain expressing chromosomally integrated dCas9, we generated and validated CRISPRi strains targeting these genes. THP-1 macrophage-like cells were exposed to ethanol-killed GBS variants, and pro-inflammatory cytokines TNF-⍺ and IL-1β were measured. Notably, knockdown of the sip gene, encoding the Surface Immunogenic Protein (Sip), significantly increased IL-1β secretion, implicating Sip in caspase-1-dependent regulation. Furthermore, Δsip mutants demonstrated impaired biofilm formation, reduced adherence to human fetal membranes, and diminished uterine persistence in a mouse colonization model. These findings suggest that Sip modulates GBS-host interactions critical for pathogenesis, underscoring its potential as a therapeutic target or vaccine component.}, }
@article {pmid40115958, year = {2025}, author = {Farinati, S and Soria Garcia, AF and Draga, S and Vannozzi, A and Palumbo, F and Scariolo, F and Gabelli, G and Barcaccia, G}, title = {Unlocking male sterility in horticultural crops through gene editing technology for precision breeding applications: presentation of a case study in tomato.}, journal = {Frontiers in plant science}, volume = {16}, number = {}, pages = {1549136}, pmid = {40115958}, issn = {1664-462X}, abstract = {Plant male sterility (MS) refers to the failure of the production of functional anthers, viable pollen grains and/or fertile sperm cells. This feature has great potential in horticultural crops for the exploitation of heterosis through the development of F1 hybrid varieties. MS in plants can occur spontaneously or can be induced artificially by exploiting biotechnological tools, such as the editing of genes involved in spore formation or pollen development. The success of such an approach strongly depends both on preliminary knowledge of the involved genes and on effective procedures for in vitro transfection/regeneration of whole plants. Furthermore, according to previous studies based on CRISPR/Cas9 technology, the efficacy of targeting and the resulting mutation profile are critically influenced by intrinsic factors, such as the CRISPR target primary sequence sites and chromatin signatures, which are often associated with varying levels of chromatin accessibility across different genomic regions. This relationship underscores the complexity of CRISPR-based genome editing and highlights the need to identify a precise suitable target. Our paper reports the results obtained for site-specific in vivo mutagenesis via a CRISPR/Cas9-mediated strategy applied to the MYB80 gene, which is a promising target for implementing male sterility in horticultural crops. We highlight the main steps that play a key role in the whole experimental pipeline, which aims at the generation of CRISPR/Cas-edited DNA-free tomato plants. This goal was achieved via protoplast-based technology and by directly delivering a ribonucleoprotein complex consisting of the Cas9 protein and in vitro synthesized single guide RNAs that can target different positions of the gene under investigation. Overall findings and insights are presented and critically discussed.}, }
@article {pmid40114706, year = {2025}, author = {Maalouf, KE and Frederick, DM and Sharma, N and Haidar, EA and Xiao, T and Han, JS and Mahamdeh, MS and Soberman, RJ and Rufino-Ramos, D and Kleinstiver, BP and Jinnah, HA and Vaine, CA and Bragg, DC and Breyne, K}, title = {Non-invasive detection of allele-specific CRISPR-SaCas9-KKH disruption of TOR1A DYT1 allele in a xenograft mouse model.}, journal = {Molecular therapy. Nucleic acids}, volume = {36}, number = {1}, pages = {102466}, pmid = {40114706}, issn = {2162-2531}, abstract = {DYT1 dystonia is a neurological movement disorder characterized by a dominant 3-base pair deletion (ΔGAG) in the TOR1A gene. This study demonstrates a gene-editing approach that selectively targets the ΔGAG mutation in the TOR1A DYT1 allele while safeguarding the wild-type (WT) TOR1A allele. We optimized an adeno-associated virus (AAV) vector-compatible variant of the Staphylococcus aureus Cas9 nuclease ortholog (SaCas9-KKH) in DYT1 patient-derived human neuronal progenitor cells (hNPCs). On-target editing of the TOR1A DYT1 allele was confirmed at the genomic level from brain tissue in a xenograft mouse model. To avoid brain biopsy for demonstrating TOR1A DYT1 editing, we developed a non-invasive monitoring method using extracellular RNA (exRNA). TOR1A exRNA was retrieved from the extracellular vesicle (EV) secretions of hNPCs and plasma samples, indicating whether the donor was a TOR1A DYT1 carrier. This technique enabled us to assess AAV-mediated disruption of the TOR1A DYT1 allele in the brains of mice using blood samples.}, }
@article {pmid40114442, year = {2025}, author = {Gregor, A and Distel, L and Ekici, AB and Kirchner, P and Uebe, S and Krumbiegel, M and Turan, S and Winner, B and Zweier, C}, title = {Proteasomal activation ameliorates neuronal phenotypes linked to FBXO11-deficiency.}, journal = {HGG advances}, volume = {6}, number = {2}, pages = {100425}, pmid = {40114442}, issn = {2666-2477}, mesh = {Humans ; Animals ; *Neurons/metabolism/cytology/drug effects ; *F-Box Proteins/genetics/metabolism ; Phenotype ; *Proteasome Endopeptidase Complex/metabolism ; Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Differentiation ; Drosophila ; CRISPR-Cas Systems ; Protein-Arginine N-Methyltransferases ; }, abstract = {Haploinsufficiency of FBXO11, encoding a ubiquitin ligase complex subunit, is associated with a variable neurodevelopmental disorder. So far, the underlying nervous system-related pathomechanisms are poorly understood, and specific therapies are lacking. Using a combined approach, we established an FBXO11-deficient human stem cell-based neuronal model using CRISPR-Cas9 and a Drosophila model using tissue-specific knockdown techniques. We performed transcriptomic analyses on iPSC-derived neurons and molecular phenotyping in both models. RNA sequencing revealed disrupted transcriptional networks related to processes important for neuronal development, such as differentiation, migration, and cell signaling. Consistently, we found that loss of FBXO11 leads to neuronal phenotypes such as impaired neuronal migration and abnormal proliferation/differentiation balance in human cultured neurons and impaired dendritic development and behavior in Drosophila. Interestingly, application of three different proteasome-activating substances could alleviate FBXO11-deficiency-associated phenotypes in both human neurons and flies. One of these substances is the long-approved drug Verapamil, opening the possibility of drug repurposing in the future. Our study shows the importance of FBXO11 for neurodevelopment and highlights the reversibility of related phenotypes, opening an avenue for potential development of therapeutic approaches through drug repurposing.}, }
@article {pmid40114372, year = {2025}, author = {Pulgarin, DV and Pelo, N and Ferrandiz, L and Tršelič, T and Nyberg, WA and Bowlin, G and Espinosa, A}, title = {Light-induced expression of gRNA allows for optogenetic gene editing of T lymphocytes in vivo.}, journal = {Nucleic acids research}, volume = {53}, number = {6}, pages = {}, pmid = {40114372}, issn = {1362-4962}, support = {200992//Cancerfonden/ ; //Whitaker Foundation/ ; //Cancerfonden/ ; F31AR072502/NH/NIH HHS/United States ; F31 AR072502/AR/NIAMS NIH HHS/United States ; //Konung Gustaf V:s och Drottning Victorias Frimurarestiftelse/ ; }, mesh = {*Gene Editing/methods ; *Optogenetics/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Animals ; CRISPR-Cas Systems ; *T-Lymphocytes/metabolism ; Light ; Mice ; Humans ; RNA, Catalytic/genetics/metabolism ; Transcription Factors/genetics/metabolism ; HEK293 Cells ; }, abstract = {There is currently a lack of tools capable of perturbing genes in both a precise and a spatiotemporal fashion. The flexibility of CRISPR (clustered regularly interspaced short palindromic repeats), coupled with light's unparalleled spatiotemporal resolution deliverable from a controllable source, makes optogenetic CRISPR a well-suited solution for precise spatiotemporal gene perturbations. Here, we present a new optogenetic CRISPR tool (Blue Light-inducible Universal VPR-Improved Production of RGRs, BLU-VIPR) that diverges from prevailing split-Cas design strategies and instead focuses on optogenetic regulation of guide RNA (gRNA) production. We engineered BLU-VIPR around a new potent blue-light activated transcription factor (VPR-EL222) and ribozyme-flanked gRNA. The BLU-VIPR design is genetically encoded and ensures precise excision of multiple gRNAs from a single messenger RNA transcript. This simplified spatiotemporal gene perturbation and allowed for several types of optogenetic CRISPR, including indels, CRISPRa, and base editing. BLU-VIPR also worked in vivo with cells previously intractable to optogenetic gene editing, achieving optogenetic gene editing in T lymphocytes in vivo.}, }
@article {pmid40113632, year = {2025}, author = {Xavier, KVM and de Oliveira Luz, AC and Silva-Junior, JW and de Melo, BST and de Aragão Batista, MV and de Albuquerque Silva, AM and de Queiroz Balbino, V and Leal-Balbino, TC}, title = {Molecular epidemiological study of Pseudomonas aeruginosa strains isolated from hospitals in Brazil by MLST and CRISPR/Cas system analysis.}, journal = {Molecular genetics and genomics : MGG}, volume = {300}, number = {1}, pages = {33}, pmid = {40113632}, issn = {1617-4623}, mesh = {*Pseudomonas aeruginosa/genetics/isolation &amp; purification/classification/pathogenicity ; Brazil/epidemiology ; Multilocus Sequence Typing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Pseudomonas Infections/microbiology/epidemiology/genetics ; Molecular Epidemiology ; Hospitals ; Phylogeny ; }, abstract = {The CRISPR/Cas system defends bacteria and archaea against invasive pathogens, such as phages, establishing an immunological memory from this interaction. Pseudomonas aeruginosa, an opportunistic pathogen, represents a significant public health concern due to its multidrug resistance. This study conducted a molecular epidemiological analysis of clinical isolates of Pseudomonas aeruginosa in Brazil using multilocus sequence typing (MLST) and characterization of CRISPR/Cas system. Most P. aeruginosa isolates harbored the type I-F CRISPR/Cas system (83%), with a subset also exhibiting the type I-E system. Additionally, some isolates presented incomplete CRISPR/Cas systems in their secondary loci. Notably, the isolate Pae93 exhibited a genetic composition rich in phage-related proteins proximal to the orphan CRISPR locus. The identification and characterization of spacer sequences, including previously undocumented ones, revealed a remarkable diversity of predatory mobile genetic elements (MGEs) among the P. aeruginosa isolates studied. The spacer sequences were incorporated into the MGE library. Additionally, the study identified the existence of prophages and anti-CRISPR genes. Two new sequence types (STs 3383 and 3384) were identified and added to the PubMLST database. No discernible correlation was established between the observed STs and the previously delineated CRISPR genotypes. However, the CRISPR system remains valuable for elucidating specific interactions between microorganisms and MGEs. The Brazilian population of clinical P. aeruginosa isolates was shown to be genetically heterogeneous with a non-clonal distribution, as revealed by MLST analysis. The presence of high-risk clones, such as ST 244 and ST 235, underscores the importance of robust epidemiological surveillance and infection control strategies for P. aeruginosa, especially in healthcare settings. This study significantly contributes to the understanding of the molecular epidemiology of these isolates in Brazil.}, }
@article {pmid40113426, year = {2025}, author = {Song, WJ and Zhang, F and Wang, ZS and Tian, JF and Niu, RF}, title = {[The application of ANXA2 gene knockout mouse models in lung cancer metastasis].}, journal = {Zhonghua zhong liu za zhi [Chinese journal of oncology]}, volume = {47}, number = {3}, pages = {254-261}, doi = {10.3760/cma.j.cn112152-20240705-00277}, pmid = {40113426}, issn = {0253-3766}, support = {81372844//National Natural Science Foundation of China/ ; }, mesh = {Animals ; *Annexin A2/genetics/metabolism ; *Lung Neoplasms/pathology/genetics/metabolism ; *Carcinoma, Lewis Lung/pathology/genetics/metabolism ; Mice, Knockout ; Mice ; *Disease Models, Animal ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; Neoplasm Metastasis ; Survival Rate ; Male ; Female ; }, abstract = {Objective: ANXA2 plays a crucial role in cancer metastasis, but its mechanism is not yet fully understood. Therefore, it is necessary to establish an ANXA2 gene knockout mouse model to provide an effective tool for subsequent studies on ANXA2-related mechanisms. Methods: A gene knockout mouse model was constructed using CRISPR/Cas9 technology. The model was validated through tissue DNA extraction followed by polymerase chain reaction (PCR), sequencing, and western blot to confirm ANXA2 genotype and protein expression. The successfully constructed models were divided into a model group and a wild-type (WT) group for the creation of a mouse tail vein injection Lewis lung carcinoma (LLC) metastasis model. Metastatic foci formation was monitored using in vivo imaging technology, and the survival rates of the two groups were compared. Results: An sgRNA sequence targeting the first exon of ANXA2 was designed, and 16 founder mice were obtained through microinjection. Through consanguineous hybridization, 30 homozygous offspring were ultimately acquired. After establishing the strains of the mouse model, mice were divided into the ANXA2 knockout group and the WT group, with 8 mice in each group. An LLC lung metastasis model was established in both groups. Compared with the WT group, the number of metastatic foci was significantly increased in the ANXA2 knockout group (7 vs. 1), and the fluorescence intensity was stronger in the WT group than in the knockout group (P=0.002). Using the GEPIA2 database to analyze ANXA2 gene expression in tumor tissues and normal tissues of lung cancer patients, it was found that ANXA2 expression levels were significantly higher in lung cancer tumor tissues compared to normal tissues (P＜0.05). The database included data from 478 lung cancer patients, and patients were stratified into high-expression and low-expression groups based on ANXA2 levels. Compared to the low-expression group, patients in the high-expression group exhibited significantly shorter disease-free survival and overall survival (P＜0.05, respectively). The survival time of mice in the ANXA2 knockout group (median survival time, 43 days) was significantly longer compared to the WT group (median survival time, 26 days; P=0.017). Additionally, ANXA2 expression is significantly associated with the prognosis of lung cancer patients (P=6.4e-14). Conclusions: ANXA2 is closely associated with cancer metastasis and holds potential as a new target for metastasis treatment. Further in-depth research will greatly facilitate the transition of ANXA2 from basic research to clinical application.}, }
@article {pmid40113237, year = {2025}, author = {Robertson, NR and Lee, S and Tafrishi, A and Wheeldon, I}, title = {Advances in CRISPR-enabled genome-wide screens in yeast.}, journal = {FEMS yeast research}, volume = {25}, number = {}, pages = {}, pmid = {40113237}, issn = {1567-1364}, support = {NSF-2323984//National Science Foundation/ ; }, mesh = {*CRISPR-Cas Systems ; *Genome, Fungal ; Metabolic Engineering/methods ; *Gene Editing/methods ; *Genomics/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Yeasts/genetics ; *Saccharomyces cerevisiae/genetics ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas genome-wide screens are powerful tools for unraveling genotype-phenotype relationships, enabling precise manipulation of genes to study and engineer industrially useful traits. Traditional genetic methods, such as random mutagenesis or RNA interference, often lack the specificity and scalability required for large-scale functional genomic screens. CRISPR systems overcome these limitations by offering precision gene targeting and manipulation, allowing for high-throughput investigations into gene function and interactions. Recent work has shown that CRISPR genome editing is widely adaptable to several yeast species, many of which have natural traits suited for industrial biotechnology. In this review, we discuss recent advances in yeast functional genomics, emphasizing advancements made with CRISPR tools. We discuss how the development and optimization of CRISPR genome-wide screens have enabled a host-first approach to metabolic engineering, which takes advantage of the natural traits of nonconventional yeast-fast growth rates, high stress tolerance, and novel metabolism-to create new production hosts. Lastly, we discuss future directions, including automation and biosensor-driven screens, to enhance high-throughput CRISPR-enabled yeast engineering.}, }
@article {pmid40113021, year = {2025}, author = {Xu, J and Xu, J and Sun, C and He, X and Shu, Y and Huangfu, Q and Meng, L and Liang, Z and Wei, J and Cai, M and Wen, J and Wang, B}, title = {Effective delivery of CRISPR/dCas9-SAM for multiplex gene activation based on mesoporous silica nanoparticles for bladder cancer therapy.}, journal = {Acta biomaterialia}, volume = {197}, number = {}, pages = {460-475}, doi = {10.1016/j.actbio.2025.03.032}, pmid = {40113021}, issn = {1878-7568}, mesh = {*Urinary Bladder Neoplasms/therapy/genetics/pathology/metabolism ; *Silicon Dioxide/chemistry ; *Nanoparticles/chemistry/therapeutic use ; Humans ; *CRISPR-Cas Systems/genetics ; Animals ; Cell Line, Tumor ; *Genetic Therapy/methods ; Porosity ; Mice ; *Gene Transfer Techniques ; Mice, Nude ; }, abstract = {The molecular complexity of bladder cancer restricts reliance on single-feature or single-gene targeted therapies, necessitating integrated individualized treatments and multi-gene interventions. In this study, we introduced the CRISPR/dCas9-SAM system to BCa treatment, known for its high specificity, low off-target effects, and reduced genetic toxicity, making it ideal for multiplexed gene activation at minimal cost-just 20 nucleotides per target. However, despite its potential in complex gene therapy and cellular engineering, challenges persist due to safety concerns associated with viral vectors and the risk of off-target effects during in vivo delivery, necessitating the development of new vectors. Herein, we reported pH-sensitive hollow mesoporous silica nanoparticles modified with PLZ4 ligands (PLZ4-Lip@AMSN/CRISPR/dCas9-SAM, PLACS NPs) for precise targeting of bladder tumors and co-delivery of CRISPR/dCas9-SAM system. With good stability and high plasmid loading capacity, they efficiently co-delivered dCas9-VP64, MS2-P65-HSF1, and sgRNA. Compared to Lipofectamine 3000, these nanoparticles exhibited superior lysosomal escape capability, significantly enhancing transfection efficiency in bladder cancer cells. Moreover, PLACS NPs simultaneously activated the expression of four target genes, inhibiting proliferation and migration, and promoting apoptosis in bladder cancer cells. In vivo, they achieved efficient gene editing at tumor sites, significantly inhibiting bladder tumor growth. Real-time imaging revealed their substantial accumulation and prolonged retention at bladder tumor sites without significant liver targeting and major organ damage, showcasing good specificity and biosafety. This study overcomes in vivo delivery challenges of multi-component CRISPR/dCas9 systems, enabling precise gene editing and anti-tumor effects, presenting an innovative strategy for targeted therapy in bladder cancer treatment. STATEMENT OF SIGNIFICANCE: This study introduces a newly-developed approach to address key challenges in bladder cancer gene therapy, namely low gene upregulation efficiency, limited targeting specificity, and inefficient nucleic acid delivery. By integrating the CRISPR/dCas9-SAM system, we achieve highly specific gene activation with minimal off-target effects, enabling the addition of treatment targets with just 20 nucleotides per target. To improve bladder cancer targeting, we developed PLACS NPs, a mesoporous silica nanoparticle system that enhances plasmid delivery, transfection efficiency, and endosomal escape. This system shows good tumor targeting and significant anti-tumor effects in bladder cancer, without significant liver targeting and major organ toxicity, offering promising therapeutic potential and broad clinical applications.}, }
@article {pmid40112818, year = {2025}, author = {Wala, J and Dalin, S and Webster, S and Shapira, O and Busanovich, J and Sarmashghi, S and Beroukhim, R and Bandopadhayay, P and Rendo, V}, title = {Recurrent breakpoints in the BRD4 locus reduce toxicity associated with gene amplification.}, journal = {Cell genomics}, volume = {5}, number = {4}, pages = {100815}, pmid = {40112818}, issn = {2666-979X}, mesh = {Humans ; *Transcription Factors/genetics/metabolism ; Cell Cycle Proteins/genetics ; Female ; Cell Line, Tumor ; *Gene Amplification/genetics ; *Nuclear Proteins/genetics/metabolism ; Ovarian Neoplasms/genetics/pathology ; Cell Proliferation/genetics ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Animals ; Cyclin E/genetics ; Protein Isoforms/genetics/metabolism ; Mice ; Bromodomain Containing Proteins ; }, abstract = {Recent work by the ICGC-PCAWG consortium identified recurrent focal deletions in the BRD4 gene, decreasing expression despite increased copy number. We show that these focal deletions occur in the context of cyclin E1 amplification in breast, ovarian, and endometrial cancers, and serve to disrupt BRD4 regulatory regions and gene expression across isoforms. We analyze open reading frame screen data and find that overexpression of BRD4 long (BRD4-L) and short isoform BRD4-S(a) impairs cell growth across cell lines. We confirm these results in OVSAHO ovarian cancer cells, where the overexpression of BRD4 isoforms significantly reduces tumor growth. Next, we mimic BRD4 focal deletions using CRISPR-Cas9 technology and show that these focal deletions rescue ovarian cancer cells from toxicity associated with BRD4 overexpression, suggesting that BRD4 levels must be fine-tuned for cancer cell proliferation. Our study provides experimental evidence for the first recurrent deletion reducing toxicity in cancer, expanding the landscape of cancer progression mechanisms.}, }
@article {pmid40112038, year = {2025}, author = {Xu, T and Li, Y and Liu, X and Yang, X and Huang, Z and Xing, J and Liang, C and Li, J and Tan, Y and Zhang, S and Qi, J and Ye, D and Li, Z and Cao, J and Tang, C and Liu, K}, title = {Rubber biosynthesis drives the biogenesis and development of rubber particles, the rubber-producing organelles.}, journal = {Plant biotechnology journal}, volume = {23}, number = {6}, pages = {2303-2316}, pmid = {40112038}, issn = {1467-7652}, support = {32201450//National Natural Science Foundation of China/ ; 32160383//National Natural Science Foundation of China/ ; 31825007//National Natural Science Foundation of China/ ; 32101543//National Natural Science Foundation of China/ ; }, mesh = {*Latex/metabolism/biosynthesis ; *Rubber/metabolism ; *Taraxacum/metabolism/genetics ; Plant Proteins/metabolism/genetics ; *Organelles/metabolism ; Transferases/metabolism/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; }, abstract = {Rubber particles (RPs) are specialized organelles for the biosynthesis and storage of natural rubber in rubber-producing plants. However, the mechanisms underlying the biogenesis and development of RPs remain unclear. In this study, two latex-specific cis-prenyltransferases (CPTs), TkCPT1 and TkCPT2, were identified in Taraxacum kok-saghyz, with almost identical orthologues retained across other Taraxacum species. For the first time, Tkcpt1 single and Tkcpt1/2 double mutants were successfully generated using the CRISPR/Cas9 system. Rubber biosynthesis was significantly depressed in Tkcpt1 mutants and completely blocked in Tkcpt1/2 mutants. The absence of RPs in the Tkcpt1/2 was confirmed using oil red O and Nile red staining, high-speed centrifugal stratification, cryo-SEM and TEM on fresh latex or laticifer cells. Transcriptomic and proteomic analyses revealed that, in the latex of Tkcpt1/2, rubber biosynthesis was blocked at the protein level, while metabolomic profiling indicated an enrichment of lipids and terpenoids. Furthermore, knockout of TkCPTL1, a latex-specific CPT-like gene that encodes a rubber transferase activator, resulted in outright disruption of rubber biosynthesis and RP ontogeny, a phenotype similar to that of Tkcpt1/2 mutants. These findings indicate that rubber biosynthesis is a driving force for the biogenesis and development of RPs, providing new insights into rubber production mechanisms.}, }
@article {pmid40111512, year = {2025}, author = {Zhang, L and Li, D and Li, X and Zong, L and Bian, H and Lu, J}, title = {CutIn: a ready-to-use construct for rapid generation of urgently needed transgenic cell lines in emerging infection research.}, journal = {Functional &amp; integrative genomics}, volume = {25}, number = {1}, pages = {67}, pmid = {40111512}, issn = {1438-7948}, support = {202303021222378//Natural Science Foundation of Young Scientists of Shanxi Province/ ; 2022A049//Changzhi People's Hospital Clinical &amp; Basic Innovative Research Project Fund/ ; }, mesh = {Humans ; DNA End-Joining Repair ; *Gene Knock-In Techniques/methods ; HCT116 Cells ; HEK293 Cells ; HeLa Cells ; Hypoxanthine Phosphoribosyltransferase/genetics ; Plasmids/genetics ; *Cell Line ; }, abstract = {Site-directed exogenous gene knock-in for stable cell line generation remains a multi-step procedure that heavily relies on expertise. Therefore, there is a need for a competent and easily manageable method, particularly when there is an urgent demand for cell lines, especially for emerging infection research. We present here a universal construct called CutIn that expresses the Cas9 protein and dual sgRNAs targeting a host cell genome locus and the ampicillin resistance (AmpR) gene of a cotransfected donor plasmid commercially available. This construct specifically induces double-strand breaks (DSBs) in cotransfected plasmids and host cell genomes, thereby facilitating whole plasmid integration through nonhomologous end joining (NHEJ) repair mechanisms. As pilot tests, adeno-associated virus integration site 1 (AAVS1) or hypoxanthine phosphoribosyl transferase (HPRT) locus was selected as host genome target, commonly used human cell lines 293T, HeLa and HCT116 were employed. CutIn was subjected for reporter plasmid knock-in in all three cell lines, either AAVS1 and AmpR or HPRT and AmpR loci were efficiently targeted. Fluorescent protein, human angiotensin-converting enzyme 2 (ACE2) and dengue virus (DENV) infection reporter transgenic cells were rapidly obtained via CutIn-mediated whole expression vector integration. This method is designed to be user-friendly and shows potential for supporting the investigation of emerging/re-emerging infectious diseases. Further validation in diverse research contexts will be necessary to fully assess its applicability and effectiveness.}, }
@article {pmid40111000, year = {2025}, author = {Zhou, X and Yang, S and Sun, B and Dong, F and Yin, M and Jiang, Y and Huang, Z and Yang, S}, title = {Implementation of RAGATH RNA-associated DNA Endonucleases as Genome Editing Tool in Escherichia coli.}, journal = {Biotechnology journal}, volume = {20}, number = {3}, pages = {e70005}, doi = {10.1002/biot.70005}, pmid = {40111000}, issn = {1860-7314}, support = {2023YFF1000200//National Key Research and Development Program of China/ ; }, mesh = {*Gene Editing/methods ; *Escherichia coli/genetics ; CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; Streptococcus pyogenes/genetics/enzymology ; DNA Transposable Elements/genetics ; Genome, Bacterial ; }, abstract = {The preferred method for Escherichia coli genome editing relies on Cas9 from Streptococcus pyogenes (SpCas9) and λ-Red recombinase. Although SpCas9 is currently the most active RNA-guided DNA endonuclease, a significant number of escapers are often observed, making it inefficient across different sites, particularly when inserting large fragments. In this study, we identified two RAGATH RNA-associated DNA endonucleases (RADs) derived from IS607 transposons. Both of them exhibited high cleavage activity in E. coli. When combined with λ-Red recombinase, they achieved editing efficiencies approaching 100%. Even at target sites where SpCas9 exhibited low editing efficiency, RADs maintained efficiencies ranging from 57% to 94%. Moreover, RADs exhibited higher efficiencies in inserting large fragments in certain cases compared to SpCas9. Taken together, these RAD-based genome editing tools provide viable alternatives to SpCas9, particularly for challenging targets and/or large fragment insertions.}, }
@article {pmid40110772, year = {2025}, author = {Yang, W and Peng, M and Wang, Y and Zhang, X and Li, W and Zhai, X and Wu, Z and Hu, P and Chen, L}, title = {Deletion of hepcidin disrupts iron homeostasis and hematopoiesis in zebrafish embryogenesis.}, journal = {Development (Cambridge, England)}, volume = {152}, number = {7}, pages = {}, doi = {10.1242/dev.204307}, pmid = {40110772}, issn = {1477-9129}, support = {32200414//National Natural Science Foundation of China/ ; }, mesh = {Animals ; *Zebrafish/embryology/genetics/metabolism ; *Hepcidins/genetics/metabolism ; *Hematopoiesis/genetics ; *Iron/metabolism ; *Homeostasis/genetics ; *Embryonic Development/genetics ; *Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Developmental ; Ferroptosis/genetics ; Gene Deletion ; Embryo, Nonmammalian/metabolism ; Gene Knockout Techniques ; GATA1 Transcription Factor/metabolism/genetics ; }, abstract = {Iron is essential for cell growth and hematopoiesis, which is regulated by hepcidin (hamp). However, the role of hamp in zebrafish hematopoiesis remains unclear. Here, we have created a stable hamp knockout zebrafish model using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 system (CRISPR/Cas9 system). Our study revealed that hamp deletion led to maternal iron overload in embryos, significantly downregulating hemoglobin genes and reducing hemoglobin content. Single-cell RNA sequencing identified abnormal expression patterns in blood progenitor cells, with a specific progenitor subtype showing increased ferroptosis and delayed development. By crossing hamp knockout zebrafish with a gata1+ line (blood cells labeled fish line), we confirmed ferroptosis in blood progenitor cells. These findings underscore the crucial role of hamp in iron regulation and hematopoiesis, offering novel insights into developmental biology and potential therapeutic targets for blood disorders.}, }
@article {pmid40110701, year = {2025}, author = {Liang, H and Li, T and Chen, Y and Wang, J and Aslam, M and Qin, H and Fan, W and Du, H and Kao, SJ and Lin, S}, title = {Urea Amidolyase as an Enzyme for Urea Utilisation in Phytoplankton: Functional Display in Chlamydomonas reinhardtii.}, journal = {Molecular ecology}, volume = {34}, number = {8}, pages = {e17734}, doi = {10.1111/mec.17734}, pmid = {40110701}, issn = {1365-294X}, support = {42206116//Natural Science Foundation of China/ ; 2024A1515011467&#xff1b;2025A1515010095//Natural Science Foundation of Guangdong Province, China/ ; 2021B1212050025//Science and Technology Plan Projects of Guangdong Province/ ; 2022KCXTD008//Program for University Innovation Team of Guangdong Province/ ; 2024-MRB-00-001//Research on Breeding Technology of Candidate Species for Guangdong Modern Marine Ranching/ ; }, mesh = {*Chlamydomonas reinhardtii/genetics/enzymology/metabolism ; *Urea/metabolism ; *Phytoplankton/genetics/enzymology/metabolism ; *Amidohydrolases/genetics/metabolism ; Nitrogen/metabolism ; Phylogeny ; CRISPR-Cas Systems ; Urease/genetics/metabolism ; Metagenome ; }, abstract = {Urea is an important source of nitrogen for many phytoplankton with the potential to stimulate harmful algal blooms, but the molecular machinery underpinning urea uptake and assimilation by algae is not fully understood. Urease (URE) is commonly regarded as the responsible enzyme, but urea amidolyase (UAL), albeit known to exist, has hardly been studied. Here, the species distribution, expression patterns and functional roles of UAL are examined. We found a widespread occurrence of UAL across six major phytoplankton lineages, along with evidence of a potential URE-independent evolutionary trajectory and lineage-specific losses. Quantitative analyses based on marine planktonic metagenomes and metatranscriptomes revealed that UAL is as prevalent as URE, but exhibits higher expression levels in phytoplankton than in bacteria, suggesting that UAL plays a crucial role in nitrogen nutrition in marine phytoplankton. Furthermore, using the CRISPR/Cas9 genome editing method and Chlamydomonas reinhardtii as the algal model, we showed that DUR2 in UAL is essential for urea utilisation, as its knockout completely abolishes the ability of algae to grow under urea as the sole nitrogen source. This study unveils an unappreciated mechanism in algae for utilising urea as a nutrient, underscores the need to consider both URE and UAL enzyme systems to model urea utilisation by algae and provides a crucial gene (DUR2) as a potential genetic marker for detecting the contribution of UAL to urea utilisation in phytoplankton.}, }
@article {pmid40110345, year = {2025}, author = {He, C and Li, Y and Liu, J and Li, Z and Li, X and Choi, JW and Li, H and Liu, S and Li, CZ}, title = {Application of CRISPR-Cas System in Human Papillomavirus Detection Using Biosensor Devices and Point-of-Care Technologies.}, journal = {BME frontiers}, volume = {6}, number = {}, pages = {0114}, pmid = {40110345}, issn = {2765-8031}, abstract = {Human papillomavirus (HPV) is the most common virus for genital tract infections. Cervical cancer ranks as the fourth most prevalent cancer globally, with over 99% of cases in women attributed to HPV infection. This infection continues to pose an ongoing threat to public health. Therefore, the development of rapid, high-throughput, and sensitive HPV detection platforms is important, especially in regions with limited access to advanced medical resources. CRISPR-based biosensors, a promising new method for nucleic acid detection, are now rapidly and widely used in basic and applied research and have received much attention in recent years for HPV diagnosis and treatment. In this review, we discuss the mechanisms and functions of the CRISPR-Cas system, focusing on its applications in HPV diagnostics. The review covers CRISPR technologies such as CRISPR-Cas9, CRISPR-Cas12, and CRISPR-Cas13, along with nucleic acid amplification methods, CRISPR-based signal output systems, and point-of-care testing (POCT) strategies. This comprehensive overview highlights the versatility and potential of CRISPR technologies in HPV detection. We also discuss the numerous CRISPR biosensors developed since the introduction of CRISPR to detect HPV. Finally, we discuss some of the challenges faced in HPV detection by the CRISPR-Cas system.}, }
@article {pmid40108777, year = {2025}, author = {Ren, C and Bao, Z}, title = {Assessment of Miniature AsCas12f1 Variants for Gene Editing and Activation.}, journal = {Biotechnology and bioengineering}, volume = {122}, number = {6}, pages = {1590-1597}, doi = {10.1002/bit.28978}, pmid = {40108777}, issn = {1097-0290}, support = {//This study was supported by the National Key R&amp;D Program of China (2023YFF1204500), the National Natural Science Foundation of China (22308316), and Fundamental Research Funds for the Central Universities (226-2022-00214 and 226-2023-00085)./ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Bacterial Proteins/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; *Transcriptional Activation ; }, abstract = {Miniature CRISPR/Cas systems possess delivery advantages for gene therapy. The type V-F Cas12f1 from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been engineered by several studies as compact genome editing tools through protein and single guide RNA (sgRNA) engineering. However, a comparative evaluation of gene editing and activation efficiencies mediated by different AsCas12f1 variants and sgRNA scaffolds is lacking. This study tested combinations of four AsCas12f1 protein variants and six sgRNA scaffolds for their gene editing and transcription activation efficiencies. The protein variant AsCas12f1-HKRA performed the best in gene editing and activation when paired with sgRNA-en_v2.1 scaffold. Furthermore, we validated a super miniature gene activator by fusing a small activation domain to AsCas12f1-HKRA. Our findings recommend using AsCas12f1-HKRA and sgRNA-en_v2.1 for gene editing and activation applications.}, }
@article {pmid40108772, year = {2025}, author = {Xie, Z and Zhao, S and Deng, R and Tang, X and Feng, L and Xie, S and Pi, Y and Chen, M and Chang, K}, title = {Logic-Measurer: A Multienzyme-Assisted Ultrasensitive Circuit for Logical Detection of Exosomal MicroRNAs.}, journal = {ACS nano}, volume = {19}, number = {12}, pages = {12222-12236}, doi = {10.1021/acsnano.5c00258}, pmid = {40108772}, issn = {1936-086X}, mesh = {*MicroRNAs/analysis/genetics ; Humans ; *Exosomes/chemistry/genetics/metabolism ; Logic ; Breast Neoplasms/diagnosis/genetics ; CRISPR-Cas Systems ; Female ; Exodeoxyribonucleases ; }, abstract = {The logic profiling of exosomal microRNAs (miRNAs) offers broad potential applications in the accurate diagnosis and staging of cancer. However, the logical detection of low-abundance exosomal miRNAs in complex clinical samples remains challenging. This study introduces a logic analysis system termed "Measurer" (a multi-enzyme-assisted ultrasensitive circuit) that offers ultrasensitive and versatile method for detecting multiple exosomal miRNAs. The Logic-Measurer comprises three modules: a stem-loop hairpin-enhanced CRISPR/Cas13a, a polymerase-driven primer exchange reaction, and an exonuclease III-mediated fluorescence output. The efficient Logic-Measurer was switched by the faster rate of trans-cleavage activity of Cas13a due to its improved affinity for hairpin RNA structures. The mechanistic model of hairpin-enhanced CRISPR/Cas13a was confirmed by molecular dynamics simulations. The Logic-Measurer accurately detected exosomal miRNA-21 or miRNA-375 down to 2.1 and 4.4 fM, with superior specificity, and enabled in situ detection of miRNA-21 and miRNA-375 in as low as 1.4 × 10[2] particles/mL exosomes via membrane fusion. In addition, this method demonstrated 87.3 and 82.1% accuracy in the diagnosis and early detection of breast cancer, respectively, among a cohort of 315 individuals. Subsequent subgroup analysis further confirmed the method's ability to accurately differentiate estrogen receptor-positive patients from healthy individuals. Therefore, the Logic-Measurer offers valuable insights into the development of a CRISPR/Cas-based enhanced diagnostic platform and the next generation of diagnostic technology based on enzyme circuits.}, }
@article {pmid40108708, year = {2025}, author = {Kaeuferle, T and Zwermann, M and Stoll, N and Ferrada-Ernst, P and Jablonowski, L and Zeidler, R and Willier, S and Stenger, D and Yassin, A and Stripecke, R and Feuchtinger, T}, title = {All-in-one CRISPR/Cas-engineered glucocorticoid-receptor knock-out EBV-gp350-CAR knock-in T cells are potent and resistant to dexamethasone.}, journal = {Experimental hematology &amp; oncology}, volume = {14}, number = {1}, pages = {40}, pmid = {40108708}, issn = {2162-3619}, support = {TI07.003//German Centre for Infection Research (DZIF)/ ; AD01.902//German Centre for Infection Research (DZIF)/ ; TTU07.836//German Centre for Infection Research (DZIF)/ ; TTU07.815//German Centre for Infection Research (DZIF)/ ; 70114234//Deutsche Krebshilfe/ ; TRR338//LETSimmun CRC (SFB), Munich-Wuerzburg, Germany/ ; }, abstract = {BACKGROUND: Epstein-Barr virus (EBV) reactivation in immunocompromised patients and post-transplantation is associated with morbidity, mortality and with the onset of a variety of malignant diseases. Adoptive T-cell therapies have emerged as promising therapeutic options, but post-transplant immunosuppression jeopardizes the protective anti-EBV immune surveillance by adoptively transferred T cells.<br><br>METHODS: Using an all-in-one CRISPR/Cas-mediated approach, we inserted an anti-EBV (gp350) CAR into the T-cell receptor (TRAC) locus and simultaneously knocked-out the glucocorticoid receptor (GR) on a good manufacturing practice (GMP)-compatible platform.<br><br>RESULTS: CAR knock-in (CAR[KI]) was confirmed in primary human T cells on genetic and on protein level with a mean efficiency of 41%. With 83%, additional GR knock-out was highly efficient in CAR[KI] cells. On a functional level CAR[KI]GR[KO] T cells showed target-specific potency in terms of cytokine secretion patterns, proliferative capacity and cytotoxic activity against gp350-expressing target cells. Further, CAR[KI]GR[KO] T cells were insensitive to dexamethasone treatment and maintained T-cell functionality. In contrast, CAR[KI]GR[KO] T cells were sensitive to the GR-independent immunosuppressant cyclosporine A (CsA), thereby providing a rescue treatment for patients in case of safety issues.<br><br>CONCLUSIONS: The study lays the proof-of-concept for virus-free all-in-one GMP-manufacturing of glucocorticoid-resistant CAR T-cell products. Further, the glucocorticoid-resistant gp350-CAR T cells can provide a future therapeutic option for high-risk post-transplant patients with EBV-reactivations or patients with EBV-associated pathologies requiring steroid treatment.}, }
@article {pmid40108474, year = {2025}, author = {Yuan, S and Sun, R and Shi, H and Chapman, NM and Hu, H and Guy, C and Rankin, S and Kc, A and Palacios, G and Meng, X and Sun, X and Zhou, P and Yang, X and Gottschalk, S and Chi, H}, title = {VDAC2 loss elicits tumour destruction and inflammation for cancer therapy.}, journal = {Nature}, volume = {640}, number = {8060}, pages = {1062-1071}, pmid = {40108474}, issn = {1476-4687}, support = {R01 AI131703/AI/NIAID NIH HHS/United States ; R01 AI150514/AI/NIAID NIH HHS/United States ; R37 AI105887/AI/NIAID NIH HHS/United States ; P30 CA021765/CA/NCI NIH HHS/United States ; U01 CA281868/CA/NCI NIH HHS/United States ; R35 CA253188/CA/NCI NIH HHS/United States ; R01 AI150241/AI/NIAID NIH HHS/United States ; R01 AI140761/AI/NIAID NIH HHS/United States ; R01 AI105887/AI/NIAID NIH HHS/United States ; }, mesh = {Animals ; Female ; Humans ; Male ; Mice ; bcl-2 Homologous Antagonist-Killer Protein/metabolism ; BH3 Interacting Domain Death Agonist Protein/metabolism ; CD8-Positive T-Lymphocytes/immunology ; Cell Line, Tumor ; CRISPR-Cas Systems/genetics ; *Immunotherapy ; *Inflammation/immunology/pathology/genetics ; Interferon-gamma/immunology/pharmacology ; Membrane Proteins/metabolism ; Mice, Inbred C57BL ; Mitochondria/metabolism/pathology ; *Neoplasms/immunology/therapy/pathology/genetics ; Nucleotidyltransferases/metabolism ; Signal Transduction ; Tumor Microenvironment ; *Voltage-Dependent Anion Channel 2/deficiency/genetics/metabolism ; }, abstract = {Tumour cells often evade immune pressure exerted by CD8[+] T cells or immunotherapies through mechanisms that are largely unclear[1,2]. Here, using complementary in vivo and in vitro CRISPR-Cas9 genetic screens to target metabolic factors, we established voltage-dependent anion channel 2 (VDAC2) as an immune signal-dependent checkpoint that curtails interferon-γ (IFNγ)-mediated tumour destruction and inflammatory reprogramming of the tumour microenvironment. Targeting VDAC2 in tumour cells enabled IFNγ-induced cell death and cGAS-STING activation, and markedly improved anti-tumour effects and immunotherapeutic responses. Using a genome-scale genetic interaction screen, we identified BAK as the mediator of VDAC2-deficiency-induced effects. Mechanistically, IFNγ stimulation increased BIM, BID and BAK expression, with VDAC2 deficiency eliciting uncontrolled IFNγ-induced BAK activation and mitochondrial damage. Consequently, mitochondrial DNA was aberrantly released into the cytosol and triggered robust activation of cGAS-STING signalling and type I IFN response. Importantly, co-deletion of STING signalling components dampened the therapeutic effects of VDAC2 depletion in tumour cells, suggesting that targeting VDAC2 integrates CD8[+] T cell- and IFNγ-mediated adaptive immunity with a tumour-intrinsic innate immune-like response. Together, our findings reveal VDAC2 as a dual-action target to overcome tumour immune evasion and establish the importance of coordinately destructing and inflaming tumours to enable efficacious cancer immunotherapy.}, }
@article {pmid40107560, year = {2025}, author = {Xiong, SS and Wang, XR and Han, KK and Tang, JQ and Lu, WT and Kong, XY and Fan, R and Sun, XL and Ji, YL and Wu, K and Guo, DD and Wan, Z and Xun, JN and Jiao, S and Zhai, H}, title = {Creation of dual-purpose soybean germplasm for grain and forage by CRISPR/Cas9-mediated targeting mutation of GmFT2a and GmFT5a.}, journal = {International journal of biological macromolecules}, volume = {307}, number = {Pt 4}, pages = {142234}, doi = {10.1016/j.ijbiomac.2025.142234}, pmid = {40107560}, issn = {1879-0003}, mesh = {*Glycine max/genetics/growth &amp; development ; *CRISPR-Cas Systems/genetics ; *Mutation ; *Plant Proteins/genetics ; *Seeds/genetics/growth &amp; development ; *Edible Grain/genetics ; Gene Editing ; Gene Expression Regulation, Plant ; Plants, Genetically Modified/genetics ; }, abstract = {Soybean [Glycine max (L.) Merr.] seeds are a rich source of high-quality protein and edible oil, and their foliage supports the rearing of the economically significant insect, 'Doudan'. This study utilized CRISPR/Cas9 to edit the GmFT2a and GmFT5a genes, resulting in soybean germplasm with enhanced grain and forage traits. Under short-day conditions, the double mutant ft2a ft5a showed significant increases in grain yield and yield-related traits, including main stem nodes, branching, and pod count per plant. Under long-day conditions, the double mutant exhibited a substantial increase in total vegetative biomass, with prolonged vegetative growth, larger leaves, and increased branching and nodes. The soluble protein and soluble sugar contents remained unchanged, while phenylalanine levels increased and tannin content decreased, which is beneficial for Doudan feeding. The ft2a ft5a double mutant is suitable for both grain and forage soybeans. We further revealed the molecular basis for the influence of GmFT2a and GmFT5a on soybean architecture. GmFT2a and GmFT5a proteins interact with each other and with Dt2 and SOC1a critical genes for branching and stem growth in soybeans. This interaction forms a complex that potently activates the downstream GmAp1s gene expression. The simultaneous mutation of GmFT2a and GmFT5a significantly downregulates GmAp1s expression, impacting soybean plant architecture. Overall, this study not only identifies the dual role of GmFT2a and GmFT5a in soybean yield and biomass but also uncovers their molecular interactions with Dt2 and SOC1a, providing a foundation for the genetic enhancement of soybean varieties tailored for both grain and forage production, underscoring the potential for improving agricultural sustainability and economic value.}, }
@article {pmid40107222, year = {2025}, author = {Yatera, K and Nishida, C and Mukae, H}, title = {Up-to-date nucleic acid assays for diagnosing respiratory infection.}, journal = {Respiratory investigation}, volume = {63}, number = {3}, pages = {383-393}, doi = {10.1016/j.resinv.2025.03.004}, pmid = {40107222}, issn = {2212-5353}, mesh = {Humans ; *COVID-19/diagnosis ; *Respiratory Tract Infections/diagnosis ; Point-of-Care Testing ; *COVID-19 Nucleic Acid Testing/methods ; SARS-CoV-2 ; Point-of-Care Systems ; Nucleic Acid Amplification Techniques/methods ; Molecular Diagnostic Techniques/methods ; }, abstract = {Nucleic acid assays have been widely used as rapid tests for diagnosing respiratory infections during and after the coronavirus disease 2019 (COVID-19) pandemic. An ideal point-of-care diagnostic must be affordable, sensitive, specific, user-friendly, rapid/robust, equipment-free and deliverable (ASSURED), and in addition to improvements to conventional methods based on polymerase chain reaction (PCR), point-of-care testing aiming for "REASSURED" are emerging through integration with microfluidic technology. Compared to conventional immunoassays, nucleic acid assays, especially rapid nucleic acid assays as point-of-care testing, contribute to improvements in various clinical outcomes, such as diagnostic yield, turnaround time, length of hospital stay, disease treatment, and infection control management. Rapid and diverse development of new nucleic acid-based molecular diagnostic technologies, such as those based on the CRISPR/Cas system or biosensor nucleic acid assays, is expected to become increasingly diverse in the future as point-of-care testing. In addition, laboratory-based DNA sequencing technology has been used to perform microbiome analyses over a wide area and is expected to shed light on the pathological mechanisms of various respiratory infectious diseases. One example of the benefits of nucleic acid amplification analysis methods is their ability to reveal the true nature of the bacterial flora in pneumonia lesions. This has been demonstrated based on the results of 16S ribosomal RNA gene sequencing analyses using bronchoalveolar lavage fluid directly obtained from pneumonia lesions in patients with pneumonia.}, }
@article {pmid40106574, year = {2025}, author = {Wiggers, CRM and Yüzügüldü, B and Tadros, NG and Heavican-Foral, TB and Cho, EY and Eisenbies, ZC and Ozdemir, M and Kulp, SB and Chae, YC and Gutierrez, A and Lohr, JG and Knoechel, B}, title = {Genome-wide CRISPR screen identifies IRF1 and TFAP4 as transcriptional regulators of Galectin-9 in T cell acute lymphoblastic leukemia.}, journal = {Science advances}, volume = {11}, number = {12}, pages = {eads8351}, pmid = {40106574}, issn = {2375-2548}, mesh = {Humans ; *Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics/metabolism/pathology ; *Galectins/genetics/metabolism ; *Interferon Regulatory Factor-1/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; *Gene Expression Regulation, Leukemic ; Promoter Regions, Genetic ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Enhancer Elements, Genetic ; Protein Binding ; Epigenesis, Genetic ; }, abstract = {Galectin-9 is overexpressed in a variety of cancers and associated with worse clinical outcome in some cancers. However, the regulators driving Galectin-9 expression are unknown. Here, we defined the transcriptional regulators and epigenetic circuitry of Galectin-9 in pediatric T cell acute lymphoblastic leukemia (T-ALL), as an example of a disease with strong Galectin-9 expression, in which higher expression was associated with lower overall survival. By performing a genome-wide CRISPR screen, we identified the transcription factors IRF1 and TFAP4 as key regulators for Galectin-9 expression by binding its regulatory elements. Whereas IRF1 was observed exclusively on the promoter, TFAP4 binding was detected at an enhancer solely in T-ALL cells associated with higher Galectin-9 levels. Together, our results show that IRF1 is responsible and indispensable for Galectin-9 expression and TFAP4 further fine-tunes its expression. Our approach, a flow-based genome-wide CRISPR screen complemented by transcription factor binding and enhancer mapping, creates innovative opportunities for understanding and manipulating epigenetic transcriptional regulation in cancer.}, }
@article {pmid40105951, year = {2025}, author = {Yang, P and Feng, J and Chen, J}, title = {Engineered S. cerevisiae construction for high-gravity ethanol production and targeted metabolomics.}, journal = {Applied microbiology and biotechnology}, volume = {109}, number = {1}, pages = {67}, pmid = {40105951}, issn = {1432-0614}, support = {2022047//Hefei Municipal Natural Science Foundation/ ; }, mesh = {*Ethanol/metabolism ; *Saccharomyces cerevisiae/genetics/metabolism ; Fermentation ; *Metabolic Engineering/methods ; *Metabolomics ; CRISPR-Cas Systems ; Hypergravity ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; Gene Knockout Techniques ; Sucrose/metabolism ; Gene Deletion ; Energy Metabolism ; }, abstract = {Strong sugar tolerance and high bioethanol yield of yeast under high-gravity fermentation have caused great attention in the bioethanol industry. In this study, Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR-Cas9) technology was used to knock out S. cerevisiae GPD2, FPS1, ADH2, DLD3, ERG5, NTH1, and AMS1 to construct engineering strain S. cerevisiae GFADENA. Under high-gravity fermentation with 400 g/L of sucrose, S. cerevisiae GFADENA produced 135 g/L ethanol, which increased 17% compared with the wild-type strain. In addition, S. cerevisiae GFADENA produced 145 g/L of ethanol by simultaneous saccharification and fermentation (SSF) using 400 g/L of corn syrup with a sugar-ethanol conversion rate of 41.1%. Further, the targeted metabolomics involving energy, amino acid, and free fatty acid metabolisms were performed to unravel its molecular mechanisms. The deletion of seven genes in S. cerevisiae GFADENA caused a more significant effect on energy metabolism compared with amino acid and free fatty acid metabolisms based on the significantly different metabolites. Two metabolites α-ketoglutaric acid and fructose-1,6-bisphosphate were the most significantly different upregulation and downregulation metabolites, respectively (p < 0.05). Functions of metabolism, environmental information processing, and genetic information processing were related to sucrose tolerance enhancement and ethanol production increase in S. cerevisiae GFADENA by the regulation of significantly different metabolites. This study provided an effective pathway to increase ethanol yield and enhance sucrose tolerance in S. cerevisiae through bioengineering modification. KEY POINTS: • S. cerevisiae GFADENA with gene deletion was constructed by the CRISPR-Cas9 approach • S. cerevisiae GFADENA could produce ethanol using high-gravity fermentation condition • The ethanol yield of 145 g/L was produced using 400 g/L corn syrup by the SSF method.}, }
@article {pmid40104595, year = {2025}, author = {Galanis, A and Papadimitriou, K and Moloney, GM}, title = {Editorial: Omics technologies and bioinformatic tools in probiotic research.}, journal = {Frontiers in microbiology}, volume = {16}, number = {}, pages = {1577852}, pmid = {40104595}, issn = {1664-302X}, }
@article {pmid40103259, year = {2025}, author = {Fonseca, A and Ishoey, T and Espinoza, C and Marshall, IPG and Nielsen, LP and Gallardo, VA}, title = {Large Filamentous Bacteria Isolated From Sulphidic Sediments Reveal Novel Species and Distinct Energy and Defence Mechanisms for Survival.}, journal = {Environmental microbiology}, volume = {27}, number = {3}, pages = {e70083}, pmid = {40103259}, issn = {1462-2920}, support = {//the Program "Doctorado en el extranjero Becas Chile" from ANID (former CONICYT)/ ; 1070552//Agencia Nacional de Investigaci&#xf3;n y Desarrollo/ ; //J. Craig Venter Institute/ ; }, mesh = {*Geologic Sediments/microbiology ; Phylogeny ; Chile ; *Bacteria/isolation &amp; purification/genetics/classification/metabolism ; Genome, Bacterial ; RNA, Ribosomal, 16S/genetics ; Deltaproteobacteria/genetics/isolation &amp; purification/classification ; Whole Genome Sequencing ; }, abstract = {Various morphotypes of large filamentous bacteria were isolated through micromanipulation from sulphidic sediment mats in the Bay of Concepción, central Chile. This study employed DNA amplification, whole-genome sequencing and bioinformatics analyses to unveil the taxonomic and genomic features of previously unidentified bacteria. The results revealed several novel genera, families and species, including three specimens belonging to Beggiatoales (Beggiatoaceae family), five to Desulfobacterales (Desulfobacteraceae family), two to the Chloroflexi phylum and one to the phylum Firmicutes. Metabolically, Beggiatoaceae bacteria exhibit a flexible and versatile genomic repertoire, enabling them to adapt to variable conditions at the sediment-water interface. All the bacteria demonstrated a mixotrophic mode, gaining energy from both inorganic and organic carbon sources. Except for the Firmicutes bacterium, all others displayed the ability to grow chemolithoautotrophically using H2 and CO2. Remarkably, the reverse tricarboxylic acid (rTCA) and Calvin-Benson-Bassham (CBB) pathways coexisted in one Beggiatoaceae bacterium. Additionally, various defence systems, such as CRISPR-Cas, along with evidence of viral interactions, have been identified. These defence mechanisms suggest that large filamentous bacteria inhabiting sulphidic sediments frequently encounter bacteriophages. Thus, robust defence mechanisms coupled with multicellularity may determine the survival or death of these large bacteria.}, }
@article {pmid40103232, year = {2025}, author = {Lin, SJ and Huang, K and Petree, C and Qin, W and Varshney, P and Varshney, GK}, title = {Optimizing gRNA selection for high-penetrance F0 CRISPR screening for interrogating disease gene function.}, journal = {Nucleic acids research}, volume = {53}, number = {5}, pages = {}, pmid = {40103232}, issn = {1362-4962}, support = {R21DC020317//US National Institutes of Health/ ; //Oklahoma Medical Research Foundation/ ; }, mesh = {Animals ; Zebrafish/genetics ; *Penetrance ; Humans ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Knockout Techniques/methods ; *Gene Editing/methods ; Phenotype ; *Genetic Testing/methods ; Epistasis, Genetic ; }, abstract = {Genes and genetic variants associated with human disease are continually being discovered, but validating their causative roles and mechanisms remains a significant challenge. CRISPR/Cas9 genome editing in model organisms like zebrafish can enable phenotypic characterization of founder generation (F0) knockouts (Crispants), but existing approaches are not amenable to high-throughput genetic screening due to high variability, cost, and low phenotype penetrance. To overcome these challenges, here we provide guide RNA (gRNA) selection rules that enable high phenotypic penetrance of up to three simultaneous knockouts in F0 animals following injection of 1-2 gRNAs per gene. We demonstrate a strong transcriptomic overlap in our F0 knockouts and stable knockout lines that take several months to generate. We systematically evaluated this approach across 324 gRNAs targeting 125 genes and demonstrated its utility in studying epistasis, characterizing paralogous genes, and validating human disease gene phenotypes across multiple tissues. Applying our approach in a high-throughput manner, we screened and identified 10 novel neurodevelopmental disorders and 50 hearing genes not previously studied in zebrafish. Altogether, our approach achieves high phenotypic penetrance using low numbers of gRNAs per gene in F0 zebrafish, offering a robust pipeline for rapidly characterizing candidate human disease genes.}, }
@article {pmid40103228, year = {2025}, author = {Tang, H and Han, S and Jie, Y and Jiang, X and Zhang, Y and Peng, J and Wang, F and Li, X and Zhou, X and Jiang, W and Weng, X}, title = {Enhanced or reversible RNA N6-methyladenosine editing by red/far-red light induction.}, journal = {Nucleic acids research}, volume = {53}, number = {5}, pages = {}, pmid = {40103228}, issn = {1362-4962}, support = {2023YFC3402200//Ministry of Science and Technology/ ; 92253202//National Natural Science Foundation of China/ ; 2042023kfyq05//Fundamental Research Funds for the Central Universities/ ; }, mesh = {*Adenosine/analogs &amp; derivatives/metabolism/genetics ; Humans ; *RNA Editing/radiation effects ; CRISPR-Cas Systems ; *Light ; HEK293 Cells ; Human Embryonic Stem Cells/metabolism ; Optogenetics/methods ; RNA, Messenger/genetics/metabolism ; *Gene Editing/methods ; Cell Differentiation/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; SOXB1 Transcription Factors/genetics/metabolism ; Red Light ; }, abstract = {The RNA N6-methyladenosine (m6A) modification is a critical regulator of various biological processes, but precise and dynamic control of m6A remains a challenge. In this work, we present a red/far-red light-inducible m6A editing system that enables efficient and reversible modulation of m6A levels with minimal off-target effects. By engineering the CRISPR dCas13 protein and sgRNA with two pairs of light-inducible heterodimerizing proteins, ΔphyA/FHY1 and Bphp1/PspR2, we achieved targeted recruitment of m6A effectors. This system significantly enhances m6A writing efficiency and allows dynamic regulation of m6A deposition and removal on specific transcripts, such as SOX2 and ACTB. Notably, reversible m6A editing was achieved through cyclic modulation at a single target site, demonstrating the ability to influence mRNA expression and modulate the differentiation state of human embryonic stem cells. This optogenetic platform offers a precise, versatile tool for cyclic and reversible m6A regulation, with broad implications for understanding RNA biology and its potential applications in research and medicine.}, }
@article {pmid40103227, year = {2025}, author = {Finocchio, G and Querques, I and Chanez, C and Speichert, KJ and Jinek, M}, title = {Structural basis of TnsC oligomerization and transposase recruitment in type I-B CRISPR-associated transposons.}, journal = {Nucleic acids research}, volume = {53}, number = {5}, pages = {}, pmid = {40103227}, issn = {1362-4962}, support = {ERC-CoG-820152/ERC_/European Research Council/International ; /HHMI/Howard Hughes Medical Institute/United States ; ALTF 296-2020//EMBO/ ; //University of Zurich/ ; }, mesh = {*Transposases/chemistry/metabolism/genetics/ultrastructure ; *DNA Transposable Elements/genetics ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; Protein Multimerization ; *Bacterial Proteins/chemistry/metabolism/genetics ; Models, Molecular ; Protein Binding ; }, abstract = {CRISPR-associated transposon (CAST) systems employ CRISPR-Cas systems as RNA-directed targeting modules for site-specific transposon DNA insertion. Among them, type I CASTs rely on the coordinated action of the guide RNA-bound Cascade complex and the transposon proteins TniQ, TnsC, and TnsAB. The interaction between the transposase TnsAB and the ATPase TnsC is crucial for transposition activity, yet the underlying molecular details have remained elusive. Here, we investigate the type I-B CAST system from Peltigera membranacea cyanobiont. Cryo-electron microscopic structures of TnsC and its complex with the C-terminal region of TnsAB reveal that TnsC forms a heptameric ring that recruits TnsAB by interacting with its C-terminal tail. In vitro binding assays indicate that TnsAB exclusively interacts with the TnsC heptamer without inducing its disassembly, in contrast to type V-K CAST systems. Mutational analysis of key structural features corroborates the significance of TnsC multimerization and TnsB interaction for transposon activity in vivo. Altogether, these findings offer detailed structural and functional insights into the molecular mechanism of type I-B CAST, with the aim of facilitating their development as genome engineering tools.}, }
@article {pmid40102632, year = {2025}, author = {Salman, A and Song, WK and Storm, T and McClements, ME and MacLaren, RE}, title = {CRISPR targeting of SNPs associated with age-related macular degeneration in ARPE-19 cells: a potential model for manipulating the complement system.}, journal = {Gene therapy}, volume = {32}, number = {2}, pages = {132-141}, pmid = {40102632}, issn = {1476-5462}, mesh = {Humans ; *Polymorphism, Single Nucleotide ; *Macular Degeneration/genetics/therapy ; *CRISPR-Cas Systems ; Retinal Pigment Epithelium/metabolism/cytology ; Cell Line ; Gene Editing/methods ; *Complement System Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Alleles ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Age-related Macular degeneration (AMD) is a major cause of vision loss and is linked to several predisposing single nucleotide polymorphisms (SNPs). CRISPR-mediated genome editing offers the potential to target negatively associated SNPs in an allele-specific manner, necessitating the need for a relevant cell model. The ARPE-19 cell line, with its stable monolayer growth and retinal pigment epithelium (RPE) characteristics, serves as an ideal model for AMD studies. Chronic inflammation and complement system dysregulation are implicated in AMD pathogenesis. Most genetic variations associated with AMD are in complement genes, suggesting their regulatory role. In this study, we conducted targeted PCRs to identify AMD-related SNPs in ARPE-19 cells and used CRISPR constructs to assess allele-specific activity. Guide RNA sequences were cloned into an EF-1-driven SpCas9 vector and packaged into lentivirus. Targeting efficiencies were evaluated with TIDE analysis, and allele-specificity was measured with NGS analysis 30 days post-transduction. Our results showed varying targeting efficiencies depending on guide RNA efficacy. For example, TIDE analysis of CFH SNPs rs1061170 and rs1410996 revealed efficiencies of 35.5% and 33.8%, respectively. CFB SNP rs4541862 showed efficiencies from 3% to 36.7%, and rs641153 ranged from 3.4% to 23.8%. Additionally, allele-specific targeting of AMD-related SNPs rs1061170, rs1410996, rs4541862, and rs641153 ranged from 48% to 52% in heterozygous differentiated ARPE-19 cells. These findings demonstrate the potential to manipulate the complement system in an AMD model by targeting disease-associated SNPs in an allele-specific manner, offering a promising therapeutic approach.}, }
@article {pmid40102626, year = {2025}, author = {Gürhan, G and Sevinç, K and Aztekin, C and Gayretli, M and Yılmaz, A and Yıldız, AB and Ervatan, EN and Morova, T and Datlı, E and Coleman, OD and Kawamura, A and Lack, NA and Syed, H and Önder, T}, title = {A chromatin-focused CRISPR screen identifies USP22 as a barrier to somatic cell reprogramming.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {454}, pmid = {40102626}, issn = {2399-3642}, mesh = {*Cellular Reprogramming/genetics ; Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems ; *Chromatin/metabolism/genetics ; *Ubiquitin Thiolesterase/genetics/metabolism ; Fibroblasts/metabolism/cytology ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Cell-autonomous barriers to reprogramming somatic cells into induced pluripotent stem cells (iPSCs) remain poorly understood. Using a focused CRISPR-Cas9 screen, we identified Ubiquitin-specific peptidase 22 (USP22) as a key chromatin-based barrier to human iPSC derivation. Suppression of USP22 significantly enhances reprogramming efficiency. Surprisingly, this effect is likely to be independent of USP22's deubiquitinase activity or its association with the SAGA complex, as shown through module-specific knockouts, and genetic rescue experiments. USP22 is not required for iPSC derivation or maintenance. Mechanistically, USP22 loss during reprogramming downregulates fibroblast-specific genes while activating pluripotency-associated genes, including DNMT3L, LIN28A, SOX2, and GDF3. Additionally, USP22 loss enhances reprogramming efficiency under naïve stem cell conditions. These findings reveal an unrecognized role for USP22 in maintaining somatic cell identity and repressing pluripotency genes, highlighting its potential as a target to improve reprogramming efficiency.}, }
@article {pmid40102313, year = {2025}, author = {Fan, R and Luo, S and He, Y and Xiao, Y and Liang, Y and Zhang, L and Li, W and Zhang, Y and Li, L}, title = {Simple and sensitive SERS platform for Staphylococcus aureus one-pot determination by photoactivated CRISPR/Cas12a cascade system and core-shell DNA tetrahedron@AuNP@Fe3O4 reporter.}, journal = {Mikrochimica acta}, volume = {192}, number = {4}, pages = {240}, pmid = {40102313}, issn = {1436-5073}, mesh = {*Staphylococcus aureus/isolation &amp; purification ; *CRISPR-Cas Systems ; *Spectrum Analysis, Raman/methods ; Gold/chemistry/radiation effects ; *Metal Nanoparticles/chemistry/radiation effects ; Limit of Detection ; Aptamers, Nucleotide/chemistry ; Humans ; Ferrosoferric Oxide/chemistry ; Biosensing Techniques/methods ; *DNA/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Staphylococcus aureus (S. aureus) is a widely prevalent Gram-positive bacteria that can cause serious infections and diseases in humans and other organisms. Timely detection and treatment in clinical settings is crucial for patient safety and public health. However, current methods for S. aureus detection still face some limitations, such as time-consuming operation, false positives, and labor-intensive available methodology with low sensitivity. Therefore, it is particularly important to develop a rapid, simple, sensitive, and cost-effective method for detecting S. aureus. We developed a SERS platform based on allosteric aptamer-triggered catalytic hairpin assembly (CHA) and photoactivated CRISPR/Cas12a reactions, combined with a multifunctional core-shell structure as the SERS reporter, enabling highly sensitive one-pot determination of S. aureus. Compared with traditional two-step and one-pot analysis methods, this strategy offers superior sensitivity and can successfully identify real samples contaminated with S. aureus. The platform utilizes light-controlled CHA and CRISPR/Cas12a reactions, effectively preventing interference between different reaction systems. Therefore, the photoactivated one-pot CHA/Cas12a strategy provides a simple, rapid, highly sensitive, specific, and cost-effective method for one-pot determination of S. aureus in clinical samples.}, }
@article {pmid40100800, year = {2025}, author = {Wu, Y and Shao, Y and Li, W and Yu, Y and Rao, X and Li, J and Waterfield, NR and Yang, G}, title = {Establishment of an RPA-CRISPR/Cas12a combined diagnostic system for Pneumocystis jirovecii pneumonia.}, journal = {PLoS neglected tropical diseases}, volume = {19}, number = {3}, pages = {e0012922}, pmid = {40100800}, issn = {1935-2735}, mesh = {Humans ; *Pneumocystis carinii/genetics/isolation &amp; purification ; *Pneumonia, Pneumocystis/diagnosis/microbiology ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Recombinases/genetics/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Pneumocystis jirovecii causes severe pneumonia in immunocompromised individuals, leading to high mortality and an economic burden. There is a need for early detection methods suitable for low-resource settings and rapid point-of-care diagnostics. This study developed a detection method using Recombinase Polymerase Amplification (RPA) followed by CRISPR/Cas12a with fluorescence detection. The RPA primers and CRISPR-derived RNAs (crRNAs) were specifically designed to target the mitochondrial small subunit rRNA (mtSSU rRNA) gene of P. jirovecii. A total of 83 clinical samples were tested using this method, including 39 confirmed and 44 suspected cases of P. jirovecii infection. The combination of crRNA5 and crRNA6 demonstrated higher sensitivity compared to the current real-time PCR detection method, with a limit of detection (LOD) of 1 copy per reaction and showed no cross-reactions with other respiratory pathogens. The concordance of this method was validated with both infected and non-infected patients. In conclusion, the method developed in this study potentially provides a highly sensitive and rapid tool suitable for the early and on-site detection of P. jirovecii pneumonia. Furthermore, this method holds potential applications for the detection of other human pathogens, representing a significant advancement in diagnostic capabilities for low-resource settings.}, }
@article {pmid40100209, year = {2025}, author = {Cheng, L}, title = {Chemical Strategies to Modulate and Manipulate RNA Epigenetic Modifications.}, journal = {Accounts of chemical research}, volume = {58}, number = {11}, pages = {1727-1741}, doi = {10.1021/acs.accounts.4c00844}, pmid = {40100209}, issn = {1520-4898}, mesh = {*Epigenesis, Genetic ; *RNA/genetics/chemistry/metabolism ; Humans ; }, abstract = {ConspectusRNA epigenetics has rapidly emerged as a key frontier in chemical biology, revealing that modifications to RNA bases and riboses can fine-tune essential cellular processes such as gene expression, translation, and metabolic homeostasis. Traditionally, researchers have relied on manipulating the "writers," "erasers," and "readers" of RNA modifications─i.e., protein cofactors─to alter and study these marks. Those enzyme-centric strategies, including small molecule inhibitors and CRISPR/Cas-based genetic perturbations, have been highly effective and are advancing in clinical applications. However, purely chemical approaches for installing, removing, or transforming RNA modifications without enzyme disturbance have offered distinct advantages, such as temporal control, reversibility, and bypassing compensatory biological feedback mechanisms that often arise with genetic or enzymatic inhibition. Every chemist should be concerned about RNA modifications, because they represent a striking intersection of molecular recognition, organic transformation, and cellular function. The ability to direct chemical reactivity at specific nucleosides in RNA can illuminate how individual modifications impact the overall gene regulation. Further, since improper RNA modification and damage patterns are implicated in cancer, metabolic disorders, and neurodegeneration, these chemical repair tools have potential as diagnostic and therapeutic interventions. Beyond medicine, agriculture also stands to benefit from chemical control of nucleoside-based plant hormones, possibly leading to improved crop productivity and resilience.In this Account, we outline several innovative chemical strategies tailored to different classes of RNA modifications. Flavin-based bioorthogonal chemistry has enabled demethylation of N[6]-methyladenosine (m[6]A) independent of endogenous demethylases, while oxidative bioorthogonal reactions can convert 5-methylcytidine (m[5]C) into distinct formyl derivatives for labeling and sequencing. Nitrogen-oxide and photochemical routes provided access for the selective removal of the side chain of N[6]-isopentenyladenosine (i[6]A), offering insights for both cell biology and plant hormone research. We also showcase how rationally designed small molecules can rewire complex RNA damage repair pathways, facilitating selective correction of vinyl-adduct lesions otherwise resistant to enzymatic repair. These purely chemical methods bypass the constraints of enzyme dependence, affording temporal precision (e.g., via light activation) and site-selective modification or labeling of RNA. By strategically engineering reactivity, we have uncovered new epitranscriptomic phenomena, such as in situ generation of non-native RNA modification, that offer fresh capabilities for cell imaging or targeted manipulation of plant callus development. Together, these discoveries signal a paradigm shift: chemical tools can complement or even surpass conventional enzyme-based methods for investigating, editing, and repairing RNA modifications. The ramifications are broad. Chemists can leverage these new reactivities to dissect the molecular underpinnings of diseases linked to epitranscriptomic dysregulation and to engineer next-generation therapeutic, diagnostic, and sequencing platforms. Plant biologists can apply the same chemical strategies to hone agronomic traits, from seed vigor to stress resilience. Ultimately, as we have deepened the mechanistic insights and refined reaction design for increased biocompatibility, purely chemical control of the RNA epigenome is poised to become one of the mainstream approaches across fields spanning chemistry, biology, and medicine─fostering deeper understanding of RNA's role in health and disease and opening new avenues for precise interventions.}, }
@article {pmid40099789, year = {2025}, author = {Huang, L and Zhou, Y and Feng, Y and Jia, S and Wang, S and Zhong, C}, title = {Tailoring Bacterial Cellulose through the CRISPR/Cas9-Mediated Gene Editing Tool in Komagataeibacter xylinus.}, journal = {ACS synthetic biology}, volume = {14}, number = {4}, pages = {1161-1172}, doi = {10.1021/acssynbio.4c00785}, pmid = {40099789}, issn = {2161-5063}, mesh = {*Gene Editing/methods ; *Cellulose/genetics/biosynthesis/metabolism/chemistry ; *CRISPR-Cas Systems/genetics ; *Gluconacetobacter xylinus/genetics/metabolism ; Metabolic Engineering/methods ; *Acetobacteraceae/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; }, abstract = {Bacterial cellulose (BC) is a nanocellulose produced by bacteria, formed by glucose units linked through β-1,4 glycosidic bonds. It features a three-dimensional network structure, superior water retention capacity, high porosity, and outstanding biocompatibility, among other notable characteristics. Komagataeibacter xylinus was the predominant strain used for BC production. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associate-protein 9)-mediated gene editing tool has been applied in various species; however, its application in K. xylinus has not been reported. To facilitate metabolic pathway engineering in K. xylinus, a CRISPR/Cas9-mediated gene editing tool specific to this strain was developed, achieving a gene editing efficiency exceeding 73%. Upon application of the CRISPR/Cas9-mediated gene editing tool in K. xylinus, the strain's ability to synthesize BC was enhanced by 23.6% (5.75 g/L), and the impact of BC synthase-correlated genes (bcsH, bcsX, bcsY, and bcsZ) on BC structure was investigated. The advancement of CRISPR/Cas9-mediated gene editing tools in K. xylinus is expected to accelerate genetic modification of this organism. This advancement has the potential to significantly improve our understanding of the genetic regulatory mechanisms that govern the structure and production of BC, thereby facilitating cost-effective synthesis of BC with tailored structural properties.}, }
@article {pmid40099627, year = {2025}, author = {Li, Y and Ren, X and Wu, D and Ma, H and Wei, Q and Ju, H and Gao, Z}, title = {CRISPR/Cas12a-powered nanoconfined biosensing platform with hybrid chain reaction cascading guanine nanowire amplification for ultrasensitive dual-mode detection of lipopolysaccharide.}, journal = {The Analyst}, volume = {150}, number = {8}, pages = {1571-1577}, doi = {10.1039/d5an00184f}, pmid = {40099627}, issn = {1364-5528}, mesh = {*Nanowires/chemistry ; *Biosensing Techniques/methods ; *Lipopolysaccharides/analysis ; *CRISPR-Cas Systems ; Limit of Detection ; *Guanine/chemistry ; Electrochemical Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Silicon Dioxide/chemistry ; Luminescent Measurements/methods ; Humans ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Traditional endotoxin detection methods face challenges in sensitivity, interference resistance, and reliability. This study develops a CRISPR/Cas12a-powered nanoconfined biosensing system that integrates mesoporous nanoengineering with a hybrid chain reaction (HCR) cascading guanine nanowire (G-wire) dual amplification strategy for ultrasensitive dual-mode detection of lipopolysaccharide (LPS). By leveraging a vertically ordered mesoporous silica membrane (VMSM) as a molecular sieve and CRISPR trans-cleavage activity modulator, the system achieves precise regulation of Ru(bpy)3[2+] adsorption via LPS-suppressed HCR assembly. This architecture enables physical confinement-mediated electrochemiluminescence (ECL) and fluorescence (FL) signal transduction, with dual-mode outputs providing mutual validation for enhanced reliability. The biosensor exhibits superior sensitivity with detection limits of 3.4 pg mL[-1] for ECL and 1.4 pg mL[-1] for FL, while also offering a broad dynamic range (0.005-100 ng mL[-1]), significantly outperforming conventional LPS assays. The CRISPR-triggered HCR cascading G-wire dual amplification synergizes with nanoconfinement of VMSM to ensure robust anti-interference performance in complex matrices, validated by recovery rates of 97.8-102.5% in real samples. By integrating CRISPR programmability with nanoengineered signal amplification, this work establishes a transformative paradigm for portable, high-precision endotoxin detection in clinical diagnostics, industrial monitoring, and environmental safety applications.}, }
@article {pmid40099511, year = {2025}, author = {Wang, B and Yu, Z and Zhang, Z and Zhu, Z and Song, Y}, title = {A dCas9/sgRNA complex-mediated competitive assay for accurate and sensitive Pseudomonas aeruginosa analysis.}, journal = {Analytical methods : advancing methods and applications}, volume = {17}, number = {13}, pages = {2791-2798}, doi = {10.1039/d4ay02194k}, pmid = {40099511}, issn = {1759-9679}, mesh = {*Pseudomonas aeruginosa/genetics/isolation &amp; purification ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Pseudomonas Infections/microbiology/diagnosis ; *CRISPR-Cas Systems ; Humans ; Limit of Detection ; DNA, Bacterial/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; }, abstract = {Pseudomonas aeruginosa (P. aeruginosa), a Gram-negative pathogenic bacterium, is one of the most common bacteria that causes severe infectious diseases. However, accurate and efficient detection of P. aeruginosa in clinical samples is a huge challenge. Therefore, in this study, we developed a Cas9 derivative (dCas9)/sgRNA-mediated competitive assay for the sensitive and precise characterization of genomic materials from P. aeruginosa. Our approach involved the identification of target genomic sequences using the dCas9/sgRNA complex, which occupied the "sensing probe" (SP) binding site, resulting in an increased availability of free SP. SP subsequently facilitated DNA polymerase/endonuclease-mediated signal cycles and signal production, enabling highly sensitive detection of P. aeruginosa. The proposed competitive assay demonstrated a robust linear response to P. aeruginosa within a concentration range from 10 CFU mL[-1] to 10[6] CFU mL[-1], leveraging numerous signal amplification processes and competitive target recognition while exhibiting robust anti-interference capacity. Compared with former strategies, the proposed competitive assay enabled the accurate detection of P. aeruginosa by directly identifying and binding genomic sequences, which could be easily extended to the detection of other bacteria by simply changing the sgRNA. In addition, the proposed approach exhibits significant clinical potential for early disease diagnosis owing to its excellent sensitivity and accuracy.}, }
@article {pmid40099180, year = {2025}, author = {Pan, Z and Xu, L and Fan, Z and Ren, F}, title = {CRIPSR-Cas for hepatitis virus: a systematic review and meta-analysis of diagnostic test accuracy studies.}, journal = {Frontiers in microbiology}, volume = {16}, number = {}, pages = {1509890}, pmid = {40099180}, issn = {1664-302X}, abstract = {BACKGROUND AND AIMS: Hepatitis viruses pose a significant global health challenge, necessitating accurate and efficient diagnostic methods. The CRISPR-Cas system, renowned for gene editing, shows potential tool in virus detection. This systematic review and meta-analysis aims to evaluate the diagnostic accuracy of CRISPR-Cas-based tests for hepatitis viruses, aiming to provide evidence for their effectiveness in clinical settings.<br><br>METHODS: Studies from Web of Science, PubMed, and CNKI were analyzed. A bivariate random-effects model was employed to compute pooled estimates for sensitivity, specificity, and the area under the summary receiver operating characteristic (SROC) curve. Additionally, the methodological quality of the studies was evaluated using the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) tool.<br><br>RESULTS: Following a rigorous screening process, 14 studies meeting our inclusion criteria were selected from an initial pool of 657 studies. The pooled sensitivity and specificity of the CRISPR-Cas system in hepatitis virus detection showed high sensitivity (0.99, 95% CI: 0.95-1.00) and specificity (0.99, 95% CI: 0.93-1.00) with SROC area 1.00 (95% CI: 0.99-1.00). However, considering the notable heterogeneity among the included studies, subgroup analyses and meta-regression were conducted. These analyses revealed that the type of hepatitis virus detected and the format of the final result presentation could be potential sources of this heterogeneity.<br><br>CONCLUSION: This systematic review and meta-analysis demonstrates the high diagnostic accuracy of CRISPR-Cas system in detecting hepatitis viruses. However, conclusions are limited by study number and quality. Therefore, more high-quality data are still needed to support this conclusion.}, }
@article {pmid40098357, year = {2025}, author = {Chong, CSC and Lau, YY and Michels, PAM and Lim, CSY}, title = {Insights into biofilm-mediated mechanisms driving last-resort antibiotic resistance in clinical ESKAPE pathogens.}, journal = {Critical reviews in microbiology}, volume = {}, number = {}, pages = {1-26}, doi = {10.1080/1040841X.2025.2473332}, pmid = {40098357}, issn = {1549-7828}, abstract = {The rise of antibiotic-resistant bacteria poses a grave threat to global health, with the ESKAPE pathogens, which comprise Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. being among the most notorious. The World Health Organization has reserved a group of last-resort antibiotics for treating multidrug-resistant bacterial infections, including those caused by ESKAPE pathogens. This situation calls for a comprehensive understanding of the resistance mechanisms as it threatens public health and hinder progress toward the Sustainable Development Goal (SDG) 3: Good Health and Well-being. The present article reviews resistance mechanisms, focusing on emerging resistance mutations in multidrug-resistant ESKAPE pathogens, particularly against last-resort antibiotics, and describes the role of biofilm formation in multidrug-resistant ESKAPE pathogens. It discusses the latest therapeutic advances, including the use of antimicrobial peptides and CRISPR-Cas systems, and the modulation of quorum sensing and iron homeostasis, which offer promising strategies for countering resistance. The integration of CRISPR-based tools and biofilm-targeted approaches provides a potential framework for managing ESKAPE infections. By highlighting the spread of current resistance mutations and biofilm-targeted approaches, the review aims to contribute significantly to advancing our understanding and strategies in combatting this pressing global health challenge.}, }
@article {pmid40097588, year = {2025}, author = {Liu, J and Zhang, J and Zhao, T and Zhao, M and Su, M and Chen, Y and Huang, Z and Wang, Y and Zhong, C and Hu, Z and Zhou, P and Tian, R and He, D}, title = {SunTag-PE: a modular prime editing system enables versatile and efficient genome editing.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {452}, pmid = {40097588}, issn = {2399-3642}, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; CRISPR-Associated Protein 9/genetics/metabolism ; Dependovirus/genetics ; }, abstract = {Prime editing (PE) holds tremendous potential in the treatment of genetic diseases because it can install any desired base substitution or local insertion/deletion. However, the full-length PE effector size (6.3-kb) is beyond the packaging capacity of adeno-associated virus (AAV), hindering its clinical translation. Various splitting strategies have been used to improve its delivery, but always accompanied by compromised PE efficiency. Here, we developed a modular and efficient SunTag-PE system that splits PE effectors into GCN4-nCas9 and single-chain variable fragment (scFv) tethered reverse transcriptase (RT). We observed that SunTag-PEs with 1×GCN4 in the N terminus of nCas9 was the most efficient configuration rather than multiple copies of GCN4. This SunTag-PE strategy achieved editing levels comparable to canonical fused-PE (nCas9 and RT are linked together) and higher than other split-PE strategies (including sPE and MS2-PE) in both PE2 and PE3 forms with no increase in insertion and deletion (indel) byproducts. Moreover, we successfully validated the modularity of SunTag-PE system in the Cas9 orthologs of SauCas9 and FrCas9. Finally, we employed dual AAVs to deliver SunTag-ePE3 and efficiently corrected the pathogenic mutation in HBB mutant cell line. Collectively, our SunTag-PE system provides an efficient modular splitting strategy for prime editing and further facilitate its transformation in clinics.}, }
@article {pmid40097290, year = {2025}, author = {Zhong, J and Li, J and Chen, S and Xu, Y and Mao, X and Xu, M and Luo, S and Yang, Y and Zhou, J and Yuan, J and Su, L and Wang, G and Zhang, X and Li, X}, title = {Rapid and efficient CRISPR-based detection of dengue virus in a single-tube.}, journal = {Journal of applied microbiology}, volume = {136}, number = {4}, pages = {}, doi = {10.1093/jambio/lxaf070}, pmid = {40097290}, issn = {1365-2672}, support = {2024XZ011//Zhejiang Shuren University Basic Scientific Research Special Funds/ ; }, mesh = {*Dengue Virus/genetics/isolation &amp; purification ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Dengue/diagnosis/virology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Nucleic Acid Amplification Techniques/methods ; Humans ; RNA, Viral/genetics ; Limit of Detection ; }, abstract = {BACKGROUND: Dengue Virus (DENV) is prevalent in tropical and subtropical regions. With the projected climate change, traditional detection methods face limitations, and there is an urgent need for more accurate and efficient diagnostic techniques.<br><br>OBJECTIVE: The aim is to integrate Recombinase-aided Amplification (recombinase-aided amplification, RAA) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to establish a special CRISPR-Cas system for rapid, convenient, high sensitivity, and high specificity typing detection of DENV.<br><br>METHODS: CRISPR RNA (crRNA) and RAA primers were designed based on the whole genome of four DENV serotypes. A single-tube assay combining RAA with CRISPR-Cas13a technology was developed after optimizing reagent concentrations.<br><br>RESULTS: The limit of detection (LoD) of DENV types 1-4 was 10&#xb3; copies&#xb7;mL -1. No cross-reaction was observed between any of the DENV serotypes and the other three flaviviruses (Zika, West Nile, and Murray Valley encephalitis). The average sensitivity of one-step method was 95.8%, and the average specificity was 96.6%. Fluorescent signal intensities demonstrated a clear dose-dependent response, with the signal increasing as the sample concentration rose. This system can effectively distinguish non-target substances. Among them, one-step method has advantages in timeliness, ease of operation, and contamination control because it runs efficiently inside a tube and does not require the lid to be removed, but its sensitivity is relatively low. The two-steps method performs well in sensitivity.<br><br>CONCLUSION: In this study, we developed a novel method for rapid typing and detection of DENV using RAA and CRISPR-Cas13a in a single-tube homogeneous system.}, }
@article {pmid40097027, year = {2025}, author = {Ahmed, S and Azli, B and Abdul Razak, M and Hair-Bejo, M and Omar, AR and Ideris, A and Mat Isa, N}, title = {Delayed nuclear localization of CRISPR/Cas9-modified fiber of fowl adenovirus serotype 8b reduces pathogenicity in Specific pathogen-free chicken embryonic liver cells.}, journal = {Microbial pathogenesis}, volume = {203}, number = {}, pages = {107482}, doi = {10.1016/j.micpath.2025.107482}, pmid = {40097027}, issn = {1096-1208}, mesh = {Animals ; *Adenoviridae Infections/veterinary/prevention &amp; control/virology ; *Poultry Diseases/virology/prevention &amp; control ; Chick Embryo ; *CRISPR-Cas Systems ; *Aviadenovirus/genetics/pathogenicity/immunology ; Chickens ; Specific Pathogen-Free Organisms ; Serogroup ; *Liver/virology/cytology ; *Capsid Proteins/genetics/immunology/metabolism ; Virulence ; Vaccines, Attenuated/immunology/genetics ; Viral Vaccines/immunology/genetics ; *Cell Nucleus/virology ; Gene Editing ; Malaysia ; }, abstract = {Fowl adenovirus (FAdV) poses incessant outbreaks to poultry production worldwide, and Inclusion body hepatitis (IBH) is a predominant FAdV infectious disease. Currently, limited vaccines are available in Malaysia to fight against the local predominant FAdV strain 8b isolate (FAdV-8b), posing a desperate demand for efficient vaccine development. The fiber protein of FAdV is one of the major constituents of the adenoviral capsid involved in the virulence of pathogens. Hence, the aim was to modify the fiber gene of FAdV-8b UPMT27 to develop a live attenuated FAdV vaccine via the gene-editing CRISPR/Cas9 technology. Primary specific pathogen-free (SPF) chicken embryonic liver cells (CELs) infected with the modified isolated (cfUPMT27) were reported with significantly reduced cytopathic effects, delayed viral localization into the nucleus, and low apoptotic rates. cfUPMT27 isolate also exhibited constant amino acid substitution of Y179D in subsequent passages. Meanwhile, the liver of cfUPMT27 inoculated-SPF chicken embryonic eggs (CEE) was observed with mild hydropericardium and reported with a delayed mortality at 6-days post-infection (dpi). This holistic, integrative study incorporating genetic, pathology, and immunology analysis proposed cfUPMT27 isolate as a candidate vaccine for FAdV infections, providing efficient future protection in chickens.}, }
@article {pmid40096954, year = {2025}, author = {Zhong, Y and Yuan, P and Dai, L and Yang, L and Xu, Z and Chen, D}, title = {First report of blaVEB-3 and blaKPC-2 coexistence with a novel blaKPC-2 transposon in Klebsiella michiganensis.}, journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases}, volume = {130}, number = {}, pages = {105740}, doi = {10.1016/j.meegid.2025.105740}, pmid = {40096954}, issn = {1567-7257}, mesh = {Humans ; Male ; *beta-Lactamases/genetics ; Middle Aged ; *Klebsiella/genetics/drug effects/enzymology/isolation &amp; purification ; *Klebsiella Infections/microbiology/drug therapy ; Anti-Bacterial Agents/pharmacology ; *DNA Transposable Elements ; Drug Resistance, Multiple, Bacterial/genetics ; Microbial Sensitivity Tests ; Whole Genome Sequencing ; *Bacterial Proteins/genetics ; Plasmids/genetics ; }, abstract = {BACKGROUND: Klebsiella michiganensis, an emerging opportunistic pathogen, poses public health risks due to its increasing multidrug resistance (MDR), especially to carbapenems.<br><br>CASE AND METHOD: A 46-year-old man with pulmonary fibrosis was hospitalized in Guangzhou, China, for worsening pneumonia. A multidrug-resistant K. michiganensis strain (YK6) was isolated from his sputum before treatment. The strain was characterized using MALDI-TOF mass spectrometry, antimicrobial susceptibility testing (AST), and whole genome sequencing (WGS). Targeted therapy guided by AST successfully resolved the infection.<br><br>RESULTS: The YK6 strain exhibited resistance to carbapenems, &#x3b2;-lactam/&#x3b2;-lactamase inhibitors, cephalosporins, aminoglycosides, and quinolones, except colistin and tigecycline. Genomic analysis revealed a 41.9-kb MDR island and an intact I-E CRISPR-Cas system on the chromosome, along with two plasmids: IncFIA/IncFII plasmid pYK6-1 carrying blaKPC-2 and IncC plasmid pYK6-2 harboring blaVEB-3. A novel blaKPC-2-transposon in pYK6-1 was identified, consisting of a non-Tn4401 element (NTE)-like structure (Tn3-ISKpn27-blaKPC-2-&#x394;ISKpn6-korC) flanked by inversely oriented ISKpn19-tnpM-tnpR elements and 31-bp inverted repeats never reported, a configuration did not reported previously. Furthermore, the blaVEB-3 genetic environment in pYK6-2 featured a unique cassette: IS26-IS6100-blaVEB-3-tnp-ISAs1-qacE&#x394;1-sul1-ISCR1. An additional ISAs1 insertion between the tnpF-like integrase and qacE&#x394;1 distinguishes it from similar blaVEB-3-harboring cassettes. The blaVEB-3 resistance region in pYK6-2 likely originated from homologous recombination mediated by IS26 and Tn5403, which flank the gene cassette.<br><br>CONCLUSIONS: To our knowledge, this is the first report of concurrent blaVEB-3 and blaKPC-2 in K. michiganensis, along with a novel blaKPC-2 transposon structure. These findings highlight the urgent need for enhanced surveillance of MDR K. michiganensis to prevent treatment failures.}, }
@article {pmid40095971, year = {2025}, author = {Pinaud, M and Zamborlini, A}, title = {Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {216}, pages = {}, doi = {10.3791/67469}, pmid = {40095971}, issn = {1940-087X}, mesh = {Humans ; *Electroporation/methods ; *Gene Knockout Techniques/methods ; *CRISPR-Cas Systems ; THP-1 Cells ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Single-Cell Analysis/methods ; }, abstract = {The human acute monocytic leukemia (AML) THP-1 cell line is widely used as a model to study the functions of human monocyte-derived macrophages, including their interplay with significant human pathogens such as the human immunodeficiency virus (HIV). Compared to other immortalized cell lines of myeloid origin, THP-1 cells retain many intact inflammatory signaling pathways and display phenotypic characteristics that more closely resemble those of primary monocytes, including the ability to differentiate into macrophages when treated with phorbol-12-myristate 13-acetate (PMA). The use of CRISPR-Cas9 technology to engineer THP-1 cells through targeted gene knockout (KO) provides a powerful approach to better characterize immune-related mechanisms, including virus-host interactions. This article describes a protocol for efficient CRISPR-Cas9-based engineering using electroporation to deliver pre-assembled Cas9:sgRNA ribonucleoproteins into the cell nucleus. Using multiple sgRNAs targeting the same locus at slightly different positions results in the deletion of large DNA fragments, thereby increasing editing efficiency, as assessed by the T7 endonuclease I assay. CRISPR-Cas9-mediated editing at the genetic level was validated by Sanger sequencing followed by Inference of CRISPR Edits (ICE) analysis. Protein depletion was confirmed by immunoblotting coupled with a functional assay. Using this protocol, up to 100% indels in the targeted locus and a decrease of over 95% in protein expression were achieved. The high editing efficiency makes it convenient to isolate single-cell clones by limiting dilution.}, }
@article {pmid40094895, year = {2025}, author = {Malik, S and Ahsan, O and Muhammad, K and Munawar, N and Waheed, Y}, title = {Phagetherapy updates: New frontiers against antibiotic resistance.}, journal = {European journal of microbiology &amp; immunology}, volume = {15}, number = {1}, pages = {1-12}, pmid = {40094895}, issn = {2062-509X}, abstract = {Antibiotic resistance is a major problem in the healthcare industry, and it presents difficulties in managing bacterial diseases worldwide. The need to find alternative antibiotic-containing methods is thus a major area for the scientific community to work on. Bacteriophage therapy is an interesting alternative that has been used in scientific research for a long time to tackle antibiotic-resistant bacteria. The purpose of this review was to compile the latest data on bacteriophages, which are progressively being used as alternatives to antibiotics, and to identify the mechanisms associated with phage therapy. The results section delves into the growing challenges posed by antibiotics and explores the potential of bacteriophages as therapeutic alternatives. This study discusses how phages can decrease antibiotic resistance, highlighting their role in modulating microbiomes and addressing various complications. This study explored the intriguing question of whether bacteriophages can combat nonbacterial diseases and examined their indirect use in pest control. In addition, this study explores the application of the CRISPR-Cas system in combating antibiotic resistance and specifically addresses phage therapy for secondary bacterial infections in COVID-19. We will further discuss whether bacteriophages are a noteworthy alternative to antibiotics by considering the evolutionary trade-offs between phages and antibiotic resistance. This section concludes by outlining future perspectives and acknowledging limitations, particularly in the context of phage and CRISPR-Cas9-mediated phage therapy. The methodology adopted for this study is a comprehensive research strategy using the Google Scholar and PubMed databases, among others. In conclusion, phage therapy is a promising strategy for tackling antibiotic-resistant bacteria, contributing to improved food production and mitigating secondary health effects. However, effective regulation requires careful selection of phages in conjunction with antibiotics to ensure judicious control of the coevolutionary dynamics between phages and antibiotics.}, }
@article {pmid40093906, year = {2025}, author = {Qin, X and Zhou, K and Dong, L and Yang, L and Li, W and Chen, Z and Shen, C and Han, L and Li, Y and Chan, AKN and Pokharel, SP and Qing, Y and Chen, M and Wang, K and Leung, K and Sau, L and Chen, CW and Deng, X and Su, R and Chen, J}, title = {CRISPR screening reveals ZNF217 as a vulnerability in high-risk B-cell acute lymphoblastic leukemia.}, journal = {Theranostics}, volume = {15}, number = {8}, pages = {3234-3256}, pmid = {40093906}, issn = {1838-7640}, support = {R37 CA292678/CA/NCI NIH HHS/United States ; }, mesh = {Humans ; Animals ; *CRISPR-Cas Systems/genetics ; Mice ; *Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics/pathology ; Cell Line, Tumor ; Fusion Proteins, bcr-abl/genetics ; *Trans-Activators/genetics/metabolism ; Xenograft Model Antitumor Assays ; Cell Survival/genetics ; }, abstract = {Rationale: Despite substantial advancement in the treatment of B-cell acute lymphoblastic leukemia (B-ALL), it remains a leading cause of cancer mortality in children due to the high relapse rate. Moreover, the long-term survival rates for adult B-ALL patients are still less than 40%. The B-ALL patients carrying MLL rearrangements or BCR-ABL fusion represent high-risk B-ALL subtypes that face particularly dismal prognoses. This study aims to identify innovative therapeutic vulnerability for high-risk B-ALL. Methods: The CRISPR-Cas9 screen was conducted to pinpoint genes essential for high-risk B-ALL cell survival/growth. Both in vitro and in vivo models were then employed to investigate the pathological role of ZNF217 in high-risk B-ALL. To characterize the downstream functionally essential targets of ZNF217, we performed RNA-seq and CUT&amp;RUN-seq, followed by integrative bioinformatics analysis and experimental validation. Results: Through the focused CRISPR-Cas9 screening, ZNF217 emerged as the most essential gene for the cell survival/growth of B-ALL driven by MLL rearrangement or BCR-ABL. Through in vitro gain- and loss-of-function assays, we demonstrated that ZNF217 is indeed required for B-ALL cell survival/growth. Moreover, we established the B-ALL xenograft model and patient-derived xenograft (PDX) model and demonstrated that ZNF217 depletion significantly suppressed B-ALL progression and substantially extended the survival of recipient mice. Through integrative multiple-omics analysis, we elucidated that ZNF217 exerts its oncogenic role in B-ALL through both CoREST-dependent and CoREST-independent mechanisms. Furthermore, we characterized FOS as a functionally essential downstream target of ZNF217, and ZNF217 inhibited FOS expression in a CoREST-independent manner. Conclusions: Our findings highlight ZNF217 as a promising therapeutic target for the treatment of high-risk B-ALL, such as those carrying MLL-rearrangements or BCR-ABL fusion.}, }
@article {pmid40093900, year = {2025}, author = {Bai, L and Pang, Y and Wang, T and Wang, S and Guo, K and Xuan, T and Zhang, Z and Liu, D and Qian, F and Zheng, Y and Jin, G and Wang, R}, title = {SPEAR: CRISPR-mediated ultrasensitive, specific and rapid one-pot detection strategy for cancer-related SNPs.}, journal = {Theranostics}, volume = {15}, number = {8}, pages = {3275-3288}, pmid = {40093900}, issn = {1838-7640}, mesh = {*Polymorphism, Single Nucleotide/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/genetics/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Point Mutation ; Molecular Diagnostic Techniques/methods ; }, abstract = {Rationale: The ultrasensitive and accurate detection of driver mutations is critical for early cancer screening and precision medicine. Current methods face challenges in balancing sensitivity, specificity, and speed, which limits their clinical utility. Therefore, a rapid, sensitive, and specific method is essential for detecting cancer-related SNPs. Methods: This study introduces SPEAR (Specific Point mutation Evaluation via CRISPR-Cas Assisted Recognition), a novel methodology combining NEAR (Nicking Enzyme Amplification Reaction) isothermal amplification with SNP-specific recognition by Cas12b RNP in a one-pot configuration to detect cancer-related single nucleotide polymorphisms (SNPs). SPEAR leverages the power of NEAR isothermal amplification to efficiently amplify target DNA, followed by Cas12b RNP for SNP-specific recognition. This integrated approach ensures a rapid and precise mutation detection system in a single reaction. Results: The method was applied to blood samples for the detection of cancer-related mutations, with results obtained in approximately 30 min. The SPEAR enables detection of gene mutations at the single-molecule level and it can detect targets at a 0.1% ratio despite strong background interference. The method exhibits single-base resolution specificity, allowing for the detection of multiple SNPs in a single reaction. It outperforms first-generation sequencing (FGS) in both convenience and sensitivity, while remaining compatible with next-generation sequencing (NGS). Conclusion: SPEAR offers a rapid, sensitive, and convenient approach to detect cancer-related SNPs, with significant potential for clinical applications, including real-time detection and molecular diagnostics in precision medicine.}, }
@article {pmid40093809, year = {2025}, author = {Li, B and Pan, Y and Wu, J and Miao, C and Wang, Z}, title = {Large-scale genomic-wide CRISPR screening revealed PRC1 as a tumor essential candidate in clear cell renal cell carcinoma.}, journal = {International journal of medical sciences}, volume = {22}, number = {7}, pages = {1658-1671}, pmid = {40093809}, issn = {1449-1907}, mesh = {Humans ; *Carcinoma, Renal Cell/genetics/pathology/mortality ; *Kidney Neoplasms/genetics/pathology/mortality ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; Cell Proliferation/genetics ; *Biomarkers, Tumor/genetics/metabolism ; Prognosis ; *Cell Cycle Proteins/genetics/metabolism ; Female ; Male ; Cell Movement/genetics ; Protein Interaction Maps/genetics ; CRISPR-Cas Systems ; }, abstract = {Background: Clear cell renal cell carcinoma (ccRCC) is a prevalent and aggressive subtype of kidney cancer, often associated with metastasis and recurrence. Identifying key genes involved in ccRCC progression is critical for improving treatment strategies and patient outcomes. Methods: We performed a large-scale genome-wide CRISPR screening to identify genes crucial to ccRCC progression using the DepMap database. For discovery and validation, we integrated multi-omics data from The Cancer Genome Atlas (TCGA), GEO, and the NJMU-ccRCC clinical cohort. Bioinformatics analyses, including differential expression, pathway enrichment, and protein-protein interaction network analysis, were conducted to elucidate the biological functions. To validate our findings, we employed immunohistochemistry, qRT-PCR, and various cellular assays to investigate the role of PRC1 in ccRCC. Results: CRISPR screening identified PRC1 as a key gene significantly overexpressed in ccRCC tissues from the DepMap database. Elevated PRC1 expression was associated with poor overall survival, disease-specific survival, and progression-free interval. Silencing PRC1 in ccRCC cell lines inhibited cell proliferation, migration, and colony formation. Functional enrichment analyses revealed that PRC1 is involved in essential processes such as cell cycle regulation, mitosis, and cytokinesis. Additionally, PRC1 expression was correlated with the activation of the Wnt/β-catenin pathway, suggesting that PRC1 plays a pivotal role in tumor progression. Conclusion: PRC1 emerges as a promising biomarker and therapeutic target for ccRCC. Elevated PRC1 expression is associated with poor prognosis, and its inhibition suppresses ccRCC cell proliferation and migration. Our findings underscore the crucial role of PRC1 in ccRCC progression and highlight the need for further investigation into its molecular mechanisms and therapeutic potential.}, }
@article {pmid40093536, year = {2025}, author = {Hershan, AA}, title = {Virology, epidemiology, transmissions, diagnostic tests, prophylaxis and treatments of human Mpox: Saudi Arabia perspective.}, journal = {Frontiers in cellular and infection microbiology}, volume = {15}, number = {}, pages = {1530900}, pmid = {40093536}, issn = {2235-2988}, mesh = {Humans ; Saudi Arabia/epidemiology ; Animals ; *Mpox, Monkeypox/epidemiology/diagnosis/prevention &amp; control/transmission/therapy/drug therapy/virology ; *Monkeypox virus/genetics/isolation &amp; purification ; Antiviral Agents/therapeutic use ; Diagnostic Tests, Routine ; }, abstract = {Mpox (Monkeypox) is a highly contagious viral disease that can be transmitted from animal-to-human or human-to-human through intimate contact, Mpox is caused by the monkeypox virus (MPXV), which is an enveloped double-stranded DNA that belongs to the genus Orthopoxvirus, Poxviridae family, and subfamily Chordopoxvirinae. Mpox cases were previously only reported in West and Central Africa, however in recent times non-endemic countries including Saudi Arabia (SA) also reported confirmed Mpox cases. The first laboratory-confirmed human Mpox case in SA was reported on 14 July 2022, since then a number of confirmed Mpox cases have been reported by WHO in SA. These confirmed Mpox cases in SA were observed among individuals with a history of visiting European Union countries. SA is not only at risk of importation of Mpox cases owing to travel to such countries, but also there are various other risk factors including geographic proximity to the African continent, trade in exotic animals, and massive inflow of tourists. Therefore, government health authorities of SA should continue to collaborate with various international health organizations including WHO to prevent, manage or monitor potential health risks at most of the entry points in SA including highways, seaports, and airports by ensuring adherence to hygiene protocols, vaccinations, and health screenings. There are a range of diagnostic tests are currently available that can be used in SA to confirm Mpox infections, including real-time PCR, loop-mediated isothermal amplification, serological testing, clustered regularly interspaced short palindromic repeat-CRISPR-associated protein (CRISPR-Cas)-based systems, whole-genome sequencing, electron microscopy, and virus isolation and culture. There is no approved treatment specifically for Mpox, however multiple approved antiviral agents for smallpox treatment were found to be useful in Mpox treatment and in the management of Mpox outbreaks, such as- trifluridine, brincidofovir, tecovirimat, and cidofovir. The aim of this review is to provide valuable insights regarding virology, pathogenesis, epidemiology, transmissions, clinical presentation, diagnostic tests, prophylactic measures and therapeutic options of Mpox from SA perspective. Moreover, a side-by-side discussion on the global trend and scenarios of Mpox has been provided for comparison and further improvement in measures against Mpox in SA.}, }
@article {pmid40092787, year = {2025}, author = {Smith, RL and Davenport, PW and Lakin, MR}, title = {A Study of CRISPR Ribonucleoprotein Displacement in Cell-Free Systems.}, journal = {ACS omega}, volume = {10}, number = {9}, pages = {9154-9164}, pmid = {40092787}, issn = {2470-1343}, abstract = {CRISPR/Cas-based transcription factors are a powerful tool for controlling gene expression in living cells and cell-free systems, as their programmable DNA-binding activity makes them a powerful tool for building and scaling up engineered genetic networks. The use of guide RNAs for targeting Cas proteins to desired binding sites opens up the possibility of using RNA engineering techniques to achieve programmable and dynamic control of CRISPR/Cas-based transcription factor activity and hence of gene expression. In this work, we investigate the use of RNA strand displacement systems to remove bound CRISPR/Cas ribonucleoprotein complexes from target DNA in cell-free systems. The binding of catalytically inactive dCas9 is monitored by using CRISPR interference to repress the expression of a reporter protein. We express an antisense RNA complementary to an extended toehold on an engineered guide RNA in an E. coli-based cell-free expression system with the goal of rapidly removing bound CRISPR/Cas ribonucleoproteins via strand displacement. We find that dCas9 appears to be surprisingly resistant to removal via this mechanism, which indicates that other strategies for dynamic removal of bound Cas proteins may prove to be more effective.}, }
@article {pmid40092730, year = {2025}, author = {Deng, Y and Xu, J and Yang, M and Huang, Y and Yang, Y}, title = {Rapid detection of the GJB2 c.235delC mutation based on CRISPR-Cas13a combined with lateral flow dipstick.}, journal = {Open life sciences}, volume = {20}, number = {1}, pages = {20251064}, pmid = {40092730}, issn = {2391-5412}, abstract = {Hereditary hearing loss, an auditory neuropathy disorder, is characterized by its high prevalence and significant impact on the quality of life of those affected. In Chinese populations, the most prevalent gap junction beta-2 (GJB2) mutation hotspot is c.235delC. Currently available genetic tests require expensive instruments and specialized technicians or have long testing cycles and high costs, and therefore cannot meet point-of-care testing (POCT) requirements. The objective of this study was to evaluate the viability of a POCT kit. In only 42 min, we successfully identified the GJB2 mutation site c.235delC by integrating CRISPR-Cas nucleic acid detection with recombinase-aided amplification (RAA) and a lateral flow dipstick (LFD) method. This method has the capacity to detect low-abundance nucleic acids (as low as 10[2] copies/μL) and low mutation frequency (20%), in addition to accurately distinguishing wild-type, homozygous, and heterozygous mutation. This approach was utilized to assess blood samples from a total of 31 deaf patients and 5 healthy volunteers. All results were subsequently confirmed through the implementation of Sanger sequencing. Our detection results were consistent with Sanger sequencing results. The diagnostic sensitivity and specificity were 100%. The combination of CRISPR-Cas13a and LFD may be a promising method for POCT of deafness genes.}, }
@article {pmid40092158, year = {2025}, author = {Sun, Y and Wu, G and Wang, Y and Jiang, J and Wang, H and Liu, F and Lu, F and Zhang, H}, title = {Application of multiple genomic-editing technologies in Streptomyces fungicidicus for improved enduracidin yield.}, journal = {Synthetic and systems biotechnology}, volume = {10}, number = {2}, pages = {564-573}, pmid = {40092158}, issn = {2405-805X}, abstract = {Streptomyces fungicidicus, an industrial strain for enduracidin production, shows significant potential as a cellular chassis for the synthesis of novel small peptides. Targeted deletion of secondary metabolite gene clusters offers a promising strategy to enhance strain performance, but is often hampered by the lack of efficient gene editing tools. In this study, we optimized the traditional homologous recombination method by integrating selection and counter-selection markers to streamline the gene editing process, and successfully deleted gene clusters of up to 54.4 kb. Recognizing the significant potential of CRISPR/Cas-based systems in Streptomyces, we evaluated the base editing efficiency of the CRISPR/cBEST system in S. fungicidicus, which enabled stop codon insertions in the targeted gene with different mutation rates depending on the applied sgRNA. Additionally, we established a CRISPR/Cas9 system in S. fungicidicus while incorporating a counter-selection marker for efficient screening, which greatly shortened the gene editing cycle. The resulting mutants with single and cumulative gene cluster deletions exhibited improved growth characteristics, including a prolonged logarithmic phase and increased biomass. Although cumulative deletions did not result in consistent yield improvements, the mutants with improved growth characteristics show potential for further strain optimization in the future. The optimized gene editing systems developed in this study provide a valuable foundation for engineering other Streptomyces species.}, }
@article {pmid40091632, year = {2025}, author = {Fu, J and Liu, X and Deng, R and Jiang, X and Cai, W and Fu, H and Shao, X}, title = {Accurate Prediction of CRISPR/Cas13a Guide Activity Using Feature Selection and Deep Learning.}, journal = {Journal of chemical information and modeling}, volume = {65}, number = {7}, pages = {3380-3387}, doi = {10.1021/acs.jcim.4c02438}, pmid = {40091632}, issn = {1549-960X}, mesh = {*Deep Learning ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Neural Networks, Computer ; }, abstract = {CRISPR/Cas13a serves as a key tool for nucleic acid tests; therefore, accurate prediction of its activity is essential for creating robust and sensitive diagnosis. In this study, we create a dual-branch neural network model that achieves high prediction accuracy and classification performance across two independent CRISPR/Cas13a data sets, outperforming previously published models relying solely on sequence features. The model integrates direct sequence encoding with descriptive features and yields 99 key descriptive features out of 1553, extracted through statistical analysis, which critically influence guide-target interactions and Cas13a guide activity. By employing Shapley Additive Explanations and Integrated Gradients for feature importance analysis, we show that sequence composition, mismatch type and frequency, and the protospacer flanking site region are primary features. These findings underscore the importance of using descriptive features as complementary inputs to deep learning-based encoding and provide valuable insights into the mechanisms underlying guide-target interaction. All in all, this study not only introduces a reliable and efficient model for Cas13a guide activity prediction but also offers a foundation for future rational design efforts.}, }
@article {pmid40091120, year = {2025}, author = {Moreno-Sánchez, I and Hernández-Huertas, L and Nahón-Cano, D and Martínez-García, PM and Treichel, AJ and Gómez-Marin, C and Tomás-Gallardo, L and da Silva Pescador, G and Kushawah, G and Egidy, R and Perera, A and Díaz-Moscoso, A and Cano-Ruiz, A and Walker, JA and Muñoz, MJ and Holden, K and Galcerán, J and Nieto, M&#xc1; and Bazzini, AA and Moreno-Mateos, MA}, title = {Enhanced RNA-targeting CRISPR-Cas technology in zebrafish.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {2591}, pmid = {40091120}, issn = {2041-1723}, support = {F31 HD110268/HD/NICHD NIH HHS/United States ; R01 GM136849/GM/NIGMS NIH HHS/United States ; R21 OD034161/OD/NIH HHS/United States ; }, mesh = {Animals ; *Zebrafish/genetics/embryology ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Gene Editing/methods ; RNA, Messenger/genetics/metabolism ; Embryo, Nonmammalian/metabolism ; }, abstract = {CRISPR-Cas13 RNA-targeting systems are widely used in basic and applied sciences. However, its application has recently generated controversy due to collateral activity in mammalian cells and mouse models. Moreover, its competence could be improved in vivo. Here, we optimized transient formulations as ribonucleoprotein complexes or mRNA-gRNA combinations to enhance the CRISPR-RfxCas13d system in zebrafish. We i) use chemically modified gRNAs to allow more penetrant loss-of-function phenotypes, ii) improve nuclear RNA targeting, and iii) compare different computational models and determine the most accurate to predict gRNA activity in vivo. Furthermore, we demonstrate that transient CRISPR-RfxCas13d can effectively deplete endogenous mRNAs in zebrafish embryos without inducing collateral effects, except when targeting extremely abundant and ectopic RNAs. Finally, we implement alternative RNA-targeting CRISPR-Cas systems such as CRISPR-Cas7-11 and CRISPR-DjCas13d. Altogether, these findings contribute to CRISPR-Cas technology optimization for RNA targeting in zebrafish through transient approaches and assist in the progression of in vivo applications.}, }
@article {pmid40089636, year = {2025}, author = {Cervera, ST and Martínez, S and Iranzo-Martínez, M and Notario, L and Melero-Fernández de Mera, RM and Alonso, J}, title = {Targeted inactivation of EWSR1 : : FLI1 gene in Ewing sarcoma via CRISPR/Cas9 driven by an Ewing-specific GGAA promoter.}, journal = {Cancer gene therapy}, volume = {32}, number = {4}, pages = {437-449}, pmid = {40089636}, issn = {1476-5500}, mesh = {*Sarcoma, Ewing/genetics/therapy/pathology ; Humans ; *CRISPR-Cas Systems ; *RNA-Binding Protein EWS/genetics ; Animals ; *Proto-Oncogene Protein c-fli-1/genetics ; *Promoter Regions, Genetic ; Cell Line, Tumor ; Mice ; Gene Editing ; *Oncogene Proteins, Fusion/genetics ; *Bone Neoplasms/genetics/therapy ; Cell Proliferation ; }, abstract = {We have recently demonstrated that genetic inactivation of EWSR1 : : FLI1 by CRISPR/Cas9, successfully blocks cell proliferation in a cell model of Ewing sarcoma. However, CRISPR/Cas9-mediated gene editing can exhibit off-target effects, and thus, precise regulation of Cas9 expression in target cells is essential to develop gene-editing strategies to inactivate EWSR1 : : FLI1 in Ewing sarcoma cells. In this study, we demonstrate that Cas9 can be specifically expressed in Ewing sarcoma cells when located downstream a promoter consisting of GGAA repeats and a consensus TATA box (GGAAprom). Under these conditions, Cas9 is selectively expressed in Ewing sarcoma cells that express EWSR1 : : FLI1 oncoproteins, but not in cells expressing wild-type FLI1. Consequently, Ewing sarcoma cells infected with GGAAprom>Cas9 and a specific gRNA designed to inactivate EWSR1 : : FLI1, showed successful EWSR1 : : FLI1 inactivation and the subsequent blockade of cell proliferation. Notably, GGAAprom>Cas9 can be efficiently delivered to Ewing sarcoma cells via adenoviral vectors both in vitro and in vivo, highlighting the potential of this approach for Ewing sarcoma treatment. Our results demonstrate that the CRISPR/Cas9 machinery is safe and specific for Ewing sarcoma cells when driven under a GGAAprom, paving the way for the development of cancer gene therapies based on the selective expression of genes with therapeutic potential.}, }
@article {pmid40088462, year = {2025}, author = {Qing, M and Huang, C and Li, Y and Yu, Q and Hu, Q and Zhou, J and Yuan, R and Bai, L}, title = {Dithiothreitol Facilitates LbCas12a with Expanded PAM Preference for Ultrasensitive Nucleic Acid Detection.}, journal = {Analytical chemistry}, volume = {97}, number = {11}, pages = {6286-6294}, doi = {10.1021/acs.analchem.5c00280}, pmid = {40088462}, issn = {1520-6882}, mesh = {*Dithiothreitol/chemistry ; *CRISPR-Cas Systems ; Humans ; *Human papillomavirus 16/genetics/isolation &amp; purification ; *Human papillomavirus 18/genetics/isolation &amp; purification ; *CRISPR-Associated Proteins/metabolism/chemistry ; Nucleic Acid Amplification Techniques ; *DNA, Viral/analysis/genetics ; Polymerase Chain Reaction ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {Clustered regularly interspaced short palindromic repeats-associated (CRISPR/Cas) proteins have been used for a growing class of in vitro molecular diagnostics due to their modularity and high specificity in targeting nucleic acid. However, the requirement of a protospacer adjacent motif (PAM) for Cas protein-catalyzed trans-cleavage poses a challenge for random nucleic acid detection. Here, we demonstrate that dithiothreitol (DTT) enables LbCas12a to adopt a relaxed preference for PAM base pairing, thereby expanding the target sequence space. Accordingly, we propose a DTT-mediated CRISPR/Cas12a toolbox (DTT-deCRISPR) that exhibits relaxed PAM specificity and is readily compatible with nucleic acid amplification techniques including recombinase polymerase amplification (RPA) and polymerase chain reaction (PCR). As a proof of concept, we integrate DTT-deCRISPR with frequently used PCR for sensitively and selectively detecting high-risk human papillomavirus (HPV) 16 and 18. The platform demonstrates the ability to detect synthesized HPV 16 and 18 plasmids down to 1 aM within 60 min. Based on the receiver operating characteristic curve analysis, the clinical sensitivities of the developed method for detecting HPV 16 and 18 are 93.75% and 80.00%, respectively. We further incorporate it into a lateral flow assay (LFA) for point-of-care detection, and the HPV 16 and HPV 18 abundances determined by LFA for clinical samples are consistent with the fluorescence analysis results. Together, this work uncovers an unexpected connection between DTT and PAM preferences of LbCas12a, promoting the universality and flexibility of CRISPR technology in molecular diagnostics.}, }
@article {pmid40088038, year = {2025}, author = {Blomme, J and Arraiza Ribera, J and De Clerck, O and Jacobs, TB}, title = {Consolidating Ulva functional genomics: gene editing and new selection systems.}, journal = {The New phytologist}, volume = {246}, number = {4}, pages = {1710-1723}, doi = {10.1111/nph.70068}, pmid = {40088038}, issn = {1469-8137}, support = {BOF/STA/202209/016//Bijzonder Onderzoeksfonds UGent/ ; BOF20/PDO/016//Bijzonder Onderzoeksfonds UGent/ ; 9329//Gordon and Betty Moore Foundation/ ; 12T3418N//Fonds Wetenschappelijk Onderzoek/ ; G015623N//Fonds Wetenschappelijk Onderzoek/ ; G0AHR24N//Fonds Wetenschappelijk Onderzoek/ ; GOH3817N//Fonds Wetenschappelijk Onderzoek/ ; I001621N//Fonds Wetenschappelijk Onderzoek/ ; }, mesh = {*Ulva/genetics/microbiology ; *Gene Editing/methods ; *Genomics/methods ; *Selection, Genetic ; CRISPR-Cas Systems/genetics ; Mutation/genetics ; }, abstract = {The green seaweed Ulva compressa is a promising model for functional biology. In addition to historical research on growth and development, -omics data and molecular tools for stable transformation are available. However, more efficient tools are needed to study gene function. Here, we expand the molecular toolkit for Ulva. We screened the survival of Ulva and its mutualistic bacteria on 14 selective agents and established that Blasticidin deaminases (BSD or bsr) can be used as selectable markers to generate stable transgenic lines. We show that Cas9 and Cas12a RNPs are suitable for targeted mutagenesis and can generate genomic deletions of up to 20 kb using the marker gene ADENINE PHOSPHORIBOSYLTRANSFERASE (APT). We demonstrate that the targeted insertion of a selectable marker via homology-directed repair or co-editing with APT is possible for nonmarker genes. We evaluated 31 vector configurations and found that the bicistronic fusion of Cas9 to a resistance marker or the incorporation of introns in Cas9 led to the most mutants. We used this to generate mutants in three nonmarker genes using a co-editing strategy. This expanded molecular toolkit now enables us to reliably make gain- and loss-of-function mutants; additional optimizations will be necessary to allow for vector-based multiplex genome editing in Ulva.}, }
@article {pmid40087793, year = {2025}, author = {Barraza-Flores, P and Moghadaszadeh, B and Lee, W and Isaac, B and Sun, L and Hickey, ET and Rockowitz, S and Sliz, P and Beggs, AH}, title = {Zebrafish and cellular models of SELENON-Congenital myopathy exhibit novel embryonic and metabolic phenotypes.}, journal = {Skeletal muscle}, volume = {15}, number = {1}, pages = {7}, pmid = {40087793}, issn = {2044-5040}, support = {P50HD105351//Eunice Kennedy Shriver National Institute of Child Health and Human Development/ ; T32NS007473/NS/NINDS NIH HHS/United States ; }, mesh = {Animals ; Zebrafish/embryology ; Disease Models, Animal ; Phenotype ; *Selenoproteins/genetics/metabolism ; *Zebrafish Proteins/genetics/metabolism ; *Muscular Diseases/genetics/metabolism ; Muscle, Skeletal/metabolism ; Myoblasts/metabolism ; CRISPR-Cas Systems ; }, abstract = {BACKGROUND: SELENON-Congenital Myopathy (SELENON-CM) is a rare congenital myopathy caused by mutations of the SELENON gene characterized by axial muscle weakness and progressive respiratory insufficiency. Muscle histopathology may be non-specific, but commonly includes multiminicores or a dystrophic pattern. The SELENON gene encodes selenoprotein N (SelN), a selenocysteine-containing redox enzyme located in the endo/sarcoplasmic reticulum membrane where it colocalizes with mitochondria-associated membranes. However, the molecular mechanism(s) by which SelN deficiency cause SELENON-CM remain poorly understood. A hurdle is the lack of cellular and animal models that show easily assayable phenotypes.<br><br>METHODS: Using CRISPR-Cas9 we generated three zebrafish models of SELENON-CM, which were then studied by spontaneous coiling, hatching, and activity assays. We also performed selenon coexpression analysis using a single cell RNAseq zebrafish embryo-atlas. SelN-deficient myoblasts were generated and assayed for glutathione, reactive oxygen species, carbonylation, and nytrosylation levels. Finally, we tested Selenon-deficient myoblasts' metabolism using a Seahorse cell respirometer.<br><br>RESULTS: We report deep-phenotyping of SelN-deficient zebrafish and muscle cells. SelN-deficient zebrafish exhibit changes in embryonic muscle function and swimming activity in larvae. Analysis of single cell RNAseq data in a zebrafish embryo-atlas revealed coexpression of selenon and genes involved in the glutathione redox pathway. SelN-deficient zebrafish and mouse myoblasts exhibit altered glutathione and redox homeostasis, as well as abnormal patterns of energy metabolism, suggesting roles for SelN in these functions.<br><br>CONCLUSIONS: These data demonstrate a role for SelN in zebrafish early development and myoblast metabolism and provide a basis for cellular and animal model assays for SELENON-CM.}, }
@article {pmid40087758, year = {2025}, author = {Peng, W and Shi, M and Hu, B and Jia, J and Li, X and Wang, N and Man, S and Ye, S and Ma, L}, title = {Nanotechnology-leveraged CRISPR/Cas systems: icebreaking in trace cancer-related nucleic acids biosensing.}, journal = {Molecular cancer}, volume = {24}, number = {1}, pages = {78}, pmid = {40087758}, issn = {1476-4598}, support = {32072309//National Natural Science Foundation of China/ ; 19JCYBJC27800//Tianjin Municipal Science and Technology Committee/ ; }, mesh = {Humans ; *CRISPR-Cas Systems ; *Neoplasms/genetics/diagnosis ; *Biosensing Techniques/methods ; *Biomarkers, Tumor/genetics ; *Nanotechnology/methods ; *Nucleic Acids/genetics ; }, abstract = {As promising noninvasive biomarkers, nucleic acids provide great potential to innovate cancer early detection methods and promote subsequent diagnosis to improve the survival rates of patient. Accurate, straightforward and sensitive detection of such nucleic acid-based cancer biomarkers in complex biological samples holds significant clinical importance. However, the low abundance creates huge challenges for their routine detection. As the next-generation diagnostic tool, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) with their high programmability, sensitivity, fidelity, single-base resolution, and precise nucleic acid positioning capabilities are extremely attractive for trace nucleic acid-based cancer biomarkers (NABCBs), permitting rapid, ultra-sensitive and specific detection. More importantly, by combing with nanotechnology, it can solve the long-lasting problems of poor sensitivity, accuracy and simplicity, as well as to achieve integrated miniaturization and portable point-of-care testing (POCT) detection. However, existing literature lacks specific emphasis on this topic. Thus, we intend to propose a timely one for the readers. This review will bridge this gap by providing insights for CRISPR/Cas-based nano-biosensing development and highlighting the current state-of-art, challenges, and prospects. We expect that it can provide better understanding and valuable insights for trace NABCBs detection, thereby facilitating advancements in early cancer screening/detection/diagnostics and win practical applications in the foreseeable future.}, }
@article {pmid40087529, year = {2025}, author = {Kim, J and Nam, Y and Jeon, D and Choi, Y and Choi, S and Hong, CP and Kim, S and Jung, H and Park, N and Sohn, Y and Rim, YA and Ju, JH}, title = {Generation of hypoimmunogenic universal iPS cells through HLA-type gene knockout.}, journal = {Experimental &amp; molecular medicine}, volume = {57}, number = {3}, pages = {686-699}, pmid = {40087529}, issn = {2092-6413}, support = {HI23C1234//Korea Health Industry Development Institute (KHIDI)/ ; 20024297//Ministry of Trade, Industry and Energy (Ministry of Trade, Industry and Energy, Korea)/ ; NRF-2023M3A9E4009811//Ministry of Education (Ministry of Education of the Republic of Korea)/ ; }, mesh = {*Induced Pluripotent Stem Cells/cytology/metabolism/immunology ; Humans ; *Gene Knockout Techniques ; CRISPR-Cas Systems ; *HLA Antigens/genetics ; Cell Differentiation ; }, abstract = {Hypoimmunogenic universal induced pluripotent stemn (iPS) cells were generated through the targeted disruption of key genes, including human leukocyte antigen (HLA)-A, HLA-B and HLA-DR alpha (DRA), using the CRISPR-Cas9 system. This approach aimed to minimize immune recognition and enhance the potential of iPS cells for allogeneic therapy. Heterozygous iPS cells were used for guide RNA design and validation to facilitate the knockout (KO) of the HLA-A, HLA-B and HLA-DRA genes. The electroporation of iPS cells using the selected guide RNAs enabled the generation of triple-KO iPS cells, followed by single-cell cloning for clone selection. Clone A7, an iPS cell with targeted KOs of the HLA-A, HLA-B and HLA-DRA genes, was identified as the final candidate. Messenger RNA analysis revealed robust expression of pluripotency markers, such as octamer-binding transcription factor 4, sex-determining region Y box 2, Krüppel-like factor 4, Lin-28 homolog A and Nanog homeobox, while protein expression assays confirmed the presence of octamer-binding transcription factor 4, stage-specific embryonic antigen 4, Nanog homeobox and tumor rejection antigen 1-60. A karyotype examination revealed no anomalies, and three-germ layer differentiation assays confirmed the differentiation potential. After interferon gamma stimulation, the gene-corrected clone A7 lacked HLA-A, HLA-B and HLA-DR protein expression. Immunogenicity testing further confirmed the hypoimmunogenicity of clone A7, which was evidenced by the absence of proliferation in central memory T cells and effector memory T cells. In conclusion, clone A7, a triple-KO iPS cell clone that demonstrates immune evasion properties, retained its intrinsic iPS cell characteristics and exhibited no immunogenicity.}, }
@article {pmid40087133, year = {2025}, author = {Della Valle, S and Orsi, E and Creutzburg, SCA and Jansen, LFM and Pentari, EN and Beisel, CL and Steel, H and Nikel, PI and Staals, RHJ and Claassens, NJ and van der Oost, J and Huang, WE and Patinios, C}, title = {Streamlined and efficient genome editing in Cupriavidus necator H16 using an optimised SIBR-Cas system.}, journal = {Trends in biotechnology}, volume = {43}, number = {6}, pages = {1470-1491}, doi = {10.1016/j.tibtech.2025.02.006}, pmid = {40087133}, issn = {1879-3096}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Cupriavidus necator/genetics ; *Riboswitch/genetics ; Introns/genetics ; Genome, Bacterial ; }, abstract = {Cupriavidus necator H16 is a promising microbial platform strain for CO2 valorisation. While C. necator is amenable to genome editing, existing tools are often inefficient or rely on lengthy protocols, hindering its rapid transition to industrial applications. In this study, we simplified and accelerated the genome editing pipeline for C. necator by harnessing the Self-splicing Intron-Based Riboswitch (SIBR) system. We used SIBR to tightly control and delay Cas9-based counterselection, achieving >80% editing efficiency at two genomic loci within 48 h after electroporation. To further increase the versatility of the genome editing toolbox, we upgraded SIBR to SIBR2.0 and used it to regulate the expression of Cas12a. SIBR2.0-Cas12a could mediate gene deletion in C. necator with ~70% editing efficiency. Overall, we streamlined the genome editing pipeline for C. necator, facilitating its potential role in the transition to a bio-based economy.}, }
@article {pmid40086675, year = {2025}, author = {Li, XH and Lu, HZ and Yao, JB and Zhang, C and Shi, TQ and Huang, H}, title = {Recent advances in the application of CRISPR/Cas-based gene editing technology in Filamentous Fungi.}, journal = {Biotechnology advances}, volume = {81}, number = {}, pages = {108561}, doi = {10.1016/j.biotechadv.2025.108561}, pmid = {40086675}, issn = {1873-1899}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Fungi/genetics ; }, abstract = {Filamentous fungi are essential industrial microorganisms that can serve as sources of enzymes, organic acids, terpenoids, and other bioactive compounds with significant applications in food, medicine, and agriculture. However, the underdevelopment of gene editing tools limits the full exploitation of filamentous fungi, which still present numerous untapped potential applications. In recent years, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats) system, a versatile genome-editing tool, has advanced significantly and been widely applied in filamentous fungi, showcasing considerable research potential. This review examines the development and mechanisms of genome-editing tools in filamentous fungi, and contrasts the CRISPR/Cas9 and CRISPR/Cpf1 systems. The transformation and delivery strategies of the CRISPR/Cas system in filamentous fungi are also examined. Additionally, recent applications of CRISPR/Cas systems in filamentous fungi are summarized, such as gene disruption, base editing, and gene regulation. Strategies to enhance editing efficiency and reduce off-target effects are also highlighted, with the aim of providing insights for the future construction and optimization of CRISPR/Cas systems in filamentous fungi.}, }
@article {pmid40086107, year = {2025}, author = {Yadav, G and Sharma, A and Hathi, UPS and Gaind, R and Singh, R}, title = {Development and optimization of multiplex PCR for rapid detection of type I-F1 and type I-F2 Cas cluster genes in Acinetobacter baumannii.}, journal = {Biologicals : journal of the International Association of Biological Standardization}, volume = {90}, number = {}, pages = {101824}, doi = {10.1016/j.biologicals.2025.101824}, pmid = {40086107}, issn = {1095-8320}, mesh = {*Acinetobacter baumannii/genetics/isolation &amp; purification ; *Multiplex Polymerase Chain Reaction/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Multigene Family ; Acinetobacter Infections/microbiology ; Sensitivity and Specificity ; }, abstract = {Polymerase chain reaction (PCR), especially the multiplex PCR assay, enables simultaneous detection of multiple genes and is highly effective for diagnostic applications. The CRISPR-associated (Cas) system consists of several genes, and complete gene clusters are essential for its activity; multiplex PCR is an excellent method for detecting these multiple genes. This study focuses on the development and validation of a multiplex PCR protocol for the specific detection of CRISPR-Cas subtypes I-F1 and I-F2 found in A. baumannii, which is classified as a critical ESKAPE pathogen. The multiplex PCR method achieved a 100 % detection rate for isolates containing Cas subtypes I-F1 and I-F2 in clinical A. baumannii isolates. Testing across various genera and Acinetobacter species confirmed the high specificity of the assay, with no false positives, establishing it as a reliable tool for large-scale clinical applications. Of the 96 clinical A. baumannii isolates analysed, 29.167 % (n = 28) were multiplex PCR positive for a CRISPR-Cas system. Among these, 71.43 % (n = 20) had subtype I-F1, while 28.57 % (n = 8) had subtype I-F2. No clear association was found between Cas subtypes and resistance to the tested antibiotics or carbapenem genes. This study provides a valuable tool for monitoring CRISPR-Cas systems and can aid in various experimental and novel strategies to manage multidrug-resistant A. baumannii.}, }
@article {pmid40084131, year = {2025}, author = {Zhang, Y and Yang, S and Zheng, X and Tan, X}, title = {Cyanobacterial type I CRISPR-Cas systems: distribution, mechanisms, and genome editing applications.}, journal = {Frontiers in bioengineering and biotechnology}, volume = {13}, number = {}, pages = {1552030}, pmid = {40084131}, issn = {2296-4185}, abstract = {Cyanobacteria, renowned for their photosynthetic capabilities, serve as efficient microbial chassis capable of converting carbon dioxide into a spectrum of bio-chemicals. However, conventional genetic manipulation strategies have proven incompatible with the precise and systematic modifications required in the field of cyanobacterial synthetic biology. Here, we present an in-depth analysis of endogenous CRISPR-Cas systems within cyanobacterial genomes, with a particular focus on the Type I systems, which are the most widely distributed. We provide a comprehensive summary of the reported DNA defense mechanisms mediated by cyanobacterial Type I CRISPR-Cas systems and their current applications in genome editing. Furthermore, we offer insights into the future applications of these systems in the context of cyanobacterial genome editing, underscoring their potential to revolutionize synthetic biology approaches.}, }
@article {pmid40083940, year = {2025}, author = {Zhu, X and Wang, S and Xue, Y and Wang, X and Hu, S and Liang, T and Liu, W}, title = {Coupling CRISPR-Cas and a personal glucose meter with an enzymatic reporter for portable detection of human papillomavirus in biological samples.}, journal = {Theranostics}, volume = {15}, number = {7}, pages = {2870-2882}, pmid = {40083940}, issn = {1838-7640}, mesh = {Humans ; *CRISPR-Cas Systems ; *Papillomavirus Infections/diagnosis/virology ; Female ; *Papillomaviridae/isolation &amp; purification/genetics ; beta-Fructofuranosidase/metabolism ; Point-of-Care Testing ; Biosensing Techniques/methods ; Sensitivity and Specificity ; Blood Glucose Self-Monitoring/methods ; Point-of-Care Systems ; Human Papillomavirus Viruses ; }, abstract = {Significant efforts and resources have been dedicated to developing CRISPR-Cas based point-of-care testing (POCT) and self-diagnosis methods for nucleic acid pathogens, aiming to complement the gold standard quantitative PCR tests, particularly in settings where centralized facilities, trained personnel, or resource-intensive equipment are unavailable. However, the reliance on stationary, high-cost readout machinery hinders their full deployment at the point of care. We aimed to develop a solid-phase invertase-labeled reporter (ILR) system that enables convenient readout of CRISPR-Cas reactions, facilitate HPV detection in a POCT-compatible manner. Methods: Through multiple chemical couplings, invertase is immobilized onto magnetic microbeads via a nucleic acid linker that responds to target nucleic acid-induced CRISPR-Cas activation. This activation releases active invertase, which then converts sucrose to glucose in proportion to the target's abundance. Enzymatic signal amplification by Cas12a/Cas13a and invertase compensates for the moderate sensitivity of personal glucose meters (PGMs). Results: When applied to human papillomavirus detection, the HPV18-targeted LAMP-Cas12a/ILR/PGM system can detect as few as 7 HPV18-positive HeLa cells out of 7,000, achieving 95.8% sensitivity and 100% specificity in cervical cell samples. Furthermore, minimal reagent adjustments allow for the rapid establishment of HPV16 and HPV52-targeted LAMP-Cas12a/ILR/PGM systems, offering satisfactory sensitivity, specificity, and cross-species detection. Conclusion: These findings demonstrate a highly efficient testing platform for a range of nucleic acid pathogens, suitable for both point-of-care and household use.}, }
@article {pmid40082762, year = {2025}, author = {Haddad, A and Golan-Lev, T and Benvenisty, N and Goldberg, M}, title = {Genome-wide screening in human embryonic stem cells identifies genes and pathways involved in the p53 pathway.}, journal = {Molecular medicine (Cambridge, Mass.)}, volume = {31}, number = {1}, pages = {97}, pmid = {40082762}, issn = {1528-3658}, support = {2054/22//The Israel Science Foundation/ ; 3605/21//The ISF-Israel Precision Medicine Partnership (IPMP) Program/ ; 20230941//The Israel Cancer Association - Robin and David Gray donation/ ; }, mesh = {Humans ; *Tumor Suppressor Protein p53/metabolism/genetics ; *Human Embryonic Stem Cells/metabolism ; *Signal Transduction/genetics ; CRISPR-Cas Systems ; Apoptosis/genetics ; Proto-Oncogene Proteins c-mdm2/metabolism ; Genome-Wide Association Study ; Piperazines/pharmacology ; Genome, Human ; Imidazoles ; }, abstract = {BACKGROUND: The tumor suppressor protein, p53, which is mutated in half of human tumors, plays a critical role in cellular responses to DNA damage and maintenance of genome stability. Therefore, increasing our understanding of the p53 pathway is essential for improving cancer treatment and diagnosis.<br><br>METHODS: This study, which aimed to identify genes and pathways that mediate resistance to p53 upregulation, used genome-wide CRISPR-Cas9 loss-of-function screening done with Nutlin-3a, which inhibits p53-MDM2 interaction, resulting in p53 accumulation and apoptotic cell death. We used bioinformatics analysis for the identification of genes and pathways that are involved in the p53 pathway and cell survival assays to validate specific genes. In addition, we used RNA-seq to identify differentially expressed p53 target genes in gene knockout (KO) cell lines.<br><br>RESULTS: Our screen revealed three significantly enriched pathways: The heparan sulfate glycosaminoglycan biosynthesis, diphthamide biosynthesis and Hippo pathway. Notably, TRIP12 was significantly enriched in our screen. We found that TRIP12 is required for the p53-dependent transcription of several pro-apoptotic genes.<br><br>CONCLUSION: Our study has identified two novel pathways that play&#xa0;a role in p53-mediated growth restriction. Moreover, we have highlighted the interaction between the Hippo and the p53 pathways. Interestingly, we have shown that TRIP12 plays an important function in the p53 pathway by selectively affecting its role as a transcription factor.}, }
@article {pmid40082754, year = {2025}, author = {Herr, S and Li, X and Wu, D and Hunter, CT and Magallanes-Lundback, M and Wood, JC and Kaczmar, N and Buell, CR and DellaPenna, D and Gore, MA}, title = {Total tocopherol levels in maize grain depend on chlorophyll biosynthesis within the embryo.}, journal = {BMC plant biology}, volume = {25}, number = {1}, pages = {328}, pmid = {40082754}, issn = {1471-2229}, support = {IOS-1546657//National Science Foundation, United States/ ; 1013641//U.S. Department of Agriculture/ ; }, mesh = {*Tocopherols/metabolism ; *Zea mays/genetics/growth &amp; development/metabolism ; *Chlorophyll/biosynthesis/metabolism ; Embryonic Structures/metabolism ; CRISPR-Cas Systems ; Oxidoreductases Acting on CH-CH Group Donors/genetics/metabolism ; Genes, Plant ; Gene Expression Regulation, Plant ; Endosperm/metabolism ; RNA, Plant/metabolism ; Gene Knockout Techniques ; }, abstract = {BACKGROUND: Tocopherols are a class of lipid-soluble compounds that have multiple functional roles in plants and exhibit vitamin E activity, an essential nutrient for human and animal health. The tocopherol biosynthetic pathway is conserved across the plant kingdom, but source of the key tocopherol pathway precursor, phytol, is unclear. Two protochlorophyllide reductases (POR1 and POR2) were previously identified as loci controlling the natural variation of total tocopherols in maize grain, a non-photosynthetic tissue. POR1 and POR2 are key genes in chlorophyll biosynthesis yet the contribution of the chlorophyll biosynthetic pathway to tocopherol biosynthesis is still not understood.<br><br>RESULTS: We took two approaches to alter the activity of these two POR genes within kernel tissue, physiological treatments and CRISPR/Cas9-mediated knockouts, to determine the role of chlorophyll biosynthesis for tocopherol content. Since light is required for POR enzymatic activity, we imposed a dark treatment on developing kernels, which reduced chlorophyll a and tocopherols levels in embryo tissue by 92-99% and 87-90%, respectively, compared to the light treatment. In CRISPR/Cas9-mediated knockouts, the levels of chlorophyll a and tocopherols in embryos of the por1 por2 double homozygous mutant were reduced by 98-100% and 76-83%, respectively, compared to WT.<br><br>CONCLUSION: These findings demonstrate that tocopherol synthesis in maize grain depends almost entirely on phytol derived from chlorophyll biosynthesis within the embryo. POR1 and POR2 activity play crucial roles in chlorophyll biosynthesis, underscoring the importance of POR alleles and their activity in the biofortification of vitamin E levels in non-photosynthetic grain of maize.}, }
@article {pmid40082732, year = {2025}, author = {Yook, S and Alper, HS}, title = {Recent advances in genetic engineering and chemical production in yeast species.}, journal = {FEMS yeast research}, volume = {25}, number = {}, pages = {}, pmid = {40082732}, issn = {1567-1364}, support = {CBET- 2133661//National Science Foundation/ ; }, mesh = {*Metabolic Engineering/methods ; *Genetic Engineering/methods ; Biofuels ; *Yeasts/genetics/metabolism ; CRISPR-Cas Systems ; Saccharomyces cerevisiae/genetics/metabolism ; Biotechnology/methods ; }, abstract = {Yeasts have emerged as well-suited microbial cell factory for the sustainable production of biofuels, organic acids, terpenoids, and specialty chemicals. This ability is bolstered by advances in genetic engineering tools, including CRISPR-Cas systems and modular cloning in both conventional (Saccharomyces cerevisiae) and non-conventional (Yarrowia lipolytica, Rhodotorula toruloides, Candida krusei) yeasts. Additionally, genome-scale metabolic models and machine learning approaches have accelerated efforts to create a broad range of compounds that help reduce dependency on fossil fuels, mitigate climate change, and offer sustainable alternatives to petrochemical-derived counterparts. In this review, we highlight the cutting-edge genetic tools driving yeast metabolic engineering and then explore the diverse applications of yeast-based platforms for producing value-added products. Collectively, this review underscores the pivotal role of yeast biotechnology in efforts to build a sustainable bioeconomy.}, }
@article {pmid40082551, year = {2025}, author = {Khalili, S and Mohseninia, A and Liu, C and Banister, CE and Heine, P and Khazan, M and Morrison, SE and Gokare, P and Cowley, GS and Weir, BA and Pocalyko, D and Bachman, KE and Buckhaults, PJ}, title = {Comprehensive genomic dependency landscape of a human colon cancer organoid.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {436}, pmid = {40082551}, issn = {2399-3642}, mesh = {Humans ; *Organoids/metabolism/pathology ; *Colonic Neoplasms/genetics/pathology/drug therapy ; CRISPR-Cas Systems ; Genomics/methods ; Mutation ; }, abstract = {Identifying genetic dependencies in human colon cancer could help identify effective treatment strategies. Genome-wide CRISPR-Cas9 dropout screens have the potential to reveal genetic dependencies, some of which could be exploited as therapeutic targets using existing drugs. In this study, we comprehensively characterized genetic dependencies present in a colon cancer organoid avatar, and validated tumor-specific selectivity of select pharmacologic agents. We conducted a genome-wide CRISPR dropout screen to elucidate the genetic dependencies that interacted with select driver somatic mutations. We found distinct genetic dependencies that interacted with WNT, MAPK, PI3K, TP53, and mismatch repair pathways and validated targets that could be exploited as treatments for this specific subtype of colon cancer. These findings demonstrate the utility of functional genomic screening in the context of personalized medicine.}, }
@article {pmid40082411, year = {2025}, author = {Liu, J and Aliaga Goltsman, DS and Alexander, LM and Khayi, KK and Hong, JH and Dunham, DT and Romano, CA and Temoche-Diaz, MM and Chadha, S and Fregoso Ocampo, R and Oki-O'Connell, J and Janson, OP and Turcios, K and Gonzalez-Osorio, L and Muysson, J and Rahman, J and Laperriere, SM and Devoto, AE and Castelle, CJ and Butterfield, CN and Cost, GJ and Brown, CT and Thomas, BC}, title = {Integration of therapeutic cargo into the human genome with programmable type V-K CAST.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {2427}, pmid = {40082411}, issn = {2041-1723}, mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Human/genetics ; *Transposases/genetics/metabolism ; HEK293 Cells ; Transgenes ; *CRISPR-Associated Proteins/genetics/metabolism ; DNA/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {CRISPR-associated (Cas) transposases (CAST) are RNA-guided systems capable of programmable integration of large segments of DNA without creating double-strand breaks. Engineered Cascade CAST function in human cells but are challenging to deploy due to the complexity of the targeting components. Unlike Cascade, which require three Cas proteins, type V-K CAST require a single Cas12k effector for targeting. Here, we show that compact type V-K CAST from uncultivated microbes are repurposable for programmable DNA integration into the genome of human cells. Engineering for nuclear localization and function enables integration of a therapeutically relevant transgene at a safe-harbor site in multiple human cell types. Notably, off-targets are rare events reproducibly found in specific genomic regions. These CAST advancements are expected to accelerate applications of genome editing to therapeutic development, biotechnology, and synthetic biology.}, }
@article {pmid40082271, year = {2025}, author = {Sharma, N and Thakur, K and Zinta, R and Mangal, V and Dalamu,  and Tiwari, JK and Sood, S and Dutt, S and Kumar, V and Singh, B and Thakur, AK}, title = {Genome editing research initiatives and regulatory landscape of genome edited crops in India.}, journal = {Transgenic research}, volume = {34}, number = {1}, pages = {13}, pmid = {40082271}, issn = {1573-9368}, support = {ICAR-Genome Editing Project, Cabin Project, AINP on Biotech Crops//Indian Council of Agricultural Research/ ; }, mesh = {*Crops, Agricultural/genetics/growth &amp; development ; *Gene Editing/legislation &amp; jurisprudence/methods ; India ; *Plants, Genetically Modified/genetics/growth &amp; development ; *Genome, Plant/genetics ; Agriculture/legislation &amp; jurisprudence ; Humans ; CRISPR-Cas Systems ; }, abstract = {Food and nutritional security are the top priorities in Indian agriculture. Exponential population growth coupled with climate change effects has become a serious challenge for sustainable agriculture. Genome editing has revolutionized the agricultural sector because of its ability to create precise, stable and predictable modifications in the genome and therefore, offers great opportunities for crop improvement in India. However, for harvesting the real benefits of this technology in agriculture sector, there is a strong need of creating awareness among the end users and development of suitable policies for regularization of genome edited products. Many regulatory agencies around the world have been modernizing their regulatory approaches to be more risk proportionate and to reflect a more science-based approach. In this article, recent research initiatives and developments undertaken by different Indian institutes/organizations for the genetic improvement of agricultural and horticultural crops via genome editing technologies are summarized. Furthermore, to benefit from this potential technology in our country, regulatory policies must be clear, science-based and proportionate. Therefore, in the present review, the regulatory policies related to the genome editing of crop products in India are discussed in detail. This review will sensitize researchers and stakeholders to the application of genome editing techniques in crop improvement and various biosafety committees involved in the development and regulation of genome edited crops.}, }
@article {pmid40081781, year = {2025}, author = {Wu, X and Wan, X and Yu, H and Liu, H}, title = {Recent advances in CRISPR-Cas system for Saccharomyces cerevisiae engineering.}, journal = {Biotechnology advances}, volume = {81}, number = {}, pages = {108557}, doi = {10.1016/j.biotechadv.2025.108557}, pmid = {40081781}, issn = {1873-1899}, mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Metabolic Engineering/methods ; }, abstract = {Yeast Saccharomyces cerevisiae (S. cerevisiae) is a crucial industrial platform for producing a wide range of chemicals, fuels, pharmaceuticals, and nutraceutical ingredients. It is also commonly used as a model organism for fundamental research. In recent years, the CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) system has become the preferred technology for genetic manipulation in S. cerevisiae owing to its high efficiency, precision, and user-friendliness. This system, along with its extensive toolbox, has significantly accelerated the construction of pathways, enzyme optimization, and metabolic engineering in S. cerevisiae. Furthermore, it has allowed researchers to accelerate phenotypic evolution and gain deeper insights into fundamental biological questions, such as genotype-phenotype relationships. In this review, we summarize the latest advancements in the CRISPR-Cas toolbox for S. cerevisiae and highlight its applications in yeast cell factory construction and optimization, enzyme and phenotypic evolution, genome-scale functional interrogation, gene drives, and the advancement of biotechnologies. Finally, we discuss the challenges and potential for further optimization and applications of the CRISPR-Cas system in S. cerevisiae.}, }
@article {pmid40081542, year = {2025}, author = {Salvesen, HA and Dearden, PK}, title = {Genome editing in hymenoptera.}, journal = {Insect biochemistry and molecular biology}, volume = {180}, number = {}, pages = {104300}, doi = {10.1016/j.ibmb.2025.104300}, pmid = {40081542}, issn = {1879-0240}, mesh = {Animals ; *Gene Editing/methods ; *Hymenoptera/genetics ; *Genome, Insect ; CRISPR-Cas Systems ; }, abstract = {The application of genome editing tools in Hymenoptera has transformative potential for functional genetics and understanding their unique biology. Hymenoptera comprise one of the most diverse Orders of animals, and the development of methods for efficiently creating precise genome modifications could have applications in conservation, pest management and agriculture. To date, sex determination, DNA methylation, taste and smell sensory systems as well as phenotypic markers have been selected for gene editing investigations. From these data, insights into eusociality, the nature of haplodiploidy and the complex communication systems that Hymenoptera possess have provided an understanding of their evolutionary history that has led them to become so diverse and successful. Insights from these functional genetics analyses have been supported by the ever-improving suite of CRIPSR tools and further expansion will allow more specific biological hypotheses to be tested and applications beyond the lab. Looking ahead, genome editing tools have potential for Hymenopteran applications in modifying biocontrol agents of agricultural pests and for use in managing invasive species through the development of technologies such as gene drives. This review provides accessibility to information regarding the status of Hymenopteran genome editing, intending to support the considered development of CRISPR tools in novel species as well as innovation and refinement of methods in species in which it has already been achieved.}, }
@article {pmid40081369, year = {2025}, author = {Binan, L and Jiang, A and Danquah, SA and Valakh, V and Simonton, B and Bezney, J and Manguso, RT and Yates, KB and Nehme, R and Cleary, B and Farhi, SL}, title = {Simultaneous CRISPR screening and spatial transcriptomics reveal intracellular, intercellular, and functional transcriptional circuits.}, journal = {Cell}, volume = {188}, number = {8}, pages = {2141-2158.e18}, pmid = {40081369}, issn = {1097-4172}, support = {R01 MH128366/MH/NIMH NIH HHS/United States ; RF1 MH121289/MH/NIMH NIH HHS/United States ; }, mesh = {Humans ; Animals ; *Transcriptome/genetics ; Induced Pluripotent Stem Cells/metabolism/cytology ; Single-Cell Analysis/methods ; Astrocytes/metabolism/cytology ; Mice ; *CRISPR-Cas Systems/genetics ; Monocytes/metabolism ; *Gene Regulatory Networks ; Autism Spectrum Disorder/genetics ; Immunity, Innate ; Gene Expression Profiling/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Lipopolysaccharides/pharmacology ; }, abstract = {Pooled optical screens have enabled the study of cellular interactions, morphology, or dynamics at massive scale, but they have not yet leveraged the power of highly plexed single-cell resolved transcriptomic readouts to inform molecular pathways. Here, we present a combination of imaging spatial transcriptomics with parallel optical detection of in situ amplified guide RNAs (Perturb-FISH). Perturb-FISH recovers intracellular effects that are consistent with single-cell RNA-sequencing-based readouts of perturbation effects (Perturb-seq) in a screen of lipopolysaccharide response in cultured monocytes, and it uncovers intercellular and density-dependent regulation of the innate immune response. Similarly, in three-dimensional xenograft models, Perturb-FISH identifies tumor-immune interactions altered by genetic knockout. When paired with a functional readout in a separate screen of autism spectrum disorder risk genes in human-induced pluripotent stem cell (hIPSC) astrocytes, Perturb-FISH shows common calcium activity phenotypes and their associated genetic interactions and dysregulated molecular pathways. Perturb-FISH is thus a general method for studying the genetic and molecular associations of spatial and functional biology at single-cell resolution.}, }
@article {pmid40080408, year = {2025}, author = {Simwela, NV and Jaecklein, E and Sassetti, CM and Russell, DG}, title = {Impaired fatty acid import or catabolism in macrophages restricts intracellular growth of Mycobacterium tuberculosis.}, journal = {eLife}, volume = {13}, number = {}, pages = {}, pmid = {40080408}, issn = {2050-084X}, support = {AI155319//National Institute of Allergy and Infectious Diseases/ ; AI162598//National Institute of Allergy and Infectious Diseases/ ; OD032135//NIH Office of the Director/ ; T32AI007349//National Institute of Allergy and Infectious Diseases/ ; }, mesh = {*Mycobacterium tuberculosis/growth &amp; development/metabolism ; *Macrophages/microbiology/metabolism ; *Fatty Acids/metabolism ; Mice ; Animals ; Lipid Metabolism ; Humans ; *Tuberculosis/microbiology ; CRISPR-Cas Systems ; Autophagy ; }, abstract = {Mycobacterium tuberculosis (Mtb) infection of macrophages reprograms cellular metabolism to promote lipid retention. While it is clearly known that intracellular Mtb utilize host-derived lipids to maintain infection, the role of macrophage lipid processing on the bacteria's ability to access the intracellular lipid pool remains undefined. We utilized a CRISPR-Cas9 genetic approach to assess the impact of sequential steps in fatty acid metabolism on the growth of intracellular Mtb. Our analyses demonstrate that macrophages that cannot either import, store, or catabolize fatty acids restrict Mtb growth by both common and divergent antimicrobial mechanisms, including increased glycolysis, increased oxidative stress, production of pro-inflammatory cytokines, enhanced autophagy, and nutrient limitation. We also show that impaired macrophage lipid droplet biogenesis is restrictive to Mtb replication, but increased induction of the same fails to rescue Mtb growth. Our work expands our understanding of how host fatty acid homeostasis impacts Mtb growth in the macrophage.}, }
@article {pmid40078539, year = {2025}, author = {Sailer, AL and Brendel, J and Chernev, A and König, S and Bischler, T and Gräfenhan, T and Urlaub, H and Gophna, U and Marchfelder, A}, title = {Internal in-frame translation generates Cas11b, which is important for effective interference in an archaeal CRISPR-Cas system.}, journal = {Frontiers in microbiology}, volume = {16}, number = {}, pages = {1543464}, pmid = {40078539}, issn = {1664-302X}, abstract = {CRISPR-Cas is a sophisticated defence system used by bacteria and archaea to fend off invaders. CRISPR-Cas systems vary in their Cas protein composition and have therefore been divided into different classes and types. Type I systems of bacteria have been shown to contain the small protein Cas11 as part of the interference complex known as Cascade. Here we show for the first time that an archaeal CRISPR-Cas type I-B system also contains a homolog of Cas11. The Cas11b protein, encoded by the cas8b gene in Haloferax volcanii, represents the first known case of an internal in-frame translation of an archaeal protein. Translation initiation at an internal methionine of the cas8b open reading frame results in synthesis of Cas11b. Cas11b is required for an effective CRISPR-Cas interference reaction, and in its absence fewer Cascade complexes are formed. Comparison of transcriptomes from wild type and a Cas11b-less strain shows that the depletion of Cas11b also results in differential transcript abundance of many genes, presumably affecting their regulation. Taken together, Cas11b is important for the defence reaction of the type I-B CRISPR-Cas system and seems to play an additional cellular role.}, }
@article {pmid40078160, year = {2025}, author = {Wu, Z and Li, J and Zhang, T and Zhang, K and Liu, X and Yang, Z and Xu, L and Han, K}, title = {One-pot synthesized three-way junction based multiple strand displacement amplification for sensitive assay of H5N1 DNA.}, journal = {The Analyst}, volume = {150}, number = {8}, pages = {1541-1552}, doi = {10.1039/d4an01586j}, pmid = {40078160}, issn = {1364-5528}, mesh = {Humans ; *Biosensing Techniques/methods ; CRISPR-Associated Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *DNA, Viral/analysis/genetics ; Endodeoxyribonucleases ; *Influenza A Virus, H5N1 Subtype/genetics/isolation &amp; purification ; Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins ; }, abstract = {The rapid and sensitive detection of H5N1, a highly pathogenic avian influenza virus, is crucial for controlling its spread and minimizing its impact on public health. In this study, we developed a novel biosensor based on strand displacement amplification (SDA) coupled with CRISPR/Cas12a for highly sensitive detection of H5N1 DNA. The biosensor utilizes a combination of a three-way junction structure, composed of three hairpins (H1, H2, H3), to initiate amplification through SDA, resulting in the production of numerous activators. These activators then trigger CRISPR/Cas12a's collateral cleavage activity, which generates a detectable fluorescence signal. The biosensor demonstrated a linear detection range from 100 fM to 800 pM, with a detection limit as low as 72.87 fM. The optimized biosensor exhibited excellent sensitivity, high specificity, and a broad dynamic range, making it a promising tool for the early detection of H5N1 DNA in complex biological samples. Additionally, the use of CRISPR/Cas12a's trans-cleavage activity significantly improved signal amplification and specificity, allowing for more reliable detection compared to traditional methods. The results highlight the advantages of the integrated SDA and CRISPR/Cas12a approach, which addresses the limitations of conventional detection methods, such as low sensitivity, lengthy analysis times, and high costs. The biosensor's ability to perform well in complex sample matrices demonstrates its potential for point-of-care diagnostics, especially in resource-limited settings. Future applications of this technology could extend to the detection of other pathogens, offering a versatile and adaptable platform for disease surveillance and management.}, }
@article {pmid40076976, year = {2025}, author = {Song, B}, title = {Efforts to Downsize Base Editors for Clinical Applications.}, journal = {International journal of molecular sciences}, volume = {26}, number = {5}, pages = {}, pmid = {40076976}, issn = {1422-0067}, support = {20231291//Soonchunhyang University/ ; 2021R1I1A1A01056885//National Research Foundation of Korea/ ; }, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; Genetic Therapy/methods ; Animals ; Dependovirus/genetics ; DNA Breaks, Double-Stranded ; }, abstract = {Since the advent of the clustered regularly interspaced short palindromic repeats (CRISPR) system in the gene editing field, diverse CRISPR-based gene editing tools have been developed for treating genetic diseases. Of these, base editors (BEs) are promising because they can carry out precise gene editing at single-nucleotide resolution without inducing DNA double-strand breaks (DSBs), which pose significant risks of genomic instability. Despite their outstanding advantages, the clinical application of BEs remains challenging due to their large size, which limits their efficient delivery, particularly in adeno-associated virus (AAV)-based systems. To address this issue, various strategies have been explored to reduce the size of BEs. These approaches include truncating the nonessential domains and replacing the bulky components with smaller substitutes without compromising the editing efficiency. In this review, we highlight the importance of downsizing BEs for therapeutic applications and introduce recent advances in size-reduction strategies. Additionally, we introduce the ongoing efforts to overcome other limitations of BEs, providing insights into their potential for improving in vivo gene editing.}, }
@article {pmid40076923, year = {2025}, author = {Ajayi, GO and Ma, A and Modarai, SR and Opdenaker, LM and Sims-Mourtada, J}, title = {CRISPR/Cas9 Targeting of Aldehyde Dehydrogenase 1A1 Reveals Heterogeneous Roles in Radiation Response and Redox Stress Across Clonal Lines in Triple-Negative Breast Cancer.}, journal = {International journal of molecular sciences}, volume = {26}, number = {5}, pages = {}, pmid = {40076923}, issn = {1422-0067}, support = {P20 GM103446/GM/NIGMS NIH HHS/United States ; P20GM103446//NIH/NIGMS/ ; }, mesh = {Humans ; *Triple Negative Breast Neoplasms/genetics/metabolism/radiotherapy/pathology ; *CRISPR-Cas Systems ; *Aldehyde Dehydrogenase 1 Family/genetics/metabolism ; Female ; Cell Line, Tumor ; *Retinal Dehydrogenase/genetics/metabolism ; *Radiation Tolerance/genetics ; Reactive Oxygen Species/metabolism ; Oxidation-Reduction ; Cell Survival/radiation effects/genetics ; Oxidative Stress ; }, abstract = {The metabolic enzyme aldehyde dehydrogenase 1A1 (ALDH1A1), a cancer stem cell marker associated with poor outcomes in breast cancer, has emerged as a promising therapeutic target in TNBC. The aim of this study was to investigate the role of ALDH1A1 in radiation resistance and redox stress in triple negative breast cancer (TNBC). Functional knockouts of ALDH1A1 were generated by the CRISPR/Cas9-mediated deletion of ALDH1A1 in the SUM159 cell line, and three distinct clonal populations were isolated. Genetic targeting was confirmed by Sanger sequencing, and the loss of ALDH1A1 protein expression was validated by Western blotting. Functional assays assessed ALDEFLUOR activity, cell viability, self-renewal capacity, and reactive oxygen species (ROS) levels with or without radiation in both the bulk population and clonal lines. Interestingly, ALDEFLUOR activity was uniformly lost across all clonal lines; however, functional effects of ALDH1A1 loss on redox stress, survival, and radiation sensitivity were observed in only one clonal population. These findings highlight significant variability in the role of ALDH1A1 among clonal populations, reflecting the complexity of tumor heterogeneity. This underscores the importance of accounting for tumor heterogeneity when targeting ALDH1A1, as certain TNBC subpopulations may rely more heavily on ALDH1A1 function. These insights are critical for developing effective ALDH1A1-targeted therapies.}, }
@article {pmid40076628, year = {2025}, author = {Mosterd, C and Moineau, S}, title = {Insight into crRNA Processing in Streptococcus mutans P42S and Application of SmutCas9 in Genome Editing.}, journal = {International journal of molecular sciences}, volume = {26}, number = {5}, pages = {}, pmid = {40076628}, issn = {1422-0067}, support = {NSERC Discovery//Natural Sciences and Engineering Research Council/ ; }, mesh = {*Streptococcus mutans/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism ; Genome, Bacterial ; RNA, Guide, CRISPR-Cas Systems/genetics ; *RNA, Bacterial/genetics/metabolism ; }, abstract = {CRISPR-Cas is an adaptive immune system found in bacteria and archaea that provides resistance against invading nucleic acids. Elements of this natural system have been harnessed to develop several genome editing tools, including CRISPR-Cas9. This technology relies on the ability of the nuclease Cas9 to cut DNA at specific locations directed by a guide RNA. In addition, the nuclease activity of Cas9 requires the presence of a short nucleotide motif (5'-NGG-3' for Cas9 from Streptococcus pyogenes) called PAM, flanking the targeted region. As the reliance on this PAM is typically strict, diverse Cas9 variants recognising different PAM motifs have been studied to target a broader range of genomic sites. In this study, we assessed the potential of Cas9 from Streptococcus mutans strain P42S (SmutCas9) in gene editing. SmutCas9 recognises the rarely targeted 5'-NAA-3' and 5'-NGAA-3' PAMs. To test its efficacy, two genes of the virulent lactococcal phage p2 were edited, thereby demonstrating the potential of SmutCas9 for gene editing purposes, particularly in AT-rich genomes. Sequencing of total RNA also revealed the RNA components of this system, allowing further molecular characterisation of the type II-A CRISPR-Cas system of S. mutans.}, }
@article {pmid40076433, year = {2025}, author = {Lynch, CRH and Drummond, RSM and Jelley, L and Baker, L and Smit, E and Fleming, R and Billington, C}, title = {Optimization and Benchmarking of RT-LAMP-CRISPR-Cas12a for the Detection of SARS-CoV-2 in Saliva.}, journal = {International journal of molecular sciences}, volume = {26}, number = {5}, pages = {}, pmid = {40076433}, issn = {1422-0067}, support = {C03X1904//Ministry of Business, Innovation and Employment/ ; ESRCBRF2022/1//Ministry of Business, Innovation and Employment/ ; }, mesh = {Humans ; *SARS-CoV-2/genetics/isolation &amp; purification ; *COVID-19/diagnosis/virology ; *Saliva/virology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; RNA, Viral/genetics ; Benchmarking ; *COVID-19 Nucleic Acid Testing/methods ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {Resource-limited settings and supply chain difficulties faced throughout the COVID-19 pandemic prompted the development of rapid and alternative methods of detecting SARS-CoV-2. These methods include reverse-transcription loop-mediated isothermal amplification (RT-LAMP), reverse-transcription recombinase polymerase amplification (RT-RPA), and CRISPR-Cas12a fluorescence detection. We describe RT-LAMP, RT-RPA, and CRISPR-Cas12a assays for the detection of the N and E-gene amplicons of SARS-CoV-2 and the optimization of various assay components, including incubation temperatures, Cas12a enzymes, reporter molecules, and the use of a lyophilized RT-LAMP master mix. We also describe the testing of a one-tube RT-LAMP-CRISPR-Cas12a assay. The one-tube assay showed promise in reducing hands-on time and improving time-to-result. We found no improvements in assay sensitivity with RT-RPA, but did achieve detection at a lower copy number with the lyophilized RT-LAMP master mix compared to liquid reagent (50 vs. 100 copies at 20 min). When used to detect the presence of SARS-CoV-2 RNA in clinical saliva samples from 75 infected patients, the discriminatory ability of the optimized RT-LAMP-CRISPR Cas12a assay was found to be comparable with RT-qPCR, with a minor reduction in sensitivity.}, }
@article {pmid40076283, year = {2025}, author = {Hofacker, DT and Kalkuhl, S and Schmid, JF and Singh, S and Stafforst, T}, title = {A Simplified Guide RNA Synthesis Protocol for SNAP- and Halo-Tag-Based RNA Editing Tools.}, journal = {Molecules (Basel, Switzerland)}, volume = {30}, number = {5}, pages = {}, pmid = {40076283}, issn = {1420-3049}, mesh = {*RNA Editing ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemical synthesis/chemistry ; Adenosine Deaminase/genetics/metabolism ; RNA-Binding Proteins/genetics/metabolism ; }, abstract = {SNAP-tag and Halo-tag have been employed to achieve targeted RNA editing by directing the deaminase domain of human ADAR to specific sites in the transcriptome. This targeting is facilitated by short guide RNAs (gRNAs) complementary to the target transcript, which are chemically modified with benzylguanine or chloroalkane moieties to enable covalent binding to the respective self-labeling enzymes. However, broad application of this approach has been limited by challenges such as low scalability, the requirement for specialized chemical expertise and equipment, and labor-intensive protocols. In this study, we introduce streamlined, efficient protocols for the synthesis and purification of these linkers, suitable for SNAP-tag and Halo-tag applications, without the need for advanced chemical equipment. Our methods enable linker coupling in a kit-like manner and support the high-yield production of modified gRNAs. We demonstrate that the newly synthesized linkers and gRNA designs perform similarly to previously published constructs with regard to RNA editing efficiency. Moreover, large-scale production of modified gRNAs facilitates their use in studies involving cellular uptake and in vivo experiments.}, }
@article {pmid40075351, year = {2025}, author = {Huang, Z and Zhao, X and Jiang, Z and Qiu, X and Sun, X and Wang, D and Zhang, H and Chen, Q and Tan, R and Shen, Y}, title = {IP6K2 mutations as a novel mechanism of resistance to oncolytic virus therapy.}, journal = {Journal of translational medicine}, volume = {23}, number = {1}, pages = {311}, pmid = {40075351}, issn = {1479-5876}, support = {U22A20326//National Natural Science Foundation of China/ ; }, mesh = {Humans ; *Oncolytic Virotherapy ; Animals ; *Mutation/genetics ; *Oncolytic Viruses/physiology ; Apoptosis/genetics ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; Cell Proliferation ; Virus Replication ; Herpesvirus 1, Human/physiology ; CRISPR-Cas Systems/genetics ; Signal Transduction ; Mice ; Neoplasms/therapy/genetics ; }, abstract = {BACKGROUND: Oncolytic virus therapy (OVT) represents a promising frontier in cancer treatment. Despite its efficacy in clinical trials, variability in patient response, particularly resistance development, highlights the need for tailored therapeutic strategies.<br><br>METHODS: The Inositol Hexakisphosphate Kinase 2 (IP6K2) gene knock out was carried by CRISPR/Cas9 system. The evaluation of biomarkers of apoptosis and relevant pathways was conducted to be assessed. Attachment assay was conducted to verify the binding ability of virus to the host cells. Cell proliferation and apoptosis was assessed. Subcutaneous xenograft model was used to evaluate IP6K2 knock out influence in vivo. cBioPortal and TCGA database were applied to analyze genomic alterations in pan-cancer.<br><br>RESULTS: IP6K2 was essential for effective Herpes Simplex Virus Type1 (HSV-1) replication and subsequent cell apoptosis, acting through the tumor Protein p53 (p53) and Cyclin-Dependent Kinase Inhibitor 1&#xa0;A (p21) signaling axis. The tumor model demonstrated that tumors lacking IP6K2 exhibited resistance to HSV-1 oncolysis, resulting in diminished therapeutic outcomes. Analysis of cBioPortal and TCGA databases corroborated the potential resistance stemming from IP6K2 mutations across various cancer types, underscoring the necessity for pre-treatment IP6K2 status assessment.<br><br>CONCLUSIONS: This study underscores the role of IP6K2 as potential markers of resistance, which opens avenues for precision medicine approaches in OVT.}, }
@article {pmid40075056, year = {2025}, author = {Yang, J and Wang, T and Huang, Y and Long, Z and Li, X and Zhang, S and Zhang, L and Liu, Z and Zhang, Q and Sun, H and Zhang, M and Yin, H and Liu, Z and Zhang, H}, title = {Insights into the compact CRISPR-Cas9d system.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {2462}, pmid = {40075056}, issn = {2041-1723}, mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry ; Cryoelectron Microscopy ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry/ultrastructure ; DNA Cleavage ; Gene Editing ; DNA/metabolism ; *Bacterial Proteins/metabolism/genetics/chemistry ; Models, Molecular ; Humans ; }, abstract = {Cas9d, the smallest known member of the Cas9 family, employs a compact domain architecture for effective target cleavage. However, the underlying mechanism remains unclear. Here, we present the cryo-EM structures of the Cas9d-sgRNA complex in both target-free and target-bound states. Biochemical assays elucidated the PAM recognition and DNA cleavage mechanisms of Cas9d. Structural comparisons revealed that at least 17 base pairs in the guide-target heteroduplex is required for nuclease activity. Beyond its typical role as an adaptor between Cas9 enzymes and targets, the sgRNA also provides structural support and functional regulation for Cas9d. A segment of the sgRNA scaffold interacts with the REC domain to form a functional target recognition module. Upon target binding, this module undergoes a coordinated conformational rearrangement, enabling heteroduplex propagation and facilitating nuclease activity. This hybrid functional module precisely monitors heteroduplex complementarity, resulting in a lower mismatch tolerance compared to SpyCas9. Moreover, structure-guided engineering in both the sgRNA and Cas9d protein led to a more compact Cas9 system with well-maintained nuclease activity. Altogether, our findings provide insights into the target recognition and cleavage mechanisms of Cas9d and shed light on the development of high-fidelity mini-CRISPR tools.}, }
@article {pmid40073844, year = {2025}, author = {Bi, K and Yates, KB}, title = {Parts and ICRAFTs: Finding new immunotherapy targets.}, journal = {Immunity}, volume = {58}, number = {3}, pages = {529-531}, doi = {10.1016/j.immuni.2025.02.016}, pmid = {40073844}, issn = {1097-4180}, mesh = {Humans ; *Immunotherapy/methods ; *Neoplasms/therapy/immunology/genetics ; Tumor Necrosis Factor alpha-Induced Protein 3/genetics ; Animals ; CRISPR-Cas Systems ; Mice ; }, abstract = {CRISPR screens are widely utilized to identify genes that regulate immune function or mediate sensitivity of cancer cells to immune attack. In this issue of Immunity, Zeng et al. present a computational framework for uncovering gene targets with dual function in both cancer and immune cells and nominate TNFAIP3 as a synergistic target whose ablation strongly elicits an antitumor response.}, }
@article {pmid40073612, year = {2025}, author = {Durazo-Martínez, K and Chaudhari, J and Sherry, LM and Webster, DA and Martins, K and Bostrom, JR and Carlson, DF and Sonstegard, TS and Vu, HLX}, title = {Modification of the splice acceptor in CD163 exon 7 of pigs is insufficient to confer resistance to PRRSV.}, journal = {Veterinary microbiology}, volume = {304}, number = {}, pages = {110450}, doi = {10.1016/j.vetmic.2025.110450}, pmid = {40073612}, issn = {1873-2542}, mesh = {Animals ; Antigens, CD/genetics ; Antigens, Differentiation, Myelomonocytic/genetics ; *CD163 Antigen ; CRISPR-Cas Systems ; Disease Resistance/genetics ; Exons/genetics ; Gene Editing ; *Porcine Reproductive and Respiratory Syndrome/genetics/immunology/virology ; *Porcine respiratory and reproductive syndrome virus/physiology ; *Receptors, Cell Surface/genetics ; *RNA Splice Sites/genetics ; RNA, Messenger/genetics ; Swine ; }, abstract = {CD163 is the primary receptor for PRRSV, and its SRCR5 domain, encoded by exon 7, is crucial for supporting PRRSV infection. Previous studies have used CRISPR/Cas9 technology to remove exon 7 from the host genome, and the edited pigs were completely resistant to PRRSV infection. In this study, we used CRISPR/Cas9 technology mimicking an adenine base editor (ABE) to edit the splice acceptor site of exon 7, rendering it nonfunctional. This alteration was intended to cause exon 6 to join directly to exon 8 during mRNA processing, resulting in a mature mRNA transcript that lacks exon 7, which encodes the SRCR5 domain. Piglets carrying the exon 7 splice site modification (CD163Ex7-ABE) were successfully generated. However, these pigs remained fully susceptible to infection with a PRRSV-2 isolate. Analysis of CD163 mRNA from the CD163Ex7-ABE pigs revealed that they predominantly expressed a mature CD163 mRNA lacking exon 7. However, due to cryptic splice sites, two additional mRNA isoforms were expressed, including an in-frame variant containing all of exon 7 and an extra 48 base pairs. This likely resulted in the expression of a full-length CD163 with a 16-amino-acid insertion upstream of the SRCR5 domain, which was sufficient to render the animals susceptible to PRRSV. Overall, our results demonstrate that merely modifying the splice acceptor site of CD163 exon 7 is not sufficient to generate PRRSV-resistant pigs.}, }
@article {pmid40073141, year = {2025}, author = {Chiang, JC and Shang, Z and Rosales, T and Cai, L and Chen, WM and Cai, F and Vu, H and Minna, JD and Ni, M and Davis, AJ and Timmerman, RD and DeBerardinis, RJ and Zhang, Y}, title = {Lipoylation inhibition enhances radiation control of lung cancer by suppressing homologous recombination DNA damage repair.}, journal = {Science advances}, volume = {11}, number = {11}, pages = {eadt1241}, pmid = {40073141}, issn = {2375-2548}, mesh = {Humans ; *Recombinational DNA Repair/drug effects/radiation effects ; *Lung Neoplasms/genetics/metabolism/radiotherapy/pathology ; Animals ; *Lipoylation/drug effects ; Cell Line, Tumor ; Mice ; DNA Damage ; *Carcinoma, Non-Small-Cell Lung/genetics/radiotherapy/metabolism/pathology ; Acyltransferases/metabolism/genetics ; Histones/metabolism ; CRISPR-Cas Systems ; Ataxia Telangiectasia Mutated Proteins/metabolism ; }, abstract = {Lung cancer exhibits altered metabolism, influencing its response to radiation. To investigate the metabolic regulation of radiation response, we conducted a comprehensive, metabolic-wide CRISPR-Cas9 loss-of-function screen using radiation as selection pressure in human non-small cell lung cancer. Lipoylation emerged as a key metabolic target for radiosensitization, with lipoyltransferase 1 (LIPT1) identified as a top hit. LIPT1 covalently conjugates mitochondrial 2-ketoacid dehydrogenases with lipoic acid, facilitating enzymatic functions involved in the tricarboxylic acid cycle. Inhibiting lipoylation, either through genetic LIPT1 knockout or a lipoylation inhibitor (CPI-613), enhanced tumor control by radiation. Mechanistically, lipoylation inhibition increased 2-hydroxyglutarate, leading to H3K9 trimethylation, disrupting TIP60 recruitment and ataxia telangiectasia mutated (ATM)-mediated DNA damage repair signaling, impairing homologous recombination repair. In summary, our findings reveal a critical role of LIPT1 in regulating DNA damage and chromosome stability and may suggest a means to enhance therapeutic outcomes with DNA-damaging agents.}, }
@article {pmid40073054, year = {2025}, author = {Butterfield, GL and Rohm, D and Roberts, A and Nethery, MA and Rizzo, AJ and Morone, DJ and Garnier, L and Iglesias, N and Barrangou, R and Gersbach, CA}, title = {Characterization of diverse Cas9 orthologs for genome and epigenome editing.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {122}, number = {11}, pages = {e2417674122}, pmid = {40073054}, issn = {1091-6490}, support = {U01AI146356//HHS | NIH (NIH)/ ; UM1HG012053//HHS | NIH (NIH)/ ; R01MH125236//HHS | NIH (NIH)/ ; RM1HG011123//HHS | NIH (NIH)/ ; EFMA-1830957//NSF (NSF)/ ; HR0011-19-2-0008//DOD | ARPA | Defense Sciences Office, DARPA (DSO)/ ; }, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Epigenome/genetics ; HEK293 Cells ; Streptococcus pyogenes/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Staphylococcus aureus/genetics ; Epigenome Editing ; }, abstract = {CRISPR-Cas9 systems have revolutionized biotechnology, creating diverse new opportunities for biomedical research and therapeutic genome and epigenome editing. Despite the abundance of bacterial CRISPR-Cas9 systems, relatively few are effective in human cells, limiting the overall potential of CRISPR technology. To expand the CRISPR-Cas toolbox, we characterized a set of type II CRISPR-Cas9 systems from select bacterial genera and species encoding diverse Cas9s. Four systems demonstrated robust and specific gene repression in human cells when used as nuclease-null dCas9s fused with a KRAB domain and were also highly active nucleases in human cells. These systems have distinct protospacer adjacent motifs (PAMs), including AT-rich motifs and sgRNA features orthogonal to the commonly used Staphylococcus aureus and Streptococcus pyogenes Cas9s. Additionally, we assessed gene activation when fused with the p300 catalytic domain. Notably, S. uberis Cas9 performed competitively against benchmarks with promising repression, activation, nuclease, and base editing activity. This study expands the CRISPR-Cas9 repertoire, enabling effective genome and epigenome editing for diverse applications.}, }
@article {pmid40071935, year = {2025}, author = {Stagno, JR and Deme, JC and Dwivedi, V and Lee, YT and Lee, HK and Yu, P and Chen, SY and Fan, L and Degenhardt, MFS and Chari, R and Young, HA and Lea, SM and Wang, YX}, title = {Structural investigation of an RNA device that regulates PD-1 expression in mammalian cells.}, journal = {Nucleic acids research}, volume = {53}, number = {5}, pages = {}, pmid = {40071935}, issn = {1362-4962}, support = {/RC/CCR NIH HHS/United States ; /CA/NCI NIH HHS/United States ; /NH/NIH HHS/United States ; HHS75N91019D00024/HH/HHS/United States ; P30GM133893/GM/NIGMS NIH HHS/United States ; KP1605010//Department of Energy's Office of Biological and Environmental Research/ ; DE-SC0012704//Department of Energy Basic Energy Sciences Program/ ; }, mesh = {*Programmed Cell Death 1 Receptor/genetics/metabolism/chemistry ; Animals ; Mice ; Tetracycline/pharmacology ; *Gene Expression Regulation/drug effects ; Humans ; Nucleic Acid Conformation ; CRISPR-Cas Systems ; Lymphocytes/metabolism/drug effects ; *RNA/chemistry/genetics ; }, abstract = {Synthetic RNA devices are engineered to control gene expression and offer great potential in both biotechnology and clinical applications. Here, we present multidisciplinary structural and biochemical data for a tetracycline (Tc)-responsive RNA device (D43) in both ligand-free and bound states, providing a structure-dynamical basis for signal transmission. Activation of self-cleavage is achieved via ligand-induced conformational and dynamical changes that stabilize the elongated bridging helix harboring the communication module, which drives proper coordination of the catalytic residues. We then show the utility of CRISPR-integrated D43 in EL4 lymphocytes to regulate programmed cell death protein 1 (PD-1), a key receptor of immune checkpoints. Treatment of these cells with Tc showed a dose-dependent reduction in PD-1 by immunostaining and a decrease in messenger RNA levels by quantitative PCR as compared with wild type. PD-1 expression was recoverable upon removal of Tc. These results provide mechanistic insight into RNA devices with potential for cancer immunotherapy or other applications.}, }
@article {pmid40070114, year = {2025}, author = {Bhargava, CN and Ashok, K and Pradhan, SK and Kumar, S and Manamohan, M and Rai, A and Asokan, R}, title = {CRISPR/Cas9 Mediated Editing of Bdtektin1 Gene Induces Sterility in Male Oriental Fruit Fly, Bactrocera dorsalis (Diptera: Tephritidae).}, journal = {Archives of insect biochemistry and physiology}, volume = {118}, number = {3}, pages = {e70043}, doi = {10.1002/arch.70043}, pmid = {40070114}, issn = {1520-6327}, support = {//The current research work has been carried out on the funding received from the ICAR-Network Project on Agricultural Bioinformatics and Computational Biology and is highly acknowledged./ ; }, mesh = {Animals ; *Tephritidae/genetics/physiology ; Male ; *CRISPR-Cas Systems ; *Insect Proteins/genetics/metabolism ; *Gene Editing ; Female ; Pest Control, Biological ; Spermatogenesis/genetics ; Infertility, Male/genetics ; }, abstract = {The Oriental fruit fly, Bactrocera dorsalis (B. dorsalis) is a highly invasive, widely distributed notorious pest restricting global fruit trade immensely. There are several approaches to managing this pest, still require newer approaches. In this regard, recently a novel approach called precision-guided sterile insect technique (pgSIT) is gaining momentum in inducing both female sex elimination or sex conversion and male sterility at one go. Developing a species-specific pgSIT system requires validation of targets such as sex determination and spermatogenesis genes. In this regard, B. dorsalis is highly amenable for area-wide pest management and in the present study, we have validated the loss-of-function of the spermatogenesis-related gene, tektin1 using the CRISPR/Cas9 ribonucleoprotein (RNP) complex. This gene was cloned from the local isolate of B. dorsalis and two promising single guide RNAs (sgRNAs) were designed and validated through in vitro restriction analysis. Injection of the RNP complex (sgRNA + Cas9 protein) into the G0 embryo resulted in three adult males carrying mutations at the target site. The phenotype of the mutants was determined through crossing studies, namely, △1♂ × WT ♀, △2♂ × WT ♀, △3♂ × WT ♀, and WT ♂ × WT ♀ and that showed hatching rates of 0%, 11.70%, 0%, and 45.12%, respectively. The mutant males had more nonviable sperm as compared to control. This study underscores the pivotal role of the Bdtektin1 gene for male fertility and is a promising candidate for further development of pgSIT system for B. dorsalis.}, }
@article {pmid40070071, year = {2025}, author = {Manchanda, D and Kumar, S and Makhija, M}, title = {Polysaccharide-Based Delivery Systems for CRISPR/Cas Gene Therapy: Overcoming Challenges and Advancing Pharmaceutical Solutions.}, journal = {Current gene therapy}, volume = {}, number = {}, pages = {}, doi = {10.2174/0115665232369121250307075817}, pmid = {40070071}, issn = {1875-5631}, abstract = {The advent of CRISPR/Cas gene-editing technology has revolutionized molecular biology, offering unprecedented precision and potential in treating genetic disorders, cancers, and other complex diseases. However, for CRISPR/Cas to be truly effective in clinical settings, one of the most significant challenges lies in the delivery of the CRISPR components, including guide RNA (gRNA) and Cas protein, into specific cells or tissues. Safe, targeted, and efficient delivery remains a critical bottleneck. Viral vectors, lipid nanoparticles, and synthetic polymers have been explored, but they come with limitations, such as immunogenicity, toxicity, and limited delivery capacity. Polysaccharide-based delivery systems, with their natural origin, biocompatibility, and versatile chemical properties, offer a promising alternative that could address these delivery challenges while advancing the pharmaceutical applications of CRISPR/Cas gene therapy.}, }
@article {pmid40069752, year = {2025}, author = {Wang, M and Fu, P and Chen, Z and Wang, X and Ma, H and Zhang, X and Gao, G}, title = {Recruitment and rejoining of remote double-strand DNA breaks for enhanced and precise chromosome editing.}, journal = {Genome biology}, volume = {26}, number = {1}, pages = {53}, pmid = {40069752}, issn = {1474-760X}, support = {2023YFC3402000//National Key R&amp;D Program of China/ ; }, mesh = {*Gene Editing/methods ; *DNA Breaks, Double-Stranded ; Humans ; CRISPR-Cas Systems ; Homologous Recombination ; DNA End-Joining Repair ; Recombinational DNA Repair ; }, abstract = {Chromosomal rearrangements, such as translocations, deletions, and inversions, underlie numerous genetic diseases and cancers, yet precise engineering of these rearrangements remains challenging. Here, we present a CRISPR-based homologous recombination-mediated rearrangement (HRMR) strategy that leverages homologous donor templates to align and repair broken chromosome ends. HRMR improves efficiency by approximately 80-fold compared to non-homologous end joining, achieving over 95% homologous recombination. Validated across multiple loci and cell lines, HRMR enables efficient and accurate chromosomal rearrangements. Live-cell imaging reveals that homologous donors mediate chromosome end proximity, enhancing rearrangement efficiency. Thus, HRMR provides a powerful tool for disease modeling, chromosomal biology, and therapeutic applications.}, }
@article {pmid40069470, year = {2025}, author = {Zhai, Z and Zhang, M and Yin, R and Zhao, S and Shen, Z and Yang, Y and Zhang, X and Wang, J and Qin, Y and Xu, D and Zhou, L and Lai, D}, title = {CRISPR/Cas9-assisted gene editing reveals that EgPKS, a polyketide synthase, is required for the biosynthesis of preussomerins in Edenia gomezpompae SV2.}, journal = {World journal of microbiology &amp; biotechnology}, volume = {41}, number = {3}, pages = {103}, pmid = {40069470}, issn = {1573-0972}, support = {2023YFD1401000//National Key Research and Development Program of China/ ; 31872623//National Natural Science Foundation of China/ ; }, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Polyketide Synthases/genetics/metabolism ; Naphthalenes/metabolism ; *Ascomycota/genetics/metabolism/enzymology ; Fungal Proteins/genetics/metabolism ; }, abstract = {Edenia gomezpompae, an endophytic fungus derived from plants, produced a diverse array of preussomerins, a type of spirobisnaphthalenes featuring two spiroketal groups, which exhibited significant antibacterial, antifungal, and cytotoxic activities. Structurally, the biosynthesis of preussomerins might be related to the biosynthesis of 1,8-dihydroxynaphthalene (DHN), a precursor of DHN-melanin. However, the absence of efficient gene-editing tools for E. gomezpompae has hindered the biosynthetic study of preussomerins. In this study, we developed a CRISPR/Cas9-based gene editing system for E. gomezpompae SV2 that was isolated from the stem of Setaria viridis, by utilizing the endogenous U6 snRNA promoter to drive sgRNA expression. Using this system, we successfully disrupted the polyketide synthase (PKS)-encoding gene, Egpks, a putative 1,3,6,8-tetrahydroxynaphthalene synthase gene involved in the biosynthesis of DHN-melanin, with an editing efficiency up to 92% and a knockout efficiency of 71% when employing the U6 snRNA-3 promoter. Furthermore, the disrupted mutant (∆Egpks) displayed white hyphae and lost the ability to produce preussomerins. These results provided a foundational tool for genetic manipulation in E. gomezpompae and revealed the role of EgPKS in the biosynthesis of preussomerin-type spirobisnaphthalenes.}, }
@article {pmid40069074, year = {2025}, author = {Yang, F and Aliyari, S and Zhu, Z and Zheng, H and Cheng, G and Zhang, S}, title = {CRISPR-Cas: a game-changer in vaccine development and the fight against viral infections.}, journal = {Trends in microbiology}, volume = {33}, number = {6}, pages = {650-664}, doi = {10.1016/j.tim.2025.02.006}, pmid = {40069074}, issn = {1878-4380}, mesh = {*CRISPR-Cas Systems ; Humans ; *Virus Diseases/prevention &amp; control/virology/immunology/therapy ; Gene Editing/methods ; *Viral Vaccines/immunology/genetics ; Animals ; *Vaccine Development/methods ; *Viruses/genetics/immunology ; Host-Pathogen Interactions/genetics ; }, abstract = {Understanding the mechanisms of virus-host interactions is crucial for advancing our knowledge of viral pathogenesis and developing effective antiviral strategies. As novel, highly virulent pathogens continue to emerge, overcoming the challenges in virus-host biology and infection control is more important than ever. The CRISPR-Cas system has revolutionized genetic engineering, offering a precise and efficient application in synthetic biology. It is also invaluable in virology research, diagnostics, and antiviral therapeutics. In this review, we explore the latest advances in CRISPR-Cas-based approaches to elucidate virus-host interactions, focusing on the identification of key receptors and host factors essential for virus infection. Furthermore, we discuss the potential of the CRISPR-Cas system as an antiviral strategy for tackling newly emerging and re-emerging viruses. Finally, we discuss the potential of CRISPR-Cas-based gene editing for the development of innovative viral vaccines.}, }
@article {pmid40068769, year = {2025}, author = {Hong, S and Moon, JS and Lee, YJ and Kim, HY}, title = {Whole-genome sequencing-based characterization of Listeria monocytogenes isolated from cattle and pig slaughterhouses.}, journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases}, volume = {130}, number = {}, pages = {105737}, doi = {10.1016/j.meegid.2025.105737}, pmid = {40068769}, issn = {1567-7257}, mesh = {Animals ; Cattle ; *Listeria monocytogenes/genetics/isolation &amp; purification/classification/drug effects ; Swine/microbiology ; *Whole Genome Sequencing ; Abattoirs ; Genome, Bacterial ; *Listeriosis/microbiology/veterinary ; Republic of Korea ; Phylogeny ; Multilocus Sequence Typing ; }, abstract = {Listeria monocytogenes is a foodborne pathogen that causes human listeriosis and may be transmitted to humans via the food chain, beginning at slaughter and extending through food production and consumption. In this study, we performed whole-genome sequencing (WGS) analysis to determine the genetic characteristics of L. monocytogenes from the carcasses and environments of cattle and pig slaughterhouses in Korea. In total, 50 L. monocytogenes isolates were collected from 46 cattle and 47 pig slaughterhouses nationwide from 2014 to 2022. They were classified into two lineages, 12 sublineages, 12 sequence types, 11 clonal complexes (CCs), and 15 core-genome multilocus sequence types. L. monocytogenes isolates were divided into two lineages: lineage I (serotypes 1/2b and 4b) and lineage II (serotypes 1/2a and 1/2c). The most frequent CCs were CC9 (46.0 %), followed by CC224 (16.0 %) and CC155 (14.0 %). Although all isolates exhibited highly conserved LIPI-1, 20.0 % and 2.0 % contained LIPI-3 or LIPI-4, respectively. Moreover, 96.0 % of the isolates had full-length inlA. Interestingly, 21 of the 23 CC9 isolates contained mutations in inlA resulting from premature stop codon (PMSC). The mdrL and Listeria genomic island-2 (LGI-2) were identified in all L. monocytogenes isolates, whereas LGI-3 was identified in 32.0 % of the isolates. The L. monocytogenes isolates contained various antimicrobial resistance genes, moreover, the plasmid-borne resistance genes tetM and mprF were also identified in 34.0 % and 100 % of the isolates, respectively. Twenty-four isolates (48.0 %) harbored one or two plasmids (pLM33, DOp1, pLGUG1, and pLM5578), and 29 isolates (58.0 %) harbored at least one insertion sequence, composite transposon, and integrative conjugative element. Four isolates showed two CRISPR-Cas types IB and II-A. In addition, phage sequences associated with the spacer constituting the CRISPR array were identified in 26 Listeria phages from 14 L. monocytogenes isolates. The genetic composition of L. monocytogenes was conserved in a collinearity relationship between each of the five L. monocytogenes isolates from the cattle and pig slaughterhouses. These findings suggest that L. monocytogenes isolated from cattle and pig slaughterhouses have the ability to cause human disease and exhibit virulent characteristics.}, }
@article {pmid40068613, year = {2025}, author = {Raftopoulou, O and Barrangou, R}, title = {Bacterial evolution: CRISPR out and virulence in.}, journal = {Current biology : CB}, volume = {35}, number = {5}, pages = {R182-R185}, doi = {10.1016/j.cub.2024.12.056}, pmid = {40068613}, issn = {1879-0445}, mesh = {Virulence/genetics ; *Xanthomonas campestris/genetics/pathogenicity ; *CRISPR-Cas Systems ; *Plant Diseases/microbiology ; Virulence Factors/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Biological Evolution ; *Brassica/microbiology ; *Evolution, Molecular ; Genome, Bacterial ; }, abstract = {The loss of CRISPR-Cas immune systems may drive bacterial evolution by increasing genome plasticity and acquisition of virulence factors. A new study explores this in Xanthomonas campestris, highlighting its adaptation into a virulent pathogenic pathovar infecting Brassica vasculature and mesophyll tissues.}, }
@article {pmid40067805, year = {2025}, author = {Nonarath, HJT and Simpson, SL and Slobodianuk, TL and Tran, H and Collery, RF and Dinculescu, A and Link, BA}, title = {The USH3A causative gene clarin1 functions in Müller glia to maintain retinal photoreceptors.}, journal = {PLoS genetics}, volume = {21}, number = {3}, pages = {e1011205}, pmid = {40067805}, issn = {1553-7404}, support = {C06 RR016511/RR/NCRR NIH HHS/United States ; R01 EY026559/EY/NEI NIH HHS/United States ; R21 EY032575/EY/NEI NIH HHS/United States ; }, mesh = {Animals ; Zebrafish/genetics ; *Ependymoglial Cells/metabolism/pathology ; *Zebrafish Proteins/genetics/metabolism ; *Usher Syndromes/genetics/pathology/metabolism ; Mutation ; Retina/metabolism/pathology ; *Membrane Proteins/genetics/metabolism ; *Photoreceptor Cells, Vertebrate/metabolism/pathology ; CRISPR-Cas Systems ; Disease Models, Animal ; }, abstract = {Mutations in CLRN1 cause Usher syndrome type IIIA (USH3A), an autosomal recessive disorder characterized by hearing and vision loss, and often accompanied by vestibular dysfunction. The identity of the cell types responsible for the pathology and mechanisms leading to vision loss in USH3A remains elusive. To address this, we employed CRISPR/Cas9 technology to delete a large region in the coding and untranslated (UTR) region of zebrafish clrn1. The retinas of clrn1 mutant larvae exhibited sensitivity to cell stress, along with age-dependent loss of function and degeneration in the photoreceptor layer. Investigation revealed disorganization in the outer retina in clrn1 mutants, including actin-based structures of the Müller glia and photoreceptor cells. To assess cell-specific contributions to USH3A pathology, we specifically re-expressed clrn1 in either Müller glia or photoreceptor cells. Müller glia re-expression of clrn1 prevented the elevated cell death observed in larval clrn1 mutant zebrafish exposed to high-intensity light. Notably, the degree of phenotypic rescue correlated with the level of Clrn1 re-expression. Surprisingly, high levels of Clrn1 expression enhanced cell death in both wild-type and clrn1 mutant animals. However, rod- or cone-specific Clrn1 re-expression did not reduce the extent of cell death. Taken together, our findings underscore three crucial insights. First, clrn1 mutant zebrafish exhibit key pathological features of USH3A; second, Clrn1 within Müller glia plays a pivotal role in photoreceptor maintenance, with its expression requiring controlled regulation; third, the reliance of photoreceptors on Müller glia suggests a structural support mechanism, possibly through direct interactions between Müller glia and photoreceptors mediated in part by Clrn1 protein.}, }
@article {pmid40067628, year = {2025}, author = {Gutierrez-Uzquiza, A and Bragado, P}, title = {Targeted Human Editing Using CRISPR Technology in Neural Stem Cells.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2899}, number = {}, pages = {233-243}, pmid = {40067628}, issn = {1940-6029}, mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Neural Stem Cells/metabolism/cytology ; Lentivirus/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genetic Vectors/genetics ; }, abstract = {Development of the programmable nuclease Ca9 has increased our understanding of the molecular basis of the physiological processes and diseases demonstrating a remarkable potential across various fields, including basic research, applied biotechnology, and biomedical research. CRISPR-Cas9 is a gene-editing technique that uses a guide RNA to direct the Cas9 enzyme to specific DNA sequences, where it creates double-strand breaks. These breaks can be repaired through either nonhomologous end joining (NHEJ), leading to gene mutations, or homology-directed repair (HDR), enabling precise edits. These characteristics render Cas9 an ideal candidate for various genome editing applications, particularly for the generation of knockout cells to study gene function either in vitro or in vivo. Delivery of the CRISPR molecular machinery is a crucial step for successful editing. Lentiviral vectors have been shown as an extremely effective method to transduce a wide range of cell types including neurons, astrocytes, adult neuronal stem cells, and oligodendrocytes. Here, we provide a useful protocol based on the lentiviral approach to develop CRISPR edition, facilitating the study of gene function in mammalian cells.}, }
@article {pmid40067591, year = {2025}, author = {Tanaka, PP and Monteiro, CJ and Duarte, MJ and Oliveira, ED and Monteleone-Cassiano, AC and Mascarenhas, RS and Vieira Machado, MC and Matos, AA and Brito, LA and Oliveira, AO and Cunha, TM and Donadi, EA and Passos, GA}, title = {The CRISPR-Cas9 System Is Used to Edit the Autoimmune Regulator Gene in Vitro and in Vivo.}, journal = {Advances in experimental medicine and biology}, volume = {1471}, number = {}, pages = {269-283}, pmid = {40067591}, issn = {0065-2598}, mesh = {Animals ; Humans ; Mice ; AIRE Protein ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Polyendocrinopathies, Autoimmune/genetics/immunology/pathology ; *Transcription Factors/genetics ; }, abstract = {Although mutations in the AIRE gene in patients with autoimmune polyendocrine syndrome type 1 (APS-1) syndrome are associated with the onset of this autoimmune disease, much of what is known about its mechanisms has been obtained through studies with Aire mutant Mus musculus mouse model or with Aire mutant medullary thymic epithelial cells (mTEC) cultured in vitro. The in vivo murine model was soon established, and ten mutant strains are currently described. Most Aire mutant mice were obtained through homologous recombination, which generated Aire knockout (KO) animals. Nevertheless, long-term cultures of mTECs from APS-1 patients or Aire mutant mice are difficult to establish. The CRISPR-Cas9 system to edit Aire in a murine mTEC line in vitro and mouse embryo has been successfully used to overcome this. The ribonucleoprotein (RNP) complexes composed of the guide RNA (gRNA), the Cas9 enzyme, and single-stranded oligonucleotides (ssODN) were designed to target Aire exons 6 and 8 separately. The CRISPR-Cas9 makes it possible to produce NHEJ-derived indels or HDR-derived mutations. Efforts are being concentrated on using RNP complex rather than plasmid vectors, as RNP makes recurrent NHEJ-derived mutations among in vitro and in vivo editions. One recurrent mutation was described in the Aire exon 6 (del 3554G) and the other in the exon 8 (del 5676_5677TG), i.e., the exon 6 mutation was kept in an mTEC clone edited in vitro and in vivo in a mouse, and the exon 8 mutation was kept in several mTEC clones in vitro. In contrast, none of the mutations obtained with the nickase system (plasmid expression vector) were recurrent, indicating the participation of the RNP complex in recurring mutation, which offers advantages, as it does not involve recombinant plasmids and does not generate a genetically modified organism but rather a mutant animal or cell.}, }
@article {pmid40066952, year = {2025}, author = {Cao, L and Wang, Z and Lei, C and Nie, Z}, title = {Engineered CRISPR/Cas Ribonucleoproteins for Enhanced Biosensing and Bioimaging.}, journal = {Analytical chemistry}, volume = {97}, number = {11}, pages = {5866-5879}, doi = {10.1021/acs.analchem.4c06789}, pmid = {40066952}, issn = {1520-6882}, mesh = {*Biosensing Techniques/methods ; *Ribonucleoproteins/genetics/metabolism/chemistry ; *CRISPR-Cas Systems/genetics ; Humans ; *Protein Engineering ; Animals ; *Molecular Imaging/methods ; }, abstract = {CRISPR-Cas systems represent a highly programmable and precise nucleic acid-targeting platform, which has been strategically engineered as a versatile toolkit for biosensing and bioimaging applications. Nevertheless, their analytical performance is constrained by inherent functional and activity limitations of natural CRISPR/Cas systems, underscoring the critical role of molecular engineering in enhancing their capabilities. This review comprehensively examines recent advancements in engineering CRISPR/Cas ribonucleoproteins (RNPs) to enhance their functional capabilities for advanced molecular detection and cellular imaging. We explore innovative strategies for developing enhanced CRISPR/Cas RNPs, including Cas protein engineering through protein mutagenesis and fusion techniques, and guide RNA engineering via chemical and structural modifications. Furthermore, we evaluate these engineered RNPs' applications in sensitive biomarker detection and live-cell genomic DNA and RNA monitoring, while analyzing the current challenges and prospective developments in CRISPR-Cas RNP engineering for advanced biosensing and bioimaging.}, }
@article {pmid40065494, year = {2025}, author = {Pei, Y and Liu, Y and Peng, J and Pei, Y and Zhang, T and Miao, P and Liu, Y and Liu, Y and Liu, J and Yang, Z and Li, F and Wang, Z}, title = {The DNA N6-adenine methylation target gene GhMAF1 promotes fiber initiation at the base of Gossypium ovules.}, journal = {The New phytologist}, volume = {246}, number = {3}, pages = {1015-1031}, doi = {10.1111/nph.70047}, pmid = {40065494}, issn = {1469-8137}, support = {KJRC2023D12//Scientific and Technological Personnel of Hainan Province/ ; 32202567//National Natural Science Foundation of China/ ; 2022YFF1001400//National Key R&amp;D Program of China/ ; SCKJ-JYRC-2023-48//Project of Sanya Yazhou Bay Science and Technology City/ ; SCKJ-JYRC-2023-51//Project of Sanya Yazhou Bay Science and Technology City/ ; 324QN359//Hainan Provincial Natural Science Foundation of China/ ; 2024A02002//Xinjiang Science and Technology Major Project of China/ ; YBXM2310//CAAS/ ; YBXM2414//CAAS/ ; }, mesh = {*Gossypium/genetics/growth &amp; development ; *Ovule/genetics ; *DNA Methylation/genetics ; Gene Expression Regulation, Plant ; *Cotton Fiber ; *Plant Proteins/metabolism/genetics ; *Adenine/metabolism ; *Genes, Plant ; Promoter Regions, Genetic/genetics ; }, abstract = {DNA methylation consists of 5-methylcytosine and N6-methyl deoxyadenosine (6mA) and is crucial in plant development. However, its specific role and potential mechanism to initiate cotton fibers remain unclear. This study employed Oxford Nanopore Technologies (ONT) sequencing to analyze DNA methylation alterations in ZM24 and ZM24 fuzzless-lintless (ZM24fl) during fiber initiation. Our results indicated that DNA 6mA methylation exhibited the most remarkable difference among ovule samples at -2, 0, and 5 d post anthesis of ZM24 and ZM24fl. Subsequently, genes with significant changes in DNA 6mA methylation and transcription during fiber initiation were screened. We found that GhMAF1 displayed significant transcriptional upregulation and 6mA enrichment in its promoter, which could serve as a potential target for DNA 6mA in fiber initiation. Further, we knocked out GhMAF1 using CRISPR-Cas technology and demonstrated that GhMAF1 specifically promotes the initiation of fiber cells at the base of the ovule by mediating the downstream JAZ2/CPC-MML3/MML4 pathway. These findings unveil a novel spatial module of fiber cell initiation on the ovule surface that involves GhMAF1. Ultimately, this work provides significant knowledge for the regulatory network of DNA 6mA modification in fiber initiation to improve fiber yield and quality.}, }
@article {pmid40065264, year = {2025}, author = {Chen, J and Wang, J and Zhao, H and Tan, X and Yan, S and Zhang, H and Wang, T and Tang, X}, title = {Molecular breeding of pigs in the genome editing era.}, journal = {Genetics, selection, evolution : GSE}, volume = {57}, number = {1}, pages = {12}, pmid = {40065264}, issn = {1297-9686}, support = {2021YFA0805901//National Key Research and Development Program of China/ ; }, mesh = {Animals ; *Gene Editing/methods ; Swine/genetics ; *Breeding/methods ; CRISPR-Cas Systems ; Animals, Genetically Modified/genetics ; Genome ; }, abstract = {BACKGROUND: To address the increasing demand for high-quality pork protein, it is essential to implement strategies that enhance diets and produce pigs with excellent production traits. Selective breeding and crossbreeding are the primary methods used for genetic improvement in modern agriculture. However, these methods face challenges due to long breeding cycles and the necessity for beneficial genetic variation associated with high-quality traits within the population. This limitation restricts the transfer of desirable alleles across different genera and species. This article systematically reviews past and current research advancements in porcine molecular breeding. It discusses the screening of clustered regularly interspaced short palindromic repeats (CRISPR) to identify resistance loci in swine and the challenges and future applications of genetically modified pigs.<br><br>MAIN BODY: The emergence of transgenic and gene editing technologies has prompted researchers to apply these methods to pig breeding. These advancements allow for alterations in the pig genome through various techniques, ranging from random integration into the genome to site-specific insertion and from target gene knockout (KO) to precise base and prime editing. As a result, numerous desirable traits, such as disease resistance, high meat yield, improved feed efficiency, reduced fat deposition, and lower environmental waste, can be achieved easily and effectively by genetic modification. These traits can serve as valuable resources to enhance swine breeding programmes.<br><br>CONCLUSION: In the era of genome editing, molecular breeding of pigs is critical to the future of agriculture. Long-term and multidomain analyses of genetically modified pigs by researchers, related policy development by regulatory agencies, and public awareness and acceptance of their safety are the keys to realizing the transition of genetically modified products from the laboratory to the market.}, }
@article {pmid40064881, year = {2025}, author = {Fu, Y and He, X and Ma, L and Gao, XD and Liu, P and Shi, H and Chai, P and Ge, S and Jia, R and Liu, DR and Fan, X and Yang, Z}, title = {In vivo prime editing rescues photoreceptor degeneration in nonsense mutant retinitis pigmentosa.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {2394}, pmid = {40064881}, issn = {2041-1723}, support = {82200961//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {Animals ; *Retinitis Pigmentosa/genetics/therapy/pathology ; *Gene Editing/methods ; *Cyclic Nucleotide Phosphodiesterases, Type 6/genetics/metabolism ; Mice ; Disease Models, Animal ; *Codon, Nonsense/genetics ; Retinal Rod Photoreceptor Cells/metabolism/pathology ; Genetic Therapy/methods ; Dependovirus/genetics ; *Retinal Degeneration/genetics/therapy ; Humans ; Female ; CRISPR-Cas Systems ; Male ; Mice, Inbred C57BL ; }, abstract = {The next-generation gene editing tool, prime editing (PE), is adept at correcting point mutations precisely with high editing efficiency and rare off-target events and shows promising therapeutic value in treating hereditary diseases. Retinitis pigmentosa (RP) is the most common type of inherited retinal dystrophy and is characterized by progressive degeneration of retinal photoreceptors and, consequently, visual decline. To date, effective treatments for RP are lacking. Herein, a PE system is designed to target the PDE6B Y347X mutation in the rd1 mouse strain, a preclinical RP model. We screen and develop the PE system with epegRNA and RT[ΔRnH], which is delivered via dual-AAV in vivo with an editing efficiency of 26.47 ± 13.35%, with negligible off-target effects confirmed by AID-Seq and PE-tag. Treatment with the PE system in vivo greatly restores PDE6B protein expression and protects rod cells from degeneration. Mouse behavioural experiments also show that compared with no treatment, prime editing inhibits vision deterioration in littermate rd1 mice. This study provides a therapeutic opportunity for the use of PE to correct mutated RPs at the genomic level.}, }
@article {pmid40064782, year = {2025}, author = {Singhal, T and Srivastava, A}, title = {CRISPR-Cas-Based Diagnosis of Geminiviruses.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2912}, number = {}, pages = {183-189}, pmid = {40064782}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems/genetics ; *Plant Diseases/virology ; *Geminiviridae/genetics/isolation &amp; purification ; *Begomovirus/genetics/isolation &amp; purification ; *Molecular Diagnostic Techniques/methods ; }, abstract = {Successful disease management relies on rapid and accurate identification of the causal agent. Begomoviruses (family Geminiviridae) cause severe economic losses and pose a serious threat to sustainable agriculture. Here, we describe a clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection method utilizing the CRISPR/Cas12a and CRISPR/Cas13a system for begomoviruses used in our laboratory. CRISPR-based diagnostic tools are very precise and sensitive and visual readout-compatible reporters do not need expensive laboratory equipment such as qRT-PCR. The assay detects the targeted begomoviruses in infected plants with high sensitivity and specificity, and can provide highly sensitive and specific detection visual readouts using a simple, low-cost blue light lamp, ideal for on-site use.}, }
@article {pmid40064677, year = {2025}, author = {Parsons, VA and Vadlamudi, S and Voos, KM and Rohy, AE and Moxley, AH and Cannon, ME and Rosen, JD and Mills, CA and Herring, LE and Broadaway, KA and Lorenzo, DN and Mohlke, KL}, title = {TBC1D30 regulates proinsulin and insulin secretion and is the target of a genomic association signal for proinsulin.}, journal = {Diabetologia}, volume = {68}, number = {6}, pages = {1169-1183}, pmid = {40064677}, issn = {1432-0428}, support = {P30 AR069619/AR/NIAMS NIH HHS/United States ; T32 GM135128/GM/NIGMS NIH HHS/United States ; F31 DK132982/DK/NIDDK NIH HHS/United States ; 1-19-JDF-081//American Diabetes Association/ ; R01 DK072193/DK/NIDDK NIH HHS/United States ; P30 CA016086/CA/NCI NIH HHS/United States ; T32 HL129982/HL/NHLBI NIH HHS/United States ; P30AR069619/NH/NIH HHS/United States ; T32HL129982/NH/NIH HHS/United States ; R01DK072193/NH/NIH HHS/United States ; UM1 DK126185/DK/NIDDK NIH HHS/United States ; UM1DK126185/NH/NIH HHS/United States ; U24 DK098085/DK/NIDDK NIH HHS/United States ; P30CA016086/NH/NIH HHS/United States ; F31DK132982/NH/NIH HHS/United States ; T32GM135128/NH/NIH HHS/United States ; }, mesh = {Humans ; *Proinsulin/metabolism/genetics ; *Insulin Secretion/genetics/physiology ; Genome-Wide Association Study ; *GTPase-Activating Proteins/genetics/metabolism ; *Insulin/metabolism ; Animals ; Cell Line, Tumor ; Insulin-Secreting Cells/metabolism ; Rats ; CRISPR-Cas Systems ; Polymorphism, Single Nucleotide ; }, abstract = {AIMS/HYPOTHESIS: Components of the insulin processing and secretion pathways remain incompletely understood. Here, we examined a genome-wide association study (GWAS) signal for plasma proinsulin levels. Lead GWAS variant rs150781447-T encodes an Arg279Cys substitution in TBC1 domain family member 30 (TBC1D30), but no role for this protein in insulin processing or secretion has been established previously. This study aimed to evaluate whether TBC1D30 drives the GWAS association signal by determining whether TBC1D30 is involved in proinsulin secretion and, if so, to examine the effects of variant alleles and potential mechanisms.<br><br>METHODS: Using CRISPR/Cas9 genome editing to create double-strand breaks and prime editing to install substitutions in INS1 832/13 insulinoma cells, we generated clonal cell lines with altered TBC1D30, as well as homozygous and heterozygous lines carrying the lead GWAS variant. We characterised lines by Sanger sequencing, quantitative PCR and ELISAs to measure glucose-stimulated proinsulin and insulin secretion. We also tested the effects of TBC1D30 knockdown on proinsulin and insulin secretion in human islets. We further assessed TBC1D30's contribution to secretory pathways by examining the effects of altered gene function on intracellular proinsulin and insulin content and insulin localisation, and by identifying potential proteins that interact with TBC1D30 using affinity purification mass spectrometry.<br><br>RESULTS: Compared with mock-edited cells, cell lines with reduced TBC1D30 expression or altered Rab GTPase-activating protein (RabGAP) domain had significantly more secreted proinsulin, 1.8- and 2.6-fold more than controls, respectively. Similarly, cells expressing the variant substitution demonstrated increased proinsulin secretion. Cell lines with a partial deletion of a critical functional domain showed 1.8-fold higher expression of Tbc1d30 and at least 2.0-fold less secreted proinsulin. Cells with altered RabGAP domain sequence also demonstrated, to a lesser extent, changes in secreted insulin levels. TBC1D30 knockdown in human islets resulted in increased insulin secretion with no significant effect on proinsulin secretion. The effects of altered TBC1D30 on mislocalisation of insulin, intracellular proinsulin and insulin content and the identities of interacting proteins are consistent with a role for TBC1D30 in proinsulin and insulin secretion.<br><br>CONCLUSIONS/INTERPRETATION: These findings suggest that effects on TBC1D30 are responsible for the GWAS signal and that TBC1D30 plays a critical role in the secretion of mature insulin.}, }
@article {pmid40063799, year = {2025}, author = {Lim, J and Van, AB and Koprowski, K and Wester, M and Valera, E and Bashir, R}, title = {Amplification-free, OR-gated CRISPR-Cascade reaction for pathogen detection in blood samples.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {122}, number = {11}, pages = {e2420166122}, pmid = {40063799}, issn = {1091-6490}, support = {P373//Jump ARCHES/ ; B7859//Dynamic Research Enterprise for Multidisciplinary Engineering Science/ ; 106709//VinUni-Illinois Smart Health Center/ ; }, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques/methods ; DNA, Bacterial/blood/genetics ; Methicillin-Resistant Staphylococcus aureus/genetics/isolation &amp; purification ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Point-of-Care Systems ; Escherichia coli/genetics/isolation &amp; purification ; Sensitivity and Specificity ; DNA, Viral/blood ; }, abstract = {Rapid and accurate detection of DNA from disease-causing pathogens is essential for controlling the spread of infections and administering timely treatments. While traditional molecular diagnostics techniques like PCR are highly sensitive, they include nucleic acid amplification and many need to be performed in centralized laboratories, limiting their utility in point-of-care settings. Recent advances in CRISPR-based diagnostics (CRISPR-Dx) have demonstrated the potential for highly specific molecular detection, but the sensitivity is often constrained by the slow trans-cleavage activity of Cas enzymes, necessitating preamplification of target nucleic acids. In this study, we present a CRISPR-Cascade assay that overcomes these limitations by integrating a positive feedback loop that enables nucleic acid amplification-free detection of pathogenic DNA at atto-molar levels and achieves a signal-to-noise ratio greater than 1.3 within just 10 min. The versatility of the assay is demonstrated through the detection of bloodstream infection pathogens, including Methicillin-Sensitive Staphylococcus aureus (MSSA), Methicillin-Resistant Staphylococcus aureus (MRSA), Escherichia coli, and Hepatitis B Virus (HBV) spiked in whole blood samples. Additionally, we introduce a multiplexing OR-function logic gate, further enhancing the potential of the CRISPR-Cascade assay for rapid and accurate diagnostics in clinical settings. Our findings highlight the ability of the CRISPR-Cascade assay to provide highly sensitive and specific molecular detection, paving the way for advanced applications in point-of-care diagnostics and beyond.}, }
@article {pmid40063648, year = {2025}, author = {Lavi, I and Bhattacharya, S and Awase, A and Orgil, O and Avital, N and Journo, G and Gurevich, V and Shamay, M}, title = {Unidirectional recruitment between MeCP2 and KSHV-encoded LANA revealed by CRISPR/Cas9 recruitment assay.}, journal = {PLoS pathogens}, volume = {21}, number = {3}, pages = {e1012972}, pmid = {40063648}, issn = {1553-7374}, mesh = {Humans ; *Herpesvirus 8, Human/genetics/metabolism ; *CRISPR-Cas Systems ; *Methyl-CpG-Binding Protein 2/metabolism/genetics ; *Nuclear Proteins/metabolism/genetics ; *Antigens, Viral/metabolism/genetics ; Virus Latency ; HEK293 Cells ; Histone Deacetylase 1/metabolism ; }, abstract = {Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8) is associated with several human malignancies. During latency, the viral genomes reside in the nucleus of infected cells as large non-integrated plasmids, known as episomes. To ensure episome maintenance, the latency protein LANA tethers the viral episomes to the cell chromosomes during cell division. Directional recruitment of protein complexes is critical for the proper function of many nuclear processes. To test for recruitment directionality between LANA and cellular proteins, we directed LANA via catalytically inactive Cas9 (dCas9) to a repeat sequence to obtain easily detectable dots. Then, the recruitment of nuclear proteins to these dots can be evaluated. We termed this assay CRISPR-PITA for Protein Interaction and Telomere Recruitment Assay. Using this protein recruitment assay, we found that LANA recruits its known interactors ORC2 and SIN3A. Interestingly, LANA was unable to recruit MeCP2, but MeCP2 recruited LANA. Both LANA and histone deacetylase 1 (HDAC1) interact with the transcriptional-repression domain (TRD) and the methyl-CpG-binding domain (MBD) of MeCP2. Similar to LANA, HDAC1 was unable to recruit MeCP2. While heterochromatin protein 1 (HP1), which interacts with the N-terminal of MeCP2, can recruit MeCP2. We propose that available interacting domains force this recruitment directionality. We hypothesized that the tandem repeats in the SunTag may force MeCP2 dimerization and mimic the form of DNA-bound MeCP2. Indeed, providing only the tandem epitopes of SunTag allows LANA to recruit MeCP2 in infected cells. Therefore, CRISPR-PITA revealed the rules of unidirectional recruitment and allowed us to break this directionality.}, }
@article {pmid40062897, year = {2025}, author = {Lin, H-C and Hsiao, W-C and Hsu, Y-C and Lin, M-C and Hsu, C-C and Zhang, MM}, title = {Highly efficient CRISPR-Cas9 base editing in Bifidobacterium with bypass of restriction modification systems.}, journal = {Applied and environmental microbiology}, volume = {91}, number = {4}, pages = {e0198524}, pmid = {40062897}, issn = {1098-5336}, support = {EX110-MG-113-SP-01,NHRI-EX113-11003BC//National Health Research Institutes/ ; MOST 112-2113-M-002-018-MY2//National Science and Technology Council/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Bifidobacterium/genetics ; Plasmids/genetics ; DNA Restriction-Modification Enzymes/genetics ; Gastrointestinal Microbiome ; Genome, Bacterial ; }, abstract = {Intestinal microbiota members of the Bifidobacterium genus are increasingly explored as probiotics and therapeutics. However, the paucity of genetic tools and the widespread restriction modification (RM) systems in Bifidobacterium limit our ability to genetically manipulate these bacteria. Here we established a CRISPR-Cas9 cytosine base editor system (cBEST) for portable genome editing in bifidobacteria. Harboring different promoters characterized in this study, these cBEST plasmids showed a range of editing efficiencies in different strains and genomic contexts, highlighting the importance of fine-tuning base editor and sgRNA expression. Additionally, we showed that disruption or bypass of RM systems dramatically improved editing efficiencies in otherwise hard-to-edit genomic loci and Bifidobacterium strains. Notably, we demonstrated the use of RM-disrupted Bifidobacterium longum strains for simultaneous assembly, amplification, and methylation of the all-in-one editing plasmids, greatly streamlining the workflow for high-efficiency base editing. Last but not least, we showed the portability of cBESTs using the same editing construct to disrupt a conserved metabolic gene in multiple Bifidobacterium species. Looking ahead, the ability to efficiently edit and engineer bifidobacterial genomes will give rise to new opportunities for research and applications toward improving human health.IMPORTANCEThe ability to genetically manipulate specific genes and biological pathways in Bifidobacterium is essential to unlocking their probiotic and therapeutic potential in human health applications. The DNA double-strand break-free CRISPR-Cas9 cytosine base editor system established in this work allows portable and efficient base editing in Bifidobacterium spp. We further showed that bypass of restriction modification systems significantly improved base editing efficiency, especially for hard-to-edit genomic loci and strains. This expanded Bifidobacterium genome editing toolbox should facilitate mechanistic investigations into the roles of Bifidobacterium in host physiology and disease.}, }
@article {pmid40062839, year = {2025}, author = {Khasa, R and Ogden, SC and Wang, Y and Mou, Z and Metzler, AD and Xie, X and Dai, X and Tang, H}, title = {A single mutation in the PrM gene of Zika virus determines AXL dependency for infection of human neural cells.}, journal = {Journal of virology}, volume = {99}, number = {4}, pages = {e0187324}, pmid = {40062839}, issn = {1098-5514}, support = {R35 GM151043/GM/NIGMS NIH HHS/United States ; R01 AI146342/AI/NIAID NIH HHS/United States ; R01AI146342, U19 AI131130//National Institute of Allergy and Infectious Diseases/ ; R35GM151043/GM/NIGMS NIH HHS/United States ; U19 AI131130/AI/NIAID NIH HHS/United States ; }, mesh = {Humans ; *Zika Virus/genetics/physiology ; *Receptor Protein-Tyrosine Kinases/genetics/metabolism ; *Proto-Oncogene Proteins/metabolism/genetics ; Axl Receptor Tyrosine Kinase ; Virus Internalization ; *Zika Virus Infection/virology/metabolism/genetics ; Mutation ; *Neurons/virology/metabolism ; *Viral Envelope Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Animals ; Cell Line, Tumor ; Gene Knockout Techniques ; }, abstract = {Zika virus (ZIKV) is spread by mosquito bites and is unique among known flaviviruses for being able to cause microcephaly. Entry factors for ZIKV are incompletely understood, but phosphatidylserine (PS) receptors, including the TAM (Tyro3, AXL, and Mer) and TIM (T-cell Ig mucin) families, can serve as cofactors for flavivirus entry in a cell type-specific manner. We identify AXL as the top hit in a CRISPR/Cas9 genome-wide screen in human glioblastoma cells and establish a definitive role of AXL, but not TYRO3 or MerTK, for ZIKV infection. Additionally, Spondweni virus also shows AXL dependency, while dengue virus infection is not affected by AXL knockout. Passage of ZIKV in AXL knockout (KO) cells generated a mutant virus capable of infection via AXL-independent mechanisms, and multiple independent selections identified a common mutation, H83R, in the prM coding region of the ZIKV genome. The mutant virus exhibits an increased infectivity rate in AXL KO cells as compared to wild-type ZIKV and is dependent upon the single H83R mutation. The mutant virus' ability to infect cells in an AXL-independent manner is unrelated to interferon signaling antagonism but likely pertains to a change in virus maturation that leads to a structural disturbance of the ZIKV virion. Our study provides evidence for a potential mechanism linking the viral structural proteins and host PS receptor usage during flavivirus infection.IMPORTANCEA major challenge in elucidating the mechanism of Zika virus (ZIKV) pathogenesis is the multitude of cell types it infects with distinct requirements. The role of phosphatidylserine (PS) receptors in ZIKV infection is cell type-specific, and the controversy surrounds their function in flavivirus entry. Here, we establish a definitive requirement of AXL for infection of human glioblastoma cells by both Zika and Spondweni virus. We then identified a single amino acid mutation (H83R) in the prM protein of ZIKV that allowed AXL-independent infection of these cells. The H83R-mediated escape of AXL requirement is independent of interferon (IFN) signaling suppression by AXL; instead, the mutation has the potential to disrupt the virus assembly and virion structure. This study reveals a previously unknown connection between the PS receptor usage and the flavivirus prM gene, which can guide detailed molecular mechanism studies of the interplay between virion assembly and virus entry.}, }
@article {pmid40060765, year = {2025}, author = {Chia, S and Guo, T and Goldys, EM and Payne, SC and Duan, W and Lovell, NH and Shivdasani, MN and Deng, F}, title = {A CRISPR mediated point-of-care assay for the detection of mucosal calprotectin in an animal model of ulcerative colitis.}, journal = {Bioengineering &amp; translational medicine}, volume = {10}, number = {2}, pages = {e10725}, pmid = {40060765}, issn = {2380-6761}, abstract = {Inflammatory bowel disease (IBD) is a chronic disorder associated with inflammation in the gastrointestinal tract, leading to a range of debilitating symptoms. Fecal calprotectin is an established biomarker for ulcerative colitis (UC), one of the main IBD diseases, which provides indications of the presence and severity of inflammation in the digestive tract. Enzyme-Linked Immunosorbent Assay (ELISA) as a gold standard approach for fecal calprotectin detection is time-consuming and impractical in point-of-care settings. Moreover, obtaining fecal samples from patients is challenging and inhibits longitudinal monitoring. To address these specific problems, we have developed a novel approach for detecting calprotectin which leverages clustered regularly interspaced short palindromic repeats (CRISPR)/Cas technology. We successfully developed a portable tube-based CRISPR/Cas assay for point-of-care testing of calprotectin. This assay showed a detection range from 1 to 10,000 ng/ml (over 4 log units), using both fluorescent and colorimetric analytical techniques. The established assay was further validated through measurements in mucosal samples obtained in an anesthetised preclinical rodent model of UC, with 2-3 times higher calprotectin concentration detected in UC rat samples compared to that of healthy control animals. This point-of-care test may provide a rapid, precise, and user-friendly approach for the diagnosis and monitoring of IBD through mucosal sample testing.}, }
@article {pmid40060553, year = {2025}, author = {Nammi, B and Jayasinghe-Arachchige, VM and Madugula, SS and Artiles, M and Radler, CN and Pham, T and Liu, J and Wang, S}, title = {CasGen: A Regularized Generative Model for CRISPR Cas Protein Design with Classification and Margin-Based Optimization.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, pmid = {40060553}, issn = {2692-8205}, support = {R21 GM144860/GM/NIGMS NIH HHS/United States ; }, abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated proteins (Cas) systems have revolutionized genome editing by providing high precision and versatility. However, most genome editing applications rely on a limited number of well-characterized Cas9 and Cas12 variants, constraining the potential for broader genome engineering applications. In this study, we extensively explored Cas9 and Cas12 proteins and developed CasGen, a novel transformer-based deep generative model with margin-based latent space regularization to enhance the quality of newly generative Cas9 and Cas12 proteins. Specifically, CasGen employs a strategies that combine classification to filter out non-Cas sequences, Bayesian optimization of the latent space to guide functionally relevant designs, and thorough structural validation using AlphaFold-based analyses to ensure robust protein generation. We collected a comprehensive dataset with 3,021 Cas9, 597 Cas12, and 597 Non-Cas protein sequences from reputable biological databases such as InterPro and PDB. To validate the generated proteins, we performed sequence alignment using the BLAST tool to ensure novelty and filter out highly similar sequences to existing Cas proteins. Structural prediction using AlphaFold2 and AlphaFold3 confirmed that the generated proteins exhibit high structural similarity to known Cas9 and Cas12 variants, with TM-scores between 0.70 and 0.85 and root-mean-square deviation (RMSD) values below 2.00 Å. Sequence identity analysis further demonstrated that the generated Cas9 orthologs exhibited 28% to 55% identity with known variants, while Cas12a variants show up to 48% identity. Our results demonstrate that the proposed Cas generative model has significant potential to expand the genome editing toolkit by designing diverse Cas proteins that retain functional integrity. The developed deep generative approach offers a promising avenue for synthetic biology and therapeutic applications, enableling the development of more precise and versatile Cas-based genome editing tools.}, }
@article {pmid40060182, year = {2025}, author = {Xia, X and Li, S and Wang, N and Cheng, P and Zhu, B and Zhang, P and Yang, D and Lin, H and Niu, L}, title = {Convenient, high-efficiency multiplex genome editing in autotetraploid alfalfa using endogenous U6 promoters and visual reporters.}, journal = {aBIOTECH}, volume = {6}, number = {1}, pages = {81-90}, pmid = {40060182}, issn = {2662-1738}, abstract = {UNLABELLED: CRISPR/Cas is a simple, robust, versatile tool for plant biology studies and precision plant breeding. However, establishing a high-efficiency gene editing system for multiplex editing of the autotetraploid crop alfalfa (Medicago sativa L.), the most important forage legume worldwide, remains a formidable challenge. Here, we systematically identified endogenous U6 promoters in alfalfa through transient expression via Agrobacterium-mediated infiltration of alfalfa leaves. We further demonstrated the efficacy of the three most active promoters for genome editing using an optimized alfalfa hairy root system. Subsequently, we established an improved CRISPR/Cas9 multiplex system containing three or four tandemly arrayed MsU6-promoter-driven polycistronic tRNA-sgRNA (PTG) expression cassettes, each consisting of three tRNA-sgRNA units, to simultaneously edit three or four alfalfa genes, coupled with the visual reporter RH1 or RUBY. This toolkit showed efficient multiplex editing in the hairy root system with visual selection. We successfully obtained regenerated, red-colored shoots resulting from the stable transformation of alfalfa. These results highlight the potential application of the visual reporter system for the stable transformation of alfalfa. Our improved CRISPR/Cas9 multiplex system enables convenient, high-efficiency multiplex genome editing in alfalfa, providing a versatile toolset to facilitate functional studies of multiple genes and gene families for basic research and the genetic improvement of alfalfa.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42994-025-00200-z.}, }
@article {pmid40059951, year = {2025}, author = {Hu, L and Jin, T and Zhang, N and Ding, J and Li, L}, title = {RNA-Based Therapies in Kidney Diseases.}, journal = {Journal of inflammation research}, volume = {18}, number = {}, pages = {3143-3160}, pmid = {40059951}, issn = {1178-7031}, abstract = {Kidney diseases are major global health challenges, affecting over 750 million people worldwide. Despite significant efforts, effective treatment strategies are still insufficient. In recent years, RNA therapeutics have made substantial progress, and an increasing number of nucleic acid-based therapies have been approved, showing potential for treating various diseases (including kidney diseases). These therapies can target proteins, transcripts, and genes that were previously considered "undruggable", allowing for the regulation of their expression and the expansion of therapeutic targets. Among RNA therapeutics, mRNA-based therapies are particularly promising because they can rapidly express therapeutic proteins, along with their design flexibility and potential to target previously inaccessible disease mechanisms. This review discussed various RNA-based strategies for developing new treatments, including antisense and RNA interference technologies, mRNA-based approaches, and CRISPR-Cas-mediated genome editing. Additionally, we highlighted the opportunities and challenges associated with the widespread application of these therapies in kidney disease treatment.}, }
@article {pmid40059750, year = {2025}, author = {Dai, W and Wang, H and Ji, H and Xiao, X and Li, Y and Jiang, D and Luo, Y and Xiao, X and Yan, B and Yu, J and Li, L}, title = {Nicking enzyme assisted amplification combined with CRISPR-Cas12a system for one-pot sensitive detection of APE1.}, journal = {The Analyst}, volume = {150}, number = {7}, pages = {1409-1418}, doi = {10.1039/d5an00009b}, pmid = {40059750}, issn = {1364-5528}, mesh = {*DNA-(Apurinic or Apyrimidinic Site) Lyase/analysis/metabolism/genetics/blood ; *CRISPR-Cas Systems ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Endodeoxyribonucleases/metabolism/genetics ; Limit of Detection ; DNA/genetics/chemistry ; *Biosensing Techniques/methods ; *Bacterial Proteins/genetics/metabolism ; CRISPR-Associated Proteins ; }, abstract = {Apurinic/apyrimidinic endonuclease 1 (APE1) is a critical enzyme in the base excision repair (BER) pathway, essential for preserving cellular equilibrium. Variations in APE1 activity within blood or tissues can provide significant insights for clinical cancer screening and disease diagnosis. Consequently, the detection of APE1 activity is critical for clinical diagnostics. However, there is currently a deficiency in rapid, straightforward, and sensitive methods for APE1 detection. To address this issue, we developed a method that integrates Nicking Enzyme Assisted Amplification (NEAA) with CRISPR-Cas12a signal amplification, enabling one-pot detection of APE1 activity. This method utilizes NEAA to produce a substantial quantity of target DNA that is complementary to the crRNA, thereby triggering the trans-cleavage activity of Cas12a. The activated Cas12a then amplifies and emits signals by cleaving the reporter probe. Our strategy allows for the swift and precise detection of APE1, in only 3 h, with a detection threshold of 1 × 10[-6] U mL[-1] and a linear detection range of 5 × 10[-6] to 0.1 U mL[-1]. It has been effectively utilized for the detection of APE1 in biological samples.}, }
@article {pmid40059372, year = {2025}, author = {Suzuki, H and Tong, G and Nath, P and Hiruta, Y and Citterio, D}, title = {Origami Paper-Based Immunoassay Device with CRISPR/Cas12a Signal Amplification.}, journal = {ACS sensors}, volume = {10}, number = {3}, pages = {1811-1821}, doi = {10.1021/acssensors.4c02733}, pmid = {40059372}, issn = {2379-3694}, mesh = {*Paper ; *CRISPR-Cas Systems ; Humans ; *Immunoglobulin G/analysis/blood/immunology ; Enzyme-Linked Immunosorbent Assay/instrumentation/methods ; *Hepatitis B Surface Antigens/analysis/blood/immunology ; Limit of Detection ; Immunoassay/instrumentation/methods ; DNA, Single-Stranded/chemistry ; CRISPR-Associated Proteins ; *Endodeoxyribonucleases ; Bacterial Proteins ; }, abstract = {In clinical diagnosis, the determination of target proteins at low concentration levels is generally performed by immunoassays, such as the enzyme-linked immunosorbent assay (ELISA), which is a time-consuming process. To date, paper-based ELISA platforms enabling faster and less expensive analysis have been developed, but their important issue for clinical applications is the limited sensitivity compared to conventional ELISA. To address this challenge, this paper introduces a simple, rapid, and highly sensitive detection method for non-nucleic acid targets achieved by integrating the CRISPR/Cas12a system into paper-based ELISA. An origami-type paper-based device enabling simple assay operation has been designed, and the detection of targets on the paper substrates is based on observing the fluorescence signal induced by the CRISPR/Cas12a enzyme cleaving a probe single-stranded DNA (ssDNA) labeled with fluorophore and quencher (FQ reporter). To enhance sensitivity, antibodies labeled with a network of multiple DNA activating the CRISPR/Cas12a enzyme have been utilized as detection antibodies. As a result, the developed device successfully boosted the detection sensitivity for both human IgG and the hepatitis B virus surface antigen (HBsAg). In particular, the limit of detection (LOD) for HBsAg was estimated to be 12 pg/mL, representing over 10-fold higher sensitivity compared with commercially available HBsAg ELISA kits (LOD: 200 pg/mL). In addition, the fluorescence response toward porcine whole blood samples containing different HBsAg concentrations was also confirmed by capturing images with a smartphone, followed by quantitative data analysis. These results demonstrate the potential applicability of the proposed platform for clinical tests at the point of care.}, }
@article {pmid40058712, year = {2025}, author = {Song, Y and Li, J and Luan, X and Li, A and Liu, N and Wu, ZH and Yang, W and Gao, W and Zheng, X and Zhang, XQ}, title = {OsCHR728 encodes a chromatin remodeling factor involved in seed size and grain chalkiness in rice.}, journal = {Gene}, volume = {951}, number = {}, pages = {149396}, doi = {10.1016/j.gene.2025.149396}, pmid = {40058712}, issn = {1879-0038}, mesh = {*Oryza/genetics/growth &amp; development/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *Chromatin Assembly and Disassembly/genetics ; *Edible Grain/genetics/growth &amp; development ; *Seeds/genetics/growth &amp; development ; *Transcription Factors/genetics/metabolism ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Adenosine Triphosphatases ; }, abstract = {The Imitation Switch (ISWI)ATP-dependent chromatin remodeling factor proteins regulate various developmental processes, spanning from flowering to stress response. However, researches on the roles of ISWI subfamily genes in rice have been limited. This study investigated the expression profile of the rice chromatin remodeler OsCHR728, encoding an ISWI protein, across various tissues and developing stages. Our findings reveal that OsCHR728 is highly accumulated during early stage of the panicle development. We generated OsCHR728 knockout (KO) lines in rice using CRISPR/Cas9 technology. These mutant lines displayed smaller grain size compared to the wild type (Zhonghua 11, ZH11). Expression analysis revealed a significant downregulation of the transcript levels of five genes associated with cell cycle regulation in KO grains compared to the wild type, consistent with the reduced cell number in the mutant grains. Additionally, total free amino acid levels were higher in the KO mutant compared to the wild type, consequently enhancing the nutritional quality of the KO mutant grains. The mature endosperm of the KO mutant exhibited a reduced percentage of chalky grains and less chalkiness, suggesting an improvement in the appearance quality of the KO mutant. These results suggest that chromatin remodeling factor OsCHR728 plays a role in grain development, potentially providing a new avenue to enhance both the appearance and nutritional quality of rice cultivars.}, }
@article {pmid40056601, year = {2025}, author = {Wu, SR and Sharpe, J and Tolliver, J and Groth, AJ and Chen, R and Guerra García, ME and Valentine, V and Williams, NT and Jacob, S and Reitman, ZJ}, title = {Combining the RCAS/tv-a retrovirus and CRISPR/Cas9 gene editing systems to generate primary mouse models of diffuse midline glioma.}, journal = {Neoplasia (New York, N.Y.)}, volume = {62}, number = {}, pages = {101139}, pmid = {40056601}, issn = {1476-5586}, mesh = {Animals ; *CRISPR-Cas Systems ; Mice ; Disease Models, Animal ; *Glioma/genetics/pathology ; *Gene Editing/methods ; *Brain Neoplasms/genetics/pathology ; Tumor Suppressor Protein p53/genetics ; Retroviridae/genetics ; Humans ; }, abstract = {Diffuse midline gliomas (DMGs) are lethal brain tumors that arise in children and young adults, resulting in a median survival of less than two years. Genetically engineered mouse models (GEMMs) are critical to studying tumorigenesis and tumor-immune interactions, which may inform new treatment approaches. However, current midline glioma GEMM approaches are limited in their ability to multiplex perturbations and/or target specific cell lineages in the brain for genetic manipulation. Here, we combined the RCAS/tv-a avian retrovirus system and CRISPR/Cas9 genetic engineering to drive midline glioma formation in mice. CRISPR/Cas9-based disruption of Trp53, a tumor suppressor that is frequently disrupted in midline gliomas, along with the oncogene PDGF-B resulted in high grade tumor formation with moderate latency (median time to tumor formation of 12 weeks). We confirmed CRISPR-mediated Trp53 disruption using next-generation sequencing (NGS) and immunohistochemistry (IHC). Next, we disrupted multiple midline glioma tumor suppressor genes (Trp53, Pten, Atm, Cdkn2a) in individual mouse brains. These mini-pooled in vivo experiments generated primary midline gliomas with decreased tumor latency (median time to tumor formation of 3.6 weeks, P < 0.0001, log-rank test compared to single-plex gRNA). Quantification of gRNA barcodes and CRISPR editing events revealed that all tumors contained cells with various disruptions of all target genes and suggested a multiclonal origin for the tumors as well as stronger selection for Trp53 disruption compared to disruption of the other genes. This mouse modeling approach will streamline midline glioma research and enable complex experiments to understand tumor evolution and therapeutics.}, }
@article {pmid40056574, year = {2025}, author = {Klompstra, TM and Yoon, KJ and Koo, BK}, title = {Evolution of organoid genetics.}, journal = {European journal of cell biology}, volume = {104}, number = {2}, pages = {151481}, doi = {10.1016/j.ejcb.2025.151481}, pmid = {40056574}, issn = {1618-1298}, mesh = {*Organoids/metabolism/cytology ; Humans ; Animals ; CRISPR-Cas Systems/genetics ; Gene Editing ; Pluripotent Stem Cells/cytology ; }, abstract = {Organoids have revolutionized in vitro research by offering three-dimensional, multicellular systems that recapitulate the structure, function, and genetics of human tissues. Initially developed from both pluripotent stem cells (PSCs) and adult stem cells (AdSCs), organoids have expanded to model nearly every major human organ, significantly advancing developmental biology, disease modeling, and therapeutic screening. This review highlights the progression of organoid technologies, emphasizing the integration of genetic tools, including CRISPR-Cas9, prime editing, and lineage tracing. These advancements have facilitated precise modeling of human-specific pathologies and drug responses, often surpassing traditional 2D cultures and animal models in accuracy. Emerging technologies, such as organoid fusion, xenografting, and optogenetics, are expected to further enhance our understanding of cellular interactions and microenvironmental dynamics. As organoid complexity and genetic engineering methods continue to evolve, they will become increasingly indispensable for personalized medicine and translational research, bridging gaps between in vitro and in vivo systems.}, }
@article {pmid40056293, year = {2025}, author = {Tan, W and Tian, J and Zhao, W and Wei, J and Xu, Y and Zhou, S and Wei, Z and Shen, Z and Wu, M and Shang, L and Li, R and Wang, Y and Qin, B}, title = {Disruption of the endoplasmic reticulum-localized fatty acyl-ACP thioesterase IPF1 caused partial male sterility in rice.}, journal = {Plant molecular biology}, volume = {115}, number = {2}, pages = {40}, pmid = {40056293}, issn = {1573-5028}, support = {2024GXNSFGA010003//Guangxi Natural Science Foundation/ ; ZY23055028//Centrally Guided Local Science and Technology Development Fund/ ; 32372023//the National Natural Science Foundation of China/ ; 31971809//the National Natural Science Foundation of China/ ; SKLCUSA-b202306//State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources/ ; YCBZ2022016//Innovation Project of Guangxi Graduate Education/ ; No.20220009//the Project of Bama County for Talents in Science and Technology/ ; }, mesh = {*Oryza/genetics/physiology/enzymology ; *Endoplasmic Reticulum/metabolism/enzymology ; *Plant Infertility/genetics ; Pollen/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *Thiolester Hydrolases/genetics/metabolism ; Mutation ; Lipid Metabolism/genetics ; CRISPR-Cas Systems ; Seeds/genetics ; }, abstract = {The fatty acyl ACP thioesterases, catalyzing the final step of fatty acid synthesis in the plastid, regulate various critical processes in plants, including seed oil accumulation, seed development, plant growth, and drought tolerance. However, their roles in male fertility have seldom been demonstrated. In this study, the function of a newly FAT, Impaired Pollen Fertility 1 (IPF1) in male fertility was investigated. IPF1 expressed prominently in microspores and tapetum. IPF1 specifically located in the endoplasmic reticulum. IPF1 knock-out mutants produced by the CRISPR/Cas9 system displayed significant reduction in seed-setting rate compared to WT. The decreased seed-setting rate in the ipf1 mutants was found to be attributed to the defects of pollen viability, not the female gamete fertility. The aborted pollen in the ipf1 mutants showed impaired pollen wall formation and diminished lipid deposition. Consistently, the expression levels of six genes critical to pollen wall formation and lipid metabolism (GPAT3, OsC6, DPW2, OsPKS1, OsPKS2, and OsSTRL2) were significantly decreased in the ipf1 mutant. Taken together, these results demonstrate that IPF1 regulates rice pollen fertility through the modulation of lipid synthesis.}, }
@article {pmid40055491, year = {2025}, author = {Tuncel, A and Pan, C and Clem, JS and Liu, D and Qi, Y}, title = {CRISPR-Cas applications in agriculture and plant research.}, journal = {Nature reviews. Molecular cell biology}, volume = {26}, number = {6}, pages = {419-441}, pmid = {40055491}, issn = {1471-0080}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome, Plant/genetics ; *Crops, Agricultural/genetics ; *Agriculture/methods ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; Genetic Engineering/methods ; }, abstract = {Growing world population and deteriorating climate conditions necessitate the development of new crops with high yields and resilience. CRISPR-Cas-mediated genome engineering presents unparalleled opportunities to engineer crop varieties cheaper, easier and faster than ever. In this Review, we discuss how the CRISPR-Cas toolbox has rapidly expanded from Cas9 and Cas12 to include different Cas orthologues and engineered variants. We present various CRISPR-Cas-based methods, including base editing and prime editing, which are used for precise genome, epigenome and transcriptome engineering, and methods used to deliver the genome editors into plants, such as bacterial-mediated and viral-mediated transformation. We then discuss how promoter editing and chromosome engineering are used in crop breeding for trait engineering and fixation, and important applications of CRISPR-Cas in crop improvement, such as de novo domestication and enhancing tolerance to abiotic stresses. We conclude with discussing future prospects of plant genome engineering.}, }
@article {pmid40055326, year = {2025}, author = {Guang, L and Ma, S and Yao, Z and Song, D and Chen, Y and Liu, S and Wang, P and Su, J and Wang, Y and Luo, L and Shyh-Chang, N}, title = {An obesogenic FTO allele causes accelerated development, growth and insulin resistance in human skeletal muscle cells.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {1645}, pmid = {40055326}, issn = {2041-1723}, mesh = {Humans ; *Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics/metabolism ; *Insulin Resistance/genetics ; *Obesity/genetics/metabolism ; Alleles ; *Muscle, Skeletal/metabolism/cytology/growth &amp; development ; RNA, Long Noncoding/genetics/metabolism ; Cell Proliferation/genetics ; Insulin-Like Growth Factor II/genetics/metabolism ; Cell Differentiation/genetics ; Insulin/metabolism ; Signal Transduction ; CRISPR-Cas Systems ; Histones/metabolism ; Diet, High-Fat/adverse effects ; Cellular Senescence/genetics ; Muscle Development/genetics ; }, abstract = {Human GWAS have shown that obesogenic FTO polymorphisms correlate with lean mass, but the mechanisms have remained unclear. It is counterintuitive because lean mass is inversely correlated with obesity and metabolic diseases. Here, we use CRISPR to knock-in FTO[rs9939609-A] into hESC-derived tissue models, to elucidate potentially hidden roles of FTO during development. We find that among human tissues, FTO[rs9939609-A] most robustly affect human muscle progenitors' proliferation, differentiation, senescence, thereby accelerating muscle developmental and metabolic aging. An edited FTO[rs9939609-A] allele over-stimulates insulin/IGF signaling via increased muscle-specific enhancer H3K27ac, FTO expression and m[6]A demethylation of H19 lncRNA and IGF2 mRNA, with excessive insulin/IGF signaling leading to insulin resistance upon replicative aging or exposure to high fat diet. This FTO-m[6]A-H19/IGF2 circuit may explain paradoxical GWAS findings linking FTO[rs9939609-A] to both leanness and obesity. Our results provide a proof-of-principle that CRISPR-hESC-tissue platforms can be harnessed to resolve puzzles in human metabolism.}, }
@article {pmid40055023, year = {2025}, author = {Ge, H and Feng, J and Huang, L and Luo, Z and Ling, H and Ma, L and Wang, M and Chen, H and Ren, L}, title = {Development of a highly sensitive, high-throughput and automated CRISPR-based device for the contamination-free pathogen detection.}, journal = {Biosensors &amp; bioelectronics}, volume = {278}, number = {}, pages = {117323}, doi = {10.1016/j.bios.2025.117323}, pmid = {40055023}, issn = {1873-4235}, mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/instrumentation ; Nucleic Acid Amplification Techniques/instrumentation ; Equipment Design ; *Lab-On-A-Chip Devices ; Humans ; Limit of Detection ; High-Throughput Screening Assays/instrumentation ; }, abstract = {Rapid, portable, and contamination-resistant nucleic acid detection methods are necessary due to the threat posed by emerging viruses to public health and agricultural output. We establish CARE (CRISPR-associated airtight real-time electronic diagnostic device), a novel platform that combines CRISPR-Cas12a with a hermetically sealed microfluidic chip to overcome the limitations of present technologies, which struggle to balance sensitivity, multiplexing, and field applicability. By combining isothermal amplification and CRISPR detection within a hermetically sealed microfluidic chip, CARE eliminates the risk of nucleic acid aerosol contamination while enabling simultaneous high-throughput analysis of seven pathogens. The device is complemented by a user-friendly nucleic acid quantification App, enabling rapid and precise analysis. The RPA-CRISPR/Cas12a system demonstrates exceptional sensitivity, detecting as few as 1 copy μL[-1] (single-plex) and 10-10[2] copies μL[-1] (multiplexed), with real-sample performance matching gold-standard methods. CARE represents a significant advancement in CRISPR-based diagnostics, offering a robust, portable solution for on-site pathogen detection in food and agricultural applications.}, }
@article {pmid40054857, year = {2025}, author = {Kougnassoukou Tchara, PE and Loehr, J and Lambert, JP}, title = {Coupling Proximity Biotinylation with Genomic Targeting to Characterize Locus-Specific Changes in Chromatin Environments.}, journal = {Journal of proteome research}, volume = {24}, number = {4}, pages = {1845-1860}, pmid = {40054857}, issn = {1535-3907}, mesh = {Humans ; *Chromatin/genetics/metabolism/chemistry ; Biotinylation/methods ; Cell Line, Tumor ; Azepines/pharmacology ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Triazoles/pharmacology ; *Proteomics/methods ; Proto-Oncogene Proteins c-myc/genetics/metabolism ; Melanoma/genetics ; Genetic Loci ; Bromodomain Containing Proteins ; Proteins ; }, abstract = {Regulating gene expression involves significant changes in the chromatin environment at the locus level, especially at regulatory sequences. However, their modulation following pharmacological treatments or pathological conditions remain mostly undetermined. Here, we report versatile locus-specific proteomics tools to address this knowledge gap, which combine the targeting ability of the CRISPR/Cas9 system and the protein-labeling capability of the highly reactive biotin ligases TurboID (in CasTurbo) and UltraID (in CasUltra). CasTurbo and CasUltra enabled rapid chromatin protein labeling at repetitive sequences like centromeres and telomeres, as well as nonamplified genes. We applied CasUltra to A375 melanoma cell lines to decipher the protein environment of the MYC promoter and characterize the molecular effects of the bromodomain inhibitor JQ1, which targets bromodomain and extra-terminal (BET) proteins that regulate MYC expression. We quantified the consequences of BET protein displacement from the MYC promoter and found that it was associated with a considerable reorganization of the chromatin composition. Additionally, BET protein retention at the MYC promoter was consistent with a model of increased JQ1 resistance. Thus, through the combination of proximity biotinylation and CRISPR/Cas9 genomic targeting, CasTurbo and CasUltra have successfully demonstrated their utility in profiling the proteome associated with a genomic locus in living cells.}, }
@article {pmid40054218, year = {2025}, author = {Zhong, J and Xu, Z and Peng, J and Guan, L and Li, J and Zhou, Z and Zhang, Y and Zhang, J and Liu, S and Yang, Y and Hao, X}, title = {A CRISPR/Cas13a system based on a dumbbell-shaped hairpin combined with DNA-PAINT to establish the DCP-platform for highly sensitive detection of Hantaan virus RNA.}, journal = {Talanta}, volume = {291}, number = {}, pages = {127852}, doi = {10.1016/j.talanta.2025.127852}, pmid = {40054218}, issn = {1873-3573}, mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Viral/analysis/genetics ; *Hantaan virus/genetics/isolation &amp; purification ; Humans ; *DNA/chemistry/genetics ; Limit of Detection ; Inverted Repeat Sequences ; }, abstract = {Rapid and sensitive detection of specific RNA sequences is crucial for clinical diagnosis, surveillance, and biotechnology applications. Currently, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the gold standard for RNA detection; however, it is associated with long processing time, complex procedures, and a high false-positive rate. To address these challenges, we developed a novel sensing platform based on CRISPR/Cas13a that incorporates a dumbbell-shaped hairpin and DNA-PAINT for rapid, highly specific, and sensitive RNA analysis. By leveraging the CRISPR/Cas13a system, this platform enables the cleavage of dumbbell-shaped hairpins, which subsequently allows the cleaved primers to initiate cyclic amplification of fluorescent signals. These signals are further enhanced by the binding and dissociation phenomena inherent to DNA-PAINT technology, ultimately achieving remarkable triple signal amplification. Additionally, the system effectively discriminates Hantaan virus RNA from Seoul virus in real samples. Importantly, the platform can be easily adapted for the detection of other RNAs by simply reconfiguring the hybridization region of crRNA. In conclusion, this platform represents a "five-in-one" RNA detection approach that integrates reliability, versatility, robustness, high specificity, and superior quantitative capabilities. It provides novel insights for direct RNA detection based on CRISPR/Cas13a and demonstrates significant potential for advancement in viral diagnostics.}, }
@article {pmid40054213, year = {2025}, author = {Du, H and Zhou, YY and Wu, JW and Wu, YY and Yan, J and Tan, XC and Feng, DF and Huang, KJ}, title = {CRISPR/Cas12a trans-cleavage cascading dual-template exponential amplification reaction for electrochemiluminescent detection of 17β-estradiol in milk.}, journal = {Talanta}, volume = {291}, number = {}, pages = {127873}, doi = {10.1016/j.talanta.2025.127873}, pmid = {40054213}, issn = {1873-3573}, mesh = {*Estradiol/analysis ; *Milk/chemistry ; *CRISPR-Cas Systems ; Animals ; *Luminescent Measurements/methods ; *Electrochemical Techniques/methods ; Metal-Organic Frameworks/chemistry ; *Biosensing Techniques/methods ; Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; }, abstract = {17β-Estradiol (E2) is a common environmental estrogen that can interfere with the endocrine systems of humans and animals, and poses a carcinogenic risk even at picomolar concentrations. In this study, a functionalized Ru(bpy)3[2+]-embedded metal-organic framework (ZnRuMOF) is synthesized, in which Ru(bpy)3[2+] served as an electrochemiluminescence (ECL) indicator and the porous structure of ZnRuMOF acts as a nanoreactor to enhance the ECL signal. Based on this, we developed an E2 detection method combining a highly specific CRISPR-Cas12a system and dual-template exponential amplification. This method utilizes the trans-cleavage activity of CRISPR-Cas12a to control a light switch, achieving precise and ultra-sensitive detection of E2. The sensing platform demonstrates excellent performance in detecting E2 concentrations ranging from 1 fg mL[-1] to 150 ng mL[-1], with a detection limit of 0.27 fg mL[-1] (S/N = 3). This study provides a reliable approach for diagnosing and treating diseases related to E2, aiming to protect environmental quality and human health.}, }
@article {pmid40054154, year = {2025}, author = {Liu, S and Yang, Y and Li, X and Choi, JW and Guo, J and Luo, H and Li, C}, title = {Development of a single-tube RPA/CRISPR-cas12a detection platform for monkeypox virus.}, journal = {Biosensors &amp; bioelectronics}, volume = {278}, number = {}, pages = {117221}, doi = {10.1016/j.bios.2025.117221}, pmid = {40054154}, issn = {1873-4235}, mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/instrumentation ; Humans ; *Monkeypox virus/isolation &amp; purification/genetics ; *Mpox, Monkeypox/diagnosis/virology ; Limit of Detection ; Point-of-Care Testing ; Animals ; Nucleic Acid Amplification Techniques ; }, abstract = {Monkeypox is a zoonotic disease caused by the monkeypox virus (MPXV), with outbreaks primarily occurring in West and Central Africa. The recent global MPXV outbreak underscores the urgent need for effective detection methods. Currently, qPCR is considered the gold standard for MPXV detection; however, it requires specialized personnel and costly equipment. This study introduces a CRISPR-Cas12a-based detection system targeting the MPXV A27L gene, achieving a detection limit as low as 10 aM. This system exhibits high specificity, with no cross-reactivity with other orthopoxviruses, and delivers results in under 40 min. To support point-of-care testing (POCT), we developed a lateral flow assay (LFA) strip for easy result visualization. The detection system was validated using six different clinical sample types, revealing that herpes fluid and saliva are the most suitable sources. The findings of this study align with qPCR results. Additionally, we lyophilized the RPA and CRISPR reagents to improve transport, storage, and field deployment. In conclusion, this study presents a reliable molecular diagnostic approach for early MPXV detection and point-of-care testing, contributing to epidemic prevention and control.}, }
@article {pmid40054045, year = {2025}, author = {Li, X and Wang, Z and Man, X and Dai, X and Zhou, Q and Zhang, S}, title = {Research advances CRISPR gene editing technology generated models in the study of epithelial ovarian carcinoma.}, journal = {Gynecologic oncology}, volume = {195}, number = {}, pages = {34-44}, doi = {10.1016/j.ygyno.2025.02.022}, pmid = {40054045}, issn = {1095-6859}, mesh = {Female ; *Carcinoma, Ovarian Epithelial/genetics/therapy ; *Gene Editing/methods ; Animals ; Humans ; *Ovarian Neoplasms/genetics/therapy ; *CRISPR-Cas Systems ; Mice ; *Disease Models, Animal ; Genetic Therapy/methods ; }, abstract = {Epithelial ovarian carcinoma (EOC), the most lethal gynecologic cancer, is often diagnosed at advanced stages, which urge us to explore the novel therapeutic strategies. Mouse models have played a crucial role in elucidating the molecular mechanisms for the development ovarian cancer and its therapeutic strategies. However, there are still various challenges in modeling the genetic drivers of ovarian cancer in animal models. Here, we provided an overview of the research advances for the molecular mechanisms underlying EOC development, therapeutic strategies, the CRISPR genome editing technology and its generated EOC models. The review also comprehensively discussed the advantages and obstacles of CRISPR in generating EOC mouse models and the promising therapeutic approach by correcting the oncogenes of EOC through in vivo delivery of gene-edited components. The development of more precise animal models, along with a deeper understanding of EOC molecular mechanisms, will dramatically benefit the investigation and treatment of EOC.}, }
@article {pmid40053823, year = {2025}, author = {Liu, Y and Li, F and Lyu, Y and Wang, F and Lee, LTO and He, S and Guo, Z and Li, J}, title = {A Semiconducting Polymer NanoCRISPR for Near-Infrared Photoactivatable Gene Editing and Cancer Gene Therapy.}, journal = {Nano letters}, volume = {25}, number = {11}, pages = {4518-4525}, doi = {10.1021/acs.nanolett.5c00285}, pmid = {40053823}, issn = {1530-6992}, mesh = {*Gene Editing/methods ; Humans ; *Genetic Therapy/methods ; *Polymers/chemistry ; *Neoplasms/therapy/genetics ; Semiconductors ; Infrared Rays ; CRISPR-Cas Systems ; Polo-Like Kinase 1 ; *Nanoparticles/chemistry ; Protein Serine-Threonine Kinases/genetics ; DNA, Single-Stranded/chemistry/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; Green Fluorescent Proteins/genetics ; }, abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR) gene editing has poor efficacy and off-target side effect concerns. We herein report a semiconducting polymer (SP)-based nanoCRISPR system to improve CRISPR delivery efficacy and allow for near-infrared (NIR) photoactivatable gene editing for cancer therapy. An amphiphilic SP acts as a photothermal converter, and its backbone is grafted with single-stranded deoxyribonucleic acid (DNA), which enables hybridization with single guide ribonucleic acid (sgRNA) via complementary base pairing to form sgRNA/SP-DNA. This sgRNA/SP-DNA nanosystem (nanoCRISPR) can effectively deliver sgRNA into cells and generate heat under NIR laser irradiation via the photothermal effect. The localized heat triggers the dissociation of single-stranded DNA and sgRNA to control the release of sgRNA, thereby achieving precise regulation of CRISPR activity. This NIR photoactivatable gene editing technology is able to precisely regulate the expression of green fluorescent protein (GFP) and polo-like kinase 1 (PLK1) gene for precision gene therapy.}, }
@article {pmid40052500, year = {2025}, author = {Lloyd, JPB and Khan, A and Lister, R}, title = {The switch-liker's guide to plant synthetic gene circuits.}, journal = {The Plant journal : for cell and molecular biology}, volume = {121}, number = {5}, pages = {e70090}, pmid = {40052500}, issn = {1365-313X}, support = {CE230100015//Australian Research Council/ ; DP240103385//Australian Research Council/ ; GNT1178460//National Health and Medical Research Council/ ; //Clifford Bradley Robertson and Gwendoline Florence Robertson fund/ ; }, mesh = {*Gene Regulatory Networks/genetics ; *Genes, Synthetic/genetics ; *Plants/genetics ; CRISPR-Cas Systems/genetics ; *Genetic Engineering/methods ; Gene Expression Regulation, Plant ; *Genes, Plant/genetics ; Synthetic Biology/methods ; Plants, Genetically Modified/genetics ; }, abstract = {Synthetic gene circuits offer powerful new approaches for engineering plant traits by enabling precise control over gene expression through programmable logical operations. Unlike simple 'always-on' transgenes, circuits can integrate multiple input signals to achieve sophisticated spatiotemporal regulation of target genes while minimising interference with host cellular processes. Recent advances have demonstrated several platforms for building plant gene circuits, including systems based on bacterial transcription factors, site-specific recombinases and CRISPR/Cas components. These diverse molecular tools allow the construction of circuits that perform Boolean logic operations to control transgene expression or modulate endogenous pathways. However, implementing synthetic gene circuits in plants faces unique challenges, including long generation times that slow design-build-test cycles, limited availability of characterised genetic parts across species and technical hurdles in stable transformation. This review examines the core principles and components of plant synthetic gene circuits, including sensors, integrators, and actuators. We discuss recent technological developments, key challenges in circuit design and implementation, and strategies to overcome them. Finally, we explore the future applications of synthetic gene circuits in agriculture and basic research, from engineering stress resistance to enabling controlled bioproduction of valuable compounds. As this technology matures, synthetic gene circuits have the potential to enable sophisticated new plant traits that respond dynamically to environmental and developmental cues.}, }
@article {pmid40052456, year = {2025}, author = {Yin, T and Tai, Y and Sun, Y and Cheng, Z and Wu, C and Sui, Y}, title = {Precise tiller angle control by manipulating TAC1 expression in rice.}, journal = {Journal of integrative plant biology}, volume = {67}, number = {6}, pages = {1444-1446}, doi = {10.1111/jipb.13877}, pmid = {40052456}, issn = {1744-7909}, support = {2024ZD04077//Biological Breeding-National Science and Technology Major Project/ ; 31801323//National Natural Science Foundation of China/ ; //Innovation Program of the Chinese Academy of Agricultural Sciences/ ; }, mesh = {*Oryza/genetics/metabolism/anatomy &amp; histology ; *Plant Proteins/metabolism/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems/genetics ; Gene Editing ; Phenotype ; }, abstract = {Tiller angle shapes plant architecture, and is one of the top traits in plant breeding. A compact plant type reduces shading between plants, especially at high planting density, but also creates a humid microenvironment often associated with a higher incidence of pathogen and pest attacks, especially under highly humid climates. However, how to precisely manipulate the tiller angle to achieve a desirable plant type has been under-approached. Here we report the creation of gradient tiller angles in indica rice by fine tuning the expression of TILLER ANGLE CONTROL1 (TAC1), a domesticated gene in cultivated rice. We edited the regions upstream and downstream of the TAC1 coding sequence using multiplex CRISPR-Cas9 technology and developed homozygous allelic lines carrying deletions/inversions of various sizes at different positions. Those lines displayed smooth gradient changes in tiller angle that aligned well with TAC1 expression levels. Additionally, changes in the TAC1 expression level had no impact on other agronomic traits examined. TAC1 is well conserved across species, including perennial fruit trees in which mutation of TAC1 orthologs leads to a broomy plant type. Thus, our results provide a guide to creating tiller angles for selection according to climate zones in rice breeding programs, this approach can be extended to diverse species for improving plant architecture.}, }
@article {pmid40052414, year = {2025}, author = {Tjallinks, G and Angeleri, N and Nguyen, QT and Mannucci, B and Arentshorst, M and Visser, J and Ram, AFJ and Fraaije, MW and Mattevi, A}, title = {Structural and Mechanistic Characterization of the Flavin-Dependent Monooxygenase and Oxidase Involved in Sorbicillinoid Biosynthesis.}, journal = {ACS chemical biology}, volume = {20}, number = {3}, pages = {646-655}, pmid = {40052414}, issn = {1554-8937}, mesh = {*Penicillium chrysogenum/enzymology/metabolism/genetics ; *Mixed Function Oxygenases/metabolism/chemistry/genetics ; Aspergillus niger/enzymology ; Catalytic Domain ; Crystallography, X-Ray ; Oxidoreductases/metabolism/chemistry/genetics ; Dinitrocresols/metabolism/chemistry ; Resorcinols/metabolism/chemistry ; Fungal Proteins/metabolism/chemistry/genetics ; Flavin-Adenine Dinucleotide/metabolism/chemistry ; Protein Conformation ; Flavins/metabolism ; Models, Molecular ; CRISPR-Cas Systems ; }, abstract = {Sorbicillinoids are yellow secondary metabolites synthesized through an elegant combination of enzymatic and spontaneous biochemical processes. The flavin-dependent monooxygenase SorC and oxidase SorD are crucial in this interplay, enabling the generation of a diverse array of functionally complex sorbicillinoids. By solving the crystal structures of SorC and SorD from Penicillium chrysogenum with sorbicillin bound in the active site, we describe the catalytically active binding conformations, crucial for attaining enantioselective and stereoselective control in these enzymatic reactions. The structure of SorC was resolved with the cofactor FAD in its out state, which allowed us to identify key residues that modulate flavin mobility and other conformational changes. Catalytic residues of SorC were also confirmed by detailed characterization of wild-type and several SorC variants. Meanwhile, using a CRISPR/Cas9-based multicopy-genome integration system, we could heterologously express the flavin-dependent oxidase SorD from P. chrysogenum in Aspergillus niger with high yields and purity. This allowed us to obtain the crystal structure of SorD with sorbicillin bound in a viable catalytic conformation. Structural analysis of the obtained complex provided insights into the substrate binding pose and highlighted potentially critical active site residues. Ultimately, having both SorC and SorD at our disposal enabled us to investigate their functions and interplays in the biosynthesis of a vast array of functionally complex sorbicillinoids.}, }
@article {pmid40052226, year = {2025}, author = {Zhang, H and Xie, L and Gao, H and Pan, H}, title = {Application of pre-amplification-based CRISPR-Cas nanostructured biosensors for bacterial detection.}, journal = {Nanomedicine (London, England)}, volume = {20}, number = {8}, pages = {903-915}, pmid = {40052226}, issn = {1748-6963}, mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Bacteria/isolation &amp; purification/genetics ; *Nanostructures/chemistry ; Humans ; *Bacterial Infections/diagnosis/microbiology ; Gene Editing ; }, abstract = {Bacterial infections are one of the primary triggers of global disease outbreaks. Traditional detection methods, such as bacterial culture and PCR, while reliable, are limited by their time-consuming procedures and operational complexity. In recent years, the CRISPR-Cas system has demonstrated significant potential in gene editing and diagnostics due to its high specificity and precision, offering innovative solutions for bacterial detection. By integrating pre-amplification techniques, the CRISPR-Cas system has substantially enhanced detection sensitivity, particularly excelling in detecting low-concentration target bacteria. This review summarizes the principles and application examples of CRISPR-Cas-based fluorescence, electrochemical, lateral flow, and colorimetric nanostructured biosensors developed over the past three years, categorizing them according to their recognition methods (e.g. bacterial genomes, aptamers, antibodies). It systematically explores the broad application prospects of these sensors in medical diagnostics, environmental monitoring, and food safety assessment. Additionally, this review discusses future research directions and potential development prospects, providing new insights and technical support for the rapid diagnosis and treatment of bacterial infections.}, }
@article {pmid40050992, year = {2025}, author = {Li, X and Zhang, W and Fang, Y and Sun, T and Chen, J and Tian, R}, title = {Large-scale CRISPRi screens link metabolic stress to glioblastoma chemoresistance.}, journal = {Journal of translational medicine}, volume = {23}, number = {1}, pages = {289}, pmid = {40050992}, issn = {1479-5876}, support = {32100766//National Natural Science Foundation of China/ ; 82171416//National Natural Science Foundation of China/ ; 2023B1515020075//Basic and Applied Basic Research Foundation of Guangdong Province/ ; JCYJ20220530112602006//Shenzhen Fundamental Research Program/ ; RCYX20221008092845052//Shenzhen Fundamental Research Program/ ; A2303039//Shenzhen Medical Research Fund/ ; }, mesh = {*Glioblastoma/genetics/drug therapy/metabolism/pathology ; Humans ; *Drug Resistance, Neoplasm/genetics/drug effects ; Cell Line, Tumor ; Temozolomide/pharmacology/therapeutic use ; *Stress, Physiological/genetics/drug effects ; Phosphoglycerate Kinase/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Animals ; *Brain Neoplasms/genetics/drug therapy/pathology/metabolism ; Gene Expression Regulation, Neoplastic/drug effects ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism ; Signal Transduction/drug effects ; Mice ; }, abstract = {BACKGROUND: Glioblastoma (GBM) patients frequently develop resistance to temozolomide (TMZ), the standard chemotherapy. While targeting cancer metabolism shows promise, the relationship between metabolic perturbation and drug resistance remains poorly understood.<br><br>METHODS: We performed high-throughput CRISPR interference screens in GBM cells to identify genes modulating TMZ sensitivity. Findings were validated using multiple GBM cell lines, patient-derived glioma stem cells, and clinical data. Molecular mechanisms were investigated through transcriptome analysis, metabolic profiling, and functional assays.<br><br>RESULTS: We identified phosphoglycerate kinase 1 (PGK1) as a key determinant of TMZ sensitivity. Paradoxically, while PGK1 inhibition suppressed tumor growth, it enhanced TMZ resistance by inducing metabolic stress. This activated AMPK and HIF-1&#x3b1; pathways, leading to enhanced DNA damage repair through 53BP1. PGK1 expression levels correlated with TMZ sensitivity across multiple GBM models and patient samples.<br><br>CONCLUSIONS: Our study reveals an unexpected link between metabolic stress and chemoresistance, demonstrating how metabolic adaptation can promote therapeutic resistance. These findings caution against single-agent metabolic targeting and suggest PGK1 as a potential biomarker for TMZ response in GBM.}, }
@article {pmid40050620, year = {2025}, author = {Benatto Perino, EH and Smolka, U and Gorzolka, K and Grützner, R and Marillonnet, S and Vahabi, K and Rosahl, S}, title = {The suberin transporter StABCG1 is required for barrier formation in potato leaves.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {7930}, pmid = {40050620}, issn = {2045-2322}, mesh = {*Solanum tuberosum/genetics/metabolism ; *Plant Leaves/metabolism/genetics ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; Cell Wall/metabolism ; *Membrane Transport Proteins/metabolism/genetics ; Gene Expression Profiling ; Lipids ; }, abstract = {Suberin is a hydrophobic biopolymer that acts as an internal and external diffusion and transpiration barrier in plants. It is involved in two phases of wound healing, i.e. initial closing layer formation and subsequent wound periderm development. Transcriptomic and metabolomic analyses of wounded potato leaf tissue revealed preferential induction of cell wall modifying processes during closing layer formation, accompanied by a highly active defense response. To address the importance of suberin in this process, we generated loss of function mutants by CRISPR-Cas9 editing the suberin transporter gene StABCG1. Both wound-induced StABCG1 transcript levels and suberin formation around wounded leaf tissue were reduced in CRISPR-lines. Moreover, wound-induced tissue damage was characterized by browning of wound-adjacent areas. Transcriptome analyses of these areas revealed up-regulation of genes encoding defense proteins and enzymes of the phenylpropanoid pathway. Levels of hydroxycinnamic acid amides, acting in defense and in cell wall reinforcement, were drastically enhanced in CRISPR compared to control plants. These results suggest that the reduction in suberin formation around wounded tissue leads to a loss of barrier function, resulting in tissue browning due to enhanced exposure to oxygen.}, }
@article {pmid40050380, year = {2025}, author = {Casco-Robles, MM and Echigoya, T and Shimazaki, T and Murakami, Y and Hirano, M and Maruo, F and Mizuno, S and Takahashi, S and Chiba, C}, title = {One-step Cre-loxP organism creation by TAx9.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {340}, pmid = {40050380}, issn = {2399-3642}, support = {24240062//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 18H04061//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 23H05483//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; }, mesh = {*Integrases/genetics/metabolism ; Animals ; Mice ; CRISPR-Cas Systems ; *Recombination, Genetic ; Escherichia coli/genetics ; Mice, Transgenic ; Plasmids/genetics ; }, abstract = {The creation of organisms with Cre-loxP conditional gene recombination systems often faces challenges, particularly when creating the initial (F0) generation with both a Cre recombinase and a DNA site flanked by loxP elements (floxed site). The primary reason is that it is difficult to synthesize a single plasmid with both the Cre gene and the floxed site, since Cre-mediated recombination spontaneously occurs when the plasmid is amplified in Escherichia coli bacterial cells. Here, we introduce an artificial nucleic acid sequence TATATATATATATATATA, named TAx9, that enables the integration of both the Cre gene and the floxed site into a single plasmid. TAx9 effectively blocks spontaneous Cre-mediated recombination in E. coli cells. Using this system, we created an F0 generation of transgenic newts and CRISPR-Cas9 knock-in mice with tissue-specific Cre recombination triggered by tamoxifen. TAx9 technology will be a powerful strategy for creating organisms capable of conditional genetic modification in the F0 generation, accelerating various life science research by reducing the time and cost for ultimately establishing and maintaining lines of genetically modified organisms.}, }
@article {pmid40049443, year = {2025}, author = {Svoboda, T and Niederdöckl-Loibl, D and Schüller, A and Hummel, K and Schlosser, S and Razzazi-Fazeli, E and Strauss, J}, title = {Locus-specific chromatin proteomics using dCas-guided proximity labelling in Aspergillus nidulans.}, journal = {Fungal genetics and biology : FG &amp; B}, volume = {178}, number = {}, pages = {103973}, doi = {10.1016/j.fgb.2025.103973}, pmid = {40049443}, issn = {1096-0937}, mesh = {*Chromatin/genetics/metabolism ; *Aspergillus nidulans/genetics/metabolism ; *Proteomics/methods ; Fungal Proteins/genetics/metabolism ; Biotinylation ; Transcription Factors/genetics ; CRISPR-Cas Systems ; Chromatin Immunoprecipitation ; Carbon-Nitrogen Ligases/genetics ; }, abstract = {Proximity labelling that uses promiscuous biotin ligases (BirA) fused to a bait protein is a powerful tool to identify protein interaction partners in vivo under different metabolic or developmental conditions. BirA can also be used to determine protein composition and interaction partners at specific chromatin locations when it is fused with enzymatically-disabled Cas9 (dCas9) and then guided to the location of interest by sgRNAs. We adapted this method (called CasID) for fungal cells using the nitrate assimilation gene cluster of A. nidulans as a model locus and estrogen-inducible expression of the dCas9-BirA fusion to improve condition-specific labelling. For method establishment, we first verified the presence of dCas-BirA and a known transcription factor at the nitrate locus by chromatin immunoprecipitation (ChIP). Results show that both dCas-BirA and the AreA transcription factor are present at the locus of interest under the conditions used for biotinylation. We then optimized the CasID procedure for efficient labelling and background reduction using the CasID-sgRNA strain and two control strains, one lacking the sgRNA and another one lacking the whole CasID system. Here we provide proof-of-concept for the suitability of the method by showing that biotinylated proteins are enriched in the CasID strains in comparison to the controls. After background reduction, 32 proteins remained in two independent experiments exclusively enriched in the Cas-ID-sgRNA strain. Among these proteins was NmrA, an AreA-interacting regulator, and we also found several chromatin-associated proteins. Overall, our results demonstrate that CasID is suitable for locus-specific labelling and identification of chromatin-associated proteins and transcription factors in A. nidulans. However, the high background of proteins that are biotinylated out of chromatin context or unspecifically attach to the affinity purification matrix needs to be addressed by implementing a set of rigorous controls. In summary, we herewith provide a detailed protocol for application of the method that proved to be useful for the identification of novel chromatin-associated proteins and their interaction partners at a specific genomic locus in divers metabolic and developmental conditions. AUTHOR SUMMARY: This study demonstrates that locus-specific proteomics can be carried out by dCas-BirA guided proximity labelling in Aspergillus nidulans. For establishment, we targeted the well-described bidirectional promoter region between niaD, a nitrate reductase, and niiA, a nitrite reductase. At this locus we could test by chromatin immunoprecipitation (ChIP) in combination with qPCR if both, the dCas9-BirA fusion as well as a central transcription factor are at the locus under the conditions of our CasID experiment. After this first control step, we considered that unspecific labelling by dCas-BirA during the time from translation to landing at the targeted chromatin locus may be one of the most relevant drawbacks of the method. Therefore, we developed a number of control strains that would allow us to clearly discriminate between background and sgRNA-dependent specific labelling at the locus. Our protein MS results validated these estimates and only considering the results of these controls enabled us to distinguish the set of locus-specific proteins from a very high general background. Finally, enrichment of biotinylated proteins through affinity purification with streptavidin resin and subsequent LC-MS/MS analysis showed that more than 800 proteins were detected in each sample, emphasizing the high background of the purification method. After background reduction of the control samples, we were able to identify 32 proteins which were exclusively detected in the test strain in two independent measurements, including several chromatin-associated proteins and NmrA, a negative regulator of the nitrate locus transcription factor AreA.}, }
@article {pmid40049338, year = {2025}, author = {Liu, K and Hu, S and Wufuer, R and Zhang, Q and Qiu, L and Zhang, Z and Wang, M and Zhang, Y}, title = {Deficiency of DDI2 suppresses liver cancer progression by worsening cell survival conditions.}, journal = {Free radical biology &amp; medicine}, volume = {232}, number = {}, pages = {200-213}, doi = {10.1016/j.freeradbiomed.2025.03.003}, pmid = {40049338}, issn = {1873-4596}, mesh = {*Liver Neoplasms/pathology/genetics/metabolism ; Humans ; Reactive Oxygen Species/metabolism ; Animals ; DNA Damage ; NF-E2-Related Factor 2/genetics/metabolism ; Mice ; Cell Survival/genetics ; Apoptosis/genetics ; Cell Proliferation ; CRISPR-Cas Systems ; Disease Progression ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; Nuclear Respiratory Factor 1/genetics/metabolism ; *Carcinoma, Hepatocellular/pathology/genetics/metabolism ; NF-E2-Related Factor 1/genetics/metabolism ; Oxidative Stress ; }, abstract = {The levels of reactive oxygen species (ROS) and the extent of ensuing DNA damage significantly influence cancer initiation and progression. Of crucial importance, the aspartate protease DDI2 has been proposed to play a pivotal role in monitoring intracellular ROS levels (to trigger oxidative eustress or distress), as well as in the oxidative DNA damage repair, through redox homeostasis-determining factor Nrf1 (encoded by NFE2L1). However, the specific role of DDI2 in the multi-step process resulting in the development and progression of liver cancer remains elusive to date. In the present study, we employed the CRISPR/Cas9 gene editing system to create two nuanced lines of DDI2 knockout (i.e., DDI2[-/-] and DDI2[insG/-]) from liver cancer cells. Subsequent experiments indicate that the knockout of DDI2 leads to increased ROS levels in hepatoma cells by downregulating two major antioxidant transcription factors Nrf1 and Nrf2 (encoded by NFE2L2), exacerbating endogenous DNA damages caused by ROS and not-yet-identified factors, thereby inhibiting cell proliferation and promoting apoptosis, and ultimately hindering in vivo malignant growth of xenograft tumor cells. Conversely, the restoration of DDI2 expression reverses the accumulation of ROS and associated DNA damage caused by DDI2 knockout, eliminating the subsequent inhibitory effects of DDI2 deficiency on both in vitro and in vivo growth of liver cancer cells. Collectively, these findings demonstrate that DDI2 deficiency impedes liver tumor growth by disrupting its survival environment, suggesting that DDI2 may serve as a novel therapeutic target for anti-cancer strategies aimed at modulating ROS or DNA damage processes.}, }
@article {pmid40049000, year = {2025}, author = {Gao, S and Guo, J and Wang, T and Xu, L}, title = {A rapid visual detection method for Sugarcane streak mosaic virus based on one-tube RPA-CRISPR/Cas12a.}, journal = {Talanta}, volume = {291}, number = {}, pages = {127888}, doi = {10.1016/j.talanta.2025.127888}, pmid = {40049000}, issn = {1873-3573}, mesh = {*Saccharum/virology ; *Nucleic Acid Amplification Techniques/methods ; *Potyviridae/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; *Recombinases/metabolism ; Plant Diseases/virology ; Plant Leaves/virology ; Potyvirus ; }, abstract = {Sugarcane is the most important crop for sugar production. Sugarcane streak mosaic virus (SCSMV) triggered sugarcane mosaic disease can lead to substantial reductions in both yield and sucrose content. In the process of disease prevention and control, target pathogen detection technology is indispensable. However, traditional detection methods are time-consuming and require expensive equipment, making them less efficient for timely disease control and unfavorable to disease resistance breeding. Here, we introduce a novel detection technology that combines recombinase polymerase amplification (RPA) with CRISPR-Cas12a. The method utilizes crude extracts from sugarcane leaves as the reaction template, significantly simplifying and expediting the preparation process. By combining RPA and CRISPR-Cas12a in a single reaction tube, the risk of aerosol contamination has decreased markedly. The entire process, from sample preparation to result interpretation, only takes 50 min, and the reaction equipment only a water bath pot, and results can be blue light spectrometer or UV flashlight assessed visually. Importantly, the method demonstrates high sensitivity, detecting a minimum of 50 copies of the plasmid, which surpasses the sensitivity of reverse transcription polymerase chain reaction (RT-PCR) and is comparable to quantitative RT-PCR (RT-qPCR). The method exhibits excellent specificity, showing no cross-reactivity with other common sugarcane viruses, including Sugarcane mosaic virus, Sugarcane yellow leaf virus, and Sorghum mosaic virus. The practicality of this technique was validated through the detection of leaf crude extracts from 40 field samples. The detection results were consistent with those obtained from RT-PCR and RT-qPCR using leaf RNA as the template, indicating its suitability for laboratory detection and field applications.}, }
@article {pmid40048486, year = {2025}, author = {Roussis, IM and Pearton, DJ and Niazi, U and Tsaknakis, G and Papadopoulos, GL and Cook, R and Saqi, M and Ragoussis, J and Strouboulis, J}, title = {A novel role for Friend of GATA1 (FOG-1) in regulating cholesterol transport in murine erythropoiesis.}, journal = {PLoS genetics}, volume = {21}, number = {3}, pages = {e1011617}, pmid = {40048486}, issn = {1553-7404}, mesh = {Animals ; *Erythropoiesis/genetics ; Mice ; *Cholesterol/metabolism ; *Transcription Factors/genetics/metabolism ; Cell Differentiation/genetics ; GATA1 Transcription Factor/genetics/metabolism ; Mice, Knockout ; Biological Transport/genetics ; ATP Binding Cassette Transporter 1/genetics/metabolism ; Sterol Regulatory Element Binding Protein 2/genetics/metabolism ; CRISPR-Cas Systems ; }, abstract = {Friend of GATA1 (FOG-1) is an essential transcriptional co-factor of the master erythroid transcription factor GATA1. The knockout of the Zfpm1 gene, coding for FOG-1, results in early embryonic lethality due to anemia in mice, similar to the embryonic lethal phenotype of the Gata1 gene knockout. However, a detailed molecular analysis of the Zfpm1 knockout phenotype in erythropoiesis is presently incomplete. To this end, we used CRISPR/Cas9 to knockout Zfpm1 in mouse erythroleukemic (MEL) cells. Phenotypic characterization of DMSO-induced terminal erythroid differentiation showed that the Zfpm1 knockout MEL cells did not progress past the proerythroblast stage of differentiation. Expression profiling of the Zfpm1 knockout MEL cells by RNAseq showed a lack of up-regulation of erythroid-related gene expression profiles. Bioinformatic analysis highlighted cholesterol transport as a pathway affected in the Zfpm1 knockout cells. Moreover, we show that the cholesterol transporters Abca1 and Ldlr fail to be repressed during erythroid differentiation in Zfpm1 knockout cells, resulting in higher intracellular lipid levels and higher membrane fluidity. We also show that in FOG-1 knockout cells, the nuclear levels of SREBP2, a key transcriptional regulator of cholesterol biosynthesis and transport, are markedly increased. On the basis of these findings we propose that FOG-1 (and, potentially, GATA1) regulate cholesterol homeostasis during erythroid differentiation directly through the down regulation of cholesterol transport genes and indirectly, through the repression of the SREBP2 transcriptional activator of cholesterol homeostasis. Taken together, our work provides a molecular basis for understanding FOG-1 functions in erythropoiesis and reveals a novel role for FOG-1 in cholesterol transport.}, }
@article {pmid40048448, year = {2025}, author = {Du, Y and Yang, Y and Zhang, Y and Zhang, F and Wu, J and Yin, J}, title = {Unraveling enhanced liver regeneration in ALPPS: Integrating multi-omics profiling and in vivo CRISPR in mouse models.}, journal = {Hepatology communications}, volume = {9}, number = {3}, pages = {}, pmid = {40048448}, issn = {2471-254X}, mesh = {Animals ; *Liver Regeneration/genetics ; Mice ; *Hepatectomy/methods ; Hepatocytes/metabolism/physiology ; MicroRNAs/genetics ; Ligation ; Portal Vein/surgery ; Mice, Knockout ; Polo-Like Kinase 1 ; Cell Proliferation/genetics ; Liver/surgery ; Cell Cycle Proteins/genetics/metabolism ; Proto-Oncogene Proteins/genetics/metabolism ; Protein Serine-Threonine Kinases/genetics/metabolism ; Male ; Disease Models, Animal ; Gene Expression Profiling ; CRISPR-Cas Systems ; Hydrolases/genetics ; Mice, Inbred C57BL ; Multiomics ; }, abstract = {BACKGROUND: Postoperative liver failure due to insufficient liver cell quantity and function remains a major cause of mortality following surgery. Hence, additional investigation and elucidation are required concerning suitable surgeries for promoting in vivo regeneration.<br><br>METHODS: We established the portal vein ligation (PVL) and associated liver partition and portal vein ligation for staged hepatectomy (ALPPS) mouse models to compare their in vivo regeneration capacity. Then, RNA-seq and microRNA-seq were conducted on the livers from both mouse models. Weighted gene co-expression network analysis algorithm was leveraged to identify crucial gene modules. ScRNA-seq analysis was used to understand the distinctions between Signature30high hepatocytes and Signature30low hepatocytes. Moreover, in vivo, validation was performed in fumarylacetoacetate hydrolase knockout mice with gene editing using the CRISPR-cas9 system. A dual luciferase report system was carried out to further identify the regulatory mechanisms.<br><br>RESULTS: RNA-seq analysis revealed that ALPPS could better promote cell proliferation compared to the sham and portal vein ligation models. Moreover, a Plk1-related 30-gene signature was identified to predict the cell state. ScRNA-seq analysis confirmed that signature30high hepatocytes had stronger proliferative ability than signature30low hepatocytes. Using microRNA-seq analysis, we identified 53 microRNAs that were time-dependently reduced after ALPPS. Finally, miR-30a-3p might be able to regulate the expression of Plk1, contributing to the liver regeneration of ALPPS.<br><br>CONCLUSIONS: ALPPS could successfully promote liver regeneration by activating hepatocytes into a proliferative state. Moreover, a Plk1-related 30-gene signature was identified to predict the cell state of hepatocytes. miR-30a-3p might be able to regulate the expression of Plk1, contributing to the liver regeneration of ALPPS.}, }
@article {pmid40047422, year = {2025}, author = {Layton, AM and McCauley, C and Redding, KE}, title = {Deletion of Re-citrate synthase allows for analysis of contributions of tricarboxylic acid cycle directionality to the growth of Heliomicrobium modesticaldum.}, journal = {Applied and environmental microbiology}, volume = {91}, number = {4}, pages = {e0177224}, pmid = {40047422}, issn = {1098-5336}, support = {18-EXO18-0080//National Aeronautics and Space Administration (NASA)/ ; }, mesh = {*Citric Acid Cycle/genetics ; *Bacterial Proteins/genetics/metabolism ; Gene Deletion ; *Firmicutes/growth &amp; development/genetics/enzymology/metabolism ; *Citrate (si)-Synthase/genetics/metabolism ; *Clostridiales/genetics/growth &amp; development/enzymology/metabolism ; Pyruvic Acid/metabolism ; }, abstract = {UNLABELLED: Heliomicrobium modesticaldum, a phototrophic member of the phylum Firmicutes and family Clostridiales, possesses most of the enzymes specific to the reductive tricarboxylic acid (rTCA) cycle, except for the key enzyme, ATP-citrate lyase. It is thought to utilize a split TCA cycle when growing on pyruvate as a carbon source, in which the oxidative TCA (oTCA) direction generates most of the 2-ketoglutarate, but some can be produced in the reductive direction. Although a typical Si-citrate synthase gene is not found in the genome, it was suggested that gene HM1_2993, annotated as homocitrate synthase, actually encodes Re-citrate synthase, which would function as the initial enzyme of the oTCA cycle. We deleted this gene to test this hypothesis and, if true, see what effect severing access to the oTCA cycle would have on this organism. The endogenous CRISPR-Cas system was used to replace the open reading frame with a selectable marker. The deletion mutants could grow on pyruvate but were unable to grow phototrophically on acetate + CO2 as carbon source. Growth on acetate could be rescued by the addition of different electron sources (formate or ascorbate), suggesting that the oTCA cycle is used to oxidize acetate to generate electrons required to drive the carboxylation of acetyl-CoA. The deletion mutants were capable of growing in acetate minimal media without additional organic supplements beyond formate, demonstrating that the rTCA cycle can be employed to support sufficient 2-ketoglutarate production in this organism, unlike citrate synthase mutants in several chemoheterotrophic organisms utilizing the oTCA cycle.<br><br>IMPORTANCE: Heliobacteria are a unique group of phototrophic bacteria that are obligate anaerobes and possess a rudimentary system to use light as a source of energy. They do not make oxygen or fix carbon dioxide. Here, we explore their fundamental carbon metabolism to understand the role and operation of the central TCA cycle. This work shows both the role and operation of this cycle under different growth modes and explains how these organisms can obtain electrons to drive their biosynthetic metabolism. This foundational knowledge will be crucial in the future when attempts are made to use this organism as a platform for oxygen-sensitive synthesis of compounds in an anaerobe that can use light as its energy source.}, }
@article {pmid40045573, year = {2025}, author = {Li, F and Tan, X and Li, S and Chen, S and Liu, L and Huang, J and Li, G and Lu, Z and Wu, J and Zeng, D and Luo, Y and Dong, X and Ma, X and Zhu, Q and Chen, L and Liu, YG and Chen, C and Xie, X}, title = {SuperDecode: An integrated toolkit for analyzing mutations induced by genome editing.}, journal = {Molecular plant}, volume = {18}, number = {4}, pages = {690-702}, doi = {10.1016/j.molp.2025.03.002}, pmid = {40045573}, issn = {1752-9867}, mesh = {*Gene Editing/methods ; *Mutation/genetics ; *Software ; CRISPR-Cas Systems/genetics ; Oryza/genetics ; *Genome, Plant/genetics ; }, abstract = {Genome editing using CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) or other systems has become a cornerstone of numerous biological and applied research fields. However, detecting the resulting mutations by analyzing sequencing data remains time consuming and inefficient. In response to this issue, we designed SuperDecode, an integrated software toolkit for analyzing editing outcomes using a range of sequencing strategies. SuperDecode comprises three modules, DSDecodeMS, HiDecode, and LaDecode, each designed to automatically decode mutations from Sanger, high-throughput short-read, and long-read sequencing data, respectively, from targeted PCR amplicons. By leveraging specific strategies for constructing sequencing libraries of pooled multiple amplicons, HiDecode and LaDecode facilitate large-scale identification of mutations induced by single or multiplex target-site editing in a cost-effective manner. We demonstrate the efficacy of SuperDecode by analyzing mutations produced using different genome editing tools (CRISPR/Cas, base editing, and prime editing) in different materials (diploid and tetraploid rice and protoplasts), underscoring its versatility in decoding genome editing outcomes across different applications. Furthermore, this toolkit can be used to analyze other genetic variations, as exemplified by its ability to estimate the C-to-U editing rate of the cellular RNA of a mitochondrial gene. SuperDecode offers both a standalone software package and a web-based version, ensuring its easy access and broad compatibility across diverse computer systems. Thus, SuperDecode provides a comprehensive platform for analyzing a wide array of mutations, advancing the utility of genome editing for scientific research and genetic engineering.}, }
@article {pmid40045211, year = {2025}, author = {Kwon, DH and Gim, GM and Yum, SY and Eom, KH and Lee, SJ and Han, SE and Kim, HS and Kim, HJ and Lee, WS and Choi, WJ and Lee, JH and Kim, DY and Jung, DJ and Kim, DH and Yi, JK and Moon, BH and Lee, WY and Jang, G}, title = {Cas9-expressing cattle using the PiggyBac transposon all-in-one system.}, journal = {BMC genomics}, volume = {26}, number = {1}, pages = {217}, pmid = {40045211}, issn = {1471-2164}, support = {NRF-2021R1A5A1033157//Brain Korea 21 FOUR Future Veterinary Medicine Leading Education and Research Center/ ; }, mesh = {Animals ; Cattle/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *DNA Transposable Elements/genetics ; Animals, Genetically Modified ; Male ; *CRISPR-Associated Protein 9/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Female ; }, abstract = {BACKGROUND: Livestock, particularly cattle, are crucial for biotechnology fields, such as genetic breeding, infectious diseases, bioreactors, and specific disease models. However, genetic engineering in cattle has lagged due to long gestation periods, single embryo pregnancies, and high rearing costs. Additionally, the slow validation of germline transmission and the absence of germline-competent embryonic stem cells hinder progress. With the development of genome editing technologies like ZFN, TALEN, and CRISPR-Cas9, recent advancements have shown that Cas9-expressing pigs and chickens have been successfully produced. We hypothesize that generating CRISPR/Cas9-expressing cattle and their resources will provide a powerful resource for bovine genome editing, advancing our understanding of bovine genetics and disease resistance.<br><br>RESULTS: In this study, two types of Cas9-expressing cattle were successfully produced: Cas9-RFP-fatty acid dehydrogenase I (FatI), Cas9-GFP-sgRNA for the prion protein (sgPRNP). Somatic cells from these cattle were induced to mutate multiple target genes when single-guide RNAs (sgRNAs) were transfected into the somatic cells. Additionally, semen from Cas9 expressing male cattle was frozen and used to fertilize wild-type oocytes, successfully transmitting the transgene (Cas9, reporter genes, FatI), and sgPRNP) to the next generation. Furthermore, the gene editing capabilities of Cas9, including knockout and high-efficiency knock-in, were confirmed in embryos derived from F1 semen through in vitro production.<br><br>CONCLUSION: These data demonstrate, for the first time, that Cas9-expressing cattle were successfully born, and this transgene was transmitted to the next-generation calves (F1) and F2 embryos. In addition, somatic and germ cells derived from F0 and F1generations were used to evaluate the potential for gene editing (knockout and knock-in) in multiple genes. PRNP-mutated F1 cattle are currently being raised as a resistance model for bovine spongiform encephalopathy. These transgenic bovine models and their derivatives will serve as a valuable resource for both in vitro and in vivo genome editing, advancing our genetic understanding of bovine genomics and diseases.}, }
@article {pmid40044943, year = {2025}, author = {García-Tenorio, EM and Álvarez, M and Gallego-Bonhomme, M and Desviat, LR and Richard, E}, title = {Novel CRISPR-Cas9 iPSC knockouts for PCCA and PCCB genes: advancing propionic acidemia research.}, journal = {Human cell}, volume = {38}, number = {3}, pages = {64}, pmid = {40044943}, issn = {1749-0774}, support = {PAF113//Propionic Acidemia Foundation/ ; PID2022-137238OB-100//Ministerio de Ciencia e Innovaci&#xf3;n/ ; }, mesh = {*Propionic Acidemia/genetics/therapy ; *Induced Pluripotent Stem Cells ; *CRISPR-Cas Systems/genetics ; Humans ; *Gene Knockout Techniques/methods ; *Methylmalonyl-CoA Decarboxylase/genetics ; *Carbon-Carbon Ligases/genetics ; Gene Editing/methods ; Cell Line ; }, abstract = {Propionic acidemia (PA) is a rare autosomal recessive metabolic disorder caused by mutations in the PCCA and PCCB genes, which encode subunits of the mitochondrial enzyme propionyl-CoA carboxylase (PCC). This enzyme deficiency leads to the accumulation of toxic metabolites, resulting in severe metabolic dysfunction. To create ideal in vitro disease models of PA with isogenic controls and provide a robust platform for therapeutic research, we generated two induced pluripotent stem cell (iPSC) lines with knockout (KO) mutations in the PCCA and PCCB genes using CRISPR-Cas9 gene editing in a healthy control iPSC line. The KO iPS cells were successfully established and characterized, confirming the presence of frameshift insertions and deletions in each target gene, as well as the loss of the corresponding transcript, protein expression, and activity. Additionally, the generated iPSC lines exhibit hallmark characteristics of pluripotency, including the potential to differentiate into all three germ layers. Our PCCA and PCCB KO iPSC models provide a valuable tool for studying the molecular mechanisms underlying PA and hold potential for advancing new therapeutic approaches.}, }
@article {pmid40044744, year = {2025}, author = {Yamada, H and Odagiri, M and Yamakita, K and Chiba, A and Ukai, A and Yasui, M and Honma, M and Sugiyama, KI and Ura, K and Sassa, A}, title = {Dual-directional epi-genotoxicity assay for assessing chemically induced epigenetic effects utilizing the housekeeping TK gene.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {7780}, pmid = {40044744}, issn = {2045-2322}, support = {21KA1001//Ministry of Health, Labor and Welfare/ ; 24KA1008//Consumer Affairs Agency/ ; 22H03748//JSPS/ ; }, mesh = {Humans ; *Epigenesis, Genetic/drug effects ; *Thymidine Kinase/genetics ; Mutagenicity Tests/methods ; Cell Line ; *Genes, Essential ; DNA Methylation/drug effects ; Tetradecanoylphorbol Acetate/toxicity ; Promoter Regions, Genetic ; Carcinogens/toxicity ; Mutation ; CRISPR-Cas Systems ; Histones/metabolism ; }, abstract = {Numerous chemicals are associated with carcinogenesis through epigenetic alterations in cells. To detect global epigenetic changes induced by carcinogens, the housekeeping gene can serve as a reporter locus, offering a baseline for identifying shifts in epigenetic marks. To investigate this potential, we developed a simple, cost-effective, and quantitative reporter system to assess chemically induced epigenetic effects, utilizing the thymidine kinase (TK) gene mutation assay as a foundation. Using a standard genotoxicity test cell line, human lymphoblast TK6, we edited the CpG promoter loci of the endogenous TK gene using the CRISPR/dCas9-SunTag-DNMT3A system. This epi-genotoxicity assay, employing modified mTK6 cells, provides a simple method for quantifying chemically induced epigenetic effects. The assay successfully detects both increased TK reversion rates induced by DNMT inhibitors, such as 5-Aza-2'-deoxycytidine and GSK-3484862, and, for the first time, a significant reduction in TK revertant frequency caused by the non-genotoxic carcinogen 12-O-tetradecanoylphorbol-13-acetate (TPA). Chromatin immunoprecipitation and western blotting analyses revealed that TPA treatment led to a global decrease in H3K27Ac levels, likely driven by TPA-mediated inflammation. These results demonstrate the utility of the epi-genotoxicity assay as a valuable tool for evaluating dual-directional epigenetic changes triggered by chemical exposure.}, }
@article {pmid40044221, year = {2025}, author = {Duan, X and Chen, C and Du, C and Guo, L and Liu, J and Hou, N and Li, P and Qi, X and Gao, F and Du, X and Song, J and Wu, S}, title = {Homozygous editing of multiple genes for accelerated generation of xenotransplantation pigs.}, journal = {Genome research}, volume = {35}, number = {5}, pages = {1167-1178}, pmid = {40044221}, issn = {1549-5469}, mesh = {Animals ; *Gene Editing/methods ; Swine/genetics ; *Transplantation, Heterologous/methods ; CRISPR-Cas Systems ; Homozygote ; Fibroblasts/metabolism ; Nuclear Transfer Techniques ; Galactosyltransferases/genetics ; Animals, Genetically Modified ; }, abstract = {Although CRISPR-Cas-based genome editing has made significant strides over the past decade, achieving simultaneous homozygous gene editing of multiple targets in primary cells remains a significant challenge. In this study, we optimized a coselection strategy to enhance homozygous gene editing rates in the genomes of primary porcine fetal fibroblasts (PFFs). The strategy utilizes the expression of a surrogate reporter (eGFP) to select for cells with the highest reporter expression, thereby improving editing efficiency. For simultaneous multigene editing, we targeted the most challenging site for selection, whereas other target sites did not require selection. Using this approach, we successfully obtained single-cell PFF clones (three of 10) with seven or more homozygously edited genes, including GGTA1, CMAH, B4GALNT2, CD46, CD47, THBD, and GHR Importantly, cells edited using this strategy can be efficiently used for somatic cell nuclear transfer (SCNT) to generate healthy xenotransplantation pigs in <5 months, a process that previously required years of breeding or multiple rounds of SCNT.}, }
@article {pmid40044139, year = {2025}, author = {Lee, HJ and Jeong, SH and Lee, SJ}, title = {Single nucleotide genome recognition and selective bacterial lysis using synthetic phages loaded with CRISPR-Cas12f1-truncated sgRNA.}, journal = {Journal of microbiology (Seoul, Korea)}, volume = {63}, number = {2}, pages = {e2501012}, doi = {10.71150/jm.2501012}, pmid = {40044139}, issn = {1976-3794}, support = {RS-2024-00342735//National Research Foundation of Korea/ ; //Ministry of Science and ICT/ ; RS-2024-00411263//Ministry of Education/ ; //Korea Health Industry Development Institute/ ; HI23C0041//Ministry of Health and Welfare/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics/virology ; *Genome, Viral ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Bacteriophage lambda/genetics/physiology ; *Bacteriolysis/genetics ; Lysogeny/genetics ; }, abstract = {Phage specificity primarily relies on host cell-surface receptors. However, integrating cas genes and guide RNAs into phage genomes could enhance their target specificity and regulatory effects. In this study, we developed a CRISPR-Cas12f1 system-equipped bacteriophage λ model capable of detecting Escherichia coli target genes. We demonstrated that synthetic λ phages carrying Cas12f1-sgRNA can effectively prevent lysogen formation. Furthermore, we showcased that truncating the 3(?)-end of sgRNA enables precise identification of single-nucleotide variations in the host genome. Moreover, infecting E. coli strains carrying various stx2 gene subtypes encoding Shiga toxin with bacteriophages harboring Cas12f1 and truncated sgRNAs resulted in the targeted elimination of strains with matching subtype genes. These findings underscore the ability of phages equipped with the CRISPR-Cas12f1 system to precisely control microbial hosts by recognizing genomic sequences with high resolution.}, }
@article {pmid40043602, year = {2025}, author = {Liu, G and Gong, H and Tang, H and Meng, Z and Wang, Z and Cui, W and Zhang, K and Chen, Y and Yang, Y}, title = {Enhanced lignocellulose degradation in Bacillus subtilis RLI2019 through CRISPR/Cas9-mediated chromosomal integration of ternary cellulase genes.}, journal = {International journal of biological macromolecules}, volume = {306}, number = {Pt 3}, pages = {141727}, doi = {10.1016/j.ijbiomac.2025.141727}, pmid = {40043602}, issn = {1879-0003}, mesh = {*Bacillus subtilis/genetics/metabolism/enzymology ; *Lignin/metabolism ; *CRISPR-Cas Systems/genetics ; *Cellulase/genetics/metabolism ; Gene Editing ; Biomass ; *Chromosomes, Bacterial/genetics ; }, abstract = {Bacillus subtilis (B. subtilis) is a crucial industrial microorganism for lignocellulose biomass degradation. However, wild-type strains from natural environments have inherent deficiencies in the composition of cellulase genes, so constructing recombinant strains through genome engineering is a generalizable strategy to overcome these shortcomings. Herein, eglS, cel48S, and bglS were integrated into the aprE, epr, and amyE loci of the B. subtilis RLI2019 chromosome, respectively, through CRISPR/Cas9-mediated genome editing, deriving the engineered strain B. subtilis AEA3. The activities of endoglucanase, exoglucanase, β-glucosidase, xylanase, and total cellulase in B. subtilis AEA3 were enhanced by 3.1-fold, 6.6-fold, 3.0-fold, 1.2-fold, and 1.8-fold, respectively, reaching 26.31 U/mL, 9.77 U/mL, 3.91 U/mL, 19.63 U/mL, and 2.42 U/mL. Notably, the engineered strain improved the saccharification efficiency of crop straws, effectively disrupting fiber structure, and significantly reducing the content of neutral and acid detergent fibers, lignocellulose and hemicellulose. In summary, this study provides a general strategy for enhancing the cellulose degradation capabilities of B. subtilis through comprehensive and systematic multi-module genetic engineering, broadening its potential application in lignocellulose biomass conversion.}, }
@article {pmid40043507, year = {2025}, author = {Xie, W and Luo, D and Wu, M and Sun, Y and Wang, Z}, title = {The evaluation of Phenylalanine-tRNA ligase beta unit (PheT), as a potential target in Mycobacterium abscessus.}, journal = {Tuberculosis (Edinburgh, Scotland)}, volume = {152}, number = {}, pages = {102626}, doi = {10.1016/j.tube.2025.102626}, pmid = {40043507}, issn = {1873-281X}, mesh = {*Mycobacterium abscessus/genetics/enzymology/drug effects/growth &amp; development ; *Mycobacterium Infections, Nontuberculous/microbiology/genetics/enzymology ; Animals ; *Bacterial Proteins/genetics/metabolism ; Humans ; Mice ; Gene Silencing ; Disease Models, Animal ; CRISPR-Cas Systems ; }, abstract = {Mycobacterium abscessus (M. abscessus) is an emerging pathogenic mycobacterium that mainly causes pulmonary infections, especially in immunocompromised patients. This bacterium shows exhibits intrinsic resistance to many anti-tuberculosis drugs, posing significant challenges for both patients and clinicians, thereby raising the need for innovative drug discovery. In this study, we selected phenylalanine-tRNA ligase beta unit (PheT) as a model target and used CRISPR interference to evaluate its essentiality as a therapeutic target against M. abscessus. The results show that genetically disruption of PheT leads to clear growth inhibitory phenotypes both in vitro and in vivo. Further transcriptome analysis revealed differential expression of host genes in response to PheT gene silencing, including genes involved in the cell cycle, apoptotic signaling, and inflammatory responses. Overall, PheT gene plays a crucial role in M. abscessus infection, and its silencing may represent a druggable therapeutic strategy for treating this infection.}, }
@article {pmid40043077, year = {2025}, author = {Zhou, Z and Xiao, J and Yin, S and Chen, Y and Yuan, Y and Zhang, J and Xiong, L and Xie, K}, title = {Cas9-Rep fusion tethers donor DNA in vivo and boosts the efficiency of HDR-mediated genome editing.}, journal = {Plant biotechnology journal}, volume = {23}, number = {6}, pages = {2006-2017}, pmid = {40043077}, issn = {1467-7652}, support = {2662023PY006//Fundamental Research Funds for the Central Universities/ ; B23YQ1516//Hainan Yazhou Bay Seed Laboratory and the China National Seed Group/ ; 2023ZD04062//Science and Technology Innovation 2030-Biological Breeding Major Projects/ ; 2023ZD0407403//Science and Technology Innovation 2030-Biological Breeding Major Projects/ ; 31821005//National Natural Science Foundation of China/ ; 32293243//National Natural Science Foundation of China/ ; }, mesh = {*Gene Editing/methods ; *Recombinational DNA Repair/genetics ; *Oryza/genetics ; Gene Knock-In Techniques ; Genome, Plant/genetics ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; *CRISPR-Associated Protein 9/metabolism/genetics ; }, abstract = {Genome editing based on the homology-directed repair (HDR) pathway enables scar-free and precise genetic manipulations. However, the low frequency of HDR hinders its application in plant genome editing. In this study, we engineered the fusion of Cas9 and a viral replication protein (Rep) as a molecular bridge to tether donor DNA in vivo, which enhances the efficiency of targeted gene insertion via the HDR pathway. This Rep-bridged knock-in (RBKI) method combines the advantages of rolling cycle replication of viral replicons and in vivo enrichment of donor DNA at the target site for HDR. Chromatin immunoprecipitation indicated that the Cas9-Rep fusion protein bound up to 66-fold more donor DNA than Cas9 did. We exemplified the RBKI method by inserting small- to middle-sized tags (33-519 bp) into 3 rice genes. Compared to Cas9, Cas9-Rep fusion increased the KI frequencies by 4-7.6-fold, and up to 72.2% of stable rice transformants carried in-frame knock-in events in the T0 generation. Whole-genome sequencing of 6 plants segregated from heterozygous KI lines indicated that the knock-in events were faithfully inherited by the progenies with neither off-target editing nor random insertions of the donor DNA fragment. Further analysis suggested that the RBKI method reduced the number of byproducts from nonhomologous end joining; however, HDR-mediated knock-in tended to accompany microhomology-mediated end joining events. Together, these findings show that the in vivo tethering of donor DNAs with Cas9-Rep is an effective strategy to increase the frequency of HDR-mediated genome editing.}, }
@article {pmid40041907, year = {2025}, author = {Mao, K and Tan, H and Cong, X and Liu, J and Xin, Y and Wang, J and Guan, M and Li, J and Zhu, G and Meng, X and Lin, G and Wang, H and Han, J and Wang, M and Yang, YG and Sun, T}, title = {Optimized lipid nanoparticles enable effective CRISPR/Cas9-mediated gene editing in dendritic cells for enhanced immunotherapy.}, journal = {Acta pharmaceutica Sinica. B}, volume = {15}, number = {1}, pages = {642-656}, pmid = {40041907}, issn = {2211-3835}, abstract = {Immunotherapy has emerged as a revolutionary approach to treat immune-related diseases. Dendritic cells (DCs) play a pivotal role in orchestrating immune responses, making them an attractive target for immunotherapeutic interventions. Modulation of gene expression in DCs using genome editing techniques, such as the CRISPR-Cas system, is important for regulating DC functions. However, the precise delivery of CRISPR-based therapies to DCs has posed a significant challenge. While lipid nanoparticles (LNPs) have been extensively studied for gene editing in tumor cells, their potential application in DCs has remained relatively unexplored. This study investigates the important role of cholesterol in regulating the efficiency of BAMEA-O16B lipid-assisted nanoparticles (BLANs) as carriers of CRISPR/Cas9 for gene editing in DCs. Remarkably, BLANs with low cholesterol density exhibit exceptional mRNA uptake, improved endosomal escape, and efficient single-guide RNA release capabilities. Administration of BLANmCas9/gPD-L1 results in substantial PD-L1 gene knockout in conventional dendritic cells (cDCs), accompanied by heightened cDC1 activation, T cell stimulation, and significant suppression of tumor growth. The study underscores the pivotal role of cholesterol density within LNPs, revealing potent influence on gene editing efficacy within DCs. This strategy holds immense promise for the field of cancer immunotherapy, offering a novel avenue for treating immune-related diseases.}, }
@article {pmid40040244, year = {2025}, author = {Cao, X and Wang, X and Chen, R and Chen, L and Liu, Y and Wang, M}, title = {Improving Bacillus subtilis as Biological Chassis Performance by the CRISPR Genetic Toolkit.}, journal = {ACS synthetic biology}, volume = {14}, number = {3}, pages = {677-688}, doi = {10.1021/acssynbio.4c00844}, pmid = {40040244}, issn = {2161-5063}, mesh = {*Bacillus subtilis/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Metabolic Engineering/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Synthetic Biology/methods ; Escherichia coli/genetics ; }, abstract = {Bacillus subtilis is the model Gram-positive and industrial chassis bacterium; it has blossomed as a robust and promising host for enzyme, biochemical, or bioflocculant production. However, synthetic biology and metabolic engineering technologies of B. subtilis have lagged behind the most widely used industrial chassis Saccharomyces cerevisiae and Escherichia coli. CRISPR (an acronym for clustered regularly interspaced short palindromic repeats) enables efficient, site-specific, and programmable DNA cleavage, which has revolutionized the manner of genome editing. In 2016, CRISPR technology was first introduced into B. subtilis and has been intensely upgraded since then. In this Review, we discuss recently developed key additions to CRISPR toolkit design in B. subtilis with gene editing, transcriptional regulation, and enzyme modulation. Second, advances in the B. subtilis chassis of efficient biochemicals and proteins with CRISPR engineering are discussed. Finally, we conclude with perspectives on the challenges and opportunities of CRISPR-based biotechnology in B. subtilis, wishing that B. subtilis can be comparable to traditional industrial microorganisms such as E. coli and S. cerevisiae someday soon.}, }
@article {pmid40038872, year = {2025}, author = {Duan, ZW and Wang, WT and Wang, Y and Wang, R and Hua, W and Shang, CY and Gao, R and Shen, HR and Li, Y and Wu, JZ and Yin, H and Wang, L and Li, JY and Xu, W and Liang, JH}, title = {SH3GL1-activated FTH1 inhibits ferroptosis and confers doxorubicin resistance in diffuse large B-cell lymphoma.}, journal = {Clinical and translational medicine}, volume = {15}, number = {3}, pages = {e70246}, pmid = {40038872}, issn = {2001-1326}, support = {82200887//National Natural Science Foundation of China/ ; 82370194//National Natural Science Foundation of China/ ; BK20220716//Jiangsu Science and Technology&#xa0;Department/ ; BE2023780//Jiangsu Science and Technology&#xa0;Department/ ; 2022M7114034//China Postdoctoral Science Foundation/ ; 2023M741463//China Postdoctoral Science Foundation/ ; }, mesh = {*Ferroptosis ; Doxorubicin/pharmacology ; *Drug Resistance, Neoplasm ; *Lymphoma, Large B-Cell, Diffuse/drug therapy/genetics/metabolism ; CRISPR-Cas Systems ; Immunohistochemistry ; Cell Proliferation ; Cell Survival ; Prognosis ; Humans ; Male ; Female ; Adult ; Middle Aged ; Aged ; Animals ; Mice ; Neoplasm Transplantation ; Gene Expression Regulation, Neoplastic ; Biomarkers, Tumor/biosynthesis ; }, abstract = {BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is predominant subtype of non-Hodgkin lymphoma and can be effectively treated. Nevertheless, a subset of patients experiences refractory or relapsed disease, highlighting the need for new therapeutic strategies.<br><br>METHODS: Depmap database based on CRISPR/Cas9 knock out analysis was employed to identify the essential gene SH3GL1, which encodes endophilin A2, as crucial for the proliferation and survival of DLBCL cells. Immunohistochemistry (IHC) staining was performed on the 126 paraffin-embedded clinical DLBCL samples to investigate the association between SH3GL1 expression levels and the prognosis. To investigate the specific mechanism modulated by SH3GL1 in the progression of DLBCL, an integrative approach was employed. This approach combined high-throughput sequencing technologies, such as Deep-DIA and LC-MS, with functional validation techniques, including CRISPR/Cas9 gene editing, xenograft models, and molecular pathway analyses.<br><br>RESULTS: Our study found that high expression levels of SH3GL1 correlate with poor prognosis in a cohort of 126 newly diagnosed DLBCL patients, underscoring its significance in disease progression. Mechanistically, we found that SH3GL1 deficiency triggers ferritin heavy chain 1 (FTH1)-mediated ferroptosis, specifically ferritinophagy-induced ferroptosis, in DLBCL cells. Additionally, high expression of SH3GL1 suppresses doxorubicin-induced ferroptosis. Cancer cells' resistance to conventional therapies is associated with increased sensitivity to ferroptosis.<br><br>CONCLUSIONS: These findings emphasise SH3GL1 as a promising prognostic biomarker and a potential therapeutic target in DLBCL, offering new avenues for treatment strategies aimed at overcoming drug resistance and improving patients' outcomes.<br><br>KEY POINTS: Elevated SH3GL1 expression in DLBCL patients was associated with a negative prognosis. SH3GL1 plays a crucial role in promoting DLBCL cell survival through the regulation of FTH1-mediated ferroptosis and doxorubicin resistance.}, }
@article {pmid40038432, year = {2025}, author = {Zhang, J and Yin, W and Jiang, Q and Mao, W and Deng, W and Jin, S and Wang, X and He, R and Qiao, J and Liu, Y}, title = {Precise amplification-free detection of highly structured RNA with an enhanced SCas12a assay.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {366}, pmid = {40038432}, issn = {2399-3642}, mesh = {*CRISPR-Cas Systems ; Humans ; *COVID-19/diagnosis/virology ; SARS-CoV-2/genetics/isolation &amp; purification ; *RNA/genetics/chemistry/analysis ; CRISPR-Associated Proteins/genetics ; Polymorphism, Single Nucleotide ; Nucleic Acid Amplification Techniques ; RNA, Viral ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {The CRISPR/Cas12a system has revolutionized molecular diagnostics, yet the direct detection of RNA, particularly those with complex structures, remains a significant challenge. Here, we present an updated SCas12a system, termed SCas12aV2, which enables precise, amplification-free detection of highly structured RNA molecules. By optimizing the length of scaffold RNA, targeting asymmetric structures, and utilizing dsDNA-ssDNA hybrid activators, we have significantly reduced steric hindrance in the detection system, thereby markedly enhancing both sensitivity and kinetics compared to traditional DNA activators. The SCas12aV2 assay achieves a detection limit of 246 aM for pooled activators and 10 pM for single-site targeting, demonstrating high specificity for single nucleotide polymorphisms (SNPs). It successfully identifies viable bacteria and SARS-CoV-2 infections in clinical samples. The assay is versatile and can be applied to various Cas12a orthologs, including thermostable CtCas12a. This work advances molecular diagnostics by improving the accuracy and efficiency of RNA detection.}, }
@article {pmid40303776, year = {2023}, author = {Xiao, X and Zhang, Q and Wu, S and Li, Y and Zhong, Z and Guo, Q and Li, J and Meng, Q and Cheng, Z and Duan, J and Wang, X and He, H and Bai, J and Lou, Y}, title = {Detection of Clostridium perfringens Using Novel Methods Based on Recombinase-Aided Amplification Assay-Assisted CRISPR/Cas12a System.}, journal = {Transboundary and emerging diseases}, volume = {2023}, number = {}, pages = {6667618}, pmid = {40303776}, issn = {1865-1682}, mesh = {*Clostridium perfringens/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; *Clostridium Infections/veterinary/diagnosis/microbiology ; Animals ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Recombinases/metabolism ; Sensitivity and Specificity ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Clostridium perfringens is a highly versatile pathogen of humans and animals. Rapid and sensitive detection methods for C. perfringens are urgently needed for the timely implementation of control. In this study, to provide novel promising methods for the detection of C. perfringens, two rapid, sensitive, and instrument-free C. perfringens detection methods based on recombinase-aided amplification (RAA) assay and clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 12a (CRISPR/Cas12a) system were developed depending on fluorescence signal (RAA-CRISPR/Cas12a-FL) and lateral flow strip (RAA-CRISPR/Cas12a-LFS), respectively. The limit of detection of the RAA-CRISPR/Cas12a-FL and RAA-CRISPR/Cas12a-LFS methods is 2 copies and 20 copies of C. perfringens genomic DNA per reaction, respectively, and the whole process can be completed in 1 hr. Moreover, these two methods show no cross-reactivity with nontarget bacteria, which were used as a negative control to evaluate the specificity of two developed methods in the detection of C. perfringens and have 100% consistent with real-time polymerase chain reaction tests for 12 clinical samples collected from 2 Chinese Milu at Beijing Milu Ecological Research Center and 6 spiked samples from human blood and stool. Overall, the constructed C. perfringens detection methods, RAA-CRISPR/Cas12a-FL and RAA-CRISPR/Cas12a-LFS, have great potential as a novel detection scheme for the early diagnosis of C. perfringens infection in humans and animals.}, }
@article {pmid40303816, year = {2023}, author = {Zhang, Y and Peng, Q and Zhang, R and Li, C and Xu, Q and Xia, L and Wang, Y and Liu, P and Pan, H}, title = {Advances in CRISPR/Cas-Based Strategies on Zoonosis.}, journal = {Transboundary and emerging diseases}, volume = {2023}, number = {}, pages = {9098445}, pmid = {40303816}, issn = {1865-1682}, mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Zoonoses/prevention &amp; control ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) has emerged as the predominant technique for gene editing technique due to its high efficiency and low cost. In the area of zoonosis, CRISPR/Cas is also widely used in different research areas. This paper reviewed the principles of CRISPR/Cas technique and its applications in zoonosis. Moreover, we analyze the shortcomings and weaknesses that currently limit its use, highlight its direction for improvement, and foresee its application prospects in the prevention and treatment of zoonosis. For the purpose of preventing and controlling zoonosis, we need to develop diagnostic method with high sensitivity and specificity, highly protective vaccines, and also better understanding of the pathogenesis. Our review aimed to promote the application and improvement of CRISPR/Cas technique in the above-mentioned areas, and provide brief and comprehensive references for CRISPR/Cas-based research. Through reviewing the advances in CRISPR/Cas-based strategies on zoonosis, we believe that CRISPR/Cas technique will provide more powerful assistance for the prevention and control of zoonosis.}, }
@article {pmid40037377, year = {2025}, author = {Emrich-Mills, TZ and Proctor, MS and Degen, GE and Jackson, PJ and Richardson, KH and Hawkings, FR and Buchert, F and Hitchcock, A and Hunter, CN and Mackinder, LCM and Hippler, M and Johnson, MP}, title = {Tethering ferredoxin-NADP+ reductase to photosystem I promotes photosynthetic cyclic electron transfer.}, journal = {The Plant cell}, volume = {37}, number = {3}, pages = {}, pmid = {40037377}, issn = {1532-298X}, support = {RPG-2019-045//Leverhulme Trust/ ; BB/V006630/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; //BBSRC White Rose DTP studentship in Mechanistic Biology/ ; //Diamond PhD studentship/ ; 854126//European Research Council Synergy/ ; HI 739/13-3//DFG/ ; HI 739/25-1//GoPMF/ ; }, mesh = {*Photosystem I Protein Complex/metabolism/genetics ; *Photosynthesis/genetics ; *Ferredoxin-NADP Reductase/metabolism/genetics ; Electron Transport ; *Chlamydomonas reinhardtii/genetics/metabolism ; CRISPR-Cas Systems ; NADP/metabolism ; }, abstract = {Fixing CO2 via photosynthesis requires ATP and NADPH, which can be generated through linear electron transfer (LET). However, depending on the environmental conditions, additional ATP may be required to fix CO2, which can be generated by cyclic electron transfer (CET). How the balance between LET and CET is determined remains largely unknown. Ferredoxin-NADP+ reductase (FNR) may act as the switch between LET and CET, channeling photosynthetic electrons to LET when it is bound to photosystem I (PSI) or to CET when it is bound to cytochrome b6f. The essential role of FNR in LET precludes the use of a direct gene knock-out to test this hypothesis. Nevertheless, we circumvented this problem using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated gene editing in Chlamydomonas reinhardtii. Through this approach, we created a chimeric form of FNR tethered to PSI via PSAF. Chimeric FNR mutants exhibited impaired photosynthetic growth and LET along with enhanced PSI acceptor side limitation relative to the wild type due to slower NADPH reduction. However, the chimeric FNR mutants also showed enhanced ΔpH production and NPQ resulting from increased CET. Overall, our results suggest that rather than promoting LET, tethering FNR to PSI promotes CET at the expense of LET and CO2 fixation.}, }
@article {pmid40036851, year = {2025}, author = {Nakamura, K and Kanou, M and Fujii, W and Kouzaki, K and Jimbo, T and Yamanouchi, K and Nakazato, K and Ueda, H and Hirata, J and Yamana, K}, title = {New selective androgen receptor modulator TEI-SARM2 improves muscle function in a Duchenne muscular dystrophy rat model.}, journal = {Human molecular genetics}, volume = {34}, number = {10}, pages = {852-863}, doi = {10.1093/hmg/ddaf028}, pmid = {40036851}, issn = {1460-2083}, support = {//Teijin Limited and Teijin Pharma Limited/ ; }, mesh = {Animals ; *Muscular Dystrophy, Duchenne/drug therapy/genetics/physiopathology/pathology/metabolism ; Rats ; *Receptors, Androgen/metabolism/genetics ; *Muscle, Skeletal/drug effects/metabolism/pathology/physiopathology ; Male ; Disease Models, Animal ; Dystrophin/genetics ; Mice, Inbred mdx ; Humans ; Mice ; }, abstract = {Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by a genetic mutation in the Dmd gene. Dystrophin mutant mice (mdx) have traditionally been used for DMD research as a disease model in the preclinical stage; however, mdx mice exhibit only very mild phenotypes to partially mimic muscle degeneration and regeneration. To overcome this limitation in preclinical studies, DMD mutant rats (DMD rats) generated by CRISPR/Cas were used as a DMD model to exhibit age-dependent progressive muscle degeneration and pathophysiological features similar to DMD patients and more severe than those displayed by mdx mice. TEI-SARM2 is a non-steroidal, orally available selective androgen receptor modulator (SARM) developed as a pharmaceutical candidate for the treatment of muscle wasting diseases based on its potent anabolic activity on skeletal muscle mass. In this study, long-term treatment of daily oral administration of TEI-SARM2 to DMD rats significantly improved muscle function (endurance and strength) assessed by grip and tetanic force measurements. TEI-SARM2 did not increase the muscle weight of hindlimbs in male DMD rats; moreover, long-term, weekly oral administration for 24 weeks improved muscle function with reduced side effects on the prostate and testes weight. Histological analysis showed that TEI-SARM2 significantly reduced adipose tissue infiltration in DMD muscle. In female DMD rats, both daily and weekly TEI-SARM2 treatment showed anabolic effects and enhanced muscle strength and endurance. Taken together, these results indicate that TEI-SARM2 has non-anabolic and anabolic effects that improve dystrophic muscle dysfunction and can be a supportive therapeutic option for DMD.}, }
@article {pmid40036525, year = {2025}, author = {Zheng, Z and Qian, Z and Huang, D and Weng, Z and Wang, J and Lin, C and Qiu, B and Lin, Z and Luo, F}, title = {Ultrasensitive Homogeneous Electrochemiluminescence Biosensor for N-Nitrosodimethylamine Detection Based on Vertically-Ordered Mesoporous Silica Film-Modified Electrode and CRISPR/Cas12a-Driven HRCA with Triple Signal Amplification.}, journal = {Analytical chemistry}, volume = {97}, number = {10}, pages = {5828-5835}, doi = {10.1021/acs.analchem.5c00555}, pmid = {40036525}, issn = {1520-6882}, mesh = {*Biosensing Techniques/methods ; *Silicon Dioxide/chemistry ; *CRISPR-Cas Systems ; Porosity ; *Electrochemical Techniques/methods ; Electrodes ; *Luminescent Measurements/methods ; *Nucleic Acid Amplification Techniques/methods ; *Dimethylnitrosamine/analysis ; DNA/chemistry ; Limit of Detection ; }, abstract = {Herein, we present an innovative electrochemiluminescence (ECL) biosensor for the ultrasensitive detection of N-nitrosodimethylamine (NDMA). The biosensor utilizes a triple signal amplification strategy, combining rolling circle amplification (RCA), CRISPR/Cas12a-driven hyperbranched rolling circle amplification (HRCA), and electrostatic repulsion with size exclusion effects from vertically ordered mesoporous silica film (VMSF)/indium tin oxide (ITO) on double-stranded DNA (dsDNA)-Ru(phen)3[2+] complexes. In this system, aptamers and circular DNA undergo RCA reactions, followed by the CRISPR/Cas12a-mediated HRCA process, producing abundant dsDNA. The electropositive ECL indicator, namely Ru(phen)3[2+], was subsequently adsorbed onto the electronegative dsDNA, forming dsDNA-Ru(phen)3[2+] complexes. These complexes are subjected to electrostatic repulsion and size exclusion by the VMSF-modified ITO electrode, resulting in a lower ECL intensity. Upon introducing NDMA, the aptamer preferentially binds to NDMA, thereby preventing the formation of long dsDNA. This process releases free Ru(phen)3[2+], which diffuses to the electrode surface through narrow mesoporous channels via electrostatic adsorption. Consequently, an enhanced and strong ECL signal is observed. The integration of VMSF enhances selectivity and sensitivity by excluding larger impurities and promoting the electrostatic repulsion of dsDNA-Ru(phen)3[2+] complexes near the electrode surface. Additionally, the CRISPR/Cas12a system eliminates the formation of primer dimers and reduces false positives through its unique cis- and trans-cleavage activities. The biosensor demonstrated excellent performance with a linear correlation between the ECL signal and NDMA concentration in the range spanning from 10 pg/mL to 10 μg/mL, achieving a low limit of detection of 5.33 pg/mL. This platform offers a reliable and robust solution for detecting NDMA in complex matrices, making it a promising tool for environmental monitoring, public health, and safety applications.}, }
@article {pmid40036508, year = {2025}, author = {Karp, H and Zoltek, M and Wasko, K and Vazquez, AL and Brim, J and Ngo, W and Schepartz, A and Doudna, JA}, title = {Packaged delivery of CRISPR-Cas9 ribonucleoproteins accelerates genome editing.}, journal = {Nucleic acids research}, volume = {53}, number = {5}, pages = {}, pmid = {40036508}, issn = {1362-4962}, support = {S10 RR026866/RR/NCRR NIH HHS/United States ; 2203903//National Science Foundation/ ; R35 GM134963/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; T32GM098218/NH/NIH HHS/United States ; //Emerson Collective/ ; PDF-578176-2023//Natural Sciences and Engineering Research Council of Canada/ ; //HHMI/ ; }, mesh = {*Ribonucleoproteins/metabolism/administration &amp; dosage/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; Electroporation ; HEK293 Cells ; CRISPR-Associated Protein 9 ; }, abstract = {Effective genome editing requires a sufficient dose of CRISPR-Cas9 ribonucleoproteins (RNPs) to enter the target cell while minimizing immune responses, off-target editing, and cytotoxicity. Clinical use of Cas9 RNPs currently entails electroporation into cells ex vivo, but no systematic comparison of this method to packaged RNP delivery has been made. Here we compared two delivery strategies, electroporation and enveloped delivery vehicles (EDVs), to investigate the Cas9 dosage requirements for genome editing. Using fluorescence correlation spectroscopy, we determined that >1300 Cas9 RNPs per nucleus are typically required for productive genome editing. EDV-mediated editing was >30-fold more efficient than electroporation, and editing occurs at least 2-fold faster for EDV delivery at comparable total Cas9 RNP doses. We hypothesize that differences in efficacy between these methods result in part from the increased duration of RNP nuclear residence resulting from EDV delivery. Our results directly compare RNP delivery strategies, showing that packaged delivery could dramatically reduce the amount of CRISPR-Cas9 RNPs required for experimental or clinical genome editing.}, }
@article {pmid40035496, year = {2025}, author = {Li, Y and Zhao, Y and Yi, Z and Han, S}, title = {A CRISPR/Cas13a-based and hybridization chain reaction coupled evanescent wave biosensor for SARS-CoV-2 gene detection.}, journal = {The Analyst}, volume = {150}, number = {7}, pages = {1367-1376}, doi = {10.1039/d4an01584c}, pmid = {40035496}, issn = {1364-5528}, mesh = {*Biosensing Techniques/methods ; *SARS-CoV-2/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; Humans ; *COVID-19/diagnosis/virology ; *RNA, Viral/genetics/analysis ; Nucleic Acid Hybridization/methods ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; }, abstract = {Timely and accurate diagnosis of RNA viruses, represented by the SARS-CoV-2, is the foundation for protecting people from health threats. Currently, direct detection of viral RNA genes remains the most accurate method. Herein, a rapid, ultrasensitive, and no heating equipment required CRISPR/Cas13a based evanescent wave fluorescence biosensing platform for quantitative detection of the SARS-CoV-2 gene is reported. The collateral effect of CRISPR/Cas13a for RNA is combined with a self-driven enzyme-free hybridization chain reaction (HCR) as a signal amplification step. When the initiator RNA strand is cleaved by Cas13a, the downstream signal amplification induced by HCR is blocked, and a multiple crRNA strategy is used to enhance the cleavage efficiency. The newly designed HCR assemblies are captured by the cDNA-modified optical fiber and generate a higher-intensity fluorescence signal induced by the evanescent field. The CRISPR/Cas13a-HCR evanescent wave fluorescence biosensing platform is capable of detection of SARS-CoV-2 with a LOD of 0.47 copies per μL and the detection time is within 35 min. The spike recovery tests in saliva and high specificity have demonstrated the potential of this method for point-of-care diagnosis. This method is also suitable for the detection of other RNA viruses, without the need to alter any design of the HCR component, and only the corresponding crRNA needs to be replaced.}, }
@article {pmid40035150, year = {2025}, author = {Zhu, H and Zhou, T and Guan, J and Li, Z and Yang, X and Li, Y and Sun, J and Xu, Q and Xuan, YH}, title = {Precise genome editing of Dense and Erect Panicle 1 promotes rice sheath blight resistance and yield production in japonica rice.}, journal = {Plant biotechnology journal}, volume = {23}, number = {5}, pages = {1832-1846}, pmid = {40035150}, issn = {1467-7652}, support = {32372482//National Natural Science Foundation of China/ ; }, mesh = {*Oryza/genetics/microbiology/growth &amp; development ; *Disease Resistance/genetics ; *Gene Editing ; *Plant Diseases/microbiology/genetics/immunology ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Rhizoctonia/physiology ; Plants, Genetically Modified ; Gene Expression Regulation, Plant ; }, abstract = {The primary goals of crop breeding are to enhance yield and improve disease resistance. However, the "trade-off" mechanism, in which signalling pathways for resistance and yield are antagonistically regulated, poses challenges for achieving both simultaneously. Previously, we demonstrated that knock-out mutants of the Dense and Erect Panicle 1 (DEP1) gene can significantly enhance rice resistance to sheath blight (ShB), and we mapped DEP1's association with panicle length. In this study, we discovered that dep1 mutants significantly reduced rice yield. Nonetheless, truncated DEP1 was able to achieve both ShB resistance and yield increase in japonica rice. To further explore the function of truncated DEP1 in promoting yield and ShB resistance, we generated CRISPR/Cas9-mediated genome editing mutants, including a full-length deletion mutant of DEP1, named dep1, and a truncated version, dep1-cys. Upon inoculation with Rhizoctonia solani, the dep1-cys mutant demonstrated stronger ShB resistance than the dep1 mutant. Additionally, dep1-cys increased yield per plant, whereas dep1 reduced it. Compared to the full DEP1 protein, the truncated DEP1 (dep1-cys) demonstrated a decreased interaction affinity with IDD14 and increased affinity with IDD10, which are known to positively and negatively regulate ShB resistance through the activation of PIN1a and ETR2, respectively. The dep1-cys mutant exhibited higher PIN1a and lower ETR2 expression than wild-type plants, suggesting that dep1-cys modulated IDD14 and IDD10 interactions to regulate PIN1a and ETR2, thereby enhancing ShB resistance. Overall, these data indicate that precise genome editing of DEP1 could simultaneously improve both ShB resistance and yield, effectively mitigating trade-off regulation in rice.}, }
@article {pmid40033995, year = {2025}, author = {Hanson, E and Kalla, N and Tharu, RJ and Demir, MM and Tok, BH and Canbaz, MA and Yigit, MV}, title = {CRISPR-Responsive Reprogrammable Label-Free Fluorescent Nanoclusters for ML-Assisted Pathogenic Genome Detection on Solid Substrates.}, journal = {Small (Weinheim an der Bergstrasse, Germany)}, volume = {21}, number = {15}, pages = {e2500784}, pmid = {40033995}, issn = {1613-6829}, support = {R35 GM156250/GM/NIGMS NIH HHS/United States ; 2022-08596//National Institute of Food and Agriculture/ ; 1R35GM156250-01/GM/NIGMS NIH HHS/United States ; 2022-08596//USDA National Institute of Food and Agriculture (NIFA)/ ; 1R35GM156250-01//National Institute of General Medical Sciences of the National Institutes of Health/ ; }, mesh = {*Salmonella/genetics ; *CRISPR-Cas Systems/genetics ; *Genome, Bacterial/genetics ; *Metal Nanoparticles/chemistry ; Silver/chemistry ; Fluorescence ; }, abstract = {The development of a paper-based genome detection assay using target-responsive DNA-templated silver nanoclusters (DFN-1) is presented. The reported nanoclusters exhibit intrinsic fluorescence, which is regulated by the cleavage of the DNA template surrounding the silver core. To enable the nanoclusters to respond to a specific genome, CRISPR-Cas12a is employed for highly specific and programmable digestion of the nanoclusters. Upon detection of the target, the DNA template is cleaved by the CRISPR-Cas12a complex, leading to a reduction in fluorescence. This assay successfully demonstrates for the detection of the Salmonella genome in the liquid phase and on 2 mm solid filter paper discs. By altering only the crRNA in the CRISPR complex, the assay is programmed to detect two different Salmonella serotypes. The selectivity of the assay is evaluated in DNA mixtures with and without the target genomic fragments. The assay also demonstrates the detection of as little as 33 copies of the full Salmonella genome by incorporating an isothermal amplification step. Furthermore, 60 unknown samples with different target content in standard 344 well plates are evaluated. The results are analyzed using custom-developed machine-learning algorithms, successfully detecting the presence of the target with 100% prediction accuracy.}, }
@article {pmid40032004, year = {2025}, author = {Kang, H and Yang, X and Jiang, R and Gao, P and Zhang, Y and Zhou, L and Ge, X and Han, J and Guo, X and Yang, H}, title = {Ultrasensitive and visual detection of pseudorabies virus based on CRISPR-Cas12b system.}, journal = {Microbial pathogenesis}, volume = {203}, number = {}, pages = {107447}, doi = {10.1016/j.micpath.2025.107447}, pmid = {40032004}, issn = {1096-1208}, mesh = {*Herpesvirus 1, Suid/isolation &amp; purification/genetics ; *Pseudorabies/diagnosis/virology ; Swine ; Animals ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Swine Diseases/diagnosis/virology ; *Nucleic Acid Amplification Techniques/methods ; Recombinases/metabolism ; *Molecular Diagnostic Techniques/methods ; Real-Time Polymerase Chain Reaction ; }, abstract = {Aujeszky's disease (AD) is an acute infectious disease that infects pigs and other animals, resulting in significant economic losses and posing a threat to human health. Reliable and rapid detection methods are essential for the prevention of AD. In this study, a RAA-Cas12b assay based on recombinase-aided amplification (RAA) and CRISPR-Cas12b system was established, optimized and evaluated for the rapid detection of wild-type Pseudorabies Virus (PRV). The results can not only be detected by real-time fluorescence readout, but also can be visualized by a portable blue light instrument. There was no cross-reaction with PRV Bartha-K61 strain or other swine infectious viruses. The analytical sensitivities of the real-time PRV RAA-Cas12b assay and visual PRV RAA-Cas12b assay were determined to be 15 copies/μL with 95 % confidence interval and 140 copies/μL with 95 % confidence interval, respectively. A total of 31 clinical samples were detected and compared with PRV qPCR assay to evaluate the diagnostic performance of the PRV RAA-Cas12b assay. The diagnostic coincidence rate of the two assays was 100 %. In summary, this convenient and reliable assay has great potential for rapid detection of wild type PRV in point-of-care testing (POCT).}, }
@article {pmid40030016, year = {2025}, author = {Le, TT and Choi, HI and Kim, JW and Yun, JH and Lee, YH and Jeon, EJ and Kwon, KK and Cho, DH and Choi, DY and Park, SB and Yoon, HR and Lee, J and Sim, EJ and Lee, YJ and Kim, HS}, title = {Cas9-mediated gene-editing frequency in microalgae is doubled by harnessing the interaction between importin α and phytopathogenic NLSs.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {122}, number = {10}, pages = {e2415072122}, pmid = {40030016}, issn = {1091-6490}, support = {RS-2024-00459155//National Research Foundation of Korea (NRF)/ ; 2021R1C1C1003425//National Research Foundation of Korea (NRF)/ ; }, mesh = {*Gene Editing/methods ; *Nuclear Localization Signals/metabolism/genetics ; *Chlamydomonas reinhardtii/genetics/metabolism ; *alpha Karyopherins/metabolism/genetics ; *Microalgae/genetics/metabolism ; *CRISPR-Associated Protein 9/metabolism/genetics ; *CRISPR-Cas Systems ; Cell Nucleus/metabolism ; Agrobacterium/genetics ; Bacterial Proteins/metabolism/genetics ; }, abstract = {Pathogen-derived nuclear localization signals (NLSs) enable vigorous nuclear invasion in the host by the virulence proteins harboring them. Herein, inspired by the robust nuclear import mechanism, we show that NLSs originating from the plant infection-associated Agrobacterium proteins VirD2 and VirE2 can be incorporated into the Cas9 system as efficient nuclear delivery enhancers, thereby improving the low gene-editing frequency in a model microalga, Chlamydomonas reinhardtii, caused by poor nuclear localization of the bulky nuclease. Prior to evaluation of the NLSs, IPA1 (Cre04.g215850) was first defined in the alga as the nuclear import-related importin alpha (Impα) that serves as a counterpart adaptor protein of the NLSs, based on extensive in silico analyses considering the protein's sequence, tertiary folding behavior, and structural basis when interacting with a well-studied SV40TAg NLS. Through precursive affinity explorations, we reproducibly found that the NLSs mediated the binding between the Cas9 and Impα with nM affinities and visually confirmed that the fusion of the NLSs strictly localized the peptide-bearing cargoes in the microalgal nucleus without compensating for their cleavage ability. When employed in a real-world application, the VirD2 NLS increases the mutation frequency (~1.12 × 10[-5]) over 2.4-fold compared to an archetypal SV40TAg NLS (~0.46 × 10[-5]) when fused with Cas9. We demonstrate the cross-species versatility of the Impα-dependent strategy by successfully applying it to an industrial alga, Chlorella Sp. HS2. This work, focused on affinity augmentation, provides insights into increasing the frequency of gene editing, which can be advantageously used in programmable mutagenesis with broad applicability.}, }
@article {pmid40029794, year = {2025}, author = {Livneh, Y and Leor-Librach, E and Agmon, D and Makov-Bouaniche, T and Tiwari, V and Shor, E and Yeselson, Y and Masci, T and Schaffer, A and Charuvi, D and Hirschberg, J and Vainstein, A}, title = {Combined enhancement of ascorbic acid, β-carotene and zeaxanthin in gene-edited lettuce.}, journal = {Plant biotechnology journal}, volume = {23}, number = {6}, pages = {1954-1967}, pmid = {40029794}, issn = {1467-7652}, support = {20-01-0209//The National Center for Genome Editing in Agriculture, Israel/ ; 7500158//Ministry of Science and Technology, Israel/ ; 12-01-0037//Ministry of Agriculture and Rural Development/ ; }, mesh = {*Zeaxanthins/metabolism ; *beta Carotene/metabolism ; *Lactuca/genetics/metabolism ; *Ascorbic Acid/metabolism ; *Gene Editing ; Intramolecular Lyases/genetics/metabolism ; CRISPR-Cas Systems ; Plants, Genetically Modified ; }, abstract = {Lettuce is widely grown and consumed but provides lower nutritional value compared to other leafy greens, particularly in the essential vitamins A and C. To address this, major control points in carotenoid and ascorbic acid (AsA) production were targeted using a viral-based CRISPR/Cas9 system in the commercial lettuce cultivar 'Noga'. Knockout of lycopene ε-cyclase (LCY-ε), the enzymatic gatekeeper opposing production of β-branch carotenoids, increased β-carotene (provitamin A) levels up to 2.7-fold and facilitated zeaxanthin accumulation up to 4.3 μg/g fresh weight. Chlorophyll fluorescence measurements revealed that photosystem II efficiency was unaffected in LCY-ε mutants, though their non-photochemical quenching (NPQ) capacity decreased at light intensities above 400 μmol m[2] s[-1]. However, the gene-edited plants exhibited normal growth and comparable plant mass, despite the absence of two major lettuce xanthophylls, lutein and lactucaxanthin. Modifications in a regulatory region in the upstream ORF of GDP-L-galactose phosphorylase 1 and 2 (uGGP1 and uGGP2), the rate-limiting enzyme in AsA production, resulted in an average 6.9-fold increase in AsA levels. The mutation in uGGP2 was found to dominantly influence AsA over-accumulation. Knockout lines that combined the mutations in LCY-ε, uGGP1, uGGP2 and in carotenoid cleavage dioxygenase 4a (CCD4a), an isozyme involved in β-carotene degradation in lettuce, exhibited significantly enhanced content of AsA, β-carotene and zeaxanthin. Our results demonstrate the potential of multi-pathway gene editing to 'supercharge' economically important crops such as lettuce as a means to address micronutrient deficiencies in modern diets.}, }
@article {pmid40029720, year = {2025}, author = {do Rêgo, RL and Neves, FPG and Miranda, FM and da Silva, AB and Cabral, AS and Dos Santos, BA and Lima, JLC and de Souza, ARV}, title = {CRISPR Elements and Their Association with Macrolide and Aminoglycoside Resistance Genes in Enterococci.}, journal = {Microbial drug resistance (Larchmont, N.Y.)}, volume = {31}, number = {3}, pages = {75-79}, doi = {10.1089/mdr.2024.0236}, pmid = {40029720}, issn = {1931-8448}, mesh = {*Aminoglycosides/pharmacology ; *Anti-Bacterial Agents/pharmacology ; *Macrolides/pharmacology ; *Enterococcus faecalis/genetics/drug effects/isolation &amp; purification ; Microbial Sensitivity Tests/methods ; *Enterococcus faecium/genetics/drug effects/isolation &amp; purification ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Drug Resistance, Bacterial/genetics ; Humans ; CRISPR-Cas Systems/genetics ; Bacterial Proteins/genetics ; Genes, Bacterial/genetics ; }, abstract = {CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems are common among enterococci and may prevent the acquisition of mobile genetic elements carrying antimicrobial resistance genes. In this study, we correlate the presence of CRISPR with genes associated with macrolide resistance and high-level resistance to aminoglycosides (HLR-A) among 216 Enterococcus faecalis and 82 Enterococcus faecium isolates. We used PCR to detect genes associated with macrolide resistance, HLR-A, and type II-A CRISPR elements. We used two-tailed Fisher's exact test to evaluate correlation between CRISPR and resistance genes. One hundred and seven (35.9%) isolates had at least one HLR-A gene; the prevalent genes were aac(6')-Ie-aph(2″)-Ia and ant(6)-Ia found in 61 (57%) and 46 (43%) isolates, respectively. The macrolide resistance genes erm(A) and erm(B) were found in 116 (38.9%) isolates. Overall, 174 (58.4%) isolates had at least one CRISPR element; the predominant one was CRISPR3-Cas (n = 117; 39.2%). The presence of three genes, two related to HLR-A [aph(2″)-Ic and ant(6)-Ia] and one macrolide resistance gene [erm(B)], was associated with the absence of CRISPR (p < 0.05), mainly in E. faecalis lacking CRISPR3-Cas. We observed the association between the absence of CRISPR and the presence of major aminoglycoside and macrolide resistance determinants, contributing to the understanding of the evolution of resistance in enterococci.}, }
@article {pmid40029713, year = {2025}, author = {Youngquist, BM and Mnguni, AT and Pungan, D and Lai, RP and Dai, G and Ng, CF and Samson, A and Abdelgaliel, Y and Lyon, CJ and Ning, B and Husain, S and Wasserman, S and Kolls, JK and Hu, TY}, title = {CRISPR-mediated detection of Pneumocystis transcripts in bronchoalveolar, oropharyngeal, and serum specimens for Pneumocystis pneumonia diagnosis.}, journal = {The Journal of clinical investigation}, volume = {135}, number = {8}, pages = {}, pmid = {40029713}, issn = {1558-8238}, support = {R35 HL139930/HL/NHLBI NIH HHS/United States ; R01 AI144168/AI/NIAID NIH HHS/United States ; R01 AI175618/AI/NIAID NIH HHS/United States ; R01 AI120033/AI/NIAID NIH HHS/United States ; R01 AI173021/AI/NIAID NIH HHS/United States ; }, mesh = {Adult ; Animals ; Child, Preschool ; Female ; Humans ; Infant ; Mice ; Bronchoalveolar Lavage Fluid/microbiology ; *CRISPR-Cas Systems ; Mice, Inbred C57BL ; Oropharynx/microbiology ; *Pneumocystis carinii/genetics/isolation &amp; purification ; *Pneumonia, Pneumocystis/diagnosis/microbiology ; Retrospective Studies ; Reverse Transcriptase Polymerase Chain Reaction ; *RNA, Fungal/blood/isolation &amp; purification ; }, abstract = {BACKGROUNDPneumocystis jirovecii pneumonia (PCP) is a leading cause of fungal pneumonia, but its diagnosis primarily relies on invasive bronchoalveolar lavage (BAL) specimens that are difficult to obtain. Oropharyngeal swabs and serum could improve the PCP diagnostic workflow, and we hypothesized that CRISPR could enhance assay sensitivity to allow robust P. jirovecii diagnosis using swabs and serum. Herein, we describe the development of an ultrasensitive RT-PCR-coupled CRISPR assay with high active-infection specificity in infant swabs and adult BAL and serum.METHODSMouse analyses employed an RT-PCR CRISPR assay to analyze P. murina transcripts in WT and Rag2-/- mouse lung RNA, BAL, and serum at 2-, 4-, and 6-weeks after infection. Human studies used an optimized RT-PCR CRISPR assay to detect P. jirovecii transcripts in infant oropharyngeal swab samples, adult serum, and adult BAL specimens from patients who were infected with P. jirovecii and those who were not.RESULTSThe P. murina assays sensitively detected Pneumocystis RNA in the serum of infected mice throughout infection. Oropharyngeal swab CRISPR assay results identified infants infected with P. jirovecii with greater sensitivity (96.3% versus 66.7%) and specificity (100% versus 90.6%) than RT-qPCR compared with mitochondrial large subunit rRNA gene (mtLSU) standard marker, and CRISPR results achieved higher sensitivity than RT-qPCR results (93.3% versus 26.7%) in adult serum specimens.CONCLUSIONSince swabs are routinely collected in pediatric patients with pneumonia and serum is easier to obtain than BAL, this assay approach could improve the accuracy and timing of pediatric and adult Pneumocystis diagnosis by achieving specificity for active infection and potentially avoiding the requirement for BAL specimens.FUNDINGThe work was supported by the NIH (R01AI120033), NHLBI (R35HL139930), the Louisiana Board of Regents Endowed Chairs for Eminent Scholars program, and by research funding provided by National Institute of Allergy and Infectious Diseases (NIAID) (R01AI144168, R01AI175618, R01AI173021). This research was also funded by the NIHR (project 134342) using UK aid from the UK government to support global health research.}, }
@article {pmid40029521, year = {2025}, author = {Berzosa, M and McShane, AN and Nanda, P and Williams, M and Malinova, D}, title = {CRISPR/Cas9 Methods for Identification and Validation of Genes Regulating BCR-Mediated Antigen Uptake.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2909}, number = {}, pages = {153-164}, pmid = {40029521}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems ; Gene Editing/methods ; *Receptors, Antigen, B-Cell/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; *Antigens/metabolism/immunology ; Animals ; B-Lymphocytes/immunology/metabolism ; Mice ; }, abstract = {Genome-wide CRISPR screens are a powerful tool to interrogate and identify gene function in a wide variety of applications and cell types. CRISPR-Cas9 technology using pooled CRISPR single guide RNA (sgRNA) libraries enables genetic editing in bulk in a large population of cells of interest. After selection of gene-edited cells, phenotyping effects can be evaluated by quantifying abundance (over- or under-representation) of individual sgRNAs using DNA sequencing. In addition to cell survival, these assays can be applied to investigations of drug sensitivity, as well as almost any cellular process with a clear phenotypic read out, for example, receptor internalization, migration, autophagy, and differentiation.Here, taking as an example the identification of molecular components governing B-cell antigen uptake through the B-cell receptor, we describe whole-genome, small-scale, and in vivo validation methods to identify and validate genes regulating BCR-mediated antigen uptake.}, }
@article {pmid40029520, year = {2025}, author = {Kumar, M and Gentner-Göbel, E and Maity, PC}, title = {Facilitating Gene Editing in Human Lymphoma Cells Using Murine Ecotropic γ-Retroviruses.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2909}, number = {}, pages = {133-151}, pmid = {40029520}, issn = {1940-6029}, mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; Animals ; Cell Line, Tumor ; Mice ; *Lymphoma/genetics ; Genetic Vectors/genetics ; Lentivirus/genetics ; *Gammaretrovirus/genetics ; *Lymphoma, B-Cell/genetics ; Transduction, Genetic ; }, abstract = {Genetic modifications using CRISPR-Cas9 have revolutionized cancer research and other preclinical studies. Exceptionally, these efficient tools are inadequate in a few disease models and cell lines due to the aberrant differentiation states and the accumulation of excessive somatic mutations that compromise the robustness of viral gene delivery and stable transduction. A couple of B lymphoma cell lines fall into this category where lentiviral transfection becomes inefficient and exhibits variable efficiency. Additionally, lentiviral delivery requires high biosafety levels. To address this challenge, we have developed a two-step strategy that supports CRISPR-Cas9 through lentivirus and murine ecotropic γ-retrovirus. By engineering B lymphoma cell lines to express Cas9 and mCat1, a specific receptor for ecotropic retroviruses, we enable efficient and safe gene editing through ecotropic γ-retrovirus. We demonstrate the efficacy of this method by generating IgM-deficient B lymphoma cell lines. This innovative approach simplifies protocols, enhances accessibility, and paves the way for standardized gene manipulation of B cell lymphoma models for molecular cell biology research.}, }
@article {pmid40029441, year = {2025}, author = {Pei, Y and Cao, W and Kong, X and Wang, S and Sun, Z and Zuo, Y and Hu, Z}, title = {CRISPR/Cas9-mediated efficient PlCYP81Q38 mutagenesis in Phryma leptostachya.}, journal = {Planta}, volume = {261}, number = {4}, pages = {73}, pmid = {40029441}, issn = {1432-2048}, support = {2023-JC-QN-0213//Natural Science Foundation of Shaanxi Province/ ; 2023YFD1700700//National Key Research and Development Program of China/ ; 2452020221//Chinese Universities Scientific Fund from Science/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Mutagenesis/genetics ; Lignans/metabolism ; Gene Editing/methods ; *Cytochrome P-450 Enzyme System/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Plant Roots/genetics/metabolism ; Dioxoles/metabolism ; Plants, Genetically Modified ; }, abstract = {Combined with hairy root transformation, the CRISPR/Cas9 system was established to initiate targeted mutagenesis of PlCYP81Q38, which influenced lignan accumulation in Phryma leptostachya. Phryma leptostachya is a traditional Chinese medicinal herb renowned for its applications in both conventional medicine and natural botanical insecticides, with lignans as the main active ingredients. During the biosynthesis of lignans, PlCYP81Q38, a P450 protein, is assumed to play a crucial role and is accountable for the production of sesamin from (+)-pinoresinol. As a cutting-edge genome editing tool, the CRISPR/Cas9 system is widely employed across diverse species for gene functional research but yet to be harnessed in P. leptostachya. This study utilized the CRISPR/Cas9 system in conjunction with hairy root transformation to initiate targeted mutagenesis in PlCYP81Q38 gene. Employing binary vectors, pYLCRISPR/Cas9Pubi-H, complemented by dual single-stranded guided RNAs (sgRNAs), enabled precise editing at two gene sites and the deletion of large fragments. This editing system resulted in mutagenesis rates surpassing 79%, achieving a notable rate of 61.9% fragment deletion mutants. Liquid chromatography/tandem mass spectrometry confirmed the impact on lignan biosynthesis by PlCYP81Q38-targeted mutagenesis, resulting in the accumulation of pinoresinol and disrupted production of sesamin, 6-demethoxy-leptostachyol acetate, and leptostachyol acetate. Furthermore, the knockout of PlCYP81Q38 up-regulated its upstream pathway genes, such as dirigent gene, cinnamoyl-CoA reductase genes, cinnamyl-alcohol dehydrogenase genes, and p-coumarate 3-hydroxylase genes, identified through gene co-expression analysis. Collectively, mediated by the CRISPR/Cas9 platform, the new biotechnology for targeted genome editing within P. leptostachya, our findings affirm the significant roles of PlCYP81Q38 in the lignan biosynthesis pathway and highlight the potential of CRISPR/Cas9 in exploring the functional genome and secondary metabolite biosynthesis of this plant species.}, }
@article {pmid40029034, year = {2025}, author = {Cheng, X and Zhao, W and Chen, D and Ren, D and Qian, T and Xia, X and Wang, X and Li, Q and Yang, J and Gu, Y and Zhang, P and Yin, K and Yu, P and Dong, W}, title = {Ultrasensitive Detection of FEN1 Activity for Cancer Diagnosis Using a CRISPR/Cas13a-Based Triple Cascade Amplification System.}, journal = {Advanced healthcare materials}, volume = {14}, number = {6}, pages = {e2404411}, doi = {10.1002/adhm.202404411}, pmid = {40029034}, issn = {2192-2659}, support = {22104090//National Natural Science Foundation of China/ ; 82102184//National Natural Science Foundation of China/ ; 82204349//National Natural Science Foundation of China/ ; 82170526//National Natural Science Foundation of China/ ; 62305207//National Natural Science Foundation of China/ ; PT21011//Shanghai High-Level Local University construction project/ ; 23ZR1436800//Shanghai 2023 "Science and Technology Innovation Action Plan" Natural Science Foundation Project/ ; ZYYZDXK-2023070//NATCM's Project of High-level Construction of Key TCM Disciplines/ ; 2023AH010073//Program for Excellent Sci-tech Innovation Teams of Universities in Anhui Province/ ; LCZX2201//Clinical Research Foundation of Huadong Hospital/ ; //the Junior Thousand Talents Program of China/ ; }, mesh = {Humans ; *Flap Endonucleases/metabolism/antagonists &amp; inhibitors ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Neoplasms/diagnosis/genetics ; Biomarkers, Tumor/metabolism ; Cell Line, Tumor ; }, abstract = {Flap endonuclease 1 (FEN1) is closely associated with tumor progression and proliferation, making it a promising biomarker for cancer diagnosis. However, developing a sensitive, reliable, and user-friendly method for quantitative FEN1 detection remains technically challenging. In this study, an ultrasensitive FEN1 biosensor is established using a target-induced cleavage-ligation-transcription-activation cascade strategy (LTACas13a) to enhance the cleavage ability of CRISPR/Cas13a. The LTACas13a method has shown excellent performance in screening FEN1 inhibitors and detecting endogenous FEN1 activity in living cells, as well as in clinical biological samples such as human serum and tissue samples. Additionally, using a universal dumbbell probe derived from FEN1, a multiplex LTACas13a strategy is developed for detecting various DNA glycosylases, including formamidopyrimidine DNA glycosylase, uracil DNA glycosylase, and human alkyl adenine DNA glycosylase. This straightforward approach provides a reliable and effective diagnostic tool for early-stage cancer detection and offers significant opportunities for FEN1 biosensing and related drug discovery.}, }
@article {pmid40027883, year = {2025}, author = {Zhang, X and Tao, C and Li, M and Zhang, S and Liang, P and Huang, Y and Liu, H and Wang, Y}, title = {Engineering of SauriCas9 with enhanced specificity.}, journal = {Molecular therapy. Nucleic acids}, volume = {36}, number = {1}, pages = {102455}, pmid = {40027883}, issn = {2162-2531}, abstract = {SauriCas9 is a compact Cas9 nuclease showing promise for in vivo therapeutic applications. However, concerns about off-target effects necessitated improvements in specificity. We addressed this by introducing mutations to eliminate polar contacts between Cas9 and the target DNA, resulting in the SauriCas9-R253A variant with enhanced specificity. To validate its efficacy, we employed SauriCas9-R253A to disrupt three genes (B2M, TRAC, and PDCD1), a strategy integral to the development of allogeneic chimeric antigen receptor T cell (CAR-T) therapies. Our results demonstrated that the most efficient single-guide RNAs for SauriCas9-R253A exhibited comparable activity to SpCas9 and showed no detectable off-target effects in the disruption of these genes, highlighting its therapeutic potential.}, }
@article {pmid40026698, year = {2025}, author = {Álvarez-Rodríguez, A and Li, Z and Jin, BK and Stijlemans, B and Geldhof, P and Magez, S}, title = {A CRISPR-Cas-based recombinase polymerase amplification assay for ultra-sensitive detection of active Trypanosoma brucei evansi infections.}, journal = {Frontiers in molecular biosciences}, volume = {12}, number = {}, pages = {1512970}, pmid = {40026698}, issn = {2296-889X}, abstract = {INTRODUCTION: Control of Trypanosoma brucei evansi (T. b. evansi) infections remains a significant challenge in managing Surra, a widespread veterinary disease affecting both wild and domestic animals. In the absence of an effective vaccine, accurate diagnosis followed by treatment is crucial for successful disease management. However, existing diagnostic methods often fail to detect active infections, particularly in field conditions. Recent advancements in CRISPR-Cas technology, combined with state-of-the-art isothermal amplification assays, offer a promising solution. This approach has led us to the development of a TevRPA-CRISPR assay, a highly sensitive and specific T. b. evansi diagnostic tool suitable for both laboratory and field settings.<br><br>METHODS: First, the TevCRISPR-Cas12b cleavage assay was developed and optimized, and its analytical sensitivity was evaluated. Next, this technology was integrated with the TevRPA to create the TevRPA-CRISPR test, with the reaction conditions being optimized and its analytical sensitivity and specificity assessed. Finally, the test's accuracy in detecting both active and cured T. b. evansi infections was evaluated.<br><br>RESULTS: The optimized TevCRISPR-Cas12b cleavage assay demonstrated the ability to detect T. b. evansi target DNA at picomolar concentrations. Integrating TevCRISPR-Cas12b with RPA in Two-Pot and One-Pot TevRPA-CRISPR tests achieved up to a 100-fold increase in analytical sensitivity over RPA alone, detecting attomolar concentrations of T. b. evansi target DNA, while maintaining analytical specificity for T. b. evansi. Both assays exhibited performance comparable to the gold standard TevPCR in experimental mouse infections, validating their effectiveness for detecting active infections and assessing treatment efficacy.<br><br>DISCUSSION: The TevRPA-CRISPR tests prove highly effective for diagnosing active infections and assessing treatment efficacy, while being adaptable for both laboratory and field use. Thus, the TevRPA-CRISPR assays emerge as a promising addition to current diagnostic tools, offering efficient and reliable detection of active T. b. evansi infections.}, }
@article {pmid40025867, year = {2025}, author = {Koodamvetty, A and Thangavel, S}, title = {Advancing Precision Medicine: Recent Innovations in Gene Editing Technologies.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {12}, number = {14}, pages = {e2410237}, pmid = {40025867}, issn = {2198-3844}, support = {/WT_/Wellcome Trust/United Kingdom ; IA/TSG/22/1/600431//DBT/Wellcome Trust India Alliance/ ; BT/PR38267/GET/119/348/2020//Department of Biotechnology, Ministry of Science and Technology, India/ ; }, mesh = {*Gene Editing/methods/trends ; Humans ; *Precision Medicine/methods/trends ; *Genetic Therapy/methods ; CRISPR-Cas Systems/genetics ; Animals ; }, abstract = {The advent of gene editing has significantly advanced the field of medicine, opening new frontiers in the treatment of genetic disorders, cancer, and infectious diseases. Gene editing technology remains a dynamic and promising area of research and development. Recent advancements in protein and RNA engineering within this field have addressed critical issues such as imprecise edits, poor editing efficiency, and off-target effects. Advancements in delivery methods have allowed the achievement of therapeutic or even selection-free gene editing efficiency with reduced toxicity in primary cells, thereby enhancing the safety and efficacy of gene manipulation. This progress paves the way for transformative changes in molecular biology, medicine, and other fields. This review provides a comprehensive overview of the advancements in gene editing techniques, focusing on prime editor proteins and their engineered variants. It also explores alternative systems that expand the toolkit for precise genomic modifications and highlights the potential of these innovations in treating hematological disorders, while also discussing the limitations and challenges that remain.}, }
@article {pmid40025280, year = {2025}, author = {Ha, TY and Chan, SW and Wang, Z and Law, PWN and Miu, KK and Lu, G and Chan, WY}, title = {SOX9 haploinsufficiency reveals SOX9-Noggin interaction in BMP-SMAD signaling pathway in chondrogenesis.}, journal = {Cellular and molecular life sciences : CMLS}, volume = {82}, number = {1}, pages = {99}, pmid = {40025280}, issn = {1420-9071}, support = {Acc 8601011//CUHK VC Discretionary Fund/ ; YFJGJS1.0//CUHK Laboratory Support Special Fund for Key Laboratory for Regenerative Medicine, Ministry of Education, China/ ; N_CUHK 428/22//NSFC/RGC Joint Grant/ ; N_CUHK 434/24//NSFC/RGC Joint Grant/ ; MHP/005/23//Innovation and Technology Commission grant (Mainland-Hong Kong Joint Funding Scheme, MHKJFS)/ ; JLFS/M-403/24//Co-funding Mechaism on Joint Laboratories with the CAS/ ; }, mesh = {Humans ; *SOX9 Transcription Factor/genetics/metabolism ; *Chondrogenesis/genetics ; *Haploinsufficiency/genetics ; *Signal Transduction ; *Bone Morphogenetic Proteins/metabolism ; *Carrier Proteins/metabolism/genetics ; Male ; Chondrocytes/metabolism/cytology ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Smad Proteins/metabolism ; Campomelic Dysplasia/genetics/pathology/metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; }, abstract = {Campomelic Dysplasia (CD) is a rare congenital disease caused by haploinsufficiency (HI) in SOX9. Patients with CD typically present with skeletal abnormalities and 75% of them have sex reversal. In this study, we use CRISPR/Cas9 to generate a human induced pluripotent stem cell (hiPSC) model from a heathy male donor, based on a previously reported SOX9 splice site mutation in a CD patients. This hiPSCs-derived chondrocytes from heterozygotes (HT) and homozygotes (HM) SOX9 mutation carriers showed significant defects in chondrogenesis. Bulk RNA profiling revealed that the BMP-SMAD signaling pathway, ribosome-related, and chromosome segregation-related gene sets were altered in the HT chondrocytes. The profile also showed significant noggin upregulation in CD chondrocytes, with ChIP-qPCR confirming that SOX9 binds to the distal regulatory element of noggin. This suggests SOX9 plays a feedback role in the BMP signaling pathway by modulating noggin expression rather than acting solely as a downstream regulator. This provides further insights into its dosage sensitivity in chondrogenesis. Overexpression of SOX9 showed promising results with improved sulfated glycosaminoglycans (GAGs) aggregation and COL2A1 expression following differentiation. We hope this finding could provide a better understanding of the dosage-dependent role of SOX9 in chondrogenesis and contribute to the development of improved therapeutic targets for CD patients.}, }
@article {pmid40024134, year = {2025}, author = {Xiong, M and Wang, Y and Lu, S and Lubanga, N and Li, T and Li, Z and He, B and Li, Y}, title = {Space-coded microchip for multiplexed respiratory virus detection via CRISPR-Cas12a and RPA.}, journal = {Talanta}, volume = {291}, number = {}, pages = {127815}, doi = {10.1016/j.talanta.2025.127815}, pmid = {40024134}, issn = {1873-3573}, mesh = {Humans ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Lab-On-A-Chip Devices ; Recombinases/metabolism ; Respiratory Tract Infections/virology/diagnosis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Multiple infections of respiratory viruses are common in patients with clinical respiratory diseases, but current detection methods still have problems such as complex equipment and long detection time. Rapid, low-cost, and on-site detection of human respiratory viruses is crucial for both clinical diagnosis and population screening. In this research, we created a space-coded microfluidic chip (SC-Chip) for the recognition of nine respiratory viruses: influenza A virus, influenza B virus, severe acute respiratory syndrome coronavirus 2, human coronavirus OC43, human coronavirus NL63, human coronavirus HKU1, human respiratory syncytial virus, human parainfluenza virus, and human metapneumovirus. For the first time, a comprehensive sequence comparison among these viruses was performed to design the recombinase polymerase amplification (RPA) primers and Cas12a-crRNAs. The SC-Chip partitions samples amplified by RPA into spatially coded wells preloaded with CRISPR-Cas12a detection reagents, enabling the identification of all nine viral targets in a single test using a single fluorescence probe. The chip-based assay displays 9 respiratory viruses in less than 40 min with a minimum detection limit at a concentration of 10[-18] M (∼1 copy/reaction). Additionally, the efficacy of the method was assessed through its application to 35 clinical patient samples identified as being at risk for respiratory virus infection, yielding a sensitivity of 90 % and a specificity of 100 %. In summary, this space-coded microfluidic CRISPR system offers several advantages, including ease of operation, cost-effectiveness, and rapid data acquisition, thereby holding great potential for multiplexed detection of nucleic acid targets in a clinical setting.}, }
@article {pmid40024074, year = {2025}, author = {Mukherjee, AG and R, K and Gopalakrishnan, AV}, title = {Recent advances in CRISPR technology in the milieu of oral squamous cell carcinoma.}, journal = {Pathology, research and practice}, volume = {269}, number = {}, pages = {155860}, doi = {10.1016/j.prp.2025.155860}, pmid = {40024074}, issn = {1618-0631}, mesh = {Humans ; *Carcinoma, Squamous Cell/genetics/therapy/pathology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genetic Therapy/methods ; *Mouth Neoplasms/genetics/therapy/pathology ; *Squamous Cell Carcinoma of Head and Neck/genetics/therapy ; }, abstract = {Oral squamous cell carcinoma (OSCC) is a widely recognized cancer that constitutes over 90 % of all oral cancer cases [1]. Thanks to advancements in cancer treatment, such as radiation, chemotherapy, and molecular target therapy, the 5-year relative survival rate for OSCC has more than doubled in the past 26 years. However, the exact cause and molecular process of OSCC have not been fully understood, and further investigation is necessary. CRISPR/Cas technology is seen as a groundbreaking tool in molecular biology, providing unparalleled accuracy in altering genes. Its use in conditions like OSCC shows excellent potential for advancing research and treatment development. OSCC, characterized by the growth of cancer cells in the lining of the mouth, presents challenges in its treatment and control. Traditional therapies like surgery, radiation, and chemotherapy often show limited success and may lead to severe side effects. The promise of CRISPR/Cas technology in tackling critical aspects of OSCC is apparent. In this correspondence, we discuss the recent advances in CRISPR technology in the OSCC milieu.}, }
@article {pmid40023418, year = {2025}, author = {Helalat, SH and Kristinsdóttir, HT and Petersen, AD and Téllez, RC and Boye, MN and Sun, Y}, title = {Enzymatic assembly for CRISPR split-Cas9 system: The emergence of a Sortase-based split-Cas9 technology.}, journal = {International journal of biological macromolecules}, volume = {306}, number = {Pt 2}, pages = {141583}, doi = {10.1016/j.ijbiomac.2025.141583}, pmid = {40023418}, issn = {1879-0003}, mesh = {*CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; HEK293 Cells ; *Aminoacyltransferases/metabolism/genetics ; *Cysteine Endopeptidases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; Jurkat Cells ; Escherichia coli/genetics ; }, abstract = {CRISPR-Cas9 has been widely used in research and medical investigations as a pioneering technology. However, challenges such as the large size of the Cas9 sequence and the need for precise control over its activity in specific cell types have impeded its widespread adoption. Various alternatives, such as split-Cas9 technology, have emerged. Split-Cas9 systems allow the large Cas9 sequence to be divided into two segments to aid in the delivery of the enzyme. Nevertheless, challenges persist in achieving precise control over the timing and location of Cas9 reassembly and activity to ensure targeted action. This study presents an enzymatic-based split-Cas9 system, introducing a new approach utilizing the Sortase enzyme for the reconstitution of the full Cas9 protein. The developed method eliminates the need for chemical or physical induction and allows for precise genome editing in specific cells through the utilization of various specific promoters or targeted drug delivery. Experimental validation of the enzymatic system was conducted in E. coli, HEK cells, and Jurkat cells, demonstrating successful assembly and activity of the assembled Cas9 enzyme. In addition, this study explored the incorporation of nuclear localization signals, the evaluation of inducible promoters, and the delivery of the system's components in mRNA or protein form. Furthermore, we investigated the potential of S/MAR minicircle technology instead of viral vectors within the system. Overall, we highlighted the feasibility and utility of the Sortase-based split-Cas9 system to enhance control and efficiency compared to traditional CRISPR-Cas9 approaches. Additionally, this study revealed the potential of using the Sortase enzyme for posttranslational modifications and protein assembly in human cells.}, }
@article {pmid40022904, year = {2025}, author = {Setyono, ESA and Rogers, NK and Hofmann, A and Lickert, H and Burtscher, I}, title = {Generation of ARX-T2A-H2B-CFP x C-PEP-mCherry-hiPSC double reporter line for monitoring of pancreatic differentiation.}, journal = {Stem cell research}, volume = {84}, number = {}, pages = {103685}, doi = {10.1016/j.scr.2025.103685}, pmid = {40022904}, issn = {1876-7753}, mesh = {*Cell Differentiation ; Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; CRISPR-Cas Systems ; Genes, Reporter ; Animals ; Red Fluorescent Protein ; Luminescent Proteins/metabolism/genetics ; Cell Line ; }, abstract = {Pancreatic islets consist of several different endocrine cell types that work in harmony. Aside from primary pancreatic islets, stem cell-derived pancreatic islets can be used as an alternative research and disease model. Here, we introduce a double reporter line of ARX-T2A-H2B-CFP x C-PEP-mCherry-hiPSC through CRISPR/Cas9-mediated insertion of mCherry in the C-terminus of C-Peptide in the previously published ARX-CFP hiPSC line. This reporter line allows live monitoring of stem cell-derived pancreatic alpha and beta cells throughout differentiation.}, }
@article {pmid40022903, year = {2025}, author = {Chen, H and Liang, Y and Chen, Y and Liang, Y and Li, X and Duan, C and Cui, Z and Gu, J and Ding, C and Sun, X and Chen, J}, title = {Generation of a human iPSC line with heterozygous PRPF8 c.5792C &gt; T, p. T1931M mutation to model retinitis pigmentosa using CRISPR/Cas9 technology.}, journal = {Stem cell research}, volume = {84}, number = {}, pages = {103689}, doi = {10.1016/j.scr.2025.103689}, pmid = {40022903}, issn = {1876-7753}, mesh = {Humans ; *Retinitis Pigmentosa/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Heterozygote ; Cell Line ; Mutation ; }, abstract = {Mutations in the PRPF8 gene frequently result in retinitis pigmentosa (RP), an autosomal dominant inherited retinal disease that can lead to nyctalopia and progressive vision loss. Currently, no effective treatment is available. In this study, we used CRISPR/Cas9 technology to introduce a heterozygous point mutation inthe PRPF8 gene of a normal induced pluripotent stem cell (iPSC) line. This mutation mirrors that found in a previously reportedRP patient-derived iPSC line (CSUASOi006-A) from our group. Establishing the PRPF8 gene mutation cell line (CSUASOi012-A-2) provides a valuable cellular resource for studying the pathogenesis of RP.}, }
@article {pmid40022609, year = {2025}, author = {Lee, WH and Bates, EA and Kipp, ZA and Pauss, SN and Martinez, GJ and Blair, CA and Hinds, TD}, title = {Insulin receptor responsiveness governs TGFβ-induced hepatic stellate cell activation: Insulin resistance instigates liver fibrosis.}, journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology}, volume = {39}, number = {5}, pages = {e70427}, pmid = {40022609}, issn = {1530-6860}, support = {F31HL170972//HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; R01 DK121797/DK/NIDDK NIH HHS/United States ; R01DA058933//HHS | NIH | National Institute on Drug Abuse (NIDA)/ ; 25PRE1374495//American Heart Association (AHA)/ ; F31 HL175979/HL/NHLBI NIH HHS/United States ; F31 HL170972/HL/NHLBI NIH HHS/United States ; R01 DA058933/DA/NIDA NIH HHS/United States ; F31HL175979//HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; R01DK121797//HHS | NIH | NIDDK | Division of Diabetes, Endocrinology, and Metabolic Diseases (DEM)/ ; }, mesh = {Cell Line ; Humans ; *Insulin Resistance ; *Receptor, Insulin/metabolism ; *Hepatic Stellate Cells/metabolism ; Transforming Growth Factor beta/metabolism ; CRISPR-Cas Systems ; *Liver Cirrhosis/metabolism ; Cell Proliferation ; Cell Movement ; Signal Transduction ; Gene Regulatory Networks ; }, abstract = {The insulin receptor (INSR) has been shown to be hyperactive in hepatic stellate cells (HSCs) in humans and rodents with liver fibrosis. To explore HSC cellular mechanisms that INSR regulates during pro-fibrotic stimulation, we used CRISPR-Cas9 technology. We knocked out a portion of the INSR gene in human LX2 HSC cells (INSR[e5-8] KO) that regulates insulin responsiveness but not the insulin-like growth factor (IGF) or transforming growth factor-β (TGFβ) signaling. The INSR[e5-8] KO HSCs had significantly higher cell growth, BrdU incorporation, and lower TP53 expression that suppresses growth, and they also exhibited increased migration compared to the Scramble control. We treated the scramble control and INSR[e5-8] KO HSCs with insulin or TGFβ and profiled hundreds of kinase activities using the PamGene PamStation kinome technology. Our analysis showed that serine/threonine kinase (STK) activities were reduced, and most of the protein-tyrosine kinase (PTK) activities were increased in the INSR[e5-8] KO compared to the Scramble control HSCs. To study gene transcripts altered in activated Scramble control and INSR[e5-8] KO HSCs, we treated them with TGFβ for 24 h. We isolated RNA for sequencing and found that the INSR[e5-8] KO cells, compared to control HSCs, had altered transcriptional responsiveness to TGFβ stimulation, collagen-activated signaling, smooth muscle cell differentiation pathways, SMAD protein signaling, collagen metabolic process, integrin-mediated cell adhesion, and notch signaling. This study demonstrates that reduced INSR responsiveness enhances HSC growth and selectively mediates TGFβ-induced HSC activation. These findings provide new insights into the development of more effective treatments for liver fibrosis.}, }
@article {pmid40022449, year = {2025}, author = {Wallace, KA and Gerstenberg, TL and Ennis, CL and Perez-Bermejo, JA and Partridge, JR and Bandoro, C and Matern, WM and Andreoletti, G and Krassovsky, K and Kabir, S and Lalisan, CD and Churi, AR and Chew, GM and Corbo, L and Vincelette, JE and Klasson, TD and Silva, BJ and Strukov, YG and Quejarro, BJ and Hill, KA and Treusch, S and Grogan, JL and Dever, DP and Porteus, MH and Wienert, B}, title = {A differentiated β-globin gene replacement strategy uses heterologous introns to restore physiological expression.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {33}, number = {4}, pages = {1407-1419}, pmid = {40022449}, issn = {1525-0024}, mesh = {*beta-Globins/genetics/metabolism ; *Introns ; Humans ; CRISPR-Cas Systems ; *beta-Thalassemia/genetics/therapy ; Animals ; *Anemia, Sickle Cell/genetics/therapy ; Mice ; Genetic Therapy/methods ; Gene Editing ; Recombinational DNA Repair ; }, abstract = {β-Hemoglobinopathies are common monogenic disorders. In sickle cell disease (SCD), a single mutation in the β-globin (HBB) gene results in dysfunctional hemoglobin protein, while in β-thalassemia, over 300 mutations distributed across the gene reduce β-globin levels and cause severe anemia. Genetic engineering replacing the whole HBB gene through homology-directed repair (HDR) is an ideal strategy to restore a benign genotype and rescue HBB expression for most genotypes. However, this is technically challenging because (1) the insert must not be homologous to the endogenous gene and (2) synonymous codon-optimized, intron-less sequences may not reconstitute adequate β-globin levels. Here, we developed an HBB gene replacement strategy using CRISPR-Cas9 that successfully addresses these challenges. We determined that a DNA donor containing a diverged HBB coding sequence and heterologous introns to avoid sequence homology provides proper physiological expression. We identified a DNA donor that uses truncated γ-globin introns, results in 34% HDR, and rescues β-globin expression in in vitro models of SCD and β-thalassemia in hematopoietic stem and progenitor cells (HSPCs). Furthermore, while HDR allele frequency dropped in vivo, it was maintained at ∼15%, demonstrating editing of long-term repopulating HSPCs. In summary, our HBB gene replacement strategy offers a differentiated approach by restoring naturally regulated adult hemoglobin expression.}, }
@article {pmid40021632, year = {2025}, author = {Gao, S and Weng, B and Wich, D and Power, L and Chen, M and Guan, H and Ye, Z and Xu, C and Xu, Q}, title = {Improving adenine base editing precision by enlarging the recognition domain of CRISPR-Cas9.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {2081}, pmid = {40021632}, issn = {2041-1723}, support = {UG3 TR002636/TR/NCATS NIH HHS/United States ; }, mesh = {*Gene Editing/methods ; *Adenine/metabolism ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism/chemistry ; Streptococcus pyogenes/genetics/enzymology ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Humans ; Protein Domains ; HEK293 Cells ; Aminohydrolases ; }, abstract = {Domain expansion contributes to diversification of RNA-guided-endonucleases including Cas9. However, it remains unclear how REC domain expansion could benefit Cas9. In this study, we identify an insertion spot that is compatible with large REC insertion and succeeds in enlarging the non-catalytic REC domain of Streptococcus pyogenes Cas9. The natural-evolution-like giant SpCas9 (GS-Cas9) is created and shows substantially improved editing precision. We further discover that enlarging the REC domain could enable regulation of the N-terminal adenine deaminase TadA8e tethered to the Cas9 scaffold, which contributes to substantially reducing unexpected editing and improving the precision of the adenine base editor ABE8e. We provide proof of concept for evolution-inspired expansion of Cas9 and offer an alternative solution for optimizing gene editors. Our study also indicates a vast potential for engineering the topological malleability of RNA-guided endonucleases and base editors.}, }
@article {pmid40021494, year = {2025}, author = {Sajjad, MW and Imran, I and Muzamil, F and Naqvi, RZ and Amin, I}, title = {AZD7648 (DNA-PKcs inhibitor): a two-edged sword for editing genomes.}, journal = {Functional &amp; integrative genomics}, volume = {25}, number = {1}, pages = {49}, pmid = {40021494}, issn = {1438-7948}, mesh = {*Gene Editing/methods ; Humans ; *DNA-Activated Protein Kinase/antagonists &amp; inhibitors ; DNA End-Joining Repair/drug effects ; CRISPR-Cas Systems ; Animals ; Recombinational DNA Repair/drug effects ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) has been the most practical technique in genome editing for the last decade. Its molecular mechanism includes steps that occur in a sequence, starting from a break in a double strand to repair. After a double-strand break in the DNA strand, the repairing of DNA done via Homology-Directed Repair (HDR) is considered important in different organisms as it is ideal for precise genome editing and the reduction of unintended mutations. Still, it is mostly dominated by the Non-Homologous End Joining (NHEJ) pathway. A recent study by Cullot et al. published in Nature Biotechnology showed interesting features of AZD7648 (a DNA-PKcs inhibitor) that increase the probability of HDR event while DNA repairing (Cullot et al. 2024).}, }
@article {pmid40020637, year = {2025}, author = {Deng, A and Mao, Z and Jin, X and Lv, W and Huang, L and Zhong, H and Wang, S and Shi, Y and Zhou, T and Zhao, J and Huang, Q and Luo, X and Ma, L and Zou, H and Fu, R and Huang, G}, title = {ID-CRISPR: A CRISPR/Cas12a platform for label-free and sensitive detection of rare mutant alleles using self-interference DNA hydrogel reporter.}, journal = {Biosensors &amp; bioelectronics}, volume = {278}, number = {}, pages = {117309}, doi = {10.1016/j.bios.2025.117309}, pmid = {40020637}, issn = {1873-4235}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Biosensing Techniques/methods ; *Hydrogels/chemistry ; Polymorphism, Single Nucleotide/genetics ; DNA/chemistry/genetics ; Alleles ; Limit of Detection ; ErbB Receptors/genetics ; DNA, Single-Stranded/chemistry/genetics ; Interferometry/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Accurate and sensitive detection of single nucleotide variants (SNVs) is paramount for cancer diagnosis and treatment. The CRISPR/Cas12a system shows promise for SNV detection due to its high sensitivity and single-base specificity. However, most CRISPR/Cas12a-based methods rely on F/Q-labeled single-stranded DNA (ssDNA) reporters, which are susceptible to fluorescence fluctuations, thereby reducing accuracy. To address these limitations, researchers have proposed using DNA hydrogels as signal transducers in CRISPR/Cas12a systems. Yet, the encapsulation of indicators into DNA hydrogels introduces additional instability, which could compromise both detection sensitivity and linearity. In this study, we integrated hyperspectral interferometry into a DNA hydrogel-based CRISPR/Cas12a detection platform (ID-CRISPR) to achieve sensitive label-free SNV detection. Using EGFR L858R SNV as a model target, we demonstrated that ID-CRISPR can detect mutant allele frequencies (MAFs) as low as 0.1% with a limit of detection (LOD) of 5 aM, while also showing its potential for quantifying SNV abundance. Its clinical utility was confirmed through analysis of lung tumor samples, with results consistent with sequencing data. Therefore, ID-CRISPR provides a sensitive, label-free, and user-friendly platform for SNV detection, offering new insights into combining optical sensing with DNA hydrogel technology in CRISPR/Cas assays.}, }
@article {pmid40020602, year = {2025}, author = {Patra, N and Barker, GC and Maiti, MK}, title = {Knockout of fatty acid elongase1 homeoalleles in amphidiploid Brassica juncea leads to undetectable erucic acid in seed oil.}, journal = {Plant physiology and biochemistry : PPB}, volume = {222}, number = {}, pages = {109679}, doi = {10.1016/j.plaphy.2025.109679}, pmid = {40020602}, issn = {1873-2690}, mesh = {*Mustard Plant/genetics/metabolism/enzymology ; *Erucic Acids/metabolism ; *Fatty Acid Elongases/genetics/metabolism ; *Seeds/metabolism/genetics ; *Plant Oils/metabolism ; Gene Knockout Techniques ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing ; }, abstract = {Indian mustard (Brassica juncea L.) is a major oilseed crop with considerable economic and nutritional importance globally. While its seed oil offers valuable dietary benefits due to a balanced ratio of human essential fatty acids, the traditional high oil-yielding varieties contain an elevated level of erucic acid (EA, C22:1) associated with adverse health effects. Therefore, developing low erucic acid (LEA) mustard cultivars is crucial for broader utilization and consumer safety. In this study, CRISPR/Cas9 genome editing tool was employed to disrupt the fatty acid elongase1 (FAE1) gene that encodes a key enzyme in EA biosynthesis in two high erucic acid (HEA) B. juncea cultivars, PCR7 (∼39% EA) and JD6 (∼45% EA). Targeted knockout (KO) of BjFAE1 homeoalleles (BjFAE1.1 and BjFAE1.2) in this amphidiploid plant species using CRISPR/Cas9 constructs, each carrying two guide RNAs led to generation of single (either fae1.1 or fae1.2) and double (fae1.1fae1.2) mutants. Best performing homozygous fae1.1fae1.2 KO lines showed a near-complete elimination of EA in both the cultivars (<0.5% in PCR7, undetectable in JD6) with a marked increase in nutritionally beneficial oleic acid (from ∼18% to ∼32% in PCR7, from ∼9% to ∼38% in JD6). Moreover, the content of essential fatty acids also increased substantially [linoleic acid (C18:2) 1.9-fold in PCR7 and 2.1-fold in JD6; linolenic acid (C18:3) 2.5-fold in PCR7 and 1.4-fold in JD6], suggesting rerouting of carbon flux from EA biosynthesis. Importantly, these LEA lines retained key agronomic traits like plant seed yield and oil content, matching the productivity of the unedited control elite cultivars. Our findings underscore the effectiveness of CRISPR/Cas9 technology for editing B. juncea genome, developing plant lines producing LEA seed oil with improved nutritional quality and broadening the utility of this important oilseed crop for food and non-food applications.}, }
@article {pmid40019808, year = {2025}, author = {Cook, AL and Moyer, AL and Boxer, L and Norris, AL}, title = {Re: Novel Off-Targeting Events Identified after Genome-Wide Analysis of CRISPR-Cas Edited Pigs.}, journal = {The CRISPR journal}, volume = {8}, number = {2}, pages = {74-76}, doi = {10.1089/crispr.2024.0091}, pmid = {40019808}, issn = {2573-1602}, }
@article {pmid40019805, year = {2025}, author = {Li, Q and Yu, H and Du, S and Li, Q}, title = {Optimizing Genome Editing in Mollusks (Crassostrea gigas) in Vitro Validation of sgRNA and Identifying Key Factors Influencing Efficiency.}, journal = {The CRISPR journal}, volume = {8}, number = {3}, pages = {205-215}, doi = {10.1089/crispr.2024.0086}, pmid = {40019805}, issn = {2573-1602}, mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Crassostrea/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Aquaculture ; Gene Knockout Techniques ; CRISPR-Associated Protein 9/genetics ; }, abstract = {CRISPR-Cas9 genome editing holds tremendous potential for accelerating genetic improvements in aquaculture. The success of the CRISPR-Cas9 system relies on the specificity and efficiency of engineered single-guide RNAs (sgRNAs). In this study, we optimized an in vitro validation protocol for sgRNAs to streamline the gene editing process, capitalizing on the limited breeding season of the Pacific oyster (Crassostrea gigas). We evaluated the efficiency of 11 sgRNAs targeting four genes both in vitro and in vivo in C. gigas. In addition, we found that Cas9 protein differs from Cas9 mRNA in gene editing efficiency at various stages of early development. Cas9 protein proved particular efficacy in achieving early and efficient gene knockout, functioning effectively during the first cell division and facilitating biallelic gene knockouts. Statistical analysis showed that in the protein group, the biallelic editing frequency ranged from 12.5% to 57.8%, and the overall editing frequency reached as high as 75-90.6%. The mRNA group exhibited a biallelic editing frequency of 3.1-14.0% and the overall editing frequency spanning 65.6-78.1%. Contrary to expectations, low-temperature incubation (20°C) of oyster embryos prolonged the time window for the first cell division but did not improve gene editing efficiency, likely due to the high temperature sensitivity of Cas9 enzyme activity. Together, this study provides a comprehensive analysis of factors affecting the efficiency of CRISPR-Cas9 gene editing in C. gigas, providing a robust framework for future gene editing endeavors in mollusks and other marine invertebrates.}, }
@article {pmid40019800, year = {2025}, author = {Major, RM and Mills, CA and Xing, L and Krantz, JL and Wolter, JM and Zylka, MJ}, title = {Exploring the Cytoplasmic Retention of CRISPR-Cas9 in Eukaryotic Cells: The Role of Nuclear Localization Signals and Ribosomal Interactions.}, journal = {The CRISPR journal}, volume = {8}, number = {2}, pages = {120-136}, doi = {10.1089/crispr.2024.0074}, pmid = {40019800}, issn = {2573-1602}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; *Ribosomes/metabolism/genetics ; *Cytoplasm/metabolism/genetics ; Cell Nucleus/metabolism/genetics ; *Nuclear Localization Signals/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics ; *Eukaryotic Cells/metabolism ; Kelch-Like ECH-Associated Protein 1/metabolism/genetics ; Gene Editing/methods ; Neurons/metabolism ; }, abstract = {Cas9 must be localized to the nucleus to access the genome of mammalian cells. For most proteins, adding a single nuclear localization signal (NLS) is sufficient to promote nuclear entry. However, Cas9 nuclear entry appears to be inefficient as multiple NLSs are typically added to Cas9. Here, we found that three different Cas9 variants interact with the ribosome in HEK293T cells, and that this interaction is RNA mediated. Following immunoprecipitation-mass spectrometry of cytoplasmic-localized Cas9-0NLS and nuclear-localized Cas9-4NLS constructs, we identified novel Cas9 interactors in postmitotic neurons, including KEAP1 and additional ribosomal subunits, the latter were enriched in Cas9-0NLS samples. Collectively, our results suggest that Cas9 is sequestered in the cytoplasm of mammalian cells, in part, via interaction with the ribosome. Increasing the number of NLSs on Cas9 and/or increasing the amount of cytoplasmic guide RNA has the potential to outcompete ribosomal RNA binding and promote efficient nuclear localization of CRISPR-Cas9 variants.}, }
@article {pmid40019770, year = {2025}, author = {Mao, X and Lu, Y and Gao, Z and Zhong, J and Xiao, A and Lin, J and Hu, J and Shu, B and Zhang, C}, title = {Modular Microfluidic Sensor Integrating Nucleic Acid Extraction, CRISPR/Cas13a, and Electrochemiluminescence for Multichannel RNA Detection.}, journal = {Analytical chemistry}, volume = {97}, number = {9}, pages = {5085-5092}, doi = {10.1021/acs.analchem.4c06197}, pmid = {40019770}, issn = {1520-6882}, mesh = {*Electrochemical Techniques ; *CRISPR-Cas Systems ; *Luminescent Measurements ; *Biosensing Techniques ; *RNA/analysis ; *Microfluidic Analytical Techniques/instrumentation ; Staphylococcus aureus/genetics ; *RNA, Bacterial/analysis/genetics ; Escherichia coli/genetics ; Humans ; *Lab-On-A-Chip Devices ; }, abstract = {Rapid and accurate screening of pathogens is crucial for disease detection. Here, a modular microfluidic sensor has been constructed for RNA detection, with integrated nucleic acid extraction, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a reaction, and electrochemiluminescence (ECL) detection. The sensor consists of nucleic acid processing and detection modules. The nucleic acid processing module is used for nucleic acid extraction, RNA distribution, and the CRISPR/Cas reaction. Specifically, immiscible filtration assisted by surface tension is employed for nucleic acid extraction, significantly reducing the extraction time. Magnetic force is utilized for RNA distribution and transportation, minimizing the need for microstructures, such as microvalves and micropumps. Multichannel CRISPR/Cas13a reactions enable biological recognition, signal amplification, and multiplex detection. The fiber material-based detection module controls fluid flow and performs dry chemistry-based ECL detection. A novel multichannel closed bipolar electrode-based ECL (MCBPE-ECL) system is employed, with simplified experimental operations and enhanced sensitivity. Together, the multichannel CRISPR/Cas13a reactions and MCBPE-ECL enable the sensor's multiplexed detection. Under optimized conditions, the sensor can complete RNA extraction and detection in 30 min, with a detection limit of 0.372 fM for Escherichia coli 16S rRNA. Furthermore, in human blood samples, the detection limit for E. coli is 63.8 cfu/mL. Notably, the sensor can simultaneously determine the growth curves of single colonies of E. coli and Staphylococcus aureus strains in the same culture medium, demonstrating its multiplexed detection capability.}, }
@article {pmid40017424, year = {2025}, author = {Song, Y and Park, H and Thirumalaraju, P and Kovilakath, N and Hardie, JM and Bigdeli, A and Bai, Y and Chang, S and Yoo, J and Kanakasabapathy, MK and Kim, S and Chun, J and Chen, H and Li, JZ and Tsibris, AM and Kuritzkes, DR and Shafiee, H}, title = {Deactivated Cas9-Engineered Magnetic Micromotors toward a Point-of-Care Digital Viral RNA Assay.}, journal = {ACS nano}, volume = {19}, number = {9}, pages = {8646-8660}, doi = {10.1021/acsnano.4c14913}, pmid = {40017424}, issn = {1936-086X}, support = {R01 AI138800/AI/NIAID NIH HHS/United States ; R01 EB033866/EB/NIBIB NIH HHS/United States ; R33 AI140489/AI/NIAID NIH HHS/United States ; U54 HL119145/HL/NHLBI NIH HHS/United States ; R01 HD115677/HD/NICHD NIH HHS/United States ; }, mesh = {*RNA, Viral/genetics/analysis ; *HIV-1/genetics/isolation &amp; purification ; Humans ; Nucleic Acid Amplification Techniques/methods ; *Point-of-Care Systems ; *CRISPR-Associated Protein 9/metabolism/chemistry ; *Molecular Diagnostic Techniques/methods ; }, abstract = {Digital nucleic acid assays, known for their high sensitivity and specificity, typically rely on fluorescent readouts and expensive and complex nanowell manufacturing, which constrain their broader use in point-of-care (POC) application. Here, we introduce an alternative digital molecular diagnostics, termed dCRISTOR, by seamlessly integrating deactivated Cas9 (dCas9)-engineered micromotors, extraction-free loop-mediated isothermal amplification (LAMP), low-cost bright field microscopy, and deep learning-enabled image processing. The micromotor, composed of a polystyrene sphere attached to a magnetic bead, incorporates a dCas9 ribonucleoprotein complex. The presence of human immunodeficiency virus-1 (HIV-1) RNA in a sample results in the formation of large-sized amplicons that can be specifically captured by the micromotors, reducing their velocity induced by an external magnetic field. The micromotor is propelled by an external magnetic field, which eliminates the need for chemical fuels, reducing system complexity, and allowing for precise control over micromotor movement, enhancing accuracy and reliability. A convolutional neural network classification-based multiobject tracking algorithm, CNN-MOT, accurately measures the change in micromotor motion, facilitating the binary digital assay format ("1" or "0") for simplified result interpretation without user bias. Incorporating an extraction-free LAMP assay streamlines the dCRISTOR workflow, enabling qualitative HIV-1 detection in spiked plasma (n = 21) that demonstrates 100% sensitivity and specificity and achieves a limit of detection (LOD) of 0.96 copies/μL. The assay also achieved 100% correlation with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in clinical patient samples (n = 9). The dCRISTOR assay, a label-free digital nucleic acid testing system that eliminates the need for fluorescence readouts, absorbance measurements, or expensive manufacturing processes, represents a substantial advancement in digital viral RNA diagnostics.}, }
@article {pmid40017406, year = {2025}, author = {Liu, G}, title = {Advancing CRISPR/Cas Biosensing with Integrated Devices.}, journal = {ACS sensors}, volume = {10}, number = {2}, pages = {575-576}, doi = {10.1021/acssensors.5c00330}, pmid = {40017406}, issn = {2379-3694}, }
@article {pmid40016920, year = {2025}, author = {Zhang, P and Feng, S and Liu, F and Han, S and Fan, T and Chen, H and Dong, X and Wang, X and Qin, Y and Chen, Y and Jiang, Y}, title = {Cascaded Strand Displacement Amplification and CRISPR/Cas12a Aptasensor Utilizing MoS2 Nanoflowers for Colorectal Cancer Biomarker Porphyromonas gingivalis Detection.}, journal = {Analytical chemistry}, volume = {97}, number = {9}, pages = {4932-4944}, doi = {10.1021/acs.analchem.4c05014}, pmid = {40016920}, issn = {1520-6882}, mesh = {*Porphyromonas gingivalis/isolation &amp; purification/genetics ; *Colorectal Neoplasms/diagnosis/microbiology ; Humans ; *CRISPR-Cas Systems ; *Disulfides/chemistry ; *Molybdenum/chemistry ; *Biomarkers, Tumor/analysis ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry/genetics ; *Nucleic Acid Amplification Techniques/methods ; Feces/microbiology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Colorectal cancer (CRC) ranks as the third most prevalent cancer globally, both in terms of diagnoses and cancer-related mortality. Increasing evidence suggests that an imbalance in intestinal flora can contribute to the progression of CRC, and fecal microbiota may serve as potential biomarkers for its screening and diagnosis. Notably, Porphyromonas gingivalis has been identified in the malignant tissues and feces of CRC patients, establishing it as a significant biomarker for early screening, diagnosis, and prognostic assessment of CRC. Current methods for detecting P. gingivalis face numerous challenges, including high costs, complex procedures, and lengthy implementation times. Therefore, developing rapid, highly specific, and sensitive detection methods for P. gingivalis is of great importance. In this study, we utilized the whole-bacterium systematic evolution of ligands by exponential enrichment method to identify highly specific and high-affinity aptamers targeting P. gingivalis through 15 selection cycles. Subsequently, we developed an aptasensor driven by MoS2 nanoflowers, which integrates strand displacement amplification and CRISPR/Cas12a double amplification for sensitive detection of P. gingivalis, achieving a limit of detection of 10 CFU/mL. Using this aptasensor, we evaluated the abundance of P. gingivalis in clinical fecal samples and observed significantly higher levels in the feces of CRC patients compared to healthy individuals, corroborating the results obtained from quantitative polymerase chain reaction. In summary, we developed a highly specific and sensitive aptasensor for the first time, representing a promising new approach for the identification of P. gingivalis, with significant potential for CRC screening and diagnosis.}, }
@article {pmid40015840, year = {2025}, author = {Chen, S and Ruan, Y and Li, Z and Zhou, C and Chen, Q and Zhou, X and Zhang, Y and Yang, C and Pan, H}, title = {CRISPR/Cas9-mediated editing of the melanization gene ebony in the 28-spotted ladybeetle, Henosepilachna vigintioctopunctata.}, journal = {Pesticide biochemistry and physiology}, volume = {208}, number = {}, pages = {106231}, doi = {10.1016/j.pestbp.2024.106231}, pmid = {40015840}, issn = {1095-9939}, mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; *Melanins/metabolism/genetics ; *Coleoptera/genetics/metabolism ; *Insect Proteins/genetics/metabolism ; Pigmentation/genetics ; RNA Interference ; }, abstract = {The melanization process, which is essential for the proper functioning of the cuticle, has been extensively investigated for its enzymatic roles and physiological effects. Henosepilachna vigintioctopunctata, a significant pest species, presents considerable economic threats. However, due to the variable efficiency of RNA interference for genetic manipulation, establishing a CRISPR/Cas9 system is crucial for providing a more precise and reliable method for functional genomics in this non-model insect. In this study, we first utilized RNAi to investigate Hvebony, which encodes N-β-alanyldopamine, a critical compound in cuticle melanization. Subsequently, we introduced CRISPR/Cas9 for the first time in H. vigintioctopunctata. RNAi experiments revealed that knockdown of Hvebony resulted in abnormal melanin accumulation and low mortality rates, indicating its involvement in cuticle tanning. A novel CRISPR/Cas9 workflow was established, successfully resulting in the knocking out of Hvebony and the creation of a stable mutant strain characterized by dark pigmentation and low fitness costs. This study establishes Hvebony as a promising molecular marker for genetic studies in H. vigintioctopunctata. Moreover, it can be utilized in the development of genome editing control strategies and for analyses of gene function in H. vigintioctopunctata.}, }
@article {pmid40015341, year = {2025}, author = {Alok, A and Chauhan, H and Rout, B and Pandey, A and Singh, K}, title = {CRISPR/dCas9-KRAB mediated transcriptional suppression of NtbHLH47 enhances tolerance to iron stress and modulates iron content in tobacco.}, journal = {Plant science : an international journal of experimental plant biology}, volume = {354}, number = {}, pages = {112449}, doi = {10.1016/j.plantsci.2025.112449}, pmid = {40015341}, issn = {1873-2259}, mesh = {*Iron/metabolism ; *Nicotiana/genetics/metabolism/physiology ; Plants, Genetically Modified/metabolism ; *CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; Homeostasis ; }, abstract = {Iron homeostasis is a multifaceted regulatory process that needs to be studied to elucidate iron distribution, uptake, and storage in plants. NtbHLH47, a homologue to AtbHLH47, is a negative regulator of iron. The current study deploys CRISPR interference-dCas9-KRAB (Krüppel-associated box) in the transcriptional suppression of NtbHLH47 and its effect on iron uptake by plants. The pHSN6I01 harbouring dCas9-KRAB and gRNA targeting NtbHHLH47 was constructed. Four gRNAs were designed, G1, G2, G3, and G4, located at + 19, + 111, + 232, and + 335 bp upstream from the ATG start codon in the promoter region of NtbHLH47. The NtbHLH47 was repressed in the developed transgenic lines of tobacco and the qRT-PCR analysis showed that target sites G1 and G2 suppressed NtbHLH47 effectively. The transgenic pHSN6I01 +G1 plants were tolerant to the elevated levels of iron, copper, zinc, and magnesium. The root Ferric chelate reductase activity of pHSN6I01 +G1 lines was reduced against wild type. The Perl staining showed high iron content in the roots of the pHSN6I01 +G1 plants. ICP-MS analysis showed increased Fe content in the roots of pHSN6I01 +G1 line suggesting that NtbHLH47 modulates it. The expression of NtbHLH38, NtbHLH100, NtbHLH101, and NtFIT was found to be upregulated in the pHSN6I01 +G1 line. This is the first report of using CRISPRi based on dCas9-KRAB in tobacco and its application in the functional validation of a gene. Using this, NtbHLH47 was transcriptionally suppressed and the generated lines expressed increased levels of iron in the roots of N. tabacum and gave insight in the iron homeostasis.}, }
@article {pmid40015249, year = {2025}, author = {Dederer, HG}, title = {Human health and genetic technology.}, journal = {Trends in biotechnology}, volume = {43}, number = {3}, pages = {522-532}, doi = {10.1016/j.tibtech.2024.12.006}, pmid = {40015249}, issn = {1879-3096}, mesh = {Humans ; Genetic Therapy ; *Organisms, Genetically Modified ; }, abstract = {The 1975 Asilomar conference contributed to the misperception that recombinant DNA (rDNA) technology is inherently risky to human health and the environment. It thus paved the way toward process-based regulation of genetically modified organisms (GMOs), such as in the EU. Initially, this regulatory approach obstructed technological uses of rDNA related to human health. However, regulators gradually softened the rules applicable to laboratories or industrial facilities. This encouraged R&amp;D and production of pharmaceuticals derived from GMOs. Nevertheless, administering pharmaceuticals containing GMOs to patients may still be confronted with burdensome process-based GMO law on the deliberate release of GMOs into the environment. On the other hand, pharmaceutical law may need to be updated regarding, for example, novel gene therapies or xenotransplantation.}, }
@article {pmid40014809, year = {2025}, author = {Li, M and Lin, Y and Cheng, Q and Wei, T}, title = {Prime Editing: A Revolutionary Technology for Precise Treatment of Genetic Disorders.}, journal = {Cell proliferation}, volume = {58}, number = {4}, pages = {e13808}, pmid = {40014809}, issn = {1365-2184}, support = {2023ZD0500601//Noncommunicable Chronic Diseases-National Science and Technology Major Project/ ; 2023IOZ0204//Initiative Scientific Research Program, Institute of Zoology, Chinese Academy of Sciences/ ; 22JCZXJC00030//Beijing-Tianjin-Hebei Basic Research Cooperation Project/ ; NCTIB2023XB02001//the "Open Competition to Select the Best Candidates" Key Technology Program for Nucleic Acid Drugs of NCTIB/ ; //the Fundamental Research Funds for the Central Universities, Peking University/ ; 2023000CC0040//the Beijing Life Science Academy/ ; 2023M740101//China Postdoctoral Science Foundation/ ; }, mesh = {Humans ; *Gene Editing/methods ; *Genetic Diseases, Inborn/therapy/genetics ; *Genetic Therapy/methods ; Animals ; CRISPR-Cas Systems/genetics ; }, abstract = {Genetic diseases have long posed significant challenges, with limited breakthroughs in treatment. Recent advances in gene editing technologies offer new possibilities in gene therapy for the treatment of inherited disorders. However, traditional gene editing methods have limitations that hinder their potential for clinical use, such as limited editing capabilities and the production of unintended byproducts. To overcome these limitations, prime editing (PE) has been developed as a powerful tool for precise and efficient genome modification. In this review, we provide an overview of the latest advancements in PE and its potential applications in the treatment of inherited disorders. Furthermore, we examine the current delivery vehicles employed for delivering PE systems in vitro and in vivo, and analyze their respective benefits and limitations. Ultimately, we discuss the challenges that need to be addressed to fully unlock the potential of PE for the remission or cure of genetic diseases.}, }
@article {pmid40014690, year = {2025}, author = {Faure, G and Saito, M and Wilkinson, ME and Quinones-Olvera, N and Xu, P and Flam-Shepherd, D and Kim, S and Reddy, N and Zhu, S and Evgeniou, L and Koonin, EV and Macrae, RK and Zhang, F}, title = {TIGR-Tas: A family of modular RNA-guided DNA-targeting systems in prokaryotes and their viruses.}, journal = {Science (New York, N.Y.)}, volume = {388}, number = {6746}, pages = {eadv9789}, pmid = {40014690}, issn = {1095-9203}, support = {/HHMI/Howard Hughes Medical Institute/United States ; }, mesh = {Humans ; *Bacterial Proteins/chemistry/genetics/metabolism ; *Bacteriophages/genetics ; *CRISPR-Cas Systems ; DNA/metabolism ; Protein Domains ; *RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Clostridiales/genetics/virology ; *Gene Editing/methods ; }, abstract = {RNA-guided systems provide remarkable versatility, enabling diverse biological functions. Through iterative structural and sequence homology-based mining starting with a guide RNA-interaction domain of Cas9, we identified a family of RNA-guided DNA-targeting proteins in phage and parasitic bacteria. Each system consists of a tandem interspaced guide RNA (TIGR) array and a TIGR-associated (Tas) protein containing a nucleolar protein (Nop) domain, sometimes fused to HNH (TasH)- or RuvC (TasR)-nuclease domains. We show that TIGR arrays are processed into 36-nucleotide RNAs (tigRNAs) that direct sequence-specific DNA binding through a tandem-spacer targeting mechanism. TasR can be reprogrammed for precise DNA cleavage, including in human cells. The structure of TasR reveals striking similarities to box C/D small nucleolar ribonucleoproteins and IS110 RNA-guided transposases, providing insights into the evolution of diverse RNA-guided systems.}, }
@article {pmid40014422, year = {2025}, author = {Bryant, EE and Gong, D and Guo, C and Garces, F and Hubert, R and Chen, I}, title = {An Arrayed CRISPR Screen Identifies Knockout Combinations Improving Antibody Productivity in HEK293 Cells.}, journal = {ACS synthetic biology}, volume = {14}, number = {3}, pages = {855-866}, doi = {10.1021/acssynbio.4c00772}, pmid = {40014422}, issn = {2161-5063}, mesh = {Humans ; HEK293 Cells ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; *Antibodies/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {Mammalian cells are used to express complex biologics, such as multispecific antibodies. While multispecifics enable promising new strategies for treating human disease, their production at high expression titer and purity can be challenging. To understand how cells respond to antibody and multispecific expression, five molecules were selected for bulk RNA sequencing (RNA-seq) early after the transfection of a human embryonic kidney 293 (HEK293) host. All five molecules shared a differential expression signature of secretory and protein folding stresses, but this signature was stronger for molecules with low titer. We then designed an arrayed CRISPR knockout screen of 206 differentially expressed target genes and 223 literature-motivated targets to identify knockouts that affect antibody productivity. Eight novel knockout targets were identified that increased expression titers by 20-80%. Notably, seven of these top eight hits were from the differentially expressed set of candidate-gene knockouts. The top knockout target, HIST2H3C, showed evidence for additivity with five other hits, including a knockout combination that increased the titer of a difficult-to-express antibody by up to 100%. Findings for both HIST2H3C and INHBE knockout targets generalized to an alternate HEK293 host expressing an additional antibody and a multispecific host with no meaningful impact on product purity. Thus, we propose HIST2H3C and INHBE disruption as a promising and novel strategy for host-cell engineering to improve antibody and multispecific productivity.}, }
@article {pmid40014250, year = {2025}, author = {Bolideei, M and Barzigar, R and Gahrouei, RB and Mohebbi, E and Haider, KH and Paul, S and Paul, MK and Mehran, MJ}, title = {Applications of Gene Editing and Nanotechnology in Stem Cell-Based Therapies for Human Diseases.}, journal = {Stem cell reviews and reports}, volume = {21}, number = {4}, pages = {905-934}, pmid = {40014250}, issn = {2629-3277}, mesh = {Humans ; *Gene Editing/methods ; *Nanotechnology/methods ; *Stem Cell Transplantation/methods ; CRISPR-Cas Systems/genetics ; Regenerative Medicine/methods ; Animals ; *Stem Cells/cytology/metabolism ; Genetic Therapy ; }, abstract = {Stem cell research is a dynamic and fast-advancing discipline with great promise for the treatment of diverse human disorders. The incorporation of gene editing technologies, including ZFNs, TALENs, and the CRISPR/Cas system, in conjunction with progress in nanotechnology, is fundamentally transforming stem cell therapy and research. These innovations not only provide a glimmer of optimism for patients and healthcare practitioners but also possess the capacity to radically reshape medical treatment paradigms. Gene editing and nanotechnology synergistically enhance stem cell-based therapies' precision, efficiency, and applicability, offering transformative potential for treating complex diseases and advancing regenerative medicine. Nevertheless, it is important to acknowledge that these technologies also give rise to ethical considerations and possible hazards, such as inadvertent genetic modifications and the development of genetically modified organisms, therefore creating a new age of designer infants. This review emphasizes the crucial significance of gene editing technologies and nanotechnology in the progress of stem cell treatments, particularly for degenerative pathologies and injuries. It emphasizes their capacity to restructure and comprehensively revolutionize medical treatment paradigms, providing fresh hope and optimism for patients and healthcare practitioners.}, }
@article {pmid40014102, year = {2025}, author = {Jiang, Y and Xue, R and Chang, Y and Cao, D and Liu, B and Li, Y}, title = {The knockout of Gγ subunit HvGS3 by CRISPR/Cas9 gene editing improves the lodging resistance of barley through dwarfing and stem strengthening.}, journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik}, volume = {138}, number = {3}, pages = {61}, pmid = {40014102}, issn = {1432-2242}, support = {2023-NK-A3//Geomatics Technology and Application Key Laboratory of Qinghai Province/ ; 077GJHZ2023028GC//Global Common Challenges Project of International Partnership Program of Chinese Academy of Sciences/ ; 2023-1-1//Qinghai Science and Technology Department/ ; }, mesh = {*Hordeum/genetics/growth &amp; development ; *CRISPR-Cas Systems ; *Gene Editing ; *Plant Proteins/genetics/metabolism ; *Plant Stems/growth &amp; development/genetics ; Gene Expression Regulation, Plant ; Gene Knockout Techniques ; Gibberellins/metabolism ; Plants, Genetically Modified ; Lignin/biosynthesis ; Phenotype ; }, abstract = {Gγ subunits participate in multiple biological processes, but their biological function in barley is unknown. Here, CRISPR/Cas9 gene editing was used to knockout HvGS3 in barley. The height of hvgs3 plants were reduced by 37.8 ~ 43.1% compared to wild type, and the culm lodging resistance index (CLRI) of the second internode of stems was increased by 76.6%. The decrease in cell length of the second internode was similar to its node length. The shorter cells may be the main reason for the declines in the internode length and plant height. The number and area of vascular bundles, the epidermal thickness, and the mechanical tissue thickness were significantly higher in hvgs3 due to the higher lignin content. Transcriptome analysis showed higher expression of structural genes related to lignin biosynthesis. Gibberellin (GA) biosynthesis was suppressed through the down-regulation of the GA3ox gene, and the application of gibberellin restored the plant height of hvgs3, indicating that plant height was altered by hindering gibberellin biosynthesis. These results shed new light on the functions of the Gγ subunit GS3 and provide a resource for breeding new lodging-resistant barley cultivars.}, }
@article {pmid40014012, year = {2025}, author = {Dort, EN and Feau, N and Hamelin, RC}, title = {Novel application of ribonucleoprotein-mediated CRISPR-Cas9 gene editing in plant pathogenic oomycete species.}, journal = {Microbiology spectrum}, volume = {13}, number = {4}, pages = {e0301224}, pmid = {40014012}, issn = {2165-0497}, support = {10106//Genome Canada (GC)/ ; CGS-D//Canadian Government | Natural Sciences and Engineering Research Council of Canada (NSERC)/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Phytophthora/genetics/pathogenicity/drug effects ; Plant Diseases/microbiology/parasitology ; *Ribonucleoproteins/genetics/metabolism ; Virulence/genetics ; Fungicides, Industrial/pharmacology ; Oomycetes/genetics ; Hydrocarbons, Fluorinated ; Pyrazoles ; }, abstract = {CRISPR-Cas9 gene editing has become an important tool for the study of plant pathogens, allowing researchers to functionally characterize specific genes involved in phytopathogenicity, virulence, and fungicide resistance. Protocols for CRISPR-Cas9 gene editing have already been developed for Phytophthoras, an important group of oomycete plant pathogens; however, these efforts have exclusively focused on agricultural pathosystems, with research lacking for forest pathosystems. We sought to develop CRISPR-Cas9 gene editing in two forest pathogenic Phytophthoras, Phytophthora cactorum and P. ramorum, using a plasmid-ribonucleoprotein (RNP) co-transformation approach. Our gene target in both species was the ortholog of PcORP1, which encodes an oxysterol-binding protein that is the target of the fungicide oxathiapiprolin in the agricultural pathogen P. capsici. We delivered liposome complexes, each containing plasmid DNA and CRISPR-Cas9 RNPs, to Phytophthora protoplasts using a polyethylene glycol-mediated transformation protocol. We obtained two ORP1 mutants in P. cactorum but were unable to obtain any mutants in P. ramorum. The two P. cactorum mutants exhibited decreased resistance to oxathiapiprolin, as measured by their radial growth relative to wild-type cultures on oxathiapiprolin-supplemented medium. Our results demonstrate the potential for RNP-mediated CRISPR-Cas9 gene editing in P. cactorum and provide a foundation for future optimization of our protocol in other forest pathogenic Phytophthora species.IMPORTANCECRISPR-Cas9 gene editing has become a valuable tool for characterizing the genetics driving virulence and pathogenicity in plant pathogens. CRISPR-Cas9 protocols are now well-established in several Phytophthora species, an oomycete genus with significant economic and ecological impact globally. These protocols, however, have been developed for agricultural Phytophthora pathogens only; CRISPR-Cas9 systems have not yet been developed for any forest pathogenic Phytophthoras. In this study, we sought to establish CRISPR-Cas9 gene editing in two forest Phytophthora pathogens that cause widespread tree mortality: P. cactorum and P. ramorum. We successfully obtained gene mutations in P. cactorum and demonstrated a decrease in fungicide resistance, a trait that could impact the pathogen's ability to cause disease. However, the same protocol did not yield any mutants in P. ramorum. The results of our study will serve as a baseline for the development of CRISPR-Cas9 gene editing in forest Phytophthoras and other oomycetes.}, }
@article {pmid40013946, year = {2025}, author = {Scaramellini, N and Panzieri, DL and Cappellini, MD}, title = {Gene therapy for sickle cell disease and thalassemia.}, journal = {Current opinion in hematology}, volume = {32}, number = {3}, pages = {120-129}, doi = {10.1097/MOH.0000000000000867}, pmid = {40013946}, issn = {1531-7048}, mesh = {Humans ; *Genetic Therapy/methods ; *Anemia, Sickle Cell/therapy/genetics ; *Thalassemia/therapy/genetics ; Gene Editing/methods ; Fetal Hemoglobin/genetics ; Animals ; CRISPR-Cas Systems ; beta-Globins/genetics ; Genetic Vectors ; }, abstract = {PURPOSE OF REVIEW: Thalassemia and sickle cell disease are among the most frequent monogenic hereditary diseases. Access to transfusions, iron chelation therapies and drugs such as hydroxyurea have improved life expectancy and quality of life. However, these diseases still cause significant disability. The first available curative therapy, bone marrow transplantation, is unfortunately not feasible for all patients. Over the past decade, numerous studies have focused on finding new curative therapies, and many clinical trials have evaluated different gene therapy approaches.<br><br>RECENT FINDINGS: The therapeutic targets focus on adding functional copies of the gene encoding &#x3b2;-globin in defective CD34 + cells, mainly using lentiviral vectors directed towards HSCs. More recently, the focus has shifted to inducing fetal hemoglobin production at therapeutic levels or repairing the underlying molecular defect, using novel gene editing techniques involving CRISPR-Cas9, transcription activation-like effector protein nucleases, zinc finger nucleases and base editing. Preclinical and clinical studies now focus on optimizing how gene therapy is performed and delivered to reduce or eliminate myeloablative treatment and its potential adverse events.<br><br>SUMMARY: In this review, we explore the potential to induce fetal hemoglobin production at therapeutic levels or to repair the underlying molecular defect that causes the disease genetically. Here, we review recent gene editing studies that are opening a new era in curative treatment for hemoglobinopathies.}, }
@article {pmid40013512, year = {2025}, author = {Gondalia, N and Quiroz, LF and Lai, L and Singh, AK and Khan, M and Brychkova, G and McKeown, PC and Chatterjee, M and Spillane, C}, title = {Harnessing promoter elements to enhance gene editing in plants: perspectives and advances.}, journal = {Plant biotechnology journal}, volume = {23}, number = {5}, pages = {1375-1395}, pmid = {40013512}, issn = {1467-7652}, mesh = {*Gene Editing/methods ; *Promoter Regions, Genetic/genetics ; Plants, Genetically Modified/genetics ; *Plants/genetics ; Gene Expression Regulation, Plant ; Genome, Plant/genetics ; CRISPR-Cas Systems ; }, abstract = {Genome-edited plants, endowed with climate-smart traits, have been promoted as tools for strengthening resilience against climate change. Successful plant gene editing (GE) requires precise regulation of the GE machinery, a process controlled by the promoters, which drives its transcription through interactions with transcription factors (TFs) and RNA polymerase. While constitutive promoters are extensively used in GE constructs, their limitations highlight the need for alternative approaches. This review emphasizes the promise of tissue/organ specific as well as inducible promoters, which enable targeted GE in a spatiotemporal manner with no effects on other tissues. Advances in synthetic biology have paved the way for the creation of synthetic promoters, offering refined control over gene expression and augmenting the potential of plant GE. The integration of these novel promoters with synthetic systems presents significant opportunities for precise and conditional genome editing. Moreover, the advent of bioinformatic tools and artificial intelligence is revolutionizing the characterization of regulatory elements, enhancing our understanding of their roles in plants. Thus, this review provides novel insights into the strategic use of promoters and promoter editing to enhance the precision, efficiency and specificity of plant GE, setting the stage for innovative crop improvement strategies.}, }
@article {pmid40011959, year = {2025}, author = {Schmidt, H and Zhang, M and Chakarov, D and Bansal, V and Mourelatos, H and Sánchez-Rivera, FJ and Lowe, SW and Ventura, A and Leslie, CS and Pritykin, Y}, title = {Genome-wide CRISPR guide RNA design and specificity analysis with GuideScan2.}, journal = {Genome biology}, volume = {26}, number = {1}, pages = {41}, pmid = {40011959}, issn = {1474-760X}, support = {T32 CA160001/CA/NCI NIH HHS/United States ; DP2 AI171161/NH/NIH HHS/United States ; P01 CA129243/NH/NIH HHS/United States ; U01 HG012103/HG/NHGRI NIH HHS/United States ; 5T32CA160001//MSKCC TROT program/ ; DP2 AI171161/AI/NIAID NIH HHS/United States ; U01 HG009395/NH/NIH HHS/United States ; CAREER 2238831//National Science Foundation/ ; }, mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; Mice ; *CRISPR-Cas Systems ; *Software ; Genome ; }, abstract = {We present GuideScan2 for memory-efficient, parallelizable construction of high-specificity CRISPR guide RNA (gRNA) databases and user-friendly design and analysis of individual gRNAs and gRNA libraries for targeting coding and non-coding regions in custom genomes. GuideScan2 analysis identifies widespread confounding effects of low-specificity gRNAs in published CRISPR screens and enables construction of a gRNA library that reduces off-target effects in a gene essentiality screen. GuideScan2 also enables the design and experimental validation of allele-specific gRNAs in a hybrid mouse genome. GuideScan2 will facilitate CRISPR experiments across a wide range of applications.}, }
@article {pmid40011813, year = {2025}, author = {Lukasiak, S and Kalinka, A and Gupta, N and Papadopoulos, A and Saeed, K and McDermott, U and Hannon, GJ and Ross-Thriepland, D and Walter, D}, title = {A benchmark comparison of CRISPRn guide-RNA design algorithms and generation of small single and dual-targeting libraries to boost screening efficiency.}, journal = {BMC genomics}, volume = {26}, number = {1}, pages = {198}, pmid = {40011813}, issn = {1471-2164}, mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Cas Systems ; *Algorithms ; Benchmarking ; *Gene Library ; Gene Editing/methods ; }, abstract = {Genome-wide CRISPR sgRNA libraries have emerged as transformative tools to systematically probe gene function. While these libraries have been iterated over time to be more efficient, their large size limits their use in some applications. Here, we benchmarked publicly available genome-wide single-targeting sgRNA libraries and evaluated dual targeting as a strategy for pooled CRISPR loss-of-function screens. We leveraged this data to design two minimal genome-wide human CRISPR-Cas9 libraries that are 50% smaller than other libraries and that preserve specificity and sensitivity, thus enabling broader deployment at scale.}, }
@article {pmid40011774, year = {2025}, author = {Cui, X and Yang, H and Cai, C and Beaman, C and Yang, X and Liu, H and Ren, X and Amador, Z and Jones, IR and Keough, KC and Zhang, M and Fair, T and Abnousi, A and Mishra, S and Ye, Z and Hu, M and Pollen, AA and Pollard, KS and Shen, Y}, title = {Comparative characterization of human accelerated regions in neurons.}, journal = {Nature}, volume = {640}, number = {8060}, pages = {991-999}, pmid = {40011774}, issn = {1476-4687}, mesh = {Animals ; Humans ; Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism ; Cell Differentiation ; CRISPR-Cas Systems ; Enhancer Elements, Genetic ; Evolution, Molecular ; Gene Expression Regulation ; Induced Pluripotent Stem Cells/cytology/metabolism ; Nerve Tissue Proteins/genetics/metabolism ; *Neurons/metabolism/cytology ; Pan troglodytes/genetics ; *Regulatory Sequences, Nucleic Acid ; Species Specificity ; Transcription Factors/metabolism ; Brain/cytology ; Genome ; Genome, Human ; }, abstract = {Human accelerated regions (HARs) are conserved genomic loci that have experienced rapid nucleotide substitutions following the divergence from chimpanzees[1,2]. HARs are enriched in candidate regulatory regions near neurodevelopmental genes, suggesting their roles in gene regulation[3]. However, their target genes and functional contributions to human brain development remain largely uncharacterized. Here we elucidate the cis-regulatory functions of HARs in human and chimpanzee induced pluripotent stem (iPS) cell-induced excitatory neurons. Using genomic[4] and chromatin looping information, we prioritized 20 HARs and their chimpanzee orthologues for functional characterization via single-cell CRISPR interference, and demonstrated their species-specific gene regulatory functions. Our findings reveal diverse functional outcomes of HAR-mediated cis-regulation in human neurons, including attenuated NPAS3 expression by altering the binding affinities of multiple transcription factors in HAR202 and maintaining iPS cell pluripotency and neuronal differentiation capacities through the upregulation of PUM2 by 2xHAR.319. Finally, we used prime editing to demonstrate differential enhancer activity caused by several HAR26;2xHAR.178 variants. In particular, we link one variant in HAR26;2xHAR.178 to elevated SOCS2 expression and increased neurite outgrowth in human neurons. Thus, our study sheds new light on the endogenous gene regulatory functions of HARs and their potential contribution to human brain evolution.}, }
@article {pmid40011704, year = {2025}, author = {Adler, BA and Al-Shimary, MJ and Patel, JR and Armbruster, EG and Colognori, D and Charles, EJ and Miller, KV and Lahiri, A and Cui, ML and Oromí-Bosch, A and Voelker, A and Trinidad, M and Lee, J and Beurnier, S and Boger, R and Nomburg, J and Barrangou, R and Mutalik, VK and Schoeniger, JS and Pogliano, JA and Savage, DF and Doudna, JA and Cress, BF}, title = {CRISPRi-ART enables functional genomics of diverse bacteriophages using RNA-binding dCas13d.}, journal = {Nature microbiology}, volume = {10}, number = {3}, pages = {694-709}, pmid = {40011704}, issn = {2058-5276}, mesh = {*Bacteriophages/genetics/classification ; *Genomics/methods ; *CRISPR-Cas Systems ; Genome, Viral ; Phylogeny ; *RNA-Binding Proteins/genetics/metabolism ; Genetic Fitness ; Bacteriophage lambda/genetics ; }, abstract = {Bacteriophages constitute one of the largest reservoirs of genes of unknown function in the biosphere. Even in well-characterized phages, the functions of most genes remain unknown. Experimental approaches to study phage gene fitness and function at genome scale are lacking, partly because phages subvert many modern functional genomics tools. Here we leverage RNA-targeting dCas13d to selectively interfere with protein translation and to measure phage gene fitness at a transcriptome-wide scale. We find CRISPR Interference through Antisense RNA-Targeting (CRISPRi-ART) to be effective across phage phylogeny, from model ssRNA, ssDNA and dsDNA phages to nucleus-forming jumbo phages. Using CRISPRi-ART, we determine a conserved role of diverse rII homologues in subverting phage Lambda RexAB-mediated immunity to superinfection and identify genes critical for phage fitness. CRISPRi-ART establishes a broad-spectrum phage functional genomics platform, revealing more than 90 previously unknown genes important for phage fitness.}, }
@article {pmid40011676, year = {2025}, author = {Cheng, ECK and Lam, JKC and Kwon, SC}, title = {Cytosolic CRISPR RNAs for efficient application of RNA-targeting CRISPR-Cas systems.}, journal = {EMBO reports}, volume = {26}, number = {7}, pages = {1891-1912}, pmid = {40011676}, issn = {1469-3178}, support = {17110223//Research Grants Council, University Grants Committee ()/ ; Start-up//University of Hong Kong (HKU)/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Promoter Regions, Genetic ; *Cytosol/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; HEK293 Cells ; *RNA/genetics/metabolism ; }, abstract = {Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) technologies have evolved rapidly over the past decade with the continuous discovery of new Cas systems. In particular, RNA-targeting CRISPR-Cas13 proteins are promising single-effector systems to regulate target mRNAs without altering genomic DNA, yet the current Cas13 systems are restrained by suboptimal efficiencies. Here, we show that U1 promoter-driven CRISPR RNAs (crRNAs) increase the efficiency of various applications, including RNA knockdown and editing, without modifying the Cas13 protein effector. We confirm that U1-driven crRNAs are exported into the cytoplasm, while conventional U6 promoter-driven crRNAs are mostly confined to the nucleus. Furthermore, we reveal that the end positions of crRNAs expressed by the U1 promoter are consistent regardless of guide sequences and lengths. We also demonstrate that U1-driven crRNAs, but not U6-driven crRNAs, can efficiently repress the translation of target genes in combination with catalytically inactive Cas13 proteins. Finally, we show that U1-driven crRNAs can counteract the inhibitory effect of miRNAs. Our simple and effective engineering enables unprecedented cytosolic RNA-targeting applications.}, }
@article {pmid40010333, year = {2025}, author = {Keith, NC and Snyder, RA and Euler, CW and Modell, JW}, title = {Bacteria exploit viral dormancy to establish CRISPR-Cas immunity.}, journal = {Cell host &amp; microbe}, volume = {33}, number = {3}, pages = {330-340.e6}, pmid = {40010333}, issn = {1934-6069}, support = {R35 GM142731/GM/NIGMS NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems/immunology ; Lysogeny ; Prophages/genetics/physiology ; *Bacteriophages/physiology/genetics/immunology ; *Bacteria/virology/immunology/genetics ; Escherichia coli/virology/genetics/immunology ; }, abstract = {CRISPR-Cas systems provide prokaryotes with adaptive immunity against foreign genetic elements, including bacteriophages, by recording DNA-based immunological memories of infection called "spacers." How cells without preexisting immunity survive a rapid lytic infection long enough to acquire a new spacer and utilize it for defense remains a mystery. Here, we show that bacteria exploit the alternative dormant or "lysogenic" life cycle of temperate phages to establish CRISPR-Cas immunity. During a phage infection, immunization rates are significantly enhanced in cells entering lysogeny compared to those undergoing lysis. Furthermore, in the absence of a foreign threat, bacteria can acquire spacers targeting prophages residing within the chromosome. In this case, self-targeting by Cas9 promotes curing of the prophage, allowing immunized cells to avoid autoimmunity. The preferred acquisition of spacers during the establishment and maintenance of lysogeny may explain why most spacers in natural bacterial isolates target temperate phages.}, }
@article {pmid40008014, year = {2025}, author = {Huang, Y and Chen, Z and Huang, H and Ding, S and Zhang, M}, title = {Important applications of DNA nanotechnology combined with CRISPR/Cas systems in biotechnology.}, journal = {RSC advances}, volume = {15}, number = {8}, pages = {6208-6230}, pmid = {40008014}, issn = {2046-2069}, abstract = {DNA nanotechnology leverages the specificity of Watson-Crick base pairing and the inherent attributes of DNA, enabling the exploitation of molecular characteristics, notably self-assembly, in nucleic acids to fabricate novel, controllable nanoscale structures and mechanisms. In the emerging field of DNA nanotechnology, DNA is not only a genetic material, but also a versatile multifunctional polymer, comprising deoxyribonucleotides, and facilitating the construction of precisely dimensioned and precise shaped two-dimensional (2D) and three-dimensional (3D) nanostructures. DNA molecules act as carriers of biological information, with notable advancements in bioimaging, biosensing, showing the profound impact. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated systems (Cas) constitute self-defense mechanisms employed by bacteria and archaea to defend against viral invasion. With the discovery and modification of various functional Cas proteins, coupled with the identification of increasingly designable and programmable CRISPR RNAs (crRNAs), the potential of the CRISPR/Cas system in the field of molecular diagnostics is steadily being realized. Structural DNA nanotechnology provides a customizable and modular platform for accurate positioning of nanoscopic materials, for e.g., biomedical uses. This addressability has just recently been applied in conjunction with the newly developed gene engineering tools to enable impactful, programmable nanotechnological applications. As of yet, self-assembled DNA nanostructures have been mainly employed to enhance and direct the delivery of CRISPR/Cas, but lately the groundwork has also been laid out for other intriguing and complex functions. These recent advances will be described in this perspective. This review explores biosensing detection methods that combine DNA nanotechnology with CRISPR/Cas systems. These techniques are used in biosensors to detect small molecules such as DNA, RNA, and etc. The combination of 2D and 3D DNA nanostructures with the CRISPR/Cas system holds significant value and great development prospects in the detection of important biomarkers, gene editing, and other biological applications in fields like biosensing.}, }
@article {pmid40007451, year = {2025}, author = {Li, D and Cheng, W and Yin, F and Yao, Y and Wang, Z and Xiang, Y}, title = {A sensitive miRNA detection method based on a split-T7 switch modulating CRISPR/Cas12a system.}, journal = {Chemical communications (Cambridge, England)}, volume = {61}, number = {23}, pages = {4555-4558}, doi = {10.1039/d5cc00170f}, pmid = {40007451}, issn = {1364-548X}, mesh = {*MicroRNAs/analysis/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; Promoter Regions, Genetic ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {This study presents a novel method for sensitive miRNA detection based on a split-T7 switch modulating CRISPR/Cas12a system. By integrating the split-T7 promoter-mediated transcription with the CRISPR/Cas12a system, this method can achieve femtomolar detection of the target miRNA within 1 h and successfully analyze miR-21 in samples from various cell lines, demonstrating its potential for clinical applications.}, }
@article {pmid40007105, year = {2025}, author = {Ebrecht, AC and Luelf, UJ and Govender, K and Opperman, DJ and Urlacher, VB and Smit, MS}, title = {Use of Whole Cells and Cell-Free Extracts of Catalase-Deficient E. coli for Peroxygenase-Catalyzed Reactions.}, journal = {Biotechnology and bioengineering}, volume = {122}, number = {6}, pages = {1376-1385}, pmid = {40007105}, issn = {1097-0290}, support = {//This work was supported by the South African Council for Scientific and Industrial Research-Industrial Biocatalysis Hub (CSIR-IBH) initiative funded by the South African Department of Science and Innovation (DSI) and the Technology Innovation Agency (TIA)./ ; }, mesh = {*Escherichia coli/genetics/enzymology/metabolism ; *Catalase/genetics/metabolism ; *Mixed Function Oxygenases/metabolism/genetics ; *Escherichia coli Proteins/genetics/metabolism ; Hydrogen Peroxide/metabolism ; Cell-Free System ; Cytochrome P-450 Enzyme System/metabolism/genetics ; }, abstract = {Unspecific peroxygenases (UPOs) and cytochrome P450 monooxygenases (CYPs) with peroxygenase activity are becoming the preferred biocatalysts for oxyfunctionalization reactions. While whole cells (WCs) or cell-free extracts (CFEs) of Escherichia coli are often preferred for cofactor-dependent monooxygenase reactions, hydrogen peroxide (H2O2) driven peroxygenase reactions are generally performed with purified enzymes, because the catalases produced by E. coli are expected to quickly degrade H2O2. We used the CRISPR/Cas system to delete the catalase encoding chromosomal genes, katG, and katE, from E. coli BL21-Gold(DE3) to obtain a catalase-deficient strain. A short UPO, DcaUPO, and two CYP peroxygenases, SscaCYP_E284A and CYP102A1_21B3, were used to compare the strains for peroxygenase expression and subsequent sulfoxidation, epoxidation, and benzylic hydroxylation activity. While 10 mM H2O2 was depleted within 10 min after addition to WCs and CFEs of the wild-type strain, at least 60% remained after 24 h in WCs and CFEs of the catalase-deficient strain. CYP peroxygenase reactions, with generally lower turnover frequencies, benefited the most from the use of the catalase-deficient strain. Comparison of purified peroxygenases in buffer versus CFEs of the catalase-deficient strain revealed that the peroxygenases in CFEs generally performed as well as the purified proteins. We also used WCs from catalase-deficient E. coli to screen three CYP peroxygenases, wild-type SscaCYP, SscaCYP_E284A, and SscaCYP_E284I for activity against 10 substrates comparing H2O2 consumption with substrate consumption and product formation. Finally, the enzyme-substrate pair with highest activity, SscaCYP_E284I, and trans-β-methylstyrene, were used in a preparative scale reaction with catalase-deficient WCs. Use of WCs or CFEs from catalase-deficient E. coli instead of purified enzymes can greatly benefit the high-throughput screening of enzyme or substrate libraries for peroxygenase activity, while they can also be used for preparative scale reactions.}, }
@article {pmid40005257, year = {2025}, author = {Yuan, Z}, title = {From Origin to the Present: Establishment, Mechanism, Evolutions and Biomedical Applications of the CRISPR/Cas-Based Macromolecular System in Brief.}, journal = {Molecules (Basel, Switzerland)}, volume = {30}, number = {4}, pages = {}, pmid = {40005257}, issn = {1420-3049}, mesh = {*CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; Animals ; }, abstract = {Advancements in biological and medical science are intricately linked to the biological central dogma. In recent years, gene editing techniques, especially CRISPR/Cas systems, have emerged as powerful tools for modifying genetic information, supplementing the central dogma and holding significant promise for disease diagnosis and treatment. Extensive research has been conducted on the continuously evolving CRISPR/Cas systems, particularly in relation to challenging diseases, such as cancer and HIV infection. Consequently, the integration of CRISPR/Cas-based techniques with contemporary medical approaches and therapies is anticipated to greatly enhance healthcare outcomes for humans. This review begins with a brief overview of the discovery of the CRISPR/Cas system. Subsequently, using CRISPR/Cas9 as an example, a clear description of the classical molecular mechanism underlying the CRISPR/Cas system was given. Additionally, the development of the CRISPR/Cas system and its applications in gene therapy and high-sensitivity disease diagnosis were discussed. Furthermore, we address the prospects for clinical applications of CRISPR/Cas-based gene therapy, highlighting the ethical considerations associated with altering genetic information. This brief review aims to enhance understanding of the CRISPR/Cas macromolecular system and provide insight into the potential of genetic macromolecular drugs for therapeutic purposes.}, }
@article {pmid40004557, year = {2025}, author = {Wang, J and Wang, H and Zhai, J and Zhu, F and Ren, Y and Zhou, J and Zhang, Z and Luo, L and Xu, W}, title = {Identification of Ziziphus jujuba cv. Dongzao DNA Demethylase ZjROS1 Gene Family and Construction of CRISPR/Cas9-Mediated Gene-Editing Vector.}, journal = {Genes}, volume = {16}, number = {2}, pages = {}, pmid = {40004557}, issn = {2073-4425}, support = {YCX24138//Graduate Innovation Program of North Minzu University/ ; 2022QCXTD04//Innovation Team for Genetic Improvement of Economic Forests/ ; 32060669//National Natural Science Foundation of China/ ; }, mesh = {*Ziziphus/genetics/growth &amp; development/enzymology ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Editing/methods ; DNA Methylation/genetics ; Fruit/genetics/growth &amp; development ; Multigene Family ; }, abstract = {DNA methylation is one of the earliest and most extensively studied epigenetic regulatory mechanisms. The ROS1 (Repressor of Silencing 1) gene was first discovered in Arabidopsis thaliana, and it is a DNA demethylase that can remove 5-methylcytosine from DNA, thereby affecting DNA methylation levels and gene expression. Objectives: The objective of this study was to investigate the role of ROS1 in the development and maturation of Ziziphus jujuba cv. "Dongzao" fruit. Methods: We cloned the ROS1 gene and conducted bioinformatics and expression characteristics analyses on it. Results: Three ROS1 genes, named ZjROS1-1~3, was identified, and each member protein was localized in the nucleus, cytoskeleton, chloroplast, and vacuole. The promoter contained cis-elements such as light response, plant hormone signal transduction, and stress response cis-elements, and it interacted with many proteins such as CMT, MET, and ZDP. The results of the real-time fluorescence quantitative PCR show that ZjROS1 has specific expression patterns in different tissues of Z. jujuba cv. Dongzao, and the expression of ZjROS1-2 in flowers and fruits is high. At the same time, CRISPR/Cas9 technology was used to construct a gene-editing vector for ZjROS1, which provided a basis for the subsequent genetic transformation. Conclusions: In this study, the biological function of ZjROS1 was clarified and a gene-editing vector was constructed, which provided a theoretical basis for the regulation mechanism of demethylase ZjROS1 in the fruit ripening and development of Z. jujuba cv. Dongzao.}, }
@article {pmid40004545, year = {2025}, author = {Shu, H and Luan, A and Ullah, H and He, J and Wang, Y and Chen, C and Wei, Q and Zhan, R and Chang, S}, title = {Utilizing Target Sequences with Multiple Flanking Protospacer Adjacent Motif (PAM) Sites Reduces Off-Target Effects of the Cas9 Enzyme in Pineapple.}, journal = {Genes}, volume = {16}, number = {2}, pages = {}, pmid = {40004545}, issn = {2073-4425}, support = {32272677//National Natural Science Foundation, China/ ; 1630032024001//Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences/ ; }, mesh = {*Ananas/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {BACKGROUND/OBJECTIVES: CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats)-associated protein 9 is now widely used in agriculture and medicine. Off-target effects can lead to unexpected results that may be harmful, and these effects are a common concern in both research and therapeutic applications.<br><br>METHODS: In this study, using pineapple as the gene-editing material, eighteen target sequences with varying numbers of PAM (Protospacer-Adjacent Motif) sites were used to construct gRNA vectors. Fifty mutant lines were generated for each target sequence, and the off-target rates were counted.<br><br>RESULTS: Selecting sequences with multiple flanking PAM sites as editing targets resulted in a lower off-target rate compared to those with a single PAM site. Target sequences with two 5'-NGG ("N" represents any nucleobase, followed by two guanine "G") PAM sites at the 3' end exhibited greater specificity and a higher probability of binding with the Cas9 protein than those only with one 5'-NGG PAM site at the 3' end. Conversely, although the target sequence with a 5'-NAG PAM site (where "N" is any nucleobase, followed by adenine "A" and guanine "G") adjacent and upstream of an NGG PAM site had a lower off-target rate compared to sequences with only an NGG PAM site, their off-target rates were still higher than those of sequences with two adjacent 5'-NAG PAM sites. Among the target sequences of pineapple mutant lines (AcACS1, AcOT5, AcCSPE6, AcPKG11A), more deletions than insertions were found.<br><br>CONCLUSIONS: We found that target sequences with multiple flanking PAM sites are more likely to bind with the Cas9 protein and induce mutations. Selecting sequences with multiple flanking PAM sites as editing targets can reduce the off-target effects of the Cas9 enzyme in pineapple. These findings provide a foundation for improving off-target prediction and engineering CRISPR-Cas9 complexes for gene editing.}, }
@article {pmid40004462, year = {2025}, author = {Lara, P and Aguilar-González, A and Martín, F and Mesas, C and Moreno, J and Rama, AR}, title = {Exploring miR-21 Knock-Out Using CRISPR/Cas as a Treatment for Lung Cancer.}, journal = {Genes}, volume = {16}, number = {2}, pages = {}, pmid = {40004462}, issn = {2073-4425}, mesh = {*MicroRNAs/genetics ; Humans ; *Lung Neoplasms/genetics/therapy/drug therapy/pathology ; *CRISPR-Cas Systems/genetics ; A549 Cells ; Cell Proliferation/genetics ; Gene Knockout Techniques ; Drug Resistance, Neoplasm/genetics ; PTEN Phosphohydrolase/genetics ; Gene Expression Regulation, Neoplastic ; Cell Movement/genetics ; Antineoplastic Agents/pharmacology ; Apoptosis Regulatory Proteins/genetics ; RNA-Binding Proteins ; }, abstract = {BACKGROUND: Lung cancer is a leading cause of cancer-related deaths worldwide. Its high incidence and poor prognosis demonstrate the need to investigate new therapies. The PI3K/AKT pathway is activated in carcinogenic processes such as invasion, proliferation, and drug resistance. MiR-21 is a microRNA overexpressed in numerous types of cancer and which activates PI3K/AKT pathway by down-regulating its main targets, PTEN and PDCD4. CRISPR is a revolutionary gene-editing technology that allows genes to be deleted. The aim of this study was to use CRISPR/Cas9 technology as an option to reduce carcinogenic and drug resistance processes by eliminating miR-21.<br><br>METHODS: CRISPR/Cas9 was used to knock out miR-21 (miR-21 KO) in A549 lung cancer cells and thus reverse the carcinogenic processes activated by miR-21 overexpression. Furthermore, the effect of miR-21 KO on drug resistance was studied, choosing the main chemotherapeutic agents used for the treatment of lung cancer: gemcitabine, carboplatin, paclitaxel, and oxaliplatin.<br><br>RESULTS: miR-21 KO A549 cells exhibited a reduction in proliferation, migration, and colony formation compared to A549 cells. In contrast, the expression of PTEN and PDCD4 increased in miR-21 KO A549 cells. Furthermore, miR-21 KO A549 cells showed a decrease in the IC50 of the drugs used for the treatment of lung cancer: gemcitabine, carboplatin, paclitaxel, and oxaliplatin.<br><br>CONCLUSIONS: Based on these results, miR-21 knock-out using CRISPR/Cas could be a promising strategy for the treatment of lung cancer.}, }
@article {pmid40004186, year = {2025}, author = {Nakamae, K and Suzuki, T and Yonezawa, S and Yamamoto, K and Kakuzaki, T and Ono, H and Naito, Y and Bono, H}, title = {Risk Prediction of RNA Off-Targets of CRISPR Base Editors in Tissue-Specific Transcriptomes Using Language Models.}, journal = {International journal of molecular sciences}, volume = {26}, number = {4}, pages = {}, pmid = {40004186}, issn = {1422-0067}, support = {030RP2022//ROIS-DS-JOINT 2022/ ; 009RP2023//ROIS-DS-JOINT 2023/ ; 012RP2024//ROIS-DS-JOINT 2024/ ; JPMJPF2010//The Center of Innovation for Bio-Digital Transformation (BioDX), an open innovation platform for industry-academia cocreation (COI-NEXT) and the Japan Science and Technology Agency (JST)/ ; }, mesh = {*Transcriptome/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Machine Learning ; *RNA Editing ; Organ Specificity/genetics ; *RNA/genetics ; Computational Biology/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Base-editing technologies, particularly cytosine base editors (CBEs), allow precise gene modification without introducing double-strand breaks; however, unintended RNA off-target effects remain a critical concern and are under studied. To address this gap, we developed the Pipeline for CRISPR-induced Transcriptome-wide Unintended RNA Editing (PiCTURE), a standardized computational pipeline for detecting and quantifying transcriptome-wide CBE-induced RNA off-target events. PiCTURE identifies both canonical ACW (W = A or T/U) motif-dependent and non-canonical RNA off-targets, revealing a broader WCW motif that underlies many unanticipated edits. Additionally, we developed two machine learning models based on the DNABERT-2 language model, termed STL and SNL, which outperformed motif-only approaches in terms of accuracy, precision, recall, and F1 score. To demonstrate the practical application of our predictive model for CBE-induced RNA off-target risk, we integrated PiCTURE outputs with the Predicting RNA Off-target compared with Tissue-specific Expression for Caring for Tissue and Organ (PROTECTiO) pipeline and estimated RNA off-target risk for each transcript showing tissue-specific expression. The analysis revealed differences among tissues: while the brain and ovaries exhibited relatively low off-target burden, the colon and lungs displayed relatively high risks. Our study provides a comprehensive framework for RNA off-target profiling, emphasizing the importance of advanced machine learning-based classifiers in CBE safety evaluations and offering valuable insights to inform the development of safer genome-editing therapies.}, }
@article {pmid40004028, year = {2025}, author = {Zeng, M and Zhang, QY and Ke, F}, title = {Characterization of Five CRISPR Systems in Microcystis aeruginosa FACHB-524 with Focus on the In Vitro Antiviral Activity of One CRISPR System.}, journal = {International journal of molecular sciences}, volume = {26}, number = {4}, pages = {}, pmid = {40004028}, issn = {1422-0067}, support = {2018YFA0903101//the National Key R&amp;D Programs of China/ ; }, mesh = {*Microcystis/genetics/virology ; *CRISPR-Cas Systems/genetics ; Escherichia coli/genetics/virology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacteriophages/genetics ; }, abstract = {Microcystis aeruginosa is an important species causing cyanobacterial blooms, which can be effectively infected and lysed by cyanophages. Several strategies have been developed by M. aeruginosa to resist cyanophage infections, including the CRISPR-Cas systems. However, detailed information on the CRISPR-Cas systems in M. aeruginosa is rare. In the present study, the CRISPR-Cas systems of M. aeruginosa FACHB-524 were analyzed by genome re-sequencing, which showed that there are two type I (Cluster 1, I-B1; Cluster 2, I-D) and three type III-B (Cluster 3/4/5) CRISPR-Cas systems in the cyanobacteria. Further comparison revealed that spacer sequences of two type III-B systems targeted several genes of the cyanophage MaMV (M. aeruginosa myovirus) strains. One of the type III systems (Cluster 4) was then cloned and expressed in Escherichia coli BL21 (DE3). Protein purification and mass spectrometry identification revealed that a Cmr-crRNA effector complex formed in the E. coli. Subsequently, T4 phage (T4) was used to infect the E. coli, expressing the Cmr-crRNA complex with or without accessory proteins. The results showed that the Cmr-crRNA effector complex exhibited anti-phage activity and the accessory protein Csx1 enhanced the immune activity of the complex. Collectively, our results comprehensively demonstrate the CRISPR systems encoded by a strain of M. aeruginosa, and for the first time, one of the CRISPR systems was constructed into E. coli, providing a foundation for further in-depth analysis of cyanobacterial CRISPR systems.}, }
@article {pmid40003981, year = {2025}, author = {Asad, M and Chang, Y and Liao, J and Yang, G}, title = {CRISPR/Cas9 Genome Editing in the Diamondback Moth: Current Progress, Challenges, and Prospects.}, journal = {International journal of molecular sciences}, volume = {26}, number = {4}, pages = {}, pmid = {40003981}, issn = {1422-0067}, support = {2023J02009//Key Project of Fujian Province Natural Science Foundation/ ; 32072425//Natural Science Foundation of China/ ; 2018NZ01010013//Special Key Project of Fujian Province, grant number/ ; KRA16001A//111" program/ ; }, mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Moths/genetics ; Insecticide Resistance/genetics ; }, abstract = {The development of site-specific genome-editing tools like CRISPR (clustered regularly interspaced short palindromic repeat) and its associated protein, Cas9, is revolutionizing genetic engineering with its highly efficient mechanism, offering the potential for effective pest management. Recently, CRISPR/Cas9 gene-editing has been extensively utilized in the management of the diamondback moth, Plutella xylostella (L.), a highly destructive pest of vegetable crops, for different purposes, such as gene function analysis and genetic control. However, the progress related to this gene-editing tool in P. xylostella has not yet been summarized. This review highlights the progress and applications of CRISPR/Cas9 in uncovering the genes critical for development, reproduction, and insecticide resistance in P. xylostella. Moreover, the progress related to the CRISPR/Cas9 gene drive for population suppression and modifications has also been discussed. In addition to the significant progress made, challenges such as low germline editing efficiency and limited homology-directed repair remain obstacles to its widespread application. To address these limitations, we have discussed the different strategies that are anticipated to improve the efficiency of CRISPR/Cas9, paving the way to it becoming a pivotal tool in sustainable pest management. Therefore, the present review will help researchers in the future enhance the efficiency of the CRISPR/Cas9 system and use it to manage the diamondback moth.}, }
@article {pmid40003953, year = {2025}, author = {Zhao, M and Yang, Z and Li, J and Ming, F and Kong, D and Xu, H and Wang, Y and Chen, P and Duan, X and Wang, M and Wang, Z}, title = {Enhancement of Dendrobine Production by CRISPR/Act3.0-Mediated Transcriptional Activation of Multiple Endogenous Genes in Dendrobium Plants.}, journal = {International journal of molecular sciences}, volume = {26}, number = {4}, pages = {}, pmid = {40003953}, issn = {1422-0067}, support = {JCYJ20210324123005016, JCYJ20230807151000001//The Science, Technology and Innovation Commission of Shenzhen Municipality/ ; }, mesh = {*Dendrobium/genetics/metabolism ; Gene Expression Regulation, Plant ; Plants, Genetically Modified/genetics/metabolism ; *CRISPR-Cas Systems ; *Transcriptional Activation ; Plant Leaves/genetics/metabolism ; Gene Editing ; }, abstract = {Dendrobine, a significant medicinal compound, typically accumulates at low concentrations within several Dendrobium species, including Dendrobium nobile, Dendrobium catenatum, and Dendrobium moniliforme. As D. nobile and D. catenatum are established ingredients in traditional Chinese medicine and have been cultivated extensively, they present ideal plant chassis for upscaling dendrobine production for industrial and research applications. This study employed two approaches: the ectopic overexpression of seven genes through multigene stacking and the activation of multiple key endogenous genes in the dendrobine synthesis pathway using CRISPR/Act3.0 in either D. nobile or D. catenatum. These methods enhanced dendrobine production in transiently infiltrated leaves by 30.1% and transgenic plants by 35.6%. The study is the first to apply CRISPR/Act3.0 to Dendrobium orchids, enhancing dendrobine production, and thus laying a solid foundation for further improvements. CRISPR/Act3.0 is a recently developed technique that demonstrates high efficiency in model plant species, including rice, maize, and Arabidopsis. By combining CRISPR with transcriptional regulatory modules, activation of multiple endogenous genes in the metabolic pathway can be achieved. The successful application in orchid molecular breeding reveals promising potential for further exploration.}, }
@article {pmid40003875, year = {2025}, author = {Ilchuk, LA and Kochegarova, KK and Baikova, IP and Safonova, PD and Bruter, AV and Kubekina, MV and Okulova, YD and Minkovskaya, TE and Kuznetsova, NA and Dolmatova, DM and Ryabinina, AY and Mozhaev, AA and Belousov, VV and Ershov, BP and Timashev, PS and Filatov, MA and Silaeva, YY}, title = {Mutations in Filamin C Associated with Both Alleles Do Not Affect the Functioning of Mice Cardiac Muscles.}, journal = {International journal of molecular sciences}, volume = {26}, number = {4}, pages = {}, pmid = {40003875}, issn = {1422-0067}, support = {075-15-2019-1661//The Ministry of Science and Higher Education of the Russian Federation/ ; }, mesh = {Animals ; *Filamins/genetics/metabolism/chemistry ; Mice ; Alleles ; *Mutation ; *Myocardium/metabolism ; Female ; Homozygote ; Muscle, Skeletal/metabolism ; Male ; CRISPR-Cas Systems ; Heterozygote ; }, abstract = {Filamin C (FLNC) is a structural protein of muscle fibers. Mutations in the FLNC gene are known to cause myopathies and cardiomyopathies in humans. Here we report the generation by a CRISPR/Cas9 editing system injected into zygote pronuclei of two mouse strains carrying filamin C mutations-one of them (AGA) has a deletion of three nucleotides at position c.7418_7420, causing E>>D substitution and N deletion at positions 2472 and 2473, respectively. The other strain carries a deletion of GA nucleotides at position c.7419_7420, leading to a frameshift and a premature stop codon. Homozygous animals (Flnc[AGA/AGA] and Flnc[GA/GA]) were embryonically lethal. We determined that Flnc[GA/GA] embryos died prior to the E12.5 stage and illustrated delayed development after the E9.5 stage. We performed histological analysis of heart tissue and skeletal muscles of heterozygous strains carrying mutations in different combinations (Flnc[GA/wt], Flnc[AGA/wt], and Flnc[GA/AGA]). By performing physiological tests (grip strength and endurance tests), we have shown that heterozygous animals of both strains (Flnc[GA/wt], Flnc[AGA/wt]) are functionally indistinguishable from wild-type animals. Interestingly, compound heterozygous mice (Flnc[GA/AGA]) are viable, develop normally, reach puberty and it was verified by ECG and Eco-CG that their cardiac muscle is functionally normal. Intriguingly, Flnc[GA/AGA] mice demonstrated better results in the grip strength physiological test in comparison to WT animals. We also propose a structural model that explains the complementary interaction of two mutant variants of filamin C.}, }
@article {pmid40003596, year = {2025}, author = {Rubí-Rangel, LM and León-Félix, J and Villicaña, C}, title = {Exploring Viral Interactions in Clavibacter Species: In Silico Analysis of Prophage Prevalence and Antiviral Defenses.}, journal = {Life (Basel, Switzerland)}, volume = {15}, number = {2}, pages = {}, pmid = {40003596}, issn = {2075-1729}, support = {P00768014//CIAD Internal Project/ ; 784//Investigadores por M&#xe9;xico CONAHCYT/ ; }, abstract = {Clavibacter is a phytopathogenic genus that causes severe diseases in economically important crops, yet the role of prophages in its evolution, pathogenicity, and adaptation remains poorly understood. In this study, we used PHASTER, Prophage Hunter, and VirSorter2 to identify prophage-like sequences in publicly available Clavibacter genomes. Prophage predictions were checked by hand to make them more accurate. We identified 353 prophages, predominantly in chromosomes, with some detected phage-plasmids. Most prophages exhibited traits of advanced domestication, such as an unimodal genome length distribution, reduced numbers of integrases, and minimal transposable elements, suggesting long-term interactions with their bacterial hosts. Comparative genomic analyses uncovered high genetic diversity, with distinct prophage clusters showing species-specific and interspecies conservation patterns. Functional annotation revealed prophage-encoded genes were involved in sugar metabolism, heavy metal resistance, virulence factors, and antibiotic resistance, highlighting their contribution to host fitness and environmental adaptation. Defense system analyses revealed that, despite lacking CRISPR-Cas, Clavibacter genomes harbor diverse antiviral systems, including PD-Lambda-1, AbiE, and MMB_gp29_gp30, some encoded within prophages. These findings underscore the pervasive presence of prophages in Clavibacter and their role in shaping bacterial adaptability and evolution.}, }
@article {pmid40003070, year = {2025}, author = {Zhao, Z and Zou, X and Zhu, Y and He, Y and Jebessa, E and Zhang, J and Ji, J and Chen, P and Luo, C}, title = {Achieving Optimal Transfection Conditions in Chicken Primordial Germ Cells Under Feeder- and Serum-Free Medium.}, journal = {Animals : an open access journal from MDPI}, volume = {15}, number = {4}, pages = {}, pmid = {40003070}, issn = {2076-2615}, support = {2022B0202110002//the Science and Technology Program of Guangdong/ ; XT202217//the Project of Collaborative Innovation Center of GDAAS/ ; ZX202401-03//the sub project of "Discipline Construction of Swine and Poultry Breeding Industry" of Special Project on Science and Technology Innovation Strategy/ ; CARS-41//the earmarked fund/ ; GDNKY-ZQQZ-K8, GDNKY-ZQQZ-K11//the Project of State Key Laboratory of Swine and Poultry Breeding Industry/ ; }, abstract = {The successful application of primordial germ cells (PGCs) is an ideal method for generating gene-edited birds. However, barriers to efficient DNA transfection in PGCs lead to low transfection efficiency, limiting the generation of genetically modified chickens. The current study utilized chemical transfection and electroporation methods to determine the optimal transfection conditions for the PGC line under feeder- and serum-free medium. Among the tested methods, the Lonza electroporation system exhibited the highest transduction efficiency, with a previously unreported rate of 71.13 ± 1.26%. Optimal transfection conditions were achieved using 4 µg of DNA and 100 µL of Entranster[TM]-E in 1 × 10[6] PGCs. Furthermore, the optimal electroporation conditions resulted in low cell death and normal expression of pluripotency-related genes, highlighting the low cytotoxicity. The resulting electroporation models were then used to deliver the enhanced green fluorescent protein (EGFP) gene to the Z chromosome with a Cas9-gRNA plasmid, achieving a 7-day insertion efficiency of 14.63 ± 1.07%. Our study highlights the vast potential of electroporation technology for the transfection of PGCs.}, }
@article {pmid40001389, year = {2025}, author = {Pandova, M and Kizheva, Y and Hristova, P}, title = {Relationship Between CRISPR-Cas Systems and Acquisition of Tetracycline Resistance in Non-Clinical Enterococcus Populations in Bulgaria.}, journal = {Antibiotics (Basel, Switzerland)}, volume = {14}, number = {2}, pages = {}, pmid = {40001389}, issn = {2079-6382}, support = {project No BG-RRP-2.004-0008//European Union-NextGenerationEU, National Recovery and Resilience Plan of the Republic of Bulgaria, SUMMIT 3.2.4./ ; }, abstract = {Non-clinical enterococci are relatively poorly studied by means of acquired antibiotic resistance to tetracycline and by the distribution, functionality and role of their CRISPR systems. Background: In our study, 72 enterococcal strains, isolated from various non-clinical origins, were investigated for their phenotypic and genotypic (tet(M), tet(O), tet(S), tet(L), tet(K), tet(T) and tet(W)) tetracycline resistance. Methods: The genetic determinants for HGT (MGEs (Int-Tn and prgW), inducible pheromones (cpd, cop and cff), aggregation substances (agg, asa1, prgB and asa373) and CRISPR-Cas systems were characterized by PCR and whole-genome sequencing. Results: Four tet genes (tetM, tetO, tetS and tetT) were detected in 39% (n = 28) of our enterococcal population, with tetM (31%) being dominant. The gene location was linked to the Tn6009 transposon. All strains that contained tet genes also had genes for HGT. No tet genes were found in E. casseliflavus and E. gilvus. In our study, 79% of all tet-positive strains correlated with non-functional CRISPR systems. The strain E. faecalis BM15 was the only one containing a combination of a functional CRISPR system (cas1, cas2, csn2 and csn1/cas9) and tet genes. The CRISPR subtype repeats II-A, III-B, IV-A2 and VI-B1 were identified among E. faecalis strains (CM4-II-A, III-B and VI-B1; BM5-IV-A2, II-A and III-B; BM12 and BM15-II-A). The subtype II-A was the most present. These repeats enclosed a great number of spacers (1-10 spacers) with lengths of 31 to 36 bp. One CRISPR locus was identified in plasmid (p.Firmicutes1 in strain E. faecalis BM5). We described the presence of CRISPR loci in the species E. pseudoavium, E. pallens and E. devriesei and their lack in E. gilvus, E. malodoratus and E. mundtii. Conclusions: Our findings generally describe the acquisition of foreign DNA as a consequence of CRISPR inactivation, and self-targeting spacers as the main cause.}, }
@article {pmid40001243, year = {2025}, author = {Liu, Z and Li, Y and Wang, S and Wang, Y and Sui, M and Liu, J and Chen, P and Wang, J and Zhang, Y and Dang, C and Hou, P}, title = {Genome-wide CRISPR screening identifies PHF8 as an effective therapeutic target for KRAS- or BRAF-mutant colorectal cancers.}, journal = {Journal of experimental &amp; clinical cancer research : CR}, volume = {44}, number = {1}, pages = {70}, pmid = {40001243}, issn = {1756-9966}, support = {82172675&#xff0c;82273036//National Natural Science Foundation of China/ ; 82303731//National Natural Science Foundation of China/ ; No. S2024-JC-QN-0868//Natural Science Basic Research Program of Shaanxi Province/ ; 2024JC-YBQN-0934//Natural Science Basic Research Program of Shaanxi Province/ ; 2022JQ-782//Natural Science Basic Research Program of Shaanxi Province/ ; }, mesh = {Humans ; *Colorectal Neoplasms/genetics/pathology/drug therapy/metabolism ; *Proto-Oncogene Proteins p21(ras)/genetics ; *Histone Demethylases/genetics/metabolism ; *Proto-Oncogene Proteins B-raf/genetics ; Mice ; Animals ; *Transcription Factors/genetics/metabolism ; *Mutation ; Cell Line, Tumor ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; Female ; }, abstract = {BACKGROUND: Mutations in KRAS and BRAF genes are prevalent in colorectal cancer (CRC), which strikingly promote tumorigenesis and lead to poor response to a variety of treatments including immunotherapy by activating the MAPK/ERK pathway. Thus, there is an urgent need to discover effective therapeutic targets and strategies.<br><br>METHODS: CRISPR-Cas9 lentiviral knockout library was used to screen the suppressors of anti-PD1 immunotherapy. Bioinformatic analysis was used to analyze the correlation between PHF8 expression and immune indicators in CRC. In vitro and in vivo experiments were utilized to determine the effects of PHF8 on the immune indexes and malignant phenotypes of CRC cells. qRT-PCR, western blotting, immunohistochemical (IHC) staining, and chromatin immunoprecipitation (ChIP)-qPCR assays were used to determine the regulatory effects of PHF8 on PD-L1, KRAS, BRAF, and c-Myc and the regulatory effect c-Myc/miR-22-3p signaling axis on PHF8 expression in CRC cells.<br><br>RESULTS: This study identified histone lysine demethylase PHF8 as a negative regulator for the efficacy of anti-PD1 therapy and found that it was highly expressed in CRCs and strongly associated with poor patient survival. Functional studies showed that PHF8 played an oncogenic role in KRAS- or BRAF-mutant CRC cells, but not in wild-type ones. Mechanistically, PHF8 up-regulated the expression of PD-L1, KRAS, BRAF, and c-Myc by increasing the levels of transcriptional activation marks H3K4me3 and H3K27ac and decreasing the levels of transcriptional repression mark H3K9me2 within their promoter regions, promoting immune escape and tumor progression. Besides, our data also demonstrated that PHF8 was up-regulated by the c-Myc/miR-22-3p signaling axis to form a positive feedback loop. Targeting PHF8 substantially improved the efficacy of anti-PD1 therapy and inhibited the malignant phenotypes of KRAS- or BRAF-mutant CRC cells.<br><br>CONCLUSION: Our data demonstrate that PHF8 may be an effective therapeutic target for KRAS- or BRAF-mutant CRCs.}, }
@article {pmid40001184, year = {2025}, author = {Nikolouli, K and Compton, A and Tu, ZJ and Bourtzis, K}, title = {Evaluation of ebony as a potential selectable marker for genetic sexing in Aedes aegypti.}, journal = {Parasites &amp; vectors}, volume = {18}, number = {1}, pages = {76}, pmid = {40001184}, issn = {1756-3305}, support = {R01 AI123338/AI/NIAID NIH HHS/United States ; AI123338//National Institute of Allergy and Infectious Diseases/ ; }, mesh = {Animals ; *Aedes/genetics ; Male ; Female ; Genetic Markers ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Mosquito Control/methods ; *Insect Proteins/genetics ; Mutation ; }, abstract = {BACKGROUND: Aedes aegypti is expected to invade previously unoccupied areas, mainly due to the climate change, the increase in travel and trade activities and the continuous transformation of the rural environment into urban areas. The sterile insect technique (SIT), which relies on the mass production and release of sterile males, is an environmentally friendly approach that can be applied for population control of Ae. aegypti. SIT programs can be greatly benefited by a genetic sexing strain (GSS) and a reliable sex sorting system to minimize any accidental female release. Visually detectable or conditionally lethal selectable markers can be used for the development of new GSSs. In this study, we evaluated the suitability and competence of a mutant Ae. aegypti ebony strain for the development of a new GSS. The ebony gene is known to be involved in the pigmentation pathway of several dipteran insects, including Ae. aegypti.<br><br>METHODS: An ebony gene knockout was developed though CRISPR/Cas9 mutagenesis. G0 individuals with the desired phenotype were crossed, and progeny were screened in every generation. PCR and sequencing were performed using gDNA from a pulled leg to determine the mutant genotype. Quality control tests, including pupae and adult recovery rates, male sex ratio and fecundity, were applied to the ebony mutant line to determine whether the mutation confers any fitness cost.<br><br>RESULTS: An Ae. aegypti ebony knockout mutant carrying a 5-bp deletion was obtained, which presented darker head and siphon phenotypes at the larval stage. However, genetic analysis revealed that this ebony mutation results in incomplete penetrance and variable expressivity. The establishment of a pure ebony mutant line was not possible because of the fitness costs conferred by the mutation.<br><br>CONCLUSIONS: In this study, the adequacy and suitability of the ebony gene as a selectable marker for the development of a GSS in Ae. aegypti were assessed. Despite its clear phenotype early in larval development, the homozygous mutant line presented phenotypic inconsistency and loss of fertility. These drawbacks clearly indicate that this particular mutation is not suitable for the development of a new GSS. Nonetheless, it cannot be excluded that a different mutation will lead to a different expression and penetrance profile and a viable homozygous mutant line.}, }
@article {pmid40000946, year = {2025}, author = {Rai, A and Skårn, MN and Elameen, A and Tengs, T and Amundsen, MR and Bjorå, OS and Haugland, LK and Yakovlev, IA and Brurberg, MB and Thorstensen, T}, title = {CRISPR-Cas9-mediated deletions of FvMYB46 in Fragaria vesca reveal its role in regulation of fruit set and phenylpropanoid biosynthesis.}, journal = {BMC plant biology}, volume = {25}, number = {1}, pages = {256}, pmid = {40000946}, issn = {1471-2229}, mesh = {*Fruit/growth &amp; development/genetics/metabolism ; *Fragaria/genetics/metabolism/growth &amp; development ; *Transcription Factors/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; Flowers/genetics/metabolism/growth &amp; development ; Flavonoids/biosynthesis ; Lignin ; Cell Wall/metabolism ; }, abstract = {The phenylpropanoid pathway, regulated by transcription factors of the MYB family, produces secondary metabolites that play important roles in fertilization and early phase of fruit development. The MYB46 transcription factor is a key regulator of secondary cell wall structure, lignin and flavonoid biosynthesis in many plants, but little is known about its activity in flowers and berries in F. vesca. For functional analysis of FvMYB46, we designed a CRISPR-Cas9 construct with an endogenous F. vesca-specific U6 promoter for efficient and specific expression of two gRNAs targeting the first exon of FvMYB46. This generated mutants with an in-frame 81-bp deletion of the first conserved MYB domain or an out-of-frame 82-bp deletion potentially knocking out gene function. In both types of mutant plants, pollen germination and fruit set were significantly reduced compared to wild type. Transcriptomic analysis of flowers revealed that FvMYB46 positively regulates the expression of genes involved in processes like xylan biosynthesis and metabolism, homeostasis of reactive oxygen species (ROS) and the phenylpropanoid pathway, including secondary cell wall biosynthesis and flavonoid biosynthesis. Genes regulating carbohydrate metabolism and signalling were also deregulated, suggesting that FvMYB46 might regulate the crosstalk between carbohydrate metabolism and phenylpropanoid biosynthesis. In the FvMYB46-mutant flowers, the flavanol and flavan-3-ol contents, especially epicatechin, quercetin-glucoside and kaempferol-3-coumaroylhexoside, were reduced, and we observed a local reduction in the lignin content in the anthers. Together, these results suggest that FvMYB46 controls fertility and efficient fruit set by regulating the cell wall structure, flavonoid biosynthesis, carbohydrate metabolism, and sugar and ROS signalling in flowers and early fruit development in F. vesca.}, }
@article {pmid40000476, year = {2025}, author = {Sun, W and Ren, X and Xiao, Y and Li, B and Pang, QA and Yang, M and Zhu, R and Guo, Z and Yu, J and Huang, J and Wang, Y and Liu, S}, title = {Fluorescent/colorimetric dual-mode for detecting of MC-LR using bidirectional RCA coupled with CdTe QDs.}, journal = {Mikrochimica acta}, volume = {192}, number = {3}, pages = {189}, pmid = {40000476}, issn = {1436-5073}, support = {ts201712048//Program for Taishan Scholar of Shandong Province/ ; 32072330//National Natural Science Foundation of China/ ; ZR2022MB066//Natural Science Foundation of Shandong Province/ ; }, mesh = {*Tellurium/chemistry ; *Cadmium Compounds/chemistry ; *Colorimetry/methods ; *Quantum Dots/chemistry ; *Biosensing Techniques/methods ; *Microcystins/analysis ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Fluorescent Dyes/chemistry ; CRISPR-Cas Systems ; Spectrometry, Fluorescence/methods ; Marine Toxins ; }, abstract = {A fluorescent/colorimetric dual-mode biosensor was designed using CdTe QDs and CRISPR/Cas for the efficient and ultrasensitive detection of microcystin-leucine-arginine (MC-LR). The biosensor mainly activates the trans-cleavage activity of Cas12a through nucleic acid amplification technology, such as bidirectional rolling circle amplification (B-RCA), to perform signal conversion, release Ag[+] from the cleaving hairpin, quench QD fluorescence, and perform signal presentation. The biosensor can perform fluorometric and colorimetric detection, enabling rapid field assays. It exhibits enhanced selectivity, increased sensitivity, and greater accuracy. The optimal conditions yield a detection range from 0.05 to 500 nM, with a minimum detectable concentration of 2.137 pM, surpassing the performance of traditional methods. The biosensor can effectively detect MC-LR in actual environmental samples. Overall, this study provided a general detection approach for the application of nucleic acid detection technology to detect trace pollutants in the environment.}, }
@article {pmid39999982, year = {2025}, author = {Chia, BS and Seah, YFS and Wang, B and Shen, K and Srivastava, D and Chew, WL}, title = {Engineering a New Generation of Gene Editors: Integrating Synthetic Biology and AI Innovations.}, journal = {ACS synthetic biology}, volume = {14}, number = {3}, pages = {636-647}, pmid = {39999982}, issn = {2161-5063}, mesh = {*Synthetic Biology/methods ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Artificial Intelligence ; Machine Learning ; Humans ; }, abstract = {CRISPR-Cas technology has revolutionized biology by enabling precise DNA and RNA edits with ease. However, significant challenges remain for translating this technology into clinical applications. Traditional protein engineering methods, such as rational design, mutagenesis screens, and directed evolution, have been used to address issues like low efficacy, specificity, and high immunogenicity. These methods are labor-intensive, time-consuming, and resource-intensive and often require detailed structural knowledge. Recently, computational strategies have emerged as powerful solutions to these limitations. Using artificial intelligence (AI) and machine learning (ML), the discovery and design of novel gene-editing enzymes can be streamlined. AI/ML models predict activity, specificity, and immunogenicity while also enhancing mutagenesis screens and directed evolution. These approaches not only accelerate rational design but also create new opportunities for developing safer and more efficient genome-editing tools, which could eventually be translated into the clinic.}, }
@article {pmid39999874, year = {2025}, author = {Carbajo, CG and Han, X and Savur, B and Upadhyaya, A and Taha, F and Tinti, M and Wheeler, RJ and Yates, PA and Tiengwe, C}, title = {A high-throughput protein tagging toolkit that retains endogenous untranslated regions for studying gene regulation in kinetoplastids.}, journal = {Open biology}, volume = {15}, number = {2}, pages = {240334}, pmid = {39999874}, issn = {2046-2441}, support = {/WT_/Wellcome Trust/United Kingdom ; R03 AI137636/AI/NIAID NIH HHS/United States ; /HI/NHLBI NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems ; *Protozoan Proteins/genetics/metabolism ; *Trypanosoma brucei brucei/genetics/metabolism ; *Gene Expression Regulation ; *Untranslated Regions ; Gene Editing ; *Kinetoplastida/genetics ; }, abstract = {Kinetoplastid parasites cause diseases that threaten human and animal health. To survive transitions between vertebrate hosts and insect vectors, these parasites rely on precise regulation of gene expression to adapt to environmental changes. Since gene regulation in kinetoplastids is primarily post-transcriptional, developing efficient genetic tools for modifying genes at their endogenous loci while preserving regulatory mRNA elements is crucial for studying their complex biology. We present a CRISPR/Cas9-based tagging system that preserves untranslated regulatory elements and uses a viral 2A peptide from Thosea asigna to generate two separate proteins from a single transcript: a drug-selectable marker and a tagged protein of interest. This dual-function design maintains native control elements, allowing discrimination between regulation of transcript abundance, translational efficiency, and post-translational events. We validate the system by tagging six Trypanosoma brucei proteins and demonstrate (i) high-efficiency positive selection and separation of drug-selectable marker and target protein, (ii) preservation of regulatory responses to environmental cues like heat shock and iron availability, and (iii) maintenance of stage-specific regulation during developmental transitions. This versatile toolkit is applicable to all kinetoplastids amenable to CRISPR/Cas9 editing, providing a powerful reverse genetic tool for studying post-transcriptional regulation and protein function in organisms where post-transcriptional control is dominant.}, }
@article {pmid39999849, year = {2025}, author = {Fallon, TK and Knouse, KA}, title = {A roadmap toward genome-wide CRISPR screening throughout the organism.}, journal = {Cell genomics}, volume = {5}, number = {3}, pages = {100777}, pmid = {39999849}, issn = {2666-979X}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Animals ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genome ; }, abstract = {Genome-wide CRISPR screening in the organism has tremendous potential to answer long-standing questions of mammalian physiology and disease. However, bringing this powerful technology in vivo presents unique challenges, including delivering a genome-wide sgRNA library to the appropriate cell type, achieving sufficient coverage of the library, and selecting for the phenotype of interest. In this review, we highlight recent advances in sgRNA delivery, library design, and phenotypic readout that can help overcome these technical challenges and thereby bring high-throughput genetic dissection to an increasing number of tissues and questions. We are excited about the potential for ongoing innovation in these areas to ultimately enable genome-wide CRISPR screening in any cell type of interest in the organism, allowing for unprecedented investigation into diverse questions of mammalian physiology and disease.}, }
@article {pmid39999224, year = {2025}, author = {Gertsenstein, M and Lintott, LG and Nutter, LMJ}, title = {Engineering Base Changes and Epitope-Tagged Alleles in Mice Using Cas9 RNA-Guided Nuclease.}, journal = {Current protocols}, volume = {5}, number = {2}, pages = {e70109}, pmid = {39999224}, issn = {2691-1299}, mesh = {Animals ; Mice ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Alleles ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Epitopes/genetics ; Zygote ; Electroporation ; }, abstract = {Mice carrying patient-associated base changes are powerful tools to define the causality of single-nucleotide variants to disease states. Epitope tags enable immuno-based studies of genes for which no antibodies are available. These alleles enable detailed and precise developmental, mechanistic, and translational research. The first step in generating these alleles is to identify within the target sequence-the orthologous sequence for base changes or the N or C terminus for epitope tags-appropriate Cas9 protospacer sequences. Subsequent steps include design and acquisition of a single-stranded oligonucleotide repair template, synthesis of a single guide RNA (sgRNA), collection of zygotes, and microinjection or electroporation of zygotes with Cas9 mRNA or protein, sgRNA, and repair template followed by screening born mice for the presence of the desired sequence change. Quality control of mouse lines includes screening for random or multicopy insertions of the repair template and, depending on sgRNA sequence, off-target sequence variation introduced by Cas9. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Single guide RNA design and synthesis Alternate Protocol 1: Single guide RNA synthesis by primer extension and in vitro transcription Basic Protocol 2: Design of oligonucleotide repair template Basic Protocol 3: Preparation of RNA mixture for microinjection Support Protocol 1: Preparation of microinjection buffer Alternate Protocol 2: Preparation of RNP complexes for electroporation Basic Protocol 4: Collection and preparation of mouse zygotes for microinjection or electroporation Basic Protocol 5: Electroporation of Cas9 RNP into zygotes using cuvettes Alternate Protocol 3: Electroporation of Cas9 RNP into zygotes using electrode slides Basic Protocol 6: Screening and quality control of derived mice Support Protocol 2: Deconvoluting multiple sequence chromatograms with DECODR.}, }
@article {pmid39998577, year = {2025}, author = {Liu, J and Wang, Y and Zhang, X and Huang, M and Li, G}, title = {Direct lysis combined with amplification-free CRISPR/Cas12a-SERS genosensor for ultrafast and on-site identification of meat authenticity.}, journal = {Mikrochimica acta}, volume = {192}, number = {3}, pages = {187}, pmid = {39998577}, issn = {1436-5073}, mesh = {*CRISPR-Cas Systems ; *Spectrum Analysis, Raman/methods ; *Food Contamination/analysis ; *Meat/analysis ; Limit of Detection ; *Biosensing Techniques/methods ; Animals ; DNA/genetics/analysis ; Polystyrenes/chemistry ; DNA, Single-Stranded/chemistry/genetics ; Nitriles/chemistry ; }, abstract = {A novel direct lysis method combined with amplification-free CRISPR/Cas12a-SERS genosensor was for the first time developed to rapidly and sensitively identify meat adulteration. Notably, polystyrene (PS) microspheres, with distinct shrinking and swelling properties, were dexterously employed to encapsulate biological-silent Raman reporter 4-mercaptobenzonitrile (4-MBN) and act as a controlled-release signal probe. Target DNA activated the trans-cleavage activity of CRISPR/Cas12a towards ssDNA linked with PS microsphere to liberate the signal probe, which was able to release numerous Raman reporters after treatment with THF solution, resulting in high signal amplification. Through this platform, trace target DNA was deftly transformed into a sensitive Raman signal and could be on-site determined through a portable Raman equipment. Under optimized conditions, this strategy displayed good linearity in the range 1-450 ng/μL (R[2] = 0.9943) and favorable sensitivity with limit of detection as low as 0.23 ng/μL without any pre-amplification. Moreover, it exhibited good applicability to on-site identification of commercial meat samples in complicated food matrix. In addition, DNA extraction by direct lysis and amplification-free detection realized ultrafast meat adulteration determination within 35 min from sampling to result. This method possessed great potential in rapid and on-site accurate determination of meat authenticity.}, }
@article {pmid39998241, year = {2025}, author = {Yang, F and Wu, Q and Zeng, X and Jiang, Q and Zhang, S and Wang, J and Zhang, Q and Li, F and Xu, D}, title = {The establishment and optimization of a Mycoplasma pneumoniae detection system based on ERA-CRISPR/Cas12a.}, journal = {Microbiology spectrum}, volume = {13}, number = {4}, pages = {e0323524}, pmid = {39998241}, issn = {2165-0497}, mesh = {*Mycoplasma pneumoniae/genetics/isolation &amp; purification ; Humans ; *Pneumonia, Mycoplasma/diagnosis/microbiology ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Molecular Diagnostic Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {UNLABELLED: Mycoplasma pneumoniae (MP) is a significant pathogen associated with community-acquired pneumonia, with considerable infectious risks posed, particularly to children and immunocompromised individuals. The current methods for detecting MP in research and clinical settings are recognized as time-consuming, instrument-dependent, and prone to non-specific cross-reactivity. Therefore, the creation of a rapid and sensitive detection method is urgently required. In this study, the MP-ERA-Cas12a system, integrating enzyme restriction amplification (ERA) with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a technology, was introduced. Three detection methods were evaluated: the two-pot system, a modified one-pot system, and a lateral flow assay (LFA) strip-based system. In the one-pot system, the amplification and detection steps were consolidated within a single reaction vessel, effectively minimizing the risk of contamination and false positives that may arise from the handling of multiple tubes. It was observed that the one-pot system generated a fluorescent signal within 1 h and produced 1.6 times higher fluorescence signal intensity compared to the two-pot system, achieving a detection limit of 1 copy/μL. In contrast, the LFA system facilitated rapid on-site screening, with visible band results appearing on the strip within 5 min of the reaction, and a detection limit of 10[2] copies/μL was achieved. High specificity for MP was demonstrated by all methods. Significant advantages, including rapid processing, the absence of complex instrumentation, and ease of use are offered by this detection system, making it particularly suitable for resource-limited clinical settings. The system is seen as an efficient tool for the early diagnosis of MP, with substantial public health and clinical relevance.<br><br>IMPORTANCE: This study successfully combined enzyme restriction amplification (ERA) with the specific detection capabilities of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a. Based on the two-pot system established before, the one-pot system and lateral flow assay (LFA) system were developed for Mycoplasma pneumoniae (MP) detection. The MP-ERA-Cas12a system eliminates the need to open the lid during the reaction, reducing aerosol contamination, and minimizing the risk of false positives. The method does not require the use of advanced instruments or equipment and shows strong specificity while not being affected by other pathogens. As a new method of MP detection, the MP-ERA-Cas12a system has an important practical application prospect.}, }
@article {pmid39998178, year = {2025}, author = {van den Berg, DF and Brouns, SJJ}, title = {Reduced prevalence of phage defense systems in Pseudomonas aeruginosa strains from cystic fibrosis patients.}, journal = {mBio}, volume = {16}, number = {4}, pages = {e0354824}, pmid = {39998178}, issn = {2150-7511}, support = {10100322//EC | European Research Council (ERC)/ ; }, mesh = {*Cystic Fibrosis/microbiology/complications ; *Pseudomonas aeruginosa/virology/isolation &amp; purification/genetics/drug effects ; Humans ; *Pseudomonas Infections/microbiology/therapy ; Phage Therapy ; *Bacteriophages/physiology ; *Pseudomonas Phages/physiology ; }, abstract = {Cystic fibrosis is a genetic disorder that affects mucus clearance, particularly of the lungs. As a result, cystic fibrosis patients often experience infections from bacteria, which contribute to the disease progression. Pseudomonas aeruginosa is one of the most common opportunistic pathogens associated with cystic fibrosis. The presence of P. aeruginosa complicates the treatment due to its high antibiotic resistance. Thus, research is ongoing to treat these infections with bacterial viruses instead, known as bacteriophages. Notably, P. aeruginosa clinical strains possess a variety of phage defense mechanisms that may limit the effectiveness of phage therapy. In this study, we compared the defense system repertoire of P. aeruginosa strains isolated from cystic fibrosis patients with those from non-cystic fibrosis patients. Our findings reveal that P. aeruginosa strains isolated from cystic fibrosis patients have fewer phage defense mechanisms per strain than from non-cystic fibrosis patients, suggesting altered phage selection pressures in strains colonizing CF patient lungs.IMPORTANCECystic fibrosis patients often experience chronic Pseudomonas aeruginosa lung infections, which are challenging to treat with antibiotics and contribute to disease progression and eventual respiratory failure. Phage therapy is being explored as an alternative treatment strategy for these infections. However, assessing strain susceptibility to phage treatment is essential for ensuring efficacy. To address this, we investigated whether CF-associated clinical P. aeruginosa strains have a distinct phage defense repertoire compared with those isolated from other lung patients. We observed that CF-associated P. aeruginosa strains have significantly fewer phage defenses, possibly affecting the susceptibility of these strains to phage infection.}, }
@article {pmid39996750, year = {2025}, author = {Mentani, A and Maresca, M and Shiriaeva, A}, title = {Prime Editing: Mechanistic Insights and DNA Repair Modulation.}, journal = {Cells}, volume = {14}, number = {4}, pages = {}, pmid = {39996750}, issn = {2073-4409}, support = {101072427//European Union's Horizon 2021/ ; }, mesh = {*Gene Editing/methods ; *DNA Repair/genetics ; Humans ; Animals ; CRISPR-Cas Systems/genetics ; DNA/genetics ; }, abstract = {Prime editing is a genome editing technique that allows precise modifications of cellular DNA without relying on donor DNA templates. Recently, several different prime editor proteins have been published in the literature, relying on single- or double-strand breaks. When prime editing occurs, the DNA undergoes one of several DNA repair pathways, and these processes can be modulated with the use of inhibitors. Firstly, this review provides an overview of several DNA repair mechanisms and their modulation by known inhibitors. In addition, we summarize different published prime editors and provide a comprehensive overview of associated DNA repair mechanisms. Finally, we discuss the delivery and safety aspects of prime editing.}, }
@article {pmid39996452, year = {2025}, author = {Tang, H and Xu, F and Sun, D and Hua, L and Xiong, J and Xu, M and Xu, J and Zhong, P}, title = {Cas9 Mouse Model of Skull Base Meningioma Driven by Combinational Gene Inactivation in Meningeal Cells.}, journal = {CNS neuroscience &amp; therapeutics}, volume = {31}, number = {2}, pages = {e70287}, pmid = {39996452}, issn = {1755-5949}, mesh = {Animals ; *Meningioma/genetics/pathology ; Mice, Transgenic ; Mice ; *Disease Models, Animal ; *Meningeal Neoplasms/genetics/pathology ; *Meninges/pathology ; Cyclin-Dependent Kinase Inhibitor p16/genetics ; CRISPR-Cas Systems/genetics ; *Skull Base Neoplasms/genetics/pathology ; *Gene Silencing ; Neurofibromin 2/genetics ; Cyclin-Dependent Kinase Inhibitor p15/genetics ; }, abstract = {INTRODUCTION: Neurofibromatosis type 2 (Nf2) gene inactivation is common in sporadic and Nf2-related meningioma. There is currently scant literature describing the development of an intracranial meningioma model in animals. Given the role of Nf2 and other gene inactivation in meningeal cells, we used Cas9 mice here as the background host to establish a new animal model of skull base meningioma in this study.<br><br>AIMS: Cas9 transgenic mice were purchased from Jackson Laboratory and raised in our institution. Subsequently, meningeal cells were obtained from the Cas9 transgenic mice, cultured in medium, and passaged in&#xa0;vitro. We then prepared lentivirus vector pLentiCre/gRNA, which could express the elements blocking the function of four genes: Nf2, P15[Ink4b], P16[Ink4a], and P19[Arf]. We infected the meningeal cells with the lentivirus vector pLentiCre/gRNA and tested the expression of these four genes in those infected meningeal cells. Next, adeno-associated virus vector pAAVCre/gRNA was injected in&#xa0;vivo into the skull base meningeal cells of the neonate Cas9 transgenic mice. These mice were observed once a week and killed 10&#x2009;months later for brain inspection and pathological analysis.<br><br>RESULTS: Twenty Cas9 transgenic mice were successfully bred. Five mice were killed so that meningeal cells could be extracted, cultured, and infected with the lentivirus vector pLentiCre/gRNA for 72&#x2009;h in&#xa0;vitro. The gene function test showed that Nf2, P15[Ink4b], P16[Ink4a], and P19[Arf] were all blocked in the infected meningeal cells, which indicated that the lentivirus vector pLentiCre/gRNA could effectively block the expression of the four genes in targeted cells. Then pAAVCre/gRNA was injected into the skull base meningeal cells of 15 mice in&#xa0;vivo, and nine mice were observed for 10&#x2009;months so that the intracranial tumor growth could be assessed. Among these nine mice, pathological analysis showed that six mice had benign meningioma subtypes similar to human meningioma, one mouse had atypical meningioma, one mouse had malignant meningioma, and one mouse had sarcoma.<br><br>CONCLUSIONS: The Cas9 mouse model of skull base meningioma generated with the Nf2 genetic defect and the combinational loss of P15[Ink4b], P16[Ink4a], and P19[Arf] could provide a new tool for investigating the pathogenesis of meningioma and the development of chemical interventions for this disease.}, }
@article {pmid39995605, year = {2025}, author = {Wang, D and Mandal, P and Rahman, MS and Yang, L}, title = {Engineering tomato disease resistance by manipulating susceptibility genes.}, journal = {Frontiers in genome editing}, volume = {7}, number = {}, pages = {1537148}, pmid = {39995605}, issn = {2673-3439}, abstract = {Various pathogens severely threaten tomato yield and quality. Advances in understanding plant-pathogen interactions have revealed the intricate roles of resistance (R) and susceptibility (S) genes in determining plant immunity. While R genes provide targeted pathogen resistance, they are often vulnerable to pathogen evolution. Conversely, S genes offer a promising avenue for developing broad-spectrum and durable resistance through targeted gene editing. Recent breakthroughs in CRISPR/Cas-based technologies have revolutionized the manipulation of plant genomes, enabling precise modification of S genes to enhance disease resistance in tomato without compromising growth or quality. However, the utilization of the full potential of this technique is challenging due to the complex plant-pathogen interactions and current technological limitations. This review highlights key advances in using gene editing tools to dissect and engineer tomato S genes for improved immunity. We discuss how S genes influence pathogen entry, immune suppression, and nutrient acquisition, and how their targeted editing has conferred resistance to bacterial, fungal, and viral pathogens. Furthermore, we address the challenges associated with growth-defense trade-offs and propose strategies, such as hormonal pathway modulation and precise regulatory edits, to overcome these limitations. This review underscores the potential of CRISPR-based approaches to transform tomato breeding, paving the way for sustainable production of disease-resistant cultivars amidst escalating global food security challenges.}, }
@article {pmid39995348, year = {2025}, author = {Chen, Q and Sun, Y and Yao, J and Lu, Y and Qiu, R and Zhou, F and Deng, Z and Sun, Y}, title = {Engineering of Peptide-Inserted Base Editors with Enhanced Accuracy and Security.}, journal = {Small (Weinheim an der Bergstrasse, Germany)}, volume = {21}, number = {14}, pages = {e2411583}, pmid = {39995348}, issn = {1613-6829}, support = {2018YFA0903200//National Key R&amp;D Program of China/ ; 31920103001//Funds for International Cooperation and Exchange of the National Natural Science Foundation of China/ ; }, mesh = {Humans ; *Peptides/chemistry ; HEK293 Cells ; *Protein Engineering/methods ; Amino Acid Sequence ; Base Sequence ; }, abstract = {Base editors are effective tools for introducing base conversions without double-strand breaks, showing broad applications in biotechnological and clinical areas. However, their non-negligible bystander mutations and off-target effects have raised extensive safety concerns. To address these issues, a novel method is developed by inserting specific peptide fragments into the substrate binding pocket of deaminases in base editors to modify these outcomes. It is validated that the composition and position of the inserted peptide can significantly impact the performance of A3A-based cytosine base editor and TadA-8e-based adenine base editor, leading to improved editing activity and precision in human HEK293T cells. Importantly, the TadA-8e variant with DPLVLRRRQ peptide inserted behind S116 residue showed a strong motif preference of Y4A5N6, which can accurately edit the A5 base in targeted protospacer with minimized bystander and off-target effects in DNA and RNA-level. By summarizing the regularity during engineering, a set of systematic procedures is established, which can potentially be used to modify other types of base editors and make them more accurate and secure. In addition, the peptide insertion strategy is also proven to be compatible with traditional amino acid changes which have been reported, exhibiting excellent compatibility.}, }
@article {pmid39995104, year = {2025}, author = {Bhatt, D and Sundaram, RK and López, KSL and Lee, T and Gueble, SE and Vasquez, JC}, title = {Development of Syngeneic Murine Glioma Models with Somatic Mismatch Repair Deficiency to Study Therapeutic Responses to Alkylating Agents and Immunotherapy.}, journal = {Current protocols}, volume = {5}, number = {2}, pages = {e70097}, doi = {10.1002/cpz1.70097}, pmid = {39995104}, issn = {2691-1299}, mesh = {Animals ; Mice ; Temozolomide/pharmacology/therapeutic use ; *Glioma/genetics/therapy/drug therapy ; *DNA Mismatch Repair/genetics ; *Disease Models, Animal ; *Brain Neoplasms/genetics/therapy/drug therapy ; *Antineoplastic Agents, Alkylating/pharmacology/therapeutic use ; *Immunotherapy/methods ; Cell Line, Tumor ; MutL Protein Homolog 1/genetics ; CRISPR-Cas Systems ; }, abstract = {Glioblastoma (GBM) carries a dismal prognosis, with a median survival of less than 15 months. Temozolomide (TMZ), the standard frontline chemotherapeutic for GBM, is an alkylating agent that generates DNA O[6]-methylguanine (O[6]MeG) lesions. Without O[6]MeG-methyltransferase (MGMT), this lesion triggers the mismatch repair (MMR) pathway and leads to cytotoxicity via futile cycling. TMZ resistance frequently arises via the somatic acquisition of MMR deficiency (MMRd). Moreover, DNA-damaging agents have been shown capable of increasing tumor immunogenicity and improving response to immune checkpoint blockade (ICB), which has had limited success in glioma. The study of how alkylating chemotherapy such as TMZ impacts antitumor immunity in glioma has been hindered by a lack of immunocompetent models that incorporate relevant DNA repair genotypes. Here, we used CRISPR/Cas9 to generate models isogenic for knockout (KO) of Mlh1 in the syngeneic SB28 murine glioma cell line. MMR KO models readily formed intracranial tumors and exhibited in vitro and in vivo resistance to TMZ. In contrast, MMR KO cells maintained sensitivity to KL-50, a newly developed alkylating compound that exerts MGMT-dependent, MMR-independent cytotoxicity. Lastly, MMR KO tumors remained resistant to ICB, mirroring the lack of response seen in patients with somatic MMRd GBM. The development of syngeneic, immunologically cold glioma models with somatic loss of MMR will facilitate future studies on the immunomodulatory effects of alkylating agents in relevant DNA repair contexts, which will be vital for optimizing combinations with ICB. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Validation of mismatch repair knockouts and in vitro sensitivity to alkylating agents Basic Protocol 2: Stereotaxic injection of isogenic SB28 cells in female C57BL/6J mice and in vivo treatment.}, }
@article {pmid39995038, year = {2025}, author = {Yin, N and Yu, H and Zhang, L and Luo, F and Wang, W and Han, X and He, Y and Zhang, Y and Wu, Y and Pu, J and Feng, T and Yang, G and Chen, T and Xie, G}, title = {Regulation of CRISPR trans-cleavage activity by an overhanging activator.}, journal = {Nucleic acids research}, volume = {53}, number = {4}, pages = {}, pmid = {39995038}, issn = {1362-4962}, support = {2022YFC2603800//National Key R&amp;D Program of China/ ; 82172369//National Natural Science Foundation of China/ ; }, mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Bacterial Proteins/metabolism/genetics/chemistry ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; }, abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system exhibits extraordinary capability in the field of molecular diagnosis and biosensing, attributed to its trans-cleavage ability. The precise modulation of performance has emerged as a significant challenge in advancing CRISPR technology to the next stage of development. Herein, we reported a CRISPR/Cas12a regulation strategy based on an overhanging activator. The presence of overhanging domains in activators creates steric hindrances that have a substantial impact on the trans-cleavage activity and activation timing of Cas12a. The trans-cleavage activity of Cas12a can be finely tuned by adjusting the position, length, and complementarity of the overhanging domains. Moreover, specific structures exhibit characteristics of automatic delayed activation. The presence of overhanging domains enables precise and timely activation of Cas12a, facilitating multifunctional applications. This system effectively accomplishes dynamic regulation, programmable release of cargo, logical operations, and multi-enzyme detection. The flexibility and versatility of this simple and powerful CRISPR regulatory strategy will pave the way for expanded applications of CRISPR/Cas in biotechnology, bioengineering, and biomedicine.}, }
@article {pmid39994790, year = {2025}, author = {Jiang, J and Cienfuegos-Gallet, AV and Long, T and Peirano, G and Chu, T and Pitout, JDD and Kreiswirth, BN and Chen, L}, title = {Correction: Intricate interplay of CRISPR-Cas systems, anti-CRISPR proteins, and antimicrobial resistance genes in a globally successful multi-drug resistant Klebsiella pneumoniae clone.}, journal = {Genome medicine}, volume = {17}, number = {1}, pages = {13}, pmid = {39994790}, issn = {1756-994X}, }
@article {pmid39994507, year = {2025}, author = {Hossain, SM and Rao, Y and Hossain, JO and Pritchard, JR and Zhao, B}, title = {goloco: a web application to create genome scale information from surprisingly small experiments.}, journal = {BMC bioinformatics}, volume = {26}, number = {1}, pages = {61}, pmid = {39994507}, issn = {1471-2105}, mesh = {*Genomics/methods ; *Software ; *Internet ; *Genome ; Humans ; CRISPR-Cas Systems ; Phenotype ; }, abstract = {BACKGROUND: Functional genomics aims to decipher gene function by observing cellular changes when specific genes are disrupted using CRISPR technology. However, these experiments are limited by scalability, as comprehensive CRISPR screens require extensive resources, involving millions of cells and thousands of sgRNAs, making large-scale studies challenging. We propose a novel approach with "CRISPR lossy compression" to reduce the complexity of CRISPR screens by focusing on key genetic nodes that can infer genome-wide phenotypes. These condensed sets, comprising 100 to 1,000 genes, enable previously impractical genome-wide screens tractable.<br><br>RESULTS: To make this approach accessible to the wider scientific community, we developed goloco, an interactive web application that allows users to explore genome-scale loss-of-function phenotypes from as few as 100 pooled measurements. The tool is complemented by a wide array of analyses, including volcano plot visualizations, regression and network analyses.<br><br>CONCLUSIONS: This tool goloco empowers researchers to conduct genome-scale functional studies with minimal experimental overhead, broadening the accessibility of large-scale functional genomics research.}, }
@article {pmid39994482, year = {2025}, author = {Huang, L and Simonian, R and Lopez, MA and Karuppasamy, M and Sanders, VM and English, KG and Fabian, L and Alexander, MS and Dowling, JJ}, title = {X-linked myopathy with excessive autophagy: characterization and therapy testing in a zebrafish model.}, journal = {EMBO molecular medicine}, volume = {17}, number = {4}, pages = {823-840}, pmid = {39994482}, issn = {1757-4684}, support = {T32 HD071866/HD/NICHD NIH HHS/United States ; U54 OD030167/OD/NIH HHS/United States ; R01 AR076227/AR/NIAMS NIH HHS/United States ; R01AG075059//HHS | NIH | National Institute on Aging (NIA)/ ; K08NS120812//HHS | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)/ ; K08 NS120812/NS/NINDS NIH HHS/United States ; R01AR076227//HHS | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)/ ; U54OD030167//HHS | NIH | NIH Office of the Director (OD)/ ; R01HD095897//HHS | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; R01 AG075059/AG/NIA NIH HHS/United States ; R01 HD095897/HD/NICHD NIH HHS/United States ; R21AR074006//HHS | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)/ ; }, mesh = {Animals ; Zebrafish/genetics ; *Autophagy ; Disease Models, Animal ; *Muscular Diseases/pathology/genetics/drug therapy ; *Genetic Diseases, X-Linked/pathology/drug therapy/genetics ; Morpholines ; CRISPR-Cas Systems ; Humans ; Zebrafish Proteins/genetics ; Lysosomal Storage Diseases ; }, abstract = {X-linked myopathy with excessive autophagy (XMEA), a rare childhood-onset autophagic vacuolar myopathy caused by mutations in VMA21, is characterized by proximal muscle weakness and progressive vacuolation. VMA21 encodes a protein chaperone of the vacuolar hydrogen ion ATPase, the loss of which leads to lysosomal neutralization and impaired function. At present, there is an incomplete understanding of XMEA, its mechanisms, consequences on other systems, and therapeutic strategies. A significant barrier to advancing knowledge and treatments is the lack of XMEA animal models. Therefore, we used CRISPR-Cas9 editing to engineer a loss-of-function mutation in zebrafish vma21. The vma21 mutant zebrafish phenocopy the human disease with impaired motor function and survival, liver dysfunction, and dysregulated autophagy indicated by lysosomal de-acidification, the presence of characteristic autophagic vacuoles in muscle fibers, altered autophagic flux, and reduced lysosomal marker staining. As proof-of-concept, we found that two drugs, edaravone and LY294002, improve swim behavior and survival. In total, we generated and characterized a novel preclinical zebrafish XMEA model and demonstrated its suitability for studying disease pathomechanisms and identifying potential therapeutic targets.}, }
@article {pmid39993805, year = {2025}, author = {Weidle, UH and Birzele, F}, title = {Prostate Cancer: De-regulated Circular RNAs With Efficacy in Preclinical In Vivo Models.}, journal = {Cancer genomics &amp; proteomics}, volume = {22}, number = {2}, pages = {136-165}, pmid = {39993805}, issn = {1790-6245}, mesh = {Male ; Humans ; *RNA, Circular/genetics ; *Prostatic Neoplasms/genetics/pathology/therapy ; Animals ; Gene Expression Regulation, Neoplastic ; Disease Models, Animal ; }, abstract = {Therapy resistance, including castration-resistance and metastasis, remains a major hurdle in the treatment of prostate cancer. In order to identify novel therapeutic targets and treatment modalities for prostate cancer, we conducted a comprehensive literature search on PubMed to identify de-regulated circular RNAs that influence treatment efficacy in preclinical prostate cancer-related in vivo models. Our analysis identified 49 circular RNAs associated with various processes, including treatment resistance, transmembrane and secreted proteins, transcription factors, signaling cascades, human antigen R, nuclear receptor binding, ubiquitination, metabolism, epigenetics and other target categories. The identified targets and circular RNAs can be further scrutinized through target validation approaches. Down-regulated circular RNAs are candidates for reconstitution therapy, while up-regulated RNAs can be inhibited using small interfering RNA (siRNA), antisense oligonucleotides (ASO) or clustered regularly interspaced short palindromic repeats/CRISPR associated (CRISPR-CAS)-related approaches.}, }
@article {pmid39993709, year = {2025}, author = {Pilz, RA and Skowronek, D and Bonde, LD and Kałużewski, T and Schamuhn, OJ and Busch, R and Gach, A and Rath, M and Steinhagen-Thiessen, E and Felbor, U}, title = {Oxford Nanopore long-read sequencing with CRISPR/Cas9-mediated target selection for accurate characterization of copy number variants in the LDLR gene.}, journal = {European journal of medical genetics}, volume = {74}, number = {}, pages = {105003}, doi = {10.1016/j.ejmg.2025.105003}, pmid = {39993709}, issn = {1878-0849}, mesh = {Humans ; *DNA Copy Number Variations ; *Receptors, LDL/genetics ; *CRISPR-Cas Systems/genetics ; *Hyperlipoproteinemia Type II/genetics/pathology ; *Nanopore Sequencing/methods ; Male ; Female ; }, abstract = {INTRODUCTION: Familial hypercholesterolemia (FH) affects around 1 in 250 people. Most FH cases are caused by pathogenic LDLR variants, with copy number variations (CNVs) accounting for about 10 %. However, short-read gene panel sequencing and multiplex ligation-dependent probe amplification (MLPA) are limited in the specification of CNV breakpoints and the identification of complex structural variants (SVs).<br><br>MATERIALS AND METHODS: We designed crRNAs for Cas9-mediated target selection of LDLR and performed long-read sequencing (LRS) on an Oxford Nanopore MinION device using high-molecular-weight (HMW) DNA or DNA from standard purification. After establishing the LRS approach, we characterized two known LDLR CNVs and tested two individuals with strong clinical evidence of FH but no pathogenic variant in short-read gene panel sequencing.<br><br>RESULTS: Complete coverage of LDLR was achieved for both HMW DNA and DNA from standard purification. LRS allowed us to specify CNV breakpoints and showed that the known LDLR deletion is 19.2&#xa0;kb in size encompassing exons 1-2 and the 5'-untranslated and promoter regions. Furthermore, LRS verified the in tandem localization of a large LDLR duplication covering exons 4-8. Both CNVs were classified as loss-of-function. Moreover, breakpoint information enabled confirmatory analysis by PCR and Sanger sequencing for both CNVs. No SVs were detected in two apparently mutation-negative FH probands using our approach.<br><br>CONCLUSIONS: Nanopore LRS with CRISPR/Cas9-mediated target selection allows for accurate characterization of CNVs and can therefore serve as a complementary method to short-read sequencing-based FH diagnostics by facilitating variant interpretation and enabling cost-effective PCR-based variant confirmation in subsequent familial analyses.}, }
@article {pmid39993558, year = {2025}, author = {Lee, J and Jeon, HH and Seo, E and Park, S and Choe, D and Cho, BK and Lee, JW}, title = {Direct mRNA-to-sgRNA conversion generates design-free ultra-dense CRISPRi libraries for systematic phenotypic screening.}, journal = {Metabolic engineering}, volume = {89}, number = {}, pages = {108-120}, doi = {10.1016/j.ymben.2025.02.011}, pmid = {39993558}, issn = {1096-7184}, mesh = {*Escherichia coli/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *RNA, Messenger/genetics ; *Gene Library ; *CRISPR-Cas Systems ; Indoles ; }, abstract = {CRISPR interference (CRISPRi) is a versatile tool for high-throughput phenotypic screening. However, rational design and synthesis of the single-guide RNA (sgRNA) library required for each genome-wide CRISPRi application is time-consuming, expensive, and unfeasible if the target organisms lack comprehensive sequencing and characterization. We developed an ultra-dense random sgRNA library generation method applicable to any organism, including those that are not well-characterized. Our method converts transcriptome-wide mRNA into 20 nt of sgRNA spacer sequences through enzymatic reactions. The generated sgRNA library selectively binds to the non-template strand of the coding sequence, leading to more efficient repression compared to binding the template strand. We then generated a genome-scale library for Escherichia coli by applying this method and identified essential and auxotrophic genes through phenotypic screening. Furthermore, we tuned the production levels of lycopene and violacein and identified new repression targets for violacein production. Our results demonstrated that a genome-scale sgRNA library can be generated without rational design and can be utilized simultaneously in a range of phenotypic screenings.}, }
@article {pmid39992915, year = {2025}, author = {Gowen, BG and Melton, K and Leong, WI and Khekare, P and McCawley, S and Chan, J and Boivin, P and Jani, V and Cantor, AJ and Tambe, A and Haak-Frendscho, M and Janatpour, MJ and Wei, SC}, title = {Systematic identification and characterization of high efficiency Cas9 guide RNAs for therapeutic targeting of ADAR.}, journal = {PloS one}, volume = {20}, number = {2}, pages = {e0317745}, pmid = {39992915}, issn = {1932-6203}, support = {R44 CA287699/CA/NCI NIH HHS/United States ; }, mesh = {*Adenosine Deaminase/genetics/metabolism ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; *RNA-Binding Proteins/genetics/antagonists &amp; inhibitors ; Gene Knockout Techniques ; CRISPR-Associated Protein 9/genetics ; HEK293 Cells ; }, abstract = {Therapeutic targeting of the adenosine deaminase ADAR has great potential in cancer and other indications; however, it remains unclear what approach can enable effective and selective therapeutic inhibition. Herein, we conduct multi-staged guide RNA screening and identify high efficiency Cas9 guide RNAs to enable a CRISPR/Cas-based approach for ADAR knockout. Through characterization in human primary immune cell systems we observe similar activity with two-part guide RNA and single guide RNA, dose responsive activity, similar guide activity rank order across different cell types, and favorable computational off-target profiles of candidate guide RNAs. We determine that knockout of ADAR using these guide RNAs induces pharmacodynamic responses primarily consisting of immunological responses such as a type I interferon response, consistent with the known function of ADAR as a key regulator of dsRNA sensing. We observe similar biological effects with targeting only the p150 isoform or both p110 and p150 isoforms of ADAR, indicating that at least in the contexts evaluated, loss of p150 ADAR mediates the primary response. These findings provide a resource of well-characterized, high efficiency ADAR-targeting Cas9 guide RNAs suitable for genomic medicines utilizing different delivery modalities and addressing different therapeutic areas.}, }
@article {pmid39992908, year = {2025}, author = {Nemeth, T and Zarnocki, A and Ladanyi, A and Papp, C and Ayaydin, F and Szebeni, GJ and Gacser, A}, title = {PCR-based CRISPR/Cas9 system for fluorescent tagging: A tool for studying Candida parapsilosis virulence.}, journal = {PloS one}, volume = {20}, number = {2}, pages = {e0312948}, doi = {10.1371/journal.pone.0312948}, pmid = {39992908}, issn = {1932-6203}, mesh = {*CRISPR-Cas Systems/genetics ; *Candida parapsilosis/pathogenicity/genetics/isolation &amp; purification ; Animals ; Mice ; Virulence/genetics ; *Polymerase Chain Reaction/methods ; Macrophages/microbiology ; Humans ; Candidiasis/microbiology ; Cell Line ; }, abstract = {Candida parapsilosis is persistent in a hospital environment hence it is often associated with nosocomial infections especially amongst low-birth weight neonates. Genetic modification is therefore important to characterise the physiological and virulence related properties of this fungus. A PCR-based CRISPR/Cas9 system has been adopted to facilitate the generation of fluorescent tagged prototroph isolates. We examined a total of eight fluorescent protein coding genes, out of which three were found to be applicable for simultaneous utilisation. We investigated three clinical isolates of C. parapsilosis in terms of their adherence to silicone and their uptake by J774.2 murine macrophages in competition assays. Interestingly, we found significant differences between them in both experiments where GA1 isolate was significantly less resistant to macrophage uptake and CDC317 was significantly more adherent to silicone material. In silico analysis of the agglutinin-like sequences (Als) exposed remarkable diversity in this protein family and additionally, the thorough analysis of the ALS genes revealed evidence of formation of a new gene by intrachromosomal recombination in the GA1 isolate. Finally, we provide a step by step protocol for the application of the PCR-based CRISPR/Cas9 system for fluorescently labelling C. parapsilosis isolates.}, }
@article {pmid39992407, year = {2025}, author = {Zhang, W and Li, S and Xu, W and Wang, Q and Zhang, H and Liu, X and Chen, X and Xu, D and Chen, H}, title = {Knocking out artificially selected gene GmAOC4[H8] improves germination in soybean.}, journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik}, volume = {138}, number = {3}, pages = {54}, pmid = {39992407}, issn = {1432-2242}, support = {32201845//National Natural Science Foundation of China/ ; 32001455//National Natural Science Foundation of China/ ; ZSBBL-KY2023-03//the Funding of Zhongshan Biological Breeding Laboratory/ ; BK20210154//Natural Science Foundation of Jiangsu Province/ ; JBGS[2021]060//the Open Competition Project of Seed Industry Revitalization of Jiangsu Province/ ; }, mesh = {*Glycine max/genetics/growth &amp; development ; *Germination/genetics ; Cyclopentanes/metabolism ; Oxylipins/metabolism ; Gene Expression Regulation, Plant ; Seeds/genetics/growth &amp; development ; *Plant Proteins/genetics/metabolism ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Genes, Plant ; }, abstract = {Seed germination is an essential stage in the life cycle of flowering plants, influencing the field emergence rates of seeds. Consequently, the role of GmAOC4 in soybean seed germination was investigated in the present study. Results suggested that the chloroplast-localized GmAOC4 exhibited high expression levels in the roots and young pods and during the seed germination stage in soybeans. It was found that GmAOC4 has been artificially selected during soybean domestication and improvement and that GmAOC4[H8] showed repressed seed germination, of which the frequency in landraces and cultivars decreased when compared with wild soybean. Knocking out GmAOC4[H8] via CRISPR/Cas9 led to enhanced germination in gmaoc4 mutants, suggesting its negative regulation on seed germination in soybeans. Additionally, decreased endogenous jasmonic acid (JA) and JA precursor, 12-oxo-phytodienoic acid, were found in gmaoc4 mutants. RNA-seq analyses revealed that 91 and 269 differentially expressed genes (DEGs) were up-regulated and down-regulated in gmaoc4 mutants, respectively. Among these DEGs, three genes were involved in JA biosynthetic and signaling pathways. Our results offer new insights into the mechanism of soybean seed germination regulation by GmAOC4.}, }
@article {pmid39992145, year = {2025}, author = {Wang, J and Zhang, F and Liu, Z and Zhou, Y and Pei, L and Yan, B}, title = {A novel biosensor for highly sensitive DNA damage detection using TdT and CRISPR-Cas12a.}, journal = {The Analyst}, volume = {150}, number = {6}, pages = {1076-1080}, doi = {10.1039/d5an00031a}, pmid = {39992145}, issn = {1364-5528}, mesh = {*Biosensing Techniques/methods ; *DNA Damage ; *CRISPR-Cas Systems ; Humans ; Limit of Detection ; Spermatozoa/chemistry/metabolism ; Male ; *Endodeoxyribonucleases/genetics/metabolism ; DNA/analysis ; *Bacterial Proteins/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism ; Spectrometry, Fluorescence ; }, abstract = {This study presents a highly sensitive fluorescence biosensor integrating TdT enzyme and CRISPR-Cas12a for precise detection of sperm DNA damage, achieving a detection limit of 0.99 pM and a linear range of 0.001-0.2 nM. Its exceptional sensitivity, simplicity, and versatility make it a transformative tool for reproductive medicine and clinical diagnostics.}, }
@article {pmid39990123, year = {2025}, author = {Lee, M and Kim, Y and Lee, HW and Park, Y and Yi, S}, title = {Complete genome sequence of psychrobacter sp. KFRI-CH2-11: A psychrotolerant bacterium with probiotic, biofortification, and antimicrobial potential for the dairy and meat industries.}, journal = {Data in brief}, volume = {59}, number = {}, pages = {111344}, pmid = {39990123}, issn = {2352-3409}, abstract = {This dataset provides the complete genome sequence of Psychrobacter sp. KFRI-CH2-11, isolated from Korean fermented anchovy, Myeolchi-jeotgal. Genomic analysis identified genes involved in Vitamin B12 biosynthesis, carbohydrate metabolism, CRISPR-Cas defense systems, and antioxidant activity, underscoring the strain's potential for use in food biotechnology. Additional genes linked to antibiotic resistance and bioremediation suggest adaptability in diverse environments, particularly cold-chain storage in the dairy and meat industry. PathogenFinder analysis confirmed the absence of pathogenicity-associated genes, validating the strain's suitability as a probiotic and biofortifying agent in food products.}, }
@article {pmid39989823, year = {2025}, author = {Li, C and Zheng, X and Xie, S and Lin, D}, title = {Exonuclease-III Assisted Signal Cycle Integrating with Self-Priming Mediated Chain Extension for Sensitive and Reliable MicroRNA Detection.}, journal = {ACS omega}, volume = {10}, number = {6}, pages = {6228-6233}, pmid = {39989823}, issn = {2470-1343}, abstract = {MicroRNA (miRNA) is pivotal in regulating pathological progression and may serve as a significant biomarker for early diagnosis, treatment, and management strategies for atherosclerosis. This study produced a self-priming amplification-accelerated CRISPR/Cas system-based method for the sensitive and selective detection of miRNA by merging Exo-III-assisted target recycling, self-priming-mediated chain extension, and the CRISPR/Cas12a system. The sensor comprises three stages: (i) the creation of a substrate template via Exo-III mediated target recycling and DNA ligase assisted ligation; (ii) the exponential isothermal reaction facilitated by DNA polymerase for signal amplification; (iii) the trans-cleavage activity of CRISPR/Cas12a after recognizing the amplification product generates signals. We employed miRNA-21 as a target. The strategy enables sensitive detection of miRNA-21 without the use of primers, and the unique design of the CRISPR/sgRNA complex efficiently mitigates background signal interference. The sensor can recognize single-base mutant homologous sequences and demonstrate a steady performance in complicated biological matrices. This sensor has been effectively employed to precisely assess miRNA-21 in engineered clinical samples, showcasing its significant potential in clinical diagnostics and of atherosclerosis.}, }
@article {pmid39989356, year = {2025}, author = {Gu, X and Ma, Z and Zhou, L and Li, N and Yu, S and Wang, F and An, R}, title = {Visual detection of HPV16 using a photoactivatable CRISPR-Cas12 system.}, journal = {Chemical communications (Cambridge, England)}, volume = {61}, number = {22}, pages = {4383-4386}, doi = {10.1039/d5cc00369e}, pmid = {39989356}, issn = {1364-548X}, mesh = {*CRISPR-Cas Systems ; *Human papillomavirus 16/isolation &amp; purification/genetics ; Humans ; Ultraviolet Rays ; Nucleic Acid Amplification Techniques ; Female ; Uterine Cervical Neoplasms/diagnosis/virology ; DNA, Viral/genetics/analysis ; }, abstract = {Human papillomavirus (HPV) screening is crucial for the diagnosis of cervical cancer. In this study, we have combined photoactivated CRISPR-Cas12a with tube-in-tube structure and recombinase polymerase amplification (RPA) to enable simple, rapid and convenient visualization detection of HPV16, facilitated by blue UV light at 302 nm. It serves as a potential tool for on-site diagnostic use, which could be beneficial in terms of portability and speed.}, }
@article {pmid39989066, year = {2025}, author = {Xuan, Q and Zhou, H and Zhu, M and Wang, J and Liang, W}, title = {[Creation of new glabrous and salt-tolerant rice germplasm along the Yellow River by CRISPR-Cas9-mediated editing of OsSPL10].}, journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology}, volume = {41}, number = {2}, pages = {706-718}, doi = {10.13345/j.cjb.240308}, pmid = {39989066}, issn = {1872-2075}, mesh = {*Oryza/genetics/physiology ; *CRISPR-Cas Systems/genetics ; *Salt Tolerance/genetics ; *Gene Editing/methods ; *Plant Proteins/genetics ; Rivers ; Plant Leaves/genetics ; Mutation ; Plants, Genetically Modified/genetics ; China ; }, abstract = {The OsSPL10 gene has previously been reported to positively regulate trichome development and negatively regulate salt and drought stress tolerance in rice. However, it is not clear whether this gene can be used for gene editing to create new germplasm of glabrous leaf and salt-tolerant rice. In this study, we created six rice mutants by CRISPR/Cas9-mediated editing of OsSPL10 from 'Xinfeng 2', 'Xinkedao 31', and 'Xindao 25', the main rice cultivars along the Yellow River. Visual observation and scanning electron microscopy verified that the mutants lacked trichomes on the leaves and glumes, and the expression of glabrous marker genes OsHL6, OsGL6, and OsWOX3B in mutants was down-regulated compared with that in the wild type. The net photosynthetic rate, stomatal conductance, and transpiration rate of flag leaves in the mutants were significantly higher than those in the wild type. In addition, the survival rates of the mutants were much higher than that of the wild type after 7 days of treatment with 200 mmol/L NaCl. The results of quantitative real-time polymerase chain reaction (qRT-PCR) further verified that compared with the wild type, the mutants demonstrated down-regulated expression of the salt stress-related gene OsGASR1 and up-regulated expression of OsNHX2 and OsIDS1. Statistical analysis of agronomic traits showed that the mutants had increased plant height and no significant changes in yield-related traits compared with the wild type. The six spl10 mutants created in this study not only had glabrous leaves and glumes but also demonstrated enhanced tolerance to salt stress, serving as new germplasm resources for directional breeding of rice along the Yellow River.}, }
@article {pmid39989063, year = {2025}, author = {Ren, Q and Lan, H and Liu, T and Zhao, H and Zhao, Y and Zhang, R and Liu, J}, title = {[Partial knockout of NtPDK1a/1b/1c/1d enhances the disease resistance of Nicotiana tabacum].}, journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology}, volume = {41}, number = {2}, pages = {670-679}, doi = {10.13345/j.cjb.240231}, pmid = {39989063}, issn = {1872-2075}, mesh = {*Nicotiana/genetics/immunology/microbiology/virology ; *Disease Resistance/genetics ; *Plant Diseases/genetics/microbiology/virology/immunology ; Plants, Genetically Modified/genetics ; Gene Knockout Techniques ; *Plant Proteins/genetics ; CRISPR-Cas Systems ; *Protein Serine-Threonine Kinases/genetics ; 3-Phosphoinositide-Dependent Protein Kinases/genetics ; Pyruvate Dehydrogenase Acetyl-Transferring Kinase ; Tobacco Mosaic Virus/pathogenicity ; }, abstract = {The protein kinase A/protein kinase G/protein kinase C-family (AGC kinase family) of eukaryotes is involved in regulating numerous biological processes. The 3-phosphoinositide- dependent protein kinase 1 (PDK1), is a conserved serine/threonine kinase in eukaryotes. To understand the roles of PDK1 homologous genes in cell death and immunity in tetraploid Nicotiana tabacum, the previuosly generated transgenic CRISPR/Cas9 lines, in which 5-7 alleles of the 4 homologous PDK1 genes (NtPDK1a/1b/1c/1d homologs) simultaneously knocked out, were used in this study. Our results showed that the hypersensitive response (HR) triggered by transient overexpression of active Pto (Pto[Y207D]) or soybean GmMEKK1 was significantly delayed, whereas the resistance to Pseudomonas syrangae pv. tomato DC3000 (Pst DC3000) and tobacco mosaic virus (TMV) was significantly elevated in these partial knockout lines. The elevated resistance to Pst DC3000 and TMV was correlated with the elevated activation of NtMPK6, NtMPK3, and NtMPK4. Taken together, our results indicated that NtPDK1s play a positive role in cell death but a positive role in disease resistance, likely through negative regulation of the MAPK signaling cascade.}, }
@article {pmid39988953, year = {2025}, author = {Nigam, D and Devkar, V and Dhiman, P and Shakoor, S and Liu, D and Patil, GB and Jiao, Y}, title = {Emerging frontiers in sorghum genetic engineering.}, journal = {The Plant journal : for cell and molecular biology}, volume = {121}, number = {4}, pages = {e17244}, doi = {10.1111/tpj.17244}, pmid = {39988953}, issn = {1365-313X}, mesh = {*Sorghum/genetics/physiology ; *Genetic Engineering/methods ; Gene Editing ; Plants, Genetically Modified/genetics ; CRISPR-Cas Systems ; Edible Grain/genetics ; }, abstract = {Sorghum, a climate-resilient cereal, is crucial for meeting the growing demand for food and feed in arid and semi-arid regions, especially amid global population growth and climate change. Despite its natural drought tolerance and adaptability, sorghum faces challenges in increasing yield, enhancing resistance to abiotic and biotic stresses, and improving grain quality. Genetic engineering has emerged as a powerful tool to address these challenges by directly modifying genes associated with desirable traits. Recent advancements have utilized morphogenic regulators to improve transformation and regeneration efficiency in sorghum. This review explores the status of genomic resources and genetic diversity in sorghum, highlighting the advancements and challenges faced in its genetic engineering efforts. Genome editing technologies, particularly CRISPR/Cas systems, have improved key agronomic traits such as stress tolerance, nutrient use efficiency, and grain quality. However, significant obstacles still need to be addressed, including low regeneration rates, high genotype dependency, and labor-intensive transformation processes. We highlight potential strategies to overcome these barriers, such as optimizing transformation protocols, exploring alternative explants, using morphogenic regulators and advancing tissue culture techniques. Additionally, we discuss the biosafety considerations and potential applications of genetically engineered sorghum in global agriculture. This review underscores the need for ongoing innovation to unlock the potential of genetically engineered sorghum in addressing global food security challenges.}, }
@article {pmid39988667, year = {2025}, author = {Bayarsaikhan, D and Bayarsaikhan, G and Lee, J and Okano, T and Kim, K and Lee, B}, title = {Development of iPSC-derived FIX-secreting hepatocyte sheet as a novel treatment tool for hemophilia B treatment.}, journal = {Stem cell research &amp; therapy}, volume = {16}, number = {1}, pages = {88}, pmid = {39988667}, issn = {1757-6512}, mesh = {*Hepatocytes/metabolism/transplantation/cytology ; *Hemophilia B/therapy/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Animals ; Humans ; *Factor IX/metabolism/genetics ; Mice ; Mice, SCID ; Cell Differentiation ; Mice, Inbred NOD ; Gene Editing ; CRISPR-Cas Systems ; Male ; }, abstract = {BACKGROUND: Hemophilia B is an inherited disorder caused by a mutation in the FIX gene, which results in insufficient blood clotting factor IX (FIX) production from hepatocytes. Currently, there are no treatments for hemophilia B patients. The patients should be continuously administrated with clotting factor concentrates 2-3 times a month to prevent bleeding. Therefore, this study aimed to develop an engineered FIX-secreting hepatocyte sheet that can release FIX for an extended period. Within this study, the engineered FIX-secreting hepatocyte sheet was developed by integrating two core technologies, including a gene editing platform to generate FIX-secreting cells and cell sheet technology to improve cell delivery efficacy.<br><br>METHODS: The human FIX gene was inserted into the APOC3 site of iPSCs by CRISPR/Cas9, which secretes the target protein after differentiation into hepatocytes. FIX-secreting hepatocyte sheets were obtained by temperature-responsive polymer grafted cell culture dishes (TRCD). Immunohistochemical and functional tests were performed for hepatocyte-like cells differentiated from FIX KI-iPSCs and wild-type iPSCs (WT-iPSCs). After validating the functional activity and secretion of FIX protein, the engineered hepatocyte-like cell sheets were transplanted to NOD/SCID mice for the in vivo experiments.<br><br>RESULTS: The insertion of the human FIX gene into the APOC3 site demonstrated a significant increase in FIX secretion in hepatocyte-like cells differentiated from FIX KI-iPSCs compared with those obtained from WT-iPSCs. Among the iPSCs to hepatocyte differentiation stages, the hepatic endoderm stage was most suitable for seeding the cells on TRCD and generating cell sheets by temperature changes from 37[o]C to room temperature when the hepatocyte-like cells have reached maturity. The engineered FIX-secreting cell sheets showed intensive expression of the FIX proteins without losing hepatocyte morphology for 20 days. Furthermore, an in vivo study showed that engineered FIX-secreting cell sheets retained their FIX secretion functions for two weeks, whereas single-cell injected traditionally were barely detected in the experimental animals.<br><br>CONCLUSIONS: The engineered FIX-secreting cell sheets fabricated from functionally improved iPSCs with practical cell delivery tools could be a promising tool for clinically treating Hemophilia B.}, }
@article {pmid39988603, year = {2025}, author = {Cocito, C and Xiang, C and Huang, M and Gongora, T and Surana, P and Davuluri, R and Dahmane, N and Greenfield, JP}, title = {Immunoglobulin superfamily 3 (Igsf3) function is dispensable for brain development.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {6526}, pmid = {39988603}, issn = {2045-2322}, support = {R01 NS111997/NS/NINDS NIH HHS/United States ; 5RO1NS111997//NIH-NINDS/ ; }, mesh = {Animals ; Mice ; Mice, Knockout ; *Brain/metabolism/growth &amp; development/embryology ; Neurons/metabolism/cytology ; Cell Movement ; Neurogenesis ; *Membrane Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Gene Expression Regulation, Developmental ; }, abstract = {The Immunoglobulin superfamily (IgSF) is a heterogeneous and conserved family of adhesion proteins crucial during the development of the central nervous system including neuronal migration and synaptogenesis. The Immunoglobulin superfamily member 3 (IGSF3) is expressed in the developing brain and has been suggested to play a role during morphological development of the granule cells neurites in the cerebellum. In addition, a role for IGSF3 in supporting glioma progression has been recently demonstrated. Remaining unexplored is the physiological role of IGSF3 in regulating brain development, including neocortical development. We generated an Igsf3 knockout (KO) mouse using a CRISPR/Cas9-based approach and explored the function of Igsf3 in regulating cortical development. We found that Igsf3 largely co-localizes with other IgSF proteins during cortical development and despite its expression being developmentally regulated in neuronal progenitors and in postmitotic neurons, Igsf3 is not essential for brain development, neuronal migration, or neuronal maturation.}, }
@article {pmid39988220, year = {2025}, author = {Marnis, H and Syahputra, K}, title = {Advancing fish disease research through CRISPR-Cas genome editing: Recent developments and future perspectives.}, journal = {Fish &amp; shellfish immunology}, volume = {160}, number = {}, pages = {110220}, doi = {10.1016/j.fsi.2025.110220}, pmid = {39988220}, issn = {1095-9947}, mesh = {Animals ; *Gene Editing/veterinary ; *CRISPR-Cas Systems ; *Fish Diseases/genetics/immunology ; Fishes/genetics ; Aquaculture ; Animals, Genetically Modified ; }, abstract = {CRISPR-Cas genome editing technology has transformed genetic research, by enabling unprecedented precision in modifying DNA sequences across various organisms, including fish. This review explores the significant advancements and potential uses of CRISPR-Cas technology in the study and management of fish diseases, which pose serious challenges to aquaculture and wild fish populations. Fish diseases cause significant economic losses and environmental impacts, therefore effective disease control a top priority. The review highlights the pivotal role of CRISPR-Cas in identifying disease-associated genes, which is critical to comprehending the genetic causes of disease susceptibility and resistance. Some studies have reported key genetic factors that influence disease outcomes, using targeted gene knockouts and modifications to pave the way for the development of disease-resistant fish strains. The creation of such genetically engineered fish holds great promise for enhancing aquaculture sustainability by reducing the reliance on antibiotics and other conventional disease control measures. In addition, CRISPR-Cas has facilitated in-depth studies of pathogen-host interactions, offering new insights into the mechanisms by which pathogens infect and proliferate within their hosts. By manipulating both host and pathogen genes, this technology provides a powerful tool for uncovering the molecular underpinnings of these interactions, leading to the development of more effective treatment strategies. While CRISPR-Cas has shown great promise in fish research, its application remains limited to a few species, primarily model organisms and some freshwater fish. In addition, challenges such as off-target effects, ecological risks, and ethical concerns regarding the release of genetically modified organisms into the environment must be carefully addressed. This review also discusses these challenges and emphasizes the need for robust regulatory frameworks and ongoing research to mitigate risks. Looking forward, the integration of CRISPR-Cas with other emerging technologies, such as multi-omics approaches, promises to further advance our understanding and management of fish diseases. This review concludes by envisioning the future directions of CRISPR-Cas applications in fish health, underscoring its potential to its growing in the field.}, }
@article {pmid39988206, year = {2025}, author = {Rashid, MHO and Kayesh, MEH and Hashem, MA and Hifumi, T and Ogawa, S and Miyoshi, N and Tanaka, Y and Kohara, M and Tsukiyama-Kohara, K}, title = {Adeno-associated virus 2 CRISPR/Cas9-mediated targeting of hepatitis B virus in tree shrews.}, journal = {Virus research}, volume = {354}, number = {}, pages = {199550}, pmid = {39988206}, issn = {1872-7492}, mesh = {Animals ; *Hepatitis B virus/genetics ; *Dependovirus/genetics ; *CRISPR-Cas Systems ; Liver/virology/pathology ; Disease Models, Animal ; Virus Replication ; Tupaiidae/virology ; *Hepatitis B/virology/therapy ; Viral Load ; Hepatocytes/virology ; Tupaia/virology ; Humans ; *Hepatitis B, Chronic/virology/therapy ; Genetic Vectors/genetics ; Hepatitis B Core Antigens ; DNA, Viral ; }, abstract = {Chronic hepatitis B virus (HBV) infection is a global health issue with limited therapeutic options given the persistence of viral episomal DNA (cccDNA). Previously, we investigated the effects of adeno-associated virus 2 (AAV2) vector-mediated delivery of three guide (g)RNAs/Cas9 selected from 16 gRNAs. AAV2/WJ11-Cas9 effectively suppressed HBV replication in vitro and in humanized chimeric mouse livers. In the present study, we examined the effect of AAV2/WJ11-Cas9 on the acute phase of HBV genotype F infection in an immunocompetent northern tree shrew (Tupaia belangeri; hereafter, "tupaia") model. AAV2/WJ11-Cas9 treatment significantly reduced the HBV viral load in serum at 1, 7, 10, and 14 days post-infection (dpi). HBV-F infection caused enlargement of hepatocytes and mild lymphocytic infiltration in the interlobular connective tissue. Thus, the virus damages hepatocytes and drives infection progression and HBV core antigen (HBcAg) accumulation, which were not observed in AAV2/WJ11-Cas9 treated and normal liver tissues. AAV2/WJ11-Cas9 treatment reduced HBV DNA and cccDNA in liver tissues, as well as serum levels of HBV surface antigen and HBV core-related antigen (HBcrAg), including HBcAg and HBeAg at 14 dpi. Anti-HBc, anti-HBs, and anti-AAV Abs production was also detected. AAV2/WJ11-Cas9 treatment suppressed inflammatory cytokines and TLR1, TLR2, TLR3, TLR4, TLR6, TLR7, and TLR9 mRNA levels. Thus, WJ11/Cas9 delivered by AAV2 vectors may provide a new therapeutic approach for inhibiting HBV infection in immunocompetent animal models, which could be developed for use in humans through further translational research.}, }
@article {pmid39988189, year = {2025}, author = {Wei, L and Hu, S and Gong, X and Ahemaiti, Y and Li, D and Ouyang, S and Huang, Y and Wang, Y and Liang, Y and Deng, Y and Liu, L and Zhao, T}, title = {Disrupted maxillofacial, cardiovascular, and nervous development in washc5 knockout Zebrafish: Insights into 3C syndrome.}, journal = {Gene}, volume = {948}, number = {}, pages = {149351}, doi = {10.1016/j.gene.2025.149351}, pmid = {39988189}, issn = {1879-0038}, mesh = {Animals ; Zebrafish/genetics ; *Zebrafish Proteins/genetics/metabolism ; Gene Knockout Techniques ; *Craniofacial Abnormalities/genetics ; *Abnormalities, Multiple/genetics ; *Cardiovascular Abnormalities/genetics ; Disease Models, Animal ; Gene Expression Regulation, Developmental ; CRISPR-Cas Systems ; }, abstract = {3C syndrome features craniofacial, nervous, and cardiovascular malformations. WASHC5 gene mutations may underline this syndrome, but the pathogenicity and underlying mechanism remain undetermined. We analyzed the expression pattern of the washc5 gene in zebrafish using whole-body in situ hybridization and generated a zebrafish model with washc5 gene knockout using CRISPR/Cas9 technology. Homozygous zebrafish exhibited high mortality, retarded growth, lighter stripes, and reduced pigmentation around the pupils. In the maxillofacial region, homozygotes displayed a shortened and tilted maxilla and delayed ossification of bones. In the heart, homozygous zebrafish showed a decreased heart rate, increased ventricular area, disorganized ventricular muscle fibers, mitochondrial swelling, Golgi lysis, and endoplasmic reticulum (ER) lysis in ventricular myocytes. The mRNA levels of nppb and myh7 were significantly increased. In the nervous system, homozygotes displayed bradykinesia and impaired neuronal development. qRT-PCR analysis revealed downregulation of col1a2, col1a1a, col1a1b, sp7, and msx2b (osteogenic factors and regulators of maxillofacial skeletal development) and abnormal expression of alpk2, alpk3b, actc2 (cardiac development factors), as well as tsen54, exosc8, and exosc9 (cerebellar development factors). Enrichment analysis of differentially expressed genes and proteins indicated involvement in ER-related processes. The washc5 knockout zebrafish model exhibits phenotypic similarities to human 3C syndrome, suggesting that mutations of this gene may play a pathogenic role in the syndrome. The mechanism of the washc5 gene in 3C syndrome may be associated with disturbances in ER homeostasis, providing insights into potential gene therapy strategies.}, }
@article {pmid39987655, year = {2025}, author = {Xu, H and Chen, Q and Meng, X and Yan, C and Yao, B and Chen, Z and Wang, Z and Chen, W}, title = {CRISPR/Cas12a-mediated cyclic signal amplification and electrochemical reporting strategy for rapid and accurate sensing of Vibrio parahaemolyticus in aquatic foods.}, journal = {Biosensors &amp; bioelectronics}, volume = {277}, number = {}, pages = {117284}, doi = {10.1016/j.bios.2025.117284}, pmid = {39987655}, issn = {1873-4235}, mesh = {*Vibrio parahaemolyticus/isolation &amp; purification/genetics/pathogenicity ; *Biosensing Techniques/methods ; Electrochemical Techniques/methods ; *CRISPR-Cas Systems/genetics ; Food Microbiology ; Limit of Detection ; Animals ; Nucleic Acid Amplification Techniques/methods ; Food Contamination/analysis ; Seafood/microbiology ; Fishes/microbiology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Rapid and accurate detection of target foodborne pathogenic bacteria is extremely important for preventing and controlling foodborne diseases. Vibrio parahaemolyticus (V. parahaemolyticus, Vp) is considered as a major cause of foodborne diseases, posing severe threat to food safety and public health. The efficiency and sensitivity of traditional protocols for Vp identification is time consuming and of poor precision. In this research, a simple electrochemical sensing method was developed for accurate detection of Vp in aquatic products. Target genes of Vp were rapid amplified with the designed recombinase polymerase amplification, which further activated the designed CRISPR/Cas12a system. The electrochemical active ssDNA probe on the sensing interface would be hydrolyzed by the activated trans-cleavage activity of Cas12a, inducing the release of active electrochemical tags from the sensing interface and the decreased sensing signals. Under the optimized conditions, this proposed RPA-mediated electrochemical-CRISPR (E-CRISPR) biosensor enabled sensitive detection of target Vp over a linear range from 10[1] to 10[6] CFU/mL, with limit of detection of 32 CFU/mL. Additionally, this E-CRISPR biosensor realized the successful determination of Vp in spiked fish samples with satisfied sensing performance. The isothermal amplification and the rapid electrochemical response of the E-CRISPR biosensor made it suitable for on-site screening. And this E-CRISPR biosensor could be well integrated with other isothermal protocols and extended to other target pathogens, showing great potential for practical applications in molecular diagnostics and other gene detection related fields.}, }
@article {pmid39987590, year = {2025}, author = {Zhang, L and Zhang, F and Yao, M}, title = {CRISPR/Cas9-mediated generation of a homozygous CRB2 knockout H1 human embryonic stem cell line.}, journal = {Stem cell research}, volume = {84}, number = {}, pages = {103677}, doi = {10.1016/j.scr.2025.103677}, pmid = {39987590}, issn = {1876-7753}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; Homozygote ; *Membrane Proteins/genetics/deficiency/metabolism ; Cell Line ; Gene Knockout Techniques ; Cell Differentiation ; *Eye Proteins/genetics ; Carrier Proteins ; }, abstract = {Mutations in the Crumbs homolog 2 (CRB2) gene cause various autosomal recessive genetic diseases, such as leber congenital amaurosis, retinitis pigmentosa and ventriculomegaly with cystic kidney disease. However, the precise roles of CRB2 in cell fate determination remains unknown. Here, we generated a homozygous CRB2 knockout (CRB2[-/-]) H1 human embryonic stem cells (hESCs) using CRISPR/Cas9 system. This cell line maintained a normal morphology and karyotype, and expressed the pluripotency makers. Importantly, the cell line has the ability to differentiate into three germ layers. The CRB2[-/-] hESCs provide valuable resources for studying the mechanisms of genetic diseases caused by CRB2 mutations.}, }
@article {pmid39987415, year = {2025}, author = {Goh, SYC and Fradera-Sola, A and Wittkopp, N and Şerifoğlu, N and Godinho Ferreira, M and Ketting, RF and Butter, F}, title = {Zbtb48 is a regulator of Mtfp1 expression in zebrafish.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {277}, pmid = {39987415}, issn = {2399-3642}, support = {Bu2996-1/3//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; }, mesh = {Animals ; *Zebrafish/genetics/metabolism ; *Zebrafish Proteins/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; Female ; Male ; Gene Expression Regulation, Developmental ; CRISPR-Cas Systems ; }, abstract = {ZBTB48 (also known as TZAP) is a transcription factor that has previously been reported to bind to telomeres and act as a negative regulator of telomere length in human cell lines. To explore whether transcription factor activity and telomere length regulation are conserved at the organismal level in vertebrates, we generate a zbtb48[-/-] zebrafish line via CRISPR‒Cas genome editing. The zbtb48[-/-] mutants display no obvious physical or behavioral abnormalities in the first two generations. We find no statistically significant changes in telomere length in first-generation adults. However, for the gene regulatory aspect of Zbtb48, similar to that in human cancer cell lines, we observe downregulation of mtfp1 at both the mRNA and protein levels in the zbtb48[-/-] mutants. This suggests that mtfp1 is an evolutionarily conserved regulatory target of Zbtb48. Further investigation of the spatiotemporal expression of zbtb48 in previously published zebrafish data reveals low transcript expression in diverse tissues, except in germline stem cells and gametocytes of the gonads. Notably, Mtfp1 protein downregulation is detected in the ovaries of 40 dpf zbtb48[-/-] mutants and in the testes of both 40 dpf and 10.5-month-old zbtb48[-/-] mutants.}, }
@article {pmid39987186, year = {2025}, author = {Yan, H and Xin, Z and Sang, Z and Li, X and Xie, J and Wu, J and Pang, S and Wen, Y and Wang, W}, title = {A rational multi-target combination strategy for synergistic improvement of non-ribosomal peptide production.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {1883}, pmid = {39987186}, issn = {2041-1723}, support = {32170095//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32100066//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32170081//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {Daptomycin/biosynthesis/metabolism ; Peptides, Cyclic/biosynthesis ; Lipopeptides/biosynthesis ; *Streptomyces/metabolism/genetics ; *Metabolic Engineering/methods ; CRISPR-Cas Systems ; Anti-Bacterial Agents/biosynthesis ; }, abstract = {Non-ribosomal peptides (NRPs) are pharmaceutically important natural products that include numerous clinical drugs. However, the biosynthesis of these NRPs is intricately regulated and improving production through manipulation of multiple regulatory targets remains largely empirical. We here develop a screening-based, multi-target rational combination strategy and demonstrate its effectiveness in enhancing the titers of three NRP drugs - daptomycin, thaxtomin A and surfactin. Initially, we devise a reliable colorimetric analog co-expression and co-biosynthesis reporter system for screening high-yielding phenotypes. Subsequently, through coupling CRISPR interference to induce genome-wide differential expression, we identify dozens of repressors that inhibit the biosynthesis of these NRPs. To address the challenge of multi-target combination, we further developed a dual-target screen approach and introduced an interplay map based on the synergy coefficient of each pairwise interaction. Employing this strategy, we engineer the final strains with multi-target synergistic combination and achieve the titer improvement of the three NRPs. Our work provides a rational multi-target combination strategy for production improvement of NRPs.}, }
@article {pmid39985663, year = {2025}, author = {Kang, B and Venkatesh, J and Lee, JH and Kim, JM and Kwon, JK and Kang, BC}, title = {CRISPR/Cas9-mediated editing of eukaryotic elongation factor 1B gamma (eEF1Bγ) reduces Tobacco etch virus accumulation in Nicotiana benthamiana.}, journal = {Plant cell reports}, volume = {44}, number = {3}, pages = {62}, pmid = {39985663}, issn = {1432-203X}, support = {RS-2024-00322053//Rural Development Administration/ ; RS-2024-00398300//Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry/ ; }, mesh = {*Nicotiana/virology/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Peptide Elongation Factor 1/genetics/metabolism ; Plant Diseases/virology/genetics ; *Plant Proteins/genetics/metabolism ; Potexvirus/physiology ; Plants, Genetically Modified ; Disease Resistance/genetics ; Plant Viruses ; }, abstract = {Tobacco etch virus accumulation declined in Nicotiana benthamiana eEF1Bγ gene-edited lines, suggesting that eEF1Bγ may be a host factor for this virus. Viruses use host factors to replicate and move from cell to cell. Therefore, the editing of genes encoding viral host factors that are not essential for plant survival enables the rapid development of plants with durable virus resistance. Eukaryotic initiation factors, such as eIF4E and eIF4G, function as host factors for viral infection, and loss-of-function mutations of these factors lead to virus resistance. Broadening the spectrum of host factor targets would help expand resources for engineering virus resistance. In this study, we tested whether editing the eukaryotic translation elongation factor gene eEF1Bγ would produce virus-resistant plants. Accordingly, we targeted the four eEF1Bγ genes in Nicotiana benthamiana for editing using virus-induced gene editing (VIGE) with Tobacco rattle virus (TRV). Although we attempted to obtain plants edited for all four eEF1Bγ homologs, we failed to identify such plants. Instead, we obtained plants with three of the four homologs knocked out, harboring 1-bp insertion/deletions resulting in premature stop codons. These eEF1Bγ-edited plants did not exhibit resistance to Potato virus X (PVX), Tobacco mosaic virus (TMV), or Tomato bushy stunt virus (TBSV) but showed reduced accumulation of Tobacco etch virus (TEV) compared to wild-type plants. These findings demonstrate the feasibility of conferring resistance in plants through gene editing of eEF1Bγ, underscoring the importance of exploring diverse host factor targets for comprehensive virus resistance.}, }
@article {pmid39985020, year = {2025}, author = {Wu, YF and Chen, JA and Jong, YJ}, title = {Treating neuromuscular diseases: unveiling gene therapy breakthroughs and pioneering future applications.}, journal = {Journal of biomedical science}, volume = {32}, number = {1}, pages = {30}, pmid = {39985020}, issn = {1423-0127}, support = {113-2326-B-001-001//National Science and Technology Council/ ; 112-2321-B-037-006//National Science and Technology Council/ ; 113-2321-B-037-008//National Science and Technology Council/ ; NHRI-EX113-11330NI//National Health Research Institutes/ ; AS-GCP-113-L02//Academia Sinica/ ; AS-BRPT-113-01//Academia Sinica/ ; }, mesh = {*Genetic Therapy/methods ; Humans ; *Neuromuscular Diseases/therapy/genetics ; *Muscular Atrophy, Spinal/therapy/genetics ; *Muscular Dystrophy, Duchenne/therapy/genetics ; Dependovirus/genetics ; Animals ; CRISPR-Cas Systems ; }, abstract = {In this review, we highlight recent advancements in adeno-associated virus (AAV)-based gene therapy for genetic neuromuscular diseases (NMDs), focusing on spinal muscular atrophy (SMA) and Duchenne muscular dystrophy (DMD). We discuss the current FDA-approved gene therapies for NMDs and provide updates on preclinical studies that demonstrate the potential of various AAV-based gene therapies to reduce SMA severity and serve as effective treatments for DMD. Additionally, we explore the transformative impact of CRISPR/Cas9 technology on the future of gene therapy for NMDs. Despite these encouraging developments, further research is required to identify robust biomarkers that can guide treatment decisions and predict outcomes. Overall, these pioneering advancements in AAV-based gene therapy lay the groundwork for future efforts aimed at curing genetic NMDs and offer a roadmap for developing gene therapies for other neurodegenerative diseases.}, }
@article {pmid39984107, year = {2025}, author = {Li, Y and Zhao, L and Ma, L and Bai, Y and Feng, F}, title = {Argonaute protein powered biosensing for pathogenic biosafety.}, journal = {International journal of biological macromolecules}, volume = {305}, number = {Pt 2}, pages = {141321}, doi = {10.1016/j.ijbiomac.2025.141321}, pmid = {39984107}, issn = {1879-0003}, mesh = {*Biosensing Techniques/methods ; *Argonaute Proteins/metabolism/genetics ; Humans ; CRISPR-Cas Systems ; Food Safety/methods ; Animals ; }, abstract = {The food safety and medical health issues caused by pathogen are particularly prominent. The development of biosensing technologies is urgent to ensure pathogenic biosafety. Argonaute system, as a promising and cutting-edged next-generation nucleic acid test technology, has the potential to address the challenges faced by CRISPR/Cas system. In this review, we focused on the current state-of-art Argonaute-powered biosensing for pathogenic biosafety. First, we introduced current methods for nucleic acid testing and programmable nucleases, followed by the working principle of Argonaute system (PfAgo, TtAgo, CbAgo, etc). Then Argonaute-medicated nucleic acid biosensing was highlighted through amplification and amplification-free manners. In addition, we summarized the application of Argonaute tools in detecting bacteria, virus, mycoplasma, etc. Finally, we pointed out the challenges and perspectives. Current pathogen methods demonstrate low sensitivity and specificity, as well as lack capabilities for multiple and point-of-care testing. Recent studies have shown that Argonaute-powered biosensing is an innovative and rapidly growing technology that could significantly enhance detection capabilities for pathogen-related issues, addressing the limitations of current methods. The application of Argonaute-powered biosensing is both promising and desirable due to the potential to offer "customized" and streamlined detection in the field of pathogenic biosafety monitoring.}, }
@article {pmid39983881, year = {2025}, author = {Shah, AA and Alwashmi, ASS and Abalkhail, A and Alkahtani, AM}, title = {Emerging challenges in Klebsiella pneumoniae: Antimicrobial resistance and novel approach.}, journal = {Microbial pathogenesis}, volume = {202}, number = {}, pages = {107399}, doi = {10.1016/j.micpath.2025.107399}, pmid = {39983881}, issn = {1096-1208}, mesh = {*Klebsiella pneumoniae/drug effects/genetics/pathogenicity ; *Klebsiella Infections/microbiology/therapy/drug therapy ; Humans ; *Anti-Bacterial Agents/pharmacology/therapeutic use ; Biofilms/drug effects/growth &amp; development ; Drug Resistance, Multiple, Bacterial ; Phage Therapy/methods ; Virulence Factors/genetics ; beta-Lactamases/metabolism/genetics ; CRISPR-Cas Systems ; Antimicrobial Peptides/therapeutic use/pharmacology ; }, abstract = {Klebsiella pneumoniae poses significant global health challenges due to its increasing antimicrobial resistance (AMR). The emergence of multidrug-resistant (MDR) and carbapenem-resistant strains has limited treatment options, making infections difficult to control. This pathogen utilizes several mechanisms, including extended-spectrum β-lactamase (ESBL) production and efflux pumps, contributing to its resistance. Novel therapeutic approaches, such as bacteriophage therapy, CRISPR-Cas systems, and antimicrobial peptides, are being explored to combat AMR. Additionally, targeting virulence factors and biofilm formation holds promise for developing alternative strategies, providing a new frontier in tackling this formidable pathogen.}, }
@article {pmid39983727, year = {2025}, author = {Meier, S and Larsen, ASG and Wanke, F and Mercado, N and Mei, A and Takacs, L and Mracsko, ES and Collin, L and Kampmann, M and Roudnicky, F and Jagasia, R}, title = {An efficient, non-viral arrayed CRISPR screening platform for iPSC-derived myeloid and microglia models.}, journal = {Stem cell reports}, volume = {20}, number = {3}, pages = {102420}, pmid = {39983727}, issn = {2213-6711}, mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; *Microglia/metabolism/cytology ; Humans ; *CRISPR-Cas Systems/genetics ; *Myeloid Cells/metabolism/cytology ; Mechanistic Target of Rapamycin Complex 1/metabolism ; Apolipoproteins E/genetics/metabolism ; Signal Transduction ; Lipid Metabolism ; Cell Differentiation ; *Models, Biological ; }, abstract = {Here, we developed a CRISPR-Cas9 arrayed screen to investigate lipid handling pathways in human induced pluripotent stem cell (iPSC)-derived microglia. We established a robust method for the nucleofection of CRISPR-Cas9 ribonucleoprotein complexes into iPSC-derived myeloid cells, enabling genetic perturbations. Using this approach, we performed a targeted screen to identify key regulators of lipid droplet formation dependent on Apolipoprotein E (APOE). We identify the Mammalian Target of Rapamycin Complex 1 (mTORC1) signaling pathway as a critical modulator of lipid storage in both APOE3 and APOE knockout microglia. This study is a proof of concept underscoring the utility of CRISPR-Cas9 technology in elucidating the molecular pathways of lipid dysregulation associated with Alzheimer's disease and neuroinflammation.}, }
@article {pmid39983483, year = {2025}, author = {Aygar, S and Daheron, L}, title = {Generation of a human iPSC line with Notch3 R133C mutation by CRISPR/Cas9: A tool for investigating CADASIL and therapeutic targets.}, journal = {Stem cell research}, volume = {84}, number = {}, pages = {103678}, doi = {10.1016/j.scr.2025.103678}, pmid = {39983483}, issn = {1876-7753}, mesh = {Humans ; *Receptor, Notch3/genetics/metabolism ; *CADASIL/genetics/pathology/metabolism ; *CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Mutation/genetics ; Cell Line ; }, abstract = {Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a rare neuro vascular disease that is caused by mutations in Notch3. Here, we developed an iPSC line harboring the R133C mutation in Notch3, which is among the most common mutations leading to CADASIL. This mutation alters the disulfide bonding pattern leading to Notch3 protein aggregation, granular osmiophilic material (GOM) formation and vascular changes. The iPSC line was generated using CRISPR/Cas9 and edits were confirmed by PCR and Sanger sequencing. This resource is a valuable tool for studying molecular mechanisms of CADASIL and enabling the development and screening of targeted therapies for Notch3-related pathologies.}, }
@article {pmid39982896, year = {2025}, author = {Sui, Z and Chen, B and Zhao, J and Yang, H and Guo, L and Xu, J}, title = {Dual-Accelerated Signal Amplification in Biosensing via Spatial Confining Catalytic Hairpin Assembly-Activated Spherical CRISPR/Cas12a System for Trans-Cleavage of Hairpin DNA Reporters.}, journal = {Analytical chemistry}, volume = {97}, number = {8}, pages = {4668-4677}, doi = {10.1021/acs.analchem.4c07111}, pmid = {39982896}, issn = {1520-6882}, mesh = {*Biosensing Techniques/methods ; *MicroRNAs/analysis/genetics ; *CRISPR-Cas Systems ; *DNA/chemistry/genetics/metabolism ; Humans ; Inverted Repeat Sequences ; Limit of Detection ; Nucleic Acid Amplification Techniques ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression and are implicated in various diseases, including cancer. Due to their critical role in diagnostics, there is a growing need for sensitive, specific, and rapid detection methods for miRNAs. In this study, we present a dual-accelerated signal amplification platform for miRNA biosensing, which integrates spatial confining catalytic hairpin assembly (SC-CHA) with spherical CRISPR/Cas12a (S-CRISPR/Cas12a) system for (SC-CHA@S-CRISPR/Cas12a) trans-cleavage of hairpin DNA reporters. The method employs a biotinylated palindrome-rich assembly sequence (PAS) to form DNA nanoballs, which serve as a scaffold for the operation of SC-CHA upon miRNA binding. The SC-CHA products bind with crRNA and Cas 12a protein, activating S-CRISPR/Cas12a system to cleave the hairpin DNA reporter and generate a detectable fluorescence signal. The uniqueness of this system lies in the combined use of DNA nanoballs and hairpin DNA reporters, both of which significantly accelerate reaction kinetics, resulting in rapid signal generation. Additionally, the spherical DNA nanostructure, integrated with the S-CRISPR/Cas12a system, greatly enhances biostability and accelerating reaction kinetics. These features enable the platform to exhibit high sensitivity, with a limit of detection (LOD) as low as 13.75 fM, and excellent specificity, successfully distinguishing miRNA-21 from other miRNAs. The assay is also biostable, demonstrating reliable performance in complex biological samples such as human serum. This dual-acceleration approach offers a promising solution for sensitive, rapid, and specific miRNA biosensing, with potential applications in early cancer diagnosis and clinical monitoring.}, }
@article {pmid39982610, year = {2025}, author = {Badwal, AK and Singh, S}, title = {Current trends in application of CRISPR/Cas9 in gene editing and diagnostics in Neglected tropical diseases (NTDs).}, journal = {Molecular biology reports}, volume = {52}, number = {1}, pages = {259}, pmid = {39982610}, issn = {1573-4978}, support = {CRG/2022/002746//Science and Engineering Research Board (SERB)/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods/trends ; *Neglected Diseases/diagnosis/genetics/parasitology ; Humans ; Tropical Medicine/methods ; Animals ; }, abstract = {Neglected tropical diseases (NTDs) include more than a dozen of diseases which despite their fatality receive less attention from the research community worldwide. High cost diagnosis of these diseases and lack of trained community which can accurately interpret them is the major drawback in the healthcare system. Nowadays, in the genetic engineering era more emphasis is given to the modern gene editing tools such as Transcription Activator-Like Effector Nucleases (TALENS), Zinc Finger Nucleases (ZFNs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) due to their unique tailoring molecular machinery. This review article details the applicability of CRISPR/Cas9 as a modern gene editing tool in case of NTD parasites such as trypanosomatids with an aim to target their virulent genes. It has been observed through a number of studies that knocking in/out virulent genes of these parasites have led to a significant decrease in infectivity, growth rates along with morphological defects. The article also mentions various advanced CRISPR/Cas based diagnostics such as Specific High-Sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) and SHERLOCK4HAT which can detect parasite concentration as low as 2 attomolar/L (aM: 10[- 18]) and 1 parasite/µL respectively. This review also enlists various regulatory and biosafety issues, for example ecological imbalance which can arise as a consequence of CRISPR/Cas based gene drives employed to target parasitic vectors. Despite its wide applications, CRISPR/Cas is associated with several limitations like off-target effects and ecological imbalance to name a few.}, }
@article {pmid39982596, year = {2025}, author = {Virgílio, MLS and Quintela, ED and Maciel, LHR and Goulart, GSS and Silva, JFAE and Cortes, MVCB}, title = {Metarhizium anisopliae engineering mediated by a CRISPR/Cas9 recyclable system.}, journal = {Folia microbiologica}, volume = {}, number = {}, pages = {}, pmid = {39982596}, issn = {1874-9356}, abstract = {The advent of CRISPR/Cas technology has revolutionized genome editing, offering simplicity, precision, and cost-effectiveness. While its application in biological control fungi has been limited, including the cosmopolitan fungus Metarhizium anisopliae, recent advancements show promise. However, integrating cas9 and selection-marker genes into fungal genomes poses challenges, including reduced efficiency, toxicity, and off-target effects. Besides, marker-free genetic engineering through a CRISPR recyclable system presents a viable solution, enabling efficient mutant generation without compromising fitness and virulence. This study pioneers the construction of marker-free strains of M. anisopliae using a CRISPR/Cas9 recyclable system. Precise deletion of albA and ku70, alongside gfp cassette insertion, confirms the system efficiency. This innovative approach holds significant potential for facilitating in-depth molecular studies, understanding their ecological roles in agricultural systems, and enhancing biocontrol efficacy against insect pests through genetic improvement.}, }
@article {pmid39982542, year = {2025}, author = {Weldemichael, MY and Gebremedhn, HM}, title = {Enhancing tiny millets through genome editing: current status and future prospects.}, journal = {Molecular genetics and genomics : MGG}, volume = {300}, number = {1}, pages = {22}, pmid = {39982542}, issn = {1617-4623}, mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Plant Breeding/methods ; *Genome, Plant ; Crops, Agricultural/genetics ; *Edible Grain/genetics/growth &amp; development ; Stress, Physiological/genetics ; }, abstract = {This study aims to address the critical need for genetic improvement of small millets, which are vital yet underutilized cereal crops cultivated in semi-arid regions of Africa and Asia. Given their high nutritional value and climate resilience, small millets hold significant potential for food security and sustainable agriculture in arid regions. However, traditional breeding methods have proven to be time-consuming and inefficient in enhancing desirable traits. This study highlights the transformative potential of genome editing technologies, particularly the CRISPR/Cas9 system, in accelerating the development of improved small millet varieties. The findings presented in this paper detail recent advancements in using CRISPR/Cas for enhancing resistance to biotic stresses, including bacterial, viral, and fungal pathogens. Additionally, we explore how genome editing can be applied to improve abiotic stress tolerance, addressing challenges such as drought, cold, heat, and herbicides in small millets. We discuss the existing challenges faced by breeders, including issues related to ploidy levels, off-target effects, and limitations in organelle genome modification. The review also suggests potential strategies for overcoming these bottlenecks, aiming to develop stress-resistant super cultivars. Overall, this paper provides an overview of the current state of genome editing research in small millets while identifying future opportunities to enhance key traits for nutrient enrichment and climate resilience, ultimately paving the way for advancements in these crucial crops.}, }
@article {pmid39981976, year = {2025}, author = {He, X and Mauki, DH and Zhao, X and Dai, S and Yang, H and Zheng, Y and Xia, Q and Wang, R and Wang, T}, title = {Targeting LncRNA-Vof16: A Novel Therapeutic Strategy for Neuropathic Pain Relief.}, journal = {CNS neuroscience &amp; therapeutics}, volume = {31}, number = {2}, pages = {e70241}, pmid = {39981976}, issn = {1755-5949}, support = {2023NSFSC1567//Natural Science Foundation of Sichuan Province of China/ ; 2023SCUH0033//Sichuan University Innovation Research Project/ ; 81471268//National Natural Science Foundation of China/ ; 2020YFS0043//Key Research and Development Program of Sichuan Science and Technology Agency/ ; }, mesh = {Animals ; *Neuralgia/metabolism/therapy/genetics ; Rats ; *RNA, Long Noncoding/genetics/metabolism ; Rats, Sprague-Dawley ; Male ; Peripheral Nerve Injuries/complications/metabolism ; Hyperalgesia ; CRISPR-Cas Systems ; Disease Models, Animal ; Spinal Cord Dorsal Horn/metabolism ; }, abstract = {AIMS: Neuropathic pain (NP) is a debilitating condition characterized by chronic pain resulting from nerve damage or lesion. Despite the ongoing efforts of clinically defining NP, its distinctive mechanisms that lead to various NP phenotypes remain unresolved.<br><br>METHODS: Using a spared nerve injury (SNI) model, we investigated the mechanisms underlying the development of NP caused by injury in the peripheral nerves. With CRISPR-Cas9-mediated knockout and virus-mediated overexpression strategies, we investigated the role of LncRNA Vof16 (abbreviated as Vof16) during SNI-induced NP.<br><br>RESULTS: Our results revealed that SNI led to the downregulation of Vof16 expression in spinal dorsal horn (SDH) of lumbar enlargement. This was evidently confirmed when we disrupted the expression of Vof16 in SNI rats of which we observed exacerbation of hyperalgesia; while overexpressing it alleviated the pain.<br><br>CONCLUSION: Our findings suggest that Vof16 plays a crucial role in maintaining normal sensory function in healthy states and a protective shield against NP following peripheral nerve injury. We therefore propose Vof16 as a new therapeutic target for alleviating NP.}, }
@article {pmid39981890, year = {2025}, author = {Gu, X and Lang, J and Chang, Y and Zhang, M}, title = {Cleavable donor-assisted CRISPR/Cas9 system significantly improves the efficiency of large DNA insertion in Physcomitrium patens.}, journal = {The Plant journal : for cell and molecular biology}, volume = {121}, number = {4}, pages = {e70020}, doi = {10.1111/tpj.70020}, pmid = {39981890}, issn = {1365-313X}, support = {32022017//National Natural Science Foundation of China/ ; 32270729//National Natural Science Foundation of China/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Bryopsida/genetics ; *Gene Editing/methods ; *Mutagenesis, Insertional/methods ; DNA, Plant/genetics ; Genome, Plant/genetics ; Protoplasts/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; }, abstract = {Precise insertion of desired fragments can be achieved by CRISPR/Cas9-based genome editing. However, a decrease in knock-in efficiency has been observed with increasing length of exogenous inserts. In this study, we developed an in vivo cleavable (IVC) donor-assisted CRISPR/Cas9 system to improve efficiency, particularly for larger inserts, in the moss Physcomitrium patens (P. patens). The IVC donor, which contains two Cas9 nuclease recognition sites flanking the homology template, enables the in vivo release of the linear template for homology-directed repair (HDR) when co-delivered with the corresponding CRISPR/Cas9 plasmid into protoplasts. In our experimental framework, two distinct sgRNAs and four different DNA inserts were evaluated. Compared with standard circular donors, IVC donors significantly enhanced the efficiency of CRISPR/Cas9-mediated precise insertion of 5.8, 7.5, and 11.1 kb DNA fragments at the PpPDV2-4 sgRNA target site, improving integration rates from 29.6 to 67.8%, from 15.0 to 72.0%, and from 12.1 to 65.6%, respectively. At an alternative sgRNA2 target site within the Pp6c18_3160 locus, the IVC donor also demonstrated a higher knock-in efficiency for a 7.4 kb fragment compared with the standard circular donor. This IVC donor-assisted CRISPR/Cas9 approach for large fragment knock-in represents a powerful tool for basic research and synthetic biology efforts in moss species. Moreover, this strategy may be potentially applicable to crops that are amenable to protoplast transformation and regeneration, facilitating the improvement of key traits.}, }
@article {pmid39980302, year = {2025}, author = {Li, Y and Li, Y and Hu, Y and Liu, R and Lv, Y}, title = {CRISPR-Cas12a/Cas13a Multiplex Bioassay for ctDNA and miRNA by Mass Spectrometry.}, journal = {Analytical chemistry}, volume = {97}, number = {9}, pages = {5049-5056}, doi = {10.1021/acs.analchem.4c05961}, pmid = {39980302}, issn = {1520-6882}, mesh = {Humans ; *CRISPR-Cas Systems ; *MicroRNAs/blood/analysis/genetics ; Mass Spectrometry/methods ; *Circulating Tumor DNA/blood/analysis/genetics ; *Biological Assay/methods ; Limit of Detection ; Biosensing Techniques ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {The CRISPR-Cas system, particularly CRISPR-Cas12a and CRISPR-Cas13a, has been widely utilized in constructing various biosensors due to their "trans-cleavage" ability as a means of signal amplification. However, this universal "trans-cleavage" characteristic also presents a challenge for realizing CRISPR-Cas multiplexed bioanalysis. Besides, potential signal cascading interference and complicated design are notable obstacles in CRISPR-Cas multiplexed bioanalysis. Herein, we propose a mass spectrometry method that leverages the CRISPR-Cas12a/13a system to achieve simultaneous detection of ctDNA and miRNA. Based on the properties of the CRISPR-Cas12a/13a system, two types of nanoparticle reporter probes have been engineered, using cancer-related biomarkers ctDNA and miR-21 as our model analytes. The nanoparticle tags, which intrinsically incorporated millions of detectable atoms, combined with the CRISPR-Cas12a/Cas13a system's "trans-cleavage" ability, allow the proposed mass spectrometry strategy to achieve fmol-level detection limits without any nucleic acid amplification procedures. The assay was successfully applied to human serum samples, demonstrating its potential for early disease diagnosis and progression tracking.}, }
@article {pmid39980205, year = {2025}, author = {Yang, K and Zhang, C and Wang, Z and Huang, Q and Qian, J and Shi, G and Sun, W and Wang, J and Ji, Y and Sun, Z and Song, Y and Han, X}, title = {CRISPR-dCas9-Mediated PTEN Activation via Tumor Cell Membrane-Coated Nanoplatform Enhances Sensitivity to Tyrosine Kinase Inhibitors in Nonsmall Cell Lung Cancer.}, journal = {ACS applied materials &amp; interfaces}, volume = {17}, number = {9}, pages = {13605-13616}, doi = {10.1021/acsami.4c21740}, pmid = {39980205}, issn = {1944-8252}, mesh = {*Carcinoma, Non-Small-Cell Lung/drug therapy/metabolism/pathology/genetics ; Humans ; *PTEN Phosphohydrolase/metabolism/genetics ; *Lung Neoplasms/drug therapy/pathology/metabolism/genetics ; *CRISPR-Cas Systems ; *Protein Kinase Inhibitors/pharmacology/chemistry/therapeutic use ; *Gefitinib/pharmacology/chemistry/therapeutic use ; Animals ; Mice ; Cell Membrane/metabolism/drug effects ; Cell Line, Tumor ; ErbB Receptors/metabolism/antagonists &amp; inhibitors/genetics ; Drug Resistance, Neoplasm/drug effects ; *Antineoplastic Agents/pharmacology/chemistry ; Mice, Nude ; Tyrosine Kinase Inhibitors ; }, abstract = {EGFR tyrosine kinase inhibitors (EGFR-TKIs) have garnered substantial clinical success in treating nonsmall cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations. However, the inevitable emergence of drug resistance, frequently attributed to activation, mutation, or deletion of multiple signaling pathways, poses a significant challenge. Notably, the loss of PTEN protein expression has emerged as a pivotal mechanism fostering resistance in EGFR mutant lung cancers. Consequently, strategies aimed at upregulating PTEN expression hold great promise for restoring drug sensitivity. Leveraging the versatility, precision, and efficacy of nuclease-deactivated Cas9 (dCas9) as a transcriptional activator, we designed a CRISPR-dCas9 system to stimulate PTEN expression. To further enhance target specificity and drug delivery efficiency, we innovatively harnessed the tumor cell membrane (CCM) as a homologous targeting surface coating for our vector, thereby creating a targeted activation nanoplatform. Comprehensive in vitro and in vivo evaluations demonstrated that the synergistic interplay between gefitinib and the CRISPR-dCas9 system significantly enhanced drug sensitivity. The finding underscores the potential of our approach in addressing the issue of lung cancer resistance, offering a promising avenue for personalized and effective cancer therapies.}, }
@article {pmid39979966, year = {2025}, author = {Zou, C and Liu, X and Wang, W and He, L and Yin, A and Cao, Z and Zhu, M and Wu, Y and Liu, X and Ma, J and He, Y and Wang, S and Zhang, W and Liu, W and Zhang, Y and Gu, J and Lin, W and Zhang, K and Li, M}, title = {Targeting GDF15 to enhance immunotherapy efficacy in glioblastoma through tumor microenvironment-responsive CRISPR-Cas9 nanoparticles.}, journal = {Journal of nanobiotechnology}, volume = {23}, number = {1}, pages = {126}, pmid = {39979966}, issn = {1477-3155}, support = {82003220//National Natural Science Foundation of China/ ; 01-SWKJYCJJ17//National Defense Biotechnology Fund for Outstanding Young Talents/ ; }, mesh = {Animals ; *Glioblastoma/therapy/immunology/genetics ; *Tumor Microenvironment/drug effects ; *CRISPR-Cas Systems/genetics ; Mice ; *Immunotherapy/methods ; *Nanoparticles/chemistry ; *Growth Differentiation Factor 15/genetics/metabolism ; Cell Line, Tumor ; Humans ; *Brain Neoplasms/therapy/immunology ; Gene Editing ; Genetic Therapy ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mice, Inbred C57BL ; }, abstract = {Despite the outstanding clinical success of immunotherapy, its therapeutic efficacy in glioblastoma (GBM) is still limited. To identify critical regulators of GBM immunity, we constructed a mouse single-guide RNA (sgRNA) library corresponding to all disease-related immune genes, and performed an in vivo CRISPR knockout (KO) screen in syngeneic GBM mouse models. We demonstrated that the deletion of GDF15 in GBM cells ameliorated the immunosuppressive tumor microenvironment (TME) and enhanced the antitumor efficacy of immune checkpoint blockade (ICB) response. Moreover, we designed unique nanoparticles for efficient encapsulation of CRISPR-Cas9, noninvasive brain delivery and tumor cell targeting, demonstrating an effective and safe strategy for GDF15 gene therapy. The CRISPR-Cas9 nanoparticles, known as ANPSS (Cas9/sgRNA), are easily created by enclosing a single Cas9/sgRNA complex in a polymer shell that is sensitive to glutathione. This shell also contains a dual-action ligand that aids in crossing the blood‒brain barrier, targeting tumor cells, and selectively releasing Cas9/sgRNA. Our encapsulating nanoparticles demonstrated promising GBM targeting, resulting in high GDF15 gene editing efficiency within brain tumors while showing minimal off-target gene editing in high-risk tissues. Treatment with ANPSS (Cas9/sgGDF15) effectively halted tumor growth, reversed immune suppression, and enhanced the efficacy of ICB therapy. These results emphasize the potential role of GDF15 in modulating the immune microenvironment and enhancing the effectiveness of current immunotherapy strategies for GBM.}, }
@article {pmid39979929, year = {2025}, author = {Zhang, S and Li, M and Zeng, J and Zhou, S and Yue, F and Chen, Z and Ma, L and Wang, Y and Wang, F and Luo, J}, title = {Somatostatin receptor-targeted polymeric nanoplatform for efficient CRISPR/Cas9 gene editing to enhance synergistic hepatocellular carcinoma therapy.}, journal = {Journal of nanobiotechnology}, volume = {23}, number = {1}, pages = {127}, pmid = {39979929}, issn = {1477-3155}, support = {2022YFA0911800//China National Key Research and Development (R&amp;D) Program/ ; 2024AFA101//Hubei Provincial Science Fund for Distinguished Young Scholars/ ; }, mesh = {Humans ; *Carcinoma, Hepatocellular/therapy/genetics/drug therapy ; *Liver Neoplasms/therapy/genetics ; *Receptors, Somatostatin/metabolism ; Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mice ; Hep G2 Cells ; *Nanoparticles/chemistry ; Genetic Therapy/methods ; Mice, Nude ; *Polymers/chemistry ; Octreotide/chemistry ; Mice, Inbred BALB C ; B7-H1 Antigen/genetics/metabolism ; Paclitaxel/pharmacology ; Cell Proliferation/drug effects ; Cell Line, Tumor ; }, abstract = {The CRISPR/Cas9 system-based gene therapy can fundamentally address the issues of cancer occurrence, development, progression, and metastasis. However, the lack of targeting and effectiveness hinders gene therapy from entering clinical application. Herein, a somatostatin receptor-targeted polymeric nanoplatform is developed for the delivery of a PD-L1-targeted CRISPR/Cas9 system and synergistic treatment of hepatocellular carcinoma. This nanoplatform can effectively incorporate the CRISPR/Cas9 system and the chemotherapeutic drug paclitaxel to simultaneously address the biological safety and packaging capacity issues of viral vectors. After the octreotide-modified polymer (LNA-PEG-OCT) guided the nanoparticle into hepatoma carcinoma cells, the nanoparticle protected the CRISPR/Cas9 ribonucleoprotein complex (RNP) and achieved lysosomal escape. Then, the RNP reached the target gene (PD-L1) under the guidance of the single guide RNA (sgRNA) in the RNP. The PD-L1 gene editing efficiency reached up to 55.8% for HepG2 cells in vitro and 46.0% for tumor tissues in vivo, leading to effective suppression of PD-L1 protein expression. Substantial inhibition of hepatocellular carcinoma cell proliferation and further 79.45% growth repression against subcutaneous xenograft tumors were achieved. Overall, this somatostatin receptor-targeted polymeric nanoplatform system not only provides a promising nanocarrier for CRISPR/Cas9 system delivery, but also expands the potential of combining gene editing and chemotherapy.}, }
@article {pmid39979478, year = {2025}, author = {Khan, N and Li, Z and Ali, A and Quan, B and Kang, J and Ullah, M and Yin, XJ and Shafiq, M}, title = {Comprehensive transcriptomic analysis of myostatin-knockout pigs: insights into muscle growth and lipid metabolism.}, journal = {Transgenic research}, volume = {34}, number = {1}, pages = {12}, pmid = {39979478}, issn = {1573-9368}, support = {31860297//National Natural Science Foundation of China/ ; }, mesh = {Animals ; *Myostatin/genetics ; *Lipid Metabolism/genetics ; Swine/genetics/growth &amp; development ; *Transcriptome/genetics ; *Muscle Development/genetics ; *Muscle, Skeletal/growth &amp; development/metabolism ; Gene Expression Profiling ; CRISPR-Cas Systems ; Animals, Genetically Modified/genetics/growth &amp; development ; Gene Knockout Techniques ; Gene Editing ; Sus scrofa/genetics/growth &amp; development ; }, abstract = {Pigs are a vital source of protein worldwide, contributing approximately 43% of global meat production. Recent genetic advancements in the myostatin (MSTN) gene have facilitated the development of double-muscling traits in livestock. In this study, we investigate the transcriptomic profiles of second-generation MSTN-knockout (MSTN[-/-]) pigs, generated through CRISPR/Cas9 gene editing and somatic cell nuclear transfer (SCNT). Using RNA sequencing, we compared the transcriptomic landscapes of muscle tissues from MSTN[-/-] pigs and wild-type (WT) counterparts. The sequencing yielded an average unique read mapping rate of 86.7% to the Sus scrofa reference genome. Our analysis revealed 15,142 differentially expressed genes (DEGs), including 121 novel genes, with 2554 genes upregulated and 1629 downregulated in the MSTN[-/-] group relative to the wild-type group. Notable transcriptomic changes were identified in genes associated with muscle development, lipid metabolism, and other physiological processes. These findings provide valuable insights into the molecular consequences of MSTN inactivation, with potential applications in the optimization of livestock breeding and advancements in biomedical research.}, }
@article {pmid39979422, year = {2025}, author = {Xu, S and Neupane, S and Wang, H and Pham, TP and Snyman, M and Huynh, TV and Wang, L}, title = {The mosaicism of Cas-induced mutations and pleiotropic effects of scarlet gene in an emerging model system.}, journal = {Heredity}, volume = {134}, number = {3-4}, pages = {221-233}, pmid = {39979422}, issn = {1365-2540}, support = {R35 GM133730/GM/NIGMS NIH HHS/United States ; MCB-2042490/2348390//National Science Foundation (NSF)/ ; R35GM133730//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; 2220695/2324639//National Science Foundation (NSF)/ ; }, mesh = {Animals ; *Mosaicism ; *CRISPR-Cas Systems ; Gene Editing/methods ; *Daphnia/genetics ; *Mutation ; *Genetic Pleiotropy ; Microinjections ; Transcriptome ; *Decorin/genetics ; }, abstract = {The effective use of CRISPR technologies in emerging model organisms faces significant challenges in efficiently generating heritable mutations and in understanding the genomic consequences of induced DNA damages and the inheritance patterns of induced mutations. This study addresses these issues by 1) developing an efficient microinjection delivery method for gene editing in the microcrustacean Daphnia pulex; 2) assessing the editing dynamics of Cas9 and Cas12a nucleases in the scarlet knock-out mutants; and 3) investigating the transcriptomes of scarlet mutants to understand the pleiotropic effects of scarlet gene. Our reengineered microinjection method results in efficient biallelic editing with both nucleases. Our data suggest site-specific DNA cleavage mostly occurs in a stepwise fashion. Indels dominate the induced mutations. A few, unexpected on-site large deletions (>1 kb) are also observed. Notably, genome-wide analyses reveal no off-target mutations. Knock-in of a stop codon cassette to the scarlet locus was successful, despite complex induced mutations surrounding the target site. Moreover, extensive germline mosaicism exists in some mutants, which unexpectedly produce different phenotypes/genotypes in their asexual progeny. Lastly, our transcriptomic analyses unveil significant gene expression changes associated with scarlet knock-out and altered swimming behavior in mutants, including several genes involved in human neurodegenerative diseases.}, }
@article {pmid39978713, year = {2025}, author = {Takahashi, S and Maehara, M and Nishihara, C and Iwata, H and Shibutani, S}, title = {A genome-wide CRISPR-Cas9 knockout screen using dynamin knockout cells identifies Nf2 and Traf3 as genes involved in dynamin-independent endocytosis.}, journal = {Experimental cell research}, volume = {446}, number = {2}, pages = {114470}, doi = {10.1016/j.yexcr.2025.114470}, pmid = {39978713}, issn = {1090-2422}, mesh = {*TNF Receptor-Associated Factor 3/genetics/metabolism ; *Endocytosis/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *Dynamins/genetics/metabolism ; *Neurofibromin 2/genetics/metabolism ; Gene Knockout Techniques ; Animals ; Clathrin/metabolism ; Mice ; }, abstract = {Endocytosis is a fundamental process by which cells take up extracellular materials, including nutrients, growth factors, and pathogens. Although several endocytic pathways, such as clathrin-mediated and caveolin-mediated endocytosis, are well-characterized, other endocytic pathways remain poorly understood. Therefore, in this study, we performed a genome-wide CRISPR-Cas9 screen to elucidate new endocytic pathways using dynamin conditional knockout cells. We identified genes that significantly reduced the cell numbers when knocked out simultaneously with dynamin. Among these, neurofibromin 2 (Nf2) and tumor necrosis factor receptor-associated factor 3 (Traf3), whose relationship with endocytosis was not well understood, were investigated for their roles in endocytosis activity. Nf2 and Traf3 knockout cells exhibited reduced non-specific fluid endocytosis in a dynamin-independent manner. However, Nf2 or Traf3 knockout did not affect the transferrin receptor-mediated endocytosis that depends on clathrin and dynamin. Moreover, Nf2 knockout cells showed reduced cholera toxin uptake in a dynamin-independent manner. Overall, this study highlights the roles of Nf2 and Traf3 in endocytosis.}, }
@article {pmid39978340, year = {2025}, author = {Sun, Y and Wu, Y and He, Z and Wang, Y and Hou, W and Cao, Y and Zhou, Q and Zhang, R}, title = {Type III CRISPR-mediated flexible RNA excision with engineered guide RNAs.}, journal = {Molecular cell}, volume = {85}, number = {5}, pages = {989-998.e4}, doi = {10.1016/j.molcel.2025.01.021}, pmid = {39978340}, issn = {1097-4164}, mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; HEK293 Cells ; Frameshift Mutation ; *RNA Editing ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Nucleic Acid Conformation ; Open Reading Frames ; }, abstract = {Current RNA editing techniques are predominantly limited to single-base edits. Here, we introduce selective cleavages and intramolecular stitches of RNA (SCISSOR) for selective cleavage and intramolecular stitches of RNA. Building on the principle that type III CRISPR complex determines target cleavage positions based on gRNA length in 6-nt increments, we hypothesized that engineering gRNAs with bulge loops could circumvent this rule, allowing for flexible RNA excision. Through systematic evaluation of gRNAs with various bulge loops, we established the rules for precise non-6-nt target cleavage and repair. We observed that the complex tolerates 1- or 2-nt bulge loops and accommodates large bulge loops ranging from 6 to 24 nt. Consequently, SCISSOR could accomplish nearly any length of short fragment excision. With its capability to modify open reading frames, we demonstrate the potential of SCISSOR in repairing frameshift mutations and introducing frameshifts to create immunogenic poly-epitopes in human cells. SCISSOR holds promise in RNA therapy and biomedical research.}, }
@article {pmid39978310, year = {2025}, author = {Chen, YE}, title = {A genetic toolbox for engineering C. acnes.}, journal = {Cell systems}, volume = {16}, number = {2}, pages = {101199}, doi = {10.1016/j.cels.2025.101199}, pmid = {39978310}, issn = {2405-4720}, mesh = {*Genetic Engineering/methods ; Humans ; *Propionibacterium acnes/genetics ; CRISPR-Cas Systems/genetics ; }, abstract = {Cutibacterium acnes is a highly prevalent and abundant skin bacterium that lives deep in the hair follicle, a unique site for host access. Thus, it is a prime target to engineer. This study introduces a genetic toolbox for C. acnes, which will enable basic science and therapeutic bioengineering.}, }
@article {pmid39978205, year = {2025}, author = {Pan, W and Yang, Y and Zhang, S and Liu, X and Hu, H and Qu, J and Liu, H}, title = {Generation of a homozygous CPAMD8 knockout human embryonic stem cell line (WAe009-A-2R) by CRISPR/Cas9 system.}, journal = {Stem cell research}, volume = {84}, number = {}, pages = {103683}, doi = {10.1016/j.scr.2025.103683}, pmid = {39978205}, issn = {1876-7753}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; Homozygote ; Cell Line ; Gene Knockout Techniques ; Retina/metabolism/cytology ; }, abstract = {CPAMD8, a constituent of the A2M/C3 (α-2-macroglobulin/complement 3) protein family, is strikingly expressed in the human fetal lens and distal neural retina. Mutations in CPAMD8 have been linked to anterior segment dysgenesis and primary congenital glaucoma. We utilized CRISPR/Cas9 technology to establish a homozygous CPAMD8 knockout human embryonic stem cell line for differentiating retinal organoids, with the intent of exploring the role of CPAMD8 in the early development of the human eye. The CPAMD8 knockout cell line exhibits normal morphology, pluripotency, and karyotype, serving as a valuable research tool for investigating the functions of CPAMD8 in ophthalmology.}, }
@article {pmid39976408, year = {2025}, author = {Zwaans, A and Seidel, S and Manceau, M and Stadler, T}, title = {A Bayesian phylodynamic inference framework for single-cell CRISPR/Cas9 lineage tracing barcode data with dependent target sites.}, journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences}, volume = {380}, number = {1919}, pages = {20230318}, pmid = {39976408}, issn = {1471-2970}, support = {/ERC_/European Research Council/International ; }, mesh = {Bayes Theorem ; *Phylogeny ; Animals ; *Cell Lineage ; *CRISPR-Cas Systems ; *Zebrafish/genetics/growth &amp; development ; *Single-Cell Analysis/methods ; Software ; *DNA Barcoding, Taxonomic/methods ; }, abstract = {Analysing single-cell lineage relationships of an organism is crucial towards understanding the fundamental cellular dynamics that drive development. Clustered regularly interspaced short palindromic repeats (CRISPR)-based dynamic lineage tracing relies on recent advances in genome editing and sequencing technologies to generate inheritable, evolving genetic barcode sequences that enable reconstruction of such cell lineage trees, also referred to as phylogenetic trees. Recent work generated custom computational strategies to produce robust tree estimates from such data. We further capitalize on these advancements and introduce GESTALT analysis using Bayesian inference (GABI), which extends the analysis of genome editing of synthetic target arrays for lineage tracing (GESTALT) data to a fully integrated Bayesian phylogenetic inference framework in software BEAST 2. This implementation allows users to represent the uncertainty in reconstructed trees and enables their scaling in absolute time. Furthermore, based on such time-scaled lineage trees, the underlying processes of growth, differentiation and apoptosis are quantified through so-called phylodynamic inference, typically relying on a birth-death or coalescent model. After validating its implementation, we demonstrate that our methodology results in robust estimates of growth dynamics characteristic of early Danio rerio development. GABI's codebase is publicly available at https://github.com/azwaans/GABI.This article is part of the theme issue '"A mathematical theory of evolution": phylogenetic models dating back 100 years'.}, }
@article {pmid39976310, year = {2025}, author = {Zhang, Y and Chen, G and Liang, C and Yang, B and Lei, X and Chen, T and Jiang, H and Xiong, W}, title = {MultiCRISPR-EGA: Optimizing Guide RNA Array Design for Multiplexed CRISPR Using the Elitist Genetic Algorithm.}, journal = {ACS synthetic biology}, volume = {14}, number = {3}, pages = {919-930}, doi = {10.1021/acssynbio.4c00860}, pmid = {39976310}, issn = {2161-5063}, mesh = {*Algorithms ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Escherichia coli/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Promoter Regions, Genetic ; Synthetic Biology/methods ; Software ; Genetic Algorithms ; }, abstract = {Multiplexed CRISPR design, which allows for the concurrent and efficient editing of multiple genomic sites, is a powerful tool for complex genetic modifications. However, designing effective multiplexed guide RNA (gRNA) arrays remains challenging due to the exponential increase in potential gRNA array candidates and the significant impact of different target site selections on efficiency and specificity. Recognizing that more stable gRNAs, characterized by lower minimum free energy (MFE), have prolonged activity and thus higher efficacy, we developed MultiCRISPR-EGA, a graphical user interface (GUI)-based tool that employs the Elitist Genetic Algorithm (EGA) to design optimized single-promoter-driven multiplexed gRNA arrays. Computational experiments on Escherichia coli gene targets demonstrate that the EGA can rapidly optimize multiplexed gRNA arrays, outperforming other intelligent optimization algorithms in CRISPR interference (CRISPRi) applications, while the GUI provides real-time design progress control and compatibility with various CRISPR-Cas systems. This tool aims to advance the multiplexed gRNA array design process, enabling more efficient and cost-effective genome editing for synthetic biology.}, }
@article {pmid39972681, year = {2025}, author = {Pan, Y and Zeng, F and Luan, X and He, G and Qin, S and Lu, Q and He, B and Han, X and Song, Y}, title = {Polyamine-Depleting Hydrogen-Bond Organic Frameworks Unleash Dendritic Cell and T Cell Vigor for Targeted CRISPR/Cas-Assisted Cancer Immunotherapy.}, journal = {Advanced materials (Deerfield Beach, Fla.)}, volume = {37}, number = {13}, pages = {e2411886}, doi = {10.1002/adma.202411886}, pmid = {39972681}, issn = {1521-4095}, support = {22477056//National Natural Science Foundation of China/ ; 82472379//National Natural Science Foundation of China/ ; 2019YFA0709200//National Key R&amp;D Program/ ; BE2021373//Jiangsu Province Key R&amp;D Program/ ; //Jiangsu Province Innovative Talents and Entrepreneurs Program/ ; 5431ZZXM2304//State Key Laboratory of Analytical Chemistry for Life Science/ ; 2024300315//Fundamental Research Funds for the Central Universities/ ; KYCX24_0277//Postgraduate Research &amp; Practice Innovation Program of Jiangsu Province/ ; }, mesh = {*CRISPR-Cas Systems ; *Dendritic Cells/immunology/drug effects ; Humans ; *Polyamines/chemistry/metabolism ; *Immunotherapy/methods ; *T-Lymphocytes/immunology/drug effects ; *Neoplasms/therapy/immunology ; Animals ; Hydrogen Bonding ; Mice ; Cell Line, Tumor ; }, abstract = {Polyamines have tantalized cancer researchers as a potential means to rein in the rampant growth of cancer cells. However, clinical trials in recent decades have disappointed in delivering notable progress. Herein, a microfluidic-assisted synthetic hydrogen-bond organic framework (HOF) as a polyamine-depleting nanoplatforms designed to unleash the vigor of both dendritic cells (DCs) and T cells for precision cancer immunotherapy is reported. Upon internalization by tumor cells, the loaded plasma amine oxidase (PAO) in HOF efficiently depletes polyamines, remolding the tumor microenvironment and alleviating T-cell immunosuppression. This process also generates acrolein and H2O2, triggering CRISPR-assisted neoantigen generation. Specifically, Acrolein induces carbonyl stress, increasing mutational burdens. Simultaneously, HOF leverages the energy from the bis[2,4,5-trichloro-6-(pentyloxycarbonyl)phenyl] oxalate (CPPO)-H2O2 reaction for CRET-triggered singlet oxygen production, leading to thioether bond cleavage and release CRISPR-Cas9. Once released, CRISPR-Cas9 knocks out the DNA mismatch repair (MMR)-related MLH1 gene, further elevating mutational burdens and generating neoantigens, ideal targets for DCs. This dual-action strategy not only corrects T-cell immunosuppression but also enhances DC efficacy, presenting a powerful approach for tumor immunotherapy.}, }
@article {pmid39971060, year = {2025}, author = {Li, H and Liu, L and Wang, X and Zhang, R and Zhu, H}, title = {Enhancing genome editing efficiency in goldfish (Carassius auratus) through utilization of CRISPR-Cas12a (Cpf1) temperature dependency.}, journal = {International journal of biological macromolecules}, volume = {305}, number = {Pt 2}, pages = {141142}, doi = {10.1016/j.ijbiomac.2025.141142}, pmid = {39971060}, issn = {1879-0003}, mesh = {Animals ; *Gene Editing/methods ; *Goldfish/genetics ; *CRISPR-Cas Systems/genetics ; *Temperature ; Monophenol Monooxygenase/genetics ; *CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {The CRISPR/Cas technology has demonstrated revolutionary potential across various fields, including agriculture, medicine, and food safety detection. However, the utility of CRISPR/Cas12a, a particularly promising gene-editing tool, is constrained by its temperature sensitivity, limiting its application in low-temperature environments. In this study, we developed a gene-editing technique based on the CRISPR/Cas12a system in the poikilothermic species goldfish Carassius auratus. We systematically evaluated the editing efficiencies of LbCas12a and AsCas12a on the tyrosinase (tyr) gene under varying temperature conditions. Our results revealed a pronounced temperature dependence of Cas12a, with elevated temperatures markedly enhancing its editing activity, particularly for AsCas12a. A brief one-hour high-temperature treatment was sufficient to achieve effective gene disruption, underscoring CRISPR/Cas12a as a rapid and efficient gene-editing tool. Temperature was utilized as a conditional trigger for Cas12a-mediated gene knockout, enabling precise modulation of gene disruption at specific embryonic developmental stages. Whole-genome resequencing of the mutants confirmed the absence of off-target effects, further emphasizing the precision of this editing process. These findings indicated that CRISPR/Cas12a represented a viable alternative to the widely utilized CRISPR/Cas9 system and could be applied in conjunction, thereby expanding the potential applications of gene-editing technologies.}, }
@article {pmid39970912, year = {2025}, author = {Zilberzwige-Tal, S and Altae-Tran, H and Kannan, S and Wilkinson, ME and Vo, SC and Strebinger, D and Edmonds, KK and Yao, CJ and Mears, KS and Shmakov, SA and Makarova, KS and Macrae, RK and Koonin, EV and Zhang, F}, title = {Reprogrammable RNA-targeting CRISPR systems evolved from RNA toxin-antitoxins.}, journal = {Cell}, volume = {188}, number = {7}, pages = {1925-1940.e20}, doi = {10.1016/j.cell.2025.01.034}, pmid = {39970912}, issn = {1097-4172}, mesh = {*CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; *Toxin-Antitoxin Systems ; Cryoelectron Microscopy ; CRISPR-Associated Proteins/metabolism/genetics/chemistry ; Evolution, Molecular ; Bacterial Proteins/metabolism/genetics/chemistry ; *Antitoxins/metabolism/chemistry/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Untranslated/metabolism/genetics ; }, abstract = {Despite ongoing efforts to study CRISPR systems, the evolutionary origins giving rise to reprogrammable RNA-guided mechanisms remain poorly understood. Here, we describe an integrated sequence/structure evolutionary tracing approach to identify the ancestors of the RNA-targeting CRISPR-Cas13 system. We find that Cas13 likely evolved from AbiF, which is encoded by an abortive infection-linked gene that is stably associated with a conserved non-coding RNA (ncRNA). We further characterize a miniature Cas13, classified here as Cas13e, which serves as an evolutionary intermediate between AbiF and other known Cas13s. Despite this relationship, we show that their functions substantially differ. Whereas Cas13e is an RNA-guided RNA-targeting system, AbiF is a toxin-antitoxin (TA) system with an RNA antitoxin. We solve the structure of AbiF using cryoelectron microscopy (cryo-EM), revealing basic structural alterations that set Cas13s apart from AbiF. Finally, we map the key structural changes that enabled a non-guided TA system to evolve into an RNA-guided CRISPR system.}, }
@article {pmid39970723, year = {2025}, author = {Cai, D and Wang, Y and Zhang, Z and Huang, E and Yang, N and Yang, X and Zhang, T and Wen, H and Wang, Y and Chen, Z and Wu, H and Liu, D}, title = {Droplet pairing-merging enabled digital RPA-CRISPR/Cas12a (DIMERIC) assay for rapid and precise quantification of Hepatitis B Virus DNA.}, journal = {Biosensors &amp; bioelectronics}, volume = {276}, number = {}, pages = {117256}, doi = {10.1016/j.bios.2025.117256}, pmid = {39970723}, issn = {1873-4235}, mesh = {*Hepatitis B virus/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *DNA, Viral/genetics/isolation &amp; purification/analysis ; Humans ; *Nucleic Acid Amplification Techniques/instrumentation ; *Biosensing Techniques/instrumentation ; *Hepatitis B/virology/diagnosis ; Lab-On-A-Chip Devices ; Recombinases/chemistry ; Equipment Design ; }, abstract = {Recombinase polymerase amplification (RPA)-CRISPR/Cas12a assays have demonstrated remarkable potential for point-of-care detection of pathogens in resource-limited settings. Nevertheless, these assays fall short in delivering direct quantitative results due to the incompatibility between the RPA and CRISPR/Cas12a systems. To overcome this limitation, we developed a droplet pairing-merging enabled digital RPA-CRISPR/Cas12a (DIMERIC) assay in this study. By leveraging a microfluidic chip with a calabash-shaped microwell array, large-volume RPA droplets and small-volume CRISPR/Cas12a droplets were sequentially and size-selectively trapped, generating one-to-one droplet pairs. This spatial separation of the droplets eliminates the inhibitory effects of the CRISPR/Cas12a chemistry on RPA. Upon the completion of RPA, the CRISPR/Cas12a system can be activated by merging the paired droplets. This temporal separation of the RPA and CRISPR/Cas reactions allows for the accumulation of sufficient amplicons to efficiently unleash the collateral cleavage activity. The DIMERIC assay offers rapid quantification of nucleic acids, with the entire procedure being accomplished within 20 min. This assay was employed for the quantitative detection of Hepatitis B virus DNA from batched clinical serum samples, demonstrating a good correlation with qPCR (R[2] = 0.92033) and ddPCR (R[2] = 0.97337) outcomes. Consequently, the developed DIMERIC assay provides a valuable tool for rapid and precise quantification of pathogenic nucleic acids.}, }
@article {pmid39970722, year = {2025}, author = {Lai, Y and Wang, J and Xie, N and Liu, G and Lacap-Bugler, DC}, title = {Identification of a novel forkhead transcription factor MtFKH1 for cellulase and xylanase gene expression in Myceliophthora thermophila (ATCC 42464).}, journal = {Microbiological research}, volume = {294}, number = {}, pages = {128097}, doi = {10.1016/j.micres.2025.128097}, pmid = {39970722}, issn = {1618-0623}, mesh = {*Cellulase/genetics/metabolism/biosynthesis ; *Gene Expression Regulation, Fungal ; *Sordariales/genetics/enzymology/metabolism/growth &amp; development ; *Endo-1,4-beta Xylanases/genetics/metabolism ; *Forkhead Transcription Factors/genetics/metabolism ; *Fungal Proteins/genetics/metabolism ; Promoter Regions, Genetic ; Gene Expression Profiling ; CRISPR-Cas Systems ; Cellulose/metabolism ; }, abstract = {Myceliophthora thermophila is a thermophilic fungus, known to produce industrially important enzymes in biorefineries. The mechanism underlying cellulase and xylanase expression in filamentous fungi is a complex regulatory network controlled by numerous transcription factors (TFs). These TFs in M. thermophila remain unclear. Here, we identified and characterised a novel cellulase and xylanase regulator MtFKH1 in M. thermophila through comparative transcriptomic and genetic analyses. Five of the eight potential TFs, which showed differential expression levels when grown on Avicel and glucose, were successfully deleted using the newly designed CRISPR/Cas9 system. This system identified the forkhead TF MtFKH1. The disruption of Mtfkh1 elevated the cellulolytic and xylanolytic enzyme activities, whereas the overexpression of Mtfkh1 led to considerable decrease in cellulase and xylanase production in M. thermophila cultivated on Avicel. The loss of Mtfkh1 also exhibited an impairment in sporulation in M. thermophila. Real-time quantitative reverse transcription PCR (RT-qPCR) and the electrophoretic mobility shift assays (EMSAs) demonstrated that MtFKH1 regulates the gene expression and specifically bind to the promoter regions of genes encoding β-glucosidase (bgl1/MYCTH_66804), cellobiohydrolase (cbh1/MYCTH_109566), and xylanase (xyn1/MYCTH_112050), respectively. Furthermore, DNase I footprinting analysis identified binding motif of MtFKH1 in the upstream region of Mtbgl1, with strongest binding affinity. Finally, transcriptomic profiling and Gene Ontology (GO) enrichment analyses of Mtfkh1 deletion mutant revealed that the regulon of MtFKH1 were significantly prevalent in hydrolase activity (acting on glycosyl bonds), polysaccharide binding, and carbohydrate metabolic process functional categories. These findings expand our knowledge on how forkhead transcription factor regulates lignocellulose degradation and provide a novel target for engineering of fungal cell factories with the hyperproduction of cellulase and xylanase.}, }
@article {pmid39970103, year = {2024}, author = {Popova, JV and Bets, VD and Omelina, ES and Boldyreva, LV and Kozhevnikova, EN}, title = {[Adapting Mouse Genome Editing Technique from Scratch Using in utero Electroporation].}, journal = {Molekuliarnaia biologiia}, volume = {58}, number = {6}, pages = {1041-1051}, pmid = {39970103}, issn = {0026-8984}, mesh = {Animals ; *Electroporation/methods ; *Gene Editing/methods ; Mice ; Female ; *CRISPR-Cas Systems ; *Interleukin-10/genetics/deficiency ; Zygote/metabolism ; Mice, Knockout ; *Gene Transfer Techniques ; Mice, Inbred C57BL ; Pregnancy ; }, abstract = {Mouse genome modification requires costly equipment and highly skilled personnel to manipulate zygotes. A number of zygote electroporation techniques were reported to be highly efficient in gene delivery. One of these methods called i-GONAD (improved Genome-editing via Oviductal Nucleic Acids Delivery) describes electroporation-based gene transfer to zygotes in utero. Here we adopted this technology to develop an easy-to-use and cost-effective pipeline enabling mouse genome-editing from scratch with minimal requirements to operator skills and animal use. We chose the CRISPR/Cas9 system as a genome editing tool and i-GONAD as a gene delivery method to produce Il10 knockout in C57BL/6 mice. Three animals out of 13 delivered pups (23%) were genetically compromised at Il10 locus suggesting the feasibility of the approach. This protocol provides detailed description of the used technical settings paired with troubleshooting tips and could be of interest to those who aim at establishing in-house mouse transgenesis pipeline at minimal equipment cost from scratch.}, }
@article {pmid39969998, year = {2025}, author = {Qiao, W and Xie, X and Shi, PY and Ooi, YS and Carette, JE}, title = {Druggable genome screens identify SPP as an antiviral host target for multiple flaviviruses.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {122}, number = {8}, pages = {e2421573122}, pmid = {39969998}, issn = {1091-6490}, support = {AI153169//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01 AI153169/AI/NIAID NIH HHS/United States ; Postdoctoral Support//SU | SOM | Stanford Maternal and Child Health Research Institute (MCHRI)/ ; AI141970//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01 AI141970/AI/NIAID NIH HHS/United States ; AI169467//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01 AI169467/AI/NIAID NIH HHS/United States ; Investigators in the Pathogenesis of Infectious Disease//Burroughs Wellcome Fund (BWF)/ ; Dean's Fellowship//SU | School of Medicine, Stanford University (SOM)/ ; }, mesh = {*Antiviral Agents/pharmacology ; Humans ; *Flavivirus/drug effects/genetics ; Virus Replication/drug effects ; *Flavivirus Infections/drug therapy/virology/genetics ; CRISPR-Cas Systems ; Zika Virus/drug effects ; Animals ; Dengue Virus/drug effects ; *Aspartic Acid Endopeptidases/genetics/metabolism/antagonists &amp; inhibitors ; West Nile virus/drug effects ; HEK293 Cells ; }, abstract = {Mosquito-borne flaviviruses, such as dengue virus (DENV), Zika virus (ZIKV), West Nile virus, and yellow fever virus, pose significant public health threats globally. Extensive efforts have led to the development of promising highly active compounds against DENV targeting viral non-structural protein 4B (NS4B) protein. However, due to the cocirculation of flaviviruses and to prepare for emerging flaviviruses, there is a need for more broadly acting antivirals. Host-directed therapy where one targets a host factor required for viral replication may be active against multiple viruses that use similar replication strategies. Here, we used a CRISPR-Cas9 library that we designed to target the druggable genome and identified signal peptide peptidase (SPP, encoded by Histocompatibility Minor 13, HM13), as a critical host factor in DENV infection. Genetic knockout or introducing mutations that disrupt the proteolytic activity of SPP markedly reduced the replication of multiple flaviviruses. Although their substrates differ, SPP has structural homology with γ-secretase, which has been pursued as a pharmacological target for Alzheimer's disease. Notably, SPP-targeting compounds exhibited potent anti-DENV activity at low nanomolar concentrations across multiple primary and disease-relevant cell types, acting specifically through SPP inhibition rather than γ-secretase inhibition. Importantly, SPP inhibitors were active at low nanomolar concentrations against flaviviruses other than DENV including ZIKV while DENV NS4B inhibitors lost activity. This study emphasizes the strong potential of SPP as a pan-flaviviral target and provides a framework for identifying host druggable targets to screen for broad-spectrum antivirals.}, }
@article {pmid39968624, year = {2025}, author = {Monfort, M and Buitink, J and Roeber, F and Nogué, F}, title = {Genome editing, an opportunity to revive soybean cultivation in Europe.}, journal = {The Plant journal : for cell and molecular biology}, volume = {121}, number = {4}, pages = {e17266}, pmid = {39968624}, issn = {1365-313X}, support = {ANR-22-PSV-002//Agence Nationale de la Recherche/ ; //Association Nationale de la Recherche et de la Technologie/ ; }, mesh = {*Glycine max/genetics/growth &amp; development ; *Gene Editing/methods ; Europe ; Plant Breeding/methods ; CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; Crops, Agricultural/genetics ; Plants, Genetically Modified ; }, abstract = {Soybean (Glycine max Merr.) is the world's most important oilseed crop and its ability to fix atmospheric nitrogen makes it a cornerstone of sustainable agriculture. Despite its importance, Europe relies heavily on imports, leading to environmental and economic vulnerabilities. To address these challenges, the European Union has implemented policies to boost local soybean production, emphasizing sustainable practices and reduced dependency on imports. However, conventional breeding methods are time-consuming and may not keep pace with the rapid environmental and consumer habit changes. Genome-editing technologies, such as CRISPR-Cas, offer precise and efficient tools for developing soybean varieties tailored to European conditions. These technologies can enhance traits related to precocity, stress responses, yield and quality that are essential for adapting to climate change and promoting ecological sustainability. This review explores the integration of genome editing (GE) in soybean breeding, highlighting its potential in advancing the agroecological transition in Europe. By having a clear regulation and enhancing breeding efforts, GE can significantly contribute to developing resilient and sustainable soybean varieties, fostering a competitive and environmentally friendly European agriculture.}, }
@article {pmid39967679, year = {2025}, author = {Hashizume, R and Wakita, S and Sawada, H and Takebayashi, SI and Kitabatake, Y and Miyagawa, Y and Hirokawa, YS and Imai, H and Kurahashi, H}, title = {Trisomic rescue via allele-specific multiple chromosome cleavage using CRISPR-Cas9 in trisomy 21 cells.}, journal = {PNAS nexus}, volume = {4}, number = {2}, pages = {pgaf022}, pmid = {39967679}, issn = {2752-6542}, abstract = {Human trisomy 21, responsible for Down syndrome, is the most prevalent genetic cause of cognitive impairment and remains a key focus for prenatal and preimplantation diagnosis. However, research directed toward eliminating supernumerary chromosomes from trisomic cells is limited. The present study demonstrates that allele-specific multiple chromosome cleavage by clustered regularly interspaced palindromic repeats Cas9 can achieve trisomy rescue by eliminating the target chromosome from human trisomy 21 induced pluripotent stem cells and fibroblasts. Unlike previously reported allele-nonspecific strategies, we have developed a comprehensive allele-specific (AS) Cas9 target sequence extraction method that efficiently removes the target chromosome. The temporary knockdown of DNA damage response genes increases the chromosome loss rate, while chromosomal rescue reversibly restores gene signatures and ameliorates cellular phenotypes. Additionally, this strategy proves effective in differentiated, nondividing cells. We anticipate that an AS approach will lay the groundwork for more sophisticated medical interventions targeting trisomy 21.}, }
@article {pmid39966887, year = {2025}, author = {Chacon Machado, L and Peters, JE}, title = {A family of Tn7-like transposons evolved to target CRISPR repeats.}, journal = {Mobile DNA}, volume = {16}, number = {1}, pages = {5}, pmid = {39966887}, issn = {1759-8753}, support = {R35 GM152260/GM/NIGMS NIH HHS/United States ; R35 GM152260/NH/NIH HHS/United States ; }, abstract = {Tn7 family transposons are mobile genetic elements known for precise target site selection, with some co-opting CRISPR-Cas systems for RNA-guided transposition. We identified a novel group of Tn7-like transposons in Cyanobacteria that preferentially target CRISPR arrays, suggesting a new functional interaction between these elements and CRISPR-Cas systems. Using bioinformatics tools, we characterized their phylogeny, target specificity, and sub-specialization. The array-targeting elements are phylogenetically close to tRNA-targeting elements. The distinct target preference coincides with loss of a C-terminal region in the TnsD protein which is responsible for recognizing target sites when compared to closely related elements. Notably, elements are found integrated into a fixed position within CRISPR spacer regions, a behavior that might minimize negative impacts on the host defense system. These transposons were identified in both plasmid and genomic CRISPR arrays, indicating that their preferred target provides a means for both safe insertion in the host chromosome and a mechanism for dissemination. Attempts to reconstitute these elements in E. coli were unsuccessful, indicating possible dependence on native host factors. Our findings expand the diversity of interactions between Tn7-like transposons and CRISPR systems.}, }
@article {pmid39966472, year = {2025}, author = {Sébastien, M and Paquette, AL and Prowse, ENP and Hendricks, AG and Brouhard, GJ}, title = {Doublecortin restricts neuronal branching by regulating tubulin polyglutamylation.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {1749}, pmid = {39966472}, issn = {2041-1723}, mesh = {*Microtubule-Associated Proteins/metabolism/genetics ; Doublecortin Protein ; *Tubulin/metabolism ; Doublecortin Domain Proteins ; *Neuropeptides/metabolism/genetics ; Humans ; *Neurons/metabolism/cytology ; Animals ; Microtubules/metabolism ; Neurites/metabolism ; *Microtubule Proteins/metabolism/genetics ; Mice ; Induced Pluripotent Stem Cells/metabolism/cytology ; CRISPR-Cas Systems ; Neurogenesis ; Peptide Synthases ; }, abstract = {Doublecortin is a neuronal microtubule-associated protein that regulates microtubule structure in neurons. Mutations in Doublecortin cause lissencephaly and subcortical band heterotopia by impairing neuronal migration. We use CRISPR/Cas9 to knock-out the Doublecortin gene in induced pluripotent stem cells and differentiate the cells into cortical neurons. DCX-KO neurons show reduced velocities of nuclear movements and an increased number of neurites early in neuronal development, consistent with previous findings. Neurite branching is regulated by a host of microtubule-associated proteins, as well as by microtubule polymerization dynamics. However, EB comet dynamics are unchanged in DCX-KO neurons. Rather, we observe a significant reduction in α-tubulin polyglutamylation in DCX-KO neurons. Polyglutamylation levels and neuronal branching are rescued by expression of Doublecortin or of TTLL11, an α-tubulin glutamylase. Using U2OS cells as an orthogonal model system, we show that DCX and TTLL11 act synergistically to promote polyglutamylation. We propose that Doublecortin acts as a positive regulator of α-tubulin polyglutamylation and restricts neurite branching. Our results indicate an unexpected role for Doublecortin in the homeostasis of the tubulin code.}, }
@article {pmid39965569, year = {2025}, author = {Wandera, KG and Schmelz, S and Migur, A and Kibe, A and Lukat, P and Achmedov, T and Caliskan, N and Blankenfeldt, W and Beisel, CL}, title = {AcrVIB1 inhibits CRISPR-Cas13b immunity by promoting unproductive crRNA binding accessible to RNase attack.}, journal = {Molecular cell}, volume = {85}, number = {6}, pages = {1162-1175.e7}, doi = {10.1016/j.molcel.2025.01.020}, pmid = {39965569}, issn = {1097-4164}, mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism/immunology/chemistry ; *Ribonucleases/metabolism/genetics ; Protein Binding ; Cryoelectron Microscopy ; *Bacterial Proteins/genetics/metabolism/immunology/chemistry ; Ribonucleoproteins/genetics/metabolism ; *RNA, Bacterial/genetics/metabolism ; }, abstract = {Anti-CRISPR proteins (Acrs) inhibit CRISPR-Cas immune defenses, with almost all known Acrs acting on the Cas nuclease-CRISPR (cr)RNA ribonucleoprotein (RNP) complex. Here, we show that AcrVIB1 from Riemerella anatipestifer, the only known Acr against Cas13b, principally acts upstream of RNP complex formation by promoting unproductive crRNA binding followed by crRNA degradation. AcrVIB1 tightly binds to Cas13b but not to the Cas13b-crRNA complex, resulting in enhanced rather than blocked crRNA binding. However, the more tightly bound crRNA does not undergo processing and fails to activate collateral RNA cleavage even with target RNA. The bound crRNA is also accessible to RNases, leading to crRNA turnover in vivo even in the presence of Cas13b. Finally, cryoelectron microscopy (cryo-EM) structures reveal that AcrVIB1 binds a helical domain of Cas13b responsible for securing the crRNA, keeping the domain untethered. These findings reveal an Acr that converts an effector nuclease into a crRNA sink to suppress CRISPR-Cas defense.}, }
@article {pmid39965366, year = {2025}, author = {Cao, Y and Yang, Q and Guo, Y and Wang, X and Li, X and Zhang, N and Lu, W and Li, J and Zhang, X and Cao, L and Gong, P}, title = {An RPA-CRISPR/Cas12a-assisted method for nucleic acid detection of Haemonchus contortus in sheep.}, journal = {Veterinary parasitology}, volume = {334}, number = {}, pages = {110421}, doi = {10.1016/j.vetpar.2025.110421}, pmid = {39965366}, issn = {1873-2550}, mesh = {Animals ; *Haemonchus/isolation &amp; purification/genetics ; Sheep ; *Haemonchiasis/veterinary/diagnosis/parasitology ; *Sheep Diseases/diagnosis/parasitology ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Feces/parasitology ; Recombinases ; }, abstract = {Haemonchus contortus (H. contortus), a highly pathogenic and blood-feeding nematode, could cause haemonchosis，resulting in tens of billions of dollars in production losses and significantly impacting the development of sheep husbandry. Rapid and accurate detection methods were particularly important for the prevention and control of haemonchosis. In this study, we developed a one-pot effective detection method that integrating recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology based on the conserved region of ITS2 of H. contortus, with readout through fluorescence signals visualized by lateral flow strips (LFS) and observable under UV or blue light. The detection procedure was successfully finished in within 1 h and demonstrated high specificity and sensitivity, with no cross-reactivity detected with nine other common ovine pathogens and a detection limit as low as 0.1 copies/μL for fluorescence and 100 copies/μL for LFS. Validation with 89 sheep fecal samples revealed a 46.07 % positivity rate, fully consistent with quantitative PCR results. In summary, the RPA-CRISPR/Cas12a method for H. contortus detection exhibited the advantages of high specificity, high sensitivity, and low device dependence, portable and visible results. The technique presented significant potential for large-scale clinical application and provided novel point-of-care testing for clinical use in remote rural and resource-constrained areas.}, }
@article {pmid39965128, year = {2025}, author = {Shen, J and Wang, Y and Liu, Y and Lan, J and Long, S and Li, Y and Chen, D and Yu, P and Zhao, J and Wang, Y and Wang, S and Yang, F}, title = {Behavioral Abnormalities, Cognitive Impairments, Synaptic Deficits, and Gene Replacement Therapy in a CRISPR Engineered Rat Model of 5p15.2 Deletion Associated With Cri du Chat Syndrome.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {12}, number = {14}, pages = {e2415224}, pmid = {39965128}, issn = {2198-3844}, support = {L222077//Beijing Natural Science Foundation/ ; IS23097//Beijing Natural Science Foundation/ ; }, mesh = {Animals ; Disease Models, Animal ; Rats ; *Cognitive Dysfunction/genetics/therapy ; *Genetic Therapy/methods ; *Cri-du-Chat Syndrome/genetics/therapy ; CRISPR-Cas Systems/genetics ; Behavior, Animal/physiology ; Male ; Chromosome Deletion ; Social Behavior ; }, abstract = {The Cri du Chat Syndrome (CdCS), a devastating genetic disorder caused by a deletion on chromosome 5p, faces challenges in finding effective treatments and accurate animal models. Using CRISPR-Cas9, a novel CdCS rat model with a 2q22 deletion is developed, mirroring a common genetic alteration in CdCS patients. This model exhibits pronounced deficits in social behavior, cognition, and anxiety, accompanied by neuronal abnormalities and immune dysregulation in key brain regions such as the hippocampus and medial prefrontal cortex (mPFC). The immunostaining and RNA-seq analyses provide new insights into CdCS pathogenesis, revealing inflammatory and immune processes. Importantly, it is demonstrated that early gene replacement therapy with AAV-Ctnnd2 alleviates cognitive impairments in CdCS rats, highlighting the potential for early intervention. However, the effectiveness of this therapy is confined to the early developmental stages and does not fully restore all CdCS symptoms. The findings deepen the understanding of CdCS pathogenesis and suggest promising therapeutic directions.}, }
@article {pmid39964768, year = {2025}, author = {Gibson, WT and Lengyell, TC and Korecki, AJ and Janssen, SM and Adair, BA and Gamu, D and Lorincz, MC and Simpson, EM}, title = {Minimally Humanized Ezh2 Exon-18 Mouse Cell Lines Validate Preclinical CRISPR/Cas9 Approach to Treat Weaver Syndrome.}, journal = {Human gene therapy}, volume = {36}, number = {5-6}, pages = {618-627}, doi = {10.1089/hum.2024.170}, pmid = {39964768}, issn = {1557-7422}, mesh = {*Enhancer of Zeste Homolog 2 Protein/genetics/metabolism ; Animals ; *CRISPR-Cas Systems ; Mice ; *Gene Editing/methods ; Humans ; *Abnormalities, Multiple/genetics/therapy ; *Congenital Hypothyroidism/genetics/therapy ; *Hand Deformities, Congenital/genetics/therapy ; *Craniofacial Abnormalities/genetics/therapy ; *Exons ; Cell Line ; *Genetic Therapy/methods ; }, abstract = {Weaver syndrome is a rare neurodevelopmental disorder that encompasses macrocephaly, tall stature, obesity, brain anomalies, intellectual disability, and increased susceptibility to cancer. This dominant monogenic disorder is caused by germline variants in enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), a key epigenetic writer. Unfortunately, there are no effective treatments for Weaver syndrome. However, preclinical results support the potential for therapeutic gains, despite the prenatal onset. Thus, for the first time, we tested whether CRISPR/Cas9 gene-editing strategies may be able to "correct" a Weaver syndrome variant at the DNA level. We initiated these preclinical studies by humanizing the region surrounding the most-common recurring patient-variant location in mouse embryonic stem cells (ESCs). Humanization ensures that DNA-binding strategies will be directly translatable to human cells and patients. We then introduced into ESCs the humanized region, but now carrying the Weaver syndrome EZH2 variant c.2035C>T p.Arg684Cys, and characterized the enzymatic properties of this missense variant. Our data showed a significant and dramatic reduction in EZH2-enzymatic activity, supporting previous cell-free studies of this variant as well as in vitro and in vivo mouse work by other teams. Intriguingly, this most-common variant does not create a complete loss-of-function, but rather is a hypomorphic allele. Together with prior reports describing hypomorphic effects of missense EZH2 variants, these results demonstrate that the etiology of Weaver syndrome does not require complete loss of EZH2 enzymatic activity. Toward therapy, we tested four CRISPR gene-editing strategies. We demonstrated that Streptococcus pyogenes Cas9 (SpCas9) showed the highest variant correction (70.5%), but unfortunately also the highest alteration of the nonvariant allele (21.1-26.2%), an important consideration for gene-editing treatment of a dominant syndrome. However, Staphylococcus aureus Cas9 (SaCas9) gave a variant correction (52.5%) that was not significantly different than SpCas9, and encouragingly the lowest alteration of the nonvariant allele (2.0%). Thus, the therapeutic strategy using the small SaCas9 enzyme, a size that allows flexibility in therapeutic delivery, was the most optimal for targeting the Weaver syndrome EZH2 variant c.2035C>T p.Arg684Cys.}, }
@article {pmid39964477, year = {2025}, author = {Zhang, K and Shen, W and Zhao, Y and Xu, X and Liu, X and Qi, Q and Huang, S and Tian, T and Zhou, X}, title = {Strategic base modifications refine RNA function and reduce CRISPR-Cas9 off-targets.}, journal = {Nucleic acids research}, volume = {53}, number = {4}, pages = {}, pmid = {39964477}, issn = {1362-4962}, support = {22177089//National Natural Science Foundation of China/ ; 2042023kf0204//Fundamental Research Funds for the Central Universities/ ; }, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *RNA/genetics/chemistry/metabolism ; Uracil/analogs &amp; derivatives/chemistry ; Humans ; *Cytosine/analogs &amp; derivatives/chemistry ; Mutation ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {In contrast to traditional RNA regulatory approaches that modify the 2'-OH group, this study explores strategic base modifications using 5-carboxylcytosine (ca5C). We developed a technique where ca5C is transformed into dihydrouracil via treatment with borane-pyridine complex or 2-picoline borane complex, leading to base mutations that directly impact RNA functionality. This innovative strategy effectively manages CRISPR-Cas9 system activities, significantly minimizing off-target effects. Our approach not only demonstrates a significant advancement in RNA manipulation but also offers a new method for the precise control of gene editing technologies, showcasing its potential for broad application in chemical biology.}, }
@article {pmid39964191, year = {2025}, author = {Chen, L and Wang, C and Zhu, Z and Yang, L}, title = {Field-Deployable Detection of Genetically Modified Organisms with an Integrated Method of Loop-Mediated Isothermal Amplification and CRISPR/FnCas12a.}, journal = {Journal of agricultural and food chemistry}, volume = {73}, number = {9}, pages = {5625-5634}, doi = {10.1021/acs.jafc.4c11373}, pmid = {39964191}, issn = {1520-5118}, mesh = {*Nucleic Acid Amplification Techniques/methods/instrumentation ; *Glycine max/genetics ; *Plants, Genetically Modified/genetics ; *Zea mays/genetics ; CRISPR-Cas Systems ; DNA, Plant/genetics ; Molecular Diagnostic Techniques ; }, abstract = {The detection of genetically modified organisms (GMOs) is crucial for regulatory compliance and consumer safety. This study presents a novel method combining loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12a cleavage, termed Cas-pfLAMP, for sensitive and specific GMO detection. We developed assays for three GM events: maize DBN9936 and MON810 and soybean GTS40-3-2. By incorporating a universal protospacer adjacent motif (PAM) sequence into LAMP primers, we overcame the limitations of PAM site dependence. The Cas-pfLAMP assays demonstrated high specificity and sensitivity, with limits of detection as low as 10-12 copies per reaction. Furthermore, we developed a point-of-care testing platform integrating rapid DNA extraction, Cas-pfLAMP, and lateral flow strips for on-site GMO detection. This platform achieved comparable sensitivity to qPCR, detecting GM contents as low as 0.1% in simulated samples within 40 min. The Cas-pfLAMP method offers the advantages of PAM site independence, high specificity and sensitivity, and suitability for field testing without specialized equipment. This approach represents a promising new generation of GMO detection methods with potential applications in various scenarios.}, }
@article {pmid39963359, year = {2024}, author = {Ahmadikhah, A and Zarabizadeh, H and Nayeri, S and Abbasi, MS}, title = {Advancements in genome editing tools for genetic studies and crop improvement.}, journal = {Frontiers in plant science}, volume = {15}, number = {}, pages = {1370675}, pmid = {39963359}, issn = {1664-462X}, abstract = {The rapid increase in global population poses a significant challenge to food security, compounded by the adverse effects of climate change, which limit crop productivity through both biotic and abiotic stressors. Despite decades of progress in plant breeding and genetic engineering, the development of new crop varieties with desirable agronomic traits remains a time-consuming process. Traditional breeding methods often fall short of addressing the urgent need for improved crop varieties. Genome editing technologies, which enable precise modifications at specific genomic loci, have emerged as powerful tools for enhancing crop traits. These technologies, including RNA interference, Meganucleases, ZFNs, TALENs, and CRISPR/Cas systems, allow for the targeted insertion, deletion, or alteration of DNA fragments, facilitating improvements in traits such as herbicide and insect resistance, nutritional quality, and stress tolerance. Among these, CRISPR/Cas9 stands out for its simplicity, efficiency, and ability to reduce off-target effects, making it a valuable tool in both agricultural biotechnology and plant functional genomics. This review examines the functional mechanisms and applications of various genome editing technologies for crop improvement, highlighting their advantages and limitations. It also explores the ethical considerations associated with genome editing in agriculture and discusses the potential of these technologies to contribute to sustainable food production in the face of growing global challenges.}, }
@article {pmid39962352, year = {2025}, author = {Helia, O and Matúšová, B and Havlová, K and Hýsková, A and Lyčka, M and Beying, N and Puchta, H and Fajkus, J and Fojtová, M}, title = {Chromosome engineering points to the cis-acting mechanism of chromosome arm-specific telomere length setting and robustness of plant phenotype, chromatin structure and gene expression.}, journal = {The Plant journal : for cell and molecular biology}, volume = {121}, number = {4}, pages = {e70024}, pmid = {39962352}, issn = {1365-313X}, support = {CZ.02.01.01/00/22_008/0004581//European Regional Development Fund/ ; 22-04364S//Grantov&#xe1; Agentura &#x10c;esk&#xe9; Republiky/ ; }, mesh = {*Arabidopsis/genetics ; *Chromosomes, Plant/genetics ; *Chromatin/genetics/metabolism ; *Telomere/genetics ; Phenotype ; Genetic Engineering ; Gene Expression Regulation, Plant ; Translocation, Genetic ; CRISPR-Cas Systems ; }, abstract = {The study investigates the impact of targeted chromosome engineering on telomere dynamics, chromatin structure, gene expression, and phenotypic stability in Arabidopsis thaliana. Using precise CRISPR/Cas-based engineering, reciprocal translocations of chromosome arms were introduced between non-homologous chromosomes. The subsequent homozygous generations of plants were assessed for phenotype, transcriptomic changes and chromatin modifications near translocation breakpoints, and telomere length maintenance. Phenotypically, translocated lines were indistinguishable from wild-type plants, as confirmed through morphological assessments and principal component analysis. Gene expression profiling detected minimal differential expression, with affected genes dispersed across the genome, indicating negligible transcriptional impact. Similarly, ChIPseq analysis showed no substantial alterations in the enrichment of key histone marks (H3K27me3, H3K4me1, H3K56ac) near junction sites or across the genome. Finally, bulk and arm-specific telomere lengths remained stable across multiple generations, except for minor variations in one translocation line. These findings highlight the remarkable genomic and phenotypic robustness of A. thaliana despite large-scale chromosomal rearrangements. The study offers insights into the cis-acting mechanisms underlying chromosome arm-specific telomere length setting and establishes the feasibility of chromosome engineering for studies of plant genome evolution and crop improvement strategies.}, }
@article {pmid39962231, year = {2025}, author = {Zakrzewski, P and Rice, CM and Fleming, K and Cela, D and Groves, SJ and Ponce-Garcia, FM and Gibbs, W and Roberts, K and Pike, T and Strathdee, D and Anderson, E and Nobbs, AH and Toye, AM and Steward, C and Amulic, B}, title = {Tafazzin regulates neutrophil maturation and inflammatory response.}, journal = {EMBO reports}, volume = {26}, number = {6}, pages = {1590-1619}, pmid = {39962231}, issn = {1469-3178}, support = {MR/R02149X/1//Medical Research Foundation (MRF)/ ; IS-BTU-1214-10032//NIHR BTRU in partnership with NHSBT/ ; WP15-05//NHS Blood and Transplant R&amp;D grant/ ; A31287//CRUK Scotland Institute core funding/ ; /WT_/Wellcome Trust/United Kingdom ; }, mesh = {Humans ; *Neutrophils/metabolism/pathology/immunology ; Cell Differentiation ; *Inflammation/metabolism/pathology/genetics ; *Barth Syndrome/genetics/pathology/metabolism/immunology ; Neutropenia/genetics ; Extracellular Traps/metabolism ; *TATA-Binding Protein Associated Factors/genetics/metabolism ; *Acyltransferases/genetics/metabolism ; Unfolded Protein Response ; Hematopoietic Stem Cells/metabolism ; CRISPR-Cas Systems ; }, abstract = {Barth syndrome (BTHS) is a rare genetic disease caused by mutations in the TAFAZZIN gene. It is characterized by neutropenia, cardiomyopathy and skeletal myopathy. Neutropenia in BTHS is associated with life-threatening infections, yet there is little understanding of the molecular and physiological causes of this phenomenon. We combined bone marrow analysis, CRISPR/Cas9 genome editing in hematopoietic stem cells and functional characterization of circulating BTHS patient neutrophils to investigate the role of TAFAZZIN in neutrophils and their progenitors. We demonstrate a partial cell intrinsic differentiation defect, along with a dysregulated neutrophil inflammatory response in BTHS, including elevated degranulation and formation of neutrophil extracellular traps (NETs) in response to calcium flux. Developmental and functional alterations in BTHS neutrophils are underpinned by perturbations in the unfolded protein response (UPR) signaling pathway, suggesting potential therapeutic avenues for targeting BTHS neutropenia.}, }
@article {pmid39961872, year = {2025}, author = {Nguyen, CX and Nguyen, TD and Dinh, TT and Nguyen, LT and Ly, LK and Chu, HH and La, TC and Do, PT}, title = {Prime editing via precise sequence insertion restores function of the recessive rc allele in rice.}, journal = {Plant cell reports}, volume = {44}, number = {3}, pages = {57}, pmid = {39961872}, issn = {1432-203X}, support = {THTNTE.01/24-25//Vietnam Academy of Science and Technology/ ; }, mesh = {*Oryza/genetics/metabolism ; *Gene Editing/methods ; Alleles ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; *Genes, Recessive/genetics ; *Mutagenesis, Insertional ; CRISPR-Cas Systems ; Genes, Plant ; Base Sequence ; }, abstract = {An improved prime editing system precisely corrected a 14-bp deletion in the rc gene of white rice, restoring the production of brown pigments.}, }
@article {pmid39961261, year = {2025}, author = {Kumar, G and Dasgupta, I}, title = {Revolutionizing viral resistance strategies in rice: Evolution from RNAi to precision genome editing.}, journal = {Virology}, volume = {604}, number = {}, pages = {110449}, doi = {10.1016/j.virol.2025.110449}, pmid = {39961261}, issn = {1096-0341}, mesh = {*Oryza/virology/genetics/immunology ; *RNA Interference ; *Gene Editing/methods ; *Plant Diseases/virology/genetics/immunology ; CRISPR-Cas Systems ; *Disease Resistance/genetics ; *Plant Viruses/genetics ; Plants, Genetically Modified ; }, abstract = {Rice viruses are a major threat to global food security, causing significant yield losses in key rice growing regions. RNA interference (RNAi) has been crucial in engineering viral resistance in rice by silencing essential viral genes. However, the advent of genome editing, especially CRISPR/Cas, has transformed this field by allowing precise alterations of viral susceptibility genes, offering more durable and targeted resistance. This review examines the advances in RNAi strategies and the shift toward CRISPR/Cas technologies, highlighting how genome editing addresses RNAi's limitations, such as broader viral strain coverage and stronger resistance. These tools, integrated with advanced breeding methods, promise to develop rice varieties with durable, broad-spectrum virus resistance, contributing to sustainable rice production and food security.}, }
@article {pmid39961250, year = {2025}, author = {Xiang, Q and Guo, F and Li, D and Xu, F and Zhou, W}, title = {Low-background CRISPR/Cas12a sensing system with circular CRISPR RNA for amplified fluorescent detection of antibody in human serum.}, journal = {Talanta}, volume = {288}, number = {}, pages = {127730}, doi = {10.1016/j.talanta.2025.127730}, pmid = {39961250}, issn = {1873-3573}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *RNA, Circular/genetics/chemistry ; *Biosensing Techniques/methods ; *Antibodies/blood ; Fluorescence ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Regular monitoring of serum antibody levels is crucial for preventing interference with therapeutic effectiveness and reducing the risk of toxicity. To address this, a CRISPR/Cas12a sensing system with circular CRISPR RNAs ([C]crRNAs) is described for highly sensitive detection of anti-digoxin (Anti-Dig) antibodies in human serum. In this work, the topology structure of [C]crRNAs effectively suppresses the function of linear crRNAs ([L]crRNAs), making them unable to regulate the cis-/trans-cleavage activity of the Cas12a system. Therefore, a low-background is obtained in the absence of targets. The target Anti-Dig antibodies trigger the assembly of the complete multicomponent nucleic acid enzyme (MNAzyme) with active enzyme activity, which can transform [C]crRNAs into [L]crRNAs. The [L]crRNAs further recover the trans-cleavage activity of the CRISPR/Cas12a system, which can degrade single-stranded reporter DNA to generate a significantly enhanced fluorescent signal. This method enables sensitive detection of Anti-Dig antibodies as low as 15 pM within 60 min and exhibits a linear detection range of 25 pM-50 nM. It also exhibits excellent selectivity against non-target antibodies and has been successfully validated in diluted serum samples, achieving a recovery rate ranging from 96.16 % to 103.08 %. This novel CRISPR/Cas12a sensing system with [C]crRNA represents a powerful and efficient tool for detecting low-abundance biomarkers in complex biological samples.}, }
@article {pmid39961165, year = {2025}, author = {Qi, X and Yao, F and Yongli, S and Xiaofen, Z}, title = {Generation of CNPY3 knock out cell line in the H1 (WA01) hESC background.}, journal = {Stem cell research}, volume = {84}, number = {}, pages = {103676}, doi = {10.1016/j.scr.2025.103676}, pmid = {39961165}, issn = {1876-7753}, mesh = {Humans ; *Human Embryonic Stem Cells/metabolism/cytology ; Cell Line ; Cell Differentiation ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Animals ; Mice ; }, abstract = {The CNPY3 gene encodes a protein that interacts with members of the toll-like receptor (TLR) protein family and functions as a chaperone, aiding in the proper folding and export of these proteins. We generated a homozygous CNPY3 knockout human embryonic stem cell (hESC) line WAe001-A-2T (H1-CNPY3[-/-]), using CRISPR/Cas9 genome editing technology. The WAe001-A-2T cell line exhibited a normal karyotype and maintained the typical characteristics of undifferentiated hESCs, including pluripotent gene expression and differentiation potential in vivo. The CNPY3 knockout cell line serves as a valuable resource for investigating the role of the CNPY3 gene in embryonic development and lineage differentiation in vitro.}, }
@article {pmid39960330, year = {2025}, author = {Zheng, S and Liu, Y and Xia, X and Xiao, J and Ma, H and Yuan, X and Zhang, Y and Chen, Z and Peng, G and Li, W and Fei, JF and Liu, Y}, title = {Sequence Context-Agnostic TadA-Derived Cytosine Base Editors for Genome-Wide Editing in Zebrafish.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {12}, number = {14}, pages = {e2411478}, pmid = {39960330}, issn = {2198-3844}, support = {32070819//National Natural Science Foundation of China/ ; 92268114//National Natural Science Foundation of China/ ; 31970782//National Natural Science Foundation of China/ ; 2021YFA0805000//National Key R&amp;D Program of China/ ; 2023YFA1800600//National Key R&amp;D Program of China/ ; DFJHBF202103//High-level Hospital Construction Project of Guangdong Provincial People's Hospital/ ; KJ012021012//High-level Hospital Construction Project of Guangdong Provincial People's Hospital/ ; }, mesh = {Animals ; *Zebrafish/genetics ; *Gene Editing/methods ; *Cytosine/metabolism ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; }, abstract = {Single-nucleotide variants (SNVs) represent a significant form of genetic variation linked to various diseases. CRISPR-mediated base editing has emerged as a powerful method for modeling diseases caused by SNVs, particularly in zebrafish, which serve as an excellent platform for investigating disease mechanisms and conducting drug screenings. However, existing cytosine base editors (CBEs) for zebrafish often have broad editing windows and strong sequence preferences, limiting their effectiveness. In this study, zebrafish (z) TadA-derived cytosine base editors, termed zTadA-CBEs, are developed by introducing key mutations into the TadA8e enzyme. These novel editors demonstrate improved efficiency and precision in cytosine base editing. Specifically, zTadA-BE4max and zTadA-BEmv offer complementary editing windows, while zTadA-SpRY-BE4max allows for PAM-flexible editing. Using zTadA-CBEs, a precise disease model for Axenfeld-Rieger syndrome is established, and created two new models for Hermansky-Pudlak syndrome. Additionally, a novel albinism model carrying two pathogenic SNVs in the F0 generation is developed. By employing specifically designed sgRNA, the fms[ts±] missense mutation is corrected back to the wild-type nucleotide (C > T), successfully restoring macrophage levels to normal. These findings underscore the potential of zTadA-CBEs to enhance genome editing techniques and their applications in developing therapies for SNV-related disorders.}, }
@article {pmid39960174, year = {2025}, author = {Stewart, C and Manriquez Roman, C and Kenderian, SS}, title = {Performing an In Vitro Genome-Wide CRISPR Knockout Screen in Chimeric Antigen Receptor T Cells.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {215}, pages = {}, doi = {10.3791/67338}, pmid = {39960174}, issn = {1940-087X}, mesh = {*Receptors, Chimeric Antigen/genetics/immunology ; Humans ; *Gene Knockout Techniques/methods ; *CRISPR-Cas Systems ; *T-Lymphocytes/immunology ; Antigens, CD19/immunology/genetics ; Immunotherapy, Adoptive/methods ; Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Chimeric antigen receptor T (CART) cell therapy is an innovative form of targeted immunotherapy that has revolutionized the treatment of cancer. However, the durable response remains limited. Recent studies have shown that the epigenetic landscape of preinfusion CART cell products can influence response to therapy, and gene editing has been proposed as a solution. However, more work needs to be done to determine the optimal gene editing strategy. Genome-wide CRISPR screens have become popular tools to both investigate mechanisms of resistance and optimize gene editing strategies. Yet their application to primary cells presents many challenges. Here we describe a method to complete a genome-wide CRISPR knockout screen in CART cells from healthy donors. As a proof-of-concept model, we designed this method to investigate the development of exhaustion in CART cells targeting the CD19 antigen. However, we believe that this approach can be used to address a variety of mechanisms of resistance to therapy in different CAR constructs and tumor models.}, }
@article {pmid39957255, year = {2025}, author = {Li, C and Liang, Y and Feng, Q}, title = {An electrochemical biosensor utilizing CRISPR/Cas12a amplification for the detection of E. coli.}, journal = {The Analyst}, volume = {150}, number = {6}, pages = {1158-1166}, doi = {10.1039/d4an01441c}, pmid = {39957255}, issn = {1364-5528}, mesh = {*Biosensing Techniques/methods ; *Escherichia coli/isolation &amp; purification/genetics ; *CRISPR-Cas Systems/genetics ; *Electrochemical Techniques/methods ; Aptamers, Nucleotide/chemistry/genetics ; Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; Electrodes ; Bacterial Proteins ; }, abstract = {Electrochemical biosensors are frequently employed to identify harmful microbes and disease indicators. However, their practical applicability is constrained by poor signal amplification efficiency and immobilization processes on the probe surface. To get over these restrictions, we here integrated roll-cycling amplification (RCA) and CRISPR/Cas12a gene editing tools with electrochemical biosensors. We constructed an electrochemical biosensor based on RCA to activate the cleavage activity of Cas12a. First, by double-stranded nucleic acid aptamer (ds Apt) specifically binding to E. coli-2571, Apt-b was competitively isolated to bind to T4 ligase and produce circular DNA. This in turn activates RCA, which in turn activates the accessory cleavage activity of CRISPR/Cas12a, which in turn cleaves DNA sequences loaded onto the electrode, changing electrochemical signals. With a linear range of 1 × 10[2]-1 × 10[7] CFU mL[-1], a detection limit of 5.28 CFU mL[-1], and experimental recoveries of 93.01-101.53%, the measured electrochemical signals were positively connected with the concentration of E. coli-2571 under the optimal experimental conditions. Therefore, by combining two approaches-RCA and CRISPR/Cas12a-our electrochemical biosensor was able to detect E. coli-2571 specifically and sensitively, providing new research opportunities for the detection of other harmful bacteria.}, }
@article {pmid39957248, year = {2025}, author = {Yin, X and Wang, R and Thackeray, A and Baehrecke, EH and Alkema, MJ}, title = {VPS13D mutations affect mitochondrial homeostasis and locomotion in Caenorhabditis elegans.}, journal = {G3 (Bethesda, Md.)}, volume = {15}, number = {4}, pages = {}, pmid = {39957248}, issn = {2160-1836}, support = {P40 OD010440/OD/NIH HHS/United States ; R35 GM131689/GM/NIGMS NIH HHS/United States ; R01 GM140480/NH/NIH HHS/United States ; }, mesh = {Animals ; *Caenorhabditis elegans/genetics/physiology ; *Mitochondria/metabolism/genetics ; *Caenorhabditis elegans Proteins/genetics/metabolism ; *Locomotion/genetics ; *Homeostasis/genetics ; *Mutation ; Unfolded Protein Response/genetics ; CRISPR-Cas Systems ; Humans ; }, abstract = {Mitochondria control cellular metabolism, serve as hubs for signaling and organelle communication, and are important for the health and survival of cells. VPS13D encodes a cytoplasmic lipid transfer protein that regulates mitochondrial morphology, mitochondria and endoplasmic reticulum contact, and quality control of mitochondria. VPS13D mutations have been reported in patients displaying ataxic and spastic gait disorders with variable age of onset. Here, we used CRISPR/Cas9 gene editing to create VPS13D-related spinocerebellar ataxia-4 missense mutations and C-terminal deletion in VPS13D's ortholog vps-13D in Caenorhabditis elegans. Consistent with SCAR4 patient movement disorders and mitochondrial dysfunction, vps-13D mutant worms exhibit locomotion defects and abnormal mitochondrial morphology. Importantly, animals with a vps-13D deletion or a N3017I missense mutation exhibited an increase in mitochondrial unfolded protein response. The cellular and behavioral changes caused by VPS13D mutations in C. elegans advance the development of animal models that are needed to study SCAR4 pathogenesis.}, }
@article {pmid39956955, year = {2025}, author = {Izzo, M and Battistini, J and Golini, E and Voellenkle, C and Provenzano, C and Orsini, T and Strimpakos, G and Scavizzi, F and Raspa, M and Baci, D and Frolova, S and Tastsoglou, S and Zaccagnini, G and Garcia-Manteiga, JM and Gourdon, G and Mandillo, S and Cardinali, B and Martelli, F and Falcone, G}, title = {Muscle-specific gene editing improves molecular and phenotypic defects in a mouse model of myotonic dystrophy type 1.}, journal = {Clinical and translational medicine}, volume = {15}, number = {2}, pages = {e70227}, pmid = {39956955}, issn = {2001-1326}, support = {23054//AFM-T&#xe9;l&#xe9;thon/ ; GGP19035//Fondazione Telethon/ ; POS T4 CAL.HUB.RIA cod. T4-AN-09//Ministero della Salute/ ; RF-2019-12368521//Ministero della Salute/ ; Ricerca Corrente 2024 1.07.128//Ministero della Salute/ ; //Fondazione Monica Stupino/ ; EU PNRR/2022/C9/MCID/I8//European Commission/ ; EU-NRRP M6C2 Inv. 2.1 PNRR-MAD-2022-12375790//European Commission/ ; PNRR-MCNT2-2023-12377983//European Commission/ ; }, mesh = {Animals ; *Myotonic Dystrophy/genetics/therapy ; *Gene Editing/methods ; Mice ; Disease Models, Animal ; Phenotype ; Myotonin-Protein Kinase/genetics ; Humans ; CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; }, abstract = {BACKGROUND: Myotonic dystrophy type 1 (DM1) is a genetic multisystemic disease, characterised by pleiotropic symptoms that exhibit notable variability in severity, nature and age of onset. The genetic cause of DM1 is the expansion of unstable CTG-repeats in the 3' untranslated region (UTR) of the DMPK gene, resulting in the accumulation of toxic CUG-transcripts that sequester RNA-binding proteins and form nuclear foci in DM1 affected tissues and, consequently, alter various cellular processes. Therapeutic gene editing for treatment of monogenic diseases is a powerful technology that could in principle remove definitively the disease-causing genetic defect. The precision and efficiency of the molecular mechanisms are still under investigation in view of a possible use in clinical practice.<br><br>METHODS: Here, we describe the application of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) strategy to remove the CTG-expansion in the DMPK gene in a mouse model carrying the human transgene from a DM1 patient. To optimise the editing efficiency in vivo, we identified new tools that allowed to improve the expression levels and the activity of the CRISPR/Cas9 machinery. Newly designed guide RNA pairs were tested in DM1-patient derived cells before in vivo application. Edited cells were analysed to assess the occurrence of off-target and the accuracy of on-target genomic events. Gene editing-dependent and -independent mechanisms leading to decreased accumulation of the mutated DMPK transcripts were also evaluated.<br><br>RESULTS AND CONCLUSION: Systemic delivery of CRISPR/Cas9 components in DM1 mice, through myotropic adeno-associated viral vectors, led to significant improvement of molecular alterations in the heart and skeletal muscle. Importantly, a persistent increase of body weight, improvement of muscle strength and body composition parameters were observed in treated animals. Accurate evaluation of CRISPR/Cas9-mediated-phenotypic recovery in vivo is a crucial preclinical step for the development of a gene therapy for DM1 patients.<br><br>KEY POINTS: In vivo application of a therapeutic gene editing strategy for permanent deletion of the pathogenetic CTG-repeat amplification in the DMPK gene that causes myotonic dystrophy type 1. Following treatment, diseased mice show a significant improvement of both molecular and phenotypic defects.}, }
@article {pmid39956852, year = {2025}, author = {Breusegem, SY and Houghton, J and Romero-Bueno, R and Fragoso-Luna, A and Kentistou, KA and Ong, KK and Janssen, AFJ and Bright, NA and Riedel, CG and Perry, JRB and Askjaer, P and Larrieu, D}, title = {A multiparametric anti-aging CRISPR screen uncovers a role for BAF in protein synthesis regulation.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {1681}, pmid = {39956852}, issn = {2041-1723}, support = {206242/Z/17/Z//Wellcome Trust (Wellcome)/ ; N/A//Federation of European Biochemical Societies (FEBS)/ ; MR/R0009015/1//RCUK | Medical Research Council (MRC)/ ; 204845/Z/16/Z//Wellcome Trust (Wellcome)/ ; MR/M010007/1//RCUK | Medical Research Council (MRC)/ ; 20 1034 Pj//Cancerfonden (Swedish Cancer Society)/ ; 2019-04868//Vetenskapsr&#xe5;det (Swedish Research Council)/ ; 2017-06088//Vetenskapsr&#xe5;det (Swedish Research Council)/ ; MC_UU_00006/2//RCUK | Medical Research Council (MRC)/ ; /WT_/Wellcome Trust/United Kingdom ; MC_UU_12015/2//RCUK | Medical Research Council (MRC)/ ; }, mesh = {Animals ; *Progeria/genetics/metabolism ; Caenorhabditis elegans/genetics/metabolism ; Humans ; Male ; *Protein Biosynthesis/genetics ; Fibroblasts/metabolism ; CRISPR-Cas Systems ; *DNA-Binding Proteins/genetics/metabolism ; Mutation ; *Aging/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Nuclear Proteins/genetics/metabolism ; }, abstract = {Progeria syndromes are very rare, incurable premature aging conditions recapitulating most aging features. Here, we report a whole genome, multiparametric CRISPR screen, identifying 43 genes that can rescue multiple cellular phenotypes associated with progeria. We implement the screen in fibroblasts from Néstor-Guillermo Progeria Syndrome male patients, carrying a homozygous A12T mutation in BAF. The hits are enriched for genes involved in protein synthesis, protein and RNA transport and osteoclast formation and are validated in a whole-organism Caenorhabditis elegans model. We further confirm that BAF A12T can disrupt protein synthesis rate and fidelity, which could contribute to premature aging in patients. This work highlights the power of multiparametric genome-wide suppressor screens to identify genes enhancing cellular resilience in premature aging and provide insights into the biology underlying progeria-associated cellular dysfunction.}, }
@article {pmid39956793, year = {2025}, author = {Uzun, S and Özcan, &#xd6; and Gök, A and Işık, A and Bakır, S and Günel-Özcan, A and Onbaşılar, &#x130; and Akyol, A}, title = {A new CRISPR-mediated Apc knockout allele leads to pyloric gland adenoma-like gastric polyps in mice with C57BL/6;FVB/N mixed background.}, journal = {Animal models and experimental medicine}, volume = {8}, number = {5}, pages = {922-929}, pmid = {39956793}, issn = {2576-2095}, support = {SBAG-215S926//The Scientific and Technological Research Council of Turkey 1001 program/ ; }, mesh = {Animals ; Mice, Inbred C57BL ; Alleles ; Mice ; *CRISPR-Cas Systems/genetics ; Mice, Knockout ; Disease Models, Animal ; *Stomach Neoplasms/genetics/pathology ; *Adenomatous Polyposis Coli Protein/genetics ; Male ; *Adenoma/genetics/pathology ; Adenomatous Polyps ; }, abstract = {Adenomatous polyposis coli (APC) mutations are the most frequently identified genetic alteration in sporadic colorectal cancer (CRC) cases, and a myriad of genetically engineered Apc-mutant CRC mouse models have been developed using various genetic manipulation techniques. The advent of the CRISPR/Cas9 system has revolutionized the field of genetic engineering and facilitated the development of new genetically engineered mouse models. In this study, we aimed to develop a novel Apc knockout allele using the CRISPR/Cas9 system and evaluate the phenotypic effects of this new allele in two different mouse strains. For this purpose, exon 16 of mouse Apc gene was targeted with a single-guide RNA, and the mouse carrying an Apc frameshift mutation at codon 750 (Δ750) was chosen as the founder. The mutant FVB-Apc[Δ750] mice were backcrossed with wild-type C57BL/6 mice, and the phenotypic effects of the knockout allele were evaluated in F8-FVB-Apc[Δ750], F4-B6;FVB-Apc[Δ750], and F1-B6;FVB-Apc[Δ750] by a macroscopic and microscopic examination of the gastrointestinal system. The result showed that the mean polyp number was significantly higher in F4-BL6;FVB-Apc[Δ750] than in F8-FVB-Apc[Δ750]. Intestinal polyposis was more prominent in F4-BL6;FVB-Apc[Δ750], whereas a higher number of colon polyps than intestinal polyps were observed in F8-FVB-Apc[Δ750]. Additionally, F1-BL6;FVB-Apc[Δ750] mixed background mice developed gastric polyps that morphologically resembled the pyloric gland adenoma of humans. In conclusion, we developed a novel CRISPR-mediated Apc knockout allele using two mouse strains. We showed that this allele can exert a strain-specific effect on the phenotype of mice and can cause gastric polyp formation.}, }
@article {pmid39956401, year = {2025}, author = {Puri, B and Majumder, S and Gaikwad, AB}, title = {CRISPR/Cas9 based knockout of lncRNA MALAT1 attenuates TGF-β1 induced Smad 2/3 mediated fibrosis during AKI-to-CKD transition.}, journal = {European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences}, volume = {207}, number = {}, pages = {107044}, doi = {10.1016/j.ejps.2025.107044}, pmid = {39956401}, issn = {1879-0720}, mesh = {Gene Knockout Techniques ; Animals ; Rats ; Cell Line ; *Acute Kidney Injury/drug therapy/genetics/metabolism/pathology ; *Renal Insufficiency, Chronic/genetics/metabolism/pathology/prevention &amp; control ; Fibrosis/genetics/metabolism/pathology ; Disease Progression ; *RNA, Long Noncoding/antagonists &amp; inhibitors/genetics/metabolism ; Transforming Growth Factor beta1/genetics/metabolism ; *Gene Expression Regulation/drug effects ; Smad2 Protein/genetics/metabolism ; Smad3 Protein/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Signal Transduction/drug effects/genetics ; *Kidney/drug effects/metabolism/pathology ; }, abstract = {Acute kidney injury (AKI) is a significant clinical issue with potential long-term consequences, as even a single episode can progress to chronic kidney disease (CKD). The AKI-to-CKD transition involves complex pathophysiology, including persistent inflammation, apoptosis, and fibrosis. Long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been recognized as a potential therapeutic target for various kidney diseases, including AKI and CKD. In our previous study, we conducted the transcriptomic analysis of lncRNAs in-vitro and animal models of AKI-to-CKD transition and found several dysregulated lncRNAs such as MALAT1, MEG3, NEAT1, MIAT, and H19 in this transition. Among these, we have selected lncRNA MALAT1 to further validate its role in AKI-to-CKD transition as a therapeutic target via a cluster regularly intercept short palindromic protein (CRISPR) associated protein 9 (Cas9)-mediated knockout approach in NRK52E cells. Guide RNAs (gRNAs) were designed to target MALAT1, and the PX459 turbo green fluorescence protein (GFP) plasmid containing MALAT1 gRNA1&amp;2 was transfected into NRK52E cells using CRISPRMAX. Results demonstrated that MALAT1 knockout significantly reduced MALAT1 expression and attenuated Smad2/3-mediated fibrosis by decreasing pSmad2, pSmad2/3, Smad4, vimentin, fibronectin, collagen-I, and α-SMA expression levels, while increasing Smad7, Smurf2, and E-cadherin levels. These findings suggest that targeting the MALAT1/Smad2/3 pathway could be a potential therapeutic target for mitigating fibrosis to prevent AKI-to-CKD transition.}, }
@article {pmid39955894, year = {2025}, author = {Kishimoto, H and Miyazaki, K and Omori, M and Higuchi, K and Shirasaka, Y and Inoue, K}, title = {Establishment of endogenous canine MUC1 knock-out MDCKII cells using CRISPR-Cas9 and evaluation of drug permeation.}, journal = {Drug metabolism and pharmacokinetics}, volume = {61}, number = {}, pages = {101051}, doi = {10.1016/j.dmpk.2025.101051}, pmid = {39955894}, issn = {1880-0920}, mesh = {Animals ; Dogs ; Madin Darby Canine Kidney Cells ; *CRISPR-Cas Systems/genetics ; *Mucin-1/genetics/metabolism ; Gene Knockout Techniques ; Cell Membrane Permeability ; ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism ; }, abstract = {Most orally administered drugs are absorbed by simple diffusion across the intestinal epithelium. Monolayers of MDCKII cells and parallel artificial membrane permeability assay are widely used to evaluate simple diffusion as an in vitro model; however, these models do not account for the contribution of mucus glycoprotein, which may play a significant role in drug permeation. We focused on the role of MUC1, a membrane-bound mucin that is found on the luminal surface of the gastrointestinal epithelium, in the simple diffusion of lipophilic drugs. We generated endogenous canine Mdr1 (cMdr1) and Muc1 (cMuc1) knock-out MDCKII cells by genomic editing and evaluated the effect of cMuc1 on the simple diffusion of various drugs. The absence of cMuc1 significantly increased the membrane permeation of lipophilic drugs, such as griseofulvin as well as paclitaxel and rhodamine 123, substrates of the MDR1 efflux transporter, which suggests that cMuc1 is one of the key factors that modulate the membrane permeation of these drugs. Taken together, we successfully established MDCKII cell lines with a complete knock-out of endogenous cMuc1 and cMdr1 expressions. This provides a novel in vitro model system for studying the mechanisms underlying drug absorption and transport, with potential applications for drug development.}, }
@article {pmid39955701, year = {2025}, author = {Sun, J and Jiang, X and Xu, F and Tian, X and Chu, J}, title = {Constructing pyrG marker by CRISPR/Cas facilities the highly-efficient precise genome editing on industrial Aspergillus niger strain.}, journal = {Bioprocess and biosystems engineering}, volume = {48}, number = {4}, pages = {679-691}, pmid = {39955701}, issn = {1615-7605}, support = {No.2022YFC2105403//the National Key Research Development Program of China/ ; No. 21276081//the National Natural Science Foundation of China/ ; QDSWZK202004//the Scientific Research Think Tank of Biological Manufacturing Industry in Qingdao/ ; JKF01211116, JKVF1211036, JKF01211708//the Fundamental Research Funds for the Central Universities/ ; }, mesh = {*Aspergillus niger/genetics ; *Gene Editing ; *CRISPR-Cas Systems ; *Genome, Fungal ; }, abstract = {To prevent the unique difficulty of hygromycin-based gene editing on industrial A. niger strain and increase the working efficiency, the local pyrG marker was removed by well-designed dual sgRNAs and repair template through Cas9-ribonucleoprotein (RNP) strategy in this study. The positive rate of the desired pyrG auxotroph construction was 100%, while no transformant was observed using the traditional methods. The complementation strain showed similar fermentation character as the starting strain. Moreover, an efficient and seamless knock out plasmid-based strategy was established, achieving positive rate at 90% and 50% for challenging Δku70 and Δku80 respectively. Further, combined hygromycin markers and miniaturization cultivation were conducted to select the poor growth strain. Finally, skillfully designed sgRNA and amdS counter-selection repair template were used to obtain ERG3[Tyr185] mutation. A highly-efficient precise strategy was established for A. niger through a diagnostic PCR method, with nearly 100% positive rate. Highly- precise desired point mutation was achieved by the developed gene toolbox.}, }
@article {pmid39955618, year = {2025}, author = {Baatz, F and Ghosh, A and Herbst, J and Polten, S and Meyer, J and Rhiel, M and Maetzig, T and Geffers, R and Rothe, M and Bastone, AL and John-Neek, P and Frühauf, J and Eiz-Vesper, B and Bonifacius, A and Falk, CS and Kaisenberg, CV and Cathomen, T and Schambach, A and van den Brink, MRM and Hust, M and Sauer, MG}, title = {Targeting BCL11B in CAR-engineered lymphoid progenitors drives NK-like cell development with prolonged anti-leukemic activity.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {33}, number = {4}, pages = {1584-1607}, pmid = {39955618}, issn = {1525-0024}, mesh = {Animals ; Humans ; Mice ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; CRISPR-Cas Systems ; *Killer Cells, Natural/immunology/metabolism/cytology ; *Lymphoid Progenitor Cells/metabolism/immunology/cytology ; *Repressor Proteins/genetics/metabolism ; *Tumor Suppressor Proteins/genetics/metabolism ; *Leukemia/therapy/immunology/genetics ; Immunotherapy, Adoptive ; Cell Differentiation ; }, abstract = {Chimeric antigen receptor (CAR)-induced suppression of the transcription factor B cell CLL/lymphoma 11B (BCL11B) propagates CAR-induced killer (CARiK) cell development from lymphoid progenitors. Here, we show that CRISPR-Cas9-mediated Bcl11b knockout in human and murine early lymphoid progenitors distinctively modulates this process either alone or in combination with a CAR. Upon adoptive transfer into hematopoietic stem cell recipients, Bcl11b-edited progenitors mediated innate-like antigen-independent anti-leukemic immune responses. With CAR expression allowing for additional antigen-specific responses, the progeny of double-edited lymphoid progenitors acquired prolonged anti-leukemic activity in vivo. These findings give important insights into how Bcl11b targeting can be used to tailor anti-leukemia functionality of CAR-engineered lymphoid progenitor cells.}, }
@article {pmid39955288, year = {2025}, author = {Zheng, W and Li, H and Liu, M and Wei, Y and Liu, B and Li, Z and Xiong, C and Huang, S and Hu, C and Ouyang, S}, title = {Molecular insights and rational engineering of a compact CRISPR-Cas effector Cas12h1 with a broad-spectrum PAM.}, journal = {Signal transduction and targeted therapy}, volume = {10}, number = {1}, pages = {66}, pmid = {39955288}, issn = {2059-3635}, support = {82225028//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82172287//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2022J01638//Natural Science Foundation of Fujian Province (Fujian Provincial Natural Science Foundation)/ ; }, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/chemistry ; DNA/genetics/chemistry ; *Deoxyribonuclease I/genetics/chemistry ; Gene Editing ; }, abstract = {Cas12h1 is a compact CRISPR-associated nuclease from functionally diverse type V CRISPR-Cas effectors and recognizes a purine-rich protospacer adjacent motif (PAM) distinct from that of other type V Cas effectors. Here, we report the nickase preference of Cas12h1, which predominantly cleaves the nontarget strand (NTS) of a double-stranded DNA (dsDNA) substrate. In addition, Cas12h1 acts as a nickase in human cells. We further determined the cryo-EM structures of Cas12h1 in the surveillance, R-loop formation, and interference states, revealing the molecular mechanisms involved in the crRNA maturation, target recognition, R-loop formation, nuclease activation and target degradation. Cas12h1 notably recognizes a broad 5'-DHR-3' PAM (D is A, G, or T; H is A, C, or T; R is A or G) both in vitro and in human cells. In addition, Cas12h1 utilizes a distinct activation mechanism that the lid motif undergoes a "flexible to stable" transition to expose the catalytic site to the substrate. A high-fidelity nucleic acid detector, Cas12h1[hf], was developed through rational engineering, which distinguishes single-base mismatches and retains comparable on-target activities. Our results shed light on the molecular mechanisms underlying Cas12h1 nickase, improve the understanding of type V Cas effectors, and expand the CRISPR toolbox for genome editing and molecular diagnosis.}, }
@article {pmid39955231, year = {2025}, author = {Oliveira, DM and Saleme, MLS and Smith, RA and Vangeel, T and Lima, MF and Chanoca, AA and Mota, TR and Vanhevel, Y and Coussens, G and Van Aelst, K and Geerts, J and Cornet, I and Vaneechoutte, D and Vandepoele, K and Pauwels, L and Goeminne, G and Morreel, K and Sels, BF and Ralph, J and Vanholme, R and Boerjan, W}, title = {CRISPR/Cas9 editing of p-COUMAROYL-CoA:MONOLIGNOL TRANSFERASE 1 in maize alters phenolic metabolism, lignin structure, and lignin-first biomass processing.}, journal = {Trends in biotechnology}, volume = {43}, number = {5}, pages = {1166-1195}, doi = {10.1016/j.tibtech.2025.01.006}, pmid = {39955231}, issn = {1879-3096}, mesh = {*Lignin/metabolism/chemistry ; *Zea mays/genetics/metabolism/enzymology ; *CRISPR-Cas Systems/genetics ; Biomass ; *Gene Editing/methods ; *Phenols/metabolism ; Plants, Genetically Modified ; *Plant Proteins/genetics/metabolism ; }, abstract = {Valorization of lignocellulosic biomass for sustainable production of high-value chemicals is challenged by the complexity of lignin, a phenolic biopolymer. Beyond the classical lignin monomers derived from p-coumaryl, coniferyl, and sinapyl alcohol, grass lignins incorporate substantial amounts of monolignol p-coumarates that are produced by p-COUMAROYL-CoA:MONOLIGNOL TRANSFERASE (PMT). Here, the CRISPR/Cas9-mediated mutation of ZmPMT1 in maize enabled the design of biomass depleted in p-coumaroylated lignin and enriched in guaiacyl lignin. Lignin-first biorefining of stem biomass from zmpmt1 mutants by reductive catalytic fractionation (RCF) generated a lignin oil depleted in carboxylates and enriched in guaiacyl-derived alcohols, which are desirable substrates for bio-based polyurethane synthesis. The reported lignin engineering in maize is a promising strategy for designing a dual-purpose crop, providing both food and feed, along with a renewable feedstock for the production of plant-based chemicals.}, }
@article {pmid39955067, year = {2025}, author = {Severino, MB and Morelli, AP and Pavan, ICB and Mancini, MCS and Góis, MM and Borges, RJ and Braga, RR and da Silva, LGS and Quintero-Ruiz, N and Costa, MM and Oliveira, WL and Bezerra, RMN and Ropelle, ER and Simabuco, FM}, title = {A CRISPR-edited isoform of the AMPK kinase LKB1 improves the response to cisplatin in A549 lung cancer cells.}, journal = {The Journal of biological chemistry}, volume = {301}, number = {3}, pages = {108308}, pmid = {39955067}, issn = {1083-351X}, mesh = {Humans ; *Cisplatin/pharmacology ; *Protein Serine-Threonine Kinases/genetics/metabolism ; AMP-Activated Protein Kinase Kinases ; A549 Cells ; *Lung Neoplasms/genetics/drug therapy/pathology/metabolism ; *CRISPR-Cas Systems ; *Gene Editing ; AMP-Activated Protein Kinases/metabolism/genetics ; *Antineoplastic Agents/pharmacology ; Autophagy/drug effects ; Drug Resistance, Neoplasm/genetics ; TOR Serine-Threonine Kinases/metabolism ; NF-E2-Related Factor 2 ; }, abstract = {Lung cancer presents the highest mortality rate in the world when compared to other cancer types and often presents chemotherapy resistance to cisplatin. The A549 nonsmall cell lung cancer line is widely used as a model for lung adenocarcinoma studies since it presents a high proliferative rate and a nonsense mutation in the STK11 gene. The LKB1 protein, encoded by the STK11 gene, is one of the major regulators of cellular metabolism through AMPK activation under nutrient deprivation. Mutation in the STK11 gene in A549 cells potentiates cancer hallmarks, such as deregulation of cellular metabolism, aside from the Warburg effect, mTOR activation, autophagy inhibition, and NRF2 and redox activation. In this study, we investigated the integration of these pathways associated with the metabolism regulation by LKB1/AMPK to improve cisplatin response in the A549 cell line. We first used the CRISPR/Cas9 system to generate cell lines with a CRISPR-edited LKB1 isoform (called Super LKB1), achieved through the introduction of a +1 adenine insertion in the first exon of the STK11 gene after NHEJ-mediated repair. This insertion led to the expression of a higher molecular weight protein containing an alternative exon described in the Peutz-Jeghers Syndrome. Through metabolic regulation by Super LKB1 expression and AMPK activation, we found an increase in autophagy flux (LC3 GFP/RFP p < 0.05), as well as a reduction in the phosphorylation of mTORC1 downstream targets (S6K2 phospho-serine 423; p < 0.05; and S6 ribosomal protein phospho-serine 240/244; p < 0.03). The NRF2 protein exhibited increased levels and more nuclear localization in A549 WT cells compared to the edited cells (p < 0.01). We also observed lower levels of H2O2 in the WT A549 cells, as a possible result of NRF2 activation, and a higher requirement of cisplatin to achieve the IC50 (WT: 10 μM; c2SL+: 5.5 μM; c3SL+: 6 μM). The data presented here suggests that the regulation of molecular pathways by the novel Super LKB1 in A549 cells related to metabolism, mTORC1, and autophagy promotes a better response of lung cancer cells to cisplatin. This NHEJ-CRISPR-based approach may be potentially used for lung cancer gene therapy.}, }
@article {pmid39954947, year = {2025}, author = {Souza, HCA and Panzenhagen, P and Dos Santos, AMP and Portes, AB and Fidelis, J and Conte-Junior, CA}, title = {Unravelling the advances of CRISPR-Cas9 as a precise antimicrobial therapy: A systematic review.}, journal = {Journal of global antimicrobial resistance}, volume = {42}, number = {}, pages = {51-60}, doi = {10.1016/j.jgar.2025.02.002}, pmid = {39954947}, issn = {2213-7173}, mesh = {*CRISPR-Cas Systems ; Humans ; *Anti-Bacterial Agents/pharmacology ; *Drug Resistance, Bacterial/genetics ; Plasmids/genetics ; Escherichia coli/genetics/drug effects ; Gene Editing ; }, abstract = {UNLABELLED: Antimicrobial resistance is a critical public health threat, compromising treatment effectiveness. The spread of resistant pathogens, facilitated by genetic variability and horizontal gene transfer, primarily through plasmids, poses significant challenges to health systems.<br><br>OBJECTIVE: This review explores the potential of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology and Cas9 nucleases in combating antimicrobial resistance.<br><br>METHODS: The literature review followed the PRISMA guidelines using PubMed, Embase, and Scopus databases until July 2023.<br><br>RESULTS: The Enterobacterales family, particularly Escherichia coli, was the main focus. The resistance genes targeted were mainly associated with &#x3b2;-lactam antibiotics, specifically bla genes, and colistin resistance linked to the mcr-1 gene. Plasmid vectors have been the primary delivery method for the CRISPR-Cas9 system, with conjugative plasmids resensitizing bacterial strains to various antimicrobials. Other delivery methods included electroporation, phage-mediated delivery, and nanoparticles. The efficacy of the CRISPR-Cas9 system in resensitizing bacterial strains ranged from 4.7% to 100%.<br><br>CONCLUSIONS: Despite challenges in delivery strategies and clinical application, studies integrating nanotechnology present promising approaches to overcome these limitations. This review highlights new perspectives for the clinical use of CRISPR-Cas9 as a specific and efficient antimicrobial agent, potentially replacing traditional broad-spectrum antimicrobials in the future.}, }
@article {pmid39954549, year = {2025}, author = {Crossman, VG and Tiong, CF and Coles, CA and Bozaoglu, K and Forbes, R and Yiu, EM and Ruparelia, AA and Currie, PD and Vlahos, K and Howden, SE and North, KN and Lamandé, SR and Houweling, PJ}, title = {Generation of an iPSC line (with isogenic control) from the PBMCs of a COL6A1 (c.1056 + 2T &gt; A) Bethlem myopathy patient.}, journal = {Stem cell research}, volume = {84}, number = {}, pages = {103673}, doi = {10.1016/j.scr.2025.103673}, pmid = {39954549}, issn = {1876-7753}, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Collagen Type VI/genetics/metabolism ; *Leukocytes, Mononuclear/metabolism/cytology ; *Contracture/genetics/pathology/metabolism ; *Muscular Dystrophies/genetics/pathology/congenital ; Cell Line ; Cell Differentiation ; CRISPR-Cas Systems ; Male ; }, abstract = {To produce an in vitro model of Bethlem myopathy, we reprogrammed the peripheral blood mononuclear cells (PBMCs) of a patient with a heterozygous COL6A1 c.1056 + 2T > A mutation at the exon/intron 14 boundary of the COL6A1 gene to induced pluripotent stem cells (iPSCs). Using CRISPR/Cas9 gene editing, we corrected the mutation to generate an isogenic control line. Both the patient and isogenic control iPSCs show a normal karyotype, express pluripotency markers and can differentiate into cell states that represent the three embryonic germ layers (endoderm, mesoderm and ectoderm). These cell lines will be differentiated and used to explore disease mechanisms and evaluate novel therapeutics for Bethlem myopathy.}, }
@article {pmid39954412, year = {2025}, author = {Liu, ML and Li, Y and Yang, WJ and Yang, WW and Zhuo, Y and He, XJ}, title = {Engineering multi-activator-encoded DNA nanonet to accelerate CRISPR-Cas12a activation for rapid and sensitive electrochemiluminescence bioassay.}, journal = {Talanta}, volume = {288}, number = {}, pages = {127724}, doi = {10.1016/j.talanta.2025.127724}, pmid = {39954412}, issn = {1873-3573}, mesh = {*CRISPR-Cas Systems ; *DNA/chemistry/genetics/metabolism ; *Biosensing Techniques/methods ; DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism/analysis ; *Luminescent Measurements/methods ; *Electrochemical Techniques/methods ; Humans ; *CRISPR-Associated Proteins/metabolism ; Limit of Detection ; *Nanostructures/chemistry ; *Endodeoxyribonucleases/metabolism ; Bacterial Proteins ; }, abstract = {Despite CRISPR-associated (Cas) nucleases have emerged as a versatile and highly programmable tool for biosensing and molecular diagnostics, the efficient manipulation of targeted CRISPR-Cas12a activation requires further improvement. Herein, we engineered a target-response DNA nanodevice called multi-activator-encoded DNA nanonet (MAIDA) which displayed efficient manipulation of CRISPR-Cas12a trans-activity for apurinic/apyrimidinic endonuclease 1 (APE1) activity monitoring. The MAIDA nanodevice was constructed by multi-activator loops (MA loops) encoded with three activator sequences and target-response loops (TR loops) encoded with three abasic sites to generate interlocked DNA nanonet. Notably, the activator sequences on MA loop were pre-hybridized with TR loop, which not only generate AP sites but also inhibit the CRISPR-Cas12a activation in the initial state. When APE1 is present, the AP sites on the MAIDA nanodevice were recognized and cleaved to the release of MA loops, which could activate the trans-cleavage of CRISPR-Cas12a and then output the signal through electrochemiluminescence (ECL) biosensor. Finally, the experimental results demonstrate that the MA loops increase the ECL response of CRISPR-Cas12a by 1.5-fold compared with the conventional single-linear activators, and the limit of detection (LOD) of APE1 by the proposed biosensor is 1.46 × 10[-10] U/μL. The MAIDA nanodevice promoted the efficient manipulation of targeted CRISPR-Cas12a activation with high sensitivity and selectivity, which provided a promising tool for enhancing DNA-based sensing and analytical applications.}, }
@article {pmid39954386, year = {2025}, author = {Vilar, LC and Rego, ACS and Miguel, MAL and Paranhos, RPDR and Laport, MS and Rossi, CC and Giambiagi-deMarval, M}, title = {Staphylococcus spp. and methicillin-resistance gene mecA dispersion in seawater: A case study of Guanabara Bay's recreational and touristic waters.}, journal = {Comparative immunology, microbiology and infectious diseases}, volume = {118}, number = {}, pages = {102326}, doi = {10.1016/j.cimid.2025.102326}, pmid = {39954386}, issn = {1878-1667}, mesh = {*Staphylococcus/genetics/isolation &amp; purification/drug effects/classification ; *Seawater/microbiology ; Brazil ; *Methicillin Resistance/genetics ; Anti-Bacterial Agents/pharmacology ; Humans ; *Bacterial Proteins/genetics ; *Bays/microbiology ; Microbial Sensitivity Tests ; *Penicillin-Binding Proteins/genetics ; Genetic Variation ; Staphylococcal Infections/microbiology ; }, abstract = {Environmental Staphylococci, particularly coagulase-negative Staphylococci (CoNS), are known reservoirs of antimicrobial resistance genes and human-animal opportunistic pathogens, yet their role within the One Health framework remains underexplored. In this study, we isolated 12 species of CoNS from two sites 10 km apart in Guanabara Bay, Rio de Janeiro, with the most frequent species being the opportunistic Staphylococcus saprophyticus (30.3 %), Staphylococcus warneri (25.7 %), and Staphylococcus epidermidis (16.7 %). GTG5-PCR fingerprinting revealed significant genetic diversity, yet identical profiles persisted across both sites throughout the year, indicating strain dispersion and persistence. Among the 66 strains analyzed, 42 exhibited resistance to clinically significant antimicrobials, including methicillin-resistant strains harboring the mecA gene. Remarkably, 22.7 % of the strains carried CRISPR-Cas systems, a frequency unusually high for Staphylococcus spp., suggesting that bacteriophage pressure in the seawater environment may drive this increase. The presence of antimicrobial-resistant CoNS in Guanabara Bay, a popular recreational area, represents a potential public health risk.}, }
@article {pmid39954255, year = {2025}, author = {Xiang, RR and Lee, SA and Tyndall, CF and Bhatia, AR and Yin, J and Singler, C and Hauk, BJ and Kipp, MP and Takeda, DY}, title = {CRISPR screening identifies regulators of enhancer-mediated androgen receptor transcription in advanced prostate cancer.}, journal = {Cell reports}, volume = {44}, number = {2}, pages = {115312}, pmid = {39954255}, issn = {2211-1247}, support = {ZIA BC011965/ImNIH/Intramural NIH HHS/United States ; }, mesh = {Male ; *Receptors, Androgen/genetics/metabolism ; Humans ; *Enhancer Elements, Genetic/genetics ; Hepatocyte Nuclear Factor 3-alpha/metabolism/genetics ; Homeodomain Proteins/metabolism/genetics ; Animals ; Cell Line, Tumor ; *CRISPR-Cas Systems/genetics ; GATA2 Transcription Factor/metabolism/genetics ; *Transcription, Genetic ; *Prostatic Neoplasms/genetics/pathology ; Gene Expression Regulation, Neoplastic ; Mice ; Transcription Factor AP-2/metabolism/genetics ; *Prostatic Neoplasms, Castration-Resistant/genetics/pathology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Amplification of the androgen receptor (AR) locus is the most frequent alteration in metastatic castration-resistant prostate cancer (CRPC). Recently, it was discovered that an enhancer of the AR is co-amplified with the AR gene body and contributes to increased AR transcription and resistance to androgen deprivation therapy. However, the mechanism of enhancer activation in advanced disease is unknown. Here, we used CRISPR-Cas9 screening to identify transcription factors that bind to the AR enhancer and modulate enhancer-mediated AR transcription. We demonstrate that HOXB13, GATA2, and TFAP2C bind the AR enhancer in patient-derived xenografts and directly impact features associated with an active chromatin state. Interestingly, the AR enhancer belongs to a set of regulatory elements that require HOXB13 to maintain FOXA1 binding, further delineating the role of HOXB13 in CRPC. This work provides a framework to functionally identify trans-acting factors required for the activation of disease-related noncoding regulatory elements.}, }
@article {pmid39953143, year = {2025}, author = {Chambial, P and Thakur, N and Bhukya, PL and Subbaiyan, A and Kumar, U}, title = {Frontiers in superbug management: innovating approaches to combat antimicrobial resistance.}, journal = {Archives of microbiology}, volume = {207}, number = {3}, pages = {60}, pmid = {39953143}, issn = {1432-072X}, mesh = {Humans ; *Anti-Bacterial Agents/pharmacology/therapeutic use ; *Bacteria/drug effects/genetics ; CRISPR-Cas Systems ; *Drug Resistance, Multiple, Bacterial ; Gene Editing ; *Drug Resistance, Bacterial ; *Bacterial Infections/drug therapy/microbiology ; }, abstract = {Anti-microbial resistance (AMR) is a global health issue causing significant mortality and economic burden. Pharmaceutical companies' discontinuation of research hinders new agents, while MDR pathogens or "superbugs" worsen the problem. Superbugs pose a threat to common infections and medical procedures, exacerbated by limited antibiotic development and rapid antibiotic resistance. The rising tide of antimicrobial resistance threatens to undermine progress in controlling infectious diseases. This review examines the global proliferation of AMR, its underlying mechanisms, and contributing factors. The study explores various methodologies, emphasizing the significance of precise and timely identification of resistant strains. We discuss recent advancements in CRISPR/Cas9, nanoparticle technology, light-based techniques, and AI-powered antibiogram analysis for combating AMR. Traditional methods often fail to effectively combat multidrug-resistant bacteria, as CRISPR-Cas9 technology offers a more effective approach by cutting specific DNA sequences, precision targeting and genome editing. AI-based smartphone applications for antibiogram analysis in resource-limited settings face challenges like internet connectivity, device compatibility, data quality, energy consumption, and algorithmic limitations. Additionally, light-based antimicrobial techniques are increasingly being used to effectively kill antibiotic-resistant microbial species and treat localized infections. This review provides an in-depth overview of AMR covering epidemiology, evolution, mechanisms, infection prevention, control measures, antibiotic access, stewardship, surveillance, challenges and emerging non-antibiotic therapeutic approaches.}, }
@article {pmid39952934, year = {2025}, author = {El Kassem, G and Hillmer, J and Boettcher, M}, title = {Evaluation of Cas13d as a tool for genetic interaction mapping.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {1631}, pmid = {39952934}, issn = {2041-1723}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; Gene Regulatory Networks ; *Endodeoxyribonucleases/genetics/metabolism ; *Chromosome Mapping/methods ; HEK293 Cells ; Bacterial Proteins ; }, abstract = {Mapping genetic interactions (GIs) is crucial for understanding genetic network complexity. In this study, we investigate the utility of Cas13d, a CRISPR system targeting RNA, for GI mapping and compare it to Cas9 and Cas12a, two DNA nucleases commonly used for GI mapping. We find that Cas13d induces faster target gene perturbation and generates more uniform cell populations with double perturbations than Cas9 or Cas12a. We then encounter Cas13d gRNA-gRNA interference when concatenating gRNAs targeting different genes into one gRNA array, which we overcome by a dual promoter gRNA expression strategy. Moreover, by concatenating three gRNAs targeting the same gene into one array, we are able to maximize the Cas13d-mediated knockdown effects. Combining these strategies enhances proliferation phenotypes while reducing library size and facilitates reproducible quantification of GIs in oncogenic signaling pathways. Our study highlights the potential of Cas13d for GI mapping, promising advancements in understanding therapeutically relevant drug response pathways.}, }
@article {pmid39952932, year = {2025}, author = {Fu, Y and Yang, X and Li, S and Ma, C and An, Y and Cheng, T and Liang, Y and Sun, S and Cheng, T and Zhao, Y and Wang, J and Wang, X and Xu, P and Yin, Y and Liang, H and Liu, N and Zou, W and Chen, B}, title = {Dynamic properties of transcriptional condensates modulate CRISPRa-mediated gene activation.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {1640}, pmid = {39952932}, issn = {2041-1723}, support = {32371507//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32171444//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {*Transcriptional Activation/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; *Biomolecular Condensates/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; E1A-Associated p300 Protein/metabolism ; }, abstract = {CRISPR activation (CRISPRa) is a powerful tool for endogenous gene activation, yet the mechanisms underlying its optimal transcriptional activation remain unclear. By monitoring real-time transcriptional bursts, we find that CRISPRa modulates both burst duration and amplitude. Our quantitative imaging reveals that CRISPR-SunTag activators, with three tandem VP64-p65-Rta (VPR), form liquid-like transcriptional condensates and exhibit high activation potency. Although visible CRISPRa condensates are associated with some RNA bursts, the overall levels of phase separation do not correlate with transcriptional bursting or activation strength in individual cells. When the number of SunTag scaffolds is increased to 10 or more, solid-like condensates form, sequestering co-activators such as p300 and MED1. These condensates display low dynamicity and liquidity, resulting in ineffective gene activation. Overall, our studies characterize various phase-separated CRISPRa systems for gene activation, highlighting the foundational principles for engineering CRISPR-based programmable synthetic condensates with appropriate properties to effectively modulate gene expression.}, }
@article {pmid39952682, year = {2025}, author = {Alhabsi, A and Ling, Y and Crespi, M and Reddy, ASN and Mahfouz, M}, title = {Alternative Splicing Dynamics in Plant Adaptive Responses to Stress.}, journal = {Annual review of plant biology}, volume = {76}, number = {1}, pages = {687-717}, doi = {10.1146/annurev-arplant-083123-090055}, pmid = {39952682}, issn = {1545-2123}, mesh = {*Alternative Splicing/genetics ; *Stress, Physiological/genetics ; *Plants/genetics ; Gene Expression Regulation, Plant ; *Adaptation, Physiological/genetics ; *Plant Physiological Phenomena/genetics ; }, abstract = {Plants thrive in dynamic environments by activating sophisticated molecular networks that fine-tune their responses to stress. A key component of these networks is gene regulation at multiple levels, including precursor messenger RNA (pre-mRNA) splicing, which shapes the transcriptome and proteome landscapes. Through the precise action of the spliceosome complex, noncoding introns are removed and coding exons are joined to produce spliced RNA transcripts. While constitutive splicing always generates the same messenger RNA (mRNA), alternative splicing (AS) produces multiple mRNA isoforms from a single pre-mRNA, enriching proteome diversity. Remarkably, 80% of multiexon genes in plants generate multiple isoforms, underscoring the importance of AS in shaping plant development and responses to abiotic and biotic stresses. Recent advances in CRISPR-Cas genome and transcriptome editing technologies offer revolutionary tools to dissect AS regulation at molecular levels, unveiling the functional significance of specific isoforms. In this review, we explore the intricate mechanisms of pre-mRNA splicing and AS in plants, with a focus on stress responses. Additionally, we examine how leveraging AS insights can unlock new opportunities to engineer stress-resilient crops, paving the way for sustainable agriculture in the face of global environmental challenges.}, }
@article {pmid39952635, year = {2025}, author = {Ahsan, M and Pindi, C and Palermo, G}, title = {Emerging Mechanisms of Metal-Catalyzed RNA and DNA Modifications.}, journal = {Annual review of physical chemistry}, volume = {76}, number = {1}, pages = {497-518}, doi = {10.1146/annurev-physchem-082423-030241}, pmid = {39952635}, issn = {1545-1593}, mesh = {*RNA/chemistry/metabolism ; *DNA/chemistry/metabolism ; CRISPR-Cas Systems ; *Metals/chemistry/metabolism ; *RNA, Catalytic/chemistry/metabolism ; Catalysis ; Gene Editing ; Humans ; }, abstract = {Metal ions play a critical role in various chemical, biological, and environmental processes. This review reports on emerging chemical mechanisms in the catalysis of DNA and RNA. We provide an overview of the metal-dependent mechanisms of DNA cleavage in CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems that are transforming life sciences through genome editing technologies, and showcase intriguing metal-dependent mechanisms of RNA cleavages. We show that newly discovered CRISPR-Cas complexes operate as protein-assisted ribozymes, highlighting RNA's versatility and the enhancement of CRISPR-Cas functions through strategic metal ion use. We demonstrate the power of computer simulations in observing chemical processes as they unfold and in advancing structural biology through innovative approaches for refining cryo-electron microscopy maps. Understanding metal ion involvement in nucleic acid catalysis is crucial for advancing genome editing, aiding therapeutic interventions for genetic disorders, and improving the editing tools' specificity and efficiency.}, }
@article {pmid39952507, year = {2025}, author = {Chang, J and Li, Q and Zhang, T and Sun, H and Jia, Z and Li, Y and Zhang, S and Sun, T and Ma, S and Cao, J}, title = {Genome-wide CRISPR screening of genes and pathways for insect cell responding to abnormal environmental pH.}, journal = {International journal of biological macromolecules}, volume = {305}, number = {Pt 1}, pages = {141000}, doi = {10.1016/j.ijbiomac.2025.141000}, pmid = {39952507}, issn = {1879-0003}, mesh = {Animals ; Hydrogen-Ion Concentration ; Signal Transduction/genetics ; *Bombyx/genetics/cytology ; Cell Line ; *CRISPR-Cas Systems ; *Genome, Insect ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Cells are bathed in the extracellular fluids in which the extracellular pH (pHe) is maintained to a narrow range, and abnormal pHe is related to multiple diseases. However, the genes and signaling pathways underlying cell response to abnormal pHe remain unclear. Identification of genes responsive to extreme pHe challenge has great value in both basic research and medicine. Here, we performed genome-wide CRISPR screening to reveal genes and pathways related to insect cell response to abnormal pHe. Cells of the Bombyx mori embryonic cell line (BmE) genome-scale CRISPR screening cell library (BmEGCKLib) were cultured in different pHe (the physiological pH 6.3 as control; pH 5.0, 5.5, 6.6 and 6.95 as abnormal pHe). In the four extreme pH groups, we identified 44 overlapped fitness genes and 24 overlapped positive selected genes respectively. We also performed Kyoto Encyclopedia of Genes and Genomes pathways enrichment analysis for the selected genes. The "phosphatidylinositol signaling system", "mRNA surveillance pathway" and "spliceosome pathway" were significantly enriched in the negative selection, suggesting that cellular signal transduction and mRNA quality play essential roles for cells to resist to abnormal pHe. This is the first time to provide insight into insect cell response to abnormal pHe on a genome-scale.}, }
@article {pmid39952463, year = {2025}, author = {van Dijk, A and Wilson, AM and Marx, B and Hough, B and Swalarsk-Parry, B and De Vos, L and Wingfield, MJ and Wingfield, BD and Steenkamp, ET}, title = {CRISPR-Cas9 genome editing reveals that the Pgs gene of Fusarium circinatum is involved in pathogenicity, growth and sporulation.}, journal = {Fungal genetics and biology : FG &amp; B}, volume = {177}, number = {}, pages = {103970}, doi = {10.1016/j.fgb.2025.103970}, pmid = {39952463}, issn = {1096-0937}, mesh = {*Fusarium/genetics/pathogenicity/growth &amp; development ; *CRISPR-Cas Systems ; Gene Editing ; Plant Diseases/microbiology ; Pinus/microbiology ; Spores, Fungal/genetics/growth &amp; development ; Virulence/genetics ; *Fungal Proteins/genetics ; Genome, Fungal ; }, abstract = {Fusarium circinatum, the causal agent of pine pitch canker, is one of the most destructive pathogens of Pinus species worldwide. Infections by this pathogen result in serious mortality of seedlings due to root and root collar disease, and growth reduction in trees due to canker formation and dieback. Although much is known about the population biology, genetics, and genomics of F. circinatum, relatively little is known regarding the molecular basis of pathogenicity in F. circinatum. In this study, a protoplast-based transformation using CRISPR-Cas9-mediated genome editing was utilized to functionally characterize a putative pathogenicity gene in three different strains of the fungus. In silico analyses suggested the gene likely encodes a small secreted protein, and all isolates in which it was deleted displayed significantly reduced vegetative growth and asexual spore production compared to the wild-type isolates. In pathogenicity tests, lesions induced by the deletion mutants on detached Pinus patula branches were significantly shorter than those produced by the wild-types. The putative pathogenicity gene was named Pgs reflecting its role in pathogenicity, growth, and sporulation. Future research will seek to explore the molecular mechanisms underlying the mutant phenotypes observed. Overall, this study represents a significant advance in F. circinatum research as the development and application of a Cas9-mediated gene deletion process opens new avenues for functional gene characterization underlying many of the pathogen's biological traits.}, }
@article {pmid39952413, year = {2025}, author = {Hamze, JG and Cambra, JM and Navarro-Serna, S and Martinez-Serrano, CA}, title = {Navigating gene editing in porcine embryos: Methods, challenges, and future perspectives.}, journal = {Genomics}, volume = {117}, number = {2}, pages = {111014}, doi = {10.1016/j.ygeno.2025.111014}, pmid = {39952413}, issn = {1089-8646}, mesh = {Animals ; *Gene Editing/methods ; Swine/genetics ; CRISPR-Cas Systems ; Nuclear Transfer Techniques ; Gene Transfer Techniques ; Male ; *Embryo, Mammalian/metabolism ; }, abstract = {Gene editing technologies, particularly CRISPR/Cas9, have emerged as transformative tools in genetic modification, significantly advancing the use of porcine embryos in biomedical and agricultural research. This review comprehensively examines the various methodologies for gene editing and delivery methods, such as somatic cell nuclear transfer (SCNT), microinjection, electroporation, and lipofection. This review, focuses on the advantages or limitations of using different biological sources (in vivo- vs. in vitro oocytes/embryos). Male germ cell manipulation using sperm-mediated gene transfer (SMGT) and testis-mediated gene transfer (TMGT) represent innovative approaches for producing genetically modified animals. Although these technologies have revolutionized the genetic engineering field, all these strategies face challenges, including high rates of off-target events and mosaicism. This review emphasizes the need to refine these methods, with a focus on reducing mosaicism and improving editing accuracy. Further advancements are essential to unlocking the full potential of gene editing for both agricultural applications and biomedical innovations.}, }
@article {pmid39951551, year = {2025}, author = {Zhang, B and Zhang, P and Wang, H and Wang, X and Hu, Z and Wang, F and Li, Z}, title = {Dual Protein Corona-Mediated Target Recognition System for Visual Detection and Single-Molecule Counting of Nucleic Acids.}, journal = {ACS nano}, volume = {19}, number = {7}, pages = {6929-6941}, doi = {10.1021/acsnano.4c13924}, pmid = {39951551}, issn = {1936-086X}, mesh = {Gold/chemistry ; Metal Nanoparticles/chemistry ; Surface Plasmon Resonance/methods ; *Protein Corona/chemistry ; Humans ; *Nucleic Acids/analysis ; CRISPR-Cas Systems ; *Single Molecule Imaging/methods ; Biosensing Techniques/methods ; Point-of-Care Systems ; }, abstract = {Rapid, highly sensitive, and specific nucleic acid detection plays a crucial role in advancing point-of-care (POC) diagnostics for pathogens and viruses, cancer monitoring, and optimizing clinical treatments. Herein, leveraging the precise recognition ability of CRISPR/dCas9 and the powerful localized surface plasmon resonance (LSPR) of gold nanoparticles (AuNPs), we report the design of a dual protein corona-mediated detection platform to simultaneously fulfill rapid POC testing and single-molecule counting of nucleic acids in a one-pot and one-step manner. This system uses guide RNA as a molecular bridge to anchor dCas9 protein onto AuNPs, forming artificial protein coronas. Upon recognizing a target, the interaction between the two protein coronas on the same nucleic acid molecule triggers cross-linked aggregation of AuNPs. Then, a target as low as 100 aM can be visually detected within 30 min, making the platform particularly well-suited for rapid POC application and the screening of emerging epidemics. Additionally, the superior LSPR properties of AuNPs increase the light-scattering signal generated during target-induced aggregation, enabling the visualization of the aggregated AuNPs as diffraction-limited spots under confocal microscopy. By counting these spots, the platform achieves unprecedented detection sensitivity, identifying a target as low as 1 aM, which is equivalent to just 6 molecules in a 10 μL system, demonstrating single-molecule detection capability. This dual protein corona-mediated detection system offers exceptional promise for large-scale screening of pathogenic viruses and the early detection of cancer, particularly in applications requiring ultrahigh sensitivity at the single-molecule level.}, }
@article {pmid39950863, year = {2025}, author = {Sun, P and Wang, S and Yan, Q and Zeng, J and Wu, Z and Qi, X}, title = {Non-nuclear localization signal-guided CRISPR/Cas9 ribonucleoproteins for translocation and gene editing via apoferritin delivery vectors.}, journal = {Nanoscale}, volume = {17}, number = {11}, pages = {6660-6675}, doi = {10.1039/d4nr04762a}, pmid = {39950863}, issn = {2040-3372}, mesh = {Humans ; *Gene Editing ; *Apoferritins/chemistry ; *CRISPR-Cas Systems ; Animals ; Mice ; HeLa Cells ; *Ribonucleoproteins/chemistry/metabolism/genetics ; *Nuclear Localization Signals ; Doxorubicin/chemistry/pharmacology ; Cell Nucleus/metabolism ; Nanoparticles/chemistry ; }, abstract = {Direct delivery of the Cas9/sgRNA ribonucleoprotein (RNP) via appropriate carriers has been proved to be an important advance for the in vivo translocation and gene editing of CRISPR/Cas9. These carriers often require the nuclear localization signal (NLS) to fuse with Cas9 or the NLS-bearing protein to form a complex with Cas9 to enter the nucleus. In this study, we introduced apoferritin nanocages as carriers and DOX as a nuclear trigger for the nuclear transport of the Cas9/sgRNA ribonucleoprotein without the NLS (RNP[-]). Our experiments showed that loading RNP[-] and DOX into 4L-FTH subunit-based apoferritin nanocages leads to efficient endocytosis and lysosomal escape. Specifically, when DOX was administered at a concentration of 1 μM, we observed the activation of cellular defense mechanisms, which effectively facilitated the translocation of 4L-HFn@RNP[-]/DOX nanoparticles into the nucleus, thereby enabling intranuclear RNP[-] delivery. This strategy has been empirically demonstrated to achieve gene editing efficiencies of approximately 33% for the Lcn2 gene in MDA-MB-231 cells and 17.9% for the copepod green fluorescent protein (copGFP) gene in HeLa.copGFP cells in vitro. Moreover, in vivo editing efficacy, as tested in a HeLa.copGFP nude mouse model, was confirmed to be 16%. This delivery system presents a novel therapeutic approach for the nuclear delivery of small molecules or nucleic acid drugs, potentially overcoming the challenges associated with nuclear entry barriers.}, }
@article {pmid39950596, year = {2025}, author = {Liu, J and An, T and Peng, J and Zhu, Q and Zhao, H and Liang, Z and Mo, K and Liu, T and Wu, K}, title = {An amplification-free digital droplet assay for influenza A viral RNA based on CRISPR/Cas13a.}, journal = {The Analyst}, volume = {150}, number = {6}, pages = {1151-1157}, doi = {10.1039/d4an01328j}, pmid = {39950596}, issn = {1364-5528}, mesh = {*RNA, Viral/analysis/genetics ; *CRISPR-Cas Systems/genetics ; *Influenza A virus/genetics/isolation &amp; purification ; Humans ; Limit of Detection ; *Microfluidic Analytical Techniques/methods ; Microspheres ; Influenza, Human/diagnosis/virology ; }, abstract = {Most of the CRISPR-based RNA detection methods are combined with amplification to improve sensitivity, which lead to some drawbacks such as aerosol pollution, complicated operation, and amplification bias. To address the above issues, we developed a digital detection method for influenza A viral RNA based on droplet microfluidics and CRISPR/Cas13a without polymerase chain reaction. We used a microsphere coupled to a capture probe to extract and concentrate the target RNA from the samples, and then restricted the target-induced CRISPR/Cas13a cleavage event to microfluidic droplets, thus enhancing the local signal intensity and enabling single-molecule detection. With a detection limit of 10 copies per μL, influenza A viral RNA can be detected in less than 1 h. Both clinical and synthetic series samples were used to validate the assay's performance. With the help of this direct RNA diagnostic method, a variety of RNA molecules can be easily and accurately detected at the single-molecule level. This research has broad prospects in clinical applications.}, }
@article {pmid39950533, year = {2025}, author = {Xue, L and Qu, P and Zhao, H and Liu, H and Huang, B and Wang, X and Zhang, Z and Dai, X and Qin, L and Dong, W and Shi, L and Zhang, X}, title = {Creation of fragrant peanut using CRISPR/Cas9.}, journal = {Journal of integrative plant biology}, volume = {67}, number = {6}, pages = {1438-1440}, pmid = {39950533}, issn = {1744-7909}, support = {2022YFD1200400//National Key Research and Development Program of China/ ; 201300111000//Key Project of Science and Technology of Henan Province/ ; 221100110300//Key Project of Science and Technology of Henan Province/ ; CARS-13//the Earmarked Fund/ ; S2012-5//Fund of Henan Province Agriculture Research System/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Arachis/genetics/metabolism ; Mutation/genetics ; Betaine-Aldehyde Dehydrogenase/genetics/metabolism ; }, abstract = {Targeted knockout of the betaine aldehyde dehydrogenase genes AhBADH1 and AhBADH2 using CRISPR/Cas9 produced peanut mutant lines with significantly elevated 2-acetyl-1-pyrroline levels and a strong aroma, marking the first creation of fragrant peanut lines.}, }
@article {pmid39950393, year = {2025}, author = {Contiliani, DF and Sretenovic, S and Dailey, M and Zhou, M and Cheng, Y and Creste, S and Xiao, S and Qi, Y}, title = {Harnessing novel cytidine deaminases from the animal kingdom for robust multiplexed base editing in rice.}, journal = {Plant biotechnology journal}, volume = {23}, number = {5}, pages = {1702-1712}, pmid = {39950393}, issn = {1467-7652}, support = {IOS-2132693//National Science Foundation/ ; IOS-2224203//National Science Foundation/ ; 2018-33522-28789//National Institute of Food and Agriculture/ ; 2024-33522-42755//National Institute of Food and Agriculture/ ; }, mesh = {*Oryza/genetics ; *Gene Editing/methods ; *Cytidine Deaminase/genetics/metabolism ; Animals ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; }, abstract = {CRISPR-Cas-based cytosine base editors (CBEs) are prominent tools that perform site-specific and precise C-to-T conversions catalysed by cytidine deaminases. However, their use is often constrained by stringent editing preferences for genomic contexts, off-target effects and restricted editing windows. To expand the repertoire of CBEs, we systematically screened 66 novel cytidine deaminases sourced from various organisms, predominantly from the animal kingdom and benchmarked them in rice protoplasts using the nCas9-BE3 configuration. After selecting candidates in rice protoplasts and further validation in transgenic rice lines, we unveiled a few cytidine deaminases exhibiting high editing efficiencies and wide editing windows. CBEs based on these cytidine deaminases also displayed minimal frequencies of indels and C-to-R (R = A/G) conversions, suggesting high purity in C-to-T base editing. Furthermore, we highlight the highly efficient cytidine deaminase OoA3GX2 derived from Orca (killer whale) for its comparable activity across GC/CC/TC/AC sites, thus broadening the targeting scope of CBEs for robust multiplexed base editing. Finally, the whole-genome sequencing analyses revealed very few sgRNA-dependent and -independent off-target effects in independent T0 lines. This study expands the cytosine base-editing toolkit with many cytidine deaminases sourced from mammals, providing better-performing CBEs that can be further leveraged for sophisticated genome engineering strategies in rice and likely in other plant species.}, }
@article {pmid39950370, year = {2025}, author = {Xiong, K and Wang, X and Feng, C and Zhang, K and Chen, D and Yang, S}, title = {Vectors in CRISPR Gene Editing for Neurological Disorders: Challenges and Opportunities.}, journal = {Advanced biology}, volume = {9}, number = {3}, pages = {e2400374}, doi = {10.1002/adbi.202400374}, pmid = {39950370}, issn = {2701-0198}, support = {81874304//National Natural Science Foundation of China/ ; 22122409//National Natural Science Foundation of China/ ; 22A350017//Key Scientific Research Projects/ ; 21HASTIT043//Programs for Science &amp;Technology Innovation Talents in Universities of Henan Province/ ; ZYQR201912191//Outstanding Young Talents in Henan Province/ ; }, mesh = {*Gene Editing/methods ; Humans ; *Nervous System Diseases/therapy/genetics ; *CRISPR-Cas Systems/genetics ; *Genetic Vectors/genetics ; *Genetic Therapy/methods ; Dependovirus/genetics ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Diseases of the nervous system are recognized as the second leading cause of death worldwide. The global prevalence of neurological diseases, such as Huntington's disease, Alzheimer's disease, and Parkinson's disease has seen a significant rise due to the increasing proportion of the aging population. The discovery of the clustered regularly interspaced short palindromic repeats (CRISPR) genome editing technique has paved way for universal neurological diseases treatment. However, finding a safe and effective method to deliver CRISPR gene-editing tools remains a main challenge for genome editing therapies in vivo. Adeno-associated virus (AAV) is currently one of the most commonly used vector systems, but some issues remain unresolved, including capsid immunogenicity, off-target mutations, and potential genotoxicity. To address these concerns, researchers are actively encouraging the development of new delivery systems, like virus-like particles and nanoparticles. These novel systems have the potential to enhance targeting efficiency, thereby offering possible solutions to the current challenges. This article reviews CRISPR delivery vectors for neurological disorders treatment and explores potential solutions to overcome limitations in vector systems. Additionally, the delivery strategies of CRISPR systems are highlighted as valuable tools for studying neurological diseases, and the challenges and opportunities that these vectors present.}, }
@article {pmid39949273, year = {2025}, author = {Li, T and Li, R and Li, Z and Li, Z and Wang, M and He, X and Zhang, G and Zhang, Y and Yang, Y and Li, Y}, title = {Unveiling a novel RNA G-triplex structure: its function and potential in CRISPR-based diagnostics.}, journal = {Chemical communications (Cambridge, England)}, volume = {61}, number = {20}, pages = {4002-4005}, doi = {10.1039/d4cc06581f}, pmid = {39949273}, issn = {1364-548X}, mesh = {*RNA/chemistry/metabolism ; *CRISPR-Cas Systems ; Benzothiazoles/chemistry ; Biosensing Techniques ; Hemin/chemistry ; Nucleic Acid Conformation ; Models, Molecular ; Humans ; }, abstract = {We report the discovery of a novel higher-order RNA structure, RNA G-triplex (rG3), formed by the TERRA sequence. Through CD spectroscopy, NMR analysis, and molecular modeling, we confirmed its stable, parallel conformation. rG3 exhibits strong binding to thioflavin T (ThT), N-methyl mesoporphyrin IX (NMM), and hemin, showcasing its potential as a biosensing element. Additionally, CRISPR-Cas13a trans-cleaves rG3, demonstrating its utility as a sensitive reporter in diagnostic applications. These findings expand the structural diversity of RNA and suggest new avenues for RNA-based biosensors and CRISPR diagnostics.}, }
@article {pmid39949262, year = {2025}, author = {Bhardwaj, P and Dhangur, P and Kalichamy, A and Singh, R}, title = {RT-RPA Assisted CRISPR/Cas12a Based One-Pot Rapid and Visual Detection of the Pan-Dengue Virus.}, journal = {Journal of medical virology}, volume = {97}, number = {2}, pages = {e70219}, doi = {10.1002/jmv.70219}, pmid = {39949262}, issn = {1096-9071}, support = {//This research was supported by the Department of Health Research, Ministry of Health &amp; Family Welfare, New Delhi funded to Pooja Bhardwaj for the Young Scientist fellowship scheme (DHR/YSS/2020/000048) and JE-AES Apex intramural project funded to Rajeev Singh by Indian Council of Medical Research, New Delhi./ ; }, mesh = {*Dengue Virus/genetics/isolation &amp; purification/classification ; *Dengue/diagnosis/virology ; Humans ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; RNA, Viral/genetics ; Serogroup ; *Molecular Diagnostic Techniques/methods ; Viral Nonstructural Proteins/genetics ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Globally ≤ 4 billion of the population are at potential risk of contracting dengue virus (DENV) infection. Seasonal outbreaks of dengue are frequently reported causing a high healthcare burden. Undiagnosed DENV can lead to severe morbidity and mortality. Early diagnosis of DENV relies on molecular methods, which are impractical in resource-constrained settings (RCSs). Dengue can be caused by any of the four distinct DENV serotypes. Therefore, a simple method for rapid diagnosis of Pan-DENV serotypes is of utmost importance at RCSs. A fluorescence detection platform for Pan-DENV using RT-RPA and CRISPR/Cas12a was developed targeting nonstructural 1 (NS1) gene for DENV-1, 2, and 3, and envelope (E) gene for DENV-2. Further, crRNA specific to DENV serotypes were designed to facilitate CRISPR/Cas12a detection. Analytical sensitivity was determined using synthetic RNA and DENV serotypes genome. Clinical validation of the assay was performed using RNA extracted from AES/AFI clinical samples. The developed CRISPR/Cas12a-based detection platform can detect all four serotypes of DENV viz 1-4 in a single pot using fluorescence detection. This assay showed the limit of detection ≥ 781 zg reaction[-] [1], ≥ 1.81 ag reaction[-1], ≥ 62.5 fg reaction[-1], and ≥ 2.5 pg reaction[-1] for synthetic DENV-1, DENV-2, DENV-3, and DENV-4 template, respectively. Our assay demonstrated the analytic sensitivity of ≥ 10 ng reaction[-1] for DENV-1 and DENV-4, and ≥ 0.5 ng reaction[-1] for DENV-3 and DENV-4 genomes. This assay showed no cross-reactivity with other related etiologies tested causing AFI/AES. With 76 clinical samples (DENV PCR positive = 16, DENV PCR negative = 60), the assay demonstrated 93.7% sensitivity and 100% specificity with an overall accuracy of 98.7% for detection of the Pan-DENV serotypes. Our assay displayed comparable results to that of RT-PCR. The ease of interpretation and rapid detection of the Pan-DENV, represents the potential of the developed assay as an ideal point-of-care test. This assay upon field-deployment could help in reducing healthcare burden, provide differential diagnosis and support initiating early and prompt treatment to patients at RCS.}, }
@article {pmid39948709, year = {2025}, author = {Homchan, A and Patchsung, M and Chantanakool, P and Wongsatit, T and Onchan, W and Muengsaen, D and Thaweeskulchai, T and Tandean, M and Sakpetch, T and Suraritdechachai, S and Aphicho, K and Panchai, C and Taiwan, S and Horthongkham, N and Sudyoadsuk, T and Reinhardt, A and Uttamapinant, C}, title = {Recombinase-Controlled Multiphase Condensates Accelerate Nucleic Acid Amplification and CRISPR-Based Diagnostics.}, journal = {Journal of the American Chemical Society}, volume = {147}, number = {12}, pages = {10088-10103}, pmid = {39948709}, issn = {1520-5126}, mesh = {*Recombinases/metabolism/chemistry ; *Nucleic Acid Amplification Techniques/methods ; Bacteriophage T4/enzymology ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Isothermal techniques for amplifying nucleic acids have found extensive applications in genotyping and diagnostic tests. These methods can be integrated with sequence-specific detection strategies, such as CRISPR-based detection, for optimal diagnostic accuracy. In particular, recombinase-based amplification uses proteins from the Escherichia virus T4 recombination system and operates effectively at moderate temperatures in field and point-of-care settings. Here, we discover that recombinase polymerase amplification (RPA) is controlled by liquid-liquid phase separation, where the condensate formation enhances the nucleic acid amplification process. While two protein components of RPA could act as scaffold proteins for condensate formation, we identify T4 UvsX recombinase as the key regulator orchestrating distinct core-shell arrangements of proteins within multiphase condensates, with the intrinsically disordered C-terminus of UvsX being crucial for phase separation. We develop volumetric imaging assays to visualize RPA condensates and the reaction progression in whole volumes, and begin to dissect how macroscopic properties such as size distribution and droplet count could contribute to the overall reaction efficiency. Spatial organization of proteins in condensates may create optimal conditions for amplification, and disruption of such structures may diminish the amplification efficiency, as we demonstrate for the case of reverse transcription-RPA. The insight that RPA functions as a multiphase condensate leads us to identify the UvsX[D274A] mutant, which has a distinct phase-separation propensity compared to the wild-type enzyme and can enhance RNA detection via RPA-coupled CRISPR-based diagnostics.}, }
@article {pmid39948378, year = {2025}, author = {Mazzaferro, E and Mujica, E and Zhang, H and Emmanouilidou, A and Jenseit, A and Evcimen, B and Metzendorf, C and Dethlefsen, O and Loos, RJ and Vienberg, SG and Larsson, A and Allalou, A and den Hoed, M}, title = {Functionally characterizing obesity-susceptibility genes using CRISPR/Cas9, in vivo imaging and deep learning.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {5408}, pmid = {39948378}, issn = {2045-2322}, mesh = {Animals ; Zebrafish/genetics ; *Deep Learning ; *Obesity/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Adipocytes/metabolism ; Lipid Metabolism/genetics ; *Genetic Predisposition to Disease ; Mutation ; Larva/genetics ; Zebrafish Proteins/genetics ; }, abstract = {Hundreds of loci have been robustly associated with obesity-related traits, but functional characterization of candidate genes remains a bottleneck. Aiming to systematically characterize candidate genes for a role in accumulation of lipids in adipocytes and other cardiometabolic traits, we developed a pipeline using CRISPR/Cas9, non-invasive, semi-automated fluorescence imaging and deep learning-based image analysis in live zebrafish larvae. Results from a dietary intervention show that 5 days of overfeeding is sufficient to increase the odds of lipid accumulation in adipocytes by 10 days post-fertilization (dpf, n = 275). However, subsequent experiments show that across 12 to 16 established obesity genes, 10 dpf is too early to detect an effect of CRISPR/Cas9-induced mutations on lipid accumulation in adipocytes (n = 1014), and effects on food intake at 8 dpf (n = 1127) are inconsistent with earlier results from mammals. Despite this, we observe effects of CRISPR/Cas9-induced mutations on ectopic accumulation of lipids in the vasculature (sh2b1 and sim1b) and liver (bdnf); as well as on body size (pcsk1, pomca, irs1); whole-body LDLc and/or total cholesterol content (irs2b and sh2b1); and pancreatic beta cell traits and/or glucose content (pcsk1, pomca, and sim1a). Taken together, our results illustrate that CRISPR/Cas9- and image-based experiments in zebrafish larvae can highlight direct effects of obesity genes on cardiometabolic traits, unconfounded by their - not yet apparent - effect on excess adiposity.}, }
@article {pmid39948336, year = {2025}, author = {Caragine, CM and Le, VT and Mustafa, M and Diaz, BJ and Morris, JA and Müller, S and Mendez-Mancilla, A and Geller, E and Liscovitch-Brauer, N and Sanjana, NE}, title = {Comprehensive dissection of cis-regulatory elements in a 2.8 Mb topologically associated domain in six human cancers.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {1611}, pmid = {39948336}, issn = {2041-1723}, support = {R01 HL168247/HL/NHLBI NIH HHS/United States ; R01 CA279135/CA/NCI NIH HHS/United States ; DP2HG10099//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01 HG012790/HG/NHGRI NIH HHS/United States ; R01 CA218668/CA/NCI NIH HHS/United States ; DP2 HG010099/HG/NHGRI NIH HHS/United States ; R01 AI176601/AI/NIAID NIH HHS/United States ; }, mesh = {Humans ; Cell Line, Tumor ; *Proto-Oncogene Proteins c-myc/genetics/metabolism ; *Neoplasms/genetics ; *Gene Expression Regulation, Neoplastic ; Enhancer Elements, Genetic/genetics ; Transcription Factors/metabolism/genetics ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; *Regulatory Sequences, Nucleic Acid/genetics ; Cell Proliferation/genetics ; }, abstract = {Cis-regulatory elements (CREs), such as enhancers and promoters, are fundamental regulators of gene expression and, across different cell types, the MYC locus utilizes a diverse regulatory architecture driven by multiple CREs. To better understand differences in CRE function, we perform pooled CRISPR inhibition (CRISPRi) screens to comprehensively probe the 2.8 Mb topologically-associated domain containing MYC in 6 human cancer cell lines with nucleotide resolution. We map 32 CREs where inhibition leads to changes in cell growth, including 8 that overlap previously identified enhancers. Targeting specific CREs decreases MYC expression by as much as 60%, and cell growth by as much as 50%. Using 3-D enhancer contact mapping, we find that these CREs almost always contact MYC but less than 10% of total MYC contacts impact growth when silenced, highlighting the utility of our approach to identify phenotypically-relevant CREs. We also detect an enrichment of lineage-specific transcription factors (TFs) at MYC CREs and, for some of these TFs, find a strong, tumor-specific correlation between TF and MYC expression not found in normal tissue. Taken together, these CREs represent systematically identified, functional regulatory regions and demonstrate how the same region of the human genome can give rise to complex, tissue-specific gene regulation.}, }
@article {pmid39947791, year = {2025}, author = {Liu, Y and Zhang, L and Lei, W and Liu, Y and Zhang, Y and Dou, Q and Zhu, Y and Zhang, L and Guo, P and Lu, P and Mao, G}, title = {Development of a rapid and sensitive RPA-CRISPR/Cas12a assay for non-invasive pre-implantation genetic testing.}, journal = {Analytica chimica acta}, volume = {1343}, number = {}, pages = {343687}, doi = {10.1016/j.aca.2025.343687}, pmid = {39947791}, issn = {1873-4324}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Genetic Testing/methods ; *Preimplantation Diagnosis/methods ; Female ; Blastocyst/metabolism ; Chromosomes, Human, Y/genetics ; Male ; }, abstract = {BACKGROUND: Pre-implantation genetic testing (PGT) is served as the primary technology for diagnosing genetic disorders. However, invasive operation may affect embryonic development, which indicates non-invasive methods might have important clinical value. Free DNA in blastocoele fluid provides the possibility for non-invasive diagnosis. The combination of RPA and CRISPR/Cas12a technology is expected to achieve analysis of free DNA in blastocoele fluid and develop an instant diagnostic platform for non-invasive PGT.<br><br>RESULTS: In this study, we collected 65 samples of day 6/7 blastocysts formed through intracytoplasmic sperm injection, and blastocysts hatched from the zona pellucida, with the corresponding blastocoele fluid, from the Center of Reproductive Medicine at the Second Affiliated Hospital of Zhengzhou University. The TSPY1 and TBC1D3 genes were analyzed using the RPA-CRISPR/Cas12a system to investigate the diagnostic potential of free DNA in the blastocoele fluid. A single-tube dual-gene assay for blastocoele fluid was successfully constructed using the RPA-CRISPR/Cas12a technology achieving specific detection of the Y chromosome and fluorescence visualization. Interpretatable results could be completed within 1h. By detecting the TSPY1 and TBC1D3 genes in 65 pairs of blastocysts, the accuracy of the Y chromosome in the interpretable results reached 95.4&#xa0;%.<br><br>SIGNIFICANCE: Free DNA in the blastocoele fluid could serve as a genetic information source for non-invasive PGT. We first established a single-tube dual-gene RPA-CRISPR/Cas12a assay to detect free DNA in blastocoele fluid and achieved rapid amplification and detection with the advantages of easy operation and fluorescence visualization, providing a rapid detection platform for the diagnosis of sex-linked disorders.}, }
@article {pmid39947471, year = {2025}, author = {Elsharkasy, OM and de Voogt, WS and Tognoli, ML and van der Werff, L and Gitz-Francois, JJ and Seinen, CW and Schiffelers, RM and de Jong, OG and Vader, P}, title = {Integrin beta 1 and fibronectin mediate extracellular vesicle uptake and functional RNA delivery.}, journal = {The Journal of biological chemistry}, volume = {301}, number = {3}, pages = {108305}, pmid = {39947471}, issn = {1083-351X}, mesh = {*Integrin beta1/metabolism/genetics ; *Extracellular Vesicles/metabolism/genetics ; Humans ; *Fibronectins/metabolism/genetics ; *RNA/metabolism/genetics ; CRISPR-Cas Systems ; Animals ; }, abstract = {Extracellular vesicles (EVs) are cell-derived vesicles secreted by all cell types into the extracellular spaces. EVs comprise a heterogenous population of vesicles that carry bioactive molecules, such as proteins, lipids, and RNAs, which they can deliver to recipient cells. Over the past few years, EVs have been recognized for their vital role in intercellular communication, and thereby in various physiological and pathological processes. In addition, EVs are increasingly being studied as potential drug delivery vehicles. It is therefore crucial to understand the mechanisms and molecular players underlying EV uptake and functional cargo delivery. Several studies have investigated various EV uptake pathways; nonetheless, molecular mechanisms governing uptake and cargo transfer remain largely lacking. Here, we show, using a CRISPR/Cas9-mediated reporter system, that integrin β1 on recipient cells plays an important role in EV uptake and EV-mediated RNA delivery. Additionally, using both RNA interference and blocking antibodies, we show that association of integrin β1 with integrin α4 is essential for this process. We demonstrate that α4β1 on recipient cells interacts with EVs through surface localized fibronectin via binding to its leucine-aspartic acid-valine motif, and that blocking of this interaction reduces both EV uptake and RNA delivery. Thus, we identify a key mechanism in EV uptake and cargo delivery which could potentially facilitate research into EV biology and pave the way for the development of novel therapeutic approaches by targeting pathways that lead to functional cargo delivery.}, }
@article {pmid39947136, year = {2025}, author = {Rohm, D and Black, JB and McCutcheon, SR and Barrera, A and Berry, SS and Morone, DJ and Nuttle, X and de Esch, CE and Tai, DJC and Talkowski, ME and Iglesias, N and Gersbach, CA}, title = {Activation of the imprinted Prader-Willi syndrome locus by CRISPR-based epigenome editing.}, journal = {Cell genomics}, volume = {5}, number = {2}, pages = {100770}, pmid = {39947136}, issn = {2666-979X}, support = {R01 DA036865/DA/NIDA NIH HHS/United States ; RM1 HG011123/HG/NHGRI NIH HHS/United States ; U01 AI146356/AI/NIAID NIH HHS/United States ; UM1 HG012053/HG/NHGRI NIH HHS/United States ; }, mesh = {*Prader-Willi Syndrome/genetics ; Humans ; *Genomic Imprinting/genetics ; Induced Pluripotent Stem Cells/metabolism ; *Gene Editing/methods ; snRNP Core Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; DNA Methylation/genetics ; Epigenesis, Genetic ; *Epigenome/genetics ; Chromosomes, Human, Pair 15/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Epigenome Editing ; }, abstract = {Epigenome editing with DNA-targeting technologies such as CRISPR-dCas9 can be used to dissect gene regulatory mechanisms and potentially treat associated disorders. For example, Prader-Willi syndrome (PWS) results from loss of paternally expressed imprinted genes on chromosome 15q11.2-q13.3, although the maternal allele is intact but epigenetically silenced. Using CRISPR repression and activation screens in human induced pluripotent stem cells (iPSCs), we identified genomic elements that control the expression of the PWS gene SNRPN from the paternal and maternal chromosomes. We showed that either targeted transcriptional activation or DNA demethylation can activate the silenced maternal SNRPN and downstream PWS transcripts. However, these two approaches function at unique regions, preferentially activating different transcript variants and involving distinct epigenetic reprogramming mechanisms. Remarkably, transient expression of the targeted demethylase leads to stable, long-term maternal SNRPN expression in PWS iPSCs. This work uncovers targeted epigenetic manipulations to reprogram a disease-associated imprinted locus and suggests possible therapeutic interventions.}, }
@article {pmid39946620, year = {2025}, author = {Kumar, K and Crobu, L and Thiam, R and Mandal, CC and Sterkers, Y and Prajapati, VK}, title = {Apoptotic proteins in Leishmania donovani: in silico screening, modeling, and validation by knock-out and gene expression analysis.}, journal = {Parasite (Paris, France)}, volume = {32}, number = {}, pages = {9}, pmid = {39946620}, issn = {1776-1042}, support = {11-LABX-0024-01//Agence Nationale de la Recherche/ ; IIRPIG-2023-0000873//Indian Council of Medical Research/ ; }, mesh = {*Leishmania donovani/genetics/physiology ; *Apoptosis/genetics ; Molecular Docking Simulation ; Molecular Dynamics Simulation ; Gene Knockout Techniques ; *Protozoan Proteins/genetics/chemistry/metabolism ; *Apoptosis Regulatory Proteins/genetics/chemistry/metabolism ; Humans ; Computer Simulation ; CRISPR-Cas Systems ; Proto-Oncogene Proteins c-bcl-2/genetics/chemistry ; Leishmaniasis, Visceral/parasitology ; Gene Expression Profiling ; }, abstract = {Visceral leishmaniasis, a life-threatening vector-borne illness that disproportionately affects children and elderly immunocompromised people, is a primary tropical neglected disease. No apoptotic partner proteins have yet been reported in Leishmania donovani, while their identification could contribute to knowledge on parasite cell death and the establishment of alternative therapeutics. We searched for mammalian Bcl-2 family protein orthologs and found one anti-apoptotic and two pro-apoptotic orthologs in L. donovani. A pro-death aquaporin protein, due to its characteristic BH3 domain known to interact with pro-apoptotic proteins in mammalian Bcl-2 family proteins, was also included in this study. Molecular docking and molecular dynamics simulations were conducted to assess protein-protein interactions between the identified apoptotic proteins and mimic mammalian intrinsic apoptotic pathways. The results showed that both pro-apoptotic proteins interacted with the hydrophobic pocket of the anti-apoptotic ortholog, forming a stable complex. This interaction may represent a critical event in an apoptotic pathway in L. donovani. To further characterise it, we used CRISPR-Cas9 approaches to target the identified proteins. Pure knocked population mutants, and episomal over-expressing mutant cells were exposed to apoptotic stimuli. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and quantitative expression profiling suggested that these proteins are involved in the parasite's apoptosis and could play a role in its survival.}, }
@article {pmid39946489, year = {2025}, author = {Du, Q and Zhang, H and Bi, Y and Wang, H and Zhou, X and Shi, P and Lv, S and Bi, S}, title = {N-Deficient B-Doped g-C3N4/CdS Heterojunction-Based PEC-FL Biosensor Assisted by CRISPR-Cas12a System for Ultrasensitive Determination of microRNA.}, journal = {Analytical chemistry}, volume = {97}, number = {7}, pages = {4049-4056}, doi = {10.1021/acs.analchem.4c05841}, pmid = {39946489}, issn = {1520-6882}, mesh = {*MicroRNAs/analysis ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Cadmium Compounds/chemistry ; *Sulfides/chemistry ; Limit of Detection ; Humans ; *Electrochemical Techniques/methods ; *Nitrogen Compounds/chemistry ; Infrared Rays ; Bacterial Proteins ; Endodeoxyribonucleases ; Graphite ; CRISPR-Associated Proteins ; }, abstract = {Near-infrared light (NIR)-driven photoelectrochemical (PEC) processes are mainly faced with the limitation of weak photocurrents. Here, N-deficient B-doped g-C3N4/CdS (NB-g-C3N4/CdS) is proposed to construct a NIR-driven PEC biosensor assisted by CRISPR-Cas12a system for the determination of microRNA-21 (miRNA-21). To promote the optical absorption as well as the separation of photogenerated electrons and holes of g-C3N4, NB-g-C3N4/CdS is constructed via engineering the electronic and band structure in terms of N defect, B doping, and heterojunction, achieving high PEC performance. To obtain the high luminescence efficiency for exciting NB-g-C3N4/CdS under NIR, the core-shell NaYF4:Yb[3+], Tm[3+]@NaYF4 upconversion nanoparticles (UCNPs) with repaired defects are prepared. Furthermore, the rolling circle amplification (RCA)-assisted CRISPR-Cas12a system is integrated to fragment the DNA on UCNPs, achieving sensitive detection of miRNA-21. On the one hand, the uncleavaged signal probes on UCNPs combined with NB-g-C3N4/CdS through π-π stacking interaction, generating photocurrents under the irradiation of NIR. On the other hand, the cleavaged signal probes which cannot link with NB-g-C3N4/CdS exhibited the fluorescence (FL) signals. The proposed PEC-FL dual-mode biosensor provides a mutual authentication of testing results and demonstrates ultrasensitivity (the detection limit of 1.1 fM for PEC mode and 7.0 fM for FL mode) and excellent specificity, which is promising in the clinical analysis of miRNA.}, }
@article {pmid39946463, year = {2025}, author = {Fagerberg, E and Attanasio, J and Dien, C and Singh, J and Kessler, EA and Abdullah, L and Shen, J and Hunt, BG and Connolly, KA and De Brouwer, E and He, J and Iyer, NR and Buck, J and Borr, ER and Damo, M and Foster, GG and Giles, JR and Huang, YH and Tsang, JS and Krishnaswamy, S and Cui, W and Joshi, NS}, title = {KLF2 maintains lineage fidelity and suppresses CD8 T cell exhaustion during acute LCMV infection.}, journal = {Science (New York, N.Y.)}, volume = {387}, number = {6735}, pages = {eadn2337}, doi = {10.1126/science.adn2337}, pmid = {39946463}, issn = {1095-9203}, support = {P30 CA016359/CA/NCI NIH HHS/United States ; S10 OD026996/OD/NIH HHS/United States ; T32 AI155387/AI/NIAID NIH HHS/United States ; R01 CA237037/CA/NCI NIH HHS/United States ; T32 AR007016/AR/NIAMS NIH HHS/United States ; R01 AI170116/AI/NIAID NIH HHS/United States ; }, mesh = {Animals ; Mice ; *CD8-Positive T-Lymphocytes/immunology/cytology ; Cell Differentiation/genetics ; Cell Lineage ; CRISPR-Cas Systems ; *Kruppel-Like Transcription Factors/genetics/metabolism ; *Lymphocytic Choriomeningitis/immunology/genetics ; *Lymphocytic choriomeningitis virus/immunology ; Mice, Inbred C57BL ; T-bet Transcription Factor/genetics/metabolism ; *T-Cell Exhaustion ; Mice, Knockout ; }, abstract = {Naïve CD8 T cells have the potential to differentiate into a spectrum of functional states during an immune response. How these developmental decisions are made and what mechanisms exist to suppress differentiation toward alternative fates remains unclear. We employed in vivo CRISPR-Cas9-based perturbation sequencing to assess the role of ~40 transcription factors (TFs) and epigenetic modulators in T cell fate decisions. Unexpectedly, we found that knockout of the TF Klf2 resulted in aberrant differentiation to exhausted-like CD8 T cells during acute infection. KLF2 was required to suppress the exhaustion-promoting TF TOX and to enable the TF TBET to drive effector differentiation. KLF2 was also necessary to maintain a polyfunctional tumor-specific progenitor state. Thus, KLF2 provides effector CD8 T cell lineage fidelity and suppresses the exhaustion program.}, }
@article {pmid39945739, year = {2025}, author = {Deaver, JW and Ryan, PJ and O'Reilly, CL and Uranga, S and López, SM and Sheffield-Moore, M and Nghiem, PP and Riechman, SE and Fluckey, JD}, title = {MCF7 breast cancer anabolic capacity reduced with CRISPR/Cas9-mediated stable overexpression of DEPTOR.}, journal = {American journal of physiology. Cell physiology}, volume = {328}, number = {2}, pages = {C670-C678}, doi = {10.1152/ajpcell.00682.2023}, pmid = {39945739}, issn = {1522-1563}, mesh = {Humans ; *Breast Neoplasms/genetics/pathology/metabolism ; Female ; *CRISPR-Cas Systems/genetics ; MCF-7 Cells ; TOR Serine-Threonine Kinases/metabolism/genetics ; *Intracellular Signaling Peptides and Proteins/genetics/metabolism ; Mechanistic Target of Rapamycin Complex 1/metabolism/genetics ; Gene Expression Regulation, Neoplastic ; Cell Proliferation ; }, abstract = {The hyperactivation of mTOR is a significant contributor to the development and progression of a number of human diseases, including a majority of human cancers. Although there have been many scientific and clinical efforts to reduce the impact of mTOR hyperactivation on downstream cellular metabolism, we aimed to mitigate this hyperactivation through a novel targeted gene edit of the intrinsic mTOR inhibitor, DEP domain containing MTOR interacting protein (DEPTOR), in MCF7 human breast cancer cells. Using publicly available bioinformatics tools, we demonstrate that DEPTOR gene expression is low in breast cancers compared with healthy tissues and that DEPTOR expression predicts overall survival, recurrence-free survival, and distant metastasis-free survival in breast cancer patients. We show that a directed overexpression of DEPTOR protein leads to significant alteration of downstream mTORC1 targets and subsequently reduces overall rates of protein synthesis. In addition, treatment of DEPTOR overexpressing cells with small-molecule DEPTOR inhibitor NSC126405 leads to a reversal of this effect, indicating a direct causal mechanism between DEPTOR protein levels and mTORC1 activation.NEW &amp; NOTEWORTHY We identify DEPTOR as a predictor of mortality in breast cancer and show that precision gene editing to restore DEPTOR expression in breast cancer slows cell growth by inhibiting mTOR activity.}, }
@article {pmid39942646, year = {2025}, author = {Lee, H and Rho, WY and Kim, YH and Chang, H and Jun, BH}, title = {CRISPR-Cas9 Gene Therapy: Non-Viral Delivery and Stimuli-Responsive Nanoformulations.}, journal = {Molecules (Basel, Switzerland)}, volume = {30}, number = {3}, pages = {}, pmid = {39942646}, issn = {1420-3049}, support = {NRF-2022R1A2C2012883//Ministry of Science and ICT/ ; 2021M3C1C3097211//Ministry of Science and ICT/ ; RS-2023-00222910//Ministry of Science and ICT/ ; }, mesh = {*CRISPR-Cas Systems ; Humans ; *Genetic Therapy/methods ; *Gene Editing/methods ; *Nanoparticles/chemistry ; *Gene Transfer Techniques ; Animals ; }, abstract = {The CRISPR-Cas9 technology, one of the groundbreaking genome editing methods for addressing genetic disorders, has emerged as a powerful, precise, and efficient tool. However, its clinical translation remains hindered by challenges in delivery efficiency and targeting specificity. This review provides a comprehensive analysis of the structural features, advantages, and potential applications of various non-viral and stimuli-responsive systems, examining recent progress to emphasize the potential to address these limitations and advance CRISPR-Cas9 therapeutics. We describe how recent reports emphasize that nonviral vectors, including lipid-based nanoparticles, extracellular vesicles, polymeric nanoparticles, gold nanoparticles, and mesoporous silica nanoparticles, can offer diverse advantages to enhance stability, cellular uptake, and biocompatibility, based on their structures and physio-chemical stability. We also summarize recent progress on stimuli-responsive nanoformulations, a type of non-viral vector, to introduce precision and control in CRISPR-Cas9 delivery. Stimuli-responsive nanoformulations are designed to respond to pH, redox states, and external triggers, facilitate controlled and targeted delivery, and minimize off-target effects. The insights in our review suggest future challenges for clinical applications of gene therapy technologies and highlight the potential of delivery systems to enhance CRISPR-Cas9's clinical efficacy, positioning them as pivotal tools for future gene-editing therapies.}, }
@article {pmid39941122, year = {2025}, author = {Zhou, S and Tian, D and Liu, H and Lu, X and Zhang, D and Chen, R and Yang, S and Wu, W and Wang, F}, title = {Editing the RR-TZF Gene Subfamily in Rice Uncovers Potential Risks of CRISPR/Cas9 for Targeted Genetic Modification.}, journal = {International journal of molecular sciences}, volume = {26}, number = {3}, pages = {}, pmid = {39941122}, issn = {1422-0067}, support = {2020NZ08017//Major science and technology projects of Fujian Province/ ; 2022J02010//the Key Program of the National Natural Science of Fujian Province/ ; 2022R1027006//Fujian Province Public Welfare Scientific Research Program/ ; }, mesh = {*Oryza/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Plant Proteins/genetics ; Plants, Genetically Modified/genetics ; Phenotype ; *Genes, Plant ; Mutation ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {The CRISPR/Cas9 system offers a powerful tool for gene editing to enhance rice productivity. In this study, we successfully edited eight RR-TZF genes in japonica rice Nipponbare using CRISPR-Cas9 technology, achieving a high editing efficiency of 73.8%. Sequencing revealed predominantly short insertions or deletions near the PAM sequence, along with multi-base deletions often flanked by identical bases. Off-target analysis identified 5 out of 31 predicted sites, suggesting the potential for off-target effects, which can be mitigated by designing gRNAs with more than three base mismatches. Notably, new mutations emerged in the progeny of several gene-edited mutants, indicating inheritable genetic mutagenicity. Phenotypic analysis of homozygous mutants revealed varied agronomic traits, even within the same gene, highlighting the complexity of gene-editing outcomes. These findings underscore the importance of backcrossing to minimize off-target and inheritable mutagenicity effects, ensuring more accurate trait evaluation. This study offers insights into CRISPR/Cas9 mechanisms and uncertain factors and may inform future strategies for rice improvement, prompting further research into CRISPR/Cas9's precision and long-term impacts.}, }
@article {pmid39941098, year = {2025}, author = {Qi, S and Wang, Y and Liu, Z and Wu, S and Zhao, Y and Li, Y and Deng, S and Yu, K and Lian, Z}, title = {Construction of a TAT-Cas9-EGFP Site-Specific Integration Eukaryotic Cell Line Using Efficient PEG10 Modification.}, journal = {International journal of molecular sciences}, volume = {26}, number = {3}, pages = {}, pmid = {39941098}, issn = {1422-0067}, support = {32072721//National Natural Science Foundation of China/ ; grant No.2022ZD04014//Biological Breeding-Major Projects/ ; }, mesh = {Humans ; HEK293 Cells ; *CRISPR-Cas Systems ; *Green Fluorescent Proteins/genetics/metabolism ; Gene Knock-In Techniques/methods ; *RNA-Binding Proteins/genetics ; DNA-Binding Proteins ; Apoptosis Regulatory Proteins ; }, abstract = {The CRISPR/Cas9 system enables precise and efficient modification of eukaryotic genomes. Among its various applications, homology-directed repair (HDR) mediated knock-in (KI) is crucial for creating human disease models, gene therapy, and agricultural genetic enhancements. Despite its potential, HDR-mediated knock-in efficiency remains relatively low. This study investigated the impact of 5' end PEG10 modification on site-specific integration of the target gene. The HEK293 cell line is considered a highly attractive expression system for the production of recombinant proteins, with the construction of site-specific integration cell lines at the AAVS1 locus enabling stable protein expression. This study investigated the impact of the 5' end PEG10 modification on the site-specific integration of the target gene at the AAVS1 locus in the 293T cell line. Utilizing this 5' end PEG10 modification resulted in a 1.9-fold increase in knock-in efficiency for a 1.8 kb target fragment, improving efficiency from 26% to 49%. An optimized system was utilized to successfully establish a high-expression, site-specific integration 293T cell line for TAT-Cas9-EGFP, providing a reliable resource of seed cells for subsequent protein production.}, }
@article {pmid39941065, year = {2025}, author = {Xia, X and Song, W and Zhang, F and Fan, Y and Zhang, B and Chen, X}, title = {ctdsp2 Knockout Induces Zebrafish Craniofacial Dysplasia via p53 Signaling Activation.}, journal = {International journal of molecular sciences}, volume = {26}, number = {3}, pages = {}, pmid = {39941065}, issn = {1422-0067}, support = {82271186 and 81974143//the General Programs of the National Natural Science Foundation of China/ ; 2022-PUMCH-C-029//the National High Level Hospital Clinical Research Funding/ ; 7232122//the Natural Science Foundation of Beijing, China/ ; }, mesh = {Animals ; *Zebrafish/genetics/metabolism/embryology ; *Tumor Suppressor Protein p53/metabolism/genetics ; *Zebrafish Proteins/genetics/metabolism ; *Signal Transduction ; *Craniofacial Abnormalities/genetics/metabolism/pathology ; Apoptosis/genetics ; Chondrocytes/metabolism/pathology ; Gene Knockout Techniques ; Neural Crest/metabolism ; Cell Proliferation ; Gene Expression Regulation, Developmental ; Cell Differentiation ; Cartilage/metabolism ; CRISPR-Cas Systems ; Disease Models, Animal ; }, abstract = {Hemifacial microsomia (HFM) is a rare congenital craniofacial deformity that significantly impacts the appearance and hearing. The genetic etiology of HFM remains largely unknown, although genetic factors are considered to be primary contributors. We previously identified CTDSP2 as a potential causative gene in HFM cases. Utilizing CRISPR/Cas9, we knocked out ctdsp2 in zebrafish and analyzed the spatiotemporal expression of ctdsp2 and neural crest cell (NCC) markers through in situ hybridization (ISH). Craniofacial cartilage and chondrocyte phenotypes were visualized using Alcian blue and wheat germ agglutinin (WGA) staining. Cell proliferation and apoptosis were assessed via immunofluorescence with PH3 and TUNEL. RNA sequencing was performed on ctdsp2[-/-] embryos and control siblings, followed by rescue experiments. Knockout of ctdsp2 in zebrafish resulted in craniofacial defects characteristic of HFM. We observed abnormalities in NCC apoptosis and proliferation in the pharyngeal arches, as well as impaired differentiation of chondrocytes in ctdsp2[-/-] embryos. RNA-Seq analysis revealed significantly higher expression of genes in the p53 signaling pathway in mutants. Furthermore, ctdsp2 mRNA injection and tp53 knockout significantly rescued pharyngeal arch cartilage dysplasia. Our findings suggest that ctdsp2 knockout induces zebrafish craniofacial dysplasia, primarily by disrupting pharyngeal chondrocyte differentiation and inhibiting NCC proliferation through p53 signaling pathway activation.}, }
@article {pmid39940922, year = {2025}, author = {Liu, Y and Elshan, M and Li, G and Han, X and Chen, X and Feng, X}, title = {Perspectives of Genome Editing Mediated Haploid Inducer Systems in Legumes.}, journal = {International journal of molecular sciences}, volume = {26}, number = {3}, pages = {}, pmid = {39940922}, issn = {1422-0067}, support = {U21A20215//National Natural Science Foundation of China/ ; }, mesh = {*Gene Editing/methods ; *Fabaceae/genetics ; *Haploidy ; *Plant Breeding/methods ; *Genome, Plant ; CRISPR-Cas Systems ; Crops, Agricultural/genetics ; Plants, Genetically Modified/genetics ; }, abstract = {Genome editing-mediated haploid inducer systems (HISs) present a promising strategy for enhancing breeding efficiency in legume crops, which are vital for sustainable agriculture due to their nutritional benefits and ability to fix nitrogen. Traditional legume breeding is often slow and complicated by the complexity of legumes' genomes and the challenges associated with tissue culture. Recent advancements have broadened the applicability of HISs in legume crops, facilitating a reduction in the duration of the breeding cycle. By integrating genome editing technology with haploid breeding systems, researchers can achieve precise genetic modifications and rapidly produce homozygous lines, thereby significantly accelerating the development of desired traits. This review explores the current status and future prospects of genome editing-mediated HISs in legumes, emphasizing the mechanisms of haploid induction; recent breakthroughs; and existing technical challenges. Furthermore, we highlight the necessity for additional research to optimize these systems across various legume species, which has the potential to greatly enhance breeding efficiency and contribute to the sustainability of legume production.}, }
@article {pmid39940839, year = {2025}, author = {Wang, H and Yang, H and Li, T and Chen, Y and Chen, J and Zhang, X and Zhang, J and Zhang, Y and Zhang, N and Ma, R and Huang, X and Liu, Q}, title = {Optimization of CRISPR/Cas9 Gene Editing System in Sheep (Ovis aries) Oocytes via Microinjection.}, journal = {International journal of molecular sciences}, volume = {26}, number = {3}, pages = {}, pmid = {39940839}, issn = {1422-0067}, support = {XDA24030205, XDA26040303//Strategic Priority Research Program of Chinese Academy of Sciences/ ; 2023ZD0406805, 2023ZD0407106//Biological Breeding- National Science and Technology Major Project/ ; XM202402//Open Project of Tianjin Key Laboratory of Animal Molecular Breeding and Biotechnology/ ; }, mesh = {Animals ; *Gene Editing/methods ; *Oocytes/metabolism ; *CRISPR-Cas Systems ; *Microinjections/methods ; Female ; Sheep/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Suppressor of Cytokine Signaling Proteins/genetics ; }, abstract = {The CRISPR/Cas9 system has become a powerful tool for molecular design breeding in livestock such as sheep. However, the efficiency of the Cas9 system combined with zygote microinjection remains suboptimal. In this study, mature sheep oocytes were used for microinjection to assess the impact of various factors on Cas9 editing efficiency. We found that the in vitro maturation efficiency of oocytes is related to environmental factors such as air temperature, pressure, and humidity. Our results indicate that high-efficiency gene editing can be achieved when targeting the SOCS2, DYA, and TBXT, using a microinjection mixture with a concentration of 10 ng/μL Cas9 and sgRNA. By optimizing the injection capillary, we significantly reduced the oocyte invalidation rate post-microinjection to 3.1-5.3%. Furthermore, we observed that using either Cas9 protein or mRNA in the microinjection process resulted in different genotypes in the edited oocytes. Importantly, parthenogenetic activation did not appear to affect the editing efficiency. Using this high-efficiency system, we successfully generated SOCS2 or DYA gene-edited sheep, with all lambs confirmed to be genetically modified. This study presents a highly efficient method for producing gene-edited sheep, potentially enabling more precise and effective strategies for livestock breeding.}, }
@article {pmid39940724, year = {2025}, author = {Danso Ofori, A and Zheng, T and Titriku, JK and Appiah, C and Xiang, X and Kandhro, AG and Ahmed, MI and Zheng, A}, title = {The Role of Genetic Resistance in Rice Disease Management.}, journal = {International journal of molecular sciences}, volume = {26}, number = {3}, pages = {}, pmid = {39940724}, issn = {1422-0067}, support = {2019YFN0010, 2020YFH0161, 22GJHZ0024//Project of the Science and Technology Department of Sichuan Province/ ; 2021-YF05-02326-SN//Chengdu Science and Technology Bureau/ ; }, mesh = {*Oryza/genetics/microbiology ; *Disease Resistance/genetics ; *Plant Diseases/genetics/microbiology ; Quantitative Trait Loci ; Xanthomonas/pathogenicity ; Gene Editing ; Host-Pathogen Interactions/genetics ; Plant Breeding ; }, abstract = {Rice (Oryza sativa) is a crucial staple crop for global food security, particularly in Asia. However, rice production faces significant challenges from various diseases that can cause substantial yield losses. This review explores the role of genetic resistance in rice disease management, focusing on the molecular mechanisms underlying plant-pathogen interactions and strategies for developing resistant varieties. The paper discusses qualitative and quantitative resistance, emphasizing the importance of resistance (R) genes, defense-regulator genes, and quantitative trait loci (QTLs) in conferring broad-spectrum disease resistance. Gene-for-gene relationships in rice-pathogen interactions are examined, particularly for Xanthomonas oryzae pv. oryzae and Magnaporthe oryzae. The review also covers recent advancements in breeding techniques, including marker-assisted selection, genetic engineering, and genome editing technologies like CRISPR-Cas. These approaches offer promising avenues for enhancing disease resistance in rice while maintaining yield potential. Understanding and exploiting genetic resistance mechanisms is crucial for developing durable and broad-spectrum disease-resistant rice varieties, essential for ensuring sustainable rice production and global food security in the face of evolving pathogen threats and changing environmental conditions.}, }
@article {pmid39940693, year = {2025}, author = {Sosnovtseva, AO and Le, TH and Karpov, DS and Vorobyev, PO and Gumennaya, YD and Alekseeva, ON and Chumakov, PM and Lipatova, AV}, title = {Establishment of a Panel of Human Cell Lines to Identify Cellular Receptors Used by Enteroviruses to Infect Cells.}, journal = {International journal of molecular sciences}, volume = {26}, number = {3}, pages = {}, pmid = {39940693}, issn = {1422-0067}, support = {075-15-2019-1660//Ministry of Science and Higher Education of the Russian Federation;/ ; 23-14-00370//the Russian Science Foundation/ ; 22-14-00377//the Russian Science Foundation/ ; }, mesh = {Humans ; *Receptors, Virus/metabolism/genetics ; HEK293 Cells ; *Virus Internalization ; *Enterovirus/physiology ; CRISPR-Cas Systems ; Cell Line ; }, abstract = {Non-pathogenic natural and recombinant strains of human Enteroviruses are the subject of ongoing study with some strains having been approved for use as anticancer agents. The efficacy of oncolytic virotherapy depends upon identifying the receptor utilized by a specific strain for cell entry, and the presence of this receptor on the surface of cancer cells. Accordingly, a rapid and straightforward approach to determining the enteroviral receptors is necessary for developing an effective patient-specific, virus-based cancer therapy. To this end, we created a panel of seven lines with double knockouts on the background of the HEK293T cell line, which lacks the IFNAR1 gene. In these lines, the main viral receptor genes, including PVR, CXADR, CD55, ITGA2, SCARB2, ICAM1, and FCGRT, were knocked out using the CRISPR/Cas9 system. The panel of lines was validated on twelve different Enteroviruses types, providing a basis for studying the molecular mechanisms of enterovirus entry into cells, and for developing new therapeutic strains.}, }
@article {pmid39939860, year = {2025}, author = {Chey, YCJ and Gierus, L and Lushington, C and Arudkumar, JC and B Geiger, A and Staker, LG and Robertson, LJ and Pfitzner, C and Kennedy, JG and Lee, RHB and Godahewa, GI and Adikusuma, F and Thomas, PQ}, title = {Optimal SpCas9- and SaCas9-mediated gene editing by enhancing gRNA transcript levels through scaffold poly-T tract reduction.}, journal = {BMC genomics}, volume = {26}, number = {1}, pages = {138}, pmid = {39939860}, issn = {1471-2164}, mesh = {*Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism ; HEK293 Cells ; Transcription, Genetic ; }, abstract = {Ensuring sufficient gRNA transcript levels is critical for obtaining optimal CRISPR-Cas9 gene editing efficiency. The standard gRNA scaffold contains a sequence of four thymine nucleotides (4T), which is known to inhibit transcription from Pol III promoters such as the U6 promoter. Our study showed that using standard plasmid transfection protocols, the presence of these 4Ts did not significantly affect editing efficiency, as most of the gRNAs tested (55 gRNAs) achieved near-perfect editing outcomes. We observed that gRNAs with lower activity were T-rich and had reduced gRNA transcript levels. However, this issue can be effectively resolved by increasing transcript levels, which can be readily achieved by shortening the 4T sequences. In this study, we demonstrated this by modifying the sequences to 3TC. Although the 3TC scaffold modification did not improve editing efficiency for already efficient gRNAs when high vector quantities were available, it proved highly beneficial under conditions of limited vector availability, where the 3TC scaffold yielded higher editing efficiency. Additionally, we demonstrated that the 3TC scaffold is compatible with SpCas9 high-fidelity variants and ABEmax base editing, enhancing their editing efficiency. Another commonly used natural Cas9 variant, SaCas9, also benefited from the 3TC scaffold sequence modification, which increased gRNA transcription and subsequently improved editing activity. This modification was applied to the EDIT-101 therapeutic strategy, where it demonstrated marked improvements in performance. This study highlights the importance of shortening the 4T sequences in the gRNA scaffold to optimize gRNA transcript expression for enhanced CRISPR-Cas9 gene editing efficiency. This optimization is particularly important for therapeutic applications, where the quantity of vector is often limited, ensuring more effective and optimal outcomes.}, }
@article {pmid39939726, year = {2025}, author = {Stewart, J and Krastev, DB and Brough, R and Zatreanu, D and Song, F and Baxter, JS and Sridhar, S and Frankum, J and Konde, A and Yang, W and Haider, S and Alexander, J and Betteridge, K and Gulati, A and Attygalle, AD and Vroobel, K and Natrajan, R and Khalique, S and Roumeliotis, TI and Choudhary, JS and Yeung, J and Wicks, AJ and Marlow, R and Banerjee, S and Pettitt, SJ and Tutt, ANJ and Lord, CJ}, title = {PPP2R1A mutations cause ATR inhibitor sensitivity in ovarian clear cell carcinoma.}, journal = {Oncogene}, volume = {44}, number = {9}, pages = {618-629}, pmid = {39939726}, issn = {1476-5594}, support = {DRCRPG-Nov21\100001//Cancer Research UK (CRUK)/ ; }, mesh = {Female ; Humans ; *Protein Phosphatase 2/genetics ; *Adenocarcinoma, Clear Cell/genetics/drug therapy/pathology ; *Ovarian Neoplasms/genetics/drug therapy/pathology ; Animals ; *Mutation ; Mice ; Cell Line, Tumor ; *Ataxia Telangiectasia Mutated Proteins/antagonists &amp; inhibitors/genetics ; Transcription Factors/genetics ; DNA-Binding Proteins/genetics ; Drug Resistance, Neoplasm/genetics ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; }, abstract = {Identification of ARID1A/ATR synthetic lethality led to ATR inhibitor phase II trials in ovarian clear cell carcinoma (OCCC), a cancer of unmet need. Using multiple CRISPR-Cas9 mutagenesis and interference screens, we show that inactivation of protein phosphatase 2A (PP2A) subunits, including PPP2R1A, enhance ATRi sensitivity in ARID1A mutant OCCC. Analysis of a new OCCC cohort indicates that 52% possess oncogenic PPP2R1A p.R183 mutations and of these, one half possessed both ARID1A as well as PPP2R1A mutations. Using CRISPR-prime editing to generate new isogenic models of PPP2R1A mutant OCCC, we found that PPP2R1A p.R183W and p.R183P mutations cause ATRi-induced S phase stress, premature mitotic entry, genomic instability and ATRi sensitivity in OCCC tumour cells. p.R183 mutation also enhanced both in vitro and in vivo ATRi sensitivity in preclinical models of ARID1A mutant OCCC. These results argue for the assessment of PPP2R1A mutations as a biomarker of ATRi sensitivity.}, }
@article {pmid39939583, year = {2025}, author = {Xiong, Y and Su, Y and He, R and Han, X and Li, S and Liu, M and Xi, X and Liu, Z and Wang, H and Xie, S and Xu, X and Li, K and Zhang, J and Xu, J and Li, X and Zhao, S and Ruan, J}, title = {EXPERT expands prime editing efficiency and range of large fragment edits.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {1592}, pmid = {39939583}, issn = {2041-1723}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; INDEL Mutation ; }, abstract = {Prime editing systems (PEs) hold great promise in modern biotechnology. However, their editing range is limited as PEs can only modify the downstream sequences of the pegRNA nick. Here, we report the development of the extended prime editor system (EXPERT) to overcome this limitation by using an extended pegRNA (ext-pegRNA) with modified 3' extension, and an additional sgRNA (ups-sgRNA) targeting the upstream region of the ext-pegRNA. We demonstrate that EXPERT can efficiently perform editing on both sides of the ext-pegRNA nick, a task that is unattainable by canonical PEs. EXPERT exhibits prominent capacity in replacing sequences up to 88 base pairs and inserting sequences up to 100 base pairs within the upstream region of the ext-pegRNA nick. Compared to canonical PEs such as PE2, the utilization of the EXPERT strategy significantly enhances the editing efficiency for large fragment edits with an average improvement of 3.12-fold, up to 122.1 times higher. Safety wise, the use of ups-sgRNA does not increase the rates of undesirable insertions and deletions (indels), as the two nicks are on the same strand. Moreover, we do not observe increased off-target editing rates genome-wide. Our work introduces EXPERT as a PE tool with significant potential in life sciences.}, }
@article {pmid39937288, year = {2025}, author = {Ren, LM and Qi, YH and Cao, FY and Zhou, EP}, title = {Study on the framework of ATP energy cycle system in Escherichia coli.}, journal = {Applied microbiology and biotechnology}, volume = {109}, number = {1}, pages = {42}, pmid = {39937288}, issn = {1432-0614}, support = {246Z2813G//Hebei Provincial Department of Science and Technology/ ; }, mesh = {*Escherichia coli/genetics/metabolism ; *Adenosine Triphosphate/metabolism ; Gene Editing/methods ; Escherichia coli Proteins/genetics/metabolism ; Genome, Bacterial ; CRISPR-Cas Systems ; *Energy Metabolism ; Promoter Regions, Genetic ; Plasmids/genetics ; }, abstract = {The high mortality rate associated with single-use CRISPR-Cas9 in Escherichia coli limits its application. Recently, new CRISPR-based techniques for E.coli gene editing have emerged. Research aims to develop a system for rapid, marker-free, multi-site, and multi-copy genome editing in E.coli to advance synthetic biology. ATP, essential for energy in living organisms, plays a crucial role in various metabolic processes. To reduce the cost of ATP-requiring reactions, it is crucial to identify and efficiently express genes in ATP synthesis pathway. This study identified a single ppk gene (No.8) capable of completing the cyclic reaction. Using MUCICAT technology, the ppk gene (No.8) was inserted into various positions and copy numbers in the E.coli genome, resulting in different activity levels. The findings suggest that the difficulty of inserting the ppk gene (No.8) into the genome follows this order: IS186 < 8array < IS186 + 8array < IS1. A single genome insertion can mimic plasmid expression level. This study explores promoter competition and offers solutions, inspiring researchers in constructing the AMP-ATP cycle system in E.coli. KEY POINTS: • The single ppk gene (No.8) can regenerate the AMP-ATP cycle, crucial for ATP-dependent reactions. • Inserting the ppk gene (No.8) into the cr5 site of the E.coli genome achieves expression levels comparable to the pET29a plasmid. • The expression level of the ppk gene (No.8) is not significantly affected by its copy number in the E.coli genome.}, }
@article {pmid39937146, year = {2025}, author = {Li, QN and Cui, YX and Dai, ZQ and Yao, ZL and Li, MY and Cai, QL and Kong, DM}, title = {Activator Strand Modifications in CRISPR/Cas12a: Unlocking the Potential for Casp-3-Targeted Biosensing and Imaging Analysis of Apoptosis.}, journal = {Analytical chemistry}, volume = {97}, number = {7}, pages = {4194-4201}, doi = {10.1021/acs.analchem.4c06591}, pmid = {39937146}, issn = {1520-6882}, mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Caspase 3/metabolism/analysis ; *Apoptosis ; Humans ; DNA/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {The CRISPR/Cas12a system has emerged as a powerful tool in biosensing due to its unique trans-cleavage activity. This study conducted an in-depth investigation of the modulatory capabilities of this system, particularly focusing on the 5'-end modifications of the activator strand, and found that introducing a hairpin structure (HP) at the 5'-end of the activator strand, which was designed based on the RESET effect, can effectively suppress the activator strand's ability to activate the trans-cleavage activity of the CRISPR/Cas12a system. This suppression is independent of the HP's relation to the activator strand and the type of linker used (DNA, RNA or peptide). Detaching the HP from the activator strand restores the system's activity. These findings enrich the development of CRISPR/Cas12a-based biosensors, and expand their application beyond DNA-based target detection to peptide sequence-based target recognition. Based on this discovery, we constructed a sensitive biosensor for caspase-3 (Casp-3), a key executor in apoptosis, by linking the HP to the activator strand with a peptide linker containing a Casp-3 recognition site. The proposed biosensor has been validated for its sensitivity and specificity in detecting Casp-3, as well as for monitoring drug-induced apoptosis through the imaging of Casp-3 in living cells, providing a valuable tool for studying the apoptotic process, screening drugs, assessing drug efficacy, and evaluating treatment outcomes. This strategy also shows promise for detecting other peptide-based targets, broadening the horizons for early disease biomarker detection and timely therapeutic interventions.}, }
@article {pmid39937081, year = {2025}, author = {Choi, J and Chen, W and Liao, H and Li, X and Shendure, J}, title = {A molecular proximity sensor based on an engineered, dual-component guide RNA.}, journal = {eLife}, volume = {13}, number = {}, pages = {}, pmid = {39937081}, issn = {2050-084X}, support = {UM1 HG011586/HG/NHGRI NIH HHS/United States ; DFS-64-24/DRCRF/Damon Runyon Cancer Research Foundation/United States ; DGE-2140004//National Science Foundation Graduate Research Fellowship Program/ ; K99 HG012973/HG/NHGRI NIH HHS/United States ; R00HG012973/HG/NHGRI NIH HHS/United States ; Cell Lineage Tracing//Allen Discovery Center/ ; P30 CA008748/CA/NCI NIH HHS/United States ; DRG-2403-20/DRCRF/Damon Runyon Cancer Research Foundation/United States ; K99HG012973/HG/NHGRI NIH HHS/United States ; R00 HG012973/HG/NHGRI NIH HHS/United States ; UM1HG011586/HG/NHGRI NIH HHS/United States ; P30CA008748/NH/NIH HHS/United States ; R01HG010632/HG/NHGRI NIH HHS/United States ; R01 HG010632/HG/NHGRI NIH HHS/United States ; }, mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Synthetic Biology/methods ; HEK293 Cells ; }, abstract = {One of the goals of synthetic biology is to enable the design of arbitrary molecular circuits with programmable inputs and outputs. Such circuits bridge the properties of electronic and natural circuits, processing information in a predictable manner within living cells. Genome editing is a potentially powerful component of synthetic molecular circuits, whether for modulating the expression of a target gene or for stably recording information to genomic DNA. However, programming molecular events such as protein-protein interactions or induced proximity as triggers for genome editing remains challenging. Here, we demonstrate a strategy termed 'P3 editing', which links protein-protein proximity to the formation of a functional CRISPR-Cas9 dual-component guide RNA. By engineering the crRNA:tracrRNA interaction, we demonstrate that various known protein-protein interactions, as well as the chemically induced dimerization of protein domains, can be used to activate prime editing or base editing in human cells. Additionally, we explore how P3 editing can incorporate outputs from ADAR-based RNA sensors, potentially allowing specific RNAs to induce specific genome edits within a larger circuit. Our strategy enhances the controllability of CRISPR-based genome editing, facilitating its use in synthetic molecular circuits deployed in living cells.}, }
@article {pmid39937074, year = {2025}, author = {Wang, Z and Zhu, X and Jiang, T and Sun, Q and Zhao, X and Suryoprabowo, S and Liu, S and Hu, Q}, title = {Alkaline Phosphatase-Regulated DNAzyme Cleavage Coupled with CRISPR/Cas12a for Quantitative Detection of Deoxynivalenol in Agricultural Crops.}, journal = {Journal of agricultural and food chemistry}, volume = {73}, number = {8}, pages = {4904-4912}, doi = {10.1021/acs.jafc.4c10262}, pmid = {39937074}, issn = {1520-5118}, mesh = {*Trichothecenes/analysis/metabolism ; *Alkaline Phosphatase/chemistry/metabolism/genetics ; Food Contamination/analysis ; CRISPR-Cas Systems ; *DNA, Catalytic/chemistry/metabolism/genetics ; *Crops, Agricultural/chemistry/genetics ; Limit of Detection ; Immunoassay/methods ; Bacterial Proteins/genetics/metabolism ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Sensitive and simplified detection of a mycotoxin such as deoxynivalenol (DON) is crucial for food safety. In recent years, the CRISPR/Cas technology has demonstrated significant potential in detecting non-nucleic acids. Herein, we present a triple enzyme-assisted fluorescence immunoassay (TEFIA) that integrates alkaline phosphatase (ALP)-regulated DNAzyme cleavage with the CRISPR/Cas12a assay for the accurate detection of mycotoxin. By employing this method for detecting DON, we exhibit a low detection limit of 0.05 ng/mL and a satisfactory linear response between 0.1 and 10 ng/mL. This performance exceeds the conventional sensitivity levels found in traditional methods. TEFIA also demonstrates a good correlation with ic-ELISA for testing DON in real samples. Thus, it offers a robust and efficient detection platform for DON in complex matrices. Furthermore, TEFIA can be employed to identify various targets of interest by merely altering the antibody-antigen pairs, indicating its great potential in a wide range of applications.}, }
@article {pmid39936934, year = {2025}, author = {Lu, C and Chen, J and Zhang, S and Tian, Y and Ying, X and Zhang, D and Luo, Z and Si, X and Li, M}, title = {Development of a vitrified CRISPR/Cas12b-based assay for rapid genotyping of SLCO1B1 SNPs without DNA amplification.}, journal = {Analytical methods : advancing methods and applications}, volume = {17}, number = {9}, pages = {2083-2093}, doi = {10.1039/d4ay02015d}, pmid = {39936934}, issn = {1759-9679}, mesh = {*Polymorphism, Single Nucleotide ; *Liver-Specific Organic Anion Transporter 1/genetics ; Humans ; *Genotyping Techniques/methods ; *CRISPR-Cas Systems/genetics ; Genotype ; }, abstract = {Two single nucleotide polymorphisms (SNPs) in the human SLCO1B1 gene, c.388A>G (rs2306283) and c.521T>C (rs4149056), are independent determinants of the efficacy and side effects of statin drugs. Multinational clinical guidelines recommend testing for SLCO1B1 genotypes before the initial use of statins. Current SLCO1B1 SNP identification methods, primarily based on quantitative fluorescence PCR, rely on expensive equipment, are time-consuming, and require cold-chain storage for reagents, making them unsuitable for use in resource-limited healthcare settings. In this study, we developed a CRISPR/Cas12b-based amplification-free genotyping technique for SLCO1B1 SNPs. Within 30 minutes of the isothermal reaction, genotyping of the c.388A>G and c.521T>C SNPs in the SLCO1B1 gene can be observed by the naked eyes under blue light. Additionally, maltodextrin was identified as an effective vitrification stabilizer for the CRISPR/Cas12b premix. A low-cost vitrification process was optimized to prepare a glass like solid reagent via room-temperature vacuum drying. The vitrified CRISPR/Cas12b reagent retained approximately 88% of its activity after 30 days of storage at 37 °C, eliminating the need for cold-chain storage and allowing for long-term preservation at room temperature. This vitrified CRISPR/Cas12b based rapid SNP detection technique is especially suitable for genotyping drug metabolism genes in primary healthcare settings, providing effective guidance for precision medicine in clinical practice.}, }
@article {pmid39936495, year = {2025}, author = {Li, L and Fu, X and Qi, X and Xiao, B and Liu, C and Wu, Q and Zhu, J and Xie, C}, title = {Harnessing haploid-inducer mediated genome editing for accelerated maize variety development.}, journal = {Plant biotechnology journal}, volume = {23}, number = {5}, pages = {1604-1614}, pmid = {39936495}, issn = {1467-7652}, support = {2023YFD1202901//National Key Research and Development Program of China/ ; 32201872//National Science Foundation of China/ ; 32301914//National Science Foundation of China/ ; YBXM2429//Nanfan special project, CAAS/ ; CARS-02-06//China Agriculture Research System/ ; }, mesh = {*Zea mays/genetics/growth &amp; development ; *Gene Editing/methods ; *Haploidy ; CRISPR-Cas Systems/genetics ; Plant Breeding/methods ; Genome, Plant/genetics ; Plants, Genetically Modified ; Mutation ; }, abstract = {The integration of haploid induction and genome editing, termed HI-Edit/IMGE, is a promising tool for generating targeted mutations for crop breeding. However, the technical components and stacking suitable for the maize seed industry have yet to be fully characterised and tested. Here, we developed and assessed three HI-Edit/IMGE maize lines: Edit[Wx], Edit[Sh], and Edit[Wx&amp;Sh], using the haploid inducer CHOI3 and lines engineered using the CRISPR-Cas9 system targeting the Waxy1 (Wx1) and Shrunken2 (Sh2) genes. We meticulously characterised the HI-Edit/IMGE systems, focusing on copy numbers and the mutant alleles mtl and dmp, which facilitate haploid induction. Using B73 and six other parental lines of major commercial varieties as recipients, HI-Edit/IMGE demonstrated maternal haploid induction efficiencies ranging from 8.55% to 20.89% and targeted mutation rates between 0.38% and 1.46%. Comprehensive assessment verified the haploid identification, target gene editing accuracy, genome background integrity, and related agronomic traits. Notably, Edit[Wx&amp;Sh] successfully combined distinct CRISPR-Cas9 systems to induce multiple desired mutations, highlighting the potential of HI-Edit/IMGE in accelerating the integration of edited traits into commercial maize varieties. Our findings underscore the importance of meticulous Cas9 copy number characterisation and highlight potential challenges related to somatic chimerism. We also validated the performance of single-cross haploids derived using the HI-Edit/IMGE process. Our results confirm the industrial applicability of generating targeted mutations through pollination and provide critical insights for further optimising this technology.}, }
@article {pmid39936449, year = {2025}, author = {Liu, C and Tan, X and Wang, J and Sun, Y and Xu, Q and Han, C and Wang, Q}, title = {Upgrading of the genetic engineering toolkit accelerated the discovery process of the virulence effect of PsGH7d on Phytophthora sojae invasion.}, journal = {Physiologia plantarum}, volume = {177}, number = {1}, pages = {e70083}, doi = {10.1111/ppl.70083}, pmid = {39936449}, issn = {1399-3054}, support = {32172049//National Natural Science Foundation of China/ ; 32172387//National Natural Science Foundation of China/ ; 32202266//National Natural Science Foundation of China/ ; ZR2024MC195//Natural Science Foundation of Shandong Province/ ; ZR2021YQ20//Natural Science Foundation of Shandong Province/ ; ZR2022MC015//Natural Science Foundation of Shandong Province/ ; tsqn202211093//Taishan Scholar Project of Shandong Province/ ; tscy20221172//Taishan Industry Leading Talents/ ; SKL81121//"811" Project of National Key Laboratory of Wheat Improvement/ ; SKL81127//"811" Project of National Key Laboratory of Wheat Improvement/ ; }, mesh = {*Phytophthora/pathogenicity/genetics ; Virulence/genetics ; *Genetic Engineering/methods ; CRISPR-Cas Systems/genetics ; Plant Diseases/microbiology/parasitology ; Gene Editing ; }, abstract = {The genus of Phytophthora includes numerous phytopathogens that have devastating impacts on agricultural production. However, the limited availability of selection markers for numerous pathogenicity pathogens of the genus Phytophthora genetic transformation hinders further research on their pathogenic functional genes. Here we report a gene of NAT I, which serves as a novel selection marker for the Phytophthora sojae transformation. Additionally, we developed a new genetic manipulation toolkit based on vectors containing NAT I, which facilitates gene editing in P. sojae. With the toolkit, the gene PsGH7d of P. sojae, which encodes a glycosyl hydrolase, was edited consecutively via the CRISPR/Cas9 system to obtain gene knockout and enzymatic active site mutation strains. The pathogenicity analysis of these transformants revealed that PsGH7d is a virulence factor dependent on its bifunctional glucanase-xylanase activities. This study develops an updated toolkit for the genus Phytophthora genetic transformation and provides initial insights into the virulence of the bifunctional enzyme PsGH7d.}, }
@article {pmid39935177, year = {2025}, author = {Cheng, Y and Zhang, J and Mu, W and Ye, S and Cheng, J and Zhu, L and Wang, G and Cao, Y and Li, D and Hu, G and Huang, L and Wang, J and Zhou, J}, title = {Dasatinib-resistant universal CAR-T cells proliferate in the presence of host immune cells and exhibit antitumor activity.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {33}, number = {4}, pages = {1535-1551}, pmid = {39935177}, issn = {1525-0024}, mesh = {*Dasatinib/pharmacology ; Animals ; Humans ; Mice ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; *Immunotherapy, Adoptive/methods ; Xenograft Model Antitumor Assays ; Cell Line, Tumor ; Cell Proliferation/drug effects ; *T-Lymphocytes/immunology/metabolism/drug effects ; *Drug Resistance, Neoplasm ; Protein Kinase Inhibitors/pharmacology ; Receptors, Antigen, T-Cell/genetics ; Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics ; CRISPR-Cas Systems ; Mutation ; }, abstract = {The universal chimeric antigen receptor T cell (UCAR-T) immunotherapy derived from healthy donors holds great promise in pan-cancer treatment. However, UCAR-T cell therapy faces a challenge in the rapid elimination of allogeneic cells by the host immune system. To address this, we introduced a T316I mutation in the leukocyte-specific protein tyrosine kinase (LCK) locus in CAR-T cells using the cytosine base editor (CBE) system. Concurrently, we disrupted endogenous T cell receptor alpha chain (TRAC) and beta-2 microglobulin (B2M) with the CRISPR-Cas9 system, along with dasatinib to overcome host immune rejection, an Src family kinase (SFK) inhibitor. The resulting LCK mutated UCAR-T (KM UCAR-T) cells exhibited normal phenotypes in activation, proliferation, differentiation, and tumor cytotoxicity in vitro. Moreover, KM UCAR-T cells demonstrated sustained expansion in mixed lymphocyte reactions (MLR) when incubated with T cells or peripheral blood mononuclear cells (PBMCs) from HLA-mismatched donors upon dasatinib treatment. Additionally, we illustrated that KM UCAR-T cells displayed antitumor activity in a xenograft murine model and verified the expansion and cytotoxicity of KM UCAR-T over traditional UCAR-T in the presence of allogeneic PBMCs when treated with dasatinib in vivo. These findings offer a novel strategy for UCAR-T cells to resist host immune rejection and achieve sustained expansion.}, }
@article {pmid39934535, year = {2025}, author = {Ghaznavi, G and Vosough, P and Ghasemian, A and Tabar, MMM and Tayebi, L and Taghizadeh, S and Savardashtaki, A}, title = {Engineering bacteriophages for targeted superbug eradication.}, journal = {Molecular biology reports}, volume = {52}, number = {1}, pages = {221}, pmid = {39934535}, issn = {1573-4978}, support = {R56 DE029191/DE/NIDCR NIH HHS/United States ; R15DE027533//Craniofacial Research of the National Institutes of Health/ ; }, mesh = {*Bacteriophages/genetics ; CRISPR-Cas Systems/genetics ; *Phage Therapy/methods ; *Genetic Engineering/methods ; Humans ; *Bacteria/virology/genetics/drug effects ; Anti-Bacterial Agents/pharmacology/therapeutic use ; *Bacterial Infections/therapy/microbiology ; Drug Resistance, Bacterial/genetics ; }, abstract = {The rise of antibiotic-resistant bacteria, termed "superbugs," presents a formidable challenge to global health. These pathogens, often responsible for persistent nosocomial infections, threaten the effectiveness of conventional antibiotic therapies. This review delves into the potential of bacteriophages, viruses specifically targeting bacteria, as a powerful tool to combat superbugs. We examined the latest developments in genetic engineering that improve the efficacy of bacteriophages, focusing on modifications in host range, lysis mechanisms, and their ability to overcome bacterial defense systems. This review article highlights the CRISPR-Cas system as a promising method for precisely manipulating phage genomes, enabling the development of novel phage therapies with enhanced efficacy and specificity. Furthermore, we discussed developing novel phage-based strategies, such as phage cocktails and phage-antibiotic combinations. We also analyzed the challenges and ethical considerations associated with phage engineering, emphasizing the need for responsible and rigorous research to ensure this technology's safe and effective deployment to combat the growing threat of antibiotic resistance.}, }
@article {pmid39933924, year = {2025}, author = {Lebon, S and Bruneel, A and Drunat, S and Albert, A and Csaba, Z and Elmaleh, M and Ntorkou, A and Ténier, Y and Fenaille, F and Gressens, P and Passemard, S and Boespflug-Tanguy, O and Dorboz, I and El Ghouzzi, V}, title = {A biallelic variant in GORASP1 causes a novel Golgipathy with glycosylation and mitotic defects.}, journal = {Life science alliance}, volume = {8}, number = {4}, pages = {}, pmid = {39933924}, issn = {2575-1077}, mesh = {Humans ; Glycosylation ; *Golgi Apparatus/metabolism/pathology/genetics ; *Golgi Matrix Proteins/genetics/metabolism ; *Mitosis/genetics ; Male ; Female ; Mutation ; Alleles ; Phenotype ; CRISPR-Cas Systems ; *Membrane Proteins/genetics ; }, abstract = {GRASP65 is a Golgi-associated peripheral protein encoded by the GORASP1 gene and required for Golgi cisternal stacking in vitro. A key role of GRASP65 in the regulation of cell division has also been suggested. However, depletion of GRASP65 in mice has little effect on the Golgi structure and the gene has not been associated with any human phenotype to date. Here, we report the identification of the first human pathogenic variant of GORASP1 (c.1170_1171del; p.Asp390Glufs*18) in a patient combining a neurodevelopmental disorder with neurosensory, neuromuscular, and skeletal abnormalities. Functional analysis revealed that the variant leads to a total absence of GRASP65. The structure of the Golgi apparatus did not show fragmentation, but glycosylation anomalies such as hyposialylation were detected. Mitosis analyses revealed an excess of prometaphases and metaphases with polar chromosomes, suggesting a delay in the cell cycle. These phenotypes were recapitulated in RPE cells in which a similar mutation was introduced by CRISPR/Cas9. These results indicate that loss of GRASP65 in humans causes a novel Golgipathy associated with defects in glycosylation and mitotic progression.}, }
@article {pmid39933697, year = {2025}, author = {Li, J and Liu, S and Kim, S and Goell, J and Drum, ZA and Flores, JP and Ma, AJ and Mahata, B and Escobar, M and Raterink, A and Ahn, JH and Terán, ER and Guerra-Resendez, RS and Zhou, Y and Yu, B and Diehl, MR and Wang, GG and Gustavsson, AK and Phanstiel, DH and Hilton, IB}, title = {Biomolecular condensation of human IDRs initiates endogenous transcription via intrachromosomal looping or high-density promoter localization.}, journal = {Nucleic acids research}, volume = {53}, number = {4}, pages = {}, pmid = {39933697}, issn = {1362-4962}, support = {R56 HG012206/HG/NHGRI NIH HHS/United States ; RR170030//Cancer Prevention &amp; Research Institute of Texas/ ; R35GM143532/NH/NIH HHS/United States ; 917025//American Heart Association predoctoral fellowship program/ ; }, mesh = {Humans ; *Promoter Regions, Genetic ; *Nuclear Pore Complex Proteins/genetics/metabolism ; *Intrinsically Disordered Proteins/genetics/metabolism/chemistry ; *Transcription, Genetic ; Oncogene Proteins, Fusion/genetics/metabolism ; Hematopoietic Stem Cells/metabolism ; Chromatin/genetics/metabolism ; }, abstract = {Protein intrinsically disordered regions (IDRs) are critical gene-regulatory components and aberrant fusions between IDRs and DNA-binding/chromatin-associating domains cause diverse human cancers. Despite this importance, how IDRs influence gene expression, and how aberrant IDR fusion proteins provoke oncogenesis, remains incompletely understood. Here we develop a series of synthetic dCas9-IDR fusions to establish that locus-specific recruitment of IDRs can be sufficient to stimulate endogenous gene expression. Using dCas9 fused to the paradigmatic leukemogenic NUP98 IDR, we also demonstrate that IDRs can activate transcription via localized biomolecular condensation and in a manner that is dependent upon overall IDR concentration, local binding density, and amino acid composition. To better clarify the oncogenic role of IDRs, we construct clinically observed NUP98 IDR fusions and show that, while generally non-overlapping, oncogenic NUP98-IDR fusions convergently drive a core leukemogenic gene expression program in donor-derived human hematopoietic stem cells. Interestingly, we find that this leukemic program arises through differing mechanistic routes based upon IDR fusion partner; either distributed intragenic binding and intrachromosomal looping, or dense binding at promoters. Altogether, our studies clarify the gene-regulatory roles of IDRs and, for the NUP98 IDR, connect this capacity to pathological cellular programs, creating potential opportunities for generalized and mechanistically tailored therapies.}, }
@article {pmid39933516, year = {2025}, author = {Paauw, M and Schravesande, WEW and Taks, NW and Rep, M and Pfeilmeier, S and van den Burg, HA}, title = {Evolution of a vascular plant pathogen is associated with the loss of CRISPR-Cas and an increase in genome plasticity and virulence genes.}, journal = {Current biology : CB}, volume = {35}, number = {5}, pages = {954-969.e5}, doi = {10.1016/j.cub.2025.01.003}, pmid = {39933516}, issn = {1879-0445}, mesh = {*CRISPR-Cas Systems/genetics ; *Xanthomonas campestris/genetics/pathogenicity ; *Virulence Factors/genetics ; Virulence/genetics ; *Plant Diseases/microbiology ; *Genome, Bacterial ; *Evolution, Molecular ; Phylogeny ; *Biological Evolution ; }, abstract = {A major question in infectious disease research is how bacteria have evolved into highly niche-adapted pathogens with efficient host infection strategies. The plant pathogenic bacterium Xanthomonas campestris is subdivided into pathovars that occupy two distinct niches of the same plant leaf: the vasculature and the mesophyll tissue. Using a pangenome comparison of 94 X. campestris isolates, we discovered that the vasculature-infecting pathovar emerged in one monophyletic clade, has lost its CRISPR-Cas system, and showed an increase in both genomic plasticity and acquisition of virulence factors, such as type III effector proteins, compared with the ancestral pathovar. In addition, we show that the CRISPR spacers of isolates belonging to the ancestral pathovar map to plasmids that circulate in Xanthomonas populations and encode high numbers of transposons and virulence factors, suggesting that CRISPR-Cas restricts gene flow toward this pathovar. Indeed, we demonstrate experimentally reduced plasmid uptake in a CRISPR-Cas-encoding isolate. Based on our data, we propose that the loss of the CRISPR-Cas system was a pivotal step in X. campestris evolution by facilitating increased genome dynamics and the emergence of the vasculature-adapted X. campestris pathovar campestris, a major pathogen of Brassica crops.}, }
@article {pmid39933068, year = {2025}, author = {Singh, MA and Chang, MM and Wang, Q and Rodgers, C and Lutz, BR and Olanrewaju, AO}, title = {Rapid Enzymatic Assay for Antiretroviral Drug Monitoring Using CRISPR-Cas12a-Enabled Readout.}, journal = {ACS synthetic biology}, volume = {14}, number = {2}, pages = {510-519}, pmid = {39933068}, issn = {2161-5063}, support = {R33 AI140460/AI/NIAID NIH HHS/United States ; U01 HL152401/HL/NHLBI NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Drug Monitoring/methods ; HIV Infections/drug therapy ; *Anti-HIV Agents/analysis ; Zidovudine/analysis ; HIV Reverse Transcriptase/metabolism/genetics ; *Enzyme Assays/methods ; Leukocytes, Mononuclear/metabolism ; DNA ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Maintaining the efficacy of human immunodeficiency virus (HIV) medications is challenging among children because of dosing difficulties, the limited number of approved drugs, and low rates of medication adherence. Drug level feedback (DLF) can support dose optimization and timely interventions to prevent treatment failure, but current tests are heavily instrumented and centralized. We developed the REverse transcriptase ACTivity crispR (REACTR) for rapid measurement of HIV drugs based on the extent of DNA synthesis by HIV reverse transcriptase. CRISPR-Cas enzymes bind to the synthesized DNA, triggering collateral cleavage of quenched reporters and generating fluorescence. We measured azidothymidine triphosphate (AZT-TP), a key drug in pediatric HIV treatment, and investigated the impact of assay time and DNA template length on REACTR's sensitivity. REACTR selectively measured clinically relevant AZT-TP concentrations in the presence of genomic DNA and peripheral blood mononuclear cell lysate. REACTR has the potential to enable rapid point-of-care HIV DLF to improve pediatric HIV care.}, }
@article {pmid39932844, year = {2025}, author = {Arshad, S and Qadir, ML and Hussain, N and Ali, Q and Han, S and Ali, D}, title = {Advances in CRISPR/Cas9 technology: shaping the future of photosynthetic microorganisms for biofuel production.}, journal = {Functional plant biology : FPB}, volume = {52}, number = {}, pages = {}, doi = {10.1071/FP24255}, pmid = {39932844}, issn = {1445-4416}, mesh = {*Biofuels ; *CRISPR-Cas Systems/genetics ; *Microalgae/genetics/metabolism ; *Photosynthesis/genetics ; Gene Editing ; }, abstract = {Use of fossil fuels causes environmental issues due to its inefficiency and and imminent depletion. This has led to interest in identifying alternative and renewable energy sources such as biofuel generation from photosynthetic organisms. A wide variety of prokaryotic and eukaryotic microorganisms, known as microalgae, have the potential to be economical and ecologically sustainable in the manufacture of biofuels such as bio-hydrogen, biodiesel, bio-oils, and bio-syngas. By using contemporary bioengineering techniques, the innate potential of algae to produce biomass of superior quality may be enhanced. In algal biotechnology, directed genome modification via RNA-guided endonucleases is a new approach. CRISPR/Cas systems have recently been frequently used to modify the genetic makeup of several aquatic and freshwater microalgae. The majority of research has used the Cas9-driven Type II system, one of two classes and six unique kinds of CRISPR systems, to specifically target desired genes in algae, and knock them out and down, or both. Using CRISPR technology to modify its genetic makeup, microalgae has produced more biomass and increased in lipid content. This review highlights the attempts made so far to target microalgae genome modification, discusses the prospects for developing the CRISPR platform for large-scale genome modification of microalgae, and identifies the opportunities and challenges in the development and distribution of CRISPR/Cas9 components.}, }
@article {pmid39930995, year = {2025}, author = {Nafian, F and Esfahani, KS and Hobabi Aghmiuni, M and Khoushab, S and Illeslamllo, T and Nafian, S and Mohamadiyan, N and Aleyasin, NS and Kamali Doust Azad, B}, title = {Emerging microfluidic technologies for CRISPR-based diagnostics: an overview.}, journal = {Analytical methods : advancing methods and applications}, volume = {17}, number = {9}, pages = {1962-1976}, doi = {10.1039/d5ay00063g}, pmid = {39930995}, issn = {1759-9679}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Microfluidic Analytical Techniques/methods/instrumentation ; Lab-On-A-Chip Devices ; Point-of-Care Testing ; }, abstract = {In recent years, CRISPR (clustered regularly interspaced short palindromic repeats) has emerged as a detection technique with high specificity and sensitivity. However, it still needs improvements in terms of reducing cost, complexity, cross-contamination, technical requirements, and lack of quantification platforms. Microfluidic strategies can advance CRISPR-based technology and be modified to a higher level in the future. This review provides an overview of CRISPR-based detection systems (CRISPR-Dx) and their mechanism. Then, it explains how they have been optimized for fast and accurate point-of-care testing (POCT) using microfluidic devices such as SHINE, CARMEN, DNAiTECH, Dμchip, MAPnavi, FAST, and ITP. We discuss their innovations, primarily focusing on how they develop CRISPR-Dx in detection throughput, quantification, simple operation, visualization, sensitivity, specificity, and anti-contamination.}, }
@article {pmid39930790, year = {2025}, author = {Huq, TB and Anil Kumar Jeeja, P and Dam, SK and Das, SS and Vivero-Escoto, JL}, title = {Recent Applications of Mesoporous Silica Nanoparticles in Gene Therapy.}, journal = {Advanced healthcare materials}, volume = {}, number = {}, pages = {e2404781}, doi = {10.1002/adhm.202404781}, pmid = {39930790}, issn = {2192-2659}, support = {R01CA263897/CA/NCI NIH HHS/United States ; }, abstract = {Gene therapy offers transformative potential for treating genetic disorders by directly addressing the molecular root causes of diseases. However, the primary challenges of gene therapy involve the efficient delivery of therapeutic genetic material to target cells, crossing biological barriers, managing toxicity and immune responses. Mesoporous silica nanoparticles (MSNs), due to their unique structural features have emerged as a promising platform to overcome these challenges. In recent years, MSNs have gained significant attention as potential nanocarriers for the efficient delivery of various nucleic acids. This review comprehensively examines the role of MSNs in gene therapy, focusing on their capabilities in the targeted delivery of siRNA, DNA, CRISPR-Cas systems, and other genetic therapeutics. This work explores the modern advancements in MSNs synthesis and functionalization strategies and the impact of structural modifications on their stability, cellular uptake, and controlled release under physiological conditions. Additionally, the review highlights the use of MSNs to develop theranostic systems, where gene delivery is combined with diagnostic imaging for real-time monitoring and personalized treatment strategies. Finally, this work discusses the future perspectives of MSNs in gene delivery, addressing regulatory challenges, enhancing clinical translation, and expanding their application for treating various genetic disorders and cancers.}, }
@article {pmid39930358, year = {2025}, author = {Zhang, Y and Newstead, S and Sarkies, P}, title = {Predicting substrates for orphan solute carrier proteins using multi-omics datasets.}, journal = {BMC genomics}, volume = {26}, number = {1}, pages = {130}, pmid = {39930358}, issn = {1471-2164}, mesh = {Humans ; *Solute Carrier Proteins/metabolism/genetics ; Algorithms ; *Computational Biology/methods ; *Genomics/methods ; Substrate Specificity ; CRISPR-Cas Systems ; Multiomics ; }, abstract = {Solute carriers (SLC) are integral membrane proteins responsible for transporting a wide variety of metabolites, signaling molecules and drugs across cellular membranes. Despite key roles in metabolism, signaling and pharmacology, around one third of SLC proteins are 'orphans' whose substrates are unknown. Experimental determination of SLC substrates is technically challenging, given the wide range of possible physiological candidates. Here, we develop a predictive algorithm to identify correlations between SLC expression levels and intracellular metabolite concentrations by leveraging existing cancer multi-omics datasets. Our predictions recovered known SLC-substrate pairs with high sensitivity and specificity compared to simulated random pairs. CRISPR-Cas9 dependency screen data and metabolic pathway adjacency data further improved the performance of our algorithm. In parallel, we combined drug sensitivity data with SLC expression profiles to predict new SLC-drug interactions. Together, we provide a novel bioinformatic pipeline to predict new substrate predictions for SLCs, offering new opportunities to de-orphanise SLCs with important implications for understanding their roles in health and disease.}, }
@article {pmid39930230, year = {2025}, author = {Léger-Charnay, E and Slembrouck-Brec, A and Goureau, O}, title = {Engineering Specific Human iPS Reporter Cell Lines to Generate Optogenetically Modified Photoreceptors.}, journal = {Advances in experimental medicine and biology}, volume = {1468}, number = {}, pages = {409-414}, pmid = {39930230}, issn = {0065-2598}, mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Optogenetics/methods ; Cell Line ; CRISPR-Cas Systems ; Genes, Reporter ; Animals ; *Photoreceptor Cells, Vertebrate/metabolism/cytology ; }, abstract = {Cell therapy, by transplantation of photoreceptors derived from induced pluripotent stem cells (iPSCs), has been proposed as a promising therapeutic approach for photoreceptor degenerative diseases. A remaining obstacle is that such transplanted cells have to develop into functional light-sensitive photoreceptors, which require outer segment formation and interaction with the underlying retinal pigmented epithelium (RPE). To overcome this limitation, a combination of cell therapy and optogenetics allows to confer light sensitivity to the donor cells thanks to the expression of a microbial opsin and therefore independently of the formation of mature outer segment or RPE contact. To ensure stable and homogenous expression of the microbial opsin in photoreceptors, we inserted the coding sequence of the red-light sensitive chloride pump Jaws under specific photoreceptor promoter into the iPSC genome, using the CRISPR/Cas9 system at the safe AAVS1 locus. We successfully generated a knock-in Jaws-EGFP iPSC line and validated its stemness and pluripotency status. These engineered iPSCs will be used to produce photoreceptors expressing Jaws that will be grafted to assess their ability to restore vision in blind animal models.}, }
@article {pmid39930201, year = {2025}, author = {Xiao, H and Marshall, R and Saxena, MT and Zhang, L}, title = {The Power of Zebrafish in Disease Modeling and Therapy Discovery for Inherited Retinal Degeneration.}, journal = {Advances in experimental medicine and biology}, volume = {1468}, number = {}, pages = {229-233}, pmid = {39930201}, issn = {0065-2598}, mesh = {Animals ; *Zebrafish/genetics ; *Retinal Degeneration/genetics/therapy/pathology ; *Disease Models, Animal ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Humans ; Gene Editing ; Drug Discovery/methods ; }, abstract = {In the research of inherited retinal degeneration (IRD), zebrafish have emerged as a powerful model system, offering profound insights into disease mechanisms and opening new therapeutic avenues. This mini-review discusses the distinctive advantages that zebrafish provide for investigating retinal degeneration. It outlines contemporary genetic tools, with a specific focus on advanced CRISPR/Cas9 gene targeting technology, utilized for genome manipulation and disease modeling in zebrafish. By emphasizing the pivotal role of zebrafish in large-scale high-throughput drug discovery and the exploration of innovative gene therapy strategies, this succinct review underscores the adaptability and significance of the zebrafish model in advancing IRD research. It establishes a robust foundation for future studies and therapeutic developments in the field.}, }
@article {pmid39930183, year = {2025}, author = {Ahmadian, M and Okan, ICT and Uyanik, G and Tschopp, M and Agca, C}, title = {Precise Gene Editing Technologies in Retinal Applications.}, journal = {Advances in experimental medicine and biology}, volume = {1468}, number = {}, pages = {119-123}, pmid = {39930183}, issn = {0065-2598}, mesh = {*Gene Editing/methods ; Humans ; *Genetic Therapy/methods ; *CRISPR-Cas Systems/genetics ; *Retinal Diseases/genetics/therapy ; Animals ; RNA Editing ; Retina/pathology/metabolism ; Mutation ; }, abstract = {Gene therapy is emerging as a promising treatment for inherited retinal diseases (IRDs). One of the first successful applications of gene therapy for IRDs was the gene replacement therapy for the RPE65 mutation. This therapy delivers a functional copy of the RPE65 gene to patients via AAV vectors, rather than targeting the mutation itself. Gene editing technologies have advanced significantly in recent years, allowing it to make precise in vivo modifications to the genetic code. After the discovery of CRISPR-Cas9, other gene editing technologies such as base editing and prime editing have been developed by modifying and combining the original CRISPR-Cas9 technology with other methods. Moreover, recently discovered CRISPR-Cas systems allow RNA editing to correct mutations at the posttranscriptional level. These technologies have potential applications in various fields, including inherited retinal diseases. This mini-review evaluates and summarizes the most current advancements in genome editing methods, including prime editing, base editing, and RNA editing, and their applications on retinal diseases.}, }
@article {pmid39930181, year = {2025}, author = {da Costa, BL and Knudsen, AS and Alves, CH and Tsang, SH and Quinn, PMJ}, title = {Megabase Deletion of the Human EYS Locus Using CRISPR/Cas9.}, journal = {Advances in experimental medicine and biology}, volume = {1468}, number = {}, pages = {107-111}, pmid = {39930181}, issn = {0065-2598}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Induced Pluripotent Stem Cells/metabolism/cytology ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Eye Proteins/genetics ; *Gene Editing/methods ; Retina/metabolism ; *Sequence Deletion ; *Gene Deletion ; *Genetic Loci ; }, abstract = {Mutations in the Eyes Shut Homolog (EYS) gene are associated with autosomal recessive retinitis pigmentosa (arRP). To date, four retinal isoforms of EYS have been identified. However, the precise retinal function of EYS is not fully understood, but it has apparent roles in retinal morphogenesis, architecture, and ciliary transport. Clustered-regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) nuclease-mediated approaches are powerful tools for genome engineering in mammalian cells. The use of paired CRISPR/Cas9-induced double-strand breaks (DSBs) using dual single guide RNAs (sgRNA) can lead to precise genomic deletions. In this study, we developed a dual sgRNA strategy to facilitate CRISPR/Cas9-mediated deletion of 1,988,210 bp of the EYS locus, removing the four currently identified human retinal EYS isoforms. This approach can be used to produce EYSdel induced pluripotent stem cell (iPSC) lines to explore the function of EYS in human iPSC-derived retinal organoids.}, }
@article {pmid39930180, year = {2025}, author = {da Costa, BL and Caruso, SM and Tsai, YT and Castillejos, DS and Sylla, M and Tsang, SH and Quinn, PMJ}, title = {Prime Editing Strategy to Install the RPE65 c.1430A&gt;G Dominant Mutation.}, journal = {Advances in experimental medicine and biology}, volume = {1468}, number = {}, pages = {101-106}, pmid = {39930180}, issn = {0065-2598}, mesh = {Humans ; *cis-trans-Isomerases/genetics ; CRISPR-Cas Systems ; *Gene Editing/methods ; Genes, Dominant ; HEK293 Cells ; Induced Pluripotent Stem Cells/metabolism ; *Mutation ; Retinal Pigment Epithelium/pathology/metabolism ; *Retinitis Pigmentosa/genetics/pathology ; }, abstract = {The retinal pigment epithelium 65-kDa protein (RPE65) is a retinal isomerase that is an essential component of the visual cycle. Mutations in RPE65 are typically associated with autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. Here, we report on a patient with RPE65-mediated autosomal dominant retinitis pigmentosa (adRP) who has widespread chorioretinal atrophy with significant macular involvement and only small areas of retinal preservation. In future studies, we plan to model the pathobiology of RPE65-mediated adRP using induced pluripotent stem cell (iPSC)-derived RPE. To effectively model rare mutations using iPSC-derived RPE and screen gene editing correction approaches, we require a strategy to install the desired mutation in wild-type iPSC and HEK293T. In this study, we developed a prime editing strategy for the installation of the pathogenic RPE65 c.1430A>G mutation underlying our patient's disease.}, }
@article {pmid39930179, year = {2025}, author = {Mittas, DM and Gavrilov, Z and Ucambarlic, E and Gandor, C and Otify, DY and Becirovic, E}, title = {CRISPR/Cas-Mediated Gene Activation as a Versatile Tool for Treatment of Inherited Retinal Dystrophies.}, journal = {Advances in experimental medicine and biology}, volume = {1468}, number = {}, pages = {95-99}, pmid = {39930179}, issn = {0065-2598}, mesh = {Humans ; *Retinal Dystrophies/genetics/therapy ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; *Gene Editing/methods ; Animals ; *Transcriptional Activation ; }, abstract = {CRISPR/Cas-mediated genome editing is an effective and attractive tool for the treatment of diseases or genes that cannot be adequately covered by gene replacement strategies. The first FDA-approved AAV-vector- and CRISPR/Cas-based clinical trials were each designed for the treatment of a subtype of an inherited retinal dystrophy, underscoring the importance of ophthalmic diseases in the field of gene (editing) therapies. This and most other pre-clinical therapeutic CRISPR/Cas approaches are typically designed for the treatment of single mutations. For the treatment of larger patient cohorts, however, mutation- or ideally gene-independent approaches appear to be more suitable. CRISPR/Cas-mediated transcriptional activation of genes (CRISPRa) is a promising approach to achieve these ambitious goals. In this minireview, focusing on inherited retinal dystrophies, we will discuss recent developments, advantages and limitations, and future prospects of CRISPRa as a therapeutic tool.}, }
@article {pmid39930152, year = {2025}, author = {Zhang, P and Zhang, Z and Wang, Y and Du, W and Song, X and Lai, W and Wang, H and Zhu, B and Xiong, J}, title = {A CRISPR-Cas9 screen reveals genetic determinants of the cellular response to decitabine.}, journal = {EMBO reports}, volume = {26}, number = {6}, pages = {1528-1565}, pmid = {39930152}, issn = {1469-3178}, support = {32288102//MOST | National Natural Science Foundation of China (NSFC)/ ; 32170607//MOST | National Natural Science Foundation of China (NSFC)/ ; 32000417//MOST | National Natural Science Foundation of China (NSFC)/ ; 2021YFC2300500//MOST | National Key Research and Development Program of China (NKPs)/ ; 2017133//CAS | Youth Innovation Promotion Association (YIPA)/ ; 2020097//CAS | Youth Innovation Promotion Association (YIPA)/ ; }, mesh = {Humans ; *CRISPR-Cas Systems ; *Decitabine/pharmacology ; DNA Damage/drug effects ; Histone Demethylases/metabolism/genetics ; Cell Line, Tumor ; DNA Methylation/drug effects ; *Antimetabolites, Antineoplastic/pharmacology ; DNA Replication/drug effects ; DNA Repair/drug effects ; Genomic Instability/drug effects ; Leukemia, Myeloid, Acute/drug therapy/genetics ; }, abstract = {Decitabine (DAC), a well-recognized DNA hypomethylating agent, has been applied to treat acute myeloid leukemia. However, clinic investigations revealed that DNA methylation reduction does not correlate with a clinical response, and relapse is prevalent. To gain a better understanding of its anti-tumor mechanism, we perform a temporally resolved CRISPR-Cas9 screen to identify factors governing the DAC response. We show that DNA damage generated by DNMT-DNA adducts and 5-aza-dUTP misincorporation through the dCMP deaminase DCTD act as drivers of DAC-induced acute cytotoxicity. The DNA damage that arises during the next S phase is dependent on DNA replication, unveiling a trans-cell cycle effect of DAC on genome stability. By exploring candidates for synthetic lethality, we unexpectedly uncover that KDM1A promotes survival after DAC treatment through interactions with ZMYM3 and CoREST, independent of its demethylase activity or regulation of viral mimicry. These findings emphasize the importance of DNA repair pathways in DAC response and provide potential biomarkers.}, }
@article {pmid39930103, year = {2025}, author = {Ling, S and Zhang, X and Dai, Y and Jiang, Z and Zhou, X and Lu, S and Qian, X and Liu, J and Selfjord, N and Satir, TM and Lundin, A and Touza, JL and Firth, M and Van Zuydam, N and Bilican, B and Akcakaya, P and Hong, J and Cai, Y}, title = {Customizable virus-like particles deliver CRISPR-Cas9 ribonucleoprotein for effective ocular neovascular and Huntington's disease gene therapy.}, journal = {Nature nanotechnology}, volume = {20}, number = {4}, pages = {543-553}, pmid = {39930103}, issn = {1748-3395}, support = {no. 31971364//National Natural Science Foundation of China (National Science Foundation of China)/ ; no. 32370148//National Natural Science Foundation of China (National Science Foundation of China)/ ; no. 81970766//National Natural Science Foundation of China (National Science Foundation of China)/ ; no. 82171102//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {*Huntington Disease/therapy/genetics ; *CRISPR-Cas Systems/genetics ; Animals ; *Genetic Therapy/methods ; Humans ; Gene Editing/methods ; *Ribonucleoproteins/genetics ; Induced Pluripotent Stem Cells/metabolism ; Huntingtin Protein/genetics ; HEK293 Cells ; *Virion/genetics ; Mice ; Disease Models, Animal ; }, abstract = {In vivo CRISPR gene editing holds enormous potential for various diseases. Ideally, CRISPR delivery should be cell type-specific and time-restricted for optimal efficacy and safety, but customizable methods are lacking. Here we develop a cell-tropism programmable CRISPR-Cas9 ribonucleoprotein delivery system (RIDE) based on virus-like particles. The efficiency of RIDE was comparable to that of adeno-associated virus and lentiviral vectors and higher than lipid nanoparticles. RIDE could be readily reprogrammed to target dendritic cells, T cells and neurons, and significantly ameliorated the disease symptoms in both ocular neovascular and Huntington's disease models via cell-specific gene editing. In addition, RIDE could efficiently edit the huntingtin gene in patients' induced pluripotent stem cell-derived neurons and was tolerated in non-human primates. This study is expected to facilitate the development of in vivo CRISPR therapeutics.}, }
@article {pmid39929897, year = {2025}, author = {Allen, A and Cooper, BH and Singh, J and Rohs, R and Qin, PZ}, title = {PAM-adjacent DNA flexibility tunes CRISPR-Cas12a off-target binding.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {4930}, pmid = {39929897}, issn = {2045-2322}, support = {R01GM124413/GM/NIGMS NIH HHS/United States ; R35 GM145341/GM/NIGMS NIH HHS/United States ; R35GM130376/GM/NIGMS NIH HHS/United States ; R35 GM130376/GM/NIGMS NIH HHS/United States ; R01 GM124413/GM/NIGMS NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics ; *DNA/metabolism/genetics/chemistry ; *Endodeoxyribonucleases/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Bacterial Proteins/metabolism/genetics ; Gene Editing ; Protein Binding ; *Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Cas12a is a class 2 type V CRISPR-associated nuclease that uses an effector complex comprised of a single protein activated by a CRISPR-encoded small RNA to cleave double-stranded DNA at specific sites. Cas12a processes unique features as compared to other CRISPR effector nucleases such as Cas9, and has been demonstrated as an effective tool for manipulating complex genomes. Prior studies have indicated that DNA flexibility at the region adjacent to the protospacer-adjacent-motif (PAM) contributes to Cas12a target recognition. Here, we adapted a SELEX-seq approach to further examine the connection between PAM-adjacent DNA flexibility and off-target binding by Cas12a. A DNA library containing DNA-DNA mismatches at PAM + 1 to + 6 positions was generated and subjected to binding in vitro with FnCas12a in the absence of pairing between the RNA guide and DNA target. The bound and unbound populations were sequenced to determine the propensity for off-target binding for each of the individual sequences. Analyzing the position and nucleotide dependency of the DNA-DNA mismatches showed that PAM-dependent Cas12a off-target binding requires unpairing of the protospacer at PAM + 1 and increases with unpairing at PAM + 2 and + 3. This revealed that PAM-adjacent DNA flexibility can tune Cas12a off-target binding. The work adds support to the notion that physical properties of the DNA modulate Cas12a target discrimination, and has implications on Cas12a-based applications.}, }
@article {pmid39929846, year = {2025}, author = {Li, S and Yu, J and Shen, Y and Xiong, B and Zhao, D and Xu, W and Zhang, S and Guan, X and Liu, Y and Shan, X and Zhu, A and Lyu, Q and Fang, Y and Chen, Z and Yin, H and Sun, L and Xu, H}, title = {Transdermal microneedle-assisted ultrasound-enhanced CRISPRa system to enable sono-gene therapy for obesity.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {1499}, pmid = {39929846}, issn = {2041-1723}, support = {82151318//National Natural Science Foundation of China (National Science Foundation of China)/ ; SHSLCZDZK03502//Foundation of Shanghai Municipal Commission of Health and Family Planning (Shanghai Municipal Commission of Health and Family Planning Foundation)/ ; }, mesh = {Animals ; *Obesity/therapy/genetics ; *Genetic Therapy/methods ; Mice ; Male ; Uncoupling Protein 1/genetics/metabolism ; Needles ; *CRISPR-Cas Systems/genetics ; Mice, Inbred C57BL ; Adipocytes/metabolism ; Drug Delivery Systems/methods ; Polyethyleneimine/chemistry ; Administration, Cutaneous ; Gene Transfer Techniques ; Polyethylene Glycols/chemistry ; Humans ; }, abstract = {Obesity, a surging global health challenge, necessitates effective, accessible and innovative therapeutic models. Here we develop a spatiotemporally controllable microneedle (MN) drug delivery platform for sono-gene therapy to fight obesity. The platform delivers the methoxy polyethylene glycol-polyethyleneimine (mPEG-PEI) modified metal-organic frameworks (MOFs) sonosensitizer and the clustered regularly interspaced short palindromic repeats-activating (CRISPRa)/CRISPRa-uncoupling protein 1 (UCP1) system intradermally to adipocytes. Overall, this therapy platform is capable of achieving two major strategies of "annihilation" and "countermeasure": one is to kill redundant white adipocytes by sonodynamic therapy, and the other is to promote the browning of white adipocytes through the controllable release of CRISPRa-UCP1 system and sonodynamic effect. Obese male mice treated with this sono-gene therapy shows significant ameliorate in glucose tolerance and insulin sensitivity, successfully achieves weight loss and restrains weight rebound. This study may enable a standard treatment paradigm for sono-gene therapy of obesity and other metabolic diseases.}, }
@article {pmid39928547, year = {2025}, author = {Hossain, KA and Nierzwicki, L and Orozco, M and Czub, J and Palermo, G}, title = {Flexibility in PAM recognition expands DNA targeting in xCas9.}, journal = {eLife}, volume = {13}, number = {}, pages = {}, pmid = {39928547}, issn = {2050-084X}, support = {R01 GM141329/GM/NIGMS NIH HHS/United States ; FG-2023-20431//Alfred P. Sloan Foundation/ ; CHE-2144823//National Science Foundation/ ; R01GM141329/NH/NIH HHS/United States ; PDI2021-122478NB-I00//Spanish Ministry of Science and Innovation/ ; TC-24-063//Camille and Henry Dreyfus Foundation/ ; 10.3030/101093290//EU-HPC, BioExcel/ ; 101094561//HORIZON EUROPE Framework Programme/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *DNA/metabolism/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; Humans ; }, abstract = {xCas9 is an evolved variant of the CRISPR-Cas9 genome editing system, engineered to improve specificity and reduce undesired off-target effects. How xCas9 expands the DNA targeting capability of Cas9 by recognising a series of alternative protospacer adjacent motif (PAM) sequences while ignoring others is unknown. Here, we elucidate the molecular mechanism underlying xCas9's expanded PAM recognition and provide critical insights for expanding DNA targeting. We demonstrate that while wild-type Cas9 enforces stringent guanine selection through the rigidity of its interacting arginine dyad, xCas9 introduces flexibility in R1335, enabling selective recognition of specific PAM sequences. This increased flexibility confers a pronounced entropic preference, which also improves recognition of the canonical TGG PAM. Furthermore, xCas9 enhances DNA binding to alternative PAM sequences during the early evolution cycles, while favouring binding to the canonical PAM in the final evolution cycle. This dual functionality highlights how xCas9 broadens PAM recognition and underscores the importance of fine-tuning the flexibility of the PAM-interacting cleft as a key strategy for expanding the DNA targeting potential of CRISPR-Cas systems. These findings deepen our understanding of DNA recognition in xCas9 and may apply to other CRISPR-Cas systems with similar PAM recognition requirements.}, }
@article {pmid39927661, year = {2025}, author = {Jin, X and Zhang, B and Sun, Y and Duan, Y and Lu, J and Liu, J and Han, J and Zhang, Y}, title = {Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {215}, pages = {}, doi = {10.3791/67616}, pmid = {39927661}, issn = {1940-087X}, mesh = {Animals ; *Zebrafish/genetics/metabolism ; *Gene Knock-In Techniques/methods ; CRISPR-Cas Systems ; *Fluorescent Dyes/chemistry ; *Zebrafish Proteins/genetics/biosynthesis/analysis ; }, abstract = {CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in vivo imaging due to its transparency during the early developmental stage. Here, we provide a detailed protocol for performing high-efficiency fluorescence gene KI, rapid screening for KI founders, and low-abundance protein tracing in zebrafish larvae, which will lay a critical foundation for subsequent physio-pathological studies in zebrafish. The current protocol includes complete steps for the sgRNA design for the gene of interest, sgRNA in vitro transcription, Cas9 mRNA in vitro transcription, in vivo sgRNA screen for the one with the highest efficiency, donor plasmid design and construction, microinjection in zebrafish larvae, KI founder screen and zebrafish live imaging. Critical steps, troubleshooting tips, quality control methods, and advantages and applications of this protocol are included and discussed. This protocol assures quick and accurate results at a low cost and has been validated by multiple trials.}, }
@article {pmid39924908, year = {2025}, author = {Gao, Y and Ang, YS and Yung, LL}, title = {CRISPR-Cas12a-Assisted DNA Circuit for Nonmicroscopic Detection of Cell Surface Receptor Clustering.}, journal = {ACS sensors}, volume = {10}, number = {2}, pages = {977-985}, doi = {10.1021/acssensors.4c02770}, pmid = {39924908}, issn = {2379-3694}, support = {U54 HG006097/HG/NHGRI NIH HHS/United States ; U54 HL127365/HL/NHLBI NIH HHS/United States ; }, mesh = {Humans ; *CRISPR-Cas Systems ; *DNA/genetics/chemistry/metabolism ; Cell Line, Tumor ; *Receptor, ErbB-2/metabolism ; *Endodeoxyribonucleases/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; Receptor, ErbB-3/metabolism ; *Bacterial Proteins/genetics/metabolism ; }, abstract = {Protein-protein interactions (PPIs) on the cell surface have been of great interest due to their high clinical relevance and significance; however, the methods for detecting PPIs heavily rely on microscopic instruments. In this work, we designed a Cas12a-assisted DNA circuit for detecting cell surface receptor clustering events without a dependence on microscopy. This nonmicroscopic approach is based on the proximity principle, where localized protein-protein interactions such as receptor clustering are converted into DNA barcodes. These barcodes can then be identified by Cas12a for signal generation in the bulk. The compatibility of the circuit with Cas12a was first experimentally verified. Several leak reactions were identified and minimized. Lastly, we implemented this design in human breast cancer cell line models to distinguish the different levels of human epidermal growth factor receptor 2 (HER2) homodimers and heterodimers with HER1 and HER3 semiquantitatively without the use of a microscope. Overall, our proposed Cas12a-assisted DNA circuit for detecting cell surface receptor clustering shows the potential for fast screening in diagnostic applications and drug discovery, demonstrating the promising use of enzymatic DNA circuits in biological applications.}, }
@article {pmid39923676, year = {2025}, author = {Li, X and Han, Z and Guo, P and Zhang, X and Hu, Y and Cao, J}, title = {LbCas12a-based DNA POCT facilitates fast genotyping on farm.}, journal = {Talanta}, volume = {287}, number = {}, pages = {127672}, doi = {10.1016/j.talanta.2025.127672}, pmid = {39923676}, issn = {1873-3573}, mesh = {Animals ; *CRISPR-Cas Systems ; *Genotyping Techniques/methods ; Polymorphism, Single Nucleotide ; Swine ; *Farms ; Sheep ; *DNA/genetics ; *CRISPR-Associated Proteins/genetics ; Genotype ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a) detection system is now widely used for nucleic acid detection and disease diagnosis. However, there are still fewer detections for single nucleotide polymorphisms (SNPs) and limited diversified detection systems for pathogen and SNP sites detection, which greatly limits their applications. Obviously, the development of a more diversified and convenient suite of detection tools is essential to unlock the full potential of CRISPR/Cas12a technology and to expand its applications across a wider range of scenarios. We have successfully developed an integrated CRISPR/Cas12a assay system. This system introduces crRNA during protein expression, reducing the number of steps and reaction time by adding only a fluorescent reporter gene and target DNA during subsequent detection. It enables on-site visualization of the assay in combination with a Recombinase polymerase amplification (RPA) reaction. Combined with the RPA reaction, we are able to rapidly detect African swine fever virus (ASFV) pathogens with high specificity. The system also enables genotyping of the SNP site of the porcine prolificacy-associated estrogen receptor (ESR) gene and the sheep prolificacy-associated Fecundity booroola (FecB) gene. Visualization is possible up to a final concentration of 3 nM, and effective differentiation of low concentrations within the concentration range of the assay. The integrated CRISPR/Cas12a assay system we developed has a robust design that ensures high-fidelity genotyping and pathogen detection are no longer restricted to the lab, allowing for rapid field analysis, which is crucial for timely interventions in agricultural and clinical settings. In addition, it has the advantages of low cost, easy operation and visualization of results.}, }
@article {pmid39922978, year = {2025}, author = {Fry, LE and Major, L and Salman, A and McDermott, LA and Yang, J and King, AJ and McClements, ME and MacLaren, RE}, title = {Comparison of CRISPR-Cas13b RNA base editing approaches for USH2A-associated inherited retinal degeneration.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {200}, pmid = {39922978}, issn = {2399-3642}, support = {/WT_/Wellcome Trust/United Kingdom ; R01 EY034524/EY/NEI NIH HHS/United States ; }, mesh = {Animals ; Humans ; Adenosine Deaminase/genetics ; *CRISPR-Cas Systems ; Dependovirus ; *Extracellular Matrix Proteins/genetics ; Mice, Knockout ; Point Mutation ; Retina/surgery ; RNA Editing ; *Targeted Gene Repair/methods ; Transgenes ; *Usher Syndromes/genetics/therapy ; Mice, Inbred C57BL ; }, abstract = {CRISPR-Cas13 systems have therapeutic promise for the precise correction of point mutations in RNA. Using adenosine deaminase acting on RNA (ADAR) effectors, A-I base conversions can be targeted using guide RNAs (gRNAs). We compare the Cas13 effectors PspCas13b and Cas13bt3 for the repair of the gene USH2A, a common cause of inherited retinal disease and Usher syndrome. In cultured cells, we demonstrate up to 80% efficiency for the repair of the common c.11864 G > A and its murine equivalent c.11840 G > A, across different gRNAs and promoters. We develop and characterize a mouse model of Usher syndrome carrying the c.11840 G > A mutation designed for the evaluation of base editors for inherited retinal disease. Finally, we compare Cas13 effectors delivered via AAV for the repair of Ush2a in photoreceptors. Mean RNA editing rates in photoreceptors across different constructs ranged from 0.32% to 2.04%, with greater efficiency in those injected with PspCas13b compared to Cas13bt3 constructs. In mice injected with PspCas13b constructs, usherin protein was successfully restored and correctly localized to the connecting cilium following RNA editing. These results support the development of transcriptome targeting gene editing therapies for retinal disease.}, }
@article {pmid39921871, year = {2025}, author = {Lv, J and Fan, Q and Zhang, Y and Zhou, X and Yu, P and Yu, X and Xin, C and Hong, J and Cheng, Y}, title = {A Serum Resistant Polymer with Exceptional Endosomal Escape and mRNA Delivery Efficacy for CRISPR Gene Therapy.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {12}, number = {13}, pages = {e2413006}, pmid = {39921871}, issn = {2198-3844}, support = {2019YFA0904500//National Key R&amp;D Program of China/ ; 2023YFA0915000//National Key R&amp;D Program of China/ ; 52373134//National Natural Science Foundation of China/ ; 82171102//National Natural Science Foundation of China/ ; 82271044//National Natural Science Foundation of China/ ; 2019YFA0904500//National Key Research and Development Program of China/ ; 2023YFA0915000//National Key Research and Development Program of China/ ; 22Y21900900//Shanghai Medical Innovation Research Program/ ; }, mesh = {Animals ; *RNA, Messenger/genetics/metabolism/administration &amp; dosage ; *Genetic Therapy/methods ; Mice ; *CRISPR-Cas Systems/genetics ; Humans ; *Endosomes/metabolism ; *Polymers/chemistry ; Gene Editing/methods ; Nanoparticles/chemistry ; *Gene Transfer Techniques ; Choroidal Neovascularization/therapy/genetics ; }, abstract = {Nanoparticle-based mRNA delivery offers a versatile platform for innovative therapies. However, most of the current delivery systems are limited by poor serum tolerance, suboptimal endosomal escape and mRNA delivery efficacy. Herein, a highly efficient mRNA-delivering material is identified from a library of fluoropolymers. The lead material FD17 shows exceptional serum stability and endosomal escape, enabling efficient mRNA delivery into various cell types, surpassing commercial mRNA delivery reagents such as Lipofectamine 3000. The formed mRNA nanoparticles adsorb abundant serum albumin on the surface, which facilitates cellular uptake via scavenger receptor-mediated endocytosis. FD17 enables the delivery of mRNAs encoding CRE, Cas9, and base editor hyCBE for efficient genome editing. The material mediates CRISPR/Cas9 gene therapy via intraocular injection effectively down-regulates vascular endothelial growth factor A in retinal pigment epithelial cells of mice, yielding promising therapeutic responses against laser-induced choroidal neovascularization. The discovered material in this study shows great promise for the development of mRNA therapeutics to combat a wide range of diseases.}, }
@article {pmid39921565, year = {2025}, author = {Harris, DT and Jan, CH}, title = {CRISPuRe-seq: pooled screening of barcoded ribonucleoprotein reporters reveals regulation of RNA polymerase III transcription by the integrated stress response via mTOR.}, journal = {Nucleic acids research}, volume = {53}, number = {4}, pages = {}, pmid = {39921565}, issn = {1362-4962}, support = {//Calico Life Sciences LLC/ ; }, mesh = {*RNA Polymerase III/genetics/metabolism ; *TOR Serine-Threonine Kinases/metabolism/genetics ; Humans ; *Ribonucleoproteins/genetics/metabolism ; *Stress, Physiological/genetics ; RNA, Transfer/genetics/metabolism/biosynthesis ; *Transcription, Genetic ; Mechanistic Target of Rapamycin Complex 1/metabolism/genetics ; CRISPR-Cas Systems ; Repressor Proteins/metabolism/genetics ; Protein Biosynthesis ; }, abstract = {Genetic screens using CRISPR (Clustered Regularly Interspaced Palindromic Repeats) provide valuable information about gene function. Nearly all pooled screening technologies rely on the cell to link genotype to phenotype, making it challenging to assay mechanistically informative, biochemically defined phenotypes. Here, we present CRISPuRe-seq (CRISPR PuRification), a novel pooled screening strategy that expands the universe of accessible phenotypes through the purification of ribonucleoprotein complexes that link genotypes to expressed RNA barcodes. While screening for regulators of the integrated stress response (ISR), we serendipitously discovered that the ISR represses transfer RNA (tRNA) production under conditions of reduced protein synthesis. This regulation is mediated through inhibition of mTORC1 and corresponding activation of the RNA polymerase III inhibitor MAF1. These data demonstrate that coherent downregulation of tRNA expression and protein synthesis is achieved through cross-talk between the ISR and mTOR, two master integrators of cell state.}, }
@article {pmid39920943, year = {2025}, author = {Li, H and Song, C and Li, Y and Zhang, T and Yang, X and Wang, H}, title = {Genome-wide CRISPR screen reveals host factors for gama- and delta-coronavirus infection in Huh7 cells.}, journal = {International journal of biological macromolecules}, volume = {304}, number = {Pt 1}, pages = {140728}, doi = {10.1016/j.ijbiomac.2025.140728}, pmid = {39920943}, issn = {1879-0003}, mesh = {Humans ; *Host-Pathogen Interactions/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Animals ; *CRISPR-Cas Systems ; *Coronavirus Infections/virology/genetics/metabolism ; *Coronavirus/physiology ; Cell Line ; Virus Internalization ; Infectious bronchitis virus/physiology ; Membrane Proteins/genetics/metabolism ; Swine ; }, abstract = {Genome-wide CRISPR screening has emerged as a powerful tool for identifying novel host factors involved in viral infections. In recent years, host factors for several Alpha- and Beta-coronaviruses have been systematically screened and characterized. However, knowledge regarding Gamma- and Delta-coronavirus infections remains limited. In this study, we conducted genome-scale CRISPR knockout (KO) screening in Huh7 cells infected with infectious bronchitis virus (IBV), a Gamma-coronavirus, and porcine deltacoronavirus (PDCoV), a Delta-coronavirus. We identified known host factors for PDCoV, including APN and TMEM41B. We confirmed that human APN does not serve as a critical host factor for IBV. Notably, SPPL3 was identified as a key factor involved in viral particle entry and S protein-induced syncytium formation through the modulation of cellular N-glycosylation. Furthermore, we performed a meta-analysis integrating all Huh7 cell-based genome-wide CRISPR screens across the four genera of coronaviruses (Alpha-, Beta-, Gamma-, and Delta-coronaviruses). Our analysis highlighted conserved host pathways, particularly those related to proteoglycans, glycoproteins, and vesicle trafficking. TMEM41B, SCAP, and FAM98A emerged as the most frequently targeted host genes. These findings provide valuable insights into the life cycles of IBV and PDCoV infections and facilitate the development of host-directed therapeutic strategies.}, }
@article {pmid39920845, year = {2025}, author = {Larrosa-Godall, M and Ang, JXD and Leftwich, PT and Gonzalez, E and Shackleford, L and Nevard, K and Noad, R and Anderson, MAE and Alphey, L}, title = {Challenges in developing a split drive targeting dsx for the genetic control of the invasive malaria vector Anopheles stephensi.}, journal = {Parasites &amp; vectors}, volume = {18}, number = {1}, pages = {46}, pmid = {39920845}, issn = {1756-3305}, support = {INV-008549/GATES/Gates Foundation/United States ; BBS/E/I/00007033, BBS/E/I/00007038, and BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; }, mesh = {Animals ; *Anopheles/genetics ; *Mosquito Vectors/genetics ; Female ; Male ; *Malaria/transmission/prevention &amp; control ; *Gene Drive Technology/methods ; *Mosquito Control/methods ; CRISPR-Cas Systems ; Animals, Genetically Modified ; Insect Proteins/genetics ; }, abstract = {BACKGROUND: Anopheles stephensi is a competent malaria vector mainly present in southern Asia and the Arabian Peninsula. Since 2012, it has invaded several countries of eastern Africa, creating an emerging risk of urban transmission. Urgent efforts are required to develop novel and more efficient strategies for targeted vector control. CRISPR/Cas9-based homing gene drives have been proposed as attractive alternative strategies. Gene drives have the potential to spread a desired trait through a population at higher rates than via normal Mendelian inheritance, even in the presence of a fitness cost. Several target genes have been suggested and tested in different mosquito vector species such as Anopheles gambiae and Aedes aegypti. Several promising suppression drives have been developed in An. gambiae that target the sex determination gene doublesex (dsx).<br><br>METHODS: In this study, a geographically confineable gene drive system targeting dsx was developed (dsx[gRNA]). Here, a transgenic line which expresses Cas9 under the control of the endogenous zpg promoter was generated. Separately a transgenic line which expresses a gRNA targeting the female specific exon of dsx was inserted into that same target site. The reproductive fitness of males and females heterozygous and homozygous for this element was determined. A series of experimental crosses was performed to combine the two elements and assess the homing rate of the dsx element in a split drive system.<br><br>RESULTS: The drive was able to home in a super-Mendelian rate comparable to those obtained by an autonomous drive in this species. Although inheritance rates as high as 99.8% were observed, potentially providing very potent gene drive, dominant effects on male and female fertility were observed, which would be sufficient to hinder spread of such a drive. Molecular analysis indicated that the gRNA expressing insertion disrupted normal splicing of dsx.<br><br>CONCLUSIONS: These results should be considered when proposing the viability of dsx as a target gene for a population suppression gene drives in Anopheles stephensi. Although high homing rates were observed, the fitness defects found in both males and females carrying the transgene would likely prohibit this drive from functioning in the field.}, }
@article {pmid39920775, year = {2025}, author = {Yousefian-Jazi, A and Kim, S and Chu, J and Choi, SH and Nguyen, PTT and Park, U and Kim, MG and Hwang, H and Lee, K and Kim, Y and Hyeon, SJ and Rhim, H and Ryu, HL and Lim, G and Stein, TD and Lim, K and Ryu, H and Lee, J}, title = {Loss of MEF2C function by enhancer mutation leads to neuronal mitochondria dysfunction and motor deficits in mice.}, journal = {Molecular neurodegeneration}, volume = {20}, number = {1}, pages = {16}, pmid = {39920775}, issn = {1750-1326}, support = {R01NS109537//NIH R01/ ; 2E30954//KIST Grant/ ; HU23C0217//Korea Dementia Research Project Grant/ ; 2022R1A2C3013138//National Research Foundation/ ; R01 NS109537/NS/NINDS NIH HHS/United States ; }, mesh = {Animals ; *Mitochondria/metabolism/genetics/pathology ; Mice ; *MEF2 Transcription Factors/genetics ; Humans ; *Motor Neurons/metabolism/pathology ; *Amyotrophic Lateral Sclerosis/genetics/metabolism ; Mutation/genetics ; HEK293 Cells ; Disease Models, Animal ; Enhancer Elements, Genetic/genetics ; }, abstract = {BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the loss of both upper and lower motor neurons, leading to progressive paralysis. Both genetic alterations and epigenetic modifications contribute to neuronal dysfunction in the pathogenesis of ALS. However, the mechanism behind genetic mutations in the non-coding region of genes that affect epigenetic modifications remains unclear.<br><br>METHODS: Convolutional neural network was used to identify an ALS-associated SNP located in the intronic region of MEF2C (rs304152), residing in a putative enhancer element. To examine the alteration of MEF2C transcription by the SNP, we generated HEK293T cells carrying the major or minor allele by CRISPR-Cas9. To verify the role of MEF2C-knockdown (MEF2C-KD) in mice, we developed AAV expressing shRNA for MEF2C based on AAV-U6 promoter vector. Neuropathological alterations of MEF2C-KD mice with mitochondrial dysfunction and motor neuronal damage were observed by confocal microscopy and transmission electron microscope (TEM). Behavioral changes of mice were examined through longitudinal study by tail suspension, inverted grid test and automated gait analysis.<br><br>RESULTS: Here, we show that enhancer mutation of MEF2C reduces own gene expression and consequently impairs mitochondrial function in motor neurons. MEF2C localizes and binds to the mitochondria DNA, and directly modulates mitochondria-encoded gene expression. CRISPR/Cas-9-induced mutation of the MEF2C enhancer decreases expression of mitochondria-encoded genes. Moreover, MEF2C mutant cells show reduction of mitochondrial membrane potential, ATP level but elevation of oxidative stress. MEF2C deficiency in the upper and lower motor neurons of mice impairs mitochondria-encoded genes, and leads to mitochondrial metabolic disruption and progressive motor behavioral deficits.<br><br>CONCLUSIONS: Together, MEF2C dysregulation by the enhancer mutation leads to mitochondrial dysfunction and oxidative stress, which are prevalent features in motor neuronal damage and ALS pathogenesis. This genetic and epigenetic crosstalk mechanism provides insights for advancing our understanding of motor neuron disease and developing effective treatments.}, }
@article {pmid39920725, year = {2025}, author = {Liu, X and Liu, X and Luo, X and Zhu, M and Liu, N and Li, J and Zhang, Q and Zou, C and Wu, Y and Cao, Z and Ma, S and Wang, W and Yang, G and Gu, J and Liu, W and Li, M and Yin, A and He, Y and Lin, W}, title = {Synergistic strategies for glioblastoma treatment: CRISPR-based multigene editing combined with immune checkpoint blockade.}, journal = {Journal of nanobiotechnology}, volume = {23}, number = {1}, pages = {94}, pmid = {39920725}, issn = {1477-3155}, support = {82003220//the National Natural Science Foundation of China/ ; }, mesh = {*Glioblastoma/therapy/genetics ; *Gene Editing/methods ; Humans ; Animals ; *Immune Checkpoint Inhibitors/pharmacology/therapeutic use ; *CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; *Brain Neoplasms/therapy/genetics ; Mice ; Extracellular Vesicles/metabolism ; Genetic Therapy/methods ; Polo-Like Kinase 1 ; Programmed Cell Death 1 Receptor/genetics/metabolism ; B7-H1 Antigen/metabolism ; Proto-Oncogene Proteins/genetics/metabolism ; CD8-Positive T-Lymphocytes/immunology ; Cell Cycle Proteins/genetics/metabolism ; }, abstract = {Glioblastoma (GBM) is a primary brain tumor known for its high levels of aggressiveness and resistance to current treatments such as radiotherapy and chemotherapy. As a result, there is a pressing need for innovative therapeutic approaches to combat GBM. Thus, we have developed an engineered multifunctional extracellular vesicle (EV) delivery system that offers an "all-in-one" strategy for GBM therapy. Our approach involved the use of genetic engineering to the long-lasting production of PD-1 and the brain-specific peptide angiopep-2 on the surface of EVs. These modified EVs were then utilized to rejuvenate exhausted CD8[+] T cells blocking PD-L1, resulting in significant therapeutic benefits for GBM treatment. Furthermore, the EVs contained Cas9 protein and sgRNA for precise and minimally invasive gene therapy, which addressing the key barriers associated with in vivo CRISPR‒Cas9 gene editing treatment. The multigene editing of EVs resulted in efficient intratumor multisite gene editing (PLK1: 58.6%, VEGF: 52.7%), leading to the successful apoptosis of tumor cells in vivo and demonstrating an antiangiogenic effect. This research introduces a promising universal platform for combining immune checkpoint blockade therapy with gene editing treatment.}, }
@article {pmid39920335, year = {2025}, author = {Skoufou-Papoutsaki, N and Adler, S and Mehmed, S and Tume, C and Olpe, C and Morrissey, E and Kemp, R and Girard, AC and Moutin, EB and Chilamakuri, CSR and Miller, JL and Lindskog, C and Werle, F and Marks, K and Perrone, F and Zilbauer, M and Tourigny, DS and Winton, DJ}, title = {Haploinsufficient phenotypes promote selection of PTEN and ARID1A-deficient clones in human colon.}, journal = {EMBO reports}, volume = {26}, number = {5}, pages = {1269-1289}, pmid = {39920335}, issn = {1469-3178}, support = {/WT_/Wellcome Trust/United Kingdom ; A24456//cambridgeuniversity | Cancer Research UK Cambridge Institute, University of Cambridge (CRUK CI)/ ; 103805/WT_/Wellcome Trust/United Kingdom ; 102160/Z/13/Z//Wellcome Trust (WT)/ ; }, mesh = {Humans ; *PTEN Phosphohydrolase/genetics/deficiency ; *Haploinsufficiency ; *DNA-Binding Proteins/genetics/deficiency ; *Transcription Factors/genetics/deficiency ; Phenotype ; *Colon/metabolism/pathology ; Mutation ; Female ; Male ; Middle Aged ; Aged ; Colorectal Neoplasms/genetics/pathology ; CRISPR-Cas Systems ; }, abstract = {Cancer driver mutations are defined by their high prevalence in cancers and presumed rarity in normal tissues. However, recent studies show that positive selection in normal epithelia can increase the prevalence of some cancer drivers. To determine their true cancer-driving potential, it is essential to evaluate how frequent these mutations are in normal tissues and what are their phenotypes. Here, we explore the bioavailability of somatic variants by quantifying age-related mutational burdens in normal human colonic epithelium using immunodetection in FFPE samples (N = 181 patients). Positive selection of variants of tumour suppressor genes PTEN and ARID1A associates with monoallelic gene loss as confirmed by CRISPR/Cas9 mutagenesis and changes in their downstream effectors. Comparison of the mutational burden in normal tissue and colorectal cancers allows quantification of cancer driver potency based on relative representation. Additionally, immune exclusion, a cancer hallmark feature, is observed within ARID1A-deficient clones in histologically normal tissue. The behaviour resulting from haploinsufficiency of PTEN and ARID1A demonstrates how somatic mosaicism of tumour suppressors arises and can predispose to cancer initiation.}, }
@article {pmid39920233, year = {2025}, author = {Liu, J and Wang, Y and Wei, J and Wang, S and Li, M and Huang, Z and Zhang, S and Liu, H and Huang, J and Wang, Y}, title = {Enhanced genome editing with a Streptococcus equinus Cas9.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {196}, pmid = {39920233}, issn = {2399-3642}, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; HEK293 Cells ; *Streptococcus/enzymology/genetics ; }, abstract = {A large number of SpCas9 orthologs has been computationally identified, but their genome editing potential remains largely unknown. In this study, a GFP-activation assay was used to screen a panel of 18 SpCas9 orthologs, ten of which demonstrated activity in human cells. Notably, these orthologs had a preference for purine-rich PAM sequences. Four of the tested orthologs displayed enhanced specificity compared to SpCas9. Of particular interest is SeqCas9, which recognizes a simple NNG PAM and displays activity and specificity comparable to SpCas9-HF1. In addition, SeqCas9 exhibits superior base editing efficiency compared to SpCas9-NG and SpCas9-NRRH at multiple endogenous loci. This research sheds light on the diversity of SpCas9 orthologs and their potential for specific and efficient genome editing, especially in cases involving base editing.}, }
@article {pmid39919720, year = {2025}, author = {Hardouin, G and Miccio, A}, title = {Two is better than one: Advancing gene therapy protocols for enhanced safety and efficacy.}, journal = {Cell stem cell}, volume = {32}, number = {2}, pages = {179-180}, doi = {10.1016/j.stem.2024.12.009}, pmid = {39919720}, issn = {1875-9777}, mesh = {*Genetic Therapy/methods/adverse effects ; Humans ; CRISPR-Cas Systems/genetics ; Gene Editing ; Animals ; }, abstract = {In this issue of Cell Stem Cell, two complementary studies from Zeng et al.[1] and Demirci et al.[2] improve the efficacy of gene therapy for β-hemoglobinopathies using multiplex CRISPR-Cas9 editing. The authors also optimize editing and conditioning protocols to reduce risks associated with current gene therapy procedures.}, }
@article {pmid39919536, year = {2025}, author = {Zhang, H and Zhong, M and Zhang, J and Chen, C}, title = {Blood cancer therapy with synthetic receptors and CRISPR technology.}, journal = {Leukemia research}, volume = {150}, number = {}, pages = {107646}, doi = {10.1016/j.leukres.2025.107646}, pmid = {39919536}, issn = {1873-5835}, mesh = {Humans ; *CRISPR-Cas Systems ; *Hematologic Neoplasms/therapy/immunology/genetics ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/genetics/immunology/therapeutic use ; Gene Editing/methods ; Animals ; }, abstract = {Chimeric antigen receptor (CAR)-T and -NK cells showed great success in treating hematological malignancies, including leukemia, lymphoma, and myeloma. CRISPR technology and other synthetic receptors (GPCR and synNotch) have helped to address some of the limitations and challenges associated with CAR-based therapies. Herein, this review aims to discuss how CAR can be integrated with other synthetic receptors and various CRISPR/Cas tools for blood cancer therapy. CAR-expressing cells equipped with other synthetic receptors can conditionally execute tumoricidal functions, prevent tumor escape from immune surveillance, and minimize non-tumor off-target toxicity. We also discussed how various CRISPR-Cas tools can be harnessed to enhance CAR cells functionality and persistence. The advances, pitfalls, and future perspectives for these synthetic receptors and CRISPR technology in blood cancer therapy are comprehensively discussed.}, }
@article {pmid39919527, year = {2025}, author = {Zhou, Y and Chen, Y and Song, X and Zhong, Z and Guo, Q and Jing, S and Ayanniyi, OO and Lu, Z and Zhang, Q and Yang, C}, title = {Rapid and sensitive detection of Trichomonas gallinae using RAA-CRISPR-Cas12a.}, journal = {Veterinary parasitology}, volume = {334}, number = {}, pages = {110412}, doi = {10.1016/j.vetpar.2025.110412}, pmid = {39919527}, issn = {1873-2550}, mesh = {Animals ; *Trichomonas/isolation &amp; purification/genetics ; *Trichomonas Infections/diagnosis/veterinary/parasitology ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Poultry Diseases/diagnosis/parasitology ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Recombinases/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Bird Diseases/diagnosis/parasitology ; Columbidae ; }, abstract = {Trichomonas gallinae (T. gallinae) is an important pathogen causing trichomoniasis in birds, especially pigeons. Rapid and sensitive detection methods for T. gallinae are urgently needed to diagnose T. gallinae early to reduce poultry industry losses. Therefore, we developed a rapid and sensitive diagnostic method based on recombinase-aided amplification (RAA) assay and clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 12a (CRISPR/Cas12a) system to detect T. gallinae. The RAA-CRISPR/Cas12a method can be divided into RAA-CRISPR/Cas12a fluorescent signal (RAA-CRISPR/Cas12a-FL) and RAA-CRISPR/Cas12a lateral flow strip (RAA-CRISPR/Cas12-LFS). Both RAA-CRISPR/Cas12a-FL and RAA-CRISPR/Cas12-LFS methods show the property of rapid. sensitive, and does not require a sophisticated instrument, and they allow the detection of T. gallinae in less than 1 hr. Meanwhile, they have satisfactory specificity and can accurately detect T. gallinae in samples of different pathogens. In summary, the RAA-CRISPR/Cas12a-FL and RAA-CRISPR/Cas12-LFS methods we constructed can be used for on-site T. gallinae detection and resource-poor areas.}, }
@article {pmid39919116, year = {2025}, author = {Juan, T and Molina, T and Xie, L and Papadopoulou, S and Cardoso, B and Jha, SG and Stainier, DYR}, title = {A recombinase-activated ribozyme to knock down endogenous gene expression in zebrafish.}, journal = {PLoS genetics}, volume = {21}, number = {2}, pages = {e1011594}, pmid = {39919116}, issn = {1553-7404}, mesh = {Animals ; *Zebrafish/genetics ; *RNA, Catalytic/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Gene Knockdown Techniques/methods ; *Recombinases/genetics/metabolism ; Introns/genetics ; RNA, Messenger/genetics ; Gene Editing/methods ; Zebrafish Proteins/genetics ; Animals, Genetically Modified ; Pigmentation/genetics ; }, abstract = {Precise regulation of gene expression is essential to understand a wide range of biological processes. Control over gene expression can be achieved using site-directed recombinases and endonucleases whose efficiency is variable and dependent on the genomic context. Here, we develop a self-cleaving ribozyme-based tool to control mRNA levels of endogenous targets in zebrafish. Using an in vivo reporter strategy, we first show that inserting the T3H48 self-cleaving ribozyme in an intron enables rapid pre-mRNA cleavage, with up to 20-fold reduction in expression, and that this ribozyme displays superior activity compared with other ribozymes. We then inserted the T3H48 ribozyme in the second intron of the albino gene using a CRISPR/Cas9 strategy and observed a pigmentation phenotype similar to that in the mutant. Using a base-editing strategy to inactivate the ribozyme, we also show that this phenotype is reversible, illustrating the specificity of the approach. In addition, we generated a Flippase- and Cre-activatable version of the T3H48 ribozyme, called RiboFlip, to control the mRNA levels of the albino gene. RiboFlip activation induced mRNA knockdown and also recapitulated the albino mutant phenotype. Furthermore, we show that a Cre- and Dre-controllable Gal4/UAS reporter in the RiboFlip cassette can label knocked-down cells independently of the expression of the target gene. Altogether, we introduce the RiboFlip cassette as a flexible tool to control endogenous gene expression in a vertebrate model and as an alternative to existing conditional knockdown strategies.}, }
@article {pmid39918307, year = {2025}, author = {Dougherty, MW and Hoffmann, RM and Hernandez, MC and Airan, Y and Gharaibeh, RZ and Herzon, SB and Yang, Y and Jobin, C}, title = {Genome-scale CRISPR/Cas9 screening reveals the role of PSMD4 in colibactin-mediated cell cycle arrest.}, journal = {mSphere}, volume = {10}, number = {3}, pages = {e0069224}, pmid = {39918307}, issn = {2379-5042}, support = {R01 CA215553/CA/NCI NIH HHS/United States ; //University of Florida Health Cancer Center/ ; //University of Florida Gatorade Fund/ ; R01CA215553//HHS | National Institutes of Health (NIH)/ ; }, mesh = {Humans ; *CRISPR-Cas Systems ; *Polyketides/pharmacology/metabolism ; *Peptides/pharmacology/metabolism ; *Cell Cycle Checkpoints/drug effects/genetics ; HEK293 Cells ; HT29 Cells ; Gene Knockout Techniques ; Cell Survival/drug effects ; }, abstract = {UNLABELLED: Colibactin is a genotoxic secondary metabolite produced by certain Enterobacteriaceae strains that populate the intestine and produces a specific mutational signature in human colonocytes. However, the host pathways involved in colibactin response remain unclear. To address this gap, we performed genome-wide CRISPR/Cas9 knockout screens and RNA sequencing utilizing live pks[+] bacteria and a synthetic colibactin analog. We identified 20 enriched genes with a MAGeCK score of >2.0 in both screens, including proteasomal subunits (e.g., PSMG4 and PSMD4), RNA processing factors (e.g., SF1 and PRPF8), and RNA polymerase III (e.g., CRCP), and validated the role of PSMD4 in colibactin sensitization. PSMD4 knockout in HEK293T and HT-29 cells promoted cell viability and ameliorated G2-M cell cycle arrest but did not affect the amount of phosphorylated H2AX foci after exposure to synthetic colibactin 742. Consistent with these observations, PSMD4[-/-] cells had a significantly higher colony formation rate and bigger colony size than control cells after 742 exposure. These findings suggest that PSMD4 regulates cell cycle arrest following colibactin-induced DNA damage and that cells with PSMD4 deficiency may continue to replicate despite DNA damage, potentially increasing the risk of malignant transformation.<br><br>IMPORTANCE: Colibactin has been implicated as a causative agent of colorectal cancer. However, colibactin-producing bacteria are also present in many healthy individuals, leading to the hypothesis that some aspects of colibactin regulation or host response dictate the molecule's carcinogenic potential. Elucidating the host-response pathways involved in dictating cell fate after colibactin intoxication has been difficult, partially due to an inability to isolate the molecule. This study provides the first high-throughput CRISPR/Cas9 screening to identify genes conferring colibactin sensitivity. Here, we utilize both bacterial infection and a synthetic colibactin analog to identify genes directly involved in colibactin response. These findings provide insight into how differences in gene expression may render certain individuals more vulnerable to colibactin-initiated tumor formation after DNA damage.}, }
@article {pmid39916610, year = {2025}, author = {Uusi-Mäkelä, M and Harjula, SE and Junno, M and Sillanpää, A and Nätkin, R and Niskanen, MT and Saralahti, AK and Nykter, M and Rämet, M}, title = {The inflammasome adaptor pycard is essential for immunity against Mycobacterium marinum infection in adult zebrafish.}, journal = {Disease models &amp; mechanisms}, volume = {18}, number = {9}, pages = {}, pmid = {39916610}, issn = {1754-8411}, support = {//Tampereen Tuberkuloosisaatio/ ; //Maud Kuistilan Muistosaatio/ ; //Tampere University Doctoral School/ ; //Suomen Kulttuurirahasto/ ; //Finnish Society for Study of Infectious Diseases/ ; 277495//The Academy of Finland/ ; //The Competitive State Research Financing of the Expert Responsibility Area of Oulu University Hospital/ ; //Tampereen Tuberkuloosis&#xe4;&#xe4;ti&#xf6;/ ; //Maud Kuistilan Muistos&#xe4;&#xe4;ti&#xf6;/ ; //V&#xe4;in&#xf6; ja Laina Kiven S&#xe4;&#xe4;ti&#xf6;/ ; //Orion Research Foundation/ ; //National Doctoral Education Pilot Based on the Immune System/ ; //Instrumentariumin Tiedes&#xe4;&#xe4;ti&#xf6;/ ; //Sigrid Jus&#xe9;liuksen S&#xe4;&#xe4;ti&#xf6;/ ; 277495//Research Council of Finland/ ; //Competitive State Research Financing of the Expert Responsibility Area of Oulun Yliopistollinen Sairaala/ ; //Tampereen Yliopisto/ ; }, mesh = {Animals ; *Zebrafish/microbiology/immunology/genetics ; *Mycobacterium marinum/physiology/immunology ; *Mycobacterium Infections, Nontuberculous/immunology/microbiology/genetics ; *Zebrafish Proteins/metabolism/genetics ; *Inflammasomes/metabolism ; Neutrophils/metabolism/immunology ; *CARD Signaling Adaptor Proteins/metabolism ; Granuloma/pathology ; *Immunity ; CRISPR-Cas Systems/genetics ; Kidney/pathology ; Gene Knockout Techniques ; Larva/microbiology ; Gene Expression Profiling ; Transcriptome/genetics ; }, abstract = {Inflammasomes regulate the host response to intracellular pathogens including mycobacteria. We have previously shown that the course of Mycobacterium marinum infection in adult zebrafish (Danio rerio) mimics the course of tuberculosis in human. To investigate the role of the inflammasome adaptor pycard in zebrafish M. marinum infection, we produced two zebrafish knockout mutant lines for the pycard gene with CRISPR/Cas9 mutagenesis. Although the zebrafish larvae lacking pycard developed normally and had unaltered resistance against M. marinum, the loss of pycard led to impaired survival and increased bacterial burden in the adult zebrafish. Based on histology, immune cell aggregates, granulomas, were larger in pycard-deficient fish than in wild-type controls. Transcriptome analysis with RNA sequencing of a zebrafish haematopoietic tissue, kidney, suggested a role for pycard in neutrophil-mediated defence, haematopoiesis and myelopoiesis during infection. Transcriptome analysis of fluorescently labelled, pycard-deficient kidney neutrophils identified genes that are associated with compromised resistance, supporting the importance of pycard for neutrophil-mediated immunity against M. marinum. Our results indicate that pycard is essential for resistance against mycobacteria in adult zebrafish.}, }
@article {pmid39916603, year = {2025}, author = {Zhou, S and Sun, H and Deng, L and Zhu, S and Chen, J and Huo, D and Hou, C}, title = {A universal two-step strategy for multiple DNA MTase activity: enhancing sensitivity through CRISPR/Cas12a-assisted hyperbranched rolling circle amplification (CA-HRCA).}, journal = {Analytical methods : advancing methods and applications}, volume = {17}, number = {8}, pages = {1870-1876}, doi = {10.1039/d4ay02143f}, pmid = {39916603}, issn = {1759-9679}, mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Humans ; DNA Methylation ; DNA ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; DNA-Cytosine Methylases ; CRISPR-Associated Proteins ; }, abstract = {DNA methyltransferase (DNA MTase) is a valuable target of genetic diseases, and detection of related DNA MTase activity is very important for drug screening, clinical diagnosis and disease treatment. Herein, a universal two-step strategy based on CRISPR/Cas12a-assisted hyperbranched rolling circle amplification (CA-HRCA) for DNA MTase activity detection is constructed, which successfully achieves the detection of Dam MTase and M.SssI MTase. In the presence of DNA MTase and restriction enzymes, the HRCA primer locked in the dumbbell probe will be released and further initiates HRCA. In the first step, DNA methylation, restriction enzyme digestion and HRCA amplification are performed simultaneously, effectively simplifying the reaction process and shortening the detection time. In the second step, the abundant HRCA products (dsDNA) act as activators to induce CRISPR/Cas12a to split fluorescent probes. Compared with ssDNA activators, dsDNA activators can cause higher collateral cleavage of CRISPR/Cas12a. As expected, this strategy presents excellent sensing performance with a detection time of 155 min. The LODs of Dam MTase and M.SssI MTase are calculated to be 7.6 × 10[-4] U mL[-1] and 1.8 × 10[-4] U mL[-1], respectively. And the proposed assay possesses extraordinary specificity and reproducibility. Moreover, the practical application ability and drug development potential are proved by the serum spiked test and inhibitor evaluation tests.}, }
@article {pmid39915901, year = {2025}, author = {Fang, Y and Meng, X and Liu, L and Li, Z and Jia, K and Liu, W}, title = {Simultaneous In Vivo Assembly and Targeted Genome Integration of Gene Clusters in Trichoderma reesei.}, journal = {ACS synthetic biology}, volume = {14}, number = {2}, pages = {575-584}, doi = {10.1021/acssynbio.4c00810}, pmid = {39915901}, issn = {2161-5063}, mesh = {CRISPR-Cas Systems/genetics ; *Hypocreales/genetics ; *Multigene Family/genetics ; *Genome, Fungal/genetics ; Fungal Proteins/genetics/metabolism ; Genetic Engineering/methods ; Homologous Recombination/genetics ; Gene Editing/methods ; Cellulase/genetics ; *Trichoderma/genetics ; }, abstract = {The saprophytic filamentous fungus Trichoderma reesei represents one of the most prolific cellulase producers and also has the potential to be developed into a tractable fungal host for biosynthesizing secondary metabolite products. To expedite the genetic engineering of filamentous fungi, efficient DNA assembly processes that can facilitate the transfer of large-sized DNA to fungal hosts, including T. reesei, are still in demand. Here, we developed a method for the simultaneous in vivo assembly and targeted genome integration of multiple DNA fragments (SATIMD) in T. reesei. While efficient orderly DNA end fusions were achieved by homologous recombination (HR) with various lengths of sequence overlaps (100-500 bp), the assembled DNA was also precisely integrated into a specific locus when combined with CRISPR/Cas9-mediated genome cutting. Specifically, we have used this method to achieve the assembly and functional expression of T. reesei key transcriptional activator Xyr1 for cellulase genes. Moreover, fusions and targeted integration of up to 10 different DNA fragments comprising the 32.7 kb sorbicillinoids biosynthetic gene cluster via a single-step transformation was demonstrated. We envision that SATIMD is a powerful tool not only useful for direct large heterologous gene cluster assembly in T. reesei but also can facilitate large-scale fungal strain genetic engineering.}, }
@article {pmid39914548, year = {2025}, author = {Liu, Y and Wang, J and Cui, G and Wang, X and Xiang, S and Huang, W and Liu, C}, title = {RNA aptamer-based CRISPR-Cas12a system for enhanced small molecule detection and point-of-care testing.}, journal = {International journal of biological macromolecules}, volume = {303}, number = {}, pages = {140675}, doi = {10.1016/j.ijbiomac.2025.140675}, pmid = {39914548}, issn = {1879-0003}, mesh = {*Aptamers, Nucleotide/genetics/chemistry ; *CRISPR-Cas Systems/genetics ; Humans ; Theophylline/analysis/blood ; *Point-of-Care Testing ; *Biosensing Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {The CRISPR-Cas12a system has emerged as a robust platform for small molecule detection. However, existing methodologies primarily emphasize DNA aptamer-based strategies. This study introduces an RNA aptamer-based CRISPR-Cas12a approach due to the fact that the majority of small molecules lack corresponding DNA aptamers. The approach employs theophylline RNA aptamer (TA) to regulate Cas12a activity through competitive inhibition of crRNA. The results demonstrate that this system effectively detects theophylline (TP) in various food, beverage, and human serum samples, exhibiting excellent selectivity and sensitivity. Additionally, a visual paper-based detection system showcases its applicability for real-time analysis in food matrices and human serum. The RNA aptamer-based CRISPR-Cas12a strategy holds significant potential for diverse biomedical applications, offering a versatile tool for future sensing applications through customized RNA aptamer designs for small molecules.}, }
@article {pmid39914388, year = {2025}, author = {Liu, S and Hamilton, MC and Cowart, T and Barrera, A and Bounds, LR and Nelson, AC and Dornbaum, SF and Riley, JW and Doty, RW and Allen, AS and Crawford, GE and Majoros, WH and Gersbach, CA}, title = {Characterization and bioinformatic filtering of ambient gRNAs in single-cell CRISPR screens using CLEANSER.}, journal = {Cell genomics}, volume = {5}, number = {2}, pages = {100766}, pmid = {39914388}, issn = {2666-979X}, support = {R01 MH125236/MH/NIMH NIH HHS/United States ; U01 HG011967/HG/NHGRI NIH HHS/United States ; UM1 HG012053/HG/NHGRI NIH HHS/United States ; R35 GM150404/GM/NIGMS NIH HHS/United States ; RM1 HG011123/HG/NHGRI NIH HHS/United States ; }, mesh = {*Single-Cell Analysis/methods ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Computational Biology/methods ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Sequence Analysis, RNA/methods ; }, abstract = {Single-cell RNA sequencing CRISPR (perturb-seq) screens enable high-throughput investigation of the genome, allowing for characterization of thousands of genomic perturbations on gene expression. Ambient gRNAs, which are contaminating gRNAs, are a major source of noise in perturb-seq experiments because they result in an excess of false-positive gRNA assignments. Here, we utilize CRISPR barnyard assays to characterize ambient gRNAs in perturb-seq screens. We use these datasets to develop CRISPR Library Evaluation and Ambient Noise Suppression for Enhanced single-cell RNA-seq (CLEANSER), a mixture model that filters ambient gRNAs. CLEANSER includes both gRNA and cell-specific normalization parameters, correcting for confounding technical factors that affect individual gRNAs and cells. The output of CLEANSER is the probability that a gRNA-cell assignment is in the native distribution over the ambient distribution. We find that ambient gRNA filtering methods impact differential gene expression analysis outcomes and that CLEANSER outperforms alternate approaches by increasing gRNA-cell assignment accuracy across multiple screen formats.}, }
@article {pmid39913998, year = {2025}, author = {Tang, Y and Aleithan, F and Madahar, SS and Mirzaesmaeili, A and Saran, S and Tang, J and Zangiabadi, S and Inman, R and Sweeney, G and Abdul-Sater, AA}, title = {Selective disruption of Traf1/cIAP2 interaction attenuates inflammatory responses and rheumatoid arthritis.}, journal = {Journal of autoimmunity}, volume = {152}, number = {}, pages = {103377}, doi = {10.1016/j.jaut.2025.103377}, pmid = {39913998}, issn = {1095-9157}, mesh = {Animals ; Humans ; *Arthritis, Rheumatoid/immunology/metabolism/genetics/pathology ; *TNF Receptor-Associated Factor 1/genetics/metabolism ; Mice ; *Baculoviral IAP Repeat-Containing 3 Protein/metabolism/genetics ; Macrophages/immunology/metabolism ; *Inflammation/immunology ; Signal Transduction ; Disease Models, Animal ; NF-kappa B/metabolism ; Protein Binding ; CRISPR-Cas Systems ; Arthritis, Experimental/immunology ; Mutation ; Cytokines/metabolism ; Ubiquitin-Protein Ligases ; Inhibitor of Apoptosis Proteins ; }, abstract = {OBJECTIVES: Tumor necrosis factor receptor-associated factor 1 (TRAF1) is an immune signaling adapter protein linked to increased susceptibility to rheumatoid arthritis (RA). TRAF1 has dual roles in regulating NF-κB and MAPK signaling: it promotes signaling through its association with cellular inhibitor of apoptosis 2 (cIAP2) downstream of certain tumor necrosis factor receptor (TNFR) family members but inhibits Toll-like receptor (TLR) signaling by limiting linear ubiquitination of key signaling proteins. In this study, we investigated whether selectively targeting TRAF1/cIAP2 interaction would lower inflammation and reduce severity of RA.<br><br>METHODS: We employed CRISPR/Cas9-mediated mediated gene editing to modify TRAF1 and specifically abrogate its interaction with cIAP2 in human macrophage cell lines and in mice. Biochemical studies were then employed to assess inflammatory signaling and cytokine production in gene edited macrophages. The collagen antibody-induced arthritis (CAIA) model of RA was used to trigger joint inflammation in mice.<br><br>RESULTS: We identify a critical mutation in TRAF1 (V203A in humans, V196A in mice) that disrupts its interaction with cIAP2, leading to a significant reduction in TLR signaling and downstream inflammation in human and murine macrophages. We demonstrate that TRAF1 is recruited to the TLR4 complex and is indispensable for the recruitment of cIAP2, facilitating TAK1 phosphorylation and the activation of NF-&#x3ba;B and MAPK signaling pathways. Remarkably, mice harboring the TRAF1 V196A mutation are protected from LPS-induced septic shock and exhibit markedly reduced joint inflammation and disease severity in the CAIA model of RA.<br><br>CONCLUSION: These findings reveal a previously unrecognized and crucial role for the TRAF1/cIAP2 axis in promoting inflammation and offer a promising foundation for the development of novel therapeutic strategies for inflammatory conditions, such as RA.}, }
@article {pmid39913574, year = {2025}, author = {Couzin-Frankel, J}, title = {A new 'mini-CRISPR' flexes its editing power in monkey muscles.}, journal = {Science (New York, N.Y.)}, volume = {387}, number = {6734}, pages = {570}, doi = {10.1126/science.adw4916}, pmid = {39913574}, issn = {1095-9203}, mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Haplorhini ; *Muscle, Skeletal/metabolism ; }, abstract = {The downsized DNA-slicing machinery may reach more tissues to take aim at more diseases.}, }
@article {pmid39912765, year = {2025}, author = {Li, Y and Hu, Q and Bai, M and Qing, M and Bai, L}, title = {CrRNA Conformation-Engineered CRISPR-Cas12a System for Robust and Ultrasensitive Nucleic Acid Detection.}, journal = {Analytical chemistry}, volume = {97}, number = {6}, pages = {3617-3624}, doi = {10.1021/acs.analchem.4c06107}, pmid = {39912765}, issn = {1520-6882}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Human papillomavirus 16/genetics/isolation &amp; purification ; Influenza A virus/genetics/isolation &amp; purification ; *CRISPR-Associated Proteins/genetics/metabolism ; Limit of Detection ; *Endodeoxyribonucleases/genetics/metabolism ; Nucleic Acid Conformation ; *RNA/genetics/chemistry ; Bacterial Proteins ; }, abstract = {Despite the widespread application of the CRISPR-Cas12a system in vitro diagnostics due to its high programmability and distinctive trans-cleavage activity, the susceptibility of its crRNA component to degradation and sensitivity to storage and working conditions poses a significant challenge to improving the practical efficacy of these diagnostic systems. Here, we show that engineered crRNA with a covalently closed circular structure (C-crRNA) can replace traditional linear crRNA to form functional complexes with Cas12a protein, significantly enhancing the anti-interference ability of the CRISPR-Cas12a system while maintaining its sensitivity and specificity. Based on this finding, a circular crRNA-mediated CRISPR molecular diagnostic (CRCD) toolkit is developed and successfully integrated with a standard nucleic acid amplification technique to detect synthesized Human Papillomavirus type 16 (HPV-16) plasmids down to 10 aM sensitivity levels. Furthermore, the CRCD system is applied for ultrasensitive detection of 40 HPV-16 and 40 influenza A viruses in clinical samples, with results consistent with those from PANTHER detection and quantitative real-time polymerase chain reaction (qRT-PCR). In conclusion, this strategy introduces a novel paradigm for engineering crRNA to program Cas12a, which has the potential to revolutionize the use of crRNA in CRISPR-based molecular diagnostics.}, }
@article {pmid39912490, year = {2025}, author = {Wu, R and Li, H and Wu, P and Yang, Q and Wan, X and Wu, Y}, title = {LRRC56 deletion causes primary ciliary dyskinesia in mice characterized by dynein arms defects.}, journal = {Biology open}, volume = {14}, number = {2}, pages = {}, pmid = {39912490}, issn = {2046-6390}, support = {82002238//National Natural Science Foundation/ ; //Central South University Third Xiangya Hospital/ ; }, mesh = {Animals ; Mice ; *Dyneins/metabolism ; Mice, Knockout ; Cilia/metabolism ; Disease Models, Animal ; Male ; *Kartagener Syndrome/genetics/metabolism ; Phenotype ; *Gene Deletion ; *Ciliary Motility Disorders/genetics/metabolism ; *Membrane Proteins/genetics ; CRISPR-Cas Systems ; }, abstract = {Leucine Rich Repeat Containing protein 56 (LRRC56), also known as DNAAF12, is a member of the LRRC superfamily, whose dysfunction is associated with mucociliary clearance and laterality defects in humans. Here, we generated LRRC56-knockout mice using the CRISPR/Cas9 nuclease system to specifically target exons 4-5 of the LRRC56 gene. We observed that homozygous LRRC56 gene deletion is definitely deleterious, as 27.8% of LRRC56-/- mice died before adulthood. Among the surviving LRRC56-/- mice, the most prominent phenotypes included hydrocephalus, situs inversus, male infertility, and bronchiectasis. Transmission electron microscopy revealed defects in dynein arms of cilia and disorganized axonemal structure in flagella. Immunofluorescence analysis similarly revealed the absence of inner and outer dynein arm markers DNALI1 and DNAI2 in the cilia. Heterozygous LRRC56+/- mice developed normally, without exhibiting any symptoms of primary ciliary dyskinesia. In conclusion, the knockout of the LRRC56 gene in mice leads to a range of conditions consistent with primary ciliary dyskinesia. The absence of DNALI1 and DNAI2 signaling in knockout mouse cilia supports the critical role of the LRRC56 gene in dynein arm assembly.}, }
@article {pmid39912395, year = {2025}, author = {Cui, T and Wang, X and Zang, R and Zhao, L and Yan, H and Li, X and Xu, Z and Wang, H and Zhou, J and Liu, Y and Yue, W and Pei, X and Xi, J}, title = {3' UTR-truncated HMGA2 promotes erythroblasts production from human embryonic stem cells.}, journal = {Stem cells translational medicine}, volume = {14}, number = {1}, pages = {}, pmid = {39912395}, issn = {2157-6580}, support = {82200690//National Natural Science Foundation of China/ ; }, mesh = {Humans ; *Erythroblasts/metabolism/cytology ; *HMGA2 Protein/genetics/metabolism ; *Human Embryonic Stem Cells/metabolism/cytology ; Cell Differentiation ; *3' Untranslated Regions/genetics ; CRISPR-Cas Systems ; Gene Editing ; Cell Line ; }, abstract = {Cultured red blood cells represent an alternative resource for blood transfusions. However, important issues such as low yields and high costs remain. Recently, gene editing of hematopoietic stem cells has been conducted to induce erythroid differentiation in vitro for producing sufficient RBCs to meet the imbalance in blood supply and demand. The differentiation and expansion of hematopoietic stem and progenitor cells are regulated by transcription factors, such as high mobility group AT-hook 2 (HMGA2). In this study, we utilized CRISPR/Cas9 to establish a doxycycline-inducible HMGA2-expressing human embryonic stem cell (hESC) line. In a defined erythroid differentiation system, HMGA2 prolonged erythroid differentiation in vitro, enabling extensive expansion of human erythroblasts. The erythroblasts derived from the HMGA2-expressing hESC line are rich in polychromatic and orthochromatic erythroblasts expressing mostly α- and γ-globin and have the capacity to differentiate into RBCs. Our findings highlight the potential of combining hematopoietic transcription factors with genome editing techniques to enhance RBC production.}, }
@article {pmid39912332, year = {2025}, author = {Bellchambers, HM and Padua, MB and Ware, SM}, title = {A CRISPR mis-insertion in the Zic3 5'UTR inhibits in vivo translation and is predicted to result in formation of an mRNA stem-loop hairpin.}, journal = {Biology open}, volume = {14}, number = {3}, pages = {}, pmid = {39912332}, issn = {2046-6390}, support = {P01 HL134599/HL/NHLBI NIH HHS/United States ; P01 HL 134599/NH/NIH HHS/United States ; //Indiana University/ ; //Indiana University School of Medicine/ ; P01 HL 134599/HL/NHLBI NIH HHS/United States ; }, mesh = {Animals ; *5' Untranslated Regions ; Mice ; *Transcription Factors/genetics/metabolism ; *RNA, Messenger/genetics/chemistry ; *Homeodomain Proteins/genetics ; *Protein Biosynthesis ; Nucleic Acid Conformation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Mutagenesis, Insertional ; Humans ; Phenotype ; Gene Editing ; *CRISPR-Cas Systems ; }, abstract = {Zic3 loss of function is associated with a range of congenital defects, including heterotaxy and isolated heart defects in humans, as well as neural tube defects, situs anomalies, and tail kinks in model organisms. Here, we describe a novel Zic3ins5V mouse line generated due to a mis-insertion during the CRISPR genome editing process, which altered the Zic3 5'UTR structure. Mice with this insertion developed similar phenotypes to Zic3LacZ null mice, including heterotaxy, isolated heart defects, neural tube defects and tail kinks. Surprisingly, gene expression analysis revealed that the novel Zic3ins5V line displays higher levels of Zic3 mRNA, but western blot analysis confirmed that levels of ZIC3 were greatly reduced in vivo. RNAfold, an RNA secondary structure prediction tool, showed that this mis-insertion may cause the formation of a large stem-loop hairpin incorporating some of the 5'UTR and first exon of Zic3, and the insertion of similar hairpins in a cell-based assay caused the loss of ZIC3 expression. Thus, this mouse line displays a loss of ZIC3 protein consistent with the inhibitory effects of 5'UTR stem-loop hairpin structures.}, }
@article {pmid39910976, year = {2025}, author = {Gou, L and Liu, D and Fan, TP and Deng, H and Cai, Y}, title = {Construction of a CRISPR-Cas9-Based Genetic Editing Tool for Serratia marcescens Using a Stationary Phase Promoter and Its Application in Putrescine Production.}, journal = {Biotechnology and bioengineering}, volume = {122}, number = {5}, pages = {1233-1244}, doi = {10.1002/bit.28949}, pmid = {39910976}, issn = {1097-0290}, support = {//The authors received no specific funding for this work./ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Serratia marcescens/genetics/metabolism ; *Promoter Regions, Genetic/genetics ; *Putrescine/biosynthesis/metabolism ; }, abstract = {Putrescine plays a significant role in green food production and agriculture by promoting plant growth and enhancing crop quality. Its application reduces the reliance on chemical fertilizers and pesticides, thereby supporting the advancement of sustainable agricultural practices. This study achieved efficient production of putrescine in Serratia marcescens. S. marcescens has been extensively used to synthesize antimicrobial substances and express proteins, but its application has been limited by the lack of efficient genome-editing tools. This study presents a CRISPR-Cas9-based tool for gene editing in S. marcescens. A dual-plasmid system was constructed, incorporating an editing template into the plasmid pEdit with target-specific sgRNA. A stationary phase promoter was used to express Cas9 from Streptococcus pyogenes protein, avoiding the need for additional inducers and ensuring efficient one-step gene knockout and integration. The tool demonstrated over 80% editing efficiency across various S. marcescens strains and enabled successful single-base mutations. Using this tool, we enhanced putrescine production in S. marcescens HBQA7, optimizing the expression of ornithine decarboxylase from Clostridium aceticum DSM1496 with the P2 promoter and identifying the optimal integration site. Putrescine production reached 8.46 g/L within 48 h. This study significantly advances S. marcescens gene editing and metabolic engineering.}, }
@article {pmid39910784, year = {2025}, author = {Pascual-Garrigos, A and Lozano-Torres, B and Das, A and Molloy, JC}, title = {Colorimetric CRISPR Biosensor: A Case Study with Salmonella Typhi.}, journal = {ACS sensors}, volume = {10}, number = {2}, pages = {717-724}, pmid = {39910784}, issn = {2379-3694}, mesh = {*Colorimetry/methods ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Salmonella typhi/genetics/isolation &amp; purification ; beta-Galactosidase/metabolism ; }, abstract = {There is a critical need to implement a sensitive and specific point-of-care (POC) biosensor that addresses the instrument limitations and manufacturing challenges faced in resource-constrained contexts. In this paper we focus on enteric fever which is a highly contagious and prevalent infection in low- and middle-income countries. Although easily treatable, its ambiguous symptoms paired with a lack of fast, accurate and affordable diagnostics lead to incorrect treatments which exacerbate the disease burden, including increasing antibiotic resistance. In this study, we develop a readout module for CRISPR-Cas12a that produces a colorimetric output that is visible to the naked eye and can act as a cascade signal amplifier in any CRISPR assay based on trans-cleavage. We achieve this by immobilizing an oligo covalently linked to a β-galactosidase (LacZ) enzyme, which is cleaved in the presence of DNA target-activated CRISPR-Cas12a. Upon cleavage, the colorimetric enzyme is released, and the supernatant transferred to an environment containing X-Gal producing an intense blue color. This method is capable of detecting amplified bacterial genomic DNA and has a lower limit of detection (LoD) to standard fluorescent assays while removing the requirement for costly equipment. Furthermore, it remained active 4 weeks after lyophilization, allowing for the possibility of shipment without cold chain, significantly reducing deployment costs.}, }
@article {pmid39910413, year = {2025}, author = {Wen, M and Zhou, M and Huang, Z and Wang, Y and Wang, M and Ding, Y and Huang, X and Wang, B and Wen, J and Chen, T and Zhang, P and Chen, M and Yang, C and Zhang, XB and Ke, G}, title = {Harnessing crRNA Transformer for Facile and Specific Nucleic Acid Detection.}, journal = {Analytical chemistry}, volume = {97}, number = {7}, pages = {3964-3971}, doi = {10.1021/acs.analchem.4c05399}, pmid = {39910413}, issn = {1520-6882}, mesh = {*CRISPR-Cas Systems ; Humans ; *Prostatic Neoplasms/diagnosis/genetics ; *RNA/analysis ; *DNA/analysis ; Male ; }, abstract = {CRISPR/Cas systems have emerged as promising tools for nucleic acid detection. However, their practical applications have been limited by poor specificity and the need for additional preprocessing steps. Inspired by the concept of transformers, we found that changing the forms of crRNA with spatial arrangement may endow CRISPR/Cas with an enhanced performance for nucleic acid detection. Specifically, we rationally designed two crRNA transformers─swap crRNA and split crRNA─and found that they direct the CRISPR/Cas system for cis- and trans- cleavage with enhanced specificity and decreased Cas binding affinity and possess both DNA and RNA detection abilities. Based on these findings, our strategy enabled the identification of clinical prostatic cancer in a one-step reaction, with a remarkable sensitivity of 90.0% and specificity of 96.0%. Our study deepens the understanding of CRISPR/Cas systems and introduces a promising strategy for simple nucleic acid detection with enhanced specificity, sensitivity, and functionality in clinical molecular diagnosis.}, }
@article {pmid39910196, year = {2025}, author = {Fernandes, LGV and Nascimento, ALTO and Nally, JE}, title = {Induced protein expression in Leptospira spp. and its application to CRISPR/Cas9 mutant generation.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {4334}, pmid = {39910196}, issn = {2045-2322}, mesh = {*CRISPR-Cas Systems/genetics ; *Leptospira/genetics/metabolism ; *Mutation ; Animals ; Leptospirosis/microbiology ; Gene Knockout Techniques ; *Bacterial Proteins/genetics ; Gene Editing/methods ; }, abstract = {Expanding the genetic toolkit for Leptospira spp. is a crucial step toward advancing our understanding of the biology and virulence of these atypical bacteria. Pathogenic Leptospira are responsible for over 1 million human leptospirosis cases annually and significantly impact domestic animals. Bovine leptospirosis causes substantial financial losses due to abortion, stillbirths, and suboptimal reproductive performance. The advent of the CRISPR/Cas9 system has marked a turning point in genetic manipulation, with applications across multiple Leptospira species. However, incorporating controlled protein expression into existing genetic tools could further expand their utility. We developed and demonstrated the functionality of IPTG-inducible heterologous protein expression in Leptospira spp. This system was applied for regulated expression of dead Cas9 (dCas9) to generate knockdown mutants, and Cas9 to produce knockout mutants by inducing double-strand breaks (DSB) into desired targets. IPTG-induced dCas9 expression enabled validation of essential genes and non-coding RNAs. Additionally, IPTG-controlled Cas9 expression combined with a constitutive non-homologous end-joining (NHEJ) system allowed for successful recovery of knockout mutants, even in the absence of IPTG. These newly controlled protein expression systems will advance studies on the basic biology and virulence of Leptospira, as well as facilitate knockout mutant generation for improved veterinary vaccines.}, }
@article {pmid39909615, year = {2025}, author = {Chignon, A and Lettre, G}, title = {Using omics data and genome editing methods to decipher GWAS loci associated with coronary artery disease.}, journal = {Atherosclerosis}, volume = {401}, number = {}, pages = {118621}, doi = {10.1016/j.atherosclerosis.2024.118621}, pmid = {39909615}, issn = {1879-1484}, mesh = {Humans ; *Genome-Wide Association Study ; *Coronary Artery Disease/genetics ; Genetic Predisposition to Disease ; *Gene Editing/methods ; Mendelian Randomization Analysis ; Quantitative Trait Loci ; Phenotype ; *Genomics/methods ; Animals ; CRISPR-Cas Systems ; Polymorphism, Single Nucleotide ; }, abstract = {Coronary artery disease (CAD) is due to atherosclerosis, a pathophysiological process that involves several cell-types and results in the accumulation of lipid-rich plaque that disrupt the normal blood flow through the coronary arteries to the heart. Genome-wide association studies have identified 1000s of genetic variants robustly associated with CAD or its traditional risk factors (e.g. blood pressure, blood lipids, type 2 diabetes, smoking). However, gaining biological insights from these genetic discoveries remain challenging because of linkage disequilibrium and the difficulty to interpret the functions of non-coding regulatory elements in the human genome. In this review, we present different statistical methods (e.g. Mendelian randomization) and molecular datasets (e.g. expression or protein quantitative trait loci) that have helped connect CAD-associated variants with genes, biological pathways, and cell-types or tissues. We emphasize that these various strategies make predictions, which need to be validated in orthologous systems. We discuss specific examples where the integration of omics data with GWAS results has prioritized causal CAD variants and genes. Finally, we review how targeted and genome-wide genome editing experiments using the CRISPR/Cas9 toolbox have been used to characterize new CAD genes in human cells. Researchers now have the statistical and bioinformatic methods, the molecular datasets, and the experimental tools to dissect comprehensively the loci that contribute to CAD risk in humans.}, }
@article {pmid39908088, year = {2025}, author = {Din, NS and Mohd Rani, F and Alattraqchi, AG and Ismail, S and A Rahman, NI and Cleary, DW and Clarke, SC and Yeo, CC}, title = {Whole-genome sequencing of Acinetobacter baumannii clinical isolates from a tertiary hospital in Terengganu, Malaysia (2011-2020), revealed the predominance of the Global Clone 2 lineage.}, journal = {Microbial genomics}, volume = {11}, number = {2}, pages = {}, pmid = {39908088}, issn = {2057-5858}, mesh = {*Acinetobacter baumannii/genetics/isolation &amp; purification/drug effects/classification ; Malaysia/epidemiology ; *Whole Genome Sequencing/methods ; Humans ; Tertiary Care Centers ; *Acinetobacter Infections/microbiology/epidemiology ; Drug Resistance, Multiple, Bacterial/genetics ; Multilocus Sequence Typing ; Microbial Sensitivity Tests ; Anti-Bacterial Agents/pharmacology ; Genome, Bacterial ; beta-Lactamases/genetics ; Carbapenems/pharmacology ; }, abstract = {Carbapenem-resistant Acinetobacter baumannii is recognized by the World Health Organization (WHO) as one of the top priority pathogens. Despite its public health importance, genomic data of clinical isolates from Malaysia remain scarce. In this study, whole-genome sequencing was performed on 126 A. baumannii isolates collected from the main tertiary hospital in the state of Terengganu, Malaysia, over a 10-year period (2011-2020). Antimicrobial susceptibilities determined for 20 antibiotics belonging to 8 classes showed that 77.0% (n=97/126) of the isolates were categorized as multidrug resistant (MDR), with all MDR isolates being carbapenem resistant. Multilocus sequence typing analysis categorized the Terengganu A. baumannii clinical isolates into 34 Pasteur and 44 Oxford sequence types (STs), with ST2Pasteur of the Global Clone 2 lineage identified as the dominant ST (n=76/126; 60.3%). The ST2Pasteur isolates could be subdivided into six Oxford STs with the majority being ST195Oxford (n=35) and ST208Oxford (n=17). Various antimicrobial resistance genes were identified with the bla OXA-23-encoded carbapenemase being the predominant acquired carbapenemase gene (n=90/126; 71.4%). Plasmid-encoded rep genes were identified in nearly all (n=122/126; 96.8%) of the isolates with the majority being Rep_3 family (n=121). Various virulence factors were identified, highlighting the pathogenic nature of this bacterium. Only 14/126 (11.1%) of the isolates were positive for the carriage of CRISPR-Cas arrays with none of the prevalent ST2Pasteur isolates harbouring them. This study provided a genomic snapshot of the A. baumannii isolates obtained from a single tertiary healthcare centre in Malaysia over a 10-year period and showed the predominance of a single closely related ST2Pasteur lineage, indicating the entrenchment of this clone in the hospital.}, }
@article {pmid39907946, year = {2025}, author = {Rachappanavar, V}, title = {Utilizing CRISPR-based genetic modification for precise control of seed dormancy: progress, obstacles, and potential directions.}, journal = {Molecular biology reports}, volume = {52}, number = {1}, pages = {204}, pmid = {39907946}, issn = {1573-4978}, mesh = {*Plant Dormancy/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Seeds/genetics/growth &amp; development ; Crops, Agricultural/genetics ; Gene Expression Regulation, Plant/genetics ; Plants, Genetically Modified/genetics ; Germination/genetics ; }, abstract = {Seed dormancy, a complex trait that is influenced by both nuclear and cytoplasmic factors, poses a significant challenge to agricultural productivity. Conventional dormancy-breaking techniques, including mechanical, physiological, and chemical methods, often yield inconsistent results, impair seed quality, and lack precision. This has necessitated exploration of more targeted and efficient approaches. CRISPR-based gene editing has emerged as a promising tool for the precise regulation of seed dormancy without compromising seed viability or sustainability. Although CRISPR has been successfully applied to modify genes that govern physiological traits in various crops, its use in dormancy regulation remains in the early stages. This review examines recent advancements in CRISPR-based approaches for modulating seed dormancy and discusses key gene targets, modification techniques, and the resulting effects. We also consider the future potential of CRISPR to enhance dormancy control across diverse crop species.}, }
@article {pmid39907302, year = {2025}, author = {Talat, A and Khan, AU}, title = {From supplements to superbugs: how probiotic patent gaps drive antimicrobial resistance and the CRISPR-Cas solutions.}, journal = {Pharmaceutical patent analyst}, volume = {}, number = {}, pages = {1-3}, doi = {10.1080/20468954.2025.2459592}, pmid = {39907302}, issn = {2046-8962}, }
@article {pmid39906987, year = {2025}, author = {Hou, Z and Ding, Z and Zhang, Y and Pang, B and Gui, L and Liu, Y and Lu, L and Roengjitd, P and Wang, H}, title = {Establishment and Application of a Rapid Detection Method of Cyprinid Herpesvirus 2 (CyHV-2) in Aquacultural Waters by Using a Novel One-Pot RAA-CRISPR/Cas12a Combined With Fe-Iron Flocculation Technology.}, journal = {Journal of fish diseases}, volume = {48}, number = {5}, pages = {e14089}, doi = {10.1111/jfd.14089}, pmid = {39906987}, issn = {1365-2761}, support = {BE2021369//Key Research and Development Plan of Jiangsu Province/ ; CARS-45-16//Earmarked Fund for China Agriculture Research System/ ; 2022-02-08-00-12-F01112//Shanghai Agriculture Applied Technology Development Program, China/ ; }, mesh = {Animals ; *Herpesviridae/isolation &amp; purification/genetics ; *Fish Diseases/virology/diagnosis ; *Herpesviridae Infections/veterinary/diagnosis/virology ; *CRISPR-Cas Systems ; Aquaculture ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *Carps ; *Goldfish ; Iron/chemistry ; *Molecular Diagnostic Techniques/veterinary/methods ; }, abstract = {Cyprinid herpesvirus 2 (CyHV-2) poses a substantial global threat to goldfish (Carassius auratus) and crucian carp (Carassius carassius). Despite the development of several sensitive molecular diagnostic techniques, there is an ongoing demand for alternative visualisation platforms to streamline the workflow, enhance safety profiles, and improve accessibility for end-users. In this study, we have integrated recombinase-aided amplification (RAA) technology with the CRISPR/Cas12a system to establish a rapid diagnostic system for CyHV-2, termed one-pot RAA-CRISPR/Cas12a. This method enables the results of detection within 60 min. The RAA-CRISPR/Cas12a platform is capable of detecting as few as 10 copies of CyHV-2 per reaction cycle without exhibiting cross-reactivity with other pathogens. The positive detection rate in clinical samples exceeds that of conventional PCR approaches, underscoring its high precision. Furthermore, the method could be used in conjunction with iron flocculation for the concentration and detection of viruses within aquaculture settings. This approach minimises the dissection of aquatic organisms, thereby maximising animal welfare and bolstering detection efficiency. Collectively, our findings validate the RAA-CRISPR/Cas12a method as a robust, specific, confirmatory, user-friendly and promising approach for on-site diagnosis of CyHV-2.}, }
@article {pmid39906792, year = {2025}, author = {Ali Agha, ASA and Al-Samydai, A and Aburjai, T}, title = {New frontiers in CRISPR: Addressing antimicrobial resistance with Cas9, Cas12, Cas13, and Cas14.}, journal = {Heliyon}, volume = {11}, number = {2}, pages = {e42013}, pmid = {39906792}, issn = {2405-8440}, abstract = {BACKGROUND: The issue of antimicrobial resistance (AMR) poses a major challenge to global health, evidenced by alarming mortality predictions and the diminishing efficiency of conventional antimicrobial drugs. The CRISPR-Cas system has proven to be a powerful tool in addressing this challenge. It originated from bacterial adaptive immune mechanisms and has gained significant recognition in the scientific community.<br><br>OBJECTIVES: This review aims to explore the applications of CRISPR-Cas technologies in combating AMR, evaluating their effectiveness, challenges, and potential for integration into current antimicrobial strategies.<br><br>METHODS: We conducted a comprehensive review of recent literature from databases such as PubMed and Web of Science, focusing on studies that employ CRISPR-Cas technologies against AMR.<br><br>CONCLUSIONS: CRISPR-Cas technologies offer a transformative approach to combat AMR, with potential applications that extend beyond traditional antimicrobial strategies. Integrating these technologies with existing methods could significantly enhance our ability to manage and potentially reverse the growing problem of antimicrobial resistance. Future research should address technical and ethical barriers to facilitate safe and effective clinical and environmental applications. This review underscores the necessity for interdisciplinary collaboration and international cooperation to harness the full potential of CRISPR-Cas technologies in the fight against superbugs.}, }
@article {pmid39906170, year = {2025}, author = {Vu, TV and Nguyen, NT and Kim, J and Vu, MH and Song, YJ and Tran, MT and Sung, YW and Kim, JY}, title = {Enhancing CRISPR-Cas-based gene targeting in tomato using a dominant-negative ku80.}, journal = {Horticulture research}, volume = {12}, number = {2}, pages = {uhae294}, pmid = {39906170}, issn = {2662-6810}, abstract = {The CRISPR-Cas-based gene targeting (GT) method has enabled precise modifications of genomic DNA ranging from single base to several kilobase scales through homologous recombination (HR). In plant somatic cells, canonical non-homologous end-joining (cNHEJ) is the predominant mechanism for repairing double-stranded breaks (DSBs), thus limiting the HR-mediated GT. In this study, we implemented an approach to shift the repair pathway preference toward HR by using a dominant-negative ku80 mutant protein (KUDN) to disrupt the initiation of cNHEJ. The employment of KUDN conferred a 1.71- to 3.55-fold improvement in GT efficiency at the callus stage. When we screened transformants, there was a more remarkable increase in GT efficiency, ranging from 1.62- to 9.84-fold, at two specific tomato loci, SlHKT1;2 and SlEPSPS1. With practical levels of efficiency, this enhanced KUDN-based GT tool successfully facilitated a 9-bp addition at an additional locus, SlCAB13. These findings provide another promising method for more efficient and precise plant breeding.}, }
@article {pmid39906167, year = {2025}, author = {Bilal, M and Geng, J and Chen, L and García-Caparros, P and Hu, T}, title = {Genome editing for grass improvement and future agriculture.}, journal = {Horticulture research}, volume = {12}, number = {2}, pages = {uhae293}, pmid = {39906167}, issn = {2662-6810}, abstract = {Grasses, including turf and forage, cover most of the earth's surface; predominantly important for land, water, livestock feed, soil, and water conservation, as well as carbon sequestration. Improved production and quality of grasses by modern molecular breeding is gaining more research attention. Recent advances in genome-editing technologies are helping to revolutionize plant breeding and also offering smart and efficient acceleration on grass improvement. Here, we reviewed all recent researches using (CRISPR)/CRISPR-associated protein (Cas)-mediated genome editing tools to enhance the growth and quality of forage and turf grasses. Furthermore, we highlighted emerging approaches aimed at advancing grass breeding program. We assessed the CRISPR-Cas effectiveness, discussed the challenges associated with its application, and explored future perspectives primarily focusing on turf and forage grasses. Despite the promising potential of genome editing in grasses, its current efficiency remains limited due to several bottlenecks, such as the absence of comprehensive reference genomes, the lack of efficient gene delivery tools, unavailability of suitable vector and delivery for grass species, high polyploidization, and multiple homoeoalleles, etc. Despite these challenges, the CRISPR-Cas system holds great potential to fully harness its benefits in grass breeding and genetics, aiming to improve and sustain the quantity and quality of turf and forage grasses.}, }
@article {pmid39905729, year = {2025}, author = {Du, W and Noyan, F and McCallion, O and Drosdek, V and Kath, J and Glaser, V and Fuster-Garcia, C and Yang, M and Stein, M and Franke, C and Pu, Y and Weber, O and Polansky, JK and Cathomen, T and Jaeckel, E and Hester, J and Issa, F and Volk, HD and Schmueck-Henneresse, M and Reinke, P and Wagner, DL}, title = {Gene editing of CD3 epsilon to redirect regulatory T cells for adoptive T cell transfer.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {33}, number = {3}, pages = {997-1013}, pmid = {39905729}, issn = {1525-0024}, mesh = {*T-Lymphocytes, Regulatory/immunology/metabolism ; Animals ; *CD3 Complex/genetics/immunology/metabolism ; Humans ; *Gene Editing/methods ; Mice ; *Adoptive Transfer/methods ; CRISPR-Cas Systems ; HLA-A2 Antigen/immunology/genetics ; Receptors, Chimeric Antigen/genetics ; Receptors, Antigen, T-Cell/genetics ; Lymphocyte Activation ; }, abstract = {Adoptive transfer of antigen-specific regulatory T cells (Tregs) is a promising strategy to combat immunopathologies in transplantation and autoimmune diseases. However, their low frequency in peripheral blood poses challenges for both manufacturing and clinical application. Chimeric antigen receptors have been used to redirect the specificity of Tregs, using retroviral vectors. However, retroviral gene transfer is costly, time consuming, and raises safety issues. Here, we explored non-viral CRISPR-Cas12a gene editing to redirect Tregs, using human leukocyte antigen (HLA)-A2-specific constructs for proof-of-concept studies in transplantation models. Knock-in of an antigen-binding domain into the N terminus of CD3 epsilon (CD3ε) gene generates Tregs expressing a chimeric CD3ε-T cell receptor fusion construct (TRuC) protein that integrates into the endogenous TCR/CD3 complex. These CD3ε-TRuC Tregs exhibit potent antigen-dependent activation while maintaining responsiveness to TCR/CD3 stimulation. This enables preferential enrichment of TRuC-redirected Tregs over CD3ε knockout Tregs via repetitive CD3/CD28 stimulation in a good manufacturing practice-compatible expansion system. CD3ε-TRuC Tregs retained their phenotypic, epigenetic, and functional identity. In a humanized mouse model, HLA-A2-specific CD3ε-TRuC Tregs demonstrate superior protection of allogeneic HLA-A2[+] skin grafts from rejection compared with polyclonal Tregs. This approach provides a pathway for developing clinical-grade CD3ε-TRuC-based Treg cell products for transplantation immunotherapy and other immunopathologies.}, }
@article {pmid39905471, year = {2025}, author = {Ye, T and Ding, W and An, Z and Zhang, H and Wei, X and Xu, J and Liu, H and Fang, H}, title = {Increased distribution of carbon metabolic flux during de novo cytidine biosynthesis via attenuation of the acetic acid metabolism pathway in Escherichia coli.}, journal = {Microbial cell factories}, volume = {24}, number = {1}, pages = {36}, pmid = {39905471}, issn = {1475-2859}, support = {31860020//National Natural Science Foundation of China/ ; 022004000010//Ningxia Hui Autonomous Region Youth Top Talent Training Project/ ; No. 2020//The Helanshan Scholars Program of Ningxia University/ ; 2023AAC02030//Natural Science Foundation of Ningxia, China/ ; }, mesh = {*Acetic Acid/metabolism ; *Escherichia coli/metabolism/genetics ; *Cytidine/biosynthesis ; *Carbon/metabolism ; Fermentation ; Escherichia coli Proteins/genetics/metabolism ; Metabolic Networks and Pathways ; CRISPR-Cas Systems ; Glucose/metabolism ; }, abstract = {Acetic acid, a by-product of cytidine synthesis, competes for carbon flux from central metabolism, which may be directed either to the tricarboxylic acid (TCA) cycle for cytidine synthesis or to overflow metabolites, such as acetic acid. In Escherichia coli, the acetic acid synthesis pathway, regulated by the poxB and pta genes, facilitates carbon consumption during cytidine production. To mitigate carbon source loss, the CRISPR-Cas9 gene-editing technique was employed to knock out the poxB and pta genes in E. coli, generating the engineered strains K12ΔpoxB and K12ΔpoxBΔpta. After 39 h of fermentation in 500 mL shake flasks, the cytidine yields of strains K12ΔpoxB and K12ΔpoxBΔpta were 1.91 ± 0.04 g/L and 18.28 ± 0.22 g/L, respectively. Disruption of the poxB and pta genes resulted in reduced acetic acid production and glucose consumption. Transcriptomic and metabolomic analyses revealed that impairing the acetic acid metabolic pathway in E. coli effectively redirected carbon flux toward cytidine biosynthesis, yielding a 5.26-fold reduction in acetate metabolism and an 11.56-fold increase in cytidine production. These findings provide novel insights into the influence of the acetate metabolic pathway on cytidine biosynthesis in E. coli.}, }
@article {pmid39905452, year = {2025}, author = {Abbasi, AF and Asim, MN and Dengel, A}, title = {Transitioning from wet lab to artificial intelligence: a systematic review of AI predictors in CRISPR.}, journal = {Journal of translational medicine}, volume = {23}, number = {1}, pages = {153}, pmid = {39905452}, issn = {1479-5876}, mesh = {Humans ; *Artificial Intelligence ; *CRISPR-Cas Systems/genetics ; Gene Editing ; RNA, Guide, CRISPR-Cas Systems/metabolism ; }, abstract = {The revolutionary CRISPR-Cas9 system leverages a programmable guide RNA (gRNA) and Cas9 proteins to precisely cleave problematic regions within DNA sequences. This groundbreaking technology holds immense potential for the development of targeted therapies for a wide range of diseases, including cancers, genetic disorders, and hereditary diseases. CRISPR-Cas9 based genome editing is a multi-step process such as designing a precise gRNA, selecting the appropriate Cas protein, and thoroughly evaluating both on-target and off-target activity of the Cas9-gRNA complex. To ensure the accuracy and effectiveness of CRISPR-Cas9 system, after the targeted DNA cleavage, the process requires careful analysis of the resultant outcomes such as indels and deletions. Following the success of artificial intelligence (AI) in various fields, researchers are now leveraging AI algorithms to catalyze and optimize the multi-step process of CRISPR-Cas9 system. To achieve this goal AI-driven applications are being integrated into each step, but existing AI predictors have limited performance and many steps still rely on expensive and time-consuming wet-lab experiments. The primary reason behind low performance of AI predictors is the gap between CRISPR and AI fields. Effective integration of AI into multi-step CRISPR-Cas9 system demands comprehensive knowledge of both domains. This paper bridges the knowledge gap between AI and CRISPR-Cas9 research. It offers a unique platform for AI researchers to grasp deep understanding of the biological foundations behind each step in the CRISPR-Cas9 multi-step process. Furthermore, it provides details of 80 available CRISPR-Cas9 system-related datasets that can be utilized to develop AI-driven applications. Within the landscape of AI predictors in CRISPR-Cas9 multi-step process, it provides insights of representation learning methods, machine and deep learning methods trends, and performance values of existing 50 predictive pipelines. In the context of representation learning methods and classifiers/regressors, a thorough analysis of existing predictive pipelines is utilized for recommendations to develop more robust and precise predictive pipelines.}, }
@article {pmid39905017, year = {2025}, author = {Moore, MM and Wekhande, S and Issner, R and Collins, A and Cruz, AJ and Liu, YV and Javed, N and Casaní-Galdón, S and Buenrostro, JD and Epstein, CB and Mattei, E and Doench, JG and Bernstein, BE and Shoresh, N and Najm, FJ}, title = {Multi-locus CRISPRi targeting with a single truncated guide RNA.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {1357}, pmid = {39905017}, issn = {2041-1723}, mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Binding Sites/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; CCCTC-Binding Factor/metabolism/genetics ; Transcription Factors/metabolism/genetics ; Gene Editing/methods ; Enhancer Elements, Genetic ; Histones/metabolism ; }, abstract = {A critical goal in functional genomics is evaluating which non-coding elements contribute to gene expression, cellular function, and disease. Functional characterization remains a challenge due to the abundance and complexity of candidate elements. Here, we develop a CRISPRi-based approach for multi-locus screening of putative transcription factor binding sites with a single truncated guide. A truncated guide with hundreds of sequence match sites can reliably disrupt enhancer activity, which expands the targeting scope of CRISPRi while maintaining repressive efficacy. We screen over 13,000 possible CTCF binding sites with 24 guides at 10 nucleotides in spacer length. These truncated guides direct CRISPRi-mediated deposition of repressive H3K9me3 marks and disrupt transcription factor binding at most sequence match target sites. This approach can be a valuable screening step for testing transcription factor binding motifs or other repeated genomic sequences and is easily implemented with existing tools.}, }
@article {pmid39904990, year = {2025}, author = {Liu, Y and Wang, L and Zhang, Q and Fu, P and Zhang, L and Yu, Y and Zhang, H and Zhu, H}, title = {Structural basis for RNA-guided DNA degradation by Cas5-HNH/Cascade complex.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {1335}, pmid = {39904990}, issn = {2041-1723}, mesh = {Cryoelectron Microscopy ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics/ultrastructure ; CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; *DNA/metabolism ; *Bacterial Proteins/metabolism/chemistry/genetics/ultrastructure ; DNA Cleavage ; Models, Molecular ; Protein Binding ; }, abstract = {Type I-E CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated proteins) system is one of the most extensively studied RNA-guided adaptive immune systems in prokaryotes, providing defense against foreign genetic elements. Unlike the previously characterized Cas3 nuclease, which exhibits progressive DNA cleavage in the typical type I-E system, a recently identified HNH-comprising Cascade system enables precise DNA cleavage. Here, we present several near-atomic cryo-electron microscopy (cryo-EM) structures of the Candidatus Cloacimonetes bacterium Cas5-HNH/Cascade complex, both in its DNA-bound and unbound states. Our analysis reveals extensive interactions between the HNH domain and adjacent subunits, including Cas6 and Cas11, with mutations in these key interactions significantly impairing enzymatic activity. Upon DNA binding, the Cas5-HNH/Cascade complex adopts a more compact conformation, with subunits converging toward the center of nuclease, leading to its activation. Notably, we also find that divalent ions such as zinc, cobalt, and nickel down-regulate enzyme activity by destabilizing the Cascade complex. Together, these findings offer structural insights into the assembly and activation of the Cas5-HNH/Cascade complex.}, }
@article {pmid39904442, year = {2025}, author = {Liu, M and Zhang, M and Huai, M and Xu, F and Meng, G and Wang, F and Dong, C}, title = {Impact of the sterol O-acyltransferase gene (Cmare2) on morphology and stability of the mushroom Cordyceps militaris.}, journal = {International journal of biological macromolecules}, volume = {302}, number = {}, pages = {140522}, doi = {10.1016/j.ijbiomac.2025.140522}, pmid = {39904442}, issn = {1879-0003}, mesh = {*Cordyceps/genetics/enzymology/growth &amp; development/cytology ; *Acyltransferases/genetics/metabolism ; *Fungal Proteins/genetics/metabolism ; Hyphae/genetics ; Fruiting Bodies, Fungal/genetics ; Phenotype ; Reactive Oxygen Species/metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Fungal ; }, abstract = {Strain degeneration in mushroom cultivation can lead to significant commercial losses, yet the genetic factors remain elusive. This study identified the sterol O-acyltransferase gene, Cmare2, which is associated with colony sectorization in Cordyceps militaris, using a T-DNA insertion-mutant library. CmARE2 is conserved across Ascomycota and Basidiomycota. Deletion of Cmare2 via CRISPR/Cas9 resulted in degenerative phenotypes, including colony sectorization, hyphal adhesion, reduced conidia production, and abnormal fruiting body development. Scanning electron microscopy revealed surface perforations, cobweb-like filaments connecting the adhering hyphae, and eventual hyphal impairment. These phenotypes may be attributed to disturbance of sterol homeostasis, which impairs cell membrane fluidity and permeability. Overexpression of Cmare2 via CRISPR/Cas9 led to a more stable strain with consistent morphology, reduced reactive oxygen species level, and robust mycelia during successive subculturing. This research integrates forward and reverse genetics to unravel the molecular mechanisms behind strain degeneration, providing valuable insights for developing stable, non-degenerative mushroom strains.}, }
@article {pmid39904299, year = {2025}, author = {Zhang, Y and Gao, L and Shi, Z and Wu, Q and Miao, X}, title = {Paper-based electrochemiluminescence telomerase activity detection using hybridization chain reaction and CRISPR/Cas12a dual signal amplification.}, journal = {Bioelectrochemistry (Amsterdam, Netherlands)}, volume = {164}, number = {}, pages = {108916}, doi = {10.1016/j.bioelechem.2025.108916}, pmid = {39904299}, issn = {1878-562X}, mesh = {*Telomerase/metabolism/analysis ; Humans ; *CRISPR-Cas Systems ; *Luminescent Measurements/instrumentation/methods ; *Nucleic Acid Hybridization ; *Paper ; *Electrochemical Techniques/instrumentation/methods ; *Biosensing Techniques/methods/instrumentation ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Limit of Detection ; Nucleic Acid Amplification Techniques ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Sensitive telomerase activity detection becomes particularly significance since the important value of it in early cancer diagnosis as a potential biomarker. Herein, we developed a paper-based analytical devices (PADs) for telomerase activity detection, using positively charged Au@luminol nanoparticles ((+)Au@luminol NPs) as electrochemiluminescence (ECL) signal probe coupling with hybridization chain reaction (HCR) and CRISPR/Cas12a dual signal amplification. Firstly, the initial strong ECL signal was obtained based on the electrostatic adsorption of (+)Au@luminol NPs onto the surface of HCR double-stranded hybrid aggregates. In the presence of telomerase, the primer was efficiently elongated with telomeric repeats of (TTAGGG)n to release activator DNA and trigger the CRISPR/Cas12a, which can prevent the happen of HCR and the adsorption of (+)Au@luminol NPs through cleaving the capture probe on the electrode surface, such results directly inducing the decrease of the ECL signal that was proportional to telomerase concentration, due to the efficient signal amplification of HCR and CRISPR/Cas12a, a low detection limit of 2.3 cells/mL for telomerase could be detected. Moreover, the sensor realized the effective application for telomerase extracts analysis in human serum samples, making it possess potential application value for telomerase activity assays in cancer diagnostics.}, }
@article {pmid39904298, year = {2025}, author = {Zhong, WJ and Yang, WG and Zhang, Y and Li, T and Su, ML and Yuan, R and Xu, S and Liang, WB}, title = {An electrochemiluminescence strategy with proximity ligation triggered multiple catalytic hairpin assembly induced CRISPR/Cas 12a system for analysis of paraquat.}, journal = {Bioelectrochemistry (Amsterdam, Netherlands)}, volume = {164}, number = {}, pages = {108915}, doi = {10.1016/j.bioelechem.2025.108915}, pmid = {39904298}, issn = {1878-562X}, mesh = {*Paraquat/analysis ; *CRISPR-Cas Systems ; *Luminescent Measurements/methods ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; Limit of Detection ; Catalysis ; *Herbicides/analysis ; Inverted Repeat Sequences ; }, abstract = {Paraquat (PQ) as a widely used non-selective herbicides has gained attention in agricultural residue detection and food safety. Herein, a novel quantitative analysis approach for PQ was proposed based on a novel kind of aggregation-induced emission electrochemiluminescence (AIECL) emitters, tetraphenylethylene-luminol (TPE-L) with a small molecule-induced multiple catalytic hairpin assembly (CHA) amplification strategy, the competitive immune reaction and CRISPR/Cas12a system. The target molecule PQ is introduced into a signal cycle, and auxiliary sensitization cycles are constructed by virtue of the cleavage characteristics of the CRISPR/Cas12a system, which realized the multiple utilization of the target by using both cis- and trans-cleavage activities. In addition, the new multiple CHA amplification strategy was attributed to cross-catalytic hairpin assembly caused by the products of the CHA cycle as the initiator chain of the next CHA cycle, realizing the efficient utilization of cyclic products and producing high-efficiency signal amplification. Thus, the ECL biosensor for ultrasensitive analysis of PQ was successfully constructed with a limit of detection of 0.7 pg/mL. Importantly, it could be easily-extended to other small molecules simply by replacing paired antibodies, providing prospects in agricultural residue detection, food safety and related medical applications.}, }
@article {pmid39903512, year = {2025}, author = {Han, HJ and Yu, D and Yu, J and Kim, J and Do Heo, W and Tark, D and Kang, SM}, title = {Targeting pseudoknots with Cas13b inhibits porcine epidemic diarrhoea virus replication.}, journal = {The Journal of general virology}, volume = {106}, number = {2}, pages = {}, pmid = {39903512}, issn = {1465-2099}, mesh = {*Porcine epidemic diarrhea virus/physiology/genetics/drug effects ; Animals ; *Virus Replication/drug effects ; *CRISPR-Cas Systems ; Swine ; *RNA, Viral/genetics/metabolism ; RNA-Dependent RNA Polymerase/genetics ; *Coronavirus Infections/virology/veterinary ; Chlorocebus aethiops ; Vero Cells ; Antiviral Agents/pharmacology ; Swine Diseases/virology ; Cell Line ; *CRISPR-Associated Proteins/genetics ; }, abstract = {Clustered regularly interspaced short palindromic repeats-associated protein 13 (CRISPR-Cas13), an RNA editing technology, has shown potential in combating RNA viruses by degrading viral RNA within mammalian cells. In this study, we demonstrate the effective inhibition of porcine epidemic diarrhoea virus (PEDV) replication and spread using CRISPR-Cas13. We analysed the sequence similarity of the pseudoknot region between PEDV and severe acute respiratory syndrome coronavirus 2, both belonging to the Coronaviridae family, as well as the similarity of the RNA-dependent RNA polymerase (RdRp) gene region among three different strains of the PED virus. Based on this analysis, we synthesized three CRISPR RNAs (crRNAs) targeting the pseudoknot region and the nonpseudoknot region, each for comparison. In cells treated with crRNA #3 targeting the pseudoknot region, RdRp gene expression decreased by 95%, membrane (M) gene expression by 89% and infectious PEDV titre within the cells reduced by over 95%. Additionally, PED viral nucleocapsid (N) and M protein expression levels decreased by 83 and 98%, respectively. The optimal concentration for high antiviral efficacy without cytotoxicity was determined. Treating cells with 1.5 µg of Cas13b mRNA and 0.5 µg of crRNA resulted in no cytotoxicity while achieving over 95% inhibition of PEDV replication. The Cas13b mRNA therapeutics approach was validated as significantly more effective through a comparative study with merafloxacin, a drug targeting the pseudoknot region of the viral genome. Our results indicate that the pseudoknot region plays a crucial role in the degradation of the PEDV genome through the CRISPR-Cas13 system. Therefore, targeting Cas13b to the pseudoknot offers a promising new approach for treating coronavirus infections.}, }
@article {pmid39902531, year = {2025}, author = {Lafi, Z and Ata, T and Asha, S}, title = {CRISPR in clinical diagnostics: bridging the gap between research and practice.}, journal = {Bioanalysis}, volume = {17}, number = {4}, pages = {281-290}, pmid = {39902531}, issn = {1757-6199}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Polymorphism, Single Nucleotide ; Biosensing Techniques/methods ; }, abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) has transformed molecular biology through its precise gene-editing capabilities. Beyond its initial applications in genetic modification, CRISPR has emerged as a powerful tool in diagnostics and biosensing. This review explores its transition from genome editing to innovative detection methods, including nucleic acid identification, single nucleotide polymorphism (SNP) analysis, and protein sensing. Advanced technologies such as SHERLOCK and DETECTR demonstrate CRISPR's potential for point-of-care diagnostics, enabling rapid and highly sensitive detection. The integration of chemical modifications, CRISPR-Chip technology, and enzymatic systems like Cas12a and Cas13a enhances signal amplification and detection efficiency. These advancements promise decentralized, real-time diagnostic solutions with significant implications for global healthcare. Furthermore, the fusion of CRISPR with artificial intelligence and digital health platforms is paving the way for more accessible, cost-effective, and scalable diagnostic approaches, ultimately revolutionizing precision medicine.}, }
@article {pmid39902289, year = {2024}, author = {Peach, LJ and Zhang, H and Weaver, BP and Boedicker, JQ}, title = {Assessing spacer acquisition rates in E. coli type I-E CRISPR arrays.}, journal = {Frontiers in microbiology}, volume = {15}, number = {}, pages = {1498959}, pmid = {39902289}, issn = {1664-302X}, abstract = {CRISPR/Cas is an adaptive defense mechanism protecting prokaryotes from viruses and other potentially harmful genetic elements. Through an adaptation process, short "spacer" sequences, captured from these elements and incorporated into a CRISPR array, provide target specificity for the immune response. CRISPR arrays and array expansion are also central to many emerging biotechnologies. The rates at which spacers integrate into native arrays within bacterial populations have not been quantified. Here, we measure naïve spacer acquisition rates in Escherichia coli Type I-E CRISPR, identify factors that affect these rates, and model this process fundamental to CRISPR/Cas defense. Prolonged Cas1-Cas2 expression produced fewer new spacers per cell on average than predicted by the model. Subsequent experiments revealed that this was due to a mean fitness reduction linked to array-expanded populations. In addition, the expression of heterologous non-homologous end-joining DNA-repair genes was found to augment spacer acquisition rates, translating to enhanced phage infection defense. Together, these results demonstrate the impact of intracellular factors that modulate spacer acquisition and identify an intrinsic fitness effect associated with array-expanded populations.}, }
@article {pmid39902056, year = {2025}, author = {Sumanto Marpaung, DS and Yap Sinaga, AO and Damayanti, D and Taharuddin, T and Gumaran, S}, title = {Current biosensing strategies based on in vitro T7 RNA polymerase reaction.}, journal = {Biotechnology notes (Amsterdam, Netherlands)}, volume = {6}, number = {}, pages = {59-66}, pmid = {39902056}, issn = {2665-9069}, abstract = {Recently, a unique behavior of T7 RNA polymerase has expanded its functionality as a biosensing platform. Various biosensors utilizing T7 RNA polymerase, combined with fluorescent aptamers, electrochemical probes, or CRISPR/Cas systems, have been developed to detect analytes, including nucleic acids and non-nucleic acid target, with high specificity and low detection limits. Each approach demonstrates unique strengths, such as real-time monitoring and minimal interference, but also presents challenges in stability, cost, and reaction optimization. This review provides an overview of T7 RNA polymerase's role in biosensing technology, highlighting its potential to advance diagnostics and molecular detection in diverse fields.}, }
@article {pmid39902050, year = {2024}, author = {Huai, G and Wang, Y and Du, J and Cheng, Z and Xie, Y and Zhou, J and Tang, H and Jiang, Y and Xing, X and Deng, S and Pan, D}, title = {The generation and evaluation of TKO/hCD55/hTM/hEPCR gene-modified pigs for clinical organ xenotransplantation.}, journal = {Frontiers in immunology}, volume = {15}, number = {}, pages = {1488552}, pmid = {39902050}, issn = {1664-3224}, mesh = {Animals ; *Transplantation, Heterologous/methods ; Swine ; Humans ; Animals, Genetically Modified ; Galactosyltransferases/genetics ; Endothelial Cells/immunology/metabolism ; Gene Knockout Techniques ; Immunoglobulin M ; Mixed Function Oxygenases/genetics ; N-Acetylgalactosaminyltransferases/genetics ; Gene Editing ; *Membrane Cofactor Protein/genetics ; CRISPR-Cas Systems ; }, abstract = {INTRODUCTION: Genetically edited pigs, modified using CRISPR-Cas9 technology, hold promise as potential sources for xenotransplantation. However, the optimal combination of genetic modifications and their expression levels for initial clinical trials remains unclear. This study investigates the generation of TKO/hCD55/hTM/hEPCR (6GE) pigs and evaluates their compatibility with human immune and coagulation systems.<br><br>METHODS: The 6GE pigs were generated through iterative genome editing and F1 generation breeding. Genotyping, flow cytometry, and immunohistochemistry confirmed the knockout of GGTA1, CMAH, and B4GALNT2. Expression levels of human genes (hCD55, hTM, hEPCR) were quantified. In vitro assays using aortic endothelial cells (pAECs) from 6GE pigs assessed human serum IgM and IgG binding, complement cytotoxicity, and thrombin-antithrombin (TAT) complex levels. Blood from gene-edited pigs was used for pathophysiological analysis.<br><br>RESULTS: Complete knockout of GGTA1, CMAH, and B4GALNT2 was confirmed in 6GE pigs. The expression of hCD55 and hTM was approximately seven and thirteen times higher than in humans, respectively, while hEPCR levels were comparable to those in humans. In vitro, 6GE pAECs showed significantly reduced binding of human IgM and IgG compared to wild-type pAECs (IgG p<0.01, IgM p<0.0001). Similar to TKO/hCD55 pAECs, 6GE pAECs exhibited a substantial reduction in complement-mediated cytotoxicity (p<0.001) compared to TKO pAECs. Co-expression of hTM and hEPCR in 6GE pigs led to a significant decrease in thrombin-antithrombin (TAT) complex levels in co-culture with human whole blood, compared to WT (p<0.0001), TKO (p<0.01), and TKO/hCD55/hTM pigs (p<0.05). Pathophysiological analysis demonstrated excellent compatibility of 6GE pig kidneys and livers with human immune and coagulation systems. However, 6GE pigs showed increased susceptibility to infection compared to other gene-edited pigs, while TKO/hCD55 pigs were considered safe when they were all bred in a general environment.<br><br>DISCUSSION: Highly expressing hCD55, along with the co-expression of hEPCR and hTM genes, is expected to effectively reduce human complement cytotoxicity and enhance anticoagulant efficacy in genetically modified pigs. The 6GE pigs exhibited robust compatibility with human physiological and immune systems, fulfilling the criteria for clinical trials. Furthermore, it is imperative to rear donor pigs in pathogen-free (DPF) facilities to mitigate infection risks and prevent the transmission of porcine pathogens to humans.}, }
@article {pmid39901848, year = {2025}, author = {Torigoe, N and Lin, Q and Liu, B and Nakayama, Y and Nakai, A and Nagahara, M and Tanihara, F and Hirata, M and Otoi, T}, title = {Effects of Electroporation Timing and Cumulus Cell Attachment on In Vitro Development and Genome Editing of Porcine Embryos.}, journal = {Reproduction in domestic animals = Zuchthygiene}, volume = {60}, number = {2}, pages = {e70011}, doi = {10.1111/rda.70011}, pmid = {39901848}, issn = {1439-0531}, support = {JP22H02499//Japan Society for the Promotion of Science/ ; JP22K19896//Japan Society for the Promotion of Science/ ; }, mesh = {Animals ; *Electroporation/veterinary/methods ; *Cumulus Cells/physiology ; *Gene Editing/veterinary/methods ; Female ; Fertilization in Vitro/veterinary ; Oocytes/physiology ; Embryonic Development ; Swine/embryology ; Zygote ; RNA, Guide, CRISPR-Cas Systems ; Blastocyst/physiology ; CRISPR-Cas Systems ; Embryo Culture Techniques/veterinary ; }, abstract = {Pig genome editing using the oviductal nucleic acid delivery (GONAD) method with electroporation would allow the efficient obtention of genetically modified pigs. However, oocytes and zygotes at various stages after ovulation must be targeted, and cumulus cell attachment and mosaic mutations are major obstacles. Therefore, we investigated whether two parameters (electroporation timing and the cumulus cell attachment) influence the effectiveness of multiplex genome editing by electroporation in porcine oocytes or zygotes. Three gRNAs targeting either GGTA1, CMAH or B4GALNT2 were introduced individually into oocytes and zygotes with and without cumulus cells at three different time points, 0 h before in vitro fertilisation (IVF) and 5 h and 10 h after IVF initiation. The introduction of gRNAs into oocytes and zygotes did not significantly affect the rates of blastocyst formation and total mutation of the resulting blastocysts irrespective of cumulus cell attachment and electroporation timing. In conclusion, the electroporation timing and the cumulus cell attachment did not interfere with the efficient delivery of the CRISPR/Cas9 system to the oocytes/zygotes, indicating that porcine genome editing in the oviduct using GONAD method may be possible.}, }
@article {pmid39901278, year = {2025}, author = {Zhang, D and Parth, F and da Silva, LM and Ha, TC and Schambach, A and Boch, J}, title = {Engineering a bacterial toxin deaminase from the DYW-family into a novel cytosine base editor for plants and mammalian cells.}, journal = {Genome biology}, volume = {26}, number = {1}, pages = {18}, pmid = {39901278}, issn = {1474-760X}, support = {BO 1496/9-1//Deutsche Forschungsgemeinschaft/ ; }, mesh = {*Gene Editing/methods ; Oryza/genetics ; *Cytosine/metabolism ; *Cytosine Deaminase/genetics/metabolism ; CRISPR-Cas Systems ; Animals ; Humans ; Hordeum/genetics ; *Bacterial Toxins/genetics/metabolism ; Mutation ; }, abstract = {Base editors are precise editing tools that employ deaminases to modify target DNA bases. The DYW-family of cytosine deaminases is structurally and phylogenetically distinct and might be harnessed for genome editing tools. We report a novel CRISPR/Cas9-cytosine base editor using SsdA, a DYW-like deaminase and bacterial toxin. A G103S mutation in SsdA enhances C-to-T editing efficiency while reducing its toxicity. Truncations result in an extraordinarily small enzyme. The SsdA-base editor efficiently converts C-to-T in rice and barley protoplasts and induces mutations in rice plants and mammalian cells. The engineered SsdA is a highly efficient genome editing tool.}, }
@article {pmid39899813, year = {2025}, author = {Zhang, C and Liu, M and Wang, X and Cheng, J and Xiang, J and Yue, M and Ning, Y and Shao, Z and Abdullah, CN and Zhou, J}, title = {De Novo Synthesis of Reticuline and Taxifolin Using Re-engineered Homologous Recombination in Yarrowia lipolytica.}, journal = {ACS synthetic biology}, volume = {14}, number = {2}, pages = {585-597}, doi = {10.1021/acssynbio.4c00853}, pmid = {39899813}, issn = {2161-5063}, mesh = {*Yarrowia/genetics/metabolism ; *Homologous Recombination/genetics ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Metabolic Engineering/methods ; Rad51 Recombinase/genetics/metabolism ; }, abstract = {Yarrowia lipolytica has been widely engineered as a eukaryotic cell factory to produce various important compounds. However, the difficulty of gene editing and the lack of efficient neutral sites make rewiring of Y. lipolytica metabolism challenging. Herein, a Cas9 system was established to redesign the Y. lipolytica homologous recombination system, which caused a more than 56-fold increase in the HR efficiency. The fusion expression of the hBrex27 sequence in the C-terminus of Cas9 recruited more Rad51 protein, and the engineered Cas9 decreased NHEJ, achieving 85% single-gene positive efficiency and 25% multigene editing efficiency. With this system, neutral sites on different chromosomes were characterized, and a deep learning model was developed for gRNA activity prediction, thus providing the corresponding integration efficiency and expression intensity. Subsequently, the tool and platform strains were validated by applying them for the de novo synthesis of (S)-reticuline and (2S)-taxifolin. The developed platform strains and tools helped transform Y. lipolytica into an easy-to-operate model cell factory, similar to Saccharomyces cerevisiae.}, }
@article {pmid39899469, year = {2025}, author = {Dong, J and Croslow, SW and Lane, ST and Castro, DC and Blanford, J and Zhou, S and Park, K and Burgess, S and Root, M and Cahoon, EB and Shanklin, J and Sweedler, JV and Zhao, H and Hudson, ME}, title = {Enhancing lipid production in plant cells through automated high-throughput genome engineering and phenotyping.}, journal = {The Plant cell}, volume = {37}, number = {2}, pages = {}, pmid = {39899469}, issn = {1532-298X}, support = {//Center for Advanced Bioenergy/ ; //U.S. Department of Energy/ ; }, mesh = {*Zea mays/genetics/metabolism ; *Gene Editing/methods ; *Phenotype ; *Nicotiana/genetics/metabolism/cytology ; *Lipids ; Plant Cells/metabolism ; Genome, Plant ; Protoplasts/metabolism ; CRISPR-Cas Systems ; Plants, Genetically Modified ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods ; Genetic Engineering/methods ; }, abstract = {Plant bioengineering is a time-consuming and labor-intensive process with no guarantee of achieving desired traits. Here, we present a fast, automated, scalable, high-throughput pipeline for plant bioengineering (FAST-PB) in maize (Zea mays) and Nicotiana benthamiana. FAST-PB enables genome editing and product characterization by integrating automated biofoundry engineering of callus and protoplast cells with single-cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We first demonstrated that FAST-PB could streamline Golden Gate cloning, with the capacity to construct 96 vectors in parallel. Using FAST-PB in protoplasts, we found that PEG2050 increased transfection efficiency by over 45%. For proof-of-concept, we established a reporter-gene-free method for CRISPR editing and phenotyping via mutation of high chlorophyll fluorescence 136. We show that diverse lipids were enhanced up to 6-fold using CRISPR activation of lipid controlling genes. In callus cells, an automated transformation platform was employed to regenerate plants with enhanced lipid traits through introducing multigene cassettes. Lastly, FAST-PB enabled high-throughput single-cell lipid profiling by integrating MALDI-MS with the biofoundry, protoplast, and callus cells, differentiating engineered and unengineered cells using single-cell lipidomics. These innovations massively increase the throughput of synthetic biology, genome editing, and metabolic engineering and change what is possible using single-cell metabolomics in plants.}, }
@article {pmid39899041, year = {2025}, author = {Lv, H and Sun, J and Guo, Y and Hang, G and Wu, Q and Sun, Z and Zhang, H}, title = {Isolation of Enterococcus hirae From Fresh White Yak Milk in Ledu District, Qinghai Province, China: A Comparative Genomic Analysis.}, journal = {Current microbiology}, volume = {82}, number = {3}, pages = {111}, pmid = {39899041}, issn = {1432-0991}, support = {U22A20540//Natural Science Foundation of China/ ; 2023KYPT0019//Inner Mongolia Science &amp; Technology planning project/ ; BR22-12-03//Basic Research Operating Expenses Program for Colleges and Universities directly under the Inner Mongolia Autonomous Region/ ; }, mesh = {Animals ; *Milk/microbiology ; Cattle ; China ; *Enterococcus hirae/genetics/isolation &amp; purification/classification ; *Genome, Bacterial ; Virulence Factors/genetics ; Genomics ; Humans ; Bacteriocins/genetics ; Food Microbiology ; Phylogeny ; Bacterial Proteins/genetics ; }, abstract = {Yak milk is a widely consumed dairy product rich in lactic acid bacteria. Although Enterococcus hirae (E. hirae) is commonly found in dairy products and other foods, there is limited information available on its genetic makeup in yak milk. In the present study, 10 E. hirae strains isolated and identified from fresh white yak milk samples, along with 442 E. hirae strains obtained from the NCBI database (totaling 452 strains), were subjected to comparative genomic analysis. The findings of this study revealed that E. hirae has an open pan-genomic structure that allows for its high adaptability and environmental plasticity. Notably, E. hirae isolates from fresh white yak milk had smaller genomes, encoded more functional genes, and had fewer copies of genes encoding carbohydrate-active enzymes involved in the degradation of oligosaccharide metabolism and autolysin synthesis (CE1, GH73, GH23, and GT4 families) than those from animal and human isolates (P < 0.05). Additionally, fresh white yak milk isolates carried only three intrinsic bacteriocins and lacked virulence factors, CRISPR-Cas systems, and resistance genes linked to pathogenicity, which may be attributed to their specialization in the milk-derived environment. This study provides new insights into the genetic and functional gene diversity of E. hirae and how it adapts to milk-derived habitats.}, }
@article {pmid39899011, year = {2025}, author = {Kim, MB and Lee, YJ}, title = {Characterisation of the CRISPR-Cas systems in Enterococcus faecalis from commercial broiler farm environments and its association with antimicrobial resistance.}, journal = {British poultry science}, volume = {}, number = {}, pages = {1-8}, doi = {10.1080/00071668.2025.2451268}, pmid = {39899011}, issn = {1466-1799}, abstract = {1. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas) systems have been highlighted for their potential applications in controlling the spread of mobile genetic elements, including antimicrobial resistance (AMR) genes. This study investigated the characteristics of CRISPR-Cas systems in E. faecalis from commercial broiler farms and assessed the impact of these systems on AMR.2. All E. faecalis isolates contained CRISPR2, and CRISPR1-Cas and CRISPR3-Cas were identified in 84 (56.4%) and 144 (96.6%) isolates. A combination of CRISPR2 and CRISPR3-Cas and a combination of CRISPR1-Cas, CRISPR2 and CRISPR3-Cas were each identified in 27 (96.4%) farms.3. There were significant differences between CRISPR-Cas systems for phenotypic AMR: CRISPR1-Cas and CRISPR3-Cas. The E. faecalis isolates without CRISPR1-Cas showed higher resistance to most antimicrobials and had a higher prevalence of multidrug resistance (MDR) than those with CRISPR1-Cas. However, the resistance rate against most antimicrobials and the prevalence of MDR did not differ significantly depending on the presence or absence of CRISPR3-Cas.4. The E. faecalis isolates without CRISPR1-Cas harboured higher levels of all AMR genes, except for tetL, than those with CRISPR1-Cas. However, the E. faecalis isolates with CRISPR3-Cas showed a significant lower prevalence of tetL gene and a significantly higher prevalence of fexA and poxtA genes.5. In the distribution of rep families, the rep9 family was predominant, followed by rep1, rep7, rep2 and rep8 families. Only prevalence of the rep7 family was significantly higher in the E. faecalis isolates without CRISPR1-Cas (15.4%) than in those with CRISPR1-Cas (0%).6. This study is the first report on the characteristics of CRISPR-Cas systems in E. faecalis isolated from commercial broiler farm environments, and the results supported the hypothesis that the development of antimicrobial strategies requires an understanding of the distinctive capabilities between CRISPR1-Cas and CRISPR3-Cas and their underlying resistance mechanisms.}, }
@article {pmid39898503, year = {2025}, author = {Fu, X and Gao, S and Zhang, H and Ma, W and Chen, Y and Luo, J and Ye, B}, title = {Multiple signal amplification strategy for ultrasensitive sensing of Mycobacterium bovis based on 8-17 DNAzyme and CRISPR-Cas13a.}, journal = {Analytical methods : advancing methods and applications}, volume = {17}, number = {8}, pages = {1825-1833}, doi = {10.1039/d4ay02141j}, pmid = {39898503}, issn = {1759-9679}, mesh = {*DNA, Catalytic/chemistry/metabolism/genetics ; *Mycobacterium bovis/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; Limit of Detection ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; Cattle ; Animals ; Tuberculosis, Bovine/diagnosis/microbiology ; }, abstract = {Bovine tuberculosis caused by Mycobacterium bovis is not only responsible for economic losses but can also seriously jeopardize human health. Therefore, for the ultra-sensitive detection of M. bovis, a novel triple-cycle amplification system was developed based on 8-17 deoxyribozyme (DNAzyme), clustered regularly interspaced short palindromic repeats-associated protein 13a (CRISPR-Cas13a)-mediated cleavage cycles and the catalytic hairpin assembly (CHA) reaction (termed as DzCCR). In the presence of the target, the A-sequence containing 8-17 DNAzyme fragments was released from A-B using a strand displacement reaction, which could specifically cleave the HX-gRNA probe, releasing the sequence of gRNA and H and realizing the first signal amplification. Then, the released gRNA could bind to the Cas13a-g complex and activate the trans-cutting ability of Cas13a to re-cut RNA bulge sequences in HX-gRNAs, achieving the second signal amplification. Moreover, the H-sequence generated by the upstream 8-17 DNAzyme and Cas13a cleavage reaction further triggered the CHA, allowing the G-quadruplex dimer to be exposed, realizing signal output by adding thioflavin T (THT), and thereby achieving the third signal amplification. Benefiting from the triple signal amplification, the DzCCR system could quantitatively detect the M. bovis target down to a concentration of 0.5 fM with a linear calibration range from 1 to 500 fM. Furthermore, we investigated the ability of this system to detect M. bovis in real samples by standard addition method, the recovery ranged from 92.6% to 107.5%, and the relative standard deviations (RSD) ranged from 1.9% to 4.1%. Owing to the constant temperature and high sensitivity, the proposed strategy could be used as a new approach for the detection of M. bovis.}, }
@article {pmid39898499, year = {2025}, author = {Deivarajan, HR and Chelliah, D and Sethupathy Ramkumar, P and Nandhakumar, D and Chacko, BM and Raghavan, A and Pandian, J and Prajna, L and Prajna, VN and Narendran, S}, title = {Clinical Utility of CRISPR-Based RID-MyC Assay in Smear and Culture-Negative Fungal Keratitis: A Case Series.}, journal = {Cornea}, volume = {44}, number = {7}, pages = {921-924}, pmid = {39898499}, issn = {1536-4798}, support = {1465//Velux Stiftung/ ; }, mesh = {Humans ; *Eye Infections, Fungal/diagnosis/microbiology/drug therapy ; Retrospective Studies ; Male ; Female ; Middle Aged ; Microscopy, Confocal ; Adult ; *Keratitis/microbiology/diagnosis ; *Fungi/genetics/isolation &amp; purification ; *DNA, Fungal/analysis/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; *Corneal Ulcer/microbiology/diagnosis ; Antifungal Agents/therapeutic use ; Aged ; }, abstract = {PURPOSE: To assess the clinical utility of the clustered regularly interspaced short palindromic repeats/Cas12a-based RID-MyC assay in diagnosing fungal keratitis (FK) in cases where conventional smear and culture methods fail to identify the causative pathogen.<br><br>METHODS: This retrospective case series included 5 patients with clinically suspected FK and negative smear and culture results who were evaluated in the Cornea Department at Aravind Eye Hospital, Coimbatore, India, between January 1, 2024, and March 31, 2024. The primary outcome was the diagnostic performance of the RID-MyC assay in detecting fungal nucleic acids in cases of suspected FK with negative smear and culture results. In vivo confocal microscopy served as a reference standard to validate the RID-MyC assay findings.<br><br>RESULTS: The RID-MyC assay successfully detected fungal nucleic acids in 3 cases, corroborated by in vivo confocal microscopy findings suggestive of fungal filaments, leading to targeted antifungal therapy and resolution of the infections. In addition, 2 cases tested negative for fungal nucleic acids, aligning with clinical and confocal evidence of nonfungal etiology, thus guiding appropriate alternative treatments that led to clinical improvement.<br><br>CONCLUSIONS: The RID-MyC assay demonstrates clinical utility in diagnosing FK in scenarios where conventional smear and culture methods prove inadequate, such as in cases with prior antifungal therapy or polymicrobial infections. This assay facilitates accurate diagnosis and timely initiation of appropriate antifungal treatment without the need for sophisticated equipment or expertise, making it particularly valuable in resource-limited settings.}, }
@article {pmid39898483, year = {2025}, author = {Guerra-Resendez, RS and Lydon, SL and Ma, AJ and Bedford, GC and Reed, DR and Kim, S and Terán, ER and Nishiguchi, T and Escobar, M and DiNardo, AR and Hilton, IB}, title = {Characterization of Rationally Designed CRISPR/Cas9-Based DNA Methyltransferases with Distinct Methyltransferase and Gene Silencing Activities in Human Cell Lines and Primary Human T Cells.}, journal = {ACS synthetic biology}, volume = {14}, number = {2}, pages = {384-397}, pmid = {39898483}, issn = {2161-5063}, support = {R01 EB036003/EB/NIBIB NIH HHS/United States ; R21 EB030772/EB/NIBIB NIH HHS/United States ; R35 GM143532/GM/NIGMS NIH HHS/United States ; }, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *DNA (Cytosine-5-)-Methyltransferases/genetics/metabolism ; DNA Methyltransferase 3A ; *T-Lymphocytes/metabolism ; DNA Methylation/genetics ; *Gene Silencing ; Gene Editing/methods ; Cell Line ; Epigenesis, Genetic ; Jurkat Cells ; HEK293 Cells ; }, abstract = {Nuclease-deactivated Cas (dCas) proteins can be used to recruit epigenetic effectors, and this class of epigenetic editing technologies has revolutionized the ability to synthetically control the mammalian epigenome and transcriptome. DNA methylation is one of the most important and well-characterized epigenetic modifications in mammals, and while many different forms of dCas-based DNA methyltransferases (dCas-DNMTs) have been developed for programmable DNA methylation, these tools are frequently poorly tolerated and/or lowly expressed in mammalian cell types. Further, the use of dCas-DNMTs has largely been restricted to cell lines, which limits mechanistic insights in karyotypically normal contexts and hampers translational utility in the longer term. Here, we extend previous insights into the rational design of the catalytic core of the mammalian DNMT3A methyltransferase and test three dCas9-DNMT3A/3L variants across different human cell lines and in primary donor-derived human T cells. We find that mutations within the catalytic core of DNMT3A stabilize the expression of dCas9-DNMT3A/3L fusion proteins in Jurkat T cells without sacrificing DNA methylation or gene-silencing performance. We also show that these rationally engineered mutations in DNMT3A alter DNA methylation profiles at loci targeted with dCas9-DNMT3A/3L in cell lines and donor-derived human T cells. Finally, we leverage the transcriptionally repressive effects of dCas9-DNMT3A/3L variants to functionally link the expression of a key immunomodulatory transcription factor to cytokine secretion in donor-derived T cells. Overall, our work expands the synthetic biology toolkit for epigenetic editing and provides a roadmap for the use of engineered dCas-based DNMTs in primary mammalian cell types.}, }
@article {pmid39895071, year = {2025}, author = {Choi, YJ and Eom, H and Nandre, R and Kim, M and Oh, YL and Kim, S and Ro, HS}, title = {Simultaneous gene editing of both nuclei in a dikaryotic strain of Ganoderma lucidum using Cas9-gRNA ribonucleoprotein.}, journal = {Journal of microbiology (Seoul, Korea)}, volume = {63}, number = {1}, pages = {e.2409006}, doi = {10.71150/jm.2409006}, pmid = {39895071}, issn = {1976-3794}, support = {2023R1A 2C1007213//National Research Foundation of Korea/ ; //Rural Development Administration/ ; RS-2024-00322425//New Breeding Technologies Development Program/ ; }, mesh = {*Gene Editing/methods ; *Cell Nucleus/genetics ; *Reishi/genetics ; *Ribonucleoproteins/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Fungal Proteins/genetics ; }, abstract = {The presence of multiple nuclei in a common cytoplasm poses a significant challenge to genetic modification in mushrooms. Here, we demonstrate successful gene editing in both nuclei of a dikaryotic strain of Ganoderma lucidum using the Cas9-gRNA ribonucleoprotein complex (RNP). The RNP targeting the pyrG gene was introduced into dikaryotic protoplasts of G. lucidum, resulting in the isolation of 31 mycelial colonies resistant to 5-fluoroorotic acid (5-FOA). Twenty-six of these isolates were confirmed as dikaryotic strains by the presence of two distinct A mating type markers, denoted as A1 and A2. All dikaryons exhibited clamp connections on their mycelial hyphae, while the remaining 5 transformants were monokaryotic. Subsequent sequence analysis of PCR amplicons targeting pyrG revealed that two dikaryons harbored disrupted pyrG in both nuclei (pyrG-/pyrG-), while 10 and 14 displayed pyrG+/pyrG- (A1/A2) and pyrG-/pyrG+ (A1/A2) configurations, respectively. The disruption was achieved through non-homologous end joining repair, involving deletion or insertion of DNA fragments at the site of the double-strand break induced by RNP. Importantly, the nuclei were stable throughout 10 serial transfers over a period of 6 months. These findings highlight the capability of RNP to target genes across multiple nuclei within the same cytoplasm.}, }
@article {pmid39895059, year = {2025}, author = {Jose, S and Sharma, H and Insan, J and Sharma, K and Arora, V and Puranapanda, S and Dhamija, S and Eid, N and Menon, MB}, title = {Kinase Inhibitor-Induced Cell-Type Specific Vacuole Formation in the Absence of Canonical ATG5-Dependent Autophagy Initiation Pathway.}, journal = {Molecular and cellular biology}, volume = {45}, number = {3}, pages = {99-115}, doi = {10.1080/10985549.2025.2454421}, pmid = {39895059}, issn = {1098-5549}, mesh = {*Vacuoles/drug effects/metabolism ; Humans ; *Autophagy/drug effects ; *Protein Kinase Inhibitors/pharmacology ; *Autophagy-Related Protein 5/metabolism/genetics ; *Pyridines/pharmacology ; *Imidazoles/pharmacology ; rab GTP-Binding Proteins/metabolism ; Cell Line, Tumor ; rab7 GTP-Binding Proteins ; CRISPR-Cas Systems ; A549 Cells ; Signal Transduction/drug effects ; }, abstract = {Pyridinyl-imidazole class p38 MAPKα/β (MAPK14/MAPK11) inhibitors including SB202190 have been shown to induce cell-type specific defective autophagy resulting in micron-scale vacuole formation, cell death, and tumor suppression. We had earlier shown that this is an off-target effect of SB202190. Here we provide evidence that this vacuole formation is independent of ATG5-mediated canonical autophagosome initiation. While SB202190 interferes with autophagic flux in many cell lines parallel to vacuolation, autophagy-deficient DU-145 cells and CRISPR/Cas9 gene-edited ATG5-knockout A549 cells also undergo vacuolation upon SB202190 treatment. Late-endosomal GTPase RAB7 colocalizes with these compartments and RAB7 GTP-binding is essential for SB202190-induced vacuolation. A screen for modulators of SB202190-induced vacuolation revealed molecules including multi-kinase inhibitor sorafenib as inhibitors of vacuolation and sorafenib co-treatment enhanced cytotoxicity of SB202190. Moreover, VE-821, an ATR inhibitor was found to phenocopy the cell-type specific vacuolation response of SB202190. To identify the factors determining the cell-type specificity of vacuolation induced by SB-compounds and VE-821, we compared the transcriptomics data from vacuole-forming and non-vacuole-forming cancer cell lines and identified a gene expression signature that may define sensitivity of cells to these small-molecules. Further analyses using small molecule tools and the gene signature discovered here, could reveal novel mechanisms regulating this interesting anti-cancer phenotype.}, }
@article {pmid39894896, year = {2025}, author = {Banday, S and Mishra, AK and Rashid, R and Ye, T and Ali, A and Li, J and Yustein, JT and Kelliher, MA and Zhu, LJ and Deibler, SK and Malonia, SK and Green, MR}, title = {The O-glycosyltransferase C1GALT1 promotes EWSR1::FLI1 expression and is a therapeutic target for Ewing sarcoma.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {1267}, pmid = {39894896}, issn = {2041-1723}, support = {R01 CA096899/CA/NCI NIH HHS/United States ; }, mesh = {Humans ; Animals ; *Sarcoma, Ewing/genetics/metabolism/drug therapy/pathology ; *Galactosyltransferases/metabolism/genetics ; Cell Line, Tumor ; *RNA-Binding Protein EWS/metabolism/genetics ; Mice ; *Proto-Oncogene Protein c-fli-1/metabolism/genetics ; Gene Expression Regulation, Neoplastic ; Signal Transduction ; *Bone Neoplasms/genetics/metabolism/drug therapy/pathology ; Oncogene Proteins, Fusion/metabolism/genetics ; CRISPR-Cas Systems ; Xenograft Model Antitumor Assays ; Hedgehog Proteins/metabolism ; }, abstract = {Ewing sarcoma (ES) is an aggressive bone cancer driven by the oncogenic fusion-protein EWSR1::FLI1, which is not present in normal cells and is therefore an attractive therapeutic target. However, as a transcription factor, EWSR1::FLI1 is considered undruggable. Factors that promote EWSR1::FLI1 expression, and thus whose inhibition would reduce EWSR1::FLI1 protein levels and function, are potential drug targets. Here, using genome-scale CRISPR/Cas9 knockout screening, we identify C1GALT1, a galactosyltransferase required for the biosynthesis of many O-glycoproteins, as a factor that promotes EWSR1::FLI1 expression. We show that C1GALT1 acts by O-glycosylating the pivotal Hedgehog (Hh) signaling component Smoothened (SMO), thereby stabilizing SMO and stimulating the Hh pathway, which we find directly activates EWSR1::FLI1 transcription. Itraconazole, an FDA-approved anti-fungal agent that is known to inhibit C1GALT1, reduces EWSR1::FLI1 levels in ES cell lines and suppresses growth of ES xenografts in mice. Our study reveals a therapeutically targetable mechanism that promotes EWSR1::FLI1 expression and ES tumor growth.}, }
@article {pmid39894446, year = {2025}, author = {Wan, L and Zhong, P and Li, P and Ren, Y and Wang, W and Yu, M and Feng, HY and Yan, Z}, title = {CRISPR-based epigenetic editing of Gad1 improves synaptic inhibition and cognitive behavior in a Tauopathy mouse model.}, journal = {Neurobiology of disease}, volume = {206}, number = {}, pages = {106826}, doi = {10.1016/j.nbd.2025.106826}, pmid = {39894446}, issn = {1095-953X}, mesh = {Animals ; *Tauopathies/genetics/metabolism/therapy ; *Glutamate Decarboxylase/genetics/metabolism ; Mice ; *Gene Editing/methods ; Humans ; *Epigenesis, Genetic ; Disease Models, Animal ; Mice, Transgenic ; Prefrontal Cortex/metabolism ; CRISPR-Cas Systems ; *Cognition/physiology ; Male ; Mice, Inbred C57BL ; *Neural Inhibition/physiology/genetics ; Synaptic Transmission/physiology ; Epigenome Editing ; }, abstract = {GABAergic signaling in the brain plays a key role in regulating synaptic transmission, neuronal excitability, and cognitive processes. Large-scale sequencing has revealed the diminished expression of GABA-related genes in Alzheimer's disease (AD), however, it is largely unclear about the epigenetic mechanisms that dysregulate the transcription of these genes in AD. We confirmed that GABA synthesizing enzymes, GAD1 and GAD2, were significantly downregulated in prefrontal cortex (PFC) of AD human postmortem tissues. A tauopathy mouse model also had the significantly reduced expression of GABA-related genes, as well as the diminished GABAergic synaptic transmission in PFC pyramidal neurons. To elevate endogenous Gad1 levels, we used the CRISPR/Cas9-based epigenome editing technology to recruit histone acetyltransferase p300 to Gad1. Cells transfected with a fusion protein consisting of the nuclease-null dCas9 protein and the catalytic core of p300 (dCas9[p300]), as well as a guide RNA targeting Gad1 promoter (gRNA[Gad1]), had significantly increased Gad1 mRNA expression and histone acetylation at Gad1 promoter. Furthermore, the tauopathy mouse model with PFC injection of dCas9[p300] and gRNA[Gad1] lentiviruses had significantly elevated GABAergic synaptic currents and improved spatial memory. These results have provided an epigenetic editing-based gene-targeting strategy to restore synaptic inhibition and cognitive function in AD and related disorders.}, }
@article {pmid39894213, year = {2025}, author = {Heidarian, Y and Fasteen, TD and Mungcal, L and Buddika, K and Mahmoudzadeh, NH and Nemkov, T and D'Alessandro, A and Tennessen, JM}, title = {Hypoxia-inducible factor 1α is required to establish the larval glycolytic program in Drosophila melanogaster.}, journal = {Molecular metabolism}, volume = {93}, number = {}, pages = {102106}, pmid = {39894213}, issn = {2212-8778}, support = {P40 OD010949/OD/NIH HHS/United States ; P40 OD018537/OD/NIH HHS/United States ; R35 GM119557/GM/NIGMS NIH HHS/United States ; U41 HG000739/HG/NHGRI NIH HHS/United States ; }, mesh = {Animals ; *Glycolysis/genetics ; Larva/metabolism/genetics ; *Hypoxia-Inducible Factor 1, alpha Subunit/metabolism/genetics ; *Drosophila melanogaster/metabolism/genetics/growth &amp; development ; *Drosophila Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Gene Expression Regulation, Developmental ; Carbohydrate Metabolism/genetics ; DNA-Binding Proteins ; }, abstract = {OBJECTIVES: The rapid growth that occurs during Drosophila larval development requires a dramatic rewiring of central carbon metabolism to support biosynthesis. Larvae achieve this metabolic state, in part, by coordinately up-regulating the expression of genes involved in carbohydrate metabolism. The resulting metabolic program exhibits hallmark characteristics of aerobic glycolysis and establishes a physiological state that supports growth. To date, the only factor known to activate the larval glycolytic program is the Drosophila Estrogen-Related Receptor (dERR). However, dERR is dynamically regulated during the onset of this metabolic switch, indicating that other factors must be involved. Here we examine the possibility that the Drosophila ortholog of Hypoxia inducible factor 1α (Hif1α) is also required to activate the larval glycolytic program.<br><br>METHODS: CRISPR/Cas9 was used to generate new loss-of-function alleles in the Drosophila gene similar (sima), which encodes the sole fly ortholog of Hif1&#x3b1;. The resulting mutant strains were analyzed using a combination of metabolomics and RNAseq for defects in carbohydrate metabolism.<br><br>RESULTS: Our studies reveal that sima mutants fail to activate aerobic glycolysis and die during larval development with metabolic phenotypes that mimic those displayed by dERR mutants. Moreover, we demonstrate that dERR and Sima/Hif1&#x3b1; protein accumulation is mutually dependent, as loss of either transcription factor results in decreased abundance of the other protein.<br><br>CONCLUSIONS: These findings demonstrate that Sima/HIF1&#x3b1; is required during embryogenesis to coordinately up-regulate carbohydrate metabolism in preparation for larval growth. Notably, our study also reveals that the Sima/HIF1&#x3b1;-dependent gene expression program shares considerable overlap with that observed in dERR mutant, suggesting that Sima/HIF1&#x3b1; and dERR cooperatively regulate embryonic and larval glycolytic gene expression.}, }
@article {pmid39894193, year = {2025}, author = {Saini, A and Dilbaghi, N and Yadav, N}, title = {CRISPR integrated biosensors: A new paradigm for cancer detection.}, journal = {Clinica chimica acta; international journal of clinical chemistry}, volume = {569}, number = {}, pages = {120179}, doi = {10.1016/j.cca.2025.120179}, pmid = {39894193}, issn = {1873-3492}, mesh = {*Biosensing Techniques/methods ; Humans ; *Neoplasms/diagnosis/genetics ; *CRISPR-Cas Systems/genetics ; Biomarkers, Tumor/genetics ; }, abstract = {Cancer remains one of the leading causes of morbidity and mortality globally, necessitating need for advancements of technologies for early therapeutics. Conventional detection methodologies often lag behind in terms of sensitivity, specificity, and cost-effectiveness, leading to delayed diagnosis and inadequate treatment. The need of advanced diagnostic techniques has considerably increased and led to the development of biosensors. Biosensing technologies offer several advantages over conventional methods hence, overcome limitations and improve diagnostic accuracy. Biosensors, particularly CRISPR-Cas based biosensors have emerged as a revolutionary technology for oncology diagnostics due to their high precision and adaptability. CRISPR-based biosensors provide remarkable precision, sensitivity, multiplexing capabilities, specificity, and rapidness for developing a cost-effective and portable point of care diagnostic device for cancer detection. In this review, we have discussed cancer pathogenicity, assessed the traditional detection techniques, and explored the advancements and advantages of biosensors, particularly CRISPR-based biosensors, in the detection of some major cancer types, namely lung, liver, colorectal, prostate, and cervical cancers. CRISPR-based biosensors represent a significant potential in cancer diagnostics, offering precise, cost-effective, and rapid detection of cancer biomarkers. The integration of CRISPR technology with biosensors holds substantial promise for enhancing early detection and improving patient outcomes in cancer diagnostics.}, }
@article {pmid39893949, year = {2025}, author = {Zhang, Y and Su, R and Zhang, Z and Jiang, Y and Miao, Y and Zhou, S and Ji, M and Hsu, CW and Xu, H and Li, Z and Wang, G}, title = {An ultrasensitive one-pot Cas13a-based microfluidic assay for rapid multiplexed detection of microRNAs.}, journal = {Biosensors &amp; bioelectronics}, volume = {274}, number = {}, pages = {117212}, doi = {10.1016/j.bios.2025.117212}, pmid = {39893949}, issn = {1873-4235}, mesh = {*MicroRNAs/genetics/isolation &amp; purification/analysis ; Humans ; *Biosensing Techniques/instrumentation ; *CRISPR-Cas Systems/genetics ; *Neoplasms/genetics/diagnosis ; *Lab-On-A-Chip Devices ; Biomarkers, Tumor/genetics ; Cell Line, Tumor ; }, abstract = {Aberrant microRNA expression is associated with tumor progression in various organs. Detecting microRNAs as clinical cancer biomarkers can facilitate early cancer diagnosis and monitoring. However, the rapid and accurate quantification of microRNAs from biological samples remains a significant challenge. Here we developed a one-pot isothermal assay utilizing a molecular circuit with CRISPR/Cas13a (CRISPR-circuit) to rapidly convert, amplify and report different microRNAs within 15 min at the attomolar (aM) level. Then the full process was performed on an active centrifugal microfluidic chip and its corresponding portable equipment for parallel detection of multiple microRNAs, including miR-21, miR-141, miR-196a, and miR-1246. We also demonstrated its application for identifying cell lines and clinical samples of cancer patients with varying microRNA levels, which showed a strong correlation with the RT-qPCR. The assay can be easily adapted for the detection of any microRNA by simply modifying the converter primer, thereby holding significant potential for accurate disease detection and clinical diagnosis.}, }
@article {pmid39893892, year = {2025}, author = {Zheng, L and Zheng, C and Wang, W and Huang, F and Jiang, Y and Lu, J and Lou, Y}, title = {A CRISPR/Cas12a-based colorimetric AuNPs biosensor for naked-eye detection of pathogenic bacteria in clinical samples.}, journal = {Colloids and surfaces. B, Biointerfaces}, volume = {250}, number = {}, pages = {114541}, doi = {10.1016/j.colsurfb.2025.114541}, pmid = {39893892}, issn = {1873-4367}, mesh = {*Biosensing Techniques/methods ; *Colorimetry/methods ; *Gold/chemistry ; *Metal Nanoparticles/chemistry ; *Pseudomonas aeruginosa/isolation &amp; purification/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; DNA, Single-Stranded/chemistry/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; }, abstract = {Pathogenic bacteria, such as Pseudomonas aeruginosa, pose significant threats to public health due to their multidrug resistance and association with severe infections. Rapid and reliable detection methods are crucial for timely treatment and effective infection control, especially in resource-limited settings. In this study, we developed a CRISPR/Cas12a-based colorimetric biosensor that leverages Cas12a's trans-cleavage activity to release left single-stranded DNA (lDNA). The released lDNA facilitates hybridization with clDNA-functionalized gold nanoparticles (AuNPs), resulting in a visible color change. The biosensor achieved a detection limit of 10[0] CFU/reaction for P. aeruginosa within 2 hours, with excellent specificity and robustness, as validated in spiked sputum and blood samples. Clinical testing using 32 blood samples (13 positive, 19 negative) confirmed its high diagnostic accuracy, achieving an AUC of 1 in ROC curve analysis. The platform's simplicity, robustness, and programmability suggest its broad potential for rapid infectious disease diagnostics, particularly in low-resource settings.}, }
@article {pmid39893465, year = {2025}, author = {Li, B and Shang, Y and Wang, L and Lv, J and Wu, Q and Wang, F and Chao, J and Mao, J and Ding, A and Wu, X and Xue, K and Chen, C and Cui, M and Sun, Y and Zhang, H and Dai, C}, title = {Efficient genome editing in dicot plants using calreticulin promoter-driven CRISPR/Cas system.}, journal = {Molecular horticulture}, volume = {5}, number = {1}, pages = {9}, pmid = {39893465}, issn = {2730-9401}, }
@article {pmid39893321, year = {2025}, author = {Gumusgoz, E and Kasiri, S and Youssef, I and Verma, M and Chopra, R and Villarreal Acha, D and Wu, J and Marriam, U and Alao, E and Chen, X and Guisso, DR and Gray, SJ and Shah, BR and Minassian, BA}, title = {Focused ultrasound widely broadens AAV-delivered Cas9 distribution and activity.}, journal = {Gene therapy}, volume = {32}, number = {3}, pages = {237-245}, pmid = {39893321}, issn = {1476-5462}, support = {P01 NS097197/NS/NINDS NIH HHS/United States ; R01 NS127900/NS/NINDS NIH HHS/United States ; P01NS097197//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; }, mesh = {*Dependovirus/genetics ; Animals ; Mice ; *Genetic Therapy/methods ; Blood-Brain Barrier/metabolism ; Hippocampus/metabolism ; Genetic Vectors/administration &amp; dosage/genetics ; *CRISPR-Associated Protein 9/genetics ; Gene Editing/methods ; CRISPR-Cas Systems ; Humans ; Gene Transfer Techniques ; Disease Models, Animal ; }, abstract = {Because children have little temporal exposure to environment and aging, most pediatric neurological diseases are inherent, i.e. genetic. Since postnatal neurons and astrocytes are mostly non-replicating, gene therapy and genome editing present enormous promise in child neurology. Unlike in other organs, which are highly permissive to adeno-associated viruses (AAV), the mature blood-brain barrier (BBB) greatly limits circulating AAV distribution to the brain. Intrathecal administration improves distribution but to no more than 20% of brain cells. Focused ultrasound (FUS) opens the BBB transiently and safely. In the present work we opened the hippocampal BBB and delivered a Cas9 gene via AAV9 intrathecally. This allowed brain first-pass, and subsequent vascular circulation and re-entry through the opened BBB. The mouse model used was of Lafora disease, a neuroinflammatory disease due to accumulations of misshapen overlong-branched glycogen. Cas9 was targeted to the gene of the glycogen branch-elongating enzyme glycogen synthase. We show that FUS dramatically (2000-fold) improved hippocampal Cas9 distribution and greatly reduced the pathogenic glycogen accumulations and hippocampal inflammation. FUS is in regular clinical use for other indications. Our work shows that it has the potential to vastly broaden gene delivery or editing along with clearance of corresponding pathologic basis of brain disease.}, }
@article {pmid39893181, year = {2025}, author = {Hu, L and Han, J and Wang, HD and Cheng, ZH and Lv, CC and Liu, DF and Yu, HQ}, title = {A universal and wide-range cytosine base editor via domain-inlaid and fidelity-optimized CRISPR-FrCas9.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {1260}, pmid = {39893181}, issn = {2041-1723}, support = {52322002//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Cytosine/metabolism ; Escherichia coli/genetics ; Pseudomonas aeruginosa/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mutation ; Shewanella/genetics ; }, abstract = {CRISPR-based base editor (BE) offer diverse editing options for genetic engineering of microorganisms, but its application is limited by protospacer adjacent motif (PAM) sequences, context preference, editing window, and off-target effects. Here, a series of iteratively improved cytosine base editors (CBEs) are constructed using the FrCas9 nickase (FrCas9n) with the unique PAM palindromic structure (NNTA) to alleviate these challenges. The deaminase domain-inlaid FrCas9n exhibits an editing range covering 38 nucleotides upstream and downstream of the palindromic PAM, without context preference, which is 6.3 times larger than that of traditional CBEs. Additionally, lower off-target editing is achieved when incorporating high-fidelity mutations at R61A and Q964A in FrCas9n, while maintaining high editing efficiency. The final CBE, HF-ID824-evoCDA-FrCas9n demonstrates broad applicability across different microbes such as Escherichia coli MG1655, Shewanella oneidensis MR-1, and Pseudomonas aeruginosa PAO1. Collectively, this tool offers robust gene editing for facilitating mechanistic studies, functional exploration, and protein evolution in microbes.}, }
@article {pmid39892116, year = {2025}, author = {Yu, L and Tang, Y and Sun, Y and Wang, H and Yi, H and Zhong, Y and Shao, Z and Zhou, S and He, S and Cao, K and Peng, L and Chen, Z}, title = {DMSO enhanced one-pot HDA-CRISPR/Cas12a biosensor for ultrasensitive detection of Monkeypox virus.}, journal = {Talanta}, volume = {287}, number = {}, pages = {127660}, doi = {10.1016/j.talanta.2025.127660}, pmid = {39892116}, issn = {1873-3573}, mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems ; *Monkeypox virus/isolation &amp; purification ; *Mpox, Monkeypox/diagnosis/virology ; *Nucleic Acid Amplification Techniques/methods ; DNA Helicases ; Dimethyl Sulfoxide ; }, abstract = {We present a dimethyl sulfoxide (DMSO)-enhanced one-pot HDA-CRISPR/Cas12a biosensor for the ultrasensitive detection of the monkeypox virus (MPXV). The MPXV B6R gene was initially amplified using DMSO-enhanced helicase-dependent amplification (HDA) in the bottom of the reaction tubes. DMSO was employed to enhance the amplification efficiency of HDA. CRISPR/Cas12a reagents, pre-added to the caps of the reaction tubes, were subsequently combined with HDA products to generate fluorescence signals. This DMSO-enhanced HDA-CRISPR/Cas12a biosensor enables the detection of synthetic B6R DNA within 1 hour, with a detection limit of 9 aM and a dynamic range of 10 aM to 100 pM. Our work demonstrated that 5% DMSO can enhance the sensitivity of the HDA -CRISPR/Cas12a assay by four orders of magnitude. For clinical applications, this approach can detect as low as 0.4 copies/μL of MPXV pseudovirus. A DMSO-enhanced HDA-CRISPR/Cas12a lateral flow biosensor (LFB) was developed for MPXV point-of-care testing (POCT), achieving a LOD of 10 fM. This method exhibits high specificity in distinguishing the monkeypox virus from closely related orthopoxviruses, including variola, vaccinia, cowpox, ectromelia, and camelpox. The assay is rapid (sample-to-answer times less than 1 h), cost-effective, and compatible with both fluorescence detection and the LFB for visual readouts.}, }
@article {pmid39891928, year = {2025}, author = {Madorsky Rowdo, FP and Martini, R and Ackermann, SE and Tang, CP and Tranquille, M and Irizarry, A and Us, I and Alawa, O and Moyer, JE and Sigouros, M and Nguyen, J and Al Assaad, M and Cheng, E and Ginter, PS and Manohar, J and Stonaker, B and Boateng, R and Oppong, JK and Adjei, EK and Awuah, B and Kyei, I and Aitpillah, FS and Adinku, MO and Ankomah, K and Osei-Bonsu, EB and Gyan, KK and Hoda, S and Newman, L and Mosquera, JM and Sboner, A and Elemento, O and Dow, LE and Davis, MB and Martin, ML}, title = {Kinome-Focused CRISPR-Cas9 Screens in African Ancestry Patient-Derived Breast Cancer Organoids Identify Essential Kinases and Synergy of EGFR and FGFR1 Inhibition.}, journal = {Cancer research}, volume = {85}, number = {3}, pages = {551-566}, pmid = {39891928}, issn = {1538-7445}, support = {R01 CA259396/CA/NCI NIH HHS/United States ; BCRF-22-191//Breast Cancer Research Foundation (BCRF)/ ; CA259396-01//National Cancer Institute (NCI)/ ; //Weill Cornell Medicine (WCM)/ ; }, mesh = {Humans ; *Organoids/drug effects/pathology/metabolism ; *Breast Neoplasms/genetics/drug therapy/pathology ; Female ; *CRISPR-Cas Systems ; *Receptor, Fibroblast Growth Factor, Type 1/antagonists &amp; inhibitors/genetics ; ErbB Receptors/antagonists &amp; inhibitors/genetics ; *Protein Kinase Inhibitors/pharmacology ; Black People/genetics ; }, abstract = {Precision medicine approaches to cancer treatment aim to exploit genomic alterations that are specific to individual patients to tailor therapeutic strategies. Yet, some targetable genes and pathways are essential for tumor cell viability even in the absence of direct genomic alterations. In underrepresented populations, the mutational landscape and determinants of response to existing therapies are poorly characterized because of limited inclusion in clinical trials and studies. One way to reveal tumor essential genes is with genetic screens. Most screens are conducted on cell lines that bear little resemblance to patient tumors, after years of culture under nonphysiologic conditions. To address this problem, we aimed to develop a CRISPR screening pipeline in three-dimensionally grown patient-derived tumor organoid (PDTO) models. A breast cancer PDTO biobank that focused on underrepresented populations, including West African patients, was established and used to conduct a negative-selection kinome-focused CRISPR screen to identify kinases essential for organoid growth and potential targets for combination therapy with EGFR or MEK inhibitors. The screen identified several previously unidentified kinase targets, and the combination of FGFR1 and EGFR inhibitors synergized to block organoid proliferation. Together, these data demonstrate the feasibility of CRISPR-based genetic screens in patient-derived tumor models, including PDTOs from underrepresented patients with cancer, and identify targets for cancer therapy. Significance: Generation of a breast cancer patient-derived tumor organoid biobank focused on underrepresented populations enabled kinome-focused CRISPR screening that identified essential kinases and potential targets for combination therapy with EGFR or MEK inhibitors. See related commentary by Trembath and Spanheimer, p. 407.}, }
@article {pmid39891927, year = {2025}, author = {Trembath, HE and Spanheimer, PM}, title = {In Search of Representative Translational Cancer Model Systems.}, journal = {Cancer research}, volume = {85}, number = {3}, pages = {407-409}, doi = {10.1158/0008-5472.CAN-24-3879}, pmid = {39891927}, issn = {1538-7445}, mesh = {Humans ; *Translational Research, Biomedical/methods ; Precision Medicine/methods ; Organoids/pathology ; Female ; *Breast Neoplasms/genetics/pathology/drug therapy ; CRISPR-Cas Systems ; *Neoplasms/genetics/pathology ; Tumor Microenvironment ; }, abstract = {Racial disparities in cancer outcomes are well documented across tumor types. For patients with breast cancer, Black women are more likely to present with more aggressive molecular features and more likely to die from disease, even after accounting for those features. Recent efforts have been aimed at developing translational model systems for precision medicine strategies, and a major focus has been on patient-derived organoids. Organoids allow for robust in vitro experimental platforms, including drug and CRISPR screens while maintaining more complex cancer and tumor microenvironment subpopulations than cell lines. For results that are broadly translationally relevant, it is important that cancer models are derived from the spectrum of human disease and humans with disease. In this issue of Cancer Research, Madorsky Rowdo and colleagues derive breast cancer organoids from patients with African ancestry and use CRISPR-Cas9 screens to identify novel therapeutic vulnerabilities. These findings demonstrate the promise of representative cancer model systems to facilitate discoveries that are most likely to translate to improved therapy for all patients. See related article by Madorsky Rowdo et al., p. 551.}, }
@article {pmid39891757, year = {2025}, author = {Roychowdhury, R and Das, SP and Das, S and Biswas, S and Patel, MK and Kumar, A and Sarker, U and Choudhary, SP and Das, R and Yogendra, K and Gangurde, SS}, title = {Advancing vegetable genetics with gene editing: a pathway to food security and nutritional resilience in climate-shifted environments.}, journal = {Functional &amp; integrative genomics}, volume = {25}, number = {1}, pages = {31}, pmid = {39891757}, issn = {1438-7948}, mesh = {*Gene Editing/methods ; *Food Security ; *Vegetables/genetics/growth &amp; development ; *Climate Change ; CRISPR-Cas Systems ; Plant Breeding ; Crops, Agricultural/genetics ; Humans ; }, abstract = {As global populations grow and climate change increasingly disrupts agricultural systems, ensuring food security and nutritional resilience has become a critical challenge. In addition to grains and legumes, vegetables are very important for both human and animals because they contain vitamins, minerals, and fibre. Enhancing the ability of vegetables to withstand climate change threats is essential; however, traditional breeding methods face challenges due to the complexity of the genomic clonal multiplication process. In the postgenomic era, gene editing (GE) has emerged as a powerful tool for improving vegetables. GE can help to increase traits such as abiotic stress tolerance, herbicide tolerance, and disease resistance; improve agricultural productivity; and improve nutritional content and shelf-life by fine-tuning key genes. GE technologies such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR-Cas9) have revolutionized vegetable breeding by enabling specific gene modifications in the genome. This review highlights recent advances in CRISPR-mediated editing across various vegetable species, highlighting successful modifications that increase their resilience to climatic stressors. Additionally, it explores the potential of GE to address malnutrition by increasing the nutrient content of vegetable crops, thereby contributing to public health and food system sustainability. Additionally, it addresses the implementation of GE-guided breeding strategies in agriculture, considering regulatory, ethical, and public acceptance issues. Enhancing vegetable genetics via GE may provide a reliable and nutritious food supply for an expanding global population under more unpredictable environmental circumstances.}, }
@article {pmid39890862, year = {2025}, author = {Nalefski, EA and Sinan, S and Cantera, JL and Kim, AG and Kooistra, RM and Rivera, RE and Janshen, JP and Bhadra, S and Bishop, JD and Ellington, AD and Finklestein, IJ and Madan, D}, title = {Room temperature CRISPR diagnostics for low-resource settings.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {3909}, pmid = {39890862}, issn = {2045-2322}, mesh = {Humans ; Temperature ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Human papillomavirus 16/genetics/isolation &amp; purification ; *Papillomavirus Infections/diagnosis/virology ; Endodeoxyribonucleases/metabolism/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; Female ; Recombinases/metabolism ; Sensitivity and Specificity ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics/metabolism ; }, abstract = {Maintaining elevated reaction temperatures and multi-step sample preparations increases the costs and complexity of diagnostics, impeding their deployment in low-resource settings. Here, we develop a one-pot, room temperature recombinase polymerase amplification (RPA)-CRISPR reaction that removes these critical challenges. We show that RPA amplification is reduced by several orders of magnitude at 25 °C as compared to 37 °C. Similarly, when coupled to RPA, the performance of multiple Cas12a orthologs, including the widely used LbCas12a, is severely compromised at temperatures below 37 °C. To mitigate these limitations, we identify the ortholog TsCas12a as a highly active nuclease at 25 °C and develop a single-protocol RPA-Cas12a detection reaction with this enzyme. A quantitative kinetic analysis reveals that fast nuclease activation is more critical than higher steady-state trans-cleavage activity for room temperature diagnostic applications. RPA-TsCas12a reactions performed at 25 °C effectively detected HPV-16 in crudely prepared cervical swab samples with high sensitivity and specificity using both optical and lateral flow readouts. The reactions developed herein reduce the complexity and equipment requirements for affordable diagnostics in low- and middle-income countries.}, }
@article {pmid39890502, year = {2025}, author = {Kamran, M and Waters, MT}, title = {Engineering crop resilience with synthetic gene circuits.}, journal = {Trends in plant science}, volume = {30}, number = {6}, pages = {582-584}, doi = {10.1016/j.tplants.2025.01.005}, pmid = {39890502}, issn = {1878-4372}, mesh = {*Crops, Agricultural/genetics/physiology ; *Genetic Engineering/methods ; *Gene Regulatory Networks ; CRISPR-Cas Systems ; *Genes, Synthetic ; Gene Expression Regulation, Plant ; Plants, Genetically Modified/genetics ; Stress, Physiological/genetics ; }, abstract = {Engineering crops to withstand environmental stresses is critical for addressing climate change and food insecurity. Recently, Khan et al. developed CRISPR interference (CRISPRi)-based synthetic gene circuits to program gene expression in plants. Their findings highlight the potential of these circuits to advance the development of stress-resilient crops.}, }
@article {pmid39889868, year = {2025}, author = {Zhao, Q and Wei, L and Chen, Y}, title = {From bench to bedside: Developing CRISPR/Cas-based therapy for ocular diseases.}, journal = {Pharmacological research}, volume = {213}, number = {}, pages = {107638}, doi = {10.1016/j.phrs.2025.107638}, pmid = {39889868}, issn = {1096-1186}, mesh = {Humans ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Eye Diseases/therapy/genetics ; *Genetic Therapy/methods ; }, abstract = {Vision-threatening disorders, including both hereditary and multifactorial ocular diseases, necessitate innovative therapeutic approaches. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has emerged as a promising tool for treating ocular diseases through gene editing and expression regulation. This system has contributed to the development of representative disease models, including animal models, organoids, and cell lines, thereby facilitating investigations into the pathogenesis of disease-related genes. Besides, therapeutic applications of CRISPR/Cas have been extensively explored in preclinical in vitro and in vivo studies, targeting various ocular conditions, such as retinitis pigmentosa, Leber congenital amaurosis, Usher syndrome, fundus neovascular diseases, glaucoma, and corneal diseases. Recent advancements have demonstrated the technology's potential to restore cellular homeostasis and alleviate disease phenotypes, thereby prompting a variety of clinical trials. To date, active trials include treatments for primary open angle glaucoma with MYOC mutations, refractory herpetic viral keratitis, CEP290-associated inherited retinal degenerations, neovascular age-related macular degeneration, and retinitis pigmentosa with RHO mutations. However, challenges remain, primarily concerning off-target effects, immunogenicity, ethical considerations, and regulatory particularity. To reach higher safety and efficiency before truly transitioning from bench to bedside, future research should concentrate on improving the specificity and efficacy of Cas proteins, optimizing delivery vectors, and broadening the applicability of therapeutic targets. This review summarizes the application strategies and delivery methods of CRISPR/Cas, discusses recent progress in CRISPR/Cas-based disease models and therapies, and provides an overview of the landscape of clinical trials. Current obstacles and future directions regarding the bench-to-bedside transition are also discussed.}, }
@article {pmid39889859, year = {2025}, author = {Fujii, T and Hino, M and Fujimoto, T and Kakino, K and Kaneko, Y and Abe, H and Lee, JM and Kusakabe, T and Shimada, T}, title = {Peroxiredoxin 6 is essential for the posttranslational activation of xanthine dehydrogenase in the uric acid synthesis of Bombyx mori.}, journal = {Insect biochemistry and molecular biology}, volume = {178}, number = {}, pages = {104264}, doi = {10.1016/j.ibmb.2025.104264}, pmid = {39889859}, issn = {1879-0240}, mesh = {Animals ; *Bombyx/genetics/metabolism/enzymology/growth &amp; development ; *Xanthine Dehydrogenase/metabolism/genetics ; *Uric Acid/metabolism ; *Insect Proteins/metabolism/genetics ; *Peroxiredoxins/metabolism/genetics ; Male ; Protein Processing, Post-Translational ; Larva/metabolism/genetics/growth &amp; development ; Female ; CRISPR-Cas Systems ; }, abstract = {We identified a novel mutant of Bombyx mori, designated as male-absent oily (genetic symbol: om). The larval integument of this mutant is translucent due to a lack of uric acid in the integument. This mutation is Z-linked, and as mutant females are infertile, it is impossible to obtain om homozygous males. Using positional cloning combined with RNA-seq analysis, we identified a 1-bp deletion in the B. mori peroxiredoxin 6 (BmPrx6) gene. CRISPR/Cas9 knockout of BmPrx6 resulted in a translucent larval integument, indicating BmPrx6 as the causative gene for the om locus. Xanthine dehydrogenase (XDH)/xanthine oxidase (XO) is a key enzyme for uric acid synthesis. Injection of bovine XO into om mutants rescued the translucent phenotype, indicating that om is a mutant with defective XDH activity. To investigate XDH in B. mori, we generated a FLAG-tagged XDH gene using the CRISPR/Cas9 knock-in approach. Western blot analysis of XDH in om mutants revealed that BmPrx6 is crucial for the posttranslational activation of XDH. The role of BmPrx6 in regulating XDH activity is discussed.}, }
@article {pmid39889213, year = {2025}, author = {Huang, D and He, Y and Xu, C and Shen, P and Li, M and Fang, M and Xu, Z and Fang, X}, title = {DNAzyme-Triggered Equilibrium Transfer with Self-Activated CRISPR-Cas12a Biosensor Enables One-Pot Diagnosis of Nucleic Acids.}, journal = {Analytical chemistry}, volume = {97}, number = {5}, pages = {3026-3035}, doi = {10.1021/acs.analchem.4c06066}, pmid = {39889213}, issn = {1520-6882}, mesh = {*Biosensing Techniques/methods ; *DNA, Catalytic/metabolism/chemistry ; *CRISPR-Cas Systems ; HIV-1/genetics ; Humans ; Nucleic Acid Amplification Techniques ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins ; }, abstract = {Integrating recombinase-polymerase amplification (RPA) with CRISPR-Cas12a holds significant potential to simplify and improve nucleic acid diagnostic procedures. However, current strategies face limitations, such as complexity, reduced efficiency, and potential compromises in Cas12a activity. In response, we developed a DNAzyme-triggered equilibrium transfer with a self-activated CRISPR-Cas12a biosensor (DESCRIBER) for integrated nucleic acid detection. This platform features varying balance points to minimize interference between RPA and Cas12a in one pot and maximize their activity at different stages. Initially, the reaction focused on RPA, while Cas12a was silenced by circular-crRNA (C-crRNA). Then, DNAzyme, the activator, was generated during the RPA process, which linearizes C-crRNA to activate Cas12a and transfer the equilibrium toward signal readout. Meanwhile, activated Cas12a can further linearize C-crRNA to promote self-activation and accelerate equilibrium transfer. According to this principle, highly sensitive detection of the HIV-1 genome, as low as 500 CPs/mL, was achieved within 1 h while maintaining universality in detecting common subtypes and specificity against opportunistic infectious pathogens. Compared with qRT-PCR, it also exhibited good accuracy in detecting 35 spiked samples. Overall, we believe that the proposed strategy will enhance existing CRISPR systems to promote their practical applications in clinical diagnosis.}, }
@article {pmid39888280, year = {2025}, author = {Rouatbi, N and Walters, AA and Zam, A and Lim, YM and Marrocu, A and Liam-Or, R and Anstee, JE and Arnold, JN and Wang, JT and Pollard, SM and Al-Jamal, KT}, title = {CD47 Knock-Out Using CRISPR-Cas9 RNA Lipid Nanocarriers Results in Reduced Mesenchymal Glioblastoma Growth In Vivo.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {12}, number = {12}, pages = {e2407262}, pmid = {39888280}, issn = {2198-3844}, support = {GN-000398//Brain Tumour Charity/ ; CRI3645//Cancer Research Institute / Wade F.B. Thompson CLIP/ ; MR/N013700/1/MRC_/Medical Research Council/United Kingdom ; WT103913/WT_/Wellcome Trust/United Kingdom ; DCRPGF&#x2216;100009/CRUK_/Cancer Research UK/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; 202021-30//Brain Research UK/ ; }, mesh = {*Glioblastoma/genetics/immunology/pathology/therapy ; Animals ; *CRISPR-Cas Systems/genetics ; *CD47 Antigen/genetics/metabolism ; Mice ; Humans ; *Nanoparticles ; *Brain Neoplasms/genetics/immunology ; Cell Line, Tumor ; Tumor Microenvironment/immunology ; Lipids ; Neoplastic Stem Cells/metabolism ; Liposomes ; }, abstract = {Immune checkpoint (ICP) blockade has shown limited effectiveness in glioblastoma (GBM), particularly in the mesenchymal subtype, where interactions between immune cells and glioblastoma cancer stem cells (GSCs) drive immunosuppression and therapy resistance. Tailoring ICPs specific to GSCs can enhance the antitumor immune response. This study proposes the use of lipid nanoparticles (LNPs) encapsulating CRISPR RNAs as an in vivo screening tool for ICPs in a syngeneic model of mesenchymal GSCs. Using PD-L1 and CD47 to validate the proof of concept, intratumoral administration of LNPs in orthotopic tumors achieved efficient editing of ICPs, leading to enhanced immune cell infiltration within the tumor microenvironment. Targeting CD47 reduced tumor growth, suggesting improved cancer cell sensitization to the immune system post-ICP editing. The study positions LNPs as a robust tool for in vivo validation of ICPs as therapeutic targets in clinically relevant GBM models. LNPs could serve as a screening tool in patient-derived xenografts to identify and optimize ICP combinations, potentially expediting ICP translation and enhancing personalized GBM immunotherapies.}, }
@article {pmid39887239, year = {2025}, author = {Chen, P-R and Wei, Y and Li, X and Yu, H-Y and Wang, S-G and Yuan, X-Z and Xia, P-F}, title = {Precision engineering of the probiotic Escherichia coli Nissle 1917 with prime editing.}, journal = {Applied and environmental microbiology}, volume = {91}, number = {2}, pages = {e0003125}, pmid = {39887239}, issn = {1098-5336}, support = {22278246//MOST | National Natural Science Foundation of China (NSFC)/ ; 2022HWYQ-017//Department of Science and Technology of Shandong Province/ ; ZR2021ME066//Natural Science Foundation of Shandong Province/ ; Qilu Young Scholar Program//Shandong University (SDU)/ ; NO. tstp20230604//Taishan Scholar Project of Shandong Province/ ; U20A20146, 22378233//MOST | National Natural Science Foundation of China (NSFC)/ ; }, mesh = {*Escherichia coli/genetics ; *Probiotics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; }, abstract = {CRISPR-Cas systems are transforming precision medicine with engineered probiotics as next-generation diagnostics and therapeutics. To promote human health and treat disease, engineering probiotic bacteria demands maximal versatility to enable non-natural functionalities while minimizing undesired genomic interferences. Here, we present a streamlined prime editing approach tailored for probiotic Escherichia coli Nissle 1917 utilizing only essential genetic modules, including Cas9 nickase from Streptococcus pyogenes, a codon-optimized reverse transcriptase, and a prime editing guide RNA, and an optimized workflow with longer induction. As a result, we achieved all types of prime editing in every individual round of experiments with efficiencies of 25.0%, 52.0%, and 66.7% for DNA deletion, insertion, and substitution, respectively. A comprehensive evaluation of off-target effects revealed a significant reduction in unintended mutations, particularly in comparison to two different base editing methods. Leveraging the prime editing system, we inserted a unique DNA sequence to barcode the edited strain and established an antibiotic-resistance-gene-free platform to enable non-natural functionalities. Our prime editing strategy presents a CRISPR-Cas system that can be readily implemented in any laboratories with the basic CRISPR setups, paving the way for future innovations in engineered probiotics.IMPORTANCEOne ultimate goal of gene editing is to introduce designed DNA variations at specific loci in living organisms with minimal unintended interferences in the genome. Achieving this goal is especially critical for creating engineered probiotics as living diagnostics and therapeutics to promote human health and treat diseases. In this endeavor, we report a customized prime editing system for precision engineering of probiotic Escherichia coli Nissle 1917. With such a system, we developed a barcoding system for tracking engineered strains, and we built an antibiotic-resistance-gene-free platform to enable non-natural functionalities. We provide not only a powerful gene editing approach for probiotic bacteria but also new insights into the advancement of innovative CRISPR-Cas systems.}, }
@article {pmid39887234, year = {2025}, author = {Xin, B and Liu, J and Li, J and Peng, Z and Gan, X and Zhang, Y and Zhong, C}, title = {CRISPR-guided base editor enables efficient and multiplex genome editing in bacterial cellulose-producing Komagataeibacter species.}, journal = {Applied and environmental microbiology}, volume = {91}, number = {2}, pages = {e0245524}, pmid = {39887234}, issn = {1098-5336}, support = {National Key R &amp; D Program of China//MOST | National Key Research and Development Program of China (NKPs)/ ; }, mesh = {*Gene Editing/methods ; *Cellulose/biosynthesis ; *Acetobacteraceae/genetics/metabolism ; *CRISPR-Cas Systems ; *Genome, Bacterial ; *Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {UNLABELLED: Bacterial cellulose (BC) is an extracellular polysaccharide produced by bacteria that has wide applications in the food industry, tissue engineering, and battery manufacturing. Genome editing of BC-producing Komagataeibacter species is expected to optimize BC production and its properties. However, the available technology can target only one gene at a time and requires foreign DNA templates, which may present a regulatory hurdle for genetically modified organisms. In this study, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-guided base editing method for Komagataeibacter species using Cas9 nickase and cytidine deaminase. Without foreign DNA templates, C-to-T conversions were performed within an 8 bp editing window with 90% efficiency. Double- and triple-gene editing was achieved with 80%-90% efficiency. Fusing uracil-DNA glycosylase with the base editor enabled C-to-G editing. The base editor worked efficiently with various Komagataeibacter species. Finally, mannitol metabolic genes were investigated using base-editing-mediated gene inactivation. This study provides a powerful tool for multiplex genome editing of Komagataeibacter species.<br><br>IMPORTANCE: Komagataeibacter, a bacterial genus belonging to the family Acetobacteraceae, has important applications in food and material biosynthesis. However, the genome editing of Komagataeibacter relies on traditional homologous recombination methods. Therefore, only one gene can be manipulated in each round using foreign DNA templates, which may present a regulatory hurdle for genetically modified organisms when microorganisms are used in the food industry. In this study, a powerful base editing technology was developed for Komagataeibacter species. C-to-T and C-to-G base conversions were efficiently implemented at up to three loci in the Komagataeibacter genome. This base editing system is expected to accelerate basic and applied research on Komagataeibacter species.}, }
@article {pmid39887213, year = {2025}, author = {Kyawe, PP and Liu, P and Jiang, Z and Bradley, ES and Cicuto, T and Trombly, MI and Silverman, N and Fitzgerald, KA and McDougall, WM and Wang, JP}, title = {CRISPR editing of candidate host factors that impact influenza A virus infection.}, journal = {Microbiology spectrum}, volume = {13}, number = {3}, pages = {e0262724}, pmid = {39887213}, issn = {2165-0497}, support = {N660011924036//U.S. Department of Defense (DOD)/ ; }, mesh = {Humans ; *Influenza A virus/physiology/genetics/pathogenicity ; A549 Cells ; Adenosine Deaminase/genetics/metabolism ; Virus Replication ; *Influenza, Human/virology/genetics ; CRISPR-Cas Systems ; *Host-Pathogen Interactions/genetics ; RNA-Binding Proteins/genetics/metabolism ; Gene Editing ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Influenza A virus (IAV) is a respiratory pathogen with a segmented negative-sense RNA genome that can cause epidemics and pandemics. The host factors required for the complete IAV infectious cycle have not been fully identified. Here, we examined three host factors for their contributions to IAV infectivity. We performed CRISPR-mediated knockout of cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS) as well as CRISPR-mediated overexpression of beta-1,4 N-acetylgalactosaminyltransferase 2 (B4GALNT2) and adenosine deaminase acting on RNA 1 (ADAR1) in the human bronchial epithelial A549 cell line and evaluated the impact on IAV and other RNA viruses. We confirmed that knockout of CMAS or overexpression of B4GALNT2 restricts IAV infection by diminishing binding to the cell surface but has no effect on vesicular stomatitis virus infection. Although ADAR1 overexpression does not significantly inhibit IAV replication, it has a pro-viral effect with coxsackie B virus (CVB) infection. This pro-viral effect is not likely secondary to reduced type I interferon (IFN) production, as the induction of the IFN-stimulated genes ISG15 and CXCL10 is negligible in both parent and ADAR1-overexpressing A549 cells following CVB challenge. In contrast, ISG15 and CXCL10 production is robust and equal for parent and ADAR1-overexpressing A549 cells challenged with IAV. Taken together, these data provide insights into how host factors can be further explored to understand the dynamics of pro- and anti-viral factors.IMPORTANCEInfluenza A virus (IAV) remains a global threat due to its ability to cause pandemics, making the identification of host factors essential for developing new antiviral strategies. In this study, we utilized CRISPR-based techniques to investigate host factors that impact IAV infectivity. Knockout of CMAS, a key enzyme in sialic acid biosynthesis, significantly reduced IAV binding and infection by disrupting sialic acid production on the cell surface. Overexpression of B4GALNT2 had similar effects, conferring resistance to IAV infection through diminished cell-surface binding. Overexpression of ADAR1, known for its role in RNA editing and immune regulation, impacted IAV replication minimally but enhanced coxsackie B virus replication. Such findings reveal the diverse roles of host factors in viral infection, offering insights for targeted therapeutic development against IAV and other pathogens.}, }
@article {pmid39887096, year = {2025}, author = {Brusson, M and Miccio, A}, title = {[A CRISPR/Cas approach to β-haemoglobinopathies].}, journal = {Medecine sciences : M/S}, volume = {41}, number = {1}, pages = {33-39}, doi = {10.1051/medsci/2024191}, pmid = {39887096}, issn = {1958-5381}, support = {ANR-10-IAHU-01//Agence nationale de la recherche/ ; 865797 DITSB//Conseil europ&#xe9;en de la recherche/ ; subvention HORIZON-RIA EDITSCD n&#xb0; 101057659//Commission europ&#xe9;enne/ ; subvention 22206//AFM-T&#xe9;l&#xe9;thon/ ; }, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods/trends ; *Gene Editing/methods ; *beta-Thalassemia/therapy/genetics ; Anemia, Sickle Cell/therapy/genetics ; *Hemoglobinopathies/therapy/genetics ; Fetal Hemoglobin/genetics ; Hematopoietic Stem Cells ; Animals ; }, abstract = {Beta-haemoglobinopathies are severe genetic anemias caused by mutations that affect adult haemoglobin production. Many therapeutic approaches aim to reactivate the expression of the fetal hemoglobin genes. To this end, the CRISPR/Cas9 system has recently been used to genetically modify patients' hematopoietic stem/progenitor cells ex vivo and reactivate fetal hemoglobin expression in their erythroid progeny. More than 70 patients with severe β-thalassemia and sickle cell disease have been treated with the Casgevy® therapy. Most have achieved a significant improvement of clinical phenotype, with high editing efficiency in hematopoietic cells associated with normal or near normal hemoglobin levels. While the long-term safety and efficacy of this powerful approach still need to be evaluated, new strategies are being developed to further improve therapeutic outcomes, reduce potential genotoxicity and lower the costs of therapy.}, }
@article {pmid39886746, year = {2025}, author = {Yuan, X and Geng, W and Ji, J and Yan, Z and Wei, J and Wu, Y and Yang, R}, title = {CRISPR/Cas13a-Programmed Cu NCs and Z-Scheme T-COF/Ag2S for Photoelectrochemical Biosensing of circRNA.}, journal = {ACS sensors}, volume = {10}, number = {2}, pages = {1270-1279}, doi = {10.1021/acssensors.4c03180}, pmid = {39886746}, issn = {2379-3694}, mesh = {*Biosensing Techniques/methods ; Humans ; *Copper/chemistry ; *RNA, Circular/blood/genetics ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; *Silver Compounds/chemistry ; Lung Neoplasms/blood ; *Metal Nanoparticles/chemistry ; Metal-Organic Frameworks/chemistry ; Photochemical Processes ; }, abstract = {Circular RNAs (circRNAs), as a class of noncoding RNA molecules with a circular structure exhibit high stability and spatiotemporal-specific expression, making them ideal cancer biomarkers for liquid biopsy. Herein, a new photoelectrochemical (PEC) biosensor for a highly sensitive circRNA assay in the whole blood of lung cancer patients was designed based on CRISPR/Cas13a-programmed Cu nanoclusters (Cu NCs) and a Z-scheme covalent organic framework/silver sulfide (T-COF/Ag2S) composite. This Z-scheme T-COF/Ag2S composite accelerates electron transfer and produces an excellent initial photocurrent. When CRISPR/Cas13a precisely targets circRNA, it nonspecifically cleaves the triple-helix molecular structure to release DNA fragments (C'/C"). After the C'/C" opens the DNA hairpin probe (HP) modified on the electrode, hybridization chain reactions are performed to produce abundant AT-rich double-stranded DNA with the addition of H1 and H2 probes. Upon the incubation of Cu[2+], Cu NCs are in situ formed via the A-Cu[2+]-T bonds and can effectively quench the photocurrent of the Z-scheme T-COF/Ag2S due to the energy transfer process. This developed PEC biosensor for the circRNA assay shows a low limit of detection of 0.5 fM, and the reusability of DNA-modified magnetic beads (MB-DNA) reduces the detection cost. Moreover, the PEC biosensor can accurately quantify the circRNA level and distinguish the circRNA expression in whole blood from healthy controls and lung cancer patients, offering strong potential in clinical diagnosis.}, }
@article {pmid39885543, year = {2025}, author = {Jiang, J and Cienfuegos-Gallet, AV and Long, T and Peirano, G and Chu, T and Pitout, JDD and Kreiswirth, BN and Chen, L}, title = {Intricate interplay of CRISPR-Cas systems, anti-CRISPR proteins, and antimicrobial resistance genes in a globally successful multi-drug resistant Klebsiella pneumoniae clone.}, journal = {Genome medicine}, volume = {17}, number = {1}, pages = {9}, pmid = {39885543}, issn = {1756-994X}, support = {R01 AI090155/AI/NIAID NIH HHS/United States ; }, mesh = {*Klebsiella pneumoniae/genetics/drug effects/classification ; *CRISPR-Cas Systems ; *Drug Resistance, Multiple, Bacterial/genetics ; Klebsiella Infections/microbiology/epidemiology ; Bacterial Proteins/genetics ; Humans ; Plasmids/genetics ; Genome, Bacterial ; beta-Lactamases/genetics ; Phylogeny ; Anti-Bacterial Agents/pharmacology ; }, abstract = {BACKGROUND: Klebsiella pneumoniae is one of the most prevalent pathogens responsible for multiple infections in healthcare settings and the community. K. pneumoniae CG147, primarily including ST147 (the founder ST), ST273, and ST392, is one of the most globally successful MDR clone linked to various carbapenemases.<br><br>METHODS: One hundred and one CG147 strains were sequenced and additional 911 publicly available CG147 genome sequences were included for analysis. The molecular epidemiology, population structure, and time phylogeny were investigated. The virulome, resistome, and mobilome were analyzed, and the recombination in the capsular region was studied. The CRISPR-Cas and anti-CRISPR were identified. The interplay between CRISPR-Cas, anti-CRISPR, and carbapenemase-encoding plasmids was analyzed and experimentally validated.<br><br>RESULTS: We analyzed 1012 global CG147 genomes, with 80.4% encoding at least one carbapenemase (NDM [529/1012, 52.3%], OXA-48-like [182/1012, 17.7%], and KPC [105/1012, 10.4%]). Surprisingly, almost all CG147 strains (99.7%, 1009/1,012) harbor a chromosomal&#xa0;type I-E CRISPR-Cas system, with 41.8% (423/1012) containing an additional plasmid-borne&#xa0;type IV-A3 CRISPR-Cas system, and both target IncF plasmids, e.g., the most prevalent KPC-encoding pKpQIL-like plasmids. We found the presence of IV-A3 CRISPR-Cas system showed a negative correlation with the presence of KPC. Interestingly, a prophage-encoding anti-CRISPR AcrIE8.1 and a plasmid-borne anti-CRISPR AcrIE9.2 were detected in 40.1% (406/1012) and 54.2% (548/1012) of strains, respectively, which displayed positive correlations with the presence of a carbapenemase. Plasmid transfer experiments confirmed that the I-E and IV-A3 CRISPR-Cas systems significantly decreased (p&#x2009;<&#x2009;0.001) KPC-encoding pKpQIL plasmid conjugation frequencies, while the AcrIE8.1 and AcrIE9.2 significantly increased (p&#x2009;<&#x2009;0.001) pKpQIL conjugation frequencies and protected plasmids from elimination by CRISPR-Cas I-E system.<br><br>CONCLUSIONS: Our results indicated a complex interplay between CRISPR-Cas, anti-CRISPR, and mobile genetic elements that shape the evolution of CG147. Our findings advance the understanding of multi-drug resistance mechanisms and will aid in preventing the emergence of future MDR clones.}, }
@article {pmid39885339, year = {2025}, author = {Low, SJ and O'Neill, M and Kerry, WJ and Wild, N and Krysiak, M and Nong, Y and Azzato, F and Hor, E and Williams, L and Taiaroa, G and Steinig, E and Pasricha, S and Williamson, DA}, title = {PathoGD: an integrative genomics approach to primer and guide RNA design for CRISPR-based diagnostics.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {147}, pmid = {39885339}, issn = {2399-3642}, support = {GA-F3791196-5514//State Government of Victoria (Victorian Government)/ ; PO4932//Department of Health, Australian Government (Department of Health)/ ; }, mesh = {*Genomics/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; *Streptococcus pyogenes/genetics/isolation &amp; purification ; *DNA Primers/genetics ; *Neisseria gonorrhoeae/genetics/isolation &amp; purification ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology/methods ; }, abstract = {Critical to the success of CRISPR-based diagnostic assays is the selection of a diagnostic target highly specific to the organism of interest, a process often requiring iterative cycles of manual selection, optimisation, and redesign. Here we present PathoGD, a bioinformatic pipeline for rapid and high-throughput design of RPA primers and gRNAs for CRISPR-Cas12a-based pathogen detection. PathoGD is fully automated, leverages publicly available sequences and is scalable to large datasets, allowing rapid continuous monitoring and validation of primer/gRNA sets to ensure ongoing assay relevance. We designed primers and gRNAs for five clinically relevant bacterial pathogens, and experimentally validated a subset of the designs for detecting Streptococcus pyogenes and/or Neisseria gonorrhoeae in assays with and without pre-amplification. We demonstrated high specificity of primers and gRNAs designed, with minimal off-target signal observed for all combinations. We anticipate PathoGD will be an important resource for assay design for current and emerging pathogens. PathoGD is available on GitHub at https://github.com/sjlow23/pathogd .}, }
@article {pmid39885211, year = {2025}, author = {Cheng, ZH and Luo, XY and Yu, SS and Min, D and Zhang, SX and Li, XF and Chen, JJ and Liu, DF and Yu, HQ}, title = {Tunable control of Cas12 activity promotes universal and fast one-pot nucleic acid detection.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {1166}, pmid = {39885211}, issn = {2041-1723}, mesh = {Humans ; SARS-CoV-2/genetics/isolation &amp; purification ; *CRISPR-Associated Proteins/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *COVID-19/diagnosis/virology ; Influenza A virus/genetics/isolation &amp; purification ; Saliva/virology ; *Endodeoxyribonucleases/metabolism/genetics ; Sensitivity and Specificity ; *Molecular Diagnostic Techniques/methods ; Heparin/chemistry ; *Bacterial Proteins/metabolism/genetics ; Wastewater/virology ; RNA, Viral ; }, abstract = {The CRISPR-based detection methods have been widely applied, yet they remain limited by the non-universal nature of one-pot diagnostic approaches. Here, we report a universal one-pot fluorescent method for the detection of epidemic pathogens, delivering results within 15-20 min. This method uses heparin sodium to precisely tunes the cis-cleavage capability of Cas12 via interference with the Cas12a-crRNA binding process, thereby generating significant fluorescence due to the accumulation of isothermal amplification products. Additionally, this universal assay accommodates both classic and suboptimal PAMs, as well as various Cas12a subtypes such as LbCas12a, AsCas12a, and AapCas12b. Such a robust method demonstrates sensitivity and specificity exceeding 95% in the detection of monkeypox pseudovirus, influenza A virus, and SARS-CoV-2 from saliva or wastewater samples, when compared with qPCR or RT-qPCR. Moreover, the cost of heparin sodium per thousand uses is $0.01 to $0.04 only. Collectively, this universal and fast one-pot approach based on heparin sodium offers potential possibilities for point-of-care testing.}, }
@article {pmid39885149, year = {2025}, author = {Jin, W and Deng, Y and La Marca, JE and Lelliott, EJ and Diepstraten, ST and König, C and Tai, L and Snetkova, V and Dorighi, KM and Hoberecht, L and Hedditch, MG and Whelan, L and Healey, G and Fayle, D and Lau, K and Potts, MA and Chen, MZ and Johnston, APR and Liao, Y and Shi, W and Kueh, AJ and Haley, B and Fortin, JP and Herold, MJ}, title = {Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {974}, pmid = {39885149}, issn = {2041-1723}, mesh = {Animals ; Mice ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Gene Knock-In Techniques/methods ; *Endodeoxyribonucleases/genetics/metabolism ; *Genetic Engineering/methods ; *Bacterial Proteins/genetics/metabolism ; Acidaminococcus/genetics/enzymology ; *CRISPR-Associated Proteins/genetics/metabolism ; Fibroblasts/metabolism ; Lymphoma/genetics ; }, abstract = {Cas12a is a next-generation gene editing tool that enables multiplexed gene targeting. Here, we present a mouse model that constitutively expresses enhanced Acidaminococcus sp. Cas12a (enAsCas12a) linked to an mCherry fluorescent reporter. We demonstrate efficient single and multiplexed gene editing in vitro, using primary and transformed cells from enAsCas12a mice. We further demonstrate successful in vivo gene editing, using normal and cancer-prone enAsCas12a stem cells to reconstitute the haematopoietic system of wild-type mice. We also present compact, genome-wide Cas12a knockout libraries, with four crRNAs per gene encoded across one (Scherzo) or two (Menuetto) vectors, and demonstrate the utility of these libraries across methodologies: in vitro enrichment and drop-out screening in lymphoma cells and immortalised fibroblasts, respectively, and in vivo screens to identify lymphoma-driving events. Finally, we demonstrate CRISPR multiplexing via simultaneous gene knockout (via Cas12a) and activation (via dCas9-SAM) using primary T cells and fibroblasts. Our enAsCas12a mouse and accompanying crRNA libraries enhance genome engineering capabilities and complement current CRISPR technologies.}, }
@article {pmid39884737, year = {2025}, author = {Jones, JD and Maloney, TD}, title = {sgRNA Single-Nucleotide Resolution by Ion-Pairing Reversed-Phase Chromatography.}, journal = {Analytical chemistry}, volume = {97}, number = {5}, pages = {2837-2844}, doi = {10.1021/acs.analchem.4c05325}, pmid = {39884737}, issn = {1520-6882}, mesh = {*Chromatography, Reverse-Phase/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; *Nucleotides/analysis ; }, abstract = {Single-stranded guide RNAs (sgRNAs) are important therapeutic modalities that facilitate selective genome editing by the CRISPR/Cas9 system. While these therapeutic modalities are synthesized through solid phase oligonucleotide synthesis similar to small interfering RNA (siRNAs) and antisense oligonucleotide (ASOs) therapeutics, their sequence length and complex secondary and tertiary structure hinder analytical characterization. The resulting current sgRNA methodologies have limited chromatographic selectivity near the FLP and limited MS compatibility. Here, we perform a systematic evaluation of chromatographic parameters to develop a highly selective ion-pairing reversed-phase chromatography separation for sgRNAs. We identify that stronger and more hydrophobic ion-pairing reagents promote the selectivity for long truncation impurities. Further, reduced flow rates and temperatures promote selectivity near the FLP. Together, this study reports the first single-nucleotide resolution chromatography method for an sgRNA modality while employing a highly MS-compatible mobile phase. This work provides further evidence that the sgRNA chromatographic selectivity is highly independent of on-column mass transfer. This methodology will enable the high-resolution characterization of sgRNA therapeutics, providing further insights into impurity profiles to facilitate toxicological studies and process development of these genetic medicines.}, }
@article {pmid39884612, year = {2025}, author = {He, D and Zhao, S and Wang, F and Wu, B and Wei, F and Zhao, Y and Wei, X and Ren, H and Zhang, M and Fan, Y and Zhang, J and Yu, S and Tang, Y and Diao, Y}, title = {H9N2 avian influenza virus diagnostics utilizing specific high-sensitivity enzymatic molecular system termed RPA-based CRISPR-Cas13a.}, journal = {International journal of biological macromolecules}, volume = {301}, number = {}, pages = {140474}, doi = {10.1016/j.ijbiomac.2025.140474}, pmid = {39884612}, issn = {1879-0003}, mesh = {*Influenza A Virus, H9N2 Subtype/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; Animals ; *Influenza in Birds/diagnosis/virology ; Humans ; Limit of Detection ; Sensitivity and Specificity ; }, abstract = {H9N2 avian influenza virus (AIV), a major pathogen causing respiratory infections in poultry, poses a significant threat to the poultry industry and human health. Early detection and control of H9N2 infections are essential for minimizing economic losses and preventing potential zoonotic transmission. A novel CRISPR-Cas family member called CRISPR-Cas13a comprises the CRISPR RNA (crRNA) and Cas13a nuclease. Through the crRNA-based reprogramming of Cas13a, a platform for sensing RNAs specifically is available. In this study, we developed a RPA-based CRISPR-Cas13a diagnostic method for rapid detection of the H9N2 AIV. The results demonstrated that at a limit of 10 copies/μL and 10[2] copies/μL could be detected within 50 min, by fluorescence detection and lateral flow strip, respectively, offering a highly sensitive method for H9N2 detection. This method exhibited excellent specificity, distinguishing H9N2 from other pathogens. Furthermore, the RPA-Cas13a-based detection system was tested on clinical samples, showing comparable performance to RT-qPCR. The detection results were visualized using either lateral flow assays or fluorescence, making it a suitable tool for on-site, field-deployable diagnostics. In a word, this RPA-Cas13a diagnostic approach offers high reliability, sensitivity, and specificity, with promising potential for rapidly detecting H9N2 and other viral pathogens in clinical and food safety applications.}, }
@article {pmid39884405, year = {2025}, author = {S, BR and Dhar, R and Devi, A}, title = {Exosomes-mediated CRISPR/Cas delivery: A cutting-edge frontier in cancer gene therapy.}, journal = {Gene}, volume = {944}, number = {}, pages = {149296}, doi = {10.1016/j.gene.2025.149296}, pmid = {39884405}, issn = {1879-0038}, mesh = {*Exosomes/genetics/metabolism ; Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/therapy/genetics ; *Genetic Therapy/methods ; *Gene Editing/methods ; Animals ; }, abstract = {Cancer is considered the second most common disease globally. In the past few decades, many approaches have been proposed for cancer treatment. One among those is targeted therapy using CRISPR/Cas system which plays a significant role in translational research through gene editing. However, due to its inability to cope with specific targeting, off-target effects, and limited tumor penetration, it is very challenging to use this approach in cancer studies. To increase its efficacy, CRISPR components are engineered into the extracellular vesicles (EVs), especially exosomes (a subpopulation of EVs). Exosomes have a significant role in cellular communication. Exosomes-based CRISPR/Cas system transport for gene editing enhances specificity, reduces off-target effects, and improves the therapeutic potential. This review highlights the role of exosomes and the CRISPR/Cas system in cancer research, exosomes-based CRISPR delivery for cancer treatment, and its future orientation.}, }
@article {pmid39883775, year = {2025}, author = {Koeppel, J and Ferreira, R and Vanderstichele, T and Riedmayr, LM and Peets, EM and Girling, G and Weller, J and Murat, P and Liberante, FG and Ellis, T and Church, GM and Parts, L}, title = {Randomizing the human genome by engineering recombination between repeat elements.}, journal = {Science (New York, N.Y.)}, volume = {387}, number = {6733}, pages = {eado3979}, doi = {10.1126/science.ado3979}, pmid = {39883775}, issn = {1095-9203}, mesh = {Humans ; *Genome, Human ; *Gene Editing/methods ; *Repetitive Sequences, Nucleic Acid ; *Recombination, Genetic ; CRISPR-Cas Systems ; Sequence Deletion ; Cell Line ; Translocation, Genetic ; }, abstract = {We lack tools to edit DNA sequences at scales necessary to study 99% of the human genome that is noncoding. To address this gap, we applied CRISPR prime editing to insert recombination handles into repetitive sequences, up to 1697 per cell line, which enables generating large-scale deletions, inversions, translocations, and circular DNA. Recombinase induction produced more than 100 stochastic megabase-sized rearrangements in each cell. We tracked these rearrangements over time to measure selection pressures, finding a preference for shorter variants that avoided essential genes. We characterized 29 clones with multiple rearrangements, finding an impact of deletions on expression of genes in the variant but not on nearby genes. This genome-scrambling strategy enables large deletions, sequence relocations, and the insertion of regulatory elements to explore genome dispensability and organization.}, }
@article {pmid39883459, year = {2025}, author = {Lin, J and Zhang, J and Zhu, X and Xia, X and Zhang, Y and Zeng, Q and Xu, Y and Deng, R and Li, J}, title = {Pyrococcus furiosus Argonaute-mediated dual recognition enables the detection of trace single-nucleotide-mutated fungicide-resistant fungal pathogens.}, journal = {Chemical communications (Cambridge, England)}, volume = {61}, number = {16}, pages = {3335-3338}, doi = {10.1039/d4cc06481j}, pmid = {39883459}, issn = {1364-548X}, mesh = {*Pyrococcus furiosus/genetics/metabolism ; *Drug Resistance, Fungal/genetics ; *Argonaute Proteins/metabolism/genetics ; *Fungicides, Industrial/pharmacology ; CRISPR-Cas Systems ; *Basidiomycota/genetics/drug effects ; Mutation ; }, abstract = {Detection of low-abundance mutations for the early discovery of fungicide-resistant fungal pathogens is highly demanded, but remains challenging. Herein, we developed a dual-recognition strategy, termed PARPA, involving Pyrococcus furiosus Argonaute (pfAgo)-mediated elimination of wild-type fungal genes and CRISPR/Cas12a-based amplicon recognition. This assay can detect fungicide-resistant Puccinia striiformis at relative abundances as low as 0.05% and has potential for achieving early screening of fungicide-resistant fungal pathogens.}, }
@article {pmid39883010, year = {2025}, author = {Zhang, W and Zhong, Y and Wang, J and Zou, G and Chen, Q and Liu, C}, title = {Direct repeat region 3' end modifications regulate Cas12a activity and expand its applications.}, journal = {Nucleic acids research}, volume = {53}, number = {3}, pages = {}, pmid = {39883010}, issn = {1362-4962}, support = {22307150//National Natural Science Foundation of China/ ; A2303012//Shenzhen Medical Research Fund/ ; 2024A1515012319//Guangdong Basic and Applied Basic Research Foundation/ ; JCYJ20230807110315032//Shenzhen Science and Technology Program/ ; }, mesh = {*CRISPR-Associated Proteins/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; Alkaline Phosphatase/metabolism ; alpha-Fetoproteins/metabolism/genetics ; *Bacterial Proteins/genetics/metabolism ; DNA/metabolism/genetics/chemistry ; Immunoglobulin G/metabolism ; }, abstract = {CRISPR-Cas12a technology has transformative potential, but as its applications grow, enhancing its inherent functionalities is essential to meet diverse demands. Here, we reveal a regulatory mechanism for LbCas12a through direct repeat (DR) region 3' end modifications and de-modifications, which can regulate LbCas12a's cis- and trans-cleavage activities. We extensively explored the effects of introducing phosphorylation, DNA, photo-cleavable linker, DNA modifications at the DR 3' end on LbCas12a's functionality. We find that the temporary inhibitory function of Cas12a can be reactivated by DR 3' end modification corresponding substances, such as alkaline phosphatase (ALP), immunoglobulin G (IgG), alpha-fetoprotein (AFP), DNA exonucleases, ultraviolet radiation, and DNA glycosylases, which greatly expand the scope of application of Cas12a. Clinical applications demonstrated promising results in ALP, AFP, and trace Epstein-Barr virus detection compared to gold standard methods. Our research provides valuable insights into regulating LbCas12a activity through direct modification of DR and significantly expands its potential clinical detection targets, paving the way for future universal clustered regularly interspaced short palindromic repeats (CRISPR) diagnostic strategies.}, }
@article {pmid39882846, year = {2025}, author = {Kanie, T and Liu, B and Love, JF and Fisher, SD and Gustavsson, AK and Jackson, PK}, title = {A hierarchical pathway for assembly of the distal appendages that organize primary cilia.}, journal = {eLife}, volume = {14}, number = {}, pages = {}, pmid = {39882846}, issn = {2050-084X}, support = {P20 GM103639/GM/NIGMS NIH HHS/United States ; R35 GM155365/GM/NIGMS NIH HHS/United States ; P20GM103447/GM/NIGMS NIH HHS/United States ; P30 CA225520/CA/NCI NIH HHS/United States ; R01 GM114276/GM/NIGMS NIH HHS/United States ; R01GM114276/GM/NIGMS NIH HHS/United States ; R35 GM151013/GM/NIGMS NIH HHS/United States ; R01GM121565/GM/NIGMS NIH HHS/United States ; S10 OD028536/OD/NIH HHS/United States ; R35GM155365/GM/NIGMS NIH HHS/United States ; C-2064-20210327//Welch Foundation/ ; RR200025//Cancer Prevention and Research Institute of Texas/ ; 1R35GM151013/GM/NIGMS NIH HHS/United States ; R00GM134187/GM/NIGMS NIH HHS/United States ; P20 GM103447/GM/NIGMS NIH HHS/United States ; R00 GM134187/GM/NIGMS NIH HHS/United States ; R01 GM121565/GM/NIGMS NIH HHS/United States ; }, mesh = {*Cilia/metabolism ; *Centrioles/metabolism ; Humans ; CRISPR-Cas Systems ; Animals ; }, abstract = {Distal appendages are ninefold symmetric blade-like structures attached to the distal end of the mother centriole. These structures are critical for the formation of the primary cilium, by regulating at least four critical steps: preciliary vesicle recruitment, recruitment and initiation of intraflagellar transport (IFT), and removal of CP110. While specific proteins that localize to the distal appendages have been identified, how exactly each protein functions to achieve the multiple roles of the distal appendages is poorly understood. Here, we comprehensively analyze known and newly discovered distal appendage proteins (CEP83, SCLT1, CEP164, TTBK2, FBF1, CEP89, KIZ, ANKRD26, PIDD1, LRRC45, NCS1, CEP15) for their precise localization, order of recruitment, and their roles in each step of cilia formation. Using CRISPR-Cas9 knockouts, we show that the order of the recruitment of the distal appendage proteins is highly interconnected and a more complex hierarchy. Our analysis highlights two protein modules, CEP83-SCLT1 and CEP164-TTBK2, as critical for structural assembly of distal appendages. Functional assays revealed that CEP89 selectively functions in the RAB34[+] vesicle recruitment, while deletion of the integral components, CEP83-SCLT1-CEP164-TTBK2, severely compromised all four steps of cilium formation. Collectively, our analyses provide a more comprehensive view of the organization and the function of the distal appendage, paving the way for molecular understanding of ciliary assembly.}, }
@article {pmid39881543, year = {2025}, author = {Tachida, Y and Manian, KV and Butcher, R and Levy, JM and Pendse, N and Hennessey, E and Liu, DR and Pierce, EA and Liu, Q and Comander, J}, title = {Systematic empirical evaluation of individual base editing targets: Validating therapeutic targets in USH2A and comparison of methods.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {33}, number = {4}, pages = {1466-1484}, pmid = {39881543}, issn = {1525-0024}, mesh = {Humans ; *Gene Editing/methods ; Animals ; *Extracellular Matrix Proteins/genetics ; Mice ; Mutation ; Genetic Therapy/methods ; *Usher Syndromes/genetics/therapy ; High-Throughput Nucleotide Sequencing ; Disease Models, Animal ; Dependovirus/genetics ; CRISPR-Cas Systems ; }, abstract = {Base editing shows promise for the correction of human mutations at a higher efficiency than other repair methods and is especially attractive for mutations in large genes that are not amenable to gene augmentation therapy. Here, we demonstrate a comprehensive workflow for in vitro screening of potential therapeutic base editing targets for the USH2A gene and empirically validate the efficiency of adenine and cytosine base editor/guide combinations for correcting 35 USH2A mutations. Editing efficiency and bystander edits are compared between different target templates (plasmids vs. transgenes) and assays (next-generation sequencing vs. Sanger), as well as comparisons between unbiased empirical results and computational predictions. Based on these observations, practical assay recommendations are discussed. Finally, a humanized knockin mouse model was created with the best-performing target, the nonsense mutation c.11864G>A p.(Trp3955∗). Split-intein AAV9 delivery of editing reagents resulted in the restoration of USH2A protein and a correction rate of 65% ± 3% at the mutant base pair and of 52% ± 3% excluding bystander amino acid changes. This efficiency is higher than that seen in a retinal gene editing program testing in a clinical trial. These results demonstrate the effectiveness of this overall strategy to identify and test base editing reagents with the potential for human therapeutic applications.}, }
@article {pmid39880951, year = {2025}, author = {Muhar, MF and Farnung, J and Cernakova, M and Hofmann, R and Henneberg, LT and Pfleiderer, MM and Denoth-Lippuner, A and Kalčic, F and Nievergelt, AS and Peters Al-Bayati, M and Sidiropoulos, ND and Beier, V and Mann, M and Jessberger, S and Jinek, M and Schulman, BA and Bode, JW and Corn, JE}, title = {C-terminal amides mark proteins for degradation via SCF-FBXO31.}, journal = {Nature}, volume = {638}, number = {8050}, pages = {519-527}, pmid = {39880951}, issn = {1476-4687}, mesh = {Humans ; *Proteolysis ; *F-Box Proteins/metabolism/chemistry/genetics ; *Amides/metabolism/chemistry ; *SKP Cullin F-Box Protein Ligases/metabolism/chemistry ; Ubiquitination ; Proteasome Endopeptidase Complex/metabolism ; HEK293 Cells ; Models, Molecular ; CRISPR-Cas Systems ; }, abstract = {During normal cellular homeostasis, unfolded and mislocalized proteins are recognized and removed, preventing the build-up of toxic byproducts[1]. When protein homeostasis is perturbed during ageing, neurodegeneration or cellular stress, proteins can accumulate several forms of chemical damage through reactive metabolites[2,3]. Such modifications have been proposed to trigger the selective removal of chemically marked proteins[3-6]; however, identifying modifications that are sufficient to induce protein degradation has remained challenging. Here, using a semi-synthetic chemical biology approach coupled to cellular assays, we found that C-terminal amide-bearing proteins (CTAPs) are rapidly cleared from human cells. A CRISPR screen identified FBXO31 as a reader of C-terminal amides. FBXO31 is a substrate receptor for the SKP1-CUL1-F-box protein (SCF) ubiquitin ligase SCF-FBXO31, which ubiquitylates CTAPs for subsequent proteasomal degradation. A conserved binding pocket enables FBXO31 to bind to almost any C-terminal peptide bearing an amide while retaining exquisite selectivity over non-modified clients. This mechanism facilitates binding and turnover of endogenous CTAPs that are formed after oxidative stress. A dominant human mutation found in neurodevelopmental disorders reverses CTAP recognition, such that non-amidated neosubstrates are now degraded and FBXO31 becomes markedly toxic. We propose that CTAPs may represent the vanguard of a largely unexplored class of modified amino acid degrons that could provide a general strategy for selective yet broad surveillance of chemically damaged proteins.}, }
@article {pmid39880939, year = {2025}, author = {Wang, W and Pan, Q and Tian, B and Yu, Z and Davidson, D and Bai, G and Akhunova, A and Trick, H and Akhunov, E}, title = {Non-additive dosage-dependent effects of TaGS3 gene editing on grain size and weight in wheat.}, journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik}, volume = {138}, number = {2}, pages = {38}, pmid = {39880939}, issn = {1432-2242}, support = {INV-004430/GATES/Gates Foundation/United States ; 2022-68013-36439//National Institute of Food and Agriculture/ ; 2021-67013-34174//National Institute of Food and Agriculture/ ; 2020-67013-30906//National Institute of Food and Agriculture/ ; }, mesh = {*Triticum/genetics/growth &amp; development ; *Gene Editing ; *Edible Grain/genetics/growth &amp; development ; Phenotype ; CRISPR-Cas Systems ; Alleles ; *Gene Dosage ; *Plant Proteins/genetics ; Loss of Function Mutation ; Oryza/genetics ; *Genes, Plant ; }, abstract = {Loss-of-function mutations induced by CRISPR-Cas9 in the TaGS3 gene homoeologs show non-additive dosage-dependent effects on grain size and weight and have potential utility for increasing grain yield in wheat. The grain size in cereals is one of the component traits contributing to yield. Previous studies showed that loss-of-function (LOF) mutations in GS3, encoding Gγ subunit of the multimeric G protein complex, increase grain size and weight in rice. While an association between allelic variation in the GS3 homologs of wheat and grain weight/size has been detected previously, the effects of LOF alleles at TaGS3 on these traits remain unknown. We used genome editing to create TaGS3 mutant lines with varying LOF homeo-allele dosages. Contrary to the results obtained in rice, editing all three TaGS3 homoeologous copies resulted in a significant decrease in grain length (4.4%), width (3.4%), grain area (7.3%) and weight (7.5%), without affecting the number of grains per spike. Compared to the wild type, the highest increase in grain weight (up to 9.6%) and area (up to 5.0%) was observed in homozygous mutants with one or two genomes carrying LOF homeo-alleles, suggesting non-additive suppressive effects of TaGS3 on grain size and weight in wheat. Our results suggest that the regulatory effects of GS3 homologs in wheat and rice have diverged. The newly developed LOF homeo-alleles of TaGS3 expand the set of CRISPR-Cas9-induced variants of yield component genes that have potential to increase grain weight in wheat.}, }
@article {pmid39880685, year = {2025}, author = {van Dongen, JE and Segerink, LI}, title = {Building the Future of Clinical Diagnostics: An Analysis of Potential Benefits and Current Barriers in CRISPR/Cas Diagnostics.}, journal = {ACS synthetic biology}, volume = {14}, number = {2}, pages = {323-331}, pmid = {39880685}, issn = {2161-5063}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Gene Editing/methods ; *Molecular Diagnostic Techniques/methods ; High-Throughput Nucleotide Sequencing ; *Pathology, Molecular/methods/trends ; }, abstract = {Advancements in molecular diagnostics, such as polymerase chain reaction and next-generation sequencing, have revolutionized disease management and prognosis. Despite these advancements in molecular diagnostics, the field faces challenges due to high operational costs and the need for sophisticated equipment and highly trained personnel besides having several technical limitations. The emergent field of CRISPR/Cas sensing technology is showing promise as a new paradigm in clinical diagnostics, although widespread clinical adoption remains limited. This perspective paper discusses specific cases where CRISPR/Cas technology can surmount the challenges of existing diagnostic methods by stressing the significant role that CRISPR/Cas technology can play in revolutionizing clinical diagnostics. It underscores the urgency and importance of addressing the technological and regulatory hurdles that must be overcome to harness this technology effectively in clinical laboratories.}, }
@article {pmid39880501, year = {2025}, author = {Tian, Y and Chen, J and Chen, F and Xu, J and Huang, L and Peng, L and Li, H and Shi, K}, title = {Multiple gRNAs-assisted CRISPR/Cas12a-based portable aptasensor enabling glucometer readout for amplification-free and quantitative detection of malathion.}, journal = {Analytica chimica acta}, volume = {1341}, number = {}, pages = {343662}, doi = {10.1016/j.aca.2025.343662}, pmid = {39880501}, issn = {1873-4324}, mesh = {*Malathion/analysis ; *Aptamers, Nucleotide/chemistry/genetics ; *Biosensing Techniques/instrumentation/methods ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; Limit of Detection ; }, abstract = {BACKGROUND: The threat of toxic malathion residues to human health has always been a serious food safety issue. The CRISPR/Cas system represents an innovative detection technology for pesticide residues, but its application to malathion detection has not been reported yet. In addition, the multiple-guide RNA (gRNA) powered-CRISPR/Cas biosensor has the advantages of being fast, sensitive and does not require pre-amplification. However, the reported multiple-gRNA CRISPR/Cas-based biosensors are largely only used for the detection of nucleic acid targets, and there are still certain challenges in detecting non-nucleic acid targets.<br><br>RESULTS: In this work, a multiplex-gRNA-assisted CRISPR/Cas12a-based portable aptasensor (MgCPA) is developed for amplification-free and quantitative detection of malathion using a glucometer. When target malathion is present in the MgCPA strategy, it specifically binds with aptamer and then activates the trans-cleavage activity of the multiplex-gRNA CRISPR/Cas12a. The activated multiple Cas12a/gRNA complexes cut invertase-HP probes on the electrode surface to obtain glucose signals with glucometer assistance. Under optimal conditions, the developed MgCPA strategy achieves satisfactory portable quantitative and sensitive detection of malathion down to 300&#xa0;fM&#xa0;(S/N&#xa0;=&#xa0;3) without pre-amplification. Moreover, the satisfactory selectivity, high reproducibility, and good stability of the proposed strategy are also obtained. Due to its excellent and robust shelf life, our developed MgCPA strategy can be practically applied in detecting malathion in orange, apple, cabbage, and spinach samples.<br><br>SIGNIFICANCE: Amplification-free, sensitive, portable quantitative and selective detection of malathion in food samples is achieved by employing our developed MgCPA strategy. This strategy not only opens up a new path for the non-nucleic-acid target detection using amplification-free methods based on multiple-gRNA-assisted CRISPR/Cas12a, but also has broad application prospects in ensuring food safety.}, }
@article {pmid39880493, year = {2025}, author = {Wang, S and Shen, X and Chen, G and Zhang, W and Tan, B}, title = {Application and development of CRISPR-Cas12a methods for the molecular diagnosis of cancer: A review.}, journal = {Analytica chimica acta}, volume = {1341}, number = {}, pages = {343603}, doi = {10.1016/j.aca.2024.343603}, pmid = {39880493}, issn = {1873-4324}, mesh = {Humans ; *Neoplasms/diagnosis/genetics ; *CRISPR-Cas Systems/genetics ; *Molecular Diagnostic Techniques/methods ; }, abstract = {Rapid, sensitive, and specific molecular detection methods are crucial for diagnosing, treating and prognosing cancer patients. With advancements in biotechnology, molecular diagnostic technology has garnered significant attention as a fast and accurate method for cancer diagnosis. CRISPR-Cas12a (Cpf1), an important CRISPR-Cas family member, has revolutionized the field of molecular diagnosis since its introduction. CRISPR-Cas technologies are a new generation of molecular tools that are widely used in the detection of pathogens, cancers, and other diseases. Liquid biopsy methods based on CRISPR-Cas12a have demonstrated remarkable success in cancer diagnosis, encompassing the detection of DNA mutations, DNA methylation, tumor-related viruses, and non-nucleic acid molecule identification. This review systematically discusses the developmental history, key technologies, and principles of CRISPR-Cas12a-based molecular diagnostic techniques and their applications in cancer diagnosis. This review has also discussed the future development directions of CRISPR-Cas12a, aiming for it to become a reliable new technology that can be used in clinical application.}, }
@article {pmid39880143, year = {2025}, author = {Li, L and Luo, M and Zhou, L and Wang, Y and Jiao, Y and Wang, C and Gong, C and Cen, X and Yao, S}, title = {Glucocorticoid pre-administration improves LNP-mRNA mediated protein replacement and genome editing therapies.}, journal = {International journal of pharmaceutics}, volume = {672}, number = {}, pages = {125282}, doi = {10.1016/j.ijpharm.2025.125282}, pmid = {39880143}, issn = {1873-3476}, mesh = {Animals ; *Gene Editing/methods ; *Glucocorticoids/administration &amp; dosage ; *RNA, Messenger/administration &amp; dosage ; Mice ; *Nanoparticles/administration &amp; dosage/chemistry ; CRISPR-Cas Systems ; *Lipids/chemistry/administration &amp; dosage ; Male ; Genetic Therapy/methods ; Mice, Inbred C57BL ; Liver/metabolism ; Humans ; Hepatocytes/metabolism ; Gene Transfer Techniques ; Macrophages/metabolism ; Liposomes ; }, abstract = {Lipid nanoparticles (LNPs) are among the most promising non-viral mRNA delivery systems for gene therapeutic applications. However, the in vivo delivery of LNP-mRNA remains challenging due to multiple intrinsic barriers that hinder LNPs from reaching their target cells. In this study, we sought to enhance LNP delivery by manipulating intrinsic regulatory mechanisms involved in their metabolism. We demonstrated that activation of the glucocorticoid pathway significantly increased the systemic delivery of LNP-mRNA in both mice and monkeys, achieving up to a fourfold improvement. This enhancement was primarily attributed to the glucocorticoid-mediated inhibition of macrophage phagocytosis in circulation and the liver, which resulted in higher LNP accumulation in hepatocytes. Consequently, glucocorticoid activation improved the therapeutic efficacy of LNP-based protein replacement and CRISPR/Cas9 genome editing therapies. Together, these findings establish a practical strategy to enhance the systemic delivery of RNA-based protein replacement and genome editing therapeutics, highlighting the potential of manipulating endogenous mechanisms to optimize exogenous gene delivery.}, }
@article {pmid39880137, year = {2025}, author = {Yang, S and Wei, Y and Quansah, E and Zhang, Z and Da, W and Wang, B and Wang, K and Sun, D and Tao, Z and Zhang, C}, title = {Cas12a is competitive for gene editing in the malaria parasites.}, journal = {Microbial pathogenesis}, volume = {200}, number = {}, pages = {107340}, doi = {10.1016/j.micpath.2025.107340}, pmid = {39880137}, issn = {1096-1208}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Plasmodium/genetics ; Malaria/parasitology ; Humans ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; Animals ; Genome, Protozoan ; Bacterial Proteins ; }, abstract = {Malaria, caused by the Plasmodium parasites, has always been one of the worst infectious diseases that threaten human health, making it necessary for us to study the genetic function and physiological mechanisms of Plasmodium parasites from the molecular level to find more effective ways of addressing the increasingly pressing threat. The CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) is an RNA-guided adaptive immune system, which has been extensively developed and used as a genome editing tool in many organisms, including Plasmodium parasites. However, due to the physiological characteristics and special genomic characteristics of Plasmodium parasites, most of the tools currently used for genome editing of Plasmodium parasites have not met expectations. CRISPR-Cas12a (also known as Cpf1), one of the CRISPR-Cas systems, has attracted considerable attention because of its characteristics of being used for biological diagnosis and multiple genome editing. Recent studies have shown that its unique properties fit the genetic makeup of Plasmodium parasites making it a promising tool for gene editing in these parasites. In this review, we have summarized the relevant content of the Cas12 family, especially the frequently used Cas12a, its advantages for gene editing, and the application prospects in Plasmodium parasites.}, }
@article {pmid39879802, year = {2025}, author = {Yuan, G and Cheng, D and Huang, J and Wang, M and Xia, X and An, H and Xie, F and Li, X and Chen, J and Tang, Y and Peng, C}, title = {An integrated and multifunctional homemade cell sensor platform based on Si-d-CQDs and CRISPR-Cas12a for CD31 detection during endothelial-to-mesenchymal transition.}, journal = {Talanta}, volume = {287}, number = {}, pages = {127612}, doi = {10.1016/j.talanta.2025.127612}, pmid = {39879802}, issn = {1873-3573}, mesh = {Humans ; *Platelet Endothelial Cell Adhesion Molecule-1/analysis/metabolism ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Electrochemical Techniques ; Aniline Compounds/chemistry ; Electrodes ; Tin Compounds/chemistry ; *Epithelial-Mesenchymal Transition ; Chitosan/chemistry ; }, abstract = {Endothelial-to-mesenchymal transition (EndMT) plays a crucial role in the initiation and progression of atherosclerosis and various disease processes. Cluster of differentiation 31 (CD31) is a significant marker in EndMT. Detecting CD31 is essential for early-stage monitoring of EndMT and diagnosing atherosclerosis. Herein, we propose a homemade electrochemical array sensor comprising four electrodes, applied for cell cultivation, electrical stimulation, and simultaneous electrochemical detection, offering a three-in-one approach for CD31 detection during EndMT. To enhance the analytical performance of the cell sensor, indium tin oxide/chitosan-MXene/polyaniline (ITO/CS-MXene/PANI) composites were synthesized. The synthesis process involved the polymerization of PANI on the surface of the CS-MXene-modified ITO electrode, resulting in the creation of highly biocompatible active sites for cell immobilization. Si-d-CQDs@acDNA-AptCD31-Fc, with exceptional photophysical and chemical properties, was integrated into the array sensor setup, which enabled the dual-mode detection of fluorescent and electrochemical signals in cultured cells. A CRISPR-Cas12a system was employed to cleave Si-d-CQDs@acDNA-AptCD31-Fc. Subsequently, the fragmented Fc molecules were enriched via electrochemistry to further amplify the electrochemical signals. Through the unique combination of programmable Si-d-CQDs@acDNA-AptCD31-Fc, the CRISPR-Cas12a system, and voltage enrichment, a novel "signal-on-off-super on" signal amplification strategy was developed. The cell sensor exhibited a wide linear range from 1 × 10[1] particles mL[-1] to 1 × 10[6] particles mL[-1] (R[2] = 0.9912) and a detection limit of 4 particles mL[-1]. The proposed strategy presents a promising approach for developing CRISPR-Cas12a-based tools for detecting various stages of EndMT and opens a new window for dual-mode analysis applications.}, }
@article {pmid39879084, year = {2025}, author = {Eom, H and Choi, YJ and Nandre, R and Kim, M and Oh, YL and Kim, S and Nakazawa, T and Honda, Y and Ro, HS}, title = {Targeted insertion of heterogenous DNA using Cas9-gRNA ribonucleoprotein-mediated gene editing in Ganoderma lucidum.}, journal = {Bioengineered}, volume = {16}, number = {1}, pages = {2458376}, pmid = {39879084}, issn = {2165-5987}, mesh = {*Gene Editing/methods ; *Reishi/genetics ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Ribonucleoproteins/genetics/metabolism ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; }, abstract = {Gene editing is emerging as a powerful tool for introducing novel functionalities in mushrooms. While CRISPR/Cas9-induced double-strand breaks (DSBs) typically rely on non-homologous end joining (NHEJ) for gene disruption, precise insertion of heterologous DNA in mushrooms is less explored. Here, we evaluated the efficacy of inserting donor DNAs (8-1008 bp) with or without homologous arms at Cas9-gRNA RNP-induced DSBs. Co-transformation of donor DNAs with RNP targeting the pyrG gene in Ganoderma lucidum yielded 184 transformants without homologous arms and 781 with 300-bp homologous arms (HR_donor DNAs). Restriction analysis and sequencing identified 122 hR_donor DNA transformants with complete donor DNA sequences, achieving 15.6% HDR efficiency (122/781), contrasting with 8 instances via NHEJ from the 184 transformants. These findings highlight the viability of HDR for precise genomic editing in mushrooms, enabling targeted modifications to enhance functionalities.}, }
@article {pmid39878871, year = {2025}, author = {Schöllkopf, AI and Almeida, L and Krammer, K and Rivero, CG and Liebl, W and Ehrenreich, A}, title = {Deletion of atypical type II restriction genes in Clostridium cellulovorans using a Cas9-based gene editing system.}, journal = {Applied microbiology and biotechnology}, volume = {109}, number = {1}, pages = {31}, pmid = {39878871}, issn = {1432-0614}, support = {161B0930//Bundesministerium f&#xfc;r Bildung und Forschung/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Gene Deletion ; *Clostridium cellulovorans/genetics/enzymology ; Promoter Regions, Genetic ; Riboswitch ; }, abstract = {The anaerobic bacterium Clostridium cellulovorans is a promising candidate for the sustainable production of biofuels and platform chemicals due to its cellulolytic properties. However, the genomic engineering of the species is hampered because of its poor genetic accessibility and the lack of genetic tools. To overcome this limitation, a protocol for triparental conjugation was established that enables the reliable transfer of vectors for markerless chromosomal modification into C. cellulovorans. The availability of reporter genes is another requirement for strain engineering and biotechnological applications. In this work, the oxygen-free fluorescence absorption-shift tag (FAST) system was used to characterize promoter strength in C. cellulovorans. Selected promoters were used to establish a CRISPR/Cas system for markerless chromosomal modifications. For stringent control of expression of Cas9, a theophylline-dependent riboswitch was used, and additionally, the anti-CRISPR protein AcrIIA4 was used to reduce the basal activity of the Cas9 in the off-state of the riboswitch. Finally, the newly established CRISPR/Cas system was used for the markerless deletion of the genes encoding two restriction endonucleases of a type II restriction-modification (RS) system from the chromosome of C. cellulovorans. In comparison to the WT, the conjugation efficiency when using the deletion mutant as the recipient strain was improved by about one order of magnitude, without the need for prior C. cellulovorans-specific in vivo methylation of the conjugative plasmid in the E. coli donor strain. KEY POINTS: • Quantification of heterologous promoters enables rational choice for genetic engineering. • CRISPR/Cas with riboswitch and anti-CRISPR allows efficient gene deletion in C. cellulovorans. • Conjugation protocol and type II REase deletion enhance genetic accessibility.}, }
@article {pmid39878690, year = {2025}, author = {I Edvard Smith, C and Zain, R and Blomberg, P}, title = {[Gene editing is changing the treatment of hereditary diseases].}, journal = {Lakartidningen}, volume = {122}, number = {}, pages = {}, pmid = {39878690}, issn = {1652-7518}, mesh = {Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; *Genetic Diseases, Inborn/therapy/genetics ; CRISPR-Cas Systems ; }, abstract = {Gene editing is a novel technology within gene therapy, which changes sequences in chromosomal DNA with precision. Even if there are alternative strategies, the Nobel Prize-winning CRISPR/Cas technology has become the dominating principle. During recent years base editing and prime editing, permitting editing without DNA double-strand breaks, have been developed. The first clinical gene editing results were reported in 2021; since then many patients have been treated, and recently the first treatment was approved as a novel therapy in the UK and later in USA and in EU. This update describes various aspects including methodological developments and safety.}, }
@article {pmid39878523, year = {2025}, author = {Xiao, X and Yang, S and Jiang, G and He, S}, title = {Current views and trends of nanomaterials as vectors for gene delivery since the 21st century: a bibliometric analysis.}, journal = {Nanomedicine (London, England)}, volume = {20}, number = {5}, pages = {439-454}, pmid = {39878523}, issn = {1748-6963}, mesh = {Humans ; *Bibliometrics ; *Gene Transfer Techniques/trends ; *Genetic Therapy/methods ; Nanoparticles/chemistry ; *Nanostructures/chemistry ; }, abstract = {BACKGROUND: Gene therapy is garnering increasing support due to its potential for a "once-delivered, lifelong benefit." The limitations of traditional gene delivery methods have spurred the advancement of bionanomaterials. Despite this progress, a thorough analysis of the evolution, current state, key contributors, focal studies, and future directions of nanomaterials in gene delivery remains absent.<br><br>METHODS: This study scrutinizes articles from the Web of Science, spanning 1 January 2 000, to 31 December 2023, employing various online tools for analysis and visualization.<br><br>RESULTS: The 21st century has witnessed consistent growth in scholarly work in this domain globally, with notable contributions from China and the US. At the same time, the Chinese Academy of Sciences (CAS), Harvard University, and Massachusetts Institute of Technology (MIT) have emerged as the most productive institutions, with CAS's academician Weihong Tan becoming the field's leading author. While drug delivery and nanoparticles (NPs) have been central themes for two decades, the research focus has shifted from modifying NPs and ultrafine particles to exploring polymer-hybrid NPs, mRNA vaccines, immune responses, green synthesis, and CRISPR/Cas tools.<br><br>CONCLUSIONS: This shift marks the transition from nanomaterials to bionanomaterials. The insights provided by this research offer a comprehensive overview of the field and valuable guidance for future investigations.}, }
@article {pmid39878334, year = {2025}, author = {Yuan, Y and Li, Y and Li, G and Lei, L and Huang, X and Li, M and Yao, Y}, title = {Intelligent Design of Lipid Nanoparticles for Enhanced Gene Therapeutics.}, journal = {Molecular pharmaceutics}, volume = {22}, number = {3}, pages = {1142-1159}, doi = {10.1021/acs.molpharmaceut.4c00925}, pmid = {39878334}, issn = {1543-8392}, mesh = {*Lipids/chemistry ; *Nanoparticles/chemistry ; Humans ; *Genetic Therapy/methods ; Animals ; Gene Transfer Techniques ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Drug Delivery Systems/methods ; Liposomes ; }, abstract = {Lipid nanoparticles (LNPs) are an effective delivery system for gene therapeutics. By optimizing their formulation, the physiochemical properties of LNPs can be tailored to improve tissue penetration, cellular uptake, and precise targeting. The application of these targeted delivery strategies within the LNP framework ensures efficient delivery of therapeutic agents to specific organs or cell types, thereby maximizing therapeutic efficacy. In the realm of genome editing, LNPs have emerged as a potent vehicle for delivering CRISPR/Cas components, offering significant advantages such as high in vivo efficacy. The incorporation of machine learning into the optimization of LNP platforms for gene therapeutics represents a significant advancement, harnessing its predictive capabilities to substantially accelerate the research and development process. This review highlights the dynamic evolution of LNP technology, which is expected to drive transformative progress in the field of gene therapy.}, }
@article {pmid39878102, year = {2025}, author = {Khosravi, S and Hinrichs, R and Rönspies, M and Haghi, R and Puchta, H and Houben, A}, title = {Epigenetic state and gene expression remain stable after CRISPR/Cas-mediated chromosomal inversions.}, journal = {The New phytologist}, volume = {245}, number = {6}, pages = {2527-2539}, pmid = {39878102}, issn = {1469-8137}, support = {EpiChrom 031B1220B//Bundesministerium f&#xfc;r Bildung und Forschung/ ; }, mesh = {*Epigenesis, Genetic ; *Arabidopsis/genetics ; *CRISPR-Cas Systems/genetics ; *Chromosome Inversion/genetics ; *Gene Expression Regulation, Plant ; DNA Methylation/genetics ; Histones/metabolism ; Chromosomes, Plant/genetics ; Genome, Plant ; }, abstract = {The epigenetic state of chromatin, gene activity and chromosomal positions are interrelated in plants. In Arabidopsis thaliana, chromosome arms are DNA-hypomethylated and enriched with the euchromatin-specific histone mark H3K4me3, while pericentromeric regions are DNA-hypermethylated and enriched with the heterochromatin-specific mark H3K9me2. We aimed to investigate how the chromosomal location affects epigenetic stability and gene expression by chromosome engineering. Two chromosomal inversions of different sizes were induced using CRISPR/Cas9 to move heterochromatic, pericentric sequences into euchromatic regions. The epigenetic status of these lines was investigated using whole-genome bisulfite sequencing and chromatin immunoprecipitation. Gene expression changes following the induction of the chromosomal inversions were studied via transcriptome analysis. Both inversions had a minimal impact on the global distribution of histone marks and DNA methylation patterns, although minor epigenetic changes were observed across the genome. Notably, the inverted chromosomal regions and their borders retained their original epigenetic profiles. Gene expression analysis showed that only 0.5-1% of genes were differentially expressed genome-wide following the induction of the inversions. CRISPR/Cas-induced chromosomal inversions minimally affect epigenetic landscape and gene expression, preserving their profiles in subsequent generations.}, }
@article {pmid39877015, year = {2024}, author = {Fatkulin, AA and Chuksina, TA and Sorokina, NP and Smykov, IT and Kuraeva, EV and Masezhnaya, ES and Smagina, KA and Shkurnikov, MY}, title = {Comparative Analysis of Spacer Targets in CRISPR-Cas Systems of Starter Cultures.}, journal = {Acta naturae}, volume = {16}, number = {4}, pages = {81-85}, pmid = {39877015}, issn = {2075-8251}, abstract = {Dairy production facilities represent a unique ecological niche for bacteriophages of lactic acid bacteria. Throughout evolution, bacteria have developed a wide range of defense mechanisms against viral infections caused by bacteriophages. The CRISPR-Cas system is of particular interest due to its adaptive nature. It allows bacteria to acquire and maintain specific resistance to certain bacteriophages. In this study, we investigated the CRISPR-Cas systems of lactic acid bacteria. Special attention was paid to the specificity of the spacers in CRISPR cassettes. CRISPR-Cas systems were found in the genomes of 43% of the lactic acid bacteria studied. Additionally, only 13.1% of the total number of CRISPR cassette spacers matched bacteriophage genomes, indicating that many predicted spacers either lack known phage targets or are directed against other types of mobile genetic elements, such as plasmids.}, }
@article {pmid39876973, year = {2024}, author = {de Mello, DC and Menezes, JM and de Oliveira, ATF and Cristovão, MM and Kimura, ET and Fuziwara, CS}, title = {Modulating gene expression as a strategy to investigate thyroid cancer biology.}, journal = {Archives of endocrinology and metabolism}, volume = {68}, number = {Spec Issue}, pages = {e240073}, pmid = {39876973}, issn = {2359-4292}, mesh = {Humans ; *Thyroid Neoplasms/genetics ; RNA Interference ; Gene Editing/methods ; RNA, Small Interfering/genetics ; *Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Animals ; }, abstract = {Modulating the expression of a coding or noncoding gene is a key tool in scientific research. This strategy has evolved methodologically due to advances in cloning approaches, modeling/algorithms in short hairpin RNA (shRNA) design for knockdown efficiency, and biochemical modifications in RNA synthesis, among other developments. Overall, these modifications have improved the ways to either reduce or induce the expression of a given gene with efficiency and facility for implementation in the lab. Allied with that, the existence of various human cell line models for cancer covering different histotypes and biological behaviors, especially for thyroid cancer, has helped improve the understanding of cancer biology. In this review, we cover the most frequently used current techniques for gene modulation in the thyroid cancer field, such as RNA interference (RNAi), short hairpin RNA (shRNA), and gene editing with CRISPR/Cas9 for inhibiting a target gene, and strategies to overexpress a gene, such as plasmid cloning and CRISPRa. Exploring the possibilities for gene modulation allows the improvement of the scientific quality of the studies and the integration of clinicians and basic scientists, leading to better development of translational research.}, }
@article {pmid39875802, year = {2025}, author = {Raudstein, M and Peñaranda, MMD and Kjærner-Semb, E and Grove, S and Morton, HC and Edvardsen, RB}, title = {Generation of IgM[+] B cell-deficient Atlantic salmon (Salmo salar) by CRISPR/Cas9-mediated IgM knockout.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {3599}, pmid = {39875802}, issn = {2045-2322}, mesh = {Animals ; *Immunoglobulin M/genetics ; *Salmo salar/genetics/immunology ; *CRISPR-Cas Systems ; *B-Lymphocytes/immunology/metabolism ; *Gene Knockout Techniques ; }, abstract = {Infectious diseases pose significant challenges to Norwegian Atlantic salmon aquaculture. Vaccines are critical for disease prevention; however, a deeper understanding of the immune system is essential to improve vaccine efficacy. Immunoglobulin M (IgM) is the main antibody involved in the systemic immune response of teleosts, including Atlantic salmon. In this study, we used CRISPR/Cas9 technology to knock out the two IgM genes in Atlantic salmon. High-throughput sequencing revealed an average mutagenesis efficiency of 97% across both loci, with a predominance of frameshift mutations (78%). Gene expression analyses demonstrated significantly reduced membrane-bound IgM mRNA levels in head kidney and spleen tissues. Flow cytometry revealed a 78% reduction in IgM[+] B cells in peripheral blood, and Western blot analyses showed decreased IgM protein levels in serum. Notably, an upregulation of IgT mRNA was observed, suggesting a potential compensatory mechanism. This work presents the first application of CRISPR/Cas9 to disrupt an immune-related gene in the F0 generation of Atlantic salmon, and lays the foundation for generating a model completely lacking IgM[+] B cells which can be used to study the role of B cells and antibodies. This study has implications for advancing immune research in teleosts and for developing strategies to improve salmon health and welfare in aquaculture.}, }
@article {pmid39874920, year = {2025}, author = {Shu, T and Yin, X and Xiong, Q and Hua, C and Bu, J and Yang, K and Zhao, J and Liu, Y and Zhu, L and Zhu, C}, title = {Lift-CM: An integrated lift-heater centrifugal microfluidic platform for point-of-care pathogen nucleic acid detection using isothermal amplification and CRISPR/Cas12a.}, journal = {Biosensors &amp; bioelectronics}, volume = {274}, number = {}, pages = {117178}, doi = {10.1016/j.bios.2025.117178}, pmid = {39874920}, issn = {1873-4235}, mesh = {*Nucleic Acid Amplification Techniques/instrumentation ; CRISPR-Cas Systems/genetics ; Humans ; *Biosensing Techniques/instrumentation ; Point-of-Care Systems ; Equipment Design ; Molecular Diagnostic Techniques/instrumentation ; Lab-On-A-Chip Devices ; }, abstract = {Pathogen nucleic acid detection technology based on isothermal amplification and CRISPR/Cas12a system offers advantages in terms of high sensitivity, high specificity, and rapidity. However, this method has not been widely applied because of its shortcomings in utilizing conventional instruments, which cannot satisfy the requirements for Point of Care Testing (POCT), such as integration, convenience, and miniaturization. In this study, we developed an integrated lift-heater centrifugal microfluidic platform (Lift-CM) to automate the processes of isothermal amplification and CRISPR/Cas12a detection. A spatially encoded centrifugal microfluidic disc (SEC-disc) was employed to physically separate the amplification and detection processes while expanding the number of targets. The design of the dual-temperature and lift-heating centrifugal mechanism of the Lift-CM platform ensures that there is no manual intervention during amplification and detection processes. A smartphone-based app enables the setting of key parameters and monitoring of the experimental process, presenting results through a generated report that includes real-time fluorescence curves. We analyzed the plasmids of the Crimean-Congo hemorrhagic fever virus, Ebola virus, and five influenza viruses using different amplification methods (RPA/LAMP) to demonstrate the good compatibility of the Lift-CM platform with various amplification schemes. In clinical validation, the detection of H3N2-positive samples was completed within 30 min, and the results were highly consistent with qPCR results. This portable and compact platform offers a novel alternative solution for both clinical and at-home pathogen nucleic acid detection in the future.}, }
@article {pmid39873911, year = {2025}, author = {Wang, W and Li, S and Yang, J and Li, J and Yan, L and Zhang, C and He, Y and Xia, L}, title = {Exploiting the efficient Exo:Cas12i3-5M fusions for robust single and multiplex gene editing in rice.}, journal = {Journal of integrative plant biology}, volume = {67}, number = {5}, pages = {1246-1253}, pmid = {39873911}, issn = {1744-7909}, support = {2023ZD04074//Biological Breeding - Major Projects/ ; 32188102//National Natural Science Foundation of China/ ; 2021YFF1000204//National Key Research and Development Program of China/ ; B23CJ0208//Hainan Seed Industry Lab/ ; //National Engineering Research Centre of Crop Molecular Breeding/ ; 32100328//National Natural Science Foundation of China/ ; }, mesh = {*Oryza/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; }, abstract = {The development of a single and multiplex gene editing system is highly desirable for either functional genomics or pyramiding beneficial alleles in crop improvement. CRISPR/Cas12i3, which belongs to the Class II Type V-I Cas system, has attracted extensive attention recently due to its smaller protein size and less restricted canonical "TTN" protospacer adjacent motif (PAM). However, due to its relatively lower editing efficiency, Cas12i3-mediated multiplex gene editing has not yet been documented in plants. Here, we fused four 5' exonucleases (Exo) including T5E, UL12, PapE, ME15 to the N terminal of an optimized Cas12i3 variant (Cas12i3-5M), respectively, and systematically evaluated the editing activities of these Exo:Cas12i3-5M fusions across six endogenous targets in rice stable lines. We demonstrated that the Exo:Cas12i3-5M fusions increased the gene editing efficiencies by up to 12.46-fold and 1.25-fold compared with Cas12i3 and Cas12i3-5M, respectively. Notably, the UL12:Cas12i3-5M fusion enabled robust single gene editing with editing efficiencies of up to 90.42%-98.61% across the six tested endogenous genes. We further demonstrated that, although all the Exo:Cas12i5-5M fusions were capable of multiplex gene editing, UL12:Cas12i3-5M exhibited a superior performance in the simultaneous editing of three, four, five or six genes with efficiencies of 82.76%, 61.36%, 52.94%, and 51.06% in rice stable lines, respectively. Together, we evaluated different Exo:Cas12i3-5M fusions systemically and established UL12:Cas12i3-5M as the more robust system for single and multiplex gene editing in rice. The development of an alternative robust single and multiplex gene editing system will enrich plant genome editing toolkits and facilitate pyramiding of agronomically important traits for crop improvement.}, }
@article {pmid39873837, year = {2025}, author = {Asa, H and Kuwabara, C and Matsumoto, K and Shigeta, R and Yamamoto, T and Masuda, Y and Yamada, T}, title = {Simultaneous site-directed mutagenesis for soybean ß-amyrin synthase genes via DNA-free CRISPR/Cas9 system using a single gRNA.}, journal = {Plant cell reports}, volume = {44}, number = {2}, pages = {40}, pmid = {39873837}, issn = {1432-203X}, support = {JPMJSP2119//JST SPRING/ ; }, mesh = {*Glycine max/genetics/enzymology ; *Mutagenesis, Site-Directed/methods ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Intramolecular Transferases/genetics/metabolism ; Plants, Genetically Modified ; Plant Proteins/genetics/metabolism ; Saponins/metabolism ; Seeds/genetics ; }, abstract = {We generated soybean mutants related to two ß-amyrin synthase genes using DNA-free site-directed mutagenesis system. Our results suggested that one of the genes is predominant in the soyasaponin biosynthesis. Soyasaponins, which are triterpenoid saponins contained in soybean [Glycine max (L.) Merril], are responsible for the astringent aftertaste of soyfood, and their complete elimination from soybean seeds is a key challenge in the development of cultivars with improved taste. While the loss of function in the ß-amyrin synthase genes (GmBAS1 and GmBAS2) has proven effective in reducing soyasaponin content in soybean seeds, the specific functional roles of these genes remain unclear. In this study, site-directed mutagenesis was performed on two GmBAS loci using a DNA-free clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system. A complex of sgRNA targeting sequences conserved in the two loci and Cas9 protein was introduced into the shoot apical meristems of soybean embryonic axes via bombardment. Cleaved amplified polymorphic sequences (CAPS) analysis conducted 1 month post-bombardment revealed that 138 seedlings out of 1,467 screened exhibited mutations at one or both GmBAS loci. CAPS and sequencing analysis in the subsequent generation identified a total of 16 plants with inheritable mutations ranging from one to ten nucleotides. High-performance liquid chromatography (HPLC) analysis showed that site-directed mutagenesis in the GmBAS1 locus resulted in the absence of soyasaponins in mature seeds, as well as in young roots, stems, and leaves. These findings demonstrate that GmBAS1 is the predominant ß-amyrin synthase gene in soybean plants. In addition, the DNA-free CRISPR/Cas9 system was shown to be highly efficient in inducing simultaneous mutagenesis at two target loci using a single gRNA.}, }
@article {pmid39873367, year = {2025}, author = {Zhang, Y and Liu, J and Li, Y and Ma, H and Ji, J and Wang, Y and Zhuang, M and Yang, L and Fang, Z and Li, J and Zhang, C and Liu, L and Lebedeva, M and Taranov, V and Zhang, Y and Lv, H}, title = {Generation of novel bpm6 and dmr6 mutants with broad-spectrum resistance using a modified CRISPR/Cas9 system in Brassica oleracea.}, journal = {Journal of integrative plant biology}, volume = {67}, number = {5}, pages = {1214-1216}, doi = {10.1111/jipb.13842}, pmid = {39873367}, issn = {1744-7909}, support = {2023YFE0111400//National Key Research and Development Program of China/ ; }, mesh = {*Brassica/genetics/microbiology ; *CRISPR-Cas Systems/genetics ; *Disease Resistance/genetics ; *Mutation/genetics ; *Plant Proteins/genetics/metabolism ; Plant Diseases/microbiology/genetics ; Plants, Genetically Modified ; Genes, Plant ; }, abstract = {Using an optimized CRISPR/Cas9 system to knock out the BTB-POZ and MATH domain gene BoBPM6 and the DOWNY MILDEW RESISTANCE 6 gene in Brassica oleracea resulted in new lines with broad-spectrum disease resistance.}, }
@article {pmid39871438, year = {2025}, author = {Guo, N and Wang, S and Whitfield, CT and Batchelor, WD and Wang, Y and Blersch, D and Higgins, BT and Feng, Y and Liles, MR and de-Bashan, LE and Wang, Y and Ma, Y}, title = {High-Efficiency CRISPR-Cas9 Genome Editing Unveils Biofilm Insights and Enhances Antimicrobial Activity in Bacillus velezensis FZB42.}, journal = {Biotechnology and bioengineering}, volume = {122}, number = {4}, pages = {983-994}, doi = {10.1002/bit.28933}, pmid = {39871438}, issn = {1097-0290}, support = {//National Institute of Food and Agriculture./ ; }, mesh = {*Bacillus/genetics/physiology ; *CRISPR-Cas Systems/genetics ; *Biofilms/growth &amp; development ; *Gene Editing/methods ; Bacterial Proteins/genetics/metabolism ; Anti-Bacterial Agents/pharmacology ; Genome, Bacterial ; }, abstract = {Bacillus velezensis FZB42 is a prominent plant growth-promoting rhizobacterium and biocontrol agent known for producing a wide array of antimicrobial compounds. The capability to genetically manipulate this strain would facilitate understanding its metabolism and enhancing its sustainable agriculture applications. In this study, we report the first successful implementation of high-efficiency CRISPR-Cas9 genome editing in B. velezensis FZB42, enabling targeted genetic modifications to gain insights into its plant growth-promotion and biocontrol properties. Deletion of the slrR gene, a key regulator of biofilm formation, resulted in significant alterations in biofilm structure and development, as demonstrated by scanning electron microscopy and quantitative biofilm assays. These findings provide valuable insights into the mechanisms of biofilm formation, which are critical for root colonization and plant growth promotion. Additionally, we overexpressed the bac gene cluster responsible for bacilysin biosynthesis by replacing its native promoter with the strong constitutive promoter P43 and integrating an additional copy of the bacG gene. This genetic manipulation led to a 2.7-fold increase in bacB gene expression and significantly enhanced antibacterial activity against Escherichia coli and Lactobacillus diolivorans. The successful implementation of the CRISPR-Cas9 system for genome editing in FZB42 provides a valuable tool for genetic engineering, with the potential to improve its biocontrol efficacy and broaden its applications in agriculture and other biotechnology areas. Our principles and procedures are broadly applicable to other agriculturally significant microorganisms.}, }
@article {pmid39871009, year = {2025}, author = {Sakthivel, K and Balasubramanian, R and Sampathrajan, V and Veerasamy, R and Appachi, SV and K K, K}, title = {Transforming tomatoes into GABA-rich functional foods through genome editing: A modern biotechnological approach.}, journal = {Functional &amp; integrative genomics}, volume = {25}, number = {1}, pages = {27}, pmid = {39871009}, issn = {1438-7948}, mesh = {*Solanum lycopersicum/genetics/metabolism ; *gamma-Aminobutyric Acid/metabolism/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Functional Food ; Humans ; Plant Breeding ; *Genome, Plant ; Plants, Genetically Modified/genetics ; Biotechnology/methods ; }, abstract = {Gamma-aminobutyric acid (GABA) functions as an inhibitory neurotransmitter which blocks the impulses between nerve cells in the brain. Due to the increasing awareness about the health promoting benefits associated with GABA, it is also artificially synthesized and consumed as a nutritional supplement by people in some regions of the world. Though among the fresh vegetables, tomato fruits do contain a comparatively higher amount of GABA (0.07 to 2.01 mg g[-1] FW), it needs to be further enhanced to fully impart its potential health benefits. Achieving this feat through classical breeding approaches is time and resource consuming, and is also associated with linkage drag. On the other hand, precise targeting of specific sites in the genome with less off- target effects is mediated by CRISPR/Cas9 genome editing tool and is widely used to overcome the barriers associated with traditional breeding approaches. Combining genome editing with speed breeding techniques can enable the rapid development of GABA-rich tomato cultivars, paving a way to unlock a new era of functional foods, where every bite contributes to cognitive well-being and holistic health. This review highlights the significance of GABA boosted functional foods and explores the potential of CRISPR/Cas9 technology for developing GABA enriched tomatoes.}, }
@article {pmid39870938, year = {2025}, author = {Yan, W and Wang, S and Zhu, L and Yu, X and Li, J}, title = {Targeted editing of CCL5 with CRISPR-Cas9 nanoparticles enhances breast cancer immunotherapy.}, journal = {Apoptosis : an international journal on programmed cell death}, volume = {30}, number = {3-4}, pages = {912-935}, pmid = {39870938}, issn = {1573-675X}, mesh = {Animals ; *Breast Neoplasms/therapy/genetics/immunology ; *CRISPR-Cas Systems/genetics ; Female ; *Nanoparticles/chemistry ; Humans ; *Chemokine CCL5/genetics ; *Gene Editing/methods ; *Immunotherapy/methods ; Mice ; Cell Line, Tumor ; Tumor Microenvironment/immunology/genetics ; CD8-Positive T-Lymphocytes/immunology ; }, abstract = {Breast cancer remains one of the leading causes of cancer-related mortality among women worldwide. Immunotherapy, a promising therapeutic approach, often faces challenges due to the immunosuppressive tumor microenvironment. This study explores the innovative use of CRISPR-Cas9 technology in conjunction with FCPCV nanoparticles to target and edit the C-C Motif Chemokine Ligand 5 (CCL5) gene, aiming to improve the efficacy of breast cancer immunotherapy. Single-cell RNA sequencing (scRNA-seq) and TCGA-BRCA data identified CCL5 as a key immune-related gene in breast cancer. Using CRISPR-Cas9, sgRNA targeting CCL5 was designed and delivered to breast cancer cells and humanized mouse models via FCPCV nanoparticles. In vitro experiments demonstrated that FCPCV nanoparticles effectively silenced CCL5, enhanced CD8[+] T cell activity, and increased the production of cytokines such as IFN-γ, TNF-α, and GZMB. In vivo studies revealed significant tumor suppression, improved immune microenvironment, and increased CD8[+]/CD4[+] ratios in treated mice, without notable toxic side effects. These findings highlight the potential of CRISPR-Cas9 nanoparticle-mediated gene editing as a novel strategy for enhancing breast cancer immunotherapy, providing a new direction for personalized and effective cancer treatment.}, }
@article {pmid39870862, year = {2025}, author = {Ramezani, M and Weisbart, E and Bauman, J and Singh, A and Yong, J and Lozada, M and Way, GP and Kavari, SL and Diaz, C and Leardini, E and Jetley, G and Pagnotta, J and Haghighi, M and Batista, TM and Pérez-Schindler, J and Claussnitzer, M and Singh, S and Cimini, BA and Blainey, PC and Carpenter, AE and Jan, CH and Neal, JT}, title = {A genome-wide atlas of human cell morphology.}, journal = {Nature methods}, volume = {22}, number = {3}, pages = {621-633}, pmid = {39870862}, issn = {1548-7105}, support = {DP2 GM146252/GM/NIGMS NIH HHS/United States ; NNF21SA0072102//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; 1DP2GM146252//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; }, mesh = {Humans ; *Genome, Human ; CRISPR-Cas Systems ; Phenotype ; *Genomics/methods ; Membrane Proteins/genetics ; Genome-Wide Association Study/methods ; }, abstract = {A key challenge of the modern genomics era is developing empirical data-driven representations of gene function. Here we present the first unbiased morphology-based genome-wide perturbation atlas in human cells, containing three genome-wide genotype-phenotype maps comprising CRISPR-Cas9-based knockouts of >20,000 genes in >30 million cells. Our optical pooled cell profiling platform (PERISCOPE) combines a destainable high-dimensional phenotyping panel (based on Cell Painting) with optical sequencing of molecular barcodes and a scalable open-source analysis pipeline to facilitate massively parallel screening of pooled perturbation libraries. This perturbation atlas comprises high-dimensional phenotypic profiles of individual cells with sufficient resolution to cluster thousands of human genes, reconstruct known pathways and protein-protein interaction networks, interrogate subcellular processes and identify culture media-specific responses. Using this atlas, we identify the poorly characterized disease-associated TMEM251/LYSET as a Golgi-resident transmembrane protein essential for mannose-6-phosphate-dependent trafficking of lysosomal enzymes. In sum, this perturbation atlas and screening platform represents a rich and accessible resource for connecting genes to cellular functions at scale.}, }
@article {pmid39870618, year = {2025}, author = {Ray-Jones, H and Sung, CK and Chan, LT and Haglund, A and Artemov, P and Della Rosa, M and Ruje, L and Burden, F and Kreuzhuber, R and Litovskikh, A and Weyenbergh, E and Brusselaers, Z and Tan, VXH and Frontini, M and Wallace, C and Malysheva, V and Bottolo, L and Vigorito, E and Spivakov, M}, title = {Genetic coupling of enhancer activity and connectivity in gene expression control.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {970}, pmid = {39870618}, issn = {2041-1723}, support = {EP/N510129/1//RCUK | Engineering and Physical Sciences Research Council (EPSRC)/ ; WT220788//Wellcome Trust (Wellcome)/ ; RE/18/1/34212//British Heart Foundation (BHF)/ ; MC-A652-5QA20//RCUK | Medical Research Council (MRC)/ ; MR/W029790/1//RCUK | Medical Research Council (MRC)/ ; FS/18/53/33863//British Heart Foundation (BHF)/ ; /WT_/Wellcome Trust/United Kingdom ; MC_UU_00002/4//RCUK | Medical Research Council (MRC)/ ; }, mesh = {*Enhancer Elements, Genetic ; *Gene Expression Regulation ; Humans ; Quantitative Trait Loci/genetics ; Monocytes/metabolism ; Cells, Cultured ; Chromatin/genetics ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Transcription Factors/metabolism ; CCCTC-Binding Factor/metabolism ; Protein Binding ; Base Sequence ; Male ; }, abstract = {Gene enhancers often form long-range contacts with promoters, but it remains unclear if the activity of enhancers and their chromosomal contacts are mediated by the same DNA sequences and recruited factors. Here, we study the effects of expression quantitative trait loci (eQTLs) on enhancer activity and promoter contacts in primary monocytes isolated from 34 male individuals. Using eQTL-Capture Hi-C and a Bayesian approach considering both intra- and inter-individual variation, we initially detect 19 eQTLs associated with enhancer-eGene promoter contacts, most of which also associate with enhancer accessibility and activity. Capitalising on these shared effects, we devise a multi-modality Bayesian strategy, identifying 629 "trimodal QTLs" jointly associated with enhancer accessibility, eGene promoter contact, and gene expression. Causal mediation analysis and CRISPR interference reveal causal relationships between these three modalities. Many detected QTLs overlap disease susceptibility loci and influence the predicted binding of myeloid transcription factors, including SPI1, GABPB and STAT3. Additionally, a variant associated with PCK2 promoter contact directly disrupts a CTCF binding motif and impacts promoter insulation from downstream enhancers. Jointly, our findings suggest an inherent genetic coupling of enhancer activity and connectivity in gene expression control relevant to human disease and highlight the regulatory role of genetically determined chromatin boundaries.}, }
@article {pmid39869590, year = {2025}, author = {Brady, DJ and Saviane, A and Battistolli, M and Varponi, I and Barca, F and Shiomi, K and Cappellozza, S and Sandrelli, F}, title = {Enhanced silk production and pupal weight in Bombyx mori through CRISPR/Cas9-mediated circadian Clock gene disruption.}, journal = {PloS one}, volume = {20}, number = {1}, pages = {e0317572}, pmid = {39869590}, issn = {1932-6203}, mesh = {Animals ; *Bombyx/genetics/growth &amp; development/metabolism ; *CRISPR-Cas Systems/genetics ; *Circadian Clocks/genetics ; *Silk/biosynthesis/genetics ; Pupa/genetics/growth &amp; development/metabolism ; Larva/genetics/growth &amp; development ; Insect Proteins/genetics/metabolism ; }, abstract = {The domesticated silkworm, Bombyx mori, is crucial for global silk production, which is a significant economic activity supporting millions of livelihoods worldwide. Beyond traditional silk production, the growing demand for insect larvae in cosmetics, biomedical products, and animal feed underscores the need to enhance B. mori productivity. This study investigates the role of the circadian clock gene Clock in B. mori using CRISPR/Cas9-mediated mutagenesis to establish the ClkΔ29 knock-out mutant strain. Dysregulation of the circadian clock in ClkΔ29 was demonstrated by altered temporal transcriptional profiles of core circadian clock genes in adult heads and disrupted circadian-controlled behaviors, including adult eclosion and egg hatching rhythms under constant darkness. By analysing larval development timing, as well as the weights of late instar larvae, pupae, and cocoon components in ClkΔ29 mutants and in ClkΔ1922 silkworms (carrying an independently generated Clk- null allele), we showed that CLK contributes to physiological processes regulating B. mori development and growth. Importantly, ClkΔ29 mutants reared on a standard sericulture diet exhibited significant increases in key economic traits, with silk production increasing by up to 7%, and pupal weight increasing by up to 25% compared to wild-type controls. This study highlights the potential of circadian clock gene manipulation to significantly enhance sericultural productivity. Future research should focus on elucidating the molecular mechanisms driving these phenotypes and determining whether they result from circadian clock functions or pleiotropic effects of B. mori Clk. These findings provide a foundation for advancing sustainable sericulture and developing new commercial applications for silkworm-derived products.}, }
@article {pmid39869587, year = {2025}, author = {Kumari, Y and Gunathilaka, N and Amarasinghe, D}, title = {A comprehensive review of biological and genetic control approaches for leishmaniasis vector sand flies; emphasis towards promoting tools for integrated vector management.}, journal = {PLoS neglected tropical diseases}, volume = {19}, number = {1}, pages = {e0012795}, pmid = {39869587}, issn = {1935-2735}, mesh = {Animals ; *Psychodidae/genetics/parasitology ; *Leishmaniasis/prevention &amp; control/transmission ; *Insect Vectors/genetics/parasitology ; *Insect Control/methods ; Humans ; Gene Editing ; CRISPR-Cas Systems ; Animals, Genetically Modified ; Gene Drive Technology ; *Pest Control, Biological/methods ; }, abstract = {BACKGROUND: Leishmaniasis is a health problem in many regions with poor health and poor life resources. According to the World Health Organization (WHO), an estimated 700,000-1 million new cases arise annually. Effective control of sand fly vector populations is crucial for reducing the transmission of this disease. Therefore, this review aims to comprehensively examine and evaluate the current methods for controlling sand fly populations, focusing on biological and gene drive techniques.<br><br>METHODS AND FINDINGS: A detailed, comprehensive literature search was carried out using databases including Google Scholar, PubMed, ScienceDirect, and the National Library of Medicine (NIH). These searches were done using specific keywords related to the field of study. This current review identified several promising methods, including genetically modified sand flies, using transgenic approaches by taking advanced gene editing tools like Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) and genetic modification of symbiotic microorganisms for controlling sand fly populations, which appeared to be proven under laboratory and field settings.<br><br>CONCLUSION: Genetic control approaches have many benefits over chemical control, including long-lasting effects on targets, high specificity, and less environmental impact. Advances in genetic engineering technologies, particularly CRISPR/Cas9, sterile insect techniques, and gene drive insect modification, offer new avenues for precise and efficient sand fly management. Future research should prioritize optimizing rearing and sterilization techniques, conducting controlled field trials, and fostering collaboration across disciplines to realize the potential of genetic control strategies in combating leishmaniasis.}, }
@article {pmid39868861, year = {2025}, author = {Shahrokhtash, A and Sivertsen, MVT and Laursen, SH and Sutherland, DS}, title = {Nanoscale Cellular Traction Force Quantification: CRISPR-Cas12a Supercharged DNA Tension Sensors in Nanoclustered Ligand Patterns.}, journal = {ACS applied materials &amp; interfaces}, volume = {17}, number = {5}, pages = {7339-7352}, pmid = {39868861}, issn = {1944-8252}, mesh = {*DNA/chemistry/metabolism ; Animals ; Mice ; *CRISPR-Cas Systems ; Ligands ; *Biosensing Techniques ; *Nanostructures/chemistry ; 3T3 Cells ; Mechanotransduction, Cellular ; *CRISPR-Associated Proteins/chemistry/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {High-throughput measurement of cellular traction forces at the nanoscale remains a significant challenge in mechanobiology, limiting our understanding of how cells interact with their microenvironment. Here, we present a novel technique for fabricating protein nanopatterns in standard multiwell microplate formats (96/384-wells), enabling the high-throughput quantification of cellular forces using DNA tension gauge tethers (TGTs) amplified by CRISPR-Cas12a. Our method employs sparse colloidal lithography to create nanopatterned surfaces with feature sizes ranging from sub 100 to 800 nm on transparent, planar, and fully PEGylated substrates. These surfaces allow for the orthogonal immobilization of two different proteins or biomolecules using click-chemistry, providing precise spatial control over cellular signaling cues. We demonstrate the robustness and versatility of this platform through imaging techniques, including total internal reflection fluorescence microscopy, confocal laser scanning microscopy, and high-throughput imaging. Applying this technology, we measured the early stage mechanical forces exerted by 3T3 fibroblasts across different nanoscale features, detecting forces ranging from 12 to 56 pN. By integrating the Mechano-Cas12a Assisted Tension Sensor (MCATS) system, we achieved rapid and high-throughput quantification of cellular traction forces, analyzing over 2 million cells within minutes. Our findings reveal that nanoscale clustering of integrin ligands significantly influences the mechanical responses of cells. This platform offers a powerful tool for mechanobiology research, facilitating the study of cellular forces and mechanotransduction pathways in a high-throughput manner compatible with standard cell culture systems.}, }
@article {pmid39868543, year = {2025}, author = {}, title = {Correction to 'DNA target binding-induced pre-crRNA processing in type II and V CRISPR-Cas systems'.}, journal = {Nucleic acids research}, volume = {53}, number = {3}, pages = {}, doi = {10.1093/nar/gkaf024}, pmid = {39868543}, issn = {1362-4962}, }
@article {pmid39868533, year = {2025}, author = {Hu, Z and Ling, S and Duan, J and Yu, Z and Che, Y and Wang, S and Zhang, S and Zhang, X and Li, Z}, title = {Proximity-activated guide RNA of CRISPR-Cas12a for programmable diagnostic detection and gene regulation.}, journal = {Nucleic acids research}, volume = {53}, number = {3}, pages = {}, pmid = {39868533}, issn = {1362-4962}, support = {2022YFF0710200//National Science and Technology Major Project of the Ministry of Science and Technology of China/ ; 22304007//National Natural Science Foundation of China/ ; 2252014//Beijing Municipal Natural Science Foundation/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism ; Escherichia coli/genetics ; Gene Editing/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; Aptamers, Nucleotide/genetics ; Humans ; *Gene Expression Regulation ; *Endodeoxyribonucleases/genetics ; Bacterial Proteins ; }, abstract = {The flexibility and programmability of CRISPR-Cas technology have made it one of the most popular tools for biomarker diagnostics and gene regulation. Especially, the CRISPR-Cas12 system has shown exceptional clinical diagnosis and gene editing capabilities. Here, we discovered that although the top loop of the 5' handle of guide RNA can undergo central splitting, deactivating CRISPR-Cas12a, the segments can dramatically restore CRISPR function through nucleic acid self-assembly or interactions with small molecules and aptamers. This discovery forms the basis of an engineered Cas12a system with a programmable proximity-activated guide RNA (PARC-Cas12a) that links targets of interest to dsDNA. Leveraging the efficient trans- and cis-cleavage of Cas12, our findings further inspired a detection platform design for RNAs or non-nucleic acid biomarkers, enabling highly sensitive and multiplexed analysis. We further demonstrated the feasibility of RNA-controllable gene knockout/knockdown in Escherichia coli. Notably, we successfully validated the gene regulatory capabilities of the PARC-Cas12a system within mammalian cell systems by utilizing the classical theophylline molecule-aptamer system. Our results introduce a programmable toolbox for precise diagnostics and cell regulation, allowing the development of versatile diagnostic tools, complex synthetic biological circuits, and cellular biosensors.}, }
@article {pmid39868490, year = {2025}, author = {Martires, LCM and Ahronian, LG and Pratt, CB and Das, NM and Zhang, X and Whittington, DA and Zhang, H and Shen, B and Come, J and McCarren, P and Liu, MS and Min, C and Feng, T and Jahic, H and Ali, JA and Aird, DR and Li, F and Andersen, JN and Huang, A and Mallender, WD and Nicholson, HE}, title = {LIG1 Is a Synthetic Lethal Target in BRCA1 Mutant Cancers.}, journal = {Molecular cancer therapeutics}, volume = {24}, number = {4}, pages = {618-627}, pmid = {39868490}, issn = {1538-8514}, mesh = {Humans ; *BRCA1 Protein/genetics ; Animals ; Mice ; *DNA Ligase ATP/genetics/metabolism/antagonists &amp; inhibitors ; Female ; *Synthetic Lethal Mutations ; *Mutation ; Xenograft Model Antitumor Assays ; Cell Line, Tumor ; CRISPR-Cas Systems ; *Breast Neoplasms/genetics/pathology ; }, abstract = {Synthetic lethality approaches in BRCA1/2-mutated cancers have focused on PARP inhibitors, which are subject to high rates of innate or acquired resistance in patients. In this study, we used CRISPR/Cas9-based screening to identify DNA ligase I (LIG1) as a novel target for synthetic lethality in BRCA1-mutated cancers. Publicly available data supported LIG1 hyperdependence of BRCA1 mutant cells across a variety of breast and ovarian cancer cell lines. We used CRISPRn, CRISPRi, RNAi, and protein degradation to confirm the lethal effect of LIG1 inactivation at the DNA, RNA, and protein level in BRCA1 mutant cells in vitro. LIG1 inactivation resulted in viability loss across multiple BRCA1-mutated cell lines, whereas no effect was observed in BRCA1/2 wild-type cell lines, demonstrating target selectivity for the BRCA1 mutant context. On-target nature of the phenotype was demonstrated through rescue of viability with exogenous wild-type LIG1 cDNA. Next, we demonstrated a concentration-dependent relationship of LIG1 protein expression and BRCA1 mutant cell viability using a titratable, degradable LIG1 fusion protein. BRCA1 mutant viability required LIG1 catalytic activity, as catalytically dead mutant LIG1K568A failed to rescue viability loss caused by endogenous LIG1 depletion. LIG1 perturbation produced proportional increases in PAR staining in BRCA1 mutant cells, indicating a mechanism consistent with the function of LIG1 in sealing ssDNA nicks. Finally, we confirmed LIG1 hyperdependence in vivo using a xenograft model in which LIG1 loss resulted in tumor stasis in all mice. Our cumulative findings demonstrate that LIG1 is a promising synthetic lethal target for development in patients with BRCA1-mutant cancers.}, }
@article {pmid39866497, year = {2025}, author = {Owaid, HA and Al-Ouqaili, MTS}, title = {Molecular characterization and genome sequencing of selected highly resistant clinical isolates of Pseudomonas aeruginosa and its association with the clustered regularly interspaced palindromic repeat/Cas system.}, journal = {Heliyon}, volume = {11}, number = {1}, pages = {e41670}, pmid = {39866497}, issn = {2405-8440}, abstract = {The presence of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system in the superbug Pseudomonas (P) aeruginosa presents a unique opportunity to precisely target and edit bacterial genomes to modify their drug resistance. The objective was to detect the prevalence of CRISPR in extensively and pan-drug-resistant Pseudomonas aeruginosa and to determine the utility of whole-genome sequencing (WGS) for the analysis of the entire genome for such strains. The antimicrobial susceptibilities of one hundred isolates were assessed using the antibiotic susceptibility test (AST) card of the VITEK system. The presence of the CRISPR/Cas system was determined via specific primers using conventional polymerase chain reaction (PCR). Further, WGS was conducted using a DNA nanoball sequencing platform via BGI-Tech for the isolates of interest. Out of 54 resistant Pseudomonas aeruginosa isolates, 33 (33.0 %) were metallo-β-lactamase producers. Cas1, Cas3, CRISPR1, and CRISPR2 were positive in 6.0 % of isolates, while incomplete CRISPR1-Cas systems alone were found in 15.0 %. Also, CRISPR2-type was found intact in 26 % of isolates. The prevalence of resistance to antimicrobials in P. aeruginosa isolates was significantly greater in the CRISPR/Cas-negative group compared to the CRISPR/Cas-positive. Significant relationships for variables were examined using Fisher's exact tests using Chi-squared and a P-value of <0.05 as a statistical threshold. Further, on examination of CRs as a collective entity, encompassing both extensive drug resistance (XDR) and pan-drug resistance (PDR), it becomes evident that the vast majority of these strains (n = 29; 87.8 %) lacked CRISPR/Cas systems. In phylogenic analysis, PDR-P. aeruginosa revealed a very close evolutionary relationship with those originating from Kazakhstan, while XDR was globally unique. Further, the entire genome showed the presence of unique virulence and resistant pseudomonal genes. The CRISPR/Cas system and drug resistance are antagonistic to one another. XDR and PDR P. aeruginosa represent a potential threat to public health and contribute to the seriousness of associated illnesses by leading to resistant infections. Further, WGS for the two strains revealed resistance to multiple antibiotics. It is important to examine specific antimicrobial resistance (AMR) pathways, which suggests that a significant number of resistant genes in these isolates indicate a loss of CRISPR genes in the two strains. Furthermore, the WGS approach can lead to a better understanding of the genomic mechanism of pseudomonal resistance to antibiotics.}, }
@article {pmid39866228, year = {2025}, author = {Lin, HW and Lee, PY and Chang, YS and Chang, MS}, title = {Loss of Arhgap39 facilitates cell migration and invasion in murine hepatocellular cancer cells.}, journal = {Oncology research}, volume = {33}, number = {2}, pages = {493-503}, pmid = {39866228}, issn = {1555-3906}, mesh = {Animals ; *Cell Movement/genetics ; *GTPase-Activating Proteins/genetics/metabolism ; Mice ; *Liver Neoplasms/pathology/genetics/metabolism ; *Carcinoma, Hepatocellular/pathology/genetics/metabolism ; Neoplasm Invasiveness/genetics ; Cell Line, Tumor ; Humans ; rhoA GTP-Binding Protein/metabolism ; CRISPR-Cas Systems ; }, abstract = {BACKGROUND: Rho GTPases are essential regulators for cellular movement and intracellular membrane trafficking. Their enzymatic activities fluctuate between active GTP-bound and inactive GDP-bound states regulated by GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). Arhgap39/Vilse/Porf-2 is a newly identified GAP. The role of Arhgap39 in migration and invasion has not been addressed thoroughly.<br><br>METHODS: The Arhgap39 gene was knocked out by Crispr-Cas9 gene editing in mouse Hepa1-6 and Hepa-1c1c7 cells to analyze the impact of Arhgap39 depletion on migration and invasion.<br><br>RESULTS: Loss of Arhgap39 noticeably increased the migration and invasive potential. Purified Arhgap39 recombinant protein facilitated the hydrolysis of GTP in RhoA and Rac1 in vitro. RNA-seq analysis revealed that matrix metalloproteinase 13 (MMP13) and Laminin subunit beta 1 (LAMB1) were increased in Arhgap39[-/-] cells. We further crossed Arhgap39fl/fl with KrasLSL-G12D and p53fl/fl mice under the control of albumin-Cre recombinase to induce the spontaneous development of hepatocellular carcinomas. Intriguingly, the expression levels of MMP13 and the overall survival in Alb-Cre_KrasLSL-G12D_p53fl/fl_Arhgap39fl/fl (KPA) mice were comparable to control Alb-Cre_KrasLSL-G12D_p53fl/fl (KP) mice. The cell migration and invasion of KPA mice were also similar to those of control KP mice.<br><br>CONCLUSION: Arhgap39 loss could modulate the migration and invasion in some hepatocellular cancer cells, but not in those isolated from KPA mice.}, }
@article {pmid39866131, year = {2025}, author = {Redel, BK and Lee, K}, title = {Response to Cook et al. re: Novel Off-Targeting Events Identified After Genome Wide Analysis of CRISPR-Cas Edited Pig.}, journal = {The CRISPR journal}, volume = {8}, number = {2}, pages = {77-78}, doi = {10.1089/crispr.2025.0003}, pmid = {39866131}, issn = {2573-1602}, }
@article {pmid39865897, year = {2025}, author = {van Belle, J and Schaart, JG and Dechesne, AC and Fei, D and Cisneros, AO and Serafini, M and Visser, RGF and van Loo, EN}, title = {Direct and indirect effects of multiplex genome editing of F5H and FAD2 in oil crop camelina.}, journal = {Plant biotechnology journal}, volume = {23}, number = {5}, pages = {1399-1412}, pmid = {39865897}, issn = {1467-7652}, support = {6635405//European Commission/ ; }, mesh = {*Gene Editing/methods ; *Fatty Acid Desaturases/genetics/metabolism ; *Brassicaceae/genetics/metabolism ; *Plant Oils/metabolism ; *Plant Proteins/genetics/metabolism ; Seeds/genetics/metabolism ; Mutation ; Fatty Acids/metabolism ; CRISPR-Cas Systems ; Plants, Genetically Modified ; }, abstract = {Mutants with simultaneous germline mutations were obtained in all three F5H genes and all three FAD2 genes (one to eleven mutated alleles) in order to improve the feed value of the seed meal and the fatty acid composition of the seed oil. In mutants with multiple mutated F5H alleles, sinapine in seed meal was reduced by up to 100%, accompanied by a sharp reduction in the S-monolignol content of lignin without causing lodging or stem break. A lower S-lignin monomer content in stems can contribute to improved stem degradability allowing new uses of stems. Mutants in all six FAD2 alleles showed an expected increase in MUFA from 8.7% to 74% and a reduction in PUFA from 53% to 13% in the fatty acids in seed oil. Remarkably, some full FAD2 mutants showed normal growth and seed production and not the dwarfing phenotype reported in previous studies. The relation between germline mutation allele dosage and phenotype was influenced by the still ongoing activity of the CRISPR/Cas9 system, leading to new somatic mutations in the leaves of flowering plants. The correlations between the total mutation frequency (germline plus new somatic mutations) for F5H with sinapine content, and FAD2 with fatty acid composition were higher than the correlations between germline mutation count and phenotypes. This shows the importance of quantifying both the germline mutations and somatic mutations when studying CRISPR/Cas9 effects in situations where the CRISPR/Cas9 system is not yet segregated out.}, }
@article {pmid39865762, year = {2025}, author = {Zhu, F and Zhao, Q}, title = {A CRISPR/Cas12a-based competitive aptasensor for ochratoxin A detection.}, journal = {Analytical methods : advancing methods and applications}, volume = {17}, number = {7}, pages = {1487-1492}, doi = {10.1039/d4ay02231a}, pmid = {39865762}, issn = {1759-9679}, mesh = {*Ochratoxins/analysis ; *Aptamers, Nucleotide/chemistry/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Food Contamination/analysis ; }, abstract = {The serious contamination of ochratoxin A (OTA) in agricultural products has promoted the development of rapid, sensitive, and selective analytical methods for OTA monitoring. We demonstrated a competitive aptasensor for OTA detection using CRISPR/Cas12a as an effective signal amplifier. OTA competes with complementary DNA of the aptamer on the microplate to bind to the aptamer. Streptavidin bridges the biotinylated aptamer and biotinylated activator to convert the OTA input into Cas12a activation, which cleaves fluorescent DNA reporters. Under optimized experimental conditions, the aptasensor was demonstrated to work well for sensitive detection of OTA, with a linear range from 0.5 nM to 62.5 nM and a detection limit of 0.5 nM. Moreover, our method not only exhibits high selectivity, but also has satisfactory anti-interference ability against complex sample matrices. Taken together, the CRISPR/Cas12a-based competitive aptasensor offers a simple and sensitive platform for OTA detection, and it holds great promise for food security monitoring.}, }
@article {pmid39865728, year = {2025}, author = {Tian, Y and Xu, Q and Pang, M and Ma, Y and Zhang, Z and Zhang, D and Guo, D and Wang, L and Li, Q and Li, Y and Zhao, F}, title = {CRISPR-Cas9 Cytidine-Base-Editor Mediated Continuous In Vivo Evolution in Aspergillus nidulans.}, journal = {ACS synthetic biology}, volume = {14}, number = {2}, pages = {621-628}, doi = {10.1021/acssynbio.4c00716}, pmid = {39865728}, issn = {2161-5063}, mesh = {*Aspergillus nidulans/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Cytidine/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Directed Molecular Evolution/methods ; Multigene Family ; }, abstract = {Filamentous fungi are important cell factories for producing chemicals, organic acids, and enzymes. Although several genome editing tools are available for filamentous fungi, few effectively enable continuous evolution for rational engineering of complex phenotype. Here, we present CRISPR-Cas9 cytidine-base-editor (CBE) assisted in vivo evolution by continuously delivering a combinatorial sgRNA library to filamentous fungi. The method was validated by targeting core genes of 46 natural product biosynthetic gene clusters in Aspergillus nidulans NRRL 8112 to eliminate fungal toxins via six rounds of evolution. NGS analysis revealed the average C-to-T conversion rates in the first, third, and sixth rounds were 2.02%, 5.25%, and 9.34%, respectively. Metabolic profiles of the evolved mutants exhibited significant changes, allowing for the isolation of clean-background strains with enhanced production of an antifungal compound Echinocandin B. This study demonstrates that CBE-mediated in vivo evolution greatly facilitates the iterative refinement of complex morphogenetic traits in filamentous fungi.}, }
@article {pmid39863930, year = {2025}, author = {Verma, SS and Sen, CK and Srivastava, R and Gnyawali, SC and Katiyar, P and Sahi, AK and Kumar, M and Rustagi, Y and Liu, S and Pandey, D and Abouhashem, AS and Fehme, LNW and Kacar, S and Mohanty, SK and Faden-McCormack, J and Murphy, MP and Roy, S and Wan, J and Yoder, MC and Singh, K}, title = {Tissue nanotransfection-based endothelial PLCγ2-targeted epigenetic gene editing rescues perfusion and diabetic ischemic wound healing.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {33}, number = {3}, pages = {950-969}, pmid = {39863930}, issn = {1525-0024}, support = {R01 DK136814/DK/NIDDK NIH HHS/United States ; }, mesh = {*Wound Healing/genetics ; *Phospholipase C gamma/genetics/metabolism ; Animals ; Humans ; Mice ; *Ischemia/genetics/therapy ; *Epigenesis, Genetic ; *Gene Editing/methods ; Promoter Regions, Genetic ; Vascular Endothelial Growth Factor A/genetics ; DNA Methylation ; Neovascularization, Physiologic/genetics ; Diabetes Mellitus, Experimental/genetics ; Endothelial Cells/metabolism ; Disease Models, Animal ; Male ; CRISPR-Cas Systems ; Transfection/methods ; }, abstract = {Diabetic wounds are complicated by underlying peripheral vasculopathy. Reliance on vascular endothelial growth factor (VEGF) therapy to improve perfusion makes logical sense, yet clinical study outcomes on rescuing diabetic wound vascularization have yielded disappointing results. Our previous work has identified that low endothelial phospholipase Cγ2 (PLCγ2) expression hinders the therapeutic effect of VEGF on the diabetic ischemic limb. In this work, guided by single-cell RNA sequencing of human wound edge, we test the efficacy of gene-targeted therapeutic demethylation intending to improve VEGF-mediated neovascularization. PLCγ2 expression was diminished in all five identified diabetic wound-edge endothelial subclusters encompassing arterial, venous, and capillary cells. Such low expression was associated with hypermethylated PLCγ2 promoter. PLCγ2 promoter was also hypermethylated at murine diabetic ischemic wound edge. To specifically demethylate endothelial PLCγ2 promoter during VEGF therapy, a CRISPR-dCas9-based demethylation cocktail was delivered to the ischemic wound edge using tissue nanotransfection (TNT) technology. Demethylation-based upregulation of PLCγ2 during VEGF therapy improved wound tissue blood flow with an increased abundance of von Willebrand factor (vWF)[+]/PLCγ2[+] vascular tissue elements by activating p44/p42-mitogen-activated protein kinase (MAPK) → hypoxia-inducible factor [HIF]-1α pathway. Taken together, TNT-based delivery of plasmids to demethylate the PLCγ2 gene promoter activity led to significant improvements in VEGF therapy for cutaneous diabetic wounds, resulting in better perfusion and accelerated wound closure.}, }
@article {pmid39863748, year = {2025}, author = {Du, Y and Yang, Y and Zheng, B and Zhang, Q and Zhou, S and Zhao, L}, title = {Finding a needle in a haystack: functional screening for novel targets in cancer immunology and immunotherapies.}, journal = {Oncogene}, volume = {44}, number = {7}, pages = {409-426}, pmid = {39863748}, issn = {1476-5594}, mesh = {Humans ; *Neoplasms/immunology/therapy/genetics ; *Immunotherapy/methods ; Animals ; CRISPR-Cas Systems ; Genetic Testing/methods ; }, abstract = {Genome-wide functional genetic screening has been widely used in the biomedicine field, which makes it possible to find a needle in a haystack at the genetic level. In cancer research, gene mutations are closely related to tumor development, metastasis, and recurrence, and the use of state-of-the-art powerful screening technologies, such as clustered regularly interspaced short palindromic repeat (CRISPR), to search for the most critical genes or coding products provides us with a new possibility to further refine the cancer mapping and provide new possibilities for the treatment of cancer patients. The use of CRISPR screening for the most critical genes or coding products has further refined the cancer atlas and provided new possibilities for the treatment of cancer patients. Immunotherapy, as a highly promising cancer treatment method, has been widely validated in the clinic, but it could only meet the needs of a small proportion of cancer patients. Finding new immunotherapy targets is the key to the future of tumor immunotherapy. Here, we revisit the application of functional screening in cancer immunology from different perspectives, from the selection of diverse in vitro and in vivo screening models to the screening of potential immune checkpoints and potentiating genes for CAR-T cells. The data will offer fresh therapeutic clues for cancer patients.}, }
@article {pmid39863179, year = {2025}, author = {Zheng, Y and Wang, S and Deng, Y and Hu, P and Xue, Q and Li, J and Lei, L and Chan, Z and Yang, J and Peng, W}, title = {Enhanced production of recombinant calf chymosin in Kluyveromyces lactis via CRISPR-Cas9 engineering.}, journal = {Bioresource technology}, volume = {419}, number = {}, pages = {132116}, doi = {10.1016/j.biortech.2025.132116}, pmid = {39863179}, issn = {1873-2976}, mesh = {*Kluyveromyces/genetics/metabolism ; *Chymosin/biosynthesis/genetics ; *Recombinant Proteins/biosynthesis/genetics ; *CRISPR-Cas Systems/genetics ; Animals ; Cattle ; Fermentation ; }, abstract = {As an important industrial enzyme, chymosin has been widely used in cheese manufacturing. Fermentation with Kluyveromyces lactis has allowed recombinant chymosin production to fit the growing global demand for cheese consumption; yet improvements can be made to allow for stable and larger-scale production. In this work, various chymosin producing (CP) strains were constructed via targeted chromosomal integration of various copies of a prochymosin expression cassette (PEC) using a CRISPR-Cas9 platform optimized for K. lactis. It enabled the demonstration that chymosin yields could be increased along with gradual chromosomal accumulation of PEC inserts within up to 3 copies. Finally, an optimal CP3i strain was constructed, and with which high yields of recombinant chymosin were attained, reaching ca. 1,200 SU/mL in shake-flask fermentation and ca. 28,000 SU/mL in batch-mode bioreaction, respectively. The activity of the product in milk-curding was observed. These findings provide direction to apply K. lactis-based platforms in the subsequent industrial-scale production of recombinant chymosin.}, }
@article {pmid39862915, year = {2025}, author = {Hou, PS and Lin, SF and Zhu, JD and Chung, CY and Tsai, SJ and Yang, AC}, title = {Local cortical structure pattern and genetic links in schizophrenia: An MRI and CRISPR/Cas9 study.}, journal = {Progress in neuro-psychopharmacology &amp; biological psychiatry}, volume = {137}, number = {}, pages = {111270}, doi = {10.1016/j.pnpbp.2025.111270}, pmid = {39862915}, issn = {1878-4216}, mesh = {*Schizophrenia/genetics/diagnostic imaging/pathology ; Humans ; Magnetic Resonance Imaging ; Male ; Female ; *Cerebral Cortex/diagnostic imaging/pathology ; Adult ; Genome-Wide Association Study ; Animals ; *CRISPR-Cas Systems/genetics ; Mice ; Polymorphism, Single Nucleotide ; Middle Aged ; Reelin Protein ; Young Adult ; }, abstract = {While the etiology of schizophrenia (SZ) remains elusive, its diverse phenotypes suggest the involvement of distinct functional cortical areas, and the heritability of SZ implies the underlying genetic factors. This study aimed to integrate imaging and molecular analyses to elucidate the genetic underpinnings of SZ. We investigated the local cortical structural pattern changes in Brodmann areas (BAs) by calculating the cortical structural pattern index (SPI) using magnetic resonance imaging analysis from 194 individuals with SZ and 330 controls. Significant local structural changes were detected in certain Brodmann areas in symmetric or asymmetric patterns, such as symmetric changes in the BA4 primary motor area and BA23 part of posterior cingulate cortex, and asymmetric changes in the BA13 insula, BA11 inferior orbitofrontal area, and BA 24, and BA 31 cingulate cortex. Following genome-wide association tests, we found genetic variants and SNP-mapped genes and verified the areal preferential expression profiles in the developing human and mouse neocortex. Finally, we performed a loss-of-function analysis using the CRISPR/Cas9 system to investigate the effects of disrupting the SZ-related SNP-mapped Morf4l1, Reep3, or Tmed3 gene on cortical cell fate to understand their roles in generating appropriate composition of cortical neurons. This study outlines a pipeline for identifying local structural changes, associated genetic causes, and potential molecular mechanisms underlying mental disorders. Additionally, these data shed light on establishing a structurally integral cerebral cortex for higher cognitive functions.}, }
@article {pmid39862805, year = {2025}, author = {Li, Y and Xu, R and Quan, F and Wu, Y and Wu, Y and Zhang, Y and Liang, Y and Zhang, Z and Gao, H and Deng, R and Zhang, K and Li, J}, title = {Topologically constrained DNA-mediated one-pot CRISPR assay for rapid detection of viral RNA with single nucleotide resolution.}, journal = {EBioMedicine}, volume = {112}, number = {}, pages = {105564}, pmid = {39862805}, issn = {2352-3964}, mesh = {*SARS-CoV-2/genetics/isolation &amp; purification ; *RNA, Viral/genetics ; Humans ; *COVID-19/diagnosis/virology ; *CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques/methods ; Genome, Viral ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Sensitivity and Specificity ; }, abstract = {BACKGROUND: The widespread and evolution of RNA viruses, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlights the importance of fast identification of virus subtypes, particularly in non-laboratory settings. Rapid and inexpensive at-home testing of viral nucleic acids with single-base resolution remains a challenge.<br><br>METHODS: Topologically constrained DNA ring is engineered as substrates for the trans-cleavage of Cas13a to yield an accelerated post isothermal amplification. The capacity of CRISPR/Cas13a for discriminating single nucleotide variant (SNV) in viral genome is leveraged by designing synthetic mismatches and hairpin structure in CRISPR RNA (crRNA), enabling robust discrimination of different SARS-CoV-2 variants. Via optimisation of CasTDR3pot to be one-pot assay, CasTDR1pot can detect Omicron and its subvariants, with only a few copies in clinical samples in less than 30&#xa0;min without pre-amplification.<br><br>FINDINGS: The detection system boasts high sensitivity (0.1 aM), single-base specificity, and the advantage of a rapid "sample-to-answer" process, which takes only 30&#xa0;min. In the detection of SARS-CoV-2 clinical samples and their variant strains, CasTDR1pot has achieved 100% accuracy. Furthermore, the design of a portable signal-reading device facilitates user-friendly result interpretation. For the detection needs of different RNA viruses, the system can be adapted simply by designing the corresponding crRNA.<br><br>INTERPRETATION: Our study provides a rapid and accurate molecular diagnostic tool for point-of-care testing, epidemiological screening, and the detection of diseases associated with other RNA biomarkers with excellent single nucleotide differentiation, high sensitivity, and simplicity.<br><br>FUNDING: National Key Research and Development Program of China (No. 2023YFB3208302), National Natural Science Foundation of China (No. 22377110, 22034004, 82402749, 82073787, 22122409), National Key Research and Development Program of China (No. 2021YFA1200104), Henan Province Fund for Cultivating Advantageous Disciplines (No. 222301420019).}, }
@article {pmid39862677, year = {2025}, author = {Pei, J and Li, L and Li, C and Li, Z and Wu, Y and Kuang, H and Ma, P and Huang, L and Liu, J and Tian, G}, title = {Dumbbell probe-bridged CRISPR/Cas13a and nicking-mediated DNA cascade reaction for highly sensitive detection of colorectal cancer-related microRNAs.}, journal = {Biosensors &amp; bioelectronics}, volume = {273}, number = {}, pages = {117190}, doi = {10.1016/j.bios.2025.117190}, pmid = {39862677}, issn = {1873-4235}, mesh = {*MicroRNAs/genetics/isolation &amp; purification ; *Colorectal Neoplasms/genetics/diagnosis ; Humans ; *Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; Limit of Detection ; Electrochemical Techniques/methods ; DNA/chemistry/genetics ; }, abstract = {Colorectal cancer (CRC) is a leading cause of cancer-related deaths globally, necessitating the development of sensitive and minimally invasive diagnostic approaches. In this study, we present a novel diagnostic strategy by integrating dumbbell probe-mediated CRISPR/Cas13a with nicking-induced DNA cascade reaction (DP-bridged Cas13a/NDCR) for highly sensitive microRNA (miRNA) detection. Target miRNA triggers Cas13a-mediated cleavage of the dumbbell probe, releasing an intermediate strand that hybridizes with a methylene blue-labeled hairpin probe on the electrode surface. Nicking enzyme cleaves the formed duplex DNA, triggering a cascade reaction that amplifies the electrochemical signal. Under optimized conditions, the method demonstrates a detection limit of 8.26 fM for miRNA-21, with reliable specificity and long-term stability. Furthermore, integration with machine learning models using multiple miRNA markers improved diagnostic accuracy, differentiating CRC from colorectal polyps and healthy controls with 100% accuracy in clinical validation cohorts. This study highlights the potential of DP-bridged Cas13a/NDCR as a sensitive and accurate diagnostic tool for CRC.}, }
@article {pmid39862216, year = {2025}, author = {Fu, R and Wang, Y and Qiao, S and Xu, P and Xianyu, Y and Zhang, J}, title = {CRISPR-Cas12a-Mediated Growth of Gold Nanoparticles for DNA Detection in Agarose Gel.}, journal = {ACS sensors}, volume = {10}, number = {2}, pages = {1429-1439}, doi = {10.1021/acssensors.4c03461}, pmid = {39862216}, issn = {2379-3694}, mesh = {*Gold/chemistry ; *Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems ; Sepharose/chemistry ; Humans ; *DNA/analysis ; BRCA1 Protein/genetics/analysis ; *Biosensing Techniques/methods ; Limit of Detection ; *CRISPR-Associated Proteins/metabolism ; DNA, Single-Stranded ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {The rapid, simple, and sensitive detection of nucleic acid biomarkers plays a significant role in clinical diagnosis. Herein, we develop a label-free and point-of-care approach for isothermal DNA detection through the trans-cleavage activity of CRISPR-Cas12 and the growth of gold nanomaterials in agarose gel. The presence of the target can activate CRISPR-Cas12a to cleave single-stranded DNA, thus modulating the length and number of DNA sequences that mediate the growth of gold nanoparticles (AuNPs) or gold nanorods (AuNRs). Due to the extraordinary plasmonic property of gold nanomaterials, they present characteristic absorption/color after the growth with unique shapes. The sensing strategy is applied to detect BRCA-1, a biomarker related to breast cancer, with limits of detection of 1.72 pM (AuNP-based) and 2.07 pM (AuNR-based). AuNPs/AuNRs can be immobilized in agarose gels that display different colors in the presence of target DNA sequences. The agarose gel-based test allows for a readout by the naked eye or the RGB value with a smartphone. The approach is isothermal and label-free without any surface modification of nanomaterials, which holds great potential for the detection of nucleic acids in clinical applications.}, }
@article {pmid39862084, year = {2025}, author = {Szabó, &#xc1; and Galla, Z and Spekker, E and Szűcs, M and Martos, D and Takeda, K and Ozaki, K and Inoue, H and Yamamoto, S and Toldi, J and Ono, E and Vécsei, L and Tanaka, M}, title = {Oxidative and Excitatory Neurotoxic Stresses in CRISPR/Cas9-Induced Kynurenine Aminotransferase Knockout Mice: A Novel Model for Despair-Based Depression and Post-Traumatic Stress Disorder.}, journal = {Frontiers in bioscience (Landmark edition)}, volume = {30}, number = {1}, pages = {25706}, doi = {10.31083/FBL25706}, pmid = {39862084}, issn = {2768-6698}, support = {K138125//National Research, Development, and Innovation Office-NKFIH/ ; 2022/5S729//SZTE SZAOK-KKA/ ; //HUN-REN Hungarian Research Network/ ; JPJSBP120203803//JSPS Joint Research Projects under Bilateral Programs/ ; }, mesh = {Animals ; *CRISPR-Cas Systems ; Mice, Knockout ; *Depression/genetics/metabolism ; Disease Models, Animal ; *Oxidative Stress ; Male ; Mice ; *Stress Disorders, Post-Traumatic/genetics/metabolism ; *Transaminases/genetics/metabolism ; Tryptophan/metabolism ; Kynurenic Acid/metabolism ; Serotonin/metabolism ; Kynurenine/metabolism ; Mice, Inbred C57BL ; }, abstract = {BACKGROUNDS: Memory and emotion are especially vulnerable to psychiatric disorders such as post-traumatic stress disorder (PTSD), which is linked to disruptions in serotonin (5-HT) metabolism. Over 90% of the 5-HT precursor tryptophan (Trp) is metabolized via the Trp-kynurenine (KYN) metabolic pathway, which generates a variety of bioactive molecules. Dysregulation of KYN metabolism, particularly low levels of kynurenic acid (KYNA), appears to be linked to neuropsychiatric disorders. The majority of KYNA is produced by the aadat (kat2) gene-encoded mitochondrial kynurenine aminotransferase (KAT) isotype 2. Little is known about the consequences of deleting the KYN enzyme gene.<br><br>METHODS: In CRISPR/Cas9-induced aadat knockout (kat2[-/-]) mice, we examined the effects on emotion, memory, motor function, Trp and its metabolite levels, enzyme activities in the plasma and urine of 8-week-old males compared to wild-type mice.<br><br>RESULTS: Transgenic mice showed more depressive-like behaviors in the forced swim test, but not in the tail suspension, anxiety, or memory tests. They also had fewer center field and corner entries, shorter walking distances, and fewer jumping counts in the open field test. Plasma metabolite levels are generally consistent with those of urine: antioxidant KYNs, 5-hydroxyindoleacetic acid, and indole-3-acetic acid levels were lower; enzyme activities in KATs, kynureninase, and monoamine oxidase/aldehyde dehydrogenase were lower, but kynurenine 3-monooxygenase was higher; and oxidative stress and excitotoxicity indices were higher. Transgenic mice displayed depression-like behavior in a learned helplessness model, emotional indifference, and motor deficits, coupled with a decrease in KYNA, a shift of Trp metabolism toward the KYN-3-hydroxykynurenine pathway, and a partial decrease in the gut microbial Trp-indole pathway metabolite.<br><br>CONCLUSIONS: This is the first evidence that deleting the aadat gene induces depression-like behaviors uniquely linked to experiences of despair, which appear to be associated with excitatory neurotoxic and oxidative stresses. This may lead to the development of a double-hit preclinical model in despair-based depression, a better understanding of these complex conditions, and more effective therapeutic strategies by elucidating the relationship between Trp metabolism and PTSD pathogenesis.}, }
@article {pmid39861899, year = {2025}, author = {Sugiokto, FG and Li, R}, title = {Targeting EBV Episome for Anti-Cancer Therapy: Emerging Strategies and Challenges.}, journal = {Viruses}, volume = {17}, number = {1}, pages = {}, pmid = {39861899}, issn = {1999-4915}, support = {R01 AI141410/AI/NIAID NIH HHS/United States ; R01AI141410//National Institute of Allergy and Infectious Diseases/ ; }, mesh = {Humans ; *Herpesvirus 4, Human/genetics ; *Neoplasms/therapy/virology ; *Epstein-Barr Virus Infections/virology/complications/therapy ; Gene Editing/methods ; Genome, Viral ; CRISPR-Cas Systems ; Animals ; }, abstract = {As a ubiquitous human pathogen, the Epstein-Barr virus (EBV) has established lifelong persistent infection in about 95% of the adult population. The EBV infection is associated with approximately 200,000 human cancer cases and 140,000 deaths per year. The presence of EBV in tumor cells provides a unique advantage in targeting the viral genome (also known as episome), to develop anti-cancer therapeutics. In this review, we summarize current strategies targeting the viral episome in cancer cells. We also highlight emerging technologies, such as clustered regularly interspersed short palindromic repeat (CRISPR)-based gene editing or activation, which offer promising avenues for selective targeting of the EBV episome for anti-cancer therapy. We discuss the challenges, limitations, and future perspectives associated with these strategies, including potential off-target effects, anti-cancer efficacy and safety.}, }
@article {pmid39861013, year = {2025}, author = {Yibar, A and Duman, M and Ay, H and Ajmi, N and Tasci, G and Gurler, F and Guler, S and Morick, D and Saticioglu, IB}, title = {Genomic Insight into Vibrio Isolates from Fresh Raw Mussels and Ready-to-Eat Stuffed Mussels.}, journal = {Pathogens (Basel, Switzerland)}, volume = {14}, number = {1}, pages = {}, pmid = {39861013}, issn = {2076-0817}, support = {TOA-2022-668 and TGA-2024-1841//Bursa Uluda&#x11f; &#xdc;ni&#x307;versi&#x307;tesi&#x307;/ ; }, mesh = {Animals ; *Vibrio/genetics/isolation &amp; purification/classification/pathogenicity ; *Bivalvia/microbiology ; *Genome, Bacterial ; Virulence Factors/genetics ; Genomics/methods ; *Food Microbiology ; Prevalence ; }, abstract = {Consuming raw or undercooked mussels can lead to gastroenteritis and septicemia due to Vibrio contamination. This study analyzed the prevalence, density, species diversity, and molecular traits of Vibrio spp. in 48 fresh raw wild mussels (FRMs) and 48 ready-to-eat stuffed mussels (RTE-SMs) through genome analysis, assessing health risks. The results showed Vibrio prevalence rates of 12.5% in FRMs and 4.2% in RTE-SMs, with V. alginolyticus as the most common species (46.7%). It was determined that the seasonal distribution of Vibrio spp. prevalence in the samples was higher in the summer months. The genome sizes of the Vibrio spp. ranged from approximately 3.9 to 6.1 Mb, with the GC contents varying between 41.9% and 50.4%. A total of 22 virulence factor (VF) classes and up to six antimicrobial resistance (AMR) genes were detected in different Vibrio species. The presence of nine different biosynthetic gene clusters (BGCs), 27 prophage regions, and eight CRISPR/Cas systems in 15 Vibrio strains provides information about their potential pathogenicity, survival strategies, and adaptation to different habitats. Overall, this study provides a comprehensive understanding of the genomic diversity of Vibrio spp. isolated from FRM and RTE-SM samples, shedding light on the prevalence, pathogenicity, and toxicity mechanisms of Vibrio-induced gastroenteritis.}, }
@article {pmid39859474, year = {2025}, author = {Wang, S and Xu, Q and Liu, W and Zhang, N and Qi, Y and Tang, F and Ge, R}, title = {Regulation of PHD2 by HIF-1α in Erythroid Cells: Insights into Erythropoiesis Under Hypoxia.}, journal = {International journal of molecular sciences}, volume = {26}, number = {2}, pages = {}, pmid = {39859474}, issn = {1422-0067}, support = {82360334//National Natural Science Foundation of China/ ; }, mesh = {*Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism/genetics ; Humans ; *Erythropoiesis/genetics ; *Hypoxia-Inducible Factor 1, alpha Subunit/metabolism/genetics ; *Erythroid Cells/metabolism/cytology ; K562 Cells ; Cell Hypoxia ; Cell Differentiation ; Gene Expression Regulation ; CRISPR-Cas Systems ; }, abstract = {The hypoxia-inducible factor (HIF) pathway has been demonstrated to play a pivotal role in the process of high-altitude adaptation. PHD2, a key regulator of the HIF pathway, has been found to be associated with erythropoiesis. However, the relationship between changes in Phd2 abundance and erythroid differentiation under hypoxic conditions remains to be elucidated. A hemin-induced K562 erythroid differentiation model was used to explore the effects of PHD2 knockdown under hypoxia. Erythroid differentiation was assessed by flow cytometry and immunofluorescence. HIF-1α's regulation of PHD2 was examined using luciferase assays and ChIP-seq. CRISPR/Cas9 was applied to knock out EGLN1 and HIF1A, and a fluorescent reporter system was developed to track PHD2 expression. PHD2 knockdown enhanced erythroid differentiation, evident by increased CD71 and CD235a expression. Reporter assays and ChIP-seq identified an HIF-1α binding site in the EGLN1 5' UTR, confirming HIF-1α as a regulator of PHD2 expression. The fluorescent reporter system provided real-time monitoring of endogenous PHD2 expression, showing that HIF-1α significantly modulates PHD2 levels under hypoxic conditions. PHD2 influences erythropoiesis under hypoxia, with HIF-1α regulating its expression. This feedback loop between HIF-1α and PHD2 sheds light on mechanisms driving erythroid differentiation under low-oxygen conditions.}, }
@article {pmid39859203, year = {2025}, author = {Petrella, S and Colombo, M and Marabese, M and Grasselli, C and Panfili, A and Chiappa, M and Sancisi, V and Craparotta, I and Barbera, MC and Cassanmagnago, GA and Bolis, M and Damia, G}, title = {Onvansertib and Navitoclax Combination as a New Therapeutic Option for Mucinous Ovarian Carcinoma.}, journal = {International journal of molecular sciences}, volume = {26}, number = {2}, pages = {}, pmid = {39859203}, issn = {1422-0067}, support = {PE00000019//Ministry of University and Research-MUR/ ; }, mesh = {Humans ; Female ; Cell Line, Tumor ; *Ovarian Neoplasms/drug therapy/genetics/metabolism/pathology ; *Sulfonamides/pharmacology ; *Aniline Compounds/pharmacology ; Protein Serine-Threonine Kinases/antagonists &amp; inhibitors ; Cell Cycle Proteins/antagonists &amp; inhibitors/metabolism ; Proto-Oncogene Proteins/antagonists &amp; inhibitors ; Polo-Like Kinase 1 ; *Carcinoma, Ovarian Epithelial/drug therapy/genetics ; Cell Proliferation/drug effects ; Cysteine Endopeptidases/genetics/metabolism ; Synthetic Lethal Mutations ; *Antineoplastic Combined Chemotherapy Protocols/pharmacology ; Gene Expression Regulation, Neoplastic/drug effects ; Cell Survival/drug effects ; CRISPR-Cas Systems ; Pteridines ; }, abstract = {Mucinous epithelial ovarian cancer (mEOC) is a rare subtype of epithelial ovarian cancer, characterized by poor responses to standard platinum-based chemotherapy. Polo-like kinase 1 (PLK1) is a key regulator of mitosis and cell cycle progression and its inhibition has been recently identified as a target in mEOC. In this study, we aimed to identify further therapeutic targets in mEOC using a CRISPR/Cas9 library targeting 3015 genes, with and without treatment with onvansertib, a PLK1 inhibitor. We identified twelve genes associated with cell survival (ZC2HC1C, RPA2, KIN17, TUBG1, SMC2, CDC26, CDC42, HOXA9, TAF10, SENP1, MRPS31, and COPS2) and three genes (JUND, CARD9, and BCL2L2) in synthetic lethality with onvansertib treatment. We validated that SENP1 downregulation is important for the growth of mEOC cells through esiRNA interference and the use of a pharmacological inhibitor Momordin Ic. The downregulation of CARD9 and BCL2L2 combined with subtoxic doses of onvansertib interfered with mEOC cell growth. Interestingly, the combination of navitoclax, an inhibitor of BcL2 family members including BCL2L2, was synergistic in all four of the mEOC cell lines tested and substantially induced cell death through apoptosis. These data support the use of a combination of navitoclax and onvansertib as a new therapeutic strategy for mEOC.}, }
@article {pmid39858940, year = {2025}, author = {Ma, J and Lu, Z}, title = {Developing a Versatile Arsenal: Novel Antimicrobials as Offensive Tools Against Pathogenic Bacteria.}, journal = {Microorganisms}, volume = {13}, number = {1}, pages = {}, pmid = {39858940}, issn = {2076-2607}, support = {31970101//the National Natural Science Foundation of China/ ; }, abstract = {The pervasive and often indiscriminate use of antibiotics has accelerated the emergence of drug-resistant bacterial strains, thus presenting an acute threat to global public health. Despite a growing acknowledgment of the severity of this crisis, the current suite of strategies to mitigate antimicrobial resistance remains markedly inadequate. This paper asserts the paramount need for the swift development of groundbreaking antimicrobial strategies and provides a comprehensive review of an array of innovative techniques currently under scrutiny. Among these, nano-antimicrobials, antimicrobials derived from ribosomal proteins, CRISPR/Cas-based systems, agents that undermine bacterial bioenergetics, and antimicrobial polysaccharides hold particular promise. This analysis gives special attention to CRISPR/Cas-based antimicrobials, scrutinizing their underlying mechanisms, exploring their potential applications, delineating their distinct advantages, and noting their likely limitations. Furthermore, we extend our exploration by proposing theoretical advancements in antimicrobial technology and evaluating feasible methods for the effective delivery of these agents. This includes leveraging these advances for broader biomedical applications, potentially revolutionizing how we confront bacterial pathogens in the future, and laying a foundation for extended research in multimodal therapeutic strategies.}, }
@article {pmid39858819, year = {2024}, author = {Zhang, Z and Wei, M and Jia, B and Yuan, Y}, title = {Recent Advances in Antimicrobial Resistance: Insights from Escherichia coli as a Model Organism.}, journal = {Microorganisms}, volume = {13}, number = {1}, pages = {}, pmid = {39858819}, issn = {2076-2607}, support = {2018YFA0903700//National Natural Science Foundation of China/ ; }, abstract = {Antimicrobial resistance (AMR) represents a critical global health threat, and a thorough understanding of resistance mechanisms in Escherichia coli is needed to guide effective treatment interventions. This review explores recent advances for investigating AMR in E. coli, including machine learning for resistance pattern analysis, laboratory evolution to generate resistant mutants, mutant library construction, and genome sequencing for in-depth characterization. Key resistance mechanisms are discussed, including drug inactivation, target modification, altered transport, and metabolic adaptation. Additionally, we highlight strategies to mitigate the spread of AMR, such as dynamic resistance monitoring, innovative therapies like phage therapy and CRISPR-Cas technology, and tighter regulation of antibiotic use in animal production systems. This review provides actionable insights into E. coli resistance mechanisms and identifies promising directions for future antibiotic development and AMR management.}, }
@article {pmid39858595, year = {2025}, author = {Dansereau, SJ and Cui, H and Dartawan, RP and Sheng, J}, title = {The Plethora of RNA-Protein Interactions Model a Basis for RNA Therapies.}, journal = {Genes}, volume = {16}, number = {1}, pages = {}, pmid = {39858595}, issn = {2073-4425}, support = {R01 GM143749/GM/NIGMS NIH HHS/United States ; 1R01GM143749-22A1/NH/NIH HHS/United States ; }, mesh = {Humans ; *RNA/genetics/therapeutic use/metabolism ; Animals ; Gene Editing/methods ; Aptamers, Nucleotide/therapeutic use/genetics ; *Genetic Therapy/methods ; RNA, Small Interfering/therapeutic use/genetics ; }, abstract = {The notion of RNA-based therapeutics has gained wide attractions in both academic and commercial institutions. RNA is a polymer of nucleic acids that has been proven to be impressively versatile, dating to its hypothesized RNA World origins, evidenced by its enzymatic roles in facilitating DNA replication, mRNA decay, and protein synthesis. This is underscored through the activities of riboswitches, spliceosomes, ribosomes, and telomerases. Given its broad range of interactions within the cell, RNA can be targeted by a therapeutic or modified as a pharmacologic scaffold for diseases such as nucleotide repeat disorders, infectious diseases, and cancer. RNA therapeutic techniques that have been researched include, but are not limited to, CRISPR/Cas gene editing, anti-sense oligonucleotides (ASOs), siRNA, small molecule treatments, and RNA aptamers. The knowledge gleaned from studying RNA-centric mechanisms will inevitably improve the design of RNA-based therapeutics. Building on this understanding, we explore the physiological diversity of RNA functions, examine specific dysfunctions, such as splicing errors and viral interactions, and discuss their therapeutic implications.}, }
@article {pmid39857007, year = {2025}, author = {Lakshmanan, K and Liu, BM}, title = {Impact of Point-of-Care Testing on Diagnosis, Treatment, and Surveillance of Vaccine-Preventable Viral Infections.}, journal = {Diagnostics (Basel, Switzerland)}, volume = {15}, number = {2}, pages = {}, pmid = {39857007}, issn = {2075-4418}, support = {P30 AI117970/AI/NIAID NIH HHS/United States ; U54 AI150225/AI/NIAID NIH HHS/United States ; P30AI117970; U54AI150225/GF/NIH HHS/United States ; }, abstract = {With the advent of a variety of vaccines against viral infections, there are multiple viruses that can be prevented via vaccination. However, breakthrough infections or uncovered strains can still cause vaccine-preventable viral infections (VPVIs). Therefore, timely diagnosis, treatment, and surveillance of these viruses is critical to patient care and public health. Point-of-care (POC) viral diagnostics tools have brought significant improvements in the detection and management of VPVIs. These cutting-edge technologies enable prompt and accurate results, enhancing patient care by facilitating timely treatment decisions. This review delves into the advancements in POC testing, including antigen/antibody detection and molecular assays, while focusing on their impact on the diagnosis, treatment, and surveillance of VPVIs such as mpox, viral hepatitis, influenza, flaviviruses (dengue, Zika, and yellow fever virus), and COVID-19. The role of POC tests in monitoring viral infection is crucial for tracking disease progression and managing outbreaks. Furthermore, the application of POC diagnostics has shown to be vital for public health strategies. In this review, we also highlight emerging POC technologies such as CRISPR-based diagnostics and smartphone-integrated POC devices, which have proven particularly beneficial in resource-limited settings. We underscore the importance of continued research to optimize these diagnostic tools for wider global use for mpox, viral hepatitis, influenza, dengue, and COVID-19, while also addressing current challenges related to their sensitivity, specificity, availability, efficiency, and more.}, }
@article {pmid39856077, year = {2025}, author = {Xu, X and Chen, J and Wang, Y and Liu, Y and Zhang, Y and Yang, J and Yang, X and Chen, B and He, Z and Champer, J}, title = {Gene drive-based population suppression in the malaria vector Anopheles stephensi.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {1007}, pmid = {39856077}, issn = {2041-1723}, support = {32302455//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {Animals ; *Anopheles/genetics ; Female ; *Mosquito Vectors/genetics ; *Malaria/transmission/prevention &amp; control ; Male ; *Gene Drive Technology/methods ; Mosquito Control/methods ; Alleles ; Heterozygote ; CRISPR-Cas Systems ; }, abstract = {Gene drives are alleles that can bias the inheritance of specific traits in target populations for the purpose of modification or suppression. Here, we construct a homing suppression drive in the major urban malaria vector Anopheles stephensi targeting the female-specific exon of doublesex, incorporating two gRNAs and a nanos-Cas9 to reduce functional resistance and improve female heterozygote fitness. Our results show that the drive was recessive sterile in both females and males, with various intersex phenotypes in drive homozygotes. Both male and female drive heterozygotes show only moderate drive conversion, indicating that the nanos promoter has lower activity in A. stephensi than in Anopheles gambiae. By amplicon sequencing, we detect a very low level of resistance allele formation. Combination of the homing suppression drive and a vasa-Cas9 line boosts the drive conversion rate of the homing drive to 100%, suggesting the use of similar systems for population suppression in a continuous release strategy with a lower release rate than SIT or fsRIDL techniques. This study contributes valuable insights to the development of more efficient and environmentally friendly pest control tools aimed at disrupting disease transmission.}, }
@article {pmid39856062, year = {2025}, author = {Pena, IA and Shi, JS and Chang, SM and Yang, J and Block, S and Adelmann, CH and Keys, HR and Ge, P and Bathla, S and Witham, IH and Sienski, G and Nairn, AC and Sabatini, DM and Lewis, CA and Kory, N and Vander Heiden, MG and Heiman, M}, title = {SLC25A38 is required for mitochondrial pyridoxal 5'-phosphate (PLP) accumulation.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {978}, pmid = {39856062}, issn = {2041-1723}, support = {P30 CA014051/CA/NCI NIH HHS/United States ; T32 GM007753/GM/NIGMS NIH HHS/United States ; P30 DA018343/DA/NIDA NIH HHS/United States ; R35 CA242379/CA/NCI NIH HHS/United States ; F31 CA228241/CA/NCI NIH HHS/United States ; F30 CA268633/CA/NCI NIH HHS/United States ; T32 GM144273/GM/NIGMS NIH HHS/United States ; R35 GM151097/GM/NIGMS NIH HHS/United States ; R00 CA241332/CA/NCI NIH HHS/United States ; F31 NS127458/NS/NINDS NIH HHS/United States ; T32 GM007287/GM/NIGMS NIH HHS/United States ; }, mesh = {*Pyridoxal Phosphate/metabolism ; Humans ; *Mitochondria/metabolism ; Vitamin B 6/metabolism ; Cell Proliferation ; K562 Cells ; *Mitochondrial Proteins/metabolism/genetics ; Glycine Hydroxymethyltransferase/metabolism ; Anemia, Sideroblastic/metabolism/genetics ; *Mitochondrial Membrane Transport Proteins/metabolism/genetics ; Metabolomics ; CRISPR-Cas Systems ; Genetic Diseases, X-Linked ; }, abstract = {Many essential proteins require pyridoxal 5'-phosphate, the active form of vitamin B6, as a cofactor for their activity. These include enzymes important for amino acid metabolism, one-carbon metabolism, polyamine synthesis, erythropoiesis, and neurotransmitter metabolism. A third of all mammalian pyridoxal 5'-phosphate-dependent enzymes are localized in the mitochondria; however, the molecular machinery involved in the regulation of mitochondrial pyridoxal 5'-phosphate levels in mammals remains unknown. In this study, we used a genome-wide CRISPR interference screen in erythroleukemia cells and organellar metabolomics to identify the mitochondrial inner membrane protein SLC25A38 as a regulator of mitochondrial pyridoxal 5'-phosphate. Loss of SLC25A38 causes depletion of mitochondrial, but not cellular, pyridoxal 5'-phosphate, and impairs cellular proliferation under both physiological and low vitamin B6 conditions. Metabolic changes associated with SLC25A38 loss suggest impaired mitochondrial pyridoxal 5'-phosphate-dependent enzymatic reactions, including serine to glycine conversion catalyzed by serine hydroxymethyltransferase-2 as well as ornithine aminotransferase. The proliferation defect of SLC25A38-null K562 cells in physiological and low vitamin B6 media can be explained by the loss of serine hydroxymethyltransferase-2-dependent production of one-carbon units and downstream de novo nucleotide synthesis. Our work points to a role for SLC25A38 in mitochondrial pyridoxal 5'-phosphate accumulation and provides insights into the pathology of congenital sideroblastic anemia.}, }
@article {pmid39856035, year = {2025}, author = {Padilla, MS and Mrksich, K and Wang, Y and Haley, RM and Li, JJ and Han, EL and El-Mayta, R and Kim, EH and Dias, S and Gong, N and Teerdhala, SV and Han, X and Chowdhary, V and Xue, L and Siddiqui, Z and Yamagata, HM and Kim, D and Yoon, IC and Wilson, JM and Radhakrishnan, R and Mitchell, MJ}, title = {Branched endosomal disruptor (BEND) lipids mediate delivery of mRNA and CRISPR-Cas9 ribonucleoprotein complex for hepatic gene editing and T cell engineering.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {996}, pmid = {39856035}, issn = {2041-1723}, support = {T90DE030854//U.S. Department of Health &amp; Human Services | NIH | National Institute of Dental and Craniofacial Research (NIDCR)/ ; T90 DE030854/DE/NIDCR NIH HHS/United States ; P30 AI045008/AI/NIAID NIH HHS/United States ; DP2 TR002776/TR/NCATS NIH HHS/United States ; P30 CA016520/CA/NCI NIH HHS/United States ; }, mesh = {*Gene Editing/methods ; *RNA, Messenger/genetics/metabolism/administration &amp; dosage ; *CRISPR-Cas Systems/genetics ; *Endosomes/metabolism ; *T-Lymphocytes/metabolism ; Humans ; *Ribonucleoproteins/genetics/metabolism ; *Lipids/chemistry ; *Liver/metabolism ; Nanoparticles/chemistry ; Animals ; Cell Engineering/methods ; Mice ; Drug Delivery Systems/methods ; CRISPR-Associated Protein 9/metabolism/genetics ; Gene Transfer Techniques ; Transfection ; Liposomes ; }, abstract = {Lipid nanoparticles (LNPs) are the preeminent non-viral drug delivery vehicle for mRNA-based therapies. Immense effort has been placed on optimizing the ionizable lipid (IL) structure, which contains an amine core conjugated to lipid tails, as small molecular adjustments can result in substantial changes in the overall efficacy of the resulting LNPs. However, despite some advancements, a major barrier for LNP delivery is endosomal escape. Here, we develop a platform for synthesizing a class of branched ILs that improve endosomal escape. These compounds incorporate terminally branched groups that increase hepatic mRNA and ribonucleoprotein complex delivery and gene editing efficiency as well as T cell transfection compared to non-branched lipids. Through an array of complementary experiments, we determine that our lipid architecture induces greater endosomal penetration and disruption. This work provides a scheme to generate a class of ILs for both mRNA and protein delivery.}, }
@article {pmid39855935, year = {2025}, author = {Perez, AR and Mavrothalassitis, O and Chen, JS and Hellman, J and Gropper, MA}, title = {CRISPR: fundamental principles and implications for anaesthesia.}, journal = {British journal of anaesthesia}, volume = {134}, number = {3}, pages = {839-852}, pmid = {39855935}, issn = {1471-6771}, support = {R38 AG070171/AG/NIA NIH HHS/United States ; }, mesh = {Humans ; *Anesthesia/methods ; *Gene Editing/methods ; *Anesthesiology/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based medical therapies are increasingly gaining regulatory approval worldwide. Consequently, patients receiving CRISPR therapy will come under the care of anaesthesiologists. An understanding of CRISPR, its technological implementations, and the characteristics of patients likely to receive this therapy will be essential to caring for this patient population. However, the role of CRISPR in anaesthesiology extends beyond simply caring for patients with prior CRISPR therapy. CRISPR has multiple direct potential applications in anaesthesia, particularly for managing chronic pain and critical illness. Additionally, given the unique skills anaesthesiologists possess, CRISPR potentially allows new roles for anaesthesiologists in the field of oncology. Consequently, CRISPR technology could enable new domains of anaesthetic practice. This review provides a primer on CRISPR for anaesthesiologists and an overview on how the technology could impact the field.}, }
@article {pmid39855050, year = {2025}, author = {Monfort-Vengut, A and Sanz-Gómez, N and Ballesteros-Sánchez, S and Ortigosa, B and Cambón, A and Ramos, M and Lorenzo, &#xc1;M and Escribano-Cebrián, M and Rosa-Rosa, JM and Martínez-López, J and Sánchez-Prieto, R and Sotillo, R and de Cárcer, G}, title = {Osmotic stress influences microtubule drug response via WNK1 kinase signaling.}, journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy}, volume = {79}, number = {}, pages = {101203}, doi = {10.1016/j.drup.2025.101203}, pmid = {39855050}, issn = {1532-2084}, mesh = {*WNK Lysine-Deficient Protein Kinase 1/metabolism/genetics/antagonists &amp; inhibitors ; Humans ; *Microtubules/drug effects/metabolism ; *Osmotic Pressure/drug effects ; Signal Transduction/drug effects ; Glycine/analogs &amp; derivatives/pharmacology ; Drug Resistance, Neoplasm/genetics/drug effects ; *Antineoplastic Agents/pharmacology ; Tubulin Modulators/pharmacology ; Cell Line, Tumor ; Mitosis/drug effects ; CRISPR-Cas Systems ; }, abstract = {Ion homeostasis is critical for numerous cellular processes, and disturbances in ionic balance underlie diverse pathological conditions, including cancer progression. Targeting ion homeostasis is even considered as a strategy to treat cancer. However, very little is known about how ion homeostasis may influence anticancer drug response. In a genome-wide CRISPR-Cas9 resistance drug screen, we identified and validated the master osmostress regulator WNK1 kinase as a modulator of the response to the mitotic inhibitor rigosertib. Osmotic stress and WNK1 inactivation lead to an altered response not only to rigosertib treatment but also to other microtubule-related drugs, minimizing the prototypical mitotic arrest produced by these compounds. This effect is due to an alteration in microtubule stability and polymerization dynamics, likely maintained by fluctuations in intracellular molecular crowding upon WNK1 inactivation. This promotes resistance to microtubule depolymerizing compounds, and increased sensitivity to microtubule stabilizing drugs. In summary, our data proposes WNK1 osmoregulation activity as an important modulator for microtubule-associated chemotherapy response.}, }
@article {pmid39854604, year = {2025}, author = {He, X and Sun, Y and Ma, H}, title = {ParSite is a multicolor DNA labeling system that allows for simultaneous imaging of triple genomic loci in living cells.}, journal = {PLoS biology}, volume = {23}, number = {1}, pages = {e3003009}, pmid = {39854604}, issn = {1545-7885}, mesh = {Humans ; *DNA/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Tumor Suppressor p53-Binding Protein 1/metabolism/genetics ; Genetic Loci ; Genome, Human ; DNA Breaks, Double-Stranded ; *Staining and Labeling/methods ; }, abstract = {The organization of the human genome in space and time is critical for transcriptional regulation and cell fate determination. However, robust methods for tracking genome organization or genomic interactions over time in living cells are lacking. Here, we developed a multicolor DNA labeling system, ParSite, to simultaneously track triple genomic loci in the U2OS cells. The tricolor ParSite system is derived from the T. thermophilus ParB/ParSc (TtParB/ParSc) system by rational design. We mutated the interface between TtParB and ParSc and generated a new pair of TtParBm and ParSm for genomic DNA labeling. The insertions of 16 base-pair palindromic ParSc and ParSm into genomic loci allow dual-color DNA imaging in living cells. A pair of genomic loci labeled by ParSite could be colocalized with p53-binding protein 1 (53BP1) in response to CRISPR/Cas9-mediated double-strand breaks (DSBs). The ParSite permits tracking promoter and terminator dynamics of the APP gene, which spans 290 kilobases in length. Intriguingly, the hybrid ParS (ParSh) of half-ParSc and half-ParSm enables for the visualization of a third locus independent of ParSc or ParSm. We simultaneously labeled 3 loci with a genomic distance of 36, 89, and 352 kilobases downstream the C3 repeat locus, respectively. In sum, the ParSite is a robust DNA labeling system for tracking multiple genomic loci in space and time in living cells.}, }
@article {pmid39854420, year = {2025}, author = {Kravets, E and Poschmann, G and Hänsch, S and Raba, V and Weidtkamp-Peters, S and Degrandi, D and Stühler, K and Pfeffer, K}, title = {mGBP2 engages Galectin-9 for immunity against Toxoplasma gondii.}, journal = {PloS one}, volume = {20}, number = {1}, pages = {e0316209}, pmid = {39854420}, issn = {1932-6203}, mesh = {*Toxoplasma/immunology ; *Galectins/metabolism/genetics/immunology ; Animals ; Mice ; *GTP-Binding Proteins/metabolism/genetics/immunology ; Humans ; *Toxoplasmosis/immunology/parasitology ; Vacuoles/metabolism/parasitology/immunology ; CRISPR-Cas Systems ; }, abstract = {Guanylate binding proteins (GBPs) are large interferon-inducible GTPases, executing essential host defense activities against Toxoplasma gondii, an invasive intracellular apicomplexan protozoan parasite of global importance. T. gondii establishes a parasitophorous vacuole (PV) which shields the parasite from the host's intracellular defense mechanisms. Murine GBPs (mGBPs) recognize T. gondii PVs and assemble into supramolecular mGBP homo- and heterocomplexes that are required for the disruption of the membrane of PVs eventually resulting in the cell-autonomous immune control of vacuole-resident pathogens. We have previously shown that mGBP2 plays an important role in T. gondii immune control. Here, to unravel mGBP2 functions, we report Galectin-9 (Gal9) as a critical mGBP2 interaction partner engaged for immunity to T. gondii. Interestingly, Gal9 also accumulates and colocalizes with mGBP2 at the T. gondii PV. Furthermore, we could prove the requirement of Gal9 for growth control of T. gondii by CRISPR/Cas9 mediated gene editing. These discoveries clearly indicate that Gal9 is a crucial factor for the mGBP2-coordinated cell-autonomous host defense mechanism against T. gondii.}, }
@article {pmid39853972, year = {2025}, author = {Fu, R and Hou, J and Wang, Z and Zhu, C and Xianyu, Y}, title = {A CRISPR-Cas and Argonaute-Driven Two-Factor Authentication Strategy for Information Security.}, journal = {ACS nano}, volume = {19}, number = {4}, pages = {4983-4992}, doi = {10.1021/acsnano.4c17560}, pmid = {39853972}, issn = {1936-086X}, mesh = {*CRISPR-Cas Systems/genetics ; *Computer Security ; *Argonaute Proteins/genetics/metabolism ; Pyrococcus furiosus/genetics ; }, abstract = {The escalating growth in computing power and the advent of quantum computing present a critical threat to the security of modern cryptography. Two-factor authentication strategies can effectively resist brute-force attacks to improve the security of access control. Herein, we proposed a two-factor and two-authentication entity strategy based on the trans-cleavage activity of CRISPR-Cas and the "dual-step" sequence-specific cleavage of Pyrococcus furiosus Argonaute. In this strategy, the output of authentication entity 1 acted as a component to operate authentication entity 2, thus enabling a role-based molecular model that implemented access control for the three roles. To further enhance information security, we designed knowledge suppression factors to constitute the command library and possession suppression factors to resist brute-force attacks. This study will promote the development of advanced molecular access control and its applications in biomedical diagnostics and data security.}, }
@article {pmid39853276, year = {2025}, author = {Shankar, S and Giraldo, D and Tauxe, GM and Spikol, ED and Li, M and Akbari, OS and Wohl, MP and McMeniman, CJ}, title = {Optimized genetic tools for neuroanatomical and functional mapping of the Aedes aegypti olfactory system.}, journal = {G3 (Bethesda, Md.)}, volume = {15}, number = {3}, pages = {}, pmid = {39853276}, issn = {2160-1836}, support = {P30 NS050274/NS/NINDS NIH HHS/United States ; T32A1007417//Molecular and Cellular Basis of Infectious Diseases/ ; S10OD016374/NH/NIH HHS/United States ; NS050274//JHU Center for Neuroscience Research Machine Shop/ ; AID-OAA-F-16-00061//USAID/ ; R01AI151004/GF/NIH HHS/United States ; 200-2017-93143/CC/CDC HHS/United States ; S10 OD023548/OD/NIH HHS/United States ; R21AI139358/GF/NIH HHS/United States ; R21AI139358/NH/NIH HHS/United States ; }, mesh = {Animals ; *Aedes/genetics/anatomy &amp; histology/physiology ; CRISPR-Cas Systems ; Olfactory Receptor Neurons/metabolism ; Animals, Genetically Modified ; *Olfactory Pathways/anatomy &amp; histology/metabolism ; Transgenes ; Insect Proteins/genetics/metabolism ; Receptors, Odorant/genetics/metabolism ; }, abstract = {The mosquito Aedes aegypti is an emerging model insect for invertebrate neurobiology. We detail the application of a dual transgenesis marker system that reports the nature of transgene integration with circular donor template for CRISPR-Cas9-mediated homology-directed repair at target mosquito chemoreceptor genes. Employing this approach, we demonstrate the establishment of cell-type-specific T2A-QF2 driver lines for the A. aegypti olfactory co-receptor genes Ir8a and orco via canonical homology-directed repair and the CO2 receptor complex gene Gr1 via noncanonical homology-directed repair involving duplication of the intended T2A-QF2 integration cassette separated by intervening donor plasmid sequence. Using Gr1+ olfactory sensory neurons as an example, we show that introgression of such T2A-QF2 driver and QUAS responder transgenes into a yellow cuticular pigmentation mutant strain facilitates transcuticular calcium imaging of CO2-evoked neural activity on the maxillary palps with enhanced sensitivity relative to wild-type mosquitoes enveloped by dark melanized cuticle. We further apply Cre-loxP excision to derive marker-free T2A-QF2 in-frame fusions to clearly map axonal projection patterns from olfactory sensory neurons expressing these 3 chemoreceptors into the A. aegypti antennal lobe devoid of background interference from 3xP3-based fluorescent transgenesis markers. The marker-free Gr1 T2A-QF2 driver facilitates clear recording of CO2-evoked responses in this central brain region using the genetically encoded calcium indicators GCaMP6s and CaMPARI2. Systematic application of these optimized methods to different chemoreceptors stands to enable mapping A. aegypti olfactory circuits at peripheral and central levels of olfactory coding at high resolution.}, }
@article {pmid39853118, year = {2025}, author = {Flick, H and Venbakkam, A and Singh, PK and Layish, B and Huang, S-W and Radhakrishnan, R and Kvaratskhelia, M and Engelman, AN and Kane, M}, title = {Interplay between the cyclophilin homology domain of RANBP2 and MX2 regulates HIV-1 capsid dependencies on nucleoporins.}, journal = {mBio}, volume = {16}, number = {3}, pages = {e0264624}, pmid = {39853118}, issn = {2150-7511}, support = {R01 AI162172/AI/NIAID NIH HHS/United States ; R01 AI162665/AI/NIAID NIH HHS/United States ; U54 AI170791/AI/NIAID NIH HHS/United States ; R01 AI150998/AI/NIAID NIH HHS/United States ; R01AI162665//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01AI052014//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01AI1621172, R01AI150988//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01 AI052014/AI/NIAID NIH HHS/United States ; U54AI170791//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; }, mesh = {*Nuclear Pore Complex Proteins/metabolism/genetics/chemistry ; Humans ; *HIV-1/physiology/genetics ; *Myxovirus Resistance Proteins/metabolism/genetics ; *Molecular Chaperones/metabolism/genetics/chemistry ; *Capsid/metabolism ; HIV Infections/virology/metabolism ; Cell Line ; *Capsid Proteins/metabolism/genetics ; *Cyclophilins/metabolism/genetics ; CRISPR-Cas Systems ; HEK293 Cells ; Active Transport, Cell Nucleus ; Virus Replication ; Protein Domains ; }, abstract = {UNLABELLED: Interlinked interactions between the viral capsid (CA), nucleoporins (Nups), and the antiviral protein myxovirus resistance 2 (MX2/MXB) influence human immunodeficiency virus 1 (HIV-1) nuclear entry and the outcome of infection. Although RANBP2/NUP358 has been repeatedly identified as a critical player in HIV-1 nuclear import and MX2 activity, the mechanism by which RANBP2 facilitates HIV-1 infection is not well understood. To explore the interactions between MX2, the viral CA, and RANBP2, we utilized CRISPR-Cas9 to generate cell lines expressing RANBP2 from its endogenous locus but lacking the C-terminal cyclophilin (Cyp) homology domain and found that both HIV-1 and HIV-2 infections were reduced significantly in RANBP2ΔCyp cells. Importantly, although MX2 still localized to the nuclear pore complex in RANBP2ΔCyp cells, antiviral activity against HIV-1 was decreased. By generating cells expressing specific point mutations in the RANBP2-Cyp domain, we determined that the effect of the RANBP2-Cyp domain on MX2 anti-HIV-1 activity is due to direct interactions between RANBP2 and CA. We further determined that CypA and RANBP2-Cyp have similar effects on HIV-1 integration targeting. Finally, we found that the Nup requirements for HIV infection and MX2 activity were altered in cells lacking the RANBP2-Cyp domain. These findings demonstrate that the RANBP2-Cyp domain affects viral infection and MX2 sensitivity by altering CA-specific interactions with cellular factors that affect nuclear import and integration targeting.<br><br>IMPORTANCE: Human immunodeficiency virus 1 (HIV-1) entry into the nucleus is an essential step in viral replication that involves complex interactions between the viral capsid (CA) and multiple cellular proteins, including nucleoporins (Nups) such as RANBP2. Nups also mediate the function of the antiviral protein myxovirus resistance 2 (MX2); however, determining the precise role of Nups in HIV infection has proved challenging due to the complex nature of the nuclear pore complex (NPC) and significant pleiotropic effects elicited by Nup depletion. We have used precise gene editing to assess the role of the cyclophilin domain of RANBP2 in HIV-1 infection and MX2 activity. We find that this domain affects viral infection, nucleoporin requirements, MX2 sensitivity, and integration targeting in a CA-specific manner, providing detailed insights into how RANBP2 contributes to HIV-1 infection.}, }
@article {pmid39851566, year = {2025}, author = {Pohl, KA and Zhang, X and Ji, JJ and Stiles, L and Sadun, AA and Yang, XJ}, title = {Derivation and Characterization of Isogenic OPA1 Mutant and Control Human Pluripotent Stem Cell Lines.}, journal = {Cells}, volume = {14}, number = {2}, pages = {}, pmid = {39851566}, issn = {2073-4409}, support = {F31 EY033242/EY/NEI NIH HHS/United States ; P30 EY000331/EY/NEI NIH HHS/United States ; R01 EY026319/EY/NEI NIH HHS/United States ; 2R01EY026319, F31EY033242, P30EY000331/GF/NIH HHS/United States ; }, mesh = {Humans ; *GTP Phosphohydrolases/genetics/metabolism ; *Mutation/genetics ; Gene Editing ; *Optic Atrophy, Autosomal Dominant/genetics/pathology ; CRISPR-Cas Systems/genetics ; Cell Line ; Induced Pluripotent Stem Cells/metabolism ; *Pluripotent Stem Cells/metabolism/cytology ; Retinal Ganglion Cells/metabolism/pathology ; Mitochondria/metabolism ; }, abstract = {Dominant optic atrophy (DOA) is the most commonly inherited optic neuropathy. The majority of DOA is caused by mutations in the OPA1 gene, which encodes a dynamin-related GTPase located to the mitochondrion. OPA1 has been shown to regulate mitochondrial dynamics and promote fusion. Within the mitochondrion, proteolytically processed OPA1 proteins form complexes to maintain membrane integrity and the respiratory chain complexity. Although OPA1 is broadly expressed, human OPA1 mutations predominantly affect retinal ganglion cells (RGCs) that are responsible for transmitting visual information from the retina to the brain. Due to the scarcity of human RGCs, DOA has not been studied in depth using the disease affected neurons. To enable studies of DOA using stem-cell-derived human RGCs, we performed CRISPR-Cas9 gene editing to generate OPA1 mutant pluripotent stem cell (PSC) lines with corresponding isogenic controls. CRISPR-Cas9 gene editing yielded both OPA1 homozygous and heterozygous mutant ESC lines from a parental control ESC line. In addition, CRISPR-mediated homology-directed repair (HDR) successfully corrected the OPA1 mutation in a DOA patient's iPSCs. In comparison to the isogenic controls, the heterozygous mutant PSCs expressed the same OPA1 protein isoforms but at reduced levels; whereas the homozygous mutant PSCs showed a loss of OPA1 protein and altered mitochondrial morphology. Furthermore, OPA1 mutant PSCs exhibited reduced rates of oxygen consumption and ATP production associated with mitochondria. These isogenic PSC lines will be valuable tools for establishing OPA1-DOA disease models in vitro and developing treatments for mitochondrial deficiency associated neurodegeneration.}, }
@article {pmid39851560, year = {2025}, author = {Bonowicz, K and Jerka, D and Piekarska, K and Olagbaju, J and Stapleton, L and Shobowale, M and Bartosiński, A and Łapot, M and Bai, Y and Gagat, M}, title = {CRISPR-Cas9 in Cardiovascular Medicine: Unlocking New Potential for Treatment.}, journal = {Cells}, volume = {14}, number = {2}, pages = {}, pmid = {39851560}, issn = {2073-4409}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Cardiovascular Diseases/therapy/genetics ; *Genetic Therapy/methods ; Animals ; }, abstract = {Cardiovascular diseases (CVDs) remain a significant global health challenge, with many current treatments addressing symptoms rather than the genetic roots of these conditions. The advent of CRISPR-Cas9 technology has revolutionized genome editing, offering a transformative approach to targeting disease-causing mutations directly. This article examines the potential of CRISPR-Cas9 in the treatment of various CVDs, including atherosclerosis, arrhythmias, cardiomyopathies, hypertension, and Duchenne muscular dystrophy (DMD). The technology's ability to correct single-gene mutations with high precision and efficiency positions it as a groundbreaking tool in cardiovascular therapy. Recent developments have extended the capabilities of CRISPR-Cas9 to include mitochondrial genome editing, a critical advancement for addressing mitochondrial dysfunctions often linked to cardiovascular disorders. Despite its promise, significant challenges remain, including off-target effects, ethical concerns, and limitations in delivery methods, which hinder its translation into clinical practice. This article also explores the ethical and regulatory considerations surrounding gene editing technologies, emphasizing the implications of somatic versus germline modifications. Future research efforts should aim to enhance the accuracy of CRISPR-Cas9, improve delivery systems for targeted tissues, and ensure the safety and efficacy of treatments in the long term. Overcoming these obstacles could enable CRISPR-Cas9 to not only treat but also potentially cure genetically driven cardiovascular diseases, heralding a new era in precision medicine for cardiovascular health.}, }
@article {pmid39849901, year = {2025}, author = {Tobias, IC and Moorthy, SD and Shchuka, VM and Langroudi, L and Cherednychenko, M and Gillespie, ZE and Duncan, AG and Tian, R and Gajewska, NA and Di Roberto, RB and Mitchell, JA}, title = {A Sox2 enhancer cluster regulates region-specific neural fates from mouse embryonic stem cells.}, journal = {G3 (Bethesda, Md.)}, volume = {15}, number = {4}, pages = {}, pmid = {39849901}, issn = {2160-1836}, support = {FRN PJT153186/CAPMC/CIHR/Canada ; //Canada Foundation for Innovation/ ; //Ontario Ministry of Research and Innovation/ ; //University of Toronto/ ; //Canadian Institutes of Health Research fellowship/ ; //Natural Science and Engineering Research Council of Canada/ ; }, mesh = {Animals ; Mice ; *SOXB1 Transcription Factors/genetics/metabolism ; *Enhancer Elements, Genetic ; *Mouse Embryonic Stem Cells/metabolism/cytology ; *Neural Stem Cells/metabolism/cytology ; Cell Differentiation/genetics ; Gene Expression Regulation, Developmental ; *Multigene Family ; Neurogenesis/genetics ; CRISPR-Cas Systems ; }, abstract = {Sex-determining region Y box 2 (Sox2) is a critical transcription factor for embryogenesis and neural stem and progenitor cell (NSPC) maintenance. While distal enhancers control Sox2 in embryonic stem cells (ESCs), enhancers closer to the gene are implicated in Sox2 transcriptional regulation in neural development. We hypothesize that a downstream enhancer cluster, termed Sox2 regulatory regions 2-18 (SRR2-18), regulates Sox2 transcription in neural stem cells and we investigate this in NSPCs derived from mouse ESCs. Using functional genomics and CRISPR-Cas9-mediated deletion analyses, we investigate the role of SRR2-18 in Sox2 regulation during neural differentiation. Transcriptome analyses demonstrate that the loss of even 1 copy of SRR2-18 disrupts the region-specific identity of NSPCs, reducing the expression of genes associated with more anterior regions of the embryonic nervous system. Homozygous deletion of this Sox2 neural enhancer cluster causes reduced SOX2 protein, less frequent interaction with transcriptional machinery, and leads to perturbed chromatin accessibility genome-wide further affecting the expression of neurodevelopmental and anterior-posterior regionalization genes. Furthermore, homozygous NSPC deletants exhibit self-renewal defects and impaired differentiation into cell types found in the brain. Altogether, our data define a cis-regulatory enhancer cluster controlling Sox2 transcription in NSPCs and highlight the sensitivity of neural differentiation processes to decreased Sox2 transcription, which causes differentiation into posterior neural fates, specifically the caudal neural tube. This study highlights the importance of precise Sox2 regulation by SRR2-18 in neural differentiation.}, }
@article {pmid39849243, year = {2025}, author = {Nawab, S and Ullah, MW and Shah, SB and Zhang, YF and Keerio, HA and Yong, YC}, title = {Recent advances in engineering non-native microorganisms for poly(3-hydroxybutyrate) production.}, journal = {World journal of microbiology &amp; biotechnology}, volume = {41}, number = {2}, pages = {48}, pmid = {39849243}, issn = {1573-0972}, support = {2023M741421//China Postdoctoral Science Foundation/ ; 2022ZB650//Jiangsu Funding Program for Excellent Postdoctoral Talent/ ; 2023ZB399//Jiangsu Funding Program for Excellent Postdoctoral Talent/ ; BK20220003//Natural Science Foundation of Jiangsu Province/ ; }, mesh = {*Hydroxybutyrates/metabolism ; *Metabolic Engineering/methods ; *Polyesters/metabolism ; Escherichia coli/metabolism/genetics ; Synthetic Biology ; *Bacteria/metabolism/genetics ; Biosynthetic Pathways ; Metabolic Networks and Pathways/genetics ; Polyhydroxyalkanoates ; CRISPR-Cas Systems ; Polyhydroxybutyrates ; }, abstract = {Poly(3-hydroxybutyrate) (PHB) is a biodegradable polymer that belongs to a group of polymers called polyhydroxyalkanoates (PHAs). PHB can be synthesized from renewable resources, making it a promising alternative to petroleum-derived plastics. It is also considered non-toxic, biodegradable, and biocompatible, which makes it suitable for various applications in the medicine and biomedicine. Many microorganisms biosynthesize and accumulate PHB naturally. However, recent advancements in metabolic engineering and synthetic biology have allowed scientists to engineer non-native microorganisms to produce PHB. This review comprehensively summarizes all non-native microbial hosts used for PHB biosynthesis and discusses different metabolic engineering approaches used to enhance PHB production. These strategies include optimizing the biosynthesis pathway through cofactor engineering, metabolic pathway reconstruction, and cell morphology engineering. Moreover, the CRISPR/Cas9 approach is also used for manipulating the genome of non-host microorganisms to enable them produce PHB. Among non-native microbial hosts, Escherichia coli has been successfully used for industrial-scale PHB production. However, further genetic engineering approaches are needed to make non-native microbial hosts more suitable for large-scale PHB production.}, }
@article {pmid39849074, year = {2025}, author = {Das, S and Unhale, T and Marinach, C and Valeriano Alegria, BDC and Roux, C and Madry, H and Mohand Oumoussa, B and Amino, R and Iwanaga, S and Briquet, S and Silvie, O}, title = {Constitutive expression of Cas9 and rapamycin-inducible Cre recombinase facilitates conditional genome editing in Plasmodium berghei.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {2949}, pmid = {39849074}, issn = {2045-2322}, support = {ANR-11-LABX-0024//Agence Nationale de la Recherche/ ; EQU201903007823//Fondation pour la recherche m&#xe9;dicale, France/ ; }, mesh = {*Plasmodium berghei/genetics/drug effects ; *Integrases/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; *Sirolimus/pharmacology ; *CRISPR-Associated Protein 9/genetics/metabolism ; Malaria/parasitology ; Mice ; Genome, Protozoan ; }, abstract = {Malaria is caused by protozoan parasites of the genus Plasmodium and remains a global health concern. The parasite has a highly adaptable life cycle comprising successive rounds of asexual replication in a vertebrate host and sexual maturation in the mosquito vector Anopheles. Genetic manipulation of the parasite has been instrumental for deciphering the function of Plasmodium genes. Conventional reverse genetic tools cannot be used to study essential genes of the asexual blood stages, thereby necessitating the development of conditional strategies. Among various such strategies, the rapamycin-inducible dimerisable Cre (DiCre) recombinase system emerged as a powerful approach for conditional editing of essential genes in human-infecting P. falciparum and in the rodent malaria model parasite P. berghei. We previously generated a DiCre-expressing P. berghei line and validated it by conditionally deleting several essential asexual stage genes, revealing their important role also in sporozoites. Another potent tool is the CRISPR/Cas9 technology, which has enabled targeted genome editing with higher accuracy and specificity and greatly advanced genome engineering in Plasmodium spp. Here, we developed new P. berghei parasite lines by integrating the DiCre cassette and a fluorescent marker in parasites constitutively expressing Cas9. Owing to the dual integration of CRISPR/Cas9 and DiCre, these new lines allow unparalleled levels of gene modification and conditional regulation simultaneously. To illustrate the versatility of this new tool, we conditionally knocked out the essential gene encoding the claudin-like apicomplexan micronemal protein (CLAMP) in P. berghei and confirmed the role of CLAMP during invasion of erythrocytes.}, }
@article {pmid39848539, year = {2025}, author = {Yang, K and Cai, L and Zhao, Y and Cheng, H and Zhou, R}, title = {Optimization of genome editing by CRISPR ribonucleoprotein for high efficiency of germline transmission of Sox9 in zebrafish.}, journal = {New biotechnology}, volume = {86}, number = {}, pages = {47-54}, doi = {10.1016/j.nbt.2025.01.009}, pmid = {39848539}, issn = {1876-4347}, mesh = {Animals ; *Zebrafish/genetics/embryology ; *Gene Editing/methods ; *SOX9 Transcription Factor/genetics ; *Germ Cells/metabolism ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/metabolism/genetics ; *Zebrafish Proteins/genetics ; Male ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Alleles ; }, abstract = {Primordial germ cells (PGCs) are the first germline stem cells to emerge during early embryonic development and are essential for the propagation and survival of species. Genome editing creates mutagenesis possibilities in vivo, but the generation of precise mutations in PGCs is still challenging. Here, we report an optimized approach for highly efficient genome editing via introducing biallelic variations in early embryos in zebrafish. We adopted an extended, GC-rich, and chemically modified sgRNA along with microinjection of the CRISPR ribonucleoprotein (RNP) complex into the yolk sac at the 1-cell stage. We found that genome editing of Sox9a generated a high proportion of heterozygotes with edited alleles in the F1 generation, indicating biallelic editing. Deep sequencing and mapping the edited cells from early embryos to future tissues revealed that the edited founder has a dominantly edited allele, sox9a M1, accounting for over 99 % of alleles in the testis. Specifically, all offspring of the founder inherited the edited allele, suggesting nearly complete editing of the alleles in early germline cells. Overall, the optimization delineates biallelic editing of sox9a in early embryos and transmission of edited alleles to offspring, thus presenting a method to create a desired genetic mutation line of Sox9a avoiding lengthy traditional crossbreeding.}, }
@article {pmid39847570, year = {2025}, author = {Al Sium, SM and Goswami, B and Chowdhury, SF and Naser, SR and Sarkar, MK and Faruq, MJ and Habib, MA and Akter, S and Banu, TA and Sarkar, MMH and Khan, MS}, title = {An insight into the genome-wide analysis of bacterial defense mechanisms in a uropathogenic Morganella morganii isolate from Bangladesh.}, journal = {PloS one}, volume = {20}, number = {1}, pages = {e0313141}, pmid = {39847570}, issn = {1932-6203}, mesh = {Bangladesh ; *Morganella morganii/genetics/isolation &amp; purification/pathogenicity ; Humans ; *Genome, Bacterial ; Phylogeny ; *Urinary Tract Infections/microbiology ; Genomic Islands ; Whole Genome Sequencing ; Virulence Factors/genetics ; }, abstract = {The gram-negative, facultative anaerobic bacterium Morganella morganii is linked to a number of illnesses, including nosocomial infections and urinary tract infections (UTIs). A clinical isolate from a UTI patient in Bangladesh was subjected to high-throughput whole genome sequencing and extensive bioinformatics analysis in order to gather knowledge about the genomic basis of bacterial defenses and pathogenicity in M. morganii. With an average nucleotide identity (ANI) of more than 97% similarity to a reference genome and phylogenetic analysis verified the isolate as M. morganii. Genome annotation identified 3,718 protein-coding sequences, including genes for metabolism, protein processing, stress response, energy, and membrane transport. The presence of biosynthetic gene clusters points to the isolate's ability to create bioactive compounds, including antibiotics. Genomic islands contained genes for metal transporters, stress proteins, toxin proteins, and genes related to horizontal gene transfer. The beta-lactam resistance gene blaDHA was found using antimicrobial resistance (AMR) gene analysis across three databases. The virulence genes kdsA and cheY, which may be involved in chemotaxis and lipopolysaccharide production, were also available in the isolate, suggesting its high pathogenicity. The genome contained mobile genetic components and defense mechanisms, such as restriction modification and CRISPR-Cas systems, indicating the bacterium's ability to defend itself against viral attacks. This thorough investigation sheds important light on M. morganii's pathogenicity and adaptive tactics by revealing its genetic characteristics, AMR, virulence components, and defense mechanisms. For the development of targeted treatments and preventing the onset of resistance in clinical care, it is essential to comprehend these genetic fingerprints.}, }
@article {pmid39847105, year = {2025}, author = {Karimi-Fard, A and Saidi, A and Tohidfar, M and Emami, SN}, title = {CRISPR-Cas-mediated adaptation of Thermus thermophilus HB8 to environmental stress conditions.}, journal = {Archives of microbiology}, volume = {207}, number = {2}, pages = {41}, pmid = {39847105}, issn = {1432-072X}, mesh = {*Thermus thermophilus/genetics/physiology ; *CRISPR-Cas Systems ; *Stress, Physiological/genetics ; *Adaptation, Physiological/genetics ; Gene Expression Regulation, Bacterial ; Bacterial Proteins/genetics/metabolism ; }, abstract = {Bacteria experience a continual array of environmental stresses, necessitating adaptive mechanisms crucial for their survival. Thermophilic bacteria, such as Thermus thermophilus, face constant environmental challenges, particularly high temperatures, which requires robust adaptive mechanisms for survival. Studying these extremophiles provides valuable insights into the intricate molecular and physiological processes used by extremophiles to adapt and survive in harsh environments. Through meta-analysis of microarray data, we revealed the key genes in T. thermophilus HB8 that respond to various environmental stresses. The analysis revealed 20 differentially expressed genes (DEGs), including 13 upregulated and seven downregulated genes, with a threshold of|log fold change| > 1 and an adjusted p-value < 0.05. Several genes identified as up-regulated in our analysis belonged to the CRISPR-associated protein (Cas) family. To validate these findings, we further evaluated the relative expression levels of TTHB188 (cas1/casA), TTHB189 (cas2/casB), TTHB190 (cas7/casC), TTHB191 (cas5/casD), TTHB192 (cas6/casE), and TTHB193 (cas1e) using RT-qPCR under H2O2 and salt stress conditions. The RT-qPCR analysis revealed significant up-regulation of transcripts, casA, casB, casC, casD, casE, and cas1e under salt stress. However, under H2O2 stress, only, casA, casB, and casC exhibited substantial increases in expression. Our findings may indicate that the CRISPR-associated proteins significantly impact the adaptive response of T. thermophilus HB8 to various environmental stresses, particularly salt stress, highlighting its significance in extremophile survival and adaptation. This research offers an important understanding of the complex strategies used by extremophiles to survive in challenging conditions.}, }
@article {pmid39846980, year = {2025}, author = {Xin, H and Strickland, LW and Hamilton, JP and Trusky, JK and Fang, C and Butler, NM and Douches, DS and Buell, CR and Jiang, J}, title = {Jan and mini-Jan, a model system for potato functional genomics.}, journal = {Plant biotechnology journal}, volume = {23}, number = {4}, pages = {1243-1256}, pmid = {39846980}, issn = {1467-7652}, support = {IS-5684-24C//United States - Israel Binational Agricultural Research and Development Fund/ ; T32GM110523/GM/NIGMS NIH HHS/United States ; T32 GM152798/GM/NIGMS NIH HHS/United States ; IS-5317-20C//United States - Israel Binational Agricultural Research and Development Fund/ ; T32GM152798/GM/NIGMS NIH HHS/United States ; T32 GM110523/GM/NIGMS NIH HHS/United States ; }, mesh = {*Solanum tuberosum/genetics/growth &amp; development ; *Genomics/methods ; *Genome, Plant/genetics ; CRISPR-Cas Systems/genetics ; Diploidy ; }, abstract = {Potato (Solanum tuberosum) is the third-most important food crop in the world. Although the potato genome has been fully sequenced, functional genomics research of potato lags behind that of other major food crops, largely due to the lack of a model experimental potato line. Here, we present a diploid potato line, 'Jan,' which possesses all essential characteristics for facile functional genomics studies. Jan exhibits a high level of homozygosity after seven generations of self-pollination. Jan is vigorous, highly fertile and produces tubers with outstanding traits. Additionally, it demonstrates high regeneration rates and excellent transformation efficiencies. We generated a chromosome-scale genome assembly for Jan, annotated its genes and identified syntelogs relative to the potato reference genome assembly DMv6.1 to facilitate functional genomics. To miniaturize plant architecture, we developed two 'mini-Jan' lines with compact and dwarf plant stature through CRISPR/Cas9-mediated mutagenesis targeting the Dwarf and Erecta genes involved in growth. One mini-Jan mutant, mini-Jan[E], is fully fertile and will permit higher-throughput studies in limited growth chamber and greenhouse space. Thus, Jan and mini-Jan offer a robust model system that can be leveraged for gene editing and functional genomics research in potato.}, }
@article {pmid39846250, year = {2025}, author = {Adjumain, S and Daniel, P and Sun, CX and Bradshaw, G and Chew, NJ and Tsui, V and Lee, H and Loi, M and Zhukova, N and Habarakada, D and Yoel, A and Vaghjiani, VG and Game, S and Ludlow, LE and Neeman, N and Sweet-Cordero, EA and Eisenstat, DD and Cain, JE and Firestein, R}, title = {Multidimensional, integrative profiling identifies BCL2L1 methylation as a predictor of MCL1 dependency in pediatric malignancies.}, journal = {JCI insight}, volume = {10}, number = {2}, pages = {}, pmid = {39846250}, issn = {2379-3708}, mesh = {Humans ; *Myeloid Cell Leukemia Sequence 1 Protein/genetics/metabolism/antagonists &amp; inhibitors ; *DNA Methylation ; Cell Line, Tumor ; Child ; *Glioma/genetics/pathology/drug therapy/metabolism ; *Brain Neoplasms/genetics/pathology/drug therapy/metabolism ; *bcl-X Protein/genetics/metabolism ; Mice ; Gene Expression Regulation, Neoplastic ; Animals ; CRISPR-Cas Systems ; Apoptosis/genetics ; Female ; }, abstract = {Pediatric high-grade gliomas (pHGGs) are the most aggressive brain tumors in children, necessitating innovative therapies to improve outcomes. Unlike adult gliomas, recent research reveals that childhood gliomas have distinct biological features, requiring specific treatment strategies. Here, we focused on deciphering unique genetic dependencies specific to childhood gliomas. Using a pooled CRISPR/Cas9 knockout screening approach on 65 pediatric and 10 adult high-grade glioma (HGG) cell lines, myeloid cell leukemia 1 (MCL1) emerged as a key antiapoptotic gene essential in pediatric but not adult gliomas. We demonstrated that MCL1 is targetable using current small molecule inhibitors, and its inhibition leads to potent anticancer activity across pediatric HGG cell lines irrespective of genotype. Employing predictive modeling approaches on a large set of childhood cancer cell lines with multiomics data features, we identified a potentially previously unreported cluster of CpG sites in the antiapoptotic BCL-xL/BCL2L1 gene, which predicted MCL1 inhibitor response. We extended these data across multiple pediatric tumor types, showing that BCL2L1 methylation is a broad predictor of MCL1 dependency in vitro and in vivo. Overall, our multidimensional, integrated genomic approach identified MCL1 as a promising therapeutic target in several BCL2L1-methylated pediatric cancers, offering a translational strategy to identify patients most likely to benefit from MCL1 inhibitor therapy.}, }
@article {pmid39846200, year = {2025}, author = {Zheng, X and Huang, CH and Yan, S and Rong, MD}, title = {Advances and applications of genome-edited animal models for severe combined immunodeficiency.}, journal = {Zoological research}, volume = {46}, number = {1}, pages = {249-260}, pmid = {39846200}, issn = {2095-8137}, mesh = {Animals ; *Severe Combined Immunodeficiency/genetics/therapy ; *Gene Editing/methods ; *Disease Models, Animal ; CRISPR-Cas Systems ; }, abstract = {Severe combined immunodeficiency disease (SCID), characterized by profound immune system dysfunction, can lead to life-threatening infections and death. Animal models play a pivotal role in elucidating biological processes and advancing therapeutic strategies. Recent advances in gene-editing technologies, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR/Cas9, and base editing, have significantly enhanced the generation of SCID models. These models have not only deepened our understanding of disease pathophysiology but have also driven progress in cancer therapy, stem cell transplantation, organ transplantation, and infectious disease management. This review provides a comprehensive overview of current SCID models generated using novel gene-editing approaches, highlighting their potential applications in translational medicine and their role in advancing biomedical research.}, }
@article {pmid39845892, year = {2024}, author = {Hu, D and Hu, L and Lu, Y and Dong, X and Cao, X and Bai, S and Zhang, L and Li, D and Sun, Y}, title = {Intein-mediated split SaCas9 for genome editing in plants.}, journal = {Frontiers in genome editing}, volume = {6}, number = {}, pages = {1506468}, pmid = {39845892}, issn = {2673-3439}, abstract = {Virus-induced genome editing (VIGE) technologies have been developed to address the limitations to plant genome editing, which heavily relies on genetic transformation and regeneration. However, the application of VIGE in plants is hampered by the challenge posed by the size of the commonly used gene editing nucleases, Cas9 and Cas12a. To overcome this challenge, we employed intein-mediated protein splicing to divide the SaCas9 transcript into two segments (Split-v1) and three segments (Split-v3). The Split-v1 system demonstrated genome editing efficiencies in transgenic plants comparable to those achieved with wild-type SaCas9, with efficiencies ranging from 70.2% to 96.1%. Additionally, we constructed barley stripe mosaic virus (BSMV)-based vectors to co-express Split-v1 SaCas9 and gRNAs targeting LcHRC, LcGW2, and LcTB1 in sheepgrass (Leymus chinensis), a Gramineae forage species known for its recalcitrance to genetic transformation. Infected leaves of sheepgrass exhibited genome editing efficiencies ranging from 10.40% to 37.03%. These results demonstrate the potential of intein-mediated split nuclease systems to broaden the applicability of VIGE in challenging plant species.}, }
@article {pmid39844455, year = {2025}, author = {Jonsdottir, TK and Paoletta, MS and Ishizaki, T and Hernandez, S and Ivanova, M and Herrera Curbelo, A and Saiki, PA and Selinger, M and Das, D and Henriksson, J and Bushell, ESC}, title = {A scalable CRISPR-Cas9 gene editing system facilitates CRISPR screens in the malaria parasite Plasmodium berghei.}, journal = {Nucleic acids research}, volume = {53}, number = {2}, pages = {}, pmid = {39844455}, issn = {1362-4962}, support = {2021-06602//Swedish Research Council/ ; 2019.0178//Knut and Alice Wallenberg Foundation/ ; 23 3102 Pj//Swedish Cancer Society/ ; 202160312//Japan Society for the Promotion of Science/ ; //Swedish Universities/ ; }, mesh = {*Plasmodium berghei/genetics ; *CRISPR-Cas Systems ; Gene Knockout Techniques ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals ; Genome, Protozoan ; Mice ; Malaria/parasitology ; }, abstract = {Many Plasmodium genes remain uncharacterized due to low genetic tractability. Previous large-scale knockout screens have only been able to target about half of the genome in the more genetically tractable rodent malaria parasite Plasmodium berghei. To overcome this limitation, we have developed a scalable CRISPR system called P. berghei high-throughput (PbHiT), which uses a single cloning step to generate targeting vectors with 100-bp homology arms physically linked to a guide RNA (gRNA) that effectively integrate into the target locus. We show that PbHiT coupled with gRNA sequencing robustly recapitulates known knockout mutant phenotypes in pooled transfections. Furthermore, we provide an online resource of knockout and tagging designs to target the entire P. berghei genome and scale-up vector production using a pooled ligation approach. This work presents for the first time a tool for high-throughput CRISPR screens in Plasmodium for studying the parasite's biology at scale.}, }
@article {pmid39844454, year = {2025}, author = {Garcia-Guerra, A and Sathyaprakash, C and de Jong, OG and Lim, WF and Vader, P and El Andaloussi, S and Bath, J and Reine, J and Aoki, Y and Turberfield, AJ and Wood, MJA and Rinaldi, C}, title = {Tissue-specific modulation of CRISPR activity by miRNA-sensing guide RNAs.}, journal = {Nucleic acids research}, volume = {53}, number = {2}, pages = {}, pmid = {39844454}, issn = {1362-4962}, support = {MR/Y009703/1/MRC_/Medical Research Council/United Kingdom ; //Kennedy's Disease Association/ ; 578222//Muscular Dystrophy Association/ ; MR/N024850/1/MRC_/Medical Research Council/United Kingdom ; 317110//Marie Curie Initial Training Network EScoDNA/ ; //xford University Press John Fell Fund/ ; LCF/BQ/EU16/11560044//La Caixa Foundation Fellowship/ ; //University of Oxford/ ; }, mesh = {*MicroRNAs/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; Muscular Dystrophy, Duchenne/genetics/therapy ; Mice ; Argonaute Proteins/metabolism/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Organ Specificity ; HEK293 Cells ; }, abstract = {Nucleic acid nanostructures offer unique opportunities for biomedical applications due to their sequence-programmable structures and functions, which enable the design of complex responses to molecular cues. Control of the biological activity of therapeutic cargoes based on endogenous molecular signatures holds the potential to overcome major hurdles in translational research: cell specificity and off-target effects. Endogenous microRNAs (miRNAs) can be used to profile cell type and cell state, and are ideal inputs for RNA nanodevices. Here, we present CRISPR MiRAGE (miRNA-activated genome editing), a tool comprising a dynamic single-guide RNA that senses miRNA complexed with Argonaute proteins and controls downstream CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) activity based on the detected miRNA signature. We study the operation of the miRNA-sensing single-guide RNA and attain muscle-specific activation of gene editing through CRISPR MiRAGE in models of Duchenne muscular dystrophy. By enabling RNA-controlled gene editing activity, this technology creates opportunities to advance tissue-specific CRISPR treatments for human diseases.}, }
@article {pmid39844444, year = {2025}, author = {Zhao, W and Zhu, X and Huang, G and Gu, H and Bi, Y and Tang, D and Ren, H}, title = {Application of Multiple Base-Editing Mediated by Polycistronic tRNA-gRNA-Processing System in Pig Cells.}, journal = {Biotechnology and bioengineering}, volume = {122}, number = {4}, pages = {779-791}, doi = {10.1002/bit.28931}, pmid = {39844444}, issn = {1097-0290}, support = {//The study was supported by the National Key Research and Development Program of China (2021YFA0805901), the National Natural Science Foundation of China (2023YFC3402004, 81900775, 82070199), the Biological Breeding-Major Projects (2023ZD0404703), the Innovation Group Project of Natural Science Foundation of Hubei Province (2024AFA027), the Guangdong Basic and Applied Basic Research Fund (2021A1515220078), and the Guangdong Provincial R&amp;D Project in Key Areas (2022B0202110002)./ ; }, mesh = {Animals ; *Gene Editing/methods ; Swine/genetics ; *RNA, Transfer/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; }, abstract = {Gene edited pigs have extensive and important application value in the fields of agriculture and biomedicine. With the increasing demand in medical research and agricultural markets, more and more application scenarios require gene edited pigs to possess two or even more advantageous phenotypes simultaneously. The current production of multi gene edited pigs is inefficient, time-consuming, and costly, and there is an urgent need to develop efficient and accurate multi gene editing application technologies. The polycistronic tRNA-gRNA-processing system (PTG), developed based on endogenous tRNA self-processing systems, has been shown to exhibit efficient multi gene editing in plants. This study aims to combine a PTG strategy with multiple gRNA production functions with an adenine base editor (ABE) to test its feasibility for efficient and precise multi gene base editing in pig cells. The results indicate that the PTG based integrated ABE plasmid can perform efficient base editing at multiple gene loci in pig cells. And while the gene editing efficiency was significantly improved, no indel and sgRNA dependent off target effects caused by DSB were detected. This work permit will provide a solid foundation for the production of multi gene edited pigs with agricultural and medical applications.}, }
@article {pmid39844394, year = {2025}, author = {Feng, S and Li, J and Yan, A and Zhu, X and Zhang, L and Tang, D and Liu, L}, title = {Application of Gene Editing in Triple-Negative Breast Cancer Research.}, journal = {Cell biochemistry and function}, volume = {43}, number = {1}, pages = {e70044}, doi = {10.1002/cbf.70044}, pmid = {39844394}, issn = {1099-0844}, support = {//This work was supported by the Scientific Research Project of Department of Education of Guangdong Province (Project Number:2023ZDZX2057) and GuangDong Basic and Applied Basic Research Foundation (Project Number:2023A1515140070)./ ; }, mesh = {Humans ; *Gene Editing ; *Triple Negative Breast Neoplasms/genetics/therapy/pathology ; Female ; Animals ; }, abstract = {With the rapid development of gene editing technology, its application in breast cancer has gradually become the focus of research. This article reviews the application of gene editing technology in the treatment of breast cancer, and discusses its challenges and future development directions. The key application areas of gene editing technology in the treatment of breast cancer will be outlined, including the discovery of new therapeutic targets and the development of drugs related to the pathway. Gene editing technology has played an important role in the discovery of new therapeutic targets. Through the use of gene editing technology, breast cancer-related genes are systematically edited to regulate key regulatory factors on related pathways or key tumor suppressor genes such as FOXC1 and BRCA, and the results are analyzed in cell or animal experiments, and the target is obtained from the experimental results, which provides important clues for the development of new drugs. This approach provides an innovative way to find more effective treatment strategies and inhibit tumor growth. In addition, gene editing technology has also promoted the personalization of breast cancer treatment. By analyzing a patient's genomic information, researchers can pinpoint key genetic mutations in a patient's tumor and design personalized treatments. This personalized treatment approach is expected to improve the therapeutic effect and reduce adverse reactions. Finally, the application of gene editing technology also provides support for the development of breast cancer immunotherapy. By editing immune cells to make them more potent against tumors, researchers are trying to develop more effective immunotherapies to bring new treatment options to breast cancer patients.}, }
@article {pmid39843658, year = {2025}, author = {Mouro Pinto, R and Murtha, R and Azevedo, A and Douglas, C and Kovalenko, M and Ulloa, J and Crescenti, S and Burch, Z and Oliver, E and Kesavan, M and Shibata, S and Vitalo, A and Mota-Silva, E and Riggs, MJ and Correia, K and Elezi, E and Demelo, B and Carroll, JB and Gillis, T and Gusella, JF and MacDonald, ME and Wheeler, VC}, title = {In vivo CRISPR-Cas9 genome editing in mice identifies genetic modifiers of somatic CAG repeat instability in Huntington's disease.}, journal = {Nature genetics}, volume = {57}, number = {2}, pages = {314-322}, pmid = {39843658}, issn = {1546-1718}, support = {NS111066//U.S. Department of Health &amp; Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)/ ; NS049206//U.S. Department of Health &amp; Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)/ ; NS091161//U.S. Department of Health &amp; Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)/ ; R01 NS091161/NS/NINDS NIH HHS/United States ; R01 NS126420/NS/NINDS NIH HHS/United States ; R01 NS049206/NS/NINDS NIH HHS/United States ; NS126420//U.S. Department of Health &amp; Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)/ ; P30 CA006516/CA/NCI NIH HHS/United States ; R21 NS111066/NS/NINDS NIH HHS/United States ; NS049206//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; }, mesh = {*Huntington Disease/genetics ; Animals ; *CRISPR-Cas Systems/genetics ; Mice ; *Gene Editing/methods ; *Trinucleotide Repeat Expansion/genetics ; Disease Models, Animal ; Humans ; *Genes, Modifier ; Huntingtin Protein/genetics ; Gene Knock-In Techniques ; Mice, Transgenic ; Genomic Instability ; }, abstract = {Huntington's disease, one of more than 50 inherited repeat expansion disorders[1], is a dominantly inherited neurodegenerative disease caused by a CAG expansion in HTT[2]. Inherited CAG repeat length is the primary determinant of age of onset, with human genetic studies underscoring that the disease is driven by the CAG length-dependent propensity of the repeat to further expand in the brain[3-9]. Routes to slowing somatic CAG expansion, therefore, hold promise for disease-modifying therapies. Several DNA repair genes, notably in the mismatch repair pathway, modify somatic expansion in Huntington's disease mouse models[10]. To identify novel modifiers of somatic expansion, we used CRISPR-Cas9 editing in Huntington's disease knock-in mice to enable in vivo screening of expansion-modifier candidates at scale. This included testing of Huntington's disease onset modifier genes emerging from human genome-wide association studies as well as interactions between modifier genes, providing insight into pathways underlying CAG expansion and potential therapeutic targets.}, }
@article {pmid39843407, year = {2025}, author = {Liu, X and Huang, L and Ye, Y and Wang, H and Tang, M and He, F and Xia, Z and Deng, S and Zhang, P and Dai, R and Liang, S}, title = {Staphylococcus aureus nt5 gene mutation through CRISPR RNA-guided base editing weakens bacterial virulence and immune evasion.}, journal = {Virulence}, volume = {16}, number = {1}, pages = {2451163}, pmid = {39843407}, issn = {2150-5608}, mesh = {*Staphylococcus aureus/genetics/pathogenicity/drug effects/immunology/enzymology ; Animals ; Virulence/genetics ; Mice ; Staphylococcal Infections/microbiology/immunology ; *Immune Evasion ; *5'-Nucleotidase/genetics ; Mutation ; Drug Resistance, Bacterial/genetics ; Anti-Bacterial Agents/pharmacology ; Daptomycin/pharmacology ; Gene Editing ; *Bacterial Proteins/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; }, abstract = {The resistance of commonly used clinical antibiotics, such as daptomycin (DAP), has become increasingly serious in the fight against Staphylococcus aureus (S. aureus) infection. It is essential to explore key pathogenicity-driven genes/proteins in bacterial infection and antibiotics resistance, which contributes to develop novel therapeutic strategies against S. aureus infections. The nt5 gene of S. aureus, encoding 5'-nucleotidase (NT5), is nearly unknown for its function in drug resistance and bacterial infection. Herein, to reveal nt5 gene role in drug resistance and infection ability of S. aureus, we performed nt5[C166T] gene mutation using a clustered regulatory interspaced short palindromic repeat ribonucleic acid (RNA)-guided base editing system to investigate the lose-of-function of NT5 protein. Subsequent transcriptome sequencing of the mutant strain revealed that nt5 inactivation caused changes in cell membrane integrity and inhibited nucleotide metabolism, suggesting the nt5 gene may be involved in bacterial drug resistance and virulence. The mutant strain exhibited enhanced tolerance to DAP treatment by attenuating cell membrane potential dissipation and slowing deoxyribonucleic acid release. Moreover, the nt5 mutation alleviated abscess degree of mouse kidneys caused by S. aureus infection byreducing the expression of IL-1β, IL-6, and IL-18. The nt5 mutant strain was easily swallowed by host immune cells, resulting in weak bacterial toxicity of the S. aureus mutant in the bacterial infection process. In summary, nt5 gene mutation confers tolerance to DAP and a lower bacterial capacity to form kidney abscesses through phagocytosis of host immune cells, which indicates the targeted inhibition of NT5 protein would offer a potential new therapeutic strategy against S. aureus infection.}, }
@article {pmid39842606, year = {2025}, author = {Zhang, K and Wang, Y and Jiang, S and Li, Y and Xiang, P and Zhang, Y and Chen, Y and Chen, M and Su, W and Liu, L and Li, S}, title = {dsDAP: An efficient method for high-abundance DNA-encoded library construction in mammalian cells.}, journal = {International journal of biological macromolecules}, volume = {298}, number = {}, pages = {140089}, doi = {10.1016/j.ijbiomac.2025.140089}, pmid = {39842606}, issn = {1879-0003}, mesh = {Humans ; HEK293 Cells ; *Gene Library ; *DNA/genetics ; CRISPR-Cas Systems/genetics ; *Polymerase Chain Reaction/methods ; }, abstract = {DNA-encoded libraries are invaluable tools for high-throughput screening and functional genomics studies. However, constructing high-abundance libraries in mammalian cells remains challenging. Here, we present dsDNA-assembly-PCR (dsDAP), a novel Gibson-assembly-PCR strategy for creating DNA-encoded libraries, offering improved flexibility and efficiency over previous methods. We demonstrated this approach by investigating the impact of translation initiation sequences (TIS) on protein expression in HEK293T cells. Both CRISPR-Cas9 and piggyBac systems were employed for genomic integration, allowing comparison of different integration methods. Our results confirmed the importance of specific nucleotides in the TIS region, particularly the preference for adenine at the -3 position in high-expression sequences. We also explored the effects of library dilution on genotype-phenotype correlations. This Gibson-assembly-PCR strategy overcomes limitations of existing methods, such as restriction enzyme dependencies, and provides a versatile tool for constructing high-abundance libraries in mammalian cells. Our approach has broad applications in functional genomics, drug discovery, and the study of gene regulation.}, }
@article {pmid39842315, year = {2025}, author = {Zhao, Y and Xie, J and Yu, S and Wu, Q and Wang, Z and Shang, Y and Wang, Z and Zhang, J and Zhai, H and Huang, Z and Ding, Y and Wang, J}, title = {A novel method of species-specific molecular target mining and accurate discrimination of Bacillus cereus sensu lato.}, journal = {International journal of food microbiology}, volume = {431}, number = {}, pages = {111068}, doi = {10.1016/j.ijfoodmicro.2025.111068}, pmid = {39842315}, issn = {1879-3460}, mesh = {Species Specificity ; Genome, Bacterial ; *Bacillus cereus/genetics/classification/isolation &amp; purification ; *Food Microbiology/methods ; Polymerase Chain Reaction/methods ; CRISPR-Cas Systems ; Food Contamination/analysis ; *Bacterial Typing Techniques/methods ; }, abstract = {Bacillus cereus, a member of the Bacillus cereus sensu lato (B. cereus s.l.), is widely distributed in nature and can contaminate a variety of foods, leading to foodborne illnesses and substantial losses in the food industry. Although culture-based methods remain the gold standard for identifying B. cereus due to their high sensitivity under specific conditions, they are often complex and labor-intensive to implement. Furthermore, the high genetic similarity among certain members of the B. cereus s.l. makes it challenging to identify species-specific molecular targets, hindering the rapid and accurate differentiation of these bacteria. In this study, we introduce a novel method, comparative analysis based on whole genome slices (CAWGS), combined with the Basic Local Alignment Search Tool (BLAST) for efficient molecular target mining. Using CAWGS-BLAST and pan-genome analysis, we successfully identified new molecular targets for B. cereus, Bacillus thuringiensis, emetic B. cereus, Bacillus anthracis, Bacillus mycoides, Bacillus weihenstephanensis, and Bacillus megaterium. Based on these newly discovered targets, we developed a PCR-CRISPR/Cas12a method for detecting B. cereus s.l. and related species. Our research not only provides a rapid and accurate approach for discriminating B. cereus s.l. and related species, but also offers a universal and valuable reference for detecting foodborne pathogens, especially those with highly similar phenotypic and genetic characteristics.}, }
@article {pmid39842078, year = {2025}, author = {Kumar, ARK and Low, J and Lim, J and Myint, B and Sun, X and Wu, L and Cheng, HS and Yip, S and Ming Cheng, CZ and Manoharan, T and Quek, YJ and Shou, Y and Tian, JS and Ng, YY and Gascoigne, NRJ and Tan, NS and Sugimura, R and Chia, G and Sze Cheung, AM and Yawata, M and Tay, A}, title = {Non-viral, high throughput genetic engineering of primary immune cells using nanostraw-mediated transfection.}, journal = {Biomaterials}, volume = {317}, number = {}, pages = {123079}, doi = {10.1016/j.biomaterials.2024.123079}, pmid = {39842078}, issn = {1878-5905}, mesh = {Humans ; *Transfection/methods ; CRISPR-Cas Systems/genetics ; *Genetic Engineering/methods ; CD8-Positive T-Lymphocytes/metabolism/cytology ; Receptors, CXCR4/genetics ; Electroporation ; Receptors, Chimeric Antigen/genetics ; CD4-Positive T-Lymphocytes/metabolism/cytology ; RNA, Messenger/genetics ; }, abstract = {Transfection of proteins, mRNA, and chimeric antigen receptor (CAR) transgenes into immune cells remains a critical bottleneck in cell manufacturing. Current methods, such as viruses and bulk electroporation, are hampered by low transfection efficiency, unintended transgene integration, and significant cell perturbation. The Nanostraw Electro-actuated Transfection (NExT) technology offers a solution by using high aspect-ratio nanostraws and localized electric fields to precisely deliver biomolecules into cells with minimal disruption. We demonstrate that NExT can deliver proteins, polysaccharides, and mRNA into primary human CD8[+] and CD4[+] T cells, and achieve CRISPR/Cas9 gene knockout of CXCR4 and TRAC in CD8[+] T cells. We showcase NExT's versatility across a range of primary human immune cells, including CD4[+] T cells, γδ-T cells, dendritic cells, NK cells, Treg cells, macrophages, and neutrophils. Finally, we developed a scalable, high-throughput multiwell NExT system capable of transfecting over 14 million cells and delivering diverse cargoes into multiple cell types from various donors simultaneously. This technology holds promise for streamlining high-throughput screening of allogeneic donors and reducing optimization costs for large-scale CAR-immune cell transfection.}, }
@article {pmid39842058, year = {2025}, author = {Jia, HY and Yao, SY and Li, YF and Ye, BC and Yin, BC}, title = {A conformational switch-controlled RNA sensor based on orthogonal dCas12a for RNA imaging in live cells.}, journal = {Biosensors &amp; bioelectronics}, volume = {273}, number = {}, pages = {117185}, doi = {10.1016/j.bios.2025.117185}, pmid = {39842058}, issn = {1873-4235}, mesh = {*Biosensing Techniques/methods ; *RNA/chemistry/genetics/analysis ; Humans ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/chemistry ; Optical Imaging/methods ; Nucleic Acid Conformation ; Fluorescent Dyes/chemistry ; *Endodeoxyribonucleases/genetics/chemistry ; Bacterial Proteins ; }, abstract = {RNA imaging technology is essential for understanding the complex RNA regulatory mechanisms and serves as a powerful tool for disease diagnosis. However, conventional RNA imaging methods often require multiple fluorescent tags for the specific labeling of individual targets, complicating both the imaging process and subsequent analysis. Herein, we develop an RNA sensor that integrates a blocked CRISPR RNA (crRNA)-based conformational switch with a controllable CRISPR activation (CRISPRa) system and apply for RNA imaging. By leveraging nuclease-inactive Cas12a (dCas12a)-mediated processing of precursor crRNA (pre-crRNA) and the orthogonality of dCas12a from different bacteria, our sensor establishes an artificial link between two unrelated RNA targets, enabling cells to sense one RNA target and image another with a single fluorescent signal. By visualizing a single target for dual-target analysis, our method significantly reduces the reliance on multiple fluorescent tags. Our sensor provides a new platform for RNA imaging, enhancing both biomedical research and the development of advanced molecular diagnostics.}, }
@article {pmid39841730, year = {2025}, author = {Khan, H and Huang, X and Raj, V and Wang, H}, title = {A versatile site-directed gene trap strategy to manipulate gene activity and control gene expression in Caenorhabditis elegans.}, journal = {PLoS genetics}, volume = {21}, number = {1}, pages = {e1011541}, pmid = {39841730}, issn = {1553-7404}, support = {K99 GM126137/GM/NIGMS NIH HHS/United States ; P40 OD010440/OD/NIH HHS/United States ; R00 GM126137/GM/NIGMS NIH HHS/United States ; R35 GM150658/GM/NIGMS NIH HHS/United States ; }, mesh = {Animals ; *Caenorhabditis elegans/genetics ; CRISPR-Cas Systems/genetics ; *Caenorhabditis elegans Proteins/genetics ; Animals, Genetically Modified ; *Gene Expression Regulation ; Transgenes ; Gene Editing/methods ; Operon ; }, abstract = {The ability to manipulate gene activity and control transgene expression is essential to study gene function. While several genetic tools for modifying genes or controlling expression separately are available for Caenorhabditis elegans, there are no genetic approaches to generate mutations that simultaneously disrupt gene function and provide genetic access to the cells expressing the disrupted gene. To achieve this, we developed a versatile gene trap strategy based on cGAL, a GAL4-UAS bipartite expression system for C. elegans. We designed a cGAL gene trap cassette and used CRISPR/Cas9 to insert it into the target gene, creating a bicistronic operon that simultaneously expresses a truncated endogenous protein and the cGAL driver in the cells expressing the target gene. We demonstrate that our cGAL gene trap strategy robustly generated loss-of-function alleles. Combining the cGAL gene trap lines with different UAS effector strains allowed us to rescue the loss-of-function phenotype, observe the gene expression pattern, and manipulate cell activity spatiotemporally. We show that, by recombinase-mediated cassette exchange (RMCE) via microinjection or genetic crossing, the cGAL gene trap lines can be further engineered in vivo to easily swap cGAL with other bipartite expression systems' drivers, including QF/QF2, Tet-On/Tet-Off, and LexA, to generate new gene trap lines with different drivers at the same genomic locus. These drivers can be combined with their corresponding effectors for orthogonal transgenic control. Thus, our cGAL-based gene trap is versatile and represents a powerful genetic tool for gene function analysis in C. elegans, which will ultimately provide new insights into how genes in the genome control the biology of an organism.}, }
@article {pmid39841710, year = {2025}, author = {Park, BJ and Heo, ST and Kim, M and Yoo, JR and Bae, EJ and Kang, SY and Park, S and Han, KR and Lee, KH and Lee, JM and Lee, H and Song, YJ}, title = {A CRISPR-Cas12a-based universal rapid scrub typhus diagnostic method targeting 16S rRNA of Orientia tsutsugamushi.}, journal = {PLoS neglected tropical diseases}, volume = {19}, number = {1}, pages = {e0012826}, pmid = {39841710}, issn = {1935-2735}, mesh = {*Scrub Typhus/diagnosis/microbiology ; *Orientia tsutsugamushi/genetics/isolation &amp; purification ; *RNA, Ribosomal, 16S/genetics ; Humans ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Molecular Diagnostic Techniques/methods ; }, abstract = {Scrub typhus is caused by Orientia tsutsugamushi infection and occurs frequently in an area called the Tsutsugamushi Triangle. Currently, there is no vaccine for O. tsutsugamushi, and its infection is treated with antibiotics such as doxycycline. Scrub typhus responds to effective treatment, and early treatment shortens the course of the disease, reduces mortality, and accelerates recovery. Therefore, it is important to rapidly diagnose O. tsutsugamushi infection to ensure successful outcomes. Here, we developed a CRISPR-Cas12a-based diagnostic method targeting the bacterial 16S rRNA to detect O. tsutsugamushi infection of all known genotypes. To reduce the possibility of contamination and increase field applicability, we designed the one-pot assay system in addition to conventional two-pot assay system. Using this method, we successfully detected up to 100 copies of in vitro transcribed O. tsutsugamushi 16S rRNA within 1 hour under isothermal conditions. In blood samples from patients confirmed to be infected with O. tsutsugamushi by nested PCR, the developed method exhibited a clinical sensitivity of 98% and high specificity. These data demonstrate that the presented method is applicable for the rapid and universal diagnosis of scrub typhus to facilitate timely and appropriate treatment.}, }
@article {pmid39841239, year = {2025}, author = {Bohra, A and Tiwari, A and Pareek, S and Joshi, R and Satheesh Naik, SJ and Kumari, K and Verma, RL and Parihar, AK and Patil, PG and Dixit, GP}, title = {Past and future of cytoplasmic male sterility and heterosis breeding in crop plants.}, journal = {Plant cell reports}, volume = {44}, number = {2}, pages = {33}, pmid = {39841239}, issn = {1432-203X}, mesh = {*Plant Infertility/genetics ; *Plant Breeding/methods ; *Crops, Agricultural/genetics/physiology ; *Hybrid Vigor/genetics ; Pollen/genetics/physiology ; *Cytoplasm/genetics ; Gene Editing ; Pollination/genetics ; }, abstract = {Plant breeding needs to embrace genetic innovations to ensure stability in crop yields under fluctuating climatic conditions. Development of commercial hybrid varieties has proven to be a sustainable and economical alternative to deliver superior yield, quality and resistance with uniformity in a number of food crops. Cytoplasmic male sterility (CMS), a maternally inherited inability to produce functional pollen, facilitates a three-line system for efficient hybrid seed production strategies in crops. The CMS system has illustrated its potential as a robust pollination control mechanism to support the billion-dollar seed industry. In plants, CMS arises due to a genomic conflict between mitochondrial open reading frames (orfs) and nuclear-encoding restoration-of-fertility (Rf) genes, leading to floral abnormalities and pollen sterility. Research on pollen sterility and fertility restoration provides deeper insights into cytoplasmic-nuclear interplay in plants and elucidates key molecular targets for hybrid breeding in crops. More recently, programmable gene editing (e.g., TALEN, CRISPR-Cas) has emerged as a promising tool to functionally validate CMS and Rf genes and obviate the need for pollen donors or Rf-genes for hybrid breeding. Modern genomic prediction models have allowed establishment of high-performing heterotic groups and patterns for sustaining long-term gain in hybrid breeding. This article reviews latest discoveries elucidating the molecular mechanisms behind CMS and fertility restoration in plants. We then present our perspective on how evolving genetic technologies are contributing to advance fundamental knowledge of the CMS-Rf genetic system for producing crop hybrids with high heterosis.}, }
@article {pmid39841016, year = {2025}, author = {Araújo, JL and Wagenblast, E and Voisin, V and McLeod, J and Gan, OI and Bansal, S and Jin, L and Mitchell, A and Gratton, B and Cutting, S and Arruda, A and Doedens, M and Travas, A and Kim, D and Capo-Chichi, JM and Abelson, S and Minden, MD and Wang, JCY and Sobrinho-Simões, MA and Pinto-do-Ó, P and Lechman, E and Dick, JE}, title = {FLT3 is genetically essential for ITD-mutated leukemic stem cells but dispensable for human hematopoietic stem cells.}, journal = {Blood}, volume = {145}, number = {20}, pages = {2361-2373}, doi = {10.1182/blood.2024025886}, pmid = {39841016}, issn = {1528-0020}, mesh = {*fms-Like Tyrosine Kinase 3/genetics/metabolism ; Humans ; *Hematopoietic Stem Cells/metabolism/pathology ; Animals ; *Neoplastic Stem Cells/pathology/metabolism ; Mice ; *Leukemia, Myeloid, Acute/genetics/pathology ; *Mutation ; *Tandem Repeat Sequences ; CRISPR-Cas Systems ; }, abstract = {Leukemic stem cells (LSCs) fuel acute myeloid leukemia (AML) growth and relapse, but therapies tailored toward eradicating LSCs without harming normal hematopoietic stem cells (HSCs) are lacking. FMS-like tyrosine kinase 3 (FLT3) is considered an important therapeutic target due to frequent mutation in AML and association with relapse. However, there has been limited clinical success with FLT3 drug targeting, suggesting either that FLT3 is not a vulnerability in LSC or that more potent inhibition is required, a scenario where HSC toxicity could become limiting. We tested these possibilities by ablating FLT3 using CRISPR/Cas9-mediated FLT3 knockout (FLT3-KO) in human LSCs and HSCs followed by functional xenograft assays. FLT3-KO in LSCs from FLT3-internal tandem duplication (ITD)-mutated but not FLT3-wild-type AMLs resulted in short-term leukemic grafts of FLT3-KO edited cells that disappeared by 12 weeks. By contrast, FLT3-KO in HSCs from the fetal liver, cord blood, and adult bone marrow did not impair multilineage hematopoiesis in primary and secondary xenografts. Our study establishes FLT3 as an ideal therapeutic target where ITD-positive LSCs are eradicated upon FLT3 deletion whereas HSCs are spared. These findings support the development of more potent FLT3-targeting drugs or gene-editing approaches for LSC eradication to improve clinical outcomes.}, }
@article {pmid39840981, year = {2025}, author = {Wu, Y and Jin, H and Yu, Q and Wei, Z and Zhu, J and Qiu, X and Luo, G and Li, J and Zhan, Y and Cai, D and Chen, S}, title = {Optimizing genome editing efficiency in Streptomyces fradiae via a CRISPR/Cas9n-mediated editing system.}, journal = {Applied and environmental microbiology}, volume = {91}, number = {2}, pages = {e0195324}, pmid = {39840981}, issn = {1098-5336}, mesh = {*Streptomyces/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Genome, Bacterial ; Bacterial Proteins/genetics ; }, abstract = {Streptomyces fradiae is an important bioresource to produce various antibacterial natural products, however, the time-consuming and labor-intensive genome editing toolkits hindered the construction and application of engineered strains, and this study aimed to establish an efficient CRISPR/Cas9n genome editing system in S. fradiae. Initially, the CRISPR/Cas9-mediated editing tool was employed to replace those awkward genome editing tools that relied on homologous recombination, while the off-target Cas9 exhibited high toxicity to S. fradiae Sf01. Therefore, the nickase mutation D10A, high-fidelity mutations including N497A, R661A, Q695A, and Q926A, and thiostrepton-induced promotor PtipA were incorporated into the Cas9 expression cassette, which reduced its toxicity. The deletion of single gene neoI and long fragment sequence (13.3 kb) were achieved with efficiencies of 77.8% and 44%, respectively. Additionally, the established tool was applied to facilitate the rapid deletion of nagB, replacement of Pfrr with PermE*, and integration of exogenous vgbS, with respective efficiencies of 77.8%, 100%, and 67.8%, and all of the above modification strategies benefited neomycin synthesis in S. fradiae. Taken together, this research established an efficient CRISPR/Cas9n-mediated genome editing toolkit in S. fradiae, paving the way for developing high-performance neomycin-producing strains and facilitating the genetic modification of Streptomyces.IMPORTANCEThis study describes the development and application of a genome editing system mediated by CRISPR/Cas9n in Streptomyces fradiae for the first time, which overcomes the challenges associated with genome editing caused by high GC content (74.5%) coupling with complex genome structure, and reduces the negative impact of "off-target effect." Our work not only provides a facile editing tool for constructing S. fradiae strains of high-yield neomycin but also offers the technical guidance for the design of a CRISPR/Cas9n mediated genome editing tool in those creatures with high GC content genomes.}, }
@article {pmid39840369, year = {2024}, author = {Moss, O and Li, X and Wang, ES and Kanagarajan, S and Guan, R and Ivarson, E and Zhu, LH}, title = {Knockout of BnaX.SGT.a caused significant sinapine reduction in transgene-free rapeseed mutants generated by protoplast-based CRISPR RNP editing.}, journal = {Frontiers in plant science}, volume = {15}, number = {}, pages = {1526941}, pmid = {39840369}, issn = {1664-462X}, abstract = {Rapeseed (Brassica napus L.) is known for its high-quality seed oil and protein content. However, its use in animal feed is restricted due to antinutritional factors present in the seedcake, with sinapine being one of the main compounds that reduces palatability. Attempts to develop rapeseed germplasm with lower sinapine levels through traditional breeding methods have shown limited progress. Genetic transformation methods could create new genotypes with reduced sinapine levels by silencing key genes involved in sinapine biosynthesis, though these methods often result in transgenic or genetically modified plants. The recent development of CRISPR-Cas technology provides a precise and efficient approach to crop improvement, with the potential to generate transgene-free mutants. In this study, we targeted the BnaX.SGT.a genes for knockout using CRISPR-Cas editing. By utilizing our newly established protoplast regeneration and transfection protocol for rapeseed, we demonstrated that DNA-free CRISPR editing via protoplast-based ribonucleoprotein (RNP) delivery was highly effective. We achieved successful knockout of the BnaX.SGT.a paralogues, with an average mutation efficiency of over 30%. Sequencing results revealed a variety of mutation types, from 1 bp insertions to 10 bp deletions, with most mutants exhibiting frameshift mutations that led to premature stop codons. The mutants displayed no visible phenotypic differences in growth patterns or flowering compared to the wild type. Importantly, sinapine content was significantly reduced in all T2 generation mutants analysed, while seed weight remained comparable between mutants and the wild type.}, }
@article {pmid39838780, year = {2025}, author = {Chen, S and Triki, M and Pinto Carneiro, S and Merkel, OM}, title = {A novel micelleplex for tumour-targeted delivery of CRISPR-Cas9 against KRAS-mutated lung cancer.}, journal = {Nanoscale}, volume = {17}, number = {11}, pages = {6604-6619}, pmid = {39838780}, issn = {2040-3372}, mesh = {Humans ; *Lung Neoplasms/genetics/pathology/metabolism/therapy ; *Micelles ; *Proto-Oncogene Proteins p21(ras)/genetics/metabolism ; *CRISPR-Cas Systems ; A549 Cells ; Gene Editing ; Mutation ; RNA, Messenger/genetics/metabolism/chemistry ; Polyethyleneimine/chemistry ; }, abstract = {CRISPR-Cas9 has emerged as a highly effective and customizable genome editing tool, holding significant promise for the treatment of KRAS mutations in lung cancer. In this study, we introduce a novel micelleplex, named C14-PEI, designed to co-deliver Cas9 mRNA and sgRNA efficiently to excise the mutated KRAS allele in lung cancer cells. C14-PEI is synthesised from 1,2-epoxytetradecane and branched PEI 600 Da via a ring-opening reaction. The resulting C14-PEI has a critical micelle concentration (CMC) of approximately 20.86 ± 0.15 mg L[-1], indicating its ability to form stable micelles at low concentrations. C14-PEI efficiently encapsulates mRNA into micelleplexes through electrostatic interactions. When the mass ratio is 8 (w/w 8), the C14-PEI formulation exhibits conducive properties, which showed encapsulation efficiency of eGFP mRNA at 99% and led to a 130-fold increase in eGFP expression in A549 cells compared to untreated cells, demonstrating the robust delivery and expression capability of the micelleplexes. Importantly, toxicity tests using intracellular reduction of a tetrazolium salt revealed no significant cytotoxicity, underscoring the biocompatibility of C14-PEI. C14-PEI also shows high efficiency in co-encapsulating Cas9 mRNA and sgRNA, as confirmed by agarose gel electrophoresis. At an sgRNA to Cas9 mRNA molar ratio of 10, the micelleplexes successfully mediate the cutting of mutated KRAS with an indel efficiency exceeding 60%, as determined by the T7 Endonuclease I (T7EI) assay. Droplet digital polymerase chain reaction (ddPCR) further demonstrates that the gene editing efficiency, measured by edited gene copies, is 48.5% in the w/w 4 group and 37.8% in the w/w 8 group. Treatment with C14-PEI micelleplexes containing Cas9 mRNA and sgRNA targeting the KRAS G12S mutation significantly impairs the migration capability of A549 cells and increases apoptosis rates. These findings suggest that C14-PEI effectively disrupts KRAS signalling pathways, leading to reduced tumor cell proliferation and enhanced cell death.}, }
@article {pmid39838754, year = {2025}, author = {Zhang, D and Zhou, Y and Li, X and Luan, Q}, title = {CRISPR/Cas13a-Enhanced Porous Hydrogel Encapsulated Photonic Barcodes for Multiplexed Detection of Virus.}, journal = {Small (Weinheim an der Bergstrasse, Germany)}, volume = {21}, number = {8}, pages = {e2408725}, doi = {10.1002/smll.202408725}, pmid = {39838754}, issn = {1613-6829}, support = {82102511//National Natural Science Foundation of China/ ; 82102181//National Natural Science Foundation of China/ ; BK20210021//National Natural Science Foundation of China/ ; BK20210009//National Natural Science Foundation of China/ ; M2021031//Research Project of&#xa0;Jiangsu Province Health Committee/ ; 2024-LCYJ-MS-15//Medical School of Nanjing University/ ; (YKK23068)//Nanjing Medical Science and Technique Development Foundation YKK23068/ ; }, mesh = {*Hydrogels/chemistry ; *SARS-CoV-2/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; Humans ; Porosity ; COVID-19/diagnosis/virology ; Photons ; RNA, Viral/genetics ; Nucleic Acid Hybridization ; }, abstract = {In this study, we present an ultrasensitive and specific multiplexed detection method for SARS-CoV-2 and influenza (Flu) utilizing CRISPR/Cas13a technology combined with a hydrogel-encapsulated photonic crystal (PhC) barcode integrated with hybridization chain reaction (HCR). The barcodes, characterized by core-shell structures, are fabricated through partial replication of periodically ordered hexagonally close-packed silicon dioxide beads. Consequently, the opal hydrogel shell of these barcodes features abundant interconnected pores that provide a substantial surface area for probe immobilization. Furthermore, the inherent structural colors remain stable during detection events due to the robust mechanical strength of the barcode cores. This integration of CRISPR/Cas13a and HCR leverages both the highly specific RNA recognition capabilities and trans-cleavage activity of Cas13a while employing HCR to enhance sensitivity. Upon encountering target RNA, Cas13a cleaves a hairpin probe, thereby initiating subsequent HCR amplification for enhanced detection sensitivity. Our method demonstrates high accuracy and sensitivity in multiplexed detection of SARS-CoV-2, Flu A and Flu B RNA with a limit-of-detection as low as 200 aM. Importantly, this assay also exhibits acceptable accuracy in repeated clinical sample testing. Thus, our platform represents a promising strategy for highly sensitive multiplexed virus detection in clinical.}, }
@article {pmid39838618, year = {2025}, author = {Su, Y and Zhao, B and Zhang, L and Shen, B and Guo, C and Xiao, H and Li, N}, title = {Quantitative Analysis of Phosphorothioate Isomers in CRISPR sgRNA at Single-Residue Resolution Using Endonuclease Digestion Coupled with Liquid Chromatography Cyclic Ion Mobility Mass Spectrometry (LC/cIMS).}, journal = {Analytical chemistry}, volume = {97}, number = {4}, pages = {2223-2231}, pmid = {39838618}, issn = {1520-6882}, mesh = {*Ion Mobility Spectrometry/methods ; *Phosphorothioate Oligonucleotides/analysis/chemistry ; Isomerism ; Mass Spectrometry/methods ; *RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism/genetics ; *Endonucleases/metabolism ; *CRISPR-Cas Systems ; Chromatography, Liquid ; *Phosphates/analysis ; }, abstract = {Phosphorothioate (PS) modifications in single-guided RNA (sgRNA) are crucial for genome editing applications using the CRISPR/Cas9 system. These modifications may enhance sgRNA stability, pharmacokinetics, and binding to targets, thereby facilitating the desired genetic alterations. Incorporating multiple PS groups at varying positions may introduce chiral centers into the sgRNA backbone, resulting in a complex mixture of constitutional- and stereoisomers that challenges current analytical capabilities for reliable identification and quantification. In this study, we developed an innovative methodology that combines endonuclease digestion of sgRNA with ion pairing reversed-phase liquid chromatography coupled with cyclic ion mobility mass spectrometry (IPRP-LC/cIMS) to fully distinguish PS-induced isomers in a complex mixture. The relative abundance of each isomer was quantified using a two-step method, wherein the ion abundance was sequentially extracted from an LC/MS ion chromatogram and LC/cIMS two-dimensional ion mobiligram. This quantification method was thoroughly evaluated, demonstrating excellent sensitivity, precision, dynamic range, repeatability, and accuracy. In addition, this method enables the investigation of the kinetics of forming PS to phosphodiester (PO) impurities in sgRNA under oxidative stress conditions, offering unprecedented insights into PS stability at a single-residue resolution. In this context, this method highlights the in-depth characterization of PS, demonstrating its capability to support biomedical research, development, and production of sgRNA products.}, }
@article {pmid39838465, year = {2025}, author = {Cheng, J and Chen, J and Chen, D and Li, B and Wei, C and Liu, T and Wang, X and Wen, Z and Jin, Y and Sun, C and Yang, G}, title = {Development of a Komagataella phaffii cell factory for sustainable production of (+)-valencene.}, journal = {Microbial cell factories}, volume = {24}, number = {1}, pages = {29}, pmid = {39838465}, issn = {1475-2859}, support = {2024SSYS0103//the major science and technology project of Zhejiang Province/ ; 2023C4S02002//the Start-up funds of Xianghu Laboratory/ ; }, mesh = {*Sesquiterpenes/metabolism ; Metabolic Engineering/methods ; CRISPR-Cas Systems ; Saccharomycetales ; }, abstract = {BACKGROUND: Sesquiterpene (+)-valencene is a characteristic aroma component from sweet orange fruit, which has a variety of biological activities and is widely used in industrial manufacturing of food, beverage and cosmetics industries. However, at present, the content in plant sources is low, and its yield and quality would be influenced by weather and land, which limit the supply of (+)-valencene. The rapid development of synthetic biology has accelerated the construction of microbial cell factories and provided an effective alternative method for the production of natural products.<br><br>RESULTS: In this study, we first introduced the (&#x2009;+)-valencene synthase into Komagataella phaffii by CRISPR/Cas9 system, and successfully constructed a (&#x2009;+)-valencene producer with the initial yield of 2.1&#xa0;mg/L. Subsequently, the (&#x2009;+)-valencene yield was increased to 8.2&#xa0;mg/L by fusing farnesyl pyrophosphate synthase with (&#x2009;+)-valencene synthase using the selected ligation linker. High expression of key genes IDI1, tHMG1, ERG12 and ERG19 enhanced metabolic flux of MVA pathway, and the yield of (&#x2009;+)-valencene was further increased by 27%. Besides, in-situ deletion of the promoter of ERG9 increased the yield of (&#x2009;+)-valencene to 48.1&#xa0;mg/L. Finally, we optimized the copy number of farnesyl pyrophosphate synthase and (&#x2009;+)-valencene synthase fusion protein, and when the copy number reached three, the yield of (&#x2009;+)-valencene achieved 173.6&#xa0;mg/L in shake flask level, which was 82-fold higher than that of the starting strain CaVAL1.<br><br>CONCLUSIONS: The results obtained here suggest that K. phaffii has the potential to efficiently synthesize other terpenoids.}, }
@article {pmid39838422, year = {2025}, author = {Hou, S and Yang, S and Bai, W}, title = {Multi-gene precision editing tool using CRISPR-Cas12a/Cpf1 system in Ogataea polymorpha.}, journal = {Microbial cell factories}, volume = {24}, number = {1}, pages = {28}, pmid = {39838422}, issn = {1475-2859}, support = {NO. XDC0120203//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; No. 306GJHZ2022010MI//CAS-NSTDA Joint Research Program/ ; TSBICIP-KJGG-020//Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Saccharomycetales/genetics/metabolism ; Homologous Recombination ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {BACKGROUND: Ogataea polymorpha, a non-conventional methylotrophic yeast, has demonstrated significant potential for heterologous protein expression and the production of high-value chemicals and biopharmaceuticals. However, the lack of precise and efficient genome editing tools severely hinders the construction of cell factories. Although the CARISP-Cas9 system has been established in Ogataea polymorpha, the gene editing efficiency, especially for multiple genes edition, needs to be further improved.<br><br>RESULTS: In this study, we developed an efficient CRISPR-Cpf1-mediated genome editing system in O. polymorpha that exhibited high editing efficiency for single gene (98.1&#x2009;&#xb1;&#x2009;1.7%), duplex genes (93.9&#x2009;&#xb1;&#x2009;2.4%), and triplex genes (94.0&#x2009;&#xb1;&#x2009;6.0%). Additionally, by knocking out non-homologous end joining (NHEJ) related genes, homologous recombination (HR) efficiency was increased from less than 30% to 90&#x2009;~&#x2009;100%, significantly enhancing precise genome editing capabilities. The increased HR rates enabled over 90% integration efficiency of triplex genes, as well as over 90% deletion rates of large DNA fragments up to 20 kb. Furthermore, using this developed CRISPR-Cpf1 system, triple genes were precisely integrated into the genome by one-step, enabling lycopene production in O. polymorpha.<br><br>CONCLUSIONS: This novel multiplexed genome-editing tool mediated by CRISPR-Cpf1 can realize the deletion and integration of multiple genes, which holds great promise for accelerating engineering efforts on this non-conventional methylotrophic yeast for metabolic engineering and genomic evolution towards its application as an industrial cell factory.}, }
@article {pmid39838098, year = {2025}, author = {Whalen, JM and Earley, J and Wisniewski, C and Mercurio, AM and Cantor, SB}, title = {Targeting BRCA1-deficient PARP inhibitor-resistant cells with nickases reveals nick resection as a cancer vulnerability.}, journal = {Nature cancer}, volume = {6}, number = {2}, pages = {278-291}, pmid = {39838098}, issn = {2662-1347}, support = {R01CA285607//U.S. Department of Health &amp; Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics)/ ; 1F32CA268524-01A1//U.S. Department of Health &amp; Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics)/ ; R01 CA285607/CA/NCI NIH HHS/United States ; F32 CA077880/CA/NCI NIH HHS/United States ; R01 CA254037/CA/NCI NIH HHS/United States ; F32 CA268524/CA/NCI NIH HHS/United States ; }, mesh = {*Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use ; Humans ; Animals ; *BRCA1 Protein/genetics/deficiency ; Mice ; *Drug Resistance, Neoplasm/genetics ; Tumor Suppressor p53-Binding Protein 1/genetics/metabolism ; Female ; *Breast Neoplasms/genetics/drug therapy/pathology ; Cell Line, Tumor ; CRISPR-Cas Systems ; DNA-Binding Proteins/genetics/metabolism ; Poly (ADP-Ribose) Polymerase-1/antagonists &amp; inhibitors/genetics ; Homologous Recombination ; }, abstract = {Tumors lacking the BRCA1 and BRCA2 (BRCA) hereditary breast cancer genes display heightened sensitivity to anti-cancer treatments, such as inhibitors of poly (ADP-ribose) polymerase 1 (PARP1). However, when resistance develops, treatments are lacking. Using CRISPR technology, we discovered that enhancing homologous recombination through increased DNA end resection in BRCA1-deficient cells by loss of the 53BP1-Shieldin complex-which is associated with resistance to PARP inhibitors-also heightens sensitivity to DNA nicks. The sensitivity is caused by hyper-resection of nicks into extensive single-stranded regions that trigger cell death. Based on these findings and that nicks limit tumor formation in mice, we propose nickases as a tool for personalized medicine. Moreover, our findings indicate that restricting nick expansion is a critical function of the 53BP1-Shieldin complex.}, }
@article {pmid39837388, year = {2025}, author = {Wang, X and Li, Y and Friess, D and Yao, L and Wang, X and He, Z and He, W and Li, M and Wang, W}, title = {Guanidyl-rich highly branched poly(β-amino ester)s for the delivery of dual CRISPR ribonucleoprotein for efficient large DNA fragment deletion.}, journal = {Journal of controlled release : official journal of the Controlled Release Society}, volume = {379}, number = {}, pages = {549-557}, doi = {10.1016/j.jconrel.2025.01.032}, pmid = {39837388}, issn = {1873-4995}, mesh = {Humans ; *Ribonucleoproteins/administration &amp; dosage/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; HeLa Cells ; *DNA/genetics ; *Polymers/chemistry/administration &amp; dosage ; *Guanidine/chemistry ; HEK293 Cells ; Transfection ; }, abstract = {Gene editing technologies, particularly clustered regularly interspersed short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, have revolutionized the ability to modify gene sequences in living cells for therapeutic purposes. Delivery of CRISPR/Cas ribonucleoprotein (RNP) is preferred over its DNA and RNA formats in terms of gene editing effectiveness and low risk of off-target events. However, the intracellular delivery of RNP poses significant challenges and necessitates the development of non-viral vectors. Our previous study has demonstrated that phenyl guanidine (PG) group modified linear poly(β-amino ester)s (PAEs) can facilitate CRISPR/Cas9 RNP mediated gene knockout in HeLa cells. Here, we further investigated the utilization of highly branched PAEs (HPAEs) with PG groups (HPAE-PG) for efficient delivery of cytosolic protein and CRISPR/Cas9 RNP complexes, while also examining the influence of branching units and branching ratios on the delivery process. The efficiency of HPAE-PG/RNP transfection for large DNA fragment deletion was assessed using a dual sgRNA-guided approach to delete exon 80 of the human COL7A1 gene, which harbors mutations associated with dystrophic epidermolysis bullosa (DEB). Our findings demonstrate that HPAE-PG/RNP successfully induced a deletion of 56 base pairs (exon 80) within COL7A1 in both HEK cells and keratinocytes derived from recessive DEB patients. This study highlights the potential of HPAE-PG as a non-viral vector for large DNA fragment deletion, emphasizing the importance of branching factors of HPAEs in optimizing CRISPR RNP delivery for therapeutic applications in genetic disorders.}, }
@article {pmid39837235, year = {2025}, author = {Ding, S and Dong, J and Shi, J and Ren, K and Cui, X and Shi, Z and Li, N and Xiang, Y and Du, F and Tang, Z}, title = {Integrating commercial personal glucose meter with peroxidase-mimic DNAzyme to develop a versatile point-of-care biosensing platform.}, journal = {Biosensors &amp; bioelectronics}, volume = {273}, number = {}, pages = {117171}, doi = {10.1016/j.bios.2025.117171}, pmid = {39837235}, issn = {1873-4235}, mesh = {*DNA, Catalytic/chemistry ; *Biosensing Techniques/instrumentation/methods ; Humans ; *SARS-CoV-2/isolation &amp; purification/genetics ; *Blood Glucose Self-Monitoring/instrumentation ; *COVID-19/diagnosis/blood/virology ; Point-of-Care Systems ; DNA, Viral/analysis ; Point-of-Care Testing ; RNA, Viral/analysis ; *Blood Glucose/analysis ; CRISPR-Cas Systems ; Peroxidase/chemistry ; Limit of Detection ; Equipment Design ; }, abstract = {The development of point-of-care testing (POCT) methods is highly desirable in molecular detection, as they enable disease diagnosis and biomarker monitoring on-site or at home. Repurposing existing POCT devices to detect diverse biomarkers is an economical way to develop new devices for POCT use. Personal glucose meter (PGM) is one of the most used off-the-shelf POCT devices that has been reused to detect non-glucose targets. However, developing a label-free, user-friendly, and cost-effective general PGM-based sensing platform remains a great challenge, primarily due to the reliance on protein enzymes in most existing signal transducing strategies. To overcome the challenges, we herein developed a DNAzyme-based signal transduction strategy that bridges non-glucose signals to PGM readouts. By integrating this strategy with CRISPR/Cas12a-mediated target sensing, we successfully established a simple and versatile platform (CaG-PGM) for biosensing. The utility of CaG-PGM in the detection of nucleic acid targets was successfully validated by detecting Monkeypox virus DNA and SARS-CoV-2 RNA with high sensitivity and specificity. We further demonstrated its generality in detecting non-nucleic acid targets including protein and small molecule. In conclusion, this study provides a cheap and effective strategy for repurposing PGM as a general biosensing platform and sheds new light on translating functional nucleic acids for POCT applications.}, }
@article {pmid39837195, year = {2025}, author = {Boismier, EC and Aboulnaga, EA and TerAvest, MA}, title = {Zymomonas mobilis: bringing an ancient human tool into the genomic era.}, journal = {Current opinion in biotechnology}, volume = {92}, number = {}, pages = {103257}, doi = {10.1016/j.copbio.2025.103257}, pmid = {39837195}, issn = {1879-0429}, mesh = {*Zymomonas/genetics/metabolism ; *Metabolic Engineering/methods ; Gene Editing ; Biofuels ; Ethanol/metabolism ; Humans ; Genomics ; CRISPR-Cas Systems ; Genome, Bacterial ; }, abstract = {Zymomonas mobilis is an ethanologenic bacterium that has been used for over 1500 years to produce alcoholic beverages. Recently, this microbe has become a top candidate for biofuel production due to its efficient metabolism. Z. mobilis is being developed to utilize lignocellulosic biomass as a feedstock and synthesize a range of valuable chemicals and fuels. Genetic and metabolic engineering strategies are crucial to reach these goals. Recent advances include genome engineering, CRISPR editing, and CRISPRi knockdown of genes. Metabolic engineering has enabled redirection of carbon from the natural product ethanol to chemicals such as 2,3-butanediol and polyhydroxybutyrate. The approaches summarized here will streamline the development of Z. mobilis as an industrial chassis for sustainable liquid fuels and chemicals.}, }
@article {pmid39836103, year = {2025}, author = {Mai, Z and Zhou, T and Lin, Z}, title = {Detecting CYP2C19 genes through an integrated CRISPR/Cas13a-assisted system.}, journal = {Analytical methods : advancing methods and applications}, volume = {17}, number = {6}, pages = {1382-1388}, doi = {10.1039/d4ay01930j}, pmid = {39836103}, issn = {1759-9679}, mesh = {*Cytochrome P-450 CYP2C19/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; Polymorphism, Single Nucleotide ; Polymerase Chain Reaction/methods ; Clopidogrel ; }, abstract = {CYP2C19 gene single nucleotide polymorphisms (SNPs) should be considered in the clinical use of clopidogrel as they have important guiding value for predicting the risk of bleeding and thrombosis after clopidogrel treatment. The CRISPR/Cas system is increasingly used for SNP detection owing to its single-nucleotide mismatch specificity. Simultaneous detection of multiple SNPs for rapid identification of the CYP2C19 genotype is important, but there is no method to detect a wide variety of CYP2C19 SNPs. This study proposes a new integrated system that integrates the PCR reaction and CRISPR/Cas detection of three CYP2C19 genes on a device, achieving rapid, sensitive, and specific detection. In our design, magnetic beads with three different sizes capture target nucleic acid from the sample, which are dragged through different areas by magnetic force, for PCR amplification reaction and CRISPR/Cas13a detection of CYP2C19*2, CYP2C19*3 and CYP2C19*17 genes. Note that magnetic beads were sorted via microporous PC membranes of different apertures. This study exhibits a broad clinical application prospect and provides a favorable tool for clinical clopidogrel administration.}, }
@article {pmid39835642, year = {2025}, author = {Hu, C and Xiang, H and Yin, Y and Li, J and Zhang, Y and Huang, X and Guo, Y and He, Y and Gao, Y and Ren, K and Han, H and Wang, W and Li, J}, title = {Electrochemiluminescence Resonance Energy Transfer Biosensor for the Human-Associated Clade of Streptococcus suis Based on Prereduction-Enhanced Yttrium MOFs.}, journal = {Analytical chemistry}, volume = {97}, number = {5}, pages = {3153-3160}, doi = {10.1021/acs.analchem.4c06560}, pmid = {39835642}, issn = {1520-6882}, mesh = {*Streptococcus suis/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; *Metal-Organic Frameworks/chemistry ; *Yttrium/chemistry ; Humans ; *Electrochemical Techniques/methods ; *Luminescent Measurements/methods ; Energy Transfer ; Gold/chemistry ; CRISPR-Cas Systems ; }, abstract = {Streptococcus suis, a significant zoonotic pathogen, annually caused substantial economic losses in the swine industry and had intensified threat to public health due to the recent emergence of human-associated clade. In this study, we discovered that the rare-earth metal-based metal-organic frameworks (Y-BTC) possessed excellent ECL capabilities. After prereduction at high voltage, its ECL intensity was enhanced by two times. Subsequently, we developed an efficient CRISPR/Cas12a-mediated electrochemiluminescence resonance energy transfer (ECL-RET) biosensor utilizing Y-BTC for the detection of the human-associated S. suis clade. Y-BTC was employed as the ECL-RET donor and ECL emitter, and the spherical nucleic acid Au NP was utilized as the ECL-RET receptor. In the presence of the target, isothermal amplification was triggered to generate a large number of amplicons, which subsequently activated the trans-cleavage activity of Cas12a. Cas12a cleaved the nucleic acid shell on the surface of Au NPs, reducing the spatial distance between Au NPs and Y-BTC due to electrostatic adsorption, thereby quenching the ECL of Y-BTC via ECL-RET. Consequently, the presence of targets can be observed by a reduced ECL signal. The sensor exhibited a detection range of 25 pM to 50 nM, with a detection limit as low as 17 pM. The practical utility was verified through actual sample testing. Our proposed ECL-RET sensing strategy provides a new avenue for the sensitive detection of S. suis. The universality has also been demonstrated using Fusobacterium nucleatum, Salmonella pullorum, and Listeria monocytogenes, holding great promise in the field of food safety and public health.}, }
@article {pmid39834214, year = {2025}, author = {Kim, J and Kim, YR and Lee, SM and Lee, J and Lee, S and Yong, D and Park, HG}, title = {Novel Isothermal Amplification Integrated with CRISPR/Cas13a and Its Applications for Ultrasensitive Detection of SARS-CoV-2.}, journal = {ACS synthetic biology}, volume = {14}, number = {2}, pages = {463-469}, doi = {10.1021/acssynbio.4c00605}, pmid = {39834214}, issn = {2161-5063}, mesh = {*SARS-CoV-2/genetics/isolation &amp; purification ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *COVID-19/diagnosis/virology ; RNA, Viral/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; Molecular Diagnostic Techniques/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *COVID-19 Nucleic Acid Testing/methods ; Sensitivity and Specificity ; Limit of Detection ; }, abstract = {We herein developed an ultrasensitive and rapid strategy to identify genomic nucleic acids by integrating a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 13a (Cas13a) into our recently developed isothermal technique, nicking and extension chain reaction system-based amplification (NESBA) reaction. In this technique, named CESBA, the NESBA reaction isothermally produces a large amount of RNA amplicons from the initial target genomic RNA (gRNA). The RNA amplicons bind to the crispr RNA (crRNA) and activate the collateral cleavage activity of Cas13a, which would then cleave the reporter probe nearby, consequently producing the final signals. Based on this design principle, we successfully detected SARS-CoV-2 gRNA as a model target very sensitively down to even a single copy (0.05 copies/μL) in both fluorescence- and lateral flow assay (LFA)-based modes with excellent specificity against other human coronaviruses (H-CoVs). We further validated the clinical applicability of CESBA by testing the 20 clinical samples with 100% clinical sensitivity and specificity. This work represents a potent and innovative strategy for the identification of genomic nucleic acids in molecular diagnostics, delivering exceptional levels of sensitivity.}, }
@article {pmid39834176, year = {2025}, author = {Tang, M}, title = {Research Status of Clustered Regulary Interspaced Short Palindromic Repeats Technology in the Treatment of Human Papillomavirus (HPV) Infection Related Diseases.}, journal = {Cancer control : journal of the Moffitt Cancer Center}, volume = {32}, number = {}, pages = {10732748241300654}, pmid = {39834176}, issn = {1526-2359}, mesh = {Humans ; *Papillomavirus Infections/therapy/virology/genetics ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; Papillomaviridae/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Uterine Cervical Neoplasms/therapy/virology ; Female ; Gene Editing/methods ; Human Papillomavirus Viruses ; }, abstract = {Background: CRISPR/Cas9 technology has rapidly advanced as a pivotal tool in cancer research, particularly in the precision targeting required for both detecting and treating malignancies. Its high specificity and low off-target effects make it exceptionally effective in applications involving Human Papillomavirus (HPV) related diseases, most notably cervical cancer. This approach offers a refined methodology for the rapid detection of viral infections and provides a robust platform for the safe and effective treatment of diseases associated with viral infections through gene therapy.Purpose: Gene therapy, within this context, involves the strategic delivery of genetic material into target cells via a vector. This is followed by the meticulous modulation of gene expression, whether through correction, addition, or suppression, specifically honed to target tumor cells while sparing healthy cells. This dual capacity to diagnose and treat at such a precise level underscores the transformative potential of CRISPR/Cas9 in contemporary medical science, particularly in oncology and virology.Research Design: This article provides an overview of the advancements made in utilizing the CRISPR-Cas9 system as a research tool for HPV-related treatments while summarizing its application status in basic research, diagnosis, and treatment of HPV.Data Collection: Furthermore, it discusses the future prospects for this technology within emerging areas of HPV research and precision medicine in clinical practice, while highlighting technical challenges and potential directions for future development.}, }
@article {pmid39833630, year = {2025}, author = {Zhang, B and Li, J and Yu, W}, title = {Integration of CRISPR/dCas9-Based methylation editing with guide positioning sequencing identifies dynamic changes of mrDEGs in breast cancer progression.}, journal = {Cellular and molecular life sciences : CMLS}, volume = {82}, number = {1}, pages = {46}, pmid = {39833630}, issn = {1420-9071}, support = {32270645//National Natural Science Foundation of China/ ; 18JC1411101//Major Special Projects of Basic Research of Shanghai Science and Technology Commission/ ; }, mesh = {Humans ; *DNA Methylation/genetics ; *Breast Neoplasms/genetics/pathology ; Female ; Disease Progression ; Cell Line, Tumor ; *CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Neoplastic ; *Gene Editing/methods ; Epigenesis, Genetic ; }, abstract = {Dynamic changes in DNA methylation are prevalent during the progression of breast cancer. However, critical alterations in aberrant methylation and gene expression patterns have not been thoroughly characterized. Here, we utilized guide positioning sequencing (GPS) to conduct whole-genome DNA methylation analysis in a unique human breast cancer progression model: MCF10 series of cell lines (representing benign/normal, atypical hyperplasia, and metastatic carcinoma). By integrating with mRNA-seq and matched clinical expression data from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO), six representative methylation-related differentially expressed genes (mrDEGs) were identified, including CAVIN2, ARL4D, DUSP1, TENT5B, P3H2, and MMP28. To validate our findings, we independently developed and optimized the dCas9-DNMT3L-DNMT3A system, achieving a high efficiency with a 98% increase in methylation at specific sites. DNA methylation levels significantly increased for the six genes, with CAVIN2 at 67.75 ± 1.05%, ARL4D at 53.29 ± 6.32%, DUSP1 at 57.63 ± 8.46%, TENT5B at 44.00 ± 5.09%, P3H2 at 58.50 ± 3.90%, and MMP28 at 49.60 ± 5.84%. RT-qPCR confirmed an inverse correlation between increased DNA methylation and gene expression. Most importantly, we mimicked tumor progression in vitro, demonstrating that transcriptional silencing of the TENT5B promotes cell proliferation in MCF10A cells owing to the crosstalk between hypermethylation and histone deacetylation. This study unveils the practical implications of DNA methylation dynamics of mrDEGs in reshaping epigenomic features during breast cancer malignant progression through integrated data analysis of the methylome and transcriptome. The application of the CRISPR/dCas9-based methylation editing technique elucidates the regulatory mechanisms and functional roles of individual genes within the DNA methylation signature, providing valuable insights for understanding breast cancer pathogenesis and facilitating potential therapeutic approaches in epigenome editing for patients with breast cancer.}, }
@article {pmid39833547, year = {2025}, author = {Dai, L and Zhang, P and Niu, X and Peng, X and Suleiman, RB and Zhang, G and Wan, X}, title = {CRISPR knock-in of a chimeric antigen receptor into GAPDH 3'UTR locus generates potent B7H3-specific NK-92MI cells.}, journal = {Cancer gene therapy}, volume = {32}, number = {2}, pages = {227-239}, pmid = {39833547}, issn = {1476-5500}, support = {2023A1515030028//Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)/ ; JCYJ20220818100806015//Shenzhen Science and Technology Innovation Commission/ ; 2019YFA0906100//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; }, mesh = {Humans ; *Receptors, Chimeric Antigen/genetics/metabolism ; *Killer Cells, Natural/immunology/metabolism ; Animals ; Mice ; *CRISPR-Cas Systems ; *3' Untranslated Regions/genetics ; *Immunotherapy, Adoptive/methods ; Cell Line, Tumor ; Gene Knock-In Techniques ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {CAR-NK therapy is becoming a promising approach to treat solid tumors. However, the random insertion of the CAR gene and inflexible CAR expression caused by common preparation methods significantly impact its efficacy and safety. Here we successfully established a novel type of CAR-NK cells by integrating CAR sequences into the GAPDH 3'UTR locus of NK-92MI cells (CRISPR-CAR-NK), achieving site-specific integration of the CAR gene and allowing endogenous regulatory components to govern CAR expression. CRISPR-CAR-NK cells had comparable growth capacity but displayed superior anti-tumor activity compared with their lentiviral counterparts. They activated and degranulated more effectively when co-cultured with tumor cells, due to increased expression of activating receptors and decreased expression of inhibitory molecules. They also enhanced the production of Granzyme B and IFN-γ, and more effectively triggered the IFN-γ pathway. Moreover, CRISPR-CAR-NK cells demonstrated distinct properties from conventional CAR-NK concerning metabolic features and signal dependence. Notably, CRISPR-CAR-NK cells exhibited lower metabolic levels without compromising antitumor activity, and their function was less reliant on the PI3K-AKT pathway, implying that the CRISPR-CAR-NK cells have significant potential for enhanced synergy with AKT inhibitors and adaptation to nutrient stress within the tumor microenvironment. These findings provide a novel potential strategy for cancer immunotherapy and an experimental foundation and paradigm for optimizing CAR-NK cells utilizing CRISPR technology, highlighting the potential of CRISPR to advance immunotherapies.}, }
@article {pmid39833501, year = {2025}, author = {Gawlig, C and Hirschberger, R and Hanci, G and Schott, S and Marandi, S and Hesse, IR and Rühl, M}, title = {Full sequencing of 100mer sgRNA via tandem mass spectrometry by targeted RNase H digestion with customized probes.}, journal = {Analytical and bioanalytical chemistry}, volume = {417}, number = {8}, pages = {1497-1506}, pmid = {39833501}, issn = {1618-2650}, mesh = {*Tandem Mass Spectrometry/methods ; *Ribonuclease H/metabolism/chemistry ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; High-Throughput Nucleotide Sequencing/methods ; Gene Editing/methods ; }, abstract = {The use of single-guide RNA (sgRNA) for gene editing using the CRISPR Cas9 system has become a powerful technique in various fields, especially with the growing interest in such molecules as therapeutic options in the last years. An important parameter for the use of these molecules is the verification of the correct sgRNA oligonucleotide sequence. Apart from next-generation sequencing protocols, mass spectrometry (MS) has been proven as a powerful technique for this purpose. The protocol and investigations presented in this work show an optimal digestion and 100% sequence coverage of sgRNA, while top-down approaches or other ribonuclease (RNase) digestion strategies obtain a sequence coverage of up to 80-90% utilizing multiple RNases. The results in this publication were obtained by utilizing DNA-RNA hybrid GAPmer-like probes and RNase H, an enzyme which specifically hydrolyzes RNA in DNA-RNA double strands. We assessed the optimal length of the DNA segment of these hybrid probes to maximize the specificity of the RNase H digestion and to achieve complete sequence confirmation by tandem MS analysis of the resulting digestion products. Furthermore, we showed that the approach is applicable for the identification of common synthesis-related impurities, like truncations and elongations. Despite the fact that the accessibility of this approach for highly modified molecules is limited to nucleotides which are not 2'-O-methylated, the optimized sequence coverage makes it a viable method.}, }
@article {pmid39833409, year = {2025}, author = {Zhang, X and Qiu, M and Han, B and Liao, L and Peng, X and Lin, J and Zhang, N and Hai, L and Liang, L and Ma, Y and Li, W and Liu, M}, title = {Generation and propagation of high fecundity gene edited fine wool sheep by CRISPR/Cas9.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {2557}, pmid = {39833409}, issn = {2045-2322}, support = {2023SNGGGCC010//the Tianshan Talent Training Program-high-level talents for the advancement of modern agriculture and animal husbandry./ ; 2023TSYCTD0007//the Scientific and Technological Innovation Team Project of Xinjiang Uygur Autonomous Region./ ; }, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Sheep/genetics ; *Fertility/genetics ; *Gene Editing/methods ; Female ; *Wool ; *Bone Morphogenetic Protein Receptors, Type I/genetics ; Litter Size/genetics ; Alleles ; }, abstract = {CRISPR/Cas9 technology has been widely utilized to enhance productive performance, increase disease resistance and generate medical models in livestock. The FecB allele in sheep is a mutation in the BMPRIB gene, recognized as the first major gene responsible for the high fecundity trait in sheep, leading to an increased ovulation rate in ewe. In this study, we employed CRISPR/Cas9-mediated homologous-directed repair (HDR) to introduce a defined point mutation (c.746 A > G) using single-stranded oligonucleotides (ssODN) and the ligase IV inhibitor (SCR7) into the BMPRIB gene of fine wool sheep. A total of nine gene-edited sheep were produced, six of which carried the targeted point mutation, with a precise base substitution efficiency (A > G) of 31.6%. Based on the six targeted founders (F0), we expanded the BMPRIB-targeted population, which included F1 heterozygous (B+) and F2 homozygous(BB) or heterozygous offspring. The average litter size of F1 ewes carrying the B + allele reached 170%, comparable to that of heterozygous native Australian Booroola sheep. Gene-edited ewes with B + and BB genotype produced 0.62 and 0.42 more lambs, respectively, compared to wide-type ewes (p < 0.01). Our results also indicated that the parity signification, our data demonstrate that highly efficient introduction of the intended base mutation into the sheep genome can be achieved by combining the CRISPR/Cas9 system with ssODN and SCR7. The offspring of BMPR/B edited sheep with the defined mutation exhibited high fecundity performance. Compared to conventional sheep breeding strategies, genetic improvement through gene editing offered significant advantages without compromising the fine wool traits of Merino sheep, which are often affected by routine cross-breeding methods.}, }
@article {pmid39833307, year = {2025}, author = {Di Cristina, G and Dirksen, E and Altenhein, B and Büschges, A and Korsching, SI}, title = {Pioneering genome editing in parthenogenetic stick insects: CRISPR/Cas9-mediated gene knockout in Medauroidea extradentata.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {2584}, pmid = {39833307}, issn = {2045-2322}, mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Gene Knockout Techniques/methods ; *Parthenogenesis/genetics ; *Insecta/genetics ; Pigmentation/genetics ; Female ; *Genome, Insect ; Insect Proteins/genetics ; Phenotype ; }, abstract = {The parthenogenetic life cycle of the stick insect Medauroidea extradentata offers unique advantages for the generation of genome-edited strains, as an isogenic and stable mutant line can in principle be achieved already in the first generation (G0). However, genetic tools for the manipulation of their genes had not been developed until now. Here, we successfully implement CRISPR/Cas9 as a technique to modify the genome of the stick insect M. extradentata. As proof-of-concept we targeted two genes involved in the ommochrome pathway of eye pigmentation (cinnabar and white, second and first exon, respectively), to generate knockout (KO) mutants. Microinjections were performed within 24 h after oviposition, to focus on the mononuclear (and haploid) stage of development. The KOs generated resulted in distinct eye and cuticle colour phenotypes for cinnabar and white. Homozygous cinnabar mutants showed pale pigmentation of eyes and cuticle. They develop into adults capable of producing viable eggs. Homozygous white KO resulted in a completely unpigmented phenotype in developing embryos that were unable to hatch. In conclusion, we show that CRISPR/Cas9 can be successfully applied to the genome of M. extradentata by creating phenotypically different and viable insects. This powerful gene editing technique can now be employed to create stable genetically modified lines using a parthenogenetic non-model organism.}, }
@article {pmid39833279, year = {2025}, author = {Bi, C and Yuan, B and Zhang, Y and Wang, M and Tian, Y and Li, M}, title = {Prevalent integration of genomic repetitive and regulatory elements and donor sequences at CRISPR-Cas9-induced breaks.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {94}, pmid = {39833279}, issn = {2399-3642}, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *Regulatory Sequences, Nucleic Acid ; *Repetitive Sequences, Nucleic Acid ; Mutagenesis, Insertional ; Recombinational DNA Repair ; }, abstract = {CRISPR-Cas9 genome editing has been extensively applied in both academia and clinical settings, but its genotoxic risks, including large insertions (LgIns), remain poorly studied due to methodological limitations. This study presents the first detailed report of unintended LgIns consistently induced by different Cas9 editing regimes using various types of donors across multiple gene loci. Among these insertions, retrotransposable elements (REs) and host genomic coding and regulatory sequences are prevalent. RE frequencies and 3D genome organization analysis suggest LgIns originate from randomly acquired genomic fragments by DNA repair mechanisms. Additionally, significant unintended full-length and concatemeric double-stranded DNA (dsDNA) donor integrations occur when donor DNA is present. We further demonstrate that phosphorylated dsDNA donors consistently reduce large insertions and deletions by almost two-fold without compromising homology-directed repair (HDR) efficiency. Taken together, our study addresses a ubiquitous and overlooked risk of unintended LgIns in Cas9 editing, contributing valuable insights for the safe use of Cas9 editing tools.}, }
@article {pmid39833194, year = {2025}, author = {Kim, DG and Gu, B and Cha, Y and Ha, J and Lee, Y and Kim, G and Cho, BK and Oh, MK}, title = {Engineered CRISPR-Cas9 for Streptomyces sp. genome editing to improve specialized metabolite production.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {874}, pmid = {39833194}, issn = {2041-1723}, support = {RS-2024-00509338//National Research Foundation of Korea (NRF)/ ; RS-2024-00357320//National Research Foundation of Korea (NRF)/ ; RS-2021-NR056596//National Research Foundation of Korea (NRF)/ ; }, mesh = {*Streptomyces/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Multigene Family ; Genome, Bacterial ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {The CRISPR-Cas9 system has frequently been used for genome editing in Streptomyces; however, cytotoxicity, caused by off-target cleavage, limits its application. In this study, we implement innovative modification to Cas9, strategically addressing challenges encountered during gene manipulation using Cas9 within strains possessing high GC content genome. The Cas9-BD, a modified Cas9 with the addition of polyaspartate to its N- and C-termini, is developed with decreased off-target binding and cytotoxicity compared with wild-type Cas9. Cas9-BD and similarly modified dCas9-BD have been successfully employed for simultaneous biosynthetic gene cluster (BGC) refactoring, multiple BGC deletions, or multiplexed gene expression modulations in Streptomyces. We also demonstrate improved secondary metabolite production using multiplexed genome editing with multiple single guide RNA libraries in several Streptomyces strains. Cas9-BD is also used to capture large BGCs using a developed in vivo cloning method. The modified CRISPR-Cas9 system is successfully applied to many Streptomyces sp., providing versatile and efficient genome editing tools for strain engineering of actinomycetes with high GC content genome.}, }
@article {pmid39833175, year = {2025}, author = {Goold, HD and Kroukamp, H and Erpf, PE and Zhao, Y and Kelso, P and Calame, J and Timmins, JJB and Wightman, ELI and Peng, K and Carpenter, AC and Llorente, B and Hawthorne, C and Clay, S and van Wyk, N and Daniel, EL and Harrison, F and Meier, F and Willows, RD and Cai, Y and Walker, RSK and Xu, X and Espinosa, MI and Stracquadanio, G and Bader, JS and Mitchell, LA and Boeke, JD and Williams, TC and Paulsen, IT and Pretorius, IS}, title = {Construction and iterative redesign of synXVI a 903 kb synthetic Saccharomyces cerevisiae chromosome.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {841}, pmid = {39833175}, issn = {2041-1723}, support = {DP200100717//Department of Education and Training | Australian Research Council (ARC)/ ; CE200100029//Department of Education and Training | Australian Research Council (ARC)/ ; }, mesh = {*Saccharomyces cerevisiae/genetics/growth &amp; development ; Genome, Fungal ; *Synthetic Biology/methods ; *Chromosomes, Fungal/genetics ; Open Reading Frames ; *Chromosomes, Artificial, Yeast/genetics ; RNA, Transfer/genetics ; CRISPR-Cas Systems ; }, abstract = {The Sc2.0 global consortium to design and construct a synthetic genome based on the Saccharomyces cerevisiae genome commenced in 2006, comprising 16 synthetic chromosomes and a new-to-nature tRNA neochromosome. In this paper we describe assembly and debugging of the 902,994-bp synthetic Saccharomyces cerevisiae chromosome synXVI of the Sc2.0 project. Application of the CRISPR D-BUGS protocol identified defective loci, which were modified to improve sporulation and recover wild-type like growth when grown on glycerol as a sole carbon source when grown at 37˚C. LoxPsym sites inserted downstream of dubious open reading frames impacted the 5' UTR of genes required for optimal growth and were identified as a systematic cause of defective growth. Based on lessons learned from analysis of Sc2.0 defects and synXVI, an in-silico redesign of the synXVI chromosome was performed, which can be used as a blueprint for future synthetic yeast genome designs. The in-silico redesign of synXVI includes reduced PCR tag frequency, modified chunk and megachunk termini, and adjustments to allocation of loxPsym sites and TAA stop codons to dubious ORFs. This redesign provides a roadmap into applications of Sc2.0 strategies in non-yeast organisms.}, }
@article {pmid39832868, year = {2025}, author = {Alarcón-Iniesta, H and de Arana, G and López-Valls, M and Pardo, D and Escalona-Noguero, C and Rodríguez, C and Castellanos, M and Cobelo, S and Martínez-Ramírez, I and Camarero, J and Heras, SL and de Vicente, J and Valera, A and Smith, W and Bernardo-Gavito, R and Cantón, R and Galán, JC and Granados, D and Miranda, R and Guerrero, H and Sot, B}, title = {CRISPR-associated Plasmonic Colorimeter Method (Ca-PCM): A real-time RGB detection system for gold nanoparticles-based nucleic acid biosensors.}, journal = {Analytica chimica acta}, volume = {1338}, number = {}, pages = {343601}, doi = {10.1016/j.aca.2024.343601}, pmid = {39832868}, issn = {1873-4324}, mesh = {*Gold/chemistry ; *Metal Nanoparticles/chemistry ; *Biosensing Techniques/methods ; *Colorimetry/methods ; *RNA/analysis/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {BACKGROUND: The detection of genetic sequences represents the gold standard procedure for species discrimination, genetic characterisation of tumours, and identification of pathogens. The development of new molecular detection methods, accessible and cost effective, is of great relevance. Biosensors based on plasmonic nanoparticles, such as gold nanoparticles (AuNPs), provide a powerful and versatile platform for highly sensitive, economic, user-friendly and label-free sensing. However, the readout techniques typically employed with such sensors lack temporal and kinetic information, which hampers the ability to perform quantitative detection.<br><br>RESULTS: In this study, a novel methodology designated the 'CRISPR-associated Plasmonic Colorimeter Method' (Ca-PCM), has been developed. This method combines RNA target recognition by CRISPR LwaCas13a, AuNPs' aggregation, and real-time colorimetric Red-Green-Blue (RGB) analysis. The system registers the AuNP's plasmonic signatures in real-time using a RGB colour sensor with 3-channel silicon photodiodes having blue, green and red sensitivities. The acquired signals are automatically analysed by an algorithm designed to distinguish between positive and negative samples and to correlate the temporal spectral patterns of aggregation with dose-dependent molecular detection of the RNA target. In addition, the combination of Ca-PCM with a previous isothermal amplification allows the target efficient detection in real clinical applications.<br><br>SIGNIFICANCE: We have shown that the combination of RGB analysis and continuous temporal measurements is a novel and promising method to characterise the behaviour of gold nanoparticle-based biosensors and to achieve dose-dependent target detection. In addition, the simplicity and cost-effectiveness of this new approach expand the possibilities of other plasmonic-based biosensors and their applicability in low-resources clinical environments.}, }
@article {pmid39832863, year = {2025}, author = {Zhang, T and Wang, X and Jiang, Y and Zhang, Y and Zhao, S and Hu, J and Hu, F and Peng, N}, title = {A miniaturized RPA-CRISPR/Cas12a-based nucleic acid diagnostic platform for rapid and simple self-testing of SARS-CoV-2.}, journal = {Analytica chimica acta}, volume = {1338}, number = {}, pages = {343593}, doi = {10.1016/j.aca.2024.343593}, pmid = {39832863}, issn = {1873-4324}, mesh = {Humans ; *SARS-CoV-2/genetics/isolation &amp; purification ; *Nucleic Acid Amplification Techniques/methods ; *COVID-19/diagnosis ; *CRISPR-Cas Systems ; Lab-On-A-Chip Devices ; *Self-Testing ; *COVID-19 Nucleic Acid Testing/methods/instrumentation ; Miniaturization ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Nucleic acid testing is the most effective detection method currently available for the diagnosis of respiratory infectious diseases. However, the conventional real-time fluorescent quantitative PCR technique, which is regarded as the gold standard method for nucleic acid detection, presents significant challenges for implementation in home self-testing and popularization in underdeveloped regions due to its rigorous experimental standards. It is therefore clear that an easy-to-use, miniaturized nucleic acid testing technology and products for nonprofessionals are of great necessity to define the pathogens and assist in controlling disease transmission. (87) RESULTS: In this study, we propose a strategy for self-testing of respiratory pathogen nucleic acid that is oriented towards the public and user-friendly. The proposed system integrates the processes of extraction-free nucleic acid release, RPA isothermal amplification, and CRISPR fluorescence detection into a compact configuration. A microfluidic testing chip actuated by air pouches and a battery/USB-powered reusable device has been developed to enable simultaneous detection of internal reference genes and viral targets in a fully enclosed condition. The system allows for sample-in, and result-out testing in less than 30 min with a detection limit of 2 copies/μL. Additionally, a straightforward signal-light-based result display method has been developed to make it easy and intuitive for users to access the results. Furthermore, freeze-drying reagent is introduced to guarantee the storage and transportation of testing chips in ambient conditions. (135) SIGNIFICANCE: This work presents a miniaturized, portable, and highly sensitive nucleic acid detection system, where simple operating procedures have been designed for unskilled users. It is our belief that the testing system developed in this work is well suited for home-based self-testing and infection diagnosis in resource-limited areas, due to the above-mentioned advantages. (52).}, }
@article {pmid39832801, year = {2025}, author = {Debnath, A and Sengupta, A and Rudrapal, S and Kumar, A and Rani, M}, title = {In-silico study of molecular adaptations in halophilic Cas9.}, journal = {Letters in applied microbiology}, volume = {78}, number = {2}, pages = {}, doi = {10.1093/lambio/ovaf006}, pmid = {39832801}, issn = {1472-765X}, support = {//UGC/ ; F.30-487/2019//BSR/ ; }, mesh = {*Bacterial Proteins/chemistry/genetics/metabolism ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; Static Electricity ; Computer Simulation ; Protein Conformation ; Adaptation, Physiological ; Amino Acid Sequence ; Models, Molecular ; }, abstract = {This study explores the structural adaptations of the CRISPR-Cas9 system in halophilic bacteria, focusing on Cas9 protein of halophilic bacterium Salicibibacter cibi. Protein sequences were analyzed using different tools such as ExPASy ProtParam for different physicochemical properties, Predictor of Natural Disordered Regions web server for disordered regions, and InterPro server and WebLogo for domains. Protein structures were generated using the AlphaFold database, and the quality of the modelled structure was checked through PROCHECK. The protein surface's amino acids and electrostatic potential were visualized using PyMOL, APBS server, and UCSF chimera. Comparative analysis revealed that halophilic Cas9 proteins possess a higher abundance of acidic residues, resulting in enhanced stability and hydration in saline conditions; halophilic Cas9 proteins also shows higher intrinsically disordered regions. Electrostatic potential maps confirmed that S. cibi Cas9 proteins maintain a highly negative surface charge, crucial for adaptation to salt-rich environments. These findings provide insights into the molecular mechanisms driving the structural and functional adaptations of Cas9 in salty environment, highlighting its potential applications in genome editing-based biotechnological approaches in extreme conditions.}, }
@article {pmid39832688, year = {2025}, author = {Harris, H and Kittur, J}, title = {Unlocking the potential of CRISPR-Cas9 for cystic fibrosis: A systematic literature review.}, journal = {Gene}, volume = {942}, number = {}, pages = {149257}, doi = {10.1016/j.gene.2025.149257}, pmid = {39832688}, issn = {1879-0038}, mesh = {*Cystic Fibrosis/genetics/therapy ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; *Genetic Therapy/methods ; Animals ; Mutation ; }, abstract = {CRISPR-Cas9 technology has revolutionized genetic engineering, offering precise and efficient genome editing capabilities. This review explores the application of CRISPR-Cas9 for cystic fibrosis (CF), particularly targeting mutations in the CFTR gene. CF is a multiorgan disease primarily affecting the lungs, gastrointestinal system (e.g., CF-related diabetes (CFRD), CF-associated liver disease (CFLD)), bones (CF-bone disease), and the reproductive system. CF, a genetic disorder characterized by defective ion transport leading to thick mucus accumulation, is often caused by mutations like ΔF508 in the CFTR gene. This review employs a systematic methodology, incorporating an extensive literature search across multiple academic databases, including PubMed, Web of Science, and ScienceDirect, to identify 40 high-quality studies focused on CRISPR-Cas9 applications for CFTR gene editing. The data collection process involved predefined inclusion criteria targeting experimental approaches, gene-editing outcomes, delivery methods, and verification techniques. Data analysis synthesized findings on editing efficiency, off-target effects, and delivery system optimization to present a comprehensive overview of the field. The review highlights the historical development of CRISPR-Cas9, its mechanism, and its transformative role in genetic engineering and medicine. A detailed examination of CRISPR-Cas9's application in CFTR gene correction emphasizes the potential for therapeutic interventions while addressing challenges such as off-target effects, delivery efficiency, and ethical considerations. Future directions include optimizing delivery systems, integrating advanced editing tools like prime and base editing, and expanding personalized medicine approaches to improve treatment outcomes. By systematically analyzing the current landscape, this review provides a foundation for advancing CRISPR-Cas9 technologies for cystic fibrosis treatment and related disorders.}, }
@article {pmid39832424, year = {2025}, author = {Choudhary, R and Ahmad, F and Kaya, C and Upadhyay, SK and Muneer, S and Kumar, V and Meena, M and Liu, H and Upadhyaya, H and Seth, CS}, title = {Decrypting proteomics, transcriptomics, genomics, and integrated omics for augmenting the abiotic, biotic, and climate change stress resilience in plants.}, journal = {Journal of plant physiology}, volume = {305}, number = {}, pages = {154430}, doi = {10.1016/j.jplph.2025.154430}, pmid = {39832424}, issn = {1618-1328}, mesh = {*Climate Change ; *Stress, Physiological/genetics ; *Proteomics/methods ; *Genomics/methods ; *Transcriptome ; *Plant Physiological Phenomena ; Metabolomics ; *Plants/genetics ; Crops, Agricultural/genetics/physiology ; }, abstract = {As our planet faces increasing environmental challenges, such as biotic pressures, abiotic stressors, and climate change, it is crucial to understand the complex mechanisms that underlie stress responses in crop plants. Over past few years, the integration of techniques of proteomics, transcriptomics, and genomics like LC-MS, IT-MS, MALDI-MS, DIGE, ESTs, SAGE, WGS, GWAS, GBS, 2D-PAGE, CRISPR-Cas, cDNA-AFLP, HLS, HRPF, MPSS, CAGE, MAS, IEF, MudPIT, SRM/MRM, SWATH-MS, ESI have significantly enhanced our ability to comprehend the molecular pathways and regulatory networks, involved in balancing the ecosystem/ecology stress adaptation. This review offers thorough synopsis of the current research on utilizing these multi-omics methods (including metabolomics, ionomics) for battling abiotic (salinity, temperature (chilling/freezing/cold/heat), flood (hypoxia), drought, heavy metals/loids), biotic (pathogens like fungi, bacteria, virus, pests, and insects (aphids, caterpillars, moths, mites, nematodes) and climate change stress (ozone, ultraviolet radiation, green house gases, carbon dioxide). These strategies can expedite crop improvement, and act as powerful tools with high throughput and instant database generation rates. They also provide a platform for interpreting intricate, systematic signalling pathways and knowing how different environmental stimuli cause phenotypic responses at cellular and molecular level by changing the expression of stress-responsive genes like RAB18, KIN1, RD29B, OsCIPK03, OsSTL, SIAGL, bZIP, SnRK, ABF. This review discusses various case studies that exemplify the successful implementation of these omics tools to enhance stress tolerance in plants. Finally, it highlights challenges and future prospects of utilizing these approaches in combating stress, emphasizing the need for interdisciplinary collaborations and bio-technological advancements for sustainable agriculture and food security.}, }
@article {pmid39832407, year = {2025}, author = {Mao, Z and Chen, R and Huang, L and Ren, S and Liu, B and Gao, Z}, title = {CRISPR analysis based on Pt@MOF dual-modal signal for multichannel fluorescence and visual detection of norovirus.}, journal = {Biosensors &amp; bioelectronics}, volume = {273}, number = {}, pages = {117153}, doi = {10.1016/j.bios.2025.117153}, pmid = {39832407}, issn = {1873-4235}, mesh = {*Biosensing Techniques/methods ; *Norovirus/isolation &amp; purification/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *Metal-Organic Frameworks/chemistry ; Platinum/chemistry ; Colorimetry/methods ; *Caliciviridae Infections/virology/diagnosis ; Fluorescence ; Limit of Detection ; Nucleic Acid Amplification Techniques ; RNA, Viral/genetics ; }, abstract = {Norovirus is a globally prevalent pathogen that causes acute viral gastroenteritis across all age groups, characterized by its high infectivity and low infectious dose. Consequently, the development of rapid, sensitive, and accurate detection technologies for norovirus presents a significant challenge. In this study, we demonstrate a combination of CRISPR-Cas-based reactions with Pt@MOF-linked immunoassay-like assays. This methodology enables both qualitative analysis and colorimetric readouts of Cas12a-mediated DNA/RNA detection at room temperature, as well as the generation of fluorescent signal readout through base deprotonation-induced Pt@MOF cleavage of a fluorogenic substrate. Furthermore, the integration of RPA amplification with noncanonical PAM-designed CRISPR significantly enhances the sensitivity and flexibility of detection, facilitating the extension of this strategy to other targets. Ultimately, the strategy was validated in spiked food samples with a 100% accuracy rate, consistent with RT-qPCR results. Collectively, this work showcases a viable approach for a dual-functional Pt@MOF-based CRISPR biosensing platform for bioanalysis and a flexible, universal strategy based on noncanonical PAM-designed gRNAs.}, }
@article {pmid39831800, year = {2025}, author = {Ju, C and Li, X and Wang, D and Wei, Z and Xu, Q and Wang, J and Zhang, W and Zhang, A}, title = {Ultrasensitive detection of microRNAs based on cascade amplification strategy of RCA-PER and Cas12a.}, journal = {The Analyst}, volume = {150}, number = {4}, pages = {692-699}, doi = {10.1039/d4an01463d}, pmid = {39831800}, issn = {1364-5528}, mesh = {*MicroRNAs/analysis/genetics/blood ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *Biosensing Techniques/methods ; Limit of Detection ; DNA, Single-Stranded/chemistry/genetics ; CRISPR-Cas Systems ; *Endodeoxyribonucleases/genetics/metabolism ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Bacterial Proteins/genetics/metabolism ; Spectrometry, Fluorescence/methods ; DNA Primers/genetics/chemistry ; }, abstract = {Since microRNAs (miRNAs) serve as markers for early cancer diagnosis, it is crucial to develop a novel biosensor to detect miRNAs quickly, sensitively and selectively. Hence, we developed a fluorescence biosensor based on target miRNA-initiated rolling circle amplification (RCA) to generate RCA products with multiple tandem catalytic hairpin DNA templates that trigger primer exchange reactions (PER) which extend short single-strand DNA (ssDNA) primers into long ssDNA. Subsequently, the long ssDNA activates the trans-cleavage activity of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system to cleave a fluorescent reporter chain, enabling ultrasensitive detection of miRNAs through the output fluorescence signal. The biosensor could quantify miRNA-141 concentrations from 10[0] to 10[5] pM, with a detection limit of 94 fM. Therefore, the biosensing strategy proposed in this study offers a robust technique for the clinical diagnosis of miRNA-141.}, }
@article {pmid39831695, year = {2025}, author = {Yan, C and Meng, H and Pei, Y and Sun, W and Zhang, J}, title = {Breeding by Design for Functional Rice with Genome Editing Technologies.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {215}, pages = {}, doi = {10.3791/67336}, pmid = {39831695}, issn = {1940-087X}, mesh = {*Oryza/genetics ; *Plant Breeding/methods ; *Genome, Plant ; *Gene Editing/methods ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {The conventional approaches to crop breeding, which rely predominantly on time-consuming and labor-intensive methods such as traditional hybridization and mutation breeding, face challenges in efficiently introducing targeted traits and generating diverse plant populations. Conversely, the emergence of genome editing technologies has ushered in a paradigm shift, enabling the precise and expedited manipulation of plant genomes to intentionally introduce desired characteristics. One of the most widespread editing tools is the CRISPR/Cas system, which has been used by researchers to study important biology-related problems. However, the precise and effective workflow of genome editing has not been well-defined in crop breeding. In this study, we demonstrated the entire process of breeding rice varieties enriched with high levels of resistant starch (RS), a functional trait that plays a crucial role in preventing diseases such as diabetes and obesity. The workflow encompassed several key steps, such as the selection of functional SBEIIb gene, designing the single-guide RNA (sgRNA), selecting an appropriate genome editing vector, determining the vector delivery method, conducting plant tissue culture, genotyping mutation and phenotypic analysis. Additionally, the time frame necessary for each stage of the process has been clearly demonstrated. This protocol not only streamlines the breeding process but also enhances the accuracy and efficiency of trait introduction, thereby accelerating the development of functional rice varieties.}, }
@article {pmid39831307, year = {2025}, author = {Fei, X and Lei, C and Ren, W and Liu, C}, title = {'Splice-at-will' Cas12a crRNA engineering enabled direct quantification of ultrashort RNAs.}, journal = {Nucleic acids research}, volume = {53}, number = {2}, pages = {}, pmid = {39831307}, issn = {1362-4962}, support = {22074088//National Natural Science Foundation of China/ ; IRT_15R43//Program for Changjiang Scholars and Innovative Research Team in University/ ; GK202305001//Fundamental Research Funds for the Central Universities/ ; //Shaanxi Normal University/ ; }, mesh = {*CRISPR-Associated Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; *RNA/genetics/analysis ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; *RNA Splicing ; DNA/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {We present a robust 'splice-at-will' CRISPR RNA (crRNA) engineering mechanism that overcomes the limitations of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system in directly detecting ultrashort RNAs. In this strategy, an intact Cas12a crRNA can be split from almost any site of the spacer region to obtain a truncated crRNA (tcrRNA) that cannot activate Cas12a even after binding an auxiliary DNA activator. While splicing tcrRNAs with a moiety of ultrashort RNA, the formed combination can work together to activate Cas12a efficiently, enabling 'splice-at-will' crRNA engineering. Importantly, the 'splice-at-will' crRNA exhibits almost the same trans-cleavage activation efficiency as that of a conventional intact crRNA. Therefore, by rationally designing a DNA auxiliary activator with a conserved tcrRNA-complementary sequence and an arbitrary short RNA-of-interest recognition domain, a general sensing system is established that directly utilizes traditional DNA-activated Cas12a to detect ultrashort RNAs. This 'splice-at-will' crRNA engineering strategy could faithfully detect ultrashort RNA sequences as short as 6-8 nt, which cannot be achieved by conventional Cas12a and Cas13a systems. Additionally, through flexible splicing site design, our method can precisely distinguish single-base differences in microRNA and other short RNA sequences. This work has significantly expanded the Cas12a-based diagnostic toolbox and opened new avenues for ultrashort RNA detection.}, }
@article {pmid39829748, year = {2025}, author = {Nayfach, S and Bhatnagar, A and Novichkov, A and Estevam, GO and Kim, N and Hill, E and Ruffolo, JA and Silverstein, R and Gallagher, J and Kleinstiver, B and Meeske, AJ and Cameron, P and Madani, A}, title = {Engineering of CRISPR-Cas PAM recognition using deep learning of vast evolutionary data.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, pmid = {39829748}, issn = {2692-8205}, support = {DP2 CA281401/CA/NCI NIH HHS/United States ; P01 HL142494/HL/NHLBI NIH HHS/United States ; }, abstract = {CRISPR-Cas enzymes must recognize a protospacer-adjacent motif (PAM) to edit a genomic site, significantly limiting the range of targetable sequences in a genome. Machine learning-based protein engineering provides a powerful solution to efficiently generate Cas protein variants tailored to recognize specific PAMs. Here, we present Protein2PAM, an evolution-informed deep learning model trained on a dataset of over 45,000 CRISPR-Cas PAMs. Protein2PAM rapidly and accurately predicts PAM specificity directly from Cas proteins across Type I, II, and V CRISPR-Cas systems. Using in silico deep mutational scanning, we demonstrate that the model can identify residues critical for PAM recognition in Cas9 without utilizing structural information. As a proof of concept for protein engineering, we employ Protein2PAM to computationally evolve Nme1Cas9, generating variants with broadened PAM recognition and up to a 50-fold increase in PAM cleavage rates compared to the wild-type under in vitro conditions. This work represents the first successful application of machine learning to achieve customization of Cas enzymes for alternate PAM recognition, paving the way for personalized genome editing.}, }
@article {pmid39829431, year = {2025}, author = {Chen, Y and Xue, Y and Jiang, Q and Jin, Y and Chen, W and Hua, M}, title = {Disruption of the FOXM1 Regulatory Region Inhibits Tumor Progression in Ovarian Cancer by CRISPR-Cas9.}, journal = {Drug development research}, volume = {86}, number = {1}, pages = {e70049}, doi = {10.1002/ddr.70049}, pmid = {39829431}, issn = {1098-2299}, support = {//This work was supported by the Nantong Social Science and Technology Plan for People's Livelihood Project (grant No. JCZ2023003) from the Science and Technology Bureau of Nantong, China, and the Research Physician Development Fund (grant No. YJXYY202204-YSC03) from the Affiliated Hospital of Nantong University, China./ ; }, mesh = {*Forkhead Box Protein M1/genetics/metabolism ; Female ; *Ovarian Neoplasms/genetics/pathology/drug therapy ; Humans ; Animals ; Cell Line, Tumor ; CRISPR-Cas Systems ; Mice ; Azepines/pharmacology ; Cell Proliferation/drug effects ; Mice, Nude ; Cell Movement/drug effects ; Gene Expression Regulation, Neoplastic/drug effects ; Disease Progression ; Xenograft Model Antitumor Assays ; Triazoles/pharmacology ; }, abstract = {Ovarian cancer is the seventh most common lethal tumor among women in the world. FOXM1 is a transcription factor implicated in the initiation and progression of ovarian cancer by regulating key oncogenic genes. The role of regulatory regions in regulating the expression of FOXM1 in ovarian cancer is not completely clarified. Treatment with bromodomain and extraterminal (BET) inhibitors JQ-1 and I-BET were explored in ovarian cancer cell lines (OVCAR3, A2780, or SKOV3) to evaluate FOXM1 expression and biological behavior by qPCR, CCK8 assay, colony formation assay, wound-healing, and transwell assays. The regulatory regions (enhancer sequence spanning promoter or exon 1) of FOXM1 were deleted using CRISPR-Cas9 in the OVCAR3 cell line. FOXM1 expression and tumor biological behavior were further assessed in FOXM1 regulatory regions deleted OVCAR3 cell line. The mouse xenograft model was assessed at the indicated time points following subcutaneous injection of enhancer-deleted cells. Treatment with the JQ-1 and I-BET reduced the expression of FOXM1, decreasing cell proliferation, migration, and invasion in a panel of ovarian cancer cell lines including OVCAR3, A2780, and SKOV3 cells. By mining the published ChIP-sequencing data (H3K27Ac) from 12 ovarian cancer cell lines, we identified a potential enhancer and promoter region. Deletion of the spanning enhancer and promoter region of FOXM1 reduced mRNA and protein expression. Similarly, cell proliferation, migration, invasion, and tumorigenesis in both cells and mouse xenograft models were significantly attenuated. Our study demonstrates that JQ-1 and I-BET can regulate the expression of the FOXM1 gene-relating network. These data also indicate that disruption of the span enhancer and promoter region activity of FOXM1 has a vital role in the anti-ovarian cancer effect, hiding a potential opportunity for the evaluation of this non-coding DNA deletion disrupts the FOXM1 transcriptional network in ovarian cancer development.}, }
@article {pmid39829104, year = {2025}, author = {Yang, L and Zhang, J and Zhang, J and Hou, T and Gao, Q and Liu, X and Li, F}, title = {CRISPR-Based Homogeneous Electrochemical Strategy for Near-Zero Background Detection of Breast Cancer Extracellular Vesicles via Fluidity-Enhanced Magnetic Capture Nanoprobe.}, journal = {Analytical chemistry}, volume = {97}, number = {4}, pages = {2176-2185}, doi = {10.1021/acs.analchem.4c05181}, pmid = {39829104}, issn = {1520-6882}, mesh = {Humans ; *Breast Neoplasms/diagnosis/pathology ; *Extracellular Vesicles/chemistry/metabolism ; *Electrochemical Techniques/methods ; Female ; *CRISPR-Cas Systems ; Biomarkers, Tumor/analysis ; Biosensing Techniques/methods ; }, abstract = {Precise identification and analysis of multiple protein biomarkers on the surface of breast cancer cell-derived extracellular vesicles (BC-EVs) are of great significance for noninvasive diagnosis of the breast cancer subtypes, but it remains a major challenge owing to their high heterogeneity and low abundance. Herein, we established a CRISPR-based homogeneous electrochemical strategy for near-zero background and ultrasensitive detection of BC-EVs. To realize the high-performance capture and isolation of BC-EVs, fluidity-enhanced magnetic nanoprobes were facilely prepared. After capturing BC-EVs, the AND logic gate-based catalytic hairpin assembly (CHA) and the trans-cleavage activity of CRISPR-Cas12a against the magnetic signal nanoprobes were triggered successively, generating a significant electrochemical signal. Notably, the as-developed metal-mediated magnetic signal nanoprobes could efficiently decrease the background signal by magnetic separation, endowing the method with a high signal-to-noise ratio. Consequently, by ingeniously integrating DNA logic gate-based CRISPR-CHA signal amplification with dual magnetic nanoprobes in a homogeneous electrochemical strategy, precise identification and ultrasensitive detection of BC-EVs was successfully achieved through simultaneous and specific recognition of dual protein markers on the BC-EVs surface. More importantly, this approach could effectively discriminate specific subgroups of BC-EVs in clinical serum samples, which may provide great opportunities for the accurate diagnosis and prognosis evaluation of breast cancer in a noninvasive manner.}, }
@article {pmid39828694, year = {2025}, author = {Matthews, MC and van der Linden, J and Robène, I and Rozsasi, S and Coetzee, B and Campa, M and Burger, J and Akwuruoha, UN and Madufor, NJ and Perold, W and Opara, UL and Viljoen, A and Mostert, D}, title = {A combined recombinase polymerase amplification CRISPR/Cas12a assay for detection of Fusarium oxysporum f. sp. cubense tropical race 4.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {2436}, pmid = {39828694}, issn = {2045-2322}, support = {138109//National Research Foundation/ ; 890856//Horizon 2020 Framework Programme/ ; }, mesh = {*Fusarium/genetics/isolation &amp; purification ; Musa/microbiology ; *CRISPR-Cas Systems ; *Plant Diseases/microbiology ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism/genetics ; Sensitivity and Specificity ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {The soilborne pathogen Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) is currently devastating banana production worldwide. Once introduced, it is not possible to eradicate the pathogen from soils where it can survive for decades. The only management option available then is to replace Foc TR4-susceptible with -resistant varieties. Timely detection of the pathogen, however, is an important strategy to prevent the introduction of Foc TR4 into new areas and prevent its spread from infested sites. In this study, a single-tube detection technique was developed by combining recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a technology (RPA-Cas12a) for detection of Foc TR4. The RPA-Cas12a assay was conducted isothermally, had a sensitivity of up to 10 fg target DNA and did not cross react with any of the 76 non-target isolates included in the specificity testing. The RPA-Cas12a assay detected Foc TR4 from naturally infected banana samples collected in the field and visualization was possible with the naked eye under LED blue light transillumination. The method can be integrated with inexpensive fluorescent or electronic detection devices to accelerate Foc TR4 in-field detection and, thereby, fast-track disease containment strategies.}, }
@article {pmid39827371, year = {2025}, author = {Almotiri, A and Abogosh, A and Abdelfattah, A and Alowaisy, D and Rodrigues, NP}, title = {Treating genetic blood disorders in the era of CRISPR-mediated genome editing.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {33}, number = {6}, pages = {2645-2662}, pmid = {39827371}, issn = {1525-0024}, mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; *Hematologic Diseases/therapy/genetics ; Animals ; Hemophilia A/genetics/therapy ; Mutation ; Hemoglobinopathies/genetics/therapy ; }, abstract = {In the setting of monogenic disease, advances made in genome editing technologies can, in principle, be deployed as a therapeutic strategy to precisely correct a specific gene mutation in an affected cell type and restore functionality. Using the β-hemoglobinopathies and hemophilia as exemplars, we review recent experimental breakthroughs using CRISPR-derived genome editing technology that have translated to significant improvements in the management of inherited hematologic disorders. Yet there are also challenges facing the use of CRISPR-mediated genome editing in these patients; we discuss possible ways to obviate those issues for furtherance of clinical benefit.}, }
@article {pmid39827180, year = {2025}, author = {Van Goethem, MW and Bezuidt, OKI and Pierneef, R and Vikram, S and Hopkins, DW and Aspray, T and Hall, G and Woodborne, S and Hogg, ID and Northen, TR and Kong, W and Daffonchio, D and Cowan, DA and Van de Peer, Y and Delgado-Baquerizo, M and Makhalanyane, TP}, title = {Novel adaptive immune systems in pristine Antarctic soils.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {2368}, pmid = {39827180}, issn = {2045-2322}, support = {118981//National Research Foundation/ ; 118981//National Research Foundation/ ; 118981//National Research Foundation/ ; 118981//National Research Foundation/ ; 118981//National Research Foundation/ ; 118981//National Research Foundation/ ; 110717//South African National Antarctic Programme/ ; 110717//South African National Antarctic Programme/ ; 110717//South African National Antarctic Programme/ ; 110717//South African National Antarctic Programme/ ; 110717//South African National Antarctic Programme/ ; 110717//South African National Antarctic Programme/ ; }, mesh = {*Soil Microbiology ; *Metagenome ; Phylogeny ; CRISPR-Cas Systems ; *Bacteria/classification/isolation &amp; purification/virology ; *Bacteriophages/classification/isolation &amp; purification ; Ice Cover/microbiology ; }, abstract = {Antarctic environments are dominated by microorganisms, which are vulnerable to viral infection. Although several studies have investigated the phylogenetic repertoire of bacteria and viruses in these poly-extreme environments with freezing temperatures, high ultra violet irradiation levels, low moisture availability and hyper-oligotrophy, the evolutionary mechanisms governing microbial immunity remain poorly understood. Using genome-resolved metagenomics, we test the hypothesis that Antarctic poly-extreme high-latitude microbiomes harbour diverse adaptive immune systems. Our analysis reveals the prevalence of prophages in bacterial genomes (Bacteroidota and Verrucomicrobiota), suggesting the significance of lysogenic infection strategies in Antarctic soils. Furthermore, we demonstrate the presence of diverse CRISPR-Cas arrays, including Class 1 arrays (Types I-B, I-C, and I-E), alongside systems exhibiting novel gene architecture among their effector cas genes. Notably, a Class 2 system featuring type V variants lacks CRISPR arrays, encodes Cas1 and Cas2 adaptation module genes. Phylogenetic analysis of Cas12 effector proteins hints at divergent evolutionary histories compared to classified type V effectors and indicates that TnpB is likely the ancestor of Cas12 nucleases. Our findings suggest substantial novelty in Antarctic cas sequences, likely driven by strong selective pressures. These results underscore the role of viral infection as a key evolutionary driver shaping polar microbiomes.}, }
@article {pmid39826761, year = {2025}, author = {Zhou, Z and Liu, S and Saleem, M and Liu, F and Hu, R and Su, H and Dong, D and Luo, Z and Wu, Y and Zhang, Y and He, Z and Wang, C}, title = {Unraveling phase-dependent variations of viral community, virus-host linkage, and functional potential during manure composting process.}, journal = {Bioresource technology}, volume = {419}, number = {}, pages = {132081}, doi = {10.1016/j.biortech.2025.132081}, pmid = {39826761}, issn = {1873-2976}, mesh = {*Composting/methods ; *Manure/microbiology/virology ; Bacteria/virology/genetics ; *Viruses/genetics ; Metagenome ; }, abstract = {The temporal dynamics of bacterial and fungal communities significantly impact the manure composting process, yet viral communities are often underexplored. Bulk metagenomes, viromes, metatranscriptomes, and metabolomes were integrated to investigate dynamics of double-stranded DNA (dsDNA) virus and virus-host interactions throughout a 63-day composting process. A total of 473 viral operational taxonomic units (vOTUs), predominantly Caudoviricetes, showed distinct phase-dependent differentiation. In phase I (initial-mesophilic), viruses targeted Gammaproteobacteria and Firmicutes, utilizing restriction-modification (RM) systems. In phase II (thermophilic-maturing), viruses infected Alphaproteobacteria, Chloroflexi, and Planctomycetes, employing CRISPR-Cas systems. Lysogenic and lytic viruses exerting differential effects on bacterial pathogens across phases. Additionally, six types of auxiliary metabolic genes (AMGs) related to galactose and cysteine metabolisms were identified. The homologous lineages of AMGs with bacterial genes, along with the significant temporal correlation observed between virus-host-metabolite interactions, underscore the critical yet often overlooked role of viral communities in modulating microbial metabolisms and pathogenesis within composting ecosystems.}, }
@article {pmid39826547, year = {2025}, author = {Hong, SH and An, SY and Park, C and Kim, Y and Kim, EH and Kim, NK and Suh, JY}, title = {Structural variants of AcrIIC5 inhibit Cas9 via divergent binding interfaces.}, journal = {Structure (London, England : 1993)}, volume = {33}, number = {3}, pages = {517-527.e5}, doi = {10.1016/j.str.2024.12.014}, pmid = {39826547}, issn = {1878-4186}, mesh = {*CRISPR-Associated Protein 9/metabolism/antagonists &amp; inhibitors/chemistry/genetics ; Protein Binding ; *Viral Proteins/chemistry/metabolism/genetics ; *Bacteriophages/metabolism/genetics/chemistry ; CRISPR-Cas Systems ; Models, Molecular ; Binding Sites ; Protein Folding ; }, abstract = {CRISPR-Cas is a bacterial defense system that employs RNA-guided endonucleases to destroy invading foreign nucleic acids. Bacteriophages produce anti-CRISPR (Acr) proteins to evade CRISPR-Cas defense during the infection. AcrIIC5, a type II-C Cas9 inhibitor, exhibits unusual variations in the local backbone fold between its orthologs. Here we investigated how the folding variations affect the inhibition of target Cas9 using AcrIIC5 orthologs. Structural comparison of free AcrIIC5Smu and AcrIIC5Nch confirmed that the folding variation correlated with characteristic indels in the helical region. Mutagenesis and biochemical assays combined with AlphaFold2 predictions identified key residues of AcrIIC5 orthologs important for Cas9 inhibition. Remarkably, AcrIIC5 orthologs employed divergent binding interfaces via folding variations to inhibit the Cas9 targets. Our study suggests that Acr proteins have evolved structural variants to diversify key interfaces for target Cas9, which could be beneficial for the adaptation of phages to evasive mutations on the Cas9 surface.}, }
@article {pmid39826349, year = {2025}, author = {Ranpura, GN and Holliday, M and Li, S and Ross, SB and Singer, ES and Fraser, ST and Bagnall, RD and Semsarian, C and Lim, S}, title = {Generation of an isogenic CRISPR/Cas9-corrected control induced pluripotent stem cell line from a patient with autosomal dominant catecholaminergic polymorphic ventricular tachycardia with a heterozygous variant in cardiac calsequestrin-2.}, journal = {Stem cell research}, volume = {83}, number = {}, pages = {103650}, doi = {10.1016/j.scr.2024.103650}, pmid = {39826349}, issn = {1876-7753}, mesh = {Female ; Humans ; Male ; *Calsequestrin/genetics/metabolism ; Cell Line ; *CRISPR-Cas Systems/genetics ; Heterozygote ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Myocytes, Cardiac/metabolism ; *Polymorphic Catecholaminergic Ventricular Tachycardia/genetics/metabolism/pathology ; }, abstract = {Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac disease characterised by adrenergic-induced arrhythmias. The leading causes of CPVT are pathogenic variants in cardiac ryanodine receptor 2 (RYR2) and rarely, in cardiac calsequestrin-2 (CASQ2) genes, which are major components of Ca[2+] handling in cardiac myocytes. This resource builds upon an established induced pluripotent stem cell line generated from a family with autosomal dominant CPVT due to a heterozygous variant in CASQ2 c.539A > G, p.Lys180Arg (CIAUi003-A) (Ross et al., 2019). The current iPSC line was genetically modified using CRISPR/Cas9 to correct the pathogenic c.539A > G variant creating a CRISPR-corrected isogenic control line (CIAUi003-A-1).}, }
@article {pmid39826273, year = {2025}, author = {Pal, T and Liu, Z and Chen, J}, title = {CIMNE-CRISPR: A novel amplification-free diagnostic for rapid early detection of African Swine Fever Virus.}, journal = {Biosensors &amp; bioelectronics}, volume = {273}, number = {}, pages = {117154}, pmid = {39826273}, issn = {1873-4235}, support = {R35 GM147069/GM/NIGMS NIH HHS/United States ; }, mesh = {*African Swine Fever Virus/isolation &amp; purification/genetics ; Animals ; Swine ; *Biosensing Techniques/methods ; *African Swine Fever/diagnosis/virology/blood ; Limit of Detection ; Magnetite Nanoparticles/chemistry ; CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques ; *DNA, Viral/genetics/isolation &amp; purification ; Silicon Dioxide/chemistry ; Reproducibility of Results ; }, abstract = {African Swine Fever Virus (ASFV) is a highly contagious pathogen with nearly 100% mortality in swine, causing severe global economic loss. Current detection methods rely on nucleic acid amplification, which requires specialized equipment and skilled operators, limiting accessibility in resource-constrained settings. To address these challenges, we developed the Covalently Immobilized Magnetic Nanoparticles Enhanced CRISPR (CIMNE-CRISPR) system. This amplification-free diagnostic system seamlessly combines target recognition, sequence-specific enrichment, and signal generation. This approach uses covalent immobilization of CRISPR-LbCas12a-crRNA complexes on Fe3O4@SiO2 core-shell magnetic nanoparticles, which improves enzyme specificity and robustness over traditional adsorption. The CIMNE-CRISPR assay reached a limit of detection (LOD) of 8.1 × 10[4] copies/μL and a limit of quantification (LOQ) of 4.2 × 10[5] copies/μL, with a dynamic range spanning 10[5] to 10[10] copies/μL and a matrix factor of 100.29% in porcine plasma. It maintained great specificity and accurately detecting 10[5] copies/μL of ASFV DNA even with high mutant concentrations (10[13] copies/μL). The method demonstrated decent reproducibility across different nanoparticle synthesis batches, with an RSD of 9.63% and recovery rates between 97% and 103%, and features rapid processing well-suited for field diagnostics. Overall, this system's cost-effectiveness, simplicity, and reliability highlight its potential to pave the way for advanced CRISPR-based diagnostics, particularly for diverse viral and bacterial targets in agricultural, environmental, and zoonotic disease contexts.}, }
@article {pmid39825389, year = {2025}, author = {Abraham, IC and Aboje, JE and Ukoaka, BM and Tom-Ayegunle, K and Amjad, M and Abdulkader, A and Agbo, CE and Akinruli, OA and Akisanmi, TR and Oyetola, EO and Olatunji, G and Kokori, E and Aderinto, N}, title = {Integrating malaria vaccine and CRISPR/Cas9 gene drive: a comprehensive strategy for accelerated malaria eradication.}, journal = {Malaria journal}, volume = {24}, number = {1}, pages = {17}, pmid = {39825389}, issn = {1475-2875}, mesh = {*Malaria Vaccines/immunology/administration &amp; dosage ; *CRISPR-Cas Systems ; *Malaria/prevention &amp; control ; *Disease Eradication/methods ; *Gene Drive Technology/methods ; Humans ; Animals ; Anopheles/genetics ; Mosquito Control/methods ; }, abstract = {Malaria remains a significant public health challenge, particularly in low- and middle-income countries, despite ongoing efforts to eradicate the disease. Recent advancements, including the rollout of malaria vaccines, such as RTS,S/AS01 and R21/Matrix-M™, offer new avenues for prevention. However, the rise of resistance to anti-malarial medications necessitates innovative strategies. This review explores the potential integration of CRISPR/Cas9 gene drive technology with malaria vaccination efforts to enhance vector control and reduce transmission. By employing gene drive mechanisms for population suppression and replacement of malaria-transmitting Anopheles mosquitoes, combined with the immunogenic properties of vaccines, a synergistic approach can be established. This paper discussed the need for integrated strategies to address the biological complexities of malaria and socio-economic factors influencing its prevalence. Challenges such as regulatory hurdles, community acceptance, ecological impacts, and sustainable funding are examined, alongside strategies for implementation within existing malaria control programmes. This integrated approach could significantly contribute to achieving the World Health Organization's targets for malaria reduction by 2030, ultimately enhancing public health outcomes and supporting broader socio-economic development.}, }
@article {pmid39825209, year = {2025}, author = {Jin, Q and Feng, X and Hong, M and Wang, K and Chen, X and Cheng, J and Kuang, Y and Si, X and Xu, M and Huang, X and Guang, S and Zhu, C}, title = {Peri-centrosomal localization of small interfering RNAs in C. elegans.}, journal = {Science China. Life sciences}, volume = {68}, number = {4}, pages = {895-911}, pmid = {39825209}, issn = {1869-1889}, mesh = {Animals ; *Caenorhabditis elegans/genetics/metabolism ; Caenorhabditis elegans Proteins/metabolism/genetics ; *Centrosome/metabolism ; *RNA, Small Interfering/metabolism/genetics ; RNA Interference ; RNA-Dependent RNA Polymerase/metabolism/genetics ; Argonaute Proteins/metabolism/genetics ; CRISPR-Cas Systems ; RNA-Binding Proteins ; }, abstract = {The centrosome is the microtubule-organizing center and a crucial part of cell division. Centrosomal RNAs (cnRNAs) have been reported to enable precise spatiotemporal control of gene expression during cell division in many species. Whether and how cnRNAs exist in C. elegans are unclear. Here, using the nuclear RNAi Argonaute protein NRDE-3 as a reporter, we observed potential peri-centrosome localized small interfering (si)RNAs in C. elegans. NRDE-3 was previously shown to associate with pre-mRNAs and pre-rRNAs via a process involving the presence of complementary siRNAs. We generated a GFP-NRDE-3 knock-in transgene through CRISPR/Cas9 technology and observed that NRDE-3 formed peri-centrosomal foci neighboring the tubulin protein TBB-2, other centriole proteins and pericentriolar material (PCM) components in C. elegans embryos. The peri-centrosomal accumulation of NRDE-3 depends on RNA-dependent RNA polymerase (RdRP)-synthesized 22G siRNAs and the PAZ domain of NRDE-3, which is essential for siRNA binding. Mutation of eri-1, ergo-1, or drh-3 significantly increased the percentage of pericentrosome-enriched NRDE-3. At the metaphase of the cell cycle, NRDE-3 was enriched in both the peri-centrosomal region and the spindle. Moreover, the integrity of centriole proteins and pericentriolar material (PCM) components is also required for the peri-centrosomal accumulation of NRDE-3. Therefore, we concluded that siRNAs could accumulate in the pericentrosomal region in C. elegans and suggested that the peri-centrosomal region may also be a platform for RNAi-mediated gene regulation.}, }
@article {pmid39824639, year = {2025}, author = {Pollitt, SL and Levy, AD and Anderson, MC and Blanpied, TA}, title = {Large Donor CRISPR for Whole-Coding Sequence Replacement of Cell Adhesion Molecule LRRTM2.}, journal = {The Journal of neuroscience : the official journal of the Society for Neuroscience}, volume = {45}, number = {7}, pages = {}, pmid = {39824639}, issn = {1529-2401}, support = {R01 MH119826/MH/NIMH NIH HHS/United States ; }, mesh = {Animals ; Rats ; Male ; Female ; Hippocampus/metabolism/cytology ; *Membrane Proteins/genetics/metabolism ; *Nerve Tissue Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Synapses/metabolism ; Receptors, AMPA/metabolism ; Cells, Cultured ; *Gene Editing/methods ; Neurons/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Disks Large Homolog 4 Protein/metabolism ; Gene Knock-In Techniques/methods ; Rats, Sprague-Dawley ; }, abstract = {The cell adhesion molecule leucine-rich repeat transmembrane neuronal protein 2 (LRRTM2) is crucial for synapse development and function. However, our understanding of its endogenous trafficking has been limited due to difficulties in manipulating its coding sequence (CDS) using standard genome editing techniques. Instead, we replaced the entire LRRTM2 CDS by adapting a two-guide CRISPR knock-in method, enabling complete control of LRRTM2. In primary rat hippocampal cultures dissociated from embryos of both sexes, N-terminally tagged, endogenous LRRTM2 was found in 80% of synapses, and synaptic LRRTM2 content correlated with PSD-95 and AMPAR levels. LRRTM2 was also enriched with AMPARs outside synapses, demonstrating the sensitivity of this method to detect relevant new biology. Finally, we leveraged total genomic control to increase the synaptic levels of LRRTM2 via simultaneous mutation of its C-terminal domain, which did not correspondingly increase AMPAR enrichment. The coding region of thousands of genes span lengths suitable for whole-CDS replacement, suggesting this simple approach will enable straightforward structure-function analysis in neurons.}, }
@article {pmid39824586, year = {2025}, author = {Arivarasan, VK and Diwakar, D and Kamarudheen, N and Loganathan, K}, title = {Current approaches in CRISPR-Cas systems for diabetes.}, journal = {Progress in molecular biology and translational science}, volume = {210}, number = {}, pages = {95-125}, doi = {10.1016/bs.pmbts.2024.08.002}, pmid = {39824586}, issn = {1878-0814}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Diabetes Mellitus/genetics/therapy ; Animals ; Gene Editing ; Genetic Therapy ; }, abstract = {In the face of advancements in health care and a shift towards healthy lifestyle, diabetes mellitus (DM) still presents as a global health challenge. This chapter explores recent advancements in the areas of genetic and molecular underpinnings of DM, addressing the revolutionary potential of CRISPR-based genome editing technologies. We delve into the multifaceted relationship between genes and molecular pathways contributing to both type1 and type 2 diabetes. We highlight the importance of how improved genetic screening and the identification of susceptibility genes are aiding in early diagnosis and risk stratification. The spotlight then shifts to CRISPR-Cas9, a robust genome editing tool capable of various applications including correcting mutations in type 1 diabetes, enhancing insulin production in T2D, modulating genes associated with metabolism of glucose and insulin sensitivity. Delivery methods for CRISPR to targeted tissues and cells are explored, including viral and non-viral vectors, alongside the exciting possibilities offered by nanocarriers. We conclude by discussing the challenges and ethical considerations surrounding CRISPR-based therapies for DM. These include potential off-target effects, ensuring long-term efficacy and safety, and navigating the ethical implications of human genome modification. This chapter offers a comprehensive perspective on how genetic and molecular insights, coupled with the transformative power of CRISPR, are paving the way for potential cures and novel therapeutic approaches for DM.}, }
@article {pmid39824585, year = {2025}, author = {Munshi, ID and Acharya, M and Mukherjee, S and Mani, I}, title = {Recent development in CRISPR-Cas systems for cardiac disease.}, journal = {Progress in molecular biology and translational science}, volume = {210}, number = {}, pages = {47-93}, doi = {10.1016/bs.pmbts.2024.08.004}, pmid = {39824585}, issn = {1878-0814}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Heart Diseases/genetics/therapy ; Gene Editing ; Genetic Therapy ; }, abstract = {The CRISPR-Cas system has emerged as a revolutionary tool in genetic research, enabling highly precise gene editing and significantly advancing the field of cardiovascular science. This chapter provides a comprehensive overview of the latest developments in utilizing CRISPR-Cas technologies to investigate and treat heart diseases. It delves into the application of CRISPR-Cas9 for creating accurate models of complex cardiac conditions, such as hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and various arrhythmias, which are essential for understanding disease mechanisms and testing potential therapies. The therapeutic potential of gene editing is also explored, with a focus on genes like PCSK9 and ANGPTL3 that play critical roles in lipid metabolism and cardiovascular health, offering promising avenues for new treatments. Furthermore, the expanding applications of CRISPR in heart tissue regeneration are examined, which could revolutionize the repair of damaged heart tissue. Cutting-edge techniques such as base editing and prime editing are discussed, highlighting their potential to further refine genetic interventions. The discussion concludes by addressing the challenges associated with delivering CRISPR components efficiently and safely, while also exploring recent innovations that may overcome these hurdles, providing insights into the future directions of CRISPR technology in cardiovascular medicine.}, }
@article {pmid39824584, year = {2025}, author = {Khoshandam, M and Soltaninejad, H and Bhia, I and Goudarzi, MTH and Hosseinkhani, S}, title = {CRISPR challenges in clinical developments.}, journal = {Progress in molecular biology and translational science}, volume = {210}, number = {}, pages = {263-279}, doi = {10.1016/bs.pmbts.2024.08.001}, pmid = {39824584}, issn = {1878-0814}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Neoplasms/therapy/genetics ; }, abstract = {CRISPR-Cas (clustered regularly interspaced short palindromic repeats and associated proteins) is a novel genome editing technology with potential applications in treating diseases. Currently, its use in humans is restricted to clinical trials, although its growth rate is significant, and some have received initial FDA approval. It is crucial to examine and address the challenges for this technology to be implemented in clinical settings. This review aims to identify and explore new research ideas to increase of CRISPR's efficiency in treating genetic diseases and cancer, as well as its future prospects. Given that a substantial amount of previous research has focused on CRISPR-Cas delivery strategies and materials, this overview introduces specific conditions and strategies. It also discusses some of the challenges and opportunities in this field, offering a unique perspective.}, }
@article {pmid39824583, year = {2025}, author = {Mishra, S and Banerjee, S and Tiwari, BS and Tiwari, AK}, title = {Recent progress in CRISPR-Cas-system for neurological disorders.}, journal = {Progress in molecular biology and translational science}, volume = {210}, number = {}, pages = {231-261}, doi = {10.1016/bs.pmbts.2024.07.017}, pmid = {39824583}, issn = {1878-0814}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Nervous System Diseases/therapy/genetics ; Animals ; Gene Editing ; Genetic Therapy ; }, abstract = {Different neurological diseases including, Parkinson's, Alzheimer's, and Huntington's diseases extant momentous global disease burdens, affecting millions of lives for imposing a heavy disease burden on the healthcare systems. Despite various treatment strategies aimed at alleviating symptoms, treatments remain elusive and ineffective due to the disease's complexity. However, recent advancements in gene therapy via the CRISPR-Cas system offer ground-breaking and targeted treatment options. Based on a bacterial immune mechanism, the CRISPR-Cas system enables precise genome editing, allowing for the alteration of different genetic mutations and the possible cure of genetic diseases. In the context of neurological disorders, the CRISPR-Cas system shows a promising avenue by allowing researchers to conduct genome-editing which is implicated in neurodegenerative disease therapeutics. This book chapter provides an updated overview of the application of the CRISPR-Cas system for addressing target-specific therapeutic approaches for neurodegenerative disorders. Furthermore, we discuss the principles of the CRISPR-Cas mechanism, its role in modeling neurological disorders, identifying molecular targets, and developing gene-based therapies. Additionally, the chapter explores the recent clinical trials and CRISPR-Cas-mediated treatments for neurological conditions. By leveraging the accuracy and versatility of the CRISPR-Cas system, scientists can more effectively handle the genetic underpinnings of neurodegenerative diseases. Furthermore, the chapter extends the critical viewpoints on ethical considerations and technical limitations related to the clinical deployment of this revolutionizing technique.}, }
@article {pmid39824582, year = {2025}, author = {Gowda, DAA and Birappa, G and Rajkumar, S and Ajaykumar, CB and Srikanth, B and Kim, SL and Singh, V and Jayachandran, A and Lee, J and Ramakrishna, S}, title = {Recent progress in CRISPR/Cas9 system for eye disorders.}, journal = {Progress in molecular biology and translational science}, volume = {210}, number = {}, pages = {21-46}, doi = {10.1016/bs.pmbts.2024.07.018}, pmid = {39824582}, issn = {1878-0814}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Eye Diseases/genetics/therapy ; Gene Editing ; Genetic Therapy ; }, abstract = {Ocular disorders encompass a broad spectrum of phenotypic and clinical symptoms resulting from several genetic variants and environmental factors. The unique anatomy and physiology of the eye facilitate validation of cutting-edge gene editing treatments. Genome editing developments have allowed researchers to treat a variety of diseases, including ocular disorders. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system holds considerable promise for therapeutic applications in the field of ophthalmology, including repair of aberrant genes and treatment of retinal illnesses related to the genome or epigenome. Application of CRISPR/Cas9 systems to the study of ocular disease and visual sciences have yielded innovations including correction of harmful mutations in patient-derived cells and gene modifications in several mammalian models of eye development and disease. In this study, we discuss the generation of several ocular disease models in mammalian cell lines and in vivo systems using a CRISPR/Cas9 system. We also provide an overview of current uses of CRISPR/Cas9 technologies for the treatment of ocular pathologies, as well as future challenges.}, }
@article {pmid39824581, year = {2025}, author = {Singh, S and Raj, D and Mathur, A and Mani, N and Kumar, D}, title = {Current approaches in CRISPR-Cas systems for hereditary diseases.}, journal = {Progress in molecular biology and translational science}, volume = {210}, number = {}, pages = {205-229}, doi = {10.1016/bs.pmbts.2024.07.015}, pmid = {39824581}, issn = {1878-0814}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Genetic Diseases, Inborn/genetics/therapy ; Animals ; Gene Editing ; Genetic Therapy ; }, abstract = {CRISPR-Cas technologies have drastically revolutionized genetic engineering and also dramatically changed the potential for treating inherited disorders. The potential to correct genetic mutations responsible for numerous hereditary disorders from single-gene disorders to complex polygenic diseases through precise DNA editing is feasible. The tactic now employed in CRISPR-Cas systems for treating inherited disorders is the usage of particular guide RNAs to target and edit disease-causing mutations in the patient's genome. Several methods such as CRISPR-Cas9, CRISPR-Cas12, and CRISPR-Cas13 are being thoroughly researched and optimized to increase effectiveness, accuracy, and safety in gene editing. Additionally, it is predicted that CRISPR-based therapies will be able to treat complex genetic illnesses such as cancer predisposition syndromes, neurological disorders, and cardiovascular conditions in addition to single-gene disorders. The available editing tools and creation of base editing technology facilitate the simultaneous correction of many mutations or accurate nucleotide changes leading to further advances in the development of multiplex editing tools and base editing technology fiction. When combined with other paradigms such as gene therapy using stem cell treatment, CRISPR-Cas promises improved efficacy. Patient treatment and lowering side effects significantly in individual genetic profiles will guide CRISPR-based treatments. These procedures will undoubtedly lead to therapies that are both efficient and curative of a wide range of genetic diseases, ushering in a new era of precision medicine. This chapter discusses about CRISPR Cas9 mechanism and its significance in the treatment of Hereditary disorders.}, }
@article {pmid39824580, year = {2025}, author = {Israr, J and Kumar, A}, title = {Current progress in CRISPR-Cas systems for rare diseases.}, journal = {Progress in molecular biology and translational science}, volume = {210}, number = {}, pages = {163-203}, doi = {10.1016/bs.pmbts.2024.07.019}, pmid = {39824580}, issn = {1878-0814}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Rare Diseases/genetics/therapy ; Animals ; Gene Editing ; Genetic Therapy ; }, abstract = {The groundbreaking CRISPR-Cas gene editing method permits exact genetic code alteration. The "CRISPR" DNA protects bacteria from viruses. CRISPR-Cas utilizes a guide RNA to steer the Cas enzyme to the genome's gene editing target. After attaching to a sequence, Cas enzymes cleave DNA to insert, delete, or modify genes. The influence of CRISPR-Cas technology on molecular biology and genetics is profound. It allows for gene function research, animal disease models, and patient genetic therapy. Gene editing has transformed biotechnology, agriculture, and customized medicine. CRISPR-Cas could revolutionize genetics and medicine. CRISPR-Cas may accurately correct genetic flaws that underlie rare diseases, improving their therapy. Gene mutations make CRISPR-Cas gene editing a viable cure for uncommon diseases. We can use CRISPR-Cas to correct genetic abnormalities at the molecular level. This strategy offers hope for remedies and disease understanding. CRISPR-Cas genome editing may enable more targeted and effective treatments for rare medical illnesses with few therapy options. By developing base- and prime-editing CRISPR technology, CRISPR-Cas allows for accurate and efficient genome editing and advanced DNA modification. This advanced method provides precise DNA alterations without double-strand breakage. These advances have improved gene editing safety and precision, reducing unfavorable effects. Lipid nanoparticles, which use viral vectors, improve therapeutic cell and tissue targeting. In rare disorders, gene therapy may be possible with CRISPR-Cas clinical trials. CRISPR-Cas research is improving gene editing, delivery, and rare disease treatment.}, }
@article {pmid39824579, year = {2025}, author = {Puri, B and Kulkarni, YA and Gaikwad, AB}, title = {Advances in CRISPR-Cas systems for kidney diseases.}, journal = {Progress in molecular biology and translational science}, volume = {210}, number = {}, pages = {149-162}, doi = {10.1016/bs.pmbts.2024.07.020}, pmid = {39824579}, issn = {1878-0814}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Kidney Diseases/genetics/therapy ; Animals ; Gene Editing ; }, abstract = {Recent advances in CRISPR-Cas systems have revolutionised the study and treatment of kidney diseases, including acute kidney injury (AKI), chronic kidney disease (CKD), diabetic kidney disease (DKD), lupus nephritis (LN), and polycystic kidney disease (PKD). CRISPR-Cas technology offers precise and versatile tools for genetic modification in monogenic kidney disorders such as PKD and Alport syndrome. Recent advances in CRISPR technology have also shown promise in addressing other kidney diseases like AKI, CKD, and DKD. CRISPR-Cas holds promise to edit genetic mutations underlying these conditions, potentially leading to more effective and long-lasting treatments. Furthermore, the adaptability of CRISPR-Cas systems allows for developing tailored therapeutic strategies that specifically target the genetic and molecular mechanisms contributing to different kidney diseases. Beyond DNA modifications, CRISPR-Cas technologies also enable editing noncoding RNA, such as lncRNAs and miRNAs, in kidney diseases. Despite these advancements, significant challenges persist, including delivery efficiency to specific kidney cells and potential off-target effects. However, the rapid progress in CRISPR-Cas technology suggests a transformative impact on the future management of kidney diseases, offering the potential for enhanced patient outcomes through personalised and precise therapeutic approaches. This chapter highlights the recent advancement of CRISPR-Cas systems and their potential applications in various kidney diseases.}, }
@article {pmid39824578, year = {2025}, author = {Ajaykumar, CB and Rajkumar, S and Suresh, B and Birappa, G and Gowda, DAA and Jayachandran, A and Kim, KS and Hong, SH and Ramakrishna, S}, title = {Advances in applications of the CRISPR/Cas9 system for respiratory diseases.}, journal = {Progress in molecular biology and translational science}, volume = {210}, number = {}, pages = {127-147}, doi = {10.1016/bs.pmbts.2024.07.014}, pmid = {39824578}, issn = {1878-0814}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Respiratory Tract Diseases/genetics/therapy ; Genetic Therapy ; Gene Editing ; }, abstract = {Genetic and environmental factors can have an impact on lung and respiratory disorders which are associated with severe symptoms and have high mortality rates. Many respiratory diseases are significantly influenced by genetic or epigenetic factors. Gene therapy offers a powerful approach providing therapeutic treatment for lung diseases. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) are promising gene modifying tool that can edit the genome. The utilization of CRISPR/Cas9 systems in the investigation of respiratory disorders has resulted in advancements such as the rectification of deleterious mutations in patient-derived cells and the alteration of genes in multiple mammalian lung disease models. New avenues of treatment for lung disorders have been opened up by advances in CRISPR/Cas9 research. In this chapter, we discuss the known genes and mutations that cause several common respiratory disorders such as COPD, asthma, IPF, and ARDS. We further review the current research using CRISPR/Cas9 in numerous respiratory disorders and possible therapeutic treatments.}, }
@article {pmid39824577, year = {2025}, author = {Arora, Y and Priya,  and Kumar, M and Kumar, D}, title = {Current approaches in CRISPR-Cas system for metabolic disorder.}, journal = {Progress in molecular biology and translational science}, volume = {210}, number = {}, pages = {1-19}, doi = {10.1016/bs.pmbts.2024.07.016}, pmid = {39824577}, issn = {1878-0814}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Metabolic Diseases/therapy/genetics ; Animals ; Genetic Therapy ; Gene Editing ; }, abstract = {A new era in genomic medicine has been brought by the development of CRISPR-Cas technology, which presents hitherto unheard-of possibilities for the treatment of metabolic illnesses. The treatment approaches used in CRISPR/Cas9-mediated gene therapy, emphasize distribution techniques such as viral vectors and their use in preclinical models of metabolic diseases like hypercholesterolemia, glycogen storage diseases, and phenylketonuria. The relevance of high-throughput CRISPR screens for target identification in discovering new genes and pathways associated with metabolic dysfunctions is an important aspect of the discovery of new approaches. With cutting-edge options for genetic correction and cellular regeneration, the combination of CRISPR-Cas technology with stem cell therapy has opened new avenues for the treatment of metabolic illnesses. The integration of stem cell therapy and CRISPR-Cas technology is an important advance in the treatment of metabolic diseases, which are difficult to treat because of their intricate genetic foundations. This chapter addresses the most recent developments in the application of stem cell therapy and CRISPR-Cas systems to treat a variety of metabolic disorders, providing fresh hope for effective and maybe curative therapies. This chapter examines techniques and developments that have been made recently to address a variety of metabolic disorders using CRISPR-Cas systems. Our chapter focuses on the foundational workings of CRISPR-Cas technology and its potential uses in gene editing, gene knockout, and activation/repression-based gene modification.}, }
@article {pmid39824412, year = {2025}, author = {Li, L and Li, J and Wang, S and Dong, Y}, title = {Dual-mode CRISPR/Cas12a-assisted fluorescent and lateral flow aptasensor based on a newly truncated aptamer for Fumonisin B1 detection.}, journal = {International journal of biological macromolecules}, volume = {298}, number = {}, pages = {139950}, doi = {10.1016/j.ijbiomac.2025.139950}, pmid = {39824412}, issn = {1879-0003}, mesh = {*Aptamers, Nucleotide/chemistry ; *Fumonisins/analysis ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Limit of Detection ; *CRISPR-Associated Proteins/chemistry ; *Endodeoxyribonucleases ; *Bacterial Proteins/chemistry ; }, abstract = {As a Group 2B carcinogen, accurate and efficient detection for Fumonisin B1 (FB1) is essential. The emergence of aptamers presents a viable solution to meet this demand. In this study, a truncated aptamer named Apt40 was developed, showcasing remarkable binding affinity to FB1. In recent years, the role of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR-associated 12a protein (Cas12a) in detection became increasingly significant, especially utilizing the trans-cleavage of Cas12a/CRISPR RNA (crRNA) complex. To further evaluate the applicability of the Apt40, a dual-mode CRISPR/Cas12a-assisted fluorescent and lateral flow aptasensor was constructed. Notably, the crRNA was designed to complementarily bind with Apt40 in its active binding sites, thus activating the trans-cleavage of Cas12a/crRNA to signal probes. Consequently, the fluorescent aptasensor exhibited a linear range of 10-1500 ng/mL with a Limit of Detection (LOD) of 0.802 ng/mL, while the lateral flow aptasensor showed a 200-3000 ng/mL linear range with a 9.031 ng/mL LOD. Both aptasensors provided high recoveries (95.11 %-106.63 %) in corn oil and starch samples, underscoring their precisions. We anticipate that this systematic strategy, from optimizing aptamer's performance to developing a novel dual-mode aptasensor, can provide a universal framework and valuable insights for the detection of other target molecules.}, }
@article {pmid39824180, year = {2025}, author = {Gui, L and Chen, K and Yan, J and Chen, P and Gao, WQ and Ma, B}, title = {Targeting the mevalonate pathway potentiates NUAK1 inhibition-induced immunogenic cell death and antitumor immunity.}, journal = {Cell reports. Medicine}, volume = {6}, number = {2}, pages = {101913}, pmid = {39824180}, issn = {2666-3791}, mesh = {*Mevalonic Acid/metabolism ; Humans ; Animals ; Reactive Oxygen Species/metabolism ; *Immunogenic Cell Death/drug effects ; Mice ; Cell Line, Tumor ; *Protein Serine-Threonine Kinases/antagonists &amp; inhibitors/metabolism/genetics ; Endoplasmic Reticulum Stress/drug effects ; Cholesterol/metabolism ; Simvastatin/pharmacology ; *Neoplasms/immunology/drug therapy ; CRISPR-Cas Systems ; Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology ; }, abstract = {The induction of immunogenic cell death (ICD) impedes tumor progression via both tumor cell-intrinsic and -extrinsic mechanisms, representing a robust therapeutic strategy. However, ICD-targeted therapy remains to be explored and optimized. Through kinome-wide CRISPR-Cas9 screen, NUAK family SNF1-like kinase 1 (NUAK1) is identified as a potential target. The ICD-provoking effect of NUAK1 inhibition depends on the production of reactive oxygen species (ROS), consequent to the downregulation of nuclear factor erythroid 2-related factor 2 (NRF2)-mediated antioxidant gene expression. Moreover, the mevalonate pathway/cholesterol biosynthesis, activated by spliced form of X-box binding protein 1 (XBP1s) downstream of ICD-induced endoplasmic reticulum (ER) stress, functions as a negative feedback mechanism. Targeting the mevalonate pathway with CRISPR knockout or the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) inhibitor simvastatin amplifies NUAK1 inhibition-mediated ICD and antitumor activity, while cholesterol dampens ROS and ICD, and therefore also dampens tumor suppression. The combination of NUAK1 inhibitor and statin enhances the efficacy of anti-PD-1 therapy. Collectively, our study unveils the promise of blocking the mevalonate-cholesterol pathway in conjunction with ICD-targeted immunotherapy.}, }
@article {pmid39823857, year = {2025}, author = {Fan, X and Gao, Z and Ling, D and Wang, D and Cui, Y and Du, H and Zhou, X}, title = {The dCas9/crRNA linked immunological assay (dCLISA) for sensitive, accurate, and facile drug resistance gene analysis.}, journal = {Biosensors &amp; bioelectronics}, volume = {273}, number = {}, pages = {117147}, doi = {10.1016/j.bios.2025.117147}, pmid = {39823857}, issn = {1873-4235}, mesh = {*Methicillin-Resistant Staphylococcus aureus/genetics/isolation &amp; purification/drug effects ; Humans ; *Biosensing Techniques/methods ; *Staphylococcal Infections/microbiology/diagnosis ; CRISPR-Cas Systems/genetics ; Immunoassay/methods ; *CRISPR-Associated Protein 9/genetics/chemistry ; *Drug Resistance, Bacterial/genetics ; }, abstract = {The rapid and reliable diagnosis of methicillin-resistant Staphylococcus aureus (MRSA) is essential for preventing the spread of MRSA infections and guiding therapeutic strategies. However, there is still a huge challenge in further simplifying MRSA detection procedures and improving detection selectivity to reduce false-positive results. In this study, we developed a derivative CRISPR-associated protein 9/CRISPR-derived RNA Linked Immunological Assay (dCLISA) for the sensitive and specific detection of MRSA. This technique utilizes two dCas9/crRNA complexes as specific targeting agents and employs a color reaction mediated by a hybridization chain reaction for signal output. The dCLISA method offers certain benefits compared to monoclonal antibodies in traditional immunoassays, primarily due to its capacity to selectively interact with target gene and its high sensitivity from the hybridization chain reaction process. Therefore, the minimum detectable concentration of dCLISA was 8.5 cfu/mL. Unlike traditional gene analysis approaches, target gene sequences in cell lysates can be directly detected by dCLISA within 60 min with high sensitivity without genomic material extraction. In addition, the absorbance intensity of the MRSA cell lysate was significantly higher than that of methicillin-susceptible S. aureus (MSSA) indicates the clinical application potential. This study demonstrates that the dCLISA is a simple, rapid, sensitive, and specific method, which can be directly used at the point of care to analyze drug resistance in bacteria, including MRSA. Moreover, dCLISA can be utilized for other bacteria detection by merely modifying the crRNA sequence.}, }
@article {pmid39823408, year = {2025}, author = {Robert, NM and Ferrier-Tarin, S and Tremblay, JJ}, title = {A New Leydig Cell-Exclusive Cre Line Allows Lineage Tracing of Fetal and Adult Leydig Cell Populations in the Mouse.}, journal = {Endocrinology}, volume = {166}, number = {2}, pages = {}, pmid = {39823408}, issn = {1945-7170}, support = {MOP-81387/CAPMC/CIHR/Canada ; //Fondation du CHU de Qu&#xe9;bec-Universit&#xe9; Laval/ ; }, mesh = {Animals ; *Leydig Cells/metabolism/cytology ; Male ; Mice ; *Cell Lineage/genetics ; *Integrases/genetics/metabolism ; Proteins/genetics/metabolism ; Insulin/genetics/metabolism ; Fetus/cytology ; Mice, Transgenic ; CRISPR-Cas Systems ; Female ; Testis/cytology/metabolism ; }, abstract = {Leydig cells produce hormones that are required for male development, fertility, and health. Two Leydig cell populations produce these hormones but at different times during development: fetal Leydig cells, which are active during fetal life, and adult Leydig cells, which are functional postnatally. Historically, our ability to understand the origin and function of Leydig cells has been made difficult by the lack of genetic models to exclusively target these cells. Taking advantage of the Leydig cell-exclusive expression pattern of the Insl3 gene, we used a CRISPR/Cas9 gene-editing strategy to knock-in iCre recombinase into the mouse Insl3 locus. To demonstrate the Leydig cell-exclusive nature of our iCre line, lineage-tracing experiments were performed by crossing Insl3iCre mice with a Rosa26LoxSTOPLox-TdTomato reporter. iCre activity was restricted to male offspring. TdTomato fluorescence was detected both in fetal and adult Leydig cells and colocalized with CYP17A1, a classic Leydig cell marker. Prior to birth, fluorescence was observed in fetal Leydig cells beginning at embryonic day 13.0. Fluorescence was also detected in adult Leydig cells starting at postnatal day 5 and continuing to the mature testis. Fluorescence was not detected in any other fetal or adult tissue examined, except for the unexpected finding that the adrenal cortex contains some Insl3-expressing Leydig-like cells. Our Leydig cell-exclusive iCre line therefore constitutes an invaluable new tool to study not only the origin of Leydig cells but also to target genes that have been long-proposed to be important for the development and functioning of these critical endocrine cells.}, }
@article {pmid39822055, year = {2025}, author = {Göritzer, K and Melnik, S and Schwestka, J and Arcalis, E and Drapal, M and Fraser, P and Ma, JK and Stoger, E}, title = {Enhancing quality and yield of recombinant secretory IgA antibodies in Nicotiana benthamiana by endoplasmic reticulum engineering.}, journal = {Plant biotechnology journal}, volume = {23}, number = {4}, pages = {1178-1189}, pmid = {39822055}, issn = {1467-7652}, support = {760331//Horizon 2020 Framework Programme/ ; J4583//Austrian Science Fund/ ; W1224//Austrian Science Fund/ ; 21EBTA/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; }, mesh = {*Nicotiana/genetics/metabolism ; *Endoplasmic Reticulum/metabolism/genetics ; Plants, Genetically Modified/metabolism ; Recombinant Proteins/metabolism/genetics/biosynthesis ; Plant Leaves/metabolism/genetics ; }, abstract = {The production of complex multimeric secretory immunoglobulins (SIgA) in Nicotiana benthamiana leaves is challenging, with significant reductions in complete protein assembly and consequently yield, being the most important difficulties. Expanding the physical dimensions of the ER to mimic professional antibody-secreting cells can help to increase yields and promote protein folding and assembly. Here, we expanded the ER in N. benthamiana leaves by targeting the enzyme CTP:phosphocholine cytidylyltransferase (CCT), which catalyses the rate-limiting step in the synthesis of the key membrane component phosphatidylcholine (PC). We used CRISPR/Cas to perform site-directed mutagenesis of each of the three endogenous CCT genes in N. benthamiana by introducing frame-shifting indels to remove the auto-inhibitory C-terminal domains. We generated stable homozygous lines of N. benthamiana containing different combinations of the edited genes, including plants where all three isofunctional CCT homologues were modified. Changes in ER morphology in the mutant plants were confirmed by in vivo confocal imaging and substantially increased the yields of two fully assembled SIgAs by prolonging the ER residence time and boosting chaperone accumulation. Through a combination of ER engineering with chaperone overexpression, we increased the yields of fully assembled SIgA by an order of magnitude, reaching almost 1 g/kg fresh leaf weight. This strategy removes a major roadblock to producing SIgA and will likely facilitate the production of other complex multimeric biopharmaceutical proteins in plants.}, }
@article {pmid39821833, year = {2025}, author = {Li, Y and Fu, Y and Li, Y and Zhang, R and Yang, J and Ma, H and Min, L and Zhang, X}, title = {Reversing anther thermotolerance by manipulating the cis-elements in the promoter of a high-temperature upregulated gene Casein Kinase I in upland cotton.}, journal = {Science China. Life sciences}, volume = {68}, number = {6}, pages = {1558-1569}, pmid = {39821833}, issn = {1869-1889}, mesh = {*Gossypium/genetics/physiology ; *Promoter Regions, Genetic/genetics ; *Thermotolerance/genetics ; Gene Expression Regulation, Plant ; *Flowers/genetics/physiology ; *Casein Kinase I/genetics/metabolism ; Gene Editing ; CRISPR-Cas Systems ; Plant Proteins/genetics/metabolism ; Hot Temperature ; Plants, Genetically Modified ; Alleles ; Up-Regulation ; Genotype ; Phenotype ; }, abstract = {High temperature (HT) stress causes male sterility, leading to reduced upland cotton yield. Previously, we identified a key gene, Casein Kinase I (GhCKI), that negatively regulates male fertility in upland cotton under HT. However, conventional genetic manipulations of GhCKI would result in male sterility, hindering its utilization in breeding programs. Here, we engineered quantitative variation for anther thermotolerance-related traits in upland cotton by creating weak promoter alleles of GhCKI genes, using CRISPR/Cas9 and CRISPR/Cpf1 genome editing. Then, we screened and identified two new upland cotton plant lines exhibiting a HT-tolerant phenotype with edited GhCKI promoters, and characterized their corresponding heat-tolerant allelic genotypes. Further research revealed that the primary reason for the HT tolerance of the GhCKI promoter editing mutants is that the trans-acting factors GhMYB73 and GhMYB4, which positively regulate GhCKI expression under HT, failed to bind and activate the expression of GhCKI. Overall, our study not only provides a rapid strategy to generate new beneficial alleles but also offers novel germplasm resources and molecular insights for crop HT tolerance breeding.}, }
@article {pmid39821831, year = {2025}, author = {Xie, Y and Liu, X and Wu, T and Luo, Y}, title = {Harnessing the Streptomyces-originating type I-E CRISPR/Cas system for efficient genome editing in Streptomyces.}, journal = {Science China. Life sciences}, volume = {68}, number = {4}, pages = {1174-1182}, pmid = {39821831}, issn = {1869-1889}, mesh = {*Streptomyces/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genome, Bacterial ; Plasmids/genetics ; Multigene Family ; }, abstract = {Since their discovery, CRISPR/Cas systems have significantly expanded the genetic toolbox, aiding in the exploration and enhanced production of natural products across various microbes. Among these, class 2 CRISPR/Cas systems are simpler and more broadly used, but they frequently fail to function effectively in many Streptomyces strains. In this study, we present an engineered class 1 type I CRISPR/Cas system derived from Streptomyces avermitilis, which enables efficient gene editing in phylogenetically distant Streptomyces strains. Through a plasmid interference assay, we identified the effective protospacer adjacent motif as 5'-AAN-3'. Utilizing this system, we achieved targeted chromosomal deletions ranging from 8 bp to 100 kb, with efficiencies exceeding 92%. We further utilized this system to insert DNA fragments into different Streptomyces genomes, facilitating the heterologous expression of exogenous genes and the activation of endogenous natural product biosynthetic gene clusters. Overall, we established a type I CRISPR/Cas-based gene-editing methodology that significantly advances the exploration of Streptomyces, known for their rich natural product resources. This is the first report of a gene editing tool developed based on the endogenous class 1 type I CRISPR/Cas system in Streptomyces spp. Our work enriches the Streptomyces gene manipulation toolbox and advances the discovery of valuable natural products within these organisms.}, }
@article {pmid39821024, year = {2025}, author = {Bu, W and Li, Y}, title = {Rat Models of Breast Cancer.}, journal = {Advances in experimental medicine and biology}, volume = {1464}, number = {}, pages = {123-148}, pmid = {39821024}, issn = {0065-2598}, mesh = {Animals ; Female ; Rats ; *Disease Models, Animal ; Humans ; *Mammary Neoplasms, Experimental/pathology/genetics/metabolism ; *Breast Neoplasms/pathology/genetics ; CRISPR-Cas Systems ; Mammary Glands, Animal/pathology/metabolism ; Mice ; }, abstract = {As the first mammal to be domesticated for research purposes, rats served as the primary animal model for various branches of biomedical research, including breast cancer studies, up until the late 1990s and early 2000s. During this time, genetic engineering of mice, but not rats, became routine, and mice gradually supplanted rats as the preferred rodent model. But recent advances in creating genetically engineered rat models, especially with the assistance of CRISPR/Cas9 technology, have rekindled the significance of rats as a critical model in exploring various facets of breast cancer research. This is particularly pronounced in the study of the formation and progression of the estrogen receptor-positive subtype, which remains challenging to model in mice. In this chapter, we embark on a historical journey through the evolution of rat models in biomedical research and provide an overview of the general and histological characteristics of rat mammary glands. Next, we critically review major rat models for breast cancer research, including those induced by carcinogens, hormones, radiation, germline transgenes, germline knockouts, and intraductal injection of retrovirus/lentivirus to deliver oncogenic drivers into mature mammary glands. We also discuss the advances in building rat models using somatic genome editing powered by CRISPR/Cas9. This chapter concludes with our forward-looking perspective on future applications of advanced rat models in critical areas of breast cancer research that have continued to challenge the mouse model community.}, }
@article {pmid39820712, year = {2025}, author = {Tian, G and Barragan, GA and Yu, H and Martinez-Amador, C and Adaikkalavan, A and Rios, X and Guo, L and Drabek, JM and Pardias, O and Xu, X and Montalbano, A and Zhang, C and Li, Y and Courtney, AN and Di Pierro, EJ and Metelitsa, LS}, title = {PRDM1 Is a Key Regulator of the NKT-cell Central Memory Program and Effector Function.}, journal = {Cancer immunology research}, volume = {13}, number = {4}, pages = {577-590}, pmid = {39820712}, issn = {2326-6074}, support = {//Kate Amato Foundation/ ; K12 CA090433/CA/NCI NIH HHS/United States ; RP210027//Cancer Prevention and Research Institute of Texas (CPRIT)/ ; 983124//Hyundai Hope On Wheels (Hope On Wheels)/ ; R01 CA262250/CA/NCI NIH HHS/United States ; RO1 CA262250//National Institutes of Health (NIH)/ ; }, mesh = {*Positive Regulatory Domain I-Binding Factor 1/genetics/metabolism ; Animals ; Humans ; *Natural Killer T-Cells/immunology/metabolism ; Mice ; *Immunologic Memory ; Cell Line, Tumor ; Immunotherapy, Adoptive/methods ; CRISPR-Cas Systems ; Xenograft Model Antitumor Assays ; Receptors, Chimeric Antigen/immunology/genetics/metabolism ; *Neuroblastoma/immunology/therapy ; }, abstract = {Natural killer T cells (NKTs) are a promising platform for cancer immunotherapy, but few genes involved in the regulation of NKT therapeutic activity have been identified. To find regulators of NKT functional fitness, we developed a CRISPR/Cas9-based mutagenesis screen that uses a guide RNA (gRNA) library targeting 1,118 immune-related genes. Unmodified NKTs and NKTs expressing a GD2-specific chimeric antigen receptor (GD2.CAR) were transduced with the gRNA library and exposed to CD1d+ leukemia or CD1d-GD2+ neuroblastoma cells, respectively, over six challenge cycles in vitro. Quantification of gRNA abundance revealed enrichment of PRDM1-specific gRNAs in both NKTs and GD2.CAR NKTs, a result that was validated through targeted PRDM1 knockout. Transcriptional, phenotypic, and functional analyses demonstrated that CAR NKTs with PRDM1 knockout underwent central memory-like differentiation and resisted exhaustion. However, these cells downregulated the cytotoxic mediator granzyme B and showed reduced in vitro cytotoxicity and only moderate in vivo antitumor activity in a xenogeneic neuroblastoma model. In contrast, short hairpin RNA-mediated PRDM1 knockdown preserved effector function while promoting central memory differentiation, resulting in GD2.CAR NKTs with potent in vivo antitumor activity. Thus, we have identified PRDM1 as a regulator of NKT memory differentiation and effector function that can be exploited to improve the efficacy of NKT-based cancer immunotherapies.}, }
@article {pmid39820420, year = {2025}, author = {Dong, Y and Zhu, J and Pan, N}, title = {Recent advances in rapid detection of Helicobacter pylori by lateral flow assay.}, journal = {Archives of microbiology}, volume = {207}, number = {2}, pages = {35}, pmid = {39820420}, issn = {1432-072X}, mesh = {*Helicobacter pylori/isolation &amp; purification/genetics/immunology ; Humans ; *Helicobacter Infections/diagnosis/microbiology ; Sensitivity and Specificity ; Point-of-Care Systems ; Enzyme-Linked Immunosorbent Assay ; Nucleic Acid Amplification Techniques/methods ; Molecular Diagnostic Techniques/methods ; }, abstract = {Infection with H. pylori (Helicobacter pylori) is the most prevalent human infection worldwide and is strongly associated with many gastrointestinal disorders, including gastric cancer. Endoscopy is mainly used to diagnose H. pylori infection in gastric biopsies. However, this approach is invasive, time-consuming and expensive. On the other hand, serology-based methods can be considered as a non-invasive approach to detecting H. pylori infection. The LFA (lateral flow assay) serves as a rapid point-of-care diagnostic tool. This paper-based platform facilitates the detection and quantification of analytes within human fluids such as blood, serum and urine. Due to ease of production, rapid results, and low costs, LFAs have a wide application in clinical laboratories and hospitals. In this comprehensive review, we examined LFA-based approaches for detection of H. pylori infection from human fluids and compare them with other high-sensitivity methods like ELISA (Enzyme-linked immunosorbent assay). Furthermore, we reviewed methods to elevate LFA sensitivity during H. pylori infection including, CRISPR/Cas system and isothermal amplification approaches. The development and optimization of novel labeling agents such as nanozyme to enhance the performance of LFA devices in detecting H. pylori were reviewed. These innovations aim to improve signal amplification and stability, thereby increasing the diagnostic accuracy of LFA devices. A combination of advances in LFA technology and molecular insight could significantly improve diagnostic accuracy, resulting in a significant improvement in clinical and remote diagnostic accuracy.}, }
@article {pmid39819332, year = {2025}, author = {Wang, C and Zhou, Y and Wang, Y and Jiao, P and Liu, S and Guan, S and Ma, Y}, title = {CRISPR-Cas9-mediated editing of ZmPL1 gene improves tolerance to drought stress in maize.}, journal = {GM crops &amp; food}, volume = {16}, number = {1}, pages = {1-16}, doi = {10.1080/21645698.2024.2448869}, pmid = {39819332}, issn = {2164-5701}, mesh = {*Zea mays/genetics/physiology ; Droughts ; Plants, Genetically Modified/genetics/physiology ; *Plant Proteins/genetics/metabolism ; *Gene Editing ; *CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; Stress, Physiological/genetics ; }, abstract = {Maize (Zea mays L.) is a widely grown food crop around the world. Drought stress seriously affects the growth and development process of plants and causes serious damage to maize yield. In the early stage, our research group conducted transcriptome sequencing analysis on the drought-resistant maize inbred line H8186 and screened out a gene with significantly down-regulated expression, Phylloplanin-like (ZmPL1). The ZmPL1 gee expression pattern was analyzed under various abiotic stresses, and the results showed that this gene was greatly affected by drought stress. Subcellular localization analysis showed that the protein was localized on the cell membrane. In order to verify the role of ZmPL1 in drought stress, we overexpressed ZmPL1 in yeast and found that the expression of ZmPL1 could significantly increase the drought sensitivity of yeast. Next, ZmPL1 transgenic plants were obtained by infecting maize callus using Agrobacterium-mediated method. Under drought stress, compared with overexpression lines, gene-edited lines had higher germination rate and seedling survival rate, lower accumulation of MDA, relative conductivity and ROS, higher antioxidant enzyme activity, and the expression levels of stress-related genes and ROS scavenging-related genes were significantly increased. Exogenous application of ABA to each lines under drought stress attenuated the damage caused by drought stress on ZmPL overexpressing plants. In summary, ZmPL1 negatively regulates drought tolerance in maize.}, }
@article {pmid39818188, year = {2025}, author = {Liu, Z and Liu, Y and Zhang, S and Wen, Y and Wang, X and Han, J and Yin, H}, title = {Crystal structure of the anti-CRISPR protein AcrIE7.}, journal = {Biochemical and biophysical research communications}, volume = {748}, number = {}, pages = {151315}, doi = {10.1016/j.bbrc.2025.151315}, pmid = {39818188}, issn = {1090-2104}, mesh = {Crystallography, X-Ray ; *CRISPR-Cas Systems ; Models, Molecular ; Protein Conformation ; Amino Acid Sequence ; *Bacterial Proteins/chemistry ; *CRISPR-Associated Proteins/chemistry ; }, abstract = {Bacterial adaptive immunity, driven by CRISPR-Cas systems, protects against foreign nucleic acids from mobile genetic elements (MGEs), like bacteriophages. The type I-E CRISPR-Cas system employs the Cascade (CRISPR-associated complex for antiviral defense) complex for target DNA cleavage, guided by crRNA. Anti-CRISPR (Acr) proteins, such as AcrIE7, counteract this defense by inhibiting Cascade activity. In this study, we characterized and determined the structure of AcrIE7, a unique member of the AcrIE family, using X-ray crystallography under two distinct crystallization conditions, achieving resolutions of 2.05 Å and 2.68 Å, respectively. Topological analysis revealed that AcrIE7 consists of seven α-helices with two distinct charge regions, likely mediating its inhibitory interactions. Structural flexibility analysis revealed notable structural stability differences between the two crystallization conditions, indicating varying rigidity of the AcrIE7 protein under different conditions. Homology searches and AlphaFold predictions reinforced the unique nature of AcrIE7, which exhibits a novel fold, underscoring its distinct role within the AcrIE family. These findings enhance our understanding of Acr proteins and provide a theoretical foundation for developing CRISPR-based gene-editing regulatory tools.}, }
@article {pmid39817779, year = {2025}, author = {Wiull, K and Haugen, LK and Eijsink, VGH and Mathiesen, G}, title = {CRISPR/Cas9-mediated genomic insertion of functional genes into Lactiplantibacillus plantarum WCFS1.}, journal = {Microbiology spectrum}, volume = {13}, number = {2}, pages = {e0202524}, pmid = {39817779}, issn = {2165-0497}, support = {//Norges Milj&#xf8;- og Biovitenskapelige Universitet (NMBU)/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Lactobacillus plantarum/genetics ; Plasmids/genetics ; Gene Editing/methods ; SARS-CoV-2/genetics ; Humans ; *Mutagenesis, Insertional/methods ; Gene Knock-In Techniques ; }, abstract = {UNLABELLED: Lactiplantibacillus plantarum, a natural inhabitant of the human body, is a promising candidate vehicle for vaccine delivery. An obstacle in developing bacterial delivery vehicles is generating a production strain that lacks antibiotic resistance genes and contains minimal foreign DNA. To deal with this obstacle, we have constructed a finetuned, inducible two-plasmid CRISPR/Cas9-system for chromosomal gene insertion in L. plantarum. The knock-in plasmid was designed with a cassette-like structure to simplify the insertion of target DNA and streamline the CRISPR/Cas9 genome editing, bringing it one step closer to becoming a routine procedure. We demonstrate that the system enables efficient insertion of expression cassettes for both inducible and constitutive production of a fluorescent reporter protein, mCherry, and for inducible production of the receptor-binding domain (RBD) of the SARS-CoV-2 virus. Two variants of RBD were successfully expressed, one directed to the cytoplasm and one directed to the cell surface. All the knock-in strains produced the target protein, although with lower yields than strains with plasmid-encoded expression.<br><br>IMPORTANCE: Genetic engineering of lactic acid bacteria, such as Lactiplantibacillus plantarum, has proven to be difficult. This study presents an inducible two-plasmid CRISPR/Cas9-system for inserting genes into the chromosome of Lactiplantibacillus plantarum. Our system successfully knock-in four expression cassettes varying in length from ~800-1,300 bp with high efficiency and insert an expression cassette encoding a SARS-CoV-2 antigen receptor-binding domain (RBD) with an anchor mediating surface display, which has not been achieved previously using CRISPR/Cas9. We demonstrate the production of the insertion genes. Importantly, the plasmid carrying the SgRNA, Cas9, and homology-directed repair template is designed for easy component exchange. These plasmids represent valuable contributions to the field as they could facilitate rapid CRISPR/Cas9 engineering of L. plantarum strains.}, }
@article {pmid39817514, year = {2025}, author = {Jungfer, K and Moravčík, &#x160; and Garcia-Doval, C and Knörlein, A and Hall, J and Jinek, M}, title = {Mechanistic determinants and dynamics of cA6 synthesis in type III CRISPR-Cas effector complexes.}, journal = {Nucleic acids research}, volume = {53}, number = {2}, pages = {}, pmid = {39817514}, issn = {1362-4962}, support = {ERC-CoG-820152/ERC_/European Research Council/International ; 51NF40-182880//SNSF/ ; ETH-24-16-2//ETH Zurich/ ; //University of Zurich/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; Cryoelectron Microscopy ; *Adenine Nucleotides/biosynthesis/chemistry ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *Bacterial Proteins/metabolism/chemistry/genetics ; Binding Sites ; *Oligoribonucleotides/biosynthesis ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Models, Molecular ; }, abstract = {Type III clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems (type III CRISPR-Cas systems) use guide RNAs to recognize RNA transcripts of foreign genetic elements, which triggers the generation of cyclic oligoadenylate (cOA) second messengers by the Cas10 subunit of the type III effector complex. In turn, cOAs bind and activate ancillary effector proteins to reinforce the host immune response. Type III systems utilize distinct cOAs, including cyclic tri- (cA3), tetra- (cA4) and hexa-adenylates (cA6). However, the molecular mechanisms dictating cOA product identity are poorly understood. Here we used cryoelectron microscopy to visualize the mechanism of cA6 biosynthesis by the Csm effector complex from Enterococcus italicus (EiCsm). We show that EiCsm synthesizes oligoadenylate nucleotides in 3'-5' direction using a set of conserved binding sites in the Cas10 Palm domains to determine the size of the nascent oligoadenylate chain. Our data also reveal that conformational dynamics induced by target RNA binding results in allosteric activation of Cas10 to trigger oligoadenylate synthesis. Mutations of a key structural element in Cas10 perturb cOA synthesis to favor cA3 and cA4 formation. Together, these results provide comprehensive insights into the dynamics of cOA synthesis in type III CRISPR-Cas systems and reveal key determinants of second messenger product selectivity, thereby illuminating potential avenues for their engineering.}, }
@article {pmid39817512, year = {2025}, author = {Zhang, L and Qiu, X and Zhou, Y and Luo, Z and Zhu, L and Shao, J and Xie, M and Wang, H}, title = {A trigger-inducible split-Csy4 architecture for programmable RNA modulation.}, journal = {Nucleic acids research}, volume = {53}, number = {2}, pages = {}, pmid = {39817512}, issn = {1362-4962}, support = {2023YFF1205400//Ministry of Science and Technology/ ; 32371498//National Natural Science Foundation of China/ ; 202209009//Westlake Laboratory of Life Sciences and Biomedicine/ ; 2024SSYS0007//'Pioneer' and 'Leading Goose' R&amp;D Program of Zhejiang/ ; }, mesh = {Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; *Endoribonucleases/genetics/metabolism/chemistry ; *RNA/genetics/metabolism ; Gene Expression Regulation ; Pyrazoles/pharmacology ; Transcriptome ; *CRISPR-Associated Proteins/genetics/metabolism ; }, abstract = {The CRISPR-derived endoribonuclease Csy4 is a popular tool for controlling transgene expression in various therapeutically relevant settings, but adverse effects potentially arising from non-specific RNA cleavage remains largely unexplored. Here, we report a split-Csy4 architecture that was carefully optimized for in vivo usage. First, we separated Csy4 into two independent protein moieties whose full catalytic activity can be restored via various constitutive or conditional protein dimerization systems. Next, we show that introduction of split-Csy4 into human cells caused a substantially reduced extent in perturbation of the endogenous transcriptome when directly compared to full-length Csy4. Inspired by these results, we went on to use such split-Csy4 module to engineer inducible CRISPR- and translation-level gene switches regulated by the FDA-approved drug grazoprevir. This work provides valuable resource for Csy4-related biomedical research and discusses important issues for the development of clinically eligible regulation tools.}, }
@article {pmid39817463, year = {2025}, author = {Zuo, D and Liu, W and Zhao, R and Zhu, K and Wang, W and Xiang, H}, title = {Drosophila CG11700 may not affect male fecundity-lifespan tradeoff as previously reported.}, journal = {Molecular biology and evolution}, volume = {42}, number = {2}, pages = {}, pmid = {39817463}, issn = {1537-1719}, mesh = {Male ; Animals ; *Fertility/genetics ; *Longevity/genetics ; *Drosophila Proteins/genetics ; CRISPR-Cas Systems ; Female ; *Drosophila/genetics/physiology ; *Drosophila melanogaster/genetics/physiology ; }, abstract = {Our recent investigations on the function of Drosophila CG11700 and CG32744 (Ubi-p5E) genes using CRISPR/Cas9 deletion technology could not repeat or confirm the results on CG11700 shown in our previous study, which was based on P-element excision assay (Zhan Z, Ding Y, Zhao R, Zhang Y, Yu H, Zhou Q, Yang S, Xiang H, Wang W. Rapid functional divergence of a newly evolved polyubiquitin gene in Drosophila and its role in the trade-off between male fecundity and lifespan. Mol Biol Evol. 2012:29(5):1407-1416. doi:10.1093/molbev/msr299). Here, by CRISPR/Cas9 editing, we generated mutants of CG32744 with the whole gene body fully deleted from the genome, and truncated mutants of CG11700 with N-terminal 103 aa deleted out of its total 301 aa peptide sequence. We carefully conducted the male fecundity assay and found that offsprings of the CG11700 mutant were not significantly more than the wild type, inconsistent with our previous report (Zhan et al. 2012). Meanwhile, we repeated the lifespan assay and did not find that the lifespan of the CG11700 mutant was significantly shorter than the wild type as reported (2012). The new results suggest that the CG11700 gene may not affect male fecundity-lifespan tradeoff as previously reported (Zhan et al. 2012). The new results are thus worthy of reporting to avoid possible misleading by the previous results to the scientific community.}, }
@article {pmid39817421, year = {2025}, author = {Debaenst, S and Jarayseh, T and De Saffel, H and Bek, JW and Boone, M and Josipovic, I and Kibleur, P and Kwon, RY and Coucke, PJ and Willaert, A}, title = {Crispant analysis in zebrafish as a tool for rapid functional screening of disease-causing genes for bone fragility.}, journal = {eLife}, volume = {13}, number = {}, pages = {}, pmid = {39817421}, issn = {2050-084X}, support = {R01 AR074417/AR/NIAMS NIH HHS/United States ; FWO.OPR.2020.0023.01//Fonds Wetenschappelijk Onderzoek/ ; }, mesh = {Animals ; *Zebrafish/genetics ; Bone Density/genetics ; Disease Models, Animal ; *Osteogenesis Imperfecta/genetics ; Bone and Bones/pathology ; CRISPR-Cas Systems ; }, abstract = {Heritable fragile bone disorders (FBDs), ranging from multifactorial to rare monogenic conditions, are characterized by an elevated fracture risk. Validating causative genes and understanding their mechanisms remain challenging. We assessed a semi-high throughput zebrafish screening platform for rapid in vivo functional testing of candidate FBD genes. Six genes linked to severe recessive osteogenesis imperfecta (OI) and four associated with bone mineral density (BMD) from genome-wide association studies were analyzed. Using CRISPR/Cas9-based crispant screening in F0 mosaic founder zebrafish, Next-generation sequencing confirmed high indel efficiency (mean 88%), mimicking stable knock-out models. Skeletal phenotyping at 7, 14, and 90 days post-fertilization (dpf) using microscopy, Alizarin Red S staining, and microCT was performed. Larval crispants showed variable osteoblast and mineralization phenotypes, while adult crispants displayed consistent skeletal defects, including malformed neural and haemal arches, vertebral fractures and fusions, and altered bone volume and density. In addition, aldh7a1 and mbtps2 crispants experienced increased mortality due to severe skeletal deformities. RT-qPCR revealed differential expression of osteogenic markers bglap and col1a1a, highlighting their biomarker potential. Our results establish zebrafish crispant screening as a robust tool for FBD gene validation, combining skeletal and molecular analyses across developmental stages to uncover novel insights into gene functions in bone biology.}, }
@article {pmid39817182, year = {2025}, author = {Xue, M and Gonzalez, DH and Osikpa, E and Gao, X and Lillehoj, PB}, title = {Rapid and automated interpretation of CRISPR-Cas13-based lateral flow assay test results using machine learning.}, journal = {Sensors &amp; diagnostics}, volume = {4}, number = {2}, pages = {171-181}, pmid = {39817182}, issn = {2635-0998}, support = {R61 AI167037/AI/NIAID NIH HHS/United States ; }, abstract = {CRISPR-Cas-based lateral flow assays (LFAs) have emerged as a promising diagnostic tool for ultrasensitive detection of nucleic acids, offering improved speed, simplicity and cost-effectiveness compared to polymerase chain reaction (PCR)-based assays. However, visual interpretation of CRISPR-Cas-based LFA test results is prone to human error, potentially leading to false-positive or false-negative outcomes when analyzing test/control lines. To address this limitation, we have developed two neural network models: one based on a fully convolutional neural network and the other on a lightweight mobile-optimized neural network for automated interpretation of CRISPR-Cas-based LFA test results. To demonstrate proof of concept, these models were applied to interpret results from a CRISPR-Cas13-based LFA for the detection of the SARS-CoV-2 N gene, a key marker for COVID-19 infection. The models were trained, evaluated, and validated using smartphone-captured images of LFA devices in various orientations with different backgrounds, lighting conditions, and image qualities. A total of 3146 images (1569 negative, 1577 positive) captured using an iPhone 13 or Samsung Galaxy A52 Android smartphone were analyzed using the trained models, which classified the LFA results within 0.2 s with 96.5% accuracy compared to the ground truth. These results demonstrate the potential of machine learning to accurately interpret test results of CRISPR-Cas-based LFAs using smartphone-captured images in real-world settings, enabling the practical use of CRISPR-Cas-based diagnostic tools for self- and at-home testing.}, }
@article {pmid39816115, year = {2024}, author = {Syahrani, RA and Wanandi, SI and Arumsari, S and Nihayah, S and Watanabe, Y and Mizuno, S and Louisa, M and Wuyung, PE}, title = {Dual sgRNA-directed knockout survivin gene expression using CRISPR/Cas9 technology for editing survivin gene in triple-negative breast cancer.}, journal = {Narra J}, volume = {4}, number = {3}, pages = {e1177}, pmid = {39816115}, issn = {2807-2618}, mesh = {*Survivin/genetics ; Humans ; *Triple Negative Breast Neoplasms/genetics ; *CRISPR-Cas Systems/genetics ; Female ; *Gene Knockout Techniques/methods ; Cell Line, Tumor ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (CRISPR/Cas9) offers a robust approach for genome manipulation, particularly in cancer therapy. Given its high expression in triple-negative breast cancer (TNBC), targeting survivin with CRISPR/Cas9 holds promise as a therapeutic strategy. The aim of this study was to design specific single guide ribonucleic acid (sgRNA) for CRISPR/Cas9 to permanently knock out the survivin gene, exploring its potential as a therapeutic approach in breast cancer while addressing potential off-target effects. Survivin gene knockout was conducted in the TNBC cell line BT549. Intron 1, exon 2, and intron 2 of the survivin gene were selected as sgRNA targets. These sgRNAs were designed in silico and then cloned into a CRISPR/Cas9 expression plasmid. The cleavage activity was assessed using an enhanced green fluorescent protein (EGFP) expression plasmid. The sgRNAs with higher cleavage activity were selected for the establishment of knockout cells. After transfecting the plasmid into the cells, the success of the survivin gene knockout was validated at the deoxyribonucleic acid (DNA) level using polymerase chain reaction (PCR) and sequencing analysis, and at the protein expression level using Western blotting. The study found that sgRNAs survin1A (targeting intron 1), survex2A (targeting intron 2), and survin2A (targeting intron 2) demonstrated higher cleavage activities compared to the other sgRNAs. However, using the single sgRNA, survex2A did not generate mutations in the survivin gene. At the protein level, survivin was still expressed, indicating that a single sgRNA was ineffective in knocking out the survivin gene. In contrast, the combination of sgRNA survin1A and sgRNA survin2A was more effective in generating mutations in the survivin gene, resulting in the deletion of the entire exon 2 and leading to a loss of survivin protein expression. In conclusion, our work provides specific sgRNAs and demonstrates the utilization of dual sgRNAs strategy in the CRISPR/Cas9 technology to knock out the survivin gene, showing potential in breast cancer therapy.}, }
@article {pmid39815622, year = {2025}, author = {Shankar, K and Zingler-Hoslet, I and Tabima, DM and Zima, S and Shi, L and Gimse, K and Forsberg, MH and Katta, V and Davis, SZ and Maldonado, D and Russell, BE and Murtaza, M and Tsai, SQ and Ayuso, JM and Capitini, CM and Saha, K}, title = {Virus-free CRISPR knockin of a chimeric antigen receptor into KLRC1 generates potent GD2-specific natural killer cells.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {33}, number = {3}, pages = {1014-1030}, pmid = {39815622}, issn = {1525-0024}, mesh = {Humans ; *Killer Cells, Natural/immunology/metabolism ; *Receptors, Chimeric Antigen/genetics/metabolism/immunology ; *CRISPR-Cas Systems ; Gene Editing ; Gene Knock-In Techniques ; Cell Line, Tumor ; Immunotherapy, Adoptive ; Gangliosides ; }, abstract = {Natural killer (NK) cells are an appealing off-the-shelf, allogeneic cellular therapy due to their cytotoxic profile. However, their activity against solid tumors remains suboptimal in part due to the upregulation of NK-inhibitory ligands, such as HLA-E, within the tumor microenvironment. Here, we utilize CRISPR-Cas9 to disrupt the KLRC1 gene (encoding the HLA-E-binding NKG2A receptor) and perform non-viral insertion of a GD2-targeting chimeric antigen receptor (CAR) within NK cells isolated from human peripheral blood. Genome editing with CRISPR-Cas9 ribonucleoprotein complexes yields efficient genomic disruption of the KLRC1 gene with 98% knockout efficiency and specific knockin of the GD2 CAR transgene as high as 23%, with minimal off-target activity as shown by CHANGE-seq, in-out PCR, amplicon sequencing, and long-read whole-genome sequencing. KLRC1-GD2 CAR NK cells display high viability and proliferation, as well as precise cellular targeting and potency against GD2[+] human tumor cells. Notably, KLRC1-GD2 CAR NK cells overcome HLA-E-based inhibition in vitro against HLA-E-expressing, GD2[+] melanoma cells. Using a single-step, virus-free genome editing workflow, this study demonstrates the feasibility of precisely disrupting inhibitory signaling within NK cells via CRISPR-Cas9 while expressing a CAR to generate potent allogeneic cell therapies against HLA-E[+] solid tumors.}, }
@article {pmid39814846, year = {2025}, author = {Shin, SW and Kim, SH and Gasselin, A and Lee, GM and Lee, JS}, title = {Comprehensive genome-scale CRISPR knockout screening of CHO cells.}, journal = {Scientific data}, volume = {12}, number = {1}, pages = {71}, pmid = {39814846}, issn = {2052-4463}, support = {RS-2024-00397714//National Research Foundation of Korea (NRF)/ ; }, mesh = {CHO Cells ; Animals ; Cricetulus ; *Gene Knockout Techniques ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome ; High-Throughput Nucleotide Sequencing ; }, abstract = {Chinese hamster ovary (CHO) cells play a pivotal role in the production of recombinant therapeutics. In the present study, we conducted a genome-scale pooled CRISPR knockout (KO) screening using a virus-free, recombinase-mediated cassette exchange-based platform in CHO-K1 host and CHO-K1 derived recombinant cells. Genome-wide guide RNA (gRNA) amplicon sequencing data were generated from cell libraries, as well as short- and long-term KO libraries, and validated through phenotypic assessment and gRNA read count distribution. Additionally, we obtained gRNA amplicon sequencing data from the highly productive recombinant cell populations. By analyzing these datasets, essential genes involved in cell fitness as well as functional target genes associated with therapeutic protein production can be identified. Collectively, our next-generation sequencing datasets, derived from a robust and reliable CRISPR screening method, provide valuable insights into CHO genomic functions, advancing the development of next-generation CHO factories.}, }
@article {pmid39814710, year = {2025}, author = {Neugebauer, E and Walter, S and Tan, J and Drayman, N and Franke, V and van Gent, M and Pennisi, S and Veratti, P and Stein, KS and Welker, I and Tay, S and Verjans, GMGM and Timmers, HTM and Akalin, A and Landthaler, M and Ensser, A and Wyler, E and Full, F}, title = {Herpesviruses mimic zygotic genome activation to promote viral replication.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {710}, pmid = {39814710}, issn = {2041-1723}, support = {En 423/5-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; DP2 AI154437/AI/NIAID NIH HHS/United States ; 01KI2017//Bundesministerium f&#xfc;r Bildung und Forschung (Federal Ministry of Education and Research)/ ; FOR5200 DEEP-DV, 443644894//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; AI154437//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R01 GM127527/GM/NIGMS NIH HHS/United States ; }, mesh = {*Virus Replication/genetics ; Humans ; Genome, Viral/genetics ; *Herpesvirus 1, Human/physiology/genetics ; *Homeodomain Proteins/metabolism/genetics ; *Zygote/metabolism/virology ; Gene Expression Regulation, Viral ; *Herpesviridae/physiology/genetics ; Animals ; CRISPR-Cas Systems ; Transcription, Genetic ; HEK293 Cells ; }, abstract = {Zygotic genome activation (ZGA) is crucial for maternal to zygotic transition at the 2-8-cell stage in order to overcome silencing of genes and enable transcription from the zygotic genome. In humans, ZGA is induced by DUX4, a pioneer factor that drives expression of downstream germline-specific genes and retroelements. Here we show that herpesviruses from all subfamilies, papillomaviruses and Merkel cell polyomavirus actively induce DUX4 expression to promote viral transcription and replication. Analysis of single-cell sequencing data sets from patients shows that viral DUX4 activation is of relevance in vivo. Herpes-simplex virus 1 (HSV-1) immediate early proteins directly induce expression of DUX4 and its target genes, which mimics zygotic genome activation. Upon HSV-1 infection, DUX4 directly binds to the viral genome and promotes viral transcription. DUX4 is functionally required for infection, since genetic depletion by CRISPR/Cas9 as well as degradation of DUX4 by nanobody constructs abrogates HSV-1 replication. Our results show that DNA viruses including herpesviruses mimic an embryonic-like transcriptional program that prevents epigenetic silencing of the viral genome and facilitates herpesviral gene expression.}, }
@article {pmid39814565, year = {2025}, author = {Chau, CCC and Weckman, NE and Thomson, EE and Actis, P}, title = {Solid-State Nanopore Real-Time Assay for Monitoring Cas9 Endonuclease Reactivity.}, journal = {ACS nano}, volume = {19}, number = {3}, pages = {3839-3851}, pmid = {39814565}, issn = {1936-086X}, mesh = {*Nanopores ; CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/metabolism ; DNA/chemistry/metabolism ; }, abstract = {The field of nanopore sensing is now moving beyond nucleic acid sequencing. An exciting avenue is the use of nanopore platforms for the monitoring of biochemical reactions. Biological nanopores have been used for this application, but solid-state nanopore approaches have lagged. This is due to the necessity of using higher salt conditions (e.g., 4 M LiCl) to improve the signal-to-noise ratio which completely abolish the activities of many biochemical reactions. We pioneered a polymer electrolyte solid-state nanopore approach that maintains a high signal-to-noise ratio even at a physiologically relevant salt concentration. Here, we report the monitoring of the restriction enzyme SwaI and CRISPR-Cas9 endonuclease activities under physiological salt conditions and in real time. We investigated the dsDNA cleavage activity of these enzymes in a range of digestion buffers and elucidated the off-target activity of CRISPR-Cas9 ribonucleoprotein endonuclease in the presence of single base pair mismatches. This approach enables the application of solid-state nanopores for the dynamic monitoring of biochemical reactions under physiological salt conditions.}, }
@article {pmid39814010, year = {2025}, author = {Li, E and Benitez, C and Boggess, SC and Koontz, M and Rose, IVL and Martinez, D and Dräger, N and Teter, OM and Samelson, AJ and Pierce, N and Ullian, EM and Kampmann, M}, title = {CRISPRi-based screens in iAssembloids to elucidate neuron-glia interactions.}, journal = {Neuron}, volume = {113}, number = {5}, pages = {701-718.e8}, pmid = {39814010}, issn = {1097-4199}, support = {F32 AG063487/AG/NIA NIH HHS/United States ; U54 NS123746/NS/NINDS NIH HHS/United States ; S10 OD028511/OD/NIH HHS/United States ; U01 AG072464/AG/NIA NIH HHS/United States ; K99 AG080116/AG/NIA NIH HHS/United States ; T32 NS115706/NS/NINDS NIH HHS/United States ; }, mesh = {*Neurons/metabolism/physiology ; *Neuroglia/metabolism/physiology ; Humans ; Coculture Techniques/methods ; *Induced Pluripotent Stem Cells/physiology/metabolism/cytology ; *Cell Communication/physiology ; *CRISPR-Cas Systems ; Oxidative Stress ; Astrocytes/metabolism ; Apolipoprotein E4/genetics/metabolism ; Glycogen Synthase Kinase 3 beta/metabolism/genetics ; NF-E2-Related Factor 2/metabolism ; Animals ; Cells, Cultured ; }, abstract = {The complexity of the human brain makes it challenging to understand the molecular mechanisms underlying brain function. Genome-wide association studies have uncovered variants associated with neurological phenotypes. Single-cell transcriptomics have provided descriptions of changes brain cells undergo during disease. However, these approaches do not establish molecular mechanism. To facilitate the scalable interrogation of causal molecular mechanisms in brain cell types, we developed a 3D co-culture system of induced pluripotent stem cell (iPSC)-derived neurons and glia, termed iAssembloids. Using iAssembloids, we ask how glial and neuronal cells interact to control neuronal death and survival. Our CRISPRi-based screens identified that GSK3β inhibits the protective NRF2-mediated oxidative stress response elicited by high neuronal activity. We then investigate the role of APOE-ε4, a risk variant for Alzheimer's disease, on neuronal survival. We find that APOE-ε4-expressing astrocytes may promote neuronal hyperactivity as compared with APOE-ε3-expressing astrocytes. This platform allows for the unbiased identification of mechanisms of neuron-glia cell interactions.}, }
@article {pmid39812375, year = {2025}, author = {Wang, C and Xu, X and Yao, W and Wang, L and Pang, X and Xu, S and Luo, X}, title = {Programmable DNA Nanoswitch-Regulated Plasmonic CRISPR/Cas12a-Gold Nanostars Reporter Platform for Nucleic Acid and Non-Nucleic Acid Biomarker Analysis Assisted by a Spatial Confinement Effect.}, journal = {Nano letters}, volume = {25}, number = {4}, pages = {1666-1672}, doi = {10.1021/acs.nanolett.4c05829}, pmid = {39812375}, issn = {1530-6992}, mesh = {*Gold/chemistry ; Humans ; *MicroRNAs/urine/analysis/genetics ; *CRISPR-Cas Systems ; *Prostate-Specific Antigen/urine/genetics ; *DNA/chemistry ; *Metal Nanoparticles/chemistry ; *Biosensing Techniques/methods ; Male ; Limit of Detection ; *CRISPR-Associated Proteins/chemistry ; Biomarkers, Tumor/urine ; Prostatic Neoplasms/diagnosis/urine ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {CRISPR/Cas 12a system based nucleic acid and non-nucleic acid targets detection faces two challenges including (1) multiple crRNAs are needed for multiple biomarkers detection and (2) insufficient sensitivity resulted from photobleaching of fluorescent dyes and the low kinetic cleavage rate for a traditional single-strand (ssDNA) reporter. To address these limitations, we developed a programmable DNA nanoswitch (NS)-regulated plasmonic CRISPR/Cas12a-gold nanostars (Au NSTs) reporter platform for detection of nucleic acid and non-nucleic acid biomarkers with the assistance of the spatial confinement effect. Through simply programming the target recognition sequence in NS, only one crRNA is required to detect both nucleic acid and non-nucleic acid biomarkers. The detection limit decreased by ∼196-fold for miRNA-375 and 122-fold for prostate-specific antigen (PSA), respectively. Moreover, versatile evaluation of miRNA-375 and PSA in clinical urine samples can also be achieved, according to which prostate cancer and healthy groups can be well identified.}, }
@article {pmid39810563, year = {2025}, author = {Wang, W and Du, H and Dai, C and Ma, H and Luo, S and Wang, X and Guo, M and Kong, D and Wei, D}, title = {Amplification-free detection of Mycobacterium tuberculosis using CRISPR-Cas12a and graphene field-effect transistors.}, journal = {Nanoscale}, volume = {17}, number = {8}, pages = {4603-4609}, doi = {10.1039/d4nr03852e}, pmid = {39810563}, issn = {2040-3372}, mesh = {*Mycobacterium tuberculosis/isolation &amp; purification/genetics ; *Graphite/chemistry ; *CRISPR-Cas Systems ; *Transistors, Electronic ; Humans ; Limit of Detection ; *Tuberculosis/diagnosis/microbiology ; Biosensing Techniques ; DNA, Bacterial/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Current molecular tests for tuberculosis (TB), such as whole genome sequencing and Xpert Mycobacterium tuberculosis/rifampicin resistance assay, exhibit limited sensitivity and necessitate the pre-amplification step of target DNA. This limitation greatly increases detection time and poses an increased risk of infection. Here, we present a graphene field-effect transistor (GFET) based on the CRISPR/Cas system for detecting Mycobacterium tuberculosis. The CRISPR/Cas12a system has the ability to specifically recognize and cleave target DNA. By integrating the system onto the FET platform and utilizing its electrical amplification capability, we achieve rapid and sensitive detection without requiring sample pre-amplification, with a limit of detection (LoD) as low as 2.42 × 10[-18] M. Cas12a-GFET devices can differentiate 30 positive cases from 56 serum samples within 5 minutes. These findings highlight its immense potential in future biological analysis and clinical diagnosis.}, }
@article {pmid39810095, year = {2025}, author = {Liu, W and Wang, D and He, Q and Cao, S and Cao, J and Zhao, H and Cui, J and Yang, F}, title = {A strategy for controlling Hypervirulent Klebsiella pneumoniae: inhibition of ClpV expression.}, journal = {BMC microbiology}, volume = {25}, number = {1}, pages = {22}, pmid = {39810095}, issn = {1471-2180}, mesh = {*Klebsiella pneumoniae/genetics/pathogenicity/drug effects/growth &amp; development ; Animals ; *Bacterial Proteins/genetics/metabolism/antagonists &amp; inhibitors ; Virulence/genetics/drug effects ; Gene Deletion ; Biofilms/growth &amp; development/drug effects ; Virulence Factors/genetics ; Gene Expression Regulation, Bacterial/drug effects ; Klebsiella Infections/microbiology/prevention &amp; control ; Moths/microbiology ; CRISPR-Cas Systems ; Larva/microbiology ; }, abstract = {The emergence and prevalence of hypervirulent Klebsiella pneumoniae (hvKP) have proposed a great challenge to control this infection. Therefore, exploring some new drugs or strategies for treating hvKP infection is an urgent issue for scientific researchers. In the present study, the clpV gene deletion strain of hvKP (ΔclpV-hvKP) was constructed using CRISPR-Cas9 technology, and the biological characteristics of ΔclpV-hvKP were investigated to explore the new targets for controlling this pathogen. The results showed that clpV gene deletion did not affect the growth ability of hvKP. However, knocking out the clpV gene markedly decreased the mucoid phenotype and the biofilm formation ability of hvKP. It reduced the interspecific competition of hvKP with Escherichia coli, Salmonella, Pseudomonas aeruginosa, and Staphylococcus aureus. The clpV deletion significantly changed the transcriptome profile of hvKP, inhibited the expression of virulence factors, and decreased the lethality of hvKP against Galleria mellonella larvae. In vitro experiments showed that lithocholic acid could inhibit the expression of the clpV gene and reduce the virulence of hvKP. Our data suggested that the clpV gene may be a potential target for decreasing hvKP infection risk.}, }
@article {pmid39809840, year = {2025}, author = {Li, XG and Zhu, GS and Cao, PJ and Huang, H and Chen, YH and Chen, C and Chen, PJ and Wu, D and Ding, C and Zhang, ZH and Zhang, RH and Hu, ZX and Zhao, WH and Liu, MH and Li, YW and Liu, HY and Chen, J}, title = {Genome-wide CRISPR-Cas9 screening identifies ITGA8 responsible for abivertinib sensitivity in lung adenocarcinoma.}, journal = {Acta pharmacologica Sinica}, volume = {46}, number = {5}, pages = {1419-1432}, pmid = {39809840}, issn = {1745-7254}, mesh = {Humans ; *CRISPR-Cas Systems ; *Lung Neoplasms/drug therapy/genetics/pathology/metabolism ; *Adenocarcinoma of Lung/drug therapy/genetics/pathology/metabolism ; Animals ; Cell Proliferation/drug effects ; Drug Resistance, Neoplasm/genetics/drug effects ; Cell Line, Tumor ; Cell Movement/drug effects ; *Protein Kinase Inhibitors/pharmacology/therapeutic use ; Mice ; *Antineoplastic Agents/pharmacology/therapeutic use ; Mice, Nude ; ErbB Receptors/antagonists &amp; inhibitors ; Mice, Inbred BALB C ; }, abstract = {The emergence of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) has improved the prognosis for lung cancer patients with EGFR-driven mutations. However, acquired resistance to EGFR-TKIs poses a significant challenge to the treatment. Overcoming the resistance has primarily focused on developing next-generation targeted therapies based on the molecular mechanisms of resistance or inhibiting the activation of bypass pathways to suppress or reverse the resistance. In this study we developed a novel approach by using CRISPR-Cas9 whole-genome library screening to identify the genes that enhance the sensitivity of lung adenocarcinoma cells to EGFR-TKIs. Through this screening, we revealed integrin subunit alpha 8 (ITGA8) as the key gene that enhanced sensitivity to abivertinib in lung adenocarcinoma. Notably, ITGA8 expression was significantly downregulated in lung adenocarcinoma tissues compared to adjacent normal tissues. Bioinformatics analyses revealed that ITGA8 was positively correlated with the sensitivity of lung adenocarcinoma to abivertinib. We showed that knockdown of ITGA8 significantly enhanced the proliferation, migration and invasion of H1975 cells. Conversely, overexpression of ITGA8 reduced the proliferation migration and invasion of H1975/ABIR cells. Furthermore, we demonstrated that ITGA8 sensitized lung adenocarcinoma cells to EGFR-TKIs by attenuating the downstream FAK/SRC/AKT/MAPK signaling pathway. In H1975 cell xenograft mouse models, knockdown of ITGA8 significantly increased tumor growth and reduced the sensitivity to abivertinib, whereas overexpression of ITGA8 markedly suppressed tumor proliferation and enhanced sensitivity to the drug. This study demonstrates that ITGA8 inhibits the proliferation, invasion and migration of lung adenocarcinoma cells, enhances the sensitivity to EGFR-TKIs, improves treatment efficacy, and delays the progression of acquired resistance. Thus, ITGA8 presents a potential therapeutic candidate for addressing acquired resistance to EGFR-TKIs from a novel perspective.}, }
@article {pmid39809780, year = {2025}, author = {Park, SJ and Ju, S and Jung, WJ and Jeong, TY and Yoon, DE and Lee, JH and Yang, J and Lee, H and Choi, J and Kim, HS and Kim, K}, title = {Robust genome editing activity and the applications of enhanced miniature CRISPR-Cas12f1.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {677}, pmid = {39809780}, issn = {2041-1723}, support = {RS-2023-00220894//National Research Foundation of Korea (NRF)/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; HEK293 Cells ; Genetic Therapy/methods ; Cell Line, Tumor ; Apoptosis/genetics ; }, abstract = {With recent advancements in gene editing technology using the CRISPR/Cas system, there is a demand for more effective gene editors. A key factor facilitating efficient gene editing is effective CRISPR delivery into cells, which is known to be associated with the size of the CRISPR system. Accordingly, compact CRISPR-Cas systems derived from various strains are discovered, among which Un1Cas12f1 is 2.6 times smaller than SpCas9, providing advantages for gene therapy research. Despite extensive engineering efforts to improve Un1Cas12f1, the editing efficiency of Un1Cas12f1 is still shown to be low depending on the target site. To overcome this limitation, we develop enhanced Cas12f1 (eCas12f1), which exhibits gene editing activity similar to SpCas9 and AsCpf1, even in gene targets where previously improved Un1Cas12f1 variants showed low gene editing efficiency. Furthermore, we demonstrate that eCas12f1 efficiently induces apoptosis in cancer cells and is compatible with base editing and regulation of gene expression, verifying its high utility and applicability in gene therapy research.}, }
@article {pmid39809757, year = {2025}, author = {Zou, J and Jiang, M and Xiao, R and Sun, H and Liu, H and Peacock, T and Tu, S and Chen, T and Guo, J and Zhao, Y and Barclay, W and Xie, S and Zhou, H}, title = {GGCX promotes Eurasian avian-like H1N1 swine influenza virus adaption to interspecies receptor binding.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {670}, pmid = {39809757}, issn = {2041-1723}, support = {32025036//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32430104//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2021CFA016//Natural Science Foundation of Hebei Province (Hebei Provincial Natural Science Foundation)/ ; }, mesh = {Animals ; *Influenza A Virus, H1N1 Subtype/physiology/genetics ; Swine ; *Receptors, Virus/metabolism ; Virus Replication/genetics ; Humans ; Hemagglutinin Glycoproteins, Influenza Virus/metabolism/genetics ; *Orthomyxoviridae Infections/virology ; CRISPR-Cas Systems ; HEK293 Cells ; Cell Line ; Virus Attachment ; Receptors, Cell Surface/metabolism ; }, abstract = {The Eurasian avian-like (EA) H1N1 swine influenza virus (SIV) possesses the capacity to instigate the next influenza pandemic, owing to its heightened affinity for the human-type α-2,6 sialic acid (SA) receptor. Nevertheless, the molecular mechanisms underlying the switch in receptor binding preferences of EA H1N1 SIV remain elusive. In this study, we conduct a comprehensive genome-wide CRISPR/Cas9 knockout screen utilizing EA H1N1 SIV in porcine kidney cells. Knocking out the enzyme gamma glutamyl carboxylase (GGCX) reduces virus replication in vitro and in vivo by inhibiting the carboxylation modification of viral haemagglutinin (HA) and the adhesion of progeny viruses, ultimately impeding the replication of EA H1N1 SIV. Furthermore, GGCX is revealed to be the determinant of the D225E substitution of EA H1N1 SIV, and GGCX-medicated carboxylation modification of HA 225E contributes to the receptor binding adaption of EA H1N1 SIV to the α-2,6 SA receptor. Taken together, our CRISPR screen has elucidated a novel function of GGCX in the support of EA H1N1 SIV adaption for binding to α-2,6 SA receptor. Consequently, GGCX emerges as a prospective antiviral target against the infection and transmission of EA H1N1 SIV.}, }
@article {pmid39809734, year = {2025}, author = {Mormile, BW and Yan, Y and Bauer, T and Wang, L and Rivero, RC and Carpenter, SCD and Danmaigona Clement, C and Cox, KL and Zhang, L and Ma, X and Wheeler, TA and Dever, JK and He, P and Bogdanove, AJ and Shan, L}, title = {Activation of three targets by a TAL effector confers susceptibility to bacterial blight of cotton.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {644}, pmid = {39809734}, issn = {2041-1723}, support = {2018-67013-28513//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2020-67013-41537//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 18-123TX//Cotton Incorporated (Cotton Inc.)/ ; IOS-2421016//National Science Foundation (NSF)/ ; }, mesh = {*Plant Diseases/microbiology/genetics ; *Xanthomonas/genetics/pathogenicity/metabolism ; *Transcription Activator-Like Effectors/genetics/metabolism ; *Gossypium/microbiology/genetics ; *Bacterial Proteins/genetics/metabolism ; Virulence/genetics ; CRISPR-Cas Systems ; Gene Editing ; Polysaccharide-Lyases/genetics/metabolism ; Gene Expression Profiling ; Host-Pathogen Interactions/genetics ; Gene Expression Regulation, Bacterial ; }, abstract = {Bacterial transcription activator-like effectors (TALEs) promote pathogenicity by activating host susceptibility (S) genes. To understand the pathogenicity and host adaptation of Xanthomonas citri pv. malvacearum (Xcm), we assemble the genome and the TALE repertoire of three recent Xcm Texas isolates. A newly evolved TALE, Tal7b, activates GhSWEET14a and GhSWEET14b, different from GhSWEET10 targeted by a TALE in an early Xcm isolate. Activation of GhSWEET14a and GhSWEET14b results in water-soaked lesions. Transcriptome profiling coupled with TALE-binding element prediction identify a pectin lyase gene as an additional Tal7b target, quantitatively contributing to Xcm virulence alongside GhSWEET14a/b. CRISPR-Cas9 gene editing supports the function of GhSWEETs in cotton bacterial blight and the promise of disrupting the TALE-binding site in S genes for disease management. Collectively, our findings elucidate the rapid evolution of TALEs in Xanthomonas field isolates and highlight the virulence mechanism wherein TALEs induce multiple S genes to promote pathogenicity.}, }
@article {pmid39807995, year = {2025}, author = {Mahdi, AK and Fitzpatrick, DS and Hagen, DE and McNabb, BR and Urbano Beach, T and Muir, WM and Werry, N and Van Eenennaam, AL and Medrano, JF and Ross, PJ}, title = {Efficient Generation of SOCS2 Knock-Out Sheep by Electroporation of CRISPR-Cas9 Ribonucleoprotein Complex with Dual-sgRNAs.}, journal = {The CRISPR journal}, volume = {8}, number = {1}, pages = {13-25}, doi = {10.1089/crispr.2024.0055}, pmid = {39807995}, issn = {2573-1602}, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Electroporation/methods ; Sheep/genetics ; *Gene Knockout Techniques/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Suppressor of Cytokine Signaling Proteins/genetics ; *Ribonucleoproteins/genetics/metabolism ; Female ; }, abstract = {In mice, naturally occurring and induced mutations in the suppressor of cytokine signaling-2 (Socs2) gene are associated with a high growth phenotype characterized by rapid post-weaning weight gain and 30-50% heavier mature body weight. In this work, we demonstrate an electroporation-based method of producing SOCS2 knock-out (KO) sheep. Electroporation of dual-guide CRISPR-Cas9 ribonucleoprotein complexes targeting SOCS2 was performed 6 h post-fertilization in sheep zygotes. Fifty-two blastocysts were transferred to 13 estrus-synchronized recipients, yielding five live lambs and one stillborn. These lambs all carried mutations predicted to result in SOCS2 KO. Three carried large deletion alleles which evaded detection in initial PCR screening. Off-target analysis using whole genome sequencing comparing the frequency of mutations in regions within 100 bp of possible sgRNA binding sites (up to 4 bp mismatches) and elsewhere in the genome showed no significant difference when comparing unedited control sheep to edited animals (p = 0.71). In conclusion, electroporation of zygotes with dual-guide CRISPR-Cas9 RNPs was effective at generating knock-out sheep with no substantial off-target activity.}, }
@article {pmid39807869, year = {2025}, author = {Sáenz, JS and Rios-Galicia, B and Seifert, J}, title = {Antiviral defense systems in the rumen microbiome.}, journal = {mSystems}, volume = {10}, number = {2}, pages = {e0152124}, pmid = {39807869}, issn = {2379-5077}, support = {327953272 (SE2059/3-1)//Deutsche Forschungsgemeinschaft (DFG)/ ; 202989534 (SE2059/2-2)//Deutsche Forschungsgemeinschaft (DFG)/ ; }, mesh = {*Rumen/microbiology/virology ; Animals ; *Bacteriophages/genetics/physiology ; Archaea/genetics/virology ; *Bacteria/genetics/virology ; *Microbiota ; *Gastrointestinal Microbiome ; Genome, Bacterial ; Host Microbial Interactions ; }, abstract = {The continuous interaction between phages and their respective hosts has resulted in the evolution of multiple bacterial immune mechanisms. However, the diversity and prevalence of antiviral defense systems in complex communities are still unknown. We therefore investigated the diversity and abundance of viral defense systems in 3,038 high-quality bacterial and archaeal genomes from the rumen. In total, 14,241 defense systems and 31,948 antiviral-related genes were identified. Those genes represented 114 unique system types grouped into 49 families. We observed a high prevalence of defense systems in the genomes. However, the number of defense systems, defense system families, and system density varied widely from genome to genome. Additionally, the number of defense system per genome correlated positively with the number of defense system families and the genome size. Restriction modification, Abi, and cas system families were the most common, but many rare systems were present in only 1% of the genomes. Antiviral defense systems are prevalent and diverse in the rumen, but only a few are dominant, indicating that most systems are rarely present. However, the collection of systems throughout the rumen may represent a pool of mechanisms that can be shared by different members of the community and modulate the phage-host interaction.IMPORTANCEPhages may act antagonistically at the cell level but have a mutualistic interaction at the microbiome level. This interaction shapes the structure of microbial communities and is mainly driven by the defense mechanism. However, the diversity of such mechanism is larger than previously thought. Because of that, we described the abundance and diversity of the antiviral defense system of a collection of genomes, metagenome-assembled genomes (MAGs) and isolates, from the rumen. While defense mechanisms seem to be prevalent among bacteria and archaea, only a few were common. This suggests that most of these defense mechanisms are not present in many rumen microbes but could be shared among different members of the microbial community. This is consistent with the "pan-immune system" model, which appears to be common across different environments.}, }
@article {pmid39807514, year = {2025}, author = {Saha, D and Panda, AK and Datta, S}, title = {Critical considerations and computational tools in plant genome editing.}, journal = {Heliyon}, volume = {11}, number = {1}, pages = {e41135}, pmid = {39807514}, issn = {2405-8440}, abstract = {Recent advances in genome editing tools and CRISPR-Cas technologies have enabled plant genome engineering reach new heights. The current regulatory exemptions for certain categories of genome edited products, such as those derived from SDN-1 and SDN-2, which are free of any transgene, have significantly accelerated genome editing research in a number of agricultural crop plants in different countries. Although CRISPR-Cas technology is becoming increasingly popular, it is still important to carefully consider a number of factors before planning and carrying conducting CRISPR-Cas studies. To attempt genome editing in a plant, a high-quality genome sequence and a repeatable tissue culture protocol for in vitro regeneration are essential. One of the most important steps in plant genome editing is the designing of a CRISPR construct, which involves selecting the appropriate Cas protein, sgRNA sequence, and appropriate regulatory sequence to trigger expression. Computational tools and algorithms play a crucial role in construct design and gRNA selection to minimize off-target effects and also to optimize their delivery techniques. Researchers may need to select appropriate software tools capable of analyzing post-editing detection of mutation events and other DNA sequence abnormalities to identify off-target effects. To fully fulfill the potential of plant genome editing, continued advances in computational biology are essential to meet the challenges it faces today.}, }
@article {pmid39806509, year = {2025}, author = {Allais-Bonnet, A and Richard, C and André, M and Gelin, V and Deloche, MC and Lamadon, A and Morin, G and Mandon-Pépin, B and Canon, E and Thépot, D and Laubier, J and Moazami-Goudarzi, K and Laffont, L and Dubois, O and Fassier, T and Congar, P and Lasserre, O and Aguirre-Lavin, T and Vilotte, JL and Pailhoux, E}, title = {CRISPR/Cas9-editing of PRNP in Alpine goats.}, journal = {Veterinary research}, volume = {56}, number = {1}, pages = {11}, pmid = {39806509}, issn = {1297-9716}, support = {101000226//Horizon 2020 Framework Programme/ ; }, mesh = {Animals ; *Goats/genetics ; *CRISPR-Cas Systems ; *Gene Editing/veterinary ; Male ; Female ; *Prion Proteins/genetics/metabolism ; Gene Knockout Techniques/veterinary ; Prion Diseases/veterinary/genetics ; *Goat Diseases/genetics ; *Prions/genetics ; }, abstract = {Misfolding of the cellular PrP (PrP[c]) protein causes prion disease, leading to neurodegenerative disorders in numerous mammalian species, including goats. A lack of PrP[c] induces complete resistance to prion disease. The aim of this work was to engineer Alpine goats carrying knockout (KO) alleles of PRNP, the PrP[c]-encoding gene, using CRISPR/Cas9-ribonucleoproteins and single-stranded donor oligonucleotides. The targeted region preceded the PRNP[Ter] mutation previously described in Norwegian goats. Genome editors were injected under the zona pellucida prior to the electroporation of 565 Alpine goat embryos/oocytes. A total of 122 two-cell-stage embryos were transferred to 46 hormonally synchronized recipient goats. Six of the goats remained pregnant and naturally gave birth to 10 offspring. Among the 10 newborns, eight founder animals carrying PRNP genome-edited alleles were obtained. Eight different mutated alleles were observed, including five inducing KO mutations. Three founders carried only genome-edited alleles and were phenotypically indistinguishable from their wild-type counterparts. Among them, one male carrying a one base pair insertion leading to a KO allele is currently used to rapidly extend a PRNP-KO line of Alpine goats for future characterization. In addition to KO alleles, a PRNP[del6] genetic variant has been identified in one-third of founder animals. This new variant will be tested for its potential properties with respect to prion disease. Future studies will also evaluate the effects of genetic background on other characters associated with PRNP KO, as previously described in the Norwegian breed or other species.}, }
@article {pmid39806441, year = {2025}, author = {Lin, Y and Li, C and Chen, Y and Gao, J and Li, J and Huang, C and Liu, Z and Wang, W and Zheng, X and Song, X and Wu, J and Wu, J and Luo, OJ and Tu, Z and Li, S and Li, XJ and Lai, L and Yan, S}, title = {RNA-Targeting CRISPR/CasRx system relieves disease symptoms in Huntington's disease models.}, journal = {Molecular neurodegeneration}, volume = {20}, number = {1}, pages = {4}, pmid = {39806441}, issn = {1750-1326}, mesh = {Animals ; *Huntington Disease/genetics/therapy ; *CRISPR-Cas Systems/genetics ; Humans ; Disease Models, Animal ; Mice ; *Huntingtin Protein/genetics ; HEK293 Cells ; Swine ; *Gene Editing/methods ; }, abstract = {BACKGROUND: HD is a devastating neurodegenerative disorder caused by the expansion of CAG repeats in the HTT. Silencing the expression of mutated proteins is a therapeutic direction to rescue HD patients, and recent advances in gene editing technology such as CRISPR/CasRx have opened up new avenues for therapeutic intervention.<br><br>METHODS: The CRISPR/CasRx system was employed to target human HTT exon 1, resulting in an efficient knockdown of HTT mRNA. This therapeutic effect was substantiated in various models: HEK 293&#xa0;T cell, the HD 140Q-KI mouse, and the HD-KI pig model. The efficiency of the knockdown was analyzed through Western blot and RT-qPCR. Additionally, neuropathological changes were examined using Western blot, immunostaining, and RNA sequencing. The impact on motor abilities was assessed via behavioral experiments, providing a comprehensive evaluation of the treatment's effectiveness.<br><br>RESULTS: CRISPR/CasRx system can significantly reduce HTT mRNA levels across various models, including HEK 293&#xa0;T cells, HD 140Q-KI mice at various disease stages, and HD-KI pigs, and resulted in decreased expression of mHTT. Utilizing the CRISPR/CasRx system to knock down HTT RNA has shown to ameliorate gliosis in HD 140Q-KI mice and delay neurodegeneration in HD pigs.<br><br>CONCLUSIONS: These findings highlight the effectiveness of the RNA-targeting CRISPR/CasRx as a potential therapeutic strategy for HD. Furthermore, the success of this approach provides valuable insights and novel avenues for the treatment of other genetic disorders caused by gene mutations.}, }
@article {pmid39806115, year = {2025}, author = {Zhang, X and Sun, R and Zheng, H and Qi, Y}, title = {Amplification-free sensitive detection of Staphylococcus aureus by spherical nucleic acid triggered CRISPR/Cas12a and Poly T-Cu reporter.}, journal = {Mikrochimica acta}, volume = {192}, number = {2}, pages = {76}, pmid = {39806115}, issn = {1436-5073}, support = {82073602//National Natural Science Foundation of China/ ; 20200201081JC//Natural Science Foundation of Jilin Province/ ; }, mesh = {*Staphylococcus aureus/isolation &amp; purification ; *CRISPR-Cas Systems ; Copper/chemistry ; *Biosensing Techniques/methods ; Limit of Detection ; Metal Nanoparticles/chemistry ; Aptamers, Nucleotide/chemistry ; *Endodeoxyribonucleases/metabolism/genetics ; Gold/chemistry ; Bacterial Proteins/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; Fluorescent Dyes/chemistry ; }, abstract = {A spherical nucleic acid (SNA, AuNPs-aptamer) into CRISPR/Cas12a system combined with poly T-template copper nanoparticles as fluorescence reporter was fabricated to establish an amplification-free sensitive method for Staphylococcus aureus (S. aureus) detection. This method, named PTCas12a, utilizes the concept that the bifunction of SNA recognizes the S. aureus and triggers the Cas12a cleavage activity. Then, the Cas12a enzyme cleaves the Poly T40 to generate a signal change in Poly T-Cu fluorescence, indicating the presence or absence of the target bacteria. The PTCas12a platform demonstrated a detection limit as low as 3.0 CFU/mL (3 N/S) in a wide response range of 1.0 × 10[1]-1.0 × 10[6] CFU/mL for S. aureus detection, which holds significant potential in ensuring food safety and preventing the spread of diseases.}, }
@article {pmid39806080, year = {2025}, author = {Li, Y and Zeng, Z and Lv, X and Jiang, H and Li, A and Liu, Y and Deng, Y and Li, X}, title = {A POCT assay based on commercial HCG strip for miRNA21 detection by integrating with RCA-HCR cascade amplification and CRISPR/Cas12a.}, journal = {Mikrochimica acta}, volume = {192}, number = {2}, pages = {73}, pmid = {39806080}, issn = {1436-5073}, mesh = {*MicroRNAs/blood/genetics/analysis ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Chorionic Gonadotropin/chemistry ; *Point-of-Care Testing ; Limit of Detection ; Nucleic Acid Hybridization ; *Reagent Strips/chemistry ; }, abstract = {A point-of-care testing (POCT) assay based on commercial HCG strip was proposed for miRNA21 detection by integrating RCA-HCR cascaded isothermal amplification with CRISPR/Cas12a. Three modules were integrated in the proposed platform: target amplification module composed of rolling circle amplification (RCA) cascaded with hybridization chain reaction (HCR), signal transduction module composed of CRISPR/Cas12a combined with HCG-agarose gel beads probes, and signal readout module composed of commercial HCG strips. The proposed RCA-HCR-CRISPR/Cas12a-HCG strip assay for miRNA21 detection had high sensitivity, and the limit of detection was as low as 37 fM. The proposed assay showed excellent specificity for miRNA21, as other miRNAs did not caused interference for detection. The recoveries of miRNA21 were ranged from 89.0 to 118.0%. The intra-batch and inter-batch coefficient of variation (CV) were 10.1-13.4% and 11.9-14.5%, respectively, which indicated a high accuracy and precision, and the serum matrix did not cause any interference. With the advantages of low-cost, high sensitivity, visualization, and easy popularization, the proposed assay is expected to provide a powerful tool for early diagnosis of tumor disease miRNA, especially in resource-limited areas.}, }
@article {pmid39806065, year = {2025}, author = {Chen, X and Ghanizada, M and Mallajosyula, V and Sola, E and Capasso, R and Kathuria, KR and Davis, MM}, title = {Differential roles of human CD4[+] and CD8[+] regulatory T cells in controlling self-reactive immune responses.}, journal = {Nature immunology}, volume = {26}, number = {2}, pages = {230-239}, pmid = {39806065}, issn = {1529-2916}, support = {U19 AI057229/AI/NIAID NIH HHS/United States ; AI057229//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; }, mesh = {Humans ; *T-Lymphocytes, Regulatory/immunology ; Forkhead Transcription Factors/genetics/metabolism ; *CD8-Positive T-Lymphocytes/immunology ; Granzymes/genetics/metabolism ; Animals ; *Autoimmunity/immunology ; Autoantigens/immunology ; Mice ; Palatine Tonsil/immunology/cytology ; *CD4-Positive T-Lymphocytes/immunology ; CRISPR-Cas Systems ; }, abstract = {Here we analyzed the relative contributions of CD4[+] regulatory T cells expressing Forkhead box protein P3 (FOXP3) and CD8[+] regulatory T cells expressing killer cell immunoglobulin-like receptors to the control of autoreactive T and B lymphocytes in human tonsil-derived immune organoids. FOXP3 and GZMB respectively encode proteins FOXP3 and granzyme B, which are critical to the suppressive functions of CD4[+] and CD8[+] regulatory T cells. Using CRISPR-Cas9 gene editing, we were able to achieve a reduction of ~90-95% in the expression of these genes. FOXP3 knockout in tonsil T cells led to production of antibodies against a variety of autoantigens and increased the affinity of influenza-specific antibodies. By contrast, GZMB knockout resulted in an increase in follicular helper T cells, consistent with the ablation of CD8[+] regulatory T cells observed in mouse models, and a marked expansion of autoreactive CD8[+] and CD4[+] T cells. These findings highlight the distinct yet complementary roles of CD8[+] and CD4[+] regulatory T cells in regulating cellular and humoral responses to prevent autoimmunity.}, }
@article {pmid39805934, year = {2025}, author = {Zhao, Y and Huang, Z and Zhou, X and Teng, W and Liu, Z and Wang, W and Tang, S and Liu, Y and Liu, J and Wang, W and Chai, L and Zhang, N and Guo, W and Liu, J and Ni, Z and Sun, Q and Wang, Y and Zong, Y}, title = {Precise deletion, replacement and inversion of large DNA fragments in plants using dual prime editing.}, journal = {Nature plants}, volume = {11}, number = {2}, pages = {191-205}, pmid = {39805934}, issn = {2055-0278}, mesh = {*Gene Editing/methods ; *Triticum/genetics ; Solanum lycopersicum/genetics ; Nicotiana/genetics ; *Genome, Plant/genetics ; *DNA, Plant/genetics ; Plants, Genetically Modified/genetics ; CRISPR-Cas Systems ; Sequence Deletion ; }, abstract = {Precise manipulation of genome structural variations holds great potential for plant trait improvement and biological research. Here we present a genome-editing approach, dual prime editing (DualPE), that efficiently facilitates precise deletion, replacement and inversion of large DNA fragments in plants. In our experiments, DualPE enabled the production of specific genomic deletions ranging from ~500 bp to 2 Mb in wheat protoplasts and plants. DualPE was effective in directly replacing wheat genomic fragments of up to 258 kb with desired sequences in the absence of donor DNA. Additionally, DualPE allowed precise DNA inversions of up to 205.4 kb in wheat plants with efficiencies of up to 51.5%. DualPE also successfully edited large DNA fragments in the dicots Nicotiana benthamiana and tomato, with editing efficiencies of up to 72.7%. DualPE thus provides a precise and efficient approach for large DNA sequence and chromosomal engineering, expanding the availability of precision genome-editing tools for crop improvement.}, }
@article {pmid39805705, year = {2025}, author = {Dahl-Jessen, M and Terkelsen, T and Bak, RO and Jensen, UB}, title = {Characterization of the role of spatial proximity of DNA double-strand breaks in the formation of CRISPR-Cas9-induced large structural variations.}, journal = {Genome research}, volume = {35}, number = {2}, pages = {231-241}, pmid = {39805705}, issn = {1549-5469}, mesh = {Humans ; *DNA Breaks, Double-Stranded ; *CRISPR-Cas Systems ; K562 Cells ; *Genomic Structural Variation ; RNA, Guide, CRISPR-Cas Systems/genetics ; DNA Repair ; Genome, Human ; Gene Editing ; }, abstract = {Structural variations (SVs) play important roles in genetic diversity, evolution, and carcinogenesis and are, as such, important for human health. However, it remains unclear how spatial proximity of double-strand breaks (DSBs) affects the formation of SVs. To investigate if spatial proximity between two DSBs affects DNA repair, we used data from 3C experiments (Hi-C, ChIA-PET, and ChIP-seq) to identify highly interacting loci on six different chromosomes. The target regions correlate with the borders of megabase-sized topologically associated domains (TADs), and we used CRISPR-Cas9 nuclease and pairs of single guide RNAs (sgRNAs) against these targets to generate DSBs in both K562 cells and H9 human embryonic stem cells (hESCs). Droplet digital PCR (ddPCR) was used to quantify the resulting recombination events, and high-throughput sequencing was used to analyze the chimeric junctions created between the two DSBs. We observe a significantly higher formation frequency of deletions and inversions with DSBs in proximity compared with deletions and inversions with DSBs not in proximity in K562 cells. Additionally, our results suggest that DSB proximity may affect the ligation of chimeric deletion junctions. Taken together, spatial proximity between DSBs is a significant predictor of large-scale deletion and inversion frequency induced by CRISPR-Cas9 in K562 cells. This finding has implications for understanding SVs in the human genome and for the future application of CRISPR-Cas9 in gene editing and the modeling of rare SVs.}, }
@article {pmid39804955, year = {2025}, author = {Weasner, BM and Weasner, BP and Cook, KR and Stinchfield, MJ and Kondo, S and Saito, K and Kumar, JP and Newfeld, SJ}, title = {A new Drosophila melanogaster research resource: CRISPR-induced mutations for clonal analysis of fourth chromosome genes.}, journal = {G3 (Bethesda, Md.)}, volume = {15}, number = {3}, pages = {}, pmid = {39804955}, issn = {2160-1836}, support = {P40 OD018537/OD/NIH HHS/United States ; R24 OD028242/OD/NIH HHS/United States ; U24 HG013300/HG/NHGRI NIH HHS/United States ; }, mesh = {Animals ; *Drosophila melanogaster/genetics ; Female ; *Mutation ; *CRISPR-Cas Systems ; *Chromosomes, Insect/genetics ; Phenotype ; Male ; *Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {As part of an ongoing effort to generate comprehensive resources for the experimental analysis of fourth chromosome genes in Drosophila melanogaster, the Fourth Chromosome Resource Project has used CRISPR mutagenesis with single guide RNAs to isolate mutations in 62 of the 80 fourth chromosome, protein-coding genes. These mutations were induced on a fourth chromosome bearing a basal FRT insertion to facilitate experimental approaches involving FLP recombinase-induced mitotic recombination. To permit straightforward comparisons among mutant stocks, most of the mutations were generated on isogenic fourth chromosomes, which were then crossed into a common genetic background. Of the 119 mutations, 84 are frameshift mutations likely to be null alleles, 29 are small, in-frame deletions, and 6 have yet to be characterized molecularly. The mutations were tested for recessive lethal, female-sterile, and visible phenotypes. Stable stocks for most of the mutations have been submitted to repositories in the United States and Japan for public distribution.}, }
@article {pmid39804945, year = {2025}, author = {Huang, Y and Pei, S and Lv, X and Yang, F and Gong, X and Li, N and Guo, Y and Feng, Y and Xiao, L}, title = {Stage-specific expression and divergent functions of two insulinase-like proteases associated with host infectivity in Cryptosporidium.}, journal = {PLoS neglected tropical diseases}, volume = {19}, number = {1}, pages = {e0012777}, pmid = {39804945}, issn = {1935-2735}, mesh = {Animals ; *Cryptosporidiosis/parasitology/pathology ; Mice ; *Cryptosporidium parvum/enzymology/genetics/pathogenicity/growth &amp; development ; Mice, Knockout ; *Protozoan Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Peptide Hydrolases/genetics/metabolism ; Female ; Humans ; }, abstract = {BACKGROUND: The determinants of differences in host infectivity among Cryptosporidium species and subtypes are poorly understood. Results from recent comparative genomic studies suggest that gains and losses of multicopy subtelomeric genes encoding insulinase-like proteases (INS-19 and INS-20 in Cryptosporidium parvum and their orthologs in closely related species) may potentially contribute to these differences.<br><br>In this study, we investigated the expression and biological function of the INS-19 and INS-20 of C. parvum. CRISPR/Cas9 was used to endogenously tag both genes with the hemagglutinin epitope. Immunofluorescence analysis revealed that INS-19 and INS-20 are expressed at different developmental stages of the pathogen. Although knockout of either had no detectable effect on the in vitro growth of C. parvum, knockout of INS-20, deletion of its multiple domains, or mutation of the active motif in the functional domain reduced the intensity of C. parvum infection in IFN-&#x3b3; knockout mice. Consistent with this, mice infected with the INS-20-deleted mutant had reduced intestinal damage and parasite burden.<br><br>CONCLUSIONS/SIGNIFICANCE: These results suggest that INS-19 and INS-20 have stage-specific expression with distinct biological functions, and that the presence of the INS-20 in zoonotic C. parvum contributes to its infectivity and fitness in mice.}, }
@article {pmid39804925, year = {2025}, author = {Ricou, A and Simon, M and Duflos, R and Azzopardi, M and Roux, F and Budar, F and Camilleri, C}, title = {Identification of novel genes responsible for a pollen killer present in local natural populations of Arabidopsis thaliana.}, journal = {PLoS genetics}, volume = {21}, number = {1}, pages = {e1011451}, pmid = {39804925}, issn = {1553-7404}, mesh = {*Arabidopsis/genetics ; *Pollen/genetics ; *Arabidopsis Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Mitochondria/genetics ; Alleles ; Genes, Plant ; }, abstract = {Gamete killers are genetic loci that distort segregation in the progeny of hybrids because the killer allele promotes the elimination of the gametes that carry the sensitive allele. They are widely distributed in eukaryotes and are important for understanding genome evolution and speciation. We had previously identified a pollen killer in hybrids between two distant natural accessions of Arabidopsis thaliana. This pollen killer involves three genetically linked genes, and we previously reported the identification of the gene encoding the antidote that protects pollen grains from the killer activity. In this study, we identified the two other genes of the pollen killer by using CRISPR-Cas9 induced mutants. These two genes are necessary for the killer activity that we demonstrated to be specific to pollen. The cellular localization of the pollen killer encoded proteins suggests that the pollen killer activity involves the mitochondria. Sequence analyses reveal predicted domains from the same families in the killer proteins. In addition, the C-terminal half of one of the killer proteins is identical to the antidote, and one amino acid, crucial for the antidote activity, is also essential for the killer function. Investigating more than 700 worldwide accessions of A. thaliana, we confirmed that the locus is subject to important structural rearrangements and copy number variation. By exploiting available de novo genomic sequences, we propose a scenario for the emergence of this pollen killer in A. thaliana. Furthermore, we report the co-occurrence and behavior of killer and sensitive genotypes in several local populations, a prerequisite for studying gamete killer evolution in the wild. This highlights the potential of the Arabidopsis model not only for functional studies of gamete killers but also for investigating their evolutionary trajectories at complementary geographical scales.}, }
@article {pmid39804665, year = {2025}, author = {Liu, X and Huang, L and Li, M and Fu, Y and Zhang, W and Zhang, S and Liang, X and Shen, Q}, title = {An Efficient and Cost-Effective Novel Strategy for Identifying CRISPR-Cas-Mediated Mutants in Plant Offspring.}, journal = {The CRISPR journal}, volume = {8}, number = {1}, pages = {26-36}, doi = {10.1089/crispr.2024.0057}, pmid = {39804665}, issn = {2573-1602}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods/economics ; Cost-Benefit Analysis ; Polymerase Chain Reaction/methods/economics ; INDEL Mutation ; Mutation ; Plants, Genetically Modified/genetics ; Arabidopsis/genetics ; Mutagenesis ; *Plants/genetics ; }, abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system has revolutionized targeted mutagenesis, but screening for mutations in large sample pools can be time-consuming and costly. We present an efficient and cost-effective polymerase chain reaction (PCR)-based strategy for identifying edited mutants in the T1 generation. Unlike previous methods, our approach addresses the challenges of large progeny populations by using T0 generation sequencing results for genotype prediction. The T1 generation plants were then divided into two scenarios: ≥4 bp indels and 1-2 bp indels. Specific primers are designed for these categories, employing dual-primers critical annealing temperature PCR for ≥4 bp indels and the derived cleaved amplified polymorphic sequences (dCAPS) method for 1-2 bp indels. This method is straightforward, cost-effective, and allows rapid and precise identification of T1 editing outcomes, distinguishing between wild-type, heterozygous, and homozygous plants. This strategy accelerates gene functional analysis in plants and beyond.}, }
@article {pmid39804663, year = {2025}, author = {Wang, C and Sun, C and Shi, L and Zhou, J and Liu, S and Bai, Y and Yu, W}, title = {Establishment of a CRISPR-Cas9-Mediated Genome Editing System in Flax.}, journal = {The CRISPR journal}, volume = {8}, number = {1}, pages = {51-59}, doi = {10.1089/crispr.2024.0064}, pmid = {39804663}, issn = {2573-1602}, mesh = {*Flax/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; Genome, Plant ; Oxidoreductases/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Engineering/methods ; Mutation ; }, abstract = {Flax is an important crop used for oil and fiber production. Although genetic engineering has been possible in flax, it is not commonly used to produce cultivars. However, the use of genome editing technology, which can produce site-specific mutations without introducing foreign genes, may be a valuable tool for creating elite cultivars that can be easily cultivated. The purpose of this study was to investigate the potential of genome editing in flax by establishing the clustered regularly interspaced short palindromic repeats (CR ISPR)-CRISPR-associated protein 9 (CRISPR-Cas9) genome editing system using the phytoene desaturase (PDS) gene, which produces albino mutants that are easily identifiable. Four sgRNAs were designed from two PDS genes of Flax (LuPDS1 and LuPDS2), and CRISPR-Cas9 genome editing vectors were constructed. After gene transformation, albino phenotypes were observed in transformed callus and regenerated plantlets on selection media. Polymerase chain reaction (PCR) amplification and sequencing of the PDS genes revealed deletions and insertions in the albino tissues, indicating successful editing of the PDS genes. Potential off-target sites were analyzed, but no off-target mutations were found, indicating the specificity of the CRISPR-Cas9 system. The establishment of a flax genome editing system using the CRISPR-Cas9 technology opens up new possibilities for the genetic engineering of flax. This study demonstrates the potential of genome editing in creating elite cultivars that can be easily cultivated, which can have significant implications for the flax industry.}, }
@article {pmid39804565, year = {2025}, author = {Gu, B and Li, M and Li, D and Huang, K}, title = {CRISPR-Cas9 Targeting PCSK9: A Promising Therapeutic Approach for Atherosclerosis.}, journal = {Journal of cardiovascular translational research}, volume = {18}, number = {2}, pages = {424-441}, pmid = {39804565}, issn = {1937-5395}, mesh = {Humans ; *Proprotein Convertase 9/genetics/metabolism ; *CRISPR-Cas Systems ; *Atherosclerosis/genetics/therapy/enzymology/blood/diagnosis ; *Gene Editing/methods ; *Genetic Therapy/methods/adverse effects ; Animals ; Biomarkers/blood ; *Cholesterol, LDL/blood ; Genetic Predisposition to Disease ; Treatment Outcome ; }, abstract = {CRISPR-Cas9 gene editing technology, as an innovative biomedical tool, holds significant potential in the prevention and treatment of atherosclerosis. By precisely editing key genes such as PCSK9, CRISPR-Cas9 offers the possibility of long-term regulation of low-density lipoprotein cholesterol (LDL-C), which may reduce the risk of cardiovascular diseases. Early clinical studies of gene editing therapies like VERVE-101 have yielded encouraging results, highlighting both the feasibility and potential efficacy of this technology. However, clinical applications still face challenges such as off-target effects, immunogenicity, and long-term safety. Future research should focus on enhancing the specificity and efficiency of gene editing, optimizing delivery systems, and improving personalized treatment strategies. Additionally, the establishment of ethical and legal regulatory frameworks will be critical for the safe adoption of this technology. With the continued advancement of gene editing technology, CRISPR-Cas9 may become an important tool for treating atherosclerosis and other complex diseases.}, }
@article {pmid39804466, year = {2025}, author = {Birling, MC and Hérault, Y and Pavlovic, G}, title = {Genome engineering with Cas9 and AAV repair templates, successes and pitfalls.}, journal = {Mammalian genome : official journal of the International Mammalian Genome Society}, volume = {36}, number = {2}, pages = {376-383}, pmid = {39804466}, issn = {1432-1777}, mesh = {*Dependovirus/genetics ; Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genetic Engineering/methods ; Genetic Vectors/genetics ; Humans ; Genome ; }, abstract = {Genome editing, in particular the CRISPR/Cas9 system, is widely used to generate new animal models. However, the generation of mutations, such as conditional knock-out or knock-in, can remain complex and inefficient, in particular because of the difficulty to deliver the donor DNA (single or double stranded) into the nucleus of fertilized oocytes. The use of recombinant adeno-associated viruses (rAAV) as donor DNA is a rapidly developing approach that promises to improve the efficiency of creation of animal models. In this mini-review, we explore the progress and challenges of using CRISPR/Cas9 in combination with rAAV for precise genome editing. We will summarise the current knowledge of rAAV transduction, data on its use in rodent embryos in combination with CRISPR/Cas9 to easily generate sequence replacements or insertions, the limitations of rAAV and the unexpected events observed to date, and the protocol optimisations already in place to facilitate its use in the generation of animal models.}, }
@article {pmid39804233, year = {2025}, author = {Wang, H and Zhao, R and Zhang, B and Xiao, Y and Yu, C and Wang, Y and Yu, C and Tang, Y and Li, Y and Lu, B and Li, B}, title = {Accurate Molecular Sensing based on a Modular and Customizable CRISPR/Cas-Assisted Nanopore Operational Nexus (CANON).}, journal = {Angewandte Chemie (International ed. in English)}, volume = {64}, number = {13}, pages = {e202423473}, doi = {10.1002/anie.202423473}, pmid = {39804233}, issn = {1521-3773}, support = {11111/NR/NINR NIH HHS/United States ; SKL202402017, SKL202302030//Science and Technology Development Plan Project of Jilin Province/ ; 22474135, 22074136//Natural Science Foundation of China/ ; 20240101005JJ//The Jilin Province Science Fund for Distinguished Young Scholars/ ; 23GZZ03//Major Project of Changchun State Key Laboratory/ ; }, mesh = {*CRISPR-Cas Systems ; *Nanopores ; *DNA/analysis/chemistry/genetics ; *Biosensing Techniques ; MicroRNAs/analysis/genetics ; }, abstract = {Solid-state nanopore is a promising single molecular detection technique, but is largely limited by relatively low resolution to small-size targets and laborious design of signaling probes. Here we establish a universal, CRISPR/Cas-Assisted Nanopore Operational Nexus (CANON), which can accurately transduce different targeting sources/species into different DNA structural probes via a "Signal-ON" mode. Target recognition activates the cleavage activity of a Cas12a/crRNA system and then completely digest the blocker of an initiator. The unblocked initiator then triggers downstream DNA assembly reaction and generate a large-size structure easy for nanopore detection. Such integration of Cas12a/crRNA with DNA assembly establishes an accurate correspondence among the input targets, output DNA structures, and the ultimate nanopore signals. We demonstrated dsDNA, long RNA (i.e., Flu virus gene), short microRNA (i.e., let-7d) and non-nucleic acids (i.e., Pb[2+]) as input paradigms. Various structural assembly reactions, such as hybridization chain reaction (HCR), G-HCR and duplex polymerization strategy (DPS), are adapted as outputs for nanopore signaling. Simultaneous assay is also verified via transferring FluA and FluB genes into HCR and G-HCR, respectively. CANON is thus a modular sensing platform holding multiple advantages such as high accuracy, high resolution and high universality, which can be easily customized into various application scenes.}, }
@article {pmid39804120, year = {2025}, author = {Libby, ARG and Rito, T and Radley, A and Briscoe, J}, title = {An in vivo CRISPR screen in chick embryos reveals a role for MLLT3 in specification of neural cells from the caudal epiblast.}, journal = {Development (Cambridge, England)}, volume = {152}, number = {3}, pages = {}, pmid = {39804120}, issn = {1477-9129}, support = {//Francis Crick Institute/ ; ALTF 149-2020//European Molecular Biology Organization/ ; /WT_/Wellcome Trust/United Kingdom ; 226633/Z/22/Z//Wellcome/ ; 742138//Horizon 2020/ ; /ERC_/European Research Council/International ; }, mesh = {Animals ; Chick Embryo ; Neural Tube/embryology/metabolism/cytology ; Gene Expression Regulation, Developmental ; *Germ Layers/cytology/metabolism/embryology ; *Neurons/cytology/metabolism ; Cell Differentiation/genetics ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Neural Stem Cells/metabolism/cytology ; }, abstract = {Tissue development relies on the coordinated differentiation of stem cells in dynamically changing environments. The formation of the vertebrate neural tube from stem cells in the caudal lateral epiblast is a well-characterized example. Despite an understanding of the signalling pathways involved, the gene regulatory mechanisms remain poorly defined. To address this, we developed a multiplexed in vivo CRISPR screening approach in chick embryos targeting genes expressed in the caudal epiblast and neural tube. This revealed a role for MLLT3, a component of the super elongation complex, in the specification of neural fate. Perturbation of MLLT3 disrupted neural tube morphology and reduced neural fate acquisition. Mutant forms of retinoic acid receptor A lacking the MLLT3 binding domain similarly reduced neural fate acquisition. Together, these findings validate an in vivo CRISPR screen strategy in chick embryos and identify a previously unreported role for MLLT3 in caudal neural tissue specification.}, }
@article {pmid39803701, year = {2025}, author = {Deniz, AH and Aydemir, E and Ekimci Gürcan, N and Dabo Jakir, A and Faruk Bayrak, &#xd6; and Şahin, F and Ayaz, F}, title = {Improved Malignancy of Colon Cancer Cells at Gene Expression Level With Constitutive Activation of the Eukaryotic Elongation Factor 2 Under Nutrition-Deficient Conditions.}, journal = {Chemistry &amp; biodiversity}, volume = {22}, number = {5}, pages = {e202402547}, doi = {10.1002/cbdv.202402547}, pmid = {39803701}, issn = {1612-1880}, support = {B&#x130;RUN&#x130;-BAP-06-2023-01-01//Biruni University/ ; BAP2023-01-01//Biruni University/ ; }, mesh = {Humans ; *Colonic Neoplasms/pathology/genetics/metabolism ; HCT116 Cells ; *Peptide Elongation Factor 2/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; }, abstract = {Regulation of protein production in response to physiological signals is achieved through precise control of eukaryotic Elongation Factor 2 (eEF2), whose distinct translocase function is crucial for cell survival. Phosphorylation of eEF2 at its Thr56 (T56) residue inactivates this function in translation. Using a genetically modified paralogue of a colon cancer cell line, HCT116 which carries a point mutation at Ser595-to-Alanine in the eEF2 gene we were able to create a constitutively active form of eEF2. Then, we compared these cells with their wild-type counterparts under complete growth and nutrient deprivation conditions. The mutant forms were created by using the CRISPR/Cas9-mediated gene cassette knock-in (KI) strategy. The samples were analyzed by a microarray-based approach. Carrying out a WikiPath analysis, we identified distinct signaling pathways and gene expression patterns that are differentially regulated in the mutant cells (HCT116[KI)], but not in wild-type HCT116 (HCT116[WT]) cells, particularly under nutrient-deprived conditions. Our results suggest that the constitutive activation of eEF2 enables an increase in the malignant gene expression in colon cancer cells compared to the wild-type counterparts under nutrient-deprived conditions.}, }
@article {pmid39803585, year = {2024}, author = {Wei, SC and Cantor, AJ and Walleshauser, J and Mepani, R and Melton, K and Bans, A and Khekare, P and Gupta, S and Wang, J and Soares, C and Kiwan, R and Lee, J and McCawley, S and Jani, V and Leong, WI and Shahi, PK and Chan, J and Boivin, P and Otoupal, P and Pattnaik, BR and Gamm, DM and Saha, K and Gowen, BG and Haak-Frendscho, M and Janatpour, MJ and Silverman, AP}, title = {Evaluation of subretinally delivered Cas9 ribonucleoproteins in murine and porcine animal models highlights key considerations for therapeutic translation of genetic medicines.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, pmid = {39803585}, issn = {2692-8205}, support = {U19 NS132296/NS/NINDS NIH HHS/United States ; }, abstract = {Genetic medicines, including CRISPR/Cas technologies, extend tremendous promise for addressing unmet medical need in inherited retinal disorders and other indications; however, there remain challenges for the development of therapeutics. Herein, we evaluate genome editing by engineered Cas9 ribonucleoproteins (eRNP) in vivo via subretinal administration using mouse and pig animal models. Subretinal administration of adenine base editor and double strand break-inducing Cas9 nuclease eRNPs mediate genome editing in both species. Editing occurs in retinal pigmented epithelium (RPE) and photoreceptor cells, with favorable tolerability in both species. Using transgenic reporter strains, we determine that editing primarily occurs close to the site of administration, within the bleb region associated with subretinal injection. Our results show that subretinal administration of eRNPs in mice mediates base editing of up to 12% of the total neural retina, with an average rate of 7% observed at the highest dose tested. In contrast, a substantially lower editing efficiency was observed in minipigs; even with direct quantification of only the treated region, a maximum base editing rate of 1.5%, with an average rate of <1%, was observed. Our data highlight the importance of species consideration in translational studies for genetic medicines targeting the eye and provide an example of a lack of translation between small and larger animal models in the context of subretinal administration of Cas9 eRNPs.}, }
@article {pmid39803448, year = {2025}, author = {Hossain, KA and Nierzwicki, L and Orozco, M and Czub, J and Palermo, G}, title = {Flexibility in PAM Recognition Expands DNA Targeting in xCas9.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, pmid = {39803448}, issn = {2692-8205}, support = {R01 GM141329/GM/NIGMS NIH HHS/United States ; }, abstract = {xCas9 is an evolved variant of the CRISPR-Cas9 genome editing system, engineered to improve specificity and reduce undesired off-target effects. How xCas9 expands the DNA targeting capability of Cas9 by recognizing a series of alternative Protospacer Adjacent Motif (PAM) sequences while ignoring others is unknown. Here, we elucidate the molecular mechanism underlying xCas9's expanded PAM recognition and provide critical insights for expanding DNA targeting. We demonstrate that while wild-type Cas9 enforces stringent guanine selection through the rigidity of its interacting arginine dyad, xCas9 introduces flexibility in R1335, enabling selective recognition of specific PAM sequences. This increased flexibility confers a pronounced entropic preference, which also improves recognition of the canonical TGG PAM. Furthermore, xCas9 enhances DNA binding to alternative PAM sequences during the early evolution cycles, while favouring binding to the canonical PAM in the final evolution cycle. This dual functionality highlights how xCas9 broadens PAM recognition and underscores the importance of fine-tuning the flexibility of the PAM-interacting cleft as a key strategy for expanding the DNA targeting potential of CRISPR-Cas systems. These findings deepen our understanding of DNA recognition in xCas9 and may apply to other CRISPR-Cas systems with similar PAM recognition requirements.}, }
@article {pmid39801754, year = {2025}, author = {Guha, S and Jagadeesan, Y and Pandey, MM and Mittal, A and Chitkara, D}, title = {Targeting the epigenome with advanced delivery strategies for epigenetic modulators.}, journal = {Bioengineering &amp; translational medicine}, volume = {10}, number = {1}, pages = {e10710}, pmid = {39801754}, issn = {2380-6761}, abstract = {Epigenetics mechanisms play a significant role in human diseases by altering DNA methylation status, chromatin structure, and/or modifying histone proteins. By modulating the epigenetic status, the expression of genes can be regulated without any change in the DNA sequence itself. Epigenetic drugs exhibit promising therapeutic efficacy against several epigenetically originated diseases including several cancers, neurodegenerative diseases, metabolic disorders, cardiovascular disorders, and so forth. Currently, a considerable amount of research is focused on discovering new drug molecules to combat the existing research gap in epigenetic drug therapy. A novel and efficient delivery system can be established as a promising approach to overcome the drawbacks associated with the current epigenetic modulators. Therefore, formulating the existing epigenetic drugs with distinct encapsulation strategies in nanocarriers, including solid lipid nanoparticles, nanogels, bio-engineered nanocarriers, liposomes, surface modified nanoparticles, and polymer-drug conjugates have been examined for therapeutic efficacy. Nonetheless, several epigenetic modulators are untouched for their therapeutic potential through different delivery strategies. This review provides a comprehensive up to date discussion on the research findings of various epigenetics mechanism, epigenetic modulators, and delivery strategies utilized to improve their therapeutic outcome. Furthermore, this review also highlights the recently emerged CRISPR tool for epigenome editing.}, }
@article {pmid39801562, year = {2025}, author = {Nguyen, T and Meleski, LWG and Belavatta, MP and Gurumoorthi, S and Zhang, C and Heins, AL and Zeng, AP}, title = {A Consecutive Genome Engineering Method Reveals a New Phenotype and Regulation of Glucose and Glycerol Utilization in Clostridium Pasteurianum.}, journal = {Engineering in life sciences}, volume = {25}, number = {1}, pages = {e202400026}, pmid = {39801562}, issn = {1618-0240}, abstract = {Clostridium pasteurianum is a microorganism for production of 1,3-propanediol (1,3-PDO) and butanol, but suffers from lacking genetic tools for metabolic engineering to improve product titers. Furthermore, previous studies of C. pasteurianum have mainly focused on single genomic modification. The aim of this work is the development and application of a method for modification of multiple gene targets in the genome of C. pasteurianum. To this end, a new approach for consecutive genome engineering is presented for the first time using a method based on endogenous CRISPR-Cas machineries. A total of three genome modifications were consecutively introduced in the same mutant and the effect of combined changes on the genome was observed by 39% decreased specific glycerol consumption rate and 29% increased 1,3-PDO yield in mixed substrate fermentations at laboratory scale in comparison to the wildtype strain. Additionally, examination of the phenotype of the generated mutant strain led to discovery of 2,3-butanediol (2,3-BDO) production of up to 0.48 g L[-1], and this metabolite was not reported to be produced by C. pasteurianum before. The developed procedure expands the genetic toolkit for C. pasteurianum and provides researchers an additional method which contributes to improved genetic accessibility of this strain.}, }
@article {pmid39800582, year = {2025}, author = {Zhang, Y and Dong, X and Jiang, C and Yu, Y and Zhang, H and Fu, J and Su, G and Liu, Y}, title = {Corrigendum to "A competitive aptamer binding-based CRISPR-cas biosensor for sensitive detection of tetracycline residues in biological samples" [Talanta, Volume 286 (2025), 127491].}, journal = {Talanta}, volume = {286}, number = {}, pages = {127558}, doi = {10.1016/j.talanta.2025.127558}, pmid = {39800582}, issn = {1873-3573}, }
@article {pmid39800499, year = {2025}, author = {Yu, F and Zhang, Q and Ma, T and Zhang, S and Wang, F and Yue, D and Liu, S and Liao, Y and Liu, LE and Wu, Y and Zang, W}, title = {Bifunctional probe propelling multipath strand displacement amplification tandem CRISPR/Cas12a for ultrasensitive and robust assay of DNA methyltransferase activity.}, journal = {Analytica chimica acta}, volume = {1337}, number = {}, pages = {343540}, doi = {10.1016/j.aca.2024.343540}, pmid = {39800499}, issn = {1873-4324}, mesh = {*CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *DNA Modification Methylases/metabolism/analysis ; *Enzyme Assays/methods ; Limit of Detection ; DNA Methylation ; *DNA Probes/chemistry ; DNA/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {BACKGROUND: DNA methylation catalyzed by various DNA methyltransferases (DNA MTases) is one of the important epigenetic regulations in both eukaryotes and prokaryotes. Therefore, the detection of DNA MTase activity is a vital target and direction in the study of methylation-related diseases.<br><br>RESULTS: In this study, an ultrasensitive and robust strategy was developed for DNA MTase activity sensing based on bifunctional probe propelling multipath strand displacement amplification and CRISPR/Cas12a techniques. First, a bifunctional hairpin probe (bHpDNA) was designed instead of a conventional single-function probe. In the presence of DNA MTase, the bHpDNA was methylated and cleaved by a restriction endonuclease into two independent primers, both of which bind with the templates to trigger strand displacement amplification and produce the active DNA of CRISPR/Cas12a. Second, annealing-assisted binding instead of free diffusion adhesion was used to improve hybridization efficiency between the primers and templates. Finally, the CRISPR/Cas12a system was used to achieve fluorescence signal output to analyze DNA MTase activity. If targets were absent, there was no signal because no primers were released from the bHpDNA. To verify the reliability of the method, two key DNA MTases, Dam and M. SssI, were analyzed, and their limits of detection were 2.458&#xa0;&#xd7;&#xa0;10[-3] and 3.820&#xa0;&#xd7;&#xa0;10[-3] U/mL, respectively, which were lower than those of most reported fluorescence methods.<br><br>SIGNIFICANCE: This method was successfully used in the evaluation of DNA MTase inhibitors and the detection of DNA MTase activity in complex biological systems with good recoveries and relative standard deviation at low spiked concentrations (0.1-1 U/mL), which all indicate that this method is an ultrasensitive and robust strategy in DNA MTase activity assay and has great potential in biomedical and clinical detection.}, }
@article {pmid39800033, year = {2025}, author = {Guo, Q and Huang, L and Liu, Y and Qi, C and Shao, H and Peng, J and Zhang, H and Zang, Y and Luo, M and Shen, X and Wang, D and Liu, L and Xu, Q and Zheng, Y and Dong, X and Xu, X}, title = {PcoCas12a: A novel CRISPR enzyme from Prevotella copri enhancing TCR-T-cell tumor suppression.}, journal = {International journal of biological macromolecules}, volume = {298}, number = {}, pages = {139740}, doi = {10.1016/j.ijbiomac.2025.139740}, pmid = {39800033}, issn = {1879-0003}, mesh = {Gene Editing/methods ; *T-Lymphocytes/immunology/metabolism ; Humans ; *CRISPR-Cas Systems ; *Prevotella/enzymology/genetics ; Animals ; *Receptors, Antigen, T-Cell/metabolism/genetics ; Cell Line, Tumor ; Mice ; *Neoplasms/immunology/therapy/genetics ; }, abstract = {Genome editing technologies have been widely utilized in cell engineering, demonstrating immense potential in cell and gene therapy. However, an optimal gene-editing enzyme for immune cell editing remains unidentified. In this study, we identified a novel gene editing enzyme, termed CRISPR/PcoCas12a, derived from Prevotella copri, which recognizes a 5'-YYN PAM sequence. We demonstrated that CRISPR/PcoCas12a offers a broader range of editing sites and superior editing efficiency at specific loci compared to AsCas12a. Furthermore, we illustrated its capability to enhance tumor suppression by targeting DGKα in TCR-T cells. DGKα functions as a negative regulator of T cell function, and its knockout significantly boosts the antitumor efficacy of TCR-T cells. The knockout efficiency and tumor suppressor ability of PcoCas12a targeting DGKα were markedly higher than those achieved with AsCas12a. Single-cell sequencing data confirmed that PcoCas12a-mediated DGKα gene knockout improves the tumor suppressive capabilities of T cells by promoting T-cell activation and strengthening immune regulatory responses. These findings establish PcoCas12a as a highly efficient enzyme for T cell editing, indicating its potential application in T-cell therapy.}, }
@article {pmid39799696, year = {2025}, author = {Jiang, Y and Li, Y and Zheng, D and Du, X and Yang, H and Wang, C and Zhao, M and Xiao, H and Zhang, L and Li, X and Shi, S}, title = {Nano-polymeric platinum activates PAR2 gene editing to suppress tumor metastasis.}, journal = {Biomaterials}, volume = {317}, number = {}, pages = {123090}, doi = {10.1016/j.biomaterials.2025.123090}, pmid = {39799696}, issn = {1878-5905}, mesh = {Animals ; Humans ; *Gene Editing/methods ; *Platinum/chemistry/pharmacology/therapeutic use ; *Neoplasm Metastasis ; CRISPR-Cas Systems/genetics ; *Receptor, PAR-2/genetics/metabolism ; Mice ; Cell Line, Tumor ; Cell Movement/drug effects ; *Polymers/chemistry ; Antineoplastic Agents/pharmacology ; Female ; }, abstract = {Metastasis as the hallmark of cancer preferentially contributes to tumor recurrence and therapy resistance, aggrandizing the lethality of patients with cancer. Despite their robust suppressions of tumor progression, chemotherapeutics failed to attenuate cancer cell migration and even triggered pro-metastatic effects. In parallel, protease-activated receptor 2 (PAR2), a member of the G protein-coupled receptor subfamily, actively participates in cancer metastasis via multiple signal transduction pathways. CRISPR/Cas9 that is a dominating genome editing tool can evoke PAR2 knockout to inhibit cancer metastasis. However, the absence of valid delivery systems largely limits its efficacy. Herein, we nanosized polymeric platinum (NanoPt) as therapeutical drug carries to deliver CRISPR/Cas9 to elicit genome editing of PAR2, which drastically augmented anti-metastatic effects and alleviated systematic toxicity of platinum-based treatment in vitro and in vivo. More importantly, the NanoPt@Cas9-PAR2 initiated PAR2 deficiency to mechanistically attenuate EMT process and ferroptosis via RAGE/ERK signalling, consequently preventing cancer cell migration. Our findings indicate that NanoPt@Cas9-PAR2 that mitigated PAR2 signalling and cytotoxic effects of platinum could be a safe and powerful all-in-one combinatorial strategy for cancer treatment.}, }
@article {pmid39799585, year = {2025}, author = {Bai, S and Cao, X and Hu, L and Hu, D and Li, D and Sun, Y}, title = {Engineering an optimized hypercompact CRISPR/Cas12j-8 system for efficient genome editing in plants.}, journal = {Plant biotechnology journal}, volume = {23}, number = {4}, pages = {1153-1164}, pmid = {39799585}, issn = {1467-7652}, support = {31800206//National Natural Science Foundation of China/ ; 32160111//National Natural Science Foundation of China/ ; 32370431//National Natural Science Foundation of China/ ; NJYT22105//Program for Young Talents of Science and Technology in Universities of Inner Mongolia Autonomous Region/ ; 2020MS03027//Natural Science Foundation of Inner Mongolia Autonomous Region/ ; 2021ZD04//Natural Science Foundation of Inner Mongolia Autonomous Region/ ; 2020ZY0005//Central Government Guiding Special Funds for the Development of Local Science and Technology/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Oryza/genetics ; Glycine max/genetics ; *Genome, Plant/genetics ; Plants, Genetically Modified ; }, abstract = {The Cas12j-8 nuclease, derived from the type V CRISPR system, is approximately half the size of Cas9 and recognizes a 5'-TTN-3' protospacer adjacent motif sequence, thus potentially having broad application in genome editing for crop improvement. However, its editing efficiency remains low in plants. In this study, we rationally engineered both the crRNA and the Cas12j-8 nuclease. The engineered crRNA and Cas12j-8 markedly improved genome editing efficiency in plants. When combined, they exhibited robust editing activity in soybean and rice, enabling the editing of target sites that were previously uneditable. Notably, for certain target sequences, the editing activity was comparable to that of SpCas9 when targeting identical sequences, and it outperformed the Cas12j-2 variant, nCas12j-2, across all tested targets. Additionally, we developed cytosine base editors based on the engineered crRNA and Cas12j-8, demonstrating an average increase of 5.36- to 6.85-fold in base-editing efficiency (C to T) compared with the unengineered system in plants, with no insertions or deletions (indels) observed. Collectively, these findings indicate that the engineered hypercompact CRISPR/Cas12j-8 system serves as an efficient tool for genome editing mediated by both nuclease cleavage and base editing in plants.}, }
@article {pmid39799577, year = {2025}, author = {Wei, XY and Pei, Y and Liu, L and Hamar, P and Pei, DS}, title = {Protocol for detecting eDNA in ecological rare fish using RPA-CRISPR-Cas12a technology.}, journal = {STAR protocols}, volume = {6}, number = {1}, pages = {103544}, pmid = {39799577}, issn = {2666-1667}, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Fishes/genetics ; *DNA, Environmental/analysis/genetics ; }, abstract = {The recombinase polymerase amplification (RPA)-CRISPR-Cas12a-FQ system enables sensitive detection of environmental DNA (eDNA) in rare fish species. Here, we present a protocol for eDNA amplification and Cas12a for target recognition using RPA. We describe steps for identifying a target site, synthesis and purification of CRISPR RNA (crRNA), and RPA isothermal amplification. We then detail procedures for constructing the eDNA CRISPR-Cas12a detection system and verifying its sensitivity. This protocol offers a high-sensitivity approach for monitoring aquatic biodiversity and conservation efforts, even in low eDNA concentrations. For complete details on the use and execution of this protocol, please refer to Wei et al.[1].}, }
@article {pmid39799395, year = {2025}, author = {Zhou, H and Dai, J and Li, D and Wang, L and Ye, M and Hu, X and LoTurco, J and Hu, J and Sun, W}, title = {Efficient gene delivery admitted by small metabolites specifically targeting astrocytes in the mouse brain.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {33}, number = {3}, pages = {1166-1179}, pmid = {39799395}, issn = {1525-0024}, mesh = {Animals ; *Astrocytes/metabolism ; Mice ; *Gene Transfer Techniques ; *Brain/metabolism ; CRISPR-Cas Systems ; Genetic Therapy/methods ; *Glioma/genetics/therapy/metabolism/pathology ; Humans ; Brain Neoplasms/genetics/therapy/metabolism/pathology ; Disease Models, Animal ; ErbB Receptors ; }, abstract = {The development of efficient and targeted methods for delivering DNA in vivo has long been a major focus of research. In this study, we introduce a gene delivery approach admitted by small metabolites (gDAM) for the efficient and targeted delivery of naked DNA into astrocytes in the adult brains of mice. gDAM uses a straightforward combination of DNA and small metabolites, including glycine, L-proline, L-serine, L-histidine, D-alanine, Gly-Gly, and Gly-Gly-Gly, to achieve astrocyte-specific delivery of naked DNA, resulting in transient and robust gene expression in these cells. Using gDAM, we successfully co-deliver the PiggyBac transposon and the CRISPR-Cas9 system to induce long-term overexpression of the oncogene EGFRvIII and knockout of tumor suppressor genes Nf1, Pten, and Trp53 in astrocytes, leading to the development of astrocyte-derived gliomas in immunocompetent mice. Furthermore, gDAM facilitates the delivery of naked DNA to peripheral glioma astrocytes. The overexpression of interferon-β and granulocyte-macrophage colony-stimulating factor in these peripheral glioma astrocytes significantly prolongs the overall survival of mice bearing 73C glioma cells. This approach offers a new perspective on developing gene delivery systems that specifically target astrocytes to meet the varied needs of both research and gene therapy. The innovative strategy behind gDAM is expected to provide fresh inspiration in the quest for DNA delivery to other tissues, such as skeletal muscle and skin.}, }
@article {pmid39799394, year = {2025}, author = {Dragon, AC and Bonifacius, A and Lienenklaus, S and Verboom, M and Gerhards, JP and Ius, F and Hinze, C and Hudecek, M and Figueiredo, C and Blasczyk, R and Eiz-Vesper, B}, title = {Depletion of alloreactive B cells by drug-resistant chimeric alloantigen receptor T cells to prevent transplant rejection.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {33}, number = {3}, pages = {1031-1047}, pmid = {39799394}, issn = {1525-0024}, mesh = {*Graft Rejection/prevention &amp; control/immunology ; Animals ; Mice ; *B-Lymphocytes/immunology/metabolism ; Humans ; *T-Lymphocytes/immunology/metabolism ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; Isoantigens/immunology ; *Receptors, Antigen, T-Cell/genetics/metabolism/immunology ; Lymphocyte Depletion ; CRISPR-Cas Systems ; }, abstract = {Antibody-mediated rejection (AMR) remains a major complication after solid organ transplantation (SOT). Current treatment options are inefficient and result in drastic impairment of the general immunity. To selectively eliminate responsible alloreactive B cells characterized by anti-donor-HLA B cell receptors (BCRs), we generated T cells overcoming rejection by antibodies (CORA-Ts) engineered with a novel chimeric receptor comprising a truncated donor-HLA molecule as antigen recognition domain. As proof-of-concept, CORA receptors based on HLA-A∗02 were developed. In co-cultures with anti-HLA-A∗02 B cell lines, CORA-Ts were specifically activated, released pro-inflammatory mediators, and exhibited strong cytotoxicity resulting in an effective reduction of anti-HLA-A∗02 antibody release. Significant reduction of growth of an anti-HLA-A∗02 B cell line could be confirmed using an in vivo mouse model. Modification of the CORA receptor effectively abrogated T cell binding, thereby avoiding T cell sensitization. Additionally, using CRISPR-Cas9-mediated knockout of the FKBP12 gene, CORA-Ts were able to resist immunosuppressive treatment with tacrolimus, thereby allowing high efficiency in transplant patients. Our results demonstrate that CORA-Ts are able to specifically eliminate alloreactive, anti-HLA B cells, thus selectively preventing anti-HLA antibody release even under immunosuppressive conditions. This suggests CORA-Ts as potent approach to combat AMR and improve long-term graft survival in SOT patients while preserving their overall B cell immunity.}, }
@article {pmid39799111, year = {2025}, author = {Su-Tobon, Q and Fan, J and Goldstein, M and Feeney, K and Ren, H and Autissier, P and Wang, P and Huang, Y and Mohanty, U and Niu, J}, title = {CRISPR-Hybrid: A CRISPR-Mediated Intracellular Directed Evolution Platform for RNA Aptamers.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {595}, pmid = {39799111}, issn = {2041-1723}, support = {1DP2HG011027-01//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; Graduate Research Fellowship//National Science Foundation (NSF)/ ; }, mesh = {*Aptamers, Nucleotide/genetics/metabolism ; Humans ; *CRISPR-Cas Systems/genetics ; *Directed Molecular Evolution/methods ; *Gene Editing/methods ; RNA-Binding Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Recent advances in gene editing and precise regulation of gene expression based on CRISPR technologies have provided powerful tools for the understanding and manipulation of gene functions. Fusing RNA aptamers to the sgRNA of CRISPR can recruit cognate RNA-binding protein (RBP) effectors to target genomic sites, and the expression of sgRNA containing different RNA aptamers permit simultaneous multiplexed and multifunctional gene regulations. Here, we report an intracellular directed evolution platform for RNA aptamers against intracellularly expressed RBPs. We optimize a bacterial CRISPR-hybrid system coupled with FACS, and identified high affinity RNA aptamers orthogonal to existing aptamer-RBP pairs. Application of orthogonal aptamer-RBP pairs in multiplexed CRISPR allows effective simultaneous transcriptional activation and repression of endogenous genes in mammalian cells.}, }
@article {pmid39798882, year = {2025}, author = {Kainov, DE and Ravlo, E and Ianevski, A}, title = {Seeking innovative concepts in development of antiviral drug combinations.}, journal = {Antiviral research}, volume = {234}, number = {}, pages = {106079}, doi = {10.1016/j.antiviral.2025.106079}, pmid = {39798882}, issn = {1872-9096}, mesh = {*Antiviral Agents/therapeutic use/pharmacology ; Humans ; *Virus Diseases/drug therapy ; Drug Therapy, Combination ; Drug Combinations ; *Drug Development ; Drug Synergism ; }, abstract = {Antiviral drugs are crucial for managing viral infections, but current treatment options remain limited, particularly for emerging viruses. These drugs can be classified based on their chemical composition, including neutralizing antibodies (nAbs), recombinant human receptors (rhRs), antiviral CRISPR/Cas systems, interferons, antiviral peptides (APs), antiviral nucleic acid polymers, and small molecules. Some of these agents target viral factors, host factors, or both. A major challenge for virus-targeted treatments is their narrow-spectrum effectiveness and the potential for drug resistance, while host-directed and virus/host-targeted therapies often suffer from significant side effects. The synergistic combination of multiple antiviral drugs holds promise for improving treatment outcomes by targeting different stages of the viral life cycle, reducing resistance, and minimizing side effects. However, developing such drug combinations presents its own set of challenges. Several drug combinations could be optimized, and new combinations developed by using AI, to more effectively treat both emerging and re-emerging viral infections.}, }
@article {pmid39798545, year = {2025}, author = {Yaremenko, AV and Khan, MM and Zhen, X and Tang, Y and Tao, W}, title = {Clinical advances of mRNA vaccines for cancer immunotherapy.}, journal = {Med (New York, N.Y.)}, volume = {6}, number = {1}, pages = {100562}, doi = {10.1016/j.medj.2024.11.015}, pmid = {39798545}, issn = {2666-6340}, mesh = {Humans ; *Cancer Vaccines/immunology/therapeutic use/administration &amp; dosage ; *Neoplasms/therapy/immunology/genetics ; *mRNA Vaccines ; *Immunotherapy/methods ; RNA, Messenger/administration &amp; dosage/genetics ; CRISPR-Cas Systems ; Antigens, Neoplasm/immunology/genetics ; Clinical Trials as Topic ; }, abstract = {The development of mRNA vaccines represents a significant advancement in cancer treatment, with more than 120 clinical trials to date demonstrating their potential across various malignancies, including lung, breast, prostate, melanoma, and more challenging cancers such as pancreatic and brain tumors. These vaccines work by encoding tumor-specific antigens and immune-stimulating molecules, effectively activating the immune system to target and eliminate cancer cells. Despite these promising advancements, significant challenges remain, particularly in achieving efficient delivery and precise regulation of the immune response. This review provides a comprehensive overview of recent clinical progress in mRNA cancer vaccines, discusses the innovative strategies being employed to overcome existing hurdles, and explores future directions, including the integration of CRISPR-Cas9 technology and advancements in mRNA design. Our aim is to provide insights into the ongoing research and clinical trials, highlighting the transformative potential of mRNA vaccines in advancing oncology and improving patient outcomes.}, }
@article {pmid39798420, year = {2025}, author = {Gao, Z and Liu, J and Zhang, Y and Xu, R and Yang, Y and Wu, L and Lei, J and Ming, T and Ren, F and Liu, L and Chen, Q}, title = {TdT combined with Cas14a for the electrochemical biosensing of NPC-derived exosomes.}, journal = {Bioelectrochemistry (Amsterdam, Netherlands)}, volume = {163}, number = {}, pages = {108900}, doi = {10.1016/j.bioelechem.2025.108900}, pmid = {39798420}, issn = {1878-562X}, mesh = {*Exosomes/metabolism/chemistry ; *Biosensing Techniques/methods ; Humans ; *DNA Nucleotidylexotransferase/metabolism/chemistry ; *Electrochemical Techniques/methods ; CRISPR-Cas Systems ; *Nasopharyngeal Carcinoma/diagnosis ; *Nasopharyngeal Neoplasms/diagnosis ; Limit of Detection ; }, abstract = {In this work, the electrochemical biosensor based on the subtle combination of terminal deoxynucleotidyl transferase (TdT), CRISPR/Cas14a, and magnetic nanoparticles (MNPs) was developed for the detection of nasopharyngeal carcinoma (NPC)-derived exosomes. Due to the synergistic effect of the following factors: the powerful elongation capacity of TdT for single-stranded DNA (ssDNA) with 3-hydroxy terminus, the outstanding trans-cleavage ability of CRISPR/Cas14a specifcally activated by the crRNA binding to target DNA, and the excellent separation ability of MNPs, the developed electrochemical biosensor exhibited high sensitivity for the detection of NPC-derived exosome, with a linear range from 6.0 × 10[2] ∼ 1.0 × 10[5] particles/mL and a limit of detection as lown as 80 particles/mL. In addition, this electrochemical biosensor successfully distinguished exosomes from NPC patients and healthy individuals. This electrochemical biosensor opens up a new pathway for the early diagnosis of NPC. Abbreviations: NPC, Nasopharyngeal carcinoma; CRISPR/Cas, Clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins system; PAMs, Protospacer adjacent motifs; RCA, Rolling circle amplification; CHA, Catalytic hairpin assembly; LAMP, Loop-mediated isothermal amplification; TdT, Terminal deoxynucleotidyl transferase; SgRNA, Single guide RNA.}, }
@article {pmid39798272, year = {2025}, author = {Saleem, A and Wei, M and Abbas, MK and Zhang, S and Fan, J and Xian, Y and Jiang, H}, title = {Generation of a PDK-1 knockout human embryonic stem cell line by CRISPR/(WAe009-A-2K) Cas9 editing.}, journal = {Stem cell research}, volume = {83}, number = {}, pages = {103642}, doi = {10.1016/j.scr.2024.103642}, pmid = {39798272}, issn = {1876-7753}, mesh = {Humans ; *Human Embryonic Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Pyruvate Dehydrogenase Acetyl-Transferring Kinase/genetics ; *Gene Editing ; Cell Line ; Gene Knockout Techniques ; Animals ; Cell Differentiation ; }, abstract = {Pyruvate Dehydrogenase Kinase1 (PDK1) belongs to the family of kinases, regulates diverse metabolic processes. PDK1 is a susceptibility locus for heart failure via thinning of ventricle walls, and enlarged atria and ventricles. We successfully developed a PDK1 knockout (PDK1[-]/[-]) human embryonic stem cell (hESC) line using an episomal vector-based CRISPR/Cas9 system explore the role of PDK in human heart development. This PDK1-KO hESC line-maintained stem cell-like morphology, pluripotency, and normal karyotype and can differentiate into all three germ layers in vivo. This cell line will be a valuable tool for future research on the role of PDK1 in heart development.}, }
@article {pmid39797940, year = {2025}, author = {Fu, Y and Zhang, P and Chen, F and Xie, Z and Xiao, S and Huang, Z and Lau, CH and Zhu, H and Luo, J}, title = {CRISPR detection of cardiac tumor-associated microRNAs.}, journal = {Molecular biology reports}, volume = {52}, number = {1}, pages = {114}, pmid = {39797940}, issn = {1573-4978}, support = {20212ACB206031//Jiangxi Provincial Natural Science Foundation/ ; 82260053//National Natural Science Foundation of China/ ; }, mesh = {Humans ; *MicroRNAs/genetics ; *Heart Neoplasms/genetics/diagnosis ; *CRISPR-Cas Systems/genetics ; Myxoma/genetics/diagnosis ; Hemangiosarcoma/genetics/diagnosis ; Biomarkers, Tumor/genetics ; }, abstract = {As multiple imaging modalities cannot reliably diagnose cardiac tumors, the molecular approach offers alternative ways to detect rare ones. One such molecular approach is CRISPR-based diagnostics (CRISPR-Dx). CRISPR-Dx enables visual readout, portable diagnostics, and rapid and multiplex detection of nucleic acids such as microRNA (miRNA). Dysregulation of miRNA expressions has been associated with cardiac tumors such as atrial myxoma and angiosarcoma. Diverse CRISPR-Dx systems have been developed to detect miRNA in recent years. These CRISPR-Dx systems are generally classified into four classes, depending on the Cas proteins used (Cas9, Cas12, Cas13, or Cas12f). CRISPR/Cas systems are integrated with various isothermal amplifications to detect low-abundance miRNAs. Amplification-free CRISPR-Dx systems have also been recently developed to detect miRNA directly. Herein, we critically discuss the advances, pitfalls, and future perspectives for these CRISPR-Dx systems in detecting miRNA, focusing on the diagnosis and prognosis of cardiac tumors.}, }
@article {pmid39797776, year = {2025}, author = {Li, Q and Zhang, Z and Wu, X and Zhao, Y and Liu, Y}, title = {Cascade-Responsive Nanoparticles for Efficient CRISPR/Cas9-Based Glioblastoma Gene Therapy.}, journal = {ACS applied materials &amp; interfaces}, volume = {17}, number = {3}, pages = {4480-4489}, doi = {10.1021/acsami.4c15671}, pmid = {39797776}, issn = {1944-8252}, mesh = {*Glioblastoma/therapy/genetics/pathology/drug therapy ; Animals ; *CRISPR-Cas Systems/genetics ; *Nanoparticles/chemistry ; Mice ; Humans ; *Genetic Therapy ; Cell Line, Tumor ; Gene Editing ; *Brain Neoplasms/therapy/genetics/pathology ; Blood-Brain Barrier/metabolism ; Temozolomide/pharmacology/therapeutic use ; }, abstract = {CRISPR/Cas9 (CRISPR, clustered regularly interspaced short palindromic repeats) gene editing technology represents great promise for treating glioblastoma (GBM) due to its potential to permanently eliminate tumor pathogenic genes. Unfortunately, delivering CRISPR to the GBM in a safe and effective manner is challenging. Herein, a glycosylated and cascade-responsive nanoparticle (GCNP) that can effectively cross the blood-brain barrier (BBB) and activate CRISPR/Cas9-based gene editing only in the GBM is designed. The GCNP possesses a cationic polyplex core and a glycosylated polymer layer that is capable of cascading response to low pH and high GSH concentration, so that the release of CRISPR/Cas9 only takes place after crossing the BBB and entering the GBM where the acidic tumor microenvironment and high concentration of glutathione (GSH) are present. By targeting the programmed death-ligand 1 (PD-L1) in GBM, GCNP effectively inhibited the tumor growth and greatly prolonged the survival time of GBM-bearing mice when combined with temozolomide (TMZ).}, }
@article {pmid39797401, year = {2025}, author = {Carrozzo, I and Maule, G and Gentile, C and Umbach, A and Ciciani, M and Guidone, D and De Santis, M and Petris, G and Vicente Galietta, LJ and Arosio, D and Cereseto, A}, title = {Functional rescue of F508del-CFTR through revertant mutations introduced by CRISPR base editing.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {33}, number = {3}, pages = {970-985}, pmid = {39797401}, issn = {1525-0024}, mesh = {*Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism/chemistry ; Humans ; *Gene Editing/methods ; *Cystic Fibrosis/genetics/therapy/metabolism ; *CRISPR-Cas Systems ; *Mutation ; Epithelial Cells/metabolism ; }, abstract = {Cystic fibrosis (CF) is a life-shortening autosomal recessive disease caused by mutations in the CFTR gene, resulting in functional impairment of the encoded ion channel. F508del mutation, a trinucleotide deletion, is the most frequent cause of CF, affecting approximately 80% of persons with CF (pwCFs). Even though current pharmacological treatments alleviate the F508del-CF disease symptoms, there is no definitive cure. Here, we leveraged revertant mutations (RMs) in cis with F508del to rescue CFTR protein folding and restore its function. We developed CRISPR base editing strategies to efficiently and precisely introduce the desired mutations in the F508del locus. Both editing and CFTR function recovery were verified in CF cellular models, including primary epithelial cells derived from pwCFs. The efficacy of the CFTR recovery strategy was validated in cultures of pseudostratified epithelia from pwCF cells showing full recovery of ion transport. Additionally, we observed an additive effect by combining our strategy with small molecules that enhance F508del activity, thus paving the way to combinatorial therapies.}, }
@article {pmid39797397, year = {2025}, author = {Hazel, K and Singh, D and He, S and Guertin, Z and Husser, MC and Helfield, B}, title = {Focused ultrasound and microbubble-mediated delivery of CRISPR-Cas9 ribonucleoprotein to human induced pluripotent stem cells.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {33}, number = {3}, pages = {986-996}, pmid = {39797397}, issn = {1525-0024}, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Microbubbles ; *CRISPR-Cas Systems ; *Ribonucleoproteins/genetics/metabolism ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Transfer Techniques ; Green Fluorescent Proteins/genetics ; Myocytes, Cardiac/metabolism ; CRISPR-Associated Protein 9 ; }, abstract = {CRISPR-Cas9 ribonucleoproteins (RNPs) have been heavily considered for gene therapy due to their high on-target efficiency, rapid activity, and lack of insertional mutagenesis relative to other CRISPR-Cas9 delivery formats. Genetic diseases such as hypertrophic cardiomyopathy currently lack effective treatment strategies and are prime targets for CRISPR-Cas9 gene editing technology. However, current in vivo delivery strategies for Cas9 pose risks of unwanted immunogenic responses. This proof-of-concept study aimed to demonstrate that focused ultrasound (FUS) in combination with microbubbles can be used to deliver Cas9-sgRNA (single-guide RNA) RNPs and functionally edit human induced pluripotent stem cells (hiPSCs) in vitro, a model system that can be expanded to cardiovascular research via hiPSC-derived cardiomyocytes. Here, we first determine acoustic conditions suitable for the viable delivery of large proteins to hiPSCs with clinical Definity microbubble agents using our customized experimental platform. From here, we delivered Cas9-sgRNA RNP complexes targeting the EGFP (enhanced green fluorescent protein) gene to EGFP-expressing hiPSCs for EGFP knockout. Simultaneous acoustic cavitation detection during treatment confirmed a strong correlation between microbubble disruption and viable FUS-mediated protein delivery in hiPSCs. This study shows for the first time the potential for an FUS-mediated technique for targeted and precise CRISPR-Cas9 gene editing in human stem cells.}, }
@article {pmid39796169, year = {2025}, author = {Rallabandi, HR and Singh, MK and Looger, LL and Nath, SK}, title = {Defining Mechanistic Links Between the Non-Coding Variant rs17673553 in CLEC16A and Lupus Susceptibility.}, journal = {International journal of molecular sciences}, volume = {26}, number = {1}, pages = {}, pmid = {39796169}, issn = {1422-0067}, support = {R01 AI172255/AI/NIAID NIH HHS/United States ; R01 AI183676/AI/NIAID NIH HHS/United States ; 5R01AI172255-02S1/NH/NIH HHS/United States ; }, mesh = {Humans ; *Lectins, C-Type/genetics/metabolism ; *Genetic Predisposition to Disease ; *Lupus Erythematosus, Systemic/genetics ; *Monosaccharide Transport Proteins/genetics/metabolism ; *Polymorphism, Single Nucleotide ; Genome-Wide Association Study ; Gene Editing ; Autophagy/genetics ; CRISPR-Cas Systems ; Alleles ; STAT3 Transcription Factor/genetics/metabolism ; B-Lymphocytes/metabolism ; }, abstract = {Systemic lupus erythematosus (SLE) is a complex autoimmune disorder characterized by widespread inflammation and autoantibody production. Its development and progression involve genetic, epigenetic, and environmental factors. Although genome-wide association studies (GWAS) have repeatedly identified a susceptibility signal at 16p13, its fine-scale source and its functional and mechanistic role in SLE remain unclear. We used bioinformatics to prioritize likely functional variants and validated the top candidate through various experimental techniques, including clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing in B cells. To assess the functional impact of the proposed causal variant in C-type lectin domain family 16, member A (CLEC16A), we compared autophagy levels between wild-type (WT) and knock-out (KO) cells. Systematic bioinformatics analysis identified the highly conserved non-coding intronic variant rs17673553, with the risk allele apparently affecting enhancer function and regulating several target genes, including CLEC16A itself. Luciferase reporter assays followed by chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) validated this enhancer activity, demonstrating that the risk allele increases the binding of enhancer histone marks (H3K27ac and H3K4me1), the CTCF-binding factor, and key immune transcription factors (GATA3 and STAT3). Knock-down of GATA3 and STAT3 via siRNA led to a significant decrease in CLEC16A expression. These regulatory effects on the target gene were further confirmed using CRISPR-based genome editing and CRISPR-dCas9-based epigenetic activation/silencing. Functionally, WT cells exhibited higher levels of starvation-induced autophagy compared to KO cells, highlighting the role of CLEC16A and the rs17673553 locus in autophagy regulation. These findings suggest that the rs17673553 locus-particularly the risk allele-drives significant allele-specific chromatin modifications and binding of multiple transcription factors, thereby mechanistically regulating the expression of target autophagy-associated genes, including CLEC16A itself. This mechanism could potentially explain the association between rs17673553 and SLE, and could underlie the signal at 16p13.}, }
@article {pmid39795970, year = {2024}, author = {Cerna-Chavez, R and Ortega-Gasco, A and Baig, HMA and Ehrenreich, N and Metais, T and Scandura, MJ and Bujakowska, K and Pierce, EA and Garita-Hernandez, M}, title = {Optimized Prime Editing of Human Induced Pluripotent Stem Cells to Efficiently Generate Isogenic Models of Mendelian Diseases.}, journal = {International journal of molecular sciences}, volume = {26}, number = {1}, pages = {}, pmid = {39795970}, issn = {1422-0067}, support = {EY012910/EY/NEI NIH HHS/United States ; EY020902/EY/NEI NIH HHS/United States ; EY014104/NH/NIH HHS/United States ; Competitive Renewal Research Grant in Pediatric Ophthalmology//Knights Templar Eye Foundation/ ; }, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Genetic Diseases, Inborn/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mutation ; }, abstract = {Prime editing (PE) is a CRISPR-based tool for genome engineering that can be applied to generate human induced pluripotent stem cell (hiPSC)-based disease models. PE technology safely introduces point mutations, small insertions, and deletions (indels) into the genome. It uses a Cas9-nickase (nCas9) fused to a reverse transcriptase (RT) as an editor and a PE guide RNA (pegRNA), which introduces the desired edit with great precision without creating double-strand breaks (DSBs). PE leads to minimal off-targets or indels when introducing single-strand breaks (SSB) in the DNA. Low efficiency can be an obstacle to its use in hiPSCs, especially when the genetic context precludes the screening of multiple pegRNAs, and other strategies must be employed to achieve the desired edit. We developed a PE platform to efficiently generate isogenic models of Mendelian disorders. We introduced the c.25G>A (p.V9M) mutation in the NMNAT1 gene with over 25% efficiency by optimizing the PE workflow. Using our optimized system, we generated other isogenic models of inherited retinal diseases (IRDs), including the c.1481C>T (p.T494M) mutation in PRPF3 and the c.6926A>C (p.H2309P) mutation in PRPF8. We modified several determinants of the hiPSC PE procedure, such as plasmid concentrations, PE component ratios, and delivery method settings, showing that our improved workflow increased the hiPSC editing efficiency.}, }
@article {pmid39795964, year = {2024}, author = {Tao, R and Zhang, J and Meng, L and Gao, J and Miao, C and Liu, W and Jin, W and Wan, Y}, title = {A Rapid Field-Visualization Detection Platform for Genetically Modified Soybean 'Zhonghuang 6106' Based on RPA-CRISPR.}, journal = {International journal of molecular sciences}, volume = {26}, number = {1}, pages = {}, pmid = {39795964}, issn = {1422-0067}, support = {2022ZD0402003//Biological Breeding-Major Projects/ ; }, mesh = {*Glycine max/genetics ; *Plants, Genetically Modified/genetics ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Gene Editing ; DNA, Plant/genetics ; }, abstract = {Genetically modified (GM) herbicide-tolerant soybean 'Zhonghuang 6106', which introduces a glyphosate-resistant gene, ensures soybean yield while allowing farmers to reduce the use of other herbicides, thereby reducing weed management costs. To protect consumer rights and facilitate government supervision, we have established a simple and rapid on-site nucleic acid detection method for GM soybean 'Zhonghuang 6106'. This method leverages the isothermal amplification characteristics of RPA technology and the high specificity of CRISPR-Cas12a to achieve high sensitivity and accuracy in detecting GM soybean components. By optimizing experimental conditions, the platform can quickly produce visual detection results, significantly reducing detection time and improving efficiency. The system can detect down to 10 copies/μL of 'Zhonghuang 6106' DNA templates, and the entire detection process takes about 1 h. The technology also has strong editing capabilities; by redesigning the primers and crRNA in the method, it can become a specific detection method for other GM samples, providing strong technical support for the regulation and safety evaluation of GM crops.}, }
@article {pmid39794549, year = {2025}, author = {Ahmed, R and Alghamdi, WN and Alharbi, FR and Alatawi, HD and Alenezi, KM and Alanazi, TF and Elsherbiny, NM}, title = {CRISPR/Cas9 System as a Promising Therapy in Thalassemia and Sickle Cell Disease: A Systematic Review of Clinical Trials.}, journal = {Molecular biotechnology}, volume = {}, number = {}, pages = {}, pmid = {39794549}, issn = {1559-0305}, abstract = {Clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system is a new gene editing tool that represents a revolution in gene therapy. This study aimed to review the clinical trials conducted to evaluate the efficacy and safety of the CRISPR/Cas9 system in treating thalassemia and sickle cell disease (SCD). We searched relevant literature using "CRISPR Cas", "thalassemia", "sickle cell" and "clinical trial" as subject terms in PubMed, Cochrane, Web of Science, and Google Scholar up to December 3rd, 2023. Following the PIO format (Patients, Intervention, Outcome), PRISMA guidelines were followed in the study selection, data extraction, and quality assessment processes. Out of 110 publications, 6 studies met our eligibility criteria with a total of 115 patients involved. CRISPR/Cas9 system was used to disrupt BCL11A gene enhancer in 4 studies and to disrupt γ-globin gene promoters in 2 studies. Patients demonstrated significant activation of fetal hemoglobin, elevated total hemoglobin, transfusion independence in thalassemia, and repression of vaso-occlusive episodes in SCD. Using CRISPR/Cas9 system to directly disrupt genes provides a safe and potential one-time functional cure for thalassemia and SCD, suggesting CRISPR/Cas9 as a potential therapeutic tool for the treatment of inherited hematological disorders.}, }
@article {pmid39793818, year = {2025}, author = {Wang, X and Li, D and Qin, Z and Wang, X and Li, J and Chen, J and Zhou, J}, title = {Anti-CRISPR proteins in Gluconobacter oxydans inactivate FnCas12a by acetylation.}, journal = {International journal of biological macromolecules}, volume = {297}, number = {}, pages = {139256}, doi = {10.1016/j.ijbiomac.2024.139256}, pmid = {39793818}, issn = {1879-0003}, mesh = {Acetylation ; *Gluconobacter oxydans/genetics/metabolism ; *Bacterial Proteins/metabolism/genetics/chemistry ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; }, abstract = {Gluconobacter oxydans is an important chassis cell for one-step production of vitamin C. Previous studies reported that CRISPR/Cas12a is naturally inactivated in G. oxydans, but the specific mechanism remains unclear. Here, we identified anti-CRISPR proteins AcrVA6, AcrVA7 and AcrVA8 in G. oxydans. They functioned as acetyltransferases to inactivate FnCas12a by respectively acetylating Lys671, Lys589 and Lys823 of FnCas12a. Lys671 and Lys823 were related residues that recognise the protospacer-adjacent motif, modification of AcrVA6 and AcrVA8 untangled the interaction between FnCas12a and dsDNA, while Lys589 played an important role in binding to the crRNA-target DNA heteroduplex, AcrVA7 prevented the formation of FnCas12a-crRNA binary complexes. In addition, histone deacetylase HDAC11 was found to prevent modification of FnCas12a by AcrVA6. Quantum mechanical calculations showed that ser37 of AcrVA6, as an intermediate between acetyl group and receptor protein, achieves acetylation through ping-pong transfer mechanism. Finally, the acetyltransferase AcrVA6 and the deacetylase HDAC11 served as photoswitches by writing and erasing acetyl groups, respectively, to achieve continuous on-off of FnCas12a. Our study reveals different mechanisms by which acetyltransferase inactivates Cas12a and successfully applies reversible acetylation to the regulation of gene editing tools, providing new insights into the function and application of acetylation.}, }
@article {pmid39793042, year = {2025}, author = {Ngo, W and Peukes, J and Baldwin, A and Xue, ZW and Hwang, S and Stickels, RR and Lin, Z and Satpathy, AT and Wells, JA and Schekman, R and Nogales, E and Doudna, JA}, title = {Mechanism-guided engineering of a minimal biological particle for genome editing.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {122}, number = {1}, pages = {e2413519121}, pmid = {39793042}, issn = {1091-6490}, support = {PDF-578176-2023//Canadian Government | Natural Sciences and Engineering Research Council of Canada (NSERC)/ ; DE-AC52-07NA27344//DOE | NNSA | Lawrence Livermore National Laboratory (LLNL)/ ; R21 HL173710/HL/NHLBI NIH HHS/United States ; 1R21HL173710-01//HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; ALTF 1031-2021//European Molecular Biology Organization (EMBO)/ ; 1R01CA248323-01//HHS | National Institutes of Health (NIH)/ ; U54 AI170792/AI/NIAID NIH HHS/United States ; R01 CA248323/CA/NCI NIH HHS/United States ; }, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; Lentivirus/genetics ; Ribonucleoproteins/metabolism/genetics ; HEK293 Cells ; CRISPR-Associated Protein 9/metabolism/genetics ; Genetic Vectors ; }, abstract = {The widespread application of genome editing to treat and cure disease requires the delivery of genome editors into the nucleus of target cells. Enveloped delivery vehicles (EDVs) are engineered virally derived particles capable of packaging and delivering CRISPR-Cas9 ribonucleoproteins (RNPs). However, the presence of lentiviral genome encapsulation and replication proteins in EDVs has obscured the underlying delivery mechanism and precluded particle optimization. Here, we show that Cas9 RNP nuclear delivery is independent of the native lentiviral capsid structure. Instead, EDV-mediated genome editing activity corresponds directly to the number of nuclear localization sequences on the Cas9 enzyme. EDV structural analysis using cryo-electron tomography and small molecule inhibitors guided the removal of ~80% of viral residues, creating a minimal EDV (miniEDV) that retains full RNP delivery capability. MiniEDVs are 25% smaller yet package equivalent amounts of Cas9 RNPs relative to the original EDVs and demonstrated increased editing in cell lines and therapeutically relevant primary human T cells. These results show that virally derived particles can be streamlined to create efficacious genome editing delivery vehicles with simpler production and manufacturing.}, }
@article {pmid39792803, year = {2025}, author = {Wu, Z and Zhao, C and Ai, H and Wang, Z and Chen, M and Lyu, Y and Tong, Q and Liu, L and Sun, H and Pu, J and Zhang, R and Hu, X and Liu, J and Ma, X and Sun, Y}, title = {A Susceptible Cell-Selective Delivery (SCSD) of mRNA-Encoded Cas13d Against Influenza Infection.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {12}, number = {9}, pages = {e2414651}, pmid = {39792803}, issn = {2198-3844}, support = {2022YFD1801005//National Key Research and Development Program of China/ ; 32171389//National Natural Science Foundation of China/ ; 32192450//National Natural Science Foundation of China/ ; WSJK2024MS235//Joint Program on Health Science &amp; Technology Innovation of Hainan Province/ ; }, mesh = {Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *RNA, Messenger/genetics ; *Influenza A virus/genetics ; Mice ; *Orthomyxoviridae Infections ; Swine ; *Influenza, Human ; Antiviral Agents ; Nanoparticles ; *Drug Delivery Systems/methods ; }, abstract = {To bolster the capacity for managing potential infectious diseases in the future, it is critical to develop specific antiviral drugs that can be rapidly designed and delivered precisely. Herein, a CRISPR/Cas13d system for broad-spectrum targeting of influenza A virus (IAV) from human, avian, and swine sources is designed, incorporating Cas13d mRNA and a tandem CRISPR RNA (crRNA) specific for the highly conserved regions of viral polymerase acidic (PA), nucleoprotein (NP), and matrix (M) gene segments, respectively. Given that the virus targets cells with specific receptors but is not limited to a single organ, a Susceptible Cell Selective Delivery (SCSD) system is developed by modifying a lipid nanoparticle with a peptide mimicking the function of the hemagglutinin of influenza virus to target sialic acid receptors. The SCSD system can precisely deliver an all-RNA-based CRISPR/Cas13d system into potentially infected cells. This drug is shown to reduce the viral load in the lungs by 2.37 log10 TCID50 mL[-1] and protect 100% of mice from lethal influenza infection. The SCSD-based CRISPR/Cas13d system shows promise for the flexible and efficient therapy of infections caused by rapidly evolving and novel viruses.}, }
@article {pmid39792571, year = {2025}, author = {Islam, MS and Habib, MA and Tonu, NS and Haque, MS and Rahman, MM}, title = {Beyond Serology: A Meta-Analysis of Advancements in Molecular Detection of Brucella spp. in Seronegative Animals and Biological Samples.}, journal = {Veterinary medicine and science}, volume = {11}, number = {1}, pages = {e70200}, pmid = {39792571}, issn = {2053-1095}, mesh = {Animals ; *Brucella/isolation &amp; purification/immunology ; *Brucellosis/veterinary/diagnosis/microbiology ; Molecular Diagnostic Techniques/veterinary/methods ; }, abstract = {BACKGROUND: Brucellosis is a zoonotic disease caused by Brucella spp., affecting various animals and humans, leading to significant economic and public health impacts. Traditional diagnostic methods, mainly serological, often fail to detect seronegative carriers, which continue to spread the infection.<br><br>OBJECTIVE: This review aims to highlight advancements in molecular diagnostics that address these limitations.<br><br>METHODS: A systematic search of PubMed, Web of Science and Scopus was conducted, focusing on studies using seronegative, PCR, qPCR and biosensor-based techniques. Data extraction and meta-analyses were performed, evaluating pooled detection rates and heterogeneity.<br><br>RESULTS: Through analysis of existing studies, we review key molecular techniques, including PCR, LAMP and biosensor-based assays, which offer high sensitivity and specificity by detecting bacterial DNA directly, thus overcoming the challenges of antibody-based tests. Meta-analysis of detection rates across different studies showed significant variability, with rates ranging from 0.96% to 100%, highlighting differences in sample types, animal species and regions. The pooled detection proportion from random-effects models was 35.08%, indicating that many seronegative animals still carry Brucella spp. A forest plot analysis further confirmed heterogeneity in detection, underlining the importance of using molecular diagnostics alongside serological tests to identify hidden carriers.<br><br>CONCLUSION: Innovations like nanoparticle-enhanced biosensors and CRISPR-Cas systems show promise for rapid, on-site diagnostics. The findings suggest that integrating molecular methods with traditional serology can improve surveillance and disease management. Future research should focus on developing portable, field-ready diagnostic devices and standardised protocols, along with exploring novel biomarkers to detect latent infections. A collaborative One Health approach, involving veterinary, public health and environmental sectors, is essential for comprehensive disease control and eradication efforts.}, }
@article {pmid39792480, year = {2025}, author = {Buyukyoruk, M and Krishna, P and Santiago-Frangos, A and Wiedenheft, B}, title = {Discovery of Diverse CRISPR Leader Motifs, Putative Functions, and Applications for Enhanced CRISPR Detection and Subtype Annotation.}, journal = {The CRISPR journal}, volume = {8}, number = {2}, pages = {137-148}, pmid = {39792480}, issn = {2573-1602}, support = {K99 GM147842/GM/NIGMS NIH HHS/United States ; R00 GM147842/GM/NIGMS NIH HHS/United States ; }, mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Nucleotide Motifs ; *Archaea/genetics ; CRISPR-Cas Systems ; *Bacteria/genetics ; Genome, Archaeal ; Genome, Bacterial ; Conserved Sequence ; }, abstract = {Bacteria and archaea acquire resistance to genetic parasites by preferentially integrating short fragments of foreign DNA at one end of a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR). "Leader" DNA upstream of CRISPR loci regulates transcription and foreign DNA integration into the CRISPR. Here, we analyze 37,477 CRISPRs from 39,277 bacterial and 556 archaeal genomes to identify conserved sequence motifs in CRISPR leaders. A global analysis of all leader sequences fails to identify universally conserved motifs. However, an analysis of leader sequences that have been grouped by 16S rRNA-based taxonomy and CRISPR subtype reveals 87 specific motifs in type I, II, III, and V CRISPR leaders. Fourteen of these leader motifs have biochemically demonstrated roles in CRISPR biology including integration, transcription, and CRISPR RNA processing. Another 28 motifs are related to DNA binding sites for proteins with functions that are consistent with regulating CRISPR activity. In addition, we show that these leader motifs can be used to improve existing CRISPR detection methods and enhance the accuracy of CRISPR classification.}, }
@article {pmid39791925, year = {2025}, author = {Guo, W and Guo, Y and Xu, H and Li, C and Zhang, X and Zou, X and Sun, Z}, title = {Ultrasensitive "On-Off" Ratiometric Fluorescence Biosensor Based on RPA-CRISPR/Cas12a for Detection of Staphylococcus aureus.}, journal = {Journal of agricultural and food chemistry}, volume = {73}, number = {3}, pages = {2167-2173}, doi = {10.1021/acs.jafc.4c12202}, pmid = {39791925}, issn = {1520-5118}, mesh = {*Biosensing Techniques/methods/instrumentation ; *Staphylococcus aureus/genetics/isolation &amp; purification ; CRISPR-Cas Systems ; Fluorescence ; *Bacterial Proteins/genetics/metabolism ; Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; Limit of Detection ; Food Contamination/analysis ; Endodeoxyribonucleases ; }, abstract = {Staphylococcus aureus (S. aureus) is a major pathogenic bacterium responsible for bacterial foodborne diseases, making its rapid, specific, and accurate detection crucial. In this study, we develop a ratiometric biosensor based on the recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats/CRISPR associated protein 12a (RPA-CRISPR/Cas12a) system and Eu-metal-organic framework (Eu-MOF) fluorescent nanomaterials for the high-sensitivity detection of S. aureus, combining with RPA for efficient isothermal amplification, this sensor enhances specificity and sensitivity by utilizing the target activation of CRISPR/Cas12a. The Eu-MOF serves a dual function, providing stable red fluorescence as a reference signal and adsorbing FAM-labeled probes for fluorescence quenching, forming a dual-signal system that significantly reduces background interference. This ratiometric design enables accurate and quantitative detection over a wide range (7.9 × 10[0] to 7.9 × 10[8] CFU/mL) with a low detection limit of 3 CFU/mL. Overall, with these merits of simplicity, rapid response, high sensitivity, and specificity, this dual-signal biosensor offers a promising method for accurately evaluating S. aureus contamination in food under complex substrate conditions.}, }
@article {pmid39789389, year = {2025}, author = {Wang, Y and Lin, RZ and Harris, M and Lavayen, B and Diwanji, N and McCreedy, B and Hofmeister, R and Getts, D}, title = {CRISPR-Enabled Autonomous Transposable Element (CREATE) for RNA-based gene editing and delivery.}, journal = {EMBO reports}, volume = {26}, number = {4}, pages = {1062-1083}, pmid = {39789389}, issn = {1469-3178}, mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Long Interspersed Nucleotide Elements/genetics ; *DNA Transposable Elements/genetics ; *Gene Transfer Techniques ; *RNA/genetics ; HEK293 Cells ; }, abstract = {To address a wide range of genetic diseases, genome editing tools that can achieve targeted delivery of large genes without causing double-strand breaks (DSBs) or requiring DNA templates are necessary. Here, we introduce CRISPR-Enabled Autonomous Transposable Element (CREATE), a genome editing system that combines the programmability and precision of CRISPR/Cas9 with the RNA-mediated gene insertion capabilities of the human LINE-1 (L1) element. CREATE employs a modified L1 mRNA to carry a payload gene, and a Cas9 nickase to facilitate targeted editing by L1-mediated reverse transcription and integration without relying on DSBs or DNA templates. Using this system, we demonstrate programmable insertion of a 1.1 kb gene expression cassette into specific genomic loci of human cell lines and primary T cells. Mechanistic studies reveal that CREATE editing is highly specific with no observed off-target events. Together, these findings establish CREATE as a programmable, RNA-based gene delivery technology with broad therapeutic potential.}, }
@article {pmid39789283, year = {2025}, author = {Hassan, YM and Mohamed, AS and Hassan, YM and El-Sayed, WM}, title = {Recent developments and future directions in point-of-care next-generation CRISPR-based rapid diagnosis.}, journal = {Clinical and experimental medicine}, volume = {25}, number = {1}, pages = {33}, pmid = {39789283}, issn = {1591-9528}, mesh = {Humans ; COVID-19/diagnosis/virology ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods/trends ; *Point-of-Care Testing/trends ; SARS-CoV-2/genetics/isolation &amp; purification ; }, abstract = {The demand for sensitive, rapid, and affordable diagnostic techniques has surged, particularly following the COVID-19 pandemic, driving the development of CRISPR-based diagnostic tools that utilize Cas effector proteins (such as Cas9, Cas12, and Cas13) as viable alternatives to traditional nucleic acid-based detection methods. These CRISPR systems, often integrated with biosensing and amplification technologies, provide precise, rapid, and portable diagnostics, making on-site testing without the need for extensive infrastructure feasible, especially in underserved or rural areas. In contrast, traditional diagnostic methods, while still essential, are often limited by the need for costly equipment and skilled operators, restricting their accessibility. As a result, developing accessible, user-friendly solutions for at-home, field, and laboratory diagnostics has become a key focus in CRISPR diagnostic innovations. This review examines the current state of CRISPR-based diagnostics and their potential applications across a wide range of diseases, including cancers (e.g., colorectal and breast cancer), genetic disorders (e.g., sickle cell disease), and infectious diseases (e.g., tuberculosis, malaria, Zika virus, and human papillomavirus). Additionally, the integration of machine learning (ML) and artificial intelligence (AI) to enhance the accuracy, scalability, and efficiency of CRISPR diagnostics is discussed, alongside the challenges of incorporating CRISPR technologies into point-of-care settings. The review also explores the potential for these cutting-edge tools to revolutionize disease diagnosis and personalized treatment in the future, while identifying the challenges and future directions necessary to address existing gaps in CRISPR-based diagnostic research.}, }
@article {pmid39789078, year = {2025}, author = {Lee, D and Muir, P and Lundberg, S and Lundholm, A and Sandegren, L and Koskiniemi, S}, title = {A CRISPR-Cas9 system protecting E. coli against acquisition of antibiotic resistance genes.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {1545}, pmid = {39789078}, issn = {2045-2322}, mesh = {*CRISPR-Cas Systems ; *Escherichia coli/genetics/drug effects ; Drug Resistance, Bacterial/genetics ; Gene Transfer, Horizontal ; Plasmids/genetics ; Anti-Bacterial Agents/pharmacology ; Conjugation, Genetic ; }, abstract = {Antimicrobial resistance (AMR) is an increasing problem worldwide, and new treatment options for bacterial infections are direly needed. Engineered probiotics show strong potential in treating or preventing bacterial infections. However, one concern with the use of live bacteria is the risk of the bacteria acquiring genes encoding for AMR or virulence factors through horizontal gene transfer (HGT), and the transformation of the probiotic into a superbug. Therefore, we developed an engineered CRISPR-Cas9 system that protects bacteria from horizontal gene transfer. We synthesized a CRISPR locus targeting eight AMR genes and cloned this with the Cas9 and transacting tracrRNA on a medium copy plasmid. We next evaluated the efficiency of the system to block HGT through transformation, transduction, and conjugation. Our results show that expression of the CRISPR-Cas9 system successfully protects E. coli MG1655 from acquiring the targeted resistance genes by transformation or transduction with 2-3 logs of protection depending on the system for transfer and the target gene. Furthermore, we show that the system blocks conjugation of a set of clinical plasmids, and that the system is also able to protect the probiotic bacterium E. coli Nissle 1917 from acquiring AMR genes.}, }
@article {pmid39789054, year = {2025}, author = {Li, W and Jiang, X and Wang, W and Hou, L and Cai, R and Li, Y and Gu, Q and Chen, Q and Ma, P and Tang, J and Guo, M and Chuai, G and Huang, X and Zhang, J and Liu, Q}, title = {Author Correction: Discovering CRISPR-Cas system with self-processing pre-crRNA capability by foundation models.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {535}, doi = {10.1038/s41467-025-55913-y}, pmid = {39789054}, issn = {2041-1723}, }
@article {pmid39788669, year = {2025}, author = {Li, X and Wang, C and Chai, J and Liu, H and Jiang, X and Li, Y and Li, Y}, title = {Structure-switchable branched inhibitors regulate the activity of CRISPR-Cas12a for nucleic acid diagnostics.}, journal = {Analytica chimica acta}, volume = {1336}, number = {}, pages = {343515}, doi = {10.1016/j.aca.2024.343515}, pmid = {39788669}, issn = {1873-4324}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; CRISPR-Associated Proteins/metabolism ; Endodeoxyribonucleases/chemistry/metabolism/antagonists &amp; inhibitors ; Nucleic Acids/chemistry ; Bacterial Proteins ; }, abstract = {BACKGROUND: In current years, the CRISPR (clustered regularly interspaced short palindromic repeats) based strategies have emerged as the most promising molecular tool in the field of gene editing, intracellular imaging, transcriptional regulation and biosensing. However, the recent CRISPR-based diagnostic technologies still require the incorporation of other amplification strategies (such as polymerase chain reaction) to improve the cis/trans cleavage activity of Cas12a, which complicates the detection workflow and lack of a uniform compatible system to respond to the target in one pot.<br><br>RESULTS: To better fully-functioning CRISPR/Cas12a, we reported a novel technique for straightforward nucleic acid detection by incorporating enzyme-responsive steric hindrance-based branched inhibitors with CRISPR/AsCas12a methodology. The construction-transferable branched inhibitors coupled with a specific overhang flap induce spatial steric effects and result in the loss of the binding ability of Cas12a, which inhibits the activity of Cas12a. Target as the input signal would trigger the site-directed APE1 enzyme incision of the inhibitors, thus transforming the conformation of the inhibitors into split activators to illumine the CRISPR/AsCas12a catalyst system. At the same time, we found that APE1 could drive the enzymatic positive feedback circuit and exhibited considerably high amplification efficiency to enhance the detection ability of nucleic acids. Besides, our method provides universal platforms and can be realized in real-time and one-pot detection of HIV-1 DNA by replacing the inhibitors and crRNA with different target recognition sequences.<br><br>SIGNIFICANCE AND NOVELTY: Overall, due to the high programmability of the nucleic acid network, this work proposed a feasible way to use the steric hindrance-based inhibitors as a switchable element, decorating the CRISPR/Cas12a-based strategy equipment for molecular diagnostics. Besides, this strategy could offer a simple tool for detecting trace nucleic acid, which opens avenues for future clinical application.}, }
@article {pmid39788657, year = {2025}, author = {Siler, T and Stanley, L and Saleem, M and Badalyan, A}, title = {A non-covalently bound redox indicator for electrochemical CRISPR-Cas12a and DNase I biosensors.}, journal = {Analytica chimica acta}, volume = {1336}, number = {}, pages = {343480}, doi = {10.1016/j.aca.2024.343480}, pmid = {39788657}, issn = {1873-4324}, mesh = {*Biosensing Techniques/methods ; *Oxidation-Reduction ; *Electrochemical Techniques ; *DNA, Single-Stranded/chemistry/metabolism ; *Deoxyribonuclease I/metabolism/chemistry ; *CRISPR-Cas Systems ; Methylene Blue/chemistry ; CRISPR-Associated Proteins/metabolism/chemistry ; }, abstract = {A rapid and accurate biosensor for detecting disease biomarkers at point-of-care is essential for early disease diagnosis and preventing pandemics. CRISPR-Cas12a is a promising recognition element for DNA biosensors due to its programmability, specificity, and deoxyribonuclease activity initiated in the presence of a biomarker. The current electrochemical CRISPR-Cas12a-based biosensors utilize the single-stranded DNA (ssDNA) self-assembled on an electrode surface and covalently modified with the redox indicator, usually methylene blue (MB). In the presence of a biomarker, the nuclease domain is activated and cleaves ssDNA, decreasing the redox indicator signal. The covalent attachment of the MB to the ssDNA implies complexity and a higher production cost. Alternatively, some redox indicators can noncovalently bind to the ssDNA. Although such indicators have been applied for electrochemical nucleic acid detection, their potential for electrochemical CRISPR-Cas-based biosensors has not been explored. In this work, a ruthenium complex, [Ru(NH3)6][3+], was investigated as a redox indicator non-covalently binding to the ssDNA. Voltammetric studies and the optimization resulted in a simple and robust electrochemical method that was tested for deoxyribonuclease I (DNase I) activity detection and applied in the CRISPR-Cas12a-based biosensor for viral DNA (HPV-16). The biosensors revealed good analytical properties and represent an alternative to reported biosensors for nuclease activity requiring a covalent attachment of the redox indicator. Moreover, the developed method offers prospects for advancement and can be transformed to operate with other Cas nucleases to detect RNA and other analytes.}, }
@article {pmid39788645, year = {2025}, author = {Chen, Y and Zhao, R and Hu, X and Wang, X}, title = {The current status and future prospects of CRISPR-based detection of monkeypox virus: A review.}, journal = {Analytica chimica acta}, volume = {1336}, number = {}, pages = {343295}, doi = {10.1016/j.aca.2024.343295}, pmid = {39788645}, issn = {1873-4324}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Monkeypox virus/genetics/isolation &amp; purification ; *Mpox (monkeypox)/diagnosis/virology ; }, abstract = {BACKGROUND: The current pandemic of 2022 global mpox (formerly known as monkeypox), caused by infection with monkeypox virus (MPXV), has now reached over 120 countries. This constitutes a critical public health issue requiring effective disease management and surveillance. Rapid and reliable diagnosis is conducive to the control of infection, early intervention, and timely treatment. Clinical laboratories use various conventional diagnostic methods for detecting MPXV, including PCR, which can be regarded as a gold-standard diagnostic method. However, the application of PCR is limited by its requirements for high-cost equipment, skilled professionals, and a laboratory setting.<br><br>RESULTS: Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic systems have provided promising prospects for the rapid, sensitive, and specific detection of infectious diseases, especially in point-of-care settings. Over the past 2 years, an increasing number of researchers have concentrated on the application of the CRISPR method to mpox diagnosis. In the majority of cases, a two-step method was chosen, with CRISPR/Cas12a and recombinase polymerase amplification (RPA) as pre-amplification methods, followed by a fluorescence readout. Different strategies have been applied to overcome the encountered limitations of CRISPR detection, but no consensus on an integrated solution has been achieved. Thus, the application of the CRISPR/Cas system in mpox detection requires further exploration and improvement.<br><br>SIGNIFICANCE: This review discusses contemporary studies on MPXV CRISPR detection systems and the strategies proposed to address the challenges faced by CRISPR diagnosis with the hope of helping the development of CRISPR detection methods and improving pathogen detection technologies.}, }
@article {pmid39788509, year = {2024}, author = {Wu, K and Xie, J and Liu, X and Yang, D and Wang, Y and Zhao, W and Shang, X and Jiang, L}, title = {Construction and biological function of Toxoplasma gondii rop41 gene knockout strain.}, journal = {Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences}, volume = {49}, number = {8}, pages = {1200-1209}, pmid = {39788509}, issn = {1672-7347}, support = {32170510//the National Natural Science Foundation of China/ ; 20240026020055//the Innovation Training Program of Central South University/ ; }, mesh = {*Toxoplasma/genetics ; *Protozoan Proteins/genetics/metabolism ; *Gene Knockout Techniques ; CRISPR-Cas Systems ; Plasmids/genetics ; Virulence Factors/genetics ; Animals ; }, abstract = {OBJECTIVES: Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii (T. gondii), which can lead to complications such as encephalitis and ocular toxoplasmosis. The disease becomes more severe when the host's immune system is compromised. Rhoptry proteins are major virulence factors that enable T. gondii to invade host cells. This study aims to construct a T. gondii rhoptry protein 41 (rop41/ROP41) gene knockout strain and preliminarily investigate the biological function of rop41.<br><br>METHODS: Using CRISPR/Cas9 technology, a specific single-guide RNA (sgRNA) for the target gene was designed and linked to a recombinant plasmid. Homologous fragments were fused with a pyrimethamine resistance gene for selection purposes. The recombinant plasmid and the homologous fragments were electroporated into T. gondii, and PCR identification was performed after drug selection and monoclonal screening. Plaque assays were used to comprehensively assess whether rop41 affected the growth and proliferation of T. gondii in host cells. Invasion and proliferation assays were conducted to evaluate the invasion ability of the knockout strain into host cells and its intracellular proliferation capacity. The STRING database was utilized to construct a protein-protein interaction (PPI) network, and functional enrichment analysis was performed to predict the signaling pathways in which ROP41 might be involved.<br><br>RESULTS: The T. gondiirop41 gene knockout strain (RH &#x394;ku80&#x394;rop41) was successfully constructed and stably inherited. Plaque assays showed that compared with the parental strain, the number of plaques formed by the rop41 gene knockout strain did not significantly decrease, but the reduction in plaque size was statistically significant (P<0.05). After the rop41 gene was knocked out, the invasion ability of T. gondii was reduced, but there was no statistically significant difference in its proliferation ability (P>0.05). The PPI network revealed that ROP41 was associated with other protein kinases and autophagy-related proteins. Enrichment analysis indicated that proteins interacting with ROP41 may be involved in signal transduction, biosynthesis, metabolism, and autophagy-related pathways and could be components of various kinase complexes and phagocytic vesicles.<br><br>CONCLUSIONS: The T. gondii RH &#x394;ku80&#x394;rop41 strain has been successfully constructed. ROP41 primarily affects the ability of T. gondii to invade host cells and may play a role in signal transduction and autophagy-related pathways between T. gondii and the host.}, }
@article {pmid39788098, year = {2025}, author = {Lin, J and Bhoobalan-Chitty, Y and Peng, X}, title = {Cad1 turns ATP into phage poison.}, journal = {Cell host &amp; microbe}, volume = {33}, number = {1}, pages = {8-10}, doi = {10.1016/j.chom.2024.12.013}, pmid = {39788098}, issn = {1934-6069}, mesh = {*Adenosine Triphosphate/metabolism ; *Bacteriophages/genetics/physiology/metabolism ; *CRISPR-Cas Systems ; Escherichia coli/genetics/virology/metabolism ; }, abstract = {Type III CRISPR-Cas executes a multifaceted anti-phage response, activating effectors such as a nuclease or membrane depolarizer. In a recent Cell paper, Baca and Majumder et al.[1] report an accessory effector, Cad1, which deaminates ATP into ITP, causing ITP accumulation and host growth arrest, thereby inhibiting phage propagation.}, }
@article {pmid39787832, year = {2025}, author = {Li, O and Zhen, Y and Sun, C and Ma, Y and Li, Q and Wen, L}, title = {Generation of a KDM5D knockout human embryonic stem cell line with CRISPR/Cas9 technology.}, journal = {Stem cell research}, volume = {83}, number = {}, pages = {103651}, doi = {10.1016/j.scr.2025.103651}, pmid = {39787832}, issn = {1876-7753}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Gene Knockout Techniques ; *Histone Demethylases/genetics/metabolism/deficiency ; Cell Line ; Male ; }, abstract = {KDM5D is a gene implicated in spermatogenic failure and sex-related differences in colon cancer progression, though its role in spermatogenesis remains unclear. We successfully generated a KDM5D knockout human embryonic stem cells using CRISPR/Cas9 technology. This knockout cell line provides a valuable model for studying KDM5D's function in spermatogenesis and its influence on sex differences in various diseases.}, }
@article {pmid39787667, year = {2025}, author = {Hall, R and Sikora, T and Suter, A and Kuah, JY and Christodoulou, J and Van Bergen, NJ}, title = {Generation and heterozygous repair of human iPSC lines from two individuals with the neurodevelopmental disorder, TRAPPC4 deficiency.}, journal = {Stem cell research}, volume = {82}, number = {}, pages = {103640}, doi = {10.1016/j.scr.2024.103640}, pmid = {39787667}, issn = {1876-7753}, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Neurodevelopmental Disorders/genetics/pathology ; Heterozygote ; *Transcription Factors/deficiency/genetics ; Male ; Cell Line ; Female ; CRISPR-Cas Systems ; Cell Differentiation ; }, abstract = {A rare neurodevelopmental disorder has been linked to a well-conserved splice site variant in the TRAPPC4 gene (c.454 + 3A > G), which causes mis-splicing of TRAPPC4 transcripts and reduced levels of TRAPPC4 protein. Patients present with severe progressive neurological symptoms including seizures, microcephaly, intellectual disability and facial dysmorphism. We have generated stem cells from fibroblasts of two individuals with the same homozygous TRAPPC4 c.454 + 3A > G pathogenic variant and used CRISPR/Cas9 editing to generate heterozygous gene-corrected isogenic controls. Clones were tested for pluripotency, differentiation potential, genotyped and karyotyped. These iPSC-based models will be used to understand disease mechanisms of TRAPPC4 disorder.}, }
@article {pmid39786576, year = {2025}, author = {Zafeer, MF and Ramzan, M and Duman, D and Mutlu, A and Seyhan, S and Kalcioglu, MT and Fitoz, S and DeRosa, BA and Guo, S and Dykxhoorn, DM and Tekin, M}, title = {Human organoids for rapid validation of gene variants linked to cochlear malformations.}, journal = {Human genetics}, volume = {144}, number = {4}, pages = {375-389}, pmid = {39786576}, issn = {1432-1203}, support = {P30 CA240139/CA/NCI NIH HHS/United States ; R01 DC009645/DC/NIDCD NIH HHS/United States ; R01 DC012836/DC/NIDCD NIH HHS/United States ; NIH R01DC009645/DC/NIDCD NIH HHS/United States ; }, mesh = {Humans ; *Organoids/metabolism/pathology ; *Cochlea/abnormalities/pathology/metabolism ; Induced Pluripotent Stem Cells/metabolism ; Male ; Female ; Polymorphism, Single Nucleotide ; Exome Sequencing ; CRISPR-Cas Systems ; Fibroblast Growth Factor 3 ; }, abstract = {Developmental anomalies of the hearing organ, the cochlea, are diagnosed in approximately one-fourth of individuals with congenital. The majority of patients with cochlear malformations remain etiologically undiagnosed due to insufficient knowledge about underlying genes or the inability to make conclusive interpretations of identified genetic variants. We used exome sequencing for the genetic evaluation of hearing loss associated with cochlear malformations in three probands from unrelated families deafness. We subsequently generated monoclonal induced pluripotent stem cell (iPSC) lines, bearing patient-specific knockins and knockouts using CRISPR/Cas9 to assess pathogenicity of candidate variants. We detected FGF3 (p.Arg165Gly) and GREB1L (p.Cys186Arg), variants of uncertain significance in two recognized genes for deafness, and PBXIP1(p.Trp574*) in a candidate gene. Upon differentiation of iPSCs towards inner ear organoids, we observed developmental aberrations in knockout lines compared to their isogenic controls. Patient-specific single nucleotide variants (SNVs) showed similar abnormalities as the knockout lines, functionally supporting their causality in the observed phenotype. Therefore, we present human inner ear organoids as a potential tool to validate the pathogenicity of DNA variants associated with cochlear malformations.}, }
@article {pmid39786412, year = {2025}, author = {Uchida, K and Fuji, Y and Tabeta, H and Akashi, T and Hirai, MY}, title = {Omics-based identification of the broader effects of 2-hydroxyisoflavanone synthase gene editing on a gene regulatory network beyond isoflavonoid loss in soybean hairy roots.}, journal = {Plant &amp; cell physiology}, volume = {66}, number = {3}, pages = {304-317}, pmid = {39786412}, issn = {1471-9053}, support = {JP19K15821//Japan Society for the Promotion of Science/ ; JPJ012287//Cross-ministerial Strategic Innovation Promotion Program/ ; //soybean protein research/ ; }, mesh = {*Glycine max/genetics/metabolism/enzymology ; *Gene Editing/methods ; *Isoflavones/metabolism ; *Plant Roots/genetics/metabolism ; *Gene Regulatory Networks/genetics ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant ; Flavonoids/metabolism ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Oxygenases ; }, abstract = {Soybean (Glycine max) is a leguminous crop cultivated worldwide that accumulates high levels of isoflavones. Although previous research has often focused on increasing the soybean isoflavone content because of the estrogen-like activity of dietary soy in humans, the rapidly increasing demand for soybean as a plant-based meat substitute has raised concerns about excessive isoflavone intake. Therefore, the production of isoflavone-free soybean has been anticipated. However, there have been no reports of an isoflavone-free soybean until now. Here, 2-hydroxyisoflavanone synthase (IFS), which is essential for isoflavone biosynthesis, was targeted for genome editing in soybean. A novel CRISPR/Cas9 system using Staphylococcus aureus Cas9 instead of the commonly used Streptococcus pyogenes Cas9 was established and customized. Through Agrobacterium rhizogenes-mediated transformation, IFS-edited hairy roots were generated in which all three IFS genes contained deletion mutations. Metabolome analyses of IFS-edited hairy roots revealed that isoflavone content significantly decreased, whereas levels of flavonoids, including a novel chalcone derivative, increased. A transcriptome analysis revealed changes in the expression levels of a large number of genes, including jasmonic acid-inducible genes. In addition, the functions of selected transcription factor genes (MYB14-L, GmbHLH112, and GmbHLH113), which were dramatically upregulated by IFS editing, were investigated by multiomics analyses of their over-expressing hairy root lines. They appear to be involved in flavonoid and triterpene saponin biosynthesis, salicylic acid metabolism, and central carbon metabolism. Overall, the results indicated that editing IFS genes caused the redirection of the metabolic flux from isoflavonoid biosynthesis to flavonoid accumulation, as well as dynamic changes in gene regulatory networks.}, }
@article {pmid39784310, year = {2025}, author = {Dai, J and Wu, B and Ai, F and Yang, Z and Lu, Y and Zinian, C and Zeng, K and Zhang, Z}, title = {Exploiting the Potential of Spherical PAM Antenna for Enhanced CRISPR-Cas12a: A Paradigm Shift toward a Universal Amplification-Free Nucleic Acid Test Platform.}, journal = {Analytical chemistry}, volume = {97}, number = {2}, pages = {1236-1245}, doi = {10.1021/acs.analchem.4c04871}, pmid = {39784310}, issn = {1520-6882}, mesh = {*CRISPR-Cas Systems ; Mycobacterium tuberculosis/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; Limit of Detection ; Humans ; *CRISPR-Associated Proteins/metabolism ; Nucleic Acid Amplification Techniques ; *Nucleic Acids/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {The CRISPR-Cas12a system has shown tremendous potential for developing efficient biosensors. Albeit important, current CRISPR-Cas system-based diagnostic technologies (CRISPR-DX) highly rely on an additional preamplification procedure to obtain high sensitivity, inevitably leading to issues such as complicated assay workflow, cross-contamination, etc. Herein, a spherical protospacer-adjacent motif (PAM)-antenna-enhanced CRISPR-Cas12a system is fabricated for universal amplification-free nucleic acid detection with a detection limit of subfemtomolar. Meanwhile, the clinical detection capability of this sensor was further verified using gold-standard real-time quantitative polymerase chain reaction through Mycobacterium tuberculosis measurement, which demonstrated its good reliability for practical applications. Importantly, its excellent sensitivity is mainly ascribed to high efficiency of target search induced by a localized PAM-enriched microenvironment and improved catalytic activity of Cas12a (up to 4 folds). Our strategy provides some new insights for rapid and sensitive detection of nucleic acids in an amplification-free fashion.}, }
@article {pmid39781718, year = {2025}, author = {Saberi, F and Yousefi-Najafabadi, Z and Shams, F and Dehghan, Z and Ahmadi, S and Pilehchi, T and Noori, E and Esmaeelzadeh, Z and Bazgiri, M and Mohammadi, R and Khani, F and Sameni, M and Moradbeigi, P and Kardar, GA and Salehi, M and Teng, Y and Jajarmi, V}, title = {CRISPR/Cas System: A Powerful Strategy to Improve Monogenic Human Diseases as Therapeutic Delivery; Current Applications and Challenges.}, journal = {Current gene therapy}, volume = {}, number = {}, pages = {}, doi = {10.2174/0115665232345516241119070150}, pmid = {39781718}, issn = {1875-5631}, abstract = {The 5,000 to 8,000 monogenic diseases are inherited disorders leading to mutations in a single gene. These diseases usually appear in childhood and sometimes lead to morbidity or premature death. Although treatments for such diseases exist, gene therapy is considered an effective and targeted method and has been used in clinics for monogenic diseases since 1989. Monogenic diseases are good candidates for novel therapeutic technologies like gene editing approaches to repair gene mutations. Clustered regularly interspaced short palindromic repeats (CRISPR)-based systems, the pioneer and effective gene editing tool, are utilized for ex vivo and in vivo treatment of monogenic diseases. The current review provides an overview of recent therapeutic applications of CRISPR-based gene editing in monogenic diseases in in vivo and ex vivo models. Furthermore, this review consolidates strategies aimed at providing new treatment options with gene therapy, thereby serving as a valuable reference for advancing the treatment landscape for patients with monogenic disorders.}, }
@article {pmid39780710, year = {2025}, author = {Elkhadragy, L and Carlino, MJ and Jordan, LR and Pennix, T and Ismail, N and Guzman, G and Samuelson, JP and Schook, LB and Schachtschneider, KM and Gaba, RC}, title = {Development of a genetically tailored implantation hepatocellular carcinoma model in Oncopigs by somatic cell CRISPR editing.}, journal = {Disease models &amp; mechanisms}, volume = {18}, number = {1}, pages = {}, pmid = {39780710}, issn = {1754-8411}, support = {R21 CA219461/CA/NCI NIH HHS/United States ; 1R21CA219461/CA/NCI NIH HHS/United States ; 1R21CA219461//NIH NCI/ ; }, mesh = {Animals ; *Carcinoma, Hepatocellular/genetics/pathology ; *Gene Editing ; *Liver Neoplasms/genetics/pathology ; *CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Cell Proliferation ; PTEN Phosphohydrolase/genetics/metabolism ; Cell Line, Tumor ; Cell Movement ; Mutation/genetics ; Humans ; Sus scrofa ; Swine ; Animals, Genetically Modified ; Cyclin-Dependent Kinase Inhibitor p16/genetics/metabolism ; }, abstract = {Hepatocellular carcinoma (HCC) is an aggressive disease with poor prognosis, necessitating preclinical models for evaluating novel therapies. Large-animal models are particularly valuable for assessing locoregional therapies, which are widely employed across HCC stages. This study aimed to develop a large-animal HCC model with tailored tumor mutations. The Oncopig, a genetically engineered pig with inducible TP53R167H and KRASG12D, was used in the study. Hepatocytes were isolated from Oncopigs and exposed to Cre recombinase in vitro to create HCC cells, and additional mutations were introduced by CRISPR/Cas9 knockout of PTEN and CDKN2A. These edits increased Oncopig HCC cell proliferation and migration. Autologous HCC cells with these CRISPR edits were implanted into Oncopigs using two approaches: ultrasound-guided percutaneous liver injections, which resulted in the development of localized intrahepatic masses, and portal vein injections, which led to multifocal tumors that regressed over time. Tumors developed by both approaches harbored PTEN and CDKN2A knockout mutations. This study demonstrates the feasibility of developing genetically tailored HCC tumors in Oncopigs using somatic cell CRISPR editing and autologous implantation, providing a valuable large-animal model for in vivo therapeutic assessment.}, }
@article {pmid39779901, year = {2025}, author = {Son, HI and Hamrick, GS and Shende, AR and Kim, K and Yang, K and Huang, TJ and You, L}, title = {Population-level amplification of gene regulation by programmable gene transfer.}, journal = {Nature chemical biology}, volume = {21}, number = {6}, pages = {939-948}, pmid = {39779901}, issn = {1552-4469}, support = {R01EB031869//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; }, mesh = {Plasmids/genetics ; *Gene Expression Regulation, Bacterial ; *Gene Transfer Techniques ; *Escherichia coli/genetics ; CRISPR-Cas Systems ; }, abstract = {Engineering cells to sense and respond to environmental cues often focuses on maximizing gene regulation at the single-cell level. Inspired by population-level control mechanisms like the immune response, we demonstrate dynamic control and amplification of gene regulation in bacterial populations using programmable plasmid-mediated gene transfer. By regulating plasmid loss rate, transfer rate and fitness effects via Cas9 endonuclease, F conjugation machinery and antibiotic selection, we modulate the fraction of plasmid-carrying cells, serving as an amplification factor for single-cell-level regulation. This approach expands the dynamic range of gene expression and allows orthogonal control across populations. Our platform offers a versatile strategy for dynamically regulating gene expression in engineered microbial communities.}, }
@article {pmid39779857, year = {2025}, author = {Huang, H and Hu, C and Na, J and Hart, SN and Gnanaolivu, RD and Abozaid, M and Rao, T and Tecleab, YA and ,  and Pesaran, T and Lyra, PCM and Karam, R and Yadav, S and Nathanson, KL and Domchek, SM and de la Hoya, M and Robson, M and Mehine, M and Bandlamudi, C and Mandelker, D and Monteiro, ANA and Iversen, ES and Boddicker, N and Chen, W and Richardson, ME and Couch, FJ}, title = {Functional evaluation and clinical classification of BRCA2 variants.}, journal = {Nature}, volume = {638}, number = {8050}, pages = {528-537}, pmid = {39779857}, issn = {1476-4687}, support = {K12 CA090628/CA/NCI NIH HHS/United States ; P50 CA116201/CA/NCI NIH HHS/United States ; R01 CA225662/CA/NCI NIH HHS/United States ; R35 CA253187/CA/NCI NIH HHS/United States ; }, mesh = {Humans ; *BRCA2 Protein/genetics/metabolism/chemistry ; Breast Neoplasms/genetics ; Female ; Ovarian Neoplasms/genetics ; Bayes Theorem ; CRISPR-Cas Systems/genetics ; Mutation, Missense/genetics ; Gene Editing ; Loss of Function Mutation/genetics ; Exons/genetics ; Genetic Testing ; Gene Knock-In Techniques ; }, abstract = {Germline BRCA2 loss-of function variants, which can be identified through clinical genetic testing, predispose to several cancers[1-5]. However, variants of uncertain significance limit the clinical utility of test results. Thus, there is a need for functional characterization and clinical classification of all BRCA2 variants to facilitate the clinical management of individuals with these variants. Here we analysed all possible single-nucleotide variants from exons 15 to 26 that encode the BRCA2 DNA-binding domain hotspot for pathogenic missense variants. To enable this, we used saturation genome editing CRISPR-Cas9-based knock-in endogenous targeting of human haploid HAP1 cells[6]. The assay was calibrated relative to nonsense and silent variants and was validated using pathogenic and benign standards from ClinVar and results from a homology-directed repair functional assay[7]. Variants (6,959 out of 6,960 evaluated) were assigned to seven categories of pathogenicity based on a VarCall Bayesian model[8]. Single-nucleotide variants that encode loss-of-function missense variants were associated with increased risks of breast cancer and ovarian cancer. The functional assay results were integrated into models from ClinGen, the American College of Medical Genetics and Genomics, and the Association for Molecular Pathology[9] for clinical classification of BRCA2 variants. Using this approach, 91% were classified as pathogenic or likely pathogenic or as benign or likely benign. These classified variants can be used to improve clinical management of individuals with a BRCA2 variant.}, }
@article {pmid39779848, year = {2025}, author = {Sahu, S and Galloux, M and Southon, E and Caylor, D and Sullivan, T and Arnaudi, M and Zanti, M and Geh, J and Chari, R and Michailidou, K and Papaleo, E and Sharan, SK}, title = {Saturation genome editing-based clinical classification of BRCA2 variants.}, journal = {Nature}, volume = {638}, number = {8050}, pages = {538-545}, pmid = {39779848}, issn = {1476-4687}, mesh = {*Gene Editing ; Humans ; *BRCA2 Protein/genetics/chemistry/metabolism ; Animals ; Mice ; CRISPR-Cas Systems/genetics ; Female ; *Polymorphism, Single Nucleotide/genetics ; Exons/genetics ; Cell Line ; Mutation, Missense/genetics ; }, abstract = {Sequencing-based genetic tests have uncovered a vast array of BRCA2 sequence variants[1]. Owing to limited clinical, familial and epidemiological data, thousands of variants are considered to be variants of uncertain significance[2-4] (VUS). Here we have utilized CRISPR-Cas9-based saturation genome editing in a humanized mouse embryonic stem cell line to determine the functional effect of VUS. We have categorized nearly all possible single nucleotide variants (SNVs) in the region that encodes the carboxylate-terminal DNA-binding domain of BRCA2. We have generated function scores for 6,551 SNVs, covering 96.4% of possible SNVs in exons 15-26 spanning BRCA2 residues 2479-3216. These variants include 1,282 SNVs that are categorized as missense VUS in the clinical variant database ClinVar, with 77.2% of these classified as benign and 20.4% classified as pathogenic using our functional score. Our assay provides evidence that 3,384 of the SNVs in the region are benign and 776 are pathogenic. Our classification aligns closely with pathogenicity data from ClinVar, orthogonal functional assays and computational meta predictors. We have integrated our embryonic stem cell-based BRCA2-saturation genome editing dataset with other available evidence and utilized the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines for clinical classification of all possible SNVs. This classification is available as a sequence-function map and serves as a valuable resource for interpreting unidentified variants in the population and for physicians and genetic counsellors to assess BRCA2 VUS in patients.}, }
@article {pmid39779704, year = {2025}, author = {Kempthorne, L and Vaizoglu, D and Cammack, AJ and Carcolé, M and Roberts, MJ and Mikheenko, A and Fisher, A and Suklai, P and Muralidharan, B and Kroll, F and Moens, TG and Yshii, L and Verschoren, S and Hölbling, BV and Moreira, FC and Katona, E and Coneys, R and de Oliveira, P and Zhang, YJ and Jansen, K and Daughrity, LM and McGown, A and Ramesh, TM and Van Den Bosch, L and Lignani, G and Rahim, AA and Coyne, AN and Petrucelli, L and Rihel, J and Isaacs, AM}, title = {Dual-targeting CRISPR-CasRx reduces C9orf72 ALS/FTD sense and antisense repeat RNAs in vitro and in vivo.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {459}, pmid = {39779704}, issn = {2041-1723}, support = {/WT_/Wellcome Trust/United Kingdom ; 217150/Z/19/Z//Wellcome Trust (Wellcome)/ ; 648716 - C9ND//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; }, mesh = {*C9orf72 Protein/genetics/metabolism ; *Amyotrophic Lateral Sclerosis/genetics/metabolism/therapy ; Humans ; *Frontotemporal Dementia/genetics/metabolism ; Animals ; *CRISPR-Cas Systems ; *RNA, Antisense/genetics ; Mice ; HEK293 Cells ; *Induced Pluripotent Stem Cells/metabolism ; DNA Repeat Expansion/genetics ; Disease Models, Animal ; Neurons/metabolism ; Genetic Therapy/methods ; }, abstract = {The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is an intronic G4C2 repeat expansion in C9orf72. The repeats undergo bidirectional transcription to produce sense and antisense repeat RNA species, which are translated into dipeptide repeat proteins (DPRs). As toxicity has been associated with both sense and antisense repeat-derived RNA and DPRs, targeting both strands may provide the most effective therapeutic strategy. CRISPR-Cas13 systems mature their own guide arrays, allowing targeting of multiple RNA species from a single construct. We show CRISPR-Cas13d variant CasRx effectively reduces overexpressed C9orf72 sense and antisense repeat transcripts and DPRs in HEK cells. In C9orf72 patient-derived iPSC-neuron lines, CRISPR-CasRx reduces endogenous sense and antisense repeat RNAs and DPRs and protects against glutamate-induced excitotoxicity. AAV delivery of CRISPR-CasRx to two distinct C9orf72 repeat mouse models significantly reduced both sense and antisense repeat-containing transcripts. This highlights the potential of RNA-targeting CRISPR systems as therapeutics for C9orf72 ALS/FTD.}, }
@article {pmid39779681, year = {2025}, author = {McCallister, TX and Lim, CKW and Singh, M and Zhang, S and Ahsan, NS and Terpstra, WM and Xiong, AY and Zeballos C, MA and Powell, JE and Drnevich, J and Kang, Y and Gaj, T}, title = {A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {460}, pmid = {39779681}, issn = {2041-1723}, support = {1R01NS123556-01A1//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; 1U01NS122102-01A1//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; 5R01GM141296//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; MDA602798//Muscular Dystrophy Association (Muscular Dystrophy Association Inc.)/ ; R01 GM141296/GM/NIGMS NIH HHS/United States ; 20-IIP-516//Amyotrophic Lateral Sclerosis Association (ALS Association)/ ; T32 EB019944/EB/NIBIB NIH HHS/United States ; U01 NS122102/NS/NINDS NIH HHS/United States ; R01 NS123556/NS/NINDS NIH HHS/United States ; }, mesh = {*Amyotrophic Lateral Sclerosis/genetics/pathology/metabolism ; *C9orf72 Protein/genetics/metabolism ; *Frontotemporal Dementia/genetics/pathology/metabolism ; *CRISPR-Cas Systems ; Humans ; Animals ; DNA Repeat Expansion/genetics ; Disease Models, Animal ; Motor Neurons/metabolism/pathology ; Mice ; }, abstract = {An abnormal expansion of a GGGGCC (G4C2) hexanucleotide repeat in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two debilitating neurodegenerative disorders driven in part by gain-of-function mechanisms involving transcribed forms of the repeat expansion. By utilizing a Cas13 variant with reduced collateral effects, we develop here a high-fidelity RNA-targeting CRISPR-based system for C9ORF72-linked ALS/FTD. When delivered to the brain of a transgenic rodent model, this Cas13-based platform curbed the expression of the G4C2 repeat-containing RNA without affecting normal C9ORF72 levels, which in turn decreased the formation of RNA foci, reduced the production of a dipeptide repeat protein, and reversed transcriptional deficits. This high-fidelity system possessed improved transcriptome-wide specificity compared to its native form and mediated targeting in motor neuron-like cells derived from a patient with ALS. These results lay the foundation for the implementation of RNA-targeting CRISPR technologies for C9ORF72-linked ALS/FTD.}, }
@article {pmid39779650, year = {2025}, author = {Aydin, A and Yerlikaya, BA and Yerlikaya, S and Yilmaz, NN and Kavas, M}, title = {CRISPR-mediated mutation of cytokinin signaling genes (SlHP2 and SlHP3) in tomato: Morphological, physiological, and molecular characterization.}, journal = {The plant genome}, volume = {18}, number = {1}, pages = {e20542}, pmid = {39779650}, issn = {1940-3372}, support = {PYO.ZRT.1904.22.006//Ondokuz Mayis &#xdc;niversitesi/ ; }, mesh = {*Solanum lycopersicum/genetics/physiology ; *Cytokinins/metabolism ; *Signal Transduction ; Mutation ; CRISPR-Cas Systems ; Droughts ; Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Stress, Physiological/genetics ; Gene Editing ; }, abstract = {Synergistic and antagonistic relationships between cytokinins and other plant growth regulators are important in response to changing environmental conditions. Our study aimed to determine the functions of SlHP2 and SlHP3, two members of cytokinin signaling in tomato, in drought stress response using CRISPR/Cas9-mediated mutagenesis. Ten distinct genome-edited lines were generated via Agrobacterium tumefaciens-mediated gene transfer and confirmed through Sanger sequencing. Stress experiments were conducted with two of these lines (slhp2,3-10 and slhp2,3-11), which harbored homozygous mutations in both genes. The responses of two lines carrying homozygous mutations in both genes under polyethylene glycol (PEG)-induced stress were examined using morphological, physiological, biochemical, and molecular methods. The genome-edited lines demonstrated enhanced water retention, reduced stomatal density, and less oxidative damage compared to the wild-type plants under PEG-induced stress. Moreover, the slhp2,3 double mutant plants exhibited improved root growth, showcasing their superior drought tolerance over wild-type plants by accessing deeper water sources and maintaining hydration in water-limited environments. To investigate the involvement of cytokinin signaling regulators and genes associated with stomatal formation and differentiation, the expression of genes (Speechless [SPCH], FAMA, MUTE, TMM, HB25, HB31, RR6, RR7, and Solyc02g080860) was assessed. The results revealed that all regulators were downregulated, with SPCH, TMM, RR7, and RR6 showing significant reductions under PEG-induced stress. These results emphasize the promise of utilizing CRISPR/Cas9 to target cytokinin signaling pathways, enhancing drought tolerance in tomatoes through improvements in water retention and root growth, along with a reduction in stomatal density and malondialdehyde content.}, }
@article {pmid39779219, year = {2025}, author = {Tang, JX and Cabrera-Orefice, A and Meisterknecht, J and Taylor, LS and Monteuuis, G and Stensland, ME and Szczepanek, A and Stals, K and Davison, J and He, L and Hopton, S and Nyman, TA and Jackson, CB and Pyle, A and Winter, M and Wittig, I and Taylor, RW}, title = {COA5 has an essential role in the early stage of mitochondrial complex IV assembly.}, journal = {Life science alliance}, volume = {8}, number = {3}, pages = {}, pmid = {39779219}, issn = {2575-1077}, support = {/WT_/Wellcome Trust/United Kingdom ; }, mesh = {Humans ; Male ; *Mitochondria/metabolism/genetics ; *Electron Transport Complex IV/metabolism/genetics ; Female ; Mitochondrial Proteins/metabolism/genetics ; Mitochondrial Diseases/genetics/metabolism/pathology ; Pedigree ; CRISPR-Cas Systems ; Fibroblasts/metabolism ; Mutation, Missense ; }, abstract = {Pathogenic variants in cytochrome c oxidase assembly factor 5 (COA5), a proposed complex IV (CIV) assembly factor, have been shown to cause clinical mitochondrial disease with two siblings affected by neonatal hypertrophic cardiomyopathy manifesting a rare, homozygous COA5 missense variant (NM_001008215.3: c.157G>C, p.Ala53Pro). The most striking observation in the affected individuals was an isolated impairment in the early stage of mitochondrial CIV assembly. In this study, we report an unrelated family in whom we have identified the same COA5 variant with patient-derived fibroblasts and skeletal muscle biopsies replicating an isolated CIV deficiency. A CRISPR/Cas9-edited homozygous COA5 knockout U2OS cell line with a similar biochemical profile was generated to interrogate the functional role of the human COA5 protein. Mitochondrial complexome profiling pinpointed a role of COA5 in early CIV assembly, more specifically, its involvement in the stage between MTCO1 maturation and the incorporation of MTCO2. We therefore propose that the COA5 protein plays an essential role in the biogenesis of MTCO2 and its integration into the early CIV assembly intermediate for downstream assembly of the functional holocomplex.}, }
@article {pmid39778581, year = {2025}, author = {Lin, DW and Zhang, L and Zhang, J and Chandrasekaran, S}, title = {Inferring metabolic objectives and trade-offs in single cells during embryogenesis.}, journal = {Cell systems}, volume = {16}, number = {1}, pages = {101164}, pmid = {39778581}, issn = {2405-4720}, support = {R35 GM137795/GM/NIGMS NIH HHS/United States ; }, mesh = {*Single-Cell Analysis/methods ; *Embryonic Development/physiology/genetics ; Animals ; Mice ; Embryonic Stem Cells/metabolism ; Cell Proliferation ; Blastocyst/metabolism ; Machine Learning ; CRISPR-Cas Systems ; Cell Cycle ; }, abstract = {While proliferating cells optimize their metabolism to produce biomass, the metabolic objectives of cells that perform non-proliferative tasks are unclear. The opposing requirements for optimizing each objective result in a trade-off that forces single cells to prioritize their metabolic needs and optimally allocate limited resources. Here, we present single-cell optimization objective and trade-off inference (SCOOTI), which infers metabolic objectives and trade-offs in biological systems by integrating bulk and single-cell omics data, using metabolic modeling and machine learning. We validated SCOOTI by identifying essential genes from CRISPR-Cas9 screens in embryonic stem cells, and by inferring the metabolic objectives of quiescent cells, during different cell-cycle phases. Applying this to embryonic cell states, we observed a decrease in metabolic entropy upon development. We further uncovered a trade-off between glutathione and biosynthetic precursors in one-cell zygote, two-cell embryo, and blastocyst cells, potentially representing a trade-off between pluripotency and proliferation. A record of this paper's transparent peer review process is included in the supplemental information.}, }
@article {pmid39778479, year = {2025}, author = {Hildebrandt, F and N Matias, A and Treeck, M}, title = {A CRISPR view on genetic screens in Toxoplasma gondii.}, journal = {Current opinion in microbiology}, volume = {83}, number = {}, pages = {102577}, doi = {10.1016/j.mib.2024.102577}, pmid = {39778479}, issn = {1879-0364}, mesh = {*Toxoplasma/genetics ; *CRISPR-Cas Systems ; Gene Editing/methods ; Animals ; *Genetic Testing/methods ; Humans ; }, abstract = {Genome editing technologies, such as CRISPR-Cas9, have revolutionised the study of genes in a variety of organisms, including unicellular parasites. Today, the CRISPR-Cas9 technology is vastly applied in high-throughput screens to investigate interactions between the Apicomplexan parasite Toxoplasma gondii and its hosts. In vitro and in vivo T. gondii screens performed in naive and restrictive conditions have led to the discovery of essential and fitness-conferring T. gondii genes, as well as factors important for virulence and dissemination. Recent studies have adapted the CRISPR-Cas9 screening technology to study T. gondii genes based on phenotypes unrelated to parasite survival. These advances were achieved by using conditional systems coupled with imaging, as well as single-cell RNA sequencing and phenotypic selection. Here, we review the state-of-the-art of CRISPR-Cas9 screening technologies with a focus on T. gondii, highlighting strengths, current limitations and future avenues for its development, including its application to other Apicomplexan species.}, }
@article {pmid39777970, year = {2025}, author = {Gao, Y and Shan Ang, Y and Lanry Yung, LY}, title = {Modulation of CRISPR-Cas9 Cleavage with an Oligo-Ribonucleoprotein Design.}, journal = {Chembiochem : a European journal of chemical biology}, volume = {26}, number = {4}, pages = {e202400821}, pmid = {39777970}, issn = {1439-7633}, support = {A-0009534-01-00//Tier 1/ ; MOE-T2EP50120-0018//Tier 2/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Ribonucleoproteins/chemistry/metabolism/genetics ; Gene Editing ; *CRISPR-Associated Protein 9/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; HEK293 Cells ; DNA/chemistry/metabolism/genetics ; *Oligonucleotides/chemistry/metabolism/genetics ; DNA Cleavage ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) associated protein Cas9 system has been widely used for genome editing. However, the editing or cleavage specificity of CRISPR Cas9 remains a major concern due to the off-target effects. The existing approaches to control or modulate CRISPR Cas9 cleavage include engineering Cas9 protein and development of anti-CRISPR proteins. There are also attempts on direct modification of sgRNA, for example, structural modification via truncation or hairpin design, or chemical modification on sgRNA such as partially replacing RNA with DNA. The above-mentioned strategies rely on extensive protein engineering and direct chemical or structural modification of sgRNA. In this study, we proposed an indirect method to modulate CRISPR Cas9 cleavage without modification on Cas9 protein or sgRNA. An oligonucleotide was used to form an RNA-DNA hybrid structure with the sgRNA spacer, creating steric hindrance during the Cas9 mediated DNA cleavage process. We first introduced a simple and robust method to assemble the oligo-ribonucleoprotein (oligo-RNP). Next, the cleavage efficiency of the assembled oligo-RNP was examined using different oligo lengths in vitro. Lastly, we showed that the oligo-RNP directly delivered into cells could also modulate Cas9 activity inside cells using three model gene targets with reduced off-target effects.}, }
@article {pmid39777467, year = {2025}, author = {Xu, X and Lv, X and Liu, Y and Li, J and Du, G and Chen, J and Ledesma-Amaro, R and Liu, L}, title = {CRISPR/Cas13X-assisted programmable and multiplexed translation regulation for controlled biosynthesis.}, journal = {Nucleic acids research}, volume = {53}, number = {1}, pages = {}, pmid = {39777467}, issn = {1362-4962}, support = {32200050//National Natural Science Foundation of China/ ; BK20221079//Natural Science Foundation of Jiangsu Province/ ; JUSRP52019A//Fundamental Research Funds for the Central Universities/ ; 2020YFA0908300//National Key Research and Development Program of China/ ; BK20233003//Jiangsu Basic Research Center for Synthetic Biology/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Bacillus subtilis/genetics/metabolism ; Protein Biosynthesis/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; *Bioreactors ; Gene Expression Regulation, Bacterial ; RNA Stability ; RNA, Messenger/genetics/metabolism ; RNA, Bacterial/genetics/metabolism ; Biosynthetic Pathways/genetics ; Riboflavin/biosynthesis/isolation &amp; purification ; Trisaccharides/biosynthesis/isolation &amp; purification ; *Metabolic Engineering/methods ; }, abstract = {Developing efficient gene regulation tools is essential for optimizing microbial cell factories, but most existing tools only modulate gene expression at the transcriptional level. Regulation at the translational level provides a faster dynamic response, whereas developing a programmable, efficient and multiplexed translational regulation tool remains a challenge. Here, we have developed CRISPRi and CRISPRa systems based on hfCas13X that can regulate gene translation in Bacillus subtilis. First, we constructed a CRISPRi system to regulate gene translation based on catalytically deactivated hfCas13X (dhfCas13X). Second, we designed unique mRNA-crRNA pairs to construct DiCRISPRa (degradation-inhibited CRISPRa) and TsCRISPRa (translation-started CRISPRa) systems, which can activate downstream gene translation by enhancing mRNA stability or initiating mRNA translation. In addition, we found that fusing dhfCas13X with the RNA-binding chaperone BHfq significantly improved the activation efficiency of the DiCRISPRa and TsCRISPRa systems (43.2-fold). Finally, we demonstrated that the constructed CRISPR systems could be used to optimize the metabolic networks of two biotechnologically relevant compounds, riboflavin and 2'-fucosyllactose, increasing their titers by 3- and 1.2-fold, respectively. The CRISPRa and CRISPRi systems developed here provide new tools for the regulation of gene expression at the translation level and offer new ideas for the construction of CRISPRa systems.}, }
@article {pmid39776254, year = {2025}, author = {Amarilla-Quintana, S and Navarro, P and Hernández, I and Ramos, A and Montero-Calle, A and Cabezas-Sainz, P and Barrero, MJ and Megías, D and Vilaplana-Martí, B and Epifano, C and Gómez-Dominguez, D and Monzón, S and Cuesta, I and Sánchez, L and Barderas, R and García-Donas, J and Martín, A and Pérez de Castro, I}, title = {CRISPR targeting of FOXL2 c.402C&gt;G mutation reduces malignant phenotype in granulosa tumor cells and identifies anti-tumoral compounds.}, journal = {Molecular oncology}, volume = {19}, number = {4}, pages = {1092-1116}, pmid = {39776254}, issn = {1878-0261}, support = {BecaGethi-Ram&#xf3;ndelasPe&#xf1;as//Grupo Espa&#xf1;ol de Tumores Huerfanos e Infrecuentes/ ; ProyectosSemillaAECC//Fundaci&#xf3;n Cient&#xed;fica Asociaci&#xf3;n Espa&#xf1;ola Contra el C&#xe1;ncer/ ; }, mesh = {*Forkhead Box Protein L2/genetics ; *Granulosa Cell Tumor/genetics/pathology/drug therapy ; Female ; Humans ; Cell Line, Tumor ; *Mutation/genetics ; Phenotype ; *Antineoplastic Agents/pharmacology ; *Ovarian Neoplasms/genetics/pathology/drug therapy ; *CRISPR-Cas Systems ; Cell Proliferation/drug effects/genetics ; Apoptosis/drug effects/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression Regulation, Neoplastic/drug effects ; }, abstract = {Forkhead box L2 (FOXL2) encodes a transcription factor essential for sex determination, and ovary development and maintenance. Mutations in this gene are implicated in syndromes involving premature ovarian failure and granulosa cell tumors (GCTs). This rare cancer accounts for less than 5% of diagnosed ovarian cancers and is causally associated with the FOXL2 c.402C>G, p.C134W mutation in 97% of the adult cases (AGCTs). In this study, we employed CRISPR technology to specifically eliminate the FOXL2 c.402C>G mutation in granulosa tumor cells. Our results show that this Cas9-mediated strategy selectively targets the mutation without affecting the wild-type allele. Granulosa cells lacking FOXL2 c.402C>G exhibit a reduced malignant phenotype, with significant changes in cell proliferation and invasion. Furthermore, these modified cells are more susceptible to dasatinib and ketoconazole. Transcriptomic and proteomic analyses reveal that CRISPR-modified granulosa tumor cells shift their expression profiles towards a wild-type-like phenotype. Additionally, this altered expression signature has led to the identification of new compounds with antiproliferative and pro-apoptotic effects on granulosa tumor cells. Our findings demonstrate the potential of CRISPR technology for the specific targeting and elimination of a mutation causing GCTs, highlighting its therapeutic promise for treating this rare ovarian cancer.}, }
@article {pmid39776154, year = {2025}, author = {Carvalho, JE and Burtin, M and Detournay, O and Amiel, AR and Röttinger, E}, title = {Optimized husbandry and targeted gene-editing for the cnidarian Nematostella vectensis.}, journal = {Development (Cambridge, England)}, volume = {152}, number = {2}, pages = {}, doi = {10.1242/dev.204387}, pmid = {39776154}, issn = {1477-9129}, support = {ANR-15-IDEX-01//Agence Nationale de la Recherche/ ; ANR-20-CE13-014//Agence Nationale de la Recherche/ ; SPF20170938703//Fondation pour la Recherche M&#xe9;dicale/ ; SPF20130526781//Fondation pour la Recherche M&#xe9;dicale/ ; N/A//R&#xe9;gion SUD/ ; DBM//Institut des sciences biologiques/ ; //Centre national de la Recherche Scientifique/ ; }, mesh = {Animals ; *Gene Editing/methods ; *Sea Anemones/genetics/growth &amp; development ; CRISPR-Cas Systems/genetics ; *Animal Husbandry/methods ; Animals, Genetically Modified ; RNA, Guide, CRISPR-Cas Systems/genetics ; Aquaculture/methods ; }, abstract = {Optimized laboratory conditions for research models are crucial for the success of scientific projects. This includes controlling the entire life cycle, having access to all developmental stages and maintaining stable physiological conditions. Reducing the life cycle of a research model can also enhance the access to biological material and speed up genetic tool development. Thus, we optimized the rearing conditions for the sea anemone Nematostella vectensis, a cnidarian research model, to study embryonic and post-metamorphic processes, such as regeneration. We adopted a semi-automated aquaculture system for N. vectensis and developed a dietary protocol optimized for the different life stages. Thereby, we increased spawning efficiencies, juvenile growth and survival rates, and considerably reduced the overall life cycle down to 2 months. To further improve the obtention of CRISPR-Cas9 mutants, we optimized the design of sgRNAs leading to full knockout animals in F0 polyps using a single sgRNA. Finally, we show that NHEJ-mediated transgene insertion is possible in N. vectensis. In summary, our study provides additional resources for the scientific community that uses or plans to use N. vectensis as a research model.}, }
@article {pmid39775780, year = {2025}, author = {Tong, G and Nath, P and Hiruta, Y and Citterio, D}, title = {Amplification-free CRISPR/Cas based dual-enzymatic colorimetric nucleic acid biosensing device.}, journal = {Lab on a chip}, volume = {25}, number = {4}, pages = {536-545}, doi = {10.1039/d4lc01039f}, pmid = {39775780}, issn = {1473-0189}, mesh = {*Colorimetry/instrumentation ; *Biosensing Techniques/instrumentation ; *CRISPR-Cas Systems ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Alkaline Phosphatase/metabolism/chemistry ; Limit of Detection ; *DNA/analysis ; }, abstract = {Nucleic acid testing (NAT) is widely considered the gold standard in analytical fields, with applications spanning environmental monitoring, forensic science and clinical diagnostics, among others. However, its widespread use is often constrained by complicated assay procedures, the need for specialized equipment, and the complexity of reagent handling. In this study, we demonstrate a fully integrated 3D-printed biosensensing device employing a CRISPR/Cas12a-based dual-enzymatic mechanism for highly sensitive and user-friendly nucleic acid detection. A plastic probe stick was designed to host small-sized gold nanoparticles, enhancing enzyme labeling density. Alkaline phosphatase (ALP) was then conjugated via single-stranded DNA, requiring only a single enzyme substrate addition to generate a simple visual signal change. This approach eliminates the need for amplification or centrifugation steps, achieving a limit of detection (LOD) as low as 10 pM - among the highest sensitivities reported for amplification-free colorimetric nucleic acid detection. Furthermore, we developed a device that incorporates this probe stick, integrates all necessary reagents, and features a smartphone-compatible accessory for quantitative analysis. This allows end-users to perform visual or quantitative DNA analysis with simple operations, achieving a visual detection limit of approximately 100 pM, comparable to other CRISPR-based non-amplified nucleic acid detection methods. Additionally, the system successfully distinguished perfectly matched from mismatched nucleic acid sequences, demonstrating its specificity and versatility. Although certain design limitations affected the sensitivity of the integrated device compared to the probe stick alone, the simplicity and portability of this device make it a promising tool for rapid nucleic acid screening in clinical diagnostics, environmental monitoring, and food safety control. This study paves the way for the development of practical biosensors for point-of-care testing (POCT) applications.}, }
@article {pmid39775585, year = {2024}, author = {Zhang, WW and Matlashewski, G}, title = {Evidence for gene essentiality in Leishmania using CRISPR.}, journal = {PloS one}, volume = {19}, number = {12}, pages = {e0316331}, pmid = {39775585}, issn = {1932-6203}, mesh = {*Genes, Essential ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Leishmania/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genes, Protozoan ; Promoter Regions, Genetic ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Ribosomal/genetics ; Plasmids/genetics ; DNA-Directed RNA Polymerases ; Viral Proteins ; }, abstract = {The ability to determine the essentiality of a gene in the protozoan parasite Leishmania is important to identify potential targets for intervention and understanding the parasite biology. CRISPR gene editing technology has significantly improved gene targeting efficiency in Leishmania. There are two commonly used CRISPR gene targeting methods in Leishmania; the stable expression of the gRNA and Cas9 using a plasmid containing a Leishmania ribosomal RNA gene promoter (rRNA-P stable protocol) and the T7 RNA polymerase based transient gRNA expression system in promastigotes stably expressing Cas9 (T7 transient protocol). There are distinct advantages with both systems. The T7 transient protocol is excellent for high throughput gene deletions and has been used to successfully delete hundreds of Leishmania genes to study mutant phenotypes and several research labs are now using this protocol to target all the genes in L. mexicana genome. The rRNA-P stable protocol stably expresses the plasmid derived gRNA and has been used to delete or disrupt single and multicopy Leishmania genes, perform single nucleotide changes and provide evidence for gene essentiality by directly observing null mutant promastigotes dying in culture. In this study, the rRNA-P stable protocol was used to target 22 Leishmania genes in which null mutants were not generated using the T7 transient protocol. Notably, the rRNA-P stable protocol was able to generate alive null mutants for 8 of the 22 genes. These results demonstrate the rRNA-P stable protocol could be used alone or in combination with the T7 transient protocol to investigate gene essentiality in Leishmania.}, }
@article {pmid39774325, year = {2025}, author = {Funk, JS and Klimovich, M and Drangenstein, D and Pielhoop, O and Hunold, P and Borowek, A and Noeparast, M and Pavlakis, E and Neumann, M and Balourdas, DI and Kochhan, K and Merle, N and Bullwinkel, I and Wanzel, M and Elmshäuser, S and Teply-Szymanski, J and Nist, A and Procida, T and Bartkuhn, M and Humpert, K and Mernberger, M and Savai, R and Soussi, T and Joerger, AC and Stiewe, T}, title = {Deep CRISPR mutagenesis characterizes the functional diversity of TP53 mutations.}, journal = {Nature genetics}, volume = {57}, number = {1}, pages = {140-153}, pmid = {39774325}, issn = {1546-1718}, support = {STI 182/15-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; STI 182/13-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; GRK2573//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 109546710 Project A10//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; SA 1923/7-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC1213 A10N//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; JO 1473/1-3//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 031L0063//Bundesministerium f&#xfc;r Bildung und Forschung (Federal Ministry of Education and Research)/ ; DZL//Bundesministerium f&#xfc;r Bildung und Forschung (Federal Ministry of Education and Research)/ ; LOEWE iCANx//Hessisches Ministerium f&#xfc;r Wissenschaft und Kunst (Hessen State Ministry of Higher Education, Research and the Arts)/ ; 65-0004//Von-Behring-R&#xf6;ntgen-Stiftung (Von Behring-R&#xf6;ntgen-Foundation)/ ; 66-LV06//Von-Behring-R&#xf6;ntgen-Stiftung (Von Behring-R&#xf6;ntgen-Foundation)/ ; 71-0012//Von-Behring-R&#xf6;ntgen-Stiftung (Von Behring-R&#xf6;ntgen-Foundation)/ ; 09 R/2018//Jos&#xe9; Carreras Leuk&#xe4;mie-Stiftung (Deutsche Jos&#xe9; Carreras Leuk&#xe4;mie-Stiftung)/ ; TACTIC//Deutsche Krebshilfe (German Cancer Aid)/ ; }, mesh = {Humans ; *Tumor Suppressor Protein p53/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Neoplasms/genetics ; Cell Line, Tumor ; Mutagenesis ; *Mutation ; Mutation, Missense/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Nonsense Mediated mRNA Decay/genetics ; }, abstract = {The mutational landscape of TP53, a tumor suppressor mutated in about half of all cancers, includes over 2,000 known missense mutations. To fully leverage TP53 mutation status for personalized medicine, a thorough understanding of the functional diversity of these mutations is essential. We conducted a deep mutational scan using saturation genome editing with CRISPR-mediated homology-directed repair to engineer 9,225 TP53 variants in cancer cells. This high-resolution approach, covering 94.5% of all cancer-associated TP53 missense mutations, precisely mapped the impact of individual mutations on tumor cell fitness, surpassing previous deep mutational scan studies in distinguishing benign from pathogenic variants. Our results revealed even subtle loss-of-function phenotypes and identified promising mutants for pharmacological reactivation. Moreover, we uncovered the roles of splicing alterations and nonsense-mediated messenger RNA decay in mutation-driven TP53 dysfunction. These findings underscore the power of saturation genome editing in advancing clinical TP53 variant interpretation for genetic counseling and personalized cancer therapy.}, }
@article {pmid39774105, year = {2025}, author = {Ocampo, RF and Bravo, JPK and Dangerfield, TL and Nocedal, I and Jirde, SA and Alexander, LM and Thomas, NC and Das, A and Nielson, S and Johnson, KA and Brown, CT and Butterfield, CN and Goltsman, DSA and Taylor, DW}, title = {DNA targeting by compact Cas9d and its resurrected ancestor.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {457}, pmid = {39774105}, issn = {2041-1723}, mesh = {*CRISPR-Associated Protein 9/chemistry ; Cryoelectron Microscopy ; *RNA, Guide, CRISPR-Cas Systems/chemistry ; Protein Domains ; R-Loop Structures ; *Gene Editing ; DNA/chemistry ; Humans ; K562 Cells ; }, abstract = {Type II CRISPR endonucleases are widely used programmable genome editing tools. Recently, CRISPR-Cas systems with highly compact nucleases have been discovered, including Cas9d (a type II-D nuclease). Here, we report the cryo-EM structures of a Cas9d nuclease (747 amino acids in length) in multiple functional states, revealing a stepwise process of DNA targeting involving a conformational switch in a REC2 domain insertion. Our structures provide insights into the intricately folded guide RNA which acts as a structural scaffold to anchor small, flexible protein domains for DNA recognition. The sgRNA can be truncated by up to ~25% yet still retain activity in vivo. Using ancestral sequence reconstruction, we generated compact nucleases capable of efficient genome editing in mammalian cells. Collectively, our results provide mechanistic insights into the evolution and DNA targeting of diverse type II CRISPR-Cas systems, providing a blueprint for future re-engineering of minimal RNA-guided DNA endonucleases.}, }
@article {pmid39774003, year = {2025}, author = {Claiborne, DT and Detwiler, Z and Docken, SS and Borland, TD and Cromer, D and Simkhovich, A and Ophinni, Y and Okawa, K and Bateson, T and Chen, T and Hudson, W and Trifonova, R and Davenport, MP and Ho, TW and Boutwell, CL and Allen, TM}, title = {High frequency CCR5 editing in human hematopoietic stem progenitor cells protects xenograft mice from HIV infection.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {446}, pmid = {39774003}, issn = {2041-1723}, support = {U19 HL129903/HL/NHLBI NIH HHS/United States ; U19 HL156247/HL/NHLBI NIH HHS/United States ; }, mesh = {*Receptors, CCR5/genetics/metabolism ; Humans ; Animals ; *Hematopoietic Stem Cells/metabolism/virology ; *HIV Infections/virology/therapy/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Hematopoietic Stem Cell Transplantation ; Mice ; Heterografts ; HIV-1/genetics ; Mice, SCID ; }, abstract = {The only cure of HIV has been achieved in a small number of people who received a hematopoietic stem cell transplant (HSCT) comprising allogeneic cells carrying a rare, naturally occurring, homozygous deletion in the CCR5 gene. The rarity of the mutation and the significant morbidity and mortality of such allogeneic transplants precludes widespread adoption of this HIV cure. Here, we show the application of CRISPR/Cas9 to achieve >90% CCR5 editing in human, mobilized hematopoietic stem progenitor cells (HSPC), resulting in a transplant that undergoes normal hematopoiesis, produces CCR5 null T cells, and renders xenograft mice refractory to HIV infection. Titration studies transplanting decreasing frequencies of CCR5 edited HSPCs demonstrate that <90% CCR5 editing confers decreasing protective benefit that becomes negligible between 54% and 26%. Our study demonstrates the feasibility of using CRISPR/Cas9/RNP to produce an HSPC transplant with high frequency CCR5 editing that is refractory to HIV replication. These results raise the potential of using CRISPR/Cas9 to produce a curative autologous HSCT and bring us closer to the development of a cure for HIV infection.}, }
@article {pmid39773393, year = {2025}, author = {Bogut, A and Kołodziejek, A and Minnich, SA and Hovde, CJ}, title = {CRISPR/Cas Systems as Diagnostic and Potential Therapeutic Tools for Enterohemorrhagic Escherichia coli.}, journal = {Archivum immunologiae et therapiae experimentalis}, volume = {73}, number = {1}, pages = {}, pmid = {39773393}, issn = {1661-4917}, mesh = {Humans ; *Enterohemorrhagic Escherichia coli/genetics ; *CRISPR-Cas Systems ; Animals ; *Escherichia coli Infections/diagnosis/therapy/immunology ; *Gene Editing/methods ; Cattle ; Virulence/genetics ; Hemolytic-Uremic Syndrome/diagnosis/therapy/immunology/microbiology ; Probiotics/therapeutic use ; Bacteriophages/genetics ; Genetic Engineering ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Following its discovery as an adaptive immune system in prokaryotes, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been developed into a multifaceted genome editing tool. This review compiles findings aimed at implementation of this technology for selective elimination or attenuation of enterohemorrhagic Escherichia coli (EHEC). EHEC are important zoonotic foodborne pathogens that cause hemorrhagic colitis and can progress to the life-threatening hemolytic uremic syndrome (HUS). Advancements in the application of CRISPR methodology include laboratory detection and identification of EHEC, genotyping, screening for pathogenic potential, and engineering probiotics to reduce microbial shedding by cattle, the primary source of human infection. Genetically engineered phages or conjugative plasmids have been designed to target and inactivate genes whose products are critical for EHEC virulence.}, }
@article {pmid39773308, year = {2025}, author = {Chen, H and Fang, HQ and Liu, JT and Chang, SY and Cheng, LB and Sun, MX and Feng, JR and Liu, ZM and Zhang, YH and Rosen, CJ and Liu, P}, title = {Atlas of Fshr expression from novel reporter mice.}, journal = {eLife}, volume = {13}, number = {}, pages = {}, pmid = {39773308}, issn = {2050-084X}, mesh = {Animals ; *Receptors, FSH/genetics/metabolism ; Mice ; *Genes, Reporter ; Male ; CRISPR-Cas Systems ; Female ; Mice, Transgenic ; }, abstract = {The FSH-FSHR pathway has been considered an essential regulator in reproductive development and fertility. But there has been emerging evidence of FSHR expression in extragonadal organs. This poses new questions and long-term debates regarding the physiological role of the FSH-FSHR, and underscores the need for reliable, in vivo analysis of FSHR expression in animal models. However, conventional methods have proven insufficient for examining FSHR expression due to several limitations. To address this challenge, we developed Fshr-ZsGreen reporter mice under the control of Fshr endogenous promoter using CRISPR-Cas9. With this novel genetic tool, we provide a reliable readout of Fshr expression at single-cell resolution level in vivo and in real time. Reporter animals were also subjected to additional analyses,to define the accurate expression profile of FSHR in gonadal and extragonadal organs/tissues. Our compelling results not only demonstrated Fshr expression in intragonadal tissues but also, strikingly, unveiled notably increased expression in Leydig cells, osteoblast lineage cells, endothelial cells in vascular structures, and epithelial cells in bronchi of the lung and renal tubes. The genetic decoding of the widespread pattern of Fshr expression highlights its physiological relevance beyond reproduction and fertility, and opens new avenues for therapeutic options for age-related disorders of the bones, lungs, kidneys, and hearts, among other tissues. Exploiting the power of the Fshr knockin reporter animals, this report provides the first comprehensive genetic record of the spatial distribution of FSHR expression, correcting a long-term misconception about Fshr expression and offering prospects for extensive exploration of FSH-FSHR biology.}, }
@article {pmid39772804, year = {2025}, author = {Cottingham, H and Judd, LM and Wisniewski, JA and Wick, RR and Stanton, TD and Vezina, B and Macesic, N and Peleg, AY and Okeke, IN and Holt, KE and Hawkey, J}, title = {Targeted sequencing of Enterobacterales bacteria using CRISPR-Cas9 enrichment and Oxford Nanopore Technologies.}, journal = {mSystems}, volume = {10}, number = {2}, pages = {e0141324}, pmid = {39772804}, issn = {2379-5077}, support = {INV-036234/GATES/Gates Foundation/United States ; OPP1210746//Bill and Melinda Gates Foundation (GF)/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Klebsiella pneumoniae/genetics/isolation &amp; purification ; Multilocus Sequence Typing/methods ; High-Throughput Nucleotide Sequencing/methods ; Feces/microbiology ; *Nanopore Sequencing/methods ; Sequence Analysis, DNA/methods ; Drug Resistance, Bacterial/genetics ; Nanopores ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {UNLABELLED: Sequencing DNA directly from patient samples enables faster pathogen characterization compared to traditional culture-based approaches, but often yields insufficient sequence data for effective downstream analysis. CRISPR-Cas9 enrichment is designed to improve the yield of low abundance sequences but has not been thoroughly explored with Oxford Nanopore Technologies (ONT) for use in clinical bacterial epidemiology. We designed CRISPR-Cas9 guide RNAs to enrich the human pathogen Klebsiella pneumoniae, by targeting multi-locus sequence type (MLST) and transfer RNA (tRNA) genes, as well as common antimicrobial resistance (AMR) genes and the resistance-associated integron gene intI1. We validated enrichment performance in 20 K. pneumoniae isolates, finding that guides generated successful enrichment across all conserved sites except for one AMR gene in two isolates. Enrichment of MLST genes led to a correct allele call in all seven loci for 8 out of 10 isolates that had depth of 30× or more in these regions. We then compared enriched and unenriched sequencing of three human fecal samples spiked with K. pneumoniae at varying abundance. Enriched sequencing generated 56× and 11.3× the number of AMR and MLST reads, respectively, compared to unenriched sequencing, and required approximately one-third of the computational storage space. Targeting the intI1 gene often led to detection of 10-20 proximal resistance genes due to the long reads produced by ONT sequencing. We demonstrated that CRISPR-Cas9 enrichment combined with ONT sequencing enabled improved genomic characterization outcomes over unenriched sequencing of patient samples. This method could be used to inform infection control strategies by identifying patients colonized with high-risk strains.<br><br>IMPORTANCE: Understanding bacteria in complex samples can be challenging due to their low abundance, which often results in insufficient data for analysis. To improve the detection of harmful bacteria, we implemented a technique aimed at increasing the amount of data from target pathogens when combined with modern DNA sequencing technologies. Our technique uses CRISPR-Cas9 to target specific gene sequences in the bacterial pathogen Klebsiella pneumoniae and improve recovery from human stool samples. We found our enrichment method to significantly outperform traditional methods, generating far more data originating from our target genes. Additionally, we developed new computational techniques to further enhance the analysis, providing a thorough method for characterizing pathogens from complex biological samples.}, }
@article {pmid39770835, year = {2024}, author = {Tuli, SR and Ali, MF and Jamal, TB and Khan, MAS and Fatima, N and Ahmed, I and Khatun, M and Sharmin, SA}, title = {Characterization and Molecular Insights of a Chromium-Reducing Bacterium Bacillus tropicus.}, journal = {Microorganisms}, volume = {12}, number = {12}, pages = {}, pmid = {39770835}, issn = {2076-2607}, abstract = {Environmental pollution from metal toxicity is a widespread concern. Certain bacteria hold promise for bioremediation via the conversion of toxic chromium compounds into less harmful forms, promoting environmental cleanup. In this study, we report the isolation and detailed characterization of a highly chromium-tolerant bacterium, Bacillus tropicus CRB14. The isolate is capable of growing on 5000 mg/L Cr (VI) in an LB (Luria Bertani) agar plate while on 900 mg/L Cr (VI) in LB broth. It shows an 86.57% reduction ability in 96 h of culture. It can also tolerate high levels of As, Cd, Co, Fe, Zn, and Pb. The isolate also shows plant growth-promoting potential as demonstrated by a significant activity of nitrogen fixation, phosphate solubilization, IAA (indole acetic acid), and siderophore production. Whole-genome sequencing revealed that the isolate lacks Cr resistance genes in their plasmids and are located on its chromosome. The presence of the chrA gene points towards Cr(VI) transport, while the absence of ycnD suggests alternative reduction pathways. The genome harbors features like genomic islands and CRISPR-Cas systems, potentially aiding adaptation and defense. Analysis suggests robust metabolic pathways, potentially involved in Cr detoxification. Notably, genes for siderophore and NRP-metallophore production were identified. Whole-genome sequencing data also provides the basis for molecular validation of various genes. Findings from this study highlight the potential application of Bacillus tropicus CRB14 for bioremediation while plant growth promotion can be utilized as an added benefit.}, }
@article {pmid39770798, year = {2024}, author = {Muriuki, R and Ndichu, M and Githigia, S and Svitek, N}, title = {CRISPR-Cas-Based Pen-Side Diagnostic Tests for Anaplasma marginale and Babesia bigemina.}, journal = {Microorganisms}, volume = {12}, number = {12}, pages = {}, pmid = {39770798}, issn = {2076-2607}, support = {7200AA20CA00022//USAID/ ; }, abstract = {Anaplasma marginale and Babesia bigemina are tick-borne pathogens, posing significant threats to the health and productivity of cattle in tropical and subtropical regions worldwide. Currently, detection of Babesia bigemina and Anaplasma marginale in infected animals relies primarily on microscopic examination of Giemsa-stained blood or organ smears, which has limited sensitivity. Molecular methods offer higher sensitivity but are costly and impractical in resource-limited settings. Following the development of a pen-side test for detecting Theileria parva infections in cattle, we have created two additional CRISPR-Cas12a assays targeting Anaplasma marginale and Babesia bigemina. The assays target the major surface protein 5 (MSP5) for A. marginale and rhoptry-associated protein 1a (RAP1a) for B. bigemina. These additional tests involve a 20 min recombinase polymerase amplification (RPA) reaction followed by a 60 min CRISPR-Cas12a detection with a lateral strip readout. Results demonstrate high specificity, with no cross-reactivity against other tick-borne parasites, and a limit of detection down to 10[2] DNA copies/µL of each target marker. The findings pave the way for sensitive and user-friendly pen-side tests to diagnose A. marginale and B. bigemina infections.}, }
@article {pmid39770347, year = {2024}, author = {de Sousa, RMP and Garcia, LS and Lemos, FS and de Campos, VS and Machado Ferreira, E and de Almeida, NAA and Maron-Gutierrez, T and de Souza, EM and de Paula, VS}, title = {CRISPR/Cas9 Eye Drop HSV-1 Treatment Reduces Brain Viral Load: A Novel Application to Prevent Neuronal Damage.}, journal = {Pathogens (Basel, Switzerland)}, volume = {13}, number = {12}, pages = {}, pmid = {39770347}, issn = {2076-0817}, support = {GM-GD 2022//National Council for Scientific and Technological Development/ ; 001//Coordena&#xe7;&#xe3;o de Aperfeicoamento de Pessoal de N&#xed;vel Superior/ ; VPGDI 024 INT 22//Funda&#xe7;&#xe3;o Oswaldo Cruz/ ; 001//Funda&#xe7;&#xe3;o Carlos Chagas Filho de Amparo &#xe0; Pesquisa do Estado do Rio de Janeiro/ ; }, mesh = {Animals ; *Herpesvirus 1, Human/drug effects/genetics/physiology/pathogenicity ; *CRISPR-Cas Systems/genetics ; Mice ; *Viral Load/drug effects ; *Mice, Inbred BALB C ; *Antiviral Agents/pharmacology ; *Brain/virology/pathology/drug effects/metabolism ; *Herpes Simplex/drug therapy/virology ; Female ; Virus Replication/drug effects ; Disease Models, Animal ; Gene Editing/methods ; }, abstract = {Herpes simplex virus-1 (HSV-1) can invade the central nervous system (CNS). However, antiviral drugs used to treat HSV-1 have significant toxicity and resistance. An alternative approach involves the use of the CRISPR/Cas9 complex as a viral replication inhibitor. Editing the UL39 gene with CRISPR/Cas9 results in >95% inhibition of HSV-1 replication in vitro; however, few studies have investigated alternative therapies in in vivo models. This study aimed to investigate the efficacy of CRISPR/Cas9 targeting the UL39 region, which was administered via the ocular route, to reduce the HSV-1 viral count in the CNS of BALB/c mice. Mice were inoculated with HSV-1 and treated using CRISPR/Cas9. The kinetics of CNS infection were assessed, and the effects of CRISPR/Cas9 were compared with those of topical acyclovir treatments. The brain viral load was analyzed, and histopathology and immunofluorescence of the nervous tissue were performed. The group treated with CRISPR/Cas9 showed a reduced viral load on the seventh day post-infection, and no brain inflammation or chromatin compaction was observed in animals that received CRISPR/Cas9 therapy. These findings suggest that CRISPR/Cas9 anti-UL39 therapy can reduce the HSV-1 viral load in brain tissue. Therefore, investigating viral detection and evaluating antiviral treatments in the brain is essential.}, }
@article {pmid39770308, year = {2024}, author = {Islam, MM and Jung, DE and Shin, WS and Oh, MH}, title = {Colistin Resistance Mechanism and Management Strategies of Colistin-Resistant Acinetobacter baumannii Infections.}, journal = {Pathogens (Basel, Switzerland)}, volume = {13}, number = {12}, pages = {}, pmid = {39770308}, issn = {2076-0817}, support = {Grant Nos. 2022R1F1A1071415 and NRF-RS-2023-00275307 and Grant No. 2019R1A6C1010033//National Research Foundation of Korea (NRF) funded by the Ministry of Education &amp; Basic Science Research Capacity Enhancement Project through Korea Basic Science Institute (National research Facilities and Equipment Center) grant funded by the Ministry o/ ; }, mesh = {*Acinetobacter baumannii/drug effects/genetics ; *Colistin/pharmacology/therapeutic use ; Humans ; *Acinetobacter Infections/drug therapy ; *Anti-Bacterial Agents/therapeutic use/pharmacology ; Drug Resistance, Multiple, Bacterial/genetics/drug effects ; Drug Resistance, Bacterial/genetics/drug effects ; Animals ; }, abstract = {The emergence of antibiotic-resistant Acinetobacter baumannii (A. baumannii) is a pressing threat in clinical settings. Colistin is currently a widely used treatment for multidrug-resistant A. baumannii, serving as the last line of defense. However, reports of colistin-resistant strains of A. baumannii have emerged, underscoring the urgent need to develop alternative medications to combat these serious pathogens. To resist colistin, A. baumannii has developed several mechanisms. These include the loss of outer membrane lipopolysaccharides (LPSs) due to mutation of LPS biosynthetic genes, modification of lipid A (a constituent of LPSs) structure through the addition of phosphoethanolamine (PEtN) moieties to the lipid A component by overexpression of chromosomal pmrCAB operon genes and eptA gene, or acquisition of plasmid-encoded mcr genes through horizontal gene transfer. Other resistance mechanisms involve alterations of outer membrane permeability through porins, the expulsion of colistin by efflux pumps, and heteroresistance. In response to the rising threat of colistin-resistant A. baumannii, researchers have developed various treatment strategies, including antibiotic combination therapy, adjuvants to potentiate antibiotic activity, repurposing existing drugs, antimicrobial peptides, nanotechnology, photodynamic therapy, CRISPR/Cas, and phage therapy. While many of these strategies have shown promise in vitro and in vivo, further clinical trials are necessary to ensure their efficacy and widen their clinical applications. Ongoing research is essential for identifying the most effective therapeutic strategies to manage colistin-resistant A. baumannii. This review explores the genetic mechanisms underlying colistin resistance and assesses potential treatment options for this challenging pathogen.}, }
@article {pmid39770154, year = {2024}, author = {Shang, Z and Liu, S and Liu, D and Pei, X and Li, S and He, Y and Tong, Y and Liu, G}, title = {CRISPR/Cas12a with Universal crRNA for Indiscriminate Virus Detection.}, journal = {Molecules (Basel, Switzerland)}, volume = {29}, number = {24}, pages = {}, pmid = {39770154}, issn = {1420-3049}, support = {No. 22004005//National Natural Science Foundation of China/ ; 223777118D//Special Project on Biomedical Innovation/ ; }, mesh = {*CRISPR-Cas Systems ; *SARS-CoV-2/genetics/isolation &amp; purification ; Humans ; *RNA, Viral/genetics/analysis ; Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/genetics ; COVID-19/diagnosis/virology ; Molecular Diagnostic Techniques/methods ; Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins/genetics ; Biosensing Techniques/methods ; }, abstract = {Viruses, known for causing widespread biological harm and even extinction, pose significant challenges to public health. Virus detection is crucial for accurate disease diagnosis and preventing the spread of infections. Recently, the outstanding analytical performance of CRISPR/Cas biosensors has shown great potential and they have been considered as augmenting methods for reverse-transcription polymerase chain reaction (RT-PCR), which was the gold standard for nucleic acid detection. We herein utilized Cas12a with universal CRISPR RNA (crRNA) for indiscriminate virus detection by attaching the target to a longer track strand for isothermal amplification. The amplified products contain a domain that is recognized by the Cas12a/crRNA complex, triggering the cleavage of surrounding reporters to produce signals, thereby escaping the target dependence of crRNA recognition. The proposed method allows the same crRNA to detect multiple viral nucleic acids with high sensitivity, including but not limited to SARS-CoV-2, human papillomaviruses (HPV), HCOV-NL63, HCOV-HKU1, and miRNA biomarkers. Taking SARS-CoV-2 and HPV16 pseudoviruses as examples, this method was proved as a versatile and sensitive platform for molecular diagnostic applications.}, }
@article {pmid39769261, year = {2024}, author = {Taki, T and Morimoto, K and Mizuno, S and Kuno, A}, title = {KOnezumi-AID: Automation Software for Efficient Multiplex Gene Knockout Using Target-AID.}, journal = {International journal of molecular sciences}, volume = {25}, number = {24}, pages = {}, pmid = {39769261}, issn = {1422-0067}, support = {JP24ama121047//Japan Agency for Medical Research and Development/ ; JPMJFR221H//Japan Science and Technology Agency/ ; 24K18045//Japan Society for the Promotion of Science/ ; }, mesh = {*Gene Knockout Techniques/methods ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals ; Mice ; Humans ; *Software ; }, abstract = {With the groundbreaking advancements in genome editing technologies, particularly CRISPR-Cas9, creating knockout mutants has become highly efficient. However, the CRISPR-Cas9 system introduces DNA double-strand breaks, increasing the risk of chromosomal rearrangements and posing a major obstacle to simultaneous multiple gene knockout. Base-editing systems, such as Target-AID, are safe alternatives for precise base modifications without requiring DNA double-strand breaks, serving as promising solutions for existing challenges. Nevertheless, the absence of adequate tools to support Target-AID-based gene knockout highlights the need for a comprehensive system to design guide RNAs (gRNAs) for the simultaneous knockout of multiple genes. Here, we aimed to develop KOnezumi-AID, a command-line tool for gRNA design for Target-AID-mediated genome editing. KOnezumi-AID facilitates gene knockout by inducing the premature termination codons or promoting exon skipping, thereby generating experiment-ready gRNA designs for mouse and human genomes. Additionally, KOnezumi-AID exhibits batch processing capacity, enabling rapid and precise gRNA design for large-scale genome editing, including CRISPR screening. In summary, KOnezumi-AID is an efficient and user-friendly tool for gRNA design, streamlining genome editing workflows and advancing gene knockout research.}, }
@article {pmid39769183, year = {2024}, author = {Kappler, M and Thielemann, L and Glaß, M and Caggegi, L and Güttler, A and Pyko, J and Blauschmidt, S and Gutschner, T and Taubert, H and Otto, S and Eckert, AW and Tavassol, F and Bache, M and Vordermark, D and Kaune, T and Rot, S}, title = {Functional and Biological Characterization of the LGR5Δ5 Splice Variant in HEK293T Cells.}, journal = {International journal of molecular sciences}, volume = {25}, number = {24}, pages = {}, pmid = {39769183}, issn = {1422-0067}, support = {NA//Dr. Kleist Stiftung (Foundation)/ ; NA//Open Access Publication Fund of the Martin Luther University Halle-Wittenberg/ ; }, mesh = {Humans ; *Receptors, G-Protein-Coupled/genetics/metabolism ; HEK293 Cells ; *Thrombospondins/genetics/metabolism ; Wnt Signaling Pathway/genetics ; Protein Isoforms/genetics/metabolism ; CRISPR-Cas Systems ; Cell Movement/genetics ; }, abstract = {The regulator of the canonical Wnt pathway, leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), is expressed in the stem cell compartment of several tissues and overexpressed in different human carcinomas. The isoform of the stem cell marker LGR5, named LGR5Δ5 and first described by our group, is associated with prognosis and metastasis in oral squamous cell carcinoma (OSCC) and soft tissue sarcoma (STS). In a proof-of-principle analysis, the function of LGR5Δ5 was investigated in HEK293T cells, a model cell line of the Wnt pathway, compared to full-length LGR5 (FL) expression. The CRISPR/CAS knockout of LGR5 and LGR4 (thereby avoiding the side effects of LGR4) resulted in a loss of Wnt activity that cannot be restored by LGR5Δ5 but by LGR5FL rescue. The ability to migrate was not affected by LGR5Δ5, but was reduced by LGR5FL overexpression. The CRISPR/CAS of LGR4 and 5 induced radiosensitization, which was enhanced by the overexpression of LGR5FL or LGR5Δ5. RNA sequencing analysis revealed a significant increase in the ligand R-spondin 1 (RSPO1) level by LGR5Δ5. Furthermore, LGR5Δ5 appears to be involved in the regulation of genes related to the cytoskeleton, extracellular matrix stiffness, and angiogenesis, while LGR5FL is associated with the regulation of collagens and histone proteins.}, }
@article {pmid39769084, year = {2024}, author = {Bairqdar, A and Karitskaya, PE and Stepanov, GA}, title = {Expanding Horizons of CRISPR/Cas Technology: Clinical Advancements, Therapeutic Applications, and Challenges in Gene Therapy.}, journal = {International journal of molecular sciences}, volume = {25}, number = {24}, pages = {}, pmid = {39769084}, issn = {1422-0067}, support = {075-15-2021-1085//Ministry of Science and Higher Education of the Russian Federation/ ; }, mesh = {Humans ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; *Gene Editing/methods ; Clinical Trials as Topic ; Animals ; }, abstract = {CRISPR-Cas technology has transformed the field of gene editing, opening new possibilities for treatment of various genetic disorders. Recent years have seen a surge in clinical trials using CRISPR-Cas-based therapies. This review examines the current landscape of CRISPR-Cas implementation in clinical trials, with data from key registries, including the Australian New Zealand Clinical Trials Registry, the Chinese Clinical Trial Register, and ClinicalTrials.gov. Emphasis is placed on the mechanism of action of tested therapies, the delivery method, and the most recent findings of each clinical trial.}, }
@article {pmid39769020, year = {2024}, author = {Sun, X and Fu, Q and Song, Y and Deng, X and Li, Y and Wu, K and Li, S and Fu, J}, title = {Research Progress and Prospects of Molecular Breeding in Bermudagrass (Cynodon dactylon).}, journal = {International journal of molecular sciences}, volume = {25}, number = {24}, pages = {}, pmid = {39769020}, issn = {1422-0067}, support = {2024LZGC00303//Key R &amp; D Plan of Shandong Province/ ; }, mesh = {*Cynodon/genetics ; Gene Editing/methods ; Genome, Plant ; Genomics/methods ; *Plant Breeding/methods ; Stress, Physiological ; *DNA Shuffling/methods ; }, abstract = {Bermudagrass (Cynodon dactylon L.) is a warm-season grass species of significant ecological and economic importance. It is widely utilized in turf management and forage production due to its resilience to drought, salt, and other environmental stresses. Recent advancements in molecular breeding, particularly through genomics technology and gene editing, have enabled the efficient identification of key genes associated with stress tolerance and turf quality. The use of techniques such as overexpression and CRISPR/Cas has enhanced resistance to drought, salt, cold, and heat, while the application of molecular markers has accelerated the development of superior varieties. The integration of multi-omics, such as genomics, transcriptomics, and proteomics, provides deeper insights into the molecular mechanisms of bermudagrass, thereby improving breeding efficiency and precision. Additionally, artificial intelligence is emerging as a powerful tool for analyzing genomic data, predicting optimal trait combinations, and accelerating breeding processes. These technologies, when combined with traditional breeding methods, hold great potential for optimizing bermudagrass varieties for both turf and forage use. Future research will focus on further integrating these tools to address the challenges of breeding posed by climate change to breeding climate-resilient turf and forage crops.}, }
@article {pmid39768143, year = {2024}, author = {Zhang, XH and Tang, FL and Trouten, AM and Morad, M}, title = {Attempts to Create Transgenic Mice Carrying the Q3924E Mutation in RyR2 Ca[2+] Binding Site.}, journal = {Cells}, volume = {13}, number = {24}, pages = {}, pmid = {39768143}, issn = {2073-4409}, support = {R01HL153504/GF/NIH HHS/United States ; R01 HL153504/HL/NHLBI NIH HHS/United States ; }, mesh = {Animals ; *Ryanodine Receptor Calcium Release Channel/genetics/metabolism ; *Mice, Transgenic ; Mice ; *Calcium/metabolism ; *Myocytes, Cardiac/metabolism ; Binding Sites ; Humans ; Mutation/genetics ; Female ; Calcium Signaling/genetics ; CRISPR-Cas Systems/genetics ; Caffeine/pharmacology ; }, abstract = {Over 200 point mutations in the ryanodine receptor (RyR2) of the cardiac sarcoplasmic reticulum (SR) are known to be associated with cardiac arrhythmia. We have already reported on the calcium signaling phenotype of a point mutation in RyR2 Ca[2+] binding site Q3925E expressed in human stem-cell-derived cardiomyocytes (hiPSC-CMs) that was found to be lethal in a 9-year-old girl. CRISPR/Cas9-gene-edited mutant cardiomyocytes carrying the RyR2-Q3925E mutation exhibited a loss of calcium-induced calcium release (CICR) and caffeine-triggered calcium release but continued to beat arrhythmically without generating significant SR Ca[2+] release, consistent with a remodeling of the calcium signaling pathway. An RNAseq heat map confirmed significant changes in calcium-associated genes, supporting the possibility of remodeling. To determine the in situ cardiac phenotype in an animal model of this mutation, we generated a knock-in mouse model of RyR2-Q3924E+/- using the CRISPR/Cas9 technique. We obtained three homozygous and one chimera mice, but they all died before reaching 3 weeks of age, preventing the establishment of germline mutation transmission in their offspring. A histo-pathological analysis of the heart showed significant cardiac hypertrophy, suggesting the Q3924E-RyR2 mutation was lethal to the mice.}, }
@article {pmid39766827, year = {2024}, author = {Yang, R and Guo, H and Sun, J and Gui, T and Li, X and Qian, H and Chen, A}, title = {The ebony Gene in Silkworm Black Pupae Significantly Affects 30 K Proteins During the Pupal Stage.}, journal = {Genes}, volume = {15}, number = {12}, pages = {}, pmid = {39766827}, issn = {2073-4425}, support = {CARS-18-ZJ0101//China Agriculture Research System of MOF and MARA/ ; BE2020418//Key R &amp; D plan of Jiangsu Province (Modern Agriculture)/ ; 2023-JC-YB-188//Basic Research Programs of the Shaanxi Provincial Science and Technology Department/ ; 2023-YBNY-134//Key Industrial Chain Projects of Shaanxi Provincial Government/ ; }, mesh = {Animals ; *Bombyx/genetics/growth &amp; development/metabolism ; *Pupa/genetics/growth &amp; development/metabolism ; *Insect Proteins/genetics/metabolism ; Gene Expression Regulation, Developmental ; Pigmentation/genetics ; CRISPR-Cas Systems ; }, abstract = {Background/Objectives: The body color and patterns of insects play important roles in foraging, evading predators, mating, thermoregulation, and environmental adaptation. During the rearing of the QiufengN silkworm strain, a mutant with black pupal cuticle (QiufengNBP) was discovered. Preliminary map-based cloning and sequence analysis indicated that the ebony gene might significantly influence the formation of the black pupa mutant and the expression of 30K proteins. This study aims to determine the function of the ebony gene and its effect on the expression of the 30K protein during the pupal stage; Methods and Results: We employed CRISPR/Cas9 gene-editing technology to knock out the ebony gene in the Nistari strain, resulting in individuals with black pupae, named Nistari Black Pupa (NisBP). This confirmed that the ebony gene plays a crucial role in black pupa formation. Two-dimensional electrophoresis (2-DE) analysis of the pupal cuticle of NisBP and its wild-type Nistari found that the ebony gene has a significant impact on the expression of 30K proteins, which are vital for embryonic development and serve as key storage proteins; Conclusions: This study is the first to demonstrate that the ebony gene affects the expression of 30K proteins, laying the foundation for further research on their functions and providing insights into the developmental mechanisms of silkworms.}, }
@article {pmid39766530, year = {2024}, author = {de la Fuente Tagarro, C and Martín-González, D and De Lucas, A and Bordel, S and Santos-Beneit, F}, title = {Current Knowledge on CRISPR Strategies Against Antimicrobial-Resistant Bacteria.}, journal = {Antibiotics (Basel, Switzerland)}, volume = {13}, number = {12}, pages = {}, pmid = {39766530}, issn = {2079-6382}, abstract = {CRISPR/Cas systems have emerged as valuable tools to approach the problem of antimicrobial resistance by either sensitizing or lysing resistant bacteria or by aiding in antibiotic development, with successful applications across diverse organisms, including bacteria and fungi. CRISPR/Cas systems can target plasmids or the bacterial chromosome of AMR-bacteria, and it is especially necessary to have an efficient entry into the target cells, which can be achieved through nanoparticles or bacteriophages. Regarding antibiotic development and production, though the use of CRISPR/Cas in this field is still modest, there is an untapped reservoir of bacterial and fungal natural products, with over 95% yet to be characterized. In Streptomyces, a key antibiotic-producing bacterial genus, CRISPR/Cas has been successfully used to activate silent biosynthetic gene clusters, leading to the discovery of new antibiotics. CRISPR/Cas is also applicable to non-model bacteria and different species of fungi, making it a versatile tool for natural products discovery. Moreover, CRISPR/Cas-based studies offer insights into metabolic regulation and biosynthetic pathways in both bacteria and fungi, highlighting its utility in understanding genetic regulation and improving industrial strains. In this work, we review ongoing innovations on ways to treat antimicrobial resistances and on antibiotic discovery using CRISPR/Cas platforms, highlighting the role of bacteria and fungi in these processes.}, }
@article {pmid39766302, year = {2024}, author = {Middlezong, W and Stinnett, V and Phan, M and Phan, B and Morsberger, L and Klausner, M and Ghabrial, J and DeMetrick, N and Zhu, J and James, T and Pallavajjala, A and Gocke, CD and Baer, MR and Zou, YS}, title = {Rapid Detection of PML::RARA Fusions in Acute Promyelocytic Leukemia: CRISPR/Cas9 Nanopore Sequencing with Adaptive Sampling.}, journal = {Biomolecules}, volume = {14}, number = {12}, pages = {}, pmid = {39766302}, issn = {2218-273X}, support = {ATIP grant//Johns Hopkins Institute for Clinical and Translational Research (ICTR)/ ; }, mesh = {Humans ; *Leukemia, Promyelocytic, Acute/genetics/diagnosis ; *Oncogene Proteins, Fusion/genetics ; *Nanopore Sequencing/methods ; *CRISPR-Cas Systems/genetics ; Retinoic Acid Receptor alpha/genetics ; Promyelocytic Leukemia Protein/genetics ; }, abstract = {Acute promyelocytic leukemia (APL) accounts for approximately 10-15% of newly diagnosed acute myeloid leukemia cases and presents with coagulopathy and bleeding. Prompt diagnosis and treatment are required to minimize early mortality in APL as initiation of all-trans retinoic acid therapy rapidly reverses coagulopathy. The PML::RARA fusion is a hallmark of APL and its rapid identification is essential for rapid initiation of specific treatment to prevent early deaths from coagulopathy and bleeding and optimize patient outcomes. Given limitations and long turnaround time of current gene fusion diagnostic strategies, we have developed a novel amplification-free nanopore sequencing-based approach with low cost, easy setup, and fast turnaround time. We termed the approach CRISPR/Cas9-enriched nanopore sequencing with adaptive sampling (CENAS). Using CENAS, we successfully sequenced breakpoints of typical and atypical PML::RARA fusions in APL patients. Compared with the standard-of-care genetic diagnostic tests, CENAS achieved good concordance in detecting PML::RARA fusions in this study. CENAS allowed for the identification of sequence information of fusion breakpoints involved in typical and atypical PML::RARA fusions and identified additional genes (ANKFN1 and JOSD1) and genomic regions (13q14.13) involving the atypical fusions. To the best of our knowledge, involvements of the ANKFN1 gene, the JOSD1 gene, and the 13q14.13 genomic region flanking with the SIAH3 and ZC3H13 genes have not been reported in the atypical PML::RARA fusions. CENAS has great potential to develop as a point-of-care test enabling immediate, low-cost bedside diagnosis of APL patients with a PML::RARA fusion. Given the early death rate in APL patients still reaches 15%, and ~10% of APL patients are resistant to initial therapy or prone to relapse, further sequencing studies of typical and atypical PML::RARA fusion might shed light on the pathophysiology of the disease and its responsiveness to treatment. Understanding the involvement of additional genes and positional effects related to the PML and RARA genes could shed light on their role in APL and may aid in the development of novel targeted therapies.}, }
@article {pmid39766265, year = {2024}, author = {Hirose, J and Aizawa, E and Yamamoto, S and Xu, M and Iwai, S and Suzuki, K}, title = {Lipid Nanoparticles Enable Efficient In Vivo DNA Knock-In via HITI-Mediated Genome Editing.}, journal = {Biomolecules}, volume = {14}, number = {12}, pages = {}, pmid = {39766265}, issn = {2218-273X}, support = {21H04811//The Japan Society for the Promotion of Science KAKENHI/ ; 22K19405//The Japan Society for the Promotion of Science KAKENHI/ ; JP23ek0109521//Japan Agency for Medical Research and Development/ ; N.A.//Daiichi Sankyo Foundation of Life Science/ ; N.A.//Osaka University Honors Program for Graduate Schools in Science, Engineering and Informatics/ ; JPMXP1224 OS1059//Ministry of Education, Culture, Sports, Science and Technology/ ; }, mesh = {*Gene Editing/methods ; Animals ; *Nanoparticles/chemistry ; Mice ; *Gene Knock-In Techniques ; *Lipids/chemistry ; *DNA/genetics/administration &amp; dosage ; Humans ; Liver/metabolism ; CRISPR-Cas Systems ; Green Fluorescent Proteins/genetics/metabolism ; Liposomes ; }, abstract = {In vivo genome editing holds great therapeutic potential for treating monogenic diseases by enabling precise gene correction or addition. However, improving the efficiency of delivery systems remains a key challenge. In this study, we investigated the use of lipid nanoparticles (LNPs) for in vivo knock-in of ectopic DNA. Our in vitro experiments demonstrated that the homology-independent targeted integration (HITI)-mediated genome-editing method achieved significantly higher knock-in efficiency at the Alb locus in hepatic cells compared to the traditional homology-directed repair (HDR)-mediated approach. By optimizing LNP composition and administration routes, we successfully achieved HITI-mediated GFP knock-in (2.1-2.7%) in the livers of mice through intravenous delivery of LNP-loaded genome editing components. Notably, repeated intravenous dosing led to a twofold increase in liver GFP knock-in efficiency (4.3-7.0%) compared to a single dose, highlighting the potential for cumulative genome editing effects. These findings provide a solid foundation for the use of LNPs in in vivo knock-in strategies, paving the way for future genome-editing therapies.}, }
@article {pmid39766243, year = {2024}, author = {Lopez-Barbera, A and Abasolo, N and Torrell, H and Canela, N and Fernández-Arroyo, S}, title = {Integrative Transcriptomic and Target Metabolite Analysis as a New Tool for Designing Metabolic Engineering in Yeast.}, journal = {Biomolecules}, volume = {14}, number = {12}, pages = {}, pmid = {39766243}, issn = {2218-273X}, support = {ACCI&#xd3;-Eurecat TRA&#xc7;A 2023-FEREMIC//Agencia per a la Competitivit de l'Empresa/ ; SGR01275-IOMICS//Ag&#xe8;ncia de Gesti&#xf3; d'Ajuts Universitaris i de Recerca/ ; Vicente L&#xf3;pez to A.L-B.//Fundaci&#xf3; Eurecat/ ; }, mesh = {*Metabolic Engineering/methods ; *Saccharomyces cerevisiae/metabolism/genetics ; *Transcriptome/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Gene Expression Profiling/methods ; Terpenes/metabolism ; Metabolomics/methods ; }, abstract = {Precision fermentation processes, especially when using edited microorganisms, demand accuracy in the bioengineering process to maximize the desired outcome and to avoid adverse effects. The selection of target sites to edit using CRISPR/Cas9 can be complex, resulting in non-controlled consequences. Therefore, the use of multi-omics strategies can help in the design, selection and efficiency of genetic editing. In this study, we present a multi-omics approach based on targeted metabolite analysis and transcriptomics for the designing of CRISPR/Cas9 in baker's yeast as a more efficient strategy to select editing regions. Multi-omics shows potential to reveal new metabolic bottlenecks and to elucidate new metabolic fluxes, which could be a key factor in minimizing the metabolic burden in edited microorganisms. In our model, we focus our attention on the isoprenoid synthesis due to their industrial interest. Targeted metabolite detection combined with a transcriptomic analysis revealed hydroxymethylglutaryl-CoA reductases (HMGs) as the best target gene to induce an increase in isoprenoid synthesis. Thus, an extra copy of HMG1 was introduced using, for the first time, the synthetic UADH1 promoter. The multi-omics analysis of the recombinant strain results in an accurate assessment of yeast behavior during the most important growth phases, highlighting the metabolic burden, Crabtree effect or the diauxic shift during culture.}, }
@article {pmid39765088, year = {2025}, author = {Yang, R and Zhao, L and Fang, M and Kong, W and Luan, Y}, title = {CRISPR-Cas12a-driven aptasensor for sensitive detection of alternariol by using a personal glucose meter.}, journal = {Talanta}, volume = {286}, number = {}, pages = {127496}, doi = {10.1016/j.talanta.2024.127496}, pmid = {39765088}, issn = {1873-3573}, mesh = {*Aptamers, Nucleotide/chemistry/genetics/metabolism ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Lactones/analysis ; Limit of Detection ; *CRISPR-Associated Proteins/metabolism/genetics ; Humans ; *Bacterial Proteins/metabolism/genetics ; *Blood Glucose Self-Monitoring/instrumentation ; beta-Fructofuranosidase/metabolism ; Endodeoxyribonucleases ; }, abstract = {Alternariol (AOH) has attracted much attention as an emerging toxin in edible herbs that can pose potential carcinogenic risks to human. However, the rapid detection of AOH to ensure food safety remains a challenge. Here, a CRISPR-Cas12a-mediated aptamer-based sensor (aptasensor) was proposed for the sensitive quantification of AOH by using a personal glucose meter. First, fluorescent probes were used to validate crRNA sequences that could activate Cas12a trans-cleavage activity. Owing to the high affinity of aptamer, different concentrations of AOH were able to release different levels of Cas12a cleavage activity to cut magnetic bead-modified invertase probes. The free invertase was collected to catalyze hydrolysis of sucrose to glucose. After optimizing a series of key parameters, the constructed aptasensor with dual signal amplification of Cas12a and invertase allowed for highly-sensitive AOH detection in a linear range of 0.45-3000 ng/mL with a detection limit of 0.048 ng/mL. In addition, this aptasensor was successfully applied in edible herb samples with satisfactory recovery rates of 92.19-102.40 %. This CRISPR-Cas12a-mediated aptasensing platform provides a simple and powerful alternative for rapid quantitative detection of AOH, which is of great significance for food safety.}, }
@article {pmid39764982, year = {2025}, author = {Yue, Y and Liu, M and Ma, M and Xu, Z and Zhang, H and Wang, Q and Liu, R}, title = {CRISPR/Cas14a integrated with DNA walker based on magnetic self-assembly for human papillomavirus type 16 oncoprotein E7 ultrasensitive detection.}, journal = {Biosensors &amp; bioelectronics}, volume = {272}, number = {}, pages = {117135}, doi = {10.1016/j.bios.2025.117135}, pmid = {39764982}, issn = {1873-4235}, mesh = {*Biosensing Techniques/methods ; *Papillomavirus E7 Proteins/blood/isolation &amp; purification/genetics ; Humans ; Animals ; CRISPR-Cas Systems/genetics ; Limit of Detection ; *Human papillomavirus 16/isolation &amp; purification/genetics ; Mice ; Electrochemical Techniques/methods ; Female ; *Papillomavirus Infections/virology/diagnosis ; Uterine Cervical Neoplasms/virology/diagnosis/blood ; Gold/chemistry ; DNA, Single-Stranded/chemistry ; }, abstract = {To enhance the biomarker diagnostics sensitivity and selectivity of human papillomavirus type 16 oncoprotein E7 (HPV16 E7) in serum, a label/enzyme-free electrochemical detection platform was developed. This platform featured a type of "Super-turn-off" nanobiosensor monitored through differential pulse voltammetry (DPV). It integrated the magnetic self-assembly property of the α-Fe2O3/Fe3O4@Au/Sub/BSA signal transport nano-medium with the high specificity of CRISPR/Cas14a and the amplification capability of the bipedal walker (DNA walker composed of two ssDNA strands), resulting in the enhanced specificity and anti-interference performance while remaining stable at 4 °C for over 30 days. The results demonstrated that the combination of walker and CRISPR yielded superior sensitivity and analytical capability compared with using either technology alone, achieving a detection limit of 67.17 fg mL[-1], a quantification limit of 0.22 pg mL[-1], and serum sample recovery rates of 98.46%-115.78%. Additionally, this platform was applied to detect serum and tissue samples from mouse models at various stages of cervical cancer, significantly improving the accuracy and effectiveness of early diagnosis and prognostic evaluation. This novel approach held promise as an efficient tool for point-of-care clinical detection of HPV16 E7, potentially reducing cervical cancer mortality.}, }
@article {pmid39764496, year = {2025}, author = {Tamaki, M and Chiku, T and Suzuki, S and Misaki, A and Funakubo, A and Matsushima, Y and Yokota, K and Igimi, S and Kajikawa, A}, title = {Application of the SpCas9 inhibitor BRD0539 for CRISPR/Cas9-based genetic tools in Lacticaseibacillus paracasei.}, journal = {Bioscience of microbiota, food and health}, volume = {44}, number = {1}, pages = {70-79}, pmid = {39764496}, issn = {2186-6953}, abstract = {Although the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system has been extensively developed since its discovery for eukaryotic and prokaryotic genome editing and other genetic manipulations, there are still areas warranting improvement, especially regarding bacteria. In this study, BRD0539, a small-molecule inhibitor of Streptococcus pyogenes Cas9 (SpCas9), was used to suppress the activity of the nuclease during genetic modification of Lacticaseibacillus paracasei, as well as to regulate CRISPR interference (CRISPRi). First, we developed and validated a CRISPR-SpCas9 system targeting the sirA gene of L. paracasei. Then BRD0539 was used for CRISPR-dependent DNA cleavage in vivo. Our results suggested that the inhibitor worked partially in both Escherichia coli and L. paracasei. Next, we designed a CRISPRi system in a L. paracasei strain by inserting an inactive SpCas9 gene into the chromosome and introducing a plasmid encoding for a single guide RNA (sgRNA) targeting the sirA gene. Expression of sirA was successfully inhibited in the recombinant strains, and CRISPRi was abolished in an inhibitor-dependent manner. Our findings may help expand the CRISPR toolbox for research on lactic acid bacteria and other microbes.}, }
@article {pmid39762743, year = {2025}, author = {Shelenkov, A and Slavokhotova, A and Mikhaylova, Y and Akimkin, V}, title = {Genomic typing, antimicrobial resistance gene, virulence factor and plasmid replicon database for the important pathogenic bacteria Klebsiella pneumoniae.}, journal = {BMC microbiology}, volume = {25}, number = {1}, pages = {3}, pmid = {39762743}, issn = {1471-2180}, support = {075-15-2019-1666//The Ministry of Science and Higher Education of the Russian Federation/ ; 075-15-2019-1666//The Ministry of Science and Higher Education of the Russian Federation/ ; 075-15-2019-1666//The Ministry of Science and Higher Education of the Russian Federation/ ; 075-15-2019-1666//The Ministry of Science and Higher Education of the Russian Federation/ ; }, mesh = {*Klebsiella pneumoniae/genetics/drug effects/pathogenicity ; *Plasmids/genetics ; *Virulence Factors/genetics ; *Genome, Bacterial/genetics ; Humans ; *Drug Resistance, Bacterial/genetics ; Klebsiella Infections/microbiology/epidemiology ; Replicon/genetics ; Multilocus Sequence Typing ; Anti-Bacterial Agents/pharmacology ; Databases, Genetic ; Genomics ; }, abstract = {BACKGROUND: The infections of bacterial origin represent a significant problem to the public healthcare worldwide both in clinical and community settings. Recent decade was marked by limiting treatment options for bacterial infections due to growing antimicrobial resistance (AMR) acquired and transferred by various bacterial species, especially the ones causing healthcare-associated infections, which has become a dangerous issue noticed by the World Health Organization. Numerous reports shown that the spread of AMR is often driven by several species-specific lineages usually called the 'global clones of high risk'. Thus, it is essential to track the isolates belonging to such clones and investigate the mechanisms of their pathogenicity and AMR acquisition. Currently, the whole genome-based analysis is more and more often used for these purposes, including the epidemiological surveillance and analysis of mobile elements involved in resistance transfer. However, in spite of the exponential growth of available bacterial genomes, their representation usually lack uniformity and availability of supporting metadata, which creates a bottleneck for such investigations.<br><br>DESCRIPTION: In this database, we provide the results of a thorough genomic analysis of 61,857 genomes of a highly dangerous bacterial pathogen Klebsiella pneumoniae. Important isolate typing information including multilocus sequence typing (MLST) types (STs), assignment of the isolates to known global clones, capsular (KL) and lipooligosaccharide (O) types, the presence of CRISPR-Cas systems, and cgMLST profiles are given, and the information regarding the presence of AMR, virulence genes and plasmid replicons within the genomes is provided.<br><br>CONCLUSION: This database is freely available under CC BY-NC-SA at https://doi.org/10.5281/zenodo.11069018 . The database will facilitate selection of the proper reference isolate sets for any types of genome-based investigations. It will be helpful for investigations in the field of K. pneumoniae genomic epidemiology, as well as antimicrobial resistance analysis and the development of prevention measures against this important pathogen.}, }
@article {pmid39762408, year = {2025}, author = {Kim, DH and Choi, SH and Sung, JJ and Kim, S and Yi, H and Park, S and Park, CW and Oh, YW and Lee, J and Kim, DS and Kim, JH and Park, CY and Kim, DW}, title = {Long-term correction of hemophilia A via integration of a functionally enhanced FVIII gene into the AAVS1 locus by nickase in patient-derived iPSCs.}, journal = {Experimental &amp; molecular medicine}, volume = {57}, number = {1}, pages = {184-192}, pmid = {39762408}, issn = {2092-6413}, support = {2022R1A2C2091165//National Research Foundation of Korea (NRF)/ ; 2022R1A2C1091800//National Research Foundation of Korea (NRF)/ ; }, mesh = {*Hemophilia A/therapy/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Factor VIII/genetics/metabolism ; Humans ; Animals ; Mice ; Genetic Therapy/methods ; Dependovirus/genetics ; CRISPR-Cas Systems ; Gene Editing ; Disease Models, Animal ; Cell Differentiation ; Endothelial Cells/metabolism ; Peptide Elongation Factor 1/genetics ; Genetic Loci ; }, abstract = {Hemophilia A (HA) is caused by mutations in coagulation factor VIII (FVIII). Genome editing in conjunction with patient-derived induced pluripotent stem cells (iPSCs) is a promising cell therapy strategy, as it replaces dysfunctional proteins resulting from genetic mutations with normal proteins. However, the low expression level and short half-life of FVIII still remain significant limiting factors in the efficacy of these approaches in HA. Here, we constructed a functionally enhanced FVIII variant, F309S/E1984V-mutated B domain-deleted (BDD)-FVIII (FE-FVIII), with increased activity and stability. We inserted FE-FVIII with a human elongation factor-1 alpha (EF1α) promoter into the AAVS1 locus of HA patient-derived iPSCs via CRISPR/Cas9 (D10A) nickase to ensure expression in any cell type. FE-FVIII was expressed not only in undifferentiated FE-FVIII-inserted (FE-KI) iPSCs but also in endothelial cells (ECs) differentiated from them in vitro. Compared with mice transplanted with wild-type BDD-FVIII-containing ECs, immunocompetent HA mice intravenously transplanted with FE-KI ECs presented a 2.12-fold increase in FVIII activity in the blood and an approximately 20% greater survival rate after hemorrhagic tail injury. For sustained efficacy, FE-KI ECs were subcutaneously transplanted into immunodeficient HA mice, resulting in amelioration of the hemophilia phenotype for more than 3 months. This strategy can improve FVIII function and may provide a universal therapeutic approach for treating HA.}, }
@article {pmid39762363, year = {2025}, author = {Kang, GH and Ko, Y and Lee, JM}, title = {Enhancing virus-mediated genome editing for cultivated tomato through low temperature.}, journal = {Plant cell reports}, volume = {44}, number = {1}, pages = {22}, pmid = {39762363}, issn = {1432-203X}, support = {RS-2024-00322217//New Breeding Technologies Development Program, Rural Development Administration/ ; 2021R1A2C2093789//National Research Foundation of Korea/ ; }, mesh = {*Solanum lycopersicum/genetics/virology ; *Gene Editing/methods ; *Cold Temperature ; *Plant Viruses/genetics ; Potexvirus/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; Genetic Vectors/genetics ; Genome, Plant/genetics ; Secoviridae/genetics ; Plant Breeding/methods ; Oxidoreductases/genetics ; Plant Leaves/genetics/virology ; }, abstract = {Viral vector-mediated gene editing is enhanced for cultivated tomato under low temperature conditions, enabling higher mutation rates, heritable, and virus-free gene editing for efficient breeding. The CRISPR/Cas system, a versatile gene-editing tool, has revolutionized plant breeding by enabling precise genetic modifications. The development of robust and efficient genome-editing tools for crops is crucial for their application in plant breeding. In this study, we highly improved virus-induced genome-editing (VIGE) system for cultivated tomato. Vectors of tobacco rattle virus (TRV) and potato virus X (PVX) were used to deliver sgRNA targeting phytoene desaturase (SlPDS), along with mobile RNA sequences of tFT or tRNA[Ileu], into Cas9-overexpressing cultivated tomato (S. lycopersicum cv. Moneymaker). Our results demonstrate that low temperature significantly enhanced viral vector-mediated gene editing efficiency in both cotyledons and systemic upper leaves. However, no mutant progeny was obtained from TRV- and PVX301-infected MM-Cas9 plants. To address this challenge, we employed tissue culture techniques and found that low-temperature incubations at the initiation stage of tissue culture lead to enhanced editing efficiency in both vectors, resulting in a higher mutation rate (> 70%) of SlPDS in regenerated plants. Heritable gene-edited and virus-free progenies were successfully identified. This study presents a straightforward approach to enhance VIGE efficiency and the expeditious production of gene-edited lines in tomato breeding.}, }
@article {pmid39762235, year = {2025}, author = {Hollingsworth, EW and Liu, TA and Alcantara, JA and Chen, CX and Jacinto, SH and Kvon, EZ}, title = {Rapid and quantitative functional interrogation of human enhancer variant activity in live mice.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {409}, pmid = {39762235}, issn = {2041-1723}, support = {T32 GM008620/GM/NIGMS NIH HHS/United States ; S10 OD021718/OD/NIH HHS/United States ; T32 NS082174/NS/NINDS NIH HHS/United States ; F30 HD110233/HD/NICHD NIH HHS/United States ; S10 OD010794/OD/NIH HHS/United States ; P30 CA062203/CA/NCI NIH HHS/United States ; S10 RR025496/RR/NCRR NIH HHS/United States ; R01 HD115268/HD/NICHD NIH HHS/United States ; }, mesh = {Animals ; *Enhancer Elements, Genetic/genetics ; Mice ; Humans ; *Otx Transcription Factors/genetics/metabolism ; MicroRNAs/genetics/metabolism ; Single-Cell Analysis ; Autism Spectrum Disorder/genetics/metabolism ; Craniofacial Abnormalities/genetics ; Genes, Reporter ; Female ; CRISPR-Cas Systems ; Genetic Variation ; Brain/metabolism ; Male ; Alleles ; }, abstract = {Functional analysis of non-coding variants associated with congenital disorders remains challenging due to the lack of efficient in vivo models. Here we introduce dual-enSERT, a robust Cas9-based two-color fluorescent reporter system which enables rapid, quantitative comparison of enhancer allele activities in live mice in less than two weeks. We use this technology to examine and measure the gain- and loss-of-function effects of enhancer variants previously linked to limb polydactyly, autism spectrum disorder, and craniofacial malformation. By combining dual-enSERT with single-cell transcriptomics, we characterise gene expression in cells where the enhancer is normally and ectopically active, revealing candidate pathways that may lead to enhancer misregulation. Finally, we demonstrate the widespread utility of dual-enSERT by testing the effects of fifteen previously uncharacterised rare and common non-coding variants linked to neurodevelopmental disorders. In doing so we identify variants that reproducibly alter the in vivo activity of OTX2 and MIR9-2 brain enhancers, implicating them in autism. Dual-enSERT thus allows researchers to go from identifying candidate enhancer variants to analysis of comparative enhancer activity in live embryos in under two weeks.}, }
@article {pmid39761726, year = {2025}, author = {Vázquez Salgado, AM and Cai, C and Lee, M and Yin, D and Chrystostome, ML and Gefre, AF and He, S and Kieckhaefer, JE and Wangensteen, KJ}, title = {In Vivo CRISPR Activation Screening Reveals Chromosome 1q Genes VPS72, GBA1, and MRPL9 Drive Hepatocellular Carcinoma.}, journal = {Cellular and molecular gastroenterology and hepatology}, volume = {19}, number = {5}, pages = {101460}, pmid = {39761726}, issn = {2352-345X}, support = {P30 DK084567/DK/NIDDK NIH HHS/United States ; R01 CA249929/CA/NCI NIH HHS/United States ; R37 CA259201/CA/NCI NIH HHS/United States ; }, mesh = {Animals ; *Carcinoma, Hepatocellular/genetics/pathology ; Humans ; *Liver Neoplasms/genetics/pathology ; Mice ; *Chromosomes, Human, Pair 1/genetics ; Gene Expression Regulation, Neoplastic ; *Vesicular Transport Proteins/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; }, abstract = {BACKGROUND &amp; AIMS: Hepatocellular carcinoma (HCC) frequently undergoes regional chromosomal amplification, resulting in elevated gene expression levels. We aimed to elucidate the role of these poorly understood genetic changes by using CRISPR activation (CRISPRa) screening in mouse livers to identify which genes within these amplified loci are cancer driver genes.<br><br>METHODS: We used data from The Cancer Genome Atlas to identify that frequently copy number-amplified and up-regulated genes all reside on human chromosomes 1q and 8q. We generated CRISPRa screening transposons that contain oncogenic Myc to drive tumor formation. We conducted CRISPRa screens in&#xa0;vivo in the liver to identify tumor driver genes. We extensively validated the findings in separate mice and performed RNA sequencing analysis to explore mechanisms driving tumorigenesis.<br><br>RESULTS: We targeted genes that frequently undergo amplification in human HCC using an in&#xa0;vivo CRISPRa screening system in mice, which induced extensive liver tumorigenesis. Human chromosome 1q genes Zbtb7b, Vps72, Gba1, and Mrpl9 emerged as drivers of liver tumorigenesis. In human HCC there is a trend in correlation between levels of MRPL9, VPS72, or GBA1 and poor survival. In validation assays, activation of Vps72, Gba1, or Mrpl9 resulted in extensive liver tumorigenesis and decreased survival in mice. RNA sequencing revealed different mechanisms driving HCC, with Mrpl9 activation altering genes functionally related to mitochondrial function, Vps72 levels altering phospholipid metabolism, and Gba1 activation enhancing endosomal-lysosomal activity, all leading to promotion of cellular proliferation. Analysis of human tumor tissues with high levels of MRPL9, VPS72, or GBA1 revealed congruent results, indicating conserved mechanisms driving HCC.<br><br>CONCLUSIONS: This study reveals chromosome 1q genes Vps72, Gba1, and Mrpl9 as drivers of HCC. Future efforts to prevent or treat HCC can focus on these new driver genes.}, }
@article {pmid39761351, year = {2025}, author = {Ansari, MA and Verma, D and Hamizan, MA and Mukherjee, MD and Mohd-Naim, NF and Ahmed, MU}, title = {Trends in Aptasensing and the Enhancement of Diagnostic Efficiency and Accuracy.}, journal = {ACS synthetic biology}, volume = {14}, number = {1}, pages = {21-40}, doi = {10.1021/acssynbio.4c00591}, pmid = {39761351}, issn = {2161-5063}, mesh = {Humans ; *Biosensing Techniques/methods/trends ; *Aptamers, Nucleotide ; Machine Learning ; Point-of-Care Systems ; Precision Medicine/methods ; Artificial Intelligence ; CRISPR-Cas Systems ; }, abstract = {The field of healthcare diagnostics is navigating complex challenges driven by evolving patient demographics and the rapid advancement of new technologies worldwide. In response to these challenges, these biosensors offer distinctive advantages over traditional diagnostic methods, such as cost-effectiveness, enhanced specificity, and adaptability, making their integration with point-of-care (POC) platforms more feasible. In recent years, aptasensors have significantly evolved in diagnostic capabilities through the integration of emerging technologies such as microfluidics, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems, wearable devices, and machine learning (ML), driving progress in precision medicine and global healthcare solutions. Moreover, these advancements not only improve diagnostic accuracy but also hold the potential to revolutionize early detection, reduce healthcare costs, and improve patient outcomes, especially in resource-limited settings. This Account examines key advancements, focusing on how scientific breakthroughs, including artificial intelligence (AI), have improved sensitivity and precision. Additionally, the integration of aptasensors with these technologies has enabled real-time monitoring and data analysis, fostering advances in personalized healthcare. Furthermore, the potential commercialization of aptasensor technologies could increase their availability in clinical settings and support their use as widespread solutions for global health challenges. Hence, this review discusses technological improvements, practical uses, and prospects while also focusing on the challenges surrounding standardization, clinical validation, and interdisciplinary collaboration for widespread application. Finally, ongoing efforts to address these challenges are key to ensure that aptasensors can be effectively implemented in diverse healthcare systems.}, }
@article {pmid39761113, year = {2025}, author = {Kardailsky, A and Durán-Vinet, B and Nester, G and Ayad, ME and Raes, EJ and Jeunen, GJ and Miller, AK and McVey, P and Corrigan, S and Fraser, M and Goncalves, P and Burnell, S and Bennett, A and Rauschert, S and Bayer, PE}, title = {Monitoring the Land and Sea: Enhancing Efficiency Through CRISPR-Cas Driven Depletion and Enrichment of Environmental DNA.}, journal = {The CRISPR journal}, volume = {8}, number = {1}, pages = {5-12}, doi = {10.1089/crispr.2024.0050}, pmid = {39761113}, issn = {2573-1602}, mesh = {*CRISPR-Cas Systems/genetics ; *DNA, Environmental/genetics ; *Metagenomics/methods ; *Environmental Monitoring/methods ; Biodiversity ; Clustered Regularly Interspaced Short Palindromic Repeats ; Ecosystem ; }, abstract = {Characterizing biodiversity using environmental DNA (eDNA) represents a paradigm shift in our capacity for biomonitoring complex environments, both aquatic and terrestrial. However, eDNA biomonitoring is limited by biases toward certain species and the low taxonomic resolution of current metabarcoding approaches. Shotgun metagenomics of eDNA enables the collection of whole ecosystem data by sequencing all molecules present, allowing characterization and identification. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated proteins (Cas)-based methods have the potential to improve the efficiency of eDNA metagenomic sequencing of low-abundant target organisms and simplify data analysis by enrichment of target species or nontarget DNA depletion before sequencing. Implementation of CRISPR-Cas in eDNA has been limited due to a lack of interest and support in the past. This perspective synthesizes current approaches of CRISPR-Cas to study underrepresented taxa and advocate for further application and optimization of depletion and enrichment methods of eDNA using CRISPR-Cas, holding promise for eDNA biomonitoring.}, }
@article {pmid39760520, year = {2025}, author = {Zhao, Z and Zhang, H and Li, W and Wang, Y and Wang, Y and Yang, H and Yin, L and Liu, X}, title = {Guanidyl-rich α-helical polypeptide enables efficient cytosolic pro-protein delivery and CRISPR-Cas9 genome editing.}, journal = {Journal of materials chemistry. B}, volume = {13}, number = {6}, pages = {1991-2002}, doi = {10.1039/d4tb02009j}, pmid = {39760520}, issn = {2050-7518}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing ; *Peptides/chemistry ; Protein Conformation, alpha-Helical ; Animals ; Cytosol/metabolism ; }, abstract = {Intracellular delivery of proteins has attracted significant interest in biological research and cancer treatment, yet it continues to face challenges due to the lack of effective delivery approaches. Herein, we developed an efficient strategy via cationic α-helical polypeptide-mediated anionic proprotein delivery. The protein was reversibly modified with adenosine triphosphate via dynamic covalent chemistry to prepare an anionic proprotein (A-protein) with abundant phosphate groups. A guanidyl-decorated α-helical polypeptide (LPP) was employed not only to encapsulate A-protein through electrostatic attraction and hydrogen bonding, forming stable nanocomplexes, but also to enhance cell membrane penetration due to its rigid α-helical conformation. Consequently, this strategy mediated the effective delivery of various proteins with different isoelectric points and molecular weights, including α-chymotrypsin, bovine serum albumin, ribonuclease A, cytochrome C, saporin, horseradish peroxidase, β-galactosidase, and anti-phospho-Akt, into cancer cells. More importantly, it enabled efficient delivery of CRISPR-Cas9 ribonucleoproteins to elicit robust polo-like kinase 1 genome editing for inhibiting cancer cell growth. This rationally designed protein delivery system may benefit the development of intracellular protein-based cancer therapy.}, }
@article {pmid39760503, year = {2025}, author = {Zhu, Z and Lu, S and Wang, H and Wang, F and Xu, W and Zhu, Y and Xue, J and Yang, L}, title = {Innovations in Transgene Integration Analysis: A Comprehensive Review of Enrichment and Sequencing Strategies in Biotechnology.}, journal = {ACS applied materials &amp; interfaces}, volume = {17}, number = {2}, pages = {2716-2735}, doi = {10.1021/acsami.4c14208}, pmid = {39760503}, issn = {1944-8252}, mesh = {*Transgenes/genetics ; *Biotechnology/methods ; Plants, Genetically Modified/genetics ; Animals ; High-Throughput Nucleotide Sequencing/methods ; Humans ; *DNA, Bacterial/genetics ; *Sequence Analysis, DNA/methods ; }, abstract = {Understanding the integration of transgene DNA (T-DNA) in transgenic crops, animals, and clinical applications is paramount for ensuring the stability and expression of inserted genes, which directly influence desired traits and therapeutic outcomes. Analyzing T-DNA integration patterns is essential for identifying potential unintended effects and evaluating the safety and environmental implications of genetically modified organisms (GMOs). This knowledge is crucial for regulatory compliance and fostering public trust in biotechnology by demonstrating transparency in genetic modifications. This review highlights recent advancements in T-DNA integration analysis, specifically focusing on targeted DNA enrichment and sequencing strategies. We examine key technologies, such as polymerase chain reaction (PCR)-based methods, hybridization capture, RNA/DNA-guided endonuclease-mediated enrichment, and high-throughput resequencing, emphasizing their contributions to enhancing precision and efficiency in transgene integration analysis. We discuss the principles, applications, and recent developments in these techniques, underscoring their critical role in advancing biotechnological products. Additionally, we address the existing challenges and future directions in the field, offering a comprehensive overview of how innovative DNA-targeted enrichment and sequencing strategies are reshaping biotechnology and genomics.}, }
@article {pmid39760438, year = {2025}, author = {Montilla-Rojo, J and Eleveld, TF and van Soest, M and Hillenius, S and Timmerman, DM and Gillis, AJM and Roelen, BAJ and Mummery, CL and Looijenga, LHJ and Salvatori, DCF}, title = {Depletion of TP53 in Human Pluripotent Stem Cells Triggers Malignant-Like Behavior.}, journal = {Advanced biology}, volume = {9}, number = {4}, pages = {e2400538}, pmid = {39760438}, issn = {2701-0198}, support = {184.034.019//Stichting Kinderen Kankervrij/ ; NNF21CC0073729//Novo Nordisk Foundation Center for Stem Cell Medicine reNEW/ ; }, mesh = {Humans ; *Tumor Suppressor Protein p53/genetics/metabolism ; *Pluripotent Stem Cells/metabolism/pathology ; CRISPR-Cas Systems ; Mutation ; Cell Proliferation/genetics ; }, abstract = {Human pluripotent stem cells (hPSCs) tend to acquire genetic aberrations upon culture in vitro. Common aberrations are mutations in the tumor suppressor TP53, suspected to confer a growth-advantage to the mutant cells. However, their full impact in the development of malignant features and safety of hPSCs for downstream applications is yet to be elucidated. Here, TP53 is knocked out in hPSCs using CRISPR-Cas9 and compared them with isogenic wild-type hPSCs and human germ cell tumor lines as models of malignancy. While no major changes in proliferation, pluripotency, and transcriptomic profiles are found, mutant lines display aberrations in some of the main chromosomal hotspots for genetic abnormalities in hPSCs. Additionally, enhanced clonogenic and anchorage-free growth, alongside resistance to chemotherapeutic compounds is observed. The results indicate that common TP53-depleting mutations in hPSCs, although potentially overlooked by standard analyses, can impact their behavior and safety in a clinical setting.}, }
@article {pmid39760089, year = {2024}, author = {Li, K and Luo, T and Zhang, Y and Li, C and Chen, H and Xia, C and Gao, C}, title = {Rapid detection of Mycoplasma hyopneumoniae by recombinase-aided amplification combined with the CRISPR/Cas12a system.}, journal = {Frontiers in cellular and infection microbiology}, volume = {14}, number = {}, pages = {1469558}, pmid = {39760089}, issn = {2235-2988}, mesh = {*Mycoplasma hyopneumoniae/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; Animals ; Swine ; *Nucleic Acid Amplification Techniques/methods ; *Pneumonia of Swine, Mycoplasmal/diagnosis/microbiology ; *Recombinases/metabolism/genetics ; Sensitivity and Specificity ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Molecular Diagnostic Techniques/methods ; Limit of Detection ; }, abstract = {Mycoplasma hyopneumoniae (M. hyopneumoniae) is one of the primary agents involved in porcine respiratory disease complex, and circulates in the swine industry worldwide. The prevention and control of M. hyopneumoniae is complicated. Thus, a recombinase-aided amplification (RAA) assay coupled with the clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas12a system was established for the detection of M. hyopneumoniae. The most suitable primer pairs and CRISPR RNA (crRNA) were screened and selected for the RAA-CRISPR/Cas12a detection system. We have achieved a detection limit of 1 copy/µL and 5 copies/µL per reaction for the RAA-CRISPR/Cas12a-fluorescence assay and RAA-CRISPR/Cas12a-lateral flow assay (LFA), respectively. Furthermore, the RAA-CRISPR/Cas12a system displayed no cross-reactivity with other respiratory pathogens. The performance of the RAA-CRISPR/Cas12a system was compared with PCR as recommended by the Chinese national standard (GB/T 35909-2018) and qPCR as recommended by the Chinese entry-exit inspection and quarantine industry standard (SN/T4104-2015) for clinical samples, and good consistency with these methods was observed. Above all, the methods shed a light on the convenient, portable, visual, highly sensitive and specific detection of M. hyopneumoniae, demonstrating a great application potential for on-site monitoring of M. hyopneumoniae in the field.}, }
@article {pmid39759869, year = {2024}, author = {Gong, Z and Wang, W and Zhao, Y and Wang, Y and Sun, R and Zhang, H and Wang, F and Lu, Y and Zhang, J}, title = {Analysis of the pathogenicity and pathological characteristics of NOTCH3 gene-sparing cysteine mutations in vitro and in vivo models.}, journal = {Frontiers in molecular neuroscience}, volume = {17}, number = {}, pages = {1391040}, pmid = {39759869}, issn = {1662-5099}, abstract = {BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is one of the most common inherited cerebral small vessel diseases caused by the NOTCH3 gene mutation. This mutation leads to the accumulation of NOTCH3 extracellular domain protein (NOTCH3[ECD]) into the cerebral arterioles, causing recurrent stroke, white matter lesions, and cognitive impairment. With the development of gene sequencing technology, cysteine-sparing mutations can also cause CADASIL disease, however, the pathogenicity and pathogenic mechanisms of cysteine-sparing mutations remain controversial.<br><br>OBJECTIVE: To analyze the pathogenicity and pathological features of cysteine-sparing mutations in both in vitro and in vivo mouse models.<br><br>METHODS: A cysteine-sparing mutant of NOTCH3[ECD] R75Q was constructed by lentiviral transfection in vitro, and the NOTCH3 R75Q knock-in mouse model was constructed by CRISPR/Cas-mediated genome engineering in vivo. A cycloheximide pulse-chase experiment was used to analyze the degradation of NOTCH3 extracellular domain proteins, and the deposition characteristics of NOTCH3[ECD] were quantitatively analyzed by immunohistochemical staining. The characteristics of the smooth muscle cells and granular osmiophilic materials were observed using electron microscopy.<br><br>RESULTS: We elucidated that the NOTCH3 R75Q mutation is pathogenic. NOTCH3[ECD] R75Q was found to be resistant to protein degradation and more likely to cause abnormal aggregation of NOTCH3[ECD], resulting in reduced cell activity in vitro. The NOTCH3 R75Q mouse model showed pathological characteristics of CADASIL, with age-dependent NOTCH3[ECD], granular osmiophilic material, and degenerated smooth muscle cells detected in the brain.<br><br>CONCLUSION: To our knowledge, this is the first study to analyze the pathogenicity of NOTCH3 R75Q cysteine-sparing mutations in both in vitro and in vivo models. We demonstrate that NOTCH3[ECD] induced by NOTCH3 R75Q mutation has toxic effects on cells and reveal the deposition characteristics of NOTCH3[ECD] in the brain. This provides a feasible model and lays the foundation for further studies on the pathogenesis and therapeutic strategies of NOTCH3 cysteine-sparing mutations.}, }
@article {pmid39758988, year = {2024}, author = {Wu, X and Xiang, R and Yang, D and He, X and Zhu, L and Sun, F and Li, H and Pi, N and Li, Y}, title = {HRP-integrated CRISPR-Cas12a biosensor for rapid point-of-care detection of Langya henipavirus.}, journal = {iScience}, volume = {27}, number = {12}, pages = {111466}, pmid = {39758988}, issn = {2589-0042}, abstract = {Global pandemic has emphasized the needs for advanced pathogen diagnosis in dealing with newly emerged infectious threats, including the Langya henipavirus (LayV). LayV, as an emerging zoonotic pathogen, has potential to cause pandemic, but lacks of rapid diagnostic tools, particularly at point-of-care level. Here, we leveraged the merits of CRISPR-Cas12a biosensing and established a highly sensitive LayV detection method. By integrating CRISPR-Cas12a with RPA, 10 copies/μL ultra-sensitive LayV RNA detection has been achieved at room temperature within 30 min. More importantly, this study developed a special horseradish peroxidase (HRP)-single-stranded DNA (ssDNA) reporter enabling CRISPR-Cas12a to detect LayV RNA without pre-amplification, achieving a sensitivity of 1,200 copies/μL detectable by the naked eye. These explorations can serve as accelerator for CRISPR-Cas biosensing toward rapid response for newly emerged biological threats, and also provide a method to realize simple, precise, and amplicon-free point-of-care pathogen screening for resource limited or underdevelopment regions.}, }
@article {pmid39756499, year = {2025}, author = {Wei, Z and Wang, C and Zhang, X and Lv, Y and Li, Y and Gao, P and Yang, X}, title = {CRISPR/Cas9-mediated knockout of Tektin 4-like gene (TEKT4L) causes male sterility of Cydia pomonella.}, journal = {Insect biochemistry and molecular biology}, volume = {177}, number = {}, pages = {104257}, doi = {10.1016/j.ibmb.2025.104257}, pmid = {39756499}, issn = {1879-0240}, mesh = {Animals ; Male ; CRISPR-Cas Systems ; *Insect Proteins/genetics/metabolism ; *Infertility, Male/genetics ; Female ; *Moths/genetics/physiology ; Gene Knockout Techniques ; }, abstract = {The sterile insect technique (SIT) is a well-established and environmentally benign method for population control. Identifying genes that regulate insect fertility while preserving growth and development is crucial for implementing a novel SIT-based pest management approach utilizing CRISPR/Cas9 to target these genes for genetic manipulation. Tektin (TEKT), an essential alpha-helical protein pivotal in sperm formation due to its role in cilia and flagella assembly, has garnered attention. In this study, we identified 7 TEKT genes in the testis of Cydia pomonella, a globally invasive fruit pest. Notably, Tektin4-like (TEKT4L) displayed the highest expression level in male adult especially the testes, suggesting its significance in reproductive processes. By utilizing CRISPR/Cas9 technology to knockout TEKT4L, male sterility was induced, showcasing dominant inherited. When wild-type (WT) females mated with TEKT4L[-/-] males, eggs laying proceeded normally, but the hatching rate was dramatically reduced, with only 15.49% progressing to the eyespot stage and 68.86% failing to develop normally. The reproductive fitness of TEKT4L[-/-] males was robust enough to facilitate the transmission of genetic modifications efficiently within the C.pomonella population, yielding a small number of viable offspring. Subsequent cage trials demonstrated the effectiveness of this population in suppressing laboratory populations of C.pomonella, achieving notable results with a relatively low release ratio (TEKT4L[-/-]♂: WT♂: WT♀ = 5:1:5). Consequently, the targeted disruption of the TEKT4L gene holds promise as a fundamental element in a novel pest control strategy against C. pomonella.}, }
@article {pmid39756423, year = {2025}, author = {Haeusser, LA and Becker, H and Kuhlburger, L and Zago, M and Walter, B and Tsiami, F and Erdmann, S and Trampert, J and Surender, S and Stahl, A and Templin, M and Wegner, E and Schmidt, T and Schmees, C and Casadei, N and Sevenich, L and Claassen, M and Nahnsen, S and Beck, S and Merk, DJ and Tabatabai, G}, title = {Genome-wide CRISPR-Cas9 screens identify BCL family members as modulators of response to regorafenib in experimental glioma.}, journal = {Neuro-oncology}, volume = {27}, number = {4}, pages = {916-931}, pmid = {39756423}, issn = {1523-5866}, support = {//Adolf Leuze Stiftung/ ; //Medical Faculty T&#xfc;bingen (Demonstratorprojekt Personalisierte Medizin and NWG program)/ ; 2019_Kolleg_14//Else Kr&#xf6;ner Forschungskolleg/ ; //Deutsche Forschungsgemeinschaft/ ; EXC 2180//Germany's Excellence Strategy, Cluster of Excellence/ ; 390900677//Germany's Excellence Strategy, Cluster of Excellence/ ; }, mesh = {Animals ; *Pyridines/pharmacology ; *Phenylurea Compounds/pharmacology ; Mice ; *Glioma/drug therapy/genetics/pathology/metabolism ; Humans ; *CRISPR-Cas Systems ; *Brain Neoplasms/drug therapy/genetics/pathology/metabolism ; Xenograft Model Antitumor Assays ; Tumor Cells, Cultured ; Apoptosis/drug effects ; *Proto-Oncogene Proteins c-bcl-2/genetics/metabolism ; Cell Proliferation/drug effects ; }, abstract = {BACKGROUND: Registered systemic treatment options for glioblastoma patients are limited. The phase II REGOMA trial suggested an improvement of median overall survival in progressive glioblastoma by the multi-tyrosine kinase inhibitor regorafenib. This has not been confirmed by GBM AGILE. So far, regorafenib has been administered as monotherapy or as an addition to standard of care in newly diagnosed glioblastoma. Rational combination therapies involving regorafenib might be a reasonable strategy. Here, we aimed at identifying functionally instructed combination therapies involving regorafenib.<br><br>METHODS: We applied a genome-wide CRISPR-Cas9-based functional genomics target discovery approach using activation and knockout screens followed by genetic, pharmacological, functional validations. Regorafenib-induced molecular alterations were assessed by RNA sequencing and DigiWest. We investigated selected functionally instructed combination therapies in three orthotopic glioma mouse models in vivo (syngeneic SMA560/VM/Dk model and two xenograft models) and performed immunohistochemistry of post-treatment brains.<br><br>RESULTS: We identified potential modifiers of regorafenib response, including BCL2, BCL2L1, ITGB3, FOXC1, SERAC1, ARAF, and PLCE1. The combination of regorafenib with Bcl-2/Bcl-xL inhibition was superior to both monotherapies alone in vitro, ex vivo, and in vivo. We identified regorafenib-induced regulations of the Bcl-2 downstream target chemokine receptor 1 (CCR1) as one potential underlying molecular mediator. Furthermore, regorafenib led to changes in the myeloid compartment of the glioma-associated microenvironment.<br><br>CONCLUSIONS: This preclinical study uses a functional genomics-based target discovery approach with subsequent validations involving regorafenib. It serves as a biological rationale for clinical translation. Particularly, an investigation of the combination of regorafenib plus navitoclax within a clinical trial is warranted.}, }
@article {pmid39756219, year = {2025}, author = {Mao, Y and Shisler, JL and Nguyen, TH}, title = {Enhanced detection for antibiotic resistance genes in wastewater samples using a CRISPR-enriched metagenomic method.}, journal = {Water research}, volume = {274}, number = {}, pages = {123056}, doi = {10.1016/j.watres.2024.123056}, pmid = {39756219}, issn = {1879-2448}, mesh = {*Wastewater/microbiology ; *Metagenomics/methods ; *Drug Resistance, Microbial/genetics ; High-Throughput Nucleotide Sequencing ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; }, abstract = {The spread of antibiotic resistance genes (ARGs) in the environment is a global public health concern. To date, over 5000 genes have been identified to express resistance to antibiotics. ARGs are usually low in abundance for wastewater samples, making them difficult to detect. Metagenomic sequencing and quantitative polymerase chain reaction (qPCR), two conventional ARG detection methods, have low sensitivity and low throughput limitations, respectively. We developed a CRISPR-Cas9-modified next-generation sequencing (NGS) method to enrich the targeted ARGs during library preparation. The false negative and false positive of this method were determined based on a mixture of bacterial isolates with known whole-genome sequences. Low values of both false negative (2/1208) and false positive (1/1208) proved the method's reliability. We compared the results obtained by this CRISPR-NGS and the conventional NGS method for six untreated wastewater samples. As compared to the ARGs detected in the same samples using the regular NGS method, the CRISPR-NGS method found up to 1189 more ARGs and up to 61 more ARG families in low abundances, including the clinically important KPC beta-lactamase genes in the six wastewater samples collected from different sources. Compared to the regular NGS method, the CRISPR-NGS method lowered the detection limit of ARGs from the magnitude of 10[-4] to 10[-5] as quantified by qPCR relative abundance. The CRISPR-NGS method is promising for ARG detection in wastewater. A similar workflow can also be applied to detect other targets that are in low abundance but of high diversity.}, }
@article {pmid39756031, year = {2025}, author = {Teng, Y and Arbogast, K and Junge, H and Chen, Z}, title = {Protocol for generating splice isoform-specific mouse mutants using CRISPR-Cas9 and a minigene splicing reporter.}, journal = {STAR protocols}, volume = {6}, number = {1}, pages = {103543}, pmid = {39756031}, issn = {2666-1667}, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Mice ; Mice, Transgenic ; Mutation ; *Genes, Reporter/genetics ; *Gene Editing/methods ; *Alternative Splicing/genetics ; *RNA Splicing/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Protein Isoforms/genetics ; }, abstract = {Here, we present a protocol to alter the production of alternatively spliced mRNA variants, without affecting the overall gene expression, through CRISPR-Cas9-engineered genomic mutations in mice. We describe steps for designing guide RNA to direct Cas9 endonuclease to consensus splice sites, producing transgenic mice through pronuclear injection, and screening for desired mutations in cultured mammalian cells using a minigene splicing reporter. Splice isoform-specific mouse mutants provide valuable tools for genetic analyses beyond loss-of-function and transgenic alleles. For complete details on the use and execution of this protocol, please refer to Dailey-Krempel et al.[1] and Johnson et al.[2].}, }
@article {pmid39755933, year = {2025}, author = {Liang, Z and Wu, Y and Deng, S and Wei, S and Zhang, K and Guo, Y}, title = {Enhanced Genome Editing Activity with Novel Chimeric ScCas9 Variants in Rice.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {12}, number = {8}, pages = {e2411549}, pmid = {39755933}, issn = {2198-3844}, support = {2022YFF1002800//National Key R&amp;D Program of China/ ; 32170410//National Natural Science Foundation of China/ ; 202204051001019//Science and Technology Innovation Young Talent Team of Shanxi Province/ ; 202203021224002//Natural Science Foundation of Shanxi Province for the Excellent Youth/ ; }, mesh = {*Oryza/genetics ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; Streptococcus/genetics ; }, abstract = {The Streptococcus canis Cas9 protein (ScCas9) recognizes the NNG protospacer adjacent motif (PAM), offering a wider range of targets than that offered by the commonly used S. pyogenes Cas9 protein (SpCas9). However, both ScCas9 and its evolved Sc++ variant still exhibit low genome editing efficiency in plants, particularly at the less preferred NTG and NCG PAM targets. In this study, a chimeric SpcRN++ variant is engineered by grafting the recognition (REC) domain of SpCas9 into the Sc++ variant, incorporating the R221K/N394K mutations, and retaining the positively charged loop of S. anginosus Cas9. The SpcRN++ variant exhibits a higher genome editing capacity and wider target range than the Sc++ variant in rice protoplasts and stable transgenic plants. Further evidence indicates that nSpcRN++-based A3A/Y130F and TadA8e exhibit enhanced cytosine and adenine editing efficiency in plants. Finally, herbicide-resistant rice germplasms are produced by targeting the OsACC gene using nSpcRN++-based adenine base editors. These results demonstrate that SpcRN++ is a powerful tool for genome editing in plants, and this integrative protein engineering strategy holds promise for engineering other Cas9 proteins.}, }
@article {pmid39755699, year = {2025}, author = {Halegua, T and Risson, V and Carras, J and Rouyer, M and Coudert, L and Jacquier, A and Schaeffer, L and Ohlmann, T and Mangeot, PE}, title = {Delivery of Prime editing in human stem cells using pseudoviral NanoScribes particles.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {397}, pmid = {39755699}, issn = {2041-1723}, mesh = {Humans ; *Gene Editing/methods ; HEK293 Cells ; CRISPR-Cas Systems ; Induced Pluripotent Stem Cells/metabolism/cytology ; Ribonucleoproteins/metabolism/genetics ; Hematopoietic Stem Cells/metabolism/cytology/virology ; Myoblasts/metabolism/cytology ; Virion/metabolism/genetics ; Stem Cells/metabolism/cytology ; }, abstract = {Prime Editing can rewrite genes in living cells by allowing point mutations, deletions, or insertion of small DNA sequences with high precision. However, its safe and efficient delivery into human stem cells remains a technical challenge. In this report, we engineer Nanoscribes, virus-like particles that encapsidate ribonucleoprotein complexes of the Prime Editing system and allow their delivery into recipient cells. We identify key features that unlock the potential of Nanoscribes, including the use of multiple fusogens, the improvement of pegRNAs structures, their encoding by a Pol II system and the optimization of Prime-Editors. Nanoscribes edit HEK293T with an efficiency of 68% at the HEK3 locus with increased fidelity over DNA-transfection and support pegRNA-multiplexing. Importantly, Nanoscribes permit editing of myoblasts, hiPSCs and hiPSCs-derived hematopoietic stem cells with an editing efficiency up to 25%. Nanoscribes is an asset for development of next generation genome editing approaches using VLPs.}, }
@article {pmid39754719, year = {2025}, author = {Chu, SN and Soupene, E and Sharma, D and Sinha, R and McCreary, T and Hernandez, B and Shen, H and Wienert, B and Bowman, C and Yin, H and Lesch, BJ and Jia, K and Romero, KA and Kostamo, Z and Zhang, Y and Tran, T and Cordero, M and Homma, S and Hampton, JP and Gardner, JM and Conklin, BR and MacKenzie, TC and Sheehan, VA and Porteus, MH and Cromer, MK}, title = {Dual α-globin-truncated erythropoietin receptor knockin restores hemoglobin production in α-thalassemia-derived erythroid cells.}, journal = {Cell reports}, volume = {44}, number = {1}, pages = {115141}, pmid = {39754719}, issn = {2211-1247}, support = {T32 AI125222/AI/NIAID NIH HHS/United States ; R01 HL130533/HL/NHLBI NIH HHS/United States ; R01 AG072052/AG/NIA NIH HHS/United States ; P01 HL146366/HL/NHLBI NIH HHS/United States ; R01 HL161291/HL/NHLBI NIH HHS/United States ; R01 HL135358/HL/NHLBI NIH HHS/United States ; }, mesh = {Humans ; *alpha-Globins/genetics/metabolism ; *Erythroid Cells/metabolism ; *Receptors, Erythropoietin/genetics/metabolism ; *alpha-Thalassemia/genetics/metabolism/pathology ; *Hemoglobins/metabolism/biosynthesis ; Gene Editing ; Hematopoietic Stem Cells/metabolism ; Gene Knock-In Techniques ; beta-Globins/genetics/metabolism ; Erythropoiesis ; CRISPR-Cas Systems ; }, abstract = {The most severe form of α-thalassemia results from loss of all four copies of α-globin. Postnatally, patients face challenges similar to β-thalassemia, including severe anemia and erythrotoxicity due to the imbalance of β-globin and α-globin chains. Despite progress in genome editing treatments for β-thalassemia, there is no analogous curative option for α-thalassemia. To address this, we designed a Cas9/AAV6-mediated genome editing strategy that integrates a functional α-globin gene into the β-globin locus in α-thalassemia patient-derived hematopoietic stem and progenitor cells (HSPCs). Incorporation of a truncated erythropoietin receptor transgene into the α-globin integration cassette significantly increased erythropoietic output from edited HSPCs and led to the most robust production of α-globin, and consequently hemoglobin tetramers. By directing edited HSPCs toward increased production of clinically relevant erythroid cells, this approach has the potential to mitigate the limitations of current treatments for the hemoglobinopathies, including low genome editing and low engraftment rates.}, }
@article {pmid39754619, year = {2025}, author = {Raina, K and Modak, K and Premkumar, C and Joshi, G and Palani, D and Nandy, K and Sivamani, Y and Velayudhan, SR and Thummer, RP}, title = {UTF1 Expression is Important for the Generation and Maintenance of Human iPSCs.}, journal = {Stem cell reviews and reports}, volume = {21}, number = {3}, pages = {859-871}, pmid = {39754619}, issn = {2629-3277}, support = {CRG/2021/004414//SERB-DST Govt of India/ ; }, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cellular Reprogramming/genetics ; Cell Differentiation/genetics ; Fibroblasts/metabolism/cytology ; *Trans-Activators/genetics/metabolism ; CRISPR-Cas Systems ; Octamer Transcription Factor-3/metabolism/genetics ; Gene Knockout Techniques ; *Nuclear Proteins/genetics/metabolism ; SOXB1 Transcription Factors/metabolism/genetics ; }, abstract = {BACKGROUND: Undifferentiated embryonic cell transcription factor 1 (UTF1) is predominantly expressed in pluripotent stem cells and plays a vital role in embryonic development and pluripotency maintenance. Despite its established importance in murine models, the role of UTF1 on human induced pluripotent stem cells (iPSCs) has not been comprehensively studied.<br><br>METHODS: This study utilized CRISPR/Cas9 gene editing to create UTF1 knockout in human fibroblasts and iPSCs. We employed episomal vectors for reprogramming UTF1 knockout fibroblasts into iPSCs and analyzed the effects of UTF1 depletion on cellular morphology, pluripotency, and viability through Western blotting, PCR, and flow cytometry. In addition, we integrated an shRNA that downregulated the expression of UTF1 for mechanistic studies to understand the impact of UTF1 depletion in iPSC pluripotency and differentiation.<br><br>RESULTS: UTF1 knockout resulted in significantly reduced reprogramming efficiency and increased spontaneous differentiation, indicating its crucial role in maintaining human iPSC identity and stability. In knockdown experiments, gradual loss of UTF1 led to change in cellular morphologies and decreased expression of core pluripotency markers OCT4 and SOX2. Interestingly, unlike complete UTF1 knockout, the gradual downregulation of UTF1 in iPSCs did not result in apoptosis, suggesting that the loss of pluripotency can occur independently of the apoptotic pathways.<br><br>CONCLUSIONS: UTF1 is essential for maintaining the pluripotency and viability of human iPSCs. Its depletion affects the fundamental properties of stem cells, underscoring the potential challenges in using UTF1-deficient cells for therapeutic applications. Future studies should explore the mechanistic pathways through which UTF1 controls pluripotency and differentiation, which could provide insights into improving iPSC stability for clinical applications.}, }
@article {pmid39754554, year = {2025}, author = {Hu, F and Liu, K and Zhang, Y and Zhao, S and Zhang, T and Yao, C and Lv, X and Wang, J and Liu, X and Peng, N}, title = {Short-Time Preamplification-Assisted One-Pot CRISPR Nucleic Acid Detection Method with Portable Self-Heating Equipment for Point-of-Care Diagnosis.}, journal = {Analytical chemistry}, volume = {97}, number = {1}, pages = {658-666}, doi = {10.1021/acs.analchem.4c05026}, pmid = {39754554}, issn = {1520-6882}, mesh = {*SARS-CoV-2/genetics/isolation &amp; purification ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *COVID-19/diagnosis ; *CRISPR-Cas Systems ; *Point-of-Care Systems ; *RNA, Viral/analysis/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Point-of-Care Testing ; *COVID-19 Nucleic Acid Testing/methods ; Limit of Detection ; }, abstract = {Infectious diseases, especially respiratory infections, have been significant threats to human health. Therefore, it is essential to develop rapid, portable, and highly sensitive diagnostic methods for their control. Herein, a short-time preamplified, one-pot clustered regularly interspaced short palindromic repeats (CRISPR) nucleic acid detection method (SPOC) is developed by combining the rapid recombinase polymerase amplification (RPA) with CRISPR-Cas12a to reduce the mutual interference and achieve facile and rapid molecular diagnosis. SPOC can reduce the detection time and stably detect up to 1 copy/μL of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA without affecting the detection sensitivity. A highly sensitive one-pot assay integrated with reverse transcription RPA is achieved by wrapping paraffin with a specific melting point on the lyophilized CRISPR reagent surface. A self-heating pack is designed based on thermodynamic principles to melt the paraffin and release CRISPR reagents, enabling low-cost and time-saving detection. Notably, the designed system, coupled with RNA extraction-free technology, can achieve "sample-in-answer-out" detection of the SARS-CoV-2 Orf1ab gene within 22 min using smartphone imaging. The developed assay is validated on 12 clinical samples, and the results 100% correlate with real-time polymerase chain reaction. SPOC is time-saving, is easy to operate, and can eliminate centrifugal and complex hardware devices, satisfying the demand for point-of-care diagnostics in resource-constrained settings.}, }
@article {pmid39754035, year = {2025}, author = {Xiang, T and Feng, H and Xing, XH and Zhang, C}, title = {GLiDe: a web-based genome-scale CRISPRi sgRNA design tool for prokaryotes.}, journal = {BMC bioinformatics}, volume = {26}, number = {1}, pages = {1}, pmid = {39754035}, issn = {1471-2105}, support = {21938004//National Natural Science Foundation of China/ ; U2032210//National Natural Science Foundation of China/ ; 2023YFC3402300//National Key Research and Development Program of China/ ; }, mesh = {*Internet ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems/genetics ; Software ; Prokaryotic Cells/metabolism ; Genomics/methods ; Genome, Bacterial ; }, abstract = {BACKGROUND: CRISPRi screening has become a powerful approach for functional genomic research. However, the off-target effects resulting from the mismatch tolerance between sgRNAs and their intended targets is a primary concern in CRISPRi applications.<br><br>RESULTS: We introduce Guide Library Designer (GLiDe), a web-based tool specifically created for the genome-scale design of sgRNA libraries tailored for CRISPRi screening in prokaryotic organisms. GLiDe incorporates a robust quality control framework, rooted in prior experimental knowledge, ensuring the accurate identification of off-target hits. It boasts an extensive built-in database, encompassing 1,397 common prokaryotic species as a comprehensive design resource. It also provides the capability to design sgRNAs for newly discovered organisms by accepting uploaded design resource. We further demonstrated that GLiDe exhibits enhanced precision in identifying off-target binding sites for the CRISPRi system.<br><br>CONCLUSIONS: We present a web server that allows the construction of genome-scale CRISPRi sgRNA libraries for prokaryotes. It mitigates off-target effects through a robust quality control framework, leveraging prior experimental knowledge within an end-to-end, user-friendly pipeline.}, }
@article {pmid39753388, year = {2025}, author = {Ahmad, N and Awan, MJA and Amin, I and Mansoor, S}, title = {Perfecting prime editing: achieving precise edits in dicots.}, journal = {Trends in plant science}, volume = {30}, number = {3}, pages = {232-234}, doi = {10.1016/j.tplants.2024.12.005}, pmid = {39753388}, issn = {1878-4372}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; Crops, Agricultural/genetics ; Plants, Genetically Modified/genetics ; }, abstract = {Prime editing (PE), a precise CRISPR-based method, has worked well in some plants but faces challenges in dicots. Vu and colleagues developed new PE tools that greatly improve PE efficiency in dicots, enabling accurate, heritable genome edits. This advance marks a breakthrough that could revolutionize crop improvement and plant biotechnology.}, }
@article {pmid39753078, year = {2025}, author = {Zhang, Y and Dong, X and Jiang, C and Yu, Y and Zhang, H and Fu, J and Su, G and Liu, Y}, title = {A competitive aptamer binding-based CRISPR-cas biosensor for sensitive detection of tetracycline residues in biological samples.}, journal = {Talanta}, volume = {286}, number = {}, pages = {127491}, doi = {10.1016/j.talanta.2024.127491}, pmid = {39753078}, issn = {1873-3573}, mesh = {*Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry/metabolism/genetics ; *Tetracycline/analysis ; Milk/chemistry ; Honey/analysis ; *CRISPR-Cas Systems ; Animals ; Food Contamination/analysis ; Limit of Detection ; Fishes ; *Drug Residues/analysis ; }, abstract = {Tetracycline (TC) is widely used in veterinary medicine and animal feed; however, TC residues in food pose a risk to human health. Thus, the sensitive and selective detection of TC is needed to ensure food safety. Herein, we developed a CRISPR-Cas12a biosensor with competitive aptamer binding to detect TC residues. The aptasensor, formed by hybridizing activator DNA with TC-specific aptamers on streptavidin-modified magnetic beads, releases activator DNA in a TC concentration-dependent manner. This activated the Cas12a-crRNA complex, which cleaved single-strand DNA reporters to generate a detectable fluorescence signal. The TC signal was amplified through a two-step incubation reaction, with a detection limit as low as 9.45 × 10[-5] μg L[-1]. The assay showed high selectivity and good recovery rates in various biological samples (e.g., honey, milk, fish), demonstrating the applicability of the biosensors in pollutant detection.}, }
@article {pmid39752901, year = {2025}, author = {Gong, Y and Zhang, J and Lu, Z and Cai, J and Song, Z and Wei, J and Zhuo, C and Tang, Q and Zhang, K and Liao, X}, title = {Dual signal amplification in ECL biosensors: A novel approach for argonaute2 detection using SAHARA CRISPR-Cas12a technology.}, journal = {Bioelectrochemistry (Amsterdam, Netherlands)}, volume = {163}, number = {}, pages = {108896}, doi = {10.1016/j.bioelechem.2024.108896}, pmid = {39752901}, issn = {1878-562X}, mesh = {*Biosensing Techniques/methods ; *Argonaute Proteins/analysis/metabolism ; *CRISPR-Cas Systems ; Humans ; *Luminescent Measurements/methods ; Limit of Detection ; *Electrochemical Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Argonaute 2 (Ago2) is a crucial enzyme in the RNA interference (RNAi) pathway, essential for gene silencing via the cleavage of target messenger RNA (mRNA) mediated by microRNA (miRNA) or small interfering RNA (siRNA). The activity of Ago2 is a significant biomarker for various diseases, including cancer and viral infections, necessitating precise monitoring techniques. Traditional methods for detecting Ago2 activity are often cumbersome and lack the necessary sensitivity and specificity for low-abundance targets in complex samples. This study presents an innovative biosensor utilizing electrochemiluminescence (ECL) technology combined with the SAHARA (Split Activator for Highly Accessible RNA Analysis) CRISPR-Cas12a system to detect Ago2 activity with high sensitivity and specificity. The introduction of Blocker RNA in the activation mechanism enhances the specificity of CRISPR-Cas12a, ensuring accurate signal generation. The dual signal amplification strategy, combining RISC-assisted and CRISPR-Cas12a-mediated cleavage, enhances the biosensor's sensitivity. The developed ECL biosensor demonstrated a remarkable limit of detection (LOD) of 0.145 aM, along with excellent precision, stability, and specificity. These attributes make it a powerful tool for detecting Ago2 activity in clinical diagnostics and research settings.}, }
@article {pmid39752253, year = {2025}, author = {Mutte, SK and Barendse, P and Ugarte, PB and Swarts, DC}, title = {Distribution of bacterial DNA repair proteins and their co-occurrence with immune systems.}, journal = {Cell reports}, volume = {44}, number = {1}, pages = {115110}, doi = {10.1016/j.celrep.2024.115110}, pmid = {39752253}, issn = {2211-1247}, mesh = {*DNA Repair ; *Bacterial Proteins/metabolism/genetics ; *Bacteria/genetics/immunology/metabolism ; *Immune System/metabolism ; Rec A Recombinases/metabolism ; }, abstract = {Bacteria encode various DNA repair pathways to maintain genome integrity. However, the high degree of homology between DNA repair proteins or their domains hampers accurate identification. Here, we describe a stringent search strategy to identify DNA repair proteins and provide a systematic analysis of taxonomic distribution and co-occurrence of DNA repair proteins involved in RecA-dependent homologous recombination. Our results reveal the widespread presence of RecA, SSB, and RecOR proteins and phyla-specific distribution for the DNA repair complexes RecBCD, AddAB, and AdnAB. Furthermore, we report co-occurrences of DNA repair proteins with immune systems, including specific CRISPR-Cas subtypes, prokaryotic Argonautes (pAgos), dGTPases, GAPS2, and Wadjet. Our results imply that while certain DNA repair proteins and immune systems might function in conjunction, no immune system strictly relies on a specific DNA repair protein. As such, these findings offer an updated perspective on the distribution of DNA repair systems and their connection to immune systems in bacteria.}, }
@article {pmid39752243, year = {2025}, author = {Riaz, A and Uzair, M and Raza, A and Inam, S and Iqbal, R and Jameel, S and Bibi, B and Khan, MR}, title = {Enhancing the productivity and resilience of rice (Oryza sativa) under environmental stress conditions using clustered regularly interspaced short palindromic repeats (CRISPR) technology.}, journal = {Functional plant biology : FPB}, volume = {52}, number = {}, pages = {}, doi = {10.1071/FP24101}, pmid = {39752243}, issn = {1445-4416}, mesh = {*Oryza/genetics ; *Stress, Physiological/genetics ; *Gene Editing ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Plants, Genetically Modified/genetics ; }, abstract = {Rice (Oryza sativa) is a crucial staple crop worldwide, providing nutrition to more than half of the global population. Nonetheless, the sustainability of grain production is increasingly jeopardized by both biotic and abiotic stressors exacerbated by climate change, which increases the crop's rvulnerability to pests and diseases. Genome-editing by clustered regularly interspaced short palindromic repeats and CRISPR-associated Protein 9 (CRISPR-Cas9) presents a potential solution for enhancing rice productivity and resilience under climatic stress. This technology can alter a plant's genetic components without the introduction of foreign DNA or genes. It has become one of the most extensively used approaches for discovering new gene functions and creating novel varieties that exhibit a higher tolerance to both abiotic and biotic stresses, herbicide resistance, and improved yield production. This study examines numerous CRISPR-Cas9-based genome-editing techniques for gene knockout, gene knock-in, multiplexing for simultaneous disruption of multiple genes, base-editing, and prime-editing. This review elucidates the application of genome-editing technologies to enhance rice production by directly targeting yield-related genes or indirectly modulating numerous abiotic and biotic stress-responsive genes. We highlight the need to integrate genetic advancements with conventional and advanced agricultural methods to create rice varieties that are resilient to stresses, thereby safeguarding food security and promoting agricultural sustainability amid climatic concerns.}, }
@article {pmid39749289, year = {2024}, author = {Bhoobalan-Chitty, Y and Stouf, M and De Paepe, M}, title = {Genetic manipulation of bacteriophage T4 utilizing the CRISPR-Cas13b system.}, journal = {Frontiers in genome editing}, volume = {6}, number = {}, pages = {1495968}, pmid = {39749289}, issn = {2673-3439}, abstract = {CRISPR-Cas type II and type V systems are inefficient in modifying bacteriophage T4 genome, due to hypermodification of its DNA. Here, we present a genome editing technique for bacteriophage T4 using the type VI CRISPR-Cas system. Using BzCas13b targeting of T4 phage, we were able to individually delete both T4 glucosyl transferase genes, α-gt and β-gt. Furthermore, we employed this method to mutate two conserved residues within the T4 DNA polymerase and to introduce the yellow fluorescent protein (YFP) coding sequence into T4 phage genome, enabling us to visualize phage infections. This T4 genome editing protocol was optimized to generate recombinant phages within a 6-hour timescale. Finally, spacers homologous to a variety of T4 genes were used to study the generality of Cas13b targeting, revealing important variability in targeting efficiency. Overall, this method constitutes a rapid and effective means of generating specific T4 phage mutants, which could be extended to other T4-like phages.}, }
@article {pmid39748374, year = {2025}, author = {Wan, X and Kong, J and Hu, X and Liu, L and Yang, Y and Li, H and Liu, G and Niu, X and Chen, F and Zhang, D and Zhu, D and Zhang, Y}, title = {SiCLAT: simultaneous imaging of chromatin loops and active transcription in living cells.}, journal = {Genome biology}, volume = {26}, number = {1}, pages = {1}, pmid = {39748374}, issn = {1474-760X}, mesh = {Animals ; *Chromatin/metabolism ; Mice ; *Transcription, Genetic ; Enhancer Elements, Genetic ; CRISPR-Cas Systems ; Promoter Regions, Genetic ; Cell Differentiation ; }, abstract = {We present SiCLAT, which introduces a dCas9-dCas13d cassette into the mouse genome. This model enables the stable expression of both dCas9 and dCas13 proteins in diverse cell populations, facilitating concurrent labeling of DNA and RNA across various cell types. Using SiCLAT, we accurately labeled chromatin loop anchor interactions and associated gene transcription during myogenic differentiation. This imaging system offers a novel means of directly observing cis-element interactions and the corresponding gene transcription in living primary cells, thus providing real-time imaging for comprehensive mechanistic investigations of dynamic enhancer-promoter or enhancer-enhancer interactions in regulating transcription activation within living cells.}, }
@article {pmid39747875, year = {2025}, author = {Raghavan, R and Friedrich, MJ and King, I and Chau-Duy-Tam Vo, S and Strebinger, D and Lash, B and Kilian, M and Platten, M and Macrae, RK and Song, Y and Nivon, L and Zhang, F}, title = {Rational engineering of minimally immunogenic nucleases for gene therapy.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {105}, pmid = {39747875}, issn = {2041-1723}, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; Animals ; CRISPR-Associated Protein 9/metabolism/genetics ; Protein Engineering/methods ; Proprotein Convertase 9/immunology/genetics/metabolism ; Mice ; HEK293 Cells ; T-Lymphocytes, Cytotoxic/immunology ; Epitopes/immunology ; Endonucleases/metabolism/genetics ; Female ; }, abstract = {Genome editing using CRISPR-Cas systems is a promising avenue for the treatment of genetic diseases. However, cellular and humoral immunogenicity of genome editing tools, which originate from bacteria, complicates their clinical use. Here we report reduced immunogenicity (Red)(i)-variants of two clinically relevant nucleases, SaCas9 and AsCas12a. Through MHC-associated peptide proteomics (MAPPs) analysis, we identify putative immunogenic epitopes on each nuclease. Using computational modeling, we rationally design these proteins to evade the immune response. SaCas9 and AsCas12a Redi variants are substantially less recognized by adaptive immune components, including reduced binding affinity to MHC molecules and attenuated generation of cytotoxic T cell responses, yet maintain wild-type levels of activity and specificity. In vivo editing of PCSK9 with SaCas9.Redi.1 is comparable in efficiency to wild-type SaCas9, but significantly reduces undesired immune responses. This demonstrates the utility of this approach in engineering proteins to evade immune detection.}, }
@article {pmid39747848, year = {2025}, author = {Escobar, H and Di Francescantonio, S and Smirnova, J and Graf, R and Müthel, S and Marg, A and Zhogov, A and Krishna, S and Metzler, E and Petkova, M and Daumke, O and Kühn, R and Spuler, S}, title = {Gene-editing in patient and humanized-mice primary muscle stem cells rescues dysferlin expression in dysferlin-deficient muscular dystrophy.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {120}, pmid = {39747848}, issn = {2041-1723}, mesh = {*Dysferlin/genetics/metabolism ; Animals ; Humans ; *Gene Editing/methods ; Mice ; *Muscle, Skeletal/metabolism ; Disease Models, Animal ; Frameshift Mutation ; CRISPR-Cas Systems ; Muscular Dystrophies/therapy/genetics/metabolism ; Exons/genetics ; Stem Cells/metabolism ; Female ; Membrane Proteins/genetics/metabolism ; Male ; Muscular Dystrophies, Limb-Girdle/therapy/genetics/metabolism ; Genetic Therapy/methods ; }, abstract = {Dystrophy-associated fer-1-like protein (dysferlin) conducts plasma membrane repair. Mutations in the DYSF gene cause a panoply of genetic muscular dystrophies. We targeted a frequent loss-of-function, DYSF exon 44, founder frameshift mutation with mRNA-mediated delivery of SpCas9 in combination with a mutation-specific sgRNA to primary muscle stem cells from two homozygous patients. We observed a consistent >60% exon 44 re-framing, rescuing a full-length and functional dysferlin protein. A new mouse model harboring a humanized Dysf exon 44 with the founder mutation, hEx44mut, recapitulates the patients' phenotype and an identical re-framing outcome in primary muscle stem cells. Finally, gene-edited murine primary muscle stem-cells are able to regenerate muscle and rescue dysferlin when transplanted back into hEx44mut hosts. These findings are the first to show that a CRISPR-mediated therapy can ameliorate dysferlin deficiency. We suggest that gene-edited primary muscle stem cells could exhibit utility, not only in treating dysferlin deficiency syndromes, but also perhaps other forms of muscular dystrophy.}, }
@article {pmid39747609, year = {2025}, author = {Motooka, Y and Tanaka, H and Maeda, Y and Katabuchi, M and Mashimo, T and Toyokuni, S}, title = {Heterozygous mutation in BRCA2 induces accelerated age-dependent decline in sperm quality with male subfertility in rats.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {447}, pmid = {39747609}, issn = {2045-2322}, support = {JP23K08883//Japan Society for the Promotion of Science/ ; JP19H0546//Japan Society for the Promotion of Science/ ; JP16H06276 [AdAMS (Aa210038)]//Japan Society for the Promotion of Science/ ; JPMJCR19H4//JST CREST/ ; }, mesh = {Male ; Animals ; *BRCA2 Protein/genetics ; Rats ; *Spermatozoa/metabolism ; *Infertility, Male/genetics ; Heterozygote ; Mutation ; Exons/genetics ; DNA Breaks, Double-Stranded ; Aging/genetics ; Humans ; Apoptosis/genetics ; Testis/pathology/metabolism ; CRISPR-Cas Systems ; }, abstract = {Tumor suppressor BRCA2 executes homologous recombination to repair DNA double-strand breaks in collaboration with RAD51, involving exon 11 and 27. Exon 11 constitutes a region where pathogenic variants (PVs) accumulate, and mutations in this region are known to contribute to carcinogenesis. However, the impact of the heterozygous PVs of BRCA2 exon 11 on the life quality beyond cancer risk, including male fertility, remains unclear. Here, we established a rat model with a frameshift on the seventh BRC repeat in Brca2 exon 11 (Brca2[+/p.T1942fs]), which is homologous to human BRCA2[+/p.T1974fs], using CRISPR/Cas9 system. Our analyses revealed that the heterozygous rats with the PV in the BRCA2 exon 11 showed increased DNA double-strand breaks and apoptosis in spermatogonia and spermatocytes, accelerated testicular germ cell loss, and deterioration in sperm quality according with aging, ultimately resulting in early male reproductive dysfunction. Of note, these alterations in testes and sperm, including DNA fragmentation in spermatozoa, were observed from completion of sexual maturation. The present findings suggest that it is crucial to consider not only cancer risk but also potential declines in reproductive capacity in men carrying BRCA2 exon 11 PVs. Further investigation is warranted to determine whether similar traits appear in humans.}, }
@article {pmid39747596, year = {2025}, author = {Glaus, AN and Brechet, M and Swinnen, G and Lebeigle, L and Iwaszkiewicz, J and Ambrosini, G and Julca, I and Zhang, J and Roberts, R and Iseli, C and Guex, N and Jiménez-Gómez, J and Glover, N and Martin, GB and Strickler, S and Soyk, S}, title = {Repairing a deleterious domestication variant in a floral regulator gene of tomato by base editing.}, journal = {Nature genetics}, volume = {57}, number = {1}, pages = {231-241}, pmid = {39747596}, issn = {1546-1718}, support = {802008//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 310030_212218//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; PCEFP3_181238//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; IOS-1546625//National Science Foundation (NSF)/ ; }, mesh = {*Solanum lycopersicum/genetics/growth &amp; development ; *Gene Editing/methods ; *Domestication ; *Plant Proteins/genetics ; Mutation ; *Flowers/genetics ; Transcription Factors/genetics ; Gene Expression Regulation, Plant ; Genome, Plant ; CRISPR-Cas Systems ; Phenotype ; }, abstract = {Crop genomes accumulate deleterious mutations-a phenomenon known as the cost of domestication. Precision genome editing has been proposed to eliminate such potentially harmful mutations; however, experimental demonstration is lacking. Here we identified a deleterious mutation in the tomato transcription factor SUPPRESSOR OF SP2 (SSP2), which became prevalent in the domesticated germplasm and diminished DNA binding to genome-wide targets. We found that the action of SSP2 is partially redundant with that of its paralog SSP in regulating shoot and inflorescence architecture. However, redundancy was compromised during tomato domestication and lost completely in the closely related species Physalis grisea, in which a single ortholog regulates shoot branching. We applied base editing to directly repair the deleterious mutation in cultivated tomato and obtained plants with compact growth that provide an early fruit yield. Our work shows how deleterious variants have sensitized modern genotypes for phenotypic tuning and illustrates how repairing deleterious mutations with genome editing may allow predictable crop improvement.}, }
@article {pmid39747369, year = {2025}, author = {Patil, S and Siji, A and Mallur, D and Kruthika, BS and Gheewalla, N and Karve, S and Kavathekar, M and Tarai, B and Naik, M and Hegde, V and Rangineni, J and Gupta, V and Chandru, V and Pradeep, BE and Arora, R}, title = {PathCrisp: an innovative molecular diagnostic tool for early detection of NDM-resistant infections.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {490}, pmid = {39747369}, issn = {2045-2322}, support = {2021 HTH 018//Rockefeller Foundation Grant/ ; 2021 HTH 018//Rockefeller Foundation Grant/ ; }, mesh = {*beta-Lactamases/genetics ; Humans ; *Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Enterobacteriaceae Infections/diagnosis/microbiology ; Carbapenem-Resistant Enterobacteriaceae/genetics/isolation &amp; purification ; Sensitivity and Specificity ; Early Diagnosis ; Anti-Bacterial Agents/pharmacology/therapeutic use ; Polymerase Chain Reaction/methods ; Microbial Sensitivity Tests ; CRISPR-Cas Systems ; }, abstract = {The rapid and early detection of infections and antibiotic resistance markers is a critical challenge in healthcare. Currently, most commercial diagnostic tools for analyzing antimicrobial resistance patterns of pathogens require elaborate culture-based testing. Our study aims to develop a rapid, accurate molecular detection system that can be used directly from culture, thereby introducing molecular testing in conjunction with culture tests to reduce turnaround time and guide therapy. PathCrisp assay, a combination of loop-mediated isothermal amplification and CRISPR-based detection, maintained at a single temperature, was designed and tested on clinical isolates. The specificity and sensitivity of the assay was analyzed, post which the assay was compared with the polymerase chain reaction (PCR) method to detect the New Delhi metallo-beta-lactamase (NDM) gene in carbapenem-resistant enterobacteriaceae clinical samples. Our PathCrisp assay demonstrated the ability to detect as few as 700 copies of the NDM gene from clinical isolates. Our assay demonstrated 100% concordance with the PCR-Sanger sequencing method, more commonly used. Additionally, the lack of the need for a kit-based DNA purification step, rather a crude extraction via heating, enables the direct use of culture samples. The PathCrisp assay is precise, specific and rapid, providing results in approximately 2 h, and operates at a constant temperature, reducing the need for complex equipment handling. In the near future, we hope that this assay can be further optimized and designed as a point-of-care test kit, facilitating its use in various healthcare settings and aiding clinicians in the choice of antibiotics for therapy.}, }
@article {pmid39747289, year = {2025}, author = {Frusteri Chiacchiera, A and Casanova, M and Bellato, M and Piazza, A and Migliavacca, R and Batt, G and Magni, P and Pasotti, L}, title = {Harnessing CRISPR interference to resensitize laboratory strains and clinical isolates to last resort antibiotics.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {261}, pmid = {39747289}, issn = {2045-2322}, support = {1139857//Regione Lombardia/ ; 20-PAMR-0010 (Seq2DiAg)//Agence Nationale de la Recherche/ ; 59576 (2021)//Fondazione Cassa di Risparmio di Padova e Rovigo/ ; INCEPTION//Institut Pasteur/ ; }, mesh = {*Anti-Bacterial Agents/pharmacology/therapeutic use ; *Escherichia coli/genetics/drug effects ; *CRISPR-Cas Systems ; Humans ; Drug Resistance, Bacterial/genetics ; Microbial Sensitivity Tests ; Mutation ; }, abstract = {The global race against antimicrobial resistance requires novel antimicrobials that are not only effective in killing specific bacteria, but also minimize the emergence of new resistances. Recently, CRISPR/Cas-based antimicrobials were proposed to address killing specificity with encouraging results. However, the emergence of target sequence mutations triggered by Cas-cleavage was identified as an escape strategy, posing the risk of generating new antibiotic-resistance gene (ARG) variants. Here, we evaluated an antibiotic re-sensitization strategy based on CRISPR interference (CRISPRi), which inhibits gene expression without damaging target DNA. The resistance to four antibiotics, including last resort drugs, was significantly reduced by individual and multi-gene targeting of ARGs in low- to high-copy numbers in recombinant E. coli. Escaper analysis confirmed the absence of mutations in target sequence, corroborating the harmless role of CRISPRi in the selection of new resistances. E. coli clinical isolates carrying ARGs of severe clinical concern were then used to assess the robustness of CRISPRi under different growth conditions. Meropenem, colistin and cefotaxime susceptibility was successfully increased in terms of MIC (up to > 4-fold) and growth delay (up to 11 h) in a medium-dependent fashion. ARG repression also worked in a pathogenic strain grown in human urine, as a demonstration of CRISPRi-mediated re-sensitization in host-mimicking media. This study laid the foundations for further leveraging CRISPRi as antimicrobial agent or research tool to selectively repress ARGs and investigate resistance mechanisms.}, }
@article {pmid39747101, year = {2025}, author = {Lin, JF and Liu, ZX and Chen, DL and Huang, RZ and Cao, F and Yu, K and Li, T and Mo, HY and Sheng, H and Liang, ZB and Liao, K and Han, Y and Li, SS and Zeng, ZL and Gao, S and Ju, HQ and Xu, RH}, title = {Nucleus-translocated GCLM promotes chemoresistance in colorectal cancer through a moonlighting function.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {263}, pmid = {39747101}, issn = {2041-1723}, support = {82303306//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82373376//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {Humans ; *Colorectal Neoplasms/drug therapy/genetics/metabolism/pathology ; *Drug Resistance, Neoplasm/genetics ; *NF-kappa B/metabolism ; *Cell Nucleus/metabolism ; Cell Line, Tumor ; *Glutamate-Cysteine Ligase/metabolism/genetics ; Phosphorylation ; Animals ; Antineoplastic Agents/therapeutic use/pharmacology ; Mice ; Gene Expression Regulation, Neoplastic ; p38 Mitogen-Activated Protein Kinases/metabolism ; Female ; CRISPR-Cas Systems ; Male ; Active Transport, Cell Nucleus ; Mice, Nude ; }, abstract = {Metabolic enzymes perform moonlighting functions during tumor progression, including the modulation of chemoresistance. However, the underlying mechanisms of these functions remain elusive. Here, utilizing a metabolic clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 knockout library screen, we observe that the loss of glutamate-cysteine ligase modifier subunit (GCLM), a rate-limiting enzyme in glutathione biosynthesis, noticeably increases the sensitivity of colorectal cancer (CRC) cells to platinum-based chemotherapy. Mechanistically, we unveil a noncanonical mechanism through which nuclear GCLM competitively interacts with NF-kappa-B (NF-κB)-repressing factor (NKRF), to promote NF-κB activity and facilitate chemoresistance. In response to platinum drug treatment, GCLM is phosphorylated by P38 MAPK at T17, resulting in its recognition by importin a5 and subsequent nuclear translocation. Furthermore, elevated expression of nuclear GCLM and phospho-GCLM correlate with an unfavorable prognosis and poor benefit from standard chemotherapy. Overall, our work highlights the essential nonmetabolic role and posttranslational regulatory mechanism of GCLM in enhancing NF-κB activity and subsequent chemoresistance.}, }
@article {pmid39747086, year = {2025}, author = {Albuquerque-Wendt, A and McCoy, C and Neish, R and Dobramysl, U and Alagöz, &#xc7; and Beneke, T and Cowley, SA and Crouch, K and Wheeler, RJ and Mottram, JC and Gluenz, E}, title = {TransLeish: Identification of membrane transporters essential for survival of intracellular Leishmania parasites in a systematic gene deletion screen.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {299}, pmid = {39747086}, issn = {2041-1723}, support = {MR/R000859/2/MRC_/Medical Research Council/United Kingdom ; 221944/A/20/Z//Wellcome Trust (Wellcome)/ ; MR/R000859/1/MRC_/Medical Research Council/United Kingdom ; MR/V000446/1/MRC_/Medical Research Council/United Kingdom ; trans-LEISHion-EU FP7, No. 798736//EC | EC Seventh Framework Programm | FP7 People: Marie-Curie Actions (FP7-PEOPLE - Specific Programme "People" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013))/ ; 211075/Z/18/Z//Wellcome Trust (Wellcome)/ ; 104111/Z/14/Z//Wellcome Trust (Wellcome)/ ; /WT_/Wellcome Trust/United Kingdom ; 15/16_MSD_836338//RCUK | Medical Research Council (MRC)/ ; 101064428 - LeishMOM//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 Marie Sk&#x142;odowska-Curie Actions (H2020 Excellent Science - Marie Sk&#x142;odowska-Curie Actions)/ ; 200807/Z/16/Z//Wellcome Trust (Wellcome)/ ; UF160661//Royal Society/ ; ALTF 727-2021//European Molecular Biology Organization (EMBO)/ ; }, mesh = {Animals ; Mice ; *Gene Deletion ; *Macrophages/parasitology/metabolism ; *CRISPR-Cas Systems ; *Protozoan Proteins/genetics/metabolism ; *Membrane Transport Proteins/genetics/metabolism ; Leishmania mexicana/genetics/metabolism ; Vacuolar Proton-Translocating ATPases/genetics/metabolism ; Female ; Mice, Inbred BALB C ; }, abstract = {For the protozoan parasite Leishmania, completion of its life cycle requires sequential adaptation of cellular physiology and nutrient scavenging mechanisms to the different environments of a sand fly alimentary tract and the acidic mammalian host cell phagolysosome. Transmembrane transporters are the gatekeepers of intracellular environments, controlling the flux of solutes and ions across membranes. To discover which transporters are vital for survival as intracellular amastigote forms, we carried out a systematic loss-of-function screen of the L. mexicana transportome. A total of 312 protein components of small molecule carriers, ion channels and pumps were identified and targeted in a CRISPR-Cas9 gene deletion screen in the promastigote form, yielding 188 viable null mutants. Forty transporter deletions caused significant loss of fitness in macrophage and mouse infections. A striking example is the Vacuolar H[+] ATPase (V-ATPase), which, unexpectedly, was dispensable for promastigote growth in vitro but essential for survival of the disease-causing amastigotes.}, }
@article {pmid39747083, year = {2025}, author = {Fei, J and Zhao, D and Pang, C and Li, J and Li, S and Qiao, W and Tan, J and Bi, C and Zhang, X}, title = {Mismatch prime editing gRNA increased efficiency and reduced indels.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {139}, pmid = {39747083}, issn = {2041-1723}, support = {32171449//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32271483//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32225031//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2022177//Youth Innovation Promotion Association of the Chinese Academy of Sciences (Youth Innovation Promotion Association CAS)/ ; }, mesh = {*Gene Editing/methods ; Humans ; *INDEL Mutation ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; HEK293 Cells ; Base Pair Mismatch ; }, abstract = {Prime editing enables precise and efficient genome editing, but its efficacy is hindered by pegRNA's 3' extension, forming secondary structures due to high complementarity with the protospacer. The continuous presence of the prime editing system also leads to unintended indel formation, raising safety concerns for therapeutic applications. To address these challenges, we develop a mismatched pegRNA (mpegRNA) strategy that introduces mismatched bases into the pegRNA protospacer, reducing complementarity and secondary structure formation, and preventing sustained activity. Our findings show that mpegRNA enhances editing efficiency by up to 2.3 times and reduces indel levels by 76.5% without compromising performance. Combining mpegRNA with epegRNA further increases efficiency up to 14-fold, or 2.4-fold in PE4max/PE5max systems, underscoring its potential in research and therapy. AlphaFold 3 analysis suggests that the optimal mpegRNA structure contributes significantly to improved editing outcomes. Overall, mpegRNA advances prime editing technology, improving efficiency while reducing indels.}, }
@article {pmid39747012, year = {2025}, author = {Tolosana, I and Willis, K and Gribble, M and Phillimore, L and Burt, A and Nolan, T and Crisanti, A and Bernardini, F}, title = {A Y chromosome-linked genome editor for efficient population suppression in the malaria vector Anopheles gambiae.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {206}, pmid = {39747012}, issn = {2041-1723}, support = {INV-006610/GATES/Gates Foundation/United States ; INV006610 "Target Malaria Phase II"//Bill and Melinda Gates Foundation (Bill &amp; Melinda Gates Foundation)/ ; }, mesh = {*Anopheles/genetics ; Animals ; Female ; *Y Chromosome/genetics ; Male ; *Mosquito Vectors/genetics ; *Malaria/prevention &amp; control/transmission ; *CRISPR-Cas Systems ; *Mosquito Control/methods ; *Gene Editing/methods ; Genome, Insect ; }, abstract = {Genetic control - the deliberate introduction of genetic traits to control a pest or vector population - offers a powerful tool to augment conventional mosquito control tools that have been successful in reducing malaria burden but that are compromised by a range of operational challenges. Self-sustaining genetic control strategies have shown great potential in laboratory settings, but hesitancy due to their invasive and persistent nature may delay their implementation. Here, instead, we describe a self-limiting strategy, designed to have geographically and temporally restricted effect, based on a Y chromosome-linked genome editor (YLE). The YLE comprises a CRISPR-Cas9 construct that is always inherited by males yet generates an autosomal dominant mutation that is transmitted to over 90% of the offspring and results in female-specific sterility. To our knowledge, our system represents a pioneering approach in the engineering of the Y chromosome to generate a genetic control strain for mosquitoes. Mathematical modelling shows that this YLE technology is up to seven times more efficient for population suppression than optimal versions of other self-limiting strategies, such as the widely used Sterile Insect Technique or the Release of Insects carrying a Dominant Lethal gene.}, }
@article {pmid39746849, year = {2025}, author = {Paialunga, E and Bagheri, N and Rossetti, M and Fabiani, L and Micheli, L and Chamorro-Garcia, A and Porchetta, A}, title = {Leveraging Synthetic Antibody-DNA Conjugates to Expand the CRISPR-Cas12a Biosensing Toolbox.}, journal = {ACS synthetic biology}, volume = {14}, number = {1}, pages = {171-178}, doi = {10.1021/acssynbio.4c00541}, pmid = {39746849}, issn = {2161-5063}, mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; SARS-CoV-2/isolation &amp; purification/immunology ; Humans ; *DNA/chemistry/metabolism/genetics ; Spike Glycoprotein, Coronavirus/analysis/immunology ; COVID-19/diagnosis/virology ; *Endodeoxyribonucleases/metabolism/genetics ; Saliva/virology ; *Antibodies/chemistry ; Limit of Detection ; Bacterial Proteins/metabolism/genetics ; CRISPR-Associated Proteins/metabolism ; Enzyme-Linked Immunosorbent Assay ; Immunoassay/methods ; }, abstract = {We report here the use of antibody-DNA conjugates (Ab-DNA) to activate the collateral cleavage activity of the CRISPR-Cas12a enzyme. Our findings demonstrate that Ab-DNA conjugates effectively trigger the collateral cleavage activity of CRISPR-Cas12a, enabling the transduction of antibody-mediated recognition events into fluorescence outputs. We developed two different immunoassays using an Ab-DNA as activator of Cas12a: the CRISPR-based immunosensing assay (CIA) for detecting SARS-CoV-2 spike S protein, which shows superior sensitivity compared with the traditional enzyme-linked immunosorbent assay (ELISA), and the CRISPR-based immunomagnetic assay (CIMA). Notably, CIMA successfully detected the SARS-CoV-2 spike S protein in undiluted saliva with a limit of detection (LOD) of 890 pM in a 2 h assay. Our results underscore the benefits of integrating Cas12a-based signal amplification with antibody detection methods. The potential of Ab-DNA conjugates, combined with CRISPR technology, offers a promising alternative to conventional enzymes used in immunoassays and could facilitate the development of versatile CRISPR analytical platforms for the detection of non-nucleic acid targets.}, }
@article {pmid39745853, year = {2025}, author = {Wei, R and Yu, Z and Ding, L and Lu, Z and Yao, K and Zhang, H and Huang, B and He, M and Ma, L}, title = {Improved split prime editors enable efficient in vivo genome editing.}, journal = {Cell reports}, volume = {44}, number = {1}, pages = {115144}, doi = {10.1016/j.celrep.2024.115144}, pmid = {39745853}, issn = {2211-1247}, mesh = {*Gene Editing/methods ; Animals ; Dependovirus/genetics ; Humans ; Mice ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; CRISPR-Associated Protein 9/metabolism ; }, abstract = {Efficient prime editor (PE) delivery in vivo is critical for realizing its full potential in disease modeling and therapeutic correction. Although PE has been divided into two halves and delivered using dual adeno-associated viruses (AAVs), the editing efficiency at different gene loci varies among split sites. Furthermore, efficient split sites within Cas9 nickase (Cas9n) are limited. Here, we verified that 1115 (Asn) is an efficient split site when delivering PEs by dual AAVs. Additionally, we utilized a feature in which reverse transcriptase could be detached from the Cas9n and designed split sites in the first half of Cas9n. We found that split-PE-367 enabled high editing efficiency with Rma intein. To test the editing efficiency in vivo, split-ePE3-367 was packaged in AAV9 and achieved 17.5% precise editing in mice. Our findings establish an alternative split-PE architecture that enables robust editing efficiency, facilitating potential utility in disease modeling and correction.}, }
@article {pmid39745650, year = {2025}, author = {Salvador-Martínez, I}, title = {Computational Methods for Lineage Reconstruction.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2886}, number = {}, pages = {355-373}, pmid = {39745650}, issn = {1940-6029}, mesh = {*Software ; *Cell Lineage/genetics ; *Algorithms ; *Phylogeny ; *Computational Biology/methods ; Animals ; CRISPR-Cas Systems ; Humans ; }, abstract = {The recent development of genetic lineage recorders, designed to register the genealogical history of cells using induced somatic mutations, has opened the possibility of reconstructing complete animal cell lineages. To reconstruct a cell lineage tree from a molecular recorder, it is crucial to use an appropriate reconstruction algorithm. Current approaches include algorithms specifically designed for cell lineage reconstruction and the repurposing of phylogenetic algorithms. These methods have, however, the same objective: to uncover the hierarchical relationships between cells and the sequence of cell divisions that have occurred during development. In this chapter, I will use the phylogenetic software FastTree to reconstruct a lineage tree, in a step-by-step manner, using data from a simulated CRISPR-Cas9 recorder. To ensure reproducibility, the code is presented as a Jupyter Notebook, available (together with the necessary input files) at https://github.com/irepansalvador/lineage_reconstruction_chapter .}, }
@article {pmid39745649, year = {2025}, author = {Espinosa-Medina, I}, title = {Design and Generation of TEMPO Reagents for Sequential Labeling and Manipulation of Vertebrate Cell Lineages.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2886}, number = {}, pages = {327-353}, pmid = {39745649}, issn = {1940-6029}, mesh = {Animals ; *Zebrafish ; Mice ; *Cell Lineage ; CRISPR-Cas Systems ; Staining and Labeling/methods ; Luminescent Proteins/genetics/metabolism ; }, abstract = {During development, cells undergo a sequence of specification events to form functional tissues and organs. To investigate complex tissue development, it is crucial to visualize how cell lineages emerge and to be able to manipulate regulatory factors with temporal control. We recently developed TEMPO (Temporal Encoding and Manipulation in a Predefined Order), a genetic tool to label with different colors and genetically manipulate consecutive cell generations in vertebrates. TEMPO relies on CRISPR to activate a cascade of fluorescent proteins which can be imaged in vivo. Here, we explain the steps to design, generate, and express TEMPO constructs in zebrafish and mice.}, }
@article {pmid39745647, year = {2025}, author = {Raj, B}, title = {Single-Cell Profiling of Lineages and Cell Types in the Vertebrate Brain.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2886}, number = {}, pages = {299-310}, pmid = {39745647}, issn = {1940-6029}, mesh = {Animals ; *Single-Cell Analysis/methods ; *Brain/cytology/metabolism ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; *Cell Lineage/genetics ; *Gene Editing/methods ; Transcriptome ; Gene Expression Profiling/methods ; }, abstract = {CRISPR-Cas tools have recently been adapted for cell lineage tracing during development. Combined with single-cell RNA sequencing, these methods enable scalable lineage tracing with single-cell resolution. Here, I describe, scGESTALTv2, which combines cumulative CRISPR-Cas9 editing of a lineage barcode array with transcriptional profiling via droplet-based single-cell RNA sequencing (scRNA-seq). The technique is applied in developing zebrafish brains to generate mutations in the barcode array during development. The recorded lineages along with cellular transcriptomes are then extracted via scRNA-seq to define cell relationships among thousands of profiled brain cells and dozens of cell types.}, }
@article {pmid39745646, year = {2025}, author = {Bowling, S and Camargo, FD}, title = {CARLIN: A Mouse Line for Simultaneous Readout of Lineage Histories and Gene Expression.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2886}, number = {}, pages = {281-298}, pmid = {39745646}, issn = {1940-6029}, mesh = {Animals ; Mice ; *Cell Lineage/genetics ; *DNA Barcoding, Taxonomic/methods ; *High-Throughput Nucleotide Sequencing/methods ; *Single-Cell Analysis/methods ; CRISPR-Cas Systems ; Gene Editing/methods ; Gene Expression/genetics ; }, abstract = {The CRISPR-activated repair lineage tracing (CARLIN) mouse line uses DNA barcoding to enable high-resolution tracing of cell lineages in vivo (Bowling et al, Cell 181, 1410-1422.e27, 2020). CARLIN mice contain expressed barcodes that allow simultaneous interrogation of lineage and gene expression information from single cells. Furthermore, barcode editing is fully inducible, resulting in cell lineage labeling that can be performed at any time point in development or adulthood. This chapter details the protocols followed for maintaining CARLIN mice, inducing barcoding, and amplifying the CARLIN barcode from DNA, RNA, and single-cell RNA-sequencing libraries for next-generation sequencing.}, }
@article {pmid39745643, year = {2025}, author = {Baron, CS and Alemany, A}, title = {Paired Single-Cell Transcriptome and DNA Barcode Detection in Zebrafish Using ScarTrace.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2886}, number = {}, pages = {221-241}, pmid = {39745643}, issn = {1940-6029}, mesh = {Animals ; *Zebrafish/genetics ; *Single-Cell Analysis/methods ; *DNA Barcoding, Taxonomic/methods ; *CRISPR-Cas Systems ; *Transcriptome/genetics ; Gene Expression Profiling/methods ; Embryo, Nonmammalian/metabolism ; }, abstract = {ScarTrace is a CRISPR/Cas9-based genetic lineage tracing method that allows for uniquely barcoding the DNA of single cells at a target GFP sequence during developing zebrafish embryos. Single cells from barcoded adult zebrafish can be isolated from various tissues (e.g., marrow, brain, eyes, fins), and their transcriptome and barcode sequences are captured by single-cell cDNA amplification and genomic DNA nested PCR, respectively. Computationally, cell type and barcode identification permit clone tracing and lineage tree reconstruction of tissues to unravel fate decisions during embryogenesis.}, }
@article {pmid39745637, year = {2025}, author = {Gentile, E and Maynard, A and He, Z and Treutlein, B}, title = {Lineage Recording in Human Brain Organoids with iTracer.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2886}, number = {}, pages = {85-101}, pmid = {39745637}, issn = {1940-6029}, mesh = {Humans ; *Organoids/cytology/metabolism ; *Brain/cytology ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Cell Lineage/genetics ; *Single-Cell Analysis/methods ; *CRISPR-Cas Systems ; Transcriptome ; Cell Differentiation ; }, abstract = {Induced pluripotent stem cell (iPSC)-derived organoids provide models to study human organ development. Single-cell transcriptomics enables highly resolved descriptions of cell states within these systems; however, approaches are needed to directly determine the lineage relationship between cells. Here we provide a detailed protocol (Fig. 1) for the application of iTracer (He Z, Maynard A, Jain A, et al., Nat Methods 19:90-99, 2022), a recently published lineage recorder that combines reporter barcodes with inducible CRISPR-Cas9 scarring and is compatible with single-cell and spatial transcriptomics. iTracer is used to explore clonality and lineage dynamics during brain organoid development. More broadly, iTracer can be adapted to any iPSC-derived culture system to dissect lineage dynamics during normal or perturbed development.}, }
@article {pmid39745602, year = {2025}, author = {Hajirnis, N}, title = {Re-arranging the Cis-regulatory Modules of Hox Complex in Drosophila via FLP-FRT and CRISPR/Cas9.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2889}, number = {}, pages = {11-24}, pmid = {39745602}, issn = {1940-6029}, mesh = {Animals ; *CRISPR-Cas Systems ; *Drosophila melanogaster/genetics/growth &amp; development ; Drosophila Proteins/genetics/metabolism ; Homeodomain Proteins/genetics/metabolism ; Genes, Homeobox/genetics ; Gene Editing/methods ; DNA Nucleotidyltransferases ; }, abstract = {FLP-FRT, a well-established technique for genome manipulation, and the revolutionary CRISPR/Cas9, known for its targeted indels, are combined in a novel approach. This unique method is applied to the Hox genes in the Drosophila melanogaster bithorax complex, which are closely located to the cis-regulatory modules that define their spatial-temporal regulation. The number and position of these genes are directly correlated to their expression pattern. This chapter unveils the exciting potential of this combinatorial use of FLP-FRT and CRISPR-Cas9 to rearrange the cis-regulatory modules of the Hox complex in Drosophila melanogaster.}, }
@article {pmid39745317, year = {2025}, author = {Hou, M and Yang, X and Gong, L and Shen, X}, title = {Surveillance of antimicrobial resistance using isothermal amplification: a review.}, journal = {Chemical communications (Cambridge, England)}, volume = {61}, number = {9}, pages = {1748-1760}, doi = {10.1039/d4cc05488a}, pmid = {39745317}, issn = {1364-548X}, mesh = {*Nucleic Acid Amplification Techniques/methods ; Humans ; *Drug Resistance, Bacterial/genetics ; *Bacteria/drug effects/genetics ; *Anti-Bacterial Agents/pharmacology ; Molecular Diagnostic Techniques ; }, abstract = {The monitoring of antibiotic resistance genes (ARGs) is crucial for understanding the level of antimicrobial resistance and the associated health burden, which in turn is essential for the control and prevention of antimicrobial resistance (AMR). Isothermal amplification, an emerging molecular biology technology, has been widely used for drug resistance detection. Furthermore, its compatibility with a range of technologies enables high-specificity, high-throughput, and portable and integrated detection in drug resistance, particularly in resource-limited areas. However, to date, reviews involved in isothermal amplification all concentrate on its technological advancements and its application in nucleic acid point-of-care testing. Few reviews have been published that focus specifically on the application of isothermal amplification in the detection of drug resistance. This review summarizes the detection principles of different isothermal amplification techniques and discusses their strengths and weaknesses as well as the applicable scenarios for drug resistance detection. It also summarizes advances in the application, challenges and prospects of isothermal amplification technologies in conjunction with different methods such as base mismatch, CRISPR-Cas, lateral flow immunoassay, sensing and microfluidic technologies for improvement of specificity, throughput and integration for drug resistance detection. It is anticipated that this review will assist scientists in comprehending the evolution of isothermal amplification in the context of drug resistance detection and provide insights into the prospective applications of isothermal amplification for highly integrated and immediate on-site detection of drug resistance.}, }
@article {pmid39744795, year = {2025}, author = {Li, X and Huang, Z and Lau, CH and Li, J and Zou, M and Wu, W and Chen, X and Li, J and Huang, Y and Wang, T and Li, Y and Xu, M and Huang, X and Zhu, H and Yang, C}, title = {One-pot isothermal CRISPR/Dx system for specific and sensitive detection of microRNA.}, journal = {Analytical methods : advancing methods and applications}, volume = {17}, number = {4}, pages = {823-833}, doi = {10.1039/d4ay01695e}, pmid = {39744795}, issn = {1759-9679}, mesh = {*MicroRNAs/genetics/analysis ; Humans ; *CRISPR-Cas Systems/genetics ; *Pancreatic Neoplasms/genetics/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Cell Line, Tumor ; }, abstract = {MicroRNA (miRNA) is a promising biomarker for the early diagnosis of pancreatic cancer. To enable sensitive and reliable miRNA detection, we have developed a one-pot isothermal CRISPR/Dx detection system by combining rolling circle amplification (RCA) and CRISPR/Cas12a. RCA and CRISPR/Cas12a reactions are carried out in a single closed tube, bypassing the transferring step. We demonstrate the feasibility of our one-pot CRISPR/Dx system in detecting pancreatic cancer by targeting miR-25, miR-191, miR-205, and miR-1246. When applied to fluorescence- and lateral flow strip paper-based detection platforms, our one-pot CRISPR/Dx system detects synthetic miR-25 at a LOD of 6.60 fM and 500 fM, respectively. It has high targeting specificity, as shown by its ability to discriminate miR-25 with a single-base mutation and highly homologous miRNA species. It is also successfully generalized to detect other pancreatic cancer-associated miRNAs, including miR-191, miR-205, and miR-1246. Importantly, our one-pot CRISPR/Dx system enables specific and sensitive detection of endogenous miR-25 in the human pancreatic cancer cell line PANC-1. We have successfully developed a one-pot isothermal CRISPR/Dx system for detecting miRNA with high specificity and sensitivity. It is highly flexible and economical, as a common crRNA can detect different miRNAs and only requires minor modifications to the locking padlock probe. Therefore, it can potentially be translated into clinical settings and POCT for the diagnosis of various human cancers.}, }
@article {pmid39744681, year = {2025}, author = {Park, M and Ryu, H and Heo, S and Kim, B and Park, J and Lim, KH and Han, SB and Park, H}, title = {Targeted demethylation of cathepsin D via epigenome editing rescues pathology in Alzheimer's disease mouse model.}, journal = {Theranostics}, volume = {15}, number = {2}, pages = {428-438}, pmid = {39744681}, issn = {1838-7640}, mesh = {Animals ; *Alzheimer Disease/genetics/therapy/metabolism ; *Disease Models, Animal ; Mice ; *Amyloid beta-Peptides/metabolism ; *Cathepsin D/metabolism/genetics ; *Gene Editing/methods ; *Mice, Transgenic ; *Epigenome ; Neurons/metabolism ; Amyloid beta-Protein Precursor/genetics/metabolism ; Humans ; Brain/metabolism/pathology ; Genetic Therapy/methods ; CRISPR-Cas Systems/genetics ; Male ; }, abstract = {Background: Cathepsin D (Ctsd) has emerged as a promising therapeutic target for Alzheimer's disease (AD) due to its role in degrading intracellular amyloid beta (Aβ). Enhancing Ctsd activity could reduce Aβ42 accumulation and restore the Aβ42/40 ratio, offering a potential AD treatment strategy. Methods: This study explored Ctsd demethylation in AD mouse models using dCas9-Tet1-mediated epigenome editing. We identified dCas9-Tet1 as an effective tool for demethylating the endogenous Ctsd gene in primary neurons and in vivo brains. Results: Treatment with Ctsd-targeted dCas9-Tet1 in primary neurons overexpressing mutant APP (mutAPP) reduced Aβ peptide levels and the Aβ42/40 ratio. Additionally, in vivo demethylation of Ctsd via dCas9-Tet1 in 5xFAD mice significantly altered Aβ levels and alleviated cognitive and behavioral deficits. Conclusion: These findings offer valuable insights into developing epigenome editing-based gene therapy strategies for AD.}, }
@article {pmid39743757, year = {2024}, author = {Yu, CH and Lai, KL}, title = {Cure the Incurable: Update of Treatment in Inherited Neuromuscular Disorders.}, journal = {Acta neurologica Taiwanica}, volume = {33(4)}, number = {}, pages = {129-135}, pmid = {39743757}, issn = {1028-768X}, mesh = {Humans ; *Genetic Therapy/methods ; *Neuromuscular Diseases/therapy/genetics ; *CRISPR-Cas Systems ; }, abstract = {Originally thought to be incurable, huge therapeutic progress has been made in recent years in the field of inherited neuromuscular disorders. Approaches aiming to rescue the underlying pathophysiology, i.e. loss-of-function or gain-of-function mutations, have been developed via end-product replacement or gene delivery/modulation, with promising results. In the review, advanced treatment in some of the inherited neuromuscular disorders will be discussed. On the other hand, it has been found more than 1000 genes are responsible for the clinical diversities in this group of diseases, and finding a way which owns the therapeutic potential to various diseases is the optimal goal. The discovery of CRISPR-Cas9 system in the last decade offers such an opportunity and is under rigorous investigation. This important issue will also be discussed. Keywords: CRISPR-Cas9, gene therapy, gene delivery/modulation, inherited neuromuscular disorders, therapeutic advances.}, }
@article {pmid39743295, year = {2024}, author = {Jiang, Y and Pan, Q and Wang, Z and Lu, K and Xia, B and Chen, T}, title = {Efficient genome editing in medaka (Oryzias latipes) using a codon-optimized SaCas9 system.}, journal = {Journal of Zhejiang University. Science. B}, volume = {25}, number = {12}, pages = {1083-1096}, pmid = {39743295}, issn = {1862-1783}, support = {32273127, 31771648 and 31672653//the National Natural Science Foundation of China/ ; ZQ2020003//the Scientific Research Foundation of Jimei University/ ; }, mesh = {Animals ; *Oryzias/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Codon ; RNA, Guide, CRISPR-Cas Systems/genetics ; Monophenol Monooxygenase/genetics ; CRISPR-Associated Protein 9/genetics ; RNA, Transfer/genetics ; Staphylococcus aureus/genetics ; PAX6 Transcription Factor/genetics ; }, abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, belonging to the type II CRISPR/Cas system, is an effective gene-editing tool widely used in different organisms, but the size of Streptococcus pyogenes Cas9 (SpCas9) is quite large (4.3 kb), which is not convenient for vector delivery. In this study, we used a codon-optimized Staphylococcus aureus Cas9 (SaCas9) system to edit the tyrosinase (tyr), oculocutaneous albinism II (oca2), and paired box 6.1 (pax6.1) genes in the fish model medaka(Oryzias latipes), in which the size of SaCas9 (3.3 kb) is much smaller and the necessary protospacer-adjacent motif (PAM) sequence is 5'-NNGRRT-3'. We also used a transfer RNA (tRNA)‍-single-guide RNA (sgRNA) system to express the functional sgRNA by transcription eitherin vivo or in vitro, and the combination of SaCas9 and tRNA-sgRNA was used to edit the tyr gene in the medaka genome. The SaCas9/sgRNA and SaCas9/tRNA-sgRNA systems were shown to edit the medaka genome effectively, while the PAM sequence is an essential part for the efficiency of editing. Besides, tRNA can improve the flexibility of the system by enabling the sgRNA to be controlled by a common promoter such as cytomegalovirus. Moreover, the all-in-one cassette cytomegalovirus (CMV)‍-SaCas9-tRNA-sgRNA-tRNA is functional in medaka gene editing. Taken together, the codon-optimized SaCas9 system provides an alternative and smaller tool to edit the medaka genome and potentially other fish genomes.}, }
@article {pmid39743283, year = {2024}, author = {He, YM and Wu, ZP and He, J and Zhang, W and Zhu, FM}, title = {[Establishment and Application of Efficient Gene Editing Method for Classical HLA-I Molecules].}, journal = {Zhongguo shi yan xue ye xue za zhi}, volume = {32}, number = {6}, pages = {1896-1902}, doi = {10.19746/j.cnki.issn.1009-2137.2024.06.040}, pmid = {39743283}, issn = {1009-2137}, mesh = {Humans ; HEK293 Cells ; *Gene Editing ; *Transfection ; *Hematopoietic Stem Cells ; RNA, Guide, CRISPR-Cas Systems ; beta 2-Microglobulin/genetics ; Histocompatibility Antigens Class I/genetics ; Flow Cytometry ; }, abstract = {OBJECTIVE: To establish an efficient gene editing method of HLA-I gene to prepare HLA-I universal hematopoietic stem cells.<br><br>METHODS: The easyedit small guide RNA(sgRNA) was designed according to the sequences of &#x3b2;2 microglobulin gene and synthesized by GenScript company. RNP complexes were formed by NLS-Cas9-NLS nuclease and Easyedit sgRNA according to different molar ratios (1&#x2236;1~1&#x2236;4). Control group and four transfection groups were performed respectively. HEK-293 cells and CD34[+] hematopoietic stem cells were nucleotransfected with RNP complex by Lonza 4D Nucleofector system. The expression of HLA-I on the surface of HEK-293 cells was detected by flow cytometry after transfection for 72 hours, the cleavage effect was determined by T7E1 enzyme digestion reaction and the presence of nested peak in the DNA sequence was identified by direct sequencing.<br><br>RESULTS: The transfection groups had different levels of HLA-I negative expression cell populations by flow cytometry after transient transfection of HEK-293 cells and CD34[+] hematopoietic stem cells with different molar concentrations of RNP complex for 72 hours. There were nested peaks proximal to the sgRNA PAM sequence in the transfection groups by direct DNA sequencing, indicating that sgRNA had obvious editing effect. In the transfection of HEK-293 cells, the highest proportion of HLA-I negative expression cells was (87.69&#xb1;0.83)% when the molar ratio of NLS-Cas9-NLS nuclease to Easyedit sgRNA was 1&#x2236;4. The cutting efficiency of T7E1 was the highest up to (38&#xb1;2.0)% when the molar ratio was 1&#x2236;3. In the transfection of CD34[+] hematopoietic stem cells, the proportion of HLA-I negative expression cells was (91.56&#xb1;3.39)% when the molar ratio was 1&#x2236;2, and the cutting efficiency of T7E1 was (64&#xb1;8.45)% when the molar ratio was 1&#x2236;1.<br><br>CONCLUSION: This study provides an efficient gene editing method for classical HLA-I molecules, which can effectively silence the expression of class HLA-I molecules on the cell surface, and is suitable for stem cell system with difficult transfection.}, }
@article {pmid39743080, year = {2025}, author = {Hao, J and Chen, L and Zhao, C and Qiao, K and Wang, N and Wang, J and Wang, Z and Ma, Q and Shi, C and Fan, S and Ma, Q}, title = {CRISPR/Cas9-mediated knockout of GhAMS11 and GhMS188 reveals key roles in tapetal development and pollen exine formation in upland cotton.}, journal = {International journal of biological macromolecules}, volume = {293}, number = {}, pages = {139362}, doi = {10.1016/j.ijbiomac.2024.139362}, pmid = {39743080}, issn = {1879-0003}, mesh = {*Gossypium/genetics/growth &amp; development ; *CRISPR-Cas Systems/genetics ; *Pollen/genetics/growth &amp; development ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; Gene Knockout Techniques ; Transcription Factors/genetics/metabolism ; *Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism ; }, abstract = {The ABORTED MICROSPORES (AMS) gene is crucial for tapetal cell development and pollen formation, but its role in Upland cotton (Gossypium hirsutum) has not been previously documented. This study identified GhAMS11 as a key transcription factor, with its high expression specifically observed during the S4-S6 stages of anther development, a critical period for tapetal activity and pollen formation. Subcellular localization confirmed that GhAMS11 was located in the nucleus. CRISPR/Cas9 knockout of GhAMS11 resulted in pollen inviability, with mutants displaying abnormal tapetal development and defective pollen exine formation. TUNEL assays highlighted GhAMS11's involvement in proper tapetal programmed cell death (PCD). Additionally, GhAMS11 was found to activate GhMS188 expression, as demonstrated by dual-luciferase assays and EMSA assays, with their interaction confirmed through LCI assays, yeast two-hybrid assays and GST pull down assays. Deletion of GhMS188 led to pollen sterility, grain collapse, and impaired pollen exine formation. Thus, this research identified the bHLH transcription factor GhAMS11, addressing a gap in AMS gene research in Upland cotton, and elucidated its key regulatory role in pollen development in cooperation with GhMS188.}, }
@article {pmid39742661, year = {2025}, author = {Huang, X and Feng, X and Yan, YH and Xu, D and Wang, K and Zhu, C and Dong, MQ and Huang, X and Guang, S and Chen, X}, title = {Compartmentalized localization of perinuclear proteins within germ granules in C. elegans.}, journal = {Developmental cell}, volume = {60}, number = {8}, pages = {1251-1270.e3}, doi = {10.1016/j.devcel.2024.12.016}, pmid = {39742661}, issn = {1878-1551}, support = {P40 OD010440/OD/NIH HHS/United States ; }, mesh = {Animals ; *Caenorhabditis elegans/metabolism/genetics ; *Caenorhabditis elegans Proteins/metabolism/genetics ; *Germ Cells/metabolism ; *Cytoplasmic Granules/metabolism ; Cell Nucleus/metabolism ; CRISPR-Cas Systems ; }, abstract = {Germ granules, or nuage, are RNA-rich condensates that are often docked on the cytoplasmic surface of germline nuclei. C. elegans perinuclear germ granules are composed of multiple subcompartments, including P granules, Mutator foci, Z granules, SIMR foci, P -bodies, and E granules. Although many perinuclear proteins have been identified, their precise localization within the subcompartments of the germ granule is still unclear. Here, we systematically labeled perinuclear proteins with fluorescent tags via CRISPR-Cas9 technology. Using this nematode strain library, we identified a series of proteins localized in Z or E granules and extended the characterization of the D granule. Finally, we found that the LOTUS domain protein MIP-1/EGGD-1 regulated the multiphase organization of the germ granule. Overall, our work identified the germ-granule architecture and redefined the compartmental localization of perinuclear proteins. Additionally, the library of genetically modified nematode strains will facilitate research on C. elegans germ granules.}, }
@article {pmid39742627, year = {2025}, author = {Wei, S and Zhen, Y and Sun, C and Ma, Y and Li, Q and Wen, L}, title = {Generation of a USP9Y knockout human embryonic stem cell line with CRISPR-Cas9 technology.}, journal = {Stem cell research}, volume = {82}, number = {}, pages = {103646}, doi = {10.1016/j.scr.2024.103646}, pmid = {39742627}, issn = {1876-7753}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; Cell Line ; *Ubiquitin Thiolesterase/genetics/metabolism/deficiency ; Male ; *Gene Knockout Techniques ; Cell Differentiation ; }, abstract = {Human embryonic stem cell (hESC) lines are vital tools for studying gene function, disease modeling, and therapy. We generated a USP9Y knockout hESC line using CRISPR-Cas9 in the male-derived H1 line. Targeted deletion of the USP9Y gene was confirmed via PCR and sequencing. The modified line retained pluripotency markers, exhibited a normal karyotype, and differentiated into all three germ layers. This model provides a valuable platform for studying USP9Y's role in human development and male infertility, offering insights into related disorders and therapeutic potential.}, }
@article {pmid39741289, year = {2024}, author = {Yazdi, ZF and Roshannezhad, S and Sharif, S and Abbaszadegan, MR}, title = {Recent progress in prompt molecular detection of liquid biopsy using Cas enzymes: innovative approaches for cancer diagnosis and analysis.}, journal = {Journal of translational medicine}, volume = {22}, number = {1}, pages = {1173}, pmid = {39741289}, issn = {1479-5876}, mesh = {Humans ; Liquid Biopsy/methods ; *Neoplasms/diagnosis/genetics/pathology ; *Neoplastic Cells, Circulating/pathology/metabolism ; CRISPR-Cas Systems/genetics ; Cell-Free Nucleic Acids/blood ; Circulating Tumor DNA/blood ; }, abstract = {Creating fast, non-invasive, precise, and specific diagnostic tests is crucial for enhancing cancer treatment outcomes. Among diagnostic methods, those relying on nucleic acid detection are highly sensitive and specific. Recent developments in diagnostic technologies, particularly those leveraging Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), are revolutionizing cancer detection, providing accurate and timely results. In clinical oncology, liquid biopsy has become a noninvasive and early-detectable alternative to traditional biopsies over the last two decades. Analyzing the nucleic acid content of liquid biopsy samples, which include Circulating Tumor Cells (CTCs), Circulating Tumor DNA (ctDNA), Circulating Cell-Free RNA (cfRNA), and tumor extracellular vesicles, provides a noninvasive method for cancer detection and monitoring. In this review, we explore how the characteristics of various Cas (CRISPR-associated) enzymes have been utilized in diagnostic assays for cancer liquid biopsy and highlight their main applications of innovative approaches in monitoring, as well as early and rapid detection of cancers.}, }
@article {pmid39741118, year = {2025}, author = {Hashimoto, T and Suenaga, H and Shin-Ya, K}, title = {Application of Cas9-Based Gene Editing to Engineering of Nonribosomal Peptide Synthetases.}, journal = {Chembiochem : a European journal of chemical biology}, volume = {26}, number = {3}, pages = {e202400765}, doi = {10.1002/cbic.202400765}, pmid = {39741118}, issn = {1439-7633}, support = {23H05474//JSPS/ ; 23&#x2005;K17983//JSPS/ ; }, mesh = {*Peptide Synthases/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Protein Engineering ; *CRISPR-Associated Protein 9/metabolism/genetics ; Multigene Family ; }, abstract = {Engineering of nonribosomal peptide synthetases (NRPSs) could transform the production of bioactive natural product derivatives. A number of recent reports have described the engineering of NRPSs without marked reductions in yield. Comparative analysis of evolutionarily related NRPSs can provide insights regarding permissive fusion sites for engineering where recombination may occur during evolutionary processes. Studies involving engineering of NRPSs using these recombination sites showed that they have great potential. Moreover, we highlight recent advances in engineering of NRPSs using CRISPR-associated protein 9 (Cas9)-based gene editing technology. The use of Cas9 facilitates the editing of even larger biosynthetic gene clusters (BGCs) close to or over 100 kb in size by precisely targeting and digesting DNA sequences at specific sites. This technology combined with growing understanding of potential fusion sites from large-scale informatics analyses will accelerate the scalable exploration of engineered NRPS assembly lines to obtain bioactive natural product derivatives in high yields.}, }
@article {pmid39741007, year = {2025}, author = {Lima, BA and Pais, AC and Dupont, J and Dias, P and Custódio, N and Sousa, AB and Carmo-Fonseca, M and Carvalho, C}, title = {Genetic modulation of RNA splicing rescues BRCA2 function in mutant cells.}, journal = {Life science alliance}, volume = {8}, number = {3}, pages = {}, pmid = {39741007}, issn = {2575-1077}, mesh = {Humans ; *BRCA2 Protein/genetics/metabolism ; *RNA Splicing/genetics ; *CRISPR-Cas Systems/genetics ; *Mutation ; Gene Editing/methods ; DNA Repair/genetics ; Nonsense Mediated mRNA Decay/genetics ; RNA, Messenger/genetics/metabolism ; Cell Line, Tumor ; Female ; BRCA1 Protein/genetics/metabolism ; Rad51 Recombinase/genetics/metabolism ; Breast Neoplasms/genetics ; }, abstract = {Variants in the hereditary cancer-associated BRCA1 and BRCA2 genes can alter RNA splicing, producing transcripts that encode internally truncated yet potentially functional proteins. However, few studies have quantitatively analyzed variant-specific splicing isoforms. Here, we investigated cells heterozygous and homozygous for the BRCA2:c.681+5G>C variant. Using droplet digital RT-PCR, we identified two variant-specific mRNA isoforms. The predominant transcript is out-of-frame, contains a premature termination codon, and is degraded via the nonsense-mediated mRNA decay pathway. In addition, we detected a novel minor isoform encoding an internally truncated protein lacking non-essential domains. Homozygous mutant cells expressed low levels of BRCA2 protein and were defective in DNA repair. Using CRISPR-Cas9 gene editing, we induced the production of in-frame transcripts in mutant cells, which resulted in increased protein expression, enhanced RAD51 focus formation, and reduced chromosomal breaks after exposure to genotoxic agents. Our findings highlight the therapeutic potential of splicing modulation to restore BRCA2 function in mutant cells, offering a promising strategy to prevent cancer development.}, }
@article {pmid39740588, year = {2025}, author = {Liu, H and Lv, MM and Li, X and Su, M and Nie, YG and Ying, ZM}, title = {Ligation-recognition triggered RPA-Cas12a cis-cleavage fluorogenic RNA aptamer for one-pot and label-free detection of MicroRNA in breast cancer.}, journal = {Biosensors &amp; bioelectronics}, volume = {272}, number = {}, pages = {117106}, doi = {10.1016/j.bios.2024.117106}, pmid = {39740588}, issn = {1873-4235}, mesh = {*MicroRNAs/genetics/isolation &amp; purification/analysis ; Humans ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry/genetics ; *Breast Neoplasms/genetics/diagnosis ; CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques/methods ; Female ; Limit of Detection ; Fluorescent Dyes/chemistry ; CRISPR-Associated Proteins/chemistry/genetics ; Endodeoxyribonucleases/chemistry/genetics ; Recombinases/chemistry ; Bacterial Proteins ; }, abstract = {"One-pot" assays which combine amplification with CRISPR/Cas12a system are in constant attracted for biosensors development. Herein, we present a one-pot isothermal assay that Ligation-recognition triggered Recombinase Polymerase Amplification (RPA)-CRISPR/Cas12a cis-cleavage (LRPA-CRISPR) fluorescent biosensor for sensitive, specific, and label-free miRNA detection. Firstly, we reveal the programmed double-stranded DNA amplicons, which utilized the ligation-recognition and polymerization to form and amplified by the RPA system. Meanwhile, we enabled exponential ligation-recognition triggered recombinase polymerase amplification of miRNA-21 sequences and exploited the cis-cleavage mechanism of Cas12a with transcription to generate functional Mango RNA for signal output. This assay can be completed within 40 min and can allow a limit of detection of 3.43 aM for miRNA-21 detection, owing to the RPA with transcription amplification and enables to product the functional Mango RNA aptamer by in vitro transcription that binds to the TO1-Biotin fluorogenic dye. Moreover, our method exhibits the advantages of self-supply crRNA, label-free, excellent specificity, and universal detection platform via the design of one-pot detection in serum and cell samples, showing tremendous potential in biomarkers diagnostics of breast cancer.}, }
@article {pmid39740568, year = {2025}, author = {Zhan, X and Jiang, Y and Lei, J and Chen, H and Liu, T and Lan, F and Ying, B and Wu, Y}, title = {DNA Tetrahedron-enhanced single-particle counting integrated with cascaded CRISPR Program for ultrasensitive dual RNAs logic sensing.}, journal = {Journal of colloid and interface science}, volume = {683}, number = {Pt 2}, pages = {521-531}, doi = {10.1016/j.jcis.2024.12.182}, pmid = {39740568}, issn = {1095-7103}, mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Humans ; *DNA/chemistry ; *RNA/analysis/genetics ; Particle Size ; Fluorescent Dyes/chemistry ; *RNA, Ribosomal, 16S/analysis/genetics ; }, abstract = {CRISPR-Cas-based technology, emerging as a leading platform for molecular assays, has been extensively researched and applied in bioanalysis. However, achieving simultaneous and highly sensitive detection of multiple nucleic acid targets remains a significant challenge for most current CRISPR-Cas systems. Herein, a CRISPR Cas12a based calibratable single particle counting-mediated biosensor was constructed for dual RNAs logic and ultra-sensitive detection in one tube based on DNA Tetrahedron (DTN)-interface supported fluorescent particle probes coupled with a novel synergistic cascaded strategy between CRISPR Cas13a system and strand displacement amplification (SDA). As expected, our platform enables dual RNA molecules intelligent detection using only one crRNA of Cas13a, achieving a sensitivity enhancement of three orders of magnitude assisted with multiple signal amplification and accurate fluorescence particle counting with DTN mediated nano-biointerface enhancement, compared to traditional bulk Cas13a assays. Moreover, the effectiveness and universality of our strategy are experimentally investigated and demonstrated through the detection of mRNAs (cervical cancer swab clinical samples and cultured cancer cells) and bacterial 16s rRNAs. This work not only proposes a highly promising avenue for designing CRISPR-based multiplex detection systems that excel in ultra-sensitivity, specificity, and clinical molecular diagnostics, but also provide new insights into the potential applications of nanotechnology in molecular diagnostics, functional surface engineering, and interface-mediated bioreactions.}, }
@article {pmid39740498, year = {2025}, author = {Yang, Y and Yang, Z and Zhang, X and Niu, B and Huang, Q and Li, Y and Yin, H and Zhang, X and Liao, M and Jia, W}, title = {Rapid detection of Pan-Avian Influenza Virus and H5, H7, H9 subtypes of Avian Influenza Virus using CRISPR/Cas13a and lateral flow assay.}, journal = {Poultry science}, volume = {104}, number = {2}, pages = {104745}, pmid = {39740498}, issn = {1525-3171}, mesh = {Animals ; *Influenza in Birds/diagnosis/virology ; *Influenza A virus/isolation &amp; purification/genetics/classification ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *Poultry Diseases/virology/diagnosis ; Sensitivity and Specificity ; *Chickens ; Real-Time Polymerase Chain Reaction/veterinary/methods ; *Molecular Diagnostic Techniques/methods/veterinary ; }, abstract = {Avian Influenza Virus (AIV) has been prevalent worldwide in recent years, resulting in substantial economic losses in the poultry industry. More importantly, AIV is capable of cross-species transmission among mammals, posing a dormant yet considerable threat to human health and safety. In this study, two rapid detection methods for AIV based on the CRISPR-Cas13a were developed. These methods can identify AIV through the M gene and differentiate the H5, H7, and H9 subtypes via the HA gene. The first method utilizes RT-RAA isothermal amplification of the target sequence in combination with the "collateral effect" of the Cas13a protein. The results are measured using a real-time quantitative PCR instrument, with a Limit of Detection (LOD) as low as 1 copy/μL. The second method combines RT-RAA with Cas13a and a lateral flow assay, allowing results to be visually observed with the naked eye, with a LOD of 10 copies/μL. Both methods demonstrated specificity and sensitivity comparable to or exceeding that of qRT-PCR, suggesting strong potential for clinical application.}, }
@article {pmid39740438, year = {2025}, author = {Shen, Y and Li, B and Hao, G and Duan, M and Zhao, Y and Liu, Z and Li, X and Jia, F}, title = {A CRISPR/Cas12a-based direct transverse relaxation time biosensor via hydrogel sol-gel transition for Salmonella detection.}, journal = {Food chemistry}, volume = {470}, number = {}, pages = {142693}, doi = {10.1016/j.foodchem.2024.142693}, pmid = {39740438}, issn = {1873-7072}, mesh = {*Biosensing Techniques/methods/instrumentation ; *Salmonella/isolation &amp; purification/genetics ; *Hydrogels/chemistry ; *CRISPR-Cas Systems ; Phase Transition ; Food Contamination/analysis ; Bacterial Proteins/genetics/metabolism ; Alkaline Phosphatase/chemistry ; }, abstract = {This research developed a magnetic relaxation switching (MRS) biosensor based on hydrogel sol-gel transition and the CRISPR/Cas12a system (MRS-CRISPR) to detect Salmonella. Herein, the alkaline phosphatase (ALP) labeled with streptavidin was captured by the biotin-modified DNA on magnetic nanoparticles (MNPs) surface, which generated an acidic environment via enzymatic reaction to release Ca[2+] and induced the transformation of alginate sol to hydrogels. In contrast, Salmonella activated the trans-cleavage activity of the CRISPR/Cas12a system, interrupting the capture of ALP and the subsequent sol-gel transition. Then, transverse relaxation time (T2), which was regulated by the hydrogelation process was measured for Salmonella detection. The MRS-CRISPR biosensor enables sensitive detection of Salmonella with a detection limit of 158 CFU/mL. It directly alters the state of water molecules, overcoming the disadvantages of traditional MRS sensors that rely on MNPs to produce T2 signals indirectly. This method offers innovative insights for the application of the MRS technology in food safety analysis.}, }
@article {pmid39740040, year = {2025}, author = {Cai, W and Yang, M and Zhao, Q and Yi, G and Lin, P and Chen, A and De, G}, title = {hURAT1 Transgenic Mouse Model for Evaluating Targeted Urate-Lowering Agents.}, journal = {International journal of rheumatic diseases}, volume = {28}, number = {1}, pages = {e70034}, doi = {10.1111/1756-185X.70034}, pmid = {39740040}, issn = {1756-185X}, support = {ZXKT23002//the Fundamental Research Funds for the Central Public Welfare Research Institutes/ ; 32273019//National Natural Science Foundation of China/ ; }, mesh = {Animals ; *Hyperuricemia/drug therapy/genetics/blood ; *Disease Models, Animal ; *Organic Anion Transporters/genetics/metabolism ; *Uric Acid/blood ; Humans ; *Organic Cation Transport Proteins/genetics/metabolism ; *Mice, Transgenic ; Uricosuric Agents/pharmacology/therapeutic use ; Gout/drug therapy/genetics/metabolism ; Mice, Inbred C57BL ; Male ; Gout Suppressants/pharmacology/therapeutic use ; Phenotype ; Gene Knock-In Techniques ; Hypoxanthine/metabolism ; Mice ; CRISPR-Cas Systems ; }, abstract = {BACKGROUND: Urate transporter 1 (URAT1) is a well-known therapeutic target for reducing urate levels in the treatment of hyperuricemia and gout. However, current pharmacological studies have failed to evaluate the efficacy of URAT1 inhibitors in non-primate animal models. We established a human URAT1 (hURAT1) transgenic knock-in (KI) mouse model to assess uricosuric agents' effectiveness and characterize URAT1-caused pathogenesis.<br><br>METHODS: We generated hURAT1 transgenic mice using CRISPR/Cas9 KI technique. mUrat1 knockout was achieved by replacing exon 1 coding sequence with a human SLC22A12 coding sequence (CDS)-pA cassette. Based on the above transgenic mice, a hyperuricemia model was further established by hypoxanthine administration.<br><br>RESULTS: The hURAT1-KI mice successfully expressed hURAT1 protein to the apical side of the kidney proximal tubule epithelium, where native human URAT1 is localized in human kidney. Upon hypoxanthine challenge, the blood uric acid (UA) level was elevated in hURAT1-KI mice (251&#x2009;&#x3bc;mol/L), showing an approximately 37% increase compared to wild-type (WT) mice (183.5&#x2009;&#x3bc;mol/L). The elevated blood UA level could be alleviated by hURAT1 inhibitor benzbromarone treatment in the hURAT1-KI mice (164.2&#x2009;&#x3bc;mol/L vs. 251&#x2009;&#x3bc;mol/L, p&#x2009;<&#x2009;0.05) whereas no response was observed in WT littermates (168.8&#x2009;&#x3bc;mol/L vs. 183.5&#x2009;&#x3bc;mol/L).<br><br>CONCLUSION: The hURAT1-KI hyperuricemia mouse model would be valuable for preclinical evaluation of gout treatment with urate-lowering drugs and for studying UA metabolic complexities in humans.}, }
@article {pmid39739998, year = {2025}, author = {Liang, H and Mu, X and Huang, Y and Zhao, S and Tian, J}, title = {Magnetic Assisted DNA Logic Gate Nanomachine Based on CRISPR/Cas12a for Recognition of Dual miRNAs.}, journal = {Chemistry, an Asian journal}, volume = {20}, number = {5}, pages = {e202401209}, doi = {10.1002/asia.202401209}, pmid = {39739998}, issn = {1861-471X}, support = {22064003//National Natural Science Foundation of China/ ; 21465007//National Natural Science Foundation of China/ ; 2015GXNSFGA139003//Guangxi Natural Science Foundation of China/ ; //Bagui Scholar of Guangxi Province/ ; //Duxiu Scholar of Guangxi Normal University/ ; CMEMR2014-A08//State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Sciences of Guangxi Normal University/ ; }, mesh = {*MicroRNAs/analysis/genetics ; *CRISPR-Cas Systems ; Humans ; *DNA/chemistry/metabolism ; *Computers, Molecular ; Biosensing Techniques ; DNA, Catalytic/metabolism/chemistry ; Logic ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {The anomalous expression of microRNA poses a serious threat to human life and health safety, and serves as an important biomarker for cancer detection. In this study, a novel magnetic-assisted DNA logic gate nanomachine triggered by miRNA-21 and miRNA-155 was designed based on the trans-cleavage activity of CRISPR/Cas12a activated by a split DNA activator, using only a single crRNA and signal probe, which simplified the detection procedure and complex nucleic acid amplification. The presence of target molecules, miRNA-21 and miRNA-155, can stimulate the DNA walker machine assembled on magnetic beads, which releases activator under the action of DNAzyme. Then the trans-cleavage activity of CRISPR/Cas12a is initiated and the system signal significantly increases. Based on this, an AND logic gate nanomachine was constructed for simultaneous analysis of miRNA-21 and miRNA-155. The detection limits of miRNA-21 and miRNA-15 were 9.00 pM and 42.00 pM, respectively, and this method was successfully applied to miRNA analysis in cell samples. This nanomachine combined the DNA walker with DNA logic circuit and CRISPR/Cas12a system, providing a new approach for simultaneous detection of multiple targets and further expanding the application of gene editing in the analysis and sensing of multiple target substances.}, }
@article {pmid39739718, year = {2024}, author = {Delgado-Nungaray, JA and Figueroa-Yáñez, LJ and Reynaga-Delgado, E and Corona-España, AM and Gonzalez-Reynoso, O}, title = {Unveiling the endogenous CRISPR-Cas system in Pseudomonas aeruginosa PAO1.}, journal = {PloS one}, volume = {19}, number = {12}, pages = {e0312783}, pmid = {39739718}, issn = {1932-6203}, mesh = {*Pseudomonas aeruginosa/genetics/drug effects ; *CRISPR-Cas Systems ; Genome, Bacterial ; Bacterial Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Multidrug resistance in Pseudomonas aeruginosa, a high-priority pathogen per the World Health Organization, poses a global threat due to carbapenem resistance and limited antibiotic treatments. Using the bioinformatic tools CRISPRCasFinder, CRISPRCasTyper, CRISPRloci, and CRISPRImmunity, we analyzed the genome of P. aeruginosa PAO1 and revealed an orphan CRISPR system, suggesting it may be a remnant of a type IV system due to the presence of the DinG protein. This system comprises two CRISPR arrays and noteworthy DinG and Cas3 proteins, supporting recent evidence about the association between type IV and I CRISPR systems. Additionally, we demonstrated a co-evolutionary relationship between the orphan CRISPR system in P. aeruginosa PAO1 and the mobile genetic element and prophages identified. One self-targeting spacer was identified, often associated with bacterial evolution and autoimmunity, and no Acr proteins. This research opens avenues for studying how these CRISPR arrays regulate pathogenicity and for developing alternative strategies using its endogenous orphan CRISPR system against carbapenem-resistant P. aeruginosa strains.}, }
@article {pmid39739523, year = {2025}, author = {Kishi, K and Nigorikawa, K and Hasegawa, Y and Ohta, Y and Matsugi, E and Matsumoto, D and Nomura, W}, title = {Cell cycle-dependent regulation of CRISPR-Cas9 repetitive activation by anti-CRISPR and Cdt1 fusion in the CRISPRa system.}, journal = {FEBS letters}, volume = {599}, number = {6}, pages = {828-837}, doi = {10.1002/1873-3468.15090}, pmid = {39739523}, issn = {1873-3468}, support = {JP22H02201//Japan Society for the Promotion of Science/ ; JP20K21253//Japan Society for the Promotion of Science/ ; JP22H04926//Japan Society for the Promotion of Science/ ; JPMJSP2132//Support for Pioneering Research Initiated by the Next Generation/ ; //Takeda Science Foundation/ ; //Naito Foundation/ ; //Uehara Memorial Foundation/ ; //Mochida Memorial Foundation/ ; //Suzuken Memorial Foundation/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Cell Cycle Proteins/genetics/metabolism ; *Cell Cycle/genetics ; Gene Editing/methods ; *CRISPR-Associated Protein 9/metabolism/genetics ; Recombinant Fusion Proteins/genetics/metabolism ; HEK293 Cells ; }, abstract = {CRISPR-Cas9 is a widely used genome-editing tool. We previously developed a method with improved homology-directed repair efficiency and reduced off-target effects by utilizing a fusion protein of AcrIIA4, a Cas9 inhibitor, and Cdt1, which accumulates in the G1 phase and activates Cas9 only in the S/G2 phase. However, it is unknown whether Cas9 inhibition by AcrIIA4 + Cdt1 occurs repeatedly in the G1 phase as the cell cycle progresses. In this study, we used the CRISPRa system to monitor changes in the interaction between Cas9 and AcrIIA4 + Cdt1 at single-cell resolution and in real time. Our findings are among the few examples of successful detection of fluctuating protein-protein interactions that oscillate over time.}, }
@article {pmid39738995, year = {2024}, author = {Argentel-Martínez, L and Peñuelas-Rubio, O and Herrera-Sepúlveda, A and González-Aguilera, J and Sudheer, S and Salim, LM and Lal, S and Pradeep, CK and Ortiz, A and Sansinenea, E and Hathurusinghe, SHK and Shin, JH and Babalola, OO and Azizoglu, U}, title = {Biotechnological advances in plant growth-promoting rhizobacteria for sustainable agriculture.}, journal = {World journal of microbiology &amp; biotechnology}, volume = {41}, number = {1}, pages = {21}, pmid = {39738995}, issn = {1573-0972}, mesh = {*Agriculture/methods ; *Bacteria/genetics/classification/metabolism ; *Biotechnology/methods ; CRISPR-Cas Systems ; *Crops, Agricultural/microbiology/growth &amp; development ; Gene Editing/methods ; *Genetic Engineering/methods ; Microbiota ; Plant Development ; Plant Roots/microbiology/growth &amp; development ; *Rhizosphere ; Soil Microbiology ; }, abstract = {The rhizosphere, the soil zone surrounding plant roots, serves as a reservoir for numerous beneficial microorganisms that enhance plant productivity and crop yield, with substantial potential for application as biofertilizers. These microbes play critical roles in ecological processes such as nutrient recycling, organic matter decomposition, and mineralization. Plant growth-promoting rhizobacteria (PGPR) represent a promising tool for sustainable agriculture, enabling green management of crop health and growth, being eco-friendly alternatives to replace chemical fertilizers and pesticides. In this sense, biotechnological advancements respecting genomics and gene editing have been crucial to develop microbiome engineering which is pivotal in developing microbial consortia to improve crop production. Genome mining, which involves comprehensive analysis of the entire genome sequence data of PGPR, is crucial for identifying genes encoding valuable bacterial enzymes and metabolites. The CRISPR-Cas system, a cutting-edge genome-editing technology, has shown significant promise in beneficial microbial species. Advances in genetic engineering, particularly CRISPR-Cas, have markedly enhanced grain output, plant biomass, resistance to pests, and the sensory and nutritional quality of crops. There has been a great advance about the use of PGPR in important crops; however, there is a need to go further studying synthetic microbial communities, microbiome engineering, and gene editing approaches in field trials. This review focuses on future research directions involving several factors and topics around the use of PGPR putting special emphasis on biotechnological advances.}, }
@article {pmid39738767, year = {2024}, author = {Lei, Z and Cai, H and Yan, Q}, title = {NF2 can mediate the expression of CAMK2A in a tissue specific manner.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {31992}, pmid = {39738767}, issn = {2045-2322}, mesh = {Humans ; *Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism/genetics ; *Gene Expression Regulation, Neoplastic ; Meningioma/genetics/metabolism/pathology ; Neurofibromin 2/metabolism/genetics ; Organ Specificity/genetics ; Meningeal Neoplasms/genetics/metabolism/pathology ; Calcium/metabolism ; }, abstract = {Meningioma is the most prevalent primary intracranial tumor, with approximately half of patients harboring NF2 alteration. The rationale behind the presence of NF2 alteration in meningiomas and its absence in non-nerve system tumors remains elusive. Therefore, meningiomas and several non-nerve system tumor types were analyzed using KEGG analysis and CRISPR/Cas 9 technology to determine the role of NF2 in regulating tissue specificity. Moreover, the different regulatory patterns of Ca[2+] and calcium/calmodulin-dependent protein kinase II alpha (CAMK2A) that play a decisive role in NF2 tissue-specific regulation were deciphered. Our results revealed that NF2 has a positive correlation in CAMK2A expression in both meningiomas and normal nervous system tissues but not in non-nervous system tumors and tissues, implying NF2 tissue-specificity is mediated by CAMK2A-related pathways. Thus, targeting CAMK2A may represent a promising strategy for drug screening and the development of therapeutics for NF2-associated meningiomas and other nervous system tumors.}, }
@article {pmid39738613, year = {2024}, author = {Koshi, D and Sugano, J and Yamasaki, F and Kawauchi, M and Nakazawa, T and Oh, M and Honda, Y}, title = {Trans-nuclei CRISPR/Cas9: safe approach for genome editing in the edible mushroom excluding foreign DNA sequences.}, journal = {Applied microbiology and biotechnology}, volume = {108}, number = {1}, pages = {548}, pmid = {39738613}, issn = {1432-0614}, support = {K-2019-002//Institute for Fermentation, Osaka/ ; 18KK0178//Japan Society for the Promotion of Science/ ; 22H00380//Japan Society for the Promotion of Science/ ; 22KK0090//Japan Society for the Promotion of Science/ ; 23KJ1317//Japan Society for the Promotion of Science/ ; PJ015554//National Institute of Horticultural and Herbal Science, Rural Development Administration/ ; PJ015554//National Institute of Horticultural and Herbal Science, Rural Development Administration/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Pleurotus/genetics ; Genome, Fungal/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Nucleus/genetics ; Plasmids/genetics ; CRISPR-Associated Protein 9/genetics ; }, abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-assisted genome editing has been applied to several major edible agaricomycetes, enabling efficient gene targeting. This method is promising for rapid and efficient breeding to isolate high-value cultivars and overcome cultivation challenges. However, the integration of foreign DNA fragments during this process raises concerns regarding genetically modified organisms (GMOs) and their regulatory restrictions. In this study, we developed a foreign-DNA-free genome editing method in Pleurotus ostreatus by transferring the Cas9/guide RNA (gRNA) complex between nuclei in the dikaryotic state. We isolated a donor monokaryotic P. ostreatus strain expressing Cas9 and gRNA targeting pyrG by introducing a recombinant plasmid, which exhibited uracil auxotrophy and 5-fluoroorotic acid (5-FOA) resistance. This strain was then crossed with a pyrG[+] recipient monokaryon, resulting in dikaryotic strains exhibiting 5-FOA resistance after mycelial growth. When these strains were de-dikaryonized into monokaryons through protoplasting, we obtained monokaryotic isolates harboring the recipient nucleus with small indels at the pyrG target site. Importantly, these isolates were confirmed to be free of foreign DNA through genomic PCR, Southern blotting, and whole-genome resequencing analyses. This is the first report of an efficient genome editing protocol in agaricomycetes that ensures no integration of exogenous DNA. This approach is expected to be applicable to other fungi with a dikaryotic life cycle, opening new possibilities for molecular breeding without the concerns associated with GMOs. KEY POINTS: • Successful genome editing via CRISPR/Cas9 trans-nuclei manner in P. ostreatus. • Recipient monokaryons from gene-edited dikaryons showed no exogenous DNA sequences. • Efficient genome editing protocol for safer molecular breeding in mushroom fungus.}, }
@article {pmid39737993, year = {2024}, author = {Yuan, Q and Zeng, H and Daniel, TC and Liu, Q and Yang, Y and Osikpa, EC and Yang, Q and Peddi, A and Abramson, LM and Zhang, B and Xu, Y and Gao, X}, title = {Orthogonal and multiplexable genetic perturbations with an engineered prime editor and a diverse RNA array.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10868}, pmid = {39737993}, issn = {2041-1723}, support = {R01 HL157714/HL/NHLBI NIH HHS/United States ; CBET-2143626//National Science Foundation (NSF)/ ; }, mesh = {Humans ; *Gene Editing/methods ; *Trans-Activators/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Homeodomain Proteins/genetics/metabolism ; Amyloid Neuropathies, Familial/genetics/therapy ; HEK293 Cells ; RNA/genetics ; Genome, Human ; CRISPR-Cas Systems ; Mutation ; RNA Interference ; Genetic Engineering/methods ; }, abstract = {Programmable and modular systems capable of orthogonal genomic and transcriptomic perturbations are crucial for biological research and treating human genetic diseases. Here, we present the minimal versatile genetic perturbation technology (mvGPT), a flexible toolkit designed for simultaneous and orthogonal gene editing, activation, and repression in human cells. The mvGPT combines an engineered compact prime editor (PE), a fusion activator MS2-p65-HSF1 (MPH), and a drive-and-process multiplex array that produces RNAs tailored to different types of genetic perturbation. mvGPT can precisely edit human genome via PE coupled with a prime editing guide RNA and a nicking guide RNA, activate endogenous gene expression using PE with a truncated single guide RNA containing MPH-recruiting MS2 aptamers, and silence endogenous gene expression via RNA interference with a short-hairpin RNA. We showcase the versatility of mvGPT by simultaneously correcting a c.3207C>A mutation in the ATP7B gene linked to Wilson's disease, upregulating the PDX1 gene expression to potentially treat Type I diabetes, and suppressing the TTR gene to manage transthyretin amyloidosis. In addition to plasmid delivery, we successfully utilize various methods to deliver the mvGPT payload, demonstrating its potential for future in vivo applications.}, }
@article {pmid39737984, year = {2024}, author = {Nagamura, R and Kujirai, T and Kato, J and Shuto, Y and Kusakizako, T and Hirano, H and Endo, M and Toki, S and Saika, H and Kurumizaka, H and Nureki, O}, title = {Structural insights into how Cas9 targets nucleosomes.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10744}, pmid = {39737984}, issn = {2041-1723}, support = {JP23fa627001//Japan Agency for Medical Research and Development (AMED)/ ; JP19am0401005//Japan Agency for Medical Research and Development (AMED)/ ; JP23ama121002//Japan Agency for Medical Research and Development (AMED)/ ; JP23ama121012//Japan Agency for Medical Research and Development (AMED)/ ; JP23ama121009//Japan Agency for Medical Research and Development (AMED)/ ; JP23H05475//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JPMJER1901//MEXT | JST | Exploratory Research for Advanced Technology (ERATO)/ ; }, mesh = {*Nucleosomes/metabolism/ultrastructure ; *Cryoelectron Microscopy ; *DNA/metabolism/chemistry ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; *RNA, Guide, CRISPR-Cas Systems/metabolism ; *Gene Editing ; Histones/metabolism/chemistry ; CRISPR-Cas Systems ; Chromatin/metabolism/chemistry ; DNA Cleavage ; Models, Molecular ; }, abstract = {The CRISPR-associated endonuclease Cas9 derived from prokaryotes is used as a genome editing, which targets specific genomic loci by single guide RNAs (sgRNAs). The eukaryotes, the target of genome editing, store their genome DNA in chromatin, in which the nucleosome is a basic unit. Despite previous structural analyses focusing on Cas9 cleaving free DNA, structural insights into Cas9 targeting of DNA within nucleosomes are limited, leading to uncertainties in understanding how Cas9 operates in the eukaryotic genome. In the present study, we perform native-polyacrylamide gel electrophoresis (PAGE) analyses and find that Cas9 targets the linker DNA and the entry-exit DNA region of the nucleosome but not the DNA tightly wrapped around the histone octamer. We further determine cryo-electron microscopy (cryo-EM) structure of the Cas9-sgRNA-nucleosome ternary complex that targets linker DNA in nucleosomes. The structure suggests interactions between Cas9 and nucleosomes at multiple sites. Mutants that reduce the interaction between nucleosomal DNA and Cas9 improve nucleosomal DNA cleavage activity in vitro, although inhibition by the interaction between Cas9 and nucleosomes is limited in vivo. These findings will contribute to the development of novel genome editing tools in chromatin.}, }
@article {pmid39737975, year = {2024}, author = {Wu, Y and Zhong, A and Sidharta, M and Kim, TW and Ramirez, B and Persily, B and Studer, L and Zhou, T}, title = {Robust and inducible genome editing via an all-in-one prime editor in human pluripotent stem cells.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10824}, pmid = {39737975}, issn = {2041-1723}, support = {P30 CA008748/CA/NCI NIH HHS/United States ; }, mesh = {Humans ; *Gene Editing/methods ; *Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems ; Cell Differentiation/genetics ; Tumor Suppressor Protein p53/genetics/metabolism ; Genome, Human ; Mutation ; Cell Line ; }, abstract = {Prime editing (PE) allows for precise genome editing in human pluripotent stem cells (hPSCs), such as introducing single nucleotide modifications, small insertions or deletions at a specific genomic locus. Here, we systematically compare a panel of prime editing conditions in hPSCs and generate a potent prime editor, "PE-Plus", through co-inhibition of mismatch repair and p53-mediated cellular stress responses. We further establish an inducible prime editing platform in hPSCs by incorporating the PE-Plus into a safe-harbor locus and demonstrated temporal control of precise editing in both hPSCs and differentiated cells. By evaluating disease-associated mutations, we show that this platform allows efficient creation of both monoallelic and biallelic disease-relevant mutations in hPSCs. In addition, this platform enables the efficient introduction of single or multiple edits in one step, demonstrating potential for multiplex editing. Our method presents an efficient and controllable multiplex prime editing tool in hPSCs and their differentiated progeny.}, }
@article {pmid39737904, year = {2024}, author = {Li, D and Zhang, S and Lin, S and Xing, W and Yang, Y and Zhu, F and Su, D and Chen, C and Liu, JG}, title = {Cas12e orthologs evolve variable structural elements to facilitate dsDNA cleavage.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10727}, pmid = {39737904}, issn = {2041-1723}, support = {32150018//National Natural Science Foundation of China (National Science Foundation of China)/ ; 21877069//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22277063//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22061160466//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32101195//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2022M711848//China Postdoctoral Science Foundation/ ; }, mesh = {*CRISPR-Cas Systems ; *DNA/metabolism/genetics/chemistry ; *DNA Cleavage ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *Gene Editing/methods ; Evolution, Molecular ; Protein Domains ; }, abstract = {Exceptionally diverse type V CRISPR-Cas systems provide numerous RNA-guided nucleases as powerful tools for DNA manipulation. Two known Cas12e nucleases, DpbCas12e and PlmCas12e, are both effective in genome editing. However, many differences exist in their in vitro dsDNA cleavage activities, reflecting the diversity in Cas12e's enzymatic properties. To comprehensively understand the Cas12e family, we identify and characterize six unreported Cas12e members that vary in their CRISPR-locus architectures, PAM preferences, and cleavage efficacies. Interestingly, among all variants, PlmCas12e exhibits the most robust trans-cleavage activity and the lowest salt sensitivity in cis-cleavage. Further structural comparisons reveal that the unique NTSB domain in PlmCas12e is beneficial to DNA unwinding at high salt concentrations, while some NTSB-lacking Cas12e proteins rely on positively charged loops for dsDNA unwinding. These findings demonstrate how divergent evolution of structural elements shapes the nuclease diversity within the Cas12e family, potentially contributing to their adaptations to varying environmental conditions.}, }
@article {pmid39736572, year = {2024}, author = {Stelmach-Wityk, K and Szymonik, K and Grzebelus, E and Kiełkowska, A}, title = {Development of an optimized protocol for protoplast-to-plant regeneration of selected varieties of Brassica oleracea L.}, journal = {BMC plant biology}, volume = {24}, number = {1}, pages = {1279}, pmid = {39736572}, issn = {1471-2229}, mesh = {*Protoplasts ; *Brassica/genetics/physiology/growth &amp; development ; *Regeneration ; Plant Shoots/genetics/growth &amp; development ; Plant Breeding/methods ; Plant Roots/genetics/growth &amp; development/physiology ; }, abstract = {BACKGROUND: Brassica oleracea L. is a key plant in the Brassicaceae family, known for popular vegetables like cabbage, broccoli, kale and collard. Collard (B. oleracea var. viridis) is a non-heading leafy vegetable grown in urban farms and community gardens in the United States and Europe. Improving collard and other Brassica germplasm can benefit from both traditional and new plant breeding technologies (NPBTs), such as CRISPR-Cas mediated transformation. An efficient transformation or protoplast fusion can only be achieved with a robust and reproducible protocol for protoplast-to-plant regeneration. This research focuses on optimizing in vitro culture conditions to enhance cell divisions, microcallus formation, and the regeneration of shoots and roots in four Brassica oleracea varieties, including collard.<br><br>RESULTS: The protocol of protoplast release, purification and immobilization was optimized to obtain a suitable number and quality of protoplasts from seven cultivars of B. oleracea. The protoplast isolation efficiency after digestion of young leaves in optimized enzyme solution reached on average 2.5&#x2009;&#xd7;&#x2009;10[6] of cells per gram of fresh weight. Protoplasts were embedded in thin alginate layers and subjected to culture in three different media. Protoplasts of all studied cultivars were viable (88.2%), underwent cell wall resynthesis and re-entered mitotic divisions in the 5th day of culture. After 30 days of culture, protoplast-derived cells of all the tested cultivars formed microcallus. Six cultivars regenerated shoots, although the shoot formation efficiency strongly depended on the genotype and composition of the regeneration medium. The regeneration medium supplemented with 1 mg&#xa0;l[-1] of NAA, 1 mg&#xa0;l[-1] of 2iP, 0.02 mg&#xa0;l[-1] GA3 and with 2% of mannitol showed the highest shoot formation efficiency for five cultivars of B. oleracea.<br><br>CONCLUSIONS: The results of this research have led to the development of a robust protoplast-to-plant regeneration protocol for four varieties of B. oleracea that could be exploited as a tool for production of transformants and somatic hybrids. Furthermore, we present the first successful regeneration of protoplast-derived plants of collard, an overlooked but valuable variety of Brassica oleracea.}, }
@article {pmid39736287, year = {2025}, author = {Cao, XX and Yuan, JJ and Bai, ZY and Zhang, M and Yun, YF and Wang, XY and Mi, CL and Sun, QL and Geng, SL and Wang, TY}, title = {Effect of CHO cell line constructed with CMAH gene-directed integration on the recombinant protein expression.}, journal = {International journal of biological macromolecules}, volume = {292}, number = {}, pages = {139274}, doi = {10.1016/j.ijbiomac.2024.139274}, pmid = {39736287}, issn = {1879-0003}, mesh = {Animals ; CHO Cells ; Cricetulus ; *Recombinant Proteins/genetics/biosynthesis ; Erythropoietin/genetics/metabolism ; *Mixed Function Oxygenases/genetics/metabolism ; Glycosylation ; Humans ; CRISPR-Cas Systems ; Gene Expression ; Green Fluorescent Proteins/genetics ; Cricetinae ; }, abstract = {Chinese hamster ovary (CHO) cells are the most widely used platform for recombinant therapeutic protein (RTP) production. Traditionally, the development of CHO cell lines has mainly depended on random integration of transgenes into the genome, which is not conducive to stable long-term expression. Cytidine monophosphate N-acetylneuraminic acid hydroxylase (CMAH) is expressed in CHO cells and produces N-hydroxyacetylneuraminic acid, which may cause a human immune response. However, the effects of transgene integration at the CMAH site on RTP expression in CHO cells remain unclear. In this study, we selected CMAH gene, which is lacking in humans, as the target site to construct recombinant CHO cell line using the CRISPR/Cas9 technique. Erythropoietin (EPO) and EGFP integration at the CMAH site resulted in more stable expression levels and lower heterogeneity than random integration. In addition, the proportion of N-glycosylation levels in the EPO glycoside of CMAH integration site also changed. In conclusion, CMAH site integration improved the stability of RTP expression in CHO cells.}, }
@article {pmid39736115, year = {2025}, author = {Masarwy, R and Breier, D and Stotsky-Oterin, L and Ad-El, N and Qassem, S and Naidu, GS and Aitha, A and Ezra, A and Goldsmith, M and Hazan-Halevy, I and Peer, D}, title = {Targeted CRISPR/Cas9 Lipid Nanoparticles Elicits Therapeutic Genome Editing in Head and Neck Cancer.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {12}, number = {7}, pages = {e2411032}, pmid = {39736115}, issn = {2198-3844}, support = {825828//European Union's Horizon 2020 research and innovation programme/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; Animals ; *Head and Neck Neoplasms/genetics/therapy ; *Nanoparticles ; Mice ; Humans ; *Gene Editing/methods ; Cell Line, Tumor ; *Squamous Cell Carcinoma of Head and Neck/genetics/therapy ; SOXB1 Transcription Factors/genetics ; Lipids ; Liposomes ; }, abstract = {Squamous cell carcinomas of the head and neck (HNSCC) originate in the upper aerodigestive tract, including the oral cavity, pharynx, and larynx. Current treatments of locally advanced HNSCC often lead to high treatment failure, and disease recurrence, resulting in poor survival rates. Advances in mRNA technologies and lipid nanoparticle (LNP) delivery systems led to several clinical trials involving LNP-CRISPR-Cas9 mRNA-based therapeutics. Despite these advances, achieving cell-type-specific extrahepatic mRNA delivery is still challenging. This study introduces a safe and effective intratumoral EGFR-targeted CRISPR-LNP delivery strategy for knocking out SOX2, which is a cancer-specific gene. To assess their therapeutic potential, it is shown that LNPs made from ionizable lipids with helper lipids co-encapsulating Cas9 mRNA and sgRNA targeting SOX2 (sgSOX2), lead to a ≈60% reduction in HNSCC cell viability in vitro. Next, using a xenograft HNSCC mouse model, targeted delivery of 𝜶EGFR- CRISPR-sgSOX2-LNPs to HNSCC cells resulted in a 90% inhibition of tumor growth and a 90% increase in survival for > 84 days, with tumor disappearance observed in 50% of the mice. These findings emphasize the potential of targeted mRNA-Cas9-LNPs in clinically accessible solid tumors, specifically in reaching tumor cells and inducing persistent therapeutic responses in tumors with high-recurrence rates like HNSCC.}, }
@article {pmid39735189, year = {2024}, author = {Lu, Z and Liang, M and Li, C and Xu, Y and Weng, S and He, J and Guo, C}, title = {Rapid, sensitive, and visual detection of mandarin fish ranavirus and infectious spleen and kidney necrosis virus using an RPA-CRISPR/Cas12a system.}, journal = {Frontiers in microbiology}, volume = {15}, number = {}, pages = {1495777}, pmid = {39735189}, issn = {1664-302X}, abstract = {Iridoviruses are large cytoplasmic icosahedral viruses that contain dsDNA. Among them, mandarin fish ranavirus (MRV) and infectious spleen and kidney necrosis virus (ISKNV) are particularly notable due to their high contagiousness and pathogenicity. These viruses pose a significant threat to fish aquaculture, resulting in substantial annual economic losses for the fish farming industry. Therefore, the development of novel, rapid virus detection technologies is essential for the prevention and control of ISKNV and MRV diseases. In this study, we developed a rapid, sensitive, and visual detection method for MRV and ISKNV using the recombinase polymerase amplification (RPA)-CRISPR/Cas12a system. This method can detect as low as 1 copy/μL of MRV and 0.1 copy/μL of ISKNV, demonstrating excellent specificity and reproducibility. The detection can be performed at a constant temperature of 37-39°C, eliminating the need for complex equipment. A 30-min RPA amplification followed by a 15-min CRISPR/Cas reaction is sufficient for detecting most samples. For low-concentration samples, extending the CRISPR/Cas reaction time to 60 min improves result visibility. The designed RPA reaction system is capable of performing reverse transcription of RNA, allowing for the detection of mRNA transcribed from the MCP gene of MRV and ISKNV in the sample. Furthermore, two probes were identified that can be observed without the need for excitation light. In conclusion, a field-suitable detection method for ISKNV and MRV has been established, providing a powerful tool for the prompt diagnosis of these aquatic pathogens and aiding in the prevention and control of ISKNV and MRV diseases.}, }
@article {pmid39734219, year = {2024}, author = {Wen, Q and Chen, J and Li, J and Dharmasiddhi, IPW and Yang, M and Xing, J and Liu, Y}, title = {A single-plasmid-based, easily curable CRISPR/Cas9 system for rapid, iterative genome editing in Pseudomonas putida KT2440.}, journal = {Microbial cell factories}, volume = {23}, number = {1}, pages = {349}, pmid = {39734219}, issn = {1475-2859}, support = {2023YFC3403601//National Key Research and Development Program of China/ ; 2023YFC3403601//National Key Research and Development Program of China/ ; 2023YFC3403601//National Key Research and Development Program of China/ ; }, mesh = {*Pseudomonas putida/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Plasmids/genetics ; Genome, Bacterial ; }, abstract = {BACKGROUND: Pseudomonas putida KT2440, a non-pathogenic soil bacterium, is a key platform strain in synthetic biology and industrial applications due to its robustness and metabolic versatility. Various systems have been developed for genome editing in P. putida, including transposon modules, integrative plasmids, recombineering systems, and CRISPR/Cas systems. However, rapid iterative genome editing is limited by complex and lengthy processes.<br><br>RESULTS: We discovered that the pBBR1MCS2 plasmid carrying the CRISPR/Cas9 module could be easily cured in P. putida KT2440 at 30 [o]C. We then developed an all-in-one CRISPR/Cas9 system for yqhD and ech-vdh-fcs deletions, respectively, and further optimized the editing efficiency by varying homology arm lengths and target sites. Sequential gene deletions of vdh and vanAB were carried out rapidly using single-round processing and easy plasmid curing. This system's user-friendliness was validated by 3 researchers from two labs for 9 deletions, 3 substitutions, and 2 insertions. Finally, iterative genome editing was used to engineer P. putida for valencene biosynthesis, achieving a 10-fold increase in yield.<br><br>CONCLUSIONS: We developed and applied a rapid all-in-one plasmid CRISPR/Cas9 system for genome editing in P. putida. This system requires less than 1.5 days for one edit due to simplified plasmid construction, electroporation and curing processes, thus accelerating the cycle of genome editing. To our knowledge, this is the fastest iterative genome editing system for P. putida. Using this system, we rapidly engineered P. putida for valencene biosynthesis for the first time, showcasing the system's potential for expanding biotechnological applications.}, }
@article {pmid39733911, year = {2025}, author = {Wang, T and Chen, T and Li, D and Hang, X and Zhang, S and Yi, H and Jiang, T and Ding, D and Zhang, X}, title = {Core-shell vector-mediated co-delivery of CRISPR/Cas9 system and hydrophobic drugs against triple-negative breast cancer stem cells.}, journal = {Journal of controlled release : official journal of the Controlled Release Society}, volume = {378}, number = {}, pages = {1080-1091}, doi = {10.1016/j.jconrel.2024.12.062}, pmid = {39733911}, issn = {1873-4995}, mesh = {*Triple Negative Breast Neoplasms/drug therapy/genetics/therapy/pathology ; Humans ; *Neoplastic Stem Cells/drug effects/metabolism ; Female ; Animals ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Doxorubicin/administration &amp; dosage ; Nanoparticles/chemistry/administration &amp; dosage ; Hydrophobic and Hydrophilic Interactions ; Mice, Nude ; Mice, Inbred BALB C ; F-Box Proteins/genetics ; Plasmids/administration &amp; dosage ; Drug Delivery Systems ; Mice ; Hyaluronic Acid/chemistry ; *Antibiotics, Antineoplastic/administration &amp; dosage ; }, abstract = {Cancer stem cells (CSCs) play an important role in the development of triple-negative breast cancer (TNBC), including metastasis, invasion, tumorigenicity, and drug resistance. Moreover, non-CSCs can spontaneously transform into CSCs in special tumor microenvironments, thereby leading to poor prognosis or even failed treatments. Therefore, reversing CSCs into normal tumor cells in a sustained-acting manner is a promising strategy. It has been reported that down-regulation of FBXO44 protein expression inhibits tumor cell stemness. Moreover, CRISPR/Cas9 technology, a well-known precise gene editing tool, was adopted to permanently block FBXO44 within the genome upon its successful implementation. Given this, a core-shell nanoparticle (NP) consisting of amphiphilic polymer core and crosslinked-hyaluronic acid shell (nDOX-PL/pFBXO44 NPs) is developed in this work to concurrently deliver FBXO44-targeted CRISPR/Cas9 plasmids (pFBXO44) and doxorubicin (DOX) for combinational CSC reprogramming and chemotherapy of TNBC, which exhibits tumor cell targeting, endosomal escape, and reduction responsiveness to release DOX and plasmids in the cytoplasma. CRISPR/Cas9-mediated downregulation of FBXO44 expression could convert CSC into normal tumor cells, and effectively inhibit tumor growth without obvious side effects in vivo after combining with chemotherapy. In summary, we developed an intelligent system to co-deliver genetic and hydrophobic drugs, achieving effective cancer stemness reversal and synergistic suppression of contractable TNBC.}, }
@article {pmid39733882, year = {2025}, author = {Jin, Z and Yi, C and Zhou, D and Wang, X and Xie, M and Zhou, H and Zhang, A}, title = {Chicken genome-wide CRISPR library screen identifies potential candidates associated with Avian influenza virus infection.}, journal = {International journal of biological macromolecules}, volume = {293}, number = {}, pages = {139267}, doi = {10.1016/j.ijbiomac.2024.139267}, pmid = {39733882}, issn = {1879-0003}, mesh = {Animals ; *Chickens/genetics/virology ; *Influenza in Birds/genetics/virology ; *CRISPR-Cas Systems ; *Influenza A virus/physiology/genetics ; Cell Line ; Gene Library ; *Genome ; Fibroblasts/virology ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {The avian influenza virus (AIV) poses a significant threat to both the poultry industry and public health. Systematic identification of host factors involved in AIV infection in chicken is critical. In this study, we developed a comprehensive chicken genome-wide sgRNA library containing 76,350 sgRNAs, with 4-6 sgRNAs designed per gene. Then, we constructed a genome-wide CRISPR/Cas9 knockout chicken fibroblasts cells (DF-1 cells) library, covering 99.9 % of the total sgRNAs. Following multiple rounds of survival selection during AIV infection, 706 potential genes were identified, including 107 genes previously associated with AIV infection. These candidate genes were primarily involved in ubiquitin-related pathways, RNA transport, endocytosis, and other cellular processes. Among these, 18 novel hits were selected and confirmed to contribute to AIV-induced cell death, with eight genes specifically implicated in AIV proliferation. Notably, RNF2 was found to negatively regulate interferon-stimulated genes (ISGs), DCP1A was suggested to influence gene expression linked to AIV proliferation, and CREB3L3 may regulate membrane cholesterol levels during AIV invasion, further validating the screening results. This study identified 599 potential chicken genes involved in AIV infection, providing a foundation for a deeper understanding of the mechanisms underlying AIV infection in avian cells.}, }
@article {pmid39732069, year = {2025}, author = {Zhang, J and Zhou, Y and Qiao, J and Liu, Y}, title = {Recent advances in spatiotemporal control of the CRISPR/Cas9 system.}, journal = {Colloids and surfaces. B, Biointerfaces}, volume = {248}, number = {}, pages = {114474}, doi = {10.1016/j.colsurfb.2024.114474}, pmid = {39732069}, issn = {1873-4367}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Humans ; Animals ; CRISPR-Associated Protein 9/metabolism/genetics ; }, abstract = {The CRISPR/Cas9 gene-editing technology, derived from the adaptive immune mechanisms of bacteria, has demonstrated remarkable advantages in fields such as gene function research and the treatment of genetic diseases due to its simplicity in design, precise targeting, and ease of use. Despite challenges such as off-target effects and cytotoxicity, effective spatiotemporal control strategies have been achieved for the CRISPR/Cas9 system through precise regulation of Cas9 protein activity as well as engineering of guide RNAs (gRNAs). This review provides a comprehensive analysis of the core components and functional mechanisms underlying the CRISPR/Cas9 system, highlights recent advancements in spatiotemporal control strategies, and discusses future directions for development.}, }
@article {pmid39731215, year = {2025}, author = {Zhou, Z and Chen, Y and Ba, Y and Xu, H and Zuo, A and Liu, S and Zhang, Y and Weng, S and Ren, Y and Luo, P and Cheng, Q and Zuo, L and Zhu, S and Zhou, X and Zhang, C and Chen, Y and Han, X and Pan, T and Liu, Z}, title = {Revolutionising Cancer Immunotherapy: Advancements and Prospects in Non-Viral CAR-NK Cell Engineering.}, journal = {Cell proliferation}, volume = {58}, number = {4}, pages = {e13791}, pmid = {39731215}, issn = {1365-2184}, support = {221100310100//Henan Provincial Science and Technology Research Project/ ; }, mesh = {Humans ; *Killer Cells, Natural/immunology/transplantation ; *Neoplasms/therapy/immunology ; *Receptors, Chimeric Antigen/immunology/genetics/metabolism ; *Cell Engineering/methods ; *Immunotherapy, Adoptive/methods ; *Immunotherapy/methods ; Gene Editing ; Animals ; CRISPR-Cas Systems ; }, abstract = {The recent advancements in cancer immunotherapy have spotlighted the potential of natural killer (NK) cells, particularly chimeric antigen receptor (CAR)-transduced NK cells. These cells, pivotal in innate immunity, offer a rapid and potent response against cancer cells and pathogens without the need for prior sensitization or recognition of peptide antigens. Although NK cell genetic modification is evolving, the viral transduction method continues to be inefficient and fraught with risks, often resulting in cytotoxic outcomes and the possibility of insertional mutagenesis. Consequently, there has been a surge in the development of non-viral transfection technologies to overcome these challenges in NK cell engineering. Non-viral approaches for CAR-NK cell generation are becoming increasingly essential. Cutting-edge techniques such as trogocytosis, electroporation, lipid nanoparticle (LNP) delivery, clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) gene editing and transposons not only enhance the efficiency and safety of CAR-NK cell engineering but also open new avenues for novel therapeutic possibilities. Additionally, the infusion of technologies already successful in CAR T-cell therapy into the CAR-NK paradigm holds immense potential for further advancements. In this review, we present an overview of the potential of NK cells in cancer immunotherapies, as well as non-viral transfection technologies for engineering NK cells.}, }
@article {pmid39730605, year = {2024}, author = {Sassone, F and Estay-Ahumada, C and Roux, MJ and Ciocca, D and Rossolillo, P and Birling, MC and Sparrow, JR and Montenegro, D and Hicks, D}, title = {Interruption of the visual cycle in a novel animal model induces progressive vision loss resembling Stargardts Disease.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {30880}, pmid = {39730605}, issn = {2045-2322}, support = {R01 EY012951/EY/NEI NIH HHS/United States ; NEI EY012951//UNADEV/ITMO Aviesan 2018-2021, Fondation de France (Association Berthe Fouassier) and USIAS (DH); and NEI EY012951 (JRS)./ ; }, mesh = {Animals ; *Disease Models, Animal ; *ATP-Binding Cassette Transporters/genetics/metabolism ; *Stargardt Disease ; Humans ; Retinal Cone Photoreceptor Cells/metabolism/pathology ; Macular Degeneration/pathology/genetics/metabolism ; Electroretinography ; CRISPR-Cas Systems ; Rats ; Retina/metabolism/pathology/physiopathology ; Tomography, Optical Coherence ; Retinal Degeneration/metabolism/pathology/genetics ; }, abstract = {Mutations in the gene ABCA4 coding for photoreceptor-specific ATP-binding cassette subfamily A member 4, are responsible for Stargardts Disease type 1 (STGD1), the most common form of inherited macular degeneration. STGD1 typically declares early in life and leads to severe visual handicap. Abca4 gene-deletion mouse models of STGD1 accumulate lipofuscin, a hallmark of the disease, but unlike the human disease show no or only moderate structural changes and no functional decline. The human macula is highly enriched in cones, and reasoning that the low cone percentage in mice retinas (< 3%) might compromise faithful modelling of human maculopathies, we performed sub-retinal injections of CRISPR/Cas9-abca4 Adeno-Associated Virus constructs into young Sand Rats (Psammomys obesus), a diurnal rodent containing > 30% cones. Compared to control injections of AAV-abca4-GFP, treated eyes exhibited extensive retinal degeneration by two months. Sanger sequencing of the CRISPR targeted sequence show a clear edition of Abca4 gene. Non-invasive fundus imaging showed widespread photoreceptor loss, confirmed by ocular coherence tomography. Functional recording by single flash and flicker electroretinography showed significant decline in photopic (cone) light responses. Post-mortem real-time PCR, immunohistochemistry and western blotting showed significant decrease of cone-specific (MW cone opsin) but not rod-specific (rhodopsin) markers. Transmission electron microscopy showed large numbers of lipid inclusions in treated but not control retinal pigmented epithelium. Finally, ultra-high performance liquid chromatography analysis of whole P. obesus eyes showed the presence of all-trans retinal-dimer, not detected in rod-rich rat eyes. In conclusion, Abca4 knockout in P. obesus results in a predominantly cone degeneration phenotype, more accurately reflecting the etiology of human STGD1, and should be valuable for characterizing pathogenic pathways and exploring treatment options.}, }
@article {pmid39730601, year = {2024}, author = {Nasrallah, A and Rezvani, HR and Kobaisi, F and Hammoud, A and Rambert, J and Smits, JPH and Sulpice, E and Rachidi, W}, title = {Generation and characterization of CRISPR-Cas9-mediated XPC gene knockout in human skin cells.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {30879}, pmid = {39730601}, issn = {2045-2322}, support = {ANR-18-CE17-0017//ANR grant PG2HEAL/ ; ANR-15-IDEX-02//the French National Research Agency in the framework of the "Investissements d'avenir" program (ANR-15-IDEX-02)./ ; }, mesh = {Humans ; *CRISPR-Cas Systems ; *DNA-Binding Proteins/genetics/metabolism ; *DNA Repair ; *Xeroderma Pigmentosum/genetics ; *Gene Knockout Techniques ; *Ultraviolet Rays/adverse effects ; Skin/metabolism/pathology/radiation effects ; DNA Damage ; Keratinocytes/metabolism ; Fibroblasts/metabolism/radiation effects ; Melanocytes/metabolism/radiation effects ; }, abstract = {Xeroderma pigmentosum group C (XPC) is a versatile protein crucial for sensing DNA damage in the global genome nucleotide excision repair (GG-NER) pathway. This pathway is vital for mammalian cells, acting as their essential approach for repairing DNA lesions stemming from interactions with environmental factors, such as exposure to ultraviolet (UV) radiation from the sun. Loss-of-function mutations in the XPC gene confer a photosensitive phenotype in XP-C patients, resulting in the accumulation of unrepaired UV-induced DNA damage. This remarkable increase in DNA damage tends to elevate by 10,000-fold the risk of developing melanoma and non-melanoma skin cancers. To date, creating accurate and reproducible models to study human XP-C disease has been an important challenge. To tackle this, we used CRISPR-Cas9 technology in order to knockout the XPC gene in various human skin cells (keratinocytes, fibroblasts, and melanocytes). After validation of the knockout in these edited skin cells, we showed that they recapitulate the major phenotypes of XPC mutations: photosensitivity and the impairment of UV-induced DNA damage repair. Moreover, these knockout cells demonstrated a reduced proliferative capacity compared to their respective controls. Finally, to better mimic the disease environment, we built a 3D reconstructed skin using these XPC knockout skin cells. This model exhibited an abnormal behavior, showing an extensive remodeling of its extracellular matrix compared to normal skin. Analyzing the composition of the fibroblast secretome revealed a significant augmented shift in the inflammatory response following XPC knockout. Our innovative "disease on a dish" approach can provide valuable insights into the molecular mechanisms underlying XP-C disease, paving the way to design novel preventive and therapeutic strategies to alleviate the disease phenotype. Also, given the high risk of skin cancer onset in XP-C disease, our new approach can serve as a link to draw novel insights into this elusive field.}, }
@article {pmid39730554, year = {2024}, author = {Cheng, Y and Zhang, L and Ke, Y and Dang, X and Miki, D}, title = {Double step screening using endogenous marker improves relative gene targeting efficiency in Arabidopsis.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {30791}, pmid = {39730554}, issn = {2045-2322}, support = {20ZR1467000//Shanghai Science and Technology Innovation Plan/ ; G202201355L//Foreign Expert Project/ ; }, mesh = {*Arabidopsis/genetics ; *CRISPR-Cas Systems ; *Gene Targeting/methods ; Genetic Markers ; Plants, Genetically Modified/genetics ; Arabidopsis Proteins/genetics/metabolism ; Genome, Plant ; Gene Editing/methods ; }, abstract = {Gene targeting (GT) is a powerful tool for manipulating endogenous genomic sequences as intended. However, its efficiency is rather low, especially in seed plants. Numerous attempts have been made to improve the efficiency of GT via the CRISPR/Cas systems in plants, but these have not been sufficiently effective to be used routinely by everyone. Here, we report a surrogate screening method that improves the relative efficiency of CRISPR/Cas9-mediated GT in Arabidopsis. Our findings indicate that simultaneous mutagenesis of the endogenous MAR1 gene, which results in kanamycin resistance, can be employed to efficiently screen for precise and heritable GT events at multiple endogenous sites in the Arabidopsis genome. In this study, we demonstrate that a double-step screening strategy can achieve up to a four-fold increase in the efficiency of GT in Arabidopsis. The principle of this surrogate system has the potential to be widely applied.}, }
@article {pmid39729269, year = {2025}, author = {Qiu, M and Chen, N and Wang, Y}, title = {CRISPR/Cas9-Mediated Gene-Knockout and In Situ Complementation System for Phytophthora sojae.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2892}, number = {}, pages = {69-82}, pmid = {39729269}, issn = {1940-6029}, mesh = {*Phytophthora/genetics ; *CRISPR-Cas Systems ; *Gene Knockout Techniques/methods ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genetic Complementation Test ; Plasmids/genetics ; Genetic Vectors/genetics ; }, abstract = {The establishment of reliable and efficient systems for genome editing in Phytophthora is very important for studying gene functions. Here, step-by-step methods for CRISPR/Cas9-based gene knockout and in situ complementation for Phytophthora sojae are presented. These steps include the sgRNA design, Cas9-sgRNA plasmid construction, homologous replacement, complementation vector construction, P. sojae transformation, and detection of mutations for both gene knockout and in situ complementation. These methods may also potentially be adapted for other Phytophthora species.}, }
@article {pmid39729268, year = {2025}, author = {Mendoza, CS and Ah-Fong, AMV and Judelson, HS}, title = {Gene Editing and Protein Tagging in the Oomycete Phytophthora infestans Using CRISPR-Cas12a.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2892}, number = {}, pages = {49-67}, pmid = {39729268}, issn = {1940-6029}, mesh = {*Gene Editing/methods ; *Phytophthora infestans/genetics ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; DNA End-Joining Repair/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Recombinational DNA Repair ; Genetic Vectors/genetics ; Endodeoxyribonucleases ; }, abstract = {Molecular genetic tools such as CRISPR-Cas gene editing systems are invaluable for understanding gene and protein function and revealing the details of a pathogen's life and disease cycles. Here we present protocols for genome editing in Phytophthora infestans, an oomycete with global importance as a pathogen of potato and tomato. Using a vector system that expresses variants of Cas12a from Lachnospiraceae bacterium and its guide RNA from a unified transcript, we first present a method for editing genes through the non-homologous end-joining (NHEJ) pathway. We then describe an application of homology-directed repair (HDR), in which Cas12a is used to fuse a protein-coding gene with a fluorescent or epitope tag. Both methods should be adaptable to many oomycetes other than P. infestans.}, }
@article {pmid39727993, year = {2024}, author = {Mascarenhas, MS and Nascimento, FDS and Schittino, LMP and Galinari, LB and Lino, LSM and Ramos, APS and Diniz, LEC and Mendes, TAO and Ferreira, CF and Santos-Serejo, JAD and Amorim, EP}, title = {Construction and Validation of CRISPR/Cas Vectors for Editing the PDS Gene in Banana (Musa spp.).}, journal = {Current issues in molecular biology}, volume = {46}, number = {12}, pages = {14422-14437}, pmid = {39727993}, issn = {1467-3045}, support = {OPP1093845//IITA - Accelerated Breeding of Better Bananas/ ; OPP1093845//Bill and Melinda Gates Foundation - Accelerated Breeding of Better Bananas/ ; }, abstract = {Bananas and plantains are important staple food crops affected by biotic and abiotic stresses. The gene editing technique via Clustered Regularly Interspaced Short Palindromic Repeats associated with the Cas protein (CRISPR/Cas) has been used as an important tool for development of cultivars with high tolerance to stresses. This study sought to develop a protocol for the construction of vectors for gene knockout. Here we use the phytoene desaturase (PDS) gene as a case study in Prata-Anã banana by the nonhomologous end junction (NHEJ) method. PDS is a key gene in the carotenoid production pathway in plants and its knockout leads to easily visualized phenotypes such as dwarfism and albinism in plants. Agrobacterium-mediated transformation delivered CRISPR/Cas9 constructs containing gRNAs were inserted into embryogenic cell suspension cultures. This is the first study to provide an effective method/protocol for constructing gene knockout vectors, demonstrating gene editing potential in a Brazilian banana variety. The constitutive (CaMV 35S) and root-specific vectors were successfully assembled and confirmed in transformed Agrobacterium by DNA extraction and PCR. The specificity of transformation protocols makes it possible to use the CRISPR-Cas9 technique to develop Prata-Anã banana plants with enhanced tolerance/resistance to major biotic and abiotic factors.}, }
@article {pmid39727898, year = {2024}, author = {Zhang, X and Huang, Z and Zhang, Y and Wang, W and Ye, Z and Liang, P and Sun, K and Kang, W and Tang, Q and Yu, X}, title = {Mitigating Antibiotic Resistance: The Utilization of CRISPR Technology in Detection.}, journal = {Biosensors}, volume = {14}, number = {12}, pages = {}, pmid = {39727898}, issn = {2079-6374}, support = {2022C02049//the "Pioneer" and "Leading Goose" R&amp;D Program of Zhejiang/ ; 2024SNJF044//Zhejiang Provincial Department of Agriculture and Rural Affairs Project/ ; 2023SNJF066//the Zhejiang Provincial Department of Agriculture and Rural Affairs Project/ ; }, mesh = {*CRISPR-Cas Systems ; *Anti-Bacterial Agents/pharmacology ; Humans ; Drug Resistance, Microbial ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Bacteria ; Biosensing Techniques ; Drug Resistance, Bacterial ; }, abstract = {Antibiotics, celebrated as some of the most significant pharmaceutical breakthroughs in medical history, are capable of eliminating or inhibiting bacterial growth, offering a primary defense against a wide array of bacterial infections. However, the rise in antimicrobial resistance (AMR), driven by the widespread use of antibiotics, has evolved into a widespread and ominous threat to global public health. Thus, the creation of efficient methods for detecting resistance genes and antibiotics is imperative for ensuring food safety and safeguarding human health. The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) systems, initially recognized as an adaptive immune defense mechanism in bacteria and archaea, have unveiled their profound potential in sensor detection, transcending their notable gene-editing applications. CRISPR/Cas technology employs Cas enzymes and guides RNA to selectively target and cleave specific DNA or RNA sequences. This review offers an extensive examination of CRISPR/Cas systems, highlighting their unique attributes and applications in antibiotic detection. It outlines the current utilization and progress of the CRISPR/Cas toolkit for identifying both nucleic acid (resistance genes) and non-nucleic acid (antibiotic micromolecules) targets within the field of antibiotic detection. In addition, it examines the current challenges, such as sensitivity and specificity, and future opportunities, including the development of point-of-care diagnostics, providing strategic insights to facilitate the curbing and oversight of antibiotic-resistance proliferation.}, }
@article {pmid39727873, year = {2024}, author = {Lu, Z and Ye, Z and Li, P and Jiang, Y and Han, S and Ma, L}, title = {An MSRE-Assisted Glycerol-Enhanced RPA-CRISPR/Cas12a Method for Methylation Detection.}, journal = {Biosensors}, volume = {14}, number = {12}, pages = {}, pmid = {39727873}, issn = {2079-6374}, support = {WDZC20200821104802001//Universities Stable Funding Key Projects/ ; 2020YFA0908900//The National Key R&amp;D Program of China/ ; n/a//State Key Laboratory of Chemical Oncogenomics/ ; }, mesh = {*DNA Methylation ; *CRISPR-Cas Systems ; Humans ; *Glycerol ; DNA Restriction Enzymes ; Biosensing Techniques ; Nasopharyngeal Carcinoma/diagnosis/genetics ; Nucleic Acid Amplification Techniques ; Endodeoxyribonucleases ; }, abstract = {BACKGROUND: Nasopharyngeal carcinoma (NPC) is a malignant tumor with high prevalence in southern China. Aberrant DNA methylation, as a hallmark of cancer, is extensively present in NPC, the detection of which facilitates early diagnosis and prognostic improvement of NPC. Conventional methylation detection methods relying on bisulfite conversion have limitations such as time-consuming, complex processes and sample degradation; thus, a more rapid and efficient method is needed.<br><br>METHODS: We propose a novel DNA methylation assay based on methylation-sensitive restriction endonuclease (MSRE) HhaI digestion and Glycerol-enhanced recombinase polymerase amplification (RPA)-CRISPR/Cas12a detection (HGRC). MSRE has a fast digestion rate, and HhaI specifically cleaves unmethylated DNA at a specific locus, leaving the methylated target intact to trigger the downstream RPA-Cas12a detection step, generating a fluorescence signal. Moreover, the detection step was supplemented with glycerol for the separation of Cas12a-containing components and RPA- and template-containing components, which avoids over-consumption of the template and, thus, enhances the amplification efficiency and detection sensitivity.<br><br>RESULTS: The HGRC method exhibits excellent performance in the detection of a CNE2-specific methylation locus with a (limit of detection) LOD of 100 aM and a linear range of 100 aM to 100 fM. It also responds well to different methylation levels and is capable of distinguishing methylation levels as low as 0.1%. Moreover, this method can distinguish NPC cells from normal cells by detecting methylation in cellular genomes. This method provides a rapid and sensitive approach for NPC detection and also holds good application prospects for other cancers and diseases featuring DNA methylation as a biomarker.}, }
@article {pmid39727179, year = {2025}, author = {Lopez, SC and Lee, Y and Zhang, K and Shipman, SL}, title = {SspA is a transcriptional regulator of CRISPR adaptation in E. coli.}, journal = {Nucleic acids research}, volume = {53}, number = {4}, pages = {}, pmid = {39727179}, issn = {1362-4962}, support = {MCB 2137692//National Science Foundation/ ; //Pew Biomedical Scholars Program/ ; //Chan Zuckerberg Biohub - San Francisco/ ; //Berkeley Fellowship for Graduate Study/ ; }, mesh = {*Escherichia coli/genetics/virology/metabolism ; *Escherichia coli Proteins/genetics/metabolism/physiology ; *CRISPR-Cas Systems/genetics ; *Gene Expression Regulation, Bacterial ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Adaptation, Physiological/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; Transcription, Genetic ; }, abstract = {The CRISPR integrases Cas1-Cas2 create immunological memories of viral infection by storing phage-derived DNA in CRISPR arrays, a process known as CRISPR adaptation. A number of host factors have been shown to influence adaptation, but the full pathway from infection to a fully integrated, phage-derived sequences in the array remains incomplete. Here, we deploy a new CRISPRi-based screen to identify putative host factors that participate in CRISPR adaptation in the Escherichia coli Type I-E system. Our screen and subsequent mechanistic characterization reveal that SspA, through its role as a global transcriptional regulator of cellular stress, is required for functional CRISPR adaptation. One target of SspA is H-NS, a known repressor of CRISPR interference proteins, but we find that the role of SspA on adaptation is not H-NS-dependent. We propose a new model of CRISPR-Cas defense that includes independent cellular control of adaptation and interference by SspA.}, }
@article {pmid39727169, year = {2025}, author = {Pujar, A and Pathania, A and Hopper, C and Pandi, A and Calderón, CR and Függer, M and Nowak, T and Kushwaha, M}, title = {Phage-mediated intercellular CRISPRi for biocomputation in bacterial consortia.}, journal = {Nucleic acids research}, volume = {53}, number = {3}, pages = {}, pmid = {39727169}, issn = {1362-4962}, support = {//igicosme working group HicDiesMeus/ ; COMBACT//Ile-de-France (IdF) region's DIM-RFSI/ ; BACON//NS2I CNRS/ ; DEPEC MODE//Universit&#xe9; Paris-Saclay's STIC department/ ; PHEMO//INRAE's MICA department/ ; ANR-21-CE48-0003//Agence Nationale de la Recherche/ ; }, mesh = {*CRISPR-Cas Systems ; *Bacteriophages/genetics ; *Microbial Consortia/genetics ; *Bacteria/genetics/virology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Regulation, Bacterial ; Escherichia coli/genetics ; }, abstract = {Coordinated actions of cells in microbial communities and multicellular organisms enable them to perform complex tasks otherwise difficult for single cells. This has inspired biological engineers to build cellular consortia for larger circuits with improved functionalities while implementing communication systems for coordination among cells. Here, we investigate the signalling dynamics of a phage-mediated synthetic DNA messaging system and couple it with CRISPR interference to build distributed circuits that perform logic gate operations in multicellular bacterial consortia. We find that growth phases of both sender and receiver cells, as well as resource competition between them, shape communication outcomes. Leveraging the easy programmability of DNA messages, we build eight orthogonal signals and demonstrate that intercellular CRISPRi (i-CRISPRi) regulates gene expression across cells. Finally, we multiplex the i-CRISPRi system to implement several multicellular logic gates that involve up to seven cells and take up to three inputs simultaneously, with single- and dual-rail encoding: NOT, YES, AND and AND-AND-NOT. The communication system developed here lays the groundwork for implementing complex biological circuits in engineered bacterial communities, using phage signals for communication.}, }
@article {pmid39726635, year = {2024}, author = {Brant, EJ and May, D and Eid, A and Altpeter, F}, title = {Comparison of genotyping assays for detection of targeted CRISPR/Cas mutagenesis in highly polyploid sugarcane.}, journal = {Frontiers in genome editing}, volume = {6}, number = {}, pages = {1505844}, pmid = {39726635}, issn = {2673-3439}, abstract = {Sugarcane (Saccharum spp.) is an important biofuel feedstock and a leading source of global table sugar. Saccharum hybrid cultivars are highly polyploid (2n = 100-130), containing large numbers of functionally redundant hom(e)ologs in their genomes. Genome editing with sequence-specific nucleases holds tremendous promise for sugarcane breeding. However, identification of plants with the desired level of co-editing within a pool of primary transformants can be difficult. While DNA sequencing provides direct evidence of targeted mutagenesis, it is cost-prohibitive as a primary screening method in sugarcane and most other methods of identifying mutant lines have not been optimized for use in highly polyploid species. In this study, non-sequencing methods of mutant screening, including capillary electrophoresis (CE), Cas9 RNP assay, and high-resolution melt analysis (HRMA), were compared to assess their potential for CRISPR/Cas9-mediated mutant screening in sugarcane. These assays were used to analyze sugarcane lines containing mutations at one or more of six sgRNA target sites. All three methods distinguished edited lines from wild type, with co-mutation frequencies ranging from 2% to 100%. Cas9 RNP assays were able to identify mutant sugarcane lines with as low as 3.2% co-mutation frequency, and samples could be scored based on undigested band intensity. CE was highlighted as the most comprehensive assay, delivering precise information on both mutagenesis frequency and indel size to a 1 bp resolution across all six targets. This represents an economical and comprehensive alternative to sequencing-based genotyping methods which could be applied in other polyploid species.}, }
@article {pmid39726634, year = {2024}, author = {Azeez, SS and Hamad, RS and Hamad, BK and Shekha, MS and Bergsten, P}, title = {Advances in CRISPR-Cas technology and its applications: revolutionising precision medicine.}, journal = {Frontiers in genome editing}, volume = {6}, number = {}, pages = {1509924}, pmid = {39726634}, issn = {2673-3439}, abstract = {CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated proteins) has undergone marked advancements since its discovery as an adaptive immune system in bacteria and archaea, emerged as a potent gene-editing tool after the successful engineering of its synthetic guide RNA (sgRNA) toward the targeting of specific DNA sequences with high accuracy. Besides its DNA editing ability, further-developed Cas variants can also edit the epigenome, rendering the CRISPR-Cas system a versatile tool for genome and epigenome manipulation and a pioneering force in precision medicine. This review explores the latest advancements in CRISPR-Cas technology and its therapeutic and biomedical applications, highlighting its transformative impact on precision medicine. Moreover, the current status of CRISPR therapeutics in clinical trials is discussed. Finally, we address the persisting challenges and prospects of CRISPR-Cas technology.}, }
@article {pmid39726403, year = {2024}, author = {Rasool, HMH and Chen, Q and Gong, X and Zhou, J}, title = {CRISPR/Cas system and its application in the diagnosis of animal infectious diseases.}, journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology}, volume = {38}, number = {24}, pages = {e70252}, pmid = {39726403}, issn = {1530-6860}, support = {2024BEG02029//The Key Research and Development Program of Ningxia Hui Autonomous Region/ ; 2022YFC2304001//MOST | National Key Research and Development Program of China (NKPs)/ ; CAAS-ZDRW202410//ASTIP | The Agricultural Science and Technology Innovation Program/ ; }, mesh = {Animals ; Humans ; *Communicable Diseases/diagnosis/genetics/veterinary ; *CRISPR-Cas Systems ; Molecular Diagnostic Techniques/methods ; Zoonoses/diagnosis/genetics ; }, abstract = {Infectious diseases are a serious threat to the existence of animals and humans' life. In the 21st century, the emergence and re-emergence of several zoonotic and non-zoonotic global pandemic diseases of socio-economic importance has affected billions of humans and animals. The need for expensive equipment and laboratories, non-availability of on-site testing abilities, with time-consuming and low sensitivity and specificity issues of currently available diagnostic techniques to identify these pathogenic micro-organisms on a large scale highlighted the need for developing cheap, portable environment friendly diagnostic methods. In recent years, these issues have been addressed by clustered regularly interspaced palindromic repeats (CRISPR)-based diagnostic platforms that have transformed the molecular diagnostic field due to their outstanding ultra-sensitive nucleic acid detecting capabilities. In this study, we highlight the types, potential of different Cas proteins, and amplification systems. We also illuminate the application of currently available CRISPR integrated setups on the diagnosis of infectious diseases, majorly in food-producing animals (pigs, ruminants, poultry, and aquaculture), domestic pets (dogs and cats), and diseases of zoonotic importance. We conclude the challenges and future perspectives of using these systems to rapidly diagnose and treat other infectious diseases and also develop control strategies to prevent the spread of pathogenic organisms.}, }
@article {pmid39725739, year = {2024}, author = {Pi, N and Xiang, R and Zhu, L and Li, Y and Wu, X}, title = {An HRP-integrated CRISPR/Cas12a biosensor towards chair-side diagnosis for Porphyromonas gingivalis.}, journal = {Biotechnology letters}, volume = {47}, number = {1}, pages = {15}, pmid = {39725739}, issn = {1573-6776}, support = {32060024//National Natural Science Foundation of China/ ; 32000088//National Natural Science Foundation of China/ ; 2022JJ40050//Natural Science Foundation of Hunan Province/ ; }, mesh = {*Porphyromonas gingivalis/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; Bacteroidaceae Infections/diagnosis/microbiology ; Periodontitis/microbiology/diagnosis ; }, abstract = {Rapid diagnostic tools for Porphyromonas gingivalis (Pg), the primary microorganism responsible for the development of periodontitis, particularly those designed for chair-side applications, could provide substantial health benefits to patients. To address this issue, we developed a CRISPR/Cas12a-based rapid Pg detection method. Dual-gRNA and hairpin reporter strategies were employed to enhance CRISPR/Cas12a reaction efficiency. By modifying the hairpin reporter with HRP, the pre-amplification-free HRP-CRISPR/Cas12a reaction was enabled to produce a colorimetric output, amplifying the detection signal. This method achieved high sensitivity (as low as 33 CFU) without the risk of aerosol contamination from pre-amplification. When testing clinical samples, the method showed high consistency with the reference RT-PCR. Furthermore, compared with RT-PCR, this method only requires room temperature operation, is simpler, and has a shorter detection time of about 35 min. In conclusion, the pre-amplification-free HRP-integrated CRISPR/Cas12a detection method requires no complex equipment, making it an ideal, end-user-friendly approach for chair-side Pg detection.}, }
@article {pmid39725167, year = {2025}, author = {Sang, N and Ma, B and Liu, H and Feng, T and Huang, X}, title = {CRISPR/Cas9-mediated GhFT-targeted mutagenesis prolongs indeterminate growth and alters plant architecture in cotton.}, journal = {Plant science : an international journal of experimental plant biology}, volume = {352}, number = {}, pages = {112374}, doi = {10.1016/j.plantsci.2024.112374}, pmid = {39725167}, issn = {1873-2259}, mesh = {*Gossypium/genetics/growth &amp; development ; *CRISPR-Cas Systems/genetics ; Flowers/growth &amp; development/genetics ; *Plant Proteins/genetics/metabolism ; Mutagenesis ; Gene Expression Regulation, Plant ; Gene Editing ; }, abstract = {The shift from vegetative to reproductive growth is an important developmental transition that affects flowering and maturation, architecture, and ecological adaptability in plants. The florigen-antiflorigen system universally controls flowering and plant architecture, and changes to the ratio of these components alter this transition and disrupt growth. The genes FT (FLOWERING LOCUS T), encoding the florigen protein FT, and CETS [CENTRORADIALIS (CEN)/TERMINAL FLOWER1 (TFL1)/SELF-PRUNING (SP)], encoding antiflorigen proteins, have opposing roles. Upland cotton (Gossypium hirsutum) is one of the world's most widely cultivated cotton varieties, and its complex allotetraploid genome contains only one homoeologous pair of FT genes (GhFT-A and GhFT-D). The functionally conserved gene GhFT promotes flowering and plays a role in plant architecture, although the molecular regulation of flowering and plant architecture in cotton remains unclear. In this study, CRISPR/Cas9 technology was used to induce mutations in the first and second exons of GhFT, respectively. G. hirsutum cv. YZ-1 was transformed with a CRISPR/Cas9-GhFT vector using Agrobacterium tumefaciens, and a diverse set of mutations was identified at the editing site. Compared with the wild type, mutant plants could not transition between vegetative and reproductive growth, and significant alterations to plant architecture were observed. Quantitative RT-PCR revealed downregulation of the homologous floral meristem identity genes APETALA1 (GhAP1) and OVEREXPRESSION OF CONSTANS 1 (GhSOC1) and upregulation of the TFL1 homologs GhTFL1-1 and GhTFL1-2. These results suggested that GhFT played a significant role in flowering time and plant architecture and that the ratio of florigen-antiflorigen components was critical to producing improved cotton varieties. This study provided a basis for future investigations of molecular breeding in cotton and guidance for the agricultural production of this crop.}, }
@article {pmid39725131, year = {2025}, author = {Compiro, P and Chomta, N and Nimnual, J and Sunantawanit, S and Payungporn, S and Rotcheewaphan, S and Keawsapsak, P}, title = {CRISPR-Cas12a-based detection and differentiation of Mycobacterium spp.}, journal = {Clinica chimica acta; international journal of clinical chemistry}, volume = {567}, number = {}, pages = {120101}, doi = {10.1016/j.cca.2024.120101}, pmid = {39725131}, issn = {1873-3492}, mesh = {*CRISPR-Cas Systems/genetics ; *Mycobacterium/genetics/isolation &amp; purification/classification ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Mycobacterium species cause several vital human diseases, including tuberculosis and non-tuberculous mycobacterial infections, which are treated with different drug regimens Therefore, accurate and rapid diagnosis is essential for effective treatment and controlling the spread of these pathogens. This study aims to develop an isothermal method combining RPA and CRISPR-Cas12a techniques, named as MyTRACK, to detect and differentiate major clinical mycobacteria at the species level. The assay has no cross-reactivity with limit of detection of 1 to 100 copies/reaction for various targeted mycobacteria. The results demonstrated 100 % specificity and 92.59 % to 100 % sensitivity in clinical isolates and were consistent with the culture technique with LPA for clinical samples. The MyTRACK assay is an effective, portable, rapid, and accurate screening method for mycobacterial detection and identification, especially in low-resource clinical settings.}, }
@article {pmid39725104, year = {2025}, author = {Sun, L and Wang, R and Ma, S and Shi, R and Zhao, P and Xia, Q}, title = {Complete BmFib-L knockout reveals its indispensable role in silk fiber formation.}, journal = {International journal of biological macromolecules}, volume = {294}, number = {}, pages = {138759}, doi = {10.1016/j.ijbiomac.2024.138759}, pmid = {39725104}, issn = {1879-0003}, mesh = {Animals ; *Bombyx/genetics/metabolism ; *Silk/genetics/biosynthesis/chemistry/metabolism ; *Fibroins/genetics/metabolism ; Gene Knockout Techniques ; *Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Phenotype ; }, abstract = {Silkworm (Bombyx mori), belonging to the order Lepidoptera, is an important model insect for economic and scientific research. The capacity of the silkworm to secrete robust silk renders it a valuable economic resource, while its biological characteristics offer insights into a number of scientific disciplines. Despite the extensive research conducted to elucidate the mechanisms of silk secretion, many aspects remain unclear. In this study, a mutant strain was generated by editing the BmFib-L gene for silk fibroin, BmFib-L was observed to terminate prematurely at 27 amino acids, using the CRISPR/Cas9 system. The BmFib-L was entirely absent in the mutant strain in comparison to the wild type (WT). On the third day of the fifth instar, the posterior silk glands of the mutant strain were dissected and found to have no significant morphological differences compared to the WT. However, the mutant cocoon phenotypes exhibited significant changes. The findings demonstrated that BmFib-L plays a pivotal role in silk formation, yet is not indispensable for silk secretion. Furthermore, BmFib-L exerts a discernible influence on the mechanical properties of silk. A comparative analysis of the transcriptome of the mutant strain and the WT revealed that the elevated expression of transporter and metabolism-related genes in the mutant strain preserved the equilibrium of the silk gland cells, thereby guaranteeing the typical development of the mutant silk gland cells. These findings offer a novel perspective for investigating the mechanism of silk secretion in silkworms.}, }
@article {pmid39724854, year = {2025}, author = {Li, X and Dong, J and Deng, L and Huo, D and Yang, M and Hou, C}, title = {CRISPR/Cas12a regulated preassembled bulb-shaped G-quadruplex signal unit for FL/CM dual-mode ultrasensitive detection of miRNA-155.}, journal = {Talanta}, volume = {286}, number = {}, pages = {127413}, doi = {10.1016/j.talanta.2024.127413}, pmid = {39724854}, issn = {1873-3573}, mesh = {*MicroRNAs/genetics/analysis/blood ; *G-Quadruplexes ; Humans ; *CRISPR-Cas Systems ; Colorimetry/methods ; Limit of Detection ; *Biosensing Techniques/methods ; Fluorescence ; Spectrometry, Fluorescence ; Nucleic Acid Amplification Techniques ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {High sensitivity and specificity in microRNA detection are of great significance for early cancer screening. This study employed a pre-assembled bulb-shaped G-quadruplex signal unit (G4MB) as a novel and efficient label-free probe. The products amplified by the miRNA-155-targeted exponential amplification reaction (EXPAR) activated the trans-cleavage activity of CRISPR/Cas12a, disrupting the G4MB structure to achieve dual-channel fluorescence/colorimetric (FL/CM) inverse signal output. Due to the strong signal amplification of EXPAR, the highly efficient cleavage by CRISPR/Cas12a, and the ultra-high response signal of the structurally stable G4MB probe, the FL mode achieved a high signal-to-noise ratio (S/N) of approximately 12.5. The CM mode, combined with smart devices for RGB curve adjustment, successfully corrected the background and provided precise and objective image data support while allowing results to be observed with the naked eye. Additionally, the sensor system exhibited high accuracy in complex human serum environments and RNA extracted from three different types of cells. Moreover, the G4MB probe required no complicated labeling, demonstrated structural stability, and had a rapid response. Most importantly, this study analyzed the advantages of the G4MB and applied it to miRNA detection for the first time, providing practical insights for biosensor construction, molecular diagnostics, and clinical applications.}, }
@article {pmid39724853, year = {2025}, author = {Zhao, L and Zhao, Z and Li, N and Wang, X}, title = {The nucleic acid detection using CRISPR/Cas biosensing system with micro-nano modality for point-of-care applications.}, journal = {Talanta}, volume = {286}, number = {}, pages = {127457}, doi = {10.1016/j.talanta.2024.127457}, pmid = {39724853}, issn = {1873-3573}, mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Point-of-Care Systems ; Humans ; *Nucleic Acids/analysis/genetics ; *Nanotechnology/methods ; }, abstract = {Nucleic acid detection is considered the golden standard for diagnosing infectious diseases caused by various pathogens, including viruses, bacteria, and parasites. PCR and other amplification-based technologies are highly sensitive and specific, allowing for accurate detection and identification of low-level causative pathogens by targeting and amplifying their unique genetic segment (DNA or RNA). However, it is important to recognize that machinery-dependent diagnostic methods may only sometimes be available or practical in resource-limited settings, where direct implementation can be challenging. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based diagnostics offer a promising alternative for nucleic acid detection. These methods provide gene sequence-specific targeting, multiplexing capability, rapid result disclosure, and ease of operation, making them suitable for point-of-care (POC) applications. CRISPR-Cas-based nucleic acid detection leverages the intrinsic gene-editing capabilities of CRISPR systems to detect specific DNA or RNA sequences with high precision, ensuring high specificity in identifying pathogens. When integrated with micro- and nano-technologies, CRISPR-based diagnostics gain additional benefits, including automated microfluidic processes, enhanced multiplexed detection, improved sensitivity through nanoparticle integration, and combined detection strategies. In this review, we analyze the motivations for tailoring the CRISPR-Cas system with microfluidic formats or nanoscale materials for nucleic acid biosensing and detection. We discuss and categorize current achievements in such systems, highlighting their differences, commonalities, and opportunities for addressing challenges, particularly for POC diagnostics. Micro- and nano-technologies can significantly enhance the practical utility of the CRISPR-Cas system, enabling more comprehensive diagnostic and surveillance capabilities. By integrating these technologies, CRISPR-based diagnostics can achieve higher levels of automation, sensitivity, and multiplexing, making them invaluable tools in the global effort to diagnose and control infectious diseases.}, }
@article {pmid39724343, year = {2025}, author = {Nakajima, T and Kuwasaki, Y and Yamamoto, S and Otabe, T and Sato, M}, title = {A Red Light-Activatable Endogenous Gene Transcription System with Red-CPTS.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2840}, number = {}, pages = {45-55}, pmid = {39724343}, issn = {1940-6029}, mesh = {Animals ; *Light ; Mice ; *CRISPR-Cas Systems ; *Optogenetics/methods ; Humans ; *Transcription, Genetic/radiation effects ; Red Light ; }, abstract = {Red light penetrates deep into mammalian tissues and has low phototoxicity. We developed a red light-activatable photoswitch (MagRed) for deep tissue optogenetics. Using MagRed, we developed a red light-activatable endogenous gene transcription system (Red-CPTS) based on CRISPR-Cas9. Here we provide a detailed protocol for endogenous gene activation using Red-CPTS in cultured mammalian cells and living mice in vivo.}, }
@article {pmid39724342, year = {2025}, author = {Renzl, C and Mayer, G}, title = {Optoribogenetic Modulation of Transcription.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2840}, number = {}, pages = {37-44}, pmid = {39724342}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems ; *Optogenetics/methods ; Humans ; Transcription, Genetic ; Light ; Transfection/methods ; HEK293 Cells ; Gene Expression Regulation ; Flow Cytometry/methods ; }, abstract = {Light can be used as a precise and reversible trigger for the activation of optogenetic tools with subcellular resolution. The interaction of the photoreceptor PAL and aptamer 53 was integrated into a CRISPR/dCas9 system, which can be applied for light-controlled activation of gene expression. Here, we describe a protocol for in vitro application of light-dependent overexpression using eBFP as a proof of concept. The experiment can be done in 3 days, which is split into cell seeding, transfection, and evaluation by flow cytometry. The method is broadly applicable including the upregulation of endogenous genes.}, }
@article {pmid39722550, year = {2025}, author = {Victor Atoki, A and Aja, PM and Shinkafi, TS and Ondari, EN and Adeniyi, AI and Fasogbon, IV and Dangana, RS and Shehu, UU and Akin-Adewumi, A}, title = {Exploring the versatility of Drosophila melanogaster as a model organism in biomedical research: a comprehensive review.}, journal = {Fly}, volume = {19}, number = {1}, pages = {2420453}, pmid = {39722550}, issn = {1933-6942}, mesh = {Animals ; *Drosophila melanogaster/genetics ; *Disease Models, Animal ; Biomedical Research ; Humans ; CRISPR-Cas Systems ; }, abstract = {Drosophila melanogaster is a highly versatile model organism that has profoundly advanced our understanding of human diseases. With more than 60% of its genes having human homologs, Drosophila provides an invaluable system for modelling a wide range of pathologies, including neurodegenerative disorders, cancer, metabolic diseases, as well as cardiac and muscular conditions. This review highlights key developments in utilizing Drosophila for disease modelling, emphasizing the genetic tools that have transformed research in this field. Technologies such as the GAL4/UAS system, RNA interference (RNAi) and CRISPR-Cas9 have enabled precise genetic manipulation, with CRISPR-Cas9 allowing for the introduction of human disease mutations into orthologous Drosophila genes. These approaches have yielded critical insights into disease mechanisms, identified novel therapeutic targets and facilitated both drug screening and toxicological studies. Articles were selected based on their relevance, impact and contribution to the field, with a particular focus on studies offering innovative perspectives on disease mechanisms or therapeutic strategies. Our findings emphasize the central role of Drosophila in studying complex human diseases, underscoring its genetic similarities to humans and its effectiveness in modelling conditions such as Alzheimer's disease, Parkinson's disease and cancer. This review reaffirms Drosophila's critical role as a model organism, highlighting its potential to drive future research and therapeutic advancements.}, }
@article {pmid39722520, year = {2024}, author = {Deng, K and Li, M and Zhang, H and Deng, Y and Qin, Y and Qin, C}, title = {[Characterization of host factors ARF4 and ARF5 upon Zika virus infection in vivo by construction of gene knockout mice].}, journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology}, volume = {40}, number = {12}, pages = {4605-4615}, doi = {10.13345/j.cjb.240307}, pmid = {39722520}, issn = {1872-2075}, mesh = {Animals ; *ADP-Ribosylation Factors/genetics/metabolism ; *Zika Virus Infection/genetics ; Mice ; *Mice, Knockout ; *Zika Virus/genetics ; *Mice, Inbred C57BL ; *CRISPR-Cas Systems ; Viral Load ; Male ; Female ; }, abstract = {The effects of host factors ADP-ribosylation factor 4 (ARF4) and ADP-ribosylation factor 5 (ARF5) upon Zika virus (ZIKV) infection in vivo were characterized by construction of gene knockout mice via CRISPR-Cas9. Firstly, ARF5 and ARF4 genes were modified by the CRISPR-Cas9 system and then microinjected into the fertilized eggs of C57BL/6JGpt mice. Fertilized eggs were transplanted to obtain ARF4 or ARF5 knockout (ARF4KO or ARF5KO) mice, and ARF4/5 double knockout mice were achieved by the mating between ARF4KO and ARF5KO mice (ARF4KO/ARF5KO). Then, the mouse genotypes were identified by PCR to identify the positive knockout mice, and RT-qPCR was employed to examine the knockout efficiency. The mice were then infected with ZIKV and the blood and tissue samples were collected after 2, 4, and 6 days. RT-qPCR was then employed to determine the virus load, and hematoxylin-eosin staining was employed to observe the pathological changes in the tissue. The results showed that expected PCR bands were detected from ARF4KO[-/+], ARF5KO[-/-], and ARF4KO[-/+]/ARF5KO[-/-] mice, respectively. The results of mRNA transcription measurement indicated the significant knockdown of ARF4 by 37.8%-50.0% but not ARF5 in ARF4KO[-/+] compared with the wild-type mice. Meanwhile, complete knockout of ARF5 and no changes in ARF4 were observed in ARF5KO[-/-] mice. Additionally, completed knockout of ARF5 and down-regulated mRNA level of ARF4 in the lung, kidney, and testis were detected in ARF4KO[-/+]/ARF5KO[-/-]mice in comparison with the wild-type mice. The virus load in the serum decreased in ARF4KO[-/+] mice, while it showed no significant change in ARF5KO[-/-] or ARF4KO[-/+]/ARF5KO[-/-] mice compared with that in the wild type. Meanwhile, ARF4KO[-/+] mice showcased no significant difference in virus load in various tissues but attenuated pathological changes in the brain and testis compared with the wild-type mice. We successfully constructed ARF4KO and ARF5KO mice by CRISPR-Cas9 in this study. ARF4 rather than ARF5 is essential for ZIKV infection in vivo. This study provided animal models for studying the roles of ARF4 and ARF5 in ZIKV infection and developing antivirals.}, }
@article {pmid39722468, year = {2025}, author = {Lohani, N and Singh, MB and Bhalla, PL}, title = {Deciphering the Vulnerability of Pollen to Heat Stress for Securing Crop Yields in a Warming Climate.}, journal = {Plant, cell &amp; environment}, volume = {48}, number = {4}, pages = {2549-2580}, doi = {10.1111/pce.15315}, pmid = {39722468}, issn = {1365-3040}, mesh = {*Pollen/physiology/growth &amp; development/genetics ; *Crops, Agricultural/physiology/growth &amp; development/genetics ; *Heat-Shock Response/physiology ; *Climate Change ; Global Warming ; Thermotolerance ; }, abstract = {Climate change is leading to more frequent and severe extreme temperature events, negatively impacting agricultural productivity and threatening global food security. Plant reproduction, the process fundamental to crop yield, is highly susceptible to heatwaves, which disrupt pollen development and ultimately affect seed-set and crop yields. Recent research has increasingly focused on understanding how pollen grains from various crops react to heat stress at the molecular and cellular levels. This surge in interest over the last decade has been driven by advances in genomic technologies, such as single-cell RNA sequencing, which holds significant potential for revealing the underlying regulatory reprogramming triggered by heat stress throughout the various stages of pollen development. This review focuses on how heat stress affects gene regulatory networks, including the heat stress response, the unfolded protein response, and autophagy, and discusses the impact of these changes on various stages of pollen development. It highlights the potential of pollen selection as a key strategy for improving heat tolerance in crops by leveraging the genetic variability among pollen grains. Additionally, genome-wide association studies and population screenings have shed light on the genetic underpinnings of traits in major crops that respond to high temperatures during male reproductive stages. Gene-editing tools like CRISPR/Cas systems could facilitate precise genetic modifications to boost pollen heat resilience. The information covered in this review is valuable for selecting traits and employing molecular genetic approaches to develop heat-tolerant genotypes.}, }
@article {pmid39721357, year = {2025}, author = {Petruškevičiūtė, A and Šimuliūnaitė, U and Polanco, CM and Rojas, B and Kuras, S and Valatkaitė-Rakštienė, B and Norvilas, R and Vijaya, AK and Muñoz, P and Neniškytė, U and Jakubauskas, A and Burokas, A and Nalvarte, I and Inzunza, J and Naumovas, D and Stoškus, M and Griškevičius, L and Baltriukienė, D and Arias, J}, title = {Generation of a genetically encoded voltage indicator MARINA reporter human iPS cell line using Cas9 (VULSCi002-A-2).}, journal = {Stem cell research}, volume = {82}, number = {}, pages = {103628}, doi = {10.1016/j.scr.2024.103628}, pmid = {39721357}, issn = {1876-7753}, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Line ; *CRISPR-Cas Systems ; *Genes, Reporter ; Gene Knock-In Techniques ; Membrane Potentials ; }, abstract = {Fluorescent protein-based Genetically Encoded Voltage Indicators (GEVI) offer a remarkable system for high-throughput screening of membrane potential phenotypes. The GEVI MARINA is a derivative from ArcLight, which conversely to ArcLight increases its fluorescence intensity alongside depolarization. Here we created knock-in reporter human iPS cell lines carrying the MARINA reporter using SpCas9 programmable nuclease and characterize a heterozygous clone.}, }
@article {pmid39721023, year = {2025}, author = {Chen, C and Liao, Y and Zhu, M and Wang, L and Yu, X and Li, M and Peng, G}, title = {Dual-nuclease single-cell lineage tracing by Cas9 and Cas12a.}, journal = {Cell reports}, volume = {44}, number = {1}, pages = {115105}, doi = {10.1016/j.celrep.2024.115105}, pmid = {39721023}, issn = {2211-1247}, mesh = {Animals ; *Cell Lineage/genetics ; *CRISPR-Cas Systems/genetics ; Mice ; *Single-Cell Analysis/methods ; Gene Editing/methods ; Embryoid Bodies/metabolism/cytology ; *CRISPR-Associated Protein 9/metabolism ; *CRISPR-Associated Proteins/metabolism/genetics ; Cell Differentiation ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {Single-cell lineage tracing based on CRISPR-Cas9 gene editing enables the simultaneous linkage of cell states and lineage history at a high resolution. Despite its immense potential in resolving the cell fate determination and genealogy within an organism, existing implementations of this technology suffer from limitations in recording capabilities and considerable barcode dropout. Here, we introduce DuTracer, a versatile tool that utilizes two orthogonal gene editing systems to record cell lineage history at single-cell resolution in an inducible manner. DuTracer shows the ability to enhance lineage recording with minimized target dropouts and potentially deeper tree depths. Applying DuTracer in mouse embryoid bodies and neuromesodermal organoids illustrates the lineage relationship of different cell types and proposes potential lineage-biased molecular drivers, showcased by identifying transcription factor Foxb1 as a modulator in the cell fate determination of neuromesodermal progenitors. Collectively, DuTracer facilitates the precise and regulatory interrogation of cellular lineages of complex biological processes.}, }
@article {pmid39721016, year = {2025}, author = {Huang, W and Wang, J and Wang, C and Liu, Y and Li, W and Chen, Q and Zhai, J and Xiang, Z and Liu, C}, title = {Expanding Cas12a Activity Control with an RNA G-Quadruplex at the 5' end of CRISPR RNA.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {12}, number = {7}, pages = {e2411305}, pmid = {39721016}, issn = {2198-3844}, support = {22307150//National Natural Science Foundation of China/ ; 82401294//National Natural Science Foundation of China/ ; A2303012//Shenzhen Medical Research Fund/ ; 2024A1515012319//Guangdong Basic and Applied Basic Research Foundation/ ; JCYJ20230807110315032//Shenzhen Science and Technology Program/ ; JCYJ20240813150427036//Shenzhen Science and Technology Program/ ; }, mesh = {*G-Quadruplexes ; *CRISPR-Associated Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; Humans ; *RNA/genetics ; *Bacterial Proteins/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Precise control of Cas12a activity is essential for the improvement of the detection limit of clinical diagnostics and the minimization of errors. This study addresses the challenge of controlling Cas12a activity, especially in the context of nucleic acid detection where the inherent incompatibility between isothermal amplification and CRISPR reactions complicates accurate diagnostics. An RNA G-quadruplex (RG4) structure at the 5' end of crRNA is introduced to modulate Cas12a activity accurately without the need for chemical modifications. The results indicate that the presence of RG4 does not significantly impact Cas12a's cleavage activity but can be controlled by RG4 stabilizers, enabling the suppression and subsequent restoration of Cas12a activity with potential for precise activity control. Moreover, the use of RG4 is expanded by incorporating it into split crRNA, introducing RG4 directly at the 5' end of the direct repeat (DR) region, enabling tailored activity regulation for different targets by matching with various Spacer regions. Additionally, a light-controlled one-pot method for activating Cas12a is developed, thereby enhancing the accuracy and sensitivity of clinical samples. This study showcases the pioneering use of RG4 in manipulating Cas12a activity, streamlining diagnostics, and paving the way for advances in clinical nucleic acid testing.}, }
@article {pmid39720925, year = {2025}, author = {Xu, Y and Chen, B and He, M and Yuan, G and Hu, B}, title = {Dual-Amplification Single-Particle ICP-MS Strategy Based on Strand Displacement Amplification-CRISPR/Cas12a Amplification for Homogeneous Detection of miRNA.}, journal = {Analytical chemistry}, volume = {97}, number = {1}, pages = {811-817}, doi = {10.1021/acs.analchem.4c05385}, pmid = {39720925}, issn = {1520-6882}, mesh = {*MicroRNAs/analysis/genetics ; Humans ; *CRISPR-Cas Systems ; Metal Nanoparticles/chemistry ; Gold/chemistry ; *Mass Spectrometry/methods ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; MCF-7 Cells ; }, abstract = {MicroRNAs (miRNAs) regulate a myriad of biological processes and thus have been regarded as useful biomarkers in biomedical research and clinical diagnosis. The specific and highly sensitive detection of miRNAs is of significant importance. Herein, a sensitive and rapid dual-amplification elemental labeling single-particle inductively coupled plasma-mass spectrometry (spICP-MS) analytical method based on strand displacement amplification (SDA) and CRISPR/Cas12a was developed for miRNA-21 detection. Taking gold nanoparticles (AuNPs) as the elemental labels, the Au NP probe initially hybridized with linker DNA, forming large aggregates. In the absence of target miRNA-21, large aggregates of AuNPs will produce high pulse signals in spICP-MS detection. In the presence of the target miRNA-21, it triggered the SDA reaction, and the SDA products activated CRISPR/Cas12a's trans-cleavage activity to cleave the linker DNA, resulting in disassembly of the AuNP aggregates. The AuNP aggregates with smaller size displayed lower pulse signals in spICP-MS detection. Under the optimal conditions, a good relationship between the average pulse signal intensity of AuNP aggregates and the concentration of miRNA-21 was obtained in the range of 0.5 fmol L[-1]-100 pmol L[-1] with a quantification limit as low as 0.5 fmol L[-1]. The developed method was successfully used for determination of miRNA-21 in human breast cancer cell lines (SK-BR-3 and MCF-7) and real blood samples from breast cancer patients. It is versatile, can be adapted to detect other targets by modifying the specific sequence of the SDA template chain that is complementary to the analytes, and offers a promising strategy for detecting various biomarkers with high sensitivity and specificity.}, }
@article {pmid39720847, year = {2025}, author = {Tang, A and Yokota, T}, title = {Is Duchenne gene therapy a suitable treatment despite its immunogenic class effect?.}, journal = {Expert opinion on drug safety}, volume = {24}, number = {4}, pages = {395-411}, doi = {10.1080/14740338.2024.2447072}, pmid = {39720847}, issn = {1744-764X}, mesh = {Humans ; *Muscular Dystrophy, Duchenne/therapy/genetics/physiopathology ; *Genetic Therapy/methods/adverse effects ; Gene Editing/methods ; CRISPR-Cas Systems ; Animals ; Dystrophin/genetics ; Genetic Vectors ; }, abstract = {INTRODUCTION: Duchenne muscular dystrophy (DMD) is a severe X-linked disorder characterized by progressive muscle weakness and eventual death due to cardiomyopathy or respiratory complications. Currently, there is no cure for DMD, with standard treatments primarily focusing on symptom management. Using immunosuppressive measures and optimized vector designs allows for gene therapies to better address the genetic cause of the disease.<br><br>AREAS COVERED: This review evaluates the efficacy and safety of emerging DMD gene therapies as of 2024. It also discusses the potential of utrophin upregulation, gene editing, and truncated dystrophin as therapeutic strategies. It highlights safety concerns associated with these therapies, including adverse events and patient deaths. A comprehensive overview of developments covers topics such as CRISPR-Cas9 therapies, micro-dystrophin, and the potential delivery of full-length dystrophin.<br><br>EXPERT OPINION: The FDA's recent approval of delandistrogene moxeparvovec (Elevidys) underscores the promise of gene replacement therapies for DMD patients. Understanding the mechanisms behind the adverse effects and excluding patients with specific pathogenic variants may enhance the safety profiles of these therapies. CRISPR/Cas9 therapies, while promising, face significant regulatory and safety challenges that hinder their clinical application. Optimal DMD therapies should target both skeletal and cardiac muscles to be effective.}, }
@article {pmid39720725, year = {2024}, author = {Wi, T and Choi, Y and Kim, J and Choi, YS and Pipkin, ME and Choi, J}, title = {Efficient gene deletion of Integrin alpha 4 in primary mouse CD4 T cells using CRISPR RNA pair-mediated fragmentation.}, journal = {Frontiers in immunology}, volume = {15}, number = {}, pages = {1445341}, pmid = {39720725}, issn = {1664-3224}, mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Integrin alpha4/genetics/metabolism/immunology ; *Gene Deletion ; CD4-Positive T-Lymphocytes/immunology/metabolism ; Th1 Cells/immunology ; Cell Differentiation/genetics/immunology ; Mice, Inbred C57BL ; Lymphocytic Choriomeningitis/immunology/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Lymphocytic choriomeningitis virus/immunology ; }, abstract = {The functional specialization of CD4 T lymphocytes into various subtypes, including TH1 and TFH cells, is crucial for effective immune responses. TFH cells facilitate B cell differentiation within germinal centers, while TH1 cells are vital for cell-mediated immunity against intracellular pathogens. Integrin α4, a cell surface adhesion molecule, plays significant roles in cell migration and co-stimulatory signaling. In this study, we investigated the role of Integrin α4 in regulating TFH and TH1 cell populations during acute viral infection using CRISPR-Cas9 gene editing. To effectively delete the Itga4 in primary mouse CD4 T cells, we selected various combinations of crRNAs and generated ribonucleoprotein complexes with fluorochrome-conjugated tracrRNAs and Cas9 proteins. These crRNA pairs enhanced gene deletion by generating deletions in the gene. By analyzing the effects of Itga4 deficiency on TFH and TH1 cell differentiation during acute LCMV infection, we found that optimized crRNA pairs significantly increased the TH1 cell population. Our results highlight the importance of selecting and combining appropriate crRNAs for effective CRISPR-Cas9 gene editing in primary CD4 T cells. Additionally, our study demonstrates the role of Integrin α4 in regulating the differentiation of CD4 T cells, suggesting the potential molecular mechanisms driving T cell subset differentiation through integrin targeting.}, }
@article {pmid39719706, year = {2025}, author = {Zhang, AN and Gaston, JM and Cárdenas, P and Zhao, S and Gu, X and Alm, EJ}, title = {CRISPR-Cas spacer acquisition is a rare event in human gut microbiome.}, journal = {Cell genomics}, volume = {5}, number = {1}, pages = {100725}, pmid = {39719706}, issn = {2666-979X}, mesh = {Humans ; *Gastrointestinal Microbiome/genetics ; *CRISPR-Cas Systems/genetics ; Gene Transfer, Horizontal/genetics ; Bacteriophages/genetics ; Bifidobacterium longum/genetics ; Metagenome/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome, Bacterial/genetics ; }, abstract = {Host-parasite relationships drive the evolution of both parties. In microbe-phage dynamics, CRISPR functions as an adaptive defense mechanism, updating immunity via spacer acquisition. Here, we investigated these interactions within the human gut microbiome, uncovering low frequencies of spacer acquisition at an average rate of one spacer every ∼2.9 point mutations using isolates' whole genomes and ∼2.7 years using metagenome time series. We identified a highly prevalent CRISPR array in Bifidobacterium longum spreading via horizontal gene transfer (HGT), with six spacers found in various genomic regions in 15 persons from the United States and Europe. These spacers, targeting two prominent Bifidobacterium phages, comprised 76% of spacer occurrence of all spacers targeting these phages in all B. longum populations. This result suggests that HGT of an entire CRISPR-Cas system introduced three times more spacers than local CRISPR-Cas acquisition in B. longum. Overall, our findings identified key ecological and evolutionary factors in prokaryote adaptive immunity.}, }
@article {pmid39719647, year = {2024}, author = {Yan, L and Song, YS and Zhou, J and Zhu, L and Shi, TW and Yu, HQ and Dong, ZQ and Wang, W and Long, T and Liu, HY and Shi, ZY and Li, JG}, title = {Expression of nicastrin, NICD1, and Hes1 in NCSTN knockout mice: implications for hidradenitis suppurativa, Alzheimer's, and liver cancer.}, journal = {European journal of medical research}, volume = {29}, number = {1}, pages = {622}, pmid = {39719647}, issn = {2047-783X}, support = {81773344//National Natural Science Foundation of China/ ; }, mesh = {Animals ; *Transcription Factor HES-1/genetics/metabolism ; *Amyloid Precursor Protein Secretases/genetics/metabolism ; *Mice, Knockout ; *Alzheimer Disease/genetics/metabolism ; Mice ; *Hidradenitis Suppurativa/genetics ; Female ; Male ; *Liver Neoplasms/genetics/pathology ; *Membrane Glycoproteins/genetics/metabolism ; Mice, Inbred C57BL ; Disease Models, Animal ; Receptor, Notch1/genetics ; }, abstract = {BACKGROUND: Nicastrin, a subunit of the γ-secretase complex, is encoded by the NCSTN gene and regulates notch signaling, it is involved in the pathogenesis of hidradenitis suppurativa (HS), Alzheimer disease (AD), and liver cancer. However, the animal models for studying HS are relatively scarce.<br><br>METHODS: CRISPR/Cas-mediated genetic engineering was used to generate targeted knockout offspring mice (C57BL/6J). Different doses (10&#xa0;mg/kg, 20&#xa0;mg/kg, and 30&#xa0;mg/kg) and injection methods (subcutaneous/intraperitoneal/gavage injection) of tamoxifen were used to induce the construction of NCSTN knockout mice (mice model). The expressions of nicastrin, NICD1, hes1 in skin, brain, and liver tissue in mice model and wild-type (WT) mice were measured by qRT-PCR and IHC.<br><br>RESULTS: The construction of mice model was successfully induced by tamoxifen, knockout efficiency was 93%, there was no difference in knockout efficiency among three doses, injection methods, genders (P&#x2009;>&#x2009;0.05). HS-like lesions appeared on the skin of NCSTN knockout mice after 1&#xa0;month of treatment with tamoxifen, male mice had a higher number of skin lesions compared to female mice (male vs female&#x2009;=&#x2009;76.5% vs 41.7%, P&#x2009;=&#x2009;0.027). Compared with WT mice, the expressions of nicastrin (skin P&#x2009;=&#x2009;0.0009, brain P&#x2009;=&#x2009;0.0194, liver P&#x2009;=&#x2009;0.0066), NICD1 (skin P&#x2009;=&#x2009;0.0115, brain P&#x2009;=&#x2009;0.0307, liver P&#x2009;=&#x2009;0.008), hes1 (skin P&#x2009;=&#x2009;0.0476, brain P&#x2009;=&#x2009;0.0143, liver P&#x2009;=&#x2009;0.0003) in mice model all decreased.<br><br>CONCLUSIONS: The NCSTN knockout mouse might be employed as HS animal model; Reducing nicastrin may affect the expression of notch1-hes1 pathway molecules in skin, brain, and liver tissues; low dose (10&#xa0;mg/kg/d) tamoxifen could be used to induce the deletion of the target gene in mice.}, }
@article {pmid39719558, year = {2024}, author = {Nadeem, I and Han, Z and Xiaoliang, H and Adzraku, SY and Kambey, PA and Kanwore, K and Peipei, M and Adekunle, AO and Adu-Amankwaah, J and Ayanlaja, AA and Zheng, Y and Dianshuai, G and Liu, X and Song, Y}, title = {Doublecortin regulates the mitochondrial-dependent apoptosis in glioma via Rho-A/Net-1/p38-MAPK signaling.}, journal = {Molecular medicine (Cambridge, Mass.)}, volume = {30}, number = {1}, pages = {272}, pmid = {39719558}, issn = {1528-3658}, support = {82273250//National Natural Science Foundation of China/ ; KYCX22_2868//Postgraduate Research &amp; Practice Innovation Program of Jiangsu Province/ ; }, mesh = {Humans ; *Glioma/metabolism/pathology/genetics ; *Apoptosis ; *Mitochondria/metabolism ; Animals ; *Doublecortin Domain Proteins ; *Doublecortin Protein ; Cell Line, Tumor ; *p38 Mitogen-Activated Protein Kinases/metabolism ; *Microtubule-Associated Proteins/metabolism/genetics ; Mice ; *Neuropeptides/metabolism/genetics ; Cell Proliferation ; Brain Neoplasms/metabolism/pathology/genetics ; Temozolomide/pharmacology ; Signal Transduction ; MAP Kinase Signaling System ; Reactive Oxygen Species/metabolism ; }, abstract = {Doublecortin (DCX) is a microtubule-associated protein known to be a key regulator of neuronal migration and differentiation during brain development. However, the role of DCX, particularly in regulating the survival and growth of glioma cells, remains unclear. In this study, we utilized CRISPR/Cas9 technology to knock down DCX in the human glioma cell line (U251). DCX depletion suppressed cell proliferation and enhanced the pro-apoptotic effects of temozolomide (TMZ) and γ-radiation treatment. DCX knockdown led to the translocation of Bax to the mitochondria and mitochondria dysfunction. Furthermore, DCX deficiency-induced apoptosis took place along with the generation of reactive oxygen species (ROS), which is crucial in triggering mitochondrial membrane depolarization, the release of cytochrome c (Cyt-c), and caspase activation. Importantly, the transcriptional inhibition of DCX downregulated Rho-A, Net-1, and activated p38-MAPK cue, critical for cell survival and proliferation. Subsequent treatment with TMZ and γ-radiation further increased p38-MAPK activity through the decreased expression of Rho-A/Net-1, resulting in a significant reduction in glioma cell migration and invasion. Additionally, intracranial xenograft tumors of DCX-modified U251 cells in nude mice demonstrated inhibited tumor growth. Tumor sections treated with TMZ and γ-radiation exhibited a higher number of TUNEL-positive cells compared to the control group, indicating increased apoptosis. Our finding suggests that DCX depletion reduces glioma cell proliferation and promotes mitochondria-dependent apoptosis by enhancing the chemo and radiotherapy response. Targeting DCX represents a potential therapeutic target for glioma treatment.}, }
@article {pmid39718835, year = {2024}, author = {Fracassi, G and Lorenzin, F and Orlando, F and Gioia, U and D'Amato, G and Casaramona, AS and Cantore, T and Prandi, D and Santer, FR and Klocker, H and d'Adda di Fagagna, F and Mateo, J and Demichelis, F}, title = {CRISPR/Cas9 screens identify LIG1 as a sensitizer of PARP inhibitors in castration-resistant prostate cancer.}, journal = {The Journal of clinical investigation}, volume = {135}, number = {4}, pages = {}, pmid = {39718835}, issn = {1558-8238}, mesh = {Male ; Humans ; *DNA Ligase ATP/genetics/metabolism ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; *CRISPR-Cas Systems ; *Prostatic Neoplasms, Castration-Resistant/genetics/drug therapy/pathology/metabolism ; Animals ; Cell Line, Tumor ; Mice ; *Neoplasm Proteins/genetics/metabolism ; Xenograft Model Antitumor Assays ; }, abstract = {PARP inhibitors (PARPi) have received regulatory approval for the treatment of several tumors, including prostate cancer (PCa), and demonstrate remarkable results in the treatment of castration-resistant prostate cancer (CRPC) patients characterized by defects in homologous recombination repair (HRR) genes. Preclinical studies showed that DNA repair genes (DRG) other than HRR genes may have therapeutic value in the context of PARPi. To this end, we performed multiple CRISPR/Cas9 screens in PCa cell lines using a custom sgRNA library targeting DRG combined with PARPi treatment. We identified DNA ligase 1 (LIG1), essential meiotic structure-specific endonuclease 1 (EME1), and Fanconi anemia core complex associated protein 24 (FAAP24) losses as PARPi sensitizers and assessed their frequencies from 3% to 6% among CRPC patients. We showed that concomitant inactivation of LIG1 and PARP induced replication stress and DNA double-strand breaks, ultimately leading to apoptosis. This synthetic lethality (SL) is conserved across multiple tumor types (e.g., lung, breast, and colorectal), and its applicability might be extended to LIG1-functional tumors through a pharmacological combinatorial approach. Importantly, the sensitivity of LIG1-deficient cells to PARPi was confirmed in vivo. Altogether, our results argue for the relevance of determining the status of LIG1 and potentially other non-HRR DRG for CRPC patient stratification and provide evidence to expand their therapeutic options.}, }
@article {pmid39718242, year = {2025}, author = {Dong, J and Hou, C and Deng, L and Gu, T and Zhu, S and Hou, J and Huo, D}, title = {CRISPR/Cas12a-Powered Electrochemical Platform for Dual-miRNA Detection via an AND Logic Circuit.}, journal = {Analytical chemistry}, volume = {97}, number = {1}, pages = {1028-1036}, doi = {10.1021/acs.analchem.4c06256}, pmid = {39718242}, issn = {1520-6882}, mesh = {*MicroRNAs/analysis/genetics ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; Humans ; *Biosensing Techniques/methods ; *Endodeoxyribonucleases/metabolism ; *CRISPR-Associated Proteins/metabolism ; Limit of Detection ; Logic ; Bacterial Proteins ; }, abstract = {The CRISPR/Cas technology shows great potential in molecular detection and diagnostics. However, it is still challenging to detect multiple targets simultaneously using the CRISPR-Cas system. Herein, we ingeniously leverage the synergistic effect of two short single-stranded DNA activators to construct a CRISPR/Cas12a-driven electrochemical sensing platform based on an AND logic circuit ("AND" LC-CRISPR) for the simultaneous detection of dual miRNAs. Specifically, the exponential amplification reaction products triggered by the dual-specific miRNAs are designed as binary inputs to bind with Cas12a/crRNA, forming an AND logic circuit and activating the trans-cleavage ability of the CRISPR-Cas12a system. Subsequently, the hairpin probe biogate on the surface of the functionalized electrochemical signal probe (MB@HP-Fe-MOF) is cleaved by activated Cas12a, leading to the release of the encapsulated electroactive signal molecule methylene blue, thereby generating a strong electrochemical signal. As a result, this "AND" LC-CRISPR sensing platform, requiring only a single crRNA assembled with Cas12a, achieves simultaneous detection of miRNA-155 and miRNA-21 at concentrations as low as 3.2 fM. Moreover, the platform allows easy adjustment of the AND logic circuit inputs according to different detection targets, allowing it to be easily expanded for the analysis and diagnosis of other multibiomarkers. This approach demonstrates promising potential for future applications in intelligent diagnostic medicine.}, }
@article {pmid39718218, year = {2025}, author = {Liu, X and Zhao, Z and Li, W and Ren, M and Zhang, H and Cao, D and Wang, Y and Yang, H and Li, Y and Zhu, M and Xie, L and Yin, L}, title = {Rationally Engineering Pro-Proteins and Membrane-Penetrating α‑Helical Polypeptides for Genome Editing Toward Choroidal Neovascularization Treatment.}, journal = {Advanced materials (Deerfield Beach, Fla.)}, volume = {37}, number = {6}, pages = {e2412366}, doi = {10.1002/adma.202412366}, pmid = {39718218}, issn = {1521-4095}, support = {BK20220245//Natural Science Foundation of Jiangsu Province/ ; 82241008//National Natural Science Foundation of China/ ; 52325305//National Natural Science Foundation of China/ ; 52273144//National Natural Science Foundation of China/ ; 52303200//National Natural Science Foundation of China/ ; BE2021642//Jiangsu Key Research and Development Plan/ ; //Collaborative Innovation Center of Suzhou Nano Science &amp; Technology/ ; //111 project/ ; //Joint International Research Laboratory of Carbon-Based FunctionalMaterials and Devices/ ; //Suzhou Key Laboratory of Nanotechnology and Biomedicine/ ; }, mesh = {*Choroidal Neovascularization/therapy/genetics/pathology ; Animals ; *Gene Editing/methods ; Humans ; Mice ; *Peptides/chemistry ; *Protein Engineering ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism/genetics ; Protein Conformation, alpha-Helical ; CRISPR-Cas Systems ; Ribonucleoproteins/chemistry/metabolism ; }, abstract = {Ribonucleoprotein (RNP)-based CRISPR/Cas9 genome editing holds great potential for the treatment of choroidal neovascularization (CNV), which however, is challenged by the lack of efficient cytosolic protein delivery tools. Herein, reversibly-phosphorylated pro-proteins (P-proteins) with conjugated adenosine triphosphate (ATP) tags are engineered and coupled with a membrane-penetrating, guanidine-enriched, α-helical polypeptide (LGP) to mediate robust and universal cytosolic delivery. LGP forms salt-stable nanocomplexes (NCs) with P-proteins via electrostatic interaction and salt bridging, and the helix-assisted, strong membrane activities of LGP enabled efficient cellular internalization and endolysosomal escape of NCs. Therefore, this approach allows efficient cytosolic delivery of a wide range of protein cargoes and maintains their bioactivities due to endolysosomal acidity-triggered traceless restoration of P-proteins. Notably, intravitreally delivered LGP/P-RNP NCs targeting hypoxia-inducible factor-1α (HIF-1α) induce pronounced gene disruption to downregulate pro-angiogenic factors and alleviate subretinal fibrosis, ultimately provoking robust therapeutic efficacy in CNV mice. Such a facile and versatile platform provides a powerful tool for cytosolic protein delivery and genome editing, and it holds promising potential for the treatment of CNV-associated diseases, such as age-related macular degeneration.}, }
@article {pmid39717575, year = {2024}, author = {Ahmad, Z and Niyazi, S and Firdoos, A and Wang, C and Manzoor, MA and Ramakrishnan, M and Upadhyay, A and Ding, Y}, title = {Enhancing plant resilience: Nanotech solutions for sustainable agriculture.}, journal = {Heliyon}, volume = {10}, number = {23}, pages = {e40735}, pmid = {39717575}, issn = {2405-8440}, abstract = {The global population growth is driving up the demand for agricultural products, while traditional farming methods like those from the Green Revolution are becoming unsustainable due to climate change. To address these challenges and ensure agricultural sustainability, innovative techniques, such as nanotechnology, are essential to meet rising food demands and enhance agricultural sustainability. Nanotechnology, which promotes a more sustainable and resilient agricultural system while enhancing food security, is a key catalyst for the Agri-tech revolution. This review offers a progressive analysis of nanotechnology's role in managing plant stress. It explores how precision agriculture, particularly via nanosensors, is enhancing our comprehension of plant stress conditions. The integration of nanotechnology with genetic engineering methods, notably CRISPR-Cas technology, is also examined. Furthermore, the review considers the potential toxicological effects of nanoparticles (NPs) on both the environment and plants. Our review has the potential to make a significant impact on human food security by enhancing food production and availability while promoting sustainable agricultural practices. By tackling these challenges, we can contribute to a more reliable and sustainable food supply for the global population.}, }
@article {pmid39716581, year = {2025}, author = {Kalaichelvan, A and Nadarajapillai, K and Sellaththurai, SR and Arachchi, UPE and Kim, MJ and Jung, S and Lee, J}, title = {CRISPR/Cas9-induced knockout of tumor necrosis factor-alpha-type I augments viral infection in zebrafish.}, journal = {Fish &amp; shellfish immunology}, volume = {157}, number = {}, pages = {110092}, doi = {10.1016/j.fsi.2024.110092}, pmid = {39716581}, issn = {1095-9947}, mesh = {Animals ; *Zebrafish/immunology/genetics ; Novirhabdovirus/physiology ; *Tumor Necrosis Factor-alpha/genetics/immunology ; CRISPR-Cas Systems ; *Hemorrhagic Septicemia, Viral/immunology/genetics ; Gene Knockout Techniques/veterinary ; *Fish Diseases/immunology/virology ; *Immunity, Innate/genetics ; *Zebrafish Proteins/genetics ; }, abstract = {Tumor necrosis factor-alpha (TNF-α) is a pleiotropic cytokine with critical roles in inflammation, cell survival, and defense. As a member of the TNF superfamily, it exerts its effects by binding to transmembrane receptors and triggering various downstream signaling pathways. Although TNF-α's involvement in antiviral responses in mammals is well-established, its role in teleost remains poorly understood. This study investigated the contribution of TNF-α1 to antiviral immunity in zebrafish using a tnf-α1[(-/-)] knockout (KO) line. We challenged both wild-type and tnf-α1[(-/-)] zebrafish with viral hemorrhagic septicemia virus (VHSV) at both embryonic and adult stages. Mortality was observed at 4 days post-infection (dpi) in tnf-α1-deficient adult fish challenged with 5 × 10[6] TCID50 (VHSV) and at 5 dpi in adult wild fish challenged with the same concentration. In addition, tnf-α1[(-/-)] KO adult fish reached the maximum mortality of 100 % at 20 dpi, whereas wild adult fish reached 54 % mortality at the same time point. This increased susceptibility to early mortality was associated with a higher viral burden and altered expression of key immune genes, including the pro-inflammatory cytokines il-6 and il-1β, the anti-inflammatory cytokine il-10, and interferon-related genes such as irf1 and ifn-γ. Our findings demonstrate the crucial role of TNF-α1 in antiviral defense mechanisms in zebrafish and provide valuable insights into the functional conservation of TNF-α signaling across vertebrate species. This knowledge may contribute to the development of strategies to combat viral diseases in fish.}, }
@article {pmid39716527, year = {2025}, author = {Liu, R and Ji, W and Jiang, M and Shen, J}, title = {CRISPR technology combined with isothermal amplification methods for the diagnosis of Candida albicans infection.}, journal = {Clinica chimica acta; international journal of clinical chemistry}, volume = {567}, number = {}, pages = {120106}, doi = {10.1016/j.cca.2024.120106}, pmid = {39716527}, issn = {1873-3492}, mesh = {*Candida albicans/genetics/isolation &amp; purification ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Candidiasis/diagnosis ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Since Candida albicans, a type of fungus, causes severe infections that pose a significant threat to human health, its rapid detection is critical in clinical antifungal therapy. Traditional fungal diagnostic approaches are largely based on the culture method. This method is time-consuming and laborious, taking about 48-72 h, and cannot identify emerging species, making it unsuitable for critically ill patients with bloodstream infections, sepsis, and so on. Other antigen or nucleic acid amplification-based methods were also found to be unsuitable for Point-of-Care Testing (POCT) diagnosis due to various limitations. Therefore, establishing a new approach for the rapid diagnosis of Candida spp is imperative. Herein, we proposed a novel diagnostic method for invasive fungi detection. Specifically, we created a new CRISPR diagnostic platform for Candida albicans-specific Internal Transcriptional Spacer 2 (ITS2) gene by combining the DNase cleavage activity of Cas12a with Recombinase Polymerase Amplification (RPA). Furthermore, to achieve rapid on-site detection under low-resource conditions, we used a transverse lateral flow strip with a single target to visualize the Cas12a single enzyme digestion product. We designated the platform as a rapid molecular detection tool that integrates RPA and the CRISPR-Cas12a technology. The entire platform can accurately identify Candida albicans within 50 minwhile remaining unaffected by other fungi or bacteria. Furthermore, the detection limit of the platform could reach 10[2] CFU/ml. Moreover, this approach offers additional benefits, including easy operation, low set-up cost, and broad applicability for Candida albicans detection across medical institutions at all levels, especially in township health centers in resource-poor regions.}, }
@article {pmid39716488, year = {2025}, author = {He, Y and Zalenski, N and Stephenson, AA and Raper, AT and Ghimire, C and Suo, Z}, title = {Conformational transitions of Streptococcus pyogenes Cas9 induced by salt and single-guide RNA binding.}, journal = {The Journal of biological chemistry}, volume = {301}, number = {2}, pages = {108120}, pmid = {39716488}, issn = {1083-351X}, support = {R01 GM122093/GM/NIGMS NIH HHS/United States ; }, mesh = {*Streptococcus pyogenes/enzymology/genetics ; *RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics ; *CRISPR-Associated Protein 9/chemistry/metabolism/genetics ; Fluorescence Resonance Energy Transfer ; *Potassium Chloride/chemistry/pharmacology ; Protein Conformation ; *Bacterial Proteins/chemistry/metabolism/genetics ; }, abstract = {Streptococcus pyogenes (Sp) Cas9 has been widely utilized to edit genomes across diverse species. To achieve high efficiency and specificity as a gene-editing enzyme, Sp Cas9 undergoes a series of sequential conformational changes during substrate binding and catalysis. Here, we employed single-molecule FRET techniques to investigate the effect of different KCl concentrations on conformational dynamics of Sp Cas9 in the presence or the absence of a single-guide RNA (sgRNA). In the absence of sgRNA and at low KCl concentrations (75 mM), apo Cas9 surprisingly exhibited two distinct conformations: a primary autoinhibited open conformation (apo Cas9 conformation [Cas9[apo]]) and a secondary sgRNA-bound-like conformation (Cas9[X]). Interestingly, increase in buffer KCl concentration led to a linear increase in the Cas9[X] population and a corresponding decrease in the Cas9[apo] population. In contrast, changes in KCl concentration exerted the opposite effects on the Cas9[X] and Cas9[apo] populations in the presence of sgRNA. Collectively, our findings by using KCl concentration as the probe suggest that Cas9 might employ a conformational sampling mechanism, in addition to the more common induced-fit mechanism established by us previously, for sgRNA binding.}, }
@article {pmid39716183, year = {2024}, author = {Cohen, S and Bergman, S and Lynn, N and Tuller, T}, title = {A tool for CRISPR-Cas9 sgRNA evaluation based on computational models of gene expression.}, journal = {Genome medicine}, volume = {16}, number = {1}, pages = {152}, pmid = {39716183}, issn = {1756-994X}, mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Software ; Computational Biology/methods ; Gene Editing/methods ; Gene Knockout Techniques ; Computer Simulation ; Models, Genetic ; Gene Expression ; Gene Expression Regulation ; }, abstract = {BACKGROUND: CRISPR is widely used to silence genes by inducing mutations expected to nullify their expression. While numerous computational tools have been developed to design single-guide RNAs (sgRNAs) with high cutting efficiency and minimal off-target effects, only a few tools focus specifically on predicting gene knockouts following CRISPR. These tools consider factors like conservation, amino acid composition, and frameshift likelihood. However, they neglect the impact of CRISPR on gene expression, which can dramatically affect the success of CRISPR-induced gene silencing attempts. Furthermore, information regarding gene expression can be useful even when the objective is not to silence a gene. Therefore, a tool that considers gene expression when predicting CRISPR outcomes is lacking.<br><br>RESULTS: We developed EXPosition, the first computational tool that combines models predicting gene knockouts after CRISPR with models that forecast gene expression, offering more accurate predictions of gene knockout outcomes. EXPosition leverages deep-learning models to predict key steps in gene expression: transcription, splicing, and translation initiation. We showed our tool performs better at predicting gene knockout than existing tools across 6 datasets, 4 cell types and ~207k sgRNAs. We also validated our gene expression models using the ClinVar dataset by showing enrichment of pathogenic mutations in high-scoring mutations according to our models.<br><br>CONCLUSIONS: We believe EXPosition will enhance both the efficiency and accuracy of genome editing projects, by directly predicting CRISPR's effect on various aspects of gene expression. EXPosition is available at http://www.cs.tau.ac.il/~tamirtul/EXPosition .&#xa0;The source code is available at https://github.com/shaicoh3n/EXPosition .}, }
@article {pmid39715634, year = {2025}, author = {Kalanithy, JC and Mingardo, E and Stegmann, JD and Dhakar, R and Dakal, TC and Rosenfeld, JA and Tan, WH and Coury, SA and Woerner, AC and Sebastian, J and Levy, PA and Fleming, LR and Waffenschmidt, L and Lindenberg, TT and Yilmaz, &#xd6; and Channab, K and Babra, BK and Christ, A and Eiberger, B and Hölzel, S and Vidic, C and Häberlein, F and Ishorst, N and Rodriguez-Gatica, JE and Pezeshkpoor, B and Kupczyk, PA and Vanakker, OM and Loddo, S and Novelli, A and Dentici, ML and Becker, A and Thiele, H and Posey, JE and Lupski, JR and Hilger, AC and Reutter, HM and Merz, WM and Dworschak, GC and Odermatt, B}, title = {TFAP2E is implicated in central nervous system, orofacial and maxillofacial anomalies.}, journal = {Journal of medical genetics}, volume = {62}, number = {2}, pages = {126-137}, pmid = {39715634}, issn = {1468-6244}, support = {UM1 HG006542/HG/NHGRI NIH HHS/United States ; R35 NS105078/NS/NINDS NIH HHS/United States ; K08 HG008986/HG/NHGRI NIH HHS/United States ; /WT_/Wellcome Trust/United Kingdom ; U01 HG011758/HG/NHGRI NIH HHS/United States ; }, mesh = {Animals ; Humans ; *Transcription Factor AP-2/genetics/chemistry/metabolism ; Zebrafish/genetics ; Female ; Male ; *Central Nervous System/metabolism/abnormalities ; *Maxillofacial Abnormalities/genetics/pathology ; Pedigree ; CRISPR-Cas Systems ; Mutation, Missense ; }, abstract = {BACKGROUND: Previous studies in mouse, Xenopus and zebrafish embryos show strong tfap2e expression in progenitor cells of neuronal and neural crest tissues suggesting its involvement in neural crest specification. However, the role of human transcription factor activator protein 2 (TFAP2E) in human embryonic central nervous system (CNS), orofacial and maxillofacial development is unknown.<br><br>METHODS: Through a collaborative work, exome survey was performed in families with congenital CNS, orofacial and maxillofacial anomalies. Exome variant prioritisation prompted TFAP2E gene for functional analysis in zebrafish embryos. Embryonic morphology and development were assessed after antisense morpholino (MO) knockdown (KD), CRISPR/Cas9 knockout and overexpression of tfap2e in fluorescent zebrafish reporter lines using in vivo microscopy. Computational structural protein modelling of the identified human variants was performed.<br><br>RESULTS: In total, exome survey identified novel or ultra-rare heterozygous missense variants in TFAP2E in seven individuals from five independent families with predominantly CNS, orofacial and maxillofacial anomalies. One variant was found de novo and another variant segregated in an affected multiplex family. Protein modelling of the identified variants indicated potential distortion of TFAP2E in the transactivation or dimerisation domain. MO KD and CRISPR/Cas9 knockout of tfap2e in zebrafish revealed hydrocephalus and a significant reduction of brain volume, consistent with a microencephaly phenotype. Furthermore, mRNA overexpression of TFAP2E indicates dosage-sensitive phenotype expression. In addition, zebrafish showed orofacial and maxillofacial anomalies following tfap2e KD, recapitulating the human phenotype.<br><br>CONCLUSION: Our human genetic data and analysis of Tfap2e manipulation in zebrafish indicate a potential role of TFAP2E in human CNS, orofacial and maxillofacial anomalies.}, }
@article {pmid39715471, year = {2025}, author = {Nguyen, NHK and Rafiee, R and Parcha, PK and Tagmount, A and Rubnitz, J and Ribeiro, R and Cao, X and Pounds, SB and Vulpe, CD and Lamba, JK}, title = {Genome-wide CRISPR/Cas9 screen identifies AraC-daunorubicin-etoposide response modulators associated with outcomes in pediatric AML.}, journal = {Blood advances}, volume = {9}, number = {5}, pages = {1078-1091}, pmid = {39715471}, issn = {2473-9537}, support = {T32 HG008958/HG/NHGRI NIH HHS/United States ; }, mesh = {Humans ; *Leukemia, Myeloid, Acute/drug therapy/genetics/mortality ; *CRISPR-Cas Systems ; Cytarabine/pharmacology/therapeutic use ; Etoposide/pharmacology/therapeutic use ; Daunorubicin/pharmacology/therapeutic use ; Child ; Drug Resistance, Neoplasm/genetics ; *Antineoplastic Combined Chemotherapy Protocols/therapeutic use ; Biomarkers, Tumor/genetics ; Female ; Male ; Prognosis ; Cell Line, Tumor ; }, abstract = {Cytarabine, daunorubicin, and etoposide (ADE) have been the standard backbone of induction chemotherapy regimen for patients with pediatric acute myeloid leukemia (pAML) for >5 decades. However, chemoresistance is still a major concern, and a significant proportion of pAML becomes resistant to ADE treatment and relapse, leading to poor survival. Therefore, there is a considerable need to identify mechanisms mediating drug resistance for overcoming chemoresistance. Herein, we performed synthetic lethal CRISPR/Cas9 screens using the ADE components to identify response markers. We further integrated significant markers in 3 independent pAML clinical cohorts treated with only an ADE regimen to identify drug response biomarkers with prognostic significance. We were able to identify several mediators that represent clinically and biologically significant marker genes for ADE treatment, such as BCL2, CLIP2, and VAV3, which are resistant markers to ADE, with high expression associated with poor outcomes in pAML treated with ADE, and GRPEL1, HCFC1, and TAF10, which are sensitive markers to ADE, with high expression showing beneficial outcomes. Notably, BCL2, CLIP2, and VAV3 knockdowns in their expression in AML cell lines sensitized the cells more to the ADE components, suggesting that these modulators should be further studied as potential therapeutic targets to overcome chemoresistance.}, }
@article {pmid39715238, year = {2025}, author = {Qiu, M and Tian, Y and Wang, H and Yang, J and Qu, B and Jiang, Y and Zhao, Q and Zhang, X and Man, C}, title = {CRISPR/Cas System Meets CLICK-17 DNAzyme: A Click Chemistry-Based Fluorescence Biosensing Platform Designed for Highly Sensitive Detection of Salmonella.}, journal = {Analytical chemistry}, volume = {97}, number = {4}, pages = {2244-2253}, doi = {10.1021/acs.analchem.4c05316}, pmid = {39715238}, issn = {1520-6882}, mesh = {*Biosensing Techniques/methods ; Click Chemistry ; *CRISPR-Cas Systems ; *DNA, Catalytic/chemistry/metabolism ; *Salmonella enterica/isolation &amp; purification/genetics ; Limit of Detection ; Copper/chemistry ; Fluorescence ; Azides/chemistry ; }, abstract = {Salmonella is one of the most dangerous and contagious foodborne pathogens, posing a significant threat to public health and food safety. In this study, we developed a click chemistry-based fluorescence biosensing platform for highly sensitive detection of Salmonella enterica (S. enterica) by integrating the trans-cleavage activity of CRISPR/Cas12a with the CLICK17-mediated copper(II)-dependent azide-alkyne cycloaddition (Cu(II)AAC) click reaction. Herein, CLICK-17 can provide binding sites for Cu ions and high redox stability for one or much catalytically vital Cu[+] within its active sites, which facilitate the click reaction. With the existence of only Cu[2+], CLICK17 still can catalyze the click reaction between 3-butyn-1-ol and 3-azido-7-hydroxycoumarin to produce a fluorescence signal. By integrating the recombinase polymerase amplification (RPA), specific recognition, and trans-cleavage ability of the CRISPR/Cas12a system and the CLICK17-catalyzed Cu(II)AAC click reaction, the established biosensor obtained high detection sensitivity. This CLICK17-assisted CRISPR/Cas12a fluorescence biosensor was used for the detection of S. enterica with a limit of detection (LOD) as low as 1 cfu/mL in a wide linear detection range of 6 × 10[1]-6 × 10[7] cfu/mL. Moreover, the developed biosensor exhibited high specificity and anti-interference capability and had a recovery of 93%-104% in detection of S. enterica in spiked milk, infant formula, orange juice, and meat samples. This study provides a promising CRISPR/Cas12a-based fluorescence biosensor for the detection of foodborne pathogens.}, }
@article {pmid39714464, year = {2024}, author = {Böck, D and Wilhelm, M and Mumenthaler, J and Carpanese, DF and Kulcsár, PI and d'Aquin, S and Cremonesi, A and Rassi, A and Häberle, J and Patriarchi, T and Schwank, G}, title = {Base editing of Ptbp1 in neurons alleviates symptoms in a mouse model of Parkinson's disease.}, journal = {eLife}, volume = {13}, number = {}, pages = {}, pmid = {39714464}, issn = {2050-084X}, support = {310030_185293/SNSF_/Swiss National Science Foundation/Switzerland ; 310030_196455/SNSF_/Swiss National Science Foundation/Switzerland ; FN20-0000000203//Novartis Foundation for Medical-Biological Research/ ; 196287/SNSF_/Swiss National Science Foundation/Switzerland ; 891959/ERC_/European Research Council/International ; }, mesh = {Animals ; *Polypyrimidine Tract-Binding Protein/metabolism/genetics ; Mice ; *Disease Models, Animal ; *Heterogeneous-Nuclear Ribonucleoproteins/metabolism/genetics ; *Parkinson Disease/genetics/metabolism ; *Astrocytes/metabolism ; Dopaminergic Neurons/metabolism ; Gene Editing ; Male ; Corpus Striatum/metabolism ; Tyrosine 3-Monooxygenase/metabolism/genetics ; Mice, Inbred C57BL ; }, abstract = {Parkinson's disease (PD) is a multifactorial disease caused by irreversible progressive loss of dopaminergic neurons (DANs). Recent studies have reported the successful conversion of astrocytes into DANs by repressing polypyrimidine tract binding protein 1 (PTBP1), which led to the rescue of motor symptoms in a chemically-induced mouse model of PD. However, follow-up studies have questioned the validity of this astrocyte-to-DAN conversion model. Here, we devised an adenine base editing strategy to downregulate PTBP1 in astrocytes and neurons in a chemically-induced PD mouse model. While PTBP1 downregulation in astrocytes had no effect, PTBP1 downregulation in neurons of the striatum resulted in the expression of the DAN marker tyrosine hydroxylase (TH) in non-dividing neurons, which was associated with an increase in striatal dopamine concentrations and a rescue of forelimb akinesia and spontaneous rotations. Phenotypic analysis using multiplexed iterative immunofluorescence imaging further revealed that most of these TH-positive cells co-expressed the dopaminergic marker DAT and the pan-neuronal marker NEUN, with the majority of these triple-positive cells being classified as mature GABAergic neurons. Additional research is needed to fully elucidate the molecular mechanisms underlying the expression of the observed markers and understand how the formation of these cells contributes to the rescue of spontaneous motor behaviors. Nevertheless, our findings support a model where downregulation of neuronal, but not astrocytic, PTBP1 can mitigate symptoms in PD mice.}, }
@article {pmid39714446, year = {2024}, author = {Petazzi, P and Ventura, T and Luongo, FP and McClafferty, H and May, A and Taylor, HA and Shipston, MJ and Romanò, N and Forrester, LM and Menendez, P and Fidanza, A}, title = {A novel human pluripotent stem cell gene activation system identifies IGFBP2 as a mediator in the production of haematopoietic progenitors in vitro.}, journal = {eLife}, volume = {13}, number = {}, pages = {}, pmid = {39714446}, issn = {2050-084X}, support = {S002219/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; EHA RAG 2021//European Hematology Association/ ; Research Global Award//American Society for Hematology/ ; Precision Medicine PhD scholarship/MRC_/Medical Research Council/United Kingdom ; Tissue Repair PhD studentship//College of Medicine and Veterinary Medicine, University of Edinburgh/ ; Traineeship Program 2016/2017//Erasmus +/ ; PERIS program//Catalan Government/ ; RTC-2018-4603-1//MINECO/ ; }, mesh = {*Insulin-Like Growth Factor Binding Protein 2/metabolism/genetics ; Humans ; *Cell Differentiation ; *Hematopoietic Stem Cells/metabolism ; Induced Pluripotent Stem Cells/metabolism ; Transcriptional Activation ; CRISPR-Cas Systems ; Transcription Factors/metabolism/genetics ; Pluripotent Stem Cells/metabolism ; }, abstract = {A major challenge in the stem cell biology field is the ability to produce fully functional cells from induced pluripotent stem cells (iPSCs) that are a valuable resource for cell therapy, drug screening, and disease modelling. Here, we developed a novel inducible CRISPR-mediated activation strategy (iCRISPRa) to drive the expression of multiple endogenous transcription factors (TFs) important for in vitro cell fate and differentiation of iPSCs to haematopoietic progenitor cells. This work has identified a key role for IGFBP2 in developing haematopoietic progenitors. We first identified nine candidate TFs that we predicted to be involved in blood cell emergence during development, then generated tagged gRNAs directed to the transcriptional start site of these TFs that could also be detected during single-cell RNA sequencing (scRNAseq). iCRISPRa activation of these endogenous TFs resulted in a significant expansion of arterial-fated endothelial cells expressing high levels of IGFBP2, and our analysis indicated that IGFBP2 is involved in the remodelling of metabolic activity during in vitro endothelial to haematopoietic transition. As well as providing fundamental new insights into the mechanisms of haematopoietic differentiation, the broader applicability of iCRISPRa provides a valuable tool for studying dynamic processes in development and for recapitulating abnormal phenotypes characterised by ectopic activation of specific endogenous gene expression in a wide range of systems.}, }
@article {pmid39714100, year = {2025}, author = {Cui, Q and Zhang, Z and Qin, L and Teng, Z and Wang, Z and Wu, W and Fan, L and Su, J and Hao, Y and Qin, J and Zhang, L and Wang, Q and Zhuang, Y and Zheng, H and Zhang, S and Geng, X and Zhu, L and Chen, Y and Lu, B and Li, Y and Zhu, X}, title = {Interleukin-37 promotes wound healing in diabetic mice by inhibiting the MAPK/NLRP3 pathway.}, journal = {Journal of diabetes investigation}, volume = {16}, number = {3}, pages = {405-413}, pmid = {39714100}, issn = {2040-1124}, support = {3030294002//Multidisciplinary Diagnosis and Treatment (MDT) Construction Project of Diabetic Foot/ ; 82400978//National Natural Science Foundation of China/ ; 82000790//National Natural Science Foundation of China/ ; 82200949//National Natural Science Foundation of China/ ; DGF501069/015//Multidisciplinary Diagnosis and Treatment (MDT) demonstration project in research hospital, Shanghai Medical College, Fudan University/ ; }, mesh = {Animals ; *Wound Healing/drug effects ; Mice ; *Interleukin-1/metabolism/genetics ; *Diabetes Mellitus, Experimental/metabolism/complications ; *NLR Family, Pyrin Domain-Containing 3 Protein/metabolism ; *Diabetic Foot/metabolism/pathology ; Humans ; Male ; Mice, Transgenic ; *MAP Kinase Signaling System/drug effects ; Mice, Inbred C57BL ; Signal Transduction ; }, abstract = {AIMS/INTRODUCTION: Diabetic foot ulcer (DFU) is a prevalent complication of diabetes characterized by heightened inflammation and impaired wound-healing processes. Interleukin-37 (IL-37) is a natural suppressor of innate inflammation. Here, we aim to investigate the potential of IL-37 in enhancing the healing process of diabetic wounds.<br><br>MATERIALS AND METHODS: The skin samples of DFU and non-diabetic patients during foot and ankle orthopedic surgery were collected. The IL-37 transgenic mice (IL-37Tg) were created using CRISPR/Cas-mediated genome engineering. Mice were administered streptozotocin (STZ, 150&#x2009;mg/kg) to induce a diabetic model. After 4&#x2009;weeks, an equidistant full-thickness excisional wound measuring 8&#x2009;mm was created on the central back of each mouse and allowed to heal naturally. Body weight and blood glucose levels were measured weekly. The wound area was measured, and skin samples were collected on Day 10 for further Quantitative polymerase chain reaction (qPCR) and WB detection and RNA sequencing analysis.<br><br>RESULTS: The proinflammation cytokines such as TNF-&#x3b1; and IL-1&#x3b2; and the MAPK signaling pathway were significantly increased in the wound margin of DFU patients. Compared with diabetic mice, diabetic IL-37Tg mice showed a significantly accelerated healing process. The enriched signaling pathways in RNA sequencing included cytokine-cytokine receptor interaction, TNF signaling pathway, and NOD-like receptor signaling pathway. Through QPCR and WB detection, we found that IL-37 could inhibit the activated MAPK and NOD-like signaling pathway, reducing TNF-&#x3b1;, IL-1&#x3b2;, and NLRP3 expression in the diabetic wound.<br><br>CONCLUSIONS: IL-37 promotes skin wound healing in diabetic mice, providing a new possible target for treating diabetic wounds.}, }
@article {pmid39713824, year = {2024}, author = {Karesh, WB}, title = {Shifting from wildlife disease threats to wildlife health.}, journal = {Revue scientifique et technique (International Office of Epizootics)}, volume = {Special Edition}, number = {}, pages = {141-144}, doi = {10.20506/rst.SE.3568}, pmid = {39713824}, issn = {0253-1933}, mesh = {Animals ; *Animals, Wild ; Humans ; Zoonoses/prevention &amp; control ; Conservation of Natural Resources ; Animal Diseases/prevention &amp; control/epidemiology ; }, abstract = {The evolution of wildlife disease management and surveillance, as documented in the World Organisation for Animal Health's Scientific and Technical Review, reflects a deepening understanding of the links between wildlife health, ecosystem integrity and human well-being. Early work, beginning with the World Assembly of Delegates in 1954, primarily focused on diseases like rabies. This focus expanded over time to include broader concerns such as the impacts of climate change, habitat loss and increased human-wildlife interactions on wildlife health. By the late 20th century, the emphasis had shifted towards improved practices for wildlife disease control and the development of advanced diagnostic methods and vaccines. Articles in the Review highlight the growing complexity of wildlife diseases and the need for holistic management strategies. The adoption in recent years of cutting-edge technologies like CRISPR-Cas systems and metagenomics points to a future of more proactive and integrated approaches to wildlife disease management. There is still a need to address not just the consequences of wildlife diseases but also their anthropogenic drivers. The latest perspectives advocate for nature-based solutions, expanded partnerships and systems-level thinking to effectively tackle 21st-century challenges in wildlife and biodiversity conservation.}, }
@article {pmid39712894, year = {2024}, author = {Wang, X and Yang, R and Tang, T and Zhou, Y and Chen, H and Jiang, Y and Zhang, S and Qin, S and Wang, M and Wang, C}, title = {One-pot MCDA-CRISPR-Cas-based detection platform for point-of-care testing of severe acute respiratory syndrome coronavirus 2.}, journal = {Frontiers in microbiology}, volume = {15}, number = {}, pages = {1503356}, pmid = {39712894}, issn = {1664-302X}, abstract = {Compared to quantitative real-time PCR (q-PCR), CRISPR-Cas-mediated technology is more suitable for point-of-care testing (POCT) and has potential for wider application in the future. Generally, the operational procedure of CRISPR-Cas-mediated diagnostic method consists of two independent steps, the reaction of signal amplification and the CRISPR-Cas-mediated signal detection. Complex multi-step procedures can easily lead to cross-contamination. To develop a convenient and rapid method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection, we propose a MCTOP method (Multiple cross displacement amplification-CRISPR-Cas12b-based testing in one-pot), which targets the open reading frame 1ab (ORF1ab) and nucleocapsid protein (N) gene of SARS-CoV-2. This method combines MCDA isothermal amplification and CRISPR-Cas-mediated sequence-specific detection into a one-pot reaction. The optimal reaction was achieved with isothermal amplification of 40 min and CRISPR-Cas-based detection of 15 min, both at 64°C. Then, the results can be visualized by the real-time fluorescence instrument and also lateral flow biosensor. The lowest detection limit of the proposed method is 10 copies of each of target sequences, and it has no cross-reactivity with non-SARS-CoV-2 templates. In a clinical test of 70 pharyngeal swab samples, MCTOP assay showed a specificity of 100% and sensitivities of 98 and 96% for the real-time fluorescence instrument and lateral flow biosensor, respectively. The MCTOP developed in this study is a rapid, convenient, highly sensitive, and specific method for SARS-CoV-2 nucleic acid detection. It can be used as an effective point-of-care testing (POCT) tool for clinical diagnosis and epidemiologic surveillance of SARS-CoV-2 infections, especially suitable for the basic, field and clinical laboratory.}, }
@article {pmid39711046, year = {2025}, author = {Yu, H and Xu, PF and Liu, Y and Jia, ZS and Li, YY and Tang, HW}, title = {LRET-Based Simultaneous Detection of Dual miRNAs via Multitrap Optical Tweezers Assisted Suspension Array Tagged by Two Different Luminescent Quenchable UCNPs Combining CRISPR/Cas12a Amplification.}, journal = {Analytical chemistry}, volume = {97}, number = {1}, pages = {602-612}, doi = {10.1021/acs.analchem.4c04895}, pmid = {39711046}, issn = {1520-6882}, mesh = {*MicroRNAs/analysis/genetics ; Humans ; *Optical Tweezers ; *CRISPR-Cas Systems ; *Nanoparticles/chemistry ; Limit of Detection ; *Fluorescence Resonance Energy Transfer ; Luminescent Measurements ; MCF-7 Cells ; Luminescence ; A549 Cells ; }, abstract = {Nowadays, optical tweezers play a vital role not only in optical manipulation but also in bioassay. As principal optical trapping objects, microbeads can combine optical tweezers with suspension array technology, with amply focused laser beams and adequately concentrated tags contributing to highly sensitive detection. In view of the inefficiency of conventional single-trap optical tweezers, multitrap systems are developed. Here, green- and blue-emitting core-shell-shell upconversion nanoparticles (UCNPs) are adopted to encode microbeads and determine dual miRNAs, with the internal shells leading the luminescence process to facilitate quenching through luminescence resonance energy transfer (LRET). Utilizing the trans cleavage of CRISPR/Cas12a, quenched luminescence signals are recovered and amplified, causing further enhanced detection sensitivity. Ultimately, limits of detection (LOD) of 17 and 22 aM are obtained with excellent specificities verified. Furthermore, dual miRNAs from MCF-7, A549, and MCF-10A cells are extracted and detected, with results consistent with those obtained by PCR. Notably, miR-155 in MCF-7 and A549 cells is detectable at the single-cell level. Thus, the differences in the measured miRNA levels between MCF-7 and MCF-10A cells imply the potential of this method to discriminate breast cancer cells from epithelial cells despite the difficulty in distinguishing different cancer cells due to similar miRNA levels.}, }
@article {pmid39710780, year = {2025}, author = {Cui, S and Wang, K and Yang, Y and Lv, X and Li, X}, title = {An integrated and paper-based microfluidic system employing LAMP-CRISPR and equipped with a portable device for simultaneous detection of pathogens.}, journal = {Analytical and bioanalytical chemistry}, volume = {417}, number = {4}, pages = {785-797}, pmid = {39710780}, issn = {1618-2650}, support = {JCKY2021602B009//Defense Industrial Technology Development Program/ ; }, mesh = {*Nucleic Acid Amplification Techniques/instrumentation/methods ; Paper ; *CRISPR-Cas Systems ; *Lab-On-A-Chip Devices ; *Molecular Diagnostic Techniques/instrumentation ; Equipment Design ; *Microfluidic Analytical Techniques/instrumentation ; Humans ; Limit of Detection ; *Bacteria/isolation &amp; purification/genetics ; }, abstract = {Point-of-care testing methods are essential for the large-scale diagnosis and monitoring of bacterial infections. This study introduces an integrated platform designed for the simultaneous detection of pathogenic bacteria. Users can simply inject samples into the system, which then conducts the entire procedure in a fully automated manner, eliminating the need for external power sources, all within 60 min or less. The innovative paper-based microfluidic system is capable of lysing bacteria and integrating loop-mediated isothermal amplification (LAMP) with the CRISPR/Cas12a system, achieving this with minimal reagent usage on a single piece of paper. The reaction reagents are pre-fabricated as freeze-dried powder on the paper, allowing for long-term storage. A portable and cost-effective detection device has been designed to provide stable temperature control and analyze fluorescent signals, complementing the paper-based microfluidic system. This compact device measures 150 × 150 × 100 mm, weighs less than 1.8 kg, has an average power consumption of under 15 W, and supports external power supply. The device utilizes non-contact QR codes for information transmission, ensuring functionality even in areas without Internet connectivity. This platform is capable of simultaneously detecting five typical pathogenic microorganisms, with a detection limit of 1 copy/μL. It boasts several advantages, including miniaturization, lightweight design, low power consumption, portability, affordability, rapid detection, and ease of operation, making it highly suitable for on-site detection.}, }
@article {pmid39710679, year = {2024}, author = {Jia, X and Yuan, B and Wang, W and Wang, K and Ling, D and Wei, M and Hu, Y and Guo, W and Chen, Z and Du, L and Jin, Y}, title = {Gene editing tool-loaded biomimetic cationic vesicles with highly efficient bacterial internalization for in vivo eradication of pathogens.}, journal = {Journal of nanobiotechnology}, volume = {22}, number = {1}, pages = {787}, pmid = {39710679}, issn = {1477-3155}, support = {7232261//Beijing Natural Science Foundation of China/ ; }, mesh = {*Gene Editing/methods ; *Pseudomonas aeruginosa/drug effects/genetics ; *CRISPR-Cas Systems ; *Plasmids/genetics ; *Acinetobacter baumannii/drug effects/genetics ; Animals ; *Cations/chemistry ; Mice ; Humans ; Pseudomonas Infections/drug therapy ; Anti-Bacterial Agents/pharmacology/chemistry ; Drug Resistance, Multiple, Bacterial/drug effects/genetics ; Acinetobacter Infections/drug therapy ; COVID-19 ; Female ; Mice, Inbred BALB C ; Biomimetics/methods ; }, abstract = {In the post-COVID-19 era, drug-resistant bacterial infections emerge as one of major death causes, where multidrug-resistant Acinetobacter baumannii (MRAB) and drug-resistant Pseudomonas aeruginosa (DRPA) represent primary pathogens. However, the classical antibiotic strategy currently faces the bottleneck of drug resistance. We develop an antimicrobial strategy that applies the selective delivery of CRISPR/Cas9 plasmids to pathogens with biomimetic cationic hybrid vesicles (BCVs), irrelevant to bacterial drug resistance. CRISPR/Cas9 plasmids were constructed, replicating in MRAB or DRPA and expressing ribonucleic proteins, leading to irreparable chromosomal lesions; however, delivering the negatively charged plasmids with extremely large molecular weight to the pathogens at the infection site became a huge challenge. We found that the BCVs integrating the bacterial out membrane vesicles and cationic lipids efficiently delivered the plasmids in vitro/in vivo to the pathogens followed by effective internalization. The BCVs were used by intratracheal or topical hydrogel application against MRAB pulmonary infection or DRPA wound infection, and both of the two pathogens were eradicated from the lung or the wound. CRISPR/Cas9 plasmid-loaded BCVs become a promising medication for drug-resistant bacteria infections.}, }
@article {pmid39709609, year = {2025}, author = {Hu, Y and Chen, Y and Zhang, Y and Liu, Z and Li, J}, title = {Protocol for conditional mutagenesis in zebrafish germ cells using Tol2 transposon and a CRISPR-Cas9-based plasmid system.}, journal = {STAR protocols}, volume = {6}, number = {1}, pages = {103516}, pmid = {39709609}, issn = {2666-1667}, mesh = {Animals ; *Zebrafish/genetics/embryology ; *CRISPR-Cas Systems/genetics ; *DNA Transposable Elements/genetics ; *Plasmids/genetics ; *Germ Cells/metabolism ; *Mutagenesis/genetics ; Microinjections/methods ; }, abstract = {Here, we present a protocol for conditional mutagenesis in zebrafish germ cells using Tol2 transposon and a CRISPR-Cas9-based plasmid system. We describe steps for conditional mutagenesis plasmid construction, zebrafish embryo microinjection, and screening for green fluorescence in the heart. This protocol is simple to execute, time efficient, and multifunctional, enabling the disruption of genes in zebrafish germ cells to be conducted with ease. For complete details on the use and execution of this protocol, please refer to Hu et al.[1].}, }
@article {pmid39709563, year = {2024}, author = {Shepelev, MV and Komkov, DS and Golubev, DS and Borovikova, SE and Mazurov, DV and Kruglova, NA}, title = {[Donor DNA Modification with Cas9 Targeting Sites Improves the Efficiency of MTC34 Knock-in into the CXCR4 Locus].}, journal = {Molekuliarnaia biologiia}, volume = {58}, number = {4}, pages = {590-600}, pmid = {39709563}, issn = {0026-8984}, mesh = {Humans ; *CRISPR-Cas Systems ; *Receptors, CXCR4/genetics/metabolism ; *Gene Knock-In Techniques/methods ; Gene Editing/methods ; Plasmids/genetics/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; DNA/genetics/metabolism ; Cell Line ; DNA Breaks, Double-Stranded ; HIV-1/genetics/metabolism ; }, abstract = {To successfully apply the genome editing technology using the CRISPR/Cas9 system in the clinic, it is necessary to achieve a high efficiency of knock-in, which is insertion of a genetic construct into a given locus of the target cell genome. One of the approaches to increase the efficiency of knock-in is to modify donor DNA with the same Cas9 targeting sites (CTS) that are used to induce double-strand breaks (DSBs) in the cell genome (the double-cut donor method). Another approach is based on introducing truncated CTS (tCTS), including a PAM site and 16 proximal nucleotides, into the donor DNA. Presumably, tCTS sites do not induce cleavage of the donor plasmid, but can support its transport into the nucleus by Cas9. However, the exact mechanisms whereby these two donor DNA modifications increase the knock-in level are unknown. In this study, the modifications were tested for effect on the knock-in efficiency of the MTC34 genetic construct encoding the HIV-1 fusion inhibitory peptide MT-C34 into the CXCR4 locus of the CEM/R5 T-cell line. When full-length CTSs were introduced into the donor plasmid DNA, the knock-in level was doubled regardless of the CTS number or position relative to the donor sequence. Modifications with tCTSs did not affect the knock-in levels. In vitro, both CTS and tCTS were efficiently cleaved by Cas9. To understand the mechanism of action of these modifications in detail, it is necessary to evaluate their cleavage both in vitro and in vivo.}, }
@article {pmid39709562, year = {2024}, author = {Golubev, DS and Komkov, DS and Shepelev, MV and Mazurov, DV and Kruglova, NA}, title = {[Methods to Increase the Efficiency of Knock-in of a Construct Encoding the HIV-1 Fusion Inhibitor, MT-C34 Peptide, into the CXCR4 Locus in the CEM/R5 T Cell Line].}, journal = {Molekuliarnaia biologiia}, volume = {58}, number = {4}, pages = {575-589}, pmid = {39709562}, issn = {0026-8984}, mesh = {*Receptors, CXCR4/genetics/metabolism ; Humans ; *Gene Knock-In Techniques ; *HIV-1/genetics/drug effects ; HIV Fusion Inhibitors/pharmacology ; CRISPR-Cas Systems ; Cell Line ; DNA End-Joining Repair/drug effects/genetics ; Gene Editing/methods ; Nuclear Localization Signals/genetics ; Plasmids/genetics/metabolism ; T-Lymphocytes/metabolism/drug effects ; NF-kappa B/metabolism/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; HIV Infections/genetics/virology/drug therapy ; }, abstract = {The low knock-in efficiency, especially in primary human cells, limits the use of the genome editing technology for therapeutic purposes, rendering it important to develop approaches for increasing the knock-in levels. In this work, the efficiencies of several approaches were studied using a model of knock-in of a construct coding for the peptide HIV fusion inhibitor MT-C34 into the human CXCR4 locus in the CEM/R5 T cell line. First, donor DNA modification was evaluated as a means to improve the efficiency of plasmid transport into the nucleus. The donor plasmid was modified to include the simian virus 40 (SV40) DNA nuclear targeting sequence (DTS) or binding sites for the transcription factor NF-κB, whose effects on the knock-in levels have not been described. The modification was ineffective in the model of MT-C34 knock-in into the CXCR4 locus. A second approach consisted in modification of Cas9 nuclease by introducing two additional nuclear localization signals (NLSs) and increased the knock-in level by 30%. Finally, blocking DNA repair via the nonhomologous end joining (NHEJ) pathway with DNA-dependent protein kinase inhibitors caused a 1.8-fold increase in knock-in. A combination of the last two approaches caused an additive effect. Thus, increasing the number of NLSs in the Cas9 protein and inhibiting DNA repair via the NHEJ pathway significantly increased the level of knock-in of the HIV-1 fusion inhibitory peptide into the clinically relevant locus CXCR4. The finding can be used to develop effective gene therapy approaches for treating HIV infection.}, }
@article {pmid39709560, year = {2024}, author = {Averina, OA and Kuznetsova, SA and Permyakov, OA and Sergiev, PV}, title = {[How to Shift the Equilibrium of DNA Break Repair in Favor of Homologous Recombination].}, journal = {Molekuliarnaia biologiia}, volume = {58}, number = {4}, pages = {525-548}, pmid = {39709560}, issn = {0026-8984}, mesh = {Humans ; Animals ; *DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; *Recombinational DNA Repair ; CRISPR-Cas Systems ; Gene Editing/methods ; Cell Cycle/genetics ; Homologous Recombination ; }, abstract = {The CRISPR/Cas technology of targeted genome editing made it possible to carry out genetic engineering manipulations with eukaryotic genomes with a high efficiency. Targeted induction of site-specific DNA breaks is one of the key stages of the technology. The cell repairs the breaks via one of the two pathways, nonhomologous end joining (NHEJ) and homology-driven repair (HDR). The choice of the DNA repair pathway is determined by the architecture of the DNA break region formed as a result of terminal resection and depends on the cell cycle phase. NHEJ is the main pathway of double-strand break (DSB) repair in mammalian cells and involves a nonspecific ligation reaction. The reaction accuracy depends on the structure of break ends, and various insertions or deletions may arise as a result in the target genome region. Integration of a necessary sequence into the genome occurs via HDR, which requires a template with homology regions flanking a DSB. Introducing a genetic construct into a particular genomic locus is an important task, but is currently intricate and laborious to perform. However, the choice of the repair pathway can be of principal importance for basic research of gene functions and construction of animal models of human diseases to develop therapies. The review summarizes and systematizes the available information on strategies designed to increase the HDR efficiency. The strategies that most efficiently shift the balance towards HDR include use of NHEJ inhibitors, regulation of the key factors of homologous recombination, control of the cell cycle and chromatin status, and construction of HDR templates.}, }
@article {pmid39709559, year = {2024}, author = {Averina, OA and Kuznetsova, SA and Permyakov, OA and Sergiev, PV}, title = {[Current Knowledge of Base Editing and Prime Editing].}, journal = {Molekuliarnaia biologiia}, volume = {58}, number = {4}, pages = {508-524}, pmid = {39709559}, issn = {0026-8984}, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; Animals ; Genetic Engineering/methods ; DNA/genetics ; }, abstract = {Modern genetic engineering technologies, such as base editing and prime editing (PE), have proven to provide the efficient and reliable genome editing tools that obviate the need for donor templates and double-strand breaks (DSBs) introduced in DNA. Relatively new, they quickly gained recognition for their accuracy, simplicity, and multiplexing capabilities. The review summarizes the new literature on the technologies and considers their architecture, methods to create editors, specificity, efficiency, and versatility. Advantages and disadvantages of the editors are discussed along with their prospective use in basic and applied research. The review may be useful for planning genome editing studies and analyzing their results to solve various problems of fundamental biology, biotechnology, medicine, and agriculture.}, }
@article {pmid39709065, year = {2025}, author = {Chen, X and Zheng, M and Lin, S and Huang, M and Chen, S and Chen, S}, title = {The application of CRISPR/Cas9-based genome-wide screening to disease research.}, journal = {Molecular and cellular probes}, volume = {79}, number = {}, pages = {102004}, doi = {10.1016/j.mcp.2024.102004}, pmid = {39709065}, issn = {1096-1194}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Genome-Wide Association Study/methods ; *Genetic Testing/methods ; Gene Editing ; Animals ; }, abstract = {High-throughput genetic screening serves as an indispensable approach for deciphering gene functions and the intricate relationships between phenotypes and genotypes. The CRISPR/Cas9 system, with its ability to precisely edit genomes on a large scale, has revolutionized the field by enabling the construction of comprehensive genomic libraries. This technology has become a cornerstone for genome-wide screenings in disease research. This review offers a comprehensive examination of how CRISPR/Cas9-based genetic screening has been leveraged to uncover genes that play a role in disease mechanisms, focusing on areas such as cancer development and viral replication processes. The insights presented in this review hold promise for the development of novel therapeutic strategies and precision medicine approaches.}, }
@article {pmid39709015, year = {2025}, author = {Bigot, S and Ouameur, AA and Roy, G and Fakhfakh, R and Ritt, JF and Légaré, D and Ouellette, M}, title = {Studies of the FBT family transporters in Leishmania infantum by gene deletion and protein localization.}, journal = {Experimental parasitology}, volume = {268}, number = {}, pages = {108880}, doi = {10.1016/j.exppara.2024.108880}, pmid = {39709015}, issn = {1090-2449}, mesh = {*Leishmania infantum/genetics/drug effects/metabolism ; *Gene Deletion ; Cell Membrane/metabolism ; Membrane Transport Proteins/genetics/metabolism ; Protozoan Proteins/genetics/metabolism ; Methotrexate/pharmacology ; Folic Acid Transporters/genetics/metabolism ; CRISPR-Cas Systems ; Animals ; Green Fluorescent Proteins/genetics/metabolism ; }, abstract = {The protozoan parasite Leishmania has a large family of major facilitator membrane proteins part of the Folate Biopterin Transporter (FBT) family. The chromosome 10 of Leishmania has a cluster of 7 FBT genes including the S-Adenosyl methionine (AdoMet) transporter and the functionally characterized folate transporters FT1 and FT5. Six of the 7 FBT proteins coded by this locus are located at the plasma membrane as determined by gene fusions with the green fluorescent protein. We deleted the whole locus of 7 genes (>30 kb) using CRISPR-Cas9 genome editing as a first step in studying the potential function of the four uncharacterized FBT genes from the locus. This knock out strain was viable, highly resistant to sinefungin (an AdoMet analogue) and to methotrexate (a folate analogue) but not to allopurinol, pentamidine or 5-fluorouracil. We similarly studied another FBT family member whose gene is encoded on chromosome 19. The protein was also located at the plasma membrane and its gene was dispensable for growth and not associated to any of the drug tested. Our work has indicated that large diploid deletion is achievable in Leishmania and the cell lines produced here will serve to better understand the function and putative substrates of these FBT proteins yet to be characterized.}, }
@article {pmid39708832, year = {2025}, author = {Kurashina, M and Snow, AW and Mizumoto, K}, title = {A modular system to label endogenous presynaptic proteins using split fluorophores in Caenorhabditis elegans.}, journal = {Genetics}, volume = {229}, number = {3}, pages = {}, pmid = {39708832}, issn = {1943-2631}, support = {P40 OD010440/OD/NIH HHS/United States ; PJT-180563//This study was supported by CIHR/ ; RGPIN-2015-04022//NSERC/ ; }, mesh = {Animals ; *Caenorhabditis elegans/genetics/metabolism ; *Caenorhabditis elegans Proteins/genetics/metabolism ; *Luminescent Proteins/genetics/metabolism ; Green Fluorescent Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Presynaptic Terminals/metabolism ; Red Fluorescent Protein ; Neurons/metabolism ; Gene Editing ; }, abstract = {Visualizing the subcellular localization of presynaptic proteins with fluorescent proteins is a powerful tool to dissect the genetic and molecular mechanisms underlying synapse formation and patterning in live animals. Here, we utilize split green and red fluorescent proteins to visualize the localization of endogenously expressed presynaptic proteins at a single-neuron resolution in Caenorhabditis elegans. By using CRISPR/Cas9 genome editing, we generated a collection of C. elegans strains in which endogenously expressed presynaptic proteins (RAB-3/Rab3, SNG-1/Synaptogyrin, CLA-1/Piccolo, SYD-2/Liprin-α, UNC-10/RIM, RIMB-1/RIM-BP, and ELKS-1/ELKS) are tagged with tandem repeats of GFP11 and/or wrmScarlet11. We show that the expression of GFP1-10 and wrmScarlet1-10 under neuron-specific promoters can robustly label presynaptic proteins in different neuron types. We believe that the combination of our knock-in strains and GFP1-10 and wrmScarlet1-10 plasmids is a versatile modular system useful for neuroscientists to examine the localization of endogenous presynaptic proteins in any neuron type in C. elegans.}, }
@article {pmid39708520, year = {2025}, author = {Balaraman, AK and Babu, MA and Moglad, E and Mandaliya, V and Rekha, MM and Gupta, S and Prasad, GVS and Kumari, M and Chauhan, AS and Ali, H and Goyal, K}, title = {Exosome-mediated delivery of CRISPR-Cas9: A revolutionary approach to cancer gene editing.}, journal = {Pathology, research and practice}, volume = {266}, number = {}, pages = {155785}, doi = {10.1016/j.prp.2024.155785}, pmid = {39708520}, issn = {1618-0631}, mesh = {Humans ; *Exosomes/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Neoplasms/genetics/therapy ; *Gene Transfer Techniques ; Animals ; *Genetic Therapy/methods ; }, abstract = {Several molecular strategies based on targeted gene delivery systems have been developed in recent years; however, the CRISPR-Cas9 technology introduced a new era of targeted gene editing, precisely modifying oncogenes, tumor suppressor genes, and other regulatory genes involved in carcinogenesis. However, efficiently and safely delivering CRISPR-Cas9 to cancer cells across the cell membrane and the nucleus is still challenging. Using viral vectors and nanoparticles presents issues of immunogenicity, off-target effects, and low targeting affinity. Naturally, extracellular vesicles called exosomes have garnered the most attention as delivery vehicles in oncology-related CRISPR-Cas9 calls due to their biocompatibility, loading capacity, and inherent targeting features. The following review discusses the current progress in using exosomes to deliver CRISPR-Cas9 components, the approaches to load the CRISPR components into exosomes, and the modification of exosomes to increase stability and tumor-targeted delivery. We discuss the latest strategies in targeting recently accomplished in the exosome field, including modifying the surface of exosomes to enhance their internalization by cancer cells, as well as the measures taken to overcome the impacts of TME on delivery efficiency. Focusing on in vitro and in vivo experimentation, this review shows that exosome-mediated CRISPR-Cas9 can potentially treat cancer types, including pancreatic, lymphoma, and leukemia, for given gene targets. This paper compares exosome-mediated delivery and conventional vectors regarding safety, immune response, and targeting ability. Last but not least, we present the major drawbacks and potential development of the seemingly promising field of exosome engineering in gene editing, with references to CRISPR technologies and applications that may help make the target exosomes therapeutic in oncology.}, }
@article {pmid39708407, year = {2025}, author = {Liu, X and Zhang, F and Chen, D and Yin, J and Bi, Z and Chen, L and Yan, J and Dong, Q and Peng, W and Xu, T and Guo, Y and Lin, H and Liu, H}, title = {Generation of INS-jGCaMP7f knock-in Ca[2+] reporter human embryonic stem cell line, GZLe001-C, using CRISPR/Cas9-based gene targeting.}, journal = {Stem cell research}, volume = {82}, number = {}, pages = {103633}, doi = {10.1016/j.scr.2024.103633}, pmid = {39708407}, issn = {1876-7753}, mesh = {Humans ; *Human Embryonic Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques ; Cell Line ; *Calcium/metabolism ; *Insulin/metabolism/genetics ; Gene Targeting ; Genes, Reporter ; }, abstract = {As a member of the single-fluorophore genetically encoded calcium indicators (GECIs), jGCaMP7f is widely applied to investigate intracellular Ca[2+] concentrations. Here, we established an INS-jGCaMP7f knock-in H1 human embryonic stem cell (hESC) line by integrating jGCaMP7f gene into insulin locus via CRISPR/Cas9 system. The reporter cell line not only effectively labelled the insulin-producing cells induced from hESC, but also reflected the cytosolic change of Ca[2+] level in response to different stimuli. This reporter cell line is a valuable research tool for studying functional maturation of hESC-derived insulin-producing cells, conducting drug screenings, and exploring the mechanisms of diabetes.}, }
@article {pmid39708170, year = {2024}, author = {Feng, R and Mao, K and Zhang, H and Zhu, H and Du, W and Yang, Z and Wang, S}, title = {Portable microfluidic devices for monitoring antibiotic resistance genes in wastewater.}, journal = {Mikrochimica acta}, volume = {192}, number = {1}, pages = {19}, pmid = {39708170}, issn = {1436-5073}, support = {42377456//the National Natural Science Foundation of China/ ; 2023415//the Youth Innovation Promotion Association CAS/ ; CAS-ANSO-FS-2024-34//CAS-ANSO Fellowship/ ; Qiankehe Jichu-ZK [2022] Yiban 565, Qiankehe Platform Talents-GCC [2023] 046//Guizhou Provincial Science and Technology Projects/ ; }, mesh = {*Wastewater/microbiology ; *Lab-On-A-Chip Devices ; Nucleic Acid Amplification Techniques/methods ; Drug Resistance, Microbial/genetics ; Genes, Bacterial ; CRISPR-Cas Systems/genetics ; Anti-Bacterial Agents ; Microfluidic Analytical Techniques/instrumentation/methods ; Drug Resistance, Bacterial/genetics ; }, abstract = {Antibiotic resistance genes (ARGs) pose serious threats to environmental and public health, and monitoring ARGs in wastewater is a growing need because wastewater is an important source. Microfluidic devices can integrate basic functional units involved in sample assays on a small chip, through the precise control and manipulation of micro/nanofluids in micro/nanoscale spaces, demonstrating the great potential of ARGs detection in wastewater. Here, we (1) summarize the state of the art in microfluidics for recognizing ARGs, (2) determine the strengths and weaknesses of portable microfluidic chips, and (3) assess the potential of portable microfluidic chips to detect ARGs in wastewater. Isothermal nucleic acid amplification and CRISPR/Cas are two commonly used identification elements for the microfluidic detection of ARGs. The former has better sensitivity due to amplification, but false positives due to inappropriate primer design and contamination; the latter has better specificity. The combination of the two can achieve complementarity to a certain extent. Compared with traditional microfluidic chips, low-cost and biocompatible paper-based microfluidics is a very attractive test for ARGs, whose fluid flow in paper does not require external force, but it is weaker in terms of repeatability and high-throughput detection. Due to that only a handful of portable microfluidics detect ARGs in wastewater, fabricating high-throughput microfluidic chips, developing and optimizing recognition techniques for the highly selective and sensitive identification and quantification of a wide range of ARGs in complex wastewater matrices are needed.}, }
@article {pmid39707712, year = {2025}, author = {Abdulsalam, L and Mordecai, J and Ahmad, I}, title = {Non-viral gene therapy for Leber's congenital amaurosis: progress and possibilities.}, journal = {Nanomedicine (London, England)}, volume = {20}, number = {3}, pages = {291-304}, pmid = {39707712}, issn = {1748-6963}, mesh = {Humans ; *Genetic Therapy/methods ; *Leber Congenital Amaurosis/therapy/genetics ; Animals ; *Gene Transfer Techniques ; Nanoparticles/chemistry ; CRISPR-Cas Systems/genetics ; Mutation ; Precision Medicine ; }, abstract = {Leber's congenital amaurosis (LCA) represents a set of rare and pervasive hereditary conditions of the retina that cause severe vision loss starting in early childhood. Targeted treatment intervention has become possible thanks to recent advances in understanding LCA genetic basis. While viral vectors have shown efficacy in gene delivery, they present challenges related to safety, low cargo capacity, and the potential for random genomic integration. Non-viral gene therapy is a safer and more flexible alternative to treating the underlying genetic mutation causing LCA. Non-viral gene delivery methods, such as inorganic nanoparticles, polymer-based delivery systems, and lipid-based nanoparticles, bypass the risks of immunogenicity and genomic integration, potentially offering a more versatile and personalized treatment for patients. This review explores the genetic background of LCA, emphasizing the mutations involved, and explores diverse non-viral gene delivery methods being developed. It also highlights recent studies on non-viral gene therapy for LCA in animal models and clinical trials. It presents future perspectives for gene therapy, including integrating emerging technologies like CRISPR-Cas9, interdisciplinary collaborations, personalized medicine, and ethical considerations.}, }
@article {pmid39707688, year = {2024}, author = {Garay-Novillo, JN and Ruiz-Masó, J&#xc1; and Del Solar, G and Barra, JL}, title = {Easy-Curing and pH-Regulated CRISPR-Cas9 Plasmids for Gene Editing and Plasmid Curing in Lactococcus cremoris.}, journal = {Microbial biotechnology}, volume = {17}, number = {12}, pages = {e70060}, pmid = {39707688}, issn = {1751-7915}, support = {CSIC 2022AEP028//Consejo Superior de Investigaciones Cient&#xed;ficas/ ; 2023/ResMi-00000018//Secretar&#xed;a de Ciencia y Tecnolog&#xed;a - Universidad Nacional de C&#xf3;rdoba and Ministerio de Ciencia y Tecnolog&#xed;a - C&#xf3;rdoba/ ; PICT Start-Up 2018-00811//Agencia Nacional de Promoci&#xf3;n Cient&#xed;fica y Tecnol&#xf3;gica/ ; CONSOLIDAR RESOL-2023-258-E-UNC-SECYT#ACTIP//Secretar&#xed;a de Ciencia y Tecnolog&#xed;a - Universidad Nacional de C&#xf3;rdoba/ ; MHE200027-EMHE-CSIC-2016-program//Consejo Superior de Investigaciones Cient&#xed;ficas/ ; }, mesh = {*Plasmids/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Genetic Vectors/genetics ; *Lactococcus/genetics ; Hydrogen-Ion Concentration ; }, abstract = {In this work, we developed a plasmid-based CRISPR-Cas9 strategy for editing Lactococcus cremoris, which allows easy generation of plasmid-free strains with the desired modification. We constructed versatile shuttle vectors based on the theta-type pAMβ1 promiscuous replicon and p15A ori, expressing both the Cas9 nuclease gene (under pH-regulated promoters derived from P170) and a single-guide RNA for specific targeting (under a strong constitutive promoter). The vectors designed for plasmid targeting were very effective for low- and high-copy-number plasmid curing in L. cremoris, and their targeting efficiency was shown to be tunable by regulating cas9 expression. For chromosome editing, we implemented a host-independent method that enhances double-homologous recombination events using plasmids expressing the genes encoding λRed-phage Redβ recombinase and Escherichia coli single-stranded DNA binding protein (EcSSB). By coupling either the endogenous recombination machinery or the Redβ-EcSSB-assisted recombination system with our novel chromosome-targeting CRISPR-Cas9 plasmids, we efficiently generated and selected thousands of gene-edited cells. Examination of the impact of the constructed CRISPR-Cas9 vectors on host fitness revealed no Cas9-associated toxicity, and, remarkably, these vectors exhibited a very high loss rate when growing the bacterial host cells in the absence of selective pressure.}, }
@article {pmid39707619, year = {2025}, author = {Liao, W and Guo, R and Li, J and Liu, N and Jiang, L and Whelan, J and Shou, H}, title = {CRISPR/Cas9-mediated mutagenesis of SEED FATTY ACID REDUCER genes significantly increased seed oil content in soybean.}, journal = {Plant &amp; cell physiology}, volume = {66}, number = {2}, pages = {273-284}, doi = {10.1093/pcp/pcae148}, pmid = {39707619}, issn = {1471-9053}, support = {2021C02057//Key Research Projects of Zhejiang Province/ ; 2021YFF1001204//Research and Development Program of China/ ; B14027//111 Project of Ministry of Education/ ; }, mesh = {*Glycine max/genetics/metabolism ; *Seeds/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *Fatty Acids/metabolism ; Mutagenesis/genetics ; *Plant Oils/metabolism ; *Plant Proteins/genetics/metabolism ; *Soybean Oil/metabolism ; Gene Editing ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; Genes, Plant ; }, abstract = {Increasing seed oil content (SOC) is an important breeding goal for soybean breeding. While significant efforts have been made to improve SOC through metabolic pathway engineering, research to increase soybean SOC by reducing lipid degradation and fatty acid (FA) decomposition during seed maturation process is limited. Seed fatty acid reducers (SFARs) are members of the GDSL enzyme family and play a crucial role in lipid metabolism. Among them, a pair of the GmSFAR4 genes is highly expressed in soybean seeds during seed desiccation and germination. In the study, GmSFAR4a/b double mutants were generated using CRISPR/Cas9-mediated gene editing technique. The seed FA content of GmSFAR4a/b double mutants was significantly increased by ∼8% compared to wild type when grown in greenhouse, and ∼17% when grown in the field, without any adverse effects on seed vitality and plant growth. Our work enriches the understanding of soybean seed oil metabolism and provides a new approach to increase soybean SOC.}, }
@article {pmid39707395, year = {2024}, author = {Xu, W and Zhang, S and Qin, H and Yao, K}, title = {From bench to bedside: cutting-edge applications of base editing and prime editing in precision medicine.}, journal = {Journal of translational medicine}, volume = {22}, number = {1}, pages = {1133}, pmid = {39707395}, issn = {1479-5876}, support = {82471107//National Natural Science Foundation of China/ ; 31970930//National Natural Science Foundation of China/ ; 2020CFA069//Natural Science Foundation of Hubei Province/ ; 2018CFB434//Natural Science Foundation of Hubei Province/ ; }, mesh = {Humans ; *Precision Medicine/methods ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Translational Research, Biomedical ; Animals ; Genetic Therapy/methods ; }, abstract = {CRISPR-based gene editing technology theoretically allows for precise manipulation of any genetic target within living cells, achieving the desired sequence modifications. This revolutionary advancement has fundamentally transformed the field of biomedicine, offering immense clinical potential for treating and correcting genetic disorders. In the treatment of most genetic diseases, precise genome editing that avoids the generation of mixed editing byproducts is considered the ideal approach. This article reviews the current progress of base editors and prime editors, elaborating on specific examples of their applications in the therapeutic field, and highlights opportunities for improvement. Furthermore, we discuss the specific performance of these technologies in terms of safety and efficacy in clinical applications, and analyze the latest advancements and potential directions that could influence the future development of genome editing technologies. Our goal is to outline the clinical relevance of this rapidly evolving scientific field and preview a roadmap for successful DNA base editing therapies for the treatment of hereditary or idiopathic diseases.}, }
@article {pmid39706434, year = {2025}, author = {Sang, S and Sun, X and Ma, T and Zhang, Y and Yao, G and Wang, X and Tan, X and Feng, L and Li, J and Ji, S and Cheng, H}, title = {Efficient promoter editing of the SBEIIb gene enables fine-tuning of the resistant starch content in rice.}, journal = {International journal of biological macromolecules}, volume = {290}, number = {}, pages = {138904}, doi = {10.1016/j.ijbiomac.2024.138904}, pmid = {39706434}, issn = {1879-0003}, mesh = {*Oryza/genetics/metabolism ; *Gene Editing/methods ; *Promoter Regions, Genetic ; CRISPR-Cas Systems ; *1,4-alpha-Glucan Branching Enzyme/genetics ; *Starch ; *Resistant Starch/metabolism ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; Mutation ; *Plant Proteins/genetics ; }, abstract = {Rice, a staple in diets, undergoes digestion post-consumption, often triggering a swift surge in blood sugar among diabetics, intensifying their health burden. Notably, resistant starch (RS) emerges as a potent ally in fostering satiety and mitigating metabolic syndrome in diabetes. The SBEIIb gene, a key orchestrator of starch branching enzymes, plays a pivotal role in starch synthesis, and its genetic alteration can dramatically boost RS content in rice. Cultivating RS-rich functional germplasms is of great significance for improving human nutrition and health. In this study, the application of CRISPR/Cas9 technology was investigated for precise multi-target editing within the SBEIIb promoter, aiming to generate valuable germplasm resources enriched with RS. The results revealed extensive mutations in the SBEIIb promoter region, resulting in a varied reduction in SBEIIb expression levels. Remarkably, the grains from the homozygous T1 mutant lines displayed a substantial elevation in RS content, ranging from 3 to 12 times greater than that of the wild-type, accompanied by notable alterations in the starch granule morphology of these grains. This study presents an effective breeding strategy for the precise enhancement of RS content and establishes a foundation for further insights into the molecular mechanisms governing cis-element regulation of RS synthesis.}, }
@article {pmid39706401, year = {2025}, author = {Yu, H and Feng, M and Liu, C and Wang, F and Pan, S and Sui, G and Jing, W and Cheng, X}, title = {CRISPR-Cas12a2-based rapid and sensitive detection system for target nucleic acid.}, journal = {International journal of biological macromolecules}, volume = {290}, number = {}, pages = {138996}, doi = {10.1016/j.ijbiomac.2024.138996}, pmid = {39706401}, issn = {1879-0003}, mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acids/genetics ; Humans ; Polymerase Chain Reaction/methods ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Infectious diseases are extremely important public health issues, where the design of effective, rapid, and convenient detection platforms is critical. In this study, we coupled SuCas12a2, a novel Cas12 family RNA-targeting nuclease, with conventional PCR and recombinase polymerase amplification (RPA), respectively, to develop novel detection approaches, named PCR-SuCas12a2 and RPA-SuCas12a2. SuCas12a2 possesses collateral cleavage activity and cuts the additional single-stranded RNA (ssRNA) added to the reaction system once the ternary complex RNA-SuCas12a2-CRISPR RNA (crRNA) is formed. SuCas12a2 is specifically activated, where the cleaved fluorescent-labeled probes release fluorescent signals, with the strength of the fluorescent signal being proportional to the concentration of nucleic acids specifically bound to crRNA. Simultaneous transcription and SuCas12a2 detection can be performed in a single tube by introducing the T7 promoter sequence into the forward primer. Entamoeba histolytica (E. histolytica) and Mycoplasma pneumoniae (M. pneumoniae) were used as proof specimens to evaluate the performance of the platform. PCR-SuCas12a2 has excellent capabilities, including high specificity with no cross-reactivity from other species and ultra-sensitivity that achieves a detection of one copy per reaction for E. histolytica and M. pneumoniae. However, the sensitivity of the RPA-SuCas12a2 assay was 10[2] copies per reaction, which was inferior to PCR-SuCas12a2. Clinical samples were obtained from suspected infection patients of E. histolytica and M. pneumoniae, and used to evaluate the systems demonstrated 100 % specificity. The technique shows robust performance and suggests great potential for point-of-care testing of other pathogens to facilitate effective management and control of the spread of diseases.}, }
@article {pmid39706269, year = {2025}, author = {Li, X and Wei, Y and Wang, SY and Wang, SG and Xia, PF}, title = {One-for-all gene inactivation via PAM-independent base editing in bacteria.}, journal = {The Journal of biological chemistry}, volume = {301}, number = {1}, pages = {108113}, pmid = {39706269}, issn = {1083-351X}, mesh = {*Gene Editing/methods ; *Escherichia coli/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Silencing ; }, abstract = {Base editing is preferable for bacterial gene inactivation without generating double-strand breaks, requiring homology recombination, or highly efficient DNA delivery capability. However, the potential of base editing is limited by the adjoined dependence on the editing window and protospacer adjacent motif. Herein, we report an unconstrained base-editing system to enable the inactivation of any genes of interest in bacteria. We employed a dCas9 derivative, dSpRY, and activation-induced cytidine deaminase to build a protospacer adjacent motif-independent base editor. Then, we programmed the base editor to exclude the START codon of a gene of interest instead of introducing premature STOP codons to obtain a universal approach for gene inactivation, namely XSTART, with an overall efficiency approaching 100%. By using XSTART, we successfully manipulated the amino acid metabolisms in Escherichia coli, generating glutamine, arginine, and aspartate auxotrophic strains. While we observed a high frequency of off-target events as a trade-off for increased efficiency, refining the regulatory system of XSTART to limit expression levels reduced off-target events by over 60% without sacrificing efficiency, aligning our results with previously reported levels. Finally, the effectiveness of XSTART was also demonstrated in probiotic E. coli Nissle 1917 and photoautotrophic cyanobacterium Synechococcus elongatus, illustrating its potential in reprogramming diverse bacteria.}, }
@article {pmid39706164, year = {2024}, author = {Cenik, BK and Aoi, Y and Iwanaszko, M and Howard, BC and Morgan, MA and Andersen, GD and Bartom, ET and Shilatifard, A}, title = {TurboCas: A method for locus-specific labeling of genomic regions and isolating their associated protein interactome.}, journal = {Molecular cell}, volume = {84}, number = {24}, pages = {4929-4944.e8}, doi = {10.1016/j.molcel.2024.11.007}, pmid = {39706164}, issn = {1097-4164}, mesh = {Humans ; *Promoter Regions, Genetic ; *Transcription Factors/metabolism/genetics ; *Chromatin/metabolism/genetics ; HEK293 Cells ; RNA Polymerase II/metabolism/genetics ; Protein Binding ; CRISPR-Cas Systems ; Cell Cycle Proteins/metabolism/genetics ; Positive Transcriptional Elongation Factor B/metabolism/genetics ; Proto-Oncogene Proteins c-myc/genetics/metabolism ; Proto-Oncogene Proteins c-fos/genetics/metabolism ; Gene Expression Regulation ; Heat-Shock Response/genetics ; Protein Interaction Maps ; Bromodomain Containing Proteins ; }, abstract = {Regulation of gene expression during development and stress response requires the concerted action of transcription factors and chromatin-binding proteins. Because this process is cell-type specific and varies with cellular conditions, mapping of chromatin factors at individual regulatory loci is crucial for understanding cis-regulatory control. Previous methods only characterize static protein binding. We present "TurboCas," a method combining a proximity-labeling (PL) enzyme, miniTurbo, with CRISPR-dCas9 that allows for efficient and site-specific labeling of chromatin factors in mammalian cells. Validating TurboCas at the FOS promoter, we identify proteins recruited upon heat shock, cross-validated via RNA polymerase II and P-TEFb immunoprecipitation. These methodologies reveal canonical and uncharacterized factors that function to activate expression of heat-shock-responsive genes. Applying TurboCas to the MYC promoter, we identify two P-TEFb coactivators, the super elongation complex (SEC) and BRD4, as MYC co-regulators. TurboCas provides a genome-specific targeting PL, with the potential to deepen our molecular understanding of transcriptional regulatory pathways in development and stress response.}, }
@article {pmid39705796, year = {2025}, author = {Bian, S and Cai, Q and Wang, S and Xie, Y and Chen, N and Song, Q and Li, H and Zhao, N and Zhang, X}, title = {Evaluation of the toxoplasma Urm1 gene deletion mutant (PruΔUrm1) as a promising vaccine candidate against toxoplasmosis in mice.}, journal = {Vaccine}, volume = {45}, number = {}, pages = {126632}, doi = {10.1016/j.vaccine.2024.126632}, pmid = {39705796}, issn = {1873-2518}, mesh = {Animals ; *Toxoplasma/genetics/immunology/pathogenicity ; *Protozoan Vaccines/immunology/genetics/administration &amp; dosage ; Mice ; Gene Deletion ; *Protozoan Proteins/genetics/immunology ; Female ; *Toxoplasmosis, Animal/prevention &amp; control/immunology ; Antibodies, Protozoan/blood ; Mice, Inbred BALB C ; Cytokines/metabolism ; *Toxoplasmosis/prevention &amp; control ; Disease Models, Animal ; CRISPR-Cas Systems ; }, abstract = {Toxoplasmosis is a significant zoonotic disease that poses a serious threat to both human and animal health. Despite ongoing research, developing an effective vaccine for toxoplasmosis remains a challenge. In this study, we evaluated the vaccine potential of the Toxoplasma Urm1 gene deletion mutant (PruΔUrm1) by assessing its pathogenicity and protective efficacy in mice. Using CRISPR/Cas9 technology, we successfully created a type II Toxoplasma gondii Pru mutant strain with a deleted Urm1 gene. Compared to the wild-type parasite, the PruΔUrm1 strain exhibited significantly reduced invasive and proliferative abilities in vitro. In in vivo studies, mice intraperitoneally infected with the parental Pru strain showed severe symptoms including emaciation, hunching, and high mortality rates. In contrast, mice infected with PruΔUrm1 tachyzoites demonstrated a 100 % survival rate, no overt symptoms, and a markedly reduced parasite burden in the liver, spleen, and lungs, indicating reduced pathogenicity. Notably, PruΔUrm1 vaccination triggered a strong immune response, characterized by significantly elevated cytokine levels, including TNF-α, IFN-γ and IL-10. Furthermore, we assessed the immunoprotective efficacy of PruΔUrm1 vaccination in mice against type I strains. Mice immunized with PruΔUrm1 were able to resist the tachyzoites of type I RH wild-type parasites, achieving a 100 % survival rate and significantly reduced parasite loads in the liver, spleen and lungs. These data demonstrate that PruΔUrm1 immunization provides effective protection against acute Toxoplasma infections and holds promise as a potential vaccine candidate for toxoplasmosis.}, }
@article {pmid39705306, year = {2024}, author = {Hou, J and Guo, P and Wang, J and Han, D and Tan, W}, title = {Artificial dynamic structure ensemble-guided rational design of a universal RNA aptamer-based sensing tag.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {121}, number = {52}, pages = {e2414793121}, pmid = {39705306}, issn = {1091-6490}, mesh = {*Aptamers, Nucleotide/chemistry ; Humans ; *Biosensing Techniques/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Gene Editing/methods ; Nucleic Acid Conformation ; RNA/chemistry ; Fluorescent Dyes/chemistry ; }, abstract = {Artificially functional RNAs, such as fluorogenic RNA aptamer (FRApt)-based biosensing tag, represent significant advancements in various biological applications but are limited by the lack of insight into dynamic structure ensembles and universal design concepts. Through the development of an artificial RNA structure ensemble, we rationally established an RNA reconstitution model, "SSPepper-Apt," to generate a universal fluorogenic RNA biosensing tag. By utilizing various target-recognizing RNA motifs, SSPepper-Apt enables the modular generation of sensing tags for low-background, highly selective imaging of metabolites, peptides, and proteins in living cells. Additionally, by employing single guide RNA (sgRNA) as the recognition RNA motif, SSPepper-Apt generates fluorescence in both CRISPR-mediated imaging and gene editing only when the Cas9-sgRNA complex is successfully assembled; therefore, it can be an effective sgRNA screening tool for gene editing. Our fluorogenic RNA-sensing tag provides a universal approach for constructing functional RNA systems, avoiding the laborious and time-consuming process of sequence combination, and expanding the application of synthetic biological tools.}, }
@article {pmid39704707, year = {2025}, author = {Deng, R and Sheng, J and Xie, Z and Yang, H and Yang, S and Xie, S and Tang, X and Zhao, S and Dong, H and Chen, M and Chang, K}, title = {miR-Cabiner: A Universal microRNA Sensing Platform Based on Self-Stacking Cascaded Bicyclic DNA Circuit-Mediated CRISPR/Cas12a.}, journal = {Analytical chemistry}, volume = {97}, number = {1}, pages = {799-810}, doi = {10.1021/acs.analchem.4c05370}, pmid = {39704707}, issn = {1520-6882}, mesh = {*MicroRNAs/analysis/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *DNA/chemistry/genetics ; Breast Neoplasms/diagnosis/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {CRISPR/Cas12a-based diagnostics have great potential for sensing nucleic acids, but their application is limited by the sequence-dependent property. A platform termed miR-Cabiner (a universal miRNA sensing platform based on self-stacking cascaded bicyclic DNA circuit-mediated CRISPR/Cas12a) is demonstrated herein that is sensitive and universal for analyzing miRNAs. This platform combines catalytic hairpin assembly (CHA) and hybrid chain reaction (HCR) into a unified circuit and finally cascades to CRISPR/Cas12a. Compared with the CHA-Cas12a and HCR-Cas12a systems, miR-Cabiner exhibits a significantly higher reaction rate. Panels of miRNAs (miR-130a, miR-10b, miR-21, and miR-1285), which are associated with diagnosis, staging, and prognosis of breast cancer, are designed to demonstrate the universality of miR-Cabiner. Four miRNAs can be detected to the fM-level by simply tuning the sequence in CHA components. Additionally, miRNA panel analysis also shows high accuracy in practical samples. This universally applicable platform for detecting miRNA may serve as an excellent tool for clinical diagnosis.}, }
@article {pmid39704190, year = {2024}, author = {Fan, Z and Xu, L and Cao, Y and Liu, T and Tian, Y and Pan, Z and Mo, Y and Wang, X and Zhu, X and Gao, Y and Zhang, X and Pan, CQ and Wang, L and Ren, F}, title = {One-Pot Assay Based on CRISPR/Cas13a Technology for HEV RNA Point-of-Care Testing.}, journal = {Journal of medical virology}, volume = {96}, number = {12}, pages = {e70115}, pmid = {39704190}, issn = {1096-9071}, support = {//This study was supported by the National Natural Science Foundation of China (82002243, 82100653), Key Projects of the Beijing Municipal Education Commission's Science and Technology Plan (KZ202010025035), Chinese Institutes for Medical Research, Beijing (Grant No. CX24PY23), Beijing Hospitals Authority Youth Programme (QML20201702), Talent Cultivation Plan of Climbing the Peak of Beijing Municipal Hospital Administration (DFL20221503), Beijing Natural Science Foundation-Changping Innovation Joint Fund (L234046), Training Fund for Open Projects at Clinical Institutes and Departments of Capital Medical University (CCMU2023ZKYXZ003), High-Level Public Health Technical Talents Project of Beijing (Subject Leaders-02-13, xuekegugan-03-48)./ ; }, mesh = {*Hepatitis E virus/genetics/isolation &amp; purification ; *Point-of-Care Testing ; *Hepatitis E/diagnosis/virology ; *RNA, Viral/genetics ; Humans ; *CRISPR-Cas Systems ; *Sensitivity and Specificity ; Animals ; Limit of Detection ; Reverse Transcriptase Polymerase Chain Reaction/methods ; Genotype ; Nucleic Acid Amplification Techniques/methods ; }, abstract = {Hepatitis E virus (HEV) poses a serious threat to both public health and animal food safety, thereby highlighting the demands for rapid, sensitive, and easy-to-use detection. This study aimed to develop a One-Pot assay using CRISPR/Cas13a for detecting HEV RNA, suitable for point-of-care testing (POCT) in resource-limited settings. CRISPR/Cas13a combined with reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription recombinase-aided amplification (RT-RAA) was applied to a One-Pot assay device. Additionally, a large cohort of HEV-infected patient (154) and animal (104) specimens was utilized for validation. The RT-PCR/RT-RAA + CRISPR/Cas13a assays for HEV RNA detection (genotypes: HEV-1, HEV-3, and HEV-4) were established, optimized, and validated, achieving a limit of detection (LoD) of 1 copy/μL and 100% specificity. In the application validation for HEV infection, the positive rates of the RT-PCR + CRISPR and RT-RAA + CRISPR assays were 98.6% and 89.6% for patients, and 96.6% and 88.8% for animals, respectively, which were superior to those of RT-qPCR. Furthermore, sample rapid lysis, reagent lyophilization, and the One-Pot device were integrated to construct a One-Pot assay with an LoD of 10[2] copies/μL. Despite slight decreases in sensitivity, the One-Pot assay significantly reduces the assay time to 35 min, making it easy to perform, minimizing contamination, and meeting the requirements for screening. We developed a One-Pot assay of HEV RNA using the CRISPR/Cas13a which effectively realizes a POCT test and maximizes the impetus for POCT implementation and shows potential as a valuable tool for detecting and monitoring HEV infection.}, }
@article {pmid39704119, year = {2025}, author = {Lv, L and Liu, S and Fu, Y and Zhang, Y and Wang, M and Sun, J and Wang, Y and Lu, Y and Niu, G}, title = {A tunable and reversible thermo-inducible bio-switch for streptomycetes.}, journal = {Nucleic acids research}, volume = {53}, number = {2}, pages = {}, pmid = {39704119}, issn = {1362-4962}, support = {2023YFD1700700//National Key Research and Development Program of China/ ; 32370077//National Natural Science Foundation of China/ ; }, mesh = {*Streptomyces/genetics/metabolism ; *Gene Expression Regulation, Bacterial ; CRISPR-Cas Systems ; Temperature ; Anthraquinones/metabolism ; Anti-Bacterial Agents/biosynthesis ; Bacterial Proteins/genetics/metabolism ; Benzoisochromanequinones ; }, abstract = {Programmable control of bacterial gene expression holds great significance for both applied and academic research. This is particularly true for Streptomyces, a genus of Gram-positive bacteria and major producers of prodigious natural products. Despite that a few inducible regulatory systems have been developed for use in Streptomyces, there is an increasing pursuit to augment the toolkit of high-performance induction systems. We herein report a robust and reversible thermo-inducible bio-switch, designated as StrepT-switch. This bio-switch enables tunable and reversible control of gene expression using physiological temperatures as stimulation inputs. It has been proven successful in highly efficient CRISPR/Cas9-mediated genome engineering, as well as programmable control of antibiotic production and morphological differentiation. The versatility of the device is also demonstrated by thermal induction of a site-specific relaxase ZouA for overproduction of actinorhodin, a blue pigmented polyketide antibiotic. This study showcases the exploration a temperature-sensing module and exemplifies its versatility for programmable control of various target genes in Streptomyces species.}, }
@article {pmid39703747, year = {2024}, author = {Fallah, T and Shafiei, M}, title = {Comprehensive Analysis of CRISPR-Cas Systems and Their Influence on Antibiotic Resistance in Salmonella enterica Strains.}, journal = {Bioinformatics and biology insights}, volume = {18}, number = {}, pages = {11779322241307984}, pmid = {39703747}, issn = {1177-9322}, abstract = {Salmonella enterica is a gram-negative bacterium that demonstrates a remarkable ability to acquire antibiotic resistance genes (ARGs). The role of the CRISPR-Cas system in influencing antibiotic resistance in S. enterica is still under investigation. This study explores the distribution and impact of CRISPR-Cas systems on antibiotic resistance by analyzing 316 S. enterica genomes. We conducted sequence alignments, phylogenetic analyses, and conservation studies on Cas genes, direct repeats (DRs), and leader sequences. Promoter predictions and RNA secondary structure analyses were also performed. ARGs were identified, and their correlation with Cas gene clusters was evaluated. Our findings revealed that 82.33% of strains possess complete CRISPR-Cas systems, while 17.66% have orphan CRISPRs. We identified 290 distinct DRs, most of which formed stable stem-loop structures, although no promoter regions were detected within the leader sequences. Most spacers were chromosome-targeting, with a smaller proportion homologous to phages and plasmids. Importantly, strains with complete CRISPR-Cas systems showed a higher incidence of ARGs compared with those with orphan or no CRISPR systems. Specifically, the incidence of ARGs was 54.3% higher in strains with complete CRISPR-Cas systems than in strains without CRISPR-Cas systems, and 15.1% higher than in strains with orphan CRISPRs. Spearman's correlation analysis confirmed a statistically significant but weak correlation between the presence of Cas genes and the frequency of ARGs (P-value = 3.892e-06). These results suggest that CRISPR-Cas systems may play a role in the acquisition of ARGs, potentially through mutations under antibiotic pressure. Future studies should investigate mutations, particularly in Cas3-the signature protein of type I CRISPR-Cas systems. In addition, experimental validation, such as culturing S. enterica strains with complete CRISPR-Cas systems under different antibiotic conditions, followed by sequencing to assess the uptake or absence of newly acquired ARGs, would help clarify the potential role of CRISPR-Cas systems in bacterial adaptation to antimicrobial pressures.}, }
@article {pmid39702810, year = {2024}, author = {Bhagat, M and Kamal, R and Sharma, J and Kaur, K and Sharma, A and Singh, TG and Bhatia, R and Awasthi, A}, title = {Gene Therapy: Towards a New Era of Medicine.}, journal = {AAPS PharmSciTech}, volume = {26}, number = {1}, pages = {17}, pmid = {39702810}, issn = {1530-9932}, mesh = {Humans ; *Genetic Therapy/methods/trends ; Animals ; *Genetic Vectors ; CRISPR-Cas Systems ; Gene Editing/methods ; Gene Transfer Techniques ; }, abstract = {Over the past years, many significant advances have been made in the field of gene therapy and shown promising results in clinical trials conducted. Gene therapy aims at modifying or replacing a defective, inefficient, or nonfunctional gene with a healthy, functional gene by administration of genome material into the cell to cure genetic diseases. Various methods have been devised to do this by using several viral and non-viral vectors which are either administered by in vivo or ex vivo technique. Viral vectors are best suitable for this therapy due to their potential to invade cells and deliver their genetic material whereas non-viral vectors are less efficient than viral vectors but possess some advantages such as less immunogenic response and large gene carrying capacity. Recent advances in biotechnology such as CRISPR-Cas9 mediated genome engineering and Cancer treatment with Chimeric antigen receptor (CAR) T-cell therapy are addressed in this review. This review article also delves into some recent research studies, gene therapy trials, and its applications, laying out future hopes for gene therapy in the treatment of various diseases namely haemophilia, Muscular dystrophy, SCID, Sickle cell disease, Familial Hypercholesterolemia, Cystic Fibrosis. Additionally, it also includes various nanoformulations and clinical trial data related to gene therapy.}, }
@article {pmid39702692, year = {2025}, author = {Du, W and Meister, LL and van Grinsven, T and Branco Dos Santos, F}, title = {Efficient Multiplex Genome Editing of the Cyanobacterium Synechocystis sp. PCC6803 via CRISPR-Cas12a.}, journal = {Biotechnology and bioengineering}, volume = {122}, number = {3}, pages = {736-743}, pmid = {39702692}, issn = {1097-0290}, mesh = {*Synechocystis/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plasmids/genetics ; *Genome, Bacterial ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Cyanobacteria have been genetically modified to convert CO2 into biochemical products, but efficient genetic engineering tools, including CRISPR-Cas systems, remain limited. This is primarily due to the polyploid nature of cyanobacteria, which hinders their effectiveness. Here, we address the latter by specifically (i) modifying the RSF1010-based replicative plasmid to simplify cloning efforts while maintaining high conjugation efficiency; (ii) improving the design of the guide RNA (gRNA) to facilitate chromosomal cleavage; (iii) introducing template DNA fragments as pure plasmids via natural transformation; and (iv) using sacB to facilitate replicative plasmid curing. With this system, the replicative plasmid containing both Cas12a and gRNA is introduced to Synechocystis sp. PCC6803 cells via conjugation to cleave the circular chromosomes. Template DNA plasmid that has meanwhile been assimilated will then repair it achieving the desired genetic modifications. This system was validated by successfully deleting various "neutral" chromosomal loci, both individually and collectively, as well as targeting an essential gene, sll1797. With the sacB-sucrose counter-selection, all deletions were simultaneously made markerless in < 4 weeks. Moreover, we also integrate YFP with various protein degradation tags into the chromosome, allowing for their characterization at the chromosomal level. We foresee this system will greatly facilitate future genome engineering in cyanobacteria.}, }
@article {pmid39702666, year = {2024}, author = {Kou, Z and Wang, S and Luo, X and Xu, J and Tomberlin, JK and Huang, Y}, title = {Wingless strain created using binary transgenic CRISPR/Cas9 alleviates concerns about mass rearing of Hermetia illucens.}, journal = {Communications biology}, volume = {7}, number = {1}, pages = {1652}, pmid = {39702666}, issn = {2399-3642}, support = {32021001//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32100381//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {Animals ; *CRISPR-Cas Systems ; *Animals, Genetically Modified ; *Diptera/genetics ; Female ; Male ; }, abstract = {Larvae of the black soldier fly Hermetia illucens have potential as a natural waste recycler and subsequent use as protein-rich feed for livestock. A common question about the insect-farming processes is, what about the concerns of mass escape of insects from large populations? Here, we present a binary transgenic CRISPR/Cas9 system to generate wingless strain with the potential to address this issue. We identified gonad-specific promoters in vivo and evaluated use of the two strongest promoters, nanos and exuperantia, to drive Cas9 expression. We found that crossing the Hiexu-Cas9 with transgenic sgRNA-expressing insects resulted in higher knockout efficiency of the marker gene white. The Hiexu-Cas9 strain exhibited a maternal deposition of Cas9 that caused more effective knockout in the progeny of female Cas9-expressing individuals. Using this system, we generated wingless mutants lacking mating ability, which can be maintained in colony through a genetic cross of two single strain. These insects are less likely to escape and would be unable to successfully mate if they did escape. Taken together, this study validates effective genetic tools that can be used for gene function studies and industrial applications in black soldier fly and provides an approach to alleviate the concern about massive rearing.}, }
@article {pmid39702333, year = {2024}, author = {Liang, QZ and Chen, W and Liu, RC and Fu, QL and Fu, GH and Cheng, LF and Chen, HM and Jiang, NS and Zhu, T and Huang, Y}, title = {CRISPR/Cas12a and recombinase polymerase amplification-based rapid on-site nucleic acid detection of duck circovirus.}, journal = {Virology journal}, volume = {21}, number = {1}, pages = {322}, pmid = {39702333}, issn = {1743-422X}, support = {ZYTS202423//the Freedom Explore Program of Fujian Academy of Agricultural Sciences/ ; 2023J01363//the Natural Science Foundation Project of Fujian Province/ ; CARS-42//the National Waterfowl Industry Technology System of Modern Agriculture for China/ ; }, mesh = {*Circovirus/genetics/isolation &amp; purification ; Animals ; *Ducks/virology ; *Circoviridae Infections/veterinary/diagnosis/virology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Sensitivity and Specificity ; Poultry Diseases/virology/diagnosis ; Recombinases/metabolism/genetics ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {BACKGROUND: Duck circovirus (DuCV) infections commonly induce immunosuppression and secondary infections in ducks, resulting in significant economic losses in the duck breeding industry. Currently, effective vaccines and treatments for DuCV have been lacking. Therefore, rapid, specific, and sensitive detection methods are crucial for preventing and controlling DuCV.<br><br>METHODS: A lateral flow strip (LFS) detection method was developed using recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a). The RPA-CRISPR/Cas12a-LFS targeted the DuCV replication protein (Rep) and was operated at 37 &#x2103; and allowed for visual interpretation without requiring sophisticated equipment.<br><br>RESULTS: The results revealed that the reaction time of RPA-CRISPR/Cas12a-LFS is only 45&#xa0;min. This method achieved a low detection limit of 2.6 gene copies. Importantly, this method demonstrated high specificity and no cross-reactivity with six other avian viruses. In a study involving 97 waterfowl samples, the Rep RPA-CRISPR/Cas12a-LFS showed 100% consistency and agreement with real-time quantitative polymerase chain reaction.<br><br>CONCLUSION: These findings underscored the potential of this user-friendly, rapid, sensitive, and accurate detection method for on-site DuCV detection.}, }
@article {pmid39701943, year = {2025}, author = {Zhao, Z and Xiong, Q and Zhu, Y and Zhang, C and Li, Z and Chen, Z and Zhang, Y and Deng, X and Tao, Y and Xu, S}, title = {CRISPR/Cas12a-Enabled Amplification-Free Colorimetric Visual Sensing Strategy for Point-of-Care Diagnostics of Biomarkers.}, journal = {Analytical chemistry}, volume = {97}, number = {1}, pages = {1019-1027}, doi = {10.1021/acs.analchem.4c06196}, pmid = {39701943}, issn = {1520-6882}, mesh = {*Colorimetry/methods ; Humans ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Point-of-Care Testing ; Gold/chemistry ; *BRCA1 Protein/genetics/blood ; Metal Nanoparticles/chemistry ; Horseradish Peroxidase/chemistry/metabolism ; *Point-of-Care Systems ; Biomarkers/analysis ; *Biomarkers, Tumor ; Benzidines/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {CRISPR/Cas12a-based biosensors have garnered significant attention in the field of point-of-care testing (POCT), yet the majority of the CRISPR-based POCT methods employ fluorescent systems as report probes. Herein, we report a new CRISPR/Cas12a-enabled multicolor visual biosensing strategy for the rapid detection of disease biomarkers. The proposed assay provided vivid color responses to enhance the accuracy of visual detection. In the existence of the target, the trans-cleavage activity of CRISPR-Cas12a was activated. The report probe modified with magnetic beads (MBs) and horseradish peroxidase (HRP) was cleaved, and HRP was released in the supernatant. As a result, HRP mediated the etching of gold nanobipyramids (AuNBPs) under hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine and generated a vivid color response. The proposed method has been verified by the detection of the breast cancer 1 gene (BRCA1) as a proof-of-principle target. According to the different colors of AuNBPs, our experimental results have demonstrated that as low as 30 pM BRCA1 can be detected with no more than 60 min. Additionally, the proposed sensor has been successfully applied in the analysis of BRCA1 in human serum samples with satisfactory results, which indicates great potential for the sensitive determination of biomarkers and the POCT area.}, }
@article {pmid39701024, year = {2025}, author = {Lee, J and Jeong, C}, title = {Single-molecule perspectives of CRISPR/Cas systems: target search, recognition, and cleavage.}, journal = {BMB reports}, volume = {58}, number = {1}, pages = {8-16}, pmid = {39701024}, issn = {1976-670X}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; DNA/genetics/metabolism ; Gene Editing/methods ; *Single Molecule Imaging/methods ; }, abstract = {CRISPR/Cas systems have emerged as powerful tools for gene editing, nucleic acid detection, and therapeutic applications. Recent advances in single-molecule techniques have provided new insights into the DNA-targeting mechanisms of CRISPR/ Cas systems, in particular, Types I, II, and V. Here, we review how single-molecule approaches have expanded our understanding of key processes, namely target search, recognition, and cleavage. Furthermore, we focus on the dynamic behavior of Cas proteins, including PAM site recognition and R-loop formation, which are crucial to ensure specificity and efficiency in gene editing. Additionally, we discuss the conformational changes and interactions that drive precise DNA cleavage by different Cas proteins. This mini review provides a comprehensive overview of CRISPR/Cas molecular dynamics, offering conclusive insights into their broader potential for genome editing and biotechnological applications. [BMB Reports 2025; 58(1): 8-16].}, }
@article {pmid39700796, year = {2025}, author = {Wang, X and Ding, H and Sun, Y and Ma, Y and Wang, G and Chen, J and Choo, J and Chen, L}, title = {CRISPR/HCR-powered ratiometric fluorescence aptasensor for ochratoxin A detection.}, journal = {Food chemistry}, volume = {468}, number = {}, pages = {142437}, doi = {10.1016/j.foodchem.2024.142437}, pmid = {39700796}, issn = {1873-7072}, mesh = {*Ochratoxins/analysis ; *Food Contamination/analysis ; *Biosensing Techniques/methods/instrumentation ; CRISPR-Cas Systems ; *Aptamers, Nucleotide/chemistry/genetics ; Limit of Detection ; Fluorescence ; Spectrometry, Fluorescence/methods ; }, abstract = {To address the need for highly sensitive and reliable detection of trace ochratoxin A (OTA) in food matrices, we developed a ratiometric fluorescent aptasensor by integrating CRISPR/Cas12a, hybridization chain reaction (HCR), and horseradish peroxidase (HRP)-induced inner filter effect (IFE). The mechanism involves OTA releasing an activator that initiates CRISPR/Cas12a trans-cleavage, blocking HCR assembly. This reduces HRP levels, limiting the conversion of o-phenylenediamine (OPD) to fluorescent 2,3-diaminophenazine (DAP) (emitting at 562 nm) while maintaining strong emission from 2-amino terephthalic acid (BDC-NH2) at 426 nm. The F426/F562 ratio serves as a "signal-on" indicator, enabling sensitive OTA detection over 0.1 pM to 10 nM, with a detection limit of 0.0417 pM. The method exhibits excellent reproducibility, with intra-day and inter-day relative standard deviations (RSDs) of 1.91 %-3.87 % and 1.79 %, respectively, along with recovery rates of 90.1 %-110.6 % in real samples. These advantages highlight its significant potential for CRISPR/Cas-based OTA detection.}, }
@article {pmid39700179, year = {2024}, author = {Bowland, K and Lai, J and Skaist, A and Zhang, Y and Teh, SSK and Roberts, NJ and Thompson, E and Wheelan, SJ and Hruban, RH and Karchin, R and Bailey, MH and Iacobuzio-Donahue, CA and Eshleman, JR}, title = {Islands of genomic stability in the face of genetically unstable metastatic cancer.}, journal = {PloS one}, volume = {19}, number = {12}, pages = {e0298490}, pmid = {39700179}, issn = {1932-6203}, support = {P30 CA006973/CA/NCI NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; P50 CA062924/CA/NCI NIH HHS/United States ; }, mesh = {Humans ; *Genomic Instability ; *Pancreatic Neoplasms/genetics/pathology ; *CRISPR-Cas Systems/genetics ; *Neoplasm Metastasis/genetics ; Carcinoma, Pancreatic Ductal/genetics/pathology/secondary ; Mutation ; Male ; Whole Genome Sequencing ; Female ; }, abstract = {INTRODUCTION: Metastatic cancer affects millions of people worldwide annually and is the leading cause of cancer-related deaths. Most patients with metastatic disease are not eligible for surgical resection, and current therapeutic regimens have varying success rates, some with 5-year survival rates below 5%. Here, we test the hypothesis that metastatic cancer can be genetically targeted by exploiting single base substitution mutations unique to individual cells that occur as part of normal aging prior to transformation. These mutations are targetable because ~10% of them form novel tumor-specific "NGG" protospacer adjacent motif (PAM) sites targetable by CRISPR-Cas9.<br><br>METHODS: Whole genome sequencing was performed on five rapid autopsy cases of patient-matched primary tumor, normal and metastatic tissue from pancreatic ductal adenocarcinoma decedents. CRISPR-Cas9 PAM targets were determined by bioinformatic tumor-normal subtraction for each patient and verified in metastatic samples by high-depth capture-based sequencing.<br><br>RESULTS: We found that 90% of PAM targets were maintained between primary carcinomas and metastases overall. We identified rules that predict PAM loss or retention, where PAMs located in heterozygous regions in the primary tumor can be lost in metastases (private LOH), but PAMs occurring in regions of loss of heterozygosity (LOH) in the primary tumor were universally conserved in metastases.<br><br>CONCLUSIONS: Regions of truncal LOH are strongly retained in the presence of genetic instability and, therefore, represent genetic vulnerabilities in pancreatic adenocarcinomas. A CRISPR-based gene therapy approach targeting these regions may be a novel way to genetically target metastatic cancer.}, }
@article {pmid39700063, year = {2025}, author = {Munusamy, S and Zheng, H and Jahani, R and Zhou, S and Chen, J and Kong, J and Guan, X}, title = {DNA-Assisted CRISPR-Cas12a Enhanced Fluorescent Assay for Protein Detection in Complicated Matrices.}, journal = {ACS applied bio materials}, volume = {8}, number = {1}, pages = {754-762}, pmid = {39700063}, issn = {2576-6422}, support = {R01 GM147247/GM/NIGMS NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems ; Humans ; *DNA/chemistry ; *Proteins/analysis ; *Endodeoxyribonucleases/genetics/metabolism ; *CRISPR-Associated Proteins ; Particle Size ; *Fluorescent Dyes/chemistry ; Bacterial Proteins ; }, abstract = {Proteins are important biological macromolecules that perform a wide variety of functions in the cell and human body, and can serve as important biomarkers for early diagnosis and prognosis of human diseases as well as monitoring the effectiveness of disease treatment. Hence, sensitive and accurate detection of proteins in human biospecimens is imperative. However, at present, there is no ideal method available for the detection of proteins in clinical samples, many of which are present at ultralow (less than 1 pM) concentrations and in complicated matrices. Herein, we report an ultrasensitive and selective DNA-assisted CRISPR-Cas12a enhanced fluorescent assay (DACEA) for protein detection with detection limits reaching as low as attomolar concentrations. The high assay sensitivity was accomplished through the combined DNA barcode amplification (by using dual-functionalized AuNPs) and CRISPR analysis, while the high selectivity and high resistance to the matrix effects of our method were accomplished via the formation of protein-antibody sandwich structure and the specific recognition of Cas12a (under the guidance of crRNA) toward the designed target ssDNA. Given its ability to accurately and sensitively detect trace amounts of proteins in complicated matrices, the DACEA protein assay platform pioneered in this work has a potential application in routine protein biomarker testing.}, }
@article {pmid39700052, year = {2025}, author = {Li, X and Chen, B and Xie, Y and Luo, Y and Zhu, D and Wang, L and Su, S}, title = {Polyvalent Aptamers Structure-Mediated Fluorescent Aptasensor for the Early Diagnosis of Alzheimer's Disease by Coupling with HCR and CRISPR-Cas System.}, journal = {Analytical chemistry}, volume = {97}, number = {1}, pages = {793-798}, doi = {10.1021/acs.analchem.4c05329}, pmid = {39700052}, issn = {1520-6882}, mesh = {*Aptamers, Nucleotide/chemistry ; *Alzheimer Disease/diagnosis ; Humans ; *tau Proteins/blood/cerebrospinal fluid/analysis ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Early Diagnosis ; *Fluorescent Dyes/chemistry ; Limit of Detection ; Nucleic Acid Hybridization ; }, abstract = {The early diagnosis of Alzheimer's disease (AD) plays a vital role in slowing the progression of AD and improving the quality of human life. However, it is still a challenge in the medical field. Herein, an ultrasensitive fluorescent aptasensor was designed for the detection of special phosphorylated tau181 (P-tau181) by coupling with polyvalent aptamers (PAs) structure, hybridization chain reaction (HCR), and the CRISPR-Cas system. Coupling with the signal amplification strategy, the specific recognition ability of the aptamer, and the high cleavage activity of Cas12a protein, the designed aptasensor showed a wide linear range (0.1-10[6] pg/mL), a low detection limit (0.069 pg/mL), high selectivity, and excellent anti-interference ability for the detection of P-tau181. Moreover, the aptasensor can efficiently analyze P-tau181 in artificial cerebro spinal fluid (aCSF) and serum, proving that it has a promising application in the early diagnosis of AD.}, }
@article {pmid39700011, year = {2025}, author = {Benz, T and Larghero, P and Meyer, C and Müller, M and Brüggmann, D and Hentrich, AE and Louwen, F and Erkner, E and Fitzel, R and Schneidawind, C and Marschalek, R}, title = {Protocol for CRISPR-Cas9-mediated induction of KMT2A rearrangements in cell line and umbilical cord blood hematopoietic stem and progenitor cells.}, journal = {STAR protocols}, volume = {6}, number = {1}, pages = {103481}, pmid = {39700011}, issn = {2666-1667}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Hematopoietic Stem Cells/metabolism/cytology ; *Fetal Blood/cytology ; *Histone-Lysine N-Methyltransferase/genetics ; *Myeloid-Lymphoid Leukemia Protein/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Rearrangement ; Translocation, Genetic/genetics ; K562 Cells ; Cell Line ; }, abstract = {KMT2A rearrangements are associated with a poor clinical outcome in infant, pediatric, and adult acute lymphoblastic and myeloid leukemia. Here, we present a protocol to reconstruct chromosomal translocations with different partner genes of KMT2A in vitro. We describe steps for patient-specific single guide RNA (sgRNA) design, optimized sgRNA in vitro transcription, detailed purification of hematopoietic stem and progenitor cells (HSPCs) from umbilical cord blood (UCB), and CRISPR-Cas9 editing of the test cell line K562 as well as UCB HSPCs. The provided methodology is donor independent.}, }
@article {pmid39699588, year = {2025}, author = {Yang, Q and Dong, MJ and Xu, J and Xing, Y and Wang, Y and Yang, J and Meng, X and Xie, T and Li, Y and Dong, H}, title = {CRISPR/RNA Aptamer System Activated by an AND Logic Gate for Biomarker-Driven Theranostics.}, journal = {Journal of the American Chemical Society}, volume = {147}, number = {1}, pages = {169-180}, doi = {10.1021/jacs.4c08719}, pmid = {39699588}, issn = {1520-5126}, mesh = {Humans ; *Aptamers, Nucleotide/chemistry ; *MicroRNAs/analysis ; *Theranostic Nanomedicine ; Photosensitizing Agents/chemistry/pharmacology ; Photochemotherapy ; CRISPR-Cas Systems ; Cell Line, Tumor ; Biomarkers, Tumor ; Nanoparticles/chemistry ; }, abstract = {The development of an engineered RNA device capable of detecting multiple biomarkers to evaluate pathological states and autonomously implement responsive therapies is urgently needed. Here, we report InCasApt, an integrated nano CRISPR Cas13a/RNA aptamer theranostic platform capable of achieving both biomarker detection and biomarker-driven therapy. Within this system, a Cas13a/crRNA complex, a hairpin reporter (HR), a dinitroaniline caged Ce6 photosensitizer (Ce6-DN), and a DN-binding RNA aptamer precursor (DNBApt) are coloaded onto dendritic mesoporous silicon nanoparticles (DMSN) in a controlled manner. While InCasApt remains inert in normal cells, its programmable theranostic capabilities are activated in tumor cells that have elevated expression of carcinogenic miRNA-155 and miRNA-21. These miRNAs act as an AND logic gate, generating fluorescence for disease condition evaluation and ROS for photodynamic therapy. This process also upregulates antioncogene BRG1 and suppresses tumor migration by inhibiting the function of miRNA-155 and miRNA-21. These effects underscore the versatility of InCasApt as an miRNA-targeting strategy for bridging the gap between diagnosis and therapy.}, }
@article {pmid39699265, year = {2025}, author = {Li, X and Wang, L and Lin, J and Gu, Y and Liu, Z and Hu, J}, title = {Detection of CRISPR‒Cas and type I R-M systems in Klebsiella pneumoniae of human and animal origins and their relationship to antibiotic resistance and virulence.}, journal = {Microbiology spectrum}, volume = {13}, number = {2}, pages = {e0000924}, pmid = {39699265}, issn = {2165-0497}, support = {No.2022YFC2303600//MOST | National Key Research and Development Program of China (NKPs)/ ; }, mesh = {*Klebsiella pneumoniae/genetics/drug effects/pathogenicity/isolation &amp; purification ; Humans ; Animals ; Virulence/genetics ; *CRISPR-Cas Systems/genetics ; *Klebsiella Infections/microbiology/veterinary ; Anti-Bacterial Agents/pharmacology ; Virulence Factors/genetics ; Genome, Bacterial ; Multilocus Sequence Typing ; *Drug Resistance, Bacterial/genetics ; Bacterial Proteins/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)‒CRISPR-associated protein (Cas) and restriction‒modification (R-M) systems are important immune systems in bacteria. Information about the distributions of these two systems in Klebsiella pneumoniae from different hosts and their mutual effect on antibiotic resistance and virulence is still limited. In this study, the whole genomes of 520 strains of K. pneumoniae from GenBank, including 325 from humans and 195 from animals, were collected for CRISPR‒Cas systems and type I R-M systems, virulence genes, antibiotic resistance genes, and multilocus sequence typing detection. The results showed that host origin had no obvious influence on the distributions of the two systems (CRISPR‒Cas systems in 29.8% and 24.1%, type I R-M systems in 9.8% and 11.8% of human-origin and animal-origin strains, respectively) in K. pneumoniae. Identical spacer sequences from different hosts demonstrated there was a risk of human-animal transmission. All virulence genes (yersiniabactin, colibactin, aerobactin, salmochelin, rmpADC, and rmpA2) detection rates were higher when only the CRISPR‒Cas systems were present but were all reduced when coexisting with type I R-M systems. However, a lower prevalence of most antibiotic-resistance genes was found when the CRISPR‒Cas systems were alone, and when type I R-M systems were coexisting, some of the antibiotic resistance gene incidence rates were even lower (quinolones, macrolides, tetracyclines and carbapenems), and some of them were higher instead (aminoglycosides, clindamycins, rifampicin-associated, sulfonamides, methotrexates, beta-lactamases and ultrabroad-spectrum beta-lactamases). The synergistic and opposed effects of the two systems on virulence and antibiotic-resistance genes need further study.IMPORTANCEK. pneumoniae is an important opportunistic pathogen responsible for both human and animal infections, and the emergence of hypervirulent and multidrug-resistant K. pneumoniae has made it difficult to control this pathogen worldwide. Here, we find that CRISPR‒Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, have synergistic and opposed effects on virulence and antibiotic resistance genes in K. pneumoniae. Moreover, this study provides insights into the distributions of the two systems in K. pneumoniae from different hosts, and there is no significant difference in the prevalence of the two systems among K. pneumoniae spp. In addition, this study also characterizes the CRISPR arrays of K. pneumoniae from different hosts, suggesting that the strains sharing the same spacer sequences have the potential to spread between humans and animals.}, }
@article {pmid39699033, year = {2025}, author = {Paggi, RA and Ferrari, MC and Cerletti, M and Giménez, MI and Schwarz, TS and Marchfelder, A and De Castro, RE}, title = {Practical laboratory class to assess gene silencing using CRISPR interference (CRISPRi) technology in the archaeon Haloferax volcanii.}, journal = {Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology}, volume = {53}, number = {2}, pages = {155-164}, doi = {10.1002/bmb.21872}, pmid = {39699033}, issn = {1539-3429}, support = {//Universidad Nacional de Mar del Plata/ ; PICT 02228//Fondo para la Investigaci&#xf3;n Cient&#xed;fica y Tecnol&#xf3;gica (FONCYT)/ ; }, mesh = {*Haloferax volcanii/genetics ; *Gene Silencing ; *CRISPR-Cas Systems ; *Laboratories ; Archaeal Proteins/genetics ; Students ; }, abstract = {Perturbation of gene expression using RNA interference (RNAi) or CRISPR interference (CRISPRi) is a useful strategy to explore the function of essential genes. In the archaeon Haloferax volcanii, the CRISPR-Cas system has been adapted as a CRISPRi tool to silence the expression of specific genes. We developed a laboratory class (LC) to conceptualize gene silencing through inactivation of the H. volcanii LonB protease gene, a negative regulator of carotenoid pigments biosynthesis, using CRISPRi. This LC has been successfully applied in the Biology and Biochemistry of Microorganisms course for undergraduate students of Biology in 2022 and 2023. The following objectives were proposed: (a) generate H. volcanii mutant strains with reduced expression of the lonB gene using CRISPRi; (b) examine the effect of lonB gene silencing on cell pigmentation and growth rate; (c) assess lonB gene repression by Western blotting (WB). This LC allows students to obtain and screen CRISPRi silenced-mutants by means of simple procedures using a non-pathogenic organism as well as handle basic microbiology, biochemistry and molecular biology protocols. Additionally, the LC fosters social actions through collaborative work (experimental work), the interpretation and discussion of data and the ability to communicate outcomes orally and in a written format (scientific report).}, }
@article {pmid39698811, year = {2025}, author = {Soczek, KM and Cofsky, JC and Tuck, OT and Shi, H and Doudna, JA}, title = {CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.}, journal = {Nucleic acids research}, volume = {53}, number = {2}, pages = {}, pmid = {39698811}, issn = {1362-4962}, support = {//UCB-Hampton University Summer Program/ ; //Innovative Genomics Institute/ ; 2334028//National Science Foundation/ ; R21 HL173710/HL/NHLBI NIH HHS/United States ; U19 NS132303/NS/NINDS NIH HHS/United States ; U01 AI142817/AI/NIAID NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; DE-AC02-05CH11231//Department of Energy/ ; //Lawrence Livermore National Laboratory/ ; U19NS132303/NS/NINDS NIH HHS/United States ; //Mr. Li Ka Shing/ ; R21HL173710/HB/NHLBI NIH HHS/United States ; //Panattoni Family Foundation/ ; U19 AI135990/AI/NIAID NIH HHS/United States ; //National Science Foundation Graduate Research Fellowship/ ; //Helen Hay Whitney Foundation/ ; //HHMI Fellow of The Jane Coffin Childs Fund for Medical Research/ ; U19 AI106754/AI/NIAID NIH HHS/United States ; //Koret-Berkeley-TAU/ ; U19AI171110/NH/NIH HHS/United States ; U54 AI170792/AI/NIAID NIH HHS/United States ; 24&#x2009;180//Apple Tree Partners/ ; }, mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *DNA/chemistry/metabolism/genetics ; Base Pairing ; RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; *Bacterial Proteins/metabolism/chemistry/genetics ; Nucleic Acid Conformation ; Gene Editing ; Cryoelectron Microscopy ; Models, Molecular ; }, abstract = {RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ∼20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a-RNA-DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA interference begins with local helix distortion by transient CRISPR-Cas protein binding.}, }
@article {pmid39698318, year = {2024}, author = {Cao, S and Ma, D and Xie, J and Wu, Z and Yan, H and Ji, S and Zhou, M and Zhu, S}, title = {Point-of-care testing diagnosis of African swine fever virus by targeting multiple genes with enzymatic recombinase amplification and CRISPR/Cas12a System.}, journal = {Frontiers in cellular and infection microbiology}, volume = {14}, number = {}, pages = {1474825}, pmid = {39698318}, issn = {2235-2988}, mesh = {*African Swine Fever Virus/genetics/isolation &amp; purification ; Animals ; *CRISPR-Cas Systems ; Swine ; *African Swine Fever/diagnosis/virology ; *Point-of-Care Testing ; *Sensitivity and Specificity ; *Recombinases/metabolism/genetics ; Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/genetics ; Endodeoxyribonucleases/genetics ; Molecular Diagnostic Techniques/methods ; Bacterial Proteins ; }, abstract = {African swine fever virus (ASFV) infection is causing devastating outbreaks globally; pig farming has suffered severe economic losses due to the ASFV. Currently, strict biosecurity control measures can mitigate the incidence of ASF. Rapid, cost-effective, and sensitive detection of ASFV can significantly reduce disease transmission and mortality. CRISPR/Cas-associated proteins can detect polymorphisms with high specificity and sensitivity, making them ideal for detecting pathogens. In this study, based on CRISPR/Cas12a integrated with enzymatic recombinase amplification (ERA) technology, a CRISPR/Cas12a detection system capable of identifying ASFV E183L, K205R, and C962R gene sequences has been developed. The ERA-CRISPR/Cas12a detection system detected ASFV precisely without cross-reactivity with other porcine pathogen templates and with a sensitivity detection limit of 10 copies per reaction; it takes 60 minutes to complete the detection process. In combination with this integrated ERA pre-amplification and Cas12a/crRNA cutting assay, it provides a rapid, straightforward, sensitive, and specific method for ASFV detection in the field.}, }
@article {pmid39696792, year = {2024}, author = {Zhao, Y and Li, Z and Li, T and Rao, R and Zhu, J and Hu, R and Xu, G and Li, Y and Yang, Y}, title = {SlipChip Enables the Integration of CRISPR-Cas12a and RPA for Fast and Stand-Alone HPV Detection.}, journal = {Analytical chemistry}, volume = {96}, number = {52}, pages = {20602-20611}, doi = {10.1021/acs.analchem.4c05290}, pmid = {39696792}, issn = {1520-6882}, mesh = {Humans ; *CRISPR-Cas Systems ; *Human papillomavirus 16/genetics/isolation &amp; purification ; *DNA, Viral/analysis/genetics ; *Human papillomavirus 18/genetics/isolation &amp; purification ; *Nucleic Acid Amplification Techniques/methods ; *Papillomavirus Infections/diagnosis/virology ; Female ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Human papillomavirus (HPV) screening is vital for the early detection and prevention of cervical cancer. However, existing methods often face challenges related to speed, simplicity, and multiplexing, especially in resource-limited settings. Here we developed a portable SlipChip-based multiplexed and rapid nucleic acid testing platform, named SMART, designed to simultaneously detect HPV16 and HPV18. SMART allows seamless integration of the RPA and Cas12a assays on the SlipChip and includes a heating membrane to regulate the on-chip assay temperatures. This allows SMART to operate as a stand-alone platform without additional control instruments. The platform also features an All-in-One imaging mode for rapid on-chip data acquisition, enhancing its performance. SMART enables sensitive detection of HPV16 and HPV18 DNA across multiple samples in just 36 min with a detection limit of approximately 6 copies per reaction. Testing of 56 clinical samples at risk of HPV infection validated SMART's performance, showing 97.7% sensitivity and 100% specificity. In summary, SMART offers a stand-alone system capable of rapidly distinguishing between the two most harmful HPV subtypes, showcasing the significant potential for rapid, multiplexed nucleic acid testing in various applications.}, }
@article {pmid39696686, year = {2024}, author = {Izadifar, M and Massumi, M and Prentice, KJ and Oussenko, T and Li, B and Elbaz, J and Puri, M and Wheeler, MB and Nagy, A}, title = {Microfluidic chip systems for characterizing glucose-responsive insulin-secreting cells equipped with FailSafe kill-switch.}, journal = {Stem cell research &amp; therapy}, volume = {15}, number = {1}, pages = {486}, pmid = {39696686}, issn = {1757-6512}, support = {143231//Canadian Institutes of Health Research Foundation Grant/ ; 950-230422//Canada Research Chairs/ ; }, mesh = {*Insulin-Secreting Cells/metabolism/drug effects ; Animals ; Mice ; *Glucose/metabolism ; *Thymidine Kinase/genetics/metabolism ; Ganciclovir/pharmacology ; CRISPR-Cas Systems ; CDC2 Protein Kinase/metabolism/genetics ; Lab-On-A-Chip Devices ; Insulin/metabolism ; Homeodomain Proteins/metabolism/genetics ; Trans-Activators ; }, abstract = {BACKGROUND: Pluripotent cell-derived islet replacement therapy offers promise for treating Type 1 diabetes (T1D), but concerns about uncontrolled cell proliferation and tumorigenicity present significant safety challenges. To address the safety concern, this study aims to establish a proof-of-concept for a glucose-responsive, insulin-secreting cell line integrated with a built-in FailSafe kill-switch.<br><br>METHOD: We generated &#x3b2; cell-induced progenitor-like cells (&#x3b2;iPLCs) from primary mouse pancreatic &#x3b2; cells through interrupted reprogramming. Then, we transcriptionally linked our FailSafe (FS) kill-switch, HSV-thymidine kinase (TK), to Cdk1 gene using&#xa0;a CRISPR/Cas9 knock-in strategy, resulting in a FailSafe &#x3b2;iPLC line, designated as FS&#x3b2;iPLCs. Subsequently we evaluated and confirmed the functionality of the drug-inducible kill-switch in FS&#x3b2;iPLCs at different ganciclovir (GCV) concentrations using our PDMS-based transcapillary microfluidic system. Finally, we assessed the functionality of FS&#x3b2;iPLCs by characterizing the dynamics of insulin secretion in response to changes in glucose concentration using our microfluidic perfusion glucose-stimulated insulin secretion (GSIS) assay-on- chip.<br><br>RESULTS: The &#x3b2;iPLCs exhibited Ins1, Pdx1 and Nkx6.1 expression, and glucose responsive insulin secretion, the essential properties of pancreatic beta cells. The &#x3b2;iPLCs were amenable to genome editing which allowed for the insertion of the kill-switch into the 3'UTR of Cdk1, confirmed by PCR genotyping. Our transcapillary microfluidic system confirmed the functionality of the drug-inducible kill-switch in FS&#x3b2;iPLCs, showing an effective cell ablation of dividing cells from a heterogeneous cell population at different ganciclovir (GCV) concentrations. The Ki67 expression assessment further confirmed that slow- or non-dividing cells in the FS&#x3b2;iPLC population were resistant to GCV. Our perfusion glucose-stimulated insulin secretion (GSIS) assay-on-chip revealed that the resistant non-dividing FS&#x3b2;iPLCs exhibited higher levels of insulin secretion and glucose responsiveness compared to their proliferating counterparts.<br><br>CONCLUSIONS: This study establishes a proof-of-concept for the integration of a FailSafe kill-switch system into a glucose-responsive, insulin-secreting cell line to address the safety concerns in stem cell-derived cell replacement treatment for T1D. The microfluidic systems provided valuable insights into the functionality and safety of these engineered cells, demonstrating the potential of the kill-switch to reduce the risk of tumorigenicity in pluripotent cell-derived insulin-secreting cells.}, }
@article {pmid39696608, year = {2024}, author = {Lin, Z and Yao, Q and Lai, K and Jiao, K and Zeng, X and Lei, G and Zhang, T and Dai, H}, title = {Cas12f1 gene drives propagate efficiently in herpesviruses and induce minimal resistance.}, journal = {Genome biology}, volume = {25}, number = {1}, pages = {311}, pmid = {39696608}, issn = {1474-760X}, mesh = {*CRISPR-Cas Systems ; *Herpesvirus 1, Human/genetics ; Gene Drive Technology/methods ; Animals ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Associated Proteins/metabolism/genetics ; Humans ; }, abstract = {BACKGROUND: Synthetic CRISPR-Cas9 gene drive has been developed to control harmful species. However, resistance to Cas9 gene drive can be acquired easily when DNA repair mechanisms patch up the genetic insults introduced by Cas9 and incorporate mutations to the sgRNA target. Although many strategies to reduce the occurrence of resistance have been developed so far, they are difficult to implement and not always effective.<br><br>RESULTS: Here, Cas12f1, a recently developed CRISPR-Cas system with minimal potential for causing mutations within target sequences, has been explored as a potential platform for yielding low-resistance in gene drives. We construct Cas9 and Cas12f1 gene drives in a fast-replicating DNA virus, HSV1. Cas9 and Cas12f1 gene drives are able to spread among the HSV1 population with specificity towards their target sites, and their transmission among HSV1 viruses is not significantly affected by the reduced fitness incurred by the viral carriers. Cas12f1 gene drives spread similarly as Cas9 gene drives at high introduction frequency but transmit more slowly than Cas9 gene drives at low introduction frequency. However, Cas12f1 gene drives outperform Cas9 gene drives because they reach higher penetration and induce lower resistance than Cas9 gene drives in all cases.<br><br>CONCLUSIONS: Due to lower resistance and higher penetration, Cas12f1 gene drives could potentially supplant Cas9 gene drives for population control.}, }
@article {pmid39696488, year = {2024}, author = {Koonin, EV and Makarova, KS}, title = {CRISPR in mobile genetic elements: counter-defense, inter-element competition and RNA-guided transposition.}, journal = {BMC biology}, volume = {22}, number = {1}, pages = {295}, pmid = {39696488}, issn = {1741-7007}, mesh = {*DNA Transposable Elements/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems ; Interspersed Repetitive Sequences ; RNA, Guide, CRISPR-Cas Systems/genetics ; Bacteria/genetics/virology ; Plasmids/genetics ; }, abstract = {CRISPR are adaptive immunity systems that protect bacteria and archaea from viruses and other mobile genetic elements (MGE) via an RNA-guided interference mechanism. However, in the course of the host-parasite co-evolution, CRISPR systems have been recruited by MGE themselves for counter-defense or other functions. Some bacteriophages encode fully functional CRISPR systems that target host defense systems, and many others recruited individual components of CRISPR systems, such as single repeat units that inhibit host CRISPR systems and CRISPR mini-arrays that target related viruses contributing to inter-virus competition. Many plasmids carry type IV or subtype V-M CRISPR systems that appear to be involved in inter-plasmid competition. Numerous Tn7-like and Mu-like transposons encode CRISPR-associated transposases (CASTs) in which interference-defective CRISPR systems of type I or type V mediate RNA-guided, site-specific transposition. The recruitment of CRISPR systems and their components by MGE is a manifestation of extensive gene shuttling between host immune systems and MGE, a major trend in the coevolution of MGE with their hosts.}, }
@article {pmid39696352, year = {2024}, author = {Zheng, S and Luo, M and Huang, H and Huang, X and Peng, Z and Zheng, S and Tan, J}, title = {New insights into the role of mitophagy related gene affecting the metastasis of osteosarcoma through scRNA-seq and CRISPR-Cas9 genome editing.}, journal = {Cell communication and signaling : CCS}, volume = {22}, number = {1}, pages = {592}, pmid = {39696352}, issn = {1478-811X}, support = {82460548//National Natural Science Foundation of China/ ; 20242BAB20368//Natural Science Foundation of Jiangxi Province of China/ ; }, mesh = {*Osteosarcoma/genetics/pathology ; *Mitophagy/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Animals ; *Bone Neoplasms/genetics/pathology/metabolism ; Neoplasm Metastasis/genetics ; Cell Line, Tumor ; Mice ; Single-Cell Analysis ; Single-Cell Gene Expression Analysis ; Mitochondrial Precursor Protein Import Complex Proteins ; }, abstract = {BACKGROUND: Osteosarcoma (OSA), the most common primary bone malignancy, poses significant challenges due to its aggressive nature and propensity for metastasis, especially in adolescents. Mitophagy analysis can help identify new therapeutic targets and combined treatment strategies.<br><br>METHODS: This study integrates single-cell sequencing (scRNA-seq) data and bulk-seq to identify mitophagy-related genes (MRGs) associated with the progression of OSA metastasis and analyze their clinical significance. scRNA-seq data elucidates the relationship between mitophagy and OSA metastasis, employing "CellChat" R package to explore intercellular communications and report on hundreds of ligand-receptor interactions. Subsequently, the combination of bulk-seq and CRISPR-Cas9 gene editing identifies mitophagy-related biomarker associated with metastatic prognosis. Finally, validation of the relationship between mitophagy and OSA metastasis is achieved through cellular biology experiments and animal studies.<br><br>RESULTS: The distinct mitophagy activity of various mitochondria manifests in diverse spatial localization, cellular developmental trajectories, and intercellular interactions. OSA tissue exhibits notable heterogeneity in mitophagy within osteoblastic OSA cells. However, high mitophagy activity correlates consistently with high metastatic potential. Subsequently, we identified three critical genes associated with mitophagy in OSA, namely RPS27A, TOMM20 and UBB. According to the aforementioned queue of genes, we have constructed a mitophagy_score (MIP_score). We observed that it consistently predicts patient prognosis in both internal and external datasets, demonstrating strong robustness and stability. Furthermore, we have found that MIP_score can also guide chemotherapy, with varying sensitivities to chemotherapeutic agents based on different MIP_score. It is noteworthy that, through the integration of CRISPR-Cas9 genome-wide screening and validation via cellular and animal experiments, we have identified RPS27A as a potential novel biomarker for OSA.<br><br>CONCLUSIONS: Our comprehensive analysis elucidated the profile of mitophagy throughout the OSA metastasis process, forming the basis for a mitophagy-related prognostic model that addresses clinical outcomes and drug sensitivity following OSA metastasis. Additionally, an online interactive platform was established to assist clinicians in decision-making (https://mip-score.shinyapps.io/labtan/). These findings lay the groundwork for developing targeted therapies aimed at improving the prognosis of OSA patients.}, }
@article {pmid39695426, year = {2024}, author = {Prado, MB and Coelho, BP and Iglesia, RP and Alves, RN and Boccacino, JM and Fernandes, CFL and Melo-Escobar, MI and Ayyadhury, S and Cruz, MC and Santos, TG and Beraldo, FH and Fan, J and Ferreira, FM and Nakaya, HI and Prado, MAM and Prado, VF and Duennwald, ML and Lopes, MH}, title = {Prion protein regulates invasiveness in glioblastoma stem cells.}, journal = {BMC cancer}, volume = {24}, number = {1}, pages = {1539}, pmid = {39695426}, issn = {1471-2407}, support = {2017/26158&#x2011;0//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 2019/14952&#x2011;0//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 2020/03714-8//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 2022/08198-3//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 2020/07450&#x2011;5//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 2019/14741&#x2011;9//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 2019/11097&#x2011;1//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 2017/20271&#x2011;0//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 101796/2020&#x2011;0//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; 409941/2021&#x2011;2//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; 03592-2021 RGPIN//Natural Sciences and Engineering Research Council of Canada/ ; }, mesh = {*Glioblastoma/pathology/metabolism/genetics ; Humans ; *Neoplastic Stem Cells/metabolism/pathology ; Cell Line, Tumor ; *Neoplasm Invasiveness ; *Brain Neoplasms/pathology/metabolism/genetics ; Cell Proliferation ; Gene Knockout Techniques ; PrPC Proteins/metabolism/genetics ; Prion Proteins/metabolism/genetics ; Gene Expression Regulation, Neoplastic ; Cell Movement ; CRISPR-Cas Systems ; }, abstract = {BACKGROUND: Glioblastoma (GBM) is an aggressive brain tumor driven by glioblastoma stem cells (GSCs), which represent an appealing target for therapeutic interventions. The cellular prion protein (PrP[C]), a scaffold protein involved in diverse cellular processes, interacts with various membrane and extracellular matrix molecules, influencing tumor biology. Herein, we investigate the impact of PrP[C] expression on GBM.<br><br>METHODS: To address this goal, we employed CRISPR-Cas9 technology to generate PrP[C] knockout (KO) glioblastoma cell lines, enabling detailed loss-of-function studies. Bulk RNA sequencing followed by differentially expressed gene and pathway enrichment analyses between U87 or U251 PrP[C]-wild-type (WT) cells and PrP[C]-knockout (KO) cells were used to identify pathways regulated by PrP[C]. Immunofluorescence assays were used to evaluate cellular morphology and protein distribution. For assessment of protein levels, Western blot and flow cytometry assays were employed. Transwell and growth curve assays were used to determine the impact of loss-of-PrP[C] in GBM invasiveness and proliferation, respectively. Single-cell RNA sequencing analysis of data from patient tumors from The Cancer Genome Atlas (TCGA) and the Broad Institute of Single-Cell Data Portal were used to evaluate the correspondence between our in vitro results and patient samples.<br><br>RESULTS: Transcriptome analysis of PrP[C]-KO GBM cell lines revealed altered expression of genes associated with crucial tumor progression pathways, including migration, proliferation, and stemness. These findings were corroborated by assays that revealed impaired invasion, migration, proliferation, and self-renewal in PrP[C]-KO GBM cells, highlighting its critical role in sustaining tumor growth. Notably, loss-of-PrP[C] disrupted the expression and localization of key stemness markers, particularly CD44. Additionally, the modulation of PrP[C] levels through CD44 overexpression further emphasizes their regulatory role in these processes.<br><br>CONCLUSIONS: These findings establish PrP[C] as a modulator of essential molecules on the cell surface of GSCs, highlighting its potential as a therapeutic target for GBM.}, }
@article {pmid39694744, year = {2025}, author = {Zhu, Y and Yu, X and Wu, J}, title = {CRISPR/Cas: a toolkit for plant disease diagnostics.}, journal = {Trends in plant science}, volume = {30}, number = {3}, pages = {245-248}, doi = {10.1016/j.tplants.2024.11.011}, pmid = {39694744}, issn = {1878-4372}, mesh = {*Plant Diseases/genetics ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Plants/genetics ; Gene Editing ; }, abstract = {Genetic factors and infectious pathogens that cause plant diseases have a major impact on agricultural production. In recent years, the potential of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system in nucleic acid analysis and plant disease diagnostics has been demonstrated. We highlight progress of CRISPR/Cas technology that is significant for monitoring plant growth and preventing diseases.}, }
@article {pmid39694477, year = {2025}, author = {Harding, KR and Malone, LM and Kyte, NAP and Jackson, SA and Smith, LM and Fineran, PC}, title = {Genome-wide identification of bacterial genes contributing to nucleus-forming jumbo phage infection.}, journal = {Nucleic acids research}, volume = {53}, number = {3}, pages = {}, pmid = {39694477}, issn = {1362-4962}, support = {//Marsden Fund/ ; //Royal Society of New Zealand/ ; //Bioprotection Aotearoa/ ; //James Cook Research Fellowship/ ; //University of Otago Doctoral Scholarships/ ; //Division of Health Sciences Career Development Postdoctoral Fellowship/ ; //EMBO Postdoctoral Fellowship/ ; }, mesh = {*Bacteriophages/genetics/physiology ; *Serratia/virology/genetics ; Genome, Viral ; Genome, Bacterial ; Cell Nucleus/virology/genetics ; *Genes, Bacterial ; CRISPR-Cas Systems ; DNA Transposable Elements ; }, abstract = {The Chimalliviridae family of bacteriophages (phages) form a proteinaceous nucleus-like structure during infection of their bacterial hosts. This phage 'nucleus' compartmentalises phage DNA replication and transcription, and shields the phage genome from DNA-targeting defence systems such as CRISPR-Cas and restriction-modification. Their insensitivity to DNA-targeting defences makes nucleus-forming jumbo phages attractive for phage therapy. However, little is known about the bacterial gene requirements during the infectious cycle of nucleus-forming phages or how phage resistance may emerge. To address this, we used the Serratia nucleus-forming jumbo phage PCH45 and exploited a combination of high-throughput transposon mutagenesis and deep sequencing (Tn-seq), and CRISPR interference (CRISPRi). We identified over 90 host genes involved in nucleus-forming phage infection, the majority of which were either involved in the biosynthesis of the primary receptor, flagella, or influenced swimming motility. In addition, the bacterial outer membrane lipopolysaccharide contributed to PCH45 adsorption. Other unrelated Serratia-flagellotropic phages used similar host genes as the nucleus-forming phage, indicating that phage resistance can lead to cross-resistance against diverse phages. Our findings demonstrate that resistance to nucleus-forming jumbo phages can readily emerge via bacterial surface receptor mutation and this should be a major factor when designing strategies for their use in phage therapy.}, }
@article {pmid39693940, year = {2025}, author = {Baranova, SV and Zhdanova, PV and Golyshev, VM and Lomzov, AA and Pestryakov, PE and Chernonosov, AA and Koval, VV}, title = {Thermodynamic parameters obtained for the formation of the Cas12a-RNA/DNA complex.}, journal = {Biochemical and biophysical research communications}, volume = {743}, number = {}, pages = {151176}, doi = {10.1016/j.bbrc.2024.151176}, pmid = {39693940}, issn = {1090-2104}, mesh = {*Thermodynamics ; *Molecular Dynamics Simulation ; *DNA/chemistry/metabolism ; *CRISPR-Associated Proteins/chemistry/metabolism ; *DNA, Single-Stranded/chemistry/metabolism ; *RNA/chemistry/metabolism ; Protein Binding ; Calorimetry ; Nucleic Acid Conformation ; Endodeoxyribonucleases/chemistry/metabolism ; Bacterial Proteins/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; }, abstract = {The thermodynamics of interactions between Cas12a, RNA, and DNA are important to understanding the molecular mechanisms governing CRISPR-Cas12a's specificity and function. In this study, we employed isothermal titration calorimetry (ITC) and molecular dynamics (MD) simulations to investigate the binding properties and energetic contributions of Cas12a-crRNA complexes with single-stranded (ssDNA) and double-stranded (dsDNA) DNA substrates. ITC analyses revealed significant thermal effects during the interaction of Cas12a-crRNA with ssDNA but no detectable effects with dsDNA. The binding to ssDNA was characterized by an enthalpy change (ΔH°) of -243 ± 18 kcal/mol and a stoichiometry of ∼0.3, indicating partial binding due to structural hindrances such as intramolecular secondary structures in RNA and DNA. MD simulations further supported these findings, highlighting the stability and dynamic behavior of Cas12a-crRNA complexes with both DNA substrates. Binding free energy calculations (MM-GBSA) revealed stronger stabilization of the Cas12a-crRNA complex by dsDNA compared to ssDNA, likely driven by additional electrostatic interactions and protein-DNA contacts. However, these interactions did not produce measurable heat effects in ITC experiments. The combined experimental and computational findings demonstrate that the CRISPR-Cas12a system's interactions with nucleic acids are predominantly governed by their structural characteristics and conformational flexibility. These results deepen our understanding of the thermodynamic and structural principles underlying Cas12a-mediated target recognition and cleavage.}, }
@article {pmid39693439, year = {2024}, author = {Wang, Q and Xu, X and Chen, S and Lu, R and Li, L and Lo, CH and Liu, Z and Ning, K and Li, T and Kowal, TJ and Wang, B and Hartnett, ME and Wang, S and Qi, LS and Sun, Y}, title = {dCasMINI-mediated therapy rescues photoreceptors degeneration in a mouse model of retinitis pigmentosa.}, journal = {Science advances}, volume = {10}, number = {51}, pages = {eadn7540}, pmid = {39693439}, issn = {2375-2548}, mesh = {Animals ; *Retinitis Pigmentosa/therapy/genetics/pathology/metabolism ; *Disease Models, Animal ; *Cyclic Nucleotide Phosphodiesterases, Type 6/genetics/metabolism ; Mice ; *Genetic Therapy/methods ; *Dependovirus/genetics ; Electroretinography ; Retinal Degeneration/therapy/genetics/pathology ; CRISPR-Cas Systems ; Humans ; Retinal Rod Photoreceptor Cells/metabolism/pathology ; Eye Proteins/metabolism/genetics ; Retinal Cone Photoreceptor Cells/metabolism/pathology ; }, abstract = {Retinitis pigmentosa (RP) is characterized by degeneration of rod and cone photoreceptors that progresses to irreversible blindness. Now, there are no mutation-agnostic approaches to treat RP. Here, we utilized a single adeno-associated virus (AAV)-based CRISPR activation system to activate phosphodiesterase 6B (Pde6b) to mitigate the severe degeneration in Pde6a[nmf363] mice. We demonstrate that transcriptional activation of Pde6b can rescue the loss of Pde6a, with preservation of retinal structure, restoration of electroretinography responses, and improvement of visual function as assessed by optokinetic response and looming-induced escape behaviors. These findings demonstrate the therapeutic potential of a dCasMINI-mediated activation strategy that provides a mutation-independent treatment for retinal degeneration. This study offers a promising therapeutic approach for RP and potentially other forms of genetic diseases.}, }
@article {pmid39693429, year = {2024}, author = {Shembrey, C and Yang, R and Casan, J and Hu, W and Chen, H and Singh, GJ and Sadras, T and Prasad, K and Shortt, J and Johnstone, RW and Trapani, JA and Ekert, PG and Fareh, M}, title = {Principles of CRISPR-Cas13 mismatch intolerance enable selective silencing of point-mutated oncogenic RNA with single-base precision.}, journal = {Science advances}, volume = {10}, number = {51}, pages = {eadl0731}, pmid = {39693429}, issn = {2375-2548}, mesh = {*CRISPR-Cas Systems ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Point Mutation ; Gene Silencing ; Gene Editing/methods ; Polymorphism, Single Nucleotide ; Proto-Oncogene Proteins p21(ras)/genetics ; Base Pair Mismatch ; Membrane Proteins/genetics ; Oncogenes ; RNA, Neoplasm/genetics/metabolism ; GTP Phosphohydrolases ; Proto-Oncogene Proteins B-raf ; }, abstract = {Single-nucleotide variants (SNVs) are extremely prevalent in human cancers, although most of these remain clinically unactionable. The programmable RNA nuclease CRISPR-Cas13 has been deployed to specifically target oncogenic RNAs. However, silencing oncogenic SNVs with single-base precision remains extremely challenging due to the intrinsic mismatch tolerance of Cas13. Here, we show that introducing synthetic mismatches at precise positions of the spacer sequence enables de novo design of guide RNAs [CRISPR RNAs (crRNAs)] with strong preferential silencing of point-mutated transcripts. We applied these design principles to effectively silence the oncogenic KRAS G12 hotspot, NRAS G12D and BRAF V600E transcripts with minimal off-target silencing of the wild-type transcripts, underscoring the adaptability of this platform to silence various SNVs. Unexpectedly, the SNV-selective crRNAs harboring mismatched nucleotides reduce the promiscuous collateral activity of the RfxCas13d ortholog. These findings demonstrate that the CRISPR-Cas13 system can be reprogrammed to target mutant transcripts with single-base precision, showcasing the tremendous potential of this tool in personalized transcriptome editing.}, }
@article {pmid39693343, year = {2024}, author = {Casagrande Raffi, G and Chen, J and Feng, X and Chen, Z and Lieftink, C and Deng, S and Mo, J and Zeng, C and Steur, M and Wang, J and Bleijerveld, OB and Hoekman, L and van der Wel, N and Wang, F and Beijersbergen, R and Zheng, J and Bernards, R and Wang, L}, title = {An antibiotic that mediates immune destruction of senescent cancer cells.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {121}, number = {52}, pages = {e2417724121}, pmid = {39693343}, issn = {1091-6490}, support = {787925//EC | ERC | HORIZON EUROPE European Research Council (ERC)/ ; LSH-TKI-LSHM20083//Health Holland/ ; 19-051-ASP//Mark Foundation For Cancer Research (The Mark Foundation for Cancer Research)/ ; 12539//Dutch Cancer Society KWF/ ; W2432050; 82372695//National Natural Science Foundation of China-Guangdong Joint Fund (NSFC)/ ; 2024A04J6484//Guangdong Basic and Applied Basic Research Foundation/ ; }, mesh = {Humans ; *Cellular Senescence/drug effects/immunology ; *Pyrans/pharmacology ; *Killer Cells, Natural/immunology/drug effects/metabolism ; Cell Line, Tumor ; Reactive Oxygen Species/metabolism ; Necroptosis/drug effects ; Neoplasms/immunology/drug therapy/metabolism ; CD8-Positive T-Lymphocytes/immunology/drug effects/metabolism ; Anti-Bacterial Agents/pharmacology ; Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism/genetics ; Pyroptosis/drug effects ; Apoptosis/drug effects ; Animals ; CRISPR-Cas Systems ; Polyether Polyketides ; }, abstract = {Drugs that eliminate senescent cells, senolytics, can be powerful when combined with prosenescence cancer therapies. Using a CRISPR/Cas9-based genetic screen, we identify here SLC25A23 as a vulnerability of senescent cancer cells. Suppressing SLC25A23 disrupts cellular calcium homeostasis, impairs oxidative phosphorylation, and interferes with redox signaling, leading to death of senescent cells. These effects can be replicated by salinomycin, a cation ionophore antibiotic. Salinomycin prompts a pyroptosis-apoptosis-necroptosis (PAN)optosis-like cell death in senescent cells, including apoptosis and two forms of immunogenic cell death: necroptosis and pyroptosis. Notably, we observed that salinomycin treatment or SLC25A23 suppression elevates reactive oxygen species, upregulating death receptor 5 via Jun N-terminal protein kinase (JNK) pathway activation. We show that a combination of a death receptor 5 (DR5) agonistic antibody and salinomycin is a robust senolytic cocktail. We provide evidence that this drug combination provokes a potent natural killer (NK) and CD8+ T cell-mediated immune destruction of senescent cancer cells, mediated by the pyroptotic cytokine interleukin 18 (IL18).}, }
@article {pmid39693337, year = {2024}, author = {Li, LL and Xiao, Y and Wang, B and Zhuang, Y and Chen, Y and Lu, J and Lou, Y and Li, R}, title = {A frameshift mutation in JAZ10 resolves the growth versus defense dilemma in rice.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {121}, number = {52}, pages = {e2413564121}, pmid = {39693337}, issn = {1091-6490}, support = {32372551//MOST | National Natural Science Foundation of China (NSFC)/ ; 32402385//MOST | National Natural Science Foundation of China (NSFC)/ ; 2021YFD1401100//MOST | National Key Research and Development Program of China (NKPs)/ ; NA//Max Planck Partner Group Program/ ; 226-2024-00159//MOE | Fundamental Research Funds for the Central Universities (Fundamental Research Fund for the Central Universities)/ ; }, mesh = {*Oryza/genetics/growth &amp; development/parasitology/metabolism ; *Frameshift Mutation ; *Cyclopentanes/metabolism ; *Plant Proteins/genetics/metabolism ; *Oxylipins/metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; Animals ; Plant Diseases/parasitology/genetics ; Hemiptera/genetics/growth &amp; development/metabolism ; Gene Editing ; Signal Transduction ; Disease Resistance/genetics ; Plants, Genetically Modified/genetics ; Gibberellins/metabolism ; }, abstract = {CRISPR-Cas9 genome editing systems have revolutionized plant gene functional studies by enabling the targeted introduction of insertion-deletions (INDELs) via the nonhomologous end-joining (NHEJ) pathway. Frameshift-inducing INDELs can introduce a premature termination codon and, in other instances, can lead to the appearance of new proteins. Here, we found that mutations in the rice jasmonate (JA) signaling gene OsJAZ10 by CRISPR-Cas9-based genome editing did not affect canonical JA signaling. However, a type of mutant with an INDEL that yielded a novel frameshift protein named FJ10 (Frameshift mutation of JAZ10), exhibited enhanced rice growth and increased resistance to brown planthopper attacks. Overexpression of FJ10 in wild-type plants phenocopies OsJAZ10 frameshift mutants. Further characterization revealed that FJ10 interacts with Slender Rice 1 (OsSLR1) and F-box/Kelch 16 (OsFBK16). These interactions disrupt the function of OsSLR1 in suppressing gibberellin-mediated growth and the function of OsFBK16 in repressing lignin-mediated defense responses, respectively. Field experiments with FJ10-expressing plants demonstrate that this protein uncouples the growth-defense tradeoff, opening broad avenues to obtain cultivars with enhanced yield without compromised defenses.}, }
@article {pmid39693330, year = {2024}, author = {Perampalam, P and McDonald, JI and Dick, FA}, title = {GO-CRISPR: A highly controlled workflow to discover gene essentiality in loss-of-function screens.}, journal = {PloS one}, volume = {19}, number = {12}, pages = {e0315923}, pmid = {39693330}, issn = {1932-6203}, mesh = {Humans ; *Genes, Essential ; *CRISPR-Cas Systems ; *Workflow ; Ovarian Neoplasms/genetics/pathology ; Female ; Software ; Cell Line, Tumor ; RNA, Guide, CRISPR-Cas Systems/genetics ; Loss of Function Mutation ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Genome-wide CRISPR screens are an effective discovery tool for genes that underlie diverse cellular mechanisms that can be scored through cell fitness. Loss-of-function screens are particularly challenging compared to gain-of-function because of the limited dynamic range of decreased sgRNA sequence detection. Here we describe Guide-Only control CRISPR (GO-CRISPR), an improved loss-of-function screening workflow, and its companion software package, Toolset for the Ranked Analysis of GO-CRISPR Screens (TRACS). We demonstrate a typical GO-CRISPR workflow in a non-proliferative 3D spheroid model of dormant high grade serous ovarian cancer and demonstrate superior performance to standard screening methods. The unique integration of the pooled sgRNA library quality and guide-only controls allows TRACS to identify novel molecular pathways that were previously unidentified in tumor dormancy and undetectable to analysis packages that lack the guide only controls. Together, GO-CRISPR and TRACS can robustly improve the discovery of essential genes in challenging biological scenarios such as growth arrested cells.}, }
@article {pmid39692461, year = {2025}, author = {Li, X and Hu, H and Wang, H and Liu, J and Jiang, W and Zhou, F and Zhang, J}, title = {DNA nanotechnology-based strategies for minimising hybridisation-dependent off-target effects in oligonucleotide therapies.}, journal = {Materials horizons}, volume = {12}, number = {5}, pages = {1388-1412}, doi = {10.1039/d4mh01158a}, pmid = {39692461}, issn = {2051-6355}, mesh = {Humans ; *Oligonucleotides/therapeutic use/chemistry ; *Nanotechnology/methods ; *Genetic Therapy/methods ; *DNA/chemistry ; Animals ; Oligonucleotides, Antisense/therapeutic use/chemistry ; Nucleic Acid Hybridization ; CRISPR-Cas Systems ; }, abstract = {Targeted therapy has emerged as a transformative breakthrough in modern medicine. Oligonucleotide drugs, such as antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), have made significant advancements in targeted therapy. Other oligonucleotide-based therapeutics like clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems are also leading a revolution in targeted gene therapy. However, hybridisation-dependent off-target effects, arising from imperfect base pairing, remain a significant and growing concern for the clinical translation of oligonucleotide-based therapeutics. These mismatches in base pairing can lead to unintended steric blocking or cleavage events in non-pathological genes, affecting the efficacy and safety of the oligonucleotide drugs. In this review, we examine recent developments in oligonucleotide-based targeted therapeutics, explore the factors influencing sequence-dependent targeting specificity, and discuss the current approaches employed to reduce the off-target side effects. The existing strategies, such as chemical modifications and oligonucleotide length optimisation, often require a trade-off between specificity and binding affinity. To further address the challenge of hybridisation-dependent off-target effects, we discuss DNA nanotechnology-based strategies that leverage the collaborative effects of nucleic acid assembly in the design of oligonucleotide-based therapies. In DNA nanotechnology, collaborative effects refer to the cooperative interactions between individual strands or nanostructures, where multiple bindings result in more stable and specific hybridisation behaviour. By requiring multiple complementary interactions to occur simultaneously, the likelihood of unintended partially complementary binding events in nucleic acid hybridisation should be reduced. And thus, with the aid of collaborative effects, DNA nanotechnology has great promise in achieving both high binding affinity and high specificity to minimise the hybridisation-dependent off-target effects of oligonucleotide-based therapeutics.}, }
@article {pmid39692298, year = {2025}, author = {Walker, RL and Ferguson, Z and Mitchell, L and Waltz, M}, title = {Enhancing Animals is "Still Genetics": Perspectives of Genome Scientists and Policymakers on Animal and Human Enhancement.}, journal = {AJOB empirical bioethics}, volume = {16}, number = {2}, pages = {94-102}, pmid = {39692298}, issn = {2329-4523}, support = {R01 HG010661/HG/NHGRI NIH HHS/United States ; }, mesh = {Humans ; *Gene Editing/ethics ; Animals ; Animal Welfare/ethics ; Policy Making ; CRISPR-Cas Systems ; Research Personnel ; Animals, Genetically Modified ; Genome ; }, abstract = {BACKGROUND: Nonhuman animals are regularly enhanced genomically with CRISPR and other gene editing tools as scientists aim at better models for biomedical research, more tractable agricultural animals, or animals that are otherwise well suited to a defined purpose. This study investigated how genome editors and policymakers perceived ethical or policy benefits and drawbacks for animal enhancement and how perceived benefits and drawbacks are alike, or differ from, those for human genome editing.<br><br>METHODS: We identified scientists through relevant literature searches as well as conference presentations. Policymakers were identified through rosters of genome editing oversight groups (e.g., International Commission on the Clinical Use of Human Germline Genome Editing, World Health Organization) or efforts aimed at influencing policy (e.g., deliberative democracy groups). Interviews covered participants' views on ethical differences between interventions affecting somatic or germline cells and distinctions between using gene editing for disease treatment, prevention, and enhancement purposes.<br><br>RESULTS: Of the 92 participants interviewed, 81 were genome editing scientists, and 33 were policymakers, with 22 interviewees being both scientists and policymakers. Multiple areas were identified in which the ethical implications of genomic enhancements for nonhuman animals differ from those for human animals including with respect to experiential welfare; germline edits; environmental sustainability; and justice.<br><br>CONCLUSIONS: Overall, respondents viewed that animal enhancement is unburdened by the ethical complexities of human enhancement. These views may be related to participant perceptions of animals' lesser moral status and because germline editing in animals is common practice.}, }
@article {pmid39692063, year = {2024}, author = {Yuan, P and Usman, M and Liu, W and Adhikari, A and Zhang, C and Njiti, V and Xia, Y}, title = {Advancements in Plant Gene Editing Technology: From Construct Design to Enhanced Transformation Efficiency.}, journal = {Biotechnology journal}, volume = {19}, number = {12}, pages = {e202400457}, pmid = {39692063}, issn = {1860-7314}, support = {OHO01511//USDA Hatch Project/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Plants, Genetically Modified/genetics ; Genome, Plant/genetics ; Transformation, Genetic ; Biotechnology/methods ; Plants/genetics ; }, abstract = {Plant gene editing technology has significantly advanced in recent years, thereby transforming both biotechnological research and agricultural practices. This review provides a comprehensive summary of recent advancements in this rapidly evolving field, showcasing significant discoveries from improved transformation efficiency to advanced construct design. The primary focus is on the maturation of the Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)9 system, which has emerged as a powerful tool for precise gene editing in plants. Through a detailed exploration, we elucidate the intricacies of integrating genetic modifications into plant genomes, shedding light on transport mechanisms, transformation techniques, and optimization strategies specific to CRISPR constructs. Furthermore, we explore the initiatives aimed at extending the frontiers of gene editing to nonmodel plant species, showcasing the growing scope of this technology. Overall, this comprehensive review highlights the significant impact of recent advancements in plant gene editing, illuminating its transformative potential in driving agricultural innovation and biotechnological progress.}, }
@article {pmid39690508, year = {2025}, author = {Wang, W and Yan, L and Li, J and Zhang, C and He, Y and Li, S and Xia, L}, title = {Engineering a robust Cas12i3 variant-mediated wheat genome editing system.}, journal = {Plant biotechnology journal}, volume = {23}, number = {3}, pages = {860-873}, pmid = {39690508}, issn = {1467-7652}, support = {2021YFF1000204//National Key Research and Development Program of China/ ; 32188102//National Natural Science Foundation of China/ ; 2023ZD04074//STI 2030-Major Projects/ ; B23CJ0208//Hainan Seed Industry Lab/ ; //National Engineering Research Centre of Crop Molecular Breeding/ ; }, mesh = {*Triticum/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; Plants, Genetically Modified ; }, abstract = {Wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) is one of the most important food crops in the world. CRISPR/Cas12i3, which belongs to the type V-I Cas system, has attracted extensive attention recently due to its smaller protein size and its less-restricted canonical 'TTN' protospacer adjacent motif (PAM). However, due to its relatively lower editing efficacy in plants and the hexaploidy complex nature of wheat, Cas12i3/Cas12i3-5M-mediated genome editing in wheat has not been documented yet. Here, we report the engineering of a robust Cas12i3-5M-mediated genome editing system in wheat through the fusion of T5 exonuclease (T5E) in combination with an optimised crRNA expression strategy (Opt). We first showed that fusion of T5E, rather than ExoI, to Cas12i3-5M increased the gene editing efficiencies by up to 1.34-fold and 3.87-fold, compared to Cas12i3-5M and Cas12i3 in HEK293T cells, respectively. However, its editing efficiency remains low in wheat. We then optimised the crRNA expression strategy and demonstrated that Opt-T5E-Cas12i3-5M could enhance the editing efficiency by 1.20- to 1.33-fold and 4.05- to 7.95-fold in wheat stable lines compared to Opt-Cas12i3-5M and Opt-Cas12i3, respectively, due to progressive 5'-end resection of the DNA strand at the cleavage site with increased deletion size. The Opt-T5E-Cas12i3-5M enabled an editing efficiency ranging from 60.71% to 90.00% across four endogenous target genes in stable lines of three elite Chinese wheat varieties. Together, the developed robust Opt-T5E-Cas12i3-5M system enriches wheat genome editing toolkits for either biological research or genetic improvement and may be extended to other important polyploidy crop species.}, }
@article {pmid39690340, year = {2024}, author = {Dixit, Y and Yadav, P and Asnani, H and Sharma, AK}, title = {CRISPR/Cas9-Engineering for Increased Amylolytic Potential of Microbes for Sustainable Wastewater Treatment: A Review.}, journal = {Current microbiology}, volume = {82}, number = {1}, pages = {44}, pmid = {39690340}, issn = {1432-0991}, mesh = {*Amylases/biosynthesis/genetics ; Bacteria/enzymology/genetics ; *CRISPR-Cas Systems ; Gene Editing/methods ; Genetic Engineering/methods ; *Wastewater/chemistry/microbiology ; Water Purification/methods ; }, abstract = {Amylases are pivotal enzymes with extensive industrial applications, including food processing, textile manufacturing, pharmaceuticals, and biofuel production. Traditional methods for enhancing amylase production in microbial strains often lack precision and efficiency. The advent of CRISPR/Cas9 technology has revolutionized genetic engineering, offering precise and targeted modifications to microbial genomes. This review explores the potential of CRISPR/Cas9 for improving amylase production, highlighting its advantages over conventional methods. This review discusses the mechanism of CRISPR/Cas9, the identification and targeting of key genes involved in amylase synthesis and regulation, and the optimization of expression systems. Additionally, current review examines case studies demonstrating successful CRISPR/Cas9 applications in various microbial hosts. The review also delves into the integration of CRISPR/Cas9 in wastewater treatment, where genetically engineered amylolytic strains enhance the degradation of complex organic pollutants. Despite the promising prospects, challenges such as off-target effects and regulatory considerations remain. This review provides a comprehensive overview of the current advancements, challenges, and future directions in the application of CRISPR/Cas9 technology for amylase production and environmental biotechnology.}, }
@article {pmid39690326, year = {2025}, author = {Barber, HM and Pater, AA and Gagnon, KT and Damha, MJ and O'Reilly, D}, title = {Chemical engineering of CRISPR-Cas systems for therapeutic application.}, journal = {Nature reviews. Drug discovery}, volume = {24}, number = {3}, pages = {209-230}, pmid = {39690326}, issn = {1474-1784}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Chemical Engineering/methods ; Animals ; *Genetic Therapy/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Drug Delivery Systems ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) technology has transformed molecular biology and the future of gene-targeted therapeutics. CRISPR systems comprise a CRISPR-associated (Cas) endonuclease and a guide RNA (gRNA) that can be programmed to guide sequence-specific binding, cleavage, or modification of complementary DNA or RNA. However, the application of CRISPR-based therapeutics is challenged by factors such as molecular size, prokaryotic or phage origins, and an essential gRNA cofactor requirement, which impact efficacy, delivery and safety. This Review focuses on chemical modification and engineering approaches for gRNAs to enhance or enable CRISPR-based therapeutics, emphasizing Cas9 and Cas12a as therapeutic paradigms. Issues that chemically modified gRNAs seek to address, including drug delivery, physiological stability, editing efficiency and off-target effects, as well as challenges that remain, are discussed.}, }
@article {pmid39689871, year = {2025}, author = {Choudhery, MS and Arif, T and Mahmood, R}, title = {Bidirectional Prime Editing: Combining Precision with Versatility for Genome Editing.}, journal = {Cellular reprogramming}, volume = {27}, number = {1}, pages = {10-23}, doi = {10.1089/cell.2024.0075}, pmid = {39689871}, issn = {2152-4998}, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; Animals ; *Genome, Human ; DNA Breaks, Double-Stranded ; }, abstract = {Genome editing techniques have potential to revolutionize the field of life sciences. Several limitations associated with traditional gene editing techniques have been resolved with the development of prime editors that precisely edit the DNA without double-strand breaks (DSBs). To further improve the efficiency, several modified versions of prime editing (PE) system have been introduced. Bi-directional PE (Bi-PE), for example, uses two PE guide RNAs enabling broad and improved editing efficiency. It has the potential to alter, delete, integrate, and replace larger genome sequences and edit multiple bases at the same time. This review aims to discuss the typical gene editing methods that offer DSB-mediated repair mechanisms, followed by the latest advances in genome editing technologies with non-DSB-mediated repair. The review specifically focuses on Bi-PE being an efficient tool to edit the human genome. In addition, the review discusses the applications, limitations, and future perspectives of Bi-PE for gene editing.}, }
@article {pmid39689711, year = {2025}, author = {Metzner, E and Southard, KM and Norman, TM}, title = {Multiome Perturb-seq unlocks scalable discovery of integrated perturbation effects on the transcriptome and epigenome.}, journal = {Cell systems}, volume = {16}, number = {1}, pages = {101161}, pmid = {39689711}, issn = {2405-4720}, support = {DP2 GM140925/GM/NIGMS NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; }, mesh = {Humans ; *Transcriptome/genetics ; *Epigenome/genetics ; Single-Cell Analysis/methods ; CRISPR-Cas Systems/genetics ; Chromatin/genetics ; Cell Line ; Chromatin Assembly and Disassembly/genetics ; Gene Expression Profiling/methods ; }, abstract = {Single-cell CRISPR screens link genetic perturbations to transcriptional states, but high-throughput methods connecting these induced changes to their regulatory foundations are limited. Here, we introduce Multiome Perturb-seq, extending single-cell CRISPR screens to simultaneously measure perturbation-induced changes in gene expression and chromatin accessibility. We apply Multiome Perturb-seq in a CRISPRi screen of 13 chromatin remodelers in human RPE-1 cells, achieving efficient assignment of sgRNA identities to single nuclei via an improved method for capturing barcode transcripts from nuclear RNA. We organize expression and accessibility measurements into coherent programs describing the integrated effects of perturbations on cell state, finding that ARID1A and SUZ12 knockdowns induce programs enriched for developmental features. Modeling of perturbation-induced heterogeneity connects accessibility changes to changes in gene expression, highlighting the value of multimodal profiling. Overall, our method provides a scalable and simply implemented system to dissect the regulatory logic underpinning cell state. A record of this paper's transparent peer review process is included in the supplemental information.}, }
@article {pmid39688838, year = {2025}, author = {Xu, X and Zhang, Y and Liu, J and Wei, S and Li, N and Yao, X and Wang, M and Su, X and Jing, G and Xu, J and Liu, Y and Lu, Y and Cheng, J and Xu, Y}, title = {Concurrent Detection of Protein and miRNA at the Single Extracellular Vesicle Level Using a Digital Dual CRISPR-Cas Assay.}, journal = {ACS nano}, volume = {19}, number = {1}, pages = {1271-1285}, doi = {10.1021/acsnano.4c13557}, pmid = {39688838}, issn = {1936-086X}, mesh = {*MicroRNAs/analysis/genetics ; *Extracellular Vesicles/chemistry/metabolism ; *CRISPR-Cas Systems/genetics ; Humans ; Breast Neoplasms/diagnosis ; Female ; *Proteins/analysis ; }, abstract = {The simultaneous detection of proteins and microRNA (miRNA) at the single extracellular vesicle (EV) level shows great promise for precise disease profiling, owing to the heterogeneity and scarcity of tumor-derived EVs. However, a highly reliable method for multiple-target analysis of single EVs remains to be developed. In this study, a digital dual CRISPR-Cas-powered Single EV Evaluation (ddSEE) system was proposed to enable the concurrent detection of surface protein and inner miRNA of EVs at the single-molecule level. By optimizing simultaneous reaction conditions of CRISPR-Cas12a and CRISPR-Cas13a, the surface protein of EVs was detected by Cas12a using antibody-DNA conjugates to transfer the signal of the protein to DNA, while the inner miRNA was analyzed by Cas13a through EV-liposome fusion. A microfluidic chip containing 188,000 microwells was used to convert the CRISPR-Cas system into a digital assay format to enable the absolute quantification of miRNA/protein-positive EVs without bias through fluorescence imaging, which can detect as few as 214 EVs/μL. Finally, a total of 31 blood samples, 21 from breast cancer patients and 10 from healthy donors, were collected and tested, achieving a diagnostic accuracy of 92% in distinguishing patients with breast cancer from healthy donors. With its absolute quantification, ease of use, and multiplexed detection capability, the ddSEE system demonstrates its great potential for both EV research and clinical applications.}, }
@article {pmid39688405, year = {2025}, author = {Allan-Blitz, L-T and Adams, G and Sanders, G and Shah, P and Ramesh, K and Jarolimova, J and Ard, KL and Branda, JA and Klausner, JD and Sabeti, PC and Lemieux, JE}, title = {Preliminary clinical performance of a Cas13a-based lateral flow assay for detecting Neisseria gonorrhoeae in urine specimens.}, journal = {mSphere}, volume = {10}, number = {1}, pages = {e0067724}, pmid = {39688405}, issn = {2379-5042}, support = {2021287//Doris Duke Charitable Foundation (DDCF)/ ; //American Sexually Transmitted Diseases Association (ASTDA)/ ; 2019123//Doris Duke Charitable Foundation (DDCF)/ ; K23 AI182453/AI/NIAID NIH HHS/United States ; U19AI110818//National Institute of Allergy and Infectious Diseases (NIAID)/ ; U19 AI110818/AI/NIAID NIH HHS/United States ; K23AI182453//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; }, mesh = {Humans ; *Neisseria gonorrhoeae/genetics/isolation &amp; purification ; Male ; *Gonorrhea/diagnosis/microbiology/urine ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; DNA, Bacterial/genetics/urine ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Machine Learning ; Smartphone ; }, abstract = {Nucleic acid amplification testing (NAAT) for N. gonorrhoeae is unavailable in resource-limited settings. We previously developed a CRISPR-based lateral flow assay for detecting N. gonorrhoeae. We aimed to pair that assay with point-of-care DNA extraction, assess performance in clinical urine specimens, and optimize assay kinetics. We collected urine specimens among men presenting with urethritis enrolling in a clinical trial at the Massachusetts General Hospital Sexual Health Clinic. We assessed the quantified DNA yield of detergent-based extractions with and without heat. We selected one detergent for extracting all specimens, paired with isothermal recombinase polymerase amplification for 90 minutes and lateral flow Cas13a detection, interpreted via pixel intensity analysis. We also trained a smartphone-based machine-learning model on 1,008 images to classify lateral flow results. We used the model to interpret lateral flow results from the clinical specimens. We also tested a modified amplification chemistry with a second forward primer lacking the T7-promoter to accelerate reaction kinetics. Extraction with 0.02% Triton X resulted in an average DNA yield of 2.6 × 10[6] copies/µL (SD ± 6.7 × 10[5]). We treated 40 urine specimens (n = 12 positive) with 0.02% Triton X, and using quantified pixel intensity analysis, the Cas13a-based assay correctly classified all specimens (100% agreement; 95% CI 91.2%-100%). The machine-learning model correctly classified 45/45 strips in the validation data set and all 40 lateral flow strips from clinical specimens. Including the second forward primer reduced incubation time to 60 minutes. Using point-of-care DNA extraction, our Cas13a-based lateral flow N. gonorrhoeae assay demonstrated promising performance among clinical urine specimens.IMPORTANCEUsing a CRISPR-based assay we previously developed for Neisseria gonorrhoeae detection, we developed new techniques to facilitate point-of-care use. We then demonstrated the promising performance of that assay in clinical specimens. Furthermore, we developed a smartphone-based machine learning application for assisting interpretation of lateral flow strip results. Such an assay has the potential to transform the care of sexually transmitted infections in low-resource settings where diagnostic tests are unavailable. A point-of-care pathogen-specific assay, paired with the connectivity offered by a smartphone application, can also support public health surveillance efforts in such areas.}, }
@article {pmid39687993, year = {2025}, author = {Chen, S and Pinto Carneiro, S and Merkel, OM}, title = {Anionic polymer coating for enhanced delivery of Cas9 mRNA and sgRNA nanoplexes.}, journal = {Biomaterials science}, volume = {13}, number = {3}, pages = {659-676}, pmid = {39687993}, issn = {2047-4849}, mesh = {Humans ; *RNA, Messenger/genetics/chemistry/administration &amp; dosage ; *Polyethylene Glycols/chemistry ; A549 Cells ; *CRISPR-Associated Protein 9/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/administration &amp; dosage ; Gene Editing ; *Polyethyleneimine/chemistry/analogs &amp; derivatives ; Proto-Oncogene Proteins p21(ras)/genetics ; Anions/chemistry ; *Polymers/chemistry ; }, abstract = {Polymeric carriers have long been recognized as some of the most effective and promising systems for nucleic acid delivery. In this study, we utilized an anionic di-block co-polymer, PEG-PLE, to enhance the performance of lipid-modified PEI (C14-PEI) nanoplexes for delivering Cas9 mRNA and sgRNA targeting KRAS G12S mutations in lung cancer cells. Our results demonstrated that PEG-PLE, when combined with C14-PEI at a weight-to-weight ratio of 0.2, produced nanoplexes with a size of approximately 140 nm, a polydispersity index (PDI) of 0.08, and a zeta potential of around -1 mV. The PEG-PLE/C14-PEI nanoplexes at this ratio were observed to be both non-cytotoxic and effective in encapsulating Cas9 mRNA and sgRNA. Confocal microscopy imaging revealed efficient endosomal escape and intracellular distribution of the RNAs. Uptake pathway inhibition studies indicated that the internalization of PEG-PLE/C14-PEI primarily involves scavenger receptors and clathrin-mediated endocytosis. Compared to C14-PEI formulations, PEG-PLE/C14-PEI demonstrated a significant increase in luciferase mRNA expression and gene editing efficiency, as confirmed by T7EI and ddPCR, in A549 cells. Sanger sequencing identified insertions and/or deletions around the PAM sequence, with a total of 69% indels observed. Post-transfection, the KRAS-ERK pathway was downregulated, resulting in significant increases in cell apoptosis and inhibition of cell migration. Taken together, this study reveals a new and promising formulation for CRISPR delivery as potential lung cancer treatment.}, }
@article {pmid39687096, year = {2024}, author = {Karpenko, A and Shelenkov, A and Petrova, L and Gusarov, V and Zamyatin, M and Mikhaylova, Y and Akimkin, V}, title = {Two multidrug-resistant Proteus mirabilis clones carrying extended spectrum beta-lactamases revealed in a single hospital department by whole genome sequencing.}, journal = {Heliyon}, volume = {10}, number = {23}, pages = {e40821}, pmid = {39687096}, issn = {2405-8440}, abstract = {Proteus mirabilis bacteria is a component of normal intestinal microflora of humans and animals, but can also be found in hospital settings causing urinary tract infections and sepsis. The problem of treating such infections is complicated by multidrug-resistant isolates producing extended spectrum beta-lactamases (ESBL), and the number of ESBL-carrying P. mirabilis strains has significantly increased recently. This study presents a detailed analysis of 12 multidrug-resistant P. mirabilis isolates obtained from the wounds of different patients in one surgical department of a multidisciplinary hospital in Moscow, Russia, using the short- and long-read whole genome sequencing. The isolates under investigation divided into two clusters (clones) C1 and C2 based on their genomic profiles and carried antimicrobial resistance (AMR) genes corresponding well with phenotypic profiles, which was the first case of reporting two different P. mirabilis clones obtained simultaneously from the same specimens at one hospital, to the best of our knowledge. Some genes, including ESBL encoding ones, were specific for either C1 or C2 (aac(6')-Ib10, ant(2″)-Ia, qnrA1, bla VEB-6 and fosA3, bla CTX -M-65 , correspondingly). Additionally, the Salmonella genomic islands 1 were found that differed in composition of multiple antibiotic resistance regions between C1 and C2 groups. CRISPR-Cas system type I-E was revealed only in C2 isolates, while the same set of virulence factors was determined for both P. mirabilis clones. Diversity of all genetic factors found in case of simultaneous existence of two clones collected from the same source at one department indicates high pathogenic potential of P. mirabilis and poses a requirement of proper spreading monitoring. The data obtained will facilitate the understanding of AMR transfer and dynamics within clinical P. mirabilis isolates and contribute to epidemiological surveillance of this pathogen.}, }
@article {pmid39686519, year = {2025}, author = {Hernandez, FJ}, title = {Nucleases: From Primitive Immune Defenders to Modern Biotechnology Tools.}, journal = {Immunology}, volume = {174}, number = {3}, pages = {279-286}, pmid = {39686519}, issn = {1365-2567}, support = {2021-05641//Vetenskapsr&#xe5;det/ ; //HORIZON-MSCA-2022-COFUND-101126600-SmartBRAIN3/ ; }, mesh = {Humans ; *Biotechnology/methods ; CRISPR-Cas Systems/immunology ; Animals ; Gene Editing ; *Bacteria/immunology/enzymology/genetics ; *Endonucleases/metabolism/genetics/immunology ; *Deoxyribonucleases/metabolism/immunology ; }, abstract = {The story of nucleases begins on the ancient battlefields of early Earth, where simple bacteria fought to survive against viral invaders. Nucleases are enzymes that degrade nucleic acids, with restriction endonucleases emerging as some of the earliest defenders, cutting foreign DNA to protect their bacteria hosts. However, bacteria sought more than just defence. They evolved the CRISPR-Cas system, an adaptive immune mechanism capable of remembering past invaders. The now-famous Cas9 nuclease, a key player in this system, has been harnessed for genome editing, revolutionising biotechnology. Over time, nucleases evolved from basic viral defence tools into complex regulators of immune function in higher organisms. In humans, DNases and RNases maintain immune balance by clearing cellular debris, preventing autoimmunity, and defending against pathogens. These enzymes have transformed from simple bacterial defenders to critical players in both human immunity and biotechnology. This review explores the evolutionary history of nucleases and their vital roles as protectors in the story of life's defence mechanisms.}, }
@article {pmid39684611, year = {2024}, author = {Pavlova, SV and Shulgina, AE and Minina, JM and Zakian, SM and Dementyeva, EV}, title = {Generation of Isogenic iPSC Lines for Studying the Effect of the p.N515del (c.1543_1545delAAC) Variant on MYBPC3 Function and Hypertrophic Cardiomyopathy Pathogenesis.}, journal = {International journal of molecular sciences}, volume = {25}, number = {23}, pages = {}, pmid = {39684611}, issn = {1422-0067}, support = {22-15-00271//Russian Science Foundation/ ; }, mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; *Cardiomyopathy, Hypertrophic/genetics/pathology/metabolism ; Humans ; *Myocytes, Cardiac/metabolism/cytology/pathology ; *Carrier Proteins/genetics/metabolism ; *Cell Differentiation/genetics ; *CRISPR-Cas Systems ; Cell Line ; Gene Editing ; }, abstract = {The clinical significance of numerous cardiovascular gene variants remains to be determined. CRISPR/Cas9 allows for the introduction and/or correction of a certain variant in induced pluripotent stem cells (iPSCs). The resulting isogenic iPSC lines can be differentiated into cardiomyocytes and used as a platform to assess the pathogenicity of the variant. In this study, isogenic iPSC lines were generated for a variant of unknown significance found previously in a patient with hypertrophic cardiomyopathy (HCM), p.N515del (c.1543_1545delAAC) in MYBPC3. The deletion was corrected with CRISPR/Cas9 in the patient-specific iPSCs. The iPSC lines with the corrected deletion in MYBPC3 maintained pluripotency and a normal karyotype and showed no off-target CRISPR/Cas9 activity. The isogenic iPSC lines, together with isogenic iPSC lines generated earlier via introducing the p.N515del (c.1543_1545delAAC) variant in MYBPC3 of iPSCs of a healthy donor, were differentiated into cardiomyocytes. The cardiomyocytes derived from both panels of the isogenic iPSCs showed an increased size in the presence of the deletion in MYBPC3, which is one of the HCM traits at the cellular level. This finding indicates the effectiveness of these iPSC lines for studying the impact of the variant on HCM development.}, }
@article {pmid39684477, year = {2024}, author = {Borovikova, SE and Shepelev, MV and Mazurov, DV and Kruglova, NA}, title = {Efficient Genome Editing Using 'NanoMEDIC' AsCas12a-VLPs Produced with Pol II-Transcribed crRNA.}, journal = {International journal of molecular sciences}, volume = {25}, number = {23}, pages = {}, pmid = {39684477}, issn = {1422-0067}, support = {22-15-00381//Russian Science Foundation/ ; 075-15-2019-1661//Ministry of Science and Higher Education of the Russian Federation/ ; }, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; *RNA Polymerase II/metabolism/genetics ; Jurkat Cells ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; }, abstract = {Virus-like particles (VLPs) are an attractive vehicle for the delivery of Cas nuclease and guide RNA ribonucleoprotein complexes (RNPs). Most VLPs are produced by packaging SpCas9 and its sgRNA, which is expressed from the RNA polymerase III (Pol III)-transcribed U6 promoter. VLPs assemble in the cytoplasm, but U6-driven sgRNA is localized in the nucleus, which hinders the efficient formation and packaging of RNPs into VLPs. In this study, using the nuclease packaging mechanism of 'NanoMEDIC' VLPs, we produced VLPs with AsCas12a and exploited its ability to process pre-crRNA. This allowed us to direct crRNA in the cytoplasm as part of a Pol II-driven transcript where AsCas12a excised mature crRNA, thus boosting RNP incorporation into VLPs. CMV-driven crRNA increased Venus and CCR5 transgene knockout levels in 293 cells from 30% to 50-90% and raised the level of endogenous CXCR4 knockout in Jurkat T cells from 1% to 20%. Changing a single crRNA to an array of three or six identical crRNAs improved CXCR4 knockout rates by up to 60-70%. Compared to SpCas9-VLPs, the editing efficiencies of AsCas12a-VLPs were higher, regardless of promoter usage. Thus, we showed that AsCas12a and CMV-driven crRNA could be efficiently packaged into VLPs and mediate high levels of gene editing. AsCas12a-VLPs are a new and promising tool for the delivery of RNPs into mammalian cells that will allow efficient target genome editing and may be useful for gene therapy applications.}, }
@article {pmid39684395, year = {2024}, author = {Xuan, Q and Wang, J and Nie, Y and Fang, C and Liang, W}, title = {Research Progress and Application of Miniature CRISPR-Cas12 System in Gene Editing.}, journal = {International journal of molecular sciences}, volume = {25}, number = {23}, pages = {}, pmid = {39684395}, issn = {1422-0067}, support = {32201248//National Natural Science Foundation of China/ ; 242102111164//Key R&amp;D and Promotion Projects in Henan Province/ ; 222301420106//Natural Science Foundation of Henan/ ; 222300420202//Natural Science Foundation of Henan/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; Plants/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; Humans ; }, abstract = {CRISPR-Cas system, a natural acquired immune system in prokaryotes that defends against exogenous DNA invasion because of its simple structure and easy operation, has been widely used in many research fields such as synthetic biology, crop genetics and breeding, precision medicine, and so on. The miniature CRISPR-Cas12 system has been an emerging genome editing tool in recent years. Compared to the commonly used CRISPR-Cas9 and CRISPR-Cas12a, the miniature CRISPR-Cas12 system has unique advantages, such as rich PAM sites, higher specificity, smaller volume, and cytotoxicity. However, the application of miniature Cas12 proteins and the methods to improve its editing efficiency have not been systematically summarized. In this review, we introduce the classification of CRISPR-Cas system and summarize the structural characteristics of type V CRISPR-Cas system and the cleavage mechanism of five miniature Cas12 proteins. The application of a miniature CRISPR-Cas12 system in the gene editing of animals, plants, and microorganisms is summarized, and the strategies to improve the editing efficiency of the miniature CRISPR-Cas12 system are discussed, aiming to provide reference for further understanding the functional mechanism and engineering modification of the miniature CRISPR-Cas12 system.}, }
@article {pmid39684387, year = {2024}, author = {Aksoy, MO and Bilinska, A and Stachowiak, M and Flisikowska, T and Szczerbal, I}, title = {Deciphering the Role of the SREBF1 Gene in the Transcriptional Regulation of Porcine Adipogenesis Using CRISPR/Cas9 Editing.}, journal = {International journal of molecular sciences}, volume = {25}, number = {23}, pages = {}, pmid = {39684387}, issn = {1422-0067}, support = {2018/29/B/NZ2/00956//National Science Center/ ; }, mesh = {Animals ; *Adipogenesis/genetics ; *Sterol Regulatory Element Binding Protein 1/genetics/metabolism ; *CRISPR-Cas Systems ; Swine ; *Gene Editing/methods ; Adipocytes/metabolism/cytology ; Gene Expression Regulation ; Lipid Metabolism/genetics ; }, abstract = {Sterol regulatory element-binding protein 1 (SREBP1) is an important transcription factor that controls lipid metabolism and adipogenesis. Two isoforms, SREBP1a and SREBP1c, are generated by alternative splicing of the first exon of the SREBF1 gene. The porcine SREBF1 gene has mainly been studied for its role in lipid metabolism in adipose tissues, but little is known about its involvement, and the role of its two isoforms, in adipogenesis. The aim of the present study was to introduce a deletion in the 5'-regulatory region of the SREBF1c gene, considered crucial for adipogenesis, using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) method. This approach allows for the evaluation of how inhibiting SREBF1c transcription affects the expression of other genes essential for adipocyte differentiation, particularly PPARG, CEBPA, CEBPB, CEBPD, GATA2, and FABP4. It was observed that disrupting the SREBF1c isoform had no effect on the GATA2 gene but did result in a decrease in the expression of the CEBPA and CEBPD genes, an increase in the expression of CEBPB, and an inhibition in the expression of the PPARG and FABP4 genes. These changes in gene expression blocked adipogenesis, as could be seen by the failure of lipid droplets to accumulate. Our results provide evidence highlighting the pivotal role of the SREBP1c isoform in the regulation of porcine adipogenesis.}, }
@article {pmid39684256, year = {2024}, author = {Yang, P and Zhang, S and Hu, D and Li, X and Guo, Y and Guo, H and Zhang, L and Ding, X}, title = {Research Progress on the Mechanism and Application of the Type I CRISPR-Cas System.}, journal = {International journal of molecular sciences}, volume = {25}, number = {23}, pages = {}, pmid = {39684256}, issn = {1422-0067}, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Archaea/genetics ; Bacteria/genetics ; Genetic Engineering/methods ; }, abstract = {The CRISPR-Cas system functions as an adaptive immune mechanism in archaea and bacteria, providing defense against the invasion of foreign nucleic acids. Most CRISPR-Cas systems are classified into class 1 or class 2, with further subdivision into several subtypes. The primary distinction between class 1 and class 2 systems lies in the assembly of their effector modules. In class 1 systems, the effector complex consists of multiple proteins with distinct functions, whereas in class 2 systems, the effector is associated with a single protein. Class 1 systems account for approximately 90% of the CRISPR-Cas repertoire and are categorized into three types (type I, type IV, and type III) and 12 subtypes. To date, various CRISPR-Cas systems have been widely employed in the field of genetic engineering as essential tools and techniques for genome editing. Type I CRISPR-Cas systems remain a valuable resource for developing sophisticated application tools. This review provides a comprehensive review of the characteristics, mechanisms of action, and applications of class 1 type I CRISPR-Cas systems, as well as transposon-associated systems, offering effective approaches and insights for future research on the mechanisms of action, as well as the subsequent development and application of type I CRISPR-Cas systems.}, }
@article {pmid39684244, year = {2024}, author = {Wattad, H and Molcho, J and Manor, R and Weil, S and Aflalo, ED and Chalifa-Caspi, V and Sagi, A}, title = {Roadmap and Considerations for Genome Editing in a Non-Model Organism: Genetic Variations and Off-Target Profiling.}, journal = {International journal of molecular sciences}, volume = {25}, number = {23}, pages = {}, pmid = {39684244}, issn = {1422-0067}, mesh = {*Gene Editing/methods ; Animals ; *CRISPR-Cas Systems ; Palaemonidae/genetics ; Genetic Variation ; Polymorphism, Single Nucleotide ; High-Throughput Nucleotide Sequencing ; Molecular Sequence Annotation ; Sex Chromosomes/genetics ; }, abstract = {The CRISPR/Cas genome editing approach in non-model organisms poses challenges that remain to be resolved. Here, we demonstrated a generalized roadmap for a de novo genome annotation approach applied to the non-model organism Macrobrachium rosenbergii. We also addressed the typical genome editing challenges arising from genetic variations, such as a high frequency of single nucleotide polymorphisms, differences in sex chromosomes, and repetitive sequences that can lead to off-target events. For the genome editing of M. rosenbergii, our laboratory recently adapted the CRISPR/Cas genome editing approach to embryos and the embryonic primary cell culture. In this continuation study, an annotation pipeline was trained to predict the gene models by leveraging the available genomic, transcriptomic, and proteomic data, and enabling accurate gene prediction and guide design for knock-outs. A next-generation sequencing analysis demonstrated a high frequency of genetic variations in genes on both autosomal and sex chromosomes, which have been shown to affect the accuracy of editing analyses. To enable future applications based on the CRISPR/Cas tool in non-model organisms, we also verified the reliability of editing efficiency and tracked off-target frequencies. Despite the lack of comprehensive information on non-model organisms, this study provides an example of the feasibility of selecting and editing specific genes with a high degree of certainty.}, }
@article {pmid39683106, year = {2024}, author = {Zhan, X and Zhang, F and Li, N and Xu, K and Wang, X and Gao, S and Yin, Y and Yuan, W and Chen, W and Ren, Z and Yao, M and Wang, F}, title = {CRISPR/Cas: An Emerging Toolbox for Engineering Virus Resistance in Plants.}, journal = {Plants (Basel, Switzerland)}, volume = {13}, number = {23}, pages = {}, pmid = {39683106}, issn = {2223-7747}, support = {2023BBB044//Fei Wang/ ; 2022BBA0060//Fei Wang/ ; HBZY2023B004-3//Minghua Yao/ ; 2023HBSTX4-06//Ning Li/ ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas have been recognized as powerful genome-editing tools in diverse eukaryotic species, including plants, and thus hold great promise for engineering virus resistance in plants. Nevertheless, further attention is required regarding various issues associated with applying new powerful technologies in the field. This mini-review focuses on the recent advances in using CRISPR/Cas9 and CRISPR/Cas13 systems to combat DNA and RNA viruses in plants. We explored the utility of CRISPR/Cas for targeting the viral genome and editing host susceptibility genes in plants. We also provide insights into the limitations and challenges of using CRISPR/Cas for plant virus interference and propose individual combinatorial solutions. In conclusion, CRISPR/Cas technology has the potential to offer innovative and highly efficient approaches for controlling viruses in important crops in the near future.}, }
@article {pmid39682777, year = {2024}, author = {Ryu, M and Yurube, T and Takeoka, Y and Kanda, Y and Tsujimoto, T and Miyazaki, K and Ohnishi, H and Matsuo, T and Kumagai, N and Kuroshima, K and Hiranaka, Y and Kuroda, R and Kakutani, K}, title = {Gene-Silencing Therapeutic Approaches Targeting PI3K/Akt/mTOR Signaling in Degenerative Intervertebral Disk Cells: An In Vitro Comparative Study Between RNA Interference and CRISPR-Cas9.}, journal = {Cells}, volume = {13}, number = {23}, pages = {}, pmid = {39682777}, issn = {2073-4409}, support = {JP21K09323//Japan Society for the Promotion of Science/ ; JP24K02565//Japan Society for the Promotion of Science/ ; }, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *TOR Serine-Threonine Kinases/metabolism ; *Proto-Oncogene Proteins c-akt/metabolism ; *Signal Transduction ; *RNA Interference ; *Intervertebral Disc Degeneration/therapy/genetics/metabolism/pathology ; Gene Silencing ; Phosphatidylinositol 3-Kinases/metabolism ; Nucleus Pulposus/metabolism/pathology ; Intervertebral Disc/metabolism/pathology ; Autophagy/genetics ; Regulatory-Associated Protein of mTOR/metabolism/genetics ; RNA, Small Interfering/metabolism/genetics ; Male ; Female ; Middle Aged ; }, abstract = {The mammalian target of rapamycin (mTOR), a serine/threonine kinase, promotes cell growth and inhibits autophagy. The following two complexes contain mTOR: mTORC1 with the regulatory associated protein of mTOR (RAPTOR) and mTORC2 with the rapamycin-insensitive companion of mTOR (RICTOR). The phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling pathway is important in the intervertebral disk, which is the largest avascular, hypoxic, low-nutrient organ in the body. To examine gene-silencing therapeutic approaches targeting PI3K/Akt/mTOR signaling in degenerative disk cells, an in vitro comparative study was designed between small interfering RNA (siRNA)-mediated RNA interference (RNAi) and clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) gene editing. Surgically obtained human disk nucleus pulposus cells were transfected with a siRNA or CRISPR-Cas9 plasmid targeting mTOR, RAPTOR, or RICTOR. Both of the approaches specifically suppressed target protein expression; however, the 24-h transfection efficiency differed by 53.8-60.3% for RNAi and 88.1-89.3% for CRISPR-Cas9 (p < 0.0001). Targeting mTOR, RAPTOR, and RICTOR all induced autophagy and inhibited apoptosis, senescence, pyroptosis, and matrix catabolism, with the most prominent effects observed with RAPTOR CRISPR-Cas9. In the time-course analysis, the 168-h suppression ratio of RAPTOR protein expression was 83.2% by CRISPR-Cas9 but only 8.8% by RNAi. While RNAi facilitates transient gene knockdown, CRISPR-Cas9 provides extensive gene knockout. Our findings suggest that RAPTOR/mTORC1 is a potential therapeutic target for degenerative disk disease.}, }
@article {pmid39681408, year = {2025}, author = {Jeong, YY and Hong, C and Han, JH and Bae, S and Seo, PJ}, title = {Development of a prime editor with improved editing efficiency in Arabidopsis.}, journal = {BMB reports}, volume = {58}, number = {2}, pages = {70-74}, pmid = {39681408}, issn = {1976-670X}, mesh = {*Arabidopsis/genetics/metabolism ; *Gene Editing/methods ; Protoplasts/metabolism ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {Prime editing is widely used in many organisms to introduce site-specific sequence modifications such as base substitutions, insertions, and deletions in genomic DNA without generating double-strand breaks. Despite wide-ranging applications of prime editing, prime editors (PEs) have low editing efficiencies, especially in dicot plants. Therefore, PEs are barely used for genome engineering in dicot plant species. Here, based on previous approaches used to improve prime editing efficiency, we generated different combinations of PE components and prime editing guide RNAs (pegRNAs) and examined their prime editing efficiencies in Arabidopsis thaliana protoplasts as a dicot model system. We found that v4e2, in which PE was fused to viral nucleocapsid (NC) protein, RNase Hdeleted M-MLV RT, and a dominant negative version of human mutL homolog 1 (hMLH1dn), showed the highest prime editing efficiency in Arabidopsis protoplasts when it was co-transfected with dual enhanced pegRNA. Our results suggest that the v4e2 PE system could be used for efficient prime editing in dicot plants. [BMB Reports 2025; 58(2): 70-74].}, }
@article {pmid39681230, year = {2025}, author = {Nafari, NB and Zamani, M and Mosayyebi, B}, title = {Recent advances in lateral flow assays for MicroRNA detection.}, journal = {Clinica chimica acta; international journal of clinical chemistry}, volume = {567}, number = {}, pages = {120096}, doi = {10.1016/j.cca.2024.120096}, pmid = {39681230}, issn = {1873-3492}, mesh = {*MicroRNAs/analysis/genetics ; Humans ; }, abstract = {Lateral flow assays (LFAs) have emerged as pivotal tools for the rapid and reliable detection of microRNAs (miRNAs). It is believed that these biomarkers are crucial for the diagnosis and prognosis of various diseases, particularly cancer. Traditional miRNA detection techniques, such as quantitative PCR, are highly sensitive but have limited efficacy due to their complexity, high cost, and technical requirements. LFAs are valuable due to their simplicity, affordability, and portability, making them ideal for point-of-care testing in low-resource environments. However, challenges remain in developing highly sensitive and accurate LFA devices for miRNA detection. This review explores recent advancements in LFAs to improve miRNA detection sensitivity and specificity. Key innovations include signal amplification using isothermal methods, the application of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas systems for direct targeting of miRNAs, and the incorporation of nanomaterials, such as gold nanoparticles and nanorods, to enhance signal intensity. Using artificial intelligence (AI) algorithms enables precise, automated, and rapid quantification of miRNAs. Moreover, this review examines the ability of LFA-based devices to detect multiple miRNAs simultaneously. One of the most significant advancements is the detection of miR-21 levels as low as 20 pM and let-7a levels as low as 40 pM within ten minutes. This highlights the potential of these devices for clinical diagnostics.}, }
@article {pmid39680881, year = {2025}, author = {Zeng, J and Huang, X and Yang, Y and Wang, J and Shi, Y and Li, H and Hu, N and Yu, B and Mu, J}, title = {Near-Infrared Optogenetic Nanosystem for Spatiotemporal Control of CRISPR-Cas9 Gene Editing and Synergistic Photodynamic Therapy.}, journal = {ACS applied materials &amp; interfaces}, volume = {17}, number = {1}, pages = {701-710}, doi = {10.1021/acsami.4c18656}, pmid = {39680881}, issn = {1944-8252}, mesh = {*Photochemotherapy ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; Humans ; Mice ; *Infrared Rays ; Nanoparticles/chemistry ; Optogenetics/methods ; Cell Line, Tumor ; Neoplasms/therapy/drug therapy/genetics ; Photosensitizing Agents/chemistry/pharmacology ; Mice, Inbred BALB C ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism ; Mice, Nude ; }, abstract = {Controlling CRISPR/Cas9 gene editing at the spatiotemporal resolution level, especially for in vivo applications, remains a great challenge. Here, we developed a near-infrared (NIR) light-activated nanophotonic system (UCPP) for controlled CRISPR-Cas9 gene editing and synergistic photodynamic therapy (PDT). Lanthanide-doped upconversion nanoparticles are not only employed as carriers for intracellular plasmid delivery but also serve as the nanotransducers to convert NIR light (980 nm) into visible light with emission at 460 and 650 nm, which could result in simultaneous activation of gene editing and PDT processes, respectively. Such unique design not only achieves light-controlled precise gene editing of hypoxia-inducible factor 1α with minimal off-target effect, which effectively ameliorates the hypoxic state at tumor sites, but also facilitates the deep-seated PDT process with synergistic antitumor effect. This optogenetically activatable CRISPR-Cas9 nanosystem holds great potential for spatially controlled in vivo gene editing and targeted cancer therapy.}, }
@article {pmid39680738, year = {2025}, author = {Yang, S and Hu, G and Wang, J and Song, J}, title = {CRISPR/Cas-Based Gene Editing Tools for Large DNA Fragment Integration.}, journal = {ACS synthetic biology}, volume = {14}, number = {1}, pages = {57-71}, doi = {10.1021/acssynbio.4c00632}, pmid = {39680738}, issn = {2161-5063}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; *DNA/genetics ; Genetic Therapy/methods ; }, abstract = {In recent years, gene editing technologies have rapidly evolved to enable precise and efficient genomic modification. These strategies serve as a crucial instrument in advancing our comprehension of genetics and treating genetic disorders. Of particular interest is the manipulation of large DNA fragments, notably the insertion of large fragments, which has emerged as a focal point of research in recent years. Nevertheless, the techniques employed to integrate larger gene fragments are frequently confronted with inefficiencies, off-target effects, and elevated costs. It is therefore imperative to develop efficient tools capable of precisely inserting kilobase-sized DNA fragments into mammalian genomes to support genetic engineering, gene therapy, and synthetic biology applications. This review provides a comprehensive overview of methods developed in the past five years for integrating large DNA fragments with a particular focus on burgeoning CRISPR-related technologies. We discuss the opportunities associated with homology-directed repair (HDR) and emerging CRISPR-transposase and CRISPR-recombinase strategies, highlighting their potential to revolutionize gene therapies for complex diseases. Additionally, we explore the challenges confronting these methodologies and outline potential future directions for their improvement with the overarching goal of facilitating the utilization and advancement of tools for large fragment gene editing.}, }
@article {pmid39680576, year = {2024}, author = {Guguin, J and Chen, TY and Cuinat, S and Besson, A and Bertiaux, E and Boutaud, L and Ardito, N and Imaz Murguiondo, M and Cabet, S and Hamel, V and Thomas, S and Pain, B and Edery, P and Putoux, A and Tang, TK and Mazoyer, S and Delous, M}, title = {A Taybi-Linder syndrome-related RTTN variant impedes neural rosette formation in human cortical organoids.}, journal = {PLoS genetics}, volume = {20}, number = {12}, pages = {e1011517}, pmid = {39680576}, issn = {1553-7404}, mesh = {Humans ; *Organoids/metabolism ; *Microcephaly/genetics ; *Neural Stem Cells/metabolism ; Induced Pluripotent Stem Cells/metabolism ; Cell Cycle Proteins/genetics/metabolism ; RNA, Small Nuclear/genetics/metabolism ; Centrosome/metabolism ; CRISPR-Cas Systems ; Mutation ; Exome Sequencing ; Cerebral Cortex/metabolism ; }, abstract = {Taybi-Linder syndrome (TALS) is a rare autosomal recessive disorder characterized by severe microcephaly with abnormal gyral pattern, severe growth retardation and bone abnormalities. It is caused by pathogenic variants in the RNU4ATAC gene. Its transcript, the small nuclear RNA U4atac, is involved in the excision of ~850 minor introns. Here, we report a patient presenting with TALS features but no pathogenic variants were found in RNU4ATAC, instead the homozygous RTTN c.2953A>G variant was detected by whole-exome sequencing. After deciphering the impact of the variant on the RTTN protein function at centrosome in engineered RTTN-depleted RPE1 cells and patient fibroblasts, we analysed neural stem cells (NSC) derived from CRISPR/Cas9-edited induced pluripotent stem cells and revealed major cell cycle and mitotic abnormalities, leading to aneuploidy, cell cycle arrest and cell death. In cortical organoids, we discovered an additional function of RTTN in the self-organisation of NSC into neural rosettes, by observing delayed apico-basal polarization of NSC. Altogether, these defects contributed to a marked delay of rosette formation in RTTN-mutated organoids, thus impeding their overall growth and shedding light on mechanisms leading to microcephaly.}, }
@article {pmid39680522, year = {2024}, author = {Zheng, J and Li, B and Jia, L and Zhang, J and Gong, Z and Le, Y and Nian, X and Li, X and Liu, B and Yu, D and Zhang, Z and Li, C}, title = {Tumorigenicity decrease in Bcl-xL deficient MDCK cells ensuring the safety for influenza vaccine production.}, journal = {PloS one}, volume = {19}, number = {12}, pages = {e0311069}, pmid = {39680522}, issn = {1932-6203}, mesh = {Animals ; Dogs ; *bcl-X Protein/genetics/metabolism ; Madin Darby Canine Kidney Cells ; *Influenza Vaccines ; Mice ; *Apoptosis ; Cell Proliferation ; CRISPR-Cas Systems ; Mice, Nude ; Carcinogenesis/genetics ; Cell Movement ; Gene Editing ; Mice, Inbred BALB C ; }, abstract = {Madin-Darby canine kidney (MDCK) cells are the recognized cell strain for influenza vaccine production. However, the tumorigenic potential of MDCK cells raises concerns about their use in biological product manufacturing. To reduce MDCK cells' tumorigenicity and ensure the safety of influenza vaccine production, a B-cell lymphoma extra-large (Bcl-xL) gene, which plays a pivotal role in apoptosis regulation, was knocked-out in original MDCK cells by CRISPR-Cas9 gene editing technology, so that a homozygous MDCK-Bcl-xL-/- cell strain was acquired and named as BY-02. Compared with original MDCK cells, the proliferation and migration ability of BY-02 were significantly reduced, while apoptosis level was significantly increased, the endogenous mitochondrial apoptotic pathway were also modulated after Bcl-xL knock-out in MDCK cells. For tumor formation assays in nude mouse tests, all ten mice injected with original MDCK cells presented tumors growth in the injection site, in contrast to only one mouse injected with BY-02 cells presented tumors growth. These findings suggest that Bcl-xL knock-down is an effective strategy to inhibit tumor formation in MDCK cells, making BY-02 a promising genetically engineered cell strain for influenza vaccine production.}, }
@article {pmid39680504, year = {2025}, author = {Schmidt, ENC and Evert, BO and Pregler, BEF and Melhem, A and Hsieh, MC and Raspe, M and Strobel, H and Roos, J and Pietsch, T and Schuss, P and Fischer-Posovszky, P and Westhoff, MA and Hölzel, M and Herrlinger, U and Vatter, H and Waha, A and Schneider, M and Potthoff, AL}, title = {Tonabersat enhances temozolomide-mediated cytotoxicity in glioblastoma by disrupting intercellular connectivity through connexin 43 inhibition.}, journal = {Molecular oncology}, volume = {19}, number = {3}, pages = {878-898}, pmid = {39680504}, issn = {1878-0261}, support = {70113307//Mildred-Scheel School of Oncology (German Cancer Aid)/ ; }, mesh = {*Glioblastoma/pathology/drug therapy/metabolism/genetics ; Humans ; *Connexin 43/metabolism/antagonists &amp; inhibitors/genetics ; *Temozolomide/pharmacology ; Cell Line, Tumor ; Gap Junctions/drug effects/metabolism ; *Brain Neoplasms/pathology/drug therapy/metabolism ; Cell Communication/drug effects ; Drug Synergism ; CRISPR-Cas Systems ; }, abstract = {Glioblastoma cells rely on connexin 43 (Cx43)-based gap junctions (GJs) for intercellular communication, enabling them to integrate into a widely branched malignant network. Although there are promising prospects for new targeted therapies, the lack of clinically feasible GJ inhibitors has impeded their adoption in clinical practice. In the present study, we investigated tonabersat (TO), a blood-brain-barrier-penetrating drug with GJ-inhibitory properties, in regard to its potential to disassemble intercellular connectivity in glioblastoma networks. Fluorescence-guided measurements of calcein cell-to-cell transfer were used to study functional intercellular connectivity. Specific DNA fragmentation rates of propidium iodide-stained nuclei were measured as a surrogate readout for cell death using flow cytometry. CRISPR/Cas9-mediated gene editing of Cx43 served as a validation tool of cellular effects related to Cx43 GJ inhibition. 3' mRNA sequencing was performed for molecular downstream analysis. We found that TO reduced intercellular GJ-mediated cytosolic traffic and yielded a significant reduction of tumor microtube (TM) length. TO-mediated inhibition of cellular tumor networks was accompanied by a synergistic effect for temozolomide-induced cell death. CRISPR/Cas9 Cx43-knockout revealed similar results, indicating that TO-mediated inhibitory effects rely on the inhibition of Cx43-based GJs. Gene set enrichment analyses found that GJ-mediated synergistic cytotoxic effects were linked to a significant upregulation of cell death signaling pathways. In conclusion, TO disrupts TM-based network connectivity via GJ inhibition and renders glioblastoma cells more susceptible to cytotoxic therapy. Given its previous use in clinical trials for migraine therapy, TO might harbor the potential of bridging the idea of a GJ-targeted therapeutic approach from bench to bedside.}, }
@article {pmid39680038, year = {2025}, author = {Van, R and Pan, X and Rostami, S and Liu, J and Agarwal, PK and Brooks, B and Rajan, R and Shao, Y}, title = {Exploring CRISPR-Cas9 HNH-Domain-Catalyzed DNA Cleavage Using Accelerated Quantum Mechanical Molecular Mechanical Free Energy Simulation.}, journal = {Biochemistry}, volume = {64}, number = {1}, pages = {289-299}, pmid = {39680038}, issn = {1520-4995}, support = {R01 GM148886/GM/NIGMS NIH HHS/United States ; R01 GM135392/GM/NIGMS NIH HHS/United States ; Z01 HL001051/ImNIH/Intramural NIH HHS/United States ; R35 GM153297/GM/NIGMS NIH HHS/United States ; R44 GM133270/GM/NIGMS NIH HHS/United States ; R44 GM096678/GM/NIGMS NIH HHS/United States ; ZIA HL001051/ImNIH/Intramural NIH HHS/United States ; P30 GM145423/GM/NIGMS NIH HHS/United States ; R21 GM144860/GM/NIGMS NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems ; *DNA Cleavage ; *Quantum Theory ; Molecular Dynamics Simulation ; Thermodynamics ; DNA/chemistry/metabolism/genetics ; Catalytic Domain ; CRISPR-Associated Protein 9/chemistry/metabolism/genetics ; }, abstract = {The target DNA (tDNA) cleavage catalyzed by the CRISPR Cas9 enzyme is a critical step in the Cas9-based genome editing technologies. Previously, the tDNA cleavage from an active SpyCas9 enzyme conformation was modeled by Palermo and co-workers (Nierzwicki et al., Nat. Catal. 2022 5, 912) using ab initio quantum mechanical molecular mechanical (ai-QM/MM) free energy simulations, where the free energy barrier was found to be more favorable than that from a pseudoactive enzyme conformation. In this work, we performed ai-QM/MM simulations based on another catalytically active conformation (PDB 7Z4J) of the Cas9 HNH domain from cryo-electron microscopy experiments. For the wildtype enzyme, we acquired a free energy profile for the tDNA cleavage that is largely consistent with the previous report. Furthermore, we explored the role of the active-site K866 residue on the catalytic efficiency by modeling the K866A mutant and found that the K866A mutation increased the reaction free energy barrier, which is consistent with the experimentally observed reduction in the enzyme activity.}, }
@article {pmid39679901, year = {2025}, author = {Zhang, W and Li, Z and Lun, X and Guo, Y}, title = {Telomerase-Responsive CRISPR System-Regulated Nanobomb for Triggering Research on Telomerase "Self-Detonation".}, journal = {ACS applied materials &amp; interfaces}, volume = {17}, number = {1}, pages = {725-738}, doi = {10.1021/acsami.4c18859}, pmid = {39679901}, issn = {1944-8252}, mesh = {*Telomerase/metabolism/genetics ; Humans ; Animals ; Mice ; MCF-7 Cells ; *Aptamers, Nucleotide/chemistry ; Female ; CRISPR-Cas Systems/genetics ; Nanoparticles/chemistry ; Mice, Nude ; Silicon Dioxide/chemistry ; Naphthalenes/chemistry/pharmacology ; Breast Neoplasms/pathology/metabolism ; Mice, Inbred BALB C ; Antineoplastic Agents/pharmacology/chemistry ; Cell Proliferation/drug effects ; Aminobenzoates ; }, abstract = {Targeting tumor markers is one of the most important approaches to tumor therapy, and the "suicide" pattern of tumor marker response is a very challenging study. Telomerase, as one of the key factors associated with human longevity and cancer progression, is considered to be an emerging biomarker for cancer diagnosis. The targeted drug delivery nanobomb─BIBR1532@HSN/FQDNA/MUC1 aptamer (B@HDA) is prepared in this study based on hollow silica nanoparticles (HSN) and CRISPR systems. Amino-modified FQDNA and amino-modified MUC1 aptamer are covalently attached to the surface of carboxyl-functionalized HSN. The modified MUC1 aptamer directs the nanobomb to specifically target breast cancer cells (MCF-7) and FQDNA sequesters the telomerase inhibitor (BIBR1532) within the HSN. Telomerase primers (TPs) is recognized by the highly expressed telomerase in MCF-7 cells and is elongated to form DNA substrates. The substrate pairs with crRNA bases to effectively activate CRISPR-Cas12a. The activated CRISPR-Cas12a precisely cut FQDNA, releasing BIBR1532, which inhibits telomerase activity. This strategy achieves telomerase "suicide". The nanobomb described above has the following advantages. (1) The "closing" effect of FQDNA contributes to reducing the nonspecific release of BIBR1532. (2) B@HDA, combined with CRISPR, regulates mitochondrial dysfunction and cell senescence in MCF-7 cells. (3) In the tumor-bearing mouse model, B@HDA, combined with CRISPR, exhibits good biocompatibility and an obvious tumor ablation effect on MCF-7 tumors, suggesting potential application prospects across a wide range of cancer cell lines. In summary, the proposed nanobomb provides a tunable switch approach for the specific inhibition of telomerase and the reduction of tumor cell growth, representing a promising avenue for promoting senescence and treating cancer.}, }
@article {pmid39678914, year = {2024}, author = {Rostampour, M and Panahi, B and Masoumi Jahandizi, R}, title = {The CRISPR-Cas system in Lactiplantibacillus plantarum strains: identification and characterization using a genome mining approach.}, journal = {Frontiers in microbiology}, volume = {15}, number = {}, pages = {1394756}, pmid = {39678914}, issn = {1664-302X}, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CAS) genes make up bacteria's adaptive immune system against bacteriophages. In this study, 675 sequences of Lactiplantibacillus plantarum isolates deposited in GenBank were analyzed in terms of diversity, occurrence, and evolution of the CRISPR-Cas system. This study investigated the presence, structural variations, phylogenetic relationships, and diversity of CRISPR-Cas systems in 675 L. plantarum strains. The analysis revealed that 143 strains harbor confirmed CRISPR-Cas systems, with subtype II-A being predominant. Moreover, targeting phages and plasmid diversity between the predicted systems were dissected. The results indicated that approximately 22% of the isolates with verified and complete CRISPR systems exhibited the coexistence of both subtypes II-A and I-E within their genomes. The results further showed that in subtype II-A, the length of the repeat sequence was 36 nucleotides, on average. In addition, the number of spacers in subtypes II-A and I-E varied between 1-24 and 3-16 spacers, respectively. The results also indicated that subtype II-A has nine protospacer adjacent motifs, which are 5'-CC-3', 5'-GAA-3', 5'-TGG-3', 5'-CTT-3', 5'-GGG-3', 5'-CAT-3', 5'-CTC-3', 5'-CCT-3', and 5'-CGG-3'. In addition, the identified systems displayed a potential for targeting Lactobacillus phages. The investigation of the relationship between the targeting of Lactobacillus phages by the antiphage system in L. plantarum species showed that subtype II-A had the highest diversity in targeting Lactobacillus phages than subtype I-E. In conclusion, current findings offer a perspective on the prevalence and evolution of the CRISPR-Cas system in L. plantarum, contributing novel insights to the expanding field of CRISPR-Cas systems within lactobacillus strains. This knowledge establishes a foundation for future applied studies focused on enhancing phage resistance in industrial fermentation, reducing contamination risks, and improving product quality. The identified targeting diversity may also foster advancements in phage therapy through the development of CRISPR-based antimicrobials.}, }
@article {pmid39677770, year = {2024}, author = {Hoelting, K and Madlmayr, A and Hoeger, B and Lewitz, D and Weng, M and Haider, T and Duggan, M and Ross, R and Horgen, FD and Sperandio, M and Dietrich, A and Gudermann, T and Zierler, S}, title = {TRPM7 activity drives human CD4 T-cell activation and differentiation in a magnesium dependent manner.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, pmid = {39677770}, issn = {2692-8205}, support = {P20 GM103466/GM/NIGMS NIH HHS/United States ; U54 GM138062/GM/NIGMS NIH HHS/United States ; }, abstract = {T lymphocyte activation is a crucial process in the regulation of innate and adaptive immune responses. The ion channel-kinase TRPM7 has previously been implicated in cellular Mg[2+] homeostasis, proliferation, and immune cell modulation. Here, we show that pharmacological and genetic silencing of TRPM7 leads to diminished human CD4 T-cell activation and proliferation following TCR mediated stimulation. In both primary human CD4 T cells and CRISPR/Cas-9 engineered Jurkat T cells, loss of TRPM7 led to altered Mg[2+] homeostasis, Ca[2+] signaling, reduced NFAT translocation, decreased IL-2 secretion and ultimately diminished proliferation and differentiation. While the activation of primary human CD4 T cells was dependent on TRPM7, polarization of naïve CD4 T cells into regulatory T cells (Treg) was not. Taken together, these results highlight TRPM7 as a key protein of cellular Mg[2+] homeostasis and CD4 T-cell activation. Its role in lymphocyte activation suggests therapeutic potential for TRPM7 in numerous T-cell mediated diseases.}, }
@article {pmid39677748, year = {2024}, author = {Shamloo, S and Schloßhauer, JL and Tiwari, S and Fischer, KD and Ghebrechristos, Y and Kratzenberg, L and Bejoy, AM and Aifantis, I and Wang, E and Imig, J}, title = {RNA Binding of GAPDH Controls Transcript Stability and Protein Translation in Acute Myeloid Leukemia.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, doi = {10.1101/2024.12.02.626357}, pmid = {39677748}, issn = {2692-8205}, abstract = {Dysregulation of RNA binding proteins (RBPs) is a hallmark in cancerous cells. In acute myeloid leukemia (AML) RBPs are key regulators of tumor proliferation. While classical RBPs have defined RNA binding domains, RNA recognition and function in AML by non-canonical RBPs (ncRBPs) remain unclear. Given the inherent complexity of targeting AML broadly, our goal was to uncover potential ncRBP candidates critical for AML survival using a CRISPR/Cas-based screening. We identified the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a pro-proliferative factor in AML cells. Based on cross-linking and immunoprecipitation (CLIP), we are defining the global targetome, detecting novel RNA targets mainly located within 5'UTRs, including GAPDH, RPL13a, and PKM. The knockdown of GAPDH unveiled genetic pathways related to ribosome biogenesis, translation initiation, and regulation. Moreover, we demonstrated a stabilizing effect through GAPDH binding to target transcripts including its own mRNA. The present findings provide new insights on the RNA functions and characteristics of GAPDH in AML.}, }
@article {pmid39676682, year = {2025}, author = {Chen, J and Lin, X and Xiang, W and Chen, Y and Zhao, Y and Huang, L and Liu, L}, title = {DNA target binding-induced pre-crRNA processing in type II and V CRISPR-Cas systems.}, journal = {Nucleic acids research}, volume = {53}, number = {3}, pages = {}, pmid = {39676682}, issn = {1362-4962}, support = {32022047//National Natural Science Foundation of China/ ; 3502Z20227020//Natural Science Foundation of Xiamen, China/ ; 2023J01023//Natural Science Foundation of Fujian Province/ ; }, mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *RNA Precursors/metabolism/genetics ; *DNA/metabolism ; DNA, Single-Stranded/metabolism ; Endodeoxyribonucleases/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Associated Protein 9/metabolism ; Bacterial Proteins/metabolism/chemistry ; }, abstract = {Precursor (pre)-CRISPR RNA (crRNA) processing can occur in both the repeat and spacer regions, leading to the removal of specific segments from the repeat and spacer sequences, thereby facilitating crRNA maturation. The processing of pre-crRNA repeat by Cas effector and ribonuclease has been observed in CRISPR-Cas9 and CRISPR-Cas12a systems. However, no evidence of pre-crRNA spacer cleavage by any enzyme has been reported in these systems. In this study, we demonstrate that DNA target binding triggers efficient cleavage of pre-crRNA spacers by type II and V Cas effectors such as Cas12a, Cas12b, Cas12i, Cas12j and Cas9. We show that the pre-crRNA spacer cleavage catalyzed by Cas12a and Cas9 has distinct characteristics. Activation of the cleavage activity in Cas12a is induced by both single-stranded DNA (ssDNA) and double-stranded DNA target binding, whereas only ssDNA target binding triggers cleavage in Cas9 toward the pre-crRNA spacer. We present a series of structures elucidating the underlying mechanisms governing conformational activation in both Cas12a and Cas9. Furthermore, leveraging the trans-cutting activity of the pre-crRNA spacer, we develop a one-step DNA detection method characterized by its simplicity, high sensitivity, and excellent specificity.}, }
@article {pmid39676667, year = {2025}, author = {Muench, P and Fiumara, M and Southern, N and Coda, D and Aschenbrenner, S and Correia, B and Gräff, J and Niopek, D and Mathony, J}, title = {A modular toolbox for the optogenetic deactivation of transcription.}, journal = {Nucleic acids research}, volume = {53}, number = {3}, pages = {}, pmid = {39676667}, issn = {1362-4962}, support = {101041570//European Union/ ; 453202693//German Research Foundation/ ; //Executive Agency/ ; //Aventis Foundation/ ; CoG 101043457//ERC/SERI/ ; //Vallee Foundation/ ; //HFSP/ ; 310 030_197752/SNSF_/Swiss National Science Foundation/Switzerland ; CoG 101043457//SERI/ ; }, mesh = {*Optogenetics/methods ; Humans ; Promoter Regions, Genetic ; *Transcriptional Activation/radiation effects ; Light ; HEK293 Cells ; *Transcription, Genetic/radiation effects ; CRISPR-Cas Systems ; Animals ; Herpes Simplex Virus Protein Vmw65/genetics/metabolism ; Gene Expression Regulation ; Trans-Activators/genetics/metabolism ; }, abstract = {Light-controlled transcriptional activation is a commonly used optogenetic strategy that allows researchers to regulate gene expression with high spatiotemporal precision. The vast majority of existing tools are, however, limited to light-triggered induction of gene expression. Here, we inverted this mode of action and created optogenetic systems capable of efficiently terminating transcriptional activation in response to blue light. First, we designed highly compact regulators by photo-controlling the VP16 (pcVP16) transactivation peptide. Then, applying a two-hybrid strategy, we engineered LOOMINA (light off-operated modular inductor of transcriptional activation), a versatile transcriptional control platform for mammalian cells that is compatible with various effector proteins. Leveraging the flexibility of CRISPR systems, we combined LOOMINA with dCas9 to control transcription with blue light from endogenous promoters with exceptionally high dynamic ranges in multiple cell lines. Functionally and mechanistically, the versatile LOOMINA platform and the exceptionally compact pcVP16 transactivator represent valuable additions to the optogenetic repertoire for transcriptional regulation.}, }
@article {pmid39676659, year = {2025}, author = {Lee, JM and Zeng, J and Liu, P and Nguyen, MA and Suchenski Loustaunau, D and Bauer, DE and Kurt Yilmaz, N and Wolfe, SA and Schiffer, CA}, title = {Direct delivery of Cas-embedded cytosine base editors as ribonucleoprotein complexes for efficient and accurate editing of clinically relevant targets.}, journal = {Nucleic acids research}, volume = {53}, number = {1}, pages = {}, pmid = {39676659}, issn = {1362-4962}, support = {R01 GM118474/GM/NIGMS NIH HHS/United States ; //University of Massachusetts Chan Medical School/ ; DDF #2022092//Doris Duke Foundation/ ; R01AI150478/NH/NIH HHS/United States ; R01 AI150478/AI/NIAID NIH HHS/United States ; }, mesh = {*Gene Editing/methods ; *Cytosine/metabolism ; *Ribonucleoproteins/genetics/metabolism ; *CRISPR-Cas Systems ; Humans ; Animals ; Mice ; Anemia, Sickle Cell/genetics/therapy ; Carrier Proteins/genetics ; HEK293 Cells ; }, abstract = {Recently, cytosine base editors (CBEs) have emerged as a promising therapeutic tool for specific editing of single nucleotide variants and disrupting specific genes associated with disease. Despite this promise, the currently available CBEs have the significant liabilities of off-target and bystander editing activities, partly due to the mechanism by which they are delivered, causing limitations in their potential applications. In this study, we engineered optimized, soluble and stable Cas-embedded CBEs (CE_CBEs) that integrate several recent advances, which were efficiently formulated for direct delivery into cells as ribonucleoprotein (RNP) complexes. Our resulting CE_CBE RNP complexes efficiently target cytosines in TC dinucleotides with minimal off-target or bystander mutations. Delivery of additional uracil glycosylase inhibitor protein in trans further increased C-to-T editing efficiency and target purity in a dose-dependent manner, minimizing indel formation. A single electroporation was sufficient to effectively edit the therapeutically relevant locus BCL11A for sickle cell disease in hematopoietic stem and progenitor cells in a dose-dependent manner without cellular toxicity. Significantly, these CE_CBE RNPs permitted highly efficient editing and engraftment of transplanted cells in mice. Thus, our designed CBE proteins provide promising reagents for RNP-based editing at disease-related sites.}, }
@article {pmid39676076, year = {2025}, author = {Gao, Z and Bak, RO}, title = {Integration of large genetic payloads using prime editing and site-specific integrases.}, journal = {Nature protocols}, volume = {20}, number = {6}, pages = {1391-1392}, pmid = {39676076}, issn = {1750-2799}, support = {101041231//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; R238-2016-3349//Lundbeckfonden (Lundbeck Foundation)/ ; 8056-00010B//Innovationsfonden (Innovation Fund Denmark)/ ; NNF21OC0071259//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; }, }
@article {pmid39675302, year = {2025}, author = {Sun, Y and Zhang, W and Zhang, H and Zhao, F and Su, L}, title = {CRISPR/Cas13a combined with reverse transcription and RPA for NoV GII.4 monitoring in water environments.}, journal = {Environment international}, volume = {195}, number = {}, pages = {109195}, doi = {10.1016/j.envint.2024.109195}, pmid = {39675302}, issn = {1873-6750}, mesh = {*Environmental Monitoring/methods ; *Norovirus/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Water Microbiology ; Reverse Transcription ; RNA, Viral/analysis ; }, abstract = {Water bodies contaminated with the norovirus (NoV) are important vectors for its transmission. Therefore, enhanced monitoring of NoV in aqueous environments plays an active role in preventing diseases. Here, we reverse transcribed viral RNA into cDNA, and then used the constructed RPA-CRISPR/Cas13a-based platform for sensitive and quantitative monitoring of NoV GII.4 in aqueous environments. The use of glycerol as a phase separator and the direct release of nucleic acids from the virus by NaOH significantly enhanced the stability of the assay and reduced its economic cost. This assay is sensitive, specific, and stable. Based on the qualitative detection method, we established a relatively accurate quantitative detection method using the plasmid as a standard. Four water samples, totaling 64 samples, were analyzed using this method and compared with the qPCR method. The results of the two methods showed 100 % concordance with no significant difference in viral load. The entire process of our established method-from viral nucleic acid extraction to the output of the results-was completed in 30 min, much less than the time required for qPCR method. This suggests that the assay can be used as an alternative to qPCR for monitoring the change of NoV GII.4 concentration in water bodies, and shows high potential for application in the immediate detection of viruses in aqueous environments and resource-limited areas.}, }
@article {pmid39675005, year = {2024}, author = {Rubio, A and Garzón, A and Moreno-Rodríguez, A and Pérez-Pulido, AJ}, title = {Biological warfare between two bacterial viruses in a defense archipelago sheds light on the spread of CRISPR-Cas systems.}, journal = {Cell reports}, volume = {43}, number = {12}, pages = {115085}, doi = {10.1016/j.celrep.2024.115085}, pmid = {39675005}, issn = {2211-1247}, mesh = {*CRISPR-Cas Systems/genetics ; Acinetobacter baumannii/genetics ; Biological Warfare ; Genome, Bacterial ; }, abstract = {CRISPR-Cas systems are adaptive immunity systems of bacteria and archaea that prevent infection by viruses and other external mobile genetic elements. It is currently known that these defense systems can be co-opted by the same viruses. We have found one of these viruses in the opportunistic pathogen Acinetobacter baumannii, and the same system has been also found in an integration hotspot of the bacterial genome that harbors other multiple defense systems. The CRISPR-Cas system appears to especially target another virus that could compete with the system itself for the same integration site. This virus is prevalent in strains of the species belonging to the so-called Global Clone 2, which causes the most frequent outbreaks worldwide. Knowledge of this viral warfare involving antiviral systems could be useful in the fight against infections caused by bacteria, and it would also shed light on how CRISPR-Cas systems expand in bacteria.}, }
@article {pmid39674517, year = {2025}, author = {Chen, X and Yao, S and Xie, L and Li, J and Xiong, L and Yang, X and Chen, Y and Cao, F and Hou, Q and You, M and Liu, Y and Gurr, GM and You, S}, title = {Disruption of the odorant receptor co-receptor (Orco) reveals its critical role in multiple olfactory behaviors of a cosmopolitan pest.}, journal = {Insect biochemistry and molecular biology}, volume = {177}, number = {}, pages = {104248}, doi = {10.1016/j.ibmb.2024.104248}, pmid = {39674517}, issn = {1879-0240}, mesh = {Animals ; *Receptors, Odorant/genetics/metabolism ; *Moths/genetics/physiology ; Female ; Male ; *Insect Proteins/genetics/metabolism ; Sexual Behavior, Animal ; Oviposition ; Sex Attractants/metabolism ; Smell ; CRISPR-Cas Systems ; }, abstract = {The olfactory system of insects plays a pivotal role in multiple, essential activities including feeding, mating, egg laying, and host localization. The capacity of odorant receptors to recognize odor molecules relies on odorant receptor co-receptors forming heterodimers. Here we report the successful engineering a homozygous mutant strain of diamondback moth (Plutella xylostella) in which the odorant receptor co-receptor PxOrco was silenced using CRISPR/Cas9. This insect is a globally important crop pest for which novel control methods are urgently required. Behavioral assays demonstrated that PxOrco knockout males exhibited abolished courtship behaviors, inability to mate, and loss of selective preference for P. xylostella's key sex pheromone components. Whilst female mating behavior and fecundity remained unaffected by PxOrco knockout, oviposition response to leaf alcohol, a key cue for normal oviposition behavior, was lost. Electroantennography revealed drastically reduced responses to sex pheromones and plant volatiles in PxOrco-deficient adults but food location by larvae was unaffected. Moreover, expression analysis of PxOrco-deficient pheromone receptors (PRs) indicated varied regulation patterns, with down-regulation observed in several PRs in both sexes. These findings underscore the critical role of PxOrco in regulating multiple olfactory aspects in P. xylostella, including feeding, mating, and host location. Our study identifies the potential of disrupting the Orco gene in this and other pest species to provide novel avenues for future pest control.}, }
@article {pmid39674516, year = {2025}, author = {Chen, JX and Zhang, CC and Sun, JW and Zhang, YB and Luo, MS and Zhang, WQ}, title = {β2-tubulin and its promoter in the brown planthopper: A versatile tool for genetic control strategies.}, journal = {Insect biochemistry and molecular biology}, volume = {177}, number = {}, pages = {104244}, doi = {10.1016/j.ibmb.2024.104244}, pmid = {39674516}, issn = {1879-0240}, mesh = {Animals ; Male ; Female ; *Hemiptera/genetics/metabolism/growth &amp; development ; *Promoter Regions, Genetic ; *Tubulin/genetics/metabolism ; *Insect Proteins/genetics/metabolism ; RNA Interference ; CRISPR-Cas Systems ; Testis/metabolism/growth &amp; development ; }, abstract = {At present, the application of CRISPR/Cas9 technology for genetic manipulation in insects is predominantly concentrated on Diptera model species, including Drosophila and mosquitoes. In contrast, non-model insects such as the brown planthoppers (BPH, Nilaparvata lugens), a major insect pest of rice, have received less attention in genetic manipulation due to insufficient tools. Here, the analysis of spatiotemporal expression patterns revealed that β2-tubulin in BPH (NlB2t) was predominantly concentrated in male adults and male testis, exhibiting high expression levels. Knockdown of NlB2t expression by using RNAi resulted in the obstruction of male testis development. Mating between the RNAi-treated males and wild-type females led to a notable reduction in the number of eggs laid and the hatching rate of those eggs by 58.2% and 50.6%, respectively. The longevity of RNAi males significantly increased, and females that had previously mated with RNAi males exhibited a diminished inclination for re-mating with wild-type males. The dual-luciferase reporter assay demonstrated robust promoter activity in the upstream 943 bp of NlB2t, capable of driving Cas9 protein expression in vivo and effectively inducing target gene knockout. These findings elucidated that NlB2t may be a key gene in BPH male testis development and reproduction, as a promising target for sterilization. Its upstream promoter serves as a germline promoter, significantly facilitating the development of genetic control tools based on CRISPR/Cas9 technology in BPH.}, }
@article {pmid39674450, year = {2025}, author = {Zhang, X and Wang, F and Guo, X and Qiao, L and Luo, X and Cui, L and Liang, Q and Liu, M and Qi, Q}, title = {Developing a RecT-assisted endogenous CRISPR/SzCas9 system for precise genome editing in Streptococcus zooepidemicus.}, journal = {International journal of biological macromolecules}, volume = {291}, number = {}, pages = {138758}, doi = {10.1016/j.ijbiomac.2024.138758}, pmid = {39674450}, issn = {1879-0003}, mesh = {*Streptococcus equi/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Hyaluronic Acid/biosynthesis/genetics ; *Genome, Bacterial ; }, abstract = {Streptococcus zooepidemicus is the most commonly utilized strain for industrial-scale hyaluronic acid (HA) production due to its capacity to produce high-quality HA. However, the lack of efficient genetic manipulation approaches has significantly hindered the industrial application of this species. In this study, we developed a RecT-assisted endogenous CRISPR/SzCas9 system to enable markerless gene deletion, gene substitution, stop codon insertion, and iterative editing in the industrially significant strain S. zooepidemicus for hyaluronic acid production. A variety of editing outcomes were achieved, including a 3186-bp deletion of the hylb gene, replacement of VHb, and insertion of stop codons into the sagA gene. Ultimately, we successfully developed an engineered strain, designated SD-3, which exhibited reduced toxicity and non-hemolytic properties. The hyaluronic acid production of SD-3 increased by 39.7%, reaching 10.8 g/L in a 5-L fermenter. This achievement represents the highest titer of high molecular weight hyaluronic acid to date, characterized by a uniform and high molecular weight of up to 2.8 × 10[6] Da.}, }
@article {pmid39674177, year = {2025}, author = {Lou, H and Li, S and Shi, Z and Zou, Y and Zhang, Y and Huang, X and Yang, D and Yang, Y and Li, Z and Xu, C}, title = {Engineering source-sink relations by prime editing confers heat-stress resilience in tomato and rice.}, journal = {Cell}, volume = {188}, number = {2}, pages = {530-549.e20}, doi = {10.1016/j.cell.2024.11.005}, pmid = {39674177}, issn = {1097-4172}, mesh = {*Oryza/genetics/physiology/metabolism ; *Solanum lycopersicum/genetics/physiology/metabolism ; *Gene Editing/methods ; *Heat-Shock Response/genetics ; Plants, Genetically Modified ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Crops, Agricultural/genetics ; }, abstract = {A 2°C climate-warming scenario is expected to further exacerbate average crop losses by 3%-13%, yet few heat-tolerant staple-crop varieties are available toward meeting future food demands. Here, we develop high-efficiency prime-editing tools to precisely knockin a 10-bp heat-shock element (HSE) into promoters of cell-wall-invertase genes (CWINs) in elite rice and tomato cultivars. HSE insertion endows CWINs with heat-responsive upregulation in both controlled and field environments to enhance carbon partitioning to grain and fruits, resulting in per-plot yield increases of 25% in rice cultivar Zhonghua11 and 33% in tomato cultivar Ailsa Craig over heat-stressed controls, without fruit quality penalties. Up to 41% of heat-induced grain losses were rescued in rice. Beyond a prime-editing system for tweaking gene expression by efficiently delivering bespoke changes into crop genomes, we demonstrate broad and robust utility for targeted knockin of cis-regulatory elements to optimize source-sink relations and boost crop climate resilience.}, }
@article {pmid39673958, year = {2025}, author = {Yang, R and Guan, X and Zhang, J and Moon, J and Guo, C and Jia, Z and Hou, C and Ganbaatar, U and Tricarico, S and Ma, AWK and Liu, C}, title = {Quencher-free CRISPR-based molecular detection using an amphiphilic DNA fluorescence probe.}, journal = {Biosensors &amp; bioelectronics}, volume = {271}, number = {}, pages = {117054}, pmid = {39673958}, issn = {1873-4235}, support = {U01 CA269147/CA/NCI NIH HHS/United States ; }, mesh = {Humans ; *Biosensing Techniques/methods ; *Fluorescent Dyes/chemistry ; *Human papillomavirus 16/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *DNA, Viral/analysis/genetics ; DNA, Single-Stranded/chemistry ; DNA Probes/chemistry/genetics ; Carbocyanines/chemistry ; Lab-On-A-Chip Devices ; Papillomavirus Infections/diagnosis/virology ; CRISPR-Associated Proteins/chemistry ; Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {Rapid, sensitive, and specific nucleic acid detection methods play crucial roles in clinical diagnostics and healthcare. Here, we report a novel amphiphilic DNA fluorescence probe for CRISPR-based nucleic acid detection. Unlike conventional fluorophore-quencher probe detection system, our amphiphilic DNA fluorescence probe features a hydrophobic Cy5 fluorophore head and a hydrophilic single-stranded DNA (ssDNA) tail. By combining the amphiphilic DNA fluorescence probe with a paper-based microfluidic device, we developed a quencher-free, CRISPR-based detection system for target nucleic acid quantification. In the presence of the target nucleic acid, the activated CRISPR-Cas12a enzyme cleaves the hydrophilic ssDNA tail of the amphiphilic DNA fluorescence probe, releasing the hydrophobic Cy5 head and altering the wettability of the CRISPR reaction solution. When the CRISPR reaction product is applied to the paper-based microfluidic device, the migration of the cleaved Cy5 head along the hydrophilic microfluidic channel is limited. The higher the target nucleic acid concentration, the shorter the fluorescence diffusion distance, enabling visual quantification of the nucleic acid target. We used human papillomavirus-16 (HPV-16) DNA as a model to evaluate the analytical performance of the system. Furthermore, we validated its clinical feasibility by testing clinical swab samples, achieving results comparable to the traditional PCR method. Our quencher-free CRISPR-based detection system shows potential for simple, affordable, and sensitive clinical diagnostics of HPV-associated cancer and other infectious diseases.}, }
@article {pmid39656986, year = {2024}, author = {Jordan, B}, title = {[New-generation editing].}, journal = {Medecine sciences : M/S}, volume = {40}, number = {11}, pages = {869-871}, doi = {10.1051/medsci/2024149}, pmid = {39656986}, issn = {1958-5381}, mesh = {Animals ; Humans ; Bacteria/genetics ; CRISPR-Cas Systems ; *Gene Editing/methods/trends ; }, abstract = {Recent work on bacterial insertion sequences reveals that some of them use an RNA sequence (called Bridge RNA or Seek RNA) to define both donor and target DNA specificity. This opens the way to easy insertion of kilobase DNA sequences at pre-defined sites in the genome, announcing a host of new possibilities. The system still needs a lot of tweaking, as it has only been demonstrated in bacteria, but it holds great promise for genome editing and engineering.}, }
@article {pmid39673702, year = {2025}, author = {Zi, H and Peng, X and Du, J and Li, J}, title = {Protocol for generating a pericyte reporter zebrafish line Ki(pdgfrb-P2A-GAL4-VP16) using a CRISPR-Cas9-mediated knockin technique.}, journal = {STAR protocols}, volume = {6}, number = {1}, pages = {103490}, pmid = {39673702}, issn = {2666-1667}, mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems/genetics ; *Pericytes/metabolism/cytology ; *Gene Knock-In Techniques/methods ; Genes, Reporter/genetics ; Animals, Genetically Modified ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {Pericytes, the mural cells that envelop small blood vessels, play crucial roles in the formation of the blood-brain barrier (BBB). Here, we present a protocol for generating a pericyte reporter zebrafish line Ki(pdgfrb-P2A-GAL4-VP16) using a CRISPR-Cas9-mediated knockin technique. We describe steps for identifying efficient single guide RNA (sgRNA), constructing donor plasmid, and generating and maintaining the knockin line. We then detail procedures for in vivo imaging of brain pericytes. This protocol is adaptable for creating other knockin lines for specific cell labeling. For complete details on the use and execution of this protocol, please refer to Zi et al.[1].}, }
@article {pmid39673524, year = {2025}, author = {Prajapat, MK and Maria, AG and Vidigal, JA}, title = {CRISPR-based dissection of miRNA binding sites using isogenic cell lines is hampered by pervasive noise.}, journal = {Nucleic acids research}, volume = {53}, number = {1}, pages = {}, pmid = {39673524}, issn = {1362-4962}, support = {ZIA BC011810/ImNIH/Intramural NIH HHS/United States ; 1ZIABC011810-02/NH/NIH HHS/United States ; /RC/CCR NIH HHS/United States ; }, mesh = {*MicroRNAs/metabolism/genetics ; Binding Sites ; Humans ; *CRISPR-Cas Systems ; Cell Line ; Animals ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Mutation ; HEK293 Cells ; }, abstract = {Non-coding regulatory sequences play essential roles in adjusting gene output to cellular needs and are thus critical to animal development and health. Numerous such sequences have been identified in mammalian genomes ranging from transcription factors binding motifs to recognition sites for RNA-binding proteins and non-coding RNAs. The advent of CRISPR has raised the possibility of assigning functionality to individual endogenous regulatory sites by facilitating the generation of isogenic cell lines that differ by a defined set of genetic modifications. Here we investigate the usefulness of this approach to assign function to individual miRNA binding sites. We find that the process of generating isogenic pairs of mammalian cell lines with CRISPR-mediated mutations introduces extensive molecular and phenotypic variability between biological replicates confounding attempts at assigning function to the binding site. Our work highlights an important consideration when employing CRISPR editing to characterize non-coding regulatory sequences in cell lines and calls for the development and adoption of alternative strategies to address this question in the future.}, }
@article {pmid39673266, year = {2024}, author = {Molina-Sánchez, MD and Martínez-Abarca, F and Millán, V and Mestre, MR and Stehantsev, P and Stetsenko, A and Guskov, A and Toro, N}, title = {Adaptive immunity of type VI CRISPR-Cas systems associated with reverse transcriptase-Cas1 fusion proteins.}, journal = {Nucleic acids research}, volume = {52}, number = {22}, pages = {14229-14243}, pmid = {39673266}, issn = {1362-4962}, support = {P20_00047//Consejeria de Transformacion Economica, industria, Conocimiento y Universidades/ ; //ERDF A way of making Europe/ ; }, mesh = {*CRISPR-Cas Systems ; *RNA-Directed DNA Polymerase/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; *Adaptive Immunity/genetics ; Bacterial Proteins/genetics/metabolism ; Gastrointestinal Microbiome/genetics/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Bacteria/genetics/immunology ; RNA Interference ; }, abstract = {Cas13-containing type VI CRISPR-Cas systems specifically target RNA; however, the mechanism of spacer acquisition remains unclear. We have previously reported the association of reverse transcriptase-Cas1 (RT-Cas1) fusion proteins with certain types of VI-A systems. Here, we show that RT-Cas1 fusion proteins are also recruited by type VI-B systems in bacteria from gut microbiomes, constituting a VI-B1 variant system that includes a CorA-encoding locus in addition to the CRISPR array and the RT-Cas1/Cas2 adaptation module. We found that type VI RT-CRISPR systems were functional for spacer acquisition, CRISPR array processing and interference activity, demonstrating that adaptive immunity mediated by these systems can function independently of other in trans systems. We provide evidence that the RT associated with these systems enables spacer acquisition from RNA molecules. We also found that CorA encoded by type VI-B1 RT-associated systems can transport divalent metal ions and downregulate Cas13b-mediated RNA interference. These findings highlight the importance of RTs in RNA-targeting CRISPR-Cas systems, potentially enabling the integration of RNA-derived spacers into CRISPR arrays as a mechanism against RNA-based invaders in specific environments.}, }
@article {pmid39672387, year = {2025}, author = {He, X and Li, P and Cao, H and Zhang, X and Zhang, M and Yu, X and Sun, Y and Ghonaim, AH and Ma, H and Li, Y and Shi, K and Zhu, H and He, Q and Li, W}, title = {Construction of a recombinant African swine fever virus with firefly luciferase and eGFP reporter genes and its application in high-throughput antiviral drug screening.}, journal = {Antiviral research}, volume = {233}, number = {}, pages = {106058}, doi = {10.1016/j.antiviral.2024.106058}, pmid = {39672387}, issn = {1872-9096}, mesh = {Animals ; *African Swine Fever Virus/genetics/drug effects ; Chlorocebus aethiops ; *Green Fluorescent Proteins/genetics ; *Antiviral Agents/pharmacology ; Vero Cells ; *Genes, Reporter ; Swine ; *Luciferases, Firefly/genetics ; *African Swine Fever/virology ; *Drug Evaluation, Preclinical/methods ; High-Throughput Screening Assays/methods ; Virus Replication/drug effects ; CRISPR-Cas Systems ; Gene Editing/methods ; Macrophages, Alveolar/virology/drug effects ; }, abstract = {African Swine Fever (ASF) is a highly lethal and contagious disease in pigs caused by African Swine Fever Virus (ASFV), which primarily infects domestic pigs and wild boars, with a mortality rate of up to 100%. Currently, there are no commercially available vaccines or drugs that are both safe and effective against ASFV. The ASFV 0428C strain was continuously passaged in Vero cells, and the adapted ASFV demonstrated efficient replication in Vero cells. The adapted ASFV was used as the parental virus, and an expression cassette encoding a dual reporter gene for firefly luciferase (Fluc) and enhanced green fluorescent protein (eGFP) was inserted into the ASFV genome using CRISPR/Cas9 gene editing technology to construct a recombinant ASFV variant (rASFV-FLuc-eGFP). rASFV-Fluc-eGFP was genetically stable, effectively infected porcine alveolar macrophages (PAM) and Vero cells, and expressed Fluc and eGFP concurrently. This study provides a tool for investigating the infection and pathogenic mechanisms of ASFV, as well as for screening essential host genes and antiviral drugs. Additionally, a high-throughput screening model of antiviral drugs was established based on rASFV-FLuc-eGFP in passaged cells, 218 compounds from the FDA-approved compound library were screened, and 5 candidate compounds with significant inhibitory effects in Vero cells were identified. The inhibitory effects on ASFV were further validated in both Vero and PAM cells, resulting in the identification of Salvianolic acid C (SAC), which demonstrated inhibitory effects and safety in both cell types. SAC is a candidate drug for the prevention and control of ASFV and shows promising application prospects.}, }
@article {pmid39672163, year = {2025}, author = {Zeng, J and Nguyen, MA and Liu, P and da Silva, LF and Levesque, S and Lin, LY and Justus, DG and Petri, K and Clement, K and Porter, SN and Verma, A and Neri, NR and Rosanwo, T and Ciuculescu, MF and Abriss, D and Mintzer, E and Maitland, SA and Demirci, S and Cha, HJ and Orkin, SH and Tisdale, JF and Williams, DA and Zhu, LJ and Pruett-Miller, SM and Pinello, L and Joung, JK and Pattanayak, V and Manis, JP and Armant, M and Pellin, D and Brendel, C and Wolfe, SA and Bauer, DE}, title = {Gene editing without ex vivo culture evades genotoxicity in human hematopoietic stem cells.}, journal = {Cell stem cell}, volume = {32}, number = {2}, pages = {191-208.e11}, pmid = {39672163}, issn = {1875-9777}, support = {R01 HL150669/HL/NHLBI NIH HHS/United States ; R35 HG010717/HG/NHGRI NIH HHS/United States ; R35 GM118158/GM/NIGMS NIH HHS/United States ; OT2 HL154984/HL/NHLBI NIH HHS/United States ; U54 DK106829/DK/NIDDK NIH HHS/United States ; P01 HL053749/HL/NHLBI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; }, mesh = {Humans ; *Gene Editing/methods ; *Hematopoietic Stem Cells/metabolism/cytology ; Animals ; CRISPR-Cas Systems/genetics ; Anemia, Sickle Cell/genetics/therapy ; Mice ; Fetal Hemoglobin/genetics/metabolism ; Repressor Proteins ; }, abstract = {Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for β-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here, we compare combined CRISPR-Cas9 editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. Dual targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two single guide RNAs (sgRNAs) resulted in superior HbF induction, including in sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. Unintended on-target outcomes of double-strand break (DSB) repair in hematopoietic stem and progenitor cells (HSPCs), such as long deletions and centromere-distal chromosome fragment loss, are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing quiescent HSPCs bypasses long deletion and micronuclei formation and preserves efficient on-target editing and engraftment function.}, }
@article {pmid39671912, year = {2025}, author = {Zhao, D and Deshpande, R and Wu, K and Tyagi, A and Sharma, S and Wu, SY and Xing, F and O'Neill, S and Ruiz, J and Lyu, F and Watabe, K}, title = {Identification of TUBB3 as an immunotherapy target in lung cancer by genome wide in vivo CRISPR screening.}, journal = {Neoplasia (New York, N.Y.)}, volume = {60}, number = {}, pages = {101100}, pmid = {39671912}, issn = {1476-5586}, support = {P30 CA012197/CA/NCI NIH HHS/United States ; R01 CA173499/CA/NCI NIH HHS/United States ; R01 CA185650/CA/NCI NIH HHS/United States ; R01 CA205067/CA/NCI NIH HHS/United States ; }, mesh = {Humans ; *Lung Neoplasms/genetics/drug therapy/immunology/therapy/metabolism/pathology ; *Tubulin/genetics/metabolism ; Animals ; *Immunotherapy/methods ; Mice ; Cell Line, Tumor ; Immune Checkpoint Inhibitors/pharmacology/therapeutic use ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic/drug effects ; Xenograft Model Antitumor Assays ; Drug Resistance, Neoplasm/genetics ; Programmed Cell Death 1 Receptor/antagonists &amp; inhibitors ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Biomarkers, Tumor ; T-Lymphocytes, Cytotoxic/immunology ; }, abstract = {Recent development of immune checkpoint inhibitors has revolutionized cancer immunotherapy. Although these drugs show dramatic effects on a subset of cancer patients, many other tumors are non-responsive and the pathological mechanism of the resistance is largely unknown. To identify genes underlying anti-PD-1 immunotherapy resistance using a systematic approach, we performed an in vivo genome wide CRISPR screening in lung cancer cells. We integrated our results with multi-omics clinical data and performed both in vitro and in vivo assays to evaluate the role of the top candidate in regulating cytotoxic T cell killing. We identified TUBB3 as a potential target to overcome the resistance and enhance the efficacy of anti-PD-1 immunotherapy. TUBB3 expression is upregulated in lung cancer patients, and its higher expression correlates with poorer patients' survival. We found that TUBB3 expression was significantly elevated in the non-responders compared to responders in our patient cohort that received immunotherapies. Importantly, the results of our preclinical experiments showed that inhibition of TUBB3 with a small molecule inhibitor synergized with anti-PD-1 treatment and enhanced tumor cell killing by cytotoxic T cells. Consistently, anti-PD-1 resistant cells showed significantly higher expression of TUBB3; however, TUBB3 inhibition rendered the resistant cells more susceptible to T cell killing. Mechanistic studies revealed that blocking TUBB3 suppressed the expression of PD-L1 through the EMT-related SNAI1 gene. Our results provide a rationale for a novel combination therapy consisting of the TUBB3 inhibition and anti-PD-1 immunotherapy for lung cancer.}, }
@article {pmid39671433, year = {2024}, author = {Taylor, T and Zhu, HV and Moorthy, SD and Khader, N and Mitchell, JA}, title = {The cells are all-right: Regulation of the Lefty genes by separate enhancers in mouse embryonic stem cells.}, journal = {PLoS genetics}, volume = {20}, number = {12}, pages = {e1011513}, pmid = {39671433}, issn = {1553-7404}, support = {R01 HG010045/HG/NHGRI NIH HHS/United States ; }, mesh = {Animals ; Mice ; *Enhancer Elements, Genetic ; *Mouse Embryonic Stem Cells/metabolism ; *Left-Right Determination Factors/genetics/metabolism ; *Chromatin/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing ; Gene Expression Regulation, Developmental ; }, abstract = {Enhancers play a critical role in regulating precise gene expression patterns essential for development and cellular identity; however, how gene-enhancer specificity is encoded within the genome is not clearly defined. To investigate how this specificity arises within topologically associated domains (TAD), we performed allele-specific genome editing of sequences surrounding the Lefty1 and Lefty2 paralogs in mouse embryonic stem cells. The Lefty genes arose from a tandem duplication event and these genes interact with each other in chromosome conformation capture assays which place these genes within the same TAD. Despite their physical proximity, we demonstrate that these genes are primarily regulated by separate enhancer elements. Through CRISPR-Cas9 mediated deletions to remove the intervening chromatin between the Lefty genes, we reveal a distance-dependent dosage effect of the Lefty2 enhancer on Lefty1 expression. These findings indicate a role for chromatin distance in insulating gene expression domains in the Lefty locus in the absence of architectural insulation.}, }
@article {pmid39671335, year = {2024}, author = {Kolodziejczyk, A and Hoffmann, J and Cubillos, P and Albert, M}, title = {Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {213}, pages = {}, doi = {10.3791/67598}, pmid = {39671335}, issn = {1940-087X}, mesh = {Humans ; *Electroporation/methods ; *Organoids/cytology/metabolism ; *CRISPR-Cas Systems ; Cerebral Cortex/cytology ; Gene Knockout Techniques/methods ; Neocortex/cytology ; }, abstract = {Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. Studies analyzing gene function by overexpression or knockout have been instrumental in animal models to provide mechanistic insights into the regulation of neocortex development. Here, we present a detailed protocol for CRISPR/Cas9-mediated acute gene knockout by electroporation of sliced human cortical organoids. The slicing of cortical organoids aids the identification of ventricle-like structures for injection and subsequent electroporation, making this a particularly well-suited model for acute genetic manipulation during human cortical development. We describe the design of guide RNAs and the validation of targeting efficiency in vitro and in cortical organoids. Electroporation of cortical organoids is performed at mid-neurogenic stages, enabling the targeting of most major cell classes in the developing neocortex, including apical radial glia, basal progenitor cells, and neurons. Taken together, the electroporation of sliced human cortical organoids represents a powerful technique to investigate gene function, gene regulation, and cell morphology during cortical development.}, }
@article {pmid39671009, year = {2024}, author = {Yu, Y and Li, Q and Yu, H and Li, Q}, title = {Comparative Analysis of Promoter Activity in Crassostrea gigas Embryos: Implications for Bivalve Gene Editing.}, journal = {Marine biotechnology (New York, N.Y.)}, volume = {27}, number = {1}, pages = {20}, pmid = {39671009}, issn = {1436-2236}, support = {42276111//National Natural Science Foundation of China/ ; ZR2022MC171//Natural Science Foundation of Shandong Province/ ; }, mesh = {Animals ; *Crassostrea/genetics/embryology ; *Promoter Regions, Genetic ; *Green Fluorescent Proteins/genetics/metabolism ; *Gene Editing/methods ; *Histones/metabolism/genetics ; Embryo, Nonmammalian/metabolism ; CRISPR-Cas Systems ; Peptide Elongation Factor 1/genetics/metabolism ; Aquaculture ; }, abstract = {In recent years, CRISPR/Cas9 gene editing technology has emerged as a powerful genetic tool with potential application in aquaculture. Crassostrea gigas, as a valuable species in aquaculture, holds promising potential for genetic enhancement and breeding through gene editing. However, the lack of efficient promoters for driving exogenous gene expression poses a major obstacle in bivalve gene editing. In this study, we isolated the promoter sequences of the β-tub and histone H3.3A genes from C. gigas. DNA expression constructs were generated by linking the promoters with the enhanced green fluorescent protein (EGFP) reporter and compared with the promoter activity of the endogenous EF-1α gene and an exogenous OsHV-1 promoter in C. gigas embryos. All four promoters effectively drive the expression of EGFP during early embryonic development in C. gigas. Among these four promoters, the β-tub promoter is the most potent promoter in driving EGFP expression in C. gigas embryos as early as 4.5 h after fertilization. The OsHV-1 promoter showed similar activity as β-tub promoter and appeared to be more active than the EF-1α and histone H3.3A promoters in C. gigas embryos. Furthermore, we assessed their performance in other three C. gigas relatives (Crassostrea ariakensis, Crassostrea nippona, and Crassostrea sikamea) and similar results were found. Collectively, these data suggest that the β-tub promoter is an effective promoter in directing gene expression in directing gene expression in oyster embryos, thus offering a potential application for gene editing in bivalves.}, }
@article {pmid39670656, year = {2025}, author = {Jung, JK and Dreyer, KS and Dray, KE and Muldoon, JJ and George, J and Shirman, S and Cabezas, MD and d'Aquino, AE and Verosloff, MS and Seki, K and Rybnicky, GA and Alam, KK and Bagheri, N and Jewett, MC and Leonard, JN and Mangan, NM and Lucks, JB}, title = {Developing, Characterizing, and Modeling CRISPR-Based Point-of-Use Pathogen Diagnostics.}, journal = {ACS synthetic biology}, volume = {14}, number = {1}, pages = {129-147}, pmid = {39670656}, issn = {2161-5063}, support = {R01 EB026510/EB/NIBIB NIH HHS/United States ; }, mesh = {*Nucleic Acid Amplification Techniques/methods ; *SARS-CoV-2/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; RNA, Viral/genetics/analysis ; *COVID-19/diagnosis/virology ; Humans ; Point-of-Care Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *COVID-19 Nucleic Acid Testing/methods ; *Molecular Diagnostic Techniques/methods ; Point-of-Care Testing ; }, abstract = {Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA-Cas13a nucleic acid detection assay. This test uses the isothermal amplification technique NASBA to amplify target viral nucleic acids, followed by the Cas13a-based detection of amplified sequences. We first demonstrate an in-house formulation of NASBA that enables the optimization of individual NASBA components. We then present design rules for NASBA primer sets and LbuCas13a guide RNAs for the fast and sensitive detection of SARS-CoV-2 viral RNA fragments, resulting in 20-200 aM sensitivity. Finally, we explore the combination of high-throughput assay condition screening with mechanistic ordinary differential equation modeling of the reaction scheme to gain a deeper understanding of the NASBA-Cas13a system. This work presents a framework for developing a mechanistic understanding of reaction performance and optimization that uses both experiments and modeling, which we anticipate will be useful in developing future nucleic acid detection technologies.}, }
@article {pmid39670632, year = {2025}, author = {Chen, W and Liu, L and Cheng, L}, title = {Conditionally Activated Cross-Linked crRNAs for CRISPR/Cas12a Based Nucleic Acid Detection.}, journal = {ACS synthetic biology}, volume = {14}, number = {1}, pages = {94-100}, doi = {10.1021/acssynbio.4c00695}, pmid = {39670632}, issn = {2161-5063}, mesh = {*CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques/methods ; Cross-Linking Reagents/chemistry ; CRISPR-Associated Proteins/genetics/metabolism ; *RNA/genetics ; *Nucleic Acids/analysis/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {CRISPR/Cas systems, particularly CRISPR/Cas12a, have revolutionized nucleic acid detection due to their exceptional specificity and sensitivity. However, CRISPR/Cas12a's cleavage activity can interfere with amplification processes, such as reverse transcription (RT) and isothermal amplification (e.g., RPA), potentially compromising detection sensitivity and accuracy. While modified CRISPR/Cas12a systems employing caging and decaging strategies have been developed to address this, these approaches typically require extensive optimization of photolabile groups and complex assay configurations. Here, we present a universal, photochemically controlled strategy for CRISPR/Cas12a-based detection that overcomes these challenges. Our approach involves cross-linking a polymeric crRNA with a photoresponsive cross-linker, effectively inactivating it during amplification and enabling rapid activation through brief light exposure to cleave the cross-linker and release active crRNA. This method obviates the need for labor-intensive optimizations and modifications, making it highly versatile and suitable for rapid, on-site detection applications. Our strategy demonstrates enhanced versatility and applicability, particularly for the immediate detection of newly emerging or unexpected nucleic acid sequences, supporting applications in pathogen detection, genetic screening, and point-of-care diagnostics.}, }
@article {pmid39670384, year = {2025}, author = {Blennemann, M and Verma, A and Bachl, S and Carnevale, J and Engelhardt, BE}, title = {Understanding TCR T cell knockout behavior using interpretable machine learning.}, journal = {Pacific Symposium on Biocomputing. Pacific Symposium on Biocomputing}, volume = {30}, number = {}, pages = {382-393}, doi = {10.1142/9789819807024_0028}, pmid = {39670384}, issn = {2335-6936}, mesh = {Humans ; *Receptors, Antigen, T-Cell/immunology/genetics ; *Machine Learning ; *Computational Biology ; *T-Lymphocytes/immunology ; *Neoplasms/immunology/genetics ; Gene Knockout Techniques ; Neural Networks, Computer ; CRISPR-Cas Systems ; }, abstract = {Genetic perturbation of T cell receptor (TCR) T cells is a promising method to unlock better TCR T cell performance to create more powerful cancer immunotherapies, but understanding the changes to T cell behavior induced by genetic perturbations remains a challenge. Prior studies have evaluated the effect of different genetic modifications with cytokine production and metabolic activity assays. Live-cell imaging is an inexpensive and robust approach to capture TCR T cell responses to cancer. Most methods to quantify T cell responses in live-cell imaging data use simple approaches to count T cells and cancer cells across time, effectively quantifying how much space in the 2D well each cell type covers, leaving actionable information unexplored. In this study, we characterize changes in TCR T cell's interactions with cancer cells from live-cell imaging data using explainable artificial intelligence (AI). We train convolutional neural networks to distinguish behaviors in TCR T cell with CRISPR knock outs of CUL5, RASA2, and a safe harbor control knockout. We use explainable AI to identify specific interaction types that define different knock-out conditions. We find that T cell and cancer cell coverage is a strong marker of TCR T cell modification when comparing similar experimental time points, but differences in cell aggregation characterize CUL5KO and RASA2KO behavior across all time points. Our pipeline for discovery in live-cell imaging data can be used for characterizing complex behaviors in arbitrary live-cell imaging datasets, and we describe best practices for this goal.}, }
@article {pmid39668248, year = {2025}, author = {Philip, R and Sharma, A and Matellan, L and Erpf, AC and Hsu, WH and Tkach, JM and Wyatt, HDM and Pelletier, L}, title = {qTAG: an adaptable plasmid scaffold for CRISPR-based endogenous tagging.}, journal = {The EMBO journal}, volume = {44}, number = {3}, pages = {947-974}, pmid = {39668248}, issn = {1460-2075}, support = {187836//Canadian Government | CIHR | Institute of Cancer Research (IC)/ ; 181763//Canadian Government | CIHR | Institute of Cancer Research (IC)/ ; 156297//Canadian Government | Canadian Institutes of Health Research (CIHR)/ ; 167279//Canadian Government | Canadian Institutes of Health Research (CIHR)/ ; N/A//Krembil Foundation/ ; }, mesh = {*Plasmids/genetics ; Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/methods ; }, abstract = {Endogenous tagging enables the study of proteins within their native regulatory context, typically using CRISPR to insert tag sequences directly into the gene sequence. Here, we introduce qTAG, a collection of repair cassettes that makes endogenous tagging more accessible. The cassettes support N- and C-terminal tagging with commonly used selectable markers and feature restriction sites for easy modification. Lox sites also enable the removal of the marker gene after successful integration. We demonstrate the utility of qTAG with a range of diverse tags for applications in fluorescence imaging, proximity labeling, epitope tagging, and targeted protein degradation. The system includes novel tags like mStayGold, offering enhanced brightness and photostability for live-cell imaging of native protein dynamics. Additionally, we explore alternative cassette designs for conditional expression tagging, selectable knockout tagging, and safe-harbor expression. The plasmid collection is available through Addgene, featuring ready-to-use constructs for common subcellular markers and tagging cassettes to target genes of interest. The qTAG system will serve as an open resource for researchers to adapt and tailor their own experiments.}, }
@article {pmid39667940, year = {2024}, author = {Yang, B and Wu, C and Teng, Y and Chou, KJ and Guarnieri, MT and Xiong, W}, title = {Tailoring microbial fitness through computational steering and CRISPRi-driven robustness regulation.}, journal = {Cell systems}, volume = {15}, number = {12}, pages = {1133-1147.e4}, doi = {10.1016/j.cels.2024.11.012}, pmid = {39667940}, issn = {2405-4720}, mesh = {*CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Computer Simulation ; Genetic Fitness/genetics ; Escherichia coli/genetics/metabolism ; Organisms, Genetically Modified ; }, abstract = {The widespread application of genetically modified microorganisms (GMMs) across diverse sectors underscores the pressing need for robust strategies to mitigate the risks associated with their potential uncontrolled escape. This study merges computational modeling with CRISPR interference (CRISPRi) to refine GMM metabolic robustness. Utilizing ensemble modeling, we achieved high-throughput in silico screening for enzymatic targets susceptible to expression alterations. Translating these insights, we developed functional CRISPRi, boosting fitness control via multiplexed gene knockdown. Our method, enhanced by an insulator-improved gRNA structure and an off-switch circuit controlling a compact Cas12m, resulted in rationally engineered strains with escape frequencies below National Institutes of Health standards. The effectiveness of this approach was confirmed under various conditions, showcasing its ability for secure GMM management. This research underscores the resilience of microbial metabolism, strategically modifying key nodes to halt growth without provoking significant resistance, thereby enabling more reliable and precise GMM control. A record of this paper's transparent peer review process is included in the supplemental information.}, }
@article {pmid39667861, year = {2025}, author = {Wu, P and Zhang, Y and Shan, Q and Wang, Z and Cheng, S and Wang, L and Liu, B and Li, W and Chen, Z and Luo, J and Liang, Y}, title = {The investigation of the mechanism underlying variations in oxidative stress tolerance of Lacticaseibacillus paracasei resulting from fermentation methods through endogenous CRISPR-Cas9 editing methodology.}, journal = {Food microbiology}, volume = {127}, number = {}, pages = {104697}, doi = {10.1016/j.fm.2024.104697}, pmid = {39667861}, issn = {1095-9998}, mesh = {*Oxidative Stress ; *Fermentation ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Lacticaseibacillus paracasei/genetics/metabolism/physiology/growth &amp; development ; Probiotics ; Bacterial Proteins/genetics/metabolism ; }, abstract = {The probiotic effects of lactic acid bacteria make them widely used in human and animal breeding industry. However, the presence of oxidative stress during the production and application process can cause bacterial damage or even death, significantly compromising the functionality of probiotics. Despite its potential for broader application scenarios that could provide a more comprehensive understanding of bacteria's internal adaptation strategies, there is a lack of research investigating oxidative stress from the perspective of culture methods. In this study, the tolerance to oxidative stress was compared between bacteria cultivated through solid-state fermentation (SSF) and liquid-state fermentation (LSF), and the physiological and transcriptional disparities between these two bacterial strains were investigated. Additionally, a novel and efficient gene editing method was developed to elucidate the genetic basis underlying these differences in tolerance. The results demonstrated a significantly higher tolerance to oxidative stress in SSF bacteria compared to LSF bacteria, along with a stronger capacity for maintaining intracellular microenvironment stability and the activity of key metabolic enzymes. It is noteworthy that the bacteria from SSF significantly enhance the transport of carbohydrate substances and facilitate intracellular metabolic flow. Gene editing experiments have confirmed the crucial role of genes glpF and glpO in regulating the glycerol metabolism pathway, which is essential for enhancing the tolerance of bacteria from SSF to oxidative stress. Based on these findings, the mechanism underlying the disparity in oxidative stress tolerance resulting from different culture methods has been summarized. Furthermore, investigation into different culture modes has revealed that moderate oxygen levels during cultivation significantly influence variation in bacterial tolerance to oxidative stress. Importantly, these variations are species-specific and depend on the ecological niche distribution of Lactobacilli. These findings elucidate a novel mechanism by which Lacticaseibacillus paracasei Zhang tolerates oxidative stress, and also suggest that distinct cultivation and processing methods should be tailored based on the specific Lactobacilli groups to achieve optimal application effects in production.}, }
@article {pmid39667860, year = {2025}, author = {Zhang, X and Chen, W and Wang, C}, title = {Regulation of citrinin biosynthesis in Monascus purpureus: Impacts on growth, morphology, and pigments production.}, journal = {Food microbiology}, volume = {127}, number = {}, pages = {104698}, doi = {10.1016/j.fm.2024.104698}, pmid = {39667860}, issn = {1095-9998}, mesh = {*Monascus/metabolism/growth &amp; development/genetics ; *Citrinin/biosynthesis/metabolism ; *Fungal Proteins/genetics/metabolism ; *Pigments, Biological/biosynthesis/metabolism ; Gene Expression Regulation, Fungal ; Mycelium/growth &amp; development/metabolism/genetics ; CRISPR-Cas Systems ; Hyphae/growth &amp; development/metabolism ; Spores, Fungal/growth &amp; development/metabolism/genetics ; Gene Knockout Techniques ; }, abstract = {Fungal hyphae self-assemble a variety of cellular macrostates, ranging from suspended mycelium to dense pellets, all inextricably linked to their productivity. In this study, using CRISPR/Cas technology, we constructed a ctnA knockout strain (ΔctnA) and an overexpression strain (A2) so as to investigate the effects of interfering with citrinin biosynthesis on the growth, morphology and pigmentation of M.purpureus. Results indicated that deletion of ctnA in M. purpureus RP2 led to increased mycelium length, delayed conidium formation, and a citrinin content of 22% of the wild-type strain. Conversely, ctnA overexpression in strain A2 resulted in delayed mycelial growth, normal conidium formation, and a citrinin content of 120% compared to the wild-type strain, with minimal effects on pigments content. Notably, the ΔctnA strain formed small, tightly structured pellets (mean diameter 1.2 ± 0.06 mm) and exhibited low citrinin content, promoting pigments production. Our findings suggest a complex interplay between citrinin biosynthesis and morphological development, providing insights for optimizing metabolite production in industrial applications.}, }
@article {pmid39667702, year = {2025}, author = {Chaudhary, N and Sharma, K and Kaur, H and Prajapati, S and Mohan, B and Taneja, N}, title = {CRISPR-Cas-assisted phage engineering for personalized antibacterial treatments.}, journal = {Indian journal of medical microbiology}, volume = {53}, number = {}, pages = {100771}, doi = {10.1016/j.ijmmb.2024.100771}, pmid = {39667702}, issn = {1998-3646}, mesh = {*CRISPR-Cas Systems ; *Bacteriophages/genetics ; Humans ; *Precision Medicine/methods ; *Anti-Bacterial Agents/therapeutic use ; Gene Editing/methods ; *Genetic Engineering/methods ; *Phage Therapy/methods ; *Bacterial Infections/therapy ; Bacteria/virology/drug effects ; Host Specificity ; }, abstract = {BACKGROUND: In the age of modern medicine, CRISPR-Cas system-aided phage engineering has emerged as a major game changer for developing personalized antibacterial treatments. Modifying genomic DNA at a specific location leads to the inactivation of target genes, the acquisition of novel genetic features, and the correction of lethal gene mutations. Phages can be modified to precisely detect and control bacteria because of the vast possibilities of CRISPR-Cas-based genetic engineering.<br><br>OBJECTIVES: The primary objective of this review is to explore the basic principles, mechanisms, limitations, and perspectives of CRISPR-Cas system-aided phage engineering in producing tailored antibacterial therapeutics. Furthermore, this study will address how editing phage genomes using CRISPR-Cas technology allows for precise bacteria targeting, broadening phage host range, and improving infection control tactics.<br><br>CONTENT: The arrival of the CRISPR-Cas system has transformed the field of phage engineering and aided in the precise modification of phag&#x435; genomes to broaden the phage host range. This novel strategy uses the accuracy of the CRISPR-Cas system to design engineered bacteriophages, giving targeted options for infection control. These recent advancements have the potential to alter the era of modern medicine.}, }
@article {pmid39667064, year = {2025}, author = {Lu, S and Chen, M and Liu, X and Li, J and Liu, H and Li, S}, title = {Generation of a BEST1 Pr-EGFP reporter human embryonic stem cell line via CRISPR/Cas9 editing.}, journal = {Stem cell research}, volume = {82}, number = {}, pages = {103625}, doi = {10.1016/j.scr.2024.103625}, pmid = {39667064}, issn = {1876-7753}, mesh = {Humans ; *Human Embryonic Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; *Green Fluorescent Proteins/metabolism/genetics ; *Bestrophins/genetics/metabolism ; Cell Line ; Cell Differentiation ; Retinal Pigment Epithelium/cytology/metabolism ; }, abstract = {The retinal pigment epithelium (RPE) cell, located between the neural retina and choriocapillaris, is vital for retinal maintenance and photoreceptor function. Human embryonic stem cells (hESCs) provide a limitless source of RPE cells for transplantation. Using CRISPR/Cas9, we inserted a fusion of the BEST1 promoter (an RPE-specific marker) and the EGFP gene into the AAVS1 locus to track differentiation in hESC-induced RPE (hESC-iRPE). The resulting gene-edited line, WAe009-A-2 M, maintained a normal karyotype, expressed pluripotency markers, and demonstrated differentiation potential, making it invaluable for RPE development and therapeutic research.}, }
@article {pmid39666857, year = {2024}, author = {Xie, F and Zhao, H and Liu, J and Yang, X and Neuber, M and Agrawal, AA and Kaur, A and Herrmann, J and Kalinina, OV and Wei, X and Müller, R and Fu, C}, title = {Autologous DNA mobilization and multiplication expedite natural products discovery from bacteria.}, journal = {Science (New York, N.Y.)}, volume = {386}, number = {6727}, pages = {eabq7333}, doi = {10.1126/science.abq7333}, pmid = {39666857}, issn = {1095-9203}, mesh = {*Biological Products/metabolism ; *Streptomyces/genetics/metabolism ; *CRISPR-Cas Systems ; *Multigene Family ; *Plasmids/genetics ; DNA, Bacterial/genetics ; Genome, Bacterial ; Drug Discovery ; Biosynthetic Pathways ; DNA Replication ; Drug Resistance, Bacterial/genetics ; }, abstract = {The transmission of antibiotic-resistance genes, comprising mobilization and relocation events, orchestrates the dissemination of antimicrobial resistance. Inspired by this evolutionarily successful paradigm, we developed ACTIMOT, a CRISPR-Cas9-based approach to unlock the vast chemical diversity concealed within bacterial genomes. ACTIMOT enables the efficient mobilization and relocation of large DNA fragments from the chromosome to replicative plasmids within the same bacterial cell. ACTIMOT circumvents the limitations of traditional molecular cloning methods involving handling and replicating large pieces of genomic DNA. Using ACTIMOT, we mobilized and activated four cryptic biosynthetic gene clusters from Streptomyces, leading to the discovery of 39 compounds across four distinct classes. This work highlights the potential of ACTIMOT for accelerating the exploration of biosynthetic pathways and the discovery of natural products.}, }
@article {pmid39666823, year = {2025}, author = {Li, Y and Li, Z and Yan, P and Hua, C and Kong, J and Wu, W and Cui, Y and Duan, Y and Li, S and Li, G and Ji, S and Chen, Y and Zhao, Y and Yang, P and Hu, C and Lu, M and Chen, M and Xiao, Y}, title = {Antiviral signaling of a type III CRISPR-associated deaminase.}, journal = {Science (New York, N.Y.)}, volume = {387}, number = {6736}, pages = {eadr0393}, doi = {10.1126/science.adr0393}, pmid = {39666823}, issn = {1095-9203}, mesh = {*Adenosine Deaminase/chemistry/metabolism/genetics ; *Adenosine Triphosphate/metabolism ; *CRISPR-Associated Proteins/chemistry/metabolism ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; Hydrolysis ; Pyrophosphatases/metabolism ; Signal Transduction ; *Verrucomicrobia/enzymology/genetics/virology ; }, abstract = {Prokaryotes have evolved diverse defense strategies against viral infection, including foreign nucleic acid degradation by CRISPR-Cas systems and DNA and RNA synthesis inhibition through nucleotide pool depletion. Here, we report an antiviral mechanism of type III CRISPR-Cas-regulated adenosine triphosphate (ATP) depletion in which ATP is converted into inosine triphosphate (ITP) by CRISPR-Cas-associated adenosine deaminase (CAAD) upon activation by either cA4 or cA6, followed by hydrolysis into inosine monophosphate (IMP) by Nudix hydrolase, ultimately resulting in cell growth arrest. The cryo-electron microscopy structures of CAAD in its apo and activated forms, together with biochemical evidence, revealed how cA4 or cA6 binds to the CRISPR-associated Rossmann fold (CARF) domain and abrogates CAAD autoinhibition, inducing substantial conformational changes that reshape the structure of CAAD and induce its deaminase activity. Our results reveal the mechanism of a CRISPR-Cas-regulated ATP depletion antiviral strategy.}, }
@article {pmid39666765, year = {2024}, author = {Shao, G and Zhu, X and Hua, R and Chen, Y and Yang, G}, title = {Development of a Copro-RPA-CRISPR/Cas12a assay to detect Echinococcus granulosus nucleic acids isolated from canine feces using NaOH-based DNA extraction method.}, journal = {PLoS neglected tropical diseases}, volume = {18}, number = {12}, pages = {e0012753}, pmid = {39666765}, issn = {1935-2735}, mesh = {Animals ; Dogs ; *Echinococcus granulosus/genetics/isolation &amp; purification ; *Feces/parasitology ; *Echinococcosis/diagnosis/veterinary/parasitology ; *Dog Diseases/diagnosis/parasitology ; *CRISPR-Cas Systems ; *DNA, Helminth/genetics/isolation &amp; purification ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/genetics/isolation &amp; purification ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {BACKGROUND: Cystic echinococcosis (CE), caused by Echinococcus granulosus sensu lato (E. granulosus s.l.), remains a significant zoonotic parasitic disease affecting both livestock and humans. It arises from the ingestion of food and water contaminated with canine feces containing E. granulosus eggs. The detection of these eggs in canine feces is essential for guiding effective preventative measures against the disease. Therefore, the development of a novel accurate, rapid, and visually interpretable point-of-care test is crucial for controlling CE.<br><br>METHODS: We combined recombinase polymerase amplification (RPA) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) with a CRISPR-associated protein 12a (Cas12a) system, forming the RPA-CRISPR/Cas12a assay. This assay targeted the E. granulosus mitochondrial nad2 gene and utilized a lateral flow strip for visual readout. To improve field applicability, we integrated a simple and cost-effective NaOH-Based DNA extraction method. Clinical validation included testing DNA extracted from eighteen canine fecal samples, followed by comparison with quantitative PCR (qPCR) and two commercial enzyme-linked immunosorbent assay (ELISA) kits.<br><br>RESULTS: The RPA-CRISPR/Cas12a assay showed a detection limit of 1 fg/&#x3bc;L DNA, without any cross-reactivity with related tapeworms such as Echinococcus multilocularis, Dipylidium caninum, Taenia hydatigera, Taenia multiceps, and Taenia pisiformis. When applied to 62 clinical fecal samples from dogs, the RPA-CRISPR/Cas12a assay demonstrated 68% sensitivity, while the developed RPA-CRISPR/Cas12a-NaOH assay exhibited 45% sensitivity. In the field performance comparison of the RPA-CRISPR/Cas12a and the RPA-CRISPR/Cas12a-NaOH assay with qPCR and two ELISA kits, the sensitivity, consistency rate, and Youden's index suggested good or fair agreement with the currently employed detection methods.<br><br>CONCLUSION: This study describes the development and validation of the RPA-CRISPR/Cas12a and RPA-CRISPR/Cas12a-NaOH assays for detecting E. granulosus in canine feces. The developed assays surpassed previous detection methods in providing enhanced diagnostic sensitivity and enabling point-of-care testing. Moreover, these assays hold potential for surveilling E. granulosus in low-income countries.}, }
@article {pmid39666677, year = {2024}, author = {Blokhina, Y and Buchwalter, A}, title = {Modeling the consequences of age-linked rDNA hypermethylation with dCas9-directed DNA methylation in human cells.}, journal = {PloS one}, volume = {19}, number = {12}, pages = {e0310626}, pmid = {39666677}, issn = {1932-6203}, support = {T32 HL007731/HL/NHLBI NIH HHS/United States ; }, mesh = {Humans ; *DNA Methylation ; *DNA, Ribosomal/genetics/metabolism ; *Promoter Regions, Genetic ; *Aging/genetics ; Pol1 Transcription Initiation Complex Proteins/metabolism/genetics ; RNA Polymerase I/metabolism/genetics ; CRISPR-Cas Systems ; Transcription, Genetic ; CRISPR-Associated Protein 9/metabolism/genetics ; Animals ; }, abstract = {Ribosomal DNA (rDNA) genes encode the structural RNAs of the ribosome and are present in hundreds of copies in mammalian genomes. Age-linked DNA hypermethylation throughout the rDNA constitutes a robust "methylation clock" that accurately reports age, yet the consequences of hypermethylation on rDNA function are unknown. We confirmed that pervasive hypermethylation of rDNA occurs during mammalian aging and senescence while rDNA copy number remains stable. We found that DNA methylation is exclusively found on the promoters and gene bodies of inactive rDNA. To model the effects of age-linked methylation on rDNA function, we directed de novo DNA methylation to the rDNA promoter or gene body with a nuclease-dead Cas9 (dCas9)-DNA methyltransferase fusion enzyme in human cells. Hypermethylation at each target site had no detectable effect on rRNA transcription, nucleolar morphology, or cellular growth rate. Instead, human UBF and Pol I remain bound to rDNA promoters in the presence of increased DNA methylation. These data suggest that promoter methylation is not sufficient to impair transcription of the human rDNA and imply that the human rDNA transcription machinery may be resilient to age-linked rDNA hypermethylation.}, }
@article {pmid39666461, year = {2024}, author = {Yan, B and Liu, Y and Cai, Y and Liu, Y and Chen, Y}, title = {Protocol for establishing CRISPR-Cas12a for efficient genome editing of Pseudomonas aeruginosa phages.}, journal = {STAR protocols}, volume = {5}, number = {4}, pages = {103488}, pmid = {39666461}, issn = {2666-1667}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Pseudomonas aeruginosa/genetics/virology ; Genome, Viral/genetics ; Pseudomonas Phages/genetics ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {We developed an efficient type V CRISPR-Cas12a system tailored specifically for Pseudomonas aeruginosa phages, showcasing its remarkable cleavage activity and the ability to precisely introduce genetic modifications, including point mutations, deletions, and insertions, into phage genomes. Here, we present a protocol for establishing CRISPR-Cas12a for genome editing of Pseudomonas aeruginosa phages. We describe steps for the construction of pCRISPR-12a plasmid and guide RNA and the utilization of the type V CRISPR-Cas12a system for precise genetic editing of phages. For complete details on the use and execution of this protocol, please refer to Chen et al.[1].}, }
@article {pmid39665756, year = {2024}, author = {Kent, K and Nozawa, K and Parkes, R and Dean, L and Daniel, F and Leng, M and Jain, A and Malovannaya, A and Matzuk, MM and X Garcia, T}, title = {Large-scale CRISPR/Cas9 deletions within the WFDC gene cluster uncover gene functionality and critical roles in mammalian reproduction.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {121}, number = {51}, pages = {e2413195121}, pmid = {39665756}, issn = {1091-6490}, support = {T32 GM139534/GM/NIGMS NIH HHS/United States ; RP210227//Cancer Prevention and Research Institute of Texas (CPRIT)/ ; S10OD026804//HHS | NIH | NIH Office of the Director (OD)/ ; Fellowship//Lalor Foundation (The Lalor Foundation)/ ; R01HD095341//HHS | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; R01HD106056//HHS | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; R01 HD088412/HD/NICHD NIH HHS/United States ; T32HD098068//HHS | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; P30 CA125123/CA/NCI NIH HHS/United States ; Fellowship//Japan Society for the Promotion of Science Overseas Research/ ; RP170005//Cancer Prevention and Research Institute of Texas (CPRIT)/ ; R01 HD106056/HD/NICHD NIH HHS/United States ; T32 HD098068/HD/NICHD NIH HHS/United States ; 2021-302//Male Contraceptive Initiative/ ; R01 HD095341/HD/NICHD NIH HHS/United States ; S10 OD026804/OD/NIH HHS/United States ; P30CA125123//HHS | NIH | National Cancer Institute (NCI)/ ; P50HD103555//HHS | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; Fellowship//A.I. &amp; Manet Schepps Discovery Foundation/ ; P50 HD103555/HD/NICHD NIH HHS/United States ; R01HD088412//HHS | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; }, mesh = {Animals ; Male ; Mice ; *Multigene Family ; *CRISPR-Cas Systems ; *Infertility, Male/genetics ; Reproduction/genetics ; Mice, Knockout ; Fertility/genetics ; Humans ; Spermatogenesis/genetics ; Female ; Proteinase Inhibitory Proteins, Secretory/genetics/metabolism ; Gene Deletion ; Spermatozoa/metabolism ; Sperm Motility/genetics ; }, abstract = {Despite 96 million years of evolution separating humans and rodents, 11 closely related reproductive tract-specific genes in humans-SPINT3, WFDC6, EPPIN, WFDC8, WFDC9, WFDC10A, WFDC11, WFDC10B, WFDC13, SPINT4, and WFDC3-and the 13 reproductive tract-specific orthologous genes in mice, form highly conserved syntenic gene clusters indicative of conserved, combined critical functions. Further, despite significant progress toward a nonhormonal male contraceptive targeting the protein encoded by one of these genes, epididymal peptidase inhibitor (EPPIN), and associations found between mutations in EPPIN and an increased risk of male infertility, neither EPPIN nor any closely related whey acidic protein four-disulfide core (WFDC) gene have been explored functionally. To clarify the involvement of WFDC genes in male fertility, we strategically used CRISPR/Cas9 to generate mice lacking 13, 10, 5, or 4 genes within the cluster and demonstrated that males with deletions of 13, 10, or 4 genes (Wfdc6a, Eppin, Wfdc8, and Wfdc6a) were sterile due to an arrest in spermatogenesis, preventing formation beyond round spermatids. In contrast, the five gene knockout (KO) males (lacking Wfdc16, Wfdc9, Wfdc10, Wfdc11, and Wfdc13), despite normal spermatogenesis and sperm counts, were infertile due to defects in sperm motility and increased sperm death. Similarly to our previously reported Spint3 single gene KO, Wfdc3 single KO mice were fertile with no obvious reproductive phenotype. Our KO mouse studies to explore the entire WFDC locus of closely related genes have clarified the functional requirements of WFDC locus genes in different aspects of male fertility. Our research has implications for improving clinical diagnoses of male infertility and identifying additional targets for nonhormonal male contraception.}, }
@article {pmid39663988, year = {2025}, author = {Xiao, X and Zhang, C and Zhang, L and Zuo, C and Wu, W and Cheng, F and Wu, D and Xie, G and Mao, X and Yang, Y}, title = {A phage amplification-assisted SEA-CRISPR/Cas12a system for viable bacteria detection.}, journal = {Journal of materials chemistry. B}, volume = {13}, number = {4}, pages = {1372-1382}, doi = {10.1039/d4tb02178a}, pmid = {39663988}, issn = {2050-7518}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Bacteriophages/genetics ; Urinary Tract Infections/diagnosis/microbiology ; DNA, Viral/genetics ; }, abstract = {Rapid and accurate detection of viable bacteria is essential for the clinical diagnosis of urinary tract infections (UTIs) and for making effective therapeutic decisions. However, most current molecular diagnostic techniques are unable to differentiate between viable and non-viable bacteria. In this study, we introduce a novel isothermal platform that integrates strand exchange amplification (SEA) with the CRISPR/Cas12a system, thereby enhancing both the sensitivity and specificity of the assay and achieving detection of phage DNA at concentrations as low as 4 × 10[2] copies per μL. Moreover, the incorporation of phages facilitates the specific recognition of viable bacteria and amplifies the initial signal through the inherent specificity and propagation properties of these phages. By employing the phage-assisted SEA-Cas12a approach, we successfully detected viable bacteria in human urine samples without the necessity of DNA extraction within 3.5 hours, achieving a detection limit of 10[3] CFU per mL. Considering its speed, accuracy, and independence from specialized equipment, this platform demonstrates significant potential as a robust tool for the rapid detection of various pathogens in resource-limited settings, thereby facilitating timely clinical management of UTI patients.}, }
@article {pmid39663809, year = {2025}, author = {Tian, Q and Zhou, H and Zhao, Z and Zhang, Y and Zhao, W and Cai, L and Guo, T}, title = {Single and dual RPA-CRISPR/Cas assays for point-of-need detection of Stewart's wilt pathogen (Pantoea stewartii subsp. stewartii) of corn and Maize dwarf mosaic virus.}, journal = {Pest management science}, volume = {81}, number = {4}, pages = {1988-1999}, pmid = {39663809}, issn = {1526-4998}, support = {//Hainan Province Key Research and Development Program grant (ZDYF2024XDNY227)/ ; //the Chinese Academy of Inspection and Quarantine Basic Research program grant (2023JK020)/ ; }, mesh = {*Zea mays/virology/microbiology ; *Plant Diseases/virology/microbiology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Pantoea/isolation &amp; purification/genetics ; Molecular Diagnostic Techniques/methods ; }, abstract = {BACKGROUND: Pantoea stewartii subsp. stewartii and Maize dwarf mosaic virus (MDMV) infections severely affect corn productivity worldwide. Rapid point-of-need diagnoses of quarantine pathogens P. stewartii subsp. stewartii and MDMV are required for early detection, timely disease management and ensuring phytosanitary regulations. Recombinase polymerase amplification (RPA) is an isothermal technique suitable for rapid diagnostics using minimally processed samples. Integrating CRISPR/Cas collateral activities with the RPA assays further enhances the specificity and sensitivity of molecular toolkits for diagnostic assays.<br><br>RESULTS: RPA-CRISPR/Cas12a assay targeting the intergenic spacer region between capsular polysaccharide genes cpsA and cpsB of P. stewartii subsp. stewartii detected 1&#x2009;&#xd7;&#x2009;10[-6]&#x2009;ng DNA &#x3bc;L[-1] using real-time fluorescence and blue light observation methods, and 1&#x2009;&#xd7;&#x2009;10[-4]&#x2009;ng DNA &#x3bc;L[-1] using the lateral flow dipstick (LFD). Likewise, RPA-CRISPR/Cas13a assay detected MDMV coat protein (CP) gene with an ultrasensitive detection limit of 3.69&#x2009;&#xd7;&#x2009;10[-7]&#x2009;ng&#x2009;&#x3bc;L[-1] using the real-time fluorescence and blue light observation methods, and 3.69&#x2009;&#xd7;&#x2009;10[-5]&#x2009;ng&#x2009;&#x3bc;L[-1] using the LFD. The dual RPA-CRISPR/Cas assays detected both pathogens without compromising the speed and/or detection sensitivity of the single assays.<br><br>CONCLUSION: The validated assays provide a useful and sensitive molecular tool for detecting two quarantine pathogens of maize within a minimal resource framework suitable for fast-tracking the containment strategies. &#xa9; 2024 The Author(s). Pest Management Science published by John Wiley &amp; Sons Ltd on behalf of Society of Chemical Industry.}, }
@article {pmid39663454, year = {2025}, author = {Arce, MM and Umhoefer, JM and Arang, N and Kasinathan, S and Freimer, JW and Steinhart, Z and Shen, H and Pham, MTN and Ota, M and Wadhera, A and Dajani, R and Dorovskyi, D and Chen, YY and Liu, Q and Zhou, Y and Swaney, DL and Obernier, K and Shy, BR and Carnevale, J and Satpathy, AT and Krogan, NJ and Pritchard, JK and Marson, A}, title = {Central control of dynamic gene circuits governs T cell rest and activation.}, journal = {Nature}, volume = {637}, number = {8047}, pages = {930-939}, pmid = {39663454}, issn = {1476-4687}, support = {K08 CA273529/CA/NCI NIH HHS/United States ; R01 HG008140/HG/NHGRI NIH HHS/United States ; L30 TR002983/TR/NCATS NIH HHS/United States ; U54 CA274502/CA/NCI NIH HHS/United States ; S10 RR028962/RR/NCRR NIH HHS/United States ; T32 AR050942/AR/NIAMS NIH HHS/United States ; K08 CA252605/CA/NCI NIH HHS/United States ; S10 OD010786/OD/NIH HHS/United States ; U01 CA260852/CA/NCI NIH HHS/United States ; }, mesh = {Humans ; *CD4-Positive T-Lymphocytes/immunology/metabolism/cytology ; CRISPR-Cas Systems/genetics ; *Gene Expression Regulation ; *Gene Regulatory Networks/genetics ; Histones/metabolism ; *Lymphocyte Activation/genetics ; Mediator Complex/metabolism/genetics ; Methylation ; Proto-Oncogene Proteins c-myc/metabolism/genetics ; Single-Cell Analysis ; T-Lymphocytes/cytology/immunology ; T-Lymphocytes, Regulatory/immunology/metabolism/cytology ; }, abstract = {The ability of cells to maintain distinct identities and respond to transient environmental signals requires tightly controlled regulation of gene networks[1-3]. These dynamic regulatory circuits that respond to extracellular cues in primary human cells remain poorly defined. The need for context-dependent regulation is prominent in T cells, where distinct lineages must respond to diverse signals to mount effective immune responses and maintain homeostasis[4-8]. Here we performed CRISPR screens in multiple primary human CD4[+] T cell contexts to identify regulators that control expression of IL-2Rα, a canonical marker of T cell activation transiently expressed by pro-inflammatory effector T cells and constitutively expressed by anti-inflammatory regulatory T cells where it is required for fitness[9-11]. Approximately 90% of identified regulators of IL-2Rα had effects that varied across cell types and/or stimulation states, including a subset that even had opposite effects across conditions. Using single-cell transcriptomics after pooled perturbation of context-specific screen hits, we characterized specific factors as regulators of overall rest or activation and constructed state-specific regulatory networks. MED12 - a component of the Mediator complex - serves as a dynamic orchestrator of key regulators, controlling expression of distinct sets of regulators in different T cell contexts. Immunoprecipitation-mass spectrometry revealed that MED12 interacts with the histone methylating COMPASS complex. MED12 was required for histone methylation and expression of genes encoding key context-specific regulators, including the rest maintenance factor KLF2 and the versatile regulator MYC. CRISPR ablation of MED12 blunted the cell-state transitions between rest and activation and protected from activation-induced cell death. Overall, this work leverages CRISPR screens performed across conditions to define dynamic gene circuits required to establish resting and activated T cell states.}, }
@article {pmid39662607, year = {2025}, author = {Guo, F and Li, J and Ma, P and Liu, M and Wu, J and Qu, H and Zheng, Y and Wang, M and Marashi, SS and Zhang, Z and Zhang, S and Fu, G and Li, P}, title = {A magnetic bead-based dual-aptamer sandwich assay for quantitative detection of ciprofloxacin using CRISPR/Cas12a.}, journal = {Molecular and cellular probes}, volume = {79}, number = {}, pages = {101998}, doi = {10.1016/j.mcp.2024.101998}, pmid = {39662607}, issn = {1096-1194}, mesh = {*Aptamers, Nucleotide/chemistry ; *Ciprofloxacin/analysis ; Milk/chemistry ; Honey/analysis ; *CRISPR-Cas Systems/genetics ; Animals ; Limit of Detection ; *Biosensing Techniques/methods ; Rivers/chemistry ; }, abstract = {Ciprofloxacin (CIP) is a broad-spectrum fluoroquinolone antibiotic, and its excessive residues in food and water sources pose potential risks to human health. Therefore, there is a need for a rapid and convenient method for its accurate quantification. The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a system has gained extensive application in signal detection and amplification due to the trans-cleavage activity of Cas12a. In this study, we devised a novel magnetic bead-based dual sandwich aptamer coupled with a CRISPR/Cas12a system for the precise quantification of CIP in milk, river water, and honey. Through the incorporation of a magnetic bead-based dual aptamer sandwich approach, the concentration of CIP in the samples was pre-enriched. Additionally, by optimizing the Fluorescence-Quencher (F-Q) probe concentration, detection aptamer (APTd) concentration, and assay duration, the limit of blank (LOB) of the system was determined as 362 nM, while the limit of detection (LOD) was determined as 403 nM. This enabled the accurate quantification of CIP within the linear range of 0.5 μM to 0.2 mM with high specificity. Moreover, the performance of this detection method was comparable to that of high-performance liquid chromatography (HPLC) in river water, milk, and honey samples.}, }
@article {pmid39662170, year = {2025}, author = {Wang, M and Chen, W and Li, M and Lin, F and Zhong, J and Ouyang, W and Cai, C and Zeng, G and Liu, H}, title = {TE-RPA: One-tube telomerase extension recombinase polymerase amplification-based electrochemical biosensor for precise diagnosis of urothelial carcinoma.}, journal = {Biosensors &amp; bioelectronics}, volume = {271}, number = {}, pages = {117042}, doi = {10.1016/j.bios.2024.117042}, pmid = {39662170}, issn = {1873-4235}, mesh = {*Telomerase/genetics/urine ; *Biosensing Techniques/methods ; Humans ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; *Electrochemical Techniques/methods ; *Recombinases/chemistry/metabolism ; Limit of Detection ; CRISPR-Cas Systems ; Urinary Bladder Neoplasms/urine/diagnosis/genetics ; Biomarkers, Tumor/urine/genetics ; Urologic Neoplasms/diagnosis/genetics/urine ; }, abstract = {Telomerase demonstrates potential as a non-invasive urinary biomarker for urothelial carcinoma (UC); however, current detection methods are either labor-intensive or exhibit suboptimal performance. There is a need for alternative approaches to enable rapid and early diagnosis of UC. In this study, we propose TE-RPA, which combines telomerase extension (TE) with recombinase polymerase amplification (RPA) for one-tube isothermal amplification. The GC content and length of the telomerase substrate were first considered during the screening process. TE-RPA exponential amplification was initiated by the addition of MgOAc along with a forward primer derived from the products of telomerase-mediated extension and a corresponding reverse primer. The amplification product from TE-RPA was subsequently detected using CRISPR-Cas12a system for trans-cleavage of signal probes on the surface of screen-printed electrode in an electrochemical biosensor, resulting in a current change that reflects the corresponding concentration of telomerase. The TE-RPA/CRISPR-Cas12a/electrochemical sensing platform achieves a limit of detection (LOD) for telomerase activity as low as a single-cell level. In addition, the platform attained an area under the curve (AUC) value of 0.9589 in a clinical evaluation involving urine samples from 43 suspected UC patients. Overall, our proposed platform not only offers an efficient method for telomerase isothermal amplification but also provides a portable and precise diagnostic tool for UC.}, }
@article {pmid39661735, year = {2025}, author = {Baysal, C and Kausch, AP and Cody, JP and Altpeter, F and Voytas, DF}, title = {Rapid and efficient in planta genome editing in sorghum using foxtail mosaic virus-mediated sgRNA delivery.}, journal = {The Plant journal : for cell and molecular biology}, volume = {121}, number = {2}, pages = {e17196}, pmid = {39661735}, issn = {1365-313X}, support = {DE-SC0018420//Biological and Environmental Research/ ; }, mesh = {*Gene Editing/methods ; *Sorghum/genetics/virology ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Potexvirus/genetics ; CRISPR-Cas Systems/genetics ; Genetic Vectors/genetics ; Genome, Plant/genetics ; Green Fluorescent Proteins/genetics/metabolism ; Plant Viruses ; }, abstract = {The requirement of in vitro tissue culture for the delivery of gene editing reagents limits the application of gene editing to commercially relevant varieties of many crop species. To overcome this bottleneck, plant RNA viruses have been deployed as versatile tools for in planta delivery of recombinant RNA. Viral delivery of single-guide RNAs (sgRNAs) to transgenic plants that stably express CRISPR-associated (Cas) endonuclease has been successfully used for targeted mutagenesis in several dicotyledonous and few monocotyledonous plants. Progress with this approach in monocotyledonous plants is limited so far by the availability of effective viral vectors. We engineered a set of foxtail mosaic virus (FoMV) and barley stripe mosaic virus (BSMV) vectors to deliver the fluorescent protein AmCyan to track viral infection and movement in Sorghum bicolor. We further used these viruses to deliver and express sgRNAs to Cas9 and Green Fluorescent Protein (GFP) expressing transgenic sorghum lines, targeting Phytoene desaturase (PDS), Magnesium-chelatase subunit I (MgCh), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, orthologs of maize Lemon white1 (Lw1) or GFP. The recombinant BSMV did neither infect sorghum nor deliver or express AmCyan and sgRNAs. In contrast, the recombinant FoMV systemically spread throughout sorghum plants and induced somatic mutations with frequencies reaching up to 60%. This mutagenesis led to visible phenotypic changes, demonstrating the potential of FoMV for in planta gene editing and functional genomics studies in sorghum.}, }
@article {pmid39661534, year = {2025}, author = {Dhinoja, S and Mary, J and Qaryoute, AA and De Maria, A and Jagadeeswaran, P}, title = {Generation and characterization of zebrafish f9l mutant confirmed that f9l is f10 like gene.}, journal = {Blood coagulation &amp; fibrinolysis : an international journal in haemostasis and thrombosis}, volume = {36}, number = {1}, pages = {26-33}, pmid = {39661534}, issn = {1473-5733}, support = {R01 HL159399/HL/NHLBI NIH HHS/United States ; R15 DK117384/DK/NIDDK NIH HHS/United States ; }, mesh = {Animals ; *Zebrafish/genetics ; CRISPR-Cas Systems ; *Zebrafish Proteins/genetics ; Mutation ; Humans ; }, abstract = {AIM: This study aimed to create an f9l mutant zebrafish using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and characterize its coagulation properties to investigate its functional similarity to human FX and explore the potential synergy between f9l and f10 .<br><br>METHODS: Three gRNAs targeting exon 8 encoded by the catalytic domain of the f9l gene were injected into 300 single-cell zebrafish embryos using CRISPR/Cas9 technology. DNA from the resulting adults was extracted from tail tips, and PCR was used to detect indels. The identified founder mutant was bred to homozygosity, and functional assays, kinetic Russel viper venom time, bleeding assay in adults, and venous laser injury on larvae were conducted to assess its hemostatic function. Additionally, f10 was knocked down in f9l homozygous embryos using f10 antisense morpholinos to study their interaction by monitoring its survival.<br><br>RESULTS: DNA from 60 adults was screened for indels, resulting in a fish with a heritable complex mutation involving one insertion and two deletions in exon 8. The f9l homozygous mutants exhibited impaired F10 activity, mild bleeding after mechanical injury, and developmental deformities in early larval stages. The caudal vein thrombosis assay showed variable occlusion times, indicating a bleeding phenotype with incomplete penetrance. Knocking down f10 in f9l homozygous embryos resulted in 50% mortality within five dpf, compared to f9l homozygous embryos injected with control morpholinos.<br><br>CONCLUSION: In summary, we generated f9l knockout and showed it is a paralog to f10. We also found a synergy between f9l and f10 genes, highlighting its importance in hemostasis.}, }
@article {pmid39661519, year = {2024}, author = {Pan, Q and Zhang, Z and Xiong, Y and Bao, Y and Chen, T and Xu, P and Liu, Z and Ma, H and Yu, Y and Zhou, Z and Wei, W}, title = {Mapping functional elements of the DNA damage response through base editor screens.}, journal = {Cell reports}, volume = {43}, number = {12}, pages = {115047}, doi = {10.1016/j.celrep.2024.115047}, pmid = {39661519}, issn = {2211-1247}, mesh = {*DNA Damage ; Humans ; *DNA Repair ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Protein Serine-Threonine Kinases/metabolism/genetics ; Cisplatin/pharmacology ; Mutation/genetics ; Replication Protein A/metabolism/genetics ; HEK293 Cells ; }, abstract = {Maintaining genomic stability is vital for cellular equilibrium. In this study, we combined CRISPR-mediated base editing with pooled screening technologies to identify numerous mutations in lysine residues and protein-coding genes. The loss of these lysine residues and genes resulted in either sensitivity or resistance to DNA-damaging agents. Among the identified variants, we characterized both loss-of-function and gain-of-function mutations in response to DNA damage. Notably, we discovered that the K494 mutation of C17orf53 disrupts its interaction with RPA proteins, leading to increased sensitivity to cisplatin. Additionally, our analysis identified STK35 as a previously unrecognized gene involved in DNA damage response (DDR) pathways, suggesting that it may play a critical role in DNA repair. We believe that this resource will offer valuable insights into the broader functions of DNA damage response genes and accelerate research on variants relevant to cancer therapy.}, }
@article {pmid39661507, year = {2024}, author = {Ordóñez, A and Ron, D and Harding, HP}, title = {Protocol for iterative enrichment of integrated sgRNAs via derivative CRISPR-Cas9 libraries from genomic DNA of sorted fixed cells.}, journal = {STAR protocols}, volume = {5}, number = {4}, pages = {103493}, pmid = {39661507}, issn = {2666-1667}, support = {/WT_/Wellcome Trust/United Kingdom ; }, mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; DNA/genetics ; Gene Library ; Lentivirus/genetics ; Gene Editing/methods ; }, abstract = {Here, we present a protocol for iterative enrichment of integrated single guide RNA (sgRNA) via derivative CRISPR-Cas9 from genomic DNA (gDNA) of phenotypically sorted fixed cells. We describe steps for high-scale lentiviral production, genome-wide screening, extracting gDNA from fixed cells, cloning of integrated sgRNAs, and high-scale transformation. This protocol introduces three key advantages: (1) applicability to fixed cells, (2) bypassing epigenetic drift, and (3) pause points lowering the contamination risk. We believe this approach will benefit researchers applying somatic cell genetics in cell biology. For complete details on the use and execution of this protocol, please refer to Ordoñez et al.[1].}, }
@article {pmid39660762, year = {2024}, author = {Zeng, T and Wu, Q and Liu, Y and Qi, Q and Shen, W and Gu, W and Zhang, Y and Xiong, W and Xie, Z and Qi, X and Tian, T and Zhou, X}, title = {Unraveling the Cleavage Reaction of Hydroxylamines with Cyclopropenones Considering Biocompatibility.}, journal = {Journal of the American Chemical Society}, volume = {146}, number = {51}, pages = {35077-35089}, doi = {10.1021/jacs.4c09757}, pmid = {39660762}, issn = {1520-5126}, mesh = {*Cyclopropanes/chemistry/pharmacology ; Humans ; HeLa Cells ; *Hydroxylamines/chemistry ; Biocompatible Materials/chemistry/pharmacology/chemical synthesis ; Density Functional Theory ; Molecular Structure ; CRISPR-Cas Systems ; Prodrugs/chemistry/pharmacology/chemical synthesis ; }, abstract = {We develop a latent biocompatible cleavage reaction involving the hitherto unexplored interaction between hydroxylamines and cyclopropenones. Our study addresses the regioselectivity challenges commonly observed in asymmetric cyclopropenone transformations, substantiated by variations in substrate, Density Functional Theory calculations, and in situ NMR analysis. This reaction is characterized by high efficiency, broad substrate scope, stability, latent biocompatibility, and mild reaction conditions. Significantly, it facilitates fluorescence activation and functions as a controlled release mechanism for prodrugs, showing great promise in biological assays. Our success in achieving the controlled release of nitrogen mustard in HeLa cells underscores its potential application in cellular contexts. Additionally, we introduce a simple and highly efficient method for synthesizing α, β-substituted pentenolides, applicable to a variety of substrates. Moreover, we extend this cleavage reaction to the CRISPR-Cas9 system, achieving precise, on-demand regulation of guide RNA activity. The introduction of this cleavage reaction offers a promising tool for biochemical research and biotechnological applications.}, }
@article {pmid39660638, year = {2025}, author = {Goh, CG and Bader, AS and Tran, TA and Belotserkovskaya, R and D'Alessandro, G and Jackson, SP}, title = {TDP1 splice-site mutation causes HAP1 cell hypersensitivity to topoisomerase I inhibition.}, journal = {Nucleic acids research}, volume = {53}, number = {2}, pages = {}, pmid = {39660638}, issn = {1362-4962}, support = {227014/Z/23/Z//Wellcome/ ; C17918/A28870//CRUK RadNet Cambridge Award/ ; //A*STAR National Science Scholarship/ ; //University of Cambridge/ ; /WT_/Wellcome Trust/United Kingdom ; DRCPGM\100005/CRUK_/Cancer Research UK/United Kingdom ; 855741//ERC Synergy Award/ ; }, mesh = {Humans ; *Phosphoric Diester Hydrolases/genetics/deficiency ; *Topoisomerase I Inhibitors/pharmacology ; Camptothecin/pharmacology ; *DNA Topoisomerases, Type I/metabolism/genetics ; *RNA Splice Sites/genetics ; Mutation ; Cell Line ; CRISPR-Cas Systems ; Gene Editing ; Exons ; }, abstract = {HAP1 is a near-haploid human cell line commonly used for mutagenesis and genome editing studies due to its hemizygous nature. We noticed an unusual hypersensitivity of HAP1 to camptothecin, an antineoplastic drug that stabilizes topoisomerase I cleavage complexes (TOP1ccs). We have attributed this hypersensitivity to a deficiency of TDP1, a key phosphodiesterase involved in resolving abortive TOP1ccs. Through whole-exome sequencing and subsequent restoration of TDP1 protein via CRISPR-Cas9 endogenous genome editing, we demonstrate that TDP1 deficiency and camptothecin hypersensitivity in HAP1 cells are a result of a splice-site mutation (TDP1 c.660-1G > A) that causes exon skipping and TDP1 loss of function. The lack of TDP1 in HAP1 cells should be considered when studying topoisomerase-associated DNA lesions and when generalizing mechanisms of DNA damage repair using HAP1 cells. Finally, we also report the generation of HAP1 STAR clones with restored TDP1 expression and function, which may be useful in further studies to probe cellular phenotypes relating to TOP1cc repair.}, }
@article {pmid39658572, year = {2024}, author = {Adachi, Y and Terakura, S and Osaki, M and Okuno, Y and Sato, Y and Sagou, K and Takeuchi, Y and Yokota, H and Imai, K and Steinberger, P and Leitner, J and Hanajiri, R and Murata, M and Kiyoi, H}, title = {Cullin-5 deficiency promotes chimeric antigen receptor T cell effector functions potentially via the modulation of JAK/STAT signaling pathway.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10376}, pmid = {39658572}, issn = {2041-1723}, mesh = {*Cullin Proteins/metabolism/genetics ; Humans ; Animals ; *Receptors, Chimeric Antigen/metabolism/immunology ; *Signal Transduction ; Mice ; *T-Lymphocytes/immunology/metabolism ; Antigens, CD19/metabolism/immunology ; Immunotherapy, Adoptive/methods ; Phosphorylation ; Cell Line, Tumor ; Cell Proliferation ; STAT3 Transcription Factor/metabolism ; CRISPR-Cas Systems ; Janus Kinases/metabolism ; STAT5 Transcription Factor/metabolism/genetics ; Xenograft Model Antitumor Assays ; STAT Transcription Factors/metabolism ; }, abstract = {Chimeric antigen receptor (CAR) T cell is a promising therapy for cancer, but factors that enhance the efficacy of CAR T cell remain elusive. Here we perform a genome-wide CRISPR screening to probe genes that regulate the proliferation and survival of CAR T cells following repetitive antigen stimulations. We find that genetic ablation of CUL5, encoding a core element of the multi-protein E3 ubiquitin-protein ligase complex, cullin-RING ligase 5, enhances human CD19 CAR T cell expansion potential and effector functions, potentially via the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway. In this regard, CUL5 knockout CD19 CAR T cells show sustained STAT3 and STAT5 phosphorylation, as well as delayed phosphorylation and degradation of JAK1 and JAK3. In vivo, shRNA-mediated knockdown of CUL5 enhances CD19 CAR T treatment outcomes in tumor-bearing mice. Our findings thus imply that targeting CUL5 in the ubiquitin system may enhance CAR T cell effector functions to enhance immunotherapy efficacy.}, }
@article {pmid39658506, year = {2024}, author = {Shen, H and Weng, Z and Zhao, H and Song, H and Wang, F and Fan, C and Song, P}, title = {Random Sanitization in DNA Information Storage Using CRISPR-Cas12a.}, journal = {Journal of the American Chemical Society}, volume = {146}, number = {51}, pages = {35155-35164}, doi = {10.1021/jacs.4c11380}, pmid = {39658506}, issn = {1520-5126}, mesh = {*CRISPR-Cas Systems/genetics ; DNA/chemistry ; Information Storage and Retrieval ; DNA, Single-Stranded/chemistry ; Thermodynamics ; }, abstract = {DNA information storage provides an excellent solution for metadata storage due to its high density, programmability, and long-term stability. However, current research primarily focuses on the processes of storing and reading data, lacking comprehensive solutions for secure metadata wiping. Herein, we present a method of random sanitization in DNA information storage using CRISPR-Cas12a (RSDISC) based on precise control of the thermodynamic energy of primer-template hybridization. We utilize the collateral cleavage (trans-activity) of single-stranded DNA (ssDNA) by CRISPR-Cas12a to achieve selective sanitization of files in metadata. This method enables ssDNA degradation with different GC contents, lengths, and secondary structures to achieve a sanitization efficiency up to 99.9% for 28,258 oligonucleotides in DNA storage within one round. We demonstrate that the number of erasable files could reach 10[12] based on a model of primer-template hybridization efficiency. Overall, RSDISC provides a random sanitization approach to set the foundation of information encryption, file classification, memory deallocation, and accurate reading in DNA storage.}, }
@article {pmid39658085, year = {2024}, author = {Hak, H and Ostendorp, S and Reza, A and Ishgur Greenberg, S and Pines, G and Kehr, J and Spiegelman, Z}, title = {Rapid on-site detection of crop RNA viruses using CRISPR/Cas13a.}, journal = {Journal of experimental botany}, volume = {}, number = {}, pages = {}, doi = {10.1093/jxb/erae495}, pmid = {39658085}, issn = {1460-2431}, abstract = {Plant viruses are destructive pathogens for various crop species. Rapid, sensitive, and specific detection is crucial for the effective containment of emerging and resistance-breaking viruses. CRISPR/Cas has been established as a new tool for plant virus identification. However, its application for direct detection of viruses in the field is still limited. In this study, we present a CRISPR/Cas13a-based method for rapid detection of different viruses directly from RNA of several crop species, including tomato, cucumber and rapeseed. This method was used to identify the emerging tomato brown rugose fruit virus (ToBRFV), a prominent pathogen in tomato cultivation, and distinguish it from closely related viruses in infected tomato plants. ToBRFV could be identified in a 100-fold dilution and early during infection, prior to the onset of viral symptoms. Finally, we developed a user-friendly, extraction-free, 15-minute protocol for on-site virus detection using a portable fluorescent viewer and a mobile phone camera. This protocol was successfully applied for ToBRFV identification in several commercial greenhouses. These results demonstrate that CRISPR/Cas13a is a robust technology for on-site detection of multiple viruses in different crop plants. This method could be swiftly adapted to identify newly emerging pests, which threaten global food security.}, }
@article {pmid39658047, year = {2025}, author = {Crawford, KD and Khan, AG and Lopez, SC and Goodarzi, H and Shipman, SL}, title = {High throughput variant libraries and machine learning yield design rules for retron gene editors.}, journal = {Nucleic acids research}, volume = {53}, number = {2}, pages = {}, pmid = {39658047}, issn = {1362-4962}, support = {R21 EB031393/EB/NIBIB NIH HHS/United States ; //W. M. Keck Foundation/ ; //UCSF Discovery Fellows Program/ ; //Pew Biomedical Scholars Program/ ; MCB 2137692//National Science Foundation/ ; R21EB031393/EB/NIBIB NIH HHS/United States ; //Gary and Eileen Morgenthaler Fund/ ; }, mesh = {Saccharomyces cerevisiae/genetics ; Gene Library ; *Machine Learning ; *Gene Editing/methods ; CRISPR-Cas Systems ; RNA, Untranslated/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {The bacterial retron reverse transcriptase system has served as an intracellular factory for single-stranded DNA in many biotechnological applications. In these technologies, a natural retron non-coding RNA (ncRNA) is modified to encode a template for the production of custom DNA sequences by reverse transcription. The efficiency of reverse transcription is a major limiting step for retron technologies, but we lack systematic knowledge of how to improve or maintain reverse transcription efficiency while changing the retron sequence for custom DNA production. Here, we test thousands of different modifications to the Retron-Eco1 ncRNA and measure DNA production in pooled variant library experiments, identifying regions of the ncRNA that are tolerant and intolerant to modification. We apply this new information to a specific application: the use of the retron to produce a precise genome editing donor in combination with a CRISPR-Cas9 RNA-guided nuclease (an editron). We use high-throughput libraries in Saccharomyces cerevisiae to additionally define design rules for editrons. We extend our new knowledge of retron DNA production and editron design rules to human genome editing to achieve the highest efficiency Retron-Eco1 editrons to date.}, }
@article {pmid39657918, year = {2025}, author = {Li, X and Zhang, S and Wang, C and Ren, B and Yan, F and Li, S and Spetz, C and Huang, J and Zhou, X and Zhou, H}, title = {Efficient in situ epitope tagging of rice genes by nuclease-mediated prime editing.}, journal = {The Plant cell}, volume = {37}, number = {2}, pages = {}, pmid = {39657918}, issn = {1532-298X}, support = {2023ZD04074//Biological Breeding-Major Projects/ ; 2023YFD1202905//National Key Research and Development Program of China/ ; YBXM2313//Nanfan special project of the Chinese Academy of Agricultural Sciences/ ; B23CJ0208//Hainan Seed Industry Laboratory/ ; //Agricultural Science and Technology Innovation Program/ ; }, mesh = {*Oryza/genetics ; *Gene Editing/methods ; *Epitopes/genetics ; *CRISPR-Cas Systems ; Plants, Genetically Modified ; Genes, Plant ; Plant Proteins/genetics/metabolism ; }, abstract = {In situ epitope tagging is crucial for probing gene expression, protein localization, and the dynamics of protein interactions within their natural cellular context. However, the practical application of this technique in plants presents considerable hurdles. Here, we comprehensively explored the potential of the CRISPR/Cas nuclease-mediated prime editing and different DNA repair pathways in epitope tagging of endogenous rice (Oryza sativa) genes. We found that a SpCas9 nuclease/microhomology-mediated end joining (MMEJ)-based prime editing (PE) strategy (termed NM-PE) facilitates more straightforward and efficient gene tagging compared to the conventional and other derivative PE methods. Furthermore, the PAM-flexible SpRY and ScCas9 nucleases-based prime editors have been engineered and implemented for the tagging of endogenous genes with diverse epitopes, significantly broadening the applicability of NM-PE in rice. Moreover, NM-PE has been successfully adopted in simultaneous tagging of the MAP kinase (MPK) genes OsMPK1 and OsMPK13 in rice plants with c-Myc and HA tags, respectively. Taken together, our results indicate great potential of the NM-PE toolkit in the targeted gene tagging for Rice Protein Tagging Project, gene function study and genetic improvement.}, }
@article {pmid39657782, year = {2025}, author = {Li, Z and Wang, X and Janssen, JM and Liu, J and Tasca, F and Hoeben, RC and Gonçalves, MAFV}, title = {Precision genome editing using combinatorial viral vector delivery of CRISPR-Cas9 nucleases and donor DNA constructs.}, journal = {Nucleic acids research}, volume = {53}, number = {2}, pages = {}, pmid = {39657782}, issn = {1362-4962}, support = {//China Scholarship Council/ ; //Prinses Beatrix Spierfonds/ ; //Dutch Duchenne Parent/ ; //Horizon Europe/ ; /NWO_/Dutch Research Council/Netherlands ; }, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Vectors/genetics ; Humans ; Dependovirus/genetics ; HEK293 Cells ; Adenoviridae/genetics ; *DNA/genetics/metabolism ; Homologous Recombination ; Transgenes ; }, abstract = {Genome editing based on programmable nucleases and donor DNA constructs permits introducing specific base-pair changes and complete transgenes or live-cell reporter tags at predefined chromosomal positions. A crucial requirement for such versatile genome editing approaches is, however, the need to co-deliver in an effective, coordinated and non-cytotoxic manner all the required components into target cells. Here, adenoviral (AdV) and adeno-associated viral (AAV) vectors are investigated as delivery agents for, respectively, engineered CRISPR-Cas9 nucleases and donor DNA constructs prone to homologous recombination (HR) or homology-mediated end joining (HMEJ) processes. Specifically, canonical single-stranded and self-complementary double-stranded AAVs served as sources of ectopic HR and HMEJ substrates, whilst second- and third-generation AdVs provided for matched CRISPR-Cas9 nucleases. We report that combining single-stranded AAV delivery of HR donors with third-generation AdV transfer of CRISPR-Cas9 nucleases results in selection-free and precise whole transgene insertion in large fractions of target-cell populations (i.e. up to 93%) and disclose that programmable nuclease-induced chromosomal breaks promote AAV transduction. Finally, besides investigating relationships between distinct AAV structures and genome-editing performance endpoints, we further report that high-fidelity CRISPR-Cas9 nucleases are critical for mitigating off-target chromosomal insertion of defective AAV genomes known to be packaged in vector particles.}, }
@article {pmid39657754, year = {2025}, author = {Kiernan, KA and Kwon, J and Merrill, BJ and Simonović, M}, title = {Structural basis of Cas9 DNA interrogation with a 5' truncated sgRNA.}, journal = {Nucleic acids research}, volume = {53}, number = {1}, pages = {}, pmid = {39657754}, issn = {1362-4962}, support = {75N91019D00024/CA/NCI NIH HHS/United States ; R01 GM139894/GM/NIGMS NIH HHS/United States ; R35 GM138348/GM/NIGMS NIH HHS/United States ; F31 GM143822/GM/NIGMS NIH HHS/United States ; F31GM143822/GM/NIGMS NIH HHS/United States ; R01 GM097042/GM/NIGMS NIH HHS/United States ; }, mesh = {*RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; *DNA/chemistry/metabolism/genetics ; *CRISPR-Associated Protein 9/chemistry/metabolism/genetics ; DNA Cleavage ; Cryoelectron Microscopy ; Models, Molecular ; Nucleic Acid Conformation ; Plasmids/genetics/metabolism ; }, abstract = {The efficiency and accuracy of CRISPR-Cas9 targeting varies considerably across genomic targets and remains a persistent issue for using this system in cells. Studies have shown that the use of 5' truncated single guide RNAs (sgRNAs) can reduce the rate of unwanted off-target recognition while still maintaining on-target specificity. However, it is not well-understood how reducing target complementarity enhances specificity or how truncation past 15 nucleotides (nts) prevents full Cas9 activation without compromising on-target binding. Here, we use biochemistry and cryogenic electron microscopy to investigate Cas9 structure and activity when bound to a 14-nt sgRNA. Our structures reveal that the shortened path of the displaced non-target strand (NTS) sterically occludes docking of the HNH L1 linker and prevents proper positioning of the nuclease domains. We show that cleavage inhibition can be alleviated by either artificially melting the protospacer adjacent motif (PAM)-distal duplex or providing a supercoiled substrate. Even though Cas9 forms a stable complex with its target, we find that plasmid cleavage is ∼1000-fold slower with a 14-nt sgRNA than with a full-length 20-nt sgRNA. Our results provide a structural basis for Cas9 target binding with 5' truncated sgRNAs and underline the importance of PAM-distal NTS availability in promoting Cas9 activation.}, }
@article {pmid39657724, year = {2025}, author = {Rasoul, A and Johnston, CR and LaChance, J and Sedbrook, JC and Alonso, AP}, title = {Propelling sustainable energy: Multi-omics analysis of pennycress FATTY ACID ELONGATION1 knockout for biofuel production.}, journal = {Plant physiology}, volume = {197}, number = {2}, pages = {}, pmid = {39657724}, issn = {1532-2548}, support = {//Department of Energy Office of Science/ ; #DE-SC0020325//Office of Biological and Environmental Research/ ; }, mesh = {*Biofuels ; Fatty Acids/metabolism ; Seeds/metabolism/genetics ; *Fatty Acid Elongases/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Plant Oils/metabolism ; Gene Knockout Techniques ; Gene Editing ; Multiomics ; }, abstract = {The aviation industry's growing interest in renewable jet fuel has encouraged the exploration of alternative oilseed crops. Replacing traditional fossil fuels with a sustainable, domestically sourced crop can substantially reduce carbon emissions, thus mitigating global climate instability. Pennycress (Thlaspi arvense L.) is an emerging oilseed intermediate crop that can be grown during the offseason between maize (Zea mays) and soybean (Glycine max) to produce renewable biofuel. Pennycress is being domesticated through breeding and mutagenesis, providing opportunities for trait enhancement. Here, we employed metabolic engineering strategies to improve seed oil composition and bolster the plant's economic competitiveness. FATTY ACID ELONGATION1 (FAE1) was targeted using CRISPR-Cas 9 gene editing to eliminate very long chain fatty acids (VLCFAs) from pennycress seed oil, thereby enhancing its cold flow properties. Through an integrated multiomics approach, we investigated the impact of eliminating VLCFAs in developing and mature plant embryos. Our findings revealed improved cold-germination efficiency in fae1, with seedling emergence occurring up to 3 d earlier at 10 °C. However, these alterations led to a tradeoff between storage oil content and composition. Additionally, these shifts in lipid biosynthesis were accompanied by broad metabolic changes, such as the accumulation of glucose and ADP-glucose quantities consistent with increased starch production. Furthermore, shifts to shorter FA chains triggered the upregulation of heat shock proteins, underscoring the importance of VLCFAs in stress signaling pathways. Overall, this research provides crucial insights for optimizing pennycress seed oil while preserving essential traits for biofuel applications.}, }
@article {pmid39656538, year = {2024}, author = {Zhang, Y and David, NL and Pesaresi, T and Andrews, RE and Kumar, GVN and Chen, H and Qiao, W and Yang, J and Patel, K and Amorim, T and Sharma, AX and Liu, S and Steinhauser, ML}, title = {Noncoding variation near UBE2E2 orchestrates cardiometabolic pathophenotypes through polygenic effectors.}, journal = {JCI insight}, volume = {10}, number = {2}, pages = {}, pmid = {39656538}, issn = {2379-3708}, support = {R01 DK120659/DK/NIDDK NIH HHS/United States ; T32 DK007052/DK/NIDDK NIH HHS/United States ; }, mesh = {Animals ; *Ubiquitin-Conjugating Enzymes/genetics/metabolism ; Mice ; *Diabetes Mellitus, Type 2/genetics/metabolism ; *Obesity/genetics ; Genome-Wide Association Study ; Humans ; Polymorphism, Single Nucleotide ; Adipogenesis/genetics ; Male ; Multifactorial Inheritance ; Mice, Inbred C57BL ; Female ; CRISPR-Cas Systems ; }, abstract = {Mechanisms underpinning signals from genome-wide association studies remain poorly understood, particularly for noncoding variation and for complex diseases such as type 2 diabetes mellitus (T2D) where pathogenic mechanisms in multiple different tissues may be disease driving. One approach is to study relevant endophenotypes, a strategy we applied to the UBE2E2 locus where noncoding single nucleotide variants (SNVs) are associated with both T2D and visceral adiposity (a pathologic endophenotype). We integrated CRISPR targeting of SNV-containing regions and unbiased CRISPR interference (CRISPRi) screening to establish candidate cis-regulatory regions, complemented by genetic loss of function in murine diet-induced obesity or ex vivo adipogenesis assays. Nomination of a single causal gene was complicated, however, because targeting of multiple genes near UBE2E2 attenuated adipogenesis in vitro; CRISPR excision of SNV-containing noncoding regions and a CRISPRi regulatory screen across the locus suggested concomitant regulation of UBE2E2, the more distant UBE2E1, and other neighborhood genes; and compound heterozygous loss of function of both Ube2e2 and Ube2e1 better replicated pathological adiposity and metabolic phenotypes compared with homozygous loss of either gene in isolation. This study advances a model whereby regulatory effects of noncoding variation not only extend beyond the nearest gene but may also drive complex diseases through polygenic regulatory effects.}, }
@article {pmid39656015, year = {2025}, author = {Hernandez-Rodriguez, Y and Bullard, AM and Busch, RJ and Marshall, A and Vargas-Muñiz, JM}, title = {Strategies for genetic manipulation of the halotolerant black yeast Hortaea werneckii: ectopic DNA integration and marker-free CRISPR/Cas9 transformation.}, journal = {Microbiology spectrum}, volume = {13}, number = {1}, pages = {e0243024}, pmid = {39656015}, issn = {2165-0497}, mesh = {*CRISPR-Cas Systems ; *Transformation, Genetic ; *Gene Editing/methods ; Ascomycota/genetics/metabolism ; DNA, Fungal/genetics ; }, abstract = {Hortaea werneckii is a halotolerant black yeast commonly found in hypersaline environments. This yeast is also the causative agent of tinea nigra, a superficial mycosis of the palm of the hand and soles of the feet of humans. In addition to their remarkable halotolerance, this black yeast exhibits an unconventional cell division cycle, alternating between fission and budding cell division. Cell density and the salt concentration in their environment regulate which cell division cycle H. werneckii uses. Although H. werneckii have been extensively studied due to their unique physiology and cell biology, deciphering the underlying mechanisms behind these remarkable phenotypes has been limited due to the lack of genetic tools available. Here, we report a new ectopic integration protocol for H. werneckii using polyethylene glycol-CaCl2 mediated protoplast transformation. This approach relies on a drug (hygromycin B) resistance gene to select for successful integration of the genetic construct. The same construct was used to express cytosolic green fluorescent protein. Finally, we developed a marker-free CRISPR/Cas9 protocol for targeted gene deletion using the melanin synthesis pathway as a visual reporter of successful transformation. These transformation strategies will allow testing hypotheses related to H. werneckii cell biology and physiology.IMPORTANCEHortaea werneckii is a remarkable yeast capable of growing in high salt concentration, and its cell division cycle alternates between fission-like and budding. For these unique attributes, H. werneckii has gathered interest in research programs studying extremophile fungi and cell division. Most of our understanding of H. werneckii biology comes from genomic analyses, the usage of drugs to target a particular pathway, or the heterologous expression of its genes in S. cerevisiae. Nonetheless, H. werneckii has remained genetically intractable. Here, we report on two strategies to transform H. werneckii: ectopic integration of a plasmid and gene deletion using CRISPR/Cas9. These approaches will be fundamental to expanding the experimental techniques available to study H. werneckii, including live-cell imaging of cellular processes and reverse genetic approaches.}, }
@article {pmid39655997, year = {2025}, author = {Li, M and Zheng, T and Zhu, J and Zhang, H and Fan, L}, title = {Cas12a/crRNA recognition initiated self-priming mediated chain extension for colorimetric cell-free DNA (cfDNA) analysis.}, journal = {The Analyst}, volume = {150}, number = {2}, pages = {258-263}, doi = {10.1039/d4an01432d}, pmid = {39655997}, issn = {1364-5528}, mesh = {Humans ; *Colorimetry/methods ; *Cell-Free Nucleic Acids/analysis/blood/genetics ; CRISPR-Cas Systems ; Limit of Detection ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; Diphosphates/chemistry ; Biosensing Techniques/methods ; Bacterial Proteins ; }, abstract = {Cell-free DNA (cfDNA) has attracted increasing attention as a promising biomarker in liquid biopsy due to its crucial role in disease diagnosis. However, previous cfDNA detection methods are commonly based on the development of target-specific primers and integrated signal amplification strategies, which may induce false-positive results. This paper presents a sensitive yet accurate method for cfDNA detection that combines phosphorothioated-terminal hairpin creation with a self-priming extension process. This approach initiates a self-priming mediated chain extension-based signal cycle following the trans-cleavage of H0@MBs when the CRISPR-Cas12a complex is activated by target cfDNA, resulting in the production of a substantial quantity of pyrophosphate. A pyrophosphate sensing probe (pp probe) was utilized, facilitating both high-efficiency and stable colorimetric signaling. This innovative technique for colorimetric detection of target cfDNA demonstrated exceptional sensitivity with a low limit of detection of 1.04 fM and greatly enhanced selectivity, with the complete detection process taking around 60 min. In addition, this technique is capable of detecting cfDNA from the culture medium of HEK293 cells, indicating its clinical application potential. Compared with the previous CRISPR-Cas system-based cfDNA method that necessitates an amplification step before detection, Cas12a was directly used to identify a target sequence that can avoid false target amplification. This technique is simple, accurate, and rapid, engineered to identify cancer-associated cfDNA via a highly sensitive colorimetric change, which is expected to be beneficial for applications requiring point-of-care cancer detection.}, }
@article {pmid39655603, year = {2024}, author = {Wu, S and Yang, Y and Lian, X and Zhang, F and Hu, C and Tsien, J and Chen, Z and Sun, Y and Vaidya, A and Kim, M and Sung, YC and Xiao, Y and Bian, X and Wang, X and Tian, Z and Guerrero, E and Robinson, J and Basak, P and Qin, T and Siegwart, DJ}, title = {Isosteric 3D Bicyclo[1.1.1]Pentane (BCP) Core-Based Lipids for mRNA Delivery and CRISPR/Cas Gene Editing.}, journal = {Journal of the American Chemical Society}, volume = {146}, number = {50}, pages = {34733-34742}, pmid = {39655603}, issn = {1520-5126}, support = {P30 CA142543/CA/NCI NIH HHS/United States ; R01 CA248736/CA/NCI NIH HHS/United States ; R01 CA269787/CA/NCI NIH HHS/United States ; R01 EB025192/EB/NIBIB NIH HHS/United States ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *RNA, Messenger/chemistry/genetics ; *Lipids/chemistry ; Animals ; Bridged Bicyclo Compounds/chemistry ; Mice ; Nanoparticles/chemistry ; Humans ; }, abstract = {Lipid nanoparticles (LNPs) are an essential component of messenger RNA (mRNA) vaccines and genome editing therapeutics. Ionizable amino lipids, which play the most crucial role in enabling mRNA to overcome delivery barriers, have, to date, been restricted to two-dimensional (2D) architectures. Inspired by improved physicochemical properties resulting from the incorporation of three-dimensionality (3D) into small-molecule drugs, we report the creation of 3D ionizable lipid designs through the introduction of bicyclo[1.1.1]pentane (BCP) core motifs. BCP-based lipids enabled efficient in vivo mRNA delivery to the liver and spleen with significantly greater performance over 2D benzene- and cyclohexane-based analogues. Notably, lead BCP-NC2-C12 LNPs mediated ∼90% reduction in the PCSK9 serum protein level via CRISPR/Cas9 gene knockout, outperforming 2D controls and clinically used DLin-MC3-DMA LNPs at the same dose. Here, we introduce BCP-based designs with superior in vivo activity, thereby expanding the chemical scope of ionizable amino lipids from 2D to 3D and offering a promising avenue to improve mRNA and gene editing efficiency for the continued development of genetic medicines.}, }
@article {pmid39655408, year = {2024}, author = {Butterfield, SP and Sizer, RE and Saunders, FL and White, RJ}, title = {Selective Recruitment of a Synthetic Histone Acetyltransferase Can Boost CHO Cell Productivity.}, journal = {Biotechnology journal}, volume = {19}, number = {12}, pages = {e202400474}, pmid = {39655408}, issn = {1860-7314}, support = {//Engineering and Physical Sciences Research Council/ ; }, mesh = {CHO Cells ; *Cricetulus ; Animals ; *Histone Acetyltransferases/metabolism/genetics ; *Promoter Regions, Genetic/genetics ; Histones/metabolism/genetics ; Antibodies, Monoclonal/genetics/metabolism ; Acetylation ; CRISPR-Cas Systems/genetics ; Epigenesis, Genetic/genetics ; Transgenes ; Cytomegalovirus/genetics ; Cricetinae ; }, abstract = {Industrial production of biologics typically involves the integration of transgenes into host cell genomes, most often Chinese hamster ovary (CHO) cells. Epigenetic control of transgene expression is a major determinant of production titers. Although the cytomegalovirus (CMV) promoter has long been used to drive industrial transgene expression, we found that its associated histones are suboptimally acetylated in CHO cells, providing an opportunity to enhance productivity through epigenetic manipulation. Expression of monoclonal antibody mRNAs increased up to 12-fold when a CRISPR-dCas9 system delivered the catalytic domain of a histone acetyltransferase to the CMV promoter. This effect was far stronger than when promoter DNA was selectively demethylated using dCas9 fused to a 5-methylcytosine dioxygenase. Mechanistically, acetylation-mediated transcriptional activation involved heightened phosphorylation and activity of RNA polymerase II, enabling it to escape promoter-proximal pausing at the transgene. This approach almost doubled the titer and specific productivity of antibody-producing CHO cells, demonstrating the potential for biomanufacturing.}, }
@article {pmid39655309, year = {2024}, author = {Singh, K and Fronza, R and Evens, H and Chuah, MK and VandenDriessche, T}, title = {Comprehensive analysis of off-target and on-target effects resulting from liver-directed CRISPR-Cas9-mediated gene targeting with AAV vectors.}, journal = {Molecular therapy. Methods &amp; clinical development}, volume = {32}, number = {4}, pages = {101365}, pmid = {39655309}, issn = {2329-0501}, abstract = {Comprehensive genome-wide studies are needed to assess the consequences of adeno-associated virus (AAV) vector-mediated gene editing. We evaluated CRISPR-Cas-mediated on-target and off-target effects and examined the integration of the AAV vectors employed to deliver the CRISPR-Cas components to neonatal mice livers. The guide RNA (gRNA) was specifically designed to target the factor IX gene (F9). On-target and off-target insertions/deletions were examined by whole-genome sequencing (WGS). Efficient F9-targeting (36.45% ± 18.29%) was apparent, whereas off-target events were rare or below the WGS detection limit since only one single putative insertion was detected out of 118 reads, based on >100 computationally predicted off-target sites. AAV integrations were identified by WGS and shearing extension primer tag selection ligation-mediated PCR (S-EPTS/LM-PCR) and occurred preferentially in CRISPR-Cas9-induced double-strand DNA breaks in the F9 locus. In contrast, AAV integrations outside F9 were not in proximity to any of ∼5,000 putative computationally predicted off-target sites (median distance of 70 kb). Moreover, without relying on such off-target prediction algorithms, analysis of DNA sequences close to AAV integrations outside the F9 locus revealed no homology to the F9-specific gRNA. This study supports the use of S-EPTS/LM-PCR for direct in vivo comprehensive, sensitive, and unbiased off-target analysis.}, }
@article {pmid39654514, year = {2025}, author = {Guo, G and Moser, M and Chifamba, L and Julian, D and Teierle, S and Rajappa, P and Miller, C and Hester, ME}, title = {CRISPR-Cas9-Mediated Correction of TSC2 Pathogenic Variants in iPSCs from Patients with Tuberous Sclerosis Complex Type 2.}, journal = {The CRISPR journal}, volume = {8}, number = {1}, pages = {60-70}, doi = {10.1089/crispr.2024.0079}, pmid = {39654514}, issn = {2573-1602}, mesh = {Humans ; *Tuberous Sclerosis Complex 2 Protein/genetics ; *CRISPR-Cas Systems/genetics ; *Tuberous Sclerosis/genetics/therapy ; *Induced Pluripotent Stem Cells/metabolism ; *Gene Editing/methods ; Exons ; Mutation ; }, abstract = {Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in either the TSC1 or TSC2 genes. Though TSC causes the formation of nonmalignant tumors throughout multiple organs, the most frequent causes of mortality and morbidity are due to neurological complications. In two-thirds of cases, TSC occurs sporadically and TSC2 pathogenic variants are approximately three times more prevalent than TSC1 pathogenic variants. Here, we utilized CRISPR-Cas9-mediated homology directed repair in patient induced pluripotent stem cells (iPSCs) to correct two types of TSC2 pathogenic variants generating two isogenic lines. In one line, we corrected a splice acceptor variant (c.2743-1G>A), which causes the skipping of coding exon 23 and subsequent frameshift and introduction of a stop codon in coding exon 25. In the second line, we corrected a missense variant in coding exon 40 within the GTPase-activating protein domain (c.5228G>A, p.R1743Q). The generation of TSC2 patient iPSCs in parallel with their corresponding CRISPR-corrected isogenic lines will be an important tool for disease modeling applications and for developing therapeutics.}, }
@article {pmid39654402, year = {2024}, author = {Torelli, F and da Fonseca, DM and Butterworth, SW and Young, JC and Treeck, M}, title = {Paracrine rescue of MYR1-deficient Toxoplasma gondii mutants reveals limitations of pooled in vivo CRISPR screens.}, journal = {eLife}, volume = {13}, number = {}, pages = {}, pmid = {39654402}, issn = {2050-084X}, support = {MR/V03314X/1/MRC_/Medical Research Council/United Kingdom ; CC0199/WT_/Wellcome Trust/United Kingdom ; 10.35802/223192/WT_/Wellcome Trust/United Kingdom ; CC2132/WT_/Wellcome Trust/United Kingdom ; TO 1349/1-1//Deutsche Forschungsgemeinschaft/ ; }, mesh = {*Toxoplasma/genetics ; Animals ; Mice ; *Protozoan Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Toxoplasmosis/parasitology/genetics ; Paracrine Communication ; Macrophages/parasitology ; Mice, Inbred C57BL ; Female ; Host-Parasite Interactions/genetics ; Mutation ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Toxoplasma gondii is an intracellular parasite that subverts host cell functions via secreted virulence factors. Up to 70% of parasite-controlled changes in the host transcriptome rely on the MYR1 protein, which is required for the translocation of secreted proteins into the host cell. Mice infected with MYR1 knock-out (KO) strains survive infection, supporting a paramount function of MYR1-dependent secreted proteins in Toxoplasma virulence and proliferation. However, we have previously shown that MYR1 mutants have no growth defect in pooled in vivo CRISPR-Cas9 screens in mice, suggesting that the presence of parasites that are wild-type at the myr1 locus in pooled screens can rescue the phenotype. Here, we demonstrate that MYR1 is not required for the survival in IFN-γ-activated murine macrophages, and that parasites lacking MYR1 are able to expand during the onset of infection. While ΔMYR1 parasites have restricted growth in single-strain murine infections, we show that the phenotype is rescued by co-infection with wild-type (WT) parasites in vivo, independent of host functional adaptive immunity or key pro-inflammatory cytokines. These data show that the major function of MYR1-dependent secreted proteins is not to protect the parasite from clearance within infected cells. Instead, MYR-dependent proteins generate a permissive niche in a paracrine manner, which rescues ΔMYR1 parasites within a pool of CRISPR mutants in mice. Our results highlight an important limitation of otherwise powerful in vivo CRISPR screens and point towards key functions for MYR1-dependent Toxoplasma-host interactions beyond the infected cell.}, }
@article {pmid39654027, year = {2024}, author = {Liang, Y and Xie, SC and Lv, YH and He, YH and Zheng, XN and Cong, W and Elsheikha, HM and Zhu, XQ}, title = {A novel single-tube LAMP-CRISPR/Cas12b method for rapid and visual detection of zoonotic Toxoplasma gondii in the environment.}, journal = {Infectious diseases of poverty}, volume = {13}, number = {1}, pages = {94}, pmid = {39654027}, issn = {2049-9957}, support = {2021YFC2300800//National Key Research and Development Program of China/ ; 2021YFC2300802//National Key Research and Development Program of China/ ; 2021YFC2300804//National Key Research and Development Program of China/ ; LXXMsxnd202101//Shanxi Provincial Agricultural and Rural Research Program/ ; RFSXIHLT202101//the Research Fund of Shanxi Province for Introduced High-level Leading Talents/ ; 2021XG001//the Special Research Fund of Shanxi Agricultural University for High-level Talents/ ; }, mesh = {*Toxoplasma/genetics/isolation &amp; purification ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Animals ; Molecular Diagnostic Techniques/methods ; Humans ; Sensitivity and Specificity ; Cats ; Zoonoses/parasitology/diagnosis ; Feces/parasitology ; Toxoplasmosis/diagnosis/parasitology ; }, abstract = {BACKGROUND: Toxoplasma gondii oocysts, excreted in cat feces, pose a significant health risk to humans through contaminated soil and water. Rapid and accurate detection of T. gondii in environmental samples is essential for public health protection.<br><br>METHODS: We developed a novel, single-tube detection method that integrates loop-mediated isothermal amplification (LAMP), the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12b system, and lateral flow immunoassay strips for rapid, visual identification of T. gondii. This method targets the T. gondii B1 gene, initially amplifies it with LAMP, directed by a single-guide RNA (sgRNA). It then recognizes the amplified target gene and activates trans-cleavage, cutting nearby single-stranded DNA (ssDNA) reporters. Fluorescence detection was performed using a 6-Carboxyfluorescein (FAM)-12N-Black Hole Quencher-1 (BHQ1) reporter, while Fluorescein Isothiocyanate (FITC)-12N-Biotin enabled visual detection on lateral flow strips. The method was tested for its ability to detect various T. gondii genotypes and related parasites, assessing its specificity and broad-spectrum applicability. It was further applied to real-world environmental samples to evaluate its practicality.<br><br>RESULTS: The LAMP-CRISPR/Cas12b method exhibited high specificity and broad-spectrum detection capability, successfully identifying nine T. gondii genotypes and distinguishing them from 11 other parasitic species. Sensitivity testing at both molecular (plasmid) and practical (oocyst) levels showed detection limits of 10 &#xa0;copies/&#x3bc;L and 0.1 oocyst, respectively. When applied to 112 environmental samples (soil, water, and cat feces), the method demonstrated 100% sensitivity, accurately reflecting known infection rates.<br><br>CONCLUSIONS: This LAMP-CRISPR/Cas12b single-tube method offers a robust, innovative approach for monitoring zoonotic T. gondii in environmental samples, with significant implications for public health surveillance.}, }
@article {pmid39652422, year = {2025}, author = {Ashkin, EL and Tang, YJ and Xu, H and Hung, KL and Belk, JA and Cai, H and Lopez, SS and Dolcen, DN and Hebert, JD and Li, R and Ruiz, PA and Keal, T and Andrejka, L and Chang, HY and Petrov, DA and Dixon, JR and Xu, Z and Winslow, MM}, title = {A STAG2-PAXIP1/PAGR1 axis suppresses lung tumorigenesis.}, journal = {The Journal of experimental medicine}, volume = {222}, number = {1}, pages = {}, pmid = {39652422}, issn = {1540-9538}, support = {F99CA284289/CA/NCI NIH HHS/United States ; F99 CA274692/CA/NCI NIH HHS/United States ; 28FT-0019//Tobacco-Related Disease Research Program/ ; //Stanford University/ ; K00 CA245784/CA/NCI NIH HHS/United States ; DGE-2146755//National Science Foundation Graduate Research Fellowship Program/ ; MFE-176568//Canadian Institute of Health Research/ ; R01 CA234349/CA/NCI NIH HHS/United States ; F99 CA284289/CA/NCI NIH HHS/United States ; P30 CA124435/CA/NCI NIH HHS/United States ; GT14928/HHMI/Howard Hughes Medical Institute/United States ; R01 CA231253/CA/NCI NIH HHS/United States ; R01-CA231253/NH/NIH HHS/United States ; PF-21-112-01-MM//American Cancer Society/ ; }, mesh = {*Lung Neoplasms/pathology/metabolism/genetics ; Humans ; Animals ; *Cell Cycle Proteins/metabolism/genetics ; Cell Line, Tumor ; Mice ; *Carcinogenesis/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Antigens, Nuclear/metabolism/genetics ; Tumor Suppressor Proteins/metabolism/genetics ; Cohesins ; }, abstract = {The cohesin complex is a critical regulator of gene expression. STAG2 is the most frequently mutated cohesin subunit across several cancer types and is a key tumor suppressor in lung cancer. Here, we coupled somatic CRISPR-Cas9 genome editing and tumor barcoding with an autochthonous oncogenic KRAS-driven lung cancer model and showed that STAG2 is uniquely tumor-suppressive among all core and auxiliary cohesin components. The heterodimeric complex components PAXIP1 and PAGR1 have highly correlated effects with STAG2 in human lung cancer cell lines, are tumor suppressors in vivo, and are epistatic to STAG2 in oncogenic KRAS-driven lung tumorigenesis in vivo. STAG2 inactivation elicits changes in gene expression, chromatin accessibility, and 3D genome conformation that impact the cancer cell state. Gene expression and chromatin accessibility similarities between STAG2- and PAXIP1-deficient neoplastic cells further relate STAG2-cohesin to PAXIP1/PAGR1. These findings reveal a STAG2-PAXIP1/PAGR1 tumor-suppressive axis and uncover novel PAXIP1-dependent and PAXIP1-independent STAG2-cohesin-mediated mechanisms of lung tumor suppression.}, }
@article {pmid39652018, year = {2025}, author = {Peña-Gutiérrez, I and Olalla-Sastre, B and Río, P and Rodríguez-Madoz, JR}, title = {Beyond precision: evaluation of off-target clustered regularly interspaced short palindromic repeats/Cas9-mediated genome editing.}, journal = {Cytotherapy}, volume = {27}, number = {3}, pages = {279-286}, doi = {10.1016/j.jcyt.2024.10.010}, pmid = {39652018}, issn = {1477-2566}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals ; *Genetic Therapy/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {The gene editing field has advanced rapidly since the development of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system because of its applicability in precisely modifying the genome. Among its multiple applications, the correction of genetic diseases has emerged as a potential curative treatment for many disorders that have eluded a cure to date. Despite its efficiency in achieving therapeutic levels of correction, the unexpected adverse effects of editing due to CRISPR/Cas9 nuclease activity are a major concern when translating these new strategies to the clinic. Multiple in silico tools and empirical methods have been developed to evaluate these off-target edits as well as other adverse alterations of the genome, including rearrangements, not only in ex vivo experiments but also in in vivo experiments. In this review, we summarize the available methods for the assessment of off-target effects of CRISPR/Cas9 systems, highlighting their advantages and limitations. Special attention will be paid to their application in pre-clinical studies and clinical trials, both in the manufacturing product and in the long-term follow-up of patients.}, }
@article {pmid39651221, year = {2024}, author = {Yun, S and Chekuri, A and Art, J and Kondabolu, K and Slaugenhaupt, SA and Zeltner, N and Kleinstiver, BP and Morini, E and Alves, CRR}, title = {Engineered CRISPR-Base Editors as a Permanent Treatment for Familial Dysautonomia.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, pmid = {39651221}, issn = {2692-8205}, support = {P01 HL142494/HL/NHLBI NIH HHS/United States ; DP2 CA281401/CA/NCI NIH HHS/United States ; R01 NS095640/NS/NINDS NIH HHS/United States ; K01 NS134784/NS/NINDS NIH HHS/United States ; R01 NS124561/NS/NINDS NIH HHS/United States ; R01 NS125353/NS/NINDS NIH HHS/United States ; }, abstract = {Familial dysautonomia (FD) is a fatal autosomal recessive congenital neuropathy caused by a T-to-C mutation in intron 20 of the Elongator acetyltransferase complex subunit 1 (ELP1) gene, which causes tissue-specific skipping of exon 20 and reduction of ELP1 protein. Here, we developed a base editor (BE) approach to precisely correct this mutation. By optimizing Cas9 variants and screening multiple gRNAs, we identified a combination that was able to promote up to 70% on-target editing in HEK293T cells harboring the ELP1 T-to-C mutation. These editing levels were sufficient to restore exon 20 inclusion in the ELP1 transcript. Moreover, we optimized an engineered dual intein-split system to deliver these constructs in vivo. Mediated by adeno-associated virus (AAV) delivery, this BE strategy effectively corrected the liver and brain ELP1 splicing defects in a humanized FD mouse model carrying the ELP1 T-to-C mutation and rescued the FD phenotype in iPSC-derived sympathetic neurons. Importantly, we observed minimal off-target editing demonstrating high levels of specificity with these optimized base editors. These findings establish a novel and highly precise BE-based therapeutic approach to correct the FD mutation and associated splicing defects and provide the foundation for the development of a transformative, permanent treatment for this devastating disease.}, }
@article {pmid39651213, year = {2024}, author = {Singh, MA and Chang, MM and Wang, Q and Rodgers, C and Lutz, BR and Olanrewaju, AO}, title = {Rapid enzymatic assay for antiretroviral drug monitoring using CRISPR-Cas12a enabled readout.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, doi = {10.1101/2024.11.25.625292}, pmid = {39651213}, issn = {2692-8205}, abstract = {Maintaining efficacy of human immunodeficiency virus (HIV) medications is challenging among children because of dosing difficulties, the limited number of approved drugs, and low rates of medication adherence. Drug level feedback (DLF) can support dose optimization and timely interventions to prevent treatment failure, but current tests are heavily instrumented and centralized. We developed the REverse-transcriptase ACTivity-crispR (REACTR) assay for rapid measurement of HIV drugs based on the extent of DNA synthesis by HIV reverse transcriptase. CRISPR-Cas enzymes bind to synthesized DNA, triggering collateral cleavage of quenched reporters and generating fluorescence. We measured azidothymidine triphosphate (AZT-TP), a key drug in pediatric HIV treatment, and investigated the impact of assay time and DNA template length on REACTR's sensitivity. REACTR selectively measured clinically relevant AZT-TP concentrations in the presence of genomic DNA and peripheral blood mononuclear cell lysate. REACTR has the potential to enable rapid point-of-care HIV DLF to improve pediatric HIV care.}, }
@article {pmid39651158, year = {2024}, author = {Kiernan, KA and Taylor, DW}, title = {Visualization of a multi-turnover Cas9 after product release.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, doi = {10.1101/2024.11.25.625307}, pmid = {39651158}, issn = {2692-8205}, abstract = {While the most widely used CRISPR-Cas enzyme is the S. pyogenes Cas9 endonuclease (Cas9), it exhibits single-turnover enzyme kinetics which leads to long residence times on product DNA. This blocks access to DNA repair machinery and acts as a major bottleneck during CRISPR-Cas9 gene editing. Although Cas9 can eventually be forcibly removed by extrinsic factors (translocating polymerases, helicases, chromatin modifying complexes, etc), the mechanisms contributing to Cas9 dissociation following cleavage remain poorly understood. Interestingly, it's been shown that Cas9 can be more easily dislodged when complexes collide with the PAM-distal region of the Cas9 complex or when the strength of Cas9 interactions in this region are weakened. Here, we employ truncated guide RNAs as a strategy to weaken PAM-distal nucleic acid interactions and still support Cas9 activity. We find that guide truncation promotes much faster Cas9 turnover and used this to capture previously uncharacterized Cas9 reaction states. Kinetics-guided cryo-EM enabled us to enrich for rare, transient states that are often difficult to capture in standard workflows. From a single dataset, we examine the entire conformational landscape of a multi-turnover Cas9, including the first detailed snapshots of Cas9 dissociating from product DNA. We discovered that while the PAM-distal product dissociates from Cas9 following cleavage, tight binding of the PAM-proximal product directly inhibits re-binding of new targets. Our work provides direct evidence as to why Cas9 acts as a single-turnover enzyme and will guide future Cas9 engineering efforts.}, }
@article {pmid39650142, year = {2024}, author = {Xiang, H and Chen, B and Wang, S and Zeng, W and Jiang, J and Kong, W and Huang, H and Mi, Q and Ni, S and Gao, Q and Li, Z}, title = {Development of an RNA virus vector for non-transgenic genome editing in tobacco and generation of berberine bridge enzyme-like mutants with reduced nicotine content.}, journal = {aBIOTECH}, volume = {5}, number = {4}, pages = {449-464}, pmid = {39650142}, issn = {2662-1738}, abstract = {UNLABELLED: Tobacco (Nicotiana tabacum) plants synthesize the psychoactive pyridine alkaloid nicotine, which has sparked growing interest in reducing nicotine levels through genome editing aiming at inactivating key biosynthetic genes. Although stable transformation-mediated genome editing is effective in tobacco, its polyploid nature complicates the complete knockout of genes and the segregation of transgenes from edited plants. In this study, we developed a non-transgenic genome editing method in tobacco by delivering the CRISPR/Cas machinery via an engineered negative-strand RNA rhabdovirus vector, followed by the regeneration of mutant plants through tissue culture. Using this method, we targeted six berberine bridge enzyme-like protein (BBL) family genes for mutagenesis, which are implicated in the last steps of pyridine alkaloid biosynthesis, in the commercial tobacco cultivar Hongda. We generated a panel of 16 mutant lines that were homozygous for mutations in various combinations of BBL genes. Alkaloid profiling revealed that lines homozygous for BBLa and BBLb mutations exhibited drastically reduced nicotine levels, while other BBL members played a minor role in nicotine synthesis. The decline of nicotine content in these lines was accompanied by reductions in anatabine and cotinine levels but increases in nornicotine and its derivative myosmine. Preliminary agronomic evaluation identified two low-nicotine lines with growth phenotypes comparable to those of wild-type plants under greenhouse and field conditions. Our work provides potentially valuable genetic materials for breeding low-nicotine tobacco and enhances our understanding of alkaloid biosynthesis.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42994-024-00188-y.}, }
@article {pmid39650138, year = {2024}, author = {Zou, J and Li, Y and Wang, K and Wang, C and Zhuo, R}, title = {Prime editing enables precise genome modification of a Populus hybrid.}, journal = {aBIOTECH}, volume = {5}, number = {4}, pages = {497-501}, pmid = {39650138}, issn = {2662-1738}, abstract = {UNLABELLED: CRISPR/Cas-based genome editing has been extensively employed in the breeding and genetic improvement of trees, yet precise editing remains challenging in these species. Prime editing (PE), a revolutionary technology for precise editing, allows for arbitrary base substitutions and the insertion/deletion of small fragments. In this study, we focused on the model tree poplar 84K (Populus alba × P. glandulosa). We used the 2 × 35S promoter to express a fusion protein of spCas9 nickase (nCas9) and engineered Moloney murine leukemia virus (MMLV), and the Arabidopsis thaliana AtU6 promoter to express an engineered PE guide RNA (epegRNA) and Nick gRNA, pioneering the establishment of the Prime Editor 3 (PE3) system in dicot poplar. Single-base substitutions, multiple-base substitutions, and small-fragment insertions/deletions were edited into three endogenous target genes. The desired edits were identified in hygromycin-resistant (transformed) calli at seven out of nine target sites, with an average editing efficiency ranging from 0.1 to 3.6%. Furthermore, stable T0 plants contained the desired edits at four out of nine targets, with editing efficiencies ranging from 3.6 to 22.2%. Establishment of the PE3 system provides a powerful tool for the precise modification of the poplar genome.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42994-024-00177-1.}, }
@article {pmid39648412, year = {2025}, author = {Wang, J and Yang, R and Wang, F and Zhang, J and Dong, Y and Wang, J and Yu, M and Xu, Y and Liu, L and Cheng, Y and Zhang, C and Yang, Y and Yang, W and Wang, J and Chen, G and Huang, Y and Tian, Y and Jian, R and Ni, B and Wu, W and Ruan, Y}, title = {CRISPR-Cas9 screening identifies the role of FER as a tumor suppressor.}, journal = {The Journal of pathology}, volume = {265}, number = {2}, pages = {158-171}, doi = {10.1002/path.6374}, pmid = {39648412}, issn = {1096-9896}, support = {2023NSCQZDX0035//Natural Science Foundation of Chongqing Municipality/ ; 82273118//National Natural Science Foundation of China/ ; CX2019JS202//New Technology Cultivation Project of Military Medicine/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; *Tumor Suppressor Proteins/genetics/metabolism ; Humans ; Cell Line ; Genome ; Genetic Association Studies ; Carcinogenesis/genetics ; Disease Progression ; Mice, Nude ; Animals ; }, abstract = {It is important to systematically identify tumor suppressor genes (TSGs) to improve our understanding of tumorigenesis and develop strategies for early diagnosis and mitigating disease progression. In the present study, we used an in vivo genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) screen and identified FPS/FES-related (FER) as a TSG. Single-cell RNA sequencing (scRNA-seq) revealed that normal cells with low FER expression exhibited elevated malignant transformation potential and stemness properties. FER knockout promoted the tumorigenic transformation, characterized by high colony-forming efficiency and suspension growth ability, acquired tumorigenicity in vivo, increased metabolic activity, dedifferentiation properties, and immune evasion. Moreover, analysis revealed that low FER expression tumors share molecular phenotypes with FER knockout cells, suggesting the consistent role of FER in tumor initiation and progression. Taken together, our findings not only provide insights into the essential role of FER as a tumor suppressor in tumor initiation and progression but also highlight its potential as a target for future clinical diagnosis. © 2024 The Pathological Society of Great Britain and Ireland.}, }
@article {pmid39647515, year = {2025}, author = {Luo, X and Germer, J and Burghardt, T and Grau, M and Lin, Y and Höhn, M and Lächelt, U and Wagner, E}, title = {Dual pH-responsive CRISPR/Cas9 ribonucleoprotein xenopeptide complexes for genome editing.}, journal = {European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences}, volume = {205}, number = {}, pages = {106983}, doi = {10.1016/j.ejps.2024.106983}, pmid = {39647515}, issn = {1879-0720}, mesh = {Humans ; *Gene Editing/methods ; *Ribonucleoproteins/chemistry/genetics/administration &amp; dosage ; *CRISPR-Cas Systems/genetics ; Hydrogen-Ion Concentration ; HeLa Cells ; *Peptides/chemistry/administration &amp; dosage ; *CRISPR-Associated Protein 9/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated (Cas) protein has been proved as a powerful tool for the treatment of genetic diseases. The Cas9 protein, when combined with single-guide RNA (sgRNA), forms a Cas9/sgRNA ribonucleoprotein (RNP) capable of targeting and editing the genome. However, the limited availability of effective carriers has restricted the broader application of CRISPR/Cas9 RNP. In this study, we evaluated dual pH-responsive amphiphilic xenopeptides (XPs) for delivering CRISPR/Cas9 RNP. These artificial lipo-XPs contain apolar cationizable lipoamino fatty acid (LAF) and polar cationizable oligoaminoethylene acid units such as succinoyl-tetraethylenepentamine (Stp) in various ratios and U-shaped topologies. The carriers were screened for functional Cas9/sgRNA RNP delivery in four different reporter cell lines, including a Duchenne muscular dystrophy (DMD) exon skipping reporter cell model. Significantly enhanced cellular uptake into HeLa cells, effective endosomal disruption in HeLa gal8-mRuby3 cells, and potent genome editing by several Cas9/sgRNA RNP complexes was observed in four different cell lines in the 5 nM sgRNA range. Comparing Cas9/sgRNA RNP complexes with Cas9 mRNA/sgRNA polyplexes in the DMD reporter cell model demonstrated similar splice site editing and high exon skipping of the two different molecular Cas9 modalities. Based on these studies, analogues of two potent U1 LAF2-Stp and LAF4-Stp2 structures were deployed, tuning the amphiphilicity of the polar Stp group by replacement with the six oligoamino acids dmGtp, chGtp, dGtp, Htp, Stt, or GEIPA. The most potent LAF2-Stp analogues (containing dGtp, chGtp or GEIPA) demonstrated further enhanced gene editing efficiency with EC50 values of 1 nM in the DMD exon skipping reporter cell line. Notably, the EC50 of LAF2-dGtp reached 0.51 nM even upon serum incubation. Another carrier (LAF4-GEIPA2) complexing Cas9/sgRNA RNP and donor DNA, facilitated up to 43 % of homology-directed repair (HDR) in HeLa eGFPd2 cells visualized by the switch from green fluorescent protein (eGFP) to blue fluorescent protein (BFP). This study presents a delivery system tunable for Cas9 RNP complexes or Cas9 RNP/donor DNA polyplexes, offering an effective and easily applicable strategy for gene editing.}, }
@article {pmid39647408, year = {2025}, author = {Zheng, L and Fan, B and Fu, Y and Wei, J and Ye, Y and Gui, Y and Zhang, S and Wei, Y and Yin, J and Li, J and Jin, M and Pang, B}, title = {Single-tube detection of a foodborne bacterial pathogen using user-friendly portable device.}, journal = {Biosensors &amp; bioelectronics}, volume = {271}, number = {}, pages = {117035}, doi = {10.1016/j.bios.2024.117035}, pmid = {39647408}, issn = {1873-4235}, mesh = {*Biosensing Techniques/instrumentation ; *Salmonella enteritidis/isolation &amp; purification/pathogenicity/genetics ; *Food Microbiology/instrumentation ; Humans ; Equipment Design ; Nucleic Acid Amplification Techniques/instrumentation ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Foodborne Diseases/microbiology ; }, abstract = {Timely and reliable detection of foodborne bacterial pathogen is crucial for reducing disease burden in low- and middle-income countries. However, laboratory-based methods are often inaccessibility in resource-limited settings. Here, we developed a single-tube assay and a low-cost palm-sized device for on-site detection of the representative foodborne bacterial pathogen, Salmonella Enteritidis. Our assay incorporates the advantages of protein-nucleic acid signal transduction, EXPonential Amplification Reaction (EXPAR), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 12a (Cas12a). After systematically investigating the compatibility of these components, we developed a "three-in-one" integration reaction, termed ST-EXPAR-CRISPR assay. This assay requires only one tube, one controlled temperature (39 °C) and simple operation, eliminating the need for bacterial isolation, nucleic acid extraction, or washing steps. ST-EXPAR-CRISPR assay is capable of detecting as few as 37 CFU/mL of target bacterium. Using our kit and portable device, untrained volunteers successfully detected contamination in food samples outdoors. The simplicity of the detection process and minimal hardware requirements make our assay highly promising for application in point-of-care and on-site scenarios. Moreover, the ST-EXPAR-CRISPR assay can be easily modified to detect other targets by changing the nucleic acid sequence with low research and development cost, potentially reducing the global disease burden.}, }
@article {pmid39645650, year = {2025}, author = {Moffa, L and Mannino, G and Bevilacqua, I and Gambino, G and Perrone, I and Pagliarani, C and Bertea, CM and Spada, A and Narduzzo, A and Zizzamia, E and Velasco, R and Chitarra, W and Nerva, L}, title = {CRISPR/Cas9-driven double modification of grapevine MLO6-7 imparts powdery mildew resistance, while editing of NPR3 augments powdery and downy mildew tolerance.}, journal = {The Plant journal : for cell and molecular biology}, volume = {122}, number = {2}, pages = {e17204}, pmid = {39645650}, issn = {1365-313X}, support = {101135088//HORIZON EUROPE European Innovation Council/ ; BIOTECH-VITECH//Ministero delle Politiche Agricole Alimentari e Forestali/ ; }, mesh = {*Vitis/genetics/microbiology/immunology ; *CRISPR-Cas Systems/genetics ; *Plant Diseases/microbiology/immunology/genetics ; Gene Editing/methods ; *Disease Resistance/genetics ; *Plant Proteins/genetics/metabolism ; *Ascomycota/physiology ; Plants, Genetically Modified ; Erysiphe/physiology ; }, abstract = {The implementation of genome editing strategies in grapevine is the easiest way to improve sustainability and resilience while preserving the original genotype. Among others, the Mildew Locus-O (MLO) genes have already been reported as good candidates to develop powdery mildew-immune plants. A never-explored grapevine target is NPR3, a negative regulator of the systemic acquired resistance. We report the exploitation of a cisgenic approach with the Cre-lox recombinase technology to generate grapevine-edited plants with the potential to be transgene-free while preserving their original genetic background. The characterization of three edited lines for each target demonstrated immunity development against Erysiphe necator in MLO6-7-edited plants. Concomitantly, a significant improvement of resilience, associated with increased leaf thickness and specific biochemical responses, was observed in defective NPR3 lines against E. necator and Plasmopara viticola. Transcriptomic analysis revealed that both MLO6-7 and NPR3 defective lines modulated their gene expression profiles, pointing to distinct though partially overlapping responses. Furthermore, targeted metabolite analysis highlighted an overaccumulation of stilbenes coupled with an improved oxidative scavenging potential in both editing targets, likely protecting the MLO6-7 mutants from detrimental pleiotropic effects. Finally, the Cre-loxP approach allowed the recovery of one MLO6-7 edited plant with the complete removal of transgene. Taken together, our achievements provide a comprehensive understanding of the molecular and biochemical adjustments occurring in double MLO-defective grape plants. In parallel, the potential of NPR3 mutants for multiple purposes has been demonstrated, raising new questions on its wide role in orchestrating biotic stress responses.}, }
@article {pmid39644903, year = {2024}, author = {Kohabir, KAV and Linthorst, J and Nooi, LO and Brouwer, R and Wolthuis, RMF and Sistermans, EA}, title = {Synthetic mismatches enable specific CRISPR-Cas12a-based detection of genome-wide SNVs tracked by ARTEMIS.}, journal = {Cell reports methods}, volume = {4}, number = {12}, pages = {100912}, pmid = {39644903}, issn = {2667-2375}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Proto-Oncogene Proteins B-raf/genetics ; Aldehyde Dehydrogenase, Mitochondrial/genetics ; Genome, Human/genetics ; Polymorphism, Single Nucleotide/genetics ; Endodeoxyribonucleases/genetics/metabolism ; CRISPR-Associated Proteins/genetics/metabolism ; BRCA2 Protein/genetics ; Neoplasms/genetics/diagnosis ; Tumor Suppressor Protein p53/genetics ; Base Pair Mismatch ; Bacterial Proteins ; }, abstract = {Detection of pathogenic DNA variants is vital in cancer diagnostics and treatment monitoring. While CRISPR-based diagnostics (CRISPRdx) offer promising avenues for cost-effective, rapid, and point-of-care testing, achieving single-nucleotide detection fidelity remains challenging. We present an in silico pipeline that scans the human genome for targeting pathogenic mutations in the seed region (ARTEMIS), the most stringent crRNA domain. ARTEMIS identified 12% of pathogenic SNVs as Cas12a recognizable, including 928 cancer-associated variants such as BRAF[V600E], BRCA2[E1953∗], TP53[V272M], and ALDH2[E504K]. Cas12a exhibited remarkable tolerance to single mismatches within the seed region. Introducing deliberate synthetic mismatches within the seed region yielded on-target activity with single-nucleotide fidelity. Both positioning and nucleobase types of mismatches influenced detection accuracy. With improved specificity, Cas12a could accurately detect and semi-quantify BRAF[V600E] in cfDNA from cell lines and patient liquid biopsies. These results provide insights toward rationalized crRNA design for high-fidelity CRISPRdx, supporting personalized and cost-efficient healthcare solutions in oncologic diagnostics.}, }
@article {pmid39644600, year = {2025}, author = {Xu, Y and Wu, H and Jiang, J and Ye, L and Hao, K and Han, K and Hu, S and Lei, W and Guo, Z}, title = {Generation and characterization of the LINC01405 knockout human embryonic stem cell line.}, journal = {Stem cell research}, volume = {82}, number = {}, pages = {103619}, doi = {10.1016/j.scr.2024.103619}, pmid = {39644600}, issn = {1876-7753}, mesh = {Humans ; *Human Embryonic Stem Cells/metabolism/cytology ; *RNA, Long Noncoding/genetics/metabolism ; Cell Line ; CRISPR-Cas Systems ; Cell Differentiation ; Gene Knockout Techniques ; }, abstract = {Long Intergenic Non-Protein Coding RNA 1405 (LINC01405), with known elevated expression in muscle, has been linked to a number of musculo-skeletal conditions. By utilizing the CRISPR/Cas9 gene editing system, we generated a LINC01405 knockout human embryonic stem cell (hESC) line. This line remains human stem cell-like morphology and pluripotency, exhibits a normal karyotype, and can differentiate into cells from all three germ layers. This cell line will be an invaluable model for the research on LINC01405's role in normal development of cardiac and skeletal muscle, and their diseases.}, }
@article {pmid39644528, year = {2025}, author = {Guo, H and Ma, P and Dong, X and Qin, M and Zhang, Y and Wang, Z}, title = {CRISPR/Cas12a-mediated fluorescent aptasensor based on DNA walker amplification for oxytetracycline detection.}, journal = {Biosensors &amp; bioelectronics}, volume = {271}, number = {}, pages = {117031}, doi = {10.1016/j.bios.2024.117031}, pmid = {39644528}, issn = {1873-4235}, mesh = {*Oxytetracycline/analysis ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry ; *Milk/chemistry ; Animals ; *CRISPR-Cas Systems ; *Honey/analysis ; Limit of Detection ; Food Contamination/analysis ; Spectrometry, Fluorescence/methods ; Anti-Bacterial Agents/analysis ; Nucleic Acid Amplification Techniques/methods ; Fluorescent Dyes/chemistry ; DNA/chemistry/genetics ; }, abstract = {Nowadays, although most oxytetracycline (OTC) are used only in animals, their leaching into the environment and residues in food lead to noticeable health problems. Fortunately, by studying the molecular dynamics of OTC aptamers and the mutations of key bases, aptamer's base fragments involved in identifying OTC was confirmed for the first time, which provided theoretical guidance for the ingenious design of aptamer detection. Thereafter, benefiting from the amplification impact by DNA walker, the disadvantage of low efficiency caused by CRISPR/Cas12a was effectively avoided. In the established delicate aptasensing platform, small quantity of OTC promoted DNA walker to produce a massive of activators, which stimulated the trans-cleavage activity of CRISPR/Cas12a. By implying nanoclusters (NCs) with covalent organic framework (COF), the sensitive fluorescence "turn-on" method for OTC detection was realized. Under the optimum conditions, the linear range of aptasensor was 0.05-50 μM, and the detection limit was 0.041 μM. In addition, the aptasensing method can be successfully used for the quantitative detection of OTC in milk and honey samples with a recovery rate of 95.67%-104.10% and 95.33%-109.70%, implying excellent analytical performance and practical application prospect.}, }
@article {pmid39644349, year = {2024}, author = {Wang, YX and Ding, ZJ and Wang, QL and Zhao, CC and Liu, SQ and Du, SL and Zhou, S and Zheng, LY and Gao, M and Shen, CC and Chen, XD}, title = {CRISPR-Cas9 screening identified novel subtypes of cutaneous melanoma based on essential cancer genes.}, journal = {Archives of dermatological research..}, volume = {317}, number = {1}, pages = {86}, pmid = {39644349}, issn = {1432-069X}, support = {MS2023007//Scientific Research Project of Nantong Municipal Health Commission/ ; ZD2022006//Key Medical Research Projects of Jiangsu Provincial Health Commission/ ; }, mesh = {Humans ; *Melanoma/genetics/immunology/pathology ; *Skin Neoplasms/genetics/immunology/pathology/diagnosis/therapy ; *CRISPR-Cas Systems/genetics ; Prognosis ; Biomarkers, Tumor/genetics ; Gene Expression Regulation, Neoplastic ; Male ; Female ; Melanoma, Cutaneous Malignant ; Cell Line, Tumor ; Databases, Genetic ; Middle Aged ; Gene Expression Profiling ; }, abstract = {Our primary objective was to identify genes critical for cutaneous melanoma (CM) and related typing, based on essential genes, to generate novel insights for clinical management and immunotherapy of patients with CM. We analyzed RNA sequencing (RNA-seq) data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx), and sequencing data of 29 CM cell line from Cancer Cell Line Encyclopedia (CCLE) databases. Combined with DepMap database, 406 CM essential cancer genes were finally obtained. Based on the expression of essential genes in cancer, the patients included in TCGA and Gene Expression Omnibus (GEO) databases were divided into three different molecular subtypes (C1, C2, and C3) by the NMF algorithm. Data analysis from TCGA and GEO datasets revealed that subtype C3 had the poorest prognosis, while subtype C1 exhibited the best prognosis. Combined with the CIBERSORT, ESTIMATE and ssGSEA algorithm, patients with different molecular subtypes can be divided into two immune subtypes (hot and cold). We found that subtype C1 was characterized by hot tumors, in contrast to subtypes C2 and C3, which were characterized by cold tumors. Then, we used univariate Cox regression, LASSO, and multifactor Cox regression analysis to select risk genes and constructed a prognostic model based on eight genes: RABIF, CDCA8, FOXM1, SPRR2E, AIP, CAP1, CTSW, and IFITM3. All patients were divided into two risk subtypes (high and low) according to the median of risk scores. We found that most hot tumor subtypes were found in the low-risk subtypes and most patients with this subtype survived for longer. Ultimately, we selected RABIF, which exhibits the highest risk coefficient, for histological and cytological verification. The results showed that RABIF was overexpressed in melanoma. Inhibition of RABIF expression could suppress the proliferation and invasion of melanoma cells and promote the apoptosis of melanoma cells. In conclusion, we used CRISPR-Cas9 screening to verify the association between molecular subtypes (C1, C2, and C3), immune subtypes (hot and cold), and risk subtypes (high and low) in patients with CM, particularly in distinguishing survival and prognosis. These findings can be used to guide clinical management and immunotherapy of patients with CM.}, }
@article {pmid39643619, year = {2024}, author = {Khan, NM and Wilderman, A and Kaiser, JM and Kamalakar, A and Goudy, SL and Cotney, J and Drissi, H}, title = {Enhanced osteogenic potential of iPSC-derived mesenchymal progenitor cells following genome editing of GWAS variants in the RUNX1 gene.}, journal = {Bone research}, volume = {12}, number = {1}, pages = {70}, pmid = {39643619}, issn = {2095-4700}, support = {R21 AR071536/AR/NIAMS NIH HHS/United States ; AR071536//U.S. Department of Health &amp; Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)/ ; }, mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; *Osteogenesis/genetics ; Humans ; *Core Binding Factor Alpha 2 Subunit/genetics/metabolism ; *Gene Editing/methods ; *Mesenchymal Stem Cells/metabolism/cytology ; *Genome-Wide Association Study ; Animals ; Cell Differentiation/genetics ; Mice ; Osteoblasts/metabolism/cytology ; Bone Density/genetics ; CRISPR-Cas Systems/genetics ; }, abstract = {Recent genome-wide association studies (GWAS) identified 518 significant loci associated with bone mineral density (BMD), including variants at the RUNX1 locus (rs13046645, rs2834676, and rs2834694). However, their regulatory impact on RUNX1 expression and bone formation remained unclear. This study utilized human induced pluripotent stem cells (iPSCs) differentiated into osteoblasts to investigate these variants' regulatory roles. CRISPR/Cas9 was employed to generate mutant (Δ) iPSC lines lacking these loci at the RUNX1 locus. Deletion lines (Δ1 and Δ2) were created in iPSCs to assess the effects of removing regions containing these loci. Deletion lines exhibited enhanced osteogenic potential, with increased expression of osteogenic marker genes and Alizarin Red staining. Circularized chromosome conformation capture (4C-Seq) was utilized to analyze interactions between BMD-associated loci and the RUNX1 promoter during osteogenesis. Analysis revealed altered chromatin interactions with multiple gene promoters including RUNX1 isoform, as well as SETD4, a histone methyltransferase, indicating their regulatory influence. Interestingly, both deletion lines notably stimulated the expression of the long isoform of RUNX1, with more modest effects on the shorter isoform. Consistent upregulation of SETD4 and other predicted targets within the Δ2 deletion suggested its removal removed a regulatory hub constraining expression of multiple genes at this locus. In vivo experiments using a bone defect model in mice demonstrated increased bone regeneration with homozygous deletion of the Δ2 region. These findings indicate that BMD-associated variants within the RUNX1 locus regulate multiple effector genes involved in osteoblast commitment, providing valuable insights into genetic regulation of bone density and potential therapeutic targets.}, }
@article {pmid39643320, year = {2025}, author = {Gao, J and Huang, S and Jiang, J and Miao, Q and Zheng, R and Kang, Y and Tang, W and Zuo, H and He, J and Xie, J}, title = {Dual-CRISPR/Cas12a-assisted RT-RAA visualization system for rapid on-site detection of nervous necrosis virus (NNV).}, journal = {Analytica chimica acta}, volume = {1335}, number = {}, pages = {343469}, doi = {10.1016/j.aca.2024.343469}, pmid = {39643320}, issn = {1873-4324}, mesh = {Aquaculture ; CRISPR-Cas Systems ; *Fish Diseases/diagnosis/virology ; *Genes, Viral ; *Nodaviridae/genetics/isolation &amp; purification ; Nucleic Acid Amplification Techniques ; Sensitivity and Specificity ; Animals ; }, abstract = {BACKGROUND: Nervous necrosis virus (NNV) poses a severe threat to the aquaculture industry, particularly infecting fish fry with devastating mortality rates and inflicting heavy economic losses. Traditional detection methods, such as cell culture and conventional RT-PCR, are not only time-consuming and require specialized laboratory facilities but also hard to eliminate contamination. Rapid and accurate on-site detection methods in aquaculture settings are crucial for effective control of NNV outbreaks in fish farms.<br><br>RESULTS: This study developed a one-tube visualization system for rapid and precise identification of NNV in a pond-side setting. This system utilizes the dual-clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-assisted reverse transcription-recombinase aided amplification (RT-RAA) detection method, employing fluorescence intensity to indicate positive results for easy interpretation by field operators. The key to this system involved the meticulous selection of RT-RAA primer sets and CRISPR RNA (crRNA) primer sets targeting two genes of NNV, the capsid protein (CP) and RNA-dependent RNA polymerase (RdRp), distributing on two particles of genomic sequences. The assay demonstrated a speed and efficiency process within 30&#xa0;min and a detection limit of 0.5 copies/&#x3bc;L, achieving 100&#xa0;% accuracy when compared to qRT-PCR. The practical utility and effectiveness were validated by using 32 field samples. The results underscored the simplicity, rapidity, and reliability of the system, confirming its potential as a robust tool for NNV diagnosis in fish farms.<br><br>SIGNIFICANCE: This study introduces the first application of a dual-CRISPR/Cas12a-assisted RT-RAA visualization system for diagnosing NNV infections. The novel approach substantially enhances on-site diagnostic capabilities, offering a rapid, reliable, and cost-effective solution for fish farm operators. This innovation not only streamlines the detection process but also ensures timely intervention, thereby mitigating the impact of NNV on aquaculture.}, }
@article {pmid39643311, year = {2025}, author = {He, H and Zhang, X and Deng, M and Zhou, Y and Pang, H and Yang, H and Lyu, J and Feng, Y and Geng, X and Guo, X and Luo, G and Guo, B}, title = {In-situ nucleic acid amplification induced by DNA self-assembly for rapid and ultrasensitive detection of miRNA.}, journal = {Analytica chimica acta}, volume = {1335}, number = {}, pages = {343457}, doi = {10.1016/j.aca.2024.343457}, pmid = {39643311}, issn = {1873-4324}, mesh = {*Nucleic Acid Amplification Techniques/methods ; *MicroRNAs/analysis/genetics ; Humans ; *DNA/chemistry/genetics ; *Limit of Detection ; Biosensing Techniques/methods ; }, abstract = {BACKGROUND: To improve the sensitivity and specificity of nucleic acid detection, coupling two or more signal amplification systems is a feasible pattern, such as nucleic acid isothermal amplification coupling genome-editing technology, and cascaded DNA self-assembly circuits. And representative signal amplification strategies include loop-mediated isothermal amplification (LAMP), clustered regularly interspaced short palindromic repeats/associated proteins (CRISPR/Cas) systems, and catalyzed hairpin assembly (CHA). However, these detection strategies often require the enrichment of intermediate products, the replacement of reaction conditions and the design of multiple probes, which may seriously affect the reliability of detection results.<br><br>RESULTS: Herein, we propose a novel nucleic acid detection system which is named as catalyzed hairpin assembly (CHA) coupled with embedded primer triggered isothermal amplification (CEA for short). DNA self-assembly probes in CEA contain a specially designed primer. When target nucleic acid (e.g., miRNA) initiates CHA reaction (the first signal amplification), the self-assembly product of CHA will expose a primer (named as embedded primer). The embedded primer will trigger a special nucleic acid isothermal amplification in situ, then generate plenty of double-stranded DNA products in 30&#xa0;min with varying lengths (the second signal amplification). Compared to that of a typical CHA reaction, the sensitivity of CEA has increased by three orders of magnitude and the detection limit is as low as 0.228&#xa0;fM. Besides, it has excellent detection performance in cancer and stem cell samples.<br><br>SIGNIFICANCE: By coupling embedded primer with DNA self-assembly system, a new nucleic acid detection system (CEA) with one-step operation and dual signal amplification has been successfully established. Compared with traditional dual signal amplification systems, CEA can not only significantly improve the reaction efficiency, but also greatly reduce the difficulty of detection system design and experimental operation.}, }
@article {pmid39643309, year = {2025}, author = {Li, X and Wang, C and Chai, J and Liu, H and Jiang, X and Li, Y and Li, Z and Li, Y}, title = {A novel all-in-one target-powered entropy-driven dynamic DNA networks to regulate the activity of CRISPR/AsCas12a for enhanced DNA detection.}, journal = {Analytica chimica acta}, volume = {1335}, number = {}, pages = {343455}, doi = {10.1016/j.aca.2024.343455}, pmid = {39643309}, issn = {1873-4324}, mesh = {*Entropy ; *CRISPR-Cas Systems/genetics ; *DNA/chemistry ; *Biosensing Techniques/methods ; Nucleic Acid Hybridization ; CRISPR-Associated Proteins/metabolism/chemistry ; Nucleic Acid Amplification Techniques ; Bacterial Proteins/chemistry/metabolism/genetics ; Endodeoxyribonucleases/chemistry/metabolism ; }, abstract = {BACKGROUND: The non-enzyme autonomous DNA nanodevices have been developed to detect various analytes through the programmability of Watson-Crick base pairing. Nevertheless, by comparison with enzymatic biosensors, the usage of enzyme-free DNA networks to create biosensors for testing low amounts of targets is still subject to the finite number of cycles. Besides, these biosensors still require the incorporation of other amplification strategies to improve the sensitivity, which complicates the detection workflow and lacks of a uniform compatible system to respond to the target in one pot.<br><br>RESULTS: Here, we put forward a novel way for rapid and sensitive DNA diagnostic via EDN (entropy-driven dynamic network) coupling with CRISPR/AsCas12a-powered amplification. In the absence of the target, the autonomous hybridization among the substrate and crRNA is kinetically hindered by enclosing complementary regions, which leads to the loss of the activation function of Cas12a. On the contrary, the target initiates the EDN, reconfiguring the activator strand from a duplex to branch construction, which provides a valid means to adjust the hybridization with crRNA, thereby controlling the indiscriminate collateral cleavage activities of the CRISPR/AsCas12a. Compared with the traditional EDN, synergistic activation between the EDN and the CRISPR catalyst could dramatically enhance the detection signal of the target in one pot. Furthermore, the proposed approach provides universal platforms through the rational functional and structural design of DNA assembly modules.<br><br>SIGNIFICANCE AND NOVELTY: Overall, this target-triggered EDN switches the activator strand to regulate the activity of AsCas12a (called TERA), which showed nearly one orders of magnitude sensitivity than the conventional Cas12a alone assay, resulting in a devisable universal CRISPR sensing platform that favours the fast, robust and one-pot detection of nucleic molecules.}, }
@article {pmid39642875, year = {2025}, author = {Rottner, AK and Lundin, A and Li, S and Firth, M and Maresca, M and Sienski, G}, title = {Optimized prime editing of the Alzheimer's disease-associated APOE4 mutation.}, journal = {Stem cell reports}, volume = {20}, number = {1}, pages = {102372}, pmid = {39642875}, issn = {2213-6711}, mesh = {Humans ; *Gene Editing/methods ; *Alzheimer Disease/genetics ; *Apolipoprotein E4/genetics ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Mutation/genetics ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {Gene editing strategies to safely and robustly modify the Alzheimer's disease-associated APOE4 isoform are still lacking. Prime editing (PE) enables the precise introduction of genetic variants with minimal unintended editing and without donor templates. However, it requires optimization for each target site and has not yet been applied to APOE4 gene editing. Here, we screened PE guide RNA (pegRNA) parameters and PE systems for introducing the APOE4 variant and applied the optimized PE strategy to generate disease-relevant human induced pluripotent stem cell models. We show that introducing a single-nucleotide difference required for APOE4 correction inhibits PE activity. To advance efficient and robust genome engineering of precise genetic variants, we further present a reliable PE enrichment strategy based on diphtheria toxin co-selection. Our work provides an optimized and reproducible genome engineering pipeline to generate APOE4 disease models and outlines novel strategies to accelerate genome editing in cellular disease model generation.}, }
@article {pmid39642411, year = {2025}, author = {Li, H and Li, Y and Zhang, T and Liu, S and Song, C and Wang, K and Yan, W and Wang, Z and Yang, Q and Yang, X and Wang, H}, title = {Genome-wide CRISPR screen reveals specific role of type I interferon signaling pathway in Newcastle disease virus establishment of persistent infection.}, journal = {Veterinary microbiology}, volume = {300}, number = {}, pages = {110288}, doi = {10.1016/j.vetmic.2024.110288}, pmid = {39642411}, issn = {1873-2542}, mesh = {*Newcastle disease virus/immunology/genetics/physiology ; *Interferon Type I/genetics/metabolism/immunology ; Humans ; *Signal Transduction ; A549 Cells ; Animals ; *Newcastle Disease/virology/immunology ; Virus Replication ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Chickens/virology ; }, abstract = {Newcastle disease virus (NDV) is a potent oncolytic agent that exhibits sensitivity to a wide range of cancer cells. Unfortunately, some cancer cells are able to resist NDV-mediated oncolysis, by developing a persistent infection. The mechanism of persistency of infection remains poorly understood. In this study, a genome-wide CRISPR screen was conducted on non-small cell lung cancer cells (A549) to identify key host factors for NDV infection. Interestingly, a persistent infection was established in the surviving cells. CRISPR high-throughput screening results showed that members of the type I interferon signaling pathway (JAK1, STAT1, STAT2 and IRF9) were identified as top hits in the surviving cells. Further studies found that the type I IFN signaling pathway is intact in A549 cells, and a violent cytokine storm was induced after NDV infection. Both NDV infection and cytokine storm can induce cell death in A549 cells. We further blocked the type I interferon signaling pathway, and impaired type I interferon signaling pathway promoted NDV replication, but it did attenuate cell death induced by cytokine storm. Furthermore, persistent infection is more easily established in type I interferon signaling pathway-impaired A549 cells than in wild-type A549 cells. These findings suggest that the type I interferon signaling pathway plays a decisive role in persistent infection by regulating the antiviral immunity and cytokine storm inducing cell death.}, }
@article {pmid39642091, year = {2025}, author = {Basol, M and Ersoz-Gulseven, E and Ozaktas, H and Kalyoncu, S and Utine, CA and Cakan-Akdogan, G}, title = {Loss of carbohydrate sulfotransferase 6 function leads to macular corneal dystrophy phenotypes and skeletal defects in zebrafish.}, journal = {The FEBS journal}, volume = {292}, number = {2}, pages = {373-390}, pmid = {39642091}, issn = {1742-4658}, support = {219S943//T&#xfc;rkiye Bilimsel ve Teknolojik Ara&#x15f;t&#x131;rma Kurumu/ ; }, mesh = {Animals ; *Zebrafish/genetics ; *Sulfotransferases/genetics/metabolism ; Disease Models, Animal ; *Zebrafish Proteins/genetics/metabolism ; *Corneal Dystrophies, Hereditary/genetics/pathology/metabolism ; Carbohydrate Sulfotransferases/genetics ; Phenotype ; Humans ; CRISPR-Cas Systems ; Keratan Sulfate/metabolism ; Cornea/metabolism/pathology ; }, abstract = {The carbohydrate sulfotransferase 6 (chst6) gene is linked to macular corneal dystrophy (MCD), a rare disease that leads to bilateral blindness due to the accumulation of opaque aggregates in the corneal stroma. chst6 encodes for a keratan sulfate proteoglycan (KSPG) specific sulfotransferase. MCD patients lose sulfated KSPGs (cKS) in the cornea and the serum. The significance of serum cKS loss has not been understood. Zebrafish cornea structure is similar to that of humans and it contains high levels of sulfated cKS in the stroma. Here, zebrafish chst6 is shown to be expressed in the cornea and head structures of the embryos. An animal model of MCD is developed by generating chst6 mutant animals with CRISPR/Cas9-mediated gene editing. The dramatic decrease in cKS epitopes in the mutants was shown with ELISA and immunofluorescence. Morphological defects or alterations of jaw cartilage were detected in a minor fraction of the mutant larvae. Loss of cKS epitopes and morphological defects was fully rescued with wild-type chst6. Mutant adult zebrafish displayed all clinical manifestations of MCD, while a fraction also displayed jaw and skeleton defects. Opaque accumulations formed in the eye, which were alcian blue positive. Loss of cKS in the corneal stroma and a decrease in corneal thickness were shown. Interestingly, alteration of transforming growth factor beta-induced (BIGH3) expression which was not described in patients was also observed. This is the first report of an MCD model in a genetically tractable organism, providing a preclinical model and insight into the importance of KSPG sulfation for proper skeletal morphogenesis.}, }
@article {pmid39641617, year = {2024}, author = {Jiang, H and Qian, C and Deng, Y and Lv, X and Liu, Y and Li, A and Li, X}, title = {Novel Multimode Assay Based on Asymmetrically Competitive CRISPR and Raman Barcode Spectra for Multiple Hepatocellular Carcinoma Biomarkers Detection.}, journal = {Analytical chemistry}, volume = {96}, number = {50}, pages = {20004-20014}, doi = {10.1021/acs.analchem.4c04593}, pmid = {39641617}, issn = {1520-6882}, mesh = {*Spectrum Analysis, Raman/methods ; *Carcinoma, Hepatocellular/diagnosis/genetics ; Humans ; *Liver Neoplasms/diagnosis/genetics ; *Biomarkers, Tumor/genetics ; *MicroRNAs/analysis ; Gold/chemistry ; Metal Nanoparticles/chemistry ; CRISPR-Cas Systems/genetics ; }, abstract = {Commercial pregnancy test strips (PTS) possess the advantages of lower price, higher stability, and better repeatability and have been popularized to integrate with novel sensing strategies to detect other disease biomarkers, which accelerates the commercialization process of those novel sensing strategies. However, the current integration of novel sensing strategies into commercial PTS still faced the problems of insufficient quantification, low sensitivity, and lack of multiple detection capabilities. Hence, we proposed the concept of "visual classification recognition, spectral signal subdivision" for multiple hepatocellular carcinoma biomarkers (miRNA122 and miRNA233) detection with dual signals based on asymmetric competitive CRISPR (acCRISPR) and surface-enhanced Raman spectroscopy coupling with PTS, named the acCRISPR-PTS-SERS assay. In this assay, acCRISPR was used as a nonamplified cascaded signal amplification method to improve the sensitivity of detection. Two AuNPs-based core-shell Raman tags, each corresponding to different miRNA biomarkers, were used to achieve both visual recognition and spectral segmentation to enhance the quantification of PTS detection and the capability for multiple detection. Under the optimal conditions, the LOD for miRNA122 and miRNA223 were 10.36 and 4.65 fM, respectively. The sensitivity was enhanced by nearly 2 orders of magnitude. In the future, simultaneous hand-held detection for fingerprint barcodes of different cancers can be achieved with the assistance of a microfluidic chip and smartphone.}, }
@article {pmid39641224, year = {2025}, author = {Yu, J and Li, S and Xiong, B and Shen, Y and Guan, X and Zhu, Y and Fang, Y and Zhang, S and Ding, S and Liu, C and Yue, W and Yin, H and Xu, H}, title = {Probiotics Bi-Enzymatic Cascade Repair System for Editing the Inflammatory Microenvironment to Boost Probiotic Therapy in Inflammatory Bowel Disease.}, journal = {Advanced materials (Deerfield Beach, Fla.)}, volume = {37}, number = {4}, pages = {e2412429}, doi = {10.1002/adma.202412429}, pmid = {39641224}, issn = {1521-4095}, support = {82151318//National Natural Science Foundation of China/ ; 82430064//National Natural Science Foundation of China/ ; 82302206//National Natural Science Foundation of China/ ; 82102050//National Natural Science Foundation of China/ ; 82402268//National Natural Science Foundation of China/ ; 82302197//National Natural Science Foundation of China/ ; 82102048//National Natural Science Foundation of China/ ; 2022ZSQD07//Scientific Research and Development Fund of Zhongshan Hospital of Fudan University/ ; 2019LJ21//Shanghai Municipal Health Commission/ ; SHSLCZDZK03502//Shanghai Municipal Health Commission/ ; 23YF1441600//Shanghai Sailing Program/ ; 2023TQ0073//China Postdoctoral Science Foundation/ ; }, mesh = {*Probiotics/therapeutic use/pharmacology ; Animals ; *Inflammatory Bowel Diseases/therapy/pathology/microbiology ; Mice ; *Gene Editing/methods ; Lacticaseibacillus rhamnosus/physiology ; Humans ; Inflammation ; Gastrointestinal Microbiome ; CRISPR-Cas Systems ; Reactive Oxygen Species/metabolism ; Mice, Inbred C57BL ; }, abstract = {Inflammatory bowel disease presents significant treatment challenges owing to its complex pathology. Although probiotics have shown promise as a therapeutic option, their effectiveness is often limited by low concentrations at sites of inflammation, exacerbated by excessive reactive oxygen species and inflammatory triggers. To address this, an innovative cascade repair system is developed to enhance probiotic therapeutic impact by modulating the intestinal microenvironment. This system uses iMXene's catalytic properties to neutralize reactive oxygen species in the gut and its capacity to deliver the CRISPR/dCas9 gene editing system to activate the NLR family pyrin domain containing 12 genes, helping suppress inflammation. By promoting the colonization of Lactobacillus rhamnosus, the system inhibits inflammation pathways and supports the restoration of a balanced intestinal flora through a cascade repair mechanism. These findings demonstrate significant therapeutic benefits in experimental models, with improvements in the overall well-being of treated mice and effective repair of intestinal inflammation damage. This pioneering approach holds promise for inflammatory bowel disease treatment and opens new avenues for managing other inflammatory conditions, offering valuable insights and guidance for future research into inflammatory diseases.}, }
@article {pmid39641183, year = {2025}, author = {Li, Y and Yang, J and Zhang, Q and Zhang, K and Xue, Q and Liu, W and Ding, X and Niu, Z}, title = {CRISPR-Cas9 Mediated Gene Editing Platform Through Callus-to-Plant Regeneration and Functional Analysis of DoALA4─DoALA6 in Dendrobium officinale.}, journal = {Plant, cell &amp; environment}, volume = {48}, number = {5}, pages = {2923-2936}, doi = {10.1111/pce.15312}, pmid = {39641183}, issn = {1365-3040}, support = {//This work was supported by grants from the National Natural Science Foundation of China (Grant No. 32470384, 31900268 and 32070353) and Agricultural Science and Technology Independent Innovation Fund Project of Jiangsu Province (CX (22)3147)./ ; }, mesh = {*Dendrobium/genetics/physiology/growth &amp; development ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Regeneration/genetics ; Plants, Genetically Modified ; *Plant Proteins/genetics/metabolism ; Genes, Plant ; }, abstract = {Dendrobium orchids are well known for their great horticultural and medicinal values; however, the CRISPR/Cas9 gene editing system for Dendrobium species still needs to be improved. Therefore, this study aims to establish a CRISPR/Cas9-based functional validation system using Dendrobium officinale as a model species for the Dendrobium genus and to validate the DoALA4─DoALA6 genes, which may relate to growth and disease resistance. We first conducted a bioinformatics analysis of the P-type ATPase gene family in D. officinale, revealing the evolutionary diversity of P-type ATPase genes in orchids. Second, we inserted the GFP gene into the vector of CRISPR/Cas9 gene editing system to enhance the selection efficiency of genome-edited plants. Comparative analysis showed that different explants exhibited varying transformation efficiencies, ranging from 5% to 46.2%. Considering the regeneration capability, survival rate and gene editing efficiency, we selected callus as the transformation explant. Third, we used this editing system to generate DoALA4─DoALA6 mutants. Phenotypic observations of the mutants and inoculation of D. officinale with Sclerotium rolfsii indicated that DoALA4─DoALA6 are crucial for the growth of D. officinale and its resistance to southern blight disease. This efficient and stable CRISPR/Cas9 platform offers a foundation for further gene studies and Dendrobium breeding.}, }
@article {pmid39640855, year = {2024}, author = {Hu, S and Gan, M and Wei, Z and Shang, P and Song, L and Feng, J and Chen, L and Niu, L and Wang, Y and Zhang, S and Shen, L and Zhu, L and Zhao, Y}, title = {Identification of host factors for livestock and poultry viruses: genome-wide screening technology based on the CRISPR system.}, journal = {Frontiers in microbiology}, volume = {15}, number = {}, pages = {1498641}, pmid = {39640855}, issn = {1664-302X}, abstract = {Genome-wide CRISPR library screening technology is a gene function research tool developed based on the CRISPR/Cas9 gene-editing system. The clustered regularly interspaced short palindromic repeats/CRISPR-associated genes (CRISPR/Cas) system, considered the third generation of gene editing after zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), is widely used for screening various viral host factors. CRISPR libraries are classified into three main categories based on the different functions of Cas9 enzymes: CRISPR knockout (CRISPR KO) library screening, CRISPR transcriptional activation (CRISPRa) library screening, and CRISPR transcriptional interference (CRISPRi) library screening. Recently, genome-wide CRISPR library screening technology has been used to identify host factors that interact with viruses at various stages, including adsorption, endocytosis, and replication. By specifically modulating the expression of these host factors, it becomes possible to cultivate disease-resistant varieties, establish disease models, and design and develop vaccines, among other applications. This review provides an overview of the development and technical processes of genome-wide CRISPR library screening, as well as its applications in identifying viral host factors in livestock and poultry.}, }
@article {pmid39639565, year = {2024}, author = {Yuan, A and Sun, T and Chen, L and Zhang, D and Xie, W and Peng, H}, title = {CRISPR/Cas12a Corona Nanomachine for Detecting Circulating Tumor Nucleic Acids in Serum.}, journal = {Analytical chemistry}, volume = {96}, number = {50}, pages = {20074-20081}, doi = {10.1021/acs.analchem.4c04993}, pmid = {39639565}, issn = {1520-6882}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gold/chemistry ; Circulating Tumor DNA/blood/genetics ; Metal Nanoparticles/chemistry ; Endodeoxyribonucleases/chemistry/metabolism ; CRISPR-Associated Proteins/chemistry/metabolism ; Biosensing Techniques ; Limit of Detection ; Bacterial Proteins ; }, abstract = {Circulating tumor nucleic acids (CTNAs), which consist of cell-free DNA or RNA released from tumor cells, are utilized as potential biomarkers for diagnosing and managing tumor prognosis. There is a significant demand for developing a highly sensitive and reliable assay for CTNAs detection. In this study, we engineered a CRISPR/Cas12a corona nanomachine capable of detecting circulating tumor DNA and RNA in serum. This nanomachine consists of a protein shell incorporating Cas12a/crRNA ribonucleoprotein complexes and a scaffold AuNP core decorated with substrate ssDNA strands. The protective CRISPR corona shields the nucleic acid core from degradation by nuclease DNase/RNase, thereby enhancing the stability of the CRISPR/Cas12a corona nanomachine in biological fluids, even tolerating up to undiluted human serum and FBS. Upon encountering target CTNAs, the CRISPR/Cas12a is activated through the sequence-specific hybridization between crRNA and CTNAs. Subsequently, the activated CRISPR/Cas12a autonomously cleaves the collateral ssDNA substrates on AuNPs, releasing the fluorophore-labeled fragment and generating an increasing fluorescent signal. The CRISPR corona nanomachine was successfully employed to detect various CTNAs, including circulating tumor (ct)DNA/RNA (EGFR L858R) and microRNA-21, achieving a limit of detection of 0.14 pM for ctDNA and 1.0 pM for RNA. This CRISPR corona nanomachine enables simultaneous detection of both DNA and RNA in complex biological samples, offering a promising tool for early diagnosis.}, }
@article {pmid39639368, year = {2024}, author = {Yu, L and Zou, J and Hussain, A and Jia, R and Fan, Y and Liu, J and Nie, X and Zhang, X and Jin, S}, title = {Systemic evaluation of various CRISPR/Cas13 orthologs for knockdown of targeted transcripts in plants.}, journal = {Genome biology}, volume = {25}, number = {1}, pages = {307}, pmid = {39639368}, issn = {1474-760X}, support = {2023ZD04074//Science and Technology Innovation 2025 Major Project of Ningbo/ ; 2022ZD0402001-04//Collaborative Innovation Center for Modern Science and Technology and Industrial Development of Jiangxi Traditional Medicine/ ; 32325039//Science Fund for Distinguished Young Scholars of Guangdong Province/ ; 32272128//Innovative Research Group Project of the National Natural Science Foundation of China/ ; 2021hszd013//Hubei Hongshan Laboratory/ ; }, mesh = {*CRISPR-Cas Systems ; *Nicotiana/genetics ; *Gene Editing ; Gene Knockdown Techniques ; Gossypium/genetics ; Plants, Genetically Modified ; RNA, Plant/genetics/metabolism ; }, abstract = {BACKGROUND: CRISPR/Cas13 system, recognized for its compact size and specificity in targeting RNA, is currently employed for RNA degradation. However, the potential of various CRISPR/Cas13 subtypes, particularly concerning the knockdown of endogenous transcripts, remains to be comprehensively characterized in plants.<br><br>RESULTS: Here we present a full spectrum of editing profiles for seven Cas13 orthologs from five distinct subtypes: VI-A (LwaCas13a), VI-B (PbuCas13b), VI-D (RfxCas13d), VI-X (Cas13x.1 and Cas13x.2), and VI-Y (Cas13y.1 and Cas13y.2). A systematic evaluation of the knockdown effects on two endogenous transcripts (GhCLA and GhPGF in cotton) as well as an RNA virus (TMV in tobacco) reveals that RfxCas13d, Cas13x.1, and Cas13x.2 exhibit enhanced stability with editing efficiencies ranging from 58 to 80%, closely followed by Cas13y.1 and Cas13y.2. Notably, both Cas13x.1 and Cas13y.1 can simultaneously degrade two endogenous transcripts through a tRNA-crRNA cassette approach, achieving editing efficiencies of up to 50%. Furthermore, different Cas13 orthologs enable varying degrees of endogenous transcript knockdown with minimal off-target effects, generating germplasms that exhibit a diverse spectrum of mutant phenotypes. Transgenic tobacco plants show significant reductions in damage, along with mild oxidative stress and minimal accumulation of viral particles after TMV infection.<br><br>CONCLUSIONS: In conclusion, our study presents an efficient and reliable platform for transcriptome editing that holds promise for plant functional research and future crop improvement.}, }
@article {pmid39639057, year = {2024}, author = {Sakurai, T and Takei, N and Wei, Y and Hayashi, M and Kamiyoshi, A and Kawate, H and Watanabe, S and Sato, M and Shindo, T}, title = {Efficient genome editing of two-cell mouse embryos via modified CRISPR/Cas electroporation.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {30347}, pmid = {39639057}, issn = {2045-2322}, mesh = {Animals ; *Gene Editing/methods ; *Electroporation/methods ; *CRISPR-Cas Systems ; Mice ; *Embryo, Mammalian/metabolism ; Female ; Gene Knock-In Techniques/methods ; Zygote/metabolism ; }, abstract = {Creating genetically modified (GM) animals using CRISPR/Cas mediated through the electroporation of two-cell stage embryos, rather than fertilized eggs, holds considerable potential. The full potential of genome editing using two-cell stage embryos is only beginning to be explored. We developed an improved electroporation method to prevent blastomere fusion in two-cell-stage embryos, enabling efficient genome editing. Using this method, we demonstrated that the indel mutation rates and ssODN knock-in (KI) efficiencies in two-cell-stage embryos are comparable to those in fertilized eggs, with a tendency for higher efficiency in long DNA KI. This study highlights the potential value of two-cell-stage embryos and provides enhanced animal model production opportunities. Furthermore, realizing genome editing in two-cell-stage embryos extends the editing timeframe from fertilized egg to two-cell-stage embryo, offering promising avenues for future research in embryo genome editing techniques.}, }
@article {pmid39638158, year = {2025}, author = {Garg, P and Singhal, G and Pareek, S and Kulkarni, P and Horne, D and Nath, A and Salgia, R and Singhal, SS}, title = {Unveiling the potential of gene editing techniques in revolutionizing Cancer treatment: A comprehensive overview.}, journal = {Biochimica et biophysica acta. Reviews on cancer}, volume = {1880}, number = {1}, pages = {189233}, doi = {10.1016/j.bbcan.2024.189233}, pmid = {39638158}, issn = {1879-2561}, support = {P30 CA033572/CA/NCI NIH HHS/United States ; }, mesh = {Humans ; *Gene Editing/methods ; *Neoplasms/genetics/therapy ; CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; Precision Medicine/methods ; Animals ; }, abstract = {Gene editing techniques have emerged as powerful tools in biomedical research, offering precise manipulation of genetic material with the potential to revolutionize cancer treatment strategies. This review provides a comprehensive overview of the current landscape of gene editing technologies, including CRISPR-Cas systems, base editing, prime editing, and synthetic gene circuits, highlighting their applications and potential in cancer therapy. It discusses the mechanisms, advantages, and limitations of each gene editing approach, emphasizing their transformative impact on targeting oncogenes, tumor suppressor genes, and drug resistance mechanisms in various cancer types. The review delves into population-level interventions and precision prevention strategies enabled by gene editing technologies, including gene drives, synthetic gene circuits, and precision prevention tools, for controlling cancer-causing genes, targeting pre-cancerous lesions, and implementing personalized preventive measures. Ethical considerations, regulatory challenges, and future directions in gene editing research for cancer treatment are also addressed. This review highlights how gene editing could revolutionize precision medicine by enhancing patient care and advancing cancer treatments with targeted, personalized methods. For these benefits to be fully realized, collaboration among researchers, doctors, regulators, and patient advocates is crucial in fighting cancer and meeting clinical needs.}, }
@article {pmid39638020, year = {2025}, author = {Qiu, Y and Chen, S and Li, J and Liu, DA and Hu, R and Xu, Y and Chen, K and Yuan, J and Zhang, X and Li, X}, title = {Crispr-cas biosensing for rapid detection of viral infection.}, journal = {Clinica chimica acta; international journal of clinical chemistry}, volume = {567}, number = {}, pages = {120071}, doi = {10.1016/j.cca.2024.120071}, pmid = {39638020}, issn = {1873-3492}, mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Virus Diseases/diagnosis ; }, abstract = {With the frequent outbreaks of viral diseases globally, accurate and rapid diagnosis of viral infections is of significant importance for disease prevention and control. The CRISPR-Cas combined biosensing strategy, as an emergent nucleic acid detection technology, exhibits notable advantages including high specificity, elevated sensitivity, operational simplicity, and cost-effectiveness, thereby demonstrating significant potential in the domain of rapid viral diagnostics. This paper summarizes the principles of the CRISPR-Cas system, the novel biotechnologies, and the latest research progress in virus detection using the combined biosensing strategy. Additionally, this paper discusses the challenges faced by CRISPR-Cas biosensing strategies and outlines future development directions, which provides a reference for further research and clinical applications in the rapid diagnosis of viral infections.}, }
@article {pmid39637860, year = {2024}, author = {Ursch, LT and Müschen, JS and Ritter, J and Klermund, J and Bernard, BE and Kolb, S and Warmuth, L and Andrieux, G and Miller, G and Jiménez-Muñoz, M and Theis, FJ and Boerries, M and Busch, DH and Cathomen, T and Schumann, K}, title = {Modulation of TCR stimulation and pifithrin-α improve the genomic safety profile of CRISPR-engineered human T cells.}, journal = {Cell reports. Medicine}, volume = {5}, number = {12}, pages = {101846}, pmid = {39637860}, issn = {2666-3791}, mesh = {Humans ; *T-Lymphocytes/immunology/drug effects ; *Receptors, Antigen, T-Cell/genetics/immunology/metabolism ; *CRISPR-Cas Systems/genetics ; *Benzothiazoles/pharmacology ; *Lymphocyte Activation/drug effects/immunology ; *Toluene/analogs &amp; derivatives/pharmacology ; *Gene Editing/methods ; Cell Proliferation/drug effects ; Receptors, Chimeric Antigen/genetics/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {CRISPR-engineered chimeric antigen receptor (CAR) T cells are at the forefront of novel cancer treatments. However, several reports describe the occurrence of CRISPR-induced chromosomal aberrations. So far, measures to increase the genomic safety of T cell products focused mainly on the components of the CRISPR-Cas9 system and less on T cell-intrinsic features, such as their massive expansion after T cell receptor (TCR) stimulation. Here, we describe driving forces of indel formation in primary human T cells. Increased T cell activation and proliferation speed correlate with larger deletions. Editing of non-activated T cells reduces the risk of large deletions with the downside of reduced knockout efficiencies. Alternatively, the addition of the small-molecule pifithrin-α limits large deletions, chromosomal translocations, and aneuploidy in a p53-independent manner while maintaining the functionality of CRISPR-engineered T cells, including CAR T cells. Controlling T cell activation and pifithrin-α treatment are easily implementable strategies to improve the genomic integrity of CRISPR-engineered T cells.}, }
@article {pmid39637703, year = {2025}, author = {Thangaraj, A and Kaul, R and Sharda, S and Kaul, T}, title = {Revolutionizing cotton cultivation: A comprehensive review of genome editing technologies and their impact on breeding and production.}, journal = {Biochemical and biophysical research communications}, volume = {742}, number = {}, pages = {151084}, doi = {10.1016/j.bbrc.2024.151084}, pmid = {39637703}, issn = {1090-2104}, mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/genetics/growth &amp; development ; *Gene Editing/methods ; Genome, Plant ; *Gossypium/genetics/growth &amp; development ; *Plant Breeding/methods ; Plants, Genetically Modified/genetics/growth &amp; development ; }, abstract = {Cotton (Gossypium hirsutum L.), a vital global cash crop, significantly impacts both the agricultural and industrial sectors, providing essential fiber for textiles and valuable byproducts such as cottonseed oil and animal feed. The cultivation of cotton supports millions of livelihoods worldwide, particularly in developing regions, making it a cornerstone of rural economies. Despite its importance, cotton production faces numerous challenges, including biotic stresses from pests and diseases, and abiotic stresses like drought, salinity, and extreme temperatures. These challenges necessitate innovative solutions to ensure sustainable production. Genome editing technologies, particularly CRISPR/Cas9, have revolutionized cotton breeding by enabling precise genetic modifications. These advancements hold promise for developing cotton varieties with enhanced resistance to pests, diseases, and environmental stresses. Early genome editing tools like ZFNs and TALENs paved the way for more precise modifications but were limited by complexity and cost. The introduction of CRISPR/Cas-based technology with its simplicity and efficiency, has dramatically transformed the field, making it the preferred tool for genome editing in crops. Improved version of the technology like CRISPR/Cas12a, CRISPR/Cas13, base and prime editing, developed from CRISPR/Cas systems, provide additional tools with distinct mechanisms, further expanding their potential applications in crop improvement. This comprehensive review explores the impact of genome editing on cotton breeding and production. It discusses the technical challenges, including off-target effects and delivery methods for genome editing components, and highlights ongoing research efforts to overcome these hurdles. The review underscores the potential of genome editing technologies to revolutionize cotton cultivation, enhancing yield, quality, and resilience, ultimately contributing to a sustainable future for the cotton industry.}, }
@article {pmid39637570, year = {2025}, author = {Ding, L and Cao, S and Bai, L and He, S and He, L and Wang, Y and Wu, Y and Yu, S}, title = {Versatile fluorescence biosensors based on CRISPR/Cas12a for determination of site-specific DNA methylation from blood and tissues.}, journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy}, volume = {329}, number = {}, pages = {125520}, doi = {10.1016/j.saa.2024.125520}, pmid = {39637570}, issn = {1873-3557}, mesh = {*DNA Methylation ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Endodeoxyribonucleases/metabolism ; *DNA/blood/genetics ; Spectrometry, Fluorescence/methods ; Bacterial Proteins ; CRISPR-Associated Proteins ; }, abstract = {The identification of DNA methylation at specific sites is crucial for the early detection of cancer since DNA methylation is intimately associated to the occurrence and development of cancer. Herein, two types of sensors that can detect site-specific DNA methylation were developed to meet practical requirements using methylation sensitive restriction endonuclease and CRISPR/Cas12a. To accomplish rapid detection of target, an AciI-mediated CRISPR/Cas12a assay was developed by coupling AciI to recognize DNA methylation with Cas12a to identify site-specific DNA. Since protospacer adjacent motif (PAM)-dependent endonuclease activity and trans-cleavage activity of Cas12a, it is possible to detect site-specific DNA methylation within 2 h with high specificity and acceptable sensitivity. To satisfy the needs of trace target detection, we developed an GlaI-strand displacement amplification (SDA) assisted CRISPR/Cas12a system. The system converts double-stranded methylated DNA to abundant single-stranded by GlaI and SDA. Then, the combination of SDA and CRISPR/Cas12a enable cascades amplification of signal. The approach can therefore be used to detect methylation at different specified sites, even those without PAM, and can increase sensitivity with a detection limit down to 8.19 fM. Importantly, the assay can distinguish between colorectal cancer and precancerous tissue, as well as identify colorectal patients and healthy people. This study provides a new avenue for the development of new biosensors for methylation analysis, and the two methods devised have the potential to meet the multiple requirements of site-specific methylation testing in various clinical settings.}, }
@article {pmid39636137, year = {2024}, author = {Nakata, M and Ueno, M and Kikuchi, Y and Iwami, M and Takayanagi-Kiya, S and Kiya, T}, title = {CRISPR/Cas9- and Single-Stranded ODN-Mediated Knock-In in Silkworm Bombyx mori.}, journal = {Zoological science}, volume = {41}, number = {6}, pages = {540-547}, doi = {10.2108/zs240019}, pmid = {39636137}, issn = {0289-0003}, mesh = {Animals ; *Bombyx/genetics ; *CRISPR-Cas Systems ; *Gene Knock-In Techniques ; Oligodeoxyribonucleotides/genetics ; Gene Editing ; }, abstract = {Although genome editing techniques have made significant progress, introducing exogenous genes into the genome through knock-in remains a challenge in many organisms. In silkworm Bombyx mori, TALEN-mediated knock-in methods have been established. However, difficulties in construction and limitations of the target sequence have hindered the application of these methods. In the present study, we verified several CRISPR/Cas9-mediated knock-in methods to expand the application of gene knock-in techniques and found that the short single-stranded oligodeoxynucleotide (ssODN)-mediated method is the most effective in silkworms. Using ssODN-mediated methods, we established knock-in silkworm strains that harbor an attP sequence, a 50 bp phiC31 integrase recognition site, at either the BmHr38 (Hormone receptor 38) or Bmdsx (doublesex) locus. Additionally, we found that the long ssODN (lsODN)-mediated method successfully introduced the GAL4 gene at the doublesex locus in embryos. The present study provides valuable information on CRISPR/Cas9-mediated knock-in methods in silkworms, expanding the utility of genome editing techniques in insects and paving the way for analyzing gene and genome function in silkworms.}, }
@article {pmid39633273, year = {2024}, author = {Peng, Y and Li, J and Niu, K and Wang, M and Chen, Y and Tong, C and Feng, Q}, title = {Bmlark is essential for embryonic development.}, journal = {BMC genomics}, volume = {25}, number = {1}, pages = {1179}, pmid = {39633273}, issn = {1471-2164}, support = {2021M691096//China&#xa0;Postdoctoral Science Foundation/ ; 32100383//Chinese National Natural Science Foundation/ ; }, mesh = {Animals ; *Bombyx/genetics/embryology/metabolism ; *Embryonic Development/genetics ; Gene Expression Regulation, Developmental ; Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Mutation ; Transcription Factors/genetics/metabolism ; Transcriptome ; }, abstract = {BACKGROUND: Transcription factor lark has been demonstrated to play multiple functions in Drosophila, but the function of this gene in embryonic development remains to be elucidated.<br><br>RESULTS: In this study, the CRISPR/Cas9 gene-editing method was used to construct a Bmlark mutant strain of Bombyx mori to investigate the roles of this gene. The results showed that the homozygous mutant Bmlark[-/-] was lethal. The Bmlark[-/-] embryos showed obvious developmental defects, such as defective sclerotization and melanization of the exoskeleton. A transcriptomic comparison of Bmlark[-/-] and wild-type embryos showed that the differentially expressed genes were mainly enriched in the structure and metabolic processes of chitin and cuticles. While the expression levels of chitin metabolism-related enzyme genes did not significantly change, in the mutant embryos compared to the wild-type embryos, the expression levels of 63 putative cuticle protein genes showed significant differences. Among which, 35 genes were downregulated and 28 genes were upregulated. The expression levels of the transcription factor BmPOUM2 and eight wing disc cuticle protein genes (WCP) also changed. BmPOUM2, WCP5, WCP9, WCP10, WCP11 were downregulated and WCP1, WCP2, WCP3, WCP6 were upregulated in Bmlark[-/-] embryos. While the expression level of TH in the tyrosine-mediated pigmentation pathway was upregulated in the mutant embryos, the expression levels of the four key pigment synthesis genes DDC, aaNAT, Laccase2A, and yellow-f2 were significantly downregulated.<br><br>CONCLUSIONS: The expression levels of 63 putative cuticle protein genes, eight WCP genes, and five pigment synthesis genes significantly changed in Bmlark mutant B. mori compared to those of the wildtype. These results suggest that Bmlark is essential for normal development of cuticle and tyrosine-mediated melanization in silkworm embryos.}, }
@article {pmid39633151, year = {2024}, author = {Wu, WY and Adiego-Pérez, B and van der Oost, J}, title = {Biology and applications of CRISPR-Cas12 and transposon-associated homologs.}, journal = {Nature biotechnology}, volume = {42}, number = {12}, pages = {1807-1821}, pmid = {39633151}, issn = {1546-1696}, mesh = {*CRISPR-Cas Systems/genetics ; *DNA Transposable Elements/genetics ; *Gene Editing/methods ; CRISPR-Associated Proteins/genetics/metabolism ; Humans ; }, abstract = {CRISPR-associated Cas12 proteins are a highly variable collection of nucleic acid-targeting proteins. All Cas12 variants use RNA guides and a single nuclease domain to target complementary DNA or, in rare cases, RNA. The high variability of Cas12 effectors can be explained by a series of independent evolution events from different transposon-associated TnpB-like ancestors. Despite basic structural and functional similarities, this has resulted in unprecedented variation of the Cas12 effector proteins in terms of size, domain composition, guide structure, target identity and interference strategy. In this Review, we compare the unique molecular features of natural and engineered Cas12 and TnpB variants. Furthermore, we provide an overview of established genome editing and diagnostic applications and discuss potential future directions.}, }
@article {pmid39633035, year = {2024}, author = {Rocha, BMO and Sabino, YNV and de Almeida, TC and Palacio, FB and Rotta, IS and Dias, VC and da Silva, VL and Diniz, CG and Azevedo, VAC and Brenig, B and Soares, SC and Paiva, AD and Medeiros, JD and Machado, ABF}, title = {Unlocking Probiotic Potential: Genomic Insights into Weissella paramesenteroides UFTM 2.6.1.}, journal = {Probiotics and antimicrobial proteins}, volume = {}, number = {}, pages = {}, pmid = {39633035}, issn = {1867-1314}, abstract = {Weissella, a genus of lactic acid bacteria, has diverse beneficial attributes including probiotic activity and biotechnological applications. Therefore, the investigation of the Weissella genus has garnered growing interest. In this study, we sequenced the complete genome of Weissella paramesenteroides UFTM 2.6.1 isolated from unpasteurized cow's milk from the Triângulo Mineiro region and performed probiogenomic analyses. Taxonomic characterization confirmed the identity of W. paramesenteroides. The genome comprises 1926 protein-coding genes, mainly related to cell metabolism, information storage and processing, and cellular processes and signaling. Ninety-nine unique genes associated with probiotic functions were identified in the genome of W. paramesenteroides UFTM 2.6.1, including genes involved in stress response, bacterial persistence in the gastrointestinal tract, and biosynthesis of vitamins. In silico analysis of bacteriocin-related genes identified Pediocin, and subsequent in vitro testing confirmed that W. paramesenteroides UFTM 2.6.1 exhibits antimicrobial activity against Listeria spp. Genomic characterization revealed the presence of the replicon pLCK4 and four prophage regions, one of which was intact. Moreover, no CRISPR-Cas array or associated Cas proteins were found, along with an absence of resistance and virulence genes, suggesting a safety aspect of the evaluated strain. Pan-genome analysis unveiled 204 exclusive genes in the genome of W. paramesenteroides UFTM 2.6.1, which includes metabolism and stress-associated genes. In general, the results indicate probiotic potential of W. paramesenteroides UFTM 2.6.1. Further studies are required to ensure the safety and beneficial effects of this bacterium in vivo, aiming for future applications in the food industry and animal and human medicine.}, }
@article {pmid39633028, year = {2025}, author = {Yin, JA and Frick, L and Scheidmann, MC and Liu, T and Trevisan, C and Dhingra, A and Spinelli, A and Wu, Y and Yao, L and Vena, DL and Knapp, B and Guo, J and De Cecco, E and Ging, K and Armani, A and Oakeley, EJ and Nigsch, F and Jenzer, J and Haegele, J and Pikusa, M and Täger, J and Rodriguez-Nieto, S and Bouris, V and Ribeiro, R and Baroni, F and Bedi, MS and Berry, S and Losa, M and Hornemann, S and Kampmann, M and Pelkmans, L and Hoepfner, D and Heutink, P and Aguzzi, A}, title = {Arrayed CRISPR libraries for the genome-wide activation, deletion and silencing of human protein-coding genes.}, journal = {Nature biomedical engineering}, volume = {9}, number = {1}, pages = {127-148}, pmid = {39633028}, issn = {2157-846X}, mesh = {Humans ; *Gene Silencing ; *Genome, Human/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Library ; *CRISPR-Cas Systems/genetics ; Plasmids/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; HEK293 Cells ; Gene Deletion ; }, abstract = {Arrayed CRISPR libraries extend the scope of gene-perturbation screens to non-selectable cell phenotypes. However, library generation requires assembling thousands of vectors expressing single-guide RNAs (sgRNAs). Here, by leveraging massively parallel plasmid-cloning methodology, we show that arrayed libraries can be constructed for the genome-wide ablation (19,936 plasmids) of human protein-coding genes and for their activation and epigenetic silencing (22,442 plasmids), with each plasmid encoding an array of four non-overlapping sgRNAs designed to tolerate most human DNA polymorphisms. The quadruple-sgRNA libraries yielded high perturbation efficacies in deletion (75-99%) and silencing (76-92%) experiments and substantial fold changes in activation experiments. Moreover, an arrayed activation screen of 1,634 human transcription factors uncovered 11 novel regulators of the cellular prion protein PrP[C], screening with a pooled version of the ablation library led to the identification of 5 novel modifiers of autophagy that otherwise went undetected, and 'post-pooling' individually produced lentiviruses eliminated template-switching artefacts and enhanced the performance of pooled screens for epigenetic silencing. Quadruple-sgRNA arrayed libraries are a powerful and versatile resource for targeted genome-wide perturbations.}, }
@article {pmid39632651, year = {2025}, author = {Xiao, L and Qin, B and Zhang, X and Yao, Y and Dou, X and Liu, Y and Duan, X}, title = {Precision Control of Cell Type-Specific Behavior via RNA Sensing and Editing.}, journal = {Small methods}, volume = {9}, number = {4}, pages = {e2400952}, doi = {10.1002/smtd.202400952}, pmid = {39632651}, issn = {2366-9608}, support = {2021YFA0911600//National Key R&amp;D Program of China/ ; 82471079//National Natural Science Foundation of China/ ; 2023TP2225//Hunan Engineering Research Center for Glaucoma with Artificial Intelligence in Diagnosis and Application of New Materials/ ; 2023JJ70014//Natural Science Foundation of Hunan Province, China/ ; kq2208495//Changsha Municipal Natural Science Foundation/ ; AR2206D5//Science and Technology Foundation of Aier Eye Hospital Group, China/ ; AIG2305D03//Science and Technology Foundation of Aier Eye Hospital Group, China/ ; RCJC20221008092723011//Shenzhen Science and Technology Innovation Program/ ; JCYJ20220818102001002//Shenzhen Science and Technology Innovation Program/ ; 2023yjlcyj006//Shenzhen High-level Hospital Construction Fund/ ; }, mesh = {Humans ; *Retinoblastoma/genetics/therapy/pathology ; *CRISPR-Cas Systems/genetics ; Animals ; *RNA/genetics/metabolism ; Cell Line, Tumor ; *Gene Editing/methods ; *RNA Editing ; Cell Proliferation/genetics ; Apoptosis/genetics ; Mice ; }, abstract = {In the realms of bioengineering and biopharmaceuticals, there exists a critical demand for advanced genetic tools that can interact with specific cell signaling pathways to accurately identify and target various cell types. This research introduces the innovative CRISPR-ADAReader system, which enables precise manipulation of cell activity through sensing target RNA. Featuring both positive and negative feedback loops, the system allows for tailored regulation across different cell types in response to various internal signals, showcasing exceptional programmability, specificity, and sensitivity. By choosing distinct RNAs as activation signals, the CRISPR-ADAReader efficiently monitors and alters targeted cell behaviors. In a case study focusing on retinoblastoma treatment, the system distinctively initiates positive feedback and self-silencing actions by detecting MCYN and Rb transcripts, thus safeguarding normal retinal pigment epithelial cells while promoting apoptosis in cancer cells. Moreover, the CRISPR-ADAReader demonstrates significant anti-tumor effectiveness in live models, markedly reducing retinoblastoma cell proliferation through the activation of several cancer-suppression pathways, outperforming the capabilities of the ADAR-sensor. Notably, the system also shows an excellent in vivo safety profile. In conclusion, the CRISPR-ADAReader system represents a groundbreaking method for the detection and editing of RNA, offering a potent instrument for the customized and precise governance of cell behavior.}, }
@article {pmid39631395, year = {2025}, author = {Kimble, MT and Sane, A and Reid, RJD and Johnson, MJ and Rothstein, R and Symington, LS}, title = {Repair of replication-dependent double-strand breaks differs between the leading and lagging strands.}, journal = {Molecular cell}, volume = {85}, number = {1}, pages = {61-77.e6}, pmid = {39631395}, issn = {1097-4164}, support = {R35 GM118180/GM/NIGMS NIH HHS/United States ; R35 GM126997/GM/NIGMS NIH HHS/United States ; T32 CA265828/CA/NCI NIH HHS/United States ; }, mesh = {*DNA Breaks, Double-Stranded ; *DNA Replication ; *Saccharomyces cerevisiae/genetics/metabolism ; Saccharomyces cerevisiae Proteins/metabolism/genetics ; DNA End-Joining Repair ; DNA Repair ; Nucleosomes/metabolism/genetics ; DNA, Fungal/genetics/metabolism ; S Phase ; Homologous Recombination ; Histones/metabolism/genetics ; Recombinational DNA Repair ; CRISPR-Associated Protein 9/metabolism/genetics ; CRISPR-Cas Systems ; }, abstract = {Single-strand breaks (SSBs) are one of the most commonly occurring endogenous lesions with the potential to give rise to cytotoxic double-strand breaks (DSBs) during DNA replication. To investigate how replication-dependent DSBs are repaired, we employed Cas9 nickase (nCas9) to generate site- and strand-specific nicks in the budding yeast genome. We found that nCas9-induced nicks are converted to mostly double-ended DSBs during S phase. Repair of replication-associated DSBs requires homologous recombination (HR) and is independent of classical non-homologous end joining. Consistent with a strong bias to repair these lesions using a sister-chromatid template, we observed minimal induction of inter-chromosomal HR by nCas9. In a genome-wide screen to identify factors necessary for the repair of replication-dependent DSBs, we recovered components of the replication-coupled nucleosome assembly (RCNA) pathway. Our findings suggest that the RCNA pathway is especially important to repair DSBs arising from nicks in the leading-strand template through acetylation of histone H3K56.}, }
@article {pmid39631148, year = {2025}, author = {Banta, AB and Cuellar, RA and Nadig, N and Davis, BC and Peters, JM}, title = {The promise of CRISPR-associated transposons for bacterial functional genomics.}, journal = {Current opinion in microbiology}, volume = {83}, number = {}, pages = {102563}, pmid = {39631148}, issn = {1879-0364}, support = {R35 GM150487/GM/NIGMS NIH HHS/United States ; T32 GM135066/GM/NIGMS NIH HHS/United States ; }, mesh = {*DNA Transposable Elements ; *Genomics/methods ; *CRISPR-Cas Systems ; *Genome, Bacterial ; Vibrio cholerae/genetics ; *Bacteria/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {CRISPR-associated transposons (CASTs) are naturally occurring amalgamations of CRISPR-Cas machinery and Tn7-like transposons that direct site-specific integration of transposon DNA via programmable guide RNAs. Although the mechanisms of CAST-based transposition have been well studied at the molecular and structural level, CASTs have yet to be broadly applied to bacterial genome engineering and systematic gene phenotyping (i.e. functional genomics) - likely due to their relatively recent discovery. Here, we describe the function and applications of CASTs, focusing on well-characterized systems, including the type I-F CAST from Vibrio cholerae (VcCAST) and type V-K CAST from Scytonema hofmanni (ShCAST). Further, we discuss the potentially transformative impact of targeted transposition on bacterial functional genomics by proposing genome-scale extensions of existing CAST tools.}, }
@article {pmid39630114, year = {2025}, author = {Guan, X and Yang, R and Zhang, J and Moon, J and Hou, C and Guo, C and Avery, L and Scarola, D and Roberts, DS and LaSala, R and Liu, C}, title = {Programmable Multiplexed Nucleic Acid Detection by Harnessing Specificity Defect of CRISPR-Cas12a.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {12}, number = {4}, pages = {e2411021}, pmid = {39630114}, issn = {2198-3844}, support = {U01 AI148306/AI/NIAID NIH HHS/United States ; U01 CA269147/CA/NCI NIH HHS/United States ; U01CA269147/NH/NIH HHS/United States ; U01AI148306/NH/NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Papillomavirus Infections/diagnosis/virology ; Sensitivity and Specificity ; Uterine Cervical Neoplasms/virology/diagnosis/genetics ; Human papillomavirus 16/genetics ; Female ; DNA, Viral/genetics ; Papillomaviridae/genetics ; *Nucleic Acids/genetics/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {CRISPR-Cas12a works like a sophisticated algorithm in nucleic acid detection, yet its challenge lies in sometimes failing to distinguish targets with mismatches due to its specificity limitations. Here, the mismatch profiles, including the quantity, location, and type of mismatches in the CRISPR-Cas12a reaction, are investigated and its various tolerances to mismatches are discovered. By harnessing the specificity defect of the CRISPR-Cas12a enzyme, a dual-mode detection strategy is designed, which includes approximate matching and precise querying of target sequences and develop a programmable multiplexed nucleic acid assay. With the assay, 14 high-risk human papillomavirus (HPV) subtypes are simultaneously detected, collectively responsible for 99% of cervical cancer cases, with attomolar sensitivity. Specifically, the assay not only distinguishes HPV16 and HPV18, the two most common subtypes but also detects 12 other high-risk pooled HPV subtypes. To enable low-cost point-of-care testing, the assay is incorporated into a paper-based microfluidic chip. Furthermore, the clinical performance of the paper-based microfluidic chip is validated by testing 75 clinical swab samples, achieving performance comparable to that of PCR. This programmable multiplexed nucleic acid assay has the potential to be widely applied for sensitive, specific, and simultaneous detection of different pathogens.}, }
@article {pmid39630060, year = {2025}, author = {Zhou, Y and Shi, L and Li, X and Wei, S and Ye, X and Gao, Y and Zhou, Y and Cheng, L and Cheng, L and Duan, F and Li, M and Zhang, H and Qian, Q and Zhou, W}, title = {Genetic engineering of RuBisCO by multiplex CRISPR editing small subunits in rice.}, journal = {Plant biotechnology journal}, volume = {23}, number = {3}, pages = {731-749}, pmid = {39630060}, issn = {1467-7652}, support = {2023ZD0407203//Biological Breeding-National Science and Technology Major Project/ ; 32330079//Program of National Natural Science Foundation of China/ ; 32472040//General&#xa0;Program&#xa0;of&#xa0;National Natural Science Foundation of China/ ; }, mesh = {*Oryza/genetics/enzymology/metabolism ; *Ribulose-Bisphosphate Carboxylase/genetics/metabolism ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Photosynthesis/genetics ; Plants, Genetically Modified ; *Genetic Engineering/methods ; Plant Proteins/genetics/metabolism ; Mutation ; }, abstract = {Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is required for photosynthetic carbon assimilation, as it catalyses the conversion of inorganic carbon into organic carbon. Despite its importance, RuBisCO is inefficient; it has a low catalytic rate and poor substrate specificity. Improving the catalytic performance of RuBisCO is one of the key routes for enhancing plant photosynthesis. As the basic subunit of RuBisCO, RbcS affects the catalytic properties and plays a key role in stabilizing the structure of holoenzyme. Yet, the understanding of functions of RbcS in crops is still largely unknown. Toward this end, we employed CRISPR-Cas9 technology to randomly edit five rbcS genes in rice (OsrbcS1-5), generating a series of knockout mutants. The mutations of predominant rbcS genes in rice photosynthetic tissues, OsrbcS2-5, conferred inhibited growth, delayed heading and reduced yield in the field conditions, accompanying with lower RuBisCO contents and activities and significantly reduced photosynthetic efficiency. The retarded phenotypes were severer caused by multiple mutations. In addition, we revealed that these mutants had fewer chloroplasts and starch grains and a lower sugar content in the shoot base, resulting in fewer rice tillers. Further structural analysis of the mutated RuBisCO enzyme in one rbcs2,3,5 mutant line uncovered no significant differences from the wild-type protein, indicating that the mutations of rbcS did not compromise the protein assembly or the structure. Our findings generated a mutant pool with genetic diversities, which offers a valuable resource and novel insights into unravelling the mechanisms of RuBisCO in rice. The multiplex genetic engineering approach of this study provides an effective and feasible strategy for RuBisCO modification in crops, further facilitate the photosynthesis improvement and sustainable crop production.}, }
@article {pmid39629326, year = {2024}, author = {Ishino, Y}, title = {A novel strategy to protect prokaryotic cells from virus infection.}, journal = {Engineering microbiology}, volume = {4}, number = {2}, pages = {100153}, pmid = {39629326}, issn = {2667-3703}, abstract = {The recent discovery of the CRISPR-Cas-mediated acquired immunity system highlights the fact that our knowledge of phage/virus defense mechanisms encoded in bacterial and archaeal genomes is far from complete. Indeed, new prokaryotic immune systems are now continually being discovered. A recent report described a novel glycosylase that recognizes α-glycosyl-hydroxymethyl cytosin (α-Glu-hmC), a modified base observed in the T4 phage genome, where it produces an abasic site, thereby inhibiting the phage propagation.}, }
@article {pmid39628591, year = {2024}, author = {Le, Y and Zhang, M and Wu, P and Wang, H and Ni, J}, title = {Biofuel production from lignocellulose via thermophile-based consolidated bioprocessing.}, journal = {Engineering microbiology}, volume = {4}, number = {4}, pages = {100174}, pmid = {39628591}, issn = {2667-3703}, abstract = {The depletion of fossil fuels and their impact on the environment have led to efforts to develop alternative sustainable fuels. While biofuel derived from lignocellulose is considered a sustainable, renewable, and green energy source, enhancing biofuel production and achieving a cost-effective bioconversion of lignocellulose at existing bio-refineries remains a challenge. Consolidated bioprocessing (CBP) using thermophiles can simplify this operation by integrating multiple processes, such as hydrolytic enzyme production, lignocellulose degradation, biofuel fermentation, and product distillation. This paper reviews recent developments in the conversion of lignocellulose to biofuel using thermophile-based CBP. First, advances in thermostable enzyme and thermophilic lignocellulolytic microorganism discovery and development for lignocellulosic biorefinery use are outlined. Then, several thermophilic CBP candidates and thermophilic microbes engineered to drive CBP of lignocellulose are reviewed. CRISPR/Cas-based genome editing tools developed for thermophiles are also highlighted. The potential applications of the Design-Build-Test-Learn (DBTL) synthetic biology strategy for designing and constructing thermophilic CBP hosts are also discussed in detail. Overall, this review illustrates how to develop highly sophisticated thermophilic CBP hosts for use in lignocellulosic biorefinery applications.}, }
@article {pmid39628447, year = {2025}, author = {Carlsen, FM and Westberg, I and Johansen, IE and Andreasson, E and Petersen, BL}, title = {Strategies and Protocols for Optimized Genome Editing in Potato.}, journal = {The CRISPR journal}, volume = {8}, number = {1}, pages = {37-50}, doi = {10.1089/crispr.2024.0068}, pmid = {39628447}, issn = {2573-1602}, mesh = {*Solanum tuberosum/genetics/growth &amp; development ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Plant ; Plants, Genetically Modified/genetics ; Protoplasts/metabolism ; Genetic Engineering/methods ; }, abstract = {The potato family includes a highly diverse cultivar repertoire and has a high potential for nutritional yield improvement and refinement but must in line with other crops be adapted to biotic and abiotic stresses, for example, accelerated by climate change and environmental demands. The combination of pluripotency, high ploidy, and relative ease of protoplast isolation, transformation, and regeneration together with clonal propagation through tubers makes potato highly suitable for precise genetic engineering. Most potato varieties are tetraploid having a very high prevalence of length polymorphisms and small nucleotide polymorphisms between alleles, often complicating CRISPR-Cas editing designs and strategies. CRISPR-Cas editing in potato can be divided into (i) characterization of target area and in silico-aided editing design, (ii) isolation and editing of protoplast cells, and (iii) the subsequent explant regeneration from single protoplast cells. Implementation of efficient CRISPR-Cas editing relies on efficient editing at the protoplast (cell pool) level and on robust high-throughput editing scoring methods at the cell pool and explant level. Gene and chromatin structure are additional features to optionally consider. Strategies and solutions for addressing key steps in genome editing of potato, including light conditions and schemes for reduced exposure to hormones during explant regeneration, which is often linked to somaclonal variation, are highlighted.}, }
@article {pmid39628369, year = {2025}, author = {Kanitchinda, S and Sritunyalucksana, K and Chaijarasphong, T}, title = {Multiplex CRISPR-Cas Assay for Rapid, Isothermal and Visual Detection of White Spot Syndrome Virus (WSSV) and Enterocytozoon hepatopenaei (EHP) in Penaeid Shrimp.}, journal = {Journal of fish diseases}, volume = {48}, number = {3}, pages = {e14059}, pmid = {39628369}, issn = {1365-2761}, support = {//Mahidol University (Fundamental Fund: fiscal year 2023 by National Science Research and Innovation Fund (NSRF)) (Grant no. FF-056/2566)/ ; }, mesh = {Animals ; *Penaeidae/virology/microbiology ; *CRISPR-Cas Systems ; *White spot syndrome virus 1/isolation &amp; purification/genetics ; *Enterocytozoon/isolation &amp; purification/genetics ; *Nucleic Acid Amplification Techniques/methods/veterinary ; Sensitivity and Specificity ; *Molecular Diagnostic Techniques/methods ; }, abstract = {White spot syndrome virus (WSSV) and Enterocytozoon hepatopenaei (EHP) represent the most economically destructive pathogens in the current shrimp industry. WSSV causes white spot disease (WSD) responsible for rapid shrimp mortality, while EHP stunts growth and therefore reduces overall productivity. Despite the importance of timely disease detection, current diagnostic methods for WSSV and EHP are typically singleplex, and those offering multiplex detection face issues such as complexity, low field compatibility and/or low sensitivity. Here, we introduce an orthogonal, multiplex CRISPR-Cas assay for concomitant detection of WSSV and EHP. This method combines recombinase polymerase amplification (RPA) for target DNA enrichment with Cas12a and Cas13a enzymes for fluorescent detection. This assay produces distinct fluorescent colours for different diagnostic outcomes, allowing naked eye visualisation without ambiguity. Further validation reveals that the assay detects as few as 20 and 200 copies of target DNA from EHP and WSSV, respectively, while producing no false positives with DNA from other shrimp pathogens. Moreover, the assay excellently agrees with established PCR methods in evaluation of clinical samples. Requiring only 37°C and less than an hour to complete, multiplex CRISPR-Cas assay presents a promising tool for onsite diagnostics, offering high accuracy while saving time and resources.}, }
@article {pmid39627841, year = {2024}, author = {Sledzinski, P and Nowaczyk, M and Smielowska, MI and Olejniczak, M}, title = {CRISPR/Cas9-induced double-strand breaks in the huntingtin locus lead to CAG repeat contraction through DNA end resection and homology-mediated repair.}, journal = {BMC biology}, volume = {22}, number = {1}, pages = {282}, pmid = {39627841}, issn = {1741-7007}, mesh = {Humans ; *DNA Breaks, Double-Stranded ; *CRISPR-Cas Systems ; *Huntingtin Protein/genetics/metabolism ; Recombinational DNA Repair ; Trinucleotide Repeats/genetics ; Trinucleotide Repeat Expansion/genetics ; DNA Repair ; }, abstract = {BACKGROUND: The expansion of CAG/CTG repeats in functionally unrelated genes is a causative factor in many inherited neurodegenerative disorders, including Huntington's disease (HD), spinocerebellar ataxias (SCAs), and myotonic dystrophy type 1 (DM1). Despite many years of research, the mechanism responsible for repeat instability is unknown, and recent findings indicate the key role of DNA repair in this process. The repair of DSBs induced by genome editing tools results in the shortening of long CAG/CTG repeats in yeast models. Understanding this mechanism is the first step in developing a therapeutic strategy based on the controlled shortening of repeats. The aim of this study was to characterize Cas9-induced DSB repair products at the endogenous HTT locus in human cells and to identify factors affecting the formation of specific types of sequences.<br><br>RESULTS: The location of the cleavage site and the surrounding sequence influence the outcome of DNA repair. DSBs within CAG repeats result in shortening of the repeats in frame in&#x2009;~&#x2009;90% of products. The mechanism of this contraction involves MRE11-CTIP and RAD51 activity and DNA end resection. We demonstrated that a DSB located upstream of CAG repeats induces polymerase theta-mediated end joining, resulting in deletion of the entire CAG tract. Furthermore, using proteomic analysis, we identified novel factors that may be involved in CAG sequence repair.<br><br>CONCLUSIONS: Our study provides new insights into the complex mechanisms of CRISPR/Cas9-induced shortening of CAG repeats in human cells.}, }
@article {pmid39627502, year = {2025}, author = {Ngoi, NYL and Gallo, D and Torrado, C and Nardo, M and Durocher, D and Yap, TA}, title = {Synthetic lethal strategies for the development of cancer therapeutics.}, journal = {Nature reviews. Clinical oncology}, volume = {22}, number = {1}, pages = {46-64}, pmid = {39627502}, issn = {1759-4782}, mesh = {Humans ; *Neoplasms/drug therapy/genetics ; *Synthetic Lethal Mutations ; Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use ; CRISPR-Cas Systems ; Antineoplastic Agents/therapeutic use ; Drug Discovery/methods ; DNA Damage/drug effects ; }, abstract = {Synthetic lethality is a genetic phenomenon whereby the simultaneous presence of two different genetic alterations impairs cellular viability. Importantly, targeting synthetic lethal interactions offers potential therapeutic strategies for cancers with alterations in pathways that might otherwise be considered undruggable. High-throughput screening methods based on modern CRISPR-Cas9 technologies have emerged and become crucial for identifying novel synthetic lethal interactions with the potential for translation into biologically rational cancer therapeutic strategies as well as associated predictive biomarkers of response capable of guiding patient selection. Spurred by the clinical success of PARP inhibitors in patients with BRCA-mutant cancers, novel agents targeting multiple synthetic lethal interactions within DNA damage response pathways are in clinical development, and rational strategies targeting synthetic lethal interactions spanning alterations in epigenetic, metabolic and proliferative pathways have also emerged and are in late preclinical and/or early clinical testing. In this Review, we provide a comprehensive overview of established and emerging technologies for synthetic lethal drug discovery and development and discuss promising therapeutic strategies targeting such interactions.}, }
@article {pmid39626969, year = {2024}, author = {Haussmann, IU and Dix, TC and McQuarrie, DWJ and Dezi, V and Hans, AI and Arnold, R and Soller, M}, title = {Structure-optimized sgRNA selection with PlatinumCRISPr for efficient Cas9 generation of knockouts.}, journal = {Genome research}, volume = {34}, number = {12}, pages = {2279-2292}, pmid = {39626969}, issn = {1549-5469}, mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; CRISPR-Associated Protein 9/metabolism/genetics ; Gene Knockout Techniques/methods ; Drosophila/genetics ; Software ; Drosophila Proteins/genetics/metabolism ; Drosophila melanogaster/genetics ; }, abstract = {A single guide RNA (sgRNA) directs Cas9 nuclease for gene-specific scission of double-stranded DNA. High Cas9 activity is essential for efficient gene editing to generate gene deletions and gene replacements by homologous recombination. However, cleavage efficiency is below 50% for more than half of randomly selected sgRNA sequences in human cell culture screens or model organisms. We used in vitro assays to determine intrinsic molecular parameters for maximal sgRNA activity including correct folding of sgRNAs and Cas9 structural information. From the comparison of over 10 data sets, we find major constraints in sgRNA design originating from defective secondary structure of the sgRNA, sequence context of the seed region, GC context, and detrimental motifs, but we also find considerable variation among different prediction tools when applied to different data sets. To aid selection of efficient sgRNAs, we developed web-based PlatinumCRISPr, an sgRNA design tool to evaluate base-pairing and sequence composition parameters for optimal design of highly efficient sgRNAs for Cas9 genome editing. We applied this tool to select sgRNAs to efficiently generate gene deletions in Drosophila Ythdc1 and Ythdf, that bind to N [6] methylated adenosines (m[6]A) in mRNA. However, we discovered that generating small deletions with sgRNAs and Cas9 leads to ectopic reinsertion of the deleted DNA fragment elsewhere in the genome. These insertions can be removed by standard genetic recombination and chromosome exchange. These new insights into sgRNA design and the mechanisms of CRISPR-Cas9 genome editing advance the efficient use of this technique for safer applications in humans.}, }
@article {pmid39626678, year = {2024}, author = {Aviram, N and Shilton, AK and Lyn, NG and Reis, BS and Brivanlou, A and Marraffini, LA}, title = {Cas10 relieves host growth arrest to facilitate spacer retention during type III-A CRISPR-Cas immunity.}, journal = {Cell host &amp; microbe}, volume = {32}, number = {12}, pages = {2050-2062.e6}, pmid = {39626678}, issn = {1934-6069}, support = {/HHMI/Howard Hughes Medical Institute/United States ; K99 GM148720/GM/NIGMS NIH HHS/United States ; R00 GM148720/GM/NIGMS NIH HHS/United States ; R01 GM149834/GM/NIGMS NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems ; *Virus Replication ; Bacterial Proteins/metabolism/genetics ; CRISPR-Associated Proteins/metabolism/genetics ; DNA, Viral/genetics ; Staphylococcus aureus/genetics/immunology ; }, abstract = {Cells from all kingdoms of life can enter growth arrest in unfavorable environmental conditions. Key to this process are mechanisms enabling recovery from this state. Staphylococcal type III-A CRISPR-Cas loci encode the Cas10 complex that uses a guide RNA to locate complementary viral transcripts and start an immune response. When the target sequence is expressed late in the viral lytic cycle, defense requires the activity of Csm6, a non-specific RNase that inhibits the growth of the infected cell. How Csm6 protects from infection and whether growth can be restored is not known. Here, we show that growth arrest provides immunity at the population level, preventing viral replication and allowing uninfected cells to propagate. In addition, the ssDNase activity of Cas10 is required for the regrowth of a subset of the arrested cells and the recovery of the infected host, presumably ending the immune response through degradation of the viral DNA.}, }
@article {pmid39626673, year = {2024}, author = {Awasthi, S and Dobrolecki, LE and Sallas, C and Zhang, X and Li, Y and Khazaei, S and Ghosh, S and Jeter, CR and Liu, J and Mills, GB and Westin, SN and Lewis, MT and Peng, W and Sood, AK and Yap, TA and Yi, SS and McGrail, DJ and Sahni, N}, title = {UBA1 inhibition sensitizes cancer cells to PARP inhibitors.}, journal = {Cell reports. Medicine}, volume = {5}, number = {12}, pages = {101834}, pmid = {39626673}, issn = {2666-3791}, mesh = {Humans ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; *Ubiquitin-Activating Enzymes/antagonists &amp; inhibitors/metabolism/genetics ; Animals ; Cell Line, Tumor ; Female ; Mice ; Xenograft Model Antitumor Assays ; Ovarian Neoplasms/drug therapy/pathology/genetics ; DNA Damage/drug effects ; CRISPR-Cas Systems/genetics ; Poly(ADP-ribose) Polymerases/metabolism/genetics ; }, abstract = {Therapeutic strategies targeting the DNA damage response, such as poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi), have revolutionized cancer treatment in tumors deficient in homologous recombination (HR). However, overcoming innate and acquired resistance to PARPi remains a significant challenge. Here, we employ a genome-wide CRISPR knockout screen and discover that the depletion of ubiquitin-activating enzyme E1 (UBA1) enhances sensitivity to PARPi in HR-proficient ovarian cancer cells. We show that silencing or pharmacological inhibition of UBA1 sensitizes multiple cell lines and organoid models to PARPi. Mechanistic studies uncover that UBA1 inhibition not only impedes HR repair to sensitize cells to PARP inhibition but also increases PARylation, which may subsequently be targeted by PARP inhibition. In vivo experiments using patient-derived xenografts demonstrate that combining PARP and UBA1 inhibition provided significant survival benefit compared to individual therapies with no detectable signs of toxicity, establishing this combination approach as a promising strategy to extend PARPi benefit.}, }
@article {pmid39626660, year = {2025}, author = {Jensen, CL and Chen, LF and Swigut, T and Crocker, OJ and Yao, D and Bassik, MC and Ferrell, JE and Boettiger, AN and Wysocka, J}, title = {Long-range regulation of transcription scales with genomic distance in a gene-specific manner.}, journal = {Molecular cell}, volume = {85}, number = {2}, pages = {347-361.e7}, pmid = {39626660}, issn = {1097-4164}, support = {R35 GM131757/GM/NIGMS NIH HHS/United States ; R35 GM131792/GM/NIGMS NIH HHS/United States ; U01 DK127419/DK/NIDDK NIH HHS/United States ; }, mesh = {Animals ; Mice ; Promoter Regions, Genetic ; *Mouse Embryonic Stem Cells/metabolism ; *Transcription, Genetic ; Transcription Factors/genetics/metabolism ; Transcriptional Activation ; Chromatin/genetics/metabolism ; Genome ; RNA, Guide, CRISPR-Cas Systems/genetics ; Enhancer Elements, Genetic ; Gene Expression Regulation ; }, abstract = {Although critical for tuning the timing and level of transcription, enhancer communication with distal promoters is not well understood. Here, we bypass the need for sequence-specific transcription factors (TFs) and recruit activators directly using a chimeric array of gRNA oligos to target dCas9 fused to the activator VP64-p65-Rta (CARGO-VPR). We show that this approach achieves effective activator recruitment to arbitrary genomic sites, even those inaccessible when targeted with a single guide. We utilize CARGO-VPR across the Prdm8-Fgf5 locus in mouse embryonic stem cells (mESCs), where neither gene is expressed. Although activator recruitment to any tested region results in the transcriptional induction of at least one gene, the expression level strongly depends on the genomic distance between the promoter and activator recruitment site. However, the expression-distance relationship for each gene scales distinctly in a manner not attributable to differences in 3D contact frequency, promoter DNA sequence, or the presence of repressive chromatin marks at the locus.}, }
@article {pmid39625871, year = {2025}, author = {Nguyen, LT and Moutesidi, P and Ziegler, J and Glasneck, A and Khosravi, S and Abel, S and Hensel, G and Krupinska, K and Humbeck, K}, title = {WHIRLY1 regulates aliphatic glucosinolate biosynthesis in early seedling development of Arabidopsis.}, journal = {The Plant journal : for cell and molecular biology}, volume = {121}, number = {1}, pages = {e17181}, pmid = {39625871}, issn = {1365-313X}, support = {400681449/GRK2498//Deutsche Forschungsgemeinschaft/ ; }, mesh = {*Arabidopsis/genetics/growth &amp; development/metabolism ; *Seedlings/genetics/growth &amp; development/metabolism ; *Glucosinolates/metabolism/biosynthesis ; *Arabidopsis Proteins/genetics/metabolism ; *Gene Expression Regulation, Plant ; Transcription Factors/genetics/metabolism ; Cotyledon/genetics/growth &amp; development/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; }, abstract = {WHIRLY1 belongs to a family of plant-specific transcription factors capable of binding DNA or RNA in all three plant cell compartments that contain genetic materials. In Arabidopsis thaliana, WHIRLY1 has been studied at the later stages of plant development, including flowering and leaf senescence, as well as in biotic and abiotic stress responses. In this study, WHIRLY1 knockout mutants of A. thaliana were prepared by CRISPR/Cas9-mediated genome editing to investigate the role of WHIRLY1 during early seedling development. The loss-of-function of WHIRLY1 in 5-day-old seedlings did not cause differences in the phenotype and the photosynthetic performance of the emerging cotyledons compared with the wild type. Nevertheless, comparative RNA sequencing analysis revealed that the knockout of WHIRLY1 affected the expression of a small but specific set of genes during this critical phase of development. About 110 genes were found to be significantly deregulated in the knockout mutant, wherein several genes involved in the early steps of aliphatic glucosinolate (GSL) biosynthesis were suppressed compared with wild-type plants. The downregulation of these genes in WHIRLY1 knockout lines led to decreased GSL contents in seedlings and in seeds. Since GSL catabolism mediated by myrosinases was not altered during seed-to-seedling transition, the results suggest that AtWHIRLY1 plays a major role in modulation of aliphatic GSL biosynthesis during early seedling development. In addition, phylogenetic analysis revealed a coincidence between the evolution of methionine-derived aliphatic GSLs and the addition of a new WHIRLY in core families of the plant order Brassicales.}, }
@article {pmid39624314, year = {2024}, author = {Panahi, B and Rostampour, M and Ghaffari, MR and Nami, Y}, title = {Genome mining approach reveals the CRISPR-Cas systems features and characteristics in Lactobacillus delbrueckii strains.}, journal = {Heliyon}, volume = {10}, number = {22}, pages = {e39920}, pmid = {39624314}, issn = {2405-8440}, abstract = {This study employed a comprehensive genome-mining approach to characterize CRISPR-Cas systems in Lactobacillus delbrueckii strains. The analysis involved retrieving 105 genome sequences to explore the variety, occurrence, and evolution of CRISPR-Cas systems within the species. Homology analysis of spacer sequences in detected CRISPR arrays was conducted to assess the variety of target phages and plasmids. The evolutionary trajectories of spaceromes in each subtype of CRISPR arrays were determined by analyzing acquisition and deletion events under the selection pressure of foreign plasmids and phages. Among the analyzed strains, 53 contained only one CRISPR-Cas locus, 11 had two loci, and 21 featured three loci with complete CRISPR-Cas systems. Subtype designation of the results of current study revealed that 56 % of these systems belong to subtypes I-E/I-C, 23 % to subtypes III-A/III-D, and 17 % to subtype II-A. Notably, certain plasmids were found to be specifically targeted by distinct CRISPR array systems. The comparison of spacer sequences with phage genomes indicated that strains containing only CRISPR-Cas type-I systems targeted a broader range of phages. In conclusion, this study highlights the diversity of the three identified CRISPR-Cas types in L. delbrueckii strains and emphasizes their significant role in defense against phage invasion.}, }
@article {pmid39623998, year = {2024}, author = {Wu, Y and Zhang, X and Li, J and Xie, J and Wang, L and Sun, H}, title = {[Progress of researches on toxoplasmosis vaccines based on the CRISPR/Cas9 technology].}, journal = {Zhongguo xue xi chong bing fang zhi za zhi = Chinese journal of schistosomiasis control}, volume = {36}, number = {5}, pages = {542-547}, doi = {10.16250/j.32.1374.2024026}, pmid = {39623998}, issn = {1005-6661}, support = {ZR2022MH271//Natural Science Foundation of Shandong Province/ ; 202301011242//Shandong Provincial Medical and Health Science and Technology Project/ ; 202101050153//Shandong Provincial Science and Technology Development Project for Medical Sciences and Health/ ; }, mesh = {*CRISPR-Cas Systems ; Humans ; Animals ; *Toxoplasma/genetics/immunology ; *Toxoplasmosis/prevention &amp; control/immunology/parasitology ; Protozoan Vaccines/immunology ; Gene Editing/methods ; }, abstract = {Toxoplasma gondii is an obligatory intracellular parasite which infects a variety of warm-blooded animals and causes toxoplasmosis. Toxoplasmosis seriously endangers human health and animal husbandry production. As one of the effective gene editing tools, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been widely used for knockout of genes in T. gondii. This review summarizes the applications of the CRISPR/Cas9 technology in vaccines against single- and double-gene deletion strains of T. gondii, so as to provide insights into development of toxoplasmosis vaccines.}, }
@article {pmid39623867, year = {2025}, author = {Giorgetti, OB and Haas-Assenbaum, A and Boehm, T}, title = {Probing TCR Specificity Using Artificial In Vivo Diversification of CDR3 Regions.}, journal = {European journal of immunology}, volume = {55}, number = {1}, pages = {e202451434}, pmid = {39623867}, issn = {1521-4141}, support = {//Max Planck Society/ ; 256073931-SFB1160//Deutsche Forschungsgemeinschaft/ ; }, mesh = {*Complementarity Determining Regions/genetics/immunology ; Animals ; Mice ; CRISPR-Cas Systems/genetics ; *Receptors, Antigen, T-Cell, alpha-beta/genetics/immunology ; Mice, Transgenic ; *T-Lymphocytes/immunology ; *Receptors, Antigen, T-Cell/genetics/immunology ; }, abstract = {The T-cell receptor sequences expressed on cells recognizing a specific peptide in the context of a given MHC molecule can be explored for common features that might explain their antigen specificity. However, despite the development of numerous experimental and bioinformatic strategies, the specificity problem remains unresolved. To address the need for additional experimental paradigms, we report here on an in vivo experimental strategy designed to artificially diversify a transgenic TCR by CRISPR/Cas9-mediated mutagenesis of Tcra and Tcrb chain genes. In this system, an initially monoclonal repertoire of known specificity is converted into an oligoclonal pool of TCRs of altered antigen reactivity. Tracking the fate of individual clonotypes during the intrathymic differentiation process illuminates the strong selective pressures that shape the repertoire of naïve T cells. Sequence analyses of the artificially diversified repertoires identify key amino acid residues in the CDR3 regions required for antigen recognition, indicating that artificial diversification of well-characterized TCR transgene sequences helps to reduce the complexities of learning the rules of antigen recognition.}, }
@article {pmid39622959, year = {2024}, author = {Fang, B and Wang, C and Yuan, Y and Liu, X and Shi, L and Li, L and Wang, Y and Dai, Y and Yang, H}, title = {Generation and characterization of genetically modified pigs with GGTA1/β4GalNT2/CMAH knockout and human CD55/CD47 expression for xenotransfusion studies.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {29870}, pmid = {39622959}, issn = {2045-2322}, support = {81970164//National Natural Science Foundation of China/ ; }, mesh = {Animals ; Humans ; *Animals, Genetically Modified ; *Galactosyltransferases/genetics/metabolism ; Swine ; *CD55 Antigens/genetics/metabolism ; *CD47 Antigen/genetics/metabolism ; *Gene Knockout Techniques/methods ; *N-Acetylgalactosaminyltransferases/genetics/metabolism ; *Mixed Function Oxygenases/genetics/metabolism ; *Transplantation, Heterologous ; CRISPR-Cas Systems ; Erythrocytes/metabolism ; Macaca fascicularis/genetics ; Immunoglobulin G ; }, abstract = {Pig red blood cells (pRBCs) represent a promising alternative to address the shortage in transfusion medicine. Nonetheless, a major obstacle to their clinical implementation is immunological rejection. In this study, we generated transgenic pigs expressing human CD47 (hCD47) and CD55 (hCD55) in α1,3-galactosyltransferase KO/β-1,4-N-acetyl-galactosaminyl transferase 2 KO/cytidine monophosphate-N-acetylneuraminic acid hydroxylase KO (TKO) pigs using CRISPR/Cas9 technology. Compared to wild-type pRBCs, TKO/hCD47/hCD55 pRBCs exhibit significantly reduced human IgG/IgM antibody binding. Moreover, when transfused into Cynomolgus monkeys, TKO/hCD47/hCD55 pRBCs remained detectable for 2 h post-transfusion, whereas wild-type pRBCs were eliminated within 20 min. This study demonstrates the potential of multi-gene edited pigs to provide immunologically compatible pRBCs.}, }
@article {pmid39622625, year = {2024}, author = {Mallozzi, A and Fusco, V and Ragazzini, F and di Bernardo, D}, title = {A Biomolecular Circuit for Automatic Gene Regulation in Mammalian Cells with CRISPR Technology.}, journal = {ACS synthetic biology}, volume = {13}, number = {12}, pages = {3917-3925}, pmid = {39622625}, issn = {2161-5063}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Gene Expression Regulation ; HEK293 Cells ; Transcription Factors/genetics/metabolism ; Plasmids/genetics/metabolism ; Gene Regulatory Networks ; Animals ; }, abstract = {We introduce a biomolecular circuit for precise control of gene expression in mammalian cells. The circuit leverages the stochiometric interaction between the artificial transcription factor VPR-dCas9 and the anti-CRISPR protein AcrIIA4, enhanced with synthetic coiled-coil domains to boost their interaction, to maintain the expression of a reporter protein constant across diverse experimental conditions, including fluctuations in protein degradation rates and plasmid concentrations, by automatically adjusting its mRNA level. This capability, known as robust perfect adaptation (RPA), is crucial for the stable functioning of biological systems and has wide-ranging implications for biotechnological applications. This system belongs to a class of biomolecular circuits named antithetic integral controllers, and it can be easily adapted to regulate any endogenous transcription factor thanks to the versatility of the CRISPR-Cas system. Finally, we show that RPA also holds in cells genomically integrated with the circuit, thus paving the way for practical applications in biotechnology that require stable cell lines.}, }
@article {pmid39622311, year = {2025}, author = {Kim, M and Hutchins, EJ}, title = {CRISPR-Cas13d as a molecular tool to achieve targeted gene expression knockdown in chick embryos.}, journal = {Developmental biology}, volume = {519}, number = {}, pages = {5-12}, pmid = {39622311}, issn = {1095-564X}, support = {R00 DE028592/DE/NIDCR NIH HHS/United States ; R35 GM150763/GM/NIGMS NIH HHS/United States ; T32 DE007306/DE/NIDCR NIH HHS/United States ; }, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Knockdown Techniques/methods ; Chick Embryo ; PAX7 Transcription Factor/genetics/metabolism ; Gene Expression Regulation, Developmental ; Neural Crest/metabolism/embryology ; Morpholinos/genetics ; }, abstract = {The chick embryo is a classical model system commonly used in developmental biology due to its amenability to gene perturbation experiments. Pairing this powerful model organism with cutting-edge technology can significantly expand the range of experiments that can be performed. Recently, the CRISPR-Cas13d system has been successfully adapted for use in zebrafish, medaka, killifish, and mouse embryos to achieve targeted gene expression knockdown. Despite its success in other animal models, no prior study has explored the potential of CRISPR-Cas13d in the chick. Here, we present an adaptation of the CRISPR-Cas13d system to achieve targeted gene expression knockdown in the chick embryo. As proof-of-principle, we demonstrate the knockdown of PAX7, an early neural crest marker. Application of this adapted CRISPR-Cas13d technique resulted in effective knockdown of PAX7 expression and function, comparable to knockdown achieved by translation-blocking morpholino. CRISPR-Cas13d complements preexisting knockdown tools such as CRISPR-Cas9 and morpholinos, thereby expanding the experimental potential and versatility of the chick model system.}, }
@article {pmid39622167, year = {2025}, author = {Maity, A and Sathyanarayanan, A and Kumar, R and Vora, J and Gawde, J and Jain, H and Bagal, B and Subramanian, PG and Sengar, M and Khattry, N and Patkar, N and Hasan, SK}, title = {RAPID-CRISPR: highly sensitive diagnostic assay for detection of PML::RARA isoforms in acute promyelocytic leukemia.}, journal = {Blood advances}, volume = {9}, number = {3}, pages = {463-472}, pmid = {39622167}, issn = {2473-9537}, mesh = {Humans ; *Leukemia, Promyelocytic, Acute/diagnosis/genetics ; *CRISPR-Cas Systems ; Protein Isoforms/genetics ; *Oncogene Proteins, Fusion/genetics ; Male ; *Promyelocytic Leukemia Protein/genetics ; Female ; Adult ; *Retinoic Acid Receptor alpha/genetics ; Sensitivity and Specificity ; Middle Aged ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Acute promyelocytic leukemia (APL), distinguished by the presence of PML::RARA fusion transcript, is a medical emergency because of its high early death rate, which is preventable when diagnosed early. Current diagnostic methods are precise and reliable but are time intensive, require sophisticated instruments, and analytical expertise. This study has redefined APL identification by CRISPR system (RAPID-CRISPR) to rapidly (<3 hours) detect PML::RARA. APL cell lines (NB4 and UF-1) and bone marrow/peripheral blood samples from 74 patients with APL (66/8, retrospective/prospective) and 48 controls were included in the study. We used a DETECTR (DNA endonuclease-targeted CRISPR transreporter) assay to identify the bcr1, bcr2, and bcr3 PML::RARA isoforms. To ensure high specificity, we used PML::RARA-specific loop-mediated isothermal amplification (LAMP) primers, synthetic protospacer-adjacent motif sites, and isoform-specific CRISPR RNAs. RAPID-CRISPR recognized APL with 100% sensitivity and 100% specificity in an ambispective cohort of patient samples. Furthermore, our blinded validation approach to detect PML::RARA in an unbiased manner provides an additional layer in the diagnostic precision of APL. RAPID-CRISPR demonstrated superior sensitivity, detecting as few as 1 copy of PML::RARA compared with 10 copies by the gold-standard reverse transcriptase qualitative and quantitative polymerase chain reaction. The nucleic acid extraction-free protocol combined with the 1-step reverse transcriptase LAMP-based DETECTR followed by lateral flow readout makes the RAPID-CRISPR assay suitable for diagnosing APL in point-of-care settings. This simple, cost-effective tool, with its easy-to-read format, is particularly valuable in underresourced regions. The assay facilitates timely diagnosis and prompt administration of lifesaving therapies such as all-trans retinoic acid and arsenic trioxide in APL.}, }
@article {pmid39621555, year = {2024}, author = {Emaldi, M and Rey-Iborra, E and Marín, &#xc1; and Mosteiro, L and Lecumberri, D and Øyjord, T and Roncier, N and Mælandsmo, GM and Angulo, JC and Errarte, P and Larrinaga, G and Pulido, R and López, JI and Nunes-Xavier, CE}, title = {Impact of B7-H3 expression on metastasis, immune exhaustion and JAK/STAT and PI3K/AKT pathways in clear cell renal cell carcinoma.}, journal = {Oncoimmunology}, volume = {13}, number = {1}, pages = {2419686}, pmid = {39621555}, issn = {2162-402X}, mesh = {*Carcinoma, Renal Cell/pathology/immunology/genetics/metabolism/drug therapy ; Humans ; *B7 Antigens/metabolism/genetics ; *Kidney Neoplasms/pathology/immunology/metabolism/genetics/drug therapy ; *Proto-Oncogene Proteins c-akt/metabolism ; *Phosphatidylinositol 3-Kinases/metabolism ; *Signal Transduction/drug effects ; Janus Kinases/metabolism ; Cell Line, Tumor ; STAT Transcription Factors/metabolism ; Male ; Female ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Middle Aged ; Neoplasm Metastasis ; Aged ; Immune System Exhaustion ; }, abstract = {Immune checkpoint inhibitors in combination with tyrosine kinase inhibitors (TKIs) are improving the response rates of advanced renal cancer patients. However, many treated patients do not respond, making novel immune checkpoint-based immunotherapies potentially clinically beneficial only for specific groups of patients. We detected high expression of the immune checkpoint protein B7-H3 in clear cell renal cell carcinomas (ccRCCs) and evaluated B7-H3 immunohistochemistry staining in tissue microarray samples from two distinct renal cancer cohorts. B7-H3 was highly expressed in approximately 50% of primary tumors and in 30% of metastatic lesions. B7-H3 expression in primary tumors correlated with tumor necrosis, sarcomatoid transformation, disease-free survival, and synchronous metastasis, while B7-H3 expression in metastasis correlated with metastases to the lymph nodes. Gene expression analysis revealed the association of B7-H3 expression with gene expression scores involved in T cell exhaustion and myeloid immune evasion, as well as with PI3K/AKT and JAK/STAT pathways. Furthermore, knocking down B7-H3 expression in renal cancer cells by siRNA and CRISPR/Cas resulted in lower 2D and 3D cell proliferation and viability as well as increased sensitivity to TKI axitinib. Together, our findings suggest a pro-oncogenic and immune evasive role for B7-H3 in ccRCC and highlight B7-H3 as an actionable novel immune checkpoint protein in combination with TKI in advanced renal cancer.}, }
@article {pmid39621264, year = {2025}, author = {Gowripalan, A and Smith, SA and Tscharke, DC}, title = {Cas9-Mediated Poxvirus Recombinant Recovery (CASPRR) for Fast Recovery of Recombinant Vaccinia Viruses.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2860}, number = {}, pages = {115-130}, pmid = {39621264}, issn = {1940-6029}, mesh = {*Vaccinia virus/genetics ; Humans ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Recombination, Genetic ; Poxviridae/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; Cell Line ; Transfection/methods ; }, abstract = {Generation of recombinant vaccinia viruses opens many avenues for poxvirus research; however current methods for virus production can be laborious. Traditional methods rely on recombination strategies that produce engineered viruses at a low frequency, which then need to be identified and isolated from a large background of parent virus. For this reason, marker and reporter genes are often included, but in many cases these require removal in subsequent steps and the entire process is relatively inefficient. Cas9-mediated selection is a technique that repurposes Cas9/guide RNA complexes to amplify chosen subsets of vaccinia viruses. We refer to this approach as Cas9-mediated poxvirus recombinant recovery (CASPRR). Transient introduction of appropriately guided Cas9 allows for recovery of marker-free recombinant viruses in just 5 days, and requires no additional virus modification. Following three rounds of purification, pure virus stocks are obtained. A newer method, stable expression of Cas9 and guide RNAs in a permissive cell line, allows for additional process streamlining, removing cell type-specific concerns related to transient transfection of Cas9. Within this chapter, the protocol for CASPRR is described in both a transient and stable expression model. These methods can be utilized to accelerate recovery of recombinant vaccinia viruses and be applied to generation of vaccinia libraries or novel therapeutic agents.}, }
@article {pmid39621263, year = {2025}, author = {Lin, YJ and Evans, DH and Noyce, RS}, title = {Rapid Generation of Recombinant Poxviruses Using CRISPR/Cas9 Gene Editing.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2860}, number = {}, pages = {97-114}, pmid = {39621263}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Vaccinia virus/genetics ; *Poxviridae/genetics ; Humans ; Genome, Viral ; DNA, Viral/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; Recombination, Genetic ; }, abstract = {The low-frequency natural recombination that is detected in poxvirus-infected cells has long been used to genetically modify poxviruses. Such recombinant poxviruses have found many applications as vaccines for preventing infectious diseases and as experimental cancer therapeutics. Unfortunately, these methods are time consuming, can leave behind "scars" or selectable markers, and many months of work may be required to generate plaque-purified recombinants bearing multiple virus gene substitutions, deletions, and/or inserted transgenes. Over the last decade, several reports have described how CRISPR/Cas9 technologies can be used to better facilitate genetic manipulation of vaccinia virus (VACV). These protocols use Cas9/gRNA complexes to introduce double-stranded breaks into specific sites in virus genomic DNA either in vivo or in vitro. Recombination-repair reactions are then employed to repair the breaks using transfected DNAs encoding the required homologies and desired mutation(s). Here we describe a method where we combine CRISPR/Cas9 genome editing in vitro, followed by Leporipoxvirus-catalyzed repair and reactivation of the cut VACV DNA using repair fragments provided in trans. This method optimizes several steps in the preparation of the CRISPR/Cas9-cut VACV DNA and can be used to introduce mutations at multiple sites without requiring selectable markers. It also provides some guidance regarding how the position of the CRISPR/Cas9-cuts can affect co-conversion of flanking markers embedded in the repair fragment. The method allows researchers to quickly generate recombinant VACV bearing multiple genetic alterations and using only a single round of reactivation and plating.}, }
@article {pmid39620587, year = {2024}, author = {Cui, Z and Yuan, P and Zhao, Z and Zhao, F and Lu, L}, title = {Retroviral CRISPR/Cas9-Mediated Gene Targeting for the Study of Th17 Differentiation in Vitro.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {213}, pages = {}, doi = {10.3791/66966}, pmid = {39620587}, issn = {1940-087X}, mesh = {Animals ; Mice ; *Th17 Cells/immunology/cytology ; *Cell Differentiation/genetics ; *CRISPR-Cas Systems/genetics ; *Retroviridae/genetics ; Gene Targeting/methods ; Flow Cytometry/methods ; }, abstract = {T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells can be stimulated with antigens and well-defined cytokine cocktails in vitro to mimic Th17 cell differentiation in vivo. Research has been conducted extensively on the Th17 differentiation regulation mechanisms using the in vitro Th17 polarization assay. Conventional Th17 polarization methods typically involve obtaining naïve CD4 T cells from genetically modified mice to study the effects of specific genes on Th17 differentiation and function. These methods can be time-consuming and costly and may be influenced by cell-extrinsic factors from the knockout animals. Thus, a protocol using retroviral transduction of guide RNA to introduce gene knockout in CRISPR/Cas9 knockin primary mouse T cells serves as a very useful alternative approach. This paper presents a protocol to differentiate naïve primary T cells into Th17 cells following retroviral-mediated gene targeting, as well as the subsequent flow cytometry analysis methods for assaying infection and differentiation efficiency.}, }
@article {pmid39620398, year = {2024}, author = {Zhao, Z and Wang, R and Yang, X and Jia, J and Zhang, Q and Ye, S and Man, S and Ma, L}, title = {Machine Learning-Assisted, Dual-Channel CRISPR/Cas12a Biosensor-In-Microdroplet for Amplification-Free Nucleic Acid Detection for Food Authenticity Testing.}, journal = {ACS nano}, volume = {18}, number = {49}, pages = {33505-33519}, doi = {10.1021/acsnano.4c10823}, pmid = {39620398}, issn = {1936-086X}, mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Machine Learning ; Food Analysis/methods ; Animals ; Meat/analysis ; Nucleic Acids/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {The development of novel detection technology for meat species authenticity is imperative. Here, we developed a machine learning-supported, dual-channel biosensor-in-microdroplet platform for meat species authenticity detection named CC-drop (CRISPR/Cas12a digital single-molecule microdroplet biosensor). This strategy allowed us to quickly identify and analyze animal-derived components in foods. This biosensor was enabled by CRISPR/Cas12a-based fluorescence lighting-up detection, and the nucleic acid signals can be recognized by a Cas12a-crRNA binary complex to trigger the trans-cleavage of any by-stander reporter single-stranded (ss) DNA, in which nucleic acid signals can be converted and amplified to fluorescent readouts. The ultralocalized microdroplet reactor was constructed by reducing the reaction volume from up to picoliter to accommodate the aforementioned reaction to further enhance the sensitivity to even render an amplification-free nucleic acid detection. Moreover, we established a smartphone App coupled with a random forest machine learning model based on parameters such as area, fluorescent intensity, and counting number to ensure the accuracy of image recording and processing. The sample-to-result time was within 80 min. Importantly, the proposed biosensor was able to accurately detect the ND1 (pork-specific) and IL-2 (duck-specific) genes in deep processed meat-derived foods that usually had truncated DNA, and the results were more robust and practical than conventional real-time polymerase chain reaction after a side-by-side comparison. All in all, the proposed biosensor can be expected to be used for rapid food authenticity and other nucleic acid detections in the future.}, }
@article {pmid39619730, year = {2024}, author = {Ahmed, MM and Kayode, HH and Okesanya, OJ and Ukoaka, BM and Eshun, G and Mourid, MR and Adigun, OA and Ogaya, JB and Mohamed, ZO and Lucero-Prisno, DE}, title = {CRISPR-Cas Systems in the Fight Against Antimicrobial Resistance: Current Status, Potentials, and Future Directions.}, journal = {Infection and drug resistance}, volume = {17}, number = {}, pages = {5229-5245}, pmid = {39619730}, issn = {1178-6973}, abstract = {BACKGROUND: Antimicrobial resistance (AMR) is a critical global health concern that threatens the efficacy of existing antibiotics and poses significant challenges to public health and the economy worldwide. This review explores the potential of CRISPR-Cas systems as a novel approach to combating AMR and examines current applications, limitations, and prospects.<br><br>METHODS: A comprehensive literature search was conducted across multiple databases, including PubMed, Google Scholar, Scopus, and Web of Science, covering publications published from 2014 to August 2024. This review focuses on CRISPR-Cas technologies and their applications in AMR.<br><br>RESULTS: CRISPR-Cas systems have demonstrated efficacy in combating antimicrobial resistance by targeting and eliminating antibiotic-resistance genes. For example, studies have shown that CRISPR-Cas9 can effectively target and eliminate colistin resistance genes in MCR-1 plasmids, restoring susceptibility to carbapenems in bacteria such as E. coli and Klebsiella pneumoniae. Further molecular findings highlight the impact of CRISPR-Cas systems on various bacterial species, such as Enterococcus faecalis, in which CRISPR systems play a crucial role in preventing the acquisition of resistance genes. The effectiveness of CRISPR-Cas in targeting these genes varies due to differences in CRISPR locus formation among bacterial species. For instance, variations in CRISPR loci influence the targeting of resistance genes in E. faecalis, and CRISPR-Cas9 successfully reduces resistance by targeting genes such as tetM and ermB.<br><br>CONCLUSION: CRISPR-Cas systems are promising for fighting AMR by targeting and eliminating antibiotic-resistant genes, as demonstrated by the effective targeting of colistin resistance genes on MCR-1 plasmids and their similar activities. However, the effectiveness of CRISPR-Cas is affected by variations in the CRISPR loci among bacterial species. Challenges persist, such as optimizing delivery methods and addressing off-target effects to ensure the safety and precision of CRISPR-Cas systems in clinical settings.}, }
@article {pmid39618347, year = {2025}, author = {Yu, C and Liu, Y and Zhang, W and Yao, X}, title = {A three-way junction probe triggered CRISPR/Cas14a1 enhanced EXPonential amplification reaction for sensitive Pseudomonas aeruginosa detection.}, journal = {Analytical methods : advancing methods and applications}, volume = {17}, number = {2}, pages = {284-290}, doi = {10.1039/d4ay01728e}, pmid = {39618347}, issn = {1759-9679}, mesh = {*Pseudomonas aeruginosa/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Humans ; Pseudomonas Infections ; Aptamers, Nucleotide/chemistry ; }, abstract = {Pseudomonas aeruginosa (P. aeruginosa, PA) is a rod-shaped Gram-negative opportunistic bacterium capable of causing nosocomial infections during nursing, such as burn wound infections and meningitis. However, sensitive and rapid PA detection remains a huge challenge. Herein, a new fluorescent biosensor was developed for the sensitive detection of PA using a three-way junction (TWJ) probe for specific identification and CRISPR/Cas14a1 for enhanced exponential amplification. The interaction between PA and its aptamer on a DNA TWJ structure probe triggered the migration of the double-stranded DNA branch, inducing DNA polymerase/endonuclease assisted chain displacement and the generation of single-stranded DNA sequences. The amplification products have the ability to activate CRISPR/Cas14a1, resulting in effective trans-cleavage and the subsequent release of fluorescence from the reporter probes. Under optimized conditions, the proposed biosensor was able to detect a wide range of bacterial concentrations, ranging from 10 to 10[5] cfu mL[-1] within 30 min. The limit of detection, which is the lowest concentration that can be reliably detected, was determined to be 3.4 cfu mL[-1] according to the 3δ rule. The results of the recovery test suggest that the biosensor shows significant potential for clinical applications. The established biosensor utilizing the TWJ probe generated multiple isothermal exponential amplification and the CRISPR/Cas14a1 biosensor is an excellent platform for rapidly detecting pathogenic bacteria in postoperative infection.}, }
@article {pmid39618159, year = {2025}, author = {Laudouze, J and Point, V and Achache, W and Crauste, C and Canaan, S and Santucci, P}, title = {Fluorescence-based CRISPR interference system for controlled genetic repression and live single-cell imaging in mycobacteria.}, journal = {FEBS letters}, volume = {599}, number = {4}, pages = {488-501}, pmid = {39618159}, issn = {1873-3468}, support = {//Institut des Sciences Biologiques/ ; //FEBS Excellence Award/ ; //Initiative d'Excellence d'Aix-Marseille Universit&#xe9; - A*MIDEX/ ; //Aix-Marseille Universit&#xe9;/ ; Project n&#xb0;ANRS0358//Agence Nationale de Recherches sur le Sida et les H&#xe9;patites Virales/ ; }, mesh = {*CRISPR-Cas Systems ; Genetic Vectors/genetics ; *Mycobacterium/genetics/cytology ; Bacterial Proteins/genetics/metabolism ; *Single-Cell Analysis/methods ; Plasmids/genetics ; Promoter Regions, Genetic ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Regulation, Bacterial ; Fluorescence ; }, abstract = {In this research letter, we report the development and validation of a new subset of fluorescence-based CRISPR interference (CRISPRi) tools for our scientific community. The pJL series is directly derived from the original pIRL CRISPRi vectors and conserves all the elements to perform inducible targeted gene repression. These vectors carry two distinct fluorescent markers under the constitutive promoter psmyc to simplify the selection of recombinant clones. We demonstrate the functionality of these vectors by targeting the expression of the glycopeptidolipid translocase mmpL4b and the essential genes rpoB and mmpL3. Finally, we describe an efficient single-step procedure to co-transform mycobacterial species with this integrative genetic tool alongside episomal vectors. Such tools and approaches should be useful to foster discovery in mycobacterial research.}, }
@article {pmid39617792, year = {2024}, author = {Hosseinpour, M and Xi, X and Liu, L and Malaver-Ortega, L and Perlaza-Jimenez, L and Joo, JE and York, HM and Beesley, J and Caldon, CE and Dugué, PA and Dowty, JG and Arumugam, S and Southey, MC and Rosenbluh, J}, title = {SAM-DNMT3A, a strategy for induction of genome-wide DNA methylation, identifies DNA methylation as a vulnerability in ER-positive breast cancers.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10449}, pmid = {39617792}, issn = {2041-1723}, support = {2011329//Department of Health | National Health and Medical Research Council (NHMRC)/ ; MCRF20035//Victorian Cancer Agency (VCA)/ ; }, mesh = {*DNA Methylation ; Humans ; *Breast Neoplasms/genetics/metabolism/drug therapy ; *DNA Methyltransferase 3A/metabolism ; Female ; *DNA (Cytosine-5-)-Methyltransferases/metabolism/genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Receptors, Estrogen/metabolism/genetics ; MCF-7 Cells ; Epigenesis, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {DNA methylation is an epigenetic mark that plays a critical role in regulating gene expression. DNA methyltransferase (DNMT) inhibitors, inhibit global DNA methylation and have been a key tool in studies of DNA methylation. A major bottleneck is the lack of tools to induce global DNA methylation. Here, we engineered a CRISPR based approach, that we initially designed, to enable site-specific DNA methylation. Using the synergistic activation mediator (SAM) system, we unexpectedly find that regardless of the targeted sequence any sgRNA induces global genome-wide DNA methylation. We term this method SAM-DNMT3A and show that induction of global DNA methylation is a unique vulnerability in ER-positive breast cancer suggesting a therapeutic approach. Our findings highlight the need of caution when using CRISPR based approaches for inducing DNA methylation and demonstrate a method for global induction of DNA methylation.}, }
@article {pmid39617755, year = {2024}, author = {Wu, Y and Huang, Z and Liu, Y and He, P and Wang, Y and Yan, L and Wang, X and Gao, S and Zhou, X and Yoon, CW and Sun, K and Situ, Y and Ho, P and Zeng, Y and Yuan, Z and Zhu, L and Zhou, Q and Zhao, Y and Liu, T and Kwong, GA and Chien, S and Liu, L and Wang, Y}, title = {Ultrasound Control of Genomic Regulatory Toolboxes for Cancer Immunotherapy.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10444}, pmid = {39617755}, issn = {2041-1723}, support = {R35 GM140929/GM/NIGMS NIH HHS/United States ; CA262815//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; GM140929//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; HD107206//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; HL121365//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01 CA262815/CA/NCI NIH HHS/United States ; EB032822//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01 CA279813/CA/NCI NIH HHS/United States ; R01 HL121365/HL/NHLBI NIH HHS/United States ; R01 EB029122/EB/NIBIB NIH HHS/United States ; K01 EB035649/EB/NIBIB NIH HHS/United States ; EB029122//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; K01EB035649//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; }, mesh = {Humans ; *Neoplasms/therapy/genetics/immunology ; Animals ; *CRISPR-Cas Systems ; *Receptors, Chimeric Antigen/metabolism/genetics ; Mice ; Cell Line, Tumor ; Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Immunotherapy, Adoptive/methods ; Dependovirus/genetics ; Immunotherapy/methods ; Genomics/methods ; Ultrasonic Waves ; }, abstract = {There remains a critical need for the precise control of CRISPR (clustered regularly interspaced short palindromic repeats)-based technologies. Here, we engineer a set of inducible CRISPR-based tools controllable by focused ultrasound (FUS), which can penetrate deep and induce localized hyperthermia for transgene activation. We demonstrate the capabilities of FUS-inducible CRISPR, CRISPR activation (CRISPRa), and CRISPR epigenetic editor (CRISPRee) in modulating the genome and epigenome. We show that FUS-CRISPR-mediated telomere disruption primes solid tumours for chimeric antigen receptor (CAR)-T cell therapy. We further deliver FUS-CRISPR in vivo using adeno-associated viruses (AAVs), followed by FUS-induced telomere disruption and the expression of a clinically validated antigen in a subpopulation of tumour cells, functioning as "training centers" to activate synthetic Notch (synNotch) CAR-T cells to produce CARs against a universal tumour antigen to exterminate neighboring tumour cells. The FUS-CRISPR(a/ee) toolbox hence allows the noninvasive and spatiotemporal control of genomic/epigenomic reprogramming for cancer treatment.}, }
@article {pmid39616960, year = {2024}, author = {Xing, W and Zhang, J and Liu, T and Wang, Y and Qian, J and Wang, B and Zhang, Y and Zhang, Q}, title = {An innovative CRISPR/Cas9 mouse model of human isolated microtia indicates the potential contribution of CNVs near HMX1 gene.}, journal = {International journal of pediatric otorhinolaryngology}, volume = {187}, number = {}, pages = {112141}, doi = {10.1016/j.ijporl.2024.112141}, pmid = {39616960}, issn = {1872-8464}, mesh = {Animals ; *Congenital Microtia/genetics ; Humans ; Mice ; *Disease Models, Animal ; *DNA Copy Number Variations ; *CRISPR-Cas Systems ; Female ; Male ; *Pedigree ; *Homeodomain Proteins/genetics ; Mutation ; Gene Editing/methods ; }, abstract = {BACKGROUND: Microtia is a prevalent congenital malformation, the precise etiology and pathogenesis of which remain elusive. Mutations in the non-coding region of the HMX1 gene have been implicated in isolated cases of microtia, emerging as a significant focus of contemporary research. Several pathogenic copy number variations (CNVs) proximal to the HMX1 gene have been documented in wild animal populations, whereas only a single large segmental duplication in this region has been identified in humans. However, the absence of a gene-edited animal model has impeded the investigation of the unclear gene function associated with HMX1 mutations in human isolated microtia. In this study, we sought to precisely identify the pathogenic mutation by analyzing three pedigrees alongside population controls. Subsequently, our objective was to develop a CRISPR/Cas9 gene-edited mouse model to elucidate the functional implications of the identified mutation.<br><br>METHODS: Genomic DNA was collected from 32 affected individuals across three pedigrees, as well as from 2000 control subjects. Comprehensive genomic analyses, including genome-wide linkage analysis, targeted capture, second-generation sequencing, and copy number analysis, were conducted to identify potential mutations associated with congenital auricle malformation. CRISPR/Cas9 gene-edited murine models were generated in response to the identified mutation. The auricular phenotypes of these gene-edited mice were systematically monitored. Small-animal Micro-CT scanning was employed to identify potential craniofacial or skeletal abnormalities. Furthermore, the expression of the HMX1 gene in the PA2 region of mouse embryos was quantified using RT-qPCR.<br><br>RESULTS: A co-segregated 600 base pair duplication located on chromosome 4 (chr4:8701900-8702500, hg19) was identified in affected individuals across three pedigrees, but was absent in healthy controls. Two types of CRISPR/Cas9 gene-edited mice were subsequently generated. The knock-in (KI) mouse model was engineered by inserting one copy of the duplicated sequence directly adjacent to the mutated site, whereas the knockout (KO) mouse model was created by excising the mutation sequence. The phenotypes of different group of CRISPR/Cas9 gene-edited mice demonstrated distinct auricular deformities. Furthermore, an increase in the copy number of the mutated sequence was associated with elevated expression levels of HMX1 in the gene-edited mouse model.<br><br>CONCLUSIONS: In this study, we further narrowed down and identified a 600 base pair copy number variation (CNV) located at chr4:8701900-8702500 (hg19), which is implicated in human bilateral, isolated microtia. Utilizing CRISPR/Cas9 technology, we developed novel mouse models harboring the identified mutation. These models serve as a robust platform for the comprehensive investigation of the underlying mechanisms of the disease.}, }
@article {pmid39616957, year = {2025}, author = {Fu, Z and Zhao, L and Guo, Y and Yang, J}, title = {Gene therapy for hereditary hearing loss.}, journal = {Hearing research}, volume = {455}, number = {}, pages = {109151}, doi = {10.1016/j.heares.2024.109151}, pmid = {39616957}, issn = {1878-5891}, mesh = {Animals ; Humans ; CRISPR-Cas Systems/genetics ; Dependovirus/genetics ; *Disease Models, Animal ; *Gene Editing/methods ; Gene Transfer Techniques ; *Genetic Therapy/methods ; *Genetic Vectors ; Hearing/genetics ; *Hearing Loss/genetics/therapy/physiopathology ; Phenotype ; Treatment Outcome ; }, abstract = {Gene therapy is a technique by which exogenous genetic material is introduced into target cells to treat or prevent diseases caused by genetic mutations. Hearing loss is the most common sensory disorder. Genetic factors contribute to approximately 50 % of all cases of profound hearing loss, and more than 150 independent genes have been reported as associated with hearing loss. Recent advances in CRISPR/Cas based gene-editing tools have facilitated the development of gene therapies for hereditary hearing loss (HHL). Viral delivery vectors, and especially adeno-associated virus (AAV) vectors, have been demonstrated as safe and efficient carriers for the delivery of transgenes into inner ear cells in animal models. More importantly, AAV-mediated gene therapy can restore hearing in some children with hereditary deafness. However, there are many different types of HHL that need to be identified and evaluated to determine appropriate gene therapy options. In the present review, we summarize recent animal model-based advances in gene therapy for HHL, as well as gene therapy strategies, gene-editing tools, delivery vectors, and administration routes. We also discuss the strengths and limitations of different gene therapy methods and describe future challenges for the eventual clinical application of gene therapy for HHL.}, }
@article {pmid39616917, year = {2024}, author = {Nassereddine, ZN and Opara, SD and Coutinho, OA and Qyteti, F and Book, R and Heinicke, MP and Napieralski, J and Tiquia-Arashiro, SM}, title = {Critical perspectives on advancing antibiotic resistant gene (ARG) detection technologies in aquatic ecosystems.}, journal = {The Science of the total environment}, volume = {957}, number = {}, pages = {177775}, doi = {10.1016/j.scitotenv.2024.177775}, pmid = {39616917}, issn = {1879-1026}, mesh = {Anti-Bacterial Agents/analysis ; Drug Resistance, Bacterial/genetics ; *Drug Resistance, Microbial/genetics ; *Ecosystem ; *Environmental Monitoring/methods ; Metagenomics/methods ; *Water Microbiology ; }, abstract = {The spread of antibiotic resistance genes (ARGs) in aquatic ecosystems poses a serious risk to environmental and public health, making advanced detection and monitoring methods essential. This review provides a fresh perspective and a critical evaluation of recent advances in detecting and monitoring ARGs in aquatic environments. It highlights the latest innovations in molecular, bioinformatic, and environmental techniques. While traditional methods like culture-based assays and polymerase chain reaction (PCR) remain important, they are increasingly being supplemented by high-throughput sequencing technologies applied to metagenomics. These technologies offer comprehensive insights into the diversity and distribution of ARGs in aquatic environments. The integration of bioinformatic tools and databases has improved the accuracy and efficiency of ARG detection, enabling the analysis of complex datasets and tracking the evolution of ARGs in aquatic settings. Additionally, new environmental monitoring methods, including novel biosensors, geographic information systems (GIS) applications, and remote sensing technologies, have emerged as powerful tools for real-time ARG surveillance in water systems. This review critically examines the challenges of standardizing these methodologies and emphasizes the need for interdisciplinary approaches to enhance ARG monitoring across different aquatic ecosystems. By assessing the strengths and limitations of various methods, this review aims to guide future research and the development of more effective strategies for managing antibiotic resistance in aquatic environments.}, }
@article {pmid39616576, year = {2025}, author = {Ng, EFY and Meitinger, F}, title = {Genetic Engineering and Screening Using Base Editing and Inducible Gene Knockout.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2872}, number = {}, pages = {167-187}, pmid = {39616576}, issn = {1940-6029}, mesh = {Humans ; *Gene Editing/methods ; *Gene Knockout Techniques/methods ; *CRISPR-Cas Systems ; Genetic Engineering/methods ; Genetic Testing/methods ; }, abstract = {Genetic engineering and screening in human cells are powerful techniques for the precise and comprehensive identification and analysis of gene and protein domain functions. Genome-wide knockout screens have been extensively utilized to discover essential genes, tumor suppressors, and genes that regulate responses to various chemicals, including antimitotic and therapeutic drugs. The advent of base editors, which facilitate the targeted mutation of single amino acids, has advanced the identification of critical and functional domains or motifs. In this context, we outline methods for creating efficient base editor and inducible knockout cell lines for targeted gene manipulation and conducting genetic screens to elucidate the roles of genes and their domains within a specific cell biological context.}, }
@article {pmid39974464, year = {2024}, author = {Yee, BJ and Ali, NA and Mohd-Naim, NFB and Ahmed, MU}, title = {Exploiting the Specificity of CRISPR/Cas System for Nucleic Acids Amplification-Free Disease Diagnostics in the Point-of-Care.}, journal = {Chem &amp; bio engineering}, volume = {1}, number = {4}, pages = {330-339}, pmid = {39974464}, issn = {2836-967X}, abstract = {Rapid and reliable molecular diagnostics employing target nucleic acids and small biomarkers are crucial strategies required for the precise detection of numerous diseases. Although diagnoses based on nucleic acid recognition are some of the most efficient and precise procedures, these tests often require expensive equipment and skilled professionals. Recent advancements in diagnostic innovations, particularly those based on clustered regularly interspaced short palindromic repeats (CRISPR), aim to provide thorough screening at homes, in clinics, and in the field. In comparison to traditional molecular techniques like PCR, CRISPR/Cas-based detection, using the single-stranded nucleic acid trans-cleavage abilities of Cas12 or Cas13, shows significant potential as a molecular diagnostic tool. It offers benefits such as attomolar-level sensitivity, single-base precision, and rapid turnover rates. Both Cas enzymes demonstrate exceptional specificity and sensitivity, holding substantial promise in disease diagnostics and beyond. Consequently, various amplification-free CRISPR/Cas-based detection methods have emerged, aiming to maintain sensitivity despite the absence of pre-amplification. This allows for the detection of non-nucleic acid targets and facilitates integration into point-of-care settings. This Review highlights current advances in amplification-free CRISPR/Cas detection systems in disease diagnostics and investigates their utility in point-of-care settings. Furthermore, the mechanisms of alternative CRISPR-based amplification-free detection of other small molecules, aside from nucleic acids, for disease diagnosis will also be briefly discussed.}, }
@article {pmid39628789, year = {2024}, author = {Lim, X and Zhang, C and Chen, X}, title = {Advances and applications of CRISPR/Cas-mediated interference in Escherichia coli.}, journal = {Engineering microbiology}, volume = {4}, number = {1}, pages = {100123}, pmid = {39628789}, issn = {2667-3703}, abstract = {The bacterium Escherichia coli (E. coli) is one of the most widely used chassis microbes employed for the biosynthesis of numerous valuable chemical compounds. In the past decade, the metabolic engineering of E. coli has undergone significant advances, although further productivity improvements will require extensive genome modification, multi-dimensional regulation, and multiple metabolic-pathway coordination. In this context, clustered regularly interspaced short palindromic repeats (CRISPR), along with CRISPR-associated protein (Cas) and its inactive variant (dCas), have emerged as notable recombination and transcriptional regulation tools that are particularly useful for multiplex metabolic engineering in E. coli. In this review, we briefly describe the CRISPR/Cas9 technology in E. coli, and then summarize the recent advances in CRISPR/dCas9 interference (CRISPRi) systems in E. coli, particularly the strategies designed to effectively regulate gene repression and overcome retroactivity during multiplexing. Moreover, we discuss recent applications of the CRISPRi system for enhancing metabolite production in E. coli, and finally highlight the major challenges and future perspectives of this technology.}, }
@article {pmid39628784, year = {2024}, author = {Gao, H and Qiu, Z and Wang, X and Zhang, X and Zhang, Y and Dai, J and Liang, Z}, title = {Recent advances in genome-scale engineering in Escherichia coli and their applications.}, journal = {Engineering microbiology}, volume = {4}, number = {1}, pages = {100115}, pmid = {39628784}, issn = {2667-3703}, abstract = {Owing to the rapid advancement of genome engineering technologies, the scale of genome engineering has expanded dramatically. Genome editing has progressed from one genomic alteration at a time that could only be employed in few species, to the simultaneous generation of multiple modifications across many genomic loci in numerous species. The development and recent advances in multiplex automated genome engineering (MAGE)-associated technologies and clustered regularly interspaced short palindromic repeats and their associated protein (CRISPR-Cas)-based approaches, together with genome-scale synthesis technologies offer unprecedented opportunities for advancing genome-scale engineering in a broader range. These approaches provide new tools to generate strains with desired phenotypes, understand the complexity of biological systems, and directly evolve a genome with novel features. Here, we review the recent major advances in genome-scale engineering tools developed for Escherichia coli, focusing on their applications in identifying essential genes, genome reduction, recoding, and beyond.}, }
@article {pmid39628916, year = {2023}, author = {Wei, J and Li, Y}, title = {CRISPR-based gene editing technology and its application in microbial engineering.}, journal = {Engineering microbiology}, volume = {3}, number = {4}, pages = {100101}, pmid = {39628916}, issn = {2667-3703}, abstract = {Gene editing technology involves the modification of a specific target gene to obtain a new function or phenotype. Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-mediated technologies have provided an efficient tool for genetic engineering of cells and organisms. Here, we review the three emerging gene editing tools (ZFNs, TALENs, and CRISPR-Cas) and briefly introduce the principle, classification, and mechanisms of the CRISPR-Cas systems. Strategies for gene editing based on endogenous and exogenous CRISPR-Cas systems, as well as the novel base editor (BE), prime editor (PE), and CRISPR-associated transposase (CAST) technologies, are described in detail. In addition, we summarize recent developments in the application of CRISPR-based gene editing tools for industrial microorganism and probiotics modifications. Finally, the potential challenges and future perspectives of CRISPR-based gene editing tools are discussed.}, }
@article {pmid39628915, year = {2023}, author = {Wu, X and Cai, P and Yao, L and Zhou, YJ}, title = {Genetic tools for metabolic engineering of Pichia pastoris.}, journal = {Engineering microbiology}, volume = {3}, number = {4}, pages = {100094}, pmid = {39628915}, issn = {2667-3703}, abstract = {The methylotrophic yeast Pichia pastoris (also known as Komagataella phaffii) is widely used as a yeast cell factory for producing heterologous proteins. Recently, it has gained attention for its potential in producing chemicals from inexpensive feedstocks, which requires efficient genetic engineering platforms. This review provides an overview of the current advances in developing genetic tools for metabolic engineering of P. pastoris. The topics cover promoters, terminators, plasmids, genome integration sites, and genetic editing systems, with a special focus on the development of CRISPR/Cas systems and their comparison to other genome editing tools. Additionally, this review highlights the prospects of multiplex genome integration, fine-tuning gene expression, and single-base editing systems. Overall, the aim of this review is to provide valuable insights into current genetic engineering and discuss potential directions for future efforts in developing efficient genetic tools in P. pastoris.}, }
@article {pmid39697359, year = {2022}, author = {Hegeman, CV and de Jong, OG and Lorenowicz, MJ}, title = {A kaleidoscopic view of extracellular vesicles in lysosomal storage disorders.}, journal = {Extracellular vesicles and circulating nucleic acids}, volume = {3}, number = {4}, pages = {393-421}, pmid = {39697359}, issn = {2767-6641}, abstract = {Extracellular vesicles (EVs) are a heterogeneous population of stable lipid membrane particles that play a critical role in the regulation of numerous physiological and pathological processes. EV cargo, which includes lipids, proteins, and RNAs including miRNAs, is affected by the metabolic status of the parental cell. Concordantly, abnormalities in the autophagic-endolysosomal pathway, as seen in lysosomal storage disorders (LSDs), can affect EV release as well as EV cargo. LSDs are a group of over 70 inheritable diseases, characterized by lysosomal dysfunction and gradual accumulation of undigested molecules. LSDs are caused by single gene mutations that lead to a deficiency of a lysosomal protein or lipid. Lysosomal dysfunction sets off a cascade of alterations in the endolysosomal pathway that can affect autophagy and alter calcium homeostasis, leading to energy imbalance, oxidative stress, and apoptosis. The pathophysiology of these diseases is very heterogenous, complex, and currently incompletely understood. LSDs lead to progressive multisystemic symptoms that often include neurological deficits. In this review, a kaleidoscopic overview will be given on the roles of EVs in LSDs, from their contribution to pathology and diagnostics to their role as drug delivery vehicles. Furthermore, EV cargo and surface engineering strategies will be discussed to show the potential of EVs in future LSD treatment, both in the context of enzyme replacement therapy, as well as future gene editing strategies like CRISPR/Cas. The use of engineered EVs as drug delivery vehicles may mask therapeutic cargo from the immune system and protect it from degradation, improving circulation time and targeted delivery.}, }
@article {pmid39872948, year = {2023}, author = {Sun, Y and Lin, W and Kaundal, R and Chi, T}, title = {iMAPping the Perturb-Atlas.}, journal = {Life medicine}, volume = {2}, number = {1}, pages = {lnac057}, pmid = {39872948}, issn = {2755-1733}, abstract = {A key objective of the research in the postgenomic era is to decipher the functions of the mammalian genome, which has remained largely enigmatic despite intensive efforts in the functional genomics field over the past two decades. To attack this problem, we have combined the CRISPR-Cas and Cre-Lox technologies to develop iMAP (inducible Mosaic Animal for Perturbation), a transformative tool for rapidly unraveling mammalian genome function in situ. Furthermore, we have used iMAP to rapidly construct a "Perturb-Atlas" profiling the functions of 90 protein-coding genes across 39 tissues in mice, which has offered rich insights into gene functions difficult to readily obtain using conventional mouse gene-targeting models. In this research highlight, we offer a brief primer on the iMAP technology, outlining its mechanism, strengths and limitations, and pointing out future directions of research in the area.}, }
@article {pmid39634158, year = {2022}, author = {Zhang, Z and Jeong, H and Zu, D and Zhao, X and Senaratne, P and Filbin, J and Silber, B and Kang, S and Gladstone, A and Lau, M and Cui, G and Park, Y and Lee, SE}, title = {Dynamic observations of CRISPR-Cas target recognition and cleavage heterogeneities.}, journal = {Nanophotonics (Berlin, Germany)}, volume = {11}, number = {19}, pages = {4419-4425}, pmid = {39634158}, issn = {2192-8614}, abstract = {CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats) have shown great potential as efficient gene editing tools in disease therapeutics. Although numerous CRISPR-Cas systems have been developed, detailed mechanisms of target recognition and DNA cleavage are still unclear. In this work, we dynamically observe the entire process of conjugation, target recognition and DNA cleavage by single particle spectroscopy of CRISPR-Cas systems on single particle surfaces (gold) with the unique advantage of extended time periods. We show the CRISPR-Cas system, comprised of Cas endonuclease and single guide RNA, is stable and functional on single particle surfaces. Owing to the photostability of single particle surfaces, we directly observe in real time the entire dynamic process of conjugation, target recognition and DNA cleavage without photobleaching. We find heterogeneity in target recognition and DNA cleavage processes in which individual spectra vary significantly from one another as well as from the ensemble. We believe an in depth understanding of heterogeneities in CRISPR-Cas systems can overcome potential barriers in precision medicine and personalized disease therapeutics.}, }
@article {pmid39629166, year = {2021}, author = {Yu, Z and She, Q}, title = {Genome editing from Cas9 to IscB: Backwards and forwards towards new breakthroughs.}, journal = {Engineering microbiology}, volume = {1}, number = {}, pages = {100004}, pmid = {39629166}, issn = {2667-3703}, }
@article {pmid39616566, year = {2025}, author = {Hatano, Y and Yonezawa, N and Tokoro, M and Yao, T and Yamagata, K}, title = {Chromosome Counting at Meiosis and Mitosis of Mouse Oocytes and Embryos Using Super-Resolution Live-Cell Imaging and CRISPR/dCas9-Mediated Live-FISH.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2872}, number = {}, pages = {21-35}, pmid = {39616566}, issn = {1940-6029}, mesh = {Animals ; Mice ; *Oocytes/cytology/metabolism ; *Meiosis/genetics ; *In Situ Hybridization, Fluorescence/methods ; *CRISPR-Cas Systems ; *Mitosis/genetics ; Female ; Embryo, Mammalian/cytology ; Microscopy, Confocal/methods ; Chromosome Segregation ; Chromosomes, Mammalian/genetics ; }, abstract = {Live cell imaging techniques are now essential for capturing chromosomal segregation in fertilized eggs. Although better spatiotemporal resolution of fluorescence observations could provide more information, higher phototoxicity may occur. Super-resolution microscopy is generally considered unsuitable for live cell imaging because of the considerable cell damage. Here, we developed a method for counting chromosomes in mouse living oocytes and early embryos using super-resolution microscopy based on disk confocal photon reassignment microscopy (OPRA). In this chapter, we describe the imaging conditions for minimally invasive, high-resolution observation of mouse oocytes and embryos and a method to count chromosomes via CRISPR/dCas-mediated live-cell fluorescence in situ hybridization.}, }
@article {pmid39615910, year = {2025}, author = {Amouzadeh Tabrizi, M and Ali, AA and Singuru, MMR and Mi, L and Bhattacharyya, P and You, M}, title = {A portable and sensitive DNA-based electrochemical sensor for detecting piconewton-scale cellular forces.}, journal = {Analytica chimica acta}, volume = {1333}, number = {}, pages = {343392}, pmid = {39615910}, issn = {1873-4324}, support = {R35 GM133507/GM/NIGMS NIH HHS/United States ; }, mesh = {*Electrochemical Techniques/instrumentation ; *DNA Probes ; *Biosensing Techniques/instrumentation ; *Smartphone ; Cell Adhesion ; HeLa Cells ; Humans ; CRISPR-Cas Systems ; }, abstract = {BACKGROUND: Cell-generated forces are a key player in cell biology, especially during cellular shape formation, migration, cancer development, and immune response. The measurement of forces exerted and experienced by cells is fundamental in understanding these mechanosensitive cellular behaviors. While cell-generated forces can now be detected based on techniques like fluorescence microscopy, atomic force microscopy, optical/magnetic tweezers, however, most of these approaches rely on complicated instruments or materials, as well as skilled operators, which could limit their potential broad applications in regular biological laboratories.<br><br>RESULTS: A new type of smartphone-based electrochemical sensor is developed here for cellular force measurement. In this system, a double-stranded DNA-based force probe, known as tension gauge tether, is attached to the surface of a gold screen-printed electrode, which is then incorporated into a portable smartphone-based electrochemical device. Cellular force-induced DNA detachment on the sensor surface results in multiple redox reporters to reach the surface of the electrode and generate enhanced electrochemical signals. To further improve the sensitivity, a CRISPR-Cas12a system has also been incorporated to cleave the remaining surface-attached anchor DNA strand. Using integrin-mediated tension as an example, piconewton-scale adhesion forces generated by&#xa0;&#x2264;&#xa0;10 HeLa cells could now be reliably detected. Meanwhile, the threshold forces of these electrochemical sensors can also be modularly tuned to detect different levels of cellular forces.<br><br>SIGNIFICANCE: These novel DNA-based highly sensitive, portable, cost-efficient, and easy-to-use electrochemical sensors can be potentially powerful tools for detecting different cell-generated molecular forces. Functioning as complementary tools with traction force microscopy and fluorescent probes, these electrochemical sensors can be straightforwardly applied in regular biological laboratories for understanding the basic mechanical principles of cell signaling and for developing novel strategies and materials in tissue engineering, regenerative medicine, and cell therapy.}, }
@article {pmid39615800, year = {2025}, author = {Mocchetti, A and De Rouck, S and Naessens, S and Dermauw, W and Van Leeuwen, T}, title = {SYNCAS based CRISPR-Cas9 gene editing in predatory mites, whiteflies and stinkbugs.}, journal = {Insect biochemistry and molecular biology}, volume = {177}, number = {}, pages = {104232}, pmid = {39615800}, issn = {1879-0240}, mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Hemiptera/genetics ; *Mites/genetics ; Insect Proteins/genetics ; Female ; }, abstract = {Despite the establishment of CRISPR-Cas9 gene editing protocols in a wide range of organisms, genetic engineering is still challenging for many organisms due to constraints including lethality of embryo injection, difficulties in egg/embryo collection or viviparous lifestyles. Recently, an efficient CRISPR-Cas9 method, termed SYNCAS, was developed to genetically modify spider mites and thrips species. The method is based on maternal injection of formulated CRISPR-Cas9 using saponin and BAPC. Here, we investigate whether the method can be used to perform gene editing in other arthropods such as the beneficial predatory mites Amblyseius swirskii and Phytoseiulus persimilis, and the pests Bemisia tabaci and Nezara viridula. For the predatory mites, Antp and SLC25A38 were used as target genes, while the ortholog of the Drosophila melanogaster ABCG transporter white was targeted in B. tabaci and N. viridula. All species were successfully edited with the highest efficiencies (up to 39%) being obtained for B. tabaci. For A. swirskii and P. persimilis no clear phenotypes could be observed, even though SLC25A38 was successfully knocked-out. The lack of a color phenotype in SLC25A38 mutants was confirmed in the spider mite Tetranychus urticae. Disruption of the target gene Antp is likely lethal in predatory mites, as no true null mutants could be recovered. For B. tabaci, KO of white resulted in orange eyes which diverges from the phenotype seen in white mutants of D. melanogaster. In the last species, N. viridula, a single phenotypic mutant could be detected having a patchy white body coloration with wild type eye coloration. Genotyping revealed a single amino acid deletion at the target site, suggesting the creation of a hypomorphic allele. To conclude, the protocols provided in this work can contribute to the genetic study of predatory mites used in biological control, as well as hemipteran pests.}, }
@article {pmid39615707, year = {2025}, author = {Kadkhoda, H and Gholizadeh, P and Ghotaslou, R and Nabizadeh, E and Pirzadeh, T and Ahangarzadeh Rezaee, M and Feizi, H and Samadi Kafil, H and Aghazadeh, M}, title = {Role of CRISPR-cas system on virulence traits and carbapenem resistance in clinical Klebsiella pneumoniae isolates.}, journal = {Microbial pathogenesis}, volume = {199}, number = {}, pages = {107151}, doi = {10.1016/j.micpath.2024.107151}, pmid = {39615707}, issn = {1096-1208}, mesh = {*Klebsiella pneumoniae/genetics/drug effects/pathogenicity/isolation &amp; purification ; Biofilms/growth &amp; development ; *CRISPR-Cas Systems/genetics ; Humans ; Bacterial Proteins/genetics ; Virulence/genetics ; Microbial Sensitivity Tests ; *Carbapenems/pharmacology ; Klebsiella Infections/microbiology ; beta-Lactamases/genetics ; Anti-Bacterial Agents/pharmacology ; Virulence Factors/genetics ; Random Amplified Polymorphic DNA Technique ; Plasmids/genetics ; Drug Resistance, Bacterial/genetics ; *Carbapenem-Resistant Enterobacteriaceae/genetics/drug effects ; }, abstract = {BACKGROUND AND OBJECTIVES: The bacterial adaptive immune system known as CRISPR-Cas (clustered regularly interspersed short palindromic repeats-CRISPR-associated protein) is engaged in defense against various mobile genetic elements (MGEs) such as plasmids and bacteriophages. The purpose of this study was to characterize the CRISPR-Cas systems in carbapenem-resistant Klebsiella pneumoniae isolates and assess any possible correlation between these systems with antibiotic susceptibility, biofilm formation, and bacterial virulence.<br><br>MATERIALS AND METHODS: A total of 156 CRKP isolates were collected from different specimens of the inpatients. Biofilm formation and antibiotic susceptibility testing were evaluated using standard methods. Furthermore, the CRISPR-Cas system subtype genes, 11 carbapenemase genes, and 17 virulence genes were identified using separate standard PCR reactions. The diversity of the isolates was determined by random amplified polymorphic DNA (RAPD)-PCR.<br><br>RESULTS: The development of biofilms and antibiotic susceptibility of several CRKP isolates were significantly correlated with the absence or presence of the CRISPR-Cas system. PCR analysis of carbapenemase genes revealed that the frequency of the blaNDM-1 gene was significantly higher in the isolates with the subtype I-E CRISPR-Cas system. Moreover, the isolates with the subtype I-E CRISPR-Cas system exhibited a propensity to possess more virulence genes such as allS, k2A, wcaG, aerobactin, rmpA, iroN, magA, rmpA2, kfu, iutA, iucB, ybtS, repA, and terW.<br><br>CONCLUSION: CRISPR-Cas systems could affect the antibiotic susceptibility, capacity for biofilm formation, and virulence of Klebsiella pneumoniae. Our findings showed that the isolates containing the CRISPR-Cas system were moderate or strong biofilm producers and had a higher frequency of virulence genes.}, }
@article {pmid39615667, year = {2025}, author = {Chen, X and Li, F and Li, X and Otto, M and Chen, Y and Siewers, V}, title = {Model-assisted CRISPRi/a library screening reveals central carbon metabolic targets for enhanced recombinant protein production in yeast.}, journal = {Metabolic engineering}, volume = {88}, number = {}, pages = {1-13}, doi = {10.1016/j.ymben.2024.11.010}, pmid = {39615667}, issn = {1096-7184}, mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *Recombinant Proteins/genetics/biosynthesis ; *alpha-Amylases/genetics/biosynthesis ; *Metabolic Engineering/methods ; *Saccharomyces cerevisiae Proteins/genetics/biosynthesis/metabolism ; *Carbon/metabolism ; *Models, Biological ; *CRISPR-Cas Systems ; Gene Library ; }, abstract = {Production of recombinant proteins is regarded as an important breakthrough in the field of biomedicine and industrial biotechnology. Due to the complexity of the protein secretory pathway and its tight interaction with cellular metabolism, the application of traditional metabolic engineering tools to improve recombinant protein production faces major challenges. A systematic approach is required to generate novel design principles for superior protein secretion cell factories. Here, we applied a proteome-constrained genome-scale protein secretory model of the yeast Saccharomyces cerevisiae (pcSecYeast) to simulate α-amylase production under limited secretory capacity and predict gene targets for downregulation and upregulation to improve α-amylase production. The predicted targets were evaluated using high-throughput screening of specifically designed CRISPR interference/activation (CRISPRi/a) libraries and droplet microfluidics screening. From each library, 200 and 190 sorted clones, respectively, were manually verified. Out of them, 50% of predicted downregulation targets and 34.6% predicted upregulation targets were confirmed to improve α-amylase production. By simultaneously fine-tuning the expression of three genes in central carbon metabolism, i.e. LPD1, MDH1, and ACS1, we were able to increase the carbon flux in the fermentative pathway and α-amylase production. This study exemplifies how model-based predictions can be rapidly validated via a high-throughput screening approach. Our findings highlight novel engineering targets for cell factories and furthermore shed light on the connectivity between recombinant protein production and central carbon metabolism.}, }
@article {pmid39615461, year = {2024}, author = {Li, CY and Chen, LW and Tsai, MC and Chou, YY and Lin, PX and Chang, YM and Hwu, WL and Chien, YH and Lin, JL and Chen, HA and Lee, NC and Su, PH and Hsieh, TC and Klinkhammer, H and Wang, YC and Huang, YT and Krawitz, PM and Lin, SH and Huang, LLH and Chiang, PM and Shih, MH and Chen, PC}, title = {Homozygous variant in translocase of outer mitochondrial membrane 7 leads to metabolic reprogramming and microcephalic osteodysplastic dwarfism with moyamoya disease.}, journal = {EBioMedicine}, volume = {110}, number = {}, pages = {105476}, pmid = {39615461}, issn = {2352-3964}, mesh = {Humans ; Animals ; *Zebrafish ; *Moyamoya Disease/genetics/metabolism/pathology ; *Microcephaly/genetics/metabolism ; Male ; *Homozygote ; Female ; Mitochondrial Precursor Protein Import Complex Proteins ; Proteomics/methods ; Mitochondria/metabolism/genetics ; Induced Pluripotent Stem Cells/metabolism ; Endothelial Cells/metabolism ; Dwarfism/genetics/metabolism/pathology ; Child ; Pedigree ; CRISPR-Cas Systems ; Child, Preschool ; Phenotype ; Disease Models, Animal ; Metabolic Reprogramming ; }, abstract = {BACKGROUND: Impaired mitochondrial protein import machinery leads to phenotypically heterogeneous diseases. Here, we report a recurrent homozygous missense variant in the gene that encodes the translocase of outer mitochondrial membrane 7 (TOMM7) in nine patients with microcephaly, short stature, facial dysmorphia, atrophic macular scarring, and moyamoya disease from seven unrelated families.<br><br>METHODS: To prove the causality of the TOMM7 variant, mitochondrial morphology, proteomics, and respiration were investigated in CRISPR/Cas9-edited iPSCs-derived endothelial cells. Cerebrovascular defects and mitochondrial respiration were also examined in CRISPR/Cas9-edited zebrafish.<br><br>FINDINGS: iPSC-derived endothelial cells with homozygous TOMM7 p.P29L showed increased TOM7 stability, enlarged mitochondria, increased senescence, and defective tube formation. In addition, proteomic analysis revealed a reduced abundance of mitochondrial proteins involved in ATP synthesis or coordinating TCA cycle and gluconeogenesis. Moreover, mitochondrial respiration was slightly decreased while ATP production from glycolysis was significantly increased. Furthermore, deletion of tomm7 in zebrafish caused craniofacial and cerebrovascular defects that recapitulated human phenotypes. Notably, homozygous iPSCs differentially expressed genes involved in glycolysis and response to hypoxia. Finally, the metabolic imbalance was evidenced by decreased oxygen consumption, increased level of hexokinase 2, and enhanced glycolysis in endothelial cells derived from the patient's iPSCs.<br><br>INTERPRETATION: These results revealed the essential role of TOMM7 in balancing cellular sources of energy production at both proteomic and transcriptomic levels and provided the molecular mechanisms through which TOMM7 p.P29L variant leads to an autosomal recessive microcephalic osteodysplastic dwarfism with moyamoya disease.<br><br>FUNDING: This work is supported by National Science and Technology Council grants and National Cheng Kung University Hospital.}, }
@article {pmid39614269, year = {2024}, author = {Wang, Y and Yu, F and Zhang, K and Shi, K and Chen, Y and Li, J and Li, X and Zhang, L}, title = {End-point RPA-CRISPR/Cas12a-based detection of Enterocytozoon bieneusi nucleic acid: rapid, sensitive and specific.}, journal = {BMC veterinary research}, volume = {20}, number = {1}, pages = {540}, pmid = {39614269}, issn = {1746-6148}, support = {22A310019//Key Scientific Research Foundation of the Higher Education Institutions of Henan Province/ ; 2023YFD1801200//National Key Research and Development Program of China/ ; 231111111500//Key Research and Development Special Project of Henan Province of China/ ; }, mesh = {*Enterocytozoon/genetics/isolation &amp; purification ; *Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Microsporidiosis/veterinary/diagnosis/microbiology ; Animals ; DNA, Fungal/genetics ; Limit of Detection ; Feces/microbiology ; Dogs ; }, abstract = {Enterocytozoon bieneusi is a common species of microsporidia that infects humans and animals. Current methods for detecting E. bieneusi infections have trade-offs in sensitivity, specificity, simplicity, cost and speed and are thus unacceptable for clinical application. We tested the effectiveness of a previously reported CRISPR/Cas12a-based method (ReCTC) when used for the nucleic acid detection of E. bieneusi. The limit of detection (LOD) and the specificity of the expanded ReCTC were evaluated using prepared target DNA, and the accuracy of the ReCTC-based detection of E. bieneusi in clinical samples was validated. The ReCTC method was successfully used for the nucleic acid detection of E. bieneusi. The sensitivity test indicated an LOD of 3.7 copies/µl for the ReCTC-based fluorescence and lateral flow strip methods. In specificity test involving other common enteric pathogens, a fluorescent signal and/or test line appeared only when the sample was positive for E. bieneusi. These results demonstrated that the ReCTC method can successfully detect E. bieneusi in clinical samples. The ReCTC method was successfully used to detect E. bieneusi nucleic acid with high sensitivity and specificity. It had excellent performance in clinical DNA samples and was superior to nested polymerase chain reaction. Furthermore, the ReCTC method demonstrated its capability for use in on-site detection.}, }
@article {pmid39614172, year = {2024}, author = {Yang, W and Zhu, JK and Jin, W}, title = {A catalog of gene editing sites and genetic variations in editing sites in model organisms.}, journal = {BMC genomics}, volume = {25}, number = {1}, pages = {1153}, pmid = {39614172}, issn = {1471-2164}, support = {2021YFA1300404//National Key R&amp;D Program of China/ ; 2021YFA0909300//National Key R&amp;D Program of China/ ; 32188102//National Natural Science Foundation of China/ ; 32170646//National Natural Science Foundation of China/ ; }, mesh = {*Gene Editing ; Animals ; Humans ; *CRISPR-Cas Systems ; *Genetic Variation ; Mice ; Receptors, CCR5/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; Oryza/genetics ; Polymorphism, Single Nucleotide ; Arabidopsis/genetics ; }, abstract = {BACKGROUND: CRISPR-Cas systems require a protospacer adjacent motif (PAM), which plays an essential role in self/non-self discrimination in their natural context, to cleave DNA for genome editing. Unfortunately, common genetic variation is distributed throughout genomes, which can block recognition of target sites by Cas proteins. However, little information is available about the distribution of editing sites in model organisms and how often common variation overlaps with those PAM sites.<br><br>RESULTS: Herein, we characterized six representative Cas proteins (Cas9, Cas12a, Cas12b, Cas12i, Cas12j and Cas12l) genomic editing sites in ten model organisms (yeast, flatworms, flies, zebrafish, mice, humans, rice, maize, Arabidopsis and tomato). We demonstrated that there were more than 34 editing sites per kilobase on average in these genomes. In each genome, 91.69-99.83% and 95.4-99.73% of genes had at least one unique editing site in exon and promoter, respectively. Depending on publicly available genomic diversity data, we identified the variations (SNPs and InDels) in editing sites in humans and rice, indicating the risk in the application of CRISPR/Cas technology. Finally, using CCR5 and BCL11A as examples, we revealed variation site was a factor that must be considered when designing sgRNA.<br><br>CONCLUSIONS: Our findings not only revealed the distribution characteristics of editing sites of six representative Cas proteins in ten model organism genomes but also shed light on the adverse effect of variation sites on target site recognition. Our current work will serve as a reminder of the risks of CRISPR application.}, }
@article {pmid39614043, year = {2025}, author = {Lei, T and Zhang, G}, title = {Generation of Cdc20 RNAi-Sensitive Cell Lines to Study Mitotic Exit.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2874}, number = {}, pages = {9-20}, pmid = {39614043}, issn = {1940-6029}, mesh = {*Cdc20 Proteins/genetics/metabolism ; *Mitosis/genetics ; *RNA Interference ; *CRISPR-Cas Systems ; Humans ; Cell Line ; Kinetochores/metabolism ; M Phase Cell Cycle Checkpoints/genetics ; }, abstract = {Accurate mitotic progression ensures the fidelity of genome passage. Cdc20 is a key mitotic regulator. It promotes mitotic exit by activating the anaphase-promoting complex or cyclosome (APC/C) and monitors kinetochore-microtubule attachment through activating the spindle assembly checkpoint (SAC). Precise characterization of Cdc20 requires efficient depletion of endogenous Cdc20, which is extremely difficult to achieve by RNA interference (RNAi). This chapter describes the methodology to generate Cdc20 RNAi-sensitive cell lines with the help of CRISPR/Cas9 technology. These cell lines are highly sensitive to Cdc20 RNAi and provide a very useful tool for Cdc20 functionality investigation without the interference of endogenous Cdc20 protein. Similar strategy could be applied to other genes.}, }
@article {pmid39612948, year = {2025}, author = {Kaul, R and Thangaraj, A and Sharda, S and Kaul, T}, title = {Optimization of tissue culture and Cas9 transgene expression in tomato: A step towards CRISPR/Cas9-based genetic improvement.}, journal = {Plant science : an international journal of experimental plant biology}, volume = {352}, number = {}, pages = {112324}, doi = {10.1016/j.plantsci.2024.112324}, pmid = {39612948}, issn = {1873-2259}, mesh = {*Solanum lycopersicum/genetics/growth &amp; development ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Tissue Culture Techniques/methods ; *Transgenes ; Gene Editing/methods ; Plant Shoots/growth &amp; development/genetics ; Plant Roots/growth &amp; development/genetics ; Regeneration ; Plant Growth Regulators/pharmacology ; Agrobacterium tumefaciens/genetics ; }, abstract = {Tomato (Solanum lycopersicum L.) is an essential source of antioxidants and a prime candidate for bioengineering experiments. Many studies have aimed to improve tomatoes using CRISPR/Cas9 technology; however, the success rate is limited due to the lack of efficient regeneration and genetic modification techniques. Here, we report an efficient regeneration and transformation procedure focused on developing efficient Cas9 gene transgenic tomato plants using the Agrobacterium tumefaciens strain LBA4404 harbouring pCRISPR/Cas9TK2-NIC binary vector. We optimized the concentrations and combinations of growth hormones to promote direct shoot and root regeneration via hypocotyl explants. We found that MS medium 2.0 mg/l Zeatin (Zn) + 1.5 mg/l Indole -3- acetic acid (IAA) + 0.3 mg/l Benzyl amino purine (BAP) was preeminent for shoot regeneration medium, and 0.5 mg/l BAP+ 0.1 mg/l IAA was appropriate for root regeneration. Cas9 transgenes in the tomato genome of putative tomato plants were validated using various methods, including polymerase chain reaction (PCR), and confirmed via Southern blotting. The developed protocol showed improved regeneration and transformation efficiencies in tomatoes of 88 % and 54 %, respectively. In this study, we successfully established a gene delivery platform for tomatoes using the CRISPR/Cas9 system.}, }
@article {pmid39612312, year = {2025}, author = {Liu, Y and Liu, Q and Yi, C and Liu, C and Shi, Q and Wang, M and Han, F}, title = {Past innovations and future possibilities in plant chromosome engineering.}, journal = {Plant biotechnology journal}, volume = {23}, number = {3}, pages = {695-708}, pmid = {39612312}, issn = {1467-7652}, support = {31991212//National Natural Science Foundation of China/ ; 2023ZD04025//Biological Breeding-National Science and Technology Major Project/ ; }, mesh = {*Chromosomes, Plant/genetics ; *Genetic Engineering/methods/trends ; Plant Breeding/methods ; Centromere/genetics ; Gene Editing ; CRISPR-Cas Systems ; }, abstract = {Plant chromosome engineering has emerged as a pivotal tool in modern plant breeding, facilitating the transfer of desirable traits through the incorporation of alien chromosome fragments into plants. Here, we provide a comprehensive overview of the past achievements, current methodologies and future prospects of plant chromosome engineering. We begin by examining the successful integration of specific examples such as the incorporation of rye chromosome segments (e.g. the 1BL/1RS translocation), Dasypyrum villosum segments (e.g. the 6VS segment for powdery mildew resistance), Thinopyrum intermedium segments (e.g. rust resistance genes) and Thinopyrum elongatum segments (e.g. Fusarium head blight resistance genes). In addition to trait transfer, advancements in plant centromere engineering have opened new possibilities for chromosomal manipulation. This includes the development of plant minichromosomes via centromere-mediated techniques, the generation of haploids through CENH3 gene editing, and the induction of aneuploidy using KaryoCreate. The advent of CRISPR/Cas technology has further revolutionized chromosome engineering, enabling large-scale chromosomal rearrangements, such as inversions and translocations, as well as enabling targeted insertion of large DNA fragments and increasing genetic recombination frequency. These advancements have significantly expanded the toolkit for genetic improvement in plants, opening new horizons for the future of plant breeding.}, }
@article {pmid39611587, year = {2024}, author = {Sayid, R and van den Hurk, AWM and Rothschild-Rodriguez, D and Herrema, H and de Jonge, PA and Nobrega, FL}, title = {Characteristics of phage-plasmids and their impact on microbial communities.}, journal = {Essays in biochemistry}, volume = {68}, number = {5}, pages = {583-592}, pmid = {39611587}, issn = {1744-1358}, support = {AUF 5188//Amsterdam University Fund/ ; PhD fellowship//Amsterdam Cardiovascular Sciences, Amsterdam University Medical Centers (ACS)/ ; //Bowel Research UK (BRUK)/ ; Aspasia premium (015.017.050)//Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)/ ; BB/T008768/1//Biotechnology and Biological Sciences Research Council (BBSRC)/ ; Black Futures Research Studentship//University of Southampton (University of Southampton UK)/ ; }, mesh = {*Bacteriophages/physiology ; *Plasmids/genetics ; *Bacteria/virology/genetics ; *Microbiota ; CRISPR-Cas Systems ; }, abstract = {Bacteria host various foreign genetic elements, most notably plasmids and bacteriophages (or phages). Historically, these two classes were seen as separate, but recent research has shown considerable interplay between them. Phage-plasmids (P-Ps) exhibit characteristics of both phages and plasmids, allowing them to exist extrachromosomally within bacterial hosts as plasmids, but also to infect and lyse bacteria as phages. This dual functionality enables P-Ps to utilize the modes of transmission of both phage and plasmids, facilitating the rapid dissemination of genetic material, including antibiotic resistance and virulence genes, throughout bacterial populations. Additionally, P-Ps have been found to encode toxin-antitoxin and CRISPR-Cas adaptive immune systems, which enhance bacterial survival under stress and provide immunity against other foreign genetic elements. Despite a growing body of literature on P-Ps, large gaps remain in our understanding of their ecological roles and environmental prevalence. This review aims to synthesise existing knowledge and identify research gaps on the impacts of P-Ps on microbial communities.}, }
@article {pmid39610939, year = {2024}, author = {Chu, LL and Sohng, JK and Bae, H and Dhakal, D}, title = {Editorial: Recent advances in application of synthetic biology for production of bioactive compounds, volume II.}, journal = {Frontiers in bioengineering and biotechnology}, volume = {12}, number = {}, pages = {1517610}, doi = {10.3389/fbioe.2024.1517610}, pmid = {39610939}, issn = {2296-4185}, }
@article {pmid39609710, year = {2025}, author = {Feng, J and Ma, Y and Zhang, D and Wang, Y}, title = {High-Efficiency Genome Editing in Naturally Isolated Aeromonas hydrophila and Edwardsiella Piscicida Using the CRISPR-Cas9 System.}, journal = {Biotechnology and bioengineering}, volume = {122}, number = {3}, pages = {606-614}, doi = {10.1002/bit.28889}, pmid = {39609710}, issn = {1097-0290}, support = {//This work was supported by a Specific Cooperative Agreement between USDA-ARS (Aquatic Animal Health Unit, Auburn, AL) and Auburn University, the USDA-NIFA Hatch project (ALA014-1017025), and the Alabama Agricultural Experiment Station./ ; }, mesh = {*Aeromonas hydrophila/genetics/isolation &amp; purification ; *Edwardsiella/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome, Bacterial ; }, abstract = {Aeromonas hydrophila and Edwardsiella piscicida are significant bacterial pathogens in aquaculture, causing severe diseases and tremendous economic losses worldwide. Additionally, both of them can act as opportunistic pathogens in humans, leading to severe infections. Efficient genome editing tools for these pathogens are essential for understanding their pathogenic mechanisms and physiological behaviors, enabling the development of targeted strategies to control and mitigate their effects. In this study, we adapted the CRISPR-Cas9 system for high-efficiency, marker-less genome editing in multiple naturally isolated strains of these two aquaculture pathogens. We developed a streamlined procedure that successfully generated deletion mutants of the aerA gene (encoding for aerolysin, a pore-forming toxin that plays a critical role in the pathogenicity) and the gfp insertion mutants in three naturally isolated A. hydrophila strains. Additionally, we deleted five putative hemolysin-encoding genes in both A. hydrophila ML10-51K and its ∆aerA derivative. The same system was also applied to the naturally isolated E. piscicida S11-285 strain, successfully deleting the ssaV gene (a component of the Type III Secretion System-a critical virulence mechanism in many pathogenic bacteria). The methodologies developed herein could be broadly applied to other pathogenic strains from natural environments, providing valuable tools for studying bacterial pathogenesis and aiding in the development of effective control strategies.}, }
@article {pmid39609594, year = {2025}, author = {Murray, JB and Harrison, PT and Scholefield, J}, title = {Prime editing: therapeutic advances and mechanistic insights.}, journal = {Gene therapy}, volume = {32}, number = {2}, pages = {83-92}, pmid = {39609594}, issn = {1476-5462}, support = {20 FFP-P-8661//Science Foundation Ireland (SFI)/ ; DRUMM22G0-COLLAB//Cystic Fibrosis Foundation (CF Foundation)/ ; }, mesh = {*Gene Editing/methods/trends ; Humans ; *Genetic Therapy/methods/trends ; CRISPR-Cas Systems ; Animals ; }, abstract = {We are often confronted with a simple question, "which gene editing technique is the best?"; the simple answer is "there isn't one". In 2021, a year after prime editing first made its mark, we evaluated the landscape of this potentially transformative advance in genome engineering towards getting treatments to the clinic [1]. Nearly 20% of the papers we cited were still in pre-print at the time which serves to indicate how early-stage the knowledge base was at that time. Now, three years later, we take a look at the landscape and ask what has been learnt to ensure this tech is broadly accessible, highlighting some key advances, especially those that push this towards the clinic. A big part of the appeal of prime editing is its ability to precisely edit DNA without double stranded breaks, and to install any of the 12 possible single-nucleotide conversion events as well as small insertions and/or deletions, or essentially any combination thereof. Over the last few decades, other transformative and Nobel prize-winning technologies that rely on Watson-Crick base-pairing such as PCR, site-directed mutagenesis, RNA interference, and one might say, "classic" CRISPR, were swiftly adopted across labs around the world because of the speed with which mechanistic rules governing their efficiency were determined. Whilst this perspective focuses on the context of gene therapy applications of prime editing, we also further look at the recent studies which have increased our understanding of the mechanism of PEs and simultaneously improved the efficiency and diversity of the PE toolbox.}, }
@article {pmid39609592, year = {2025}, author = {Geuverink, WP and Houtman, D and Retel Helmrich, IRA and Kist, JD and Henneman, L and Cornel, MC and Riedijk, SR and , }, title = {A decade of public engagement regarding human germline gene editing: a systematic scoping review.}, journal = {European journal of human genetics : EJHG}, volume = {33}, number = {5}, pages = {570-579}, pmid = {39609592}, issn = {1476-5438}, mesh = {Humans ; *Gene Editing/ethics ; *Germ Cells/metabolism ; *Community Participation ; CRISPR-Cas Systems ; }, abstract = {Following the discovery of the CRISPR-Cas technology in 2012, there has been a growing global call for public engagement regarding the potential use of human germline gene editing (HGGE). In this systematic scoping review, we aim to evaluate public engagement studies considering the following questions based on three points of attention: 1) Inclusion of underrepresented groups: who have been engaged? 2) Gathering values: what output has been reported? 3) Reaching societal impact: what objectives of public engagement have been reported? A systematic literature search from 2012 to 2023 identified 3464 articles reporting on public engagement studies regarding HGGE retrieved from 12 databases. After screening, 52 full-text articles were assessed for eligibility, resulting in 36 articles that cover 31 public engagement studies. We conclude that co-created efforts are needed to engage underrepresented groups as well as to yield values rather than acceptance levels, and to concretise how engagement might result in societal impact.}, }
@article {pmid39609561, year = {2025}, author = {Hołubowicz, R and Du, SW and Felgner, J and Smidak, R and Choi, EH and Palczewska, G and Menezes, CR and Dong, Z and Gao, F and Medani, O and Yan, AL and Hołubowicz, MW and Chen, PZ and Bassetto, M and Risaliti, E and Salom, D and Workman, JN and Kiser, PD and Foik, AT and Lyon, DC and Newby, GA and Liu, DR and Felgner, PL and Palczewski, K}, title = {Safer and efficient base editing and prime editing via ribonucleoproteins delivered through optimized lipid-nanoparticle formulations.}, journal = {Nature biomedical engineering}, volume = {9}, number = {1}, pages = {57-78}, pmid = {39609561}, issn = {2157-846X}, support = {F30 EY033642/EY/NEI NIH HHS/United States ; FENG.02.01-IP.05-T005/23//Fundacja na rzecz Nauki Polskiej (Foundation for Polish Science)/ ; R01 EY032948/EY/NEI NIH HHS/United States ; R01EY032948, R21NS113264//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R00 HL163805/HL/NHLBI NIH HHS/United States ; R21 NS113264/NS/NINDS NIH HHS/United States ; R01 EY030873/EY/NEI NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; UG3AI150551, U01AI142756, R35GM118062, RM1HG009490//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01 EY034501/EY/NEI NIH HHS/United States ; N66001-21-C-4013//United States Department of Defense | Defense Threat Reduction Agency (DTRA)/ ; T32GM008620, F30EY033642//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; T32GM148383//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; P30EY034070//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; I01BX004939//U.S. Department of Veterans Affairs (Department of Veterans Affairs)/ ; UG3 AI150551/AI/NIAID NIH HHS/United States ; 75N93022C00054/AI/NIAID NIH HHS/United States ; R01EY009339, R01EY030873, P30EY034070, P30CA062203//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01 EY009339/EY/NEI NIH HHS/United States ; P30 EY034070/EY/NEI NIH HHS/United States ; T32 GM008620/GM/NIGMS NIH HHS/United States ; R00HL163805//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; I01 BX004939/BX/BLRD VA/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; T32 GM148383/GM/NIGMS NIH HHS/United States ; P30 CA062203/CA/NCI NIH HHS/United States ; 2022/47/B/NZ5/03023, 2020/39/D/NZ4/01881, 2019/34/E/NZ5/00434//Narodowe Centrum Nauki (National Science Centre)/ ; }, mesh = {*Gene Editing/methods ; *Ribonucleoproteins/genetics/chemistry/metabolism ; *Nanoparticles/chemistry ; *Lipids/chemistry ; Humans ; Animals ; Mice ; Cell-Penetrating Peptides/chemistry ; HEK293 Cells ; CRISPR-Cas Systems ; Liposomes ; }, abstract = {Delivering ribonucleoproteins (RNPs) for in vivo genome editing is safer than using viruses encoding for Cas9 and its respective guide RNA. However, transient RNP activity does not typically lead to optimal editing outcomes. Here we show that the efficiency of delivering RNPs can be enhanced by cell-penetrating peptides (covalently fused to the protein or as excipients) and that lipid nanoparticles (LNPs) encapsulating RNPs can be optimized for enhanced RNP stability, delivery efficiency and editing potency. Specifically, after screening for suitable ionizable cationic lipids and by optimizing the concentration of the synthetic lipid DMG-PEG 2000, we show that the encapsulation, via microfluidic mixing, of adenine base editor and prime editor RNPs within LNPs using the ionizable lipid SM102 can result in in vivo editing-efficiency enhancements larger than 300-fold (with respect to the delivery of the naked RNP) without detectable off-target edits. We believe that chemically defined LNP formulations optimized for RNP-encapsulation stability and delivery efficiency will lead to safer genome editing.}, }
@article {pmid39609418, year = {2024}, author = {Miskalis, A and Shirguppe, S and Winter, J and Elias, G and Swami, D and Nambiar, A and Stilger, M and Woods, WS and Gosstola, N and Gapinske, M and Zeballos, A and Moore, H and Maslov, S and Gaj, T and Perez-Pinera, P}, title = {SPLICER: a highly efficient base editing toolbox that enables in vivo therapeutic exon skipping.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10354}, pmid = {39609418}, issn = {2041-1723}, support = {R01 GM131272/GM/NIGMS NIH HHS/United States ; MDA602798//Muscular Dystrophy Association (Muscular Dystrophy Association Inc.)/ ; UL1 TR001422/TR/NCATS NIH HHS/United States ; 17SDG33650087//American Heart Association (American Heart Association, Inc.)/ ; 1U01NS122102//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; 1R01NS123556//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; 1R01GM127497//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01 GM141296/GM/NIGMS NIH HHS/United States ; 1R01GM141296//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01 GM127497/GM/NIGMS NIH HHS/United States ; U01 NS122102/NS/NINDS NIH HHS/United States ; R01 NS123556/NS/NINDS NIH HHS/United States ; T32 EB019944/EB/NIBIB NIH HHS/United States ; 1R01GM131272//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; }, mesh = {*Exons/genetics ; Animals ; *Gene Editing/methods ; Humans ; Mice ; *Alzheimer Disease/genetics/therapy/metabolism ; Disease Models, Animal ; HEK293 Cells ; CRISPR-Cas Systems ; Mice, Transgenic ; RNA Splice Sites/genetics ; Amyloid beta-Peptides/metabolism/genetics ; RNA Splicing ; }, abstract = {Exon skipping technologies enable exclusion of targeted exons from mature mRNA transcripts, which have broad applications in medicine and biotechnology. Existing techniques including antisense oligonucleotides, targetable nucleases, and base editors, while effective for specific applications, remain hindered by transient effects, genotoxicity, and inconsistent exon skipping. To overcome these limitations, here we develop SPLICER, a toolbox of next-generation base editors containing near-PAMless Cas9 nickase variants fused to adenosine or cytosine deaminases for the simultaneous editing of splice acceptor (SA) and splice donor (SD) sequences. Synchronized SA and SD editing improves exon skipping, reduces aberrant splicing, and enables skipping of exons refractory to single splice site editing. To demonstrate the therapeutic potential of SPLICER, we target APP exon 17, which encodes amino acids that are cleaved to form Aβ plaques in Alzheimer's disease. SPLICER reduces the formation of Aβ42 peptides in vitro and enables efficient exon skipping in a mouse model of Alzheimer's disease. Overall, SPLICER is a widely applicable and efficient exon skipping toolbox.}, }
@article {pmid39609269, year = {2024}, author = {Li, C and Xiao, Y and Zhou, J and Liu, S and Zhang, L and Song, X and Guo, X and Song, Q and Zhao, J and Deng, N}, title = {Knockout of onecut2 inhibits proliferation and promotes apoptosis of tumor cells through SKP2-mediated p53 acetylation in hepatocellular carcinoma.}, journal = {Cellular and molecular life sciences : CMLS}, volume = {81}, number = {1}, pages = {469}, pmid = {39609269}, issn = {1420-9071}, support = {81972705//National Natural Science Foundation of China/ ; }, mesh = {*S-Phase Kinase-Associated Proteins/metabolism/genetics ; *Carcinoma, Hepatocellular/pathology/metabolism/genetics ; Humans ; *Liver Neoplasms/pathology/metabolism/genetics ; *Apoptosis/genetics ; *Cell Proliferation/genetics ; Acetylation ; Animals ; *Tumor Suppressor Protein p53/metabolism/genetics ; Mice ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Mice, Nude ; Gene Knockout Techniques ; CRISPR-Cas Systems/genetics ; }, abstract = {Onecut2 (OC2) plays a vital regulatory role in tumor growth, metastasis and angiogenesis. In this study, we report the regulatory role and specific molecular mechanism of OC2 in the apoptosis of hepatocellular carcinoma (HCC) cells. We found that OC2 knockout via the CRISPR/CAS9 system not only significantly inhibited the proliferation and angiogenesis of HCC cells but also significantly promoted apoptosis. The apoptosis rate of the OC2 knockout HCC cell line reached 30.514%. In a mouse model, the proliferation inhibition rate of tumor cells reached 98.8%. To explore the mechanism of apoptosis, ChIP-Seq and dual-luciferase reporter assays were carried out. The results showed that OC2 could directly bind to the promotor of SKP2 and regulate its expression. Moreover, downregulating the expression of OC2 and SKP2 could release p300, promote the acetylation of p53, increase the expression of p21 and p27, and promote the apoptosis of HCC cells. Moreover, the overexpression of OC2 or SKP2 in the knockout HCC cell line clearly inhibited the acetylation level of p53 and reduced cell apoptosis. This study revealed that OC2 could regulate the apoptosis of HCC cells through the SKP2/p53/p21 axis, which may provide some therapeutic targets for HCC in the clinic.}, }
@article {pmid39608317, year = {2025}, author = {Nguyen, HV and Hwang, S and Lee, SW and Jin, E and Lee, MH}, title = {Detection of HPV 16 and 18 L1 genes by a nucleic acid amplification-free electrochemical biosensor powered by CRISPR/Cas9.}, journal = {Bioelectrochemistry (Amsterdam, Netherlands)}, volume = {162}, number = {}, pages = {108861}, doi = {10.1016/j.bioelechem.2024.108861}, pmid = {39608317}, issn = {1878-562X}, mesh = {*Biosensing Techniques/methods ; *Human papillomavirus 16/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *Human papillomavirus 18/genetics ; *Electrochemical Techniques/methods ; Humans ; *Oncogene Proteins, Viral/genetics ; Capsid Proteins/genetics ; Limit of Detection ; Papillomavirus Infections/diagnosis/virology ; Nucleic Acid Amplification Techniques/methods ; Female ; }, abstract = {Cervical cancer, closely linked to Human Papillomavirus (HPV) infection, remains a significant health threat for women worldwide. Conventional HPV detection methods, such as reverse transcription polymerase chain reaction (RT-PCR), rely on nucleic acid amplification (NAA), which can be costly and time-consuming. This study introduces an NAA-free electrochemical Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based biosensor designed to detect HPV 16 and HPV 18 L1 genes simultaneously. The system utilizes a Cas9-single guided RNA complex to initiate a selective cleavage reaction, releasing Methylene blue or Ferrocene-labeled fragments correlate to L1 gene concentrations. These fragments then interact with modified gold electrodes immobilized with a complementary probe, allowing precise electrochemical signal measurement during hybridization. The biosensor offers a wide detection range from 1 fM to 10 nM, with detection limits as low as 0.4 fM for HPV 16 L1 and 0.51 fM for HPV 18 L1, providing a sensitive and efficient solution for L1 gene detection. Additionally, its specificity and sensitivity closely match RT-PCR results in clinical testing, highlighting its potential for molecular diagnostics and point-of-care applications.}, }
@article {pmid39607706, year = {2024}, author = {Deng, C and Xin, R and Li, X and Zhang, J and Fan, L and Qiu, Y and Zhao, L}, title = {Optogenetic control of Corynebacterium glutamicum gene expression.}, journal = {Nucleic acids research}, volume = {52}, number = {22}, pages = {14260-14276}, pmid = {39607706}, issn = {1362-4962}, support = {32301214//National Natural Science Foundation of China/ ; //Shanghai Municipal Education Commission/ ; }, mesh = {*Corynebacterium glutamicum/genetics/metabolism ; *Gene Expression Regulation, Bacterial ; *Optogenetics/methods ; Light ; CRISPR-Cas Systems ; Transcription Factors/metabolism/genetics ; Bioreactors ; Bacterial Proteins/metabolism/genetics ; Oligosaccharides/metabolism/biosynthesis ; Metabolic Engineering/methods ; }, abstract = {Corynebacterium glutamicum is a key industrial workhorse for producing amino acids and high-value chemicals. Balancing metabolic flow between cell growth and product synthesis is crucial for enhancing production efficiency. Developing dynamic, broadly applicable, and minimally toxic gene regulation tools for C. glutamicum remains challenging, as optogenetic tools ideal for dynamic regulatory strategies have not yet been developed. This study introduces an advanced light-controlled gene expression system using light-controlled RNA-binding proteins (RBP), a first for Corynebacterium glutamicum. We established a gene expression regulation system, 'LightOnC.glu', utilizing the light-controlled RBP to construct light-controlled transcription factors in C. glutamicum. Simultaneously, we developed a high-performance light-controlled gene interference system using CRISPR/Cpf1 tools. The metabolic flow in the synthesis network was designed to enable the production of chitin oligosaccharides (CHOSs) and chondroitin sulphate oligosaccharides A (CSA) for the first time in C. glutamicum. Additionally, a light-controlled bioreactor was constructed, achieving a CHOSs production concentration of 6.2 g/L, the highest titer recorded for CHOSs biosynthesis to date. Herein, we have established a programmable light-responsive genetic circuit in C. glutamicum, advancing the theory of dynamic regulation based on light signaling. This breakthrough has potential applications in optimizing metabolic modules in other chassis cells and synthesizing other compounds.}, }
@article {pmid39607705, year = {2024}, author = {Kadam, A and Shilo, S and Naor, H and Wainstein, A and Brilon, Y and Feldman, T and Minden, M and Kaushansky, N and Chapal-Ilani, N and Shlush, L}, title = {Utilizing insights of DNA repair machinery to discover MMEJ deletions and novel mechanisms.}, journal = {Nucleic acids research}, volume = {52}, number = {22}, pages = {e106}, pmid = {39607705}, issn = {1362-4962}, support = {isf-nsfc 2427/18//Rising Tide Foundation/ ; //Steven B. Rubenstein research fund/ ; //Applebaum Foundation/ ; //Ernest and Bonnie Beutler Research Program/ ; //Sagol Institute for Longevity Research/ ; //Barry and Eleanore Reznik Family Cancer Research fund/ ; 1123/21//ISF-IPMP-Israel Precision Medicine Program/ ; }, mesh = {Humans ; *Sequence Deletion ; *DNA End-Joining Repair ; Algorithms ; DNA Repair/genetics ; Exome/genetics ; CRISPR-Cas Systems ; Leukemia, Myeloid, Acute/genetics ; Female ; G-Quadruplexes ; Breast Neoplasms/genetics ; }, abstract = {We developed Del-read, an algorithm targeting medium-sized deletions (6-100 bp) in short-reads, which are challenging for current variant callers relying on alignment. Our focus was on Micro-Homolog mediated End Joining deletions (MMEJ-dels), prevalent in myeloid malignancies. MMEJ-dels follow a distinct pattern, occurring between two homologies, allowing us to generate a comprehensive list of MMEJ-dels in the exome. Using Del-read, we identified numerous novel germline and somatic MMEJ-dels in BEAT-AML and TCGA-breast datasets. Validation in 672 healthy individuals confirmed their presence. These novel MMEJ-dels were linked to genomic features associated with replication stress, like G-quadruplexes and minisatellite. Additionally, we observed a new category of MMEJ-dels with an imperfect-match at the flanking sequences of the homologies, suggesting a mechanism involving mispairing in homology alignment. We demonstrated robustness of the repair system despite CRISPR/Cas9-induced mismatches in the homologies. Further analysis of the canonical ASXL1 deletion revealed a diverse array of these imperfect-matches. This suggests a potentially more flexible and error-prone MMEJ repair system than previously understood. Our findings highlight Del-read's potential in uncovering previously undetected deletions and deepen our understanding of repair mechanisms.}, }
@article {pmid39607301, year = {2024}, author = {Du, B and Chen, M and Chang, L and Zhang, X and Zhang, X and Wang, X and Gong, P and Zhang, N and Zhang, X and Li, X and Li, J}, title = {Immunization with the NcMYR1 gene knockout strain effectively protected C57BL/6 mice and their pups against the Neospora caninum challenge.}, journal = {Virulence}, volume = {15}, number = {1}, pages = {2427844}, pmid = {39607301}, issn = {2150-5608}, mesh = {Animals ; *Neospora/genetics/immunology/pathogenicity ; *Coccidiosis/prevention &amp; control/immunology/parasitology/veterinary ; *Mice, Inbred C57BL ; Mice ; *Protozoan Proteins/genetics/immunology ; *Protozoan Vaccines/immunology/genetics ; *Vaccines, Attenuated/immunology/genetics/administration &amp; dosage ; *Gene Knockout Techniques ; Female ; Immunization ; Virulence ; CRISPR-Cas Systems ; }, abstract = {Neospora caninum is an important protozoan parasite that causes abortion in cattle and nervous system dysfunction in dogs. No effective drugs and vaccines for neosporosis are available. Further elucidation of proteins related to N. caninum virulence will provide potential candidates for vaccine development against neosporosis. In the present study, N. caninum c-Myc regulatory protein (NcMYR1) gene knockout strains (ΔNcMYR1-1, ΔNcMYR1-2, and ΔNcMYR1-3) were generated using the CRISPR-Cas9 gene editing system to investigate phenotype changes and the potential of the ΔNcMYR1-1 strain as an attenuated vaccine, and this is the first time of using the N. caninum CRISPR-Cas9 gene knockout strain as an attenuated vaccine. NcMYR1 was determined to be a cytoplasmic protein in N. caninum tachyzoites. The deficiency of NcMYR1 decreased the plaque area and the rate of invasion, replication, and egression of the parasites. ΔNcMYR1-1 strain-infected C57BL/6 mice had 100% survival rate, reduced parasite burden, and alleviated pathological changes in tissues compared with those in Nc-1 strain-infected mice. Immunization with ΔNcMYR1-1 tachyzoites increased the productions of cytokines in mice, with a survival rate reaching 80%, and the parasite burdens in the liver and spleen were greatly reduced when challenged with the Nc-1 strain with a lethal dose after 40 days of ΔNcMYR1-1 tachyzoite immunization. ΔNcMYR1 immunization could decrease the abortion rate of female mice from 71.4% to 12.5% and increase the survival rate of pups from 12.5% to 83.3% against the N. caninum challenge. Above all, NcMYR1 is a virulence factor and the ΔNcMYR1-1 strain could be used as a candidate vaccine against N. caninum infection and vertical transmission.}, }
@article {pmid39606944, year = {2024}, author = {Gautier, LJ}, title = {CRISPR in 3D: Innovations in Disease Modelling and Personalized Medicine.}, journal = {BioTechniques}, volume = {76}, number = {10}, pages = {473-478}, doi = {10.1080/07366205.2024.2418748}, pmid = {39606944}, issn = {1940-9818}, mesh = {Animals ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Models, Biological ; *Precision Medicine/methods ; }, abstract = {The CRISPR-Cas system of genetic engineering has had a significant impact on science and society since its advent in 2013. CRISPR integration with 3D culture systems such as organ-on-a-chips, as well as fast emerging commercial technologies, has encouraged translation of more complex pathophysiological modelling and personalized medicine.}, }
@article {pmid39606390, year = {2024}, author = {Orosco, C and Rananaware, SR and Huang, B and Hanna, MP and Ahmadimashhadi, MR and Lewis, JG and Baugh, MP and Bodin, AP and Flannery, SJ and Lange, IH and Fang, ZR and Karalkar, VN and Meister, KS and Jain, PK}, title = {DNA-guided CRISPR/Cas12 for RNA targeting.}, journal = {medRxiv : the preprint server for health sciences}, volume = {}, number = {}, pages = {}, pmid = {39606390}, support = {R21 AI156321/AI/NIAID NIH HHS/United States ; R21 AI168795/AI/NIAID NIH HHS/United States ; R35 GM147788/GM/NIGMS NIH HHS/United States ; R61 AI181016/AI/NIAID NIH HHS/United States ; }, abstract = {CRISPR-Cas nucleases are transforming genome editing, RNA editing, and diagnostics but have been limited to RNA-guided systems. We present ΨDNA, a DNA-based guide for Cas12 enzymes, engineered for specific and efficient RNA targeting. ΨDNA mimics a crRNA but with a reverse orientation, enabling stable Cas12-RNA assembly and activating trans-cleavage without RNA components. ΨDNAs are effective in sensing short and long RNAs and demonstrated 100% accuracy for detecting HCV RNA in clinical samples. We discovered that ΨDNAs can guide certain Cas12 enzymes for RNA targeting in cells, enhancing mRNA degradation via ribosome stalling and enabling multiplex knockdown of multiple RNA transcripts. This study establishes ΨDNA as a robust alternative to RNA guides, augmenting the potential of CRISPR-Cas12 for diagnostic applications and targeted RNA modulation in cellular environments.}, }
@article {pmid39605323, year = {2024}, author = {Alves, CRR and Das, S and Krishnan, V and Ha, LL and Fox, LR and Stutzman, HE and Shamber, CE and Kalailingam, P and McCarthy, S and Lino Cardenas, CL and Fong, CE and Imai, T and Mitra, S and Yun, S and Wood, RK and Benning, FMC and Lawton, J and Kim, N and Silverstein, RA and da Silva, JF and de la Cruz, D and Richa, R and Malhotra, R and Chung, DY and Chao, LH and Tsai, SQ and Maguire, CA and Lindsay, ME and Kleinstiver, BP and Musolino, PL}, title = {In vivo Treatment of a Severe Vascular Disease via a Bespoke CRISPR-Cas9 Base Editor.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, pmid = {39605323}, issn = {2692-8205}, support = {R35 GM142553/GM/NIGMS NIH HHS/United States ; R01 DC017117/DC/NIDCD NIH HHS/United States ; R01 HL162928/HL/NHLBI NIH HHS/United States ; K08 NS112601/NS/NINDS NIH HHS/United States ; P01 HL142494/HL/NHLBI NIH HHS/United States ; DP2 CA281401/CA/NCI NIH HHS/United States ; R01 NS125353/NS/NINDS NIH HHS/United States ; K01 NS134784/NS/NINDS NIH HHS/United States ; U01 AI176470/AI/NIAID NIH HHS/United States ; U01 AI176471/AI/NIAID NIH HHS/United States ; }, abstract = {Genetic vascular disorders are prevalent diseases that have diverse etiologies and few treatment options. Pathogenic missense mutations in the alpha actin isotype 2 gene (ACTA2) primarily affect smooth muscle cell (SMC) function and cause multisystemic smooth muscle dysfunction syndrome (MSMDS), a genetic vasculopathy that is associated with stroke, aortic dissection, and death in childhood. Here, we explored genome editing to correct the most common MSMDS-causative mutation ACTA2 R179H. In a first-in-kind approach, we performed mutation-specific protein engineering to develop a bespoke CRISPR-Cas9 enzyme with enhanced on-target activity against the R179H sequence. To directly correct the R179H mutation, we screened dozens of configurations of base editors (comprised of Cas9 enzymes, deaminases, and gRNAs) to develop a highly precise corrective A-to-G edit with minimal deleterious bystander editing that is otherwise prevalent when using wild-type SpCas9 base editors. We then created a murine model of MSMDS that exhibits phenotypes consistent with human patients, including vasculopathy and premature death, to explore the in vivo therapeutic potential of this base editing strategy. Delivery of the customized base editor via an engineered SMC-tropic adeno-associated virus (AAV-PR) vector substantially prolonged survival and rescued systemic phenotypes across the lifespan of MSMDS mice, including in the vasculature, aorta, and brain. Together, our optimization of a customized base editor highlights how bespoke CRISPR-Cas enzymes can enhance on-target correction while minimizing bystander edits, culminating in a precise editing approach that may enable a long-lasting treatment for patients with MSMDS.}, }
@article {pmid39605316, year = {2024}, author = {Weber, R and Vasella, F and Klimko, A and Silginer, M and Lamfers, M and Neidert, MC and Regli, L and Schwank, G and Weller, M}, title = {Targeting the IDH1 [R132H] mutation in gliomas by CRISPR/Cas precision base editing.}, journal = {Neuro-oncology advances}, volume = {6}, number = {1}, pages = {vdae182}, pmid = {39605316}, issn = {2632-2498}, abstract = {BACKGROUND: Gliomas, the most frequent malignant primary brain tumors, lack curative treatments. Understanding glioma-specific molecular alterations is crucial to develop novel therapies. Among them, the biological consequences of the isocitrate dehydrogenase 1 gene mutation (IDH1 [R132H]) remain inconclusive despite its early occurrence and widespread expression.<br><br>METHODS: We thus employed CRISPR/Cas adenine base editors, which allow precise base pair alterations with minimal undesirable effects, to correct the IDH1 [R132H] mutation.<br><br>RESULTS: Successful correction of the IDH1 [R132H] mutation in primary patient-derived cell models led to reduced IDH1 [R132H] protein levels and decreased production of 2-hydroxyglutarate, but increased proliferation. A dual adeno-associated virus split intein system was used to successfully deliver the base editor in vitro and in vivo.<br><br>CONCLUSIONS: Taken together, our study provides a strategy for a precise genetic intervention to target the IDH1 [R132H] mutation, enabling the development of accurate models to study its impact on glioma biology and serving as a framework for an in vivo gene therapy.}, }
@article {pmid39604746, year = {2024}, author = {Saha, UB and Dixit, KK and Jadhav, SV and Pathak, KN and Gupta, NS and Saroj, SD}, title = {Genomic Insights of Multidrug-Resistant Enterococcus faecalis and Acinetobacter baumannii Isolated from a Sepsis Patient with Pauci-Immune Crescentic Glomerulonephritis, India.}, journal = {Current microbiology}, volume = {82}, number = {1}, pages = {16}, pmid = {39604746}, issn = {1432-0991}, support = {SR/PURSE/2023/181//Department of Science and Technology, Ministry of Science and Technology, India/ ; }, mesh = {Humans ; Acinetobacter baumannii/genetics/drug effects/isolation &amp; purification ; *Acinetobacter Infections/microbiology ; *Anti-Bacterial Agents/pharmacology ; Bacteremia/microbiology ; Coinfection/microbiology ; *Drug Resistance, Multiple, Bacterial/genetics ; Enterococcus faecalis/genetics/drug effects/isolation &amp; purification ; *Genome, Bacterial ; Genomics ; *Glomerulonephritis/microbiology/genetics ; *Gram-Positive Bacterial Infections/microbiology ; India ; Microbial Sensitivity Tests ; *Sepsis/microbiology ; Whole Genome Sequencing ; }, abstract = {Acinetobacter baumannii and Enterococcus faecalis are opportunistic bacteria frequently associated with hospital-acquired infections. A. baumannii nosocomial infections in intensive care units are a worldwide problem, with high mortality rates. It may also develop rapidly multidrug resistance (MDR), extensive drug resistance (XDR), and even pan-drug resistance (PDR). Colistin resistance which is an example of pan-drug resistance, is highly alarming as it's used as a last-line antibiotic. Microbes capable of crossing epithelial barriers such as E. faecalis have developed novel strategies to counter antimicrobial agents and cause bacteremia in immunocompromised patients. However, the coinfection of these bacteria in the same patient is unusual. Here, we report a genomic investigation of the extensively drug-resistant E. faecalis and A. baumannii isolated from the blood sample of a patient diagnosed with pauci-immune crescentic glomerulonephritis (PICGN). Identification of cultures isolated from blood sample was carried out using whole-genome sequencing and resistome profiles were mapped. Whole genome sequencing revealed that E. faecalis SVJ-EF01 had a genome size of 2,935,226 bp and GC content of 37.4%, whereas A. baumannii SVJ-AC01 had a genome size of 3,730,857 bp and GC content of 39%. Draft genomes were functionally annotated demonstrating that the organism harbors multiple virulence factors and antimicrobial-resistant mechanisms including MDR efflux pumps. A. baumannii genome possessed a CRISPR-Cas system which might contribute to antimicrobial resistance. This highlights the significance of polymicrobial nature in ESKAPE pathogenesis research. This genomic investigation helps to gain insights into the virulence, resistance profile, and functional potential of these pathogens.}, }
@article {pmid39604418, year = {2024}, author = {Qiao, L and Niu, L and Wang, M and Wang, Z and Kong, D and Yu, G and Ye, H}, title = {A sensitive red/far-red photoswitch for controllable gene therapy in mouse models of metabolic diseases.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10310}, pmid = {39604418}, issn = {2041-1723}, support = {no.32430064//National Natural Science Foundation of China (National Science Foundation of China)/ ; no.32261160373//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {Animals ; *Genetic Therapy/methods ; *Optogenetics/methods ; Mice ; *Dependovirus/genetics ; Humans ; *Disease Models, Animal ; Metabolic Diseases/therapy/genetics ; Light ; Insulin/metabolism ; Cytokines/metabolism ; Obesity/therapy/genetics/metabolism ; Diabetes Mellitus, Type 1/therapy/genetics/metabolism ; CRISPR-Cas Systems ; HEK293 Cells ; Male ; Mice, Inbred C57BL ; Blood Glucose/metabolism ; Gene Expression Regulation ; Tumor Necrosis Factor Ligand Superfamily Member 14 ; }, abstract = {Red light optogenetic systems are in high demand for the precise control of gene expression for gene- and cell-based therapies. Here, we report a red/far-red light-inducible photoswitch (REDLIP) system based on the chimeric photosensory protein FnBphP (Fn-REDLIP) or PnBphP (Pn-REDLIP) and their interaction partner LDB3, which enables efficient dynamic regulation of gene expression with a timescale of seconds without exogenous administration of a chromophore in mammals. We use the REDLIP system to establish the REDLIP-mediated CRISPR-dCas9 (REDLIPcas) system, enabling optogenetic activation of endogenous target genes in mammalian cells and mice. The REDLIP system is small enough to support packaging into adeno-associated viruses (AAVs), facilitating its therapeutic application. Demonstrating its capacity to treat metabolic diseases, we show that an AAV-delivered Fn-REDLIP system achieved optogenetic control of insulin expression to effectively lower blood glucose levels in type 1 diabetes model mice and control an anti-obesity therapeutic protein (thymic stromal lymphopoietin, TSLP) to reduce body weight in obesity model mice. REDLIP is a compact and sensitive optogenetic tool for reversible and non-invasive control that can facilitate basic biological and biomedical research.}, }
@article {pmid39604381, year = {2024}, author = {Goldberg, GW and Kogenaru, M and Keegan, S and Haase, MAB and Kagermazova, L and Arias, MA and Onyebeke, K and Adams, S and Beyer, DK and Fenyö, D and Noyes, MB and Boeke, JD}, title = {Engineered transcription-associated Cas9 targeting in eukaryotic cells.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10287}, pmid = {39604381}, issn = {2041-1723}, support = {F32GM137482//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01 GM133936/GM/NIGMS NIH HHS/United States ; RM1HG009491//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; RM1 HG009491/HG/NHGRI NIH HHS/United States ; R01 GM118851/GM/NIGMS NIH HHS/United States ; F32 GM137482/GM/NIGMS NIH HHS/United States ; K99 GM147604/GM/NIGMS NIH HHS/United States ; R01GM118851//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; K99GM147604//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; }, mesh = {Humans ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics ; *Transcription, Genetic ; *Saccharomyces cerevisiae/genetics/metabolism ; Gene Editing/methods ; DNA/metabolism/genetics ; Eukaryotic Cells/metabolism ; HEK293 Cells ; Genetic Engineering/methods ; }, abstract = {DNA targeting Class 2 CRISPR-Cas effector nucleases, including the well-studied Cas9 proteins, evolved protospacer-adjacent motif (PAM) and guide RNA interactions that sequentially license their binding and cleavage activities at protospacer target sites. Both interactions are nucleic acid sequence specific but function constitutively; thus, they provide intrinsic spatial control over DNA targeting activities but naturally lack temporal control. Here we show that engineered Cas9 fusion proteins which bind to nascent RNAs near a protospacer can facilitate spatiotemporal coupling between transcription and DNA targeting at that protospacer: Transcription-associated Cas9 Targeting (TraCT). Engineered TraCT is enabled in eukaryotic yeast or human cells when suboptimal PAM interactions limit basal activity and when one or more nascent RNA substrates are still tethered to the actively transcribed target DNA in cis. Using yeast, we further show that this phenomenon can be applied for selective editing at one of two identical targets in distinct gene loci, or, in diploid allelic loci that are differentially transcribed. Our work demonstrates that temporal control over Cas9's targeting activity at specific DNA sites may be engineered without modifying Cas9's core domains and guide RNA components or their expression levels. More broadly, it establishes co-transcriptional RNA binding as a cis-acting mechanism that can conditionally stimulate CRISPR-Cas DNA targeting in eukaryotic cells.}, }
@article {pmid39604306, year = {2024}, author = {Kim, Y and Lee, E and Kang, BC}, title = {Etiolation promotes protoplast transfection and genome editing efficiency.}, journal = {Physiologia plantarum}, volume = {176}, number = {6}, pages = {e14637}, doi = {10.1111/ppl.14637}, pmid = {39604306}, issn = {1399-3054}, support = {RS-2022-NR072144//National Research Foundation of Korea (NRF)/ ; }, mesh = {*Protoplasts/metabolism ; *Gene Editing/methods ; *Lactuca/genetics/growth &amp; development ; *Transfection/methods ; Brassica/genetics/growth &amp; development ; Genome, Plant/genetics ; CRISPR-Cas Systems ; Green Fluorescent Proteins/metabolism/genetics ; }, abstract = {In plants, DNA-free genome editing using preassembled clustered regularly interspaced short palindromic repeats (CRISPR)-ribonucleoprotein (RNP) has the advantage of avoiding transgene integration and limiting off-target effects. The efficiency of this gene editing strategy can vary, so optimization of protoplast transfection conditions is necessary to achieve maximum yield. In this study, we examined the effects of etiolation, or increased exposure to darkness during cultivation, on the transfection efficiency of protoplasts from lettuce and Chinese cabbage. Seedlings were grown under three different conditions: non-etiolated, etiolated, and de-etiolated. First, we tested PEG-mediated transfection after etiolation using a plasmid DNA for green fluorescent protein (GFP)-expression. Etiolated protoplasts had the highest percentage of GFP-expressing cells, with a 3.1-fold and 4.8-fold improvement in lettuce and Chinese cabbage, respectively, compared with non-etiolated protoplasts. We also assessed gene editing of endogenous genes after etiolation using CRISPR-RNP. Using targeted deep sequencing, we observed the highest editing efficiency in etiolated protoplasts from both plant species, for the LsPDS and LsFT genes in lettuce, this led to an 8.7-fold and 4.4-fold improvement compared with non-etiolated protoplasts, respectively. These results suggest that etiolation during seedling growth can improve transfection efficiency and DNA-free gene editing in protoplasts.}, }
@article {pmid39603754, year = {2024}, author = {Maladan, Y and Retnaningrum, E and Daryono, BS and Sarassari, R and Sari, RF and Balqis, SA and Wahid, GA and Safari, D}, title = {A New Serotyping Method of Streptococcus pneumoniae Based on CRISPR/Cas9-Targeted Sequencing.}, journal = {The Journal of molecular diagnostics : JMD}, volume = {26}, number = {12}, pages = {1045-1054}, doi = {10.1016/j.jmoldx.2024.08.009}, pmid = {39603754}, issn = {1943-7811}, mesh = {*Streptococcus pneumoniae/genetics/classification ; *CRISPR-Cas Systems/genetics ; *Serotyping/methods ; Humans ; High-Throughput Nucleotide Sequencing/methods ; Sequence Analysis, DNA/methods ; Pneumococcal Infections/microbiology/genetics ; Genome, Bacterial ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) application for targeted sequencing has made a breakthrough in the genomic research era. High diversity in the capsular polysaccharide (cps) locus of Streptococcus pneumoniae has hampered identification of the serotype. This study developed a new serotyping method for S. pneumoniae using CRISPR/Cas9-targeted sequencing with the Oxford Nanopore Technologies platform. A probe was designed at the position of the cps locus using an excision approach on two sides flanking genes between the dexB and aliA genes with approximately 20 kb. A native barcoding method was used for multiplexing. The probe will attach to a specific side followed by attachment of CRISPR/Cas9 to cut the recognition area. The study used de novo assembly to reconstruct sequence reads, which were analyzed using PneumoCRISPR, a new serotyping pipeline for Oxford Nanopore Technologies sequencing data output. Four CRISPR/Cas9 probes have been designed and recognize the cps locus of S. pneumoniae. Serotyping results align precisely with serotyping data from whole-genome sequencing. This serotyping method also allows researchers to use multiple samples in a single run. The new serotyping method based on CRISPR/Cas9-targeted sequencing holds immense promise for serotype identification of S. pneumoniae.}, }
@article {pmid39603242, year = {2024}, author = {Pahl, V and Lubrano, P and Troßmann, F and Petras, D and Link, H}, title = {Intact protein barcoding enables one-shot identification of CRISPRi strains and their metabolic state.}, journal = {Cell reports methods}, volume = {4}, number = {12}, pages = {100908}, pmid = {39603242}, issn = {2667-2375}, mesh = {*Mass Spectrometry/methods ; Ubiquitin/metabolism/genetics ; CRISPR-Cas Systems/genetics ; Escherichia coli/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Metabolome ; }, abstract = {Detecting strain-specific barcodes with mass spectrometry can facilitate the screening of genetically engineered bacterial libraries. Here, we introduce intact protein barcoding, a method to measure protein-based library barcodes and metabolites using flow injection mass spectrometry (FI-MS). Protein barcodes are based on ubiquitin with N-terminal tags of six amino acids. We demonstrate that FI-MS detects intact ubiquitin proteins and identifies the mass of N-terminal barcodes. In the same analysis, we measured relative concentrations of primary metabolites. We constructed six ubiquitin-barcoded CRISPR interference (CRISPRi) strains targeting metabolic enzymes and analyzed their metabolic profiles and ubiquitin barcodes. FI-MS detected barcodes and distinct metabolome changes in CRISPRi-targeted pathways. We demonstrate the scalability of intact protein barcoding by measuring 132 ubiquitin barcodes in microtiter plates. These results show that intact protein barcoding enables fast and simultaneous detection of library barcodes and intracellular metabolites, opening up new possibilities for mass spectrometry-based barcoding.}, }
@article {pmid39603211, year = {2025}, author = {Yan, J and Yin, B and Zhang, Q and Li, C and Chen, J and Huang, Y and Hao, J and Yi, C and Zhang, Y and Wong, SHD and Yang, M}, title = {A CRISPR-Cas12a-mediated dual-mode luminescence and colorimetric nucleic acid biosensing platform based on upconversion nanozyme.}, journal = {Biosensors &amp; bioelectronics}, volume = {270}, number = {}, pages = {116963}, doi = {10.1016/j.bios.2024.116963}, pmid = {39603211}, issn = {1873-4235}, mesh = {*Biosensing Techniques/methods ; *Colorimetry/methods ; *CRISPR-Cas Systems ; *Graphite/chemistry ; DNA, Single-Stranded/chemistry ; Limit of Detection ; Silicon Dioxide/chemistry ; Cerium/chemistry ; CRISPR-Associated Proteins/chemistry ; Luminescent Measurements/methods ; Benzidines/chemistry ; Bacterial Proteins/chemistry/genetics ; Luminescence ; Endodeoxyribonucleases ; }, abstract = {In this study, a CRISPR-Cas12a-mediated dual-mode upconversion luminescence/colorimetric nucleic acid biosensing platform is developed based on UCNP@SiO2/CeO2 (UNSC) nanozyme. Here, UNSC is conjugated with single-stranded DNA (ssDNA) probes used as both peroxidase-like nanozyme and upconversion luminescence donors. When no target nucleic acid is present, ssDNA-conjugated UNSC attaches on magnetic graphene oxide (MGO) via pi-pi stacking force, resulting in upconversion luminescence quenching (OFF) and no color change after magnetic removal of nanozymes attached on the MGO. In the presence of target nucleic acid, Cas12a is specifically activated by targeted nucleic acid and indiscriminately cleaves the ssDNA probes on UNSCs. UNSCs then detach from the MGO surface due to the weakening of binding force, leading to upconversion luminescence recovery (ON) and colorimetric change due to the existence of free nanozyme in the 3,3',5,5'-tetramethyl-benzidine assay. As a proof-of-concept, this biosensing platform shows a limit of detection of around 320 fM in the upconversion luminescence mode and ∼28.4 pM in the colorimetric mode for nucleic acid detection, respectively. This UNSC nanozyme-based CRISPR-Cas12a dual-mode biosensing system also demonstrates high selectivity, good repeatability, and facile operation, which allows easy adaption to other nucleic acid-based detection only by redesigning the sequence of CRISPR RNA.}, }
@article {pmid39603170, year = {2025}, author = {Ali, N and Singh, S and Garg, R}, title = {Unlocking crops' genetic potential: Advances in genome and epigenome editing of regulatory regions.}, journal = {Current opinion in plant biology}, volume = {83}, number = {}, pages = {102669}, doi = {10.1016/j.pbi.2024.102669}, pmid = {39603170}, issn = {1879-0356}, mesh = {*Crops, Agricultural/genetics ; *Gene Editing/methods ; *Genome, Plant/genetics ; *Epigenesis, Genetic ; *Epigenome/genetics ; CRISPR-Cas Systems ; Plant Breeding ; Gene Expression Regulation, Plant ; DNA Methylation ; Epigenome Editing ; }, abstract = {Genome editing tools could precisely and efficiently target plant genomes leading to the development of improved crops. Besides editing the coding regions, researchers can employ editing technologies to target specific gene regulatory elements or modify epigenetic marks associated with distal regulatory regions, thereby regulating gene expression in crops. This review outlines several prominent genome editing technologies, including CRISPR-Cas9, TALENs, and ZFNs and recent advancements. The applications for genome and epigenome editing especially of regulatory regions in crop plants is also discussed, including efforts to enhance abiotic stress tolerance, yield, disease resistance and plant phenotype. Additionally, the review addresses the potential of epigenetic modifications, such as DNA methylation and histone modifications, to alter gene expression for crop improvement. Finally, the limitations and future scope of utilizing various genome editing tools to manipulate regulatory elements for gene regulation to unlock the full potential of these tools in plant breeding has been discussed.}, }
@article {pmid39603092, year = {2024}, author = {Zhang, S and Li, J and Pan, W and Ren, Q and Zhou, Y and Chen, M and Qu, J and Li, S}, title = {Establishment of a CPAMD8-GFP reporter human embryonic stem cell line, IBBDe001-B, using CRISPR/Cas9 editing.}, journal = {Stem cell research}, volume = {81}, number = {}, pages = {103615}, doi = {10.1016/j.scr.2024.103615}, pmid = {39603092}, issn = {1876-7753}, mesh = {Humans ; *CRISPR-Cas Systems ; *Human Embryonic Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *Green Fluorescent Proteins/metabolism/genetics ; Cell Line ; Cell Differentiation ; Genes, Reporter ; }, abstract = {The human CPAMD8 gene encodes proteins in the A2M/C3 (alpha-2-macroglobulin/complement 3) family, predominantly expressed in the distal tips of the retinal neuroepithelium that form the iris and ciliary body. Mutations in CPAMD8 have been linked to anterior segment dysplasia and congenital glaucoma. Using CRISPR/Cas9 editing, we inserted a 3*EAAAK-EGFP fluorescent tag into the CPAMD8 gene, enabling real-time observation of its expression and providing insights into its biological functions. The resulting gene-edited cell line retained normal stem cell morphology and karyotype, expressed essential pluripotency markers, and exhibited differentiation potential.}, }
@article {pmid39602282, year = {2025}, author = {Lim, RM and Lu, A and Chuang, BM and Anaraki, C and Chu, B and Halbrook, CJ and Edinger, AL}, title = {CARMIL1-AA selectively inhibits macropinocytosis while sparing autophagy.}, journal = {Molecular biology of the cell}, volume = {36}, number = {1}, pages = {ar4}, pmid = {39602282}, issn = {1939-4586}, support = {P30 CA062203/CA/NCI NIH HHS/United States ; R37 CA283575/CA/NCI NIH HHS/United States ; }, mesh = {*Pinocytosis/physiology ; Humans ; *Autophagy/physiology ; Cell Line, Tumor ; Sodium-Hydrogen Exchangers/metabolism/genetics ; Amiloride/pharmacology/analogs &amp; derivatives ; Cell Proliferation ; CRISPR-Cas Systems ; }, abstract = {Macropinocytosis is reported to fuel tumor growth and drug resistance by allowing cancer cells to scavenge extracellular macromolecules. However, accurately defining the role of macropinocytosis in cancer depends on our ability to selectively block this process. 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) is widely used to inhibit macropinocytosis but affects multiple Na[+]/H[+] exchangers (NHE) that regulate cytoplasmic and organellar pH. Consistent with this, we report that EIPA slows proliferation to a greater extent than can be accounted for by macropinocytosis inhibition and triggers conjugation of ATG8 to single membranes (CASM). Knocking down only NHE1 would not avoid macropinocytosis-independent effects on pH. Moreover, contrary to published reports, NHE1 loss did not block macropinocytosis in multiple cell lines. Knocking down CARMIL1 with CRISPR-Cas9 editing limited macropinocytosis, but only by 50%. In contrast, expressing the CARMIL1-AA mutant inhibits macropinocytosis induced by a wide range of macropinocytic stimuli to a similar extent as EIPA. CARMIL1-AA expression did not inhibit proliferation, highlighting the shortcomings of EIPA as a macropinocytosis inhibitor. Importantly, autophagy, another actin dependent, nutrient-producing process, was not affected by CARMIL1-AA expression. In sum, constitutive or inducible CARMIL1-AA expression reduced macropinocytosis without affecting proliferation, RAC activation, or autophagy, other processes that drive tumor initiation and progression.}, }
@article {pmid39601843, year = {2025}, author = {Cao, L and Chen, W and Kang, W and Lei, C and Nie, Z}, title = {Engineering stimuli-responsive CRISPR-Cas systems for versatile biosensing.}, journal = {Analytical and bioanalytical chemistry}, volume = {417}, number = {9}, pages = {1699-1711}, pmid = {39601843}, issn = {1618-2650}, support = {22034002//National Natural Science Foundation of China/ ; 92253304//National Natural Science Foundation of China/ ; 2020YFA0907500//National Key Research and Development Program of China/ ; 2021YFA0910100//National Key Research and Development Program of China/ ; }, mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Protein Engineering/methods ; }, abstract = {The precise target recognition and nuclease-mediated effective signal amplification capacities of CRISPR-Cas systems have attracted considerable research interest within the biosensing field. Guided by insights into their structural and biochemical mechanisms, researchers have endeavored to engineer the key biocomponents of CRISPR-Cas systems with stimulus-responsive functionalities. By the incorporation of protein/nucleic acid engineering techniques, a variety of conditional CRISPR-Cas systems whose activities depend on the presence of target triggers have been established for the efficient detection of diverse types of non-nucleic acid analytes. In this review, we summarized recent research progress in engineering Cas proteins, guide RNA, and substrate nucleic acids to possess target analyte-responsive abilities for diverse biosensing applications. Furthermore, we also discussed the challenges and future possibilities of the stimulus-responsive CRISPR-Cas systems in versatile biosensing.}, }
@article {pmid39601618, year = {2024}, author = {Liu, X and Peng, H and Gong, L and Zhang, H and Zhao, C and Lai, W and An, G and Zhao, X}, title = {Reliable and precise lipoprotein detection based on a self-priming hairpin-triggered Cas12a/crRNA based signaling strategy.}, journal = {The Analyst}, volume = {150}, number = {1}, pages = {46-54}, doi = {10.1039/d4an01167h}, pmid = {39601618}, issn = {1364-5528}, mesh = {Humans ; *Limit of Detection ; *Lipoproteins, LDL/blood/analysis ; Lipoproteins, HDL/blood/analysis ; Antibodies, Immobilized/immunology ; CRISPR-Cas Systems ; CRISPR-Associated Proteins/metabolism ; Lipoproteins/chemistry/blood ; }, abstract = {Cardiovascular disease, intimately linked to dyslipidemia, is one of the leading global causes of mortality. Dyslipidaemia often presents as an elevated concentration of low-density lipoprotein (LDL) and a decreased concentration of high-density lipoprotein (HDL). Therefore, accurately measuring the levels of LDL and HDL particles is crucial for assessing the risk of developing cardiovascular diseases. However, conventional approaches can commonly quantify HDL/LDL particles by detecting cholesterol or protein molecules within them, which may fail to reflect the number of intact particles. In addition, these approaches are sometimes tedious and time-consuming, highlighting the need for a novel method for precise and effective identification of intact HDL and LDL particles. We have devised a technique that allows accurately and sensitively determining the levels of intact HDL and LDL in a sample without the need for isolation. This method relies on antibody-based immobilization and a self-priming hairpin-triggered Cas12a/crRNA signaling strategy. Based on the elegant design, this technique can be employed to directly and precisely measure the concentration of "actual" HDL and LDL particles, rather than the cholesterol content inside HDL and LDL. The approach has detection limits of 12.3 mg dL[-1] and 5.4 mg dL[-1] for HDL and LDL, respectively, and is also suitable for analyzing lipoproteins in clinical samples. Hence, this platform exhibits immense potential for clinical applications and health management.}, }
@article {pmid39601564, year = {2025}, author = {Zhang, X and Hao, S and Feng, Z and Ning, K and Aksu Kuz, C and McFarlin, S and Richart, D and Cheng, F and Zhang-Chen, A and McFarlane, R and Yan, Z and Qiu, J}, title = {Identification of SLC35A1 as an essential host factor for the transduction of multi-serotype recombinant adeno-associated virus (AAV) vectors.}, journal = {mBio}, volume = {16}, number = {1}, pages = {e0326824}, pmid = {39601564}, issn = {2150-7511}, support = {AI166293//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R21 AI166293/AI/NIAID NIH HHS/United States ; R01 HL174593/HL/NHLBI NIH HHS/United States ; P30 GM103326/GM/NIGMS NIH HHS/United States ; S10 OD032207/OD/NIH HHS/United States ; S10 OD023625/OD/NIH HHS/United States ; YAN23G0//Cystic Fibrosis Foundation (CFF)/ ; HL174593//HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; AI150877//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; P30 CA168524/CA/NCI NIH HHS/United States ; R21 AI180416/AI/NIAID NIH HHS/United States ; AI180416//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; AI156448//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01 AI150877/AI/NIAID NIH HHS/United States ; R21 AI156448/AI/NIAID NIH HHS/United States ; }, mesh = {*Dependovirus/genetics ; Humans ; *Nucleotide Transport Proteins/genetics/metabolism ; *Genetic Vectors/genetics/metabolism ; *Transduction, Genetic ; HEK293 Cells ; CRISPR-Cas Systems ; Golgi Apparatus/metabolism ; Serogroup ; Gene Knockout Techniques ; }, abstract = {We conducted a genome-wide CRISPR/Cas9 screen in suspension 293 F cells transduced with rAAV5. The highly selected genes revealed after two rounds of screening included the previously reported KIAA0319L, TM9SF2, and RNF121, along with a cluster of genes involved in glycan biogenesis, Golgi apparatus localization, and endoplasmic reticulum penetration. In this report, we focused on solute carrier family 35 member A1 (SLC35A1), a Golgi apparatus-localized cytidine 5'-monophosphate-sialic acid (CMP-SIA) transporter. We confirmed that SLC35A1 knockout (KO) significantly decreased rAAV5 transduction to a level lower than that observed in KIAA0319L or TM9SF2 KO cells. Although SLC35A1 KO drastically reduced the expression of α2,6-linked SIA on the cell surface, the expression of α2,3-linked SIA, as well as the cell binding and internalization of rAAV5, was only moderately affected. Moreover, SLC35A1 KO significantly diminished the transduction of AAV multi-serotypes, including rAAV2 and rAAV3, which do not utilize SIAs for primary attachment. Notably, the SLC35A1 KO markedly increased transduction of rAAV9 and rAAV11, which primarily attach to cells via binding to galactose. Further analyses revealed that SLC35A1 KO significantly decreased vector nuclear import. More importantly, although the C-terminal cytoplasmic tail deletion (∆C Tail) mutant of SLC35A1 did not drastically decrease SIA expression, it significantly decreased rAAV transduction, as well as vector nuclear import, suggesting that the C-tail is critical in these processes. Furthermore, the T128A mutant significantly decreased SIA expression but still supported rAAV transduction and nuclear import. These findings highlight the involvement of the CMP-SIA transporter in the intracellular trafficking of rAAV vectors post-internalization.IMPORTANCErAAV is an essential tool for gene delivery in the treatment of genetic disorders; however, the mechanisms of rAAV transduction remain partially understood. GPR108 is vital for the transduction of most rAAV vectors, but not for rAAV5. We aimed to identify host factors that impact AAV5 transduction akin to GPR108. Using a genome-wide CRISPR/Cas9 screen in 293 F cells, we identified SLC35A1, a Golgi apparatus-localized CMP-sialic acid transporter that transports CMP-sialic acid from the cytoplasm into the Golgi apparatus for sialylation, is essential to rAAV transduction. Further studies across various AAV serotypes showed SLC35A1 significantly affects vector nuclear import post-internalization. These results underscore the crucial role of SLC35A1 in intracellular trafficking beyond the initial cell attachment of rAAV.}, }
@article {pmid39601181, year = {2024}, author = {Bamundo, M and Palumbo, S and D'Auria, L and Missero, C and Di Girolamo, D}, title = {CRISPR/Cas9 Ribonucleoprotein Nucleofection for Genome Editing in Primary Human Keratinocytes: Knockouts, Deletions, and Homology-Directed Repair Mutagenesis.}, journal = {Current protocols}, volume = {4}, number = {11}, pages = {e70056}, pmid = {39601181}, issn = {2691-1299}, mesh = {Humans ; *Keratinocytes/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Gene Knockout Techniques/methods ; Ribonucleoproteins/genetics/metabolism ; Transfection/methods ; Recombinational DNA Repair ; Cells, Cultured ; Mutagenesis ; }, abstract = {Keratinocytes are the most abundant cell type in the human epidermis, the outermost layer of the skin. For years, primary human keratinocytes (HKs) have been used as a crucial tool for studying the pathogenesis of a wide range of skin-related diseases. To mimic the physiological and pathological behavior of human skin, organotypic 3D skin models can be generated by in vitro differentiation of HKs. However, manipulation of HKs is notoriously difficult. Liposome-mediated gene delivery often results in low transfection rates, and conventional electroporation results in high mortality, is difficult to optimize, and requires high cell numbers without necessarily achieving maximum efficiency. Additionally, HKs have a short lifespan in vitro, with a limited number of cell divisions before senescence, even when cultured on a feeder layer. Therefore, the possibility to use an efficient CRISPR/Cas9 system in HKs is not without challenge in terms of transfection technology and clonal selection. In this article, we provide detailed protocols to perform efficient gene knock-out (KO) or genomic deletion in a small number of HKs without clonal selection of edited cells. By nucleofecting ribonucleoprotein complexes, we efficiently generate KO cells as well as deletion of specific genomic regions. Moreover, we describe an optimized protocol for generating site-specific mutations in immortalized keratinocytes (N/TERT2G) by exploiting the homology-directed repair system combined with rapid single-clone screening. These methods can also be applied to other immortalized cells and tumoral cells of epithelial origin. Together, these protocols provide a comprehensive and powerful tool that can be used to better understand the molecular mechanisms underlying different skin diseases. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Knock-out generation by indel mutation in primary human keratinocytes using nucleofection of ribonucleoprotein (RNP) complex Basic Protocol 2: Deletion of specific genomic region using RNPs via nucleofection Basic Protocol 3: Use of homology-directed repair system to introduce site-specific mutations.}, }
@article {pmid39599550, year = {2024}, author = {Mikhaylova, Y and Tyumentseva, M and Karbyshev, K and Tyumentsev, A and Slavokhotova, A and Smirnova, S and Akinin, A and Shelenkov, A and Akimkin, V}, title = {Interrelation Between Pathoadaptability Factors and Crispr-Element Patterns in the Genomes of Escherichia coli Isolates Collected from Healthy Puerperant Women in Ural Region, Russia.}, journal = {Pathogens (Basel, Switzerland)}, volume = {13}, number = {11}, pages = {}, pmid = {39599550}, issn = {2076-0817}, support = {075-15-2019-1666//Ministry of Science and Higher Education of Russian Federation/ ; }, mesh = {*Escherichia coli/genetics/isolation &amp; purification/drug effects ; Female ; Humans ; *Genome, Bacterial/genetics ; Russia/epidemiology ; *Escherichia coli Infections/microbiology/epidemiology ; *Virulence Factors/genetics ; *Phylogeny ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Anti-Bacterial Agents/pharmacology ; Whole Genome Sequencing ; Genotype ; Postpartum Period ; Drug Resistance, Bacterial/genetics ; Plasmids/genetics ; Microbial Sensitivity Tests ; }, abstract = {Escherichia coli is a commensal and opportunistic bacterium widely distributed around the world in different niches including intestinal of humans and animals, and its extraordinary genome plasticity led to the emergence of pathogenic strains causing a wide range of diseases. E. coli is one of the monitored species in maternity hospitals, being the main etiological agent of urogenital infections, endometriosis, puerperal sepsis, and neonatal diseases. This study presents a comprehensive analysis of E. coli isolates obtained from the maternal birth canal of healthy puerperant women 3-4 days after labor. According to whole genome sequencing data, 31 sequence types and six phylogenetic groups characterized the collection containing 53 isolates. The majority of the isolates belonged to the B2 phylogroup. The data also includes phenotypic and genotypic antibiotic resistance profiles, virulence factors, and plasmid replicons. Phenotypic and genotypic antibiotic resistance testing did not demonstrate extensive drug resistance traits except for two multidrug-resistant E. coli isolates. The pathogenic factors revealed in silico were assessed with respect to CRISPR-element patterns. Multiparametric and correlation analyses were conducted to study the interrelation of different pathoadaptability factors, including antimicrobial resistance and virulence genomic determinants carried by the isolates under investigation. The data presented will serve as a valuable addition to further scientific investigations in the field of bacterial pathoadaptability, especially in studying the role of CRISPR/Cas systems in the E. coli genome plasticity and evolution.}, }
@article {pmid39597141, year = {2024}, author = {Xie, S and Yue, Y and Yang, F}, title = {Recent Advances in CRISPR/Cas System-Based Biosensors for the Detection of Foodborne Pathogenic Microorganisms.}, journal = {Micromachines}, volume = {15}, number = {11}, pages = {}, pmid = {39597141}, issn = {2072-666X}, support = {32102633//National Natural Science Foundation of China/ ; }, abstract = {Foodborne pathogens pose significant risks to food safety. Conventional biochemical detection techniques are facing a series of challenges. In recent years, with the gradual development of CRISPR (clustered regularly interspaced short palindromic repeats) technology, CRISPR/Cas system-based biosensors, a newly emerging technology, have received much attention from researchers because of their supreme flexibility, sensitivity, and specificity. While numerous CRISPR-based biosensors have a broad application in the field of environmental monitoring, food safety, and point-of-care diagnosis, they remain in high demand to summarize recent advances in CRISPR/Cas system-based biosensors for foodborne pathogen detection. In this paper, we briefly classify and discuss the working principles of CRISPR/Cas systems with trans-cleavage activity in applications for the detection of foodborne pathogenic microorganisms. We highlight the current status, the unique feature of each CRISPR system and CRISPR-based biosensing platforms, and the integration of CRISPR-Cas with other techniques, concluding with a discussion of the advantages, disadvantages, and future directions.}, }
@article {pmid39596692, year = {2024}, author = {Chen, J and Miao, Z and Kong, D and Zhang, A and Wang, F and Liu, G and Yu, X and Luo, L and Liu, Y}, title = {Application of CRISPR/Cas9 Technology in Rice Germplasm Innovation and Genetic Improvement.}, journal = {Genes}, volume = {15}, number = {11}, pages = {}, pmid = {39596692}, issn = {2073-4425}, support = {23N11900100//Shanghai Municipal Commission of Science and technology/ ; INV-033236-3//The Bill and Melinda Gates Foundation/ ; }, mesh = {*Oryza/genetics/growth &amp; development ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Plant Breeding/methods ; Plants, Genetically Modified/genetics ; Crops, Agricultural/genetics ; Genome, Plant ; }, abstract = {Improving the efficiency of germplasm innovation has always been the aim of rice breeders. Traditional hybrid breeding methods for variety selection rarely meet the practical needs of rice production. The emergence of genome-editing technologies, such as CRISPR/Cas9, provides a new approach to the genetic improvement of crops such as rice. The number of published scientific papers related to "gene editing" and "CRISPR/Cas9" retrievable on websites both from China and other countries exhibited an increasing trend, year by year, from 2014 to 2023. Research related to gene editing in rice accounts for 33.4% and 12.3% of all the literature on gene editing published in China and other countries, respectively, much higher than that on maize and wheat. This article reviews recent research on CRISPR/Cas9 gene-editing technology in rice, especially germplasm innovation and genetic improvement of commercially promoted varieties with improved traits such as disease, insect, and herbicide resistance, salt tolerance, quality, nutrition, and safety. The aim is to provide a reference for the precise and efficient development of new rice cultivars that meet market demand.}, }
@article {pmid39596534, year = {2024}, author = {Kim, JY and Lee, YJ and Lee, HJ and Go, JY and Lee, HM and Park, JS and Cho, YG and Jung, YJ and Kang, KK}, title = {Knockout of OsGAPDHC7 Gene Encoding Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase Affects Energy Metabolism in Rice Seeds.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596534}, issn = {1422-0067}, mesh = {*Oryza/genetics/metabolism/growth &amp; development/enzymology ; *Glyceraldehyde-3-Phosphate Dehydrogenases/genetics/metabolism ; *Energy Metabolism/genetics ; *Gene Expression Regulation, Plant ; *Seeds/metabolism/genetics ; Plant Proteins/genetics/metabolism ; Gene Knockout Techniques ; Cytosol/metabolism ; CRISPR-Cas Systems ; Starch/metabolism ; Carbohydrate Metabolism/genetics ; }, abstract = {Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a major glycolytic enzyme that plays an important role in several cellular processes, including plant hormone signaling, plant development, and transcriptional regulation. In this study, we divided it into four groups through structural analysis of eight GAPDH genes identified in the rice genome. Among them, the expression level of five genes of cytosolic GAPDH was shown to be different for each organ. The mutation induction of the GAPDHC7 gene by the CRISPR/Cas9 system revealed that the 7 bp and 2 bp deletion, early end codon, was used in protein production. In addition, the selected mutants showed lower plant heights compared to the wild-type plants. To investigate the effect on carbohydrate metabolism, the expression of the genes of starch-branched enzyme I (SbeI), sucrose synthase (SS), and 3-phosphoglycer phosphokinase (PGK) increased the expression of the SBeI gene threefold in the knockout lines compared to the wild-type (WT) plant, while the expression of the SS and PGK genes decreased significantly. And the starch and soluble sugar content of the knockout lines increased by more than 60% compared to the WT plant. Also, the free amino acid content was significantly increased in the Gln and Asn contents of the knockout lines compared to the WT plants, while the contents of Gly and Ser were decreased. Our results suggest that OsGAPDHC7 has a great influence on energy metabolism, such as pre-harvested sprouting and amino acid content.}, }
@article {pmid39596521, year = {2024}, author = {Sheveleva, O and Protasova, E and Grigor'eva, E and Butorina, N and Kuziaeva, V and Antonov, D and Melnikova, V and Medvedev, S and Lyadova, I}, title = {The Generation of Genetically Engineered Human Induced Pluripotent Stem Cells Overexpressing IFN-β for Future Experimental and Clinically Oriented Studies.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596521}, issn = {1422-0067}, support = {&#x2116;075-15-2021-1075//Ministry of Science and Higher Education of the Russian Federation/ ; }, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Interferon-beta/metabolism/genetics ; *Cell Differentiation/genetics ; *CRISPR-Cas Systems ; Genetic Engineering/methods ; Embryoid Bodies/metabolism/cytology ; Cell Line ; Gene Editing/methods ; }, abstract = {Induced pluripotent stem cells (iPSCs) can be generated from various adult cells, genetically modified and differentiated into diverse cell populations. Type I interferons (IFN-Is) have multiple immunotherapeutic applications; however, their systemic administration can lead to severe adverse outcomes. One way of overcoming the limitation is to introduce cells able to enter the site of pathology and to produce IFN-Is locally. As a first step towards the generation of such cells, here, we aimed to generate human iPSCs overexpressing interferon-beta (IFNB, IFNB-iPSCs). IFNB-iPSCs were obtained by CRISPR/Cas9 editing of the previously generated iPSC line K7-4Lf. IFNB-iPSCs overexpressed IFNB RNA and produced a functionally active IFN-β. The cells displayed typical iPSC morphology and expressed pluripotency markers. Following spontaneous differentiation, IFNB-iPSCs formed embryoid bodies and upregulated endoderm, mesoderm, and some ectoderm markers. However, an upregulation of key neuroectoderm markers, PAX6 and LHX2, was compromised. A negative effect of IFN-β on iPSC neuroectoderm differentiation was confirmed in parental iPSCs differentiated in the presence of a recombinant IFN-β. The study describes new IFN-β-producing iPSC lines suitable for the generation of various types of IFN-β-producing cells for future experimental and clinical applications, and it unravels an inhibitory effect of IFN-β on stem cell neuroectoderm differentiation.}, }
@article {pmid39596457, year = {2024}, author = {Sakovina, L and Vokhtantsev, I and Akhmetova, E and Vorobyeva, M and Vorobjev, P and Zharkov, DO and Novopashina, D}, title = {Photocleavable Guide crRNAs for a Light-Controllable CRISPR/Cas9 System.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596457}, issn = {1422-0067}, support = {22-14-00294//Russian Science Foundation/ ; 121031300042-1//Russian state-funded project/ ; }, mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; CRISPR-Associated Protein 9/metabolism/genetics ; Humans ; Ultraviolet Rays ; Photolysis ; Light ; }, abstract = {The design of controllable and precise RNA-targeted CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) systems is an important problem of modern molecular biology and genetic technology. Herein, we have designed a series of photocleavable guide CRISPR RNAs (crRNA) and their 2'-modified (2'-fluoro and locked nucleic acid) analogs containing one or two 1-(2-nitrophenyl)-1,2-ethanediol photolabile linkers (PL). We have demonstrated that these crRNAs can be destroyed by relatively mild UVA irradiation with the rate constants 0.24-0.77 min[-1] and that the photocleavage markedly slows down the action of Cas9 nuclease in the model in vitro system. Two PLs provide more rapid crRNA destruction than a single linker. PLs in the crRNA structure improve the specificity of DNA cleavage by Cas9 nuclease for the fully complementary target. The application of photocleavable crRNA in CRISPR/Cas9 genome editing permits the system to be switched off in a spatiotemporally controlled manner, thus alleviating its off-target effects.}, }
@article {pmid39596448, year = {2024}, author = {Chang, H and Sha, H and Gao, S and Liu, Q and Liu, Y and Ma, C and Shi, B and Nie, S}, title = {A Novel Gene, OsRLCK191, Involved in Culm Strength Improving Lodging Resistance in Rice.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596448}, issn = {1422-0067}, support = {LH2021C092, CZKYF2023-1-A005, CXSTOP2021005, HTRC202209//Heilongjiang Provincial Natural Science Foundation of China, Fundamental Research Funds for the Research Institutes of Heilongjiang Province, the Opening Foundation of the Key Laboratory of Germplasm Enhancement, Physiology and Ecology of Food Crops in Co/ ; }, mesh = {*Oryza/genetics/growth &amp; development ; *Plant Proteins/genetics/metabolism ; *Gene Expression Regulation, Plant ; Cell Wall/metabolism/genetics ; CRISPR-Cas Systems ; Genes, Plant ; Mutation ; }, abstract = {Lodging is one of the major problems in rice production. However, few genes that can explain the culm strength within the temperate japonica subspecies have been identified. In this study, we identified OsRLCK191, which encodes receptor-like cytoplasmic kinase and plays critical roles in culm strength. OsRLCK191 mutants were produced by the CRISPR-Cas9 DNA-editing system. Compared with wild types (WTs), the bending moment of the whole plant (WP), the bending moment at breaking (BM), and the section modulus (SM) were decreased in rlck191 significantly. Although there is no significant decrease in the culm length of rlck191 compared with the WT; in the mutant, except the length of the fourth internode being significantly increased, the lengths of other internodes are significantly shortened. In addition, the yield traits of panicle length, thousand-seed weight, and seed setting rate decreased significantly in rlck191. Moreover, RNA-seq experiments were performed at an early stage of rice panicle differentiation in shoot apex. The differentially expressed genes (DEGs) are mainly involved in cell wall biogenesis, cell wall polysaccharide metabolic processes, cellar component biogenesis, and DNA-binding transcription factors. Transcriptome analysis of the cell wall biological process pathways showed that major genes that participated in the cytokinin oxidase/dehydrogenase family, cellulose synthase catalytic subunit genes, and ethylene response factor family transcription factor were related to culm strength. Our research provides an important theoretical basis for analyzing the lodging resistance mechanism and lodging resistance breeding of temperate japonica.}, }
@article {pmid39596447, year = {2024}, author = {Meng, L and Zhang, J and Clarke, N}, title = {A Critical Review of Recent Advances in Maize Stress Molecular Biology.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596447}, issn = {1422-0067}, support = {C200937//Natural Science Foundation of Heilongjiang Province/ ; }, mesh = {*Zea mays/genetics/metabolism ; *Stress, Physiological/genetics ; *Gene Expression Regulation, Plant ; Genome-Wide Association Study ; Quantitative Trait Loci ; Droughts ; }, abstract = {With the intensification of global climate change and environmental stress, research on abiotic and biotic stress resistance in maize is particularly important. High temperatures and drought, low temperatures, heavy metals, salinization, and diseases are widespread stress factors that can reduce maize yields and are a focus of maize-breeding research. Molecular biology provides new opportunities for the study of maize and other plants. This article reviews the physiological and biochemical responses of maize to high temperatures and drought, low temperatures, heavy metals, salinization, and diseases, as well as the molecular mechanisms associated with them. Special attention is given to key transcription factors in signal transduction pathways and their roles in regulating maize stress adaptability. In addition, the application of transcriptomics, genome-wide association studies (GWAS), and QTL technology provides new strategies for the identification of molecular markers and genes for maize-stress-resistance traits. Crop genetic improvements through gene editing technologies such as the CRISPR/Cas system provide a new avenue for the development of new stress-resistant varieties. These studies not only help to understand the molecular basis of maize stress responses but also provide important scientific evidence for improving crop tolerance through molecular biological methods.}, }
@article {pmid39596426, year = {2024}, author = {Niu, D and Zhao, Q and Xu, L and Lin, K}, title = {Physiological and Molecular Mechanisms of Lepidopteran Insects: Genomic Insights and Applications of Genome Editing for Future Research.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596426}, issn = {1422-0067}, support = {2022YFD1400600//Research on Mechanisms of Grassland Moth Catastrophes and Sustainable Control Technologies/ ; }, mesh = {Animals ; *Gene Editing/methods ; *Lepidoptera/genetics ; *Genomics/methods ; *Insecticide Resistance/genetics ; CRISPR-Cas Systems ; Genome, Insect ; Insecticides/pharmacology ; }, abstract = {Lepidopteran insects are a major threat to global agriculture, causing significant crop losses and economic damage. Traditional pest control methods are becoming less effective due to the rapid evolution of insecticide resistance. This study explores the current status and genomic characteristics of 1315 Lepidopteran records, alongside an overview of relevant research, utilizing advanced functional genomics techniques, including RNA-seq and CRISPR/Cas9 gene-editing technologies to uncover the molecular mechanisms underlying insecticide resistance. Our genomic analysis revealed significant variability in genome size, assembly quality, and chromosome number, which may influence species' biology and resistance mechanisms. We identified key resistance-associated genes and pathways, including detoxification and metabolic pathways, which help these insects evade chemical control. By employing CRISPR/Cas9 gene-editing techniques, we directly manipulated resistance-associated genes to confirm their roles in resistance, demonstrating their potential for targeted interventions in pest management. These findings emphasize the value of integrating genomic data into the development of effective and sustainable pest control strategies, reducing reliance on chemical insecticides and promoting environmentally friendly integrated pest management (IPM) approaches. Our study highlights the critical role of functional genomics in IPM and its potential to provide long-term solutions to the growing challenge of Lepidopteran resistance.}, }
@article {pmid39596412, year = {2024}, author = {Sosnovtseva, AO and Demidova, NA and Klimova, RR and Kovalev, MA and Kushch, AA and Starodubova, ES and Latanova, AA and Karpov, DS}, title = {Control of HSV-1 Infection: Directions for the Development of CRISPR/Cas-Based Therapeutics and Diagnostics.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596412}, issn = {1422-0067}, support = {22-14-00377//Russian Science Foundation/ ; 075-15-2019-1660//Ministry of Science and Higher Education of the Russian Federation/ ; }, mesh = {*CRISPR-Cas Systems ; Humans ; *Herpesvirus 1, Human/genetics ; *Herpes Simplex/diagnosis/therapy/virology ; Animals ; Antiviral Agents/therapeutic use/pharmacology ; Gene Editing/methods ; Drug Resistance, Viral/genetics ; Genetic Therapy/methods ; }, abstract = {It is estimated that nearly all individuals have been infected with herpesviruses, with herpes simplex virus type 1 (HSV-1) representing the most prevalent virus. In most cases, HSV-1 causes non-life-threatening skin damage in adults. However, in patients with compromised immune systems, it can cause serious diseases, including death. The situation is further complicated by the emergence of strains that are resistant to both traditional and novel antiviral drugs. It is, therefore, imperative that new methods of combating HSV-1 and other herpesviruses be developed without delay. CRISPR/Cas systems may prove an effective means of controlling herpesvirus infections. This review presents the current understanding of the underlying molecular mechanisms of HSV-1 infection and discusses four potential applications of CRISPR/Cas systems in the fight against HSV-1 infections. These include the search for viral and cellular genes that may serve as effective targets, the optimization of anti-HSV-1 activity of CRISPR/Cas systems in vivo, the development of CRISPR/Cas-based HSV-1 diagnostics, and the validation of HSV-1 drug resistance mutations.}, }
@article {pmid39596282, year = {2024}, author = {Khmeleva, SA and Kurbatov, LK and Ptitsyn, KG and Timoshenko, OS and Morozova, DD and Suprun, EV and Radko, SP and Lisitsa, AV}, title = {Detection of Potato Pathogen Clavibacter sepedonicus by CRISPR/Cas13a Analysis of NASBA Amplicons.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596282}, issn = {1422-0067}, support = {075-15-2021-1345, unique identifier RF-193021X0012//Ministry of Science and Higher Education of the Russian Federation/ ; }, mesh = {*Solanum tuberosum/microbiology ; *CRISPR-Cas Systems ; *Clavibacter/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Plant Diseases/microbiology ; RNA, Ribosomal, 16S/genetics ; Plant Tubers/microbiology ; Molecular Diagnostic Techniques ; }, abstract = {The ring rot of potato caused by the bacterial pathogen Clavibacter sepedonicus is a quarantine disease posing a threat to the potato industry worldwide. The sensitive and selective detection of C. sepedonicus is of a high importance for its effective control. Here, the detection system is reported to determine viable bacteria of C. sepedonicus in potato tubers, based on the coupling of CRISPR/Cas13a nuclease with NASBA (Nucleic Acid Sequence Based Amplification)-the method of isothermal amplification of RNA. Detection can be conducted using both instrumental and non-instrumental (visual inspection of test tubes under blue light) modes. When NASBA and Cas13a analyses were carried out in separate test tubes, the limit of detection (LOD) for the system was 1000 copies of purified target 16S rRNA per NASBA reaction or about 24 colony-forming units (CFUs) of C. sepedonicus per 1 g of tuber tissue. The testing can also be conducted in the "one-pot" format (a single test tube), though with lower sensitivity: LOD was 10,000 copies of target RNA or about 100 CFU per 1 g of tuber tissue for both instrumental and visual detection modes. The overall time of NASBA/Cas13a analysis did not exceed 2 h. The developed NASBA/Cas13a detection system has the potential to be employed as a routine test of C. sepedonicus, especially for on-site testing.}, }
@article {pmid39596246, year = {2024}, author = {Shmelev, ME and Pilnik, AA and Shved, NA and Penkova, AO and Gulaia, VS and Kumeiko, VV}, title = {IDH1 R132H and TP53 R248Q Mutations Modulate Glioma Cell Migration and Adhesion on Different ECM Components.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596246}, issn = {1422-0067}, support = {20-15-00378&#x43f;//Russian Science Foundation/ ; }, mesh = {Humans ; *Isocitrate Dehydrogenase/genetics ; *Cell Movement/genetics ; *Tumor Suppressor Protein p53/genetics/metabolism ; *Extracellular Matrix/metabolism/genetics ; Cell Line, Tumor ; *Cell Adhesion/genetics ; *Glioma/genetics/pathology/metabolism ; Mutation ; Brain Neoplasms/genetics/pathology/metabolism ; }, abstract = {Mutations in IDH1 and TP53 have a significant impact on glioma prognosis and progression; however, their roles in tumor cell invasion in terms of interactions with particular components of the extracellular matrix (ECM) are still unclear. Using gene editing protocol based on CRISPR-Cas 9 with cytidine deaminase, we introduced point mutations into U87MG glioblastoma cells to establish modified cell lines with heterozygous IDH1 R132H, homozygous TP53 R248Q and heterozygous IDH1 R132H, homozygous TP53 R248Q genotypes. A comparative study of cell migration on major ECM components was carried out by high-content microscopy. IDH1 R132H mutation introduced to U87MG glioblastoma cells was shown to decrease the migration speed on Matrigel and collagen IV substrates compared to the wild-type. This data were supported by cell adhesion quantification via the lateral shift assay performed by atomic force microscopy (AFM). TP53 R248Q mutation increased cell adhesion to various substrates and significantly promoted cell migration on hyaluronic acid and chondroitin sulfate but did not change the migration rates on laminin and collagens IV and I. A double-mutant genotype produced by consequently introducing IDH1 R132H and TP53 R248Q to parental glioblastoma cells was characterized by the highest migration among all the cell lines, with particularly faster motility on chondroitin sulfate. These findings underscore the complex interactions between glioma cells, with the most important driver mutations and specific ECM components regulating cancer cell migration, offering valuable insights for potential therapeutic targets in glioma treatment.}, }
@article {pmid39596176, year = {2024}, author = {Wang, Z and Wang, C and Zhai, Y and Bai, Y and Wang, H and Rong, X}, title = {Loss of Brcc3 in Zebrafish Embryos Increases Their Susceptibility to DNA Damage Stress.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596176}, issn = {1422-0067}, support = {2022YFC2804400//the National Key R &amp; D Program of China/ ; }, mesh = {Animals ; *Zebrafish/genetics/embryology ; *Zebrafish Proteins/genetics/metabolism ; *DNA Damage ; Tumor Suppressor Protein p53/genetics/metabolism ; Ultraviolet Rays/adverse effects ; Apoptosis/genetics ; CRISPR-Cas Systems ; Embryo, Nonmammalian/metabolism ; DNA Breaks, Double-Stranded ; DNA Repair ; Etoposide/pharmacology ; }, abstract = {DNA double-strand breaks (DSBs) represent one of the most severe forms of genetic damage in organisms, yet vertebrate models capable of monitoring DSBs in real-time remain scarce. BRCA1/BRCA2-containing complex subunit 3 (BRCC3), also known as BRCC36, functions within various multiprotein complexes to mediate diverse biological processes. However, the physiological role of BRCC3 in vertebrates, as well as the underlying mechanisms that govern its activity, are not well understood. To explore these questions, we generated brcc3-knockout zebrafish using CRISPR/Cas9 gene-editing technology. While brcc3 mutant zebrafish appear phenotypically normal and remain fertile, they exhibit significantly increased rates of mortality and deformity following exposure to DNA damage. Furthermore, embryos lacking Brcc3 display heightened p53 signaling, elevated γ-H2AX levels, and increased apoptosis in response to DNA-damaging agents such as ultraviolet (UV) light and Etoposide (ETO). Notably, genetic inactivation of p53 or pharmacological inhibition of Ataxia-telangiectasia mutated (ATM) activity rescues the hypersensitivity to UV and ETO observed in Brcc3-deficient embryos. These findings suggest that Brcc3 plays a critical role in DNA damage response (DDR), promoting cell survival during embryogenesis. Additionally, brcc3-null mutant zebrafish offer a promising vertebrate model for real-time monitoring of DSBs.}, }
@article {pmid39596160, year = {2024}, author = {de Jonge, AV and Csikós, T and Eken, M and Bulthuis, EP and Poddighe, PJ and Roemer, MGM and Chamuleau, MED and Mutis, T}, title = {Delineating MYC-Mediated Escape Mechanisms from Conventional and T Cell-Redirecting Therapeutic Antibodies.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596160}, issn = {1422-0067}, mesh = {Humans ; *T-Lymphocytes/immunology/metabolism ; Cell Line, Tumor ; Proto-Oncogene Proteins c-myc/genetics/metabolism/immunology ; Killer Cells, Natural/immunology/metabolism ; Antibodies, Bispecific/pharmacology ; Apoptosis ; Rituximab/pharmacology/therapeutic use ; Multiple Myeloma/therapy/immunology/genetics/pathology ; CRISPR-Cas Systems ; Tumor Escape/drug effects ; Antigens, CD20/immunology/metabolism/genetics ; Antibodies, Monoclonal/pharmacology/therapeutic use ; }, abstract = {In B-cell malignancies, the overexpression of MYC is associated with poor prognosis, but its mechanism underlying resistance to immunochemotherapy remains less clear. In further investigations of this issue, we show here that the pharmacological inhibition of MYC in various lymphoma and multiple myeloma cell lines, as well as patient-derived primary tumor cells, enhances their susceptibility to NK cell-mediated cytotoxicity induced by conventional antibodies targeting CD20 (rituximab) and CD38 (daratumumab), as well as T cell-mediated cytotoxicity induced by the CD19-targeting bispecific T-cell engager blinatumomab. This was associated with upregulation of the target antigen only for rituximab, suggesting additional escape mechanisms. To investigate these mechanisms, we targeted the MYC gene in OCI-LY18 cells using CRISPR-Cas9 gene-editing technology. CRISPR-Cas9-mediated MYC targeting not only upregulated CD20 but also triggered broader apoptotic pathways, upregulating pro-apoptotic PUMA and downregulating anti-apoptotic proteins BCL-2, XIAP, survivin and MCL-1, thereby rendering tumor cells more prone to apoptosis, a key tumor-lysis mechanism employed by T-cells and NK-cells. Moreover, MYC downregulation boosted T-cell activation and cytokine release in response to blinatumomab, revealing a MYC-mediated T-cell suppression mechanism. In conclusion, MYC overexpressing tumor cells mitigated the efficacy of therapeutic antibodies through several non-overlapping mechanisms. Given the challenges associated with direct MYC inhibition due to toxicity, successful modulation of MYC-mediated immune evasion mechanisms may improve the outcome of immunotherapeutic approaches in B-cell malignancies.}, }
@article {pmid39596103, year = {2024}, author = {Begovic, M and Schneider, L and Zhou, X and Hamdani, N and Akin, I and El-Battrawy, I}, title = {The Role of Human-Induced Pluripotent Stem Cells in Studying Cardiac Channelopathies.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596103}, issn = {1422-0067}, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Channelopathies/genetics/pathology ; *Myocytes, Cardiac/metabolism/pathology ; Brugada Syndrome/genetics/pathology/metabolism ; Gene Editing ; Animals ; Tachycardia, Ventricular/genetics/pathology ; CRISPR-Cas Systems ; Polymorphic Catecholaminergic Ventricular Tachycardia ; }, abstract = {Cardiac channelopathies are inherited diseases that increase the risk of sudden cardiac death. While different genes have been associated with inherited channelopathies, there are still subtypes, e.g., catecholaminergic polymorphic ventricular tachycardia and Brugada syndrome, where the genetic cause remains unknown. Various models, including animal models, heterologous expression systems, and the human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSCs-CMs) model, have been used to study the pathophysiological mechanisms of channelopathies. Recently, researchers have focused on using hiPSCs-CMs to understand the genotype-phenotype correlation and screen drugs. By combining innovative techniques such as Clustered Regularly Interspaced Short Palindromic Repeats/Clustered Regularly Interspaced Short Palindromic Repeats associated protein 9 (CRISPR/Cas9)-mediated genome editing, and three-dimensional (3D) engineered heart tissues, we can gain new insights into the pathophysiological mechanisms of channelopathies. This approach holds promise for improving personalized drug treatment. This review highlights the role of hiPSCs-CMs in understanding the pathomechanism of Brugada syndrome and catecholaminergic polymorphic ventricular tachycardia and how these models can be utilized for drug screening.}, }
@article {pmid39595965, year = {2024}, author = {Fernández, JP and Petersen, B and Hassel, P and Lucas Hahn, A and Kielau, P and Geibel, J and Kues, WA}, title = {Comparison Between Electroporation at Different Voltage Levels and Microinjection to Generate Porcine Embryos with Multiple Xenoantigen Knock-Outs.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39595965}, issn = {1422-0067}, support = {TRR 127//Deutsche Forschungsgemeinschaft/ ; }, mesh = {Animals ; *Electroporation/methods ; *Microinjections/methods ; Swine ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Gene Knockout Techniques/methods ; Animals, Genetically Modified ; Antigens, Heterophile/genetics/immunology ; Galactosyltransferases/genetics ; Embryo, Mammalian/metabolism ; Mosaicism ; Blastocyst/metabolism ; Zygote/metabolism ; Mixed Function Oxygenases ; }, abstract = {In the context of xenotransplantation, the production of genetically modified pigs is essential. For several years, knock-out pigs were generated through somatic cell nuclear transfer employing donor cells with the desired genetic modifications, which resulted in a lengthy and cumbersome procedure. The CRISPR/Cas9 system enables direct targeting of specific genes in zygotes directly through microinjection or electroporation. However, these techniques require improvement to minimize mosaicism and low mutation rates without compromising embryo survival. This study aimed to determine the gene editing potential of these two techniques to deliver multiplexed ribonucleotide proteins (RNPs) to generate triple-knock-out porcine embryos with a multi-transgenic background. We designed RNP complexes targeting the major porcine xenoantigens GGTA1, CMAH, and B4GALNT2. We then compared the development of mosaicism and gene editing efficiencies between electroporation and microinjection. Our results indicated a significant effect of voltage increase on molecule intake in electroporated embryos, without it notably affecting the blastocyst formation rate. Our gene editing analysis revealed differences among delivery approaches and gene loci. Notably, employing electroporation at 35 V yielded the highest frequency of biallelic disruptions. However, mosaicism was the predominant genetic variant in all RNP delivery methods, underscoring the need for further research to optimize multiplex genome editing in porcine zygotes.}, }
@article {pmid39595944, year = {2024}, author = {Li, P and Shang, H and Xu, X and Gong, J and Wu, JL and Zhang, X}, title = {A Novel Single Base Mutation in OsSPL42 Leads to the Formation of Leaf Lesions in Rice.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39595944}, issn = {1422-0067}, support = {LZ24C130004//the Zhejiang Provincial Natural Science Foundation of China/ ; LQ24C130008//the Zhejiang Provincial Natural Science Foundation of China/ ; 2022YFF1003301//The National Key Research and Development Program of China/ ; }, mesh = {*Oryza/genetics/metabolism ; *Plant Leaves/genetics/metabolism ; *Plant Diseases/genetics/microbiology ; *Plant Proteins/genetics/metabolism ; *Disease Resistance/genetics ; *Plants, Genetically Modified ; Mutation ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; Cloning, Molecular ; Xanthomonas/pathogenicity ; }, abstract = {Rice spotted-leaf mutants serve as valuable resources for studying plant programmed cell death (PCD) and disease resistance mechanisms, making them crucial for research on disease resistance in rice. Map-based cloning was used to identify and clone the spotted-leaf gene OsSPL42. Then, functional complementation and CRISPR/Cas9 techniques were also employed to further validate the function of this gene. By applying leaf clippings for bacterial blight (BB) inoculation, the BB resistance of different rice lines was assessed. The results in this study were as follows: The OsSPL42 behaved as a recessive nuclear gene and was narrowed down to a 111 kb region on chromosome 8. All T0 transgenic rice plants in the complementation experiments exhibited a wild-type phenotype, without any lesion spots on the rice leaves. This suggests that the LOC_Os08g06100 encoding O-methyltransferase is the candidate gene for the mutant spl42. The OsSpl42 is widely expressed and the OsSPL42-GFP protein is mainly localized in the cytoplasm. OsSPL42 overexpression lines are more susceptible to BBs, which indicates that OsSPL42 may act as a negative regulator of rice resistance to BB. In summary, we speculate that OsSPL42 plays an important role in the regulation of pathogen response, providing new insights into plant defense mechanisms.}, }
@article {pmid39595588, year = {2024}, author = {Benati, D and Cattin, E and Corradi, F and Ferrari, T and Pedrazzoli, E and Patrizi, C and Marchionni, M and Bertorelli, R and De Sanctis, V and Merlini, L and Ferlini, A and Sabatelli, P and Gualandi, F and Recchia, A}, title = {Restored Collagen VI Microfilaments Network in the Extracellular Matrix of CRISPR-Edited Ullrich Congenital Muscular Dystrophy Fibroblasts.}, journal = {Biomolecules}, volume = {14}, number = {11}, pages = {}, pmid = {39595588}, issn = {2218-273X}, support = {Million Dollar Bike Ride Award 2018//Cure CMD/ ; }, mesh = {*Collagen Type VI/genetics/metabolism ; Humans ; *Fibroblasts/metabolism ; *Gene Editing ; *Extracellular Matrix/metabolism ; *Muscular Dystrophies/genetics/metabolism/therapy/pathology ; *CRISPR-Cas Systems/genetics ; Sclerosis/genetics ; Mutation ; }, abstract = {Collagen VI is an essential component of the extracellular matrix (ECM) composed by α1, α2 and α3 chains and encoded by COL6A1, COL6A2 and COL6A3 genes. Dominant negative pathogenic variants in COL6A genes result in defects in collagen VI protein and are implicated in the pathogenesis of muscular diseases, including Ullrich congenital muscular dystrophy (UCMD). Here, we designed a CRISPR genome editing strategy to tackle a dominant heterozygous deletion c.824_838del in exon 9 of the COL6A1 gene, causing a lack of secreted collagen VI in a patient's dermal fibroblasts. The evaluation of efficiency and specificity of gene editing in treating patient's fibroblasts revealed the 32% efficiency of editing the mutated allele but negligible editing of the wild-type allele. CRISPR-treated UCMD skin fibroblasts rescued the secretion of collagen VI in the ECM, which restored the ultrastructure of the collagen VI microfibril network. By using normal melanocytes as surrogates of muscle cells, we found that collagen VI secreted by the corrected patient's skin fibroblasts recovered the anchorage to the cell surface, pointing to a functional improvement of the protein properties. These results support the application of the CRISPR editing approach to knock out COL6A1 mutated alleles and rescue the UCMD phenotype in patient-derived fibroblasts.}, }
@article {pmid39595585, year = {2024}, author = {Fu, B and Ma, H and Huo, X and Zhu, Y and Liu, D}, title = {CRISPR Technology Acts as a Dual-Purpose Tool in Pig Breeding: Enhancing Both Agricultural Productivity and Biomedical Applications.}, journal = {Biomolecules}, volume = {14}, number = {11}, pages = {}, pmid = {39595585}, issn = {2218-273X}, support = {CZKYF2024-1-B006//Heilongjiang Provincial Research Institutes Research Business Fund Project/ ; U20A2052//National Natural Science Foundation of China/ ; }, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Swine/genetics ; *Gene Editing/methods ; *Agriculture/methods ; Breeding/methods ; Animals, Genetically Modified/genetics ; Humans ; }, abstract = {Pigs have long been integral to human society for their roles in agriculture and medicine. Consequently, there is an urgent need for genetic improvement of pigs to meet human dual needs for medicine and food. In agriculture, gene editing can improve productivity traits, such as growth rate and disease resistance, which could lower farming costs and benefit consumers through enhanced meat quality. In biomedical research, gene-edited pigs offer invaluable resources as disease models and in xenotransplantation, providing organs compatible with human physiology. Currently, with CRISPR technology, especially the CRISPR/Cas9 system emerging as a transformative force in modern genetics, pigs are not only sources of sustenance but also cornerstones of biomedical innovation. This review aims to summarize the applications of CRISPR/Cas9 technology in developing pigs that serve dual roles in agriculture and biomedical applications. Compared to ZFNs and TALENs, the CRISPR/Cas9 system offers several advantages, including higher efficiency, greater specificity, ease of design and implementation, and the capability to target multiple genes simultaneously, significantly streamlining the process of genetic modifications in complex genomes. Therefore, CRISPR technology supports the enhancement of traits beneficial for agricultural productivity and facilitates applications in medicine. Furthermore, we must acknowledge the inherent deficiencies and technical challenges of the CRISPR/Cas9 technology while also anticipating emerging technologies poised to surpass CRISPR/Cas9 as the next milestones in gene editing. We hypothesize that with the continuous advancements in gene editing technologies and successful integration of traits beneficial to both agricultural productivity and medical applications, the goal of developing dual-purpose pigs for both agricultural and medical use can ultimately be achieved.}, }
@article {pmid39593080, year = {2024}, author = {Lam, S and Thomas, JC and Jackson, SP}, title = {Genome-aware annotation of CRISPR guides validates targets in variant cell lines and enhances discovery in screens.}, journal = {Genome medicine}, volume = {16}, number = {1}, pages = {139}, pmid = {39593080}, issn = {1756-994X}, mesh = {Humans ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Molecular Sequence Annotation ; Genome, Human ; Cell Line ; Algorithms ; Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Exome ; }, abstract = {BACKGROUND: CRISPR-Cas9 technology has revolutionised genetic screens and can inform on gene essentiality and chemo-genetic interactions. It is easily deployed and widely supported with many pooled CRISPR libraries available commercially. However, discrepancies between the reference genomes used in the design of those CRISPR libraries and the cell line under investigation can lead to loss of signal or introduction of bias. The problem is particularly acute when dealing with variant cell lines such as cancer cell lines.<br><br>RESULTS: Here, we present an algorithm, EXOme-guided Re-annotation of nuCleotIde SEquences (Exorcise), which uses sequence search to detect and correct mis-annotations in CRISPR libraries. Exorcise verifies the presence of CRISPR targets in the target genome and applies corrections to CRISPR libraries using existing exome annotations. We applied Exorcise to re-annotate guides in pooled CRISPR libraries available on Addgene and found that libraries designed on a more permissive reference sequence had more mis-annotations. In simulated CRISPR screens, we modelled common mis-annotations and found that they adversely affect discovery of hits in the intermediate range. We then confirmed this by applying Exorcise on datasets from Dependency Map (DepMap) and the DNA Damage Response CRISPR Screen Viewer (DDRcs), where we found improved discovery power upon Exorcise while retaining the strongest hits.<br><br>CONCLUSIONS: Pooled CRISPR libraries map guide sequences to genes and these mappings might not be ready to use due to permissive library design or investigating a variant cell line. By re-annotating CRISPR guides, Exorcise focuses CRISPR experiments towards the genome of the cell line under investigation. Exorcise can be applied at the library design stage or the analysis stage and allows post hoc re-analysis of completed screens. It is available under a Creative Commons Zero v1.0 Universal licence at https://github.com/SimonLammmm/exorcise .}, }
@article {pmid39592946, year = {2024}, author = {Bovet, L and Battey, J and Lu, J and Sierro, N and Dewey, RE and Goepfert, S}, title = {Nitrate assimilation pathway is impacted in young tobacco plants overexpressing a constitutively active nitrate reductase or displaying a defective CLCNt2.}, journal = {BMC plant biology}, volume = {24}, number = {1}, pages = {1132}, pmid = {39592946}, issn = {1471-2229}, mesh = {*Nitrates/metabolism ; *Nitrate Reductase/metabolism/genetics ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; Chloride Channels/metabolism/genetics ; Plants, Genetically Modified/genetics ; Plant Leaves/genetics/metabolism ; Tobacco Products ; }, abstract = {BACKGROUND: We have previously shown that the expression of a constitutively active nitrate reductase variant and the suppression of CLCNt2 gene function (belonging to the chloride channel (CLC) gene family) in field-grown tobacco reduces tobacco-specific nitrosamines (TSNA) accumulation in cured leaves and cigarette smoke. In both cases, TSNA reductions resulted from a strong diminution of free nitrate in the leaf, as nitrate is a precursor of the TSNA-producing nitrosating agents formed during tobacco curing and smoking. These nitrosating agents modify tobacco alkaloids to produce TSNAs, the most problematic of which are NNN (N-nitrosonornicotine) and NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone). The expression of a deregulated nitrate reductase enzyme (DNR) that is no longer responsive to light regulation is believed to diminish free nitrate pools by immediately channeling incoming nitrate into the nitrate assimilation pathway. The reduction in nitrate observed when the two tobacco gene copies encoding the vacuolar nitrate transporter CLCNt2 were down-regulated by RNAi-mediated suppression or knocked out using the CRISPR-Cas technology was mechanistically distinct; likely attributable to the inability of the tobacco cell to efficiently sequester nitrate into the vacuole where this metabolite is protected from further assimilation. In this study, we used transcriptomic and metabolomic analyses to compare the nitrate assimilation response in tobacco plants either expressing DNR or lacking CLCNt2 function.<br><br>RESULTS: When grown in a controlled environment, both DNR and CLCNt2-KO (CLCKO) plants exhibited (1) reduced nitrate content in the leaf; (2) increased N-assimilation into the amino acids Gln and Asn; and (3) a similar pattern of differential regulation of several genes controlling stress responses, including water stress, and cell wall metabolism in comparison to wild-type plants. Differences in gene regulation were also observed between DNR and CLCKO plants, including genes encoding nitrite reductase and asparagine synthetase.<br><br>CONCLUSIONS: Our data suggest that even though both DNR and CLCKO plants display common characteristics with respect to nitrate assimilation, cellular responses, water stress, and cell wall remodeling, notable differences in gene regulatory patterns between the two low nitrate plants are also observed. These findings open new avenues in using plants fixing more nitrogen into amino acids for plant improvement or nutrition perspectives.}, }
@article {pmid39592573, year = {2024}, author = {Pallaseni, A and Peets, EM and Girling, G and Crepaldi, L and Kuzmin, I and Moor, M and Muñoz-Subirana, N and Schimmel, J and Serçin, &#xd6; and Mardin, BR and Tijsterman, M and Peterson, H and Kosicki, M and Parts, L}, title = {The interplay of DNA repair context with target sequence predictably biases Cas9-generated mutations.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10271}, pmid = {39592573}, issn = {2041-1723}, support = {/WT_/Wellcome Trust/United Kingdom ; R01 HG003988/HG/NHGRI NIH HHS/United States ; 220540/Z/20/A//Wellcome Trust (Wellcome)/ ; }, mesh = {Mice ; Animals ; *DNA Repair/genetics ; *CRISPR-Cas Systems ; *DNA-Activated Protein Kinase/genetics/metabolism ; *Mutation ; DNA Breaks, Double-Stranded ; Mouse Embryonic Stem Cells/metabolism ; CRISPR-Associated Protein 9/metabolism/genetics ; Cell Line ; DNA-Directed DNA Polymerase/metabolism/genetics ; Nuclear Proteins/genetics/metabolism ; Gene Editing/methods ; DNA-Binding Proteins ; }, abstract = {Repair of double-stranded breaks generated by CRISPR/Cas9 is highly dependent on the flanking DNA sequence. To learn about interactions between DNA repair and target sequence, we measure frequencies of over 236,000 distinct Cas9-generated mutational outcomes at over 2800 synthetic target sequences in 18 DNA repair deficient mouse embryonic stem cells lines. We classify the outcomes in an unbiased way, finding a specialised role for Prkdc (DNA-PKcs protein) and Polm in creating 1 bp insertions matching the nucleotide on the protospacer-adjacent motif side of the break, a variable involvement of Nbn and Polq in the creation of different deletion outcomes, and uni-directional deletions dependent on both end-protection and end-resection. Using our dataset, we build predictive models of the mutagenic outcomes of Cas9 scission that outperform the current standards. This work improves our understanding of DNA repair gene function, and provides avenues for more precise modulation of Cas9-generated mutations.}, }
@article {pmid39592077, year = {2024}, author = {Shen, JY and Mao, FH and Wang, Q and Ou, PP and Liu, JK and Zhao, Q and He, QL}, title = {Efficient genome editing using CRISPR/Cas9 technology and its application for identifying Sesquiterpene synthases involved in the biosynthesis of Steperoxides in Steccherinum ochraceum.}, journal = {Fungal genetics and biology : FG &amp; B}, volume = {175}, number = {}, pages = {103944}, doi = {10.1016/j.fgb.2024.103944}, pmid = {39592077}, issn = {1096-0937}, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Sesquiterpenes/metabolism ; Promoter Regions, Genetic/genetics ; Alkyl and Aryl Transferases/genetics ; Fungal Proteins/genetics/metabolism ; Agaricales/genetics/enzymology ; }, abstract = {CRISPR technology has been widely used for gene editing in various species，but the genetic manipulation in basidiomycete mushrooms is still notoriously difficult for unknown endogenous promoters and inefficient DNA delivery. Steccherinum ochraceum is a white rot basidiomycete fungus with abundant secondary metabolites and plays an important ecological role worldwide. To facilitate the study of gene function in S. ochraceum, an effective CRISPR/Cas9 system was successfully developed by identifying highly efficient endogenous promoters, and utilizing the Agrobacterium-transformation method. Two efficient endogenous RNA polymerase II promoters (Psogpd and Psotef1) and one efficient RNA polymerase III promoter (Pu6-d) were identified and characterized, with an editing efficiency of 61.5 % at the ura3 locus. Using this optimized system, the sesquiterpene gene A0064, which could produce 10 possible sesquiterpenes in the heterologous expression system of A. oryzae, was knocked out to obtain A0064 knockout strain S. ochraceum (∆A0064). Steperoxide A could not be detected in S. ochraceum (∆A0064), demonstrating that A0064 was the only enzyme responsible for the biosynthesis of β-chamigrene (the sesquiterpene skeleton of steperoxide A) in S. ochraceum. This efficient system will enable precise targeting and multiplex editing of S. ochraceum genes, facilitating functional studies of genes involved in lignin degradation and natural product biosynthesis in S. ochraceum, and providing some valuable guidance for gene editing in tens of thousands of macrofungi.}, }
@article {pmid39592070, year = {2025}, author = {Donaka, R and Zheng, H and Ackert-Bicknell, CL and Karasik, D}, title = {Early life lipid overload in Native American Myopathy is phenocopied by stac3 knockout in zebrafish.}, journal = {Gene}, volume = {936}, number = {}, pages = {149123}, doi = {10.1016/j.gene.2024.149123}, pmid = {39592070}, issn = {1879-0038}, mesh = {Animals ; *Zebrafish/genetics ; *Zebrafish Proteins/genetics/metabolism ; Disease Models, Animal ; Muscle, Skeletal/metabolism/pathology ; Gene Knockout Techniques ; Muscular Diseases/genetics/pathology/metabolism ; Humans ; Calcium/metabolism ; MyoD Protein/genetics/metabolism ; Muscle Development/genetics ; CRISPR-Cas Systems ; Myogenic Regulatory Factor 5/genetics/metabolism ; Actins/metabolism/genetics ; Larva/genetics ; Lipid Metabolism/genetics ; Adaptor Proteins, Signal Transducing ; }, abstract = {Understanding the early stages of human congenital myopathies is critical for proposing strategies for improving musculoskeletal muscle performance, such as restoring the functional integrity of the cytoskeleton. SH3 and cysteine-rich domain 3 (STAC3) are proteins involved in nutrient regulation and are an essential component of the excitation-contraction (EC) coupling machinery for Ca[2+] releasing. A mutation in STAC3 causes debilitating Native American Myopathy (NAM) in humans, while loss of this gene in mice and zebrafish (ZF) results in premature death. Clinically, NAM patients demonstrated increased lipids in skeletal muscle, but it is unclear if neutral lipids are associated with altered muscle function in NAM. Using a CRISPR/Cas9 induced stac3[-/-] knockout (KO) zebrafish model, we determined that loss of stac3 leads to delayed larval hatching which corresponds with muscle weakness and decreased whole-body Ca[2+] level during early skeletal development. Specifically, we observed defects in the cytoskeleton in F-actin and slow muscle fibers at 5 and 7 days post-fertilizations (dpf). Myogenesis regulators such as myoD and myf5, mstnb were significantly altered in stac3[-/-] larvae. These muscle alterations were associated with elevated neutral lipid levels starting at 5 dpf and persisting beyond 7 dpf. Larva lacking stac3 had reduced viability with no larva knockouts surviving past 11 dpf. This data suggests that our stac3[-/-] zebrafish serve as an alternative model to study the diminished muscle function seen in NAM patients. The data gathered from this new model over time supports a mechanistic view of lipotoxicity as a critical part of the pathology of NAM and the associated loss of function in muscle.}, }
@article {pmid39591548, year = {2024}, author = {Wu, R and Cao, Y and Liu, Y and Zhou, Y and Chen, Z and Zhu, JJ}, title = {Alkynyl Ligands Templated Assemblies of Silver Nanoclusters with Exceptional Electrochemiluminescence Activity for Pancreatic Cancer Specific tsRNAs Measurement.}, journal = {Analytical chemistry}, volume = {96}, number = {49}, pages = {19396-19403}, doi = {10.1021/acs.analchem.4c03758}, pmid = {39591548}, issn = {1520-6882}, mesh = {*Silver/chemistry ; *Pancreatic Neoplasms ; Ligands ; *Metal Nanoparticles/chemistry ; Humans ; *Electrochemical Techniques ; *Alkynes/chemistry ; *Luminescent Measurements ; Biosensing Techniques/methods ; RNA, Small Interfering/chemistry ; }, abstract = {Proper manipulation of the ligand complex on the motifs of metal nanoclusters (MNCs) to form an ordered self-assembly is an effective approach to enhance the electrochemiluminescence (ECL) emission of MNCs. We report a facile approach for the preparation of self-assembled AgNCs (AgNCsAssy) induced by alkynyl ligands with enhanced ECL and stability. The formation of these AgNCsAssy was simultaneously driven by the diverse coordination modes of alkynyl ligands with Ag and intercluster interactions, for which it was found that the para-substituted alkynyl ligands exhibited apparently irregular nanoparticles, while the monosubstituted counterparts were present in the form of ribbons. The calculations revealed that the energy gap between the highest occupied molecular orbitals (HOMOs) and the lowest unoccupied molecular orbitals (LUMOs) played a crucial role in their ECL emissions because of the substituent effects, especially, the low-lying LUMO levels could help to enhance the ECL emission. Moreover, mechanistic studies revealed that both the coreactant and alkynyl ligands made significant contributions to the ECL performance. Concurrently, the CRISPR-associated proteins (CRISPR-Cas) 12a system shows great potential in biosensing applications due to the advantages of easy design and precise targeting. As a proof of concept, we integrated the cascade amplification of catalytic hairpin assembly (CHA) circuit and the collateral cleavage activity of CRISPR-Cas12a to construct an ultrasensitive ECL biosensor for pancreatic cancer (PC)-specific tsRNAs, with a detection limit of 3.33 fM. This work is not only instructive for the synthesis of self-assembled MNCs with high ECL activities but also contributes to the understanding of the ECL mechanism of self-assembled MNCs.}, }
@article {pmid39590667, year = {2024}, author = {Leal, K and Rojas, E and Madariaga, D and Contreras, MJ and Nuñez-Montero, K and Barrientos, L and Goméz-Espinoza, O and Iturrieta-González, I}, title = {Unlocking Fungal Potential: The CRISPR-Cas System as a Strategy for Secondary Metabolite Discovery.}, journal = {Journal of fungi (Basel, Switzerland)}, volume = {10}, number = {11}, pages = {}, pmid = {39590667}, issn = {2309-608X}, support = {11230475//FONDECYT Iniciaci&#xf3;n/ ; 3230521//ANID Fondecyt Postdoctorado/ ; }, abstract = {Natural products (NPs) are crucial for the development of novel antibiotics, anticancer agents, and immunosuppressants. To highlight the ability of fungi to produce structurally diverse NPs, this article focuses on the impact of genome mining and CRISPR-Cas9 technology in uncovering and manipulating the biosynthetic gene clusters (BGCs) responsible for NP synthesis. The CRISPR-Cas9 system, originally identified as a bacterial adaptive immune mechanism, has been adapted for precise genome editing in fungi, enabling targeted modifications, such as gene deletions, insertions, and transcription modulation, without altering the genomic sequence. This review elaborates on various CRISPR-Cas9 systems used in fungi, notably the Streptococcus pyogenes type II Cas9 system, and explores advancements in different Cas proteins for fungal genome editing. This review discusses the methodologies employed in CRISPR-Cas9 genome editing of fungi, including guide RNA design, delivery methods, and verification of edited strains. The application of CRISPR-Cas9 has led to enhanced production of secondary metabolites in filamentous fungi, showcasing the potential of this system in biotechnology, medical mycology, and plant pathology. Moreover, this article emphasizes the integration of multi-omics data (genomics, transcriptomics, proteomics, and metabolomics) to validate CRISPR-Cas9 editing effects in fungi. This comprehensive approach aids in understanding molecular changes, identifying off-target effects, and optimizing the editing protocols. Statistical and machine learning techniques are also crucial for analyzing multi-omics data, enabling the development of predictive models and identification of key molecular pathways affected by CRISPR-Cas9 editing. In conclusion, CRISPR-Cas9 technology is a powerful tool for exploring fungal NPs with the potential to accelerate the discovery of novel bioactive compounds. The integration of CRISPR-Cas9 with multi-omics approaches significantly enhances our ability to understand and manipulate fungal genomes for the production of valuable secondary metabolites and for promising new applications in medicine and industry.}, }
@article {pmid39590528, year = {2024}, author = {Kaupbayeva, B and Tsoy, A and Safarova Yantsen, Y and Nurmagambetova, A and Murata, H and Matyjaszewski, K and Askarova, S}, title = {Unlocking Genome Editing: Advances and Obstacles in CRISPR/Cas Delivery Technologies.}, journal = {Journal of functional biomaterials}, volume = {15}, number = {11}, pages = {}, pmid = {39590528}, issn = {2079-4983}, support = {AP14871448//Kazakhstan MSHE/ ; }, abstract = {CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats associated with protein 9) was first identified as a component of the bacterial adaptive immune system and subsequently engineered into a genome-editing tool. The key breakthrough in this field came with the realization that CRISPR/Cas9 could be used in mammalian cells to enable transformative genetic editing. This technology has since become a vital tool for various genetic manipulations, including gene knockouts, knock-in point mutations, and gene regulation at both transcriptional and post-transcriptional levels. CRISPR/Cas9 holds great potential in human medicine, particularly for curing genetic disorders. However, despite significant innovation and advancement in genome editing, the technology still possesses critical limitations, such as off-target effects, immunogenicity issues, ethical considerations, regulatory hurdles, and the need for efficient delivery methods. To overcome these obstacles, efforts have focused on creating more accurate and reliable Cas9 nucleases and exploring innovative delivery methods. Recently, functional biomaterials and synthetic carriers have shown great potential as effective delivery vehicles for CRISPR/Cas9 components. In this review, we attempt to provide a comprehensive survey of the existing CRISPR-Cas9 delivery strategies, including viral delivery, biomaterials-based delivery, synthetic carriers, and physical delivery techniques. We underscore the urgent need for effective delivery systems to fully unlock the power of CRISPR/Cas9 technology and realize a seamless transition from benchtop research to clinical applications.}, }
@article {pmid39590000, year = {2024}, author = {Guermonprez, P and Nioche, P and Renaud, L and Battaglini, N and Sanaur, S and Krejci, E and Piro, B}, title = {CRISPR-Cas Systems Associated with Electrolyte-Gated Graphene-Based Transistors: How They Work and How to Combine Them.}, journal = {Biosensors}, volume = {14}, number = {11}, pages = {}, pmid = {39590000}, issn = {2079-6374}, support = {ANR-21-CE19-0039.//ANR/ ; PhD scholarship//Universit&#xe9; Paris Cit&#xe9;/ ; }, mesh = {*Graphite/chemistry ; *Transistors, Electronic ; *Biosensing Techniques ; *CRISPR-Cas Systems ; Humans ; SARS-CoV-2 ; COVID-19 ; Electrolytes ; RNA, Viral ; }, abstract = {In this review, recent advances in the combination of CRISPR-Cas systems with graphene-based electrolyte-gated transistors are discussed in detail. In the first part, the functioning of CRISPR-Cas systems is briefly explained, as well as the most common ways to convert their molecular activity into measurable signals. Other than optical means, conventional electrochemical transducers are also developed. However, it seems that the incorporation of CRISPR/Cas systems into transistor devices could be extremely powerful, as the former provides molecular amplification, while the latter provides electrical amplification; combined, the two could help to advance in terms of sensitivity and compete with conventional PCR assays. Today, organic transistors suffer from poor stability in biological media, whereas graphene materials perform better by being extremely sensitive to their chemical environment and being stable. The need for fast and inexpensive sensors to detect viral RNA arose on the occasion of the COVID-19 crisis, but many other RNA viruses are of interest, such as dengue, hepatitis C, hepatitis E, West Nile fever, Ebola, and polio, for which detection means are needed.}, }
@article {pmid39589995, year = {2024}, author = {Yang, JW and Kim, H and Hyeon, LS and Yoo, JS and Kang, S}, title = {Development of a Recombinase Polymerase Amplification-Coupled CRISPR/Cas12a Platform for Rapid Detection of Antimicrobial-Resistant Genes in Carbapenem-Resistant Enterobacterales.}, journal = {Biosensors}, volume = {14}, number = {11}, pages = {}, pmid = {39589995}, issn = {2079-6374}, support = {2022-NI-002-00//National Institute of Health/ ; 2024-NI-023-00//National Institute of Health/ ; }, mesh = {*CRISPR-Cas Systems ; *Recombinases ; *Bacterial Proteins/genetics ; beta-Lactamases/genetics ; Humans ; Carbapenem-Resistant Enterobacteriaceae/genetics ; Nucleic Acid Amplification Techniques ; Carbapenems/pharmacology ; Biosensing Techniques ; Enterobacteriaceae/genetics ; }, abstract = {The worldwide spread of carbapenemase-producing Enterobacterales (CPE) represents a significant threat owing to the high mortality and morbidity rates. Traditional diagnostic methods are often too slow and complex for rapid point-of-care testing. Therefore, we developed a recombinase polymerase amplification (RPA)-coupled CRISPR/Cas12a system (RCCS), a rapid, accurate, and simple diagnostic platform for detecting antimicrobial-resistant genes. The RCCS detected carbapenemase genes (blaKPC and blaNDM) within 50 min, including 10 min for DNA extraction and 30-40 min for RCCS reaction (a 20 min RPA reaction with a 10-20-min CRISPR/Cas12a assay). Fluorescence signals obtained from the RCCS platform were visualized using lateral-flow test strips (LFSs) and real-time and endpoint fluorescence. The LFS clearly displayed test lines while detecting carbapenemase genes. Furthermore, the RCCS platform demonstrated high sensitivity by successfully detecting blaKPC and blaNDM at the attomolar and picomolar levels, respectively. The accuracy of the RCCS platform was validated with clinical isolates of Klebsiella pneumoniae and Escherichia coli; a 100% detection accuracy was achieved, which has not been reported when using conventional PCR. Overall, these findings indicate that the RCCS platform is a powerful tool for rapid and reliable detection of carbapenemase-encoding genes, with significant potential for implementation in point-of-care settings and resource-limited environments.}, }
@article {pmid39589913, year = {2024}, author = {Wang, Q and Yang, G and Jia, R and Wang, F and Wang, G and Xu, Z and Li, J and Li, B and Yu, L and Zhang, Y and Alariqi, M and Cao, J and Liang, S and Zhang, X and Nie, X and Jin, S}, title = {Utilizing the mutant library to investigate the functional characterization of GhGLR3.4 regulating jasmonic acid to defense pest infestation.}, journal = {The Plant journal : for cell and molecular biology}, volume = {120}, number = {6}, pages = {2889-2903}, doi = {10.1111/tpj.17152}, pmid = {39589913}, issn = {1365-313X}, support = {32272128//National Natural Science Foundation of China/ ; 32401780//National Natural Science Foundation of China/ ; 027Y2022-021//Science and Technology Project of Hubei Province of China National Tobacco Corporation/ ; 2022YFF1001403//National Key Research and Development Program of China/ ; 32325039//National Natural Science Fund of China for Distinguished Young Scholars/ ; }, mesh = {*Cyclopentanes/metabolism ; *Oxylipins/metabolism ; *Gossypium/genetics/parasitology ; *Plant Proteins/genetics/metabolism ; Animals ; Receptors, Glutamate/genetics/metabolism ; Gene Expression Regulation, Plant ; Signal Transduction ; Plant Growth Regulators/metabolism ; Arabidopsis/genetics/physiology ; Mutation ; CRISPR-Cas Systems ; Plant Diseases/parasitology/genetics/immunology ; }, abstract = {The glutamate receptor (GLR) serves as a ligand-gated ion channel that plays a vital role in plant growth, development, and stress response. Nevertheless, research on GLRs in cotton is still very limited. The present study conducted a comprehensive analysis of GLRs gene family in cotton. In total, 41 members of the GLR family were identified in cotton unveiling distinct subgroups in comparison to Arabidopsis. Among these members, the third subgroup highlights its pivotal role in cotton's defense against insect infestation. Furthermore, the CRISPR/Cas9 system was utilized to create a mutant library of GLR members, which consisted of a total of 135 independent mutant lines, resulting in the production of novel cotton materials with valuable breeding potential for pest control. Further, this study elucidates the influence of GhGLR3.4 on jasmonic acid (JA) pathway signal transduction and demonstrated its participation in the influx of intracellular Ca[2+], which regulates "calcium transients" following stimulation, thereby influencing multiple intracellular reactions. The study also found that GhGLR3.4 influences the synthesis of the JA pathway and actively partakes in long-distance signal transmission among plants, facilitating the transfer of defense signals to neighbor leaves and thereby triggering systemic defense. Consequently, this research advances our knowledge of plants' comprehensive defense mechanism against insect pest infestation.}, }
@article {pmid39589873, year = {2024}, author = {Chen, X and Koo, J and Kumar Arya, S and Palli, SR}, title = {Chronologically inappropriate morphogenesis (Chinmo) is required for maintenance of larval stages of fall armyworm.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {121}, number = {49}, pages = {e2411286121}, pmid = {39589873}, issn = {1091-6490}, support = {2019-67013-29351//U.S. Department of Agriculture (USDA)/ ; 2353057000//U.S. Department of Agriculture (USDA)/ ; }, mesh = {Animals ; *Larva/growth &amp; development/metabolism/genetics ; *Metamorphosis, Biological ; *Gene Expression Regulation, Developmental ; Drosophila Proteins/genetics/metabolism ; Morphogenesis/genetics ; Transcription Factors/genetics/metabolism ; Pupa/growth &amp; development/genetics/metabolism ; CRISPR-Cas Systems ; Drosophila melanogaster/genetics/growth &amp; development ; Signal Transduction ; Kruppel-Like Transcription Factors ; }, abstract = {Broad complex (Br-C) and eip93F (E93) transcription factors promote insect metamorphosis from larva to pupa and from pupa to adult, respectively. Recently, chronologically inappropriate morphogenesis (Chinmo) has been proposed as a larval specifier in Drosophila melanogaster. However, whether Chinmo is required for larval maintenance in lepidopteran insects, the underlying mechanisms involved in maintaining the larval stage, and its interactions with the JH signaling pathway are not well understood. Here, we used a binary transgenic CRISPR/Cas9 system to knockout Chinmo and Kr-h1 (primary response gene in the JH signaling pathway) in the fall armyworm (FAW). Kr-h1 knockout induced premature metamorphosis only after L5 (penultimate), whereas Chinmo and Kr-h1 double knockout induced premature metamorphosis in L3. Sequencing and differential gene expression (DEG) analysis of RNA isolated from mutants and single-cell multiome ATAC analysis of Chinmo, Kr-h1, and Chinmo and Kr-h1 double knockout Sf9 cells revealed that Chinmo participates in chromatin modifications that prevent the promoter accessibility and expression of metamorphosis promoting genes. These results suggest that Chinmo is a larval specifier that plays a major role in preventing metamorphosis in early larval stages by controlling chromatin accessibility near the promoters of genes such as Br-C and E93 required for pupal and adult development.}, }
@article {pmid39588774, year = {2024}, author = {Zhang, J and Guan, X and Moon, J and Zhang, S and Jia, Z and Yang, R and Hou, C and Guo, C and Pei, M and Liu, C}, title = {Interpreting CRISPR-Cas12a enzyme kinetics through free energy change of nucleic acids.}, journal = {Nucleic acids research}, volume = {52}, number = {22}, pages = {14077-14092}, pmid = {39588774}, issn = {1362-4962}, support = {U01 CA269147/CA/NCI NIH HHS/United States ; R33AI154642/NH/NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems ; Kinetics ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *Thermodynamics ; Endodeoxyribonucleases/metabolism/chemistry/genetics ; DNA/metabolism/chemistry/genetics ; Bacterial Proteins/metabolism/genetics/chemistry ; DNA, Single-Stranded/metabolism/chemistry ; Machine Learning ; }, abstract = {While CRISPR has revolutionized biotechnology, predicting CRISPR-Cas nuclease activity remains a challenge. Herein, through the trans-cleavage feature of CRISPR-Cas12a, we investigate the correlation between CRISPR enzyme kinetics and the free energy change of crRNA and DNA targets from their initial thermodynamic states to a presumed transition state before hybridization. By subjecting computationally designed CRISPR RNAs (crRNAs), we unravel a linear correlation between the trans-cleavage kinetics of Cas12a and the energy barrier for crRNA spacer and single-stranded DNA target unwinding. This correlation shifts to a parabolic relationship with the energy consumption required for double-stranded DNA target separation. We further validate these correlations using ∼100 randomly selected crRNA/DNA pairs from viral genomes. Through machine learning methods, we reveal the synergistic effect of free energy change of crRNA and DNA on categorizing Cas12a activity on a two-dimensional map. Furthermore, by examining other potential factors, we find that the free energy change is the predominant factor governing Cas12a kinetics. This study will not only empower sequence design for numerous applications of CRISPR-Cas12a systems, but can also extend to activity prediction for a variety of enzymatic reactions driven by nucleic acid dynamics.}, }
@article {pmid39588656, year = {2024}, author = {Jiang, Y and Wang, Y and Luo, W and Luan, X and Zhang, Z and Pan, Y and He, B and Gao, Y and Song, Y}, title = {Detecting telomerase activity at the single-cell level using a CRISPR-Cas12a-based chip.}, journal = {Lab on a chip}, volume = {25}, number = {1}, pages = {49-56}, doi = {10.1039/d4lc00619d}, pmid = {39588656}, issn = {1473-0189}, mesh = {*Telomerase/metabolism ; *CRISPR-Cas Systems ; Humans ; *Single-Cell Analysis/instrumentation ; Lab-On-A-Chip Devices ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {The intimate association between telomerase activity and cancer has driven the exploration of diverse methodologies for its precise detection. However, detecting telomerase activity at the single-cell level remains a significant challenge. Herein, we present a MOF-DNA barcode-amplified CRISPR-Cas12a strategy integrated with a single-cell microfluidic chip for ultrasensitive detection of telomerase activity. DNA-functionalized UiO-66 nanoparticles act as signal transducers, effectively converting telomerase activity into DNA activation strands, which subsequently trigger the trans-cleavage activity of CRISPR-Cas12a. This amplification-based assay could be integrated with a microfluidic chip to enable highly sensitive detection of telomerase activity at the single-cell level, offering promising advancements in early cancer diagnosis.}, }
@article {pmid39588089, year = {2024}, author = {Rasmussen, SK and Jain, SM}, title = {Editorial: Mutational breeding: from induced mutations to site-directed mutagenesis.}, journal = {Frontiers in plant science}, volume = {15}, number = {}, pages = {1511363}, pmid = {39588089}, issn = {1664-462X}, }
@article {pmid39587072, year = {2024}, author = {Montepeloso, A and Mattioli, D and Pellin, D and Peviani, M and Genovese, P and Biffi, A}, title = {Haploinsufficiency at the CX3CR1 locus of hematopoietic stem cells favors the appearance of microglia-like cells in the central nervous system of transplant recipients.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10192}, pmid = {39587072}, issn = {2041-1723}, mesh = {*CX3C Chemokine Receptor 1/metabolism/genetics ; Animals ; *Microglia/metabolism ; *Hematopoietic Stem Cells/metabolism/cytology ; *Hematopoietic Stem Cell Transplantation ; Humans ; *Haploinsufficiency ; Mice ; *Central Nervous System/metabolism ; CRISPR-Cas Systems ; Female ; Male ; Genetic Therapy/methods ; Genetic Loci ; }, abstract = {Transplantation of engineered hematopoietic stem/progenitor cells (HSPCs) showed curative potential in patients affected by neurometabolic diseases treated in early stage. Favoring the engraftment and maturation of the engineered HSPCs in the central nervous system (CNS) could allow enhancing further the therapeutic potential of this approach. Here we unveil that HSPCs haplo-insufficient at the Cx3cr1 (Cx3cr1[-/+]) locus are favored in central nervous system (CNS) engraftment and generation of microglia-like progeny cells (MLCs) as compared to wild type (Cx3cr1[+/+]) HSPCs upon transplantation in mice. Based on this evidence, we have developed a CRISPR-based targeted gene addition strategy at the human CX3CR1 locus resulting in an enhanced ability of the edited human HSPCs to generate mature MLCs upon transplantation in immunodeficient mice, and in lineage specific, regulated and robust transgene expression. This approach, which benefits from the modulation of pathways involved in microglia maturation and migration in haplo-insufficient cells, may broaden the application of HSPC gene therapy to a larger spectrum of neurometabolic and neurodegenerative diseases.}, }
@article {pmid39587061, year = {2024}, author = {Qu, J and Wang, Y and Xiong, C and Wang, M and He, X and Jia, W and Li, CY and Zhang, T and Wang, Z and Li, W and Kuang, BY and Shi, P}, title = {In vivo gene editing of T-cells in lymph nodes for enhanced cancer immunotherapy.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10218}, pmid = {39587061}, issn = {2041-1723}, mesh = {Animals ; *Gene Editing/methods ; *Lymph Nodes/immunology ; Mice ; *T-Lymphocytes/immunology ; *CRISPR-Cas Systems/genetics ; *Immunotherapy/methods ; *Programmed Cell Death 1 Receptor/metabolism/genetics/antagonists &amp; inhibitors ; Mice, Inbred C57BL ; Cell Line, Tumor ; Immune Checkpoint Inhibitors/therapeutic use/pharmacology ; Humans ; Female ; Melanoma, Experimental/therapy/immunology/genetics ; Melanoma/therapy/immunology/genetics ; Electroporation/methods ; }, abstract = {Immune checkpoint blockade (ICB) therapy, while promising for cancer treatment, faces challenges like unexpected side effects and limited objective responses. Here, we develop an in vivo gene-editing strategy for improving ICB cancer therapy in a lastingly effective manner. The approach uses a conductive hydrogel-based electroporation system to enable nucleofection of programmed cell death protein 1 (PD1) targeted CRISPR-Cas9 DNAs into T-cells directly within the lymph nodes, and subsequently produces PD1-deficient T-cells to combat tumor growth, metastasis and recurrence in different melanoma models in mice. Following in vivo gene editing, animals show enhanced cellular and humoral immune responses along with multi-fold increases of effector T-cells infiltration to the solid tumors, preventing tumor recurrence and prolonging their survival. These findings provide a proof-of-concept for direct in vivo T-cell engineering via localized gene-editing for enhanced cancer immunotherapy, and also unlock the possibilities of using this method to treat more complex human diseases.}, }
@article {pmid39586398, year = {2025}, author = {Tachibana, Y and Yamamoto, M}, title = {Recent advances in identifying and characterizing secretory proteins of Toxoplasma gondii by CRISPR-based screening.}, journal = {Parasitology international}, volume = {105}, number = {}, pages = {102997}, doi = {10.1016/j.parint.2024.102997}, pmid = {39586398}, issn = {1873-0329}, mesh = {Animals ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; Host-Parasite Interactions ; *Protozoan Proteins/genetics/metabolism ; *Toxoplasma/genetics/metabolism/pathogenicity ; }, abstract = {The apicomplexan parasite, Toxoplasma gondii, develops unique secretory organelles, such as micronemes, rhoptries, and dense granules, which do not exist in other well-studied eukaryotic organisms. These secretory organelles are key features of apicomplexan parasites and discharge various proteins that are essential for invasion, replication, egress, host-parasite interactions, and virulence. Many studies have therefore focused on identifying and characterizing the proteins secreted by T. gondii that play essential roles in pathology and that can be targeted for therapeutics and vaccine development. The recent development of functional genetic screens based on CRISPR/Cas9 technology has revolutionized this field and has enabled the identification of genes that contribute to parasite fitness in vitro and in vivo. Consequently, characterization of genes identified by unbiased CRISPR screens has revealed novel aspects of apicomplexan biology. In this review, we describe the development of CRIPSR-based screening technology for T. gondii, and recent advances in our understanding of secretory proteins identified and characterized by CRISPR-based screening.}, }
@article {pmid39586143, year = {2025}, author = {Jiang, H and Zhu, X and Jiao, J and Yan, C and Liu, K and Chen, W and Qin, P}, title = {CRISPR/dCas9-based hotspot self-assembling SERS biosensor integrated with a smartphone for simultaneous, ultrasensitive and robust detection of multiple pathogens.}, journal = {Biosensors &amp; bioelectronics}, volume = {270}, number = {}, pages = {116974}, doi = {10.1016/j.bios.2024.116974}, pmid = {39586143}, issn = {1873-4235}, mesh = {*Biosensing Techniques/instrumentation ; *Spectrum Analysis, Raman ; *Smartphone ; *Gold/chemistry ; *Pseudomonas aeruginosa/isolation &amp; purification/genetics ; *Metal Nanoparticles/chemistry ; *Staphylococcus aureus/isolation &amp; purification/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Escherichia coli O157/isolation &amp; purification/genetics ; *Nucleic Acid Amplification Techniques/instrumentation ; DNA, Bacterial/genetics/analysis ; Limit of Detection ; Molecular Diagnostic Techniques ; }, abstract = {Accurate detection of multiple pathogens in the early stages of infection is critical for guiding treatment and saving patients' lives, but current methods are still challenged by low sensitivity, poor robustness and long turnaround times. Here, we report a CRISPR/dCas9-based hotspot self-assembling surface-enhanced Raman scattering (SERS) biosensor (called dCasSERS) and its integration with a smartphone to address these challenges. In this design, bacterial DNA was pre-amplified by loop-mediated isothermal amplification (LAMP), and the repeat sequences of the amplicons were recognized by CRISPR/dCas9, providing abundant sites for the assembly of gold nanoparticles (AuNPs) and forming numerous hotspots for SERS analysis. Using AuNPs labeled with different Raman molecules as reporters, the presence of Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli O157:H7 (E. coli O157:H7) could be dexterously converted into distinguishable SERS signals. CRISPR/dCas9-based amplicon-specific recognition and SERS hotspot self-assembly improved the specificity and sensitivity, enabling the biosensor to simultaneously detect three target pathogens down to 1 CFU/mL without any cross-reactivity. By introducing a rapid extraction procedure and a smartphone-integrated handheld Raman spectrometer, rapid on-site analysis of multiple pathogens could be achieved in less than 50 min. The accuracy and robustness of the biosensor were demonstrated by 500 real urine specimens. This study displays a new paradigm for CRISPR/Cas-based self-assembly of SERS hotspots and provides insight into the future development of pathogen screening tools.}, }
@article {pmid39585990, year = {2024}, author = {Li, L and Scott, WS and Khristich, AN and Armenia, JF and Mirkin, SM}, title = {Recurrent DNA nicks drive massive expansions of (GAA)n repeats.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {121}, number = {49}, pages = {e2413298121}, pmid = {39585990}, issn = {1091-6490}, support = {R35 GM130322/GM/NIGMS NIH HHS/United States ; R35GM130322//HHS | NIH | National Institute of General Medical Sciences (NIGMS)/ ; 2153071//NSF | BIO | Division of Molecular and Cellular Biosciences (MCB)/ ; }, mesh = {Humans ; *Trinucleotide Repeat Expansion/genetics ; Friedreich Ataxia/genetics ; CRISPR-Cas Systems ; DNA/genetics ; DNA Breaks, Single-Stranded ; }, abstract = {Over 50 hereditary degenerative disorders are caused by expansions of short tandem DNA repeats (STRs). (GAA)n repeat expansions are responsible for Friedreich's ataxia as well as late-onset cerebellar ataxias (LOCAs). Thus, the mechanisms of (GAA)n repeat expansions attract broad scientific attention. To investigate the role of DNA nicks in this process, we utilized a CRISPR-Cas9 nickase system to introduce targeted nicks adjacent to the (GAA)n repeat tract. We found that DNA nicks 5' of the (GAA)100 run led to a dramatic increase in both the rate and scale of its expansion in dividing cells. Strikingly, they also promoted large-scale expansions of carrier- and large normal-size (GAA)n repeats, recreating, in a model system, the expansion events that occur in human pedigrees. DNA nicks 3' of the (GAA)100 repeat led to a smaller but significant increase in the expansion rate as well. Our genetic analysis implies that in dividing cells, conversion of nicks into double-strand breaks (DSBs) during DNA replication followed by DSB or fork repair leads to repeat expansions. Finally, we showed that 5' GAA-strand nicks increase expansion frequency in nondividing yeast cells, albeit to a lesser extent than in dividing cells.}, }
@article {pmid39585742, year = {2025}, author = {Tan, YY and Liew, YY and Lee, RRQ and Castel, B and Chan, NM and Wan, WL and Zhang, Y and Hu, D and Chan, P and Kim, ST and Chae, E}, title = {Generation of Inheritable A-to-G Transitions Using Adenine Base Editing and NG-PAM Cas9 in Arabidopsis thaliana.}, journal = {Molecular plant-microbe interactions : MPMI}, volume = {38}, number = {1}, pages = {30-42}, doi = {10.1094/MPMI-10-24-0127-TA}, pmid = {39585742}, issn = {0894-0282}, mesh = {*Arabidopsis/genetics ; *Gene Editing/methods ; *Adenine/metabolism ; *CRISPR-Cas Systems/genetics ; Arabidopsis Proteins/genetics/metabolism ; *CRISPR-Associated Protein 9/genetics/metabolism ; Plants, Genetically Modified ; Adenosine Deaminase/genetics/metabolism ; }, abstract = {Towards precise genome editing, base editors have been developed by fusing catalytically compromised Cas9 with deaminase components, mediating C-to-T (cytosine base editors) or A-to-G (adenine base editors) transition. We developed a set of vectors consisting of a 5'-NG-3' PAM-recognizing variant of SpCas9 with adenosine deaminases TadA7.10 or TadA8e. Using a phenotype-based screen in Arabidopsis thaliana targeting multiple PDS3 intron splice sites, we achieved up to 81% somatic A-to-G editing in primary transformants at a splice acceptor site with NGG PAM, while 35% was achieved for the same target adenine with NGA PAM. Among tested vectors, pECNUS4 (Addgene #184887), carrying TadA8e, showed the highest adenine base editor (ABE) efficiency. With pECNUS4, we recreated a naturally occurring allele of DANGEROUS MIX3 (DM3) in two generations, transgene-free, for NGC PAM. We also simultaneously base-edited four redundant DM1/SSI4 homologs, encoding nucleotide-binding leucine-rich repeat (NLR) proteins, using a single gRNA with NGA PAM targeting the conserved yet functionally crucial P-loop motif of NLR proteins. We found fixation of A-to-G in three NLR genes for all three possible adenine sites within base-editing window 3-9, as the edited genes segregate in T2. Multigene targeting succeeded in rescuing the previously reported autoimmune phenotype in two generations. Mediating desired ABE on seven NLR genes simultaneously was successful as well; above 77% editing was achieved in six of the seven possible targets in a T1 plant, with the remaining having a moderately high (32%) editing. ABE application to specifically inactivate functional motifs is anticipated to expedite the discovery of novel roles for proteins. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.}, }
@article {pmid39585465, year = {2024}, author = {Qiu, J and Liu, C and Zhu, Y}, title = {Rolling circle amplification cooperating crRNA switch for direct and sensitive methicillin-resistant Staphylococcus aureus (MRSA) analysis.}, journal = {Biotechnology letters}, volume = {47}, number = {1}, pages = {4}, pmid = {39585465}, issn = {1573-6776}, mesh = {*Methicillin-Resistant Staphylococcus aureus/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Staphylococcal Infections/microbiology/diagnosis ; Exodeoxyribonucleases/genetics/metabolism ; RNA, Bacterial/genetics ; Sensitivity and Specificity ; }, abstract = {Evaluating the methicillin resistance of Staphylococcus aureus (S. aureus) is highly important for adapting nursing strategies. Nevertheless, the identification of methicillin-resistant S. aureus (MRSA) that is both sensitive and reliable continues to pose a significant obstacle. This study describes a method for detecting MRSA using a combination of fixed rolling circle amplification (RCA) and the exonuclease-iii (Exo-iii) assisted CRISPR-Cas12a system for signal amplification. When MRSA is present, the interaction between the "b" chain in the capture probe and MRSA allows the "a" chain to be exposed. This "a" chain acts as a primer to initiate the fixed RCA process. The H probe, which includes the crRNA segment, forms a bond with the RCA product and then releases the crRNA segment with the aid of Exo-iii. The Cas12a protein, when combined with the crRNA, generates an activated CRISPR-Cas12a system that cleaves the "Reporter" probe, resulting in the production of fluorescent signals. Furthermore, this fluorescent test has been utilized for the examination of clinical samples with a satisfactory rate of retrieval. Based on the elegant design, the proposed method exhibited a low detection limit of 4.6 cfu/mL, while maintaining a high specificity for MRSA even from a mixture of several interfering bacteria. Due to its cost-effectiveness, simplicity, and adaptability, the sensing system shows potential as a platform for detecting MRSA and evaluating postoperative nursing for stomach cancer patients.}, }
@article {pmid39585363, year = {2025}, author = {Guo, Y and Zhao, X}, title = {CRISPR-based genetic screens in human pluripotent stem cells derived neurons and brain organoids.}, journal = {Cell and tissue research}, volume = {399}, number = {1}, pages = {1-8}, pmid = {39585363}, issn = {1432-0878}, support = {P50HD105353//National Institute of Child Health and Human Development/ ; R01 MH136152/MH/NIMH NIH HHS/United States ; R01 NS138268/NS/NINDS NIH HHS/United States ; P50 HD105353/HD/NICHD NIH HHS/United States ; R01 MH118827/MH/NIMH NIH HHS/United States ; R01MH118827/MH/NIMH NIH HHS/United States ; }, mesh = {Humans ; *Organoids/metabolism/cytology ; *Neurons/metabolism/cytology ; *Brain/metabolism/cytology ; *Pluripotent Stem Cells/cytology/metabolism ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Genetic Testing/methods ; Neurogenesis/genetics ; }, abstract = {Recent large-scale genome-wide association and single-cell RNA sequencing (scRNA-seq) studies have uncovered disease-associated genetic risk factors and cell type-specific genetic alterations. However, our understanding of how these genetic variants cause diseases and the underlying mechanisms remains largely unknown. Functional genomics screens using CRISPR-based technologies offer an effective tool for studying genes relevant to disease phenotypes. Here, we summarize recent CRISPR-based functional genomics screen approaches applied to human pluripotent stem cell (hPSC)-derived neurons and brain organoids. These screens have identified genes crucial for neurogenesis, neuronal survival, morphological development, and migration. Combining CRISPR-based genetic screens with scRNA-seq, researchers have revealed downstream genes and cellular pathways impacted by these genetic variants in human neural cells, providing new insights into the pathogenesis of neurodevelopmental disorders, such as microcephaly and autism spectrum disorders. Finally, we discuss current challenges and future directions for using CRISPR-based screens in furthering our understanding of neurological diseases and developing potential therapeutic strategies. Despite challenges, CRISPR-based screens have enormous potential for advancing the therapeutic development of many diseases.}, }
@article {pmid39584421, year = {2024}, author = {Wang, S and Hu, Y and Deng, Z and Liu, R and Lv, Y}, title = {CRISPR/Cas12a-enhanced DNA nanomachine for multiple respiratory pathogens detection.}, journal = {Chemical communications (Cambridge, England)}, volume = {60}, number = {99}, pages = {14814-14817}, doi = {10.1039/d4cc05639f}, pmid = {39584421}, issn = {1364-548X}, mesh = {*CRISPR-Cas Systems/genetics ; *SARS-CoV-2/genetics/isolation &amp; purification ; Humans ; *Influenza A Virus, H1N1 Subtype/isolation &amp; purification/genetics ; *Mycoplasma pneumoniae/genetics/isolation &amp; purification ; DNA/chemistry ; COVID-19/virology/diagnosis ; Limit of Detection ; Nanostructures/chemistry ; Mass Spectrometry ; CRISPR-Associated Proteins/metabolism/genetics ; Respiratory Tract Infections/virology/microbiology/diagnosis ; }, abstract = {Respiratory infection caused by pathogens is among the most prevalent health issues affecting people worldwide. Accurate and rapid screening of respiratory pathogens is crucial for selecting appropriate treatments to control epidemics. However, it is often challenged by two aspects: first, the low concentration of pathogens in the early stages of infection; second, the difficulty of analyzing multiple pathogens. Herein, we report a mass spectrometry strategy combining the CRISPR/Cas12a system with DNA nanomachines for respiratory pathogens detection. Thanks to the high sensitivity of the CRISPR/Cas12a-enhanced DNA nanomachine and the multiple analysis of elemental mass spectrometry, the proposed method was successfully applied for clinical sample analysis with a low detection limit of 28 amol, 30 amol, and 38 amol for SARS-CoV-2, influenza A virus subtype H1N1, and Mycoplasma pneumoniae, respectively.}, }
@article {pmid39584349, year = {2024}, author = {Chen, Z and Wu, Y and Zhang, Y and Gao, Y}, title = {[Knockout of RIG-I in HEK293 cells by CRISPR/Cas9].}, journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology}, volume = {40}, number = {11}, pages = {4254-4265}, doi = {10.13345/j.cjb.240130}, pmid = {39584349}, issn = {1872-2075}, mesh = {Humans ; HEK293 Cells ; *CRISPR-Cas Systems ; *DEAD Box Protein 58/genetics/metabolism ; *Signal Transduction ; *Receptors, Immunologic/genetics/metabolism ; Gene Knockout Techniques ; Transfection ; DEAD-box RNA Helicases/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Interferon-Induced Helicase, IFIH1/genetics/metabolism ; Transcription Factor RelA/genetics/metabolism ; Interferon-beta/genetics/metabolism ; }, abstract = {We knocked out the retinoic acid-inducible gene I (RIG-I) in HEK293 cells via CRISPR/Cas9 to reveal the effects of RIG-I knockout on the key factors in the type I interferon signaling pathway. Three single guide RNAs (sgRNAs) targeting RIG-I were designed, and the recombination vectors were constructed on the basis of the pX459 vector and used to transfect HEK293 cells, which were screened by puromycin subsequently. Furthermore, a mimic of virus, poly I: C, was used to transfect the cells screened out. RIG-I knockout was checked by sequencing, real-time quantitative PCR, Western blotting, and immunofluorescence assay. Meanwhile, the expression levels of key factors of type I interferon signaling pathway such as melanoma differentiation-associated gene 5 (MDA5), interferonβ1 (IFNβ1), and nuclear factor-kappa B p65 [NF-κB(p65)], as well as cell viability, were determined. The results showed that two HEK293 cell lines (S1 and S3) with RIG-I knockout were obtained, which exhibited lower mRNA and protein levels of RIG-I than the wild type HEK293 cells (P < 0.05). The mRNA levels of MDA5 and IFNβ1 in S1 and S3 cells and the protein level of NF-κB(p65) in S3 cells were lower than those in the wild type (P < 0.05). More extranuclear NF-κB(p65) protein was detected in S1 cells than in the wild type after transfection with poly I: C. Plus, the wild-type and S1 cells transfected with poly I: C for 48 h showcased reduced viability (P < 0.05), while S3 cells did not display the reduction in cell viability. In summary, the present study obtained two HEK293 cell lines with RIG-I knockout via CRISPR/Cas9, which provided a stable cell model for exploring the mechanism of type I interferon signaling pathway.}, }
@article {pmid39584322, year = {2024}, author = {Hu, Y and Chen, Z and Zhang, S and Ge, S}, title = {[Advances in visual detection of pathogen nucleic acids by CRISPR-Cas].}, journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology}, volume = {40}, number = {11}, pages = {3872-3887}, doi = {10.13345/j.cjb.240111}, pmid = {39584322}, issn = {1872-2075}, mesh = {*CRISPR-Cas Systems ; Humans ; Nucleic Acids/analysis ; Point-of-Care Testing ; }, abstract = {Visual detection is a technique for evaluating the results through visual judgment without relying on complex optical detection systems. It obtains results quickly based on signals, such as visible light, changes in air pressure, and migration distance, that can be directly observed by naked eyes, being widely used in the in vitro diagnostics industry. The CRISPR-Cas system has the potential to be used in the development of point of care testing (POCT) technologies due to the advantages of mild reaction conditions, no need for thermal cycling or other control measures, and a robust signal amplification capability. In recent years, the combination of visual detection and CRISPR-Cas has significantly reduced the need for laboratory infrastructures, precision instruments, and specialized personnel for nucleic acid detection. This has promoted the development of POCT technology and methods for nucleic acids. This article summarizes the signal output modes and characteristics of the visual detection of nucleic acid by CRISPR-Cas and discusses the issues in the application. Finally, its future clinical translation is envisioned with a view to informing the development of CRISPR-Cas visualization assays.}, }
@article {pmid39582866, year = {2024}, author = {Amiri, M and Moaveni, AK and Majidi Zolbin, M and Shademan, B and Nourazarian, A}, title = {Optimizing cancer treatment: the synergistic potential of CAR-T cell therapy and CRISPR/Cas9.}, journal = {Frontiers in immunology}, volume = {15}, number = {}, pages = {1462697}, pmid = {39582866}, issn = {1664-3224}, mesh = {Humans ; *CRISPR-Cas Systems ; *Immunotherapy, Adoptive/methods ; *Neoplasms/therapy/immunology/genetics ; *Gene Editing/methods ; Animals ; Receptors, Chimeric Antigen/genetics/immunology ; Combined Modality Therapy ; }, abstract = {Optimizing cancer treatment has become a pivotal goal in modern oncology, with advancements in immunotherapy and genetic engineering offering promising avenues. CAR-T cell therapy, a revolutionary approach that harnesses the body's own immune cells to target and destroy cancer cells, has shown remarkable success, particularly in treating acute lymphoblastic leukemia (ALL), and in treating other hematologic malignancies. While CAR-T cell therapy has shown promise, challenges such as high cost and manufacturing complexity remain. However, its efficacy in solid tumors remains limited. The integration of CRISPR/Cas9 technology, a powerful and precise genome-editing tool, also raises safety concerns regarding unintended edits and off-target effects, offers a synergistic potential to overcome these limitations. CRISPR/Cas9 can enhance CAR-T cell therapy by improving the specificity and persistence of CAR-T cells, reducing off-target effects, and engineering resistance to tumor-induced immunosuppression. This combination can also facilitate the knockout of immune checkpoint inhibitors, boosting the anti-tumor activity of CAR-T cells. Recent studies have demonstrated that CRISPR/Cas9-edited CAR-T cells can target previously untreatable cancer types, offering new hope for patients with refractory cancers. This synergistic approach not only enhances the efficacy of cancer treatment but also paves the way for personalized therapies tailored to individual genetic profiles. This review highlights the ongoing research efforts to refine this approach and explores its potential to revolutionize cancer treatment across a broader range of malignancies. As research progresses, the integration of CAR-T cell therapy and CRISPR/Cas9 holds the promise of transforming cancer treatment, making it more effective and accessible. This review explores the current advancements, challenges, and future prospects of this innovative therapeutic strategy.}, }
@article {pmid39581190, year = {2025}, author = {Egawa, M and Uno, N and Komazaki, R and Ohkame, Y and Yamazaki, K and Yoshimatsu, C and Ishizu, Y and Okano, Y and Miyamoto, H and Osaki, M and Suzuki, T and Hosomichi, K and Aizawa, Y and Kazuki, Y and Tomizuka, K}, title = {Generation of Monosomy 21q Human iPS Cells by CRISPR/Cas9-Mediated Interstitial Megabase Deletion.}, journal = {Genes to cells : devoted to molecular &amp; cellular mechanisms}, volume = {30}, number = {1}, pages = {e13184}, pmid = {39581190}, issn = {1365-2443}, support = {21-101//Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences/ ; JP24bm1123038//Japan Agency for Medical Research and Development/ ; JP24ama121046//Japan Agency for Medical Research and Development/ ; JP24gm0010010//Japan Agency for Medical Research and Development/ ; JPMJCR18S4//CREST, Japan Science and Technology Agency/ ; JPMJSP2134//SPRING, Japan Science and Technology Agency/ ; }, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems ; *Chromosomes, Human, Pair 21/genetics ; *Chromosome Deletion ; Monosomy/genetics ; Cell Line ; }, abstract = {Missing an entire chromosome or chromosome arm in normal diploid cells has a deleterious impact on cell viability, which may contribute to the development of specific birth defects. Nevertheless, the effects of chromosome loss in human cells have remained unexplored due to the lack of suitable model systems. Here, we developed an efficient, selection-free approach to generate partial monosomy in human induced pluripotent stem cells (iPSCs). The introduction of Cas9 proteins and a pair of gRNAs induces over megabase-sized interstitial chromosomal deletions. Using human chromosome 21 (HSA21) as a model, partial monosomy 21q (PM21q) iPSC lines with deletions ranging from 4.5 to 27.9 Mb were isolated. A 33.6 Mb deletion, encompassing all protein-coding genes on 21q, was also achieved, establishing the first 21q monosomy human iPSC line. Transcriptome and proteome analyses revealed that the abundances of mRNA and protein encoded by the majority of genes in the monosomic regions are half of the diploid expression level, indicating an absence of dosage compensation. The ability to generate customized partial monosomy cell lines on an isogenic, karyotypically normal background should facilitate the gain of novel insights into the impact of chromosome loss on cellular fitness.}, }
@article {pmid39581026, year = {2025}, author = {Wang, W and Sun, J and Gao, Y and Jia, XX and Ye, Y and Ren, S and Peng, Y and Han, D and Zhou, H and Gao, Z and Sun, X}, title = {Ultra-sensitive detection of norovirus using a three-in-one CRISPR platform based on a DNA hydrogel and composite functional nanomaterials.}, journal = {Journal of hazardous materials}, volume = {482}, number = {}, pages = {136523}, doi = {10.1016/j.jhazmat.2024.136523}, pmid = {39581026}, issn = {1873-3336}, mesh = {*Norovirus/isolation &amp; purification ; *Hydrogels/chemistry ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Colorimetry ; *DNA/chemistry ; Limit of Detection ; *Nanostructures/chemistry ; Nucleic Acid Amplification Techniques ; }, abstract = {The ultrasensitive sensor with three optical response mechanisms was proposed for the determination of trace amounts of norovirus using a 3-in-1 GCSNAs (a gap-containing spherical nucleic acid nanoparticles) probe. A simple and highly sensitive three-mode biosensor with Raman, colorimetric, and fluorescence functions was proposed and implemented using the GCSNAs probe and a DNA hydrogel for norovirus detection. When the virus exists, the trans-cleavage activity of CRISPR-Cas12a was activated by double-stranded dsDNA (dsDNA) generated by reverse transcription and recombinase polymerase isothermal amplification (RT-RPA) to degrade the DNA hydrogel/GCSNA composition and release the three-in-one (3-in-1) probe-GCSNA, realising the triple ultrasensitive detection of norovirus. The colorimetric sensing mode allows for semi-quantitative on-site detection, which is visible to the naked eye and the quantitative detection can be achieved by conducting grayscale analysis using the "Colour Grab" function of a smartphone. This new triple sensor achieved the successful quantification of norovirus at concentrations as low as the femtomolar scale with an excellent selectivity and accuracy. Considering the colorimetric properties of rolling circle amplification (RCA)-based DNA hydrogels and GCSNAs, the proposed method has a broad application prospect in virus on-site detection in food. It should be applicable for virus detection in a wide range of fields such, as environmental analysis, medical diagnosis, and food safety. It is anticipated that this mechanism will open new avenues for the development of multimodal analyses and multifunctional sensing platforms for various applications. We anticipate that this sensing mechanism will open up a new way for the development of food safety detection.}, }
@article {pmid39580667, year = {2025}, author = {Kim, B and Seo, HW and Lee, K and Yong, D and Park, YK and Lee, Y and Lee, S and Kim, DW and Kim, D and Ryu, CM}, title = {Lipid Nanoparticle-Mediated CRISPR-Cas13a Delivery for the Control of Bacterial Infection.}, journal = {Advanced healthcare materials}, volume = {14}, number = {7}, pages = {e2403281}, pmid = {39580667}, issn = {2192-2659}, support = {KGM9942421//Korea Research Institute of Bioscience and Biotechnology/ ; RS-2023-00219213//Ministry of Science and ICT, South Korea/ ; }, mesh = {Animals ; *Nanoparticles/chemistry ; *CRISPR-Cas Systems/genetics ; Mice ; Escherichia coli/genetics ; Plasmids/genetics ; *Bacterial Infections ; *Lipids/chemistry ; Humans ; Gene Transfer Techniques ; Liposomes ; }, abstract = {Lipid nanoparticles (LNPs) can assist in the delivery of nucleic acid inside animal cells, as demonstrated by their use in COVID-19 vaccine development. However, LNPs applicable to bacteria have not been reported. Here, the screening of 511 LNPs containing random combinations of different lipid components identified two LNPs, LNP 496 and LNP 470, that efficiently delivered plasmids into Escherichia coli BW25113. Since Gram-negative bacteria have lipid bilayers, the bacteria are pretreated with LNP-helper that weakens the bacterial membrane. The cationic lipid DOTAP improved delivery of LNP-encapsulated plasmid DNA when present at a molar ratio of 10-25 mol% in the LNP. LNP encapsulation of the Cas13a/gRNA expression vector controlled infection by a clinical Escherichia strain in Galleria mellonela larvae and mouse infection models when used in combination with non-cytotoxic concentrations of polymyxin B, a bacterial membrane disruptor. Together, the results show that LNPs can be useful as a delivery platform for agents that counteract pathogenic bacterial infections.}, }
@article {pmid39579910, year = {2025}, author = {Chen, C and Li, YW and Zheng, YY and Li, XJ and Wu, N and Guo, Q and Shi, TQ and Huang, H}, title = {Expanding the frontiers of genome engineering: A comprehensive review of CRISPR-associated transposons.}, journal = {Biotechnology advances}, volume = {78}, number = {}, pages = {108481}, doi = {10.1016/j.biotechadv.2024.108481}, pmid = {39579910}, issn = {1873-1899}, mesh = {Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *DNA Transposable Elements/genetics ; *Gene Editing/methods ; }, abstract = {Genome engineering is extensively utilized in diverse scientific disciplines, advancing human welfare and addressing various challenges. Numerous genome engineering tools have been developed to modify genomic sequences. Among these, the CRISPR-Cas system has transformed the field and remains the most commonly employed genome-editing tool. However, the CRISPR-Cas system relies on induced double-strand breaks, with editing efficiency often limited by factors such as cell type and homologous recombination, impeding further progress. CRISPR-associated transposons (CASTs) represent programmable mobile genetic elements. CASTs identified as active were developed as CAST systems, which can perform RNA-guided DNA integration and are featured by high precision, programmability, and kilobase-level payload capacity. Moreover, CAST system allows for precise genome modifications independent of host DNA repair mechanisms, addressing the constraints of conventional CRISPR-Cas systems. It expands the genome engineering toolkit and is poised to become a representative of next-generation genome editing tools. This review thoroughly examines the research progress on CASTs, highlighting the current challenges faced in genome engineering based on CASTs, and offering insights into the ongoing development of this transformative technology.}, }
@article {pmid39579237, year = {2024}, author = {Kumari, K and Gusain, S and Joshi, R}, title = {Engineering cold resilience: implementing gene editing tools for plant cold stress tolerance.}, journal = {Planta}, volume = {261}, number = {1}, pages = {2}, pmid = {39579237}, issn = {1432-2048}, mesh = {*Gene Editing/methods ; *Cold-Shock Response/genetics ; *CRISPR-Cas Systems ; Crops, Agricultural/genetics ; Cold Temperature ; Acclimatization/genetics ; Plants/genetics ; Genetic Engineering/methods ; Genome, Plant/genetics ; Plant Breeding/methods ; }, abstract = {This paper highlights the need for innovative approaches to enhance cold tolerance. It underscores how genome-editing tools can deepen our understanding of genes involved in cold stress. Cold stress is a significant abiotic factor in high-altitude regions, adversely affecting plant growth and limiting crop productivity. Plants have evolved various mechanisms in response to low temperatures that enable resistance at both physiological and molecular levels during chilling and freezing stress. Several cold-inducible genes have been isolated and characterized, with most playing key roles in providing tolerance against low-temperature stress. However, many plants fail to survive at low temperatures due to the absence of cold acclimatization mechanisms. Conventional breeding techniques, such as inter-specific or inter-genic hybridization, have had limited effectiveness in enhancing the cold resistance of essential crops. Thus, it is crucial to develop crops with improved adaptability, high yields and resistance to cold stress using advanced genomic approaches. The current availability of gene editing tools offers the opportunity to introduce targeted modifications in plant genomes efficiently, thereby developing cold-tolerant varieties. This review discusses advancements in gene editing tools, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)/Cas12a(Cpf1), prime editing (PE) and retron library recombineering (RLR). We focus specifically on the CRISPR/Cas system, which has garnered significant attention in recent years as a groundbreaking tool for genome editing across various species. These techniques will enhance our understanding of molecular interactions under low-temperature stress response and highlight the progress of genome editing in designing future climate-resilient crops.}, }
@article {pmid39579214, year = {2024}, author = {Karmakar, S and Panda, D and Behera, D and Saha, R and Baig, MJ and Molla, KA}, title = {Adaptation of bacterial natural single guide RNA (tracr-L) for efficient plant genome editing.}, journal = {Plant cell reports}, volume = {43}, number = {12}, pages = {291}, pmid = {39579214}, issn = {1432-203X}, mesh = {*Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; *Streptococcus pyogenes/genetics ; RNA, Bacterial/genetics ; Oryza/genetics/microbiology ; Protoplasts ; DNA Breaks, Double-Stranded ; }, abstract = {A long tracrRNA (tracr-L), which naturally act as single guide RNA, and its truncated version, Δtracr-L, from S. pyogenes, efficiently induce Cas9-mediated double-strand breaks (DSBs) in plant genomic loci, as demonstrated by in vitro cleavage assay and protoplast transfection. CRISPR-Cas system provides a form of immune memory in prokaryotes and archaea, protecting them against viruses and foreign genetic elements. In Streptococcus pyogenes, this system includes the pre-crRNA along with another non-coding RNA, tracrRNA, which aids in CRISPR-based immunity. In S. pyogenes, two distinct tracrRNAs are produced: a long form (tracr-L) and a short form (tracr-S). The tracr-S regulates crRNA biogenesis and Cas9 cleavage, while tracr-L suppresses CRISPR-Cas expression by targeting the Cas9 promoter to prevent autoimmunity. Deleting 79 nucleotides from tracr-L results in Δtracr-L, which retains similar functionality in gene repression. This study investigates, for the first time, the effectiveness of tracr-L, and Δtracr-L in genome editing within plant systems. In vitro cleavage assays using purified Cas9 and synthesized sgRNAs targeting the Cas9 gene, OsPDS, and the OsSWEET11 promoter revealed that across all target sites, tracr-S demonstrated the highest cleavage efficiency compared to tracr-L and Δtracr-L. For in vivo genome editing, we transfected rice protoplasts with tracr-L, Δtracr-L, and tracr-S, targeting three rice genes: OsPDS, OsSPL14, and the promoter of OsSWEET14. Amplicon deep sequencing revealed various types of indels at the target regions across all three tracrRNA versions, indicating comparable levels of efficiency. This study establishes the utility of both the long-form tracrRNA (tracr-L) and its truncated variant (Δtracr-L) in eukaryote genome editing. These two new forms of tracrRNA provide proof of concept and expand the CRISPR-Cas toolkit for plant genome editing applications, and for eukaryotes more broadly.}, }
@article {pmid39578577, year = {2024}, author = {Ingle, H and Molleston, JM and Hall, PD and Bui, D and Wang, L and Bhatt, KD and Foster, L and Antia, A and Ding, S and Lee, S and Fremont, DH and Baldridge, MT}, title = {The neonatal Fc receptor is a cellular receptor for human astrovirus.}, journal = {Nature microbiology}, volume = {9}, number = {12}, pages = {3321-3331}, pmid = {39578577}, issn = {2058-5276}, support = {R01 AI141478/AI/NIAID NIH HHS/United States ; R01 AI139314/AI/NIAID NIH HHS/United States ; R01 AI127552/AI/NIAID NIH HHS/United States ; T32 DK007130/DK/NIDDK NIH HHS/United States ; R01AI181955//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; F30AI181285//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; T32DK007130//U.S. Department of Health &amp; Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes &amp; Digestive &amp; Kidney Diseases)/ ; U19 AI116484/AI/NIAID NIH HHS/United States ; P30 DK052574/DK/NIDDK NIH HHS/United States ; T32AI106688//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; P20 GM109035/GM/NIGMS NIH HHS/United States ; F30 AI181285/AI/NIAID NIH HHS/United States ; Pew Biomedical Scholars Program//Pew Charitable Trusts/ ; 75N93022C00035/AI/NIAID NIH HHS/United States ; T32DK077653//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; U19AI116484//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01AI139314//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; T32 DK077653/DK/NIDDK NIH HHS/United States ; T32 AI106688/AI/NIAID NIH HHS/United States ; Pathogenesis of Infectious Disease Program//Burroughs Wellcome Fund (BWF)/ ; R01 AI181955/AI/NIAID NIH HHS/United States ; R01AI141478//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01 AI150796/AI/NIAID NIH HHS/United States ; R01AI127552//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R00 AI141683/AI/NIAID NIH HHS/United States ; R01AI150796//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; }, mesh = {Humans ; *Receptors, Fc/metabolism/genetics ; *Histocompatibility Antigens Class I/metabolism/genetics ; *Mamastrovirus/metabolism/genetics/physiology ; *Virus Internalization ; *Dipeptidyl Peptidase 4/metabolism/genetics ; Astroviridae Infections/virology/metabolism ; CRISPR-Cas Systems ; Cell Line ; Receptors, Virus/metabolism ; HEK293 Cells ; }, abstract = {Human astroviruses (HAstV) are major causes of gastroenteritis, especially in children, and there are no vaccines or antivirals currently available. Little is known about host factors required for their cellular entry. Here we utilized complementary CRISPR-Cas9-based knockout and activation screens to identify neonatal Fc receptor (FcRn) and dipeptidyl-peptidase IV (DPP4) as entry factors for HAstV infection in vitro. Disruption of FcRn or DPP4 reduced HAstV infection in permissive cells and, reciprocally, overexpression of these factors in non-permissive cells was sufficient to promote infection. We observed direct binding of FcRn, but not DPP4, with HAstV virions and the purified spike protein. This suggests that FcRn is a receptor for HAstVs while DPP4 is a cofactor for entry. Inhibitors for DPP4 and FcRn currently in clinical use prevented HAstV infection in cell lines and human enteroids. Our results reveal mechanisms of HAstV entry as well as druggable targets to limit HAstV infection.}, }
@article {pmid39578174, year = {2025}, author = {Rosenberg-Mogilevsky, A and Siegfried, Z and Karni, R}, title = {Generation of tumor neoantigens by RNA splicing perturbation.}, journal = {Trends in cancer}, volume = {11}, number = {1}, pages = {12-24}, doi = {10.1016/j.trecan.2024.10.008}, pmid = {39578174}, issn = {2405-8025}, mesh = {Humans ; *Neoplasms/immunology/genetics/therapy ; *Antigens, Neoplasm/genetics/immunology ; *RNA Splicing/genetics/drug effects ; *Immunotherapy/methods ; Oligonucleotides, Antisense ; Nonsense Mediated mRNA Decay/drug effects ; RNA, Small Interfering/genetics ; RNA Splicing Factors/genetics ; Animals ; Alternative Splicing ; Mutation ; CRISPR-Cas Systems ; }, abstract = {Immunotherapy has revolutionized cancer treatment, but the limited availability of tumor-specific neoantigens still remains a challenge. The potential of alternative mRNA splicing-derived neoantigens as a source of new immunotherapy targets has gained significant attention. Tumors exhibit unique splicing changes and splicing factor mutations which are prevalent in various cancers and play a crucial role in neoantigen production. We present advances in splicing modulation approaches, including small-molecule drugs, decoy and splice-switching antisense oligonucleotides (SSOs), CRISPR, small interfering RNAs (siRNAs), and nonsense-mediated RNA decay (NMD) inhibition, that can be adapted to enhance antitumor immune responses. Finally, we explore the clinical implications of these approaches, highlighting their potential to transform cancer immunotherapy and broaden its efficacy.}, }
@article {pmid39578074, year = {2025}, author = {Ferdigg, A and Hopp, AK and Wolf, G and Superti-Furga, G}, title = {Membrane transporters modulating the toxicity of arsenic, cadmium, and mercury in human cells.}, journal = {Life science alliance}, volume = {8}, number = {2}, pages = {}, pmid = {39578074}, issn = {2575-1077}, mesh = {Humans ; *Cadmium/toxicity/metabolism ; *Arsenic/toxicity/metabolism ; *Mercury/toxicity/metabolism ; *Multidrug Resistance-Associated Proteins/metabolism/genetics ; Cation Transport Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Membrane Transport Proteins/metabolism/genetics ; Cell Line ; Zinc/metabolism ; HEK293 Cells ; }, abstract = {Non-essential metals are extremely toxic to living organisms, posing significant health risks, particularly in developing nations where they are a major contributor to illness and death. Although their toxicity is widely acknowledged, the mechanisms by which they are regulated within human cells remain incompletely understood. Specifically, the role of membrane transporters in mediating heavy metal toxicity is not well comprehended. Our study demonstrates how specific transporters can modulate the toxicity of cadmium, mercury, and the metalloid arsenic in human cells. Using CRISPR/Cas9 loss-of-function screens, we found that the multidrug resistance protein MRP1/ABCC1 provided protection against toxicity induced by arsenic and mercury. In addition, we found that SLC39A14 and SLC30A1 increased cellular sensitivity to cadmium. Using a reporter cell line to monitor cellular metal accumulation and performing a cDNA gain-of-function screen, we were able to clarify the function of SLC30A1 in controlling cadmium toxicity through the modulation of intracellular zinc levels. This transporter-wide approach provides new insights into the complex roles of membrane transporters in influencing the toxicity of arsenic, cadmium, and mercury in human cell lines.}, }
@article {pmid39577309, year = {2024}, author = {Tran, HD and Denman, CR and Shin, MK and Jeon, D and Kuhn, B and Jo, J}, title = {Establishment of TH-EGFP human embryonic stem cell line for specific labeling of dopaminergic neurons.}, journal = {Stem cell research}, volume = {81}, number = {}, pages = {103613}, doi = {10.1016/j.scr.2024.103613}, pmid = {39577309}, issn = {1876-7753}, mesh = {Humans ; *Dopaminergic Neurons/metabolism/cytology ; *Human Embryonic Stem Cells/metabolism/cytology ; *Green Fluorescent Proteins/metabolism/genetics ; Cell Differentiation ; Cell Line ; Tyrosine 3-Monooxygenase/metabolism/genetics ; CRISPR-Cas Systems ; }, abstract = {Dopaminergic (DA) neurons play critical roles in various neurological processes and disorders, particularly Parkinson's disease. To enable precise visualization and tracking of DA neurons, we generated TH-EGFP, a tyrosine hydroxylase (TH)-driven enhanced green fluorescent protein (EGFP)-expressing knock-in cell line, by employing CRISPR/Cas9 technology. We introduced EGFP into the targeted genomic region of human embryonic stem cells (hESCs) and successfully established a TH-EGFP hESC line. Differentiation of TH-EGFP hESCs into human midbrain organoids confirmed the accurate integration of EGFP into TH-positive cells. The TH-EGFP hESC line serves as a valuable reporter for studying the development, maturation, and function of DA neurons.}, }
@article {pmid39577180, year = {2025}, author = {Dai, F and Zhang, T and Pang, F and Jiao, T and Wang, K and Zhang, Z and Wang, N and Xie, Z and Zhang, Y and Wang, Z and Chen, Z and Yu, M and Wei, H and Song, J}, title = {A compact, palm-sized isothermal fluorescent diagnostic intelligent IoT device for personal health monitoring and beyond via one-tube/one-step LAMP-CRISPR assay.}, journal = {Biosensors &amp; bioelectronics}, volume = {270}, number = {}, pages = {116945}, doi = {10.1016/j.bios.2024.116945}, pmid = {39577180}, issn = {1873-4235}, mesh = {*Biosensing Techniques/instrumentation/methods ; *Nucleic Acid Amplification Techniques/instrumentation/methods ; Humans ; *COVID-19/diagnosis/virology ; *SARS-CoV-2/isolation &amp; purification/genetics ; Molecular Diagnostic Techniques/instrumentation ; CRISPR-Cas Systems ; Smartphone ; Animals ; Equipment Design ; Fluorescence ; }, abstract = {The demand for accurate, user-friendly, and sensitive at-home nucleic acid testing solutions is rising due to occasional outbreaks of various infectious diseases and a growing desire for an improved quality of life. In response, we developed the WeD-mini, a compact, palm-sized isothermal fluorescent diagnostic IoT device that weighs just 61 g. The WeD-mini features a uniquely designed, highly sensitive optical sensing system, ultra-low power consumption, a minimalist industrial design, and an intelligent operating algorithm. It integrates real-time fluorescence detection and automatic result interpretation via a smartphone, with results seamlessly uploaded to the 'EzDx Cloud' for comprehensive health management and spatio-temporal disease mapping. The device supports various assays that operate at different temperatures and with varying fluorescence emission intensities, such as RPA (39 °C, low intensity), LAMP (65 °C, high intensity), and LAMP-PfAgo (65/95 °C, high intensity), while maintaining precise temperature control and exceptional fluorescence detection sensitivity. Additionally, we engineered a more thermostable AapCRISPR-Cas12b variant that operates effectively at 63 °C, enhancing compatibility with LAMP to create a robust One-Tube/One-Step LAMP-CRISPR assay. Adaptable for at-home testing of SARS-CoV-2 and influenza viruses, the WeD-mini achieved 100% sensitivity and specificity with the newly established One-Tube/One-Step LAMP-CRISPR assay. Furthermore, the WeD-mini shows potential applications in detecting meat adulteration, monitoring respiratory diseases in pets, and conducting wastewater surveillance, making it suitable for a wide range of personal and public health use cases.}, }
@article {pmid39577179, year = {2025}, author = {Liu, C and Liu, Q and Chen, X and Guo, M and Chen, Z and Zhao, J and Chen, H and Guo, S and Cen, H and Yao, G and Chen, L and Wang, Y and Yang, PC and Wang, L and Chen, F}, title = {A novel label-free biosensor for myocardial ischemia biomarker detection via CRISPR/12a.}, journal = {Biosensors &amp; bioelectronics}, volume = {270}, number = {}, pages = {116954}, doi = {10.1016/j.bios.2024.116954}, pmid = {39577179}, issn = {1873-4235}, mesh = {*Biosensing Techniques/instrumentation/methods ; *Myocardial Ischemia/diagnosis/genetics ; Animals ; *Biomarkers ; Mice ; Humans ; *CRISPR-Cas Systems ; Adenosine Triphosphate/analysis ; Myocytes, Cardiac ; G-Quadruplexes ; }, abstract = {Myocardial infarction (MI) is the leading cause of death worldwide. Here, we present a novel, label-free biosensor for detecting myocardial ischemia biomarkers via CRISPR/Cas12a. This system utilizes the unique properties of CRISPR/Cas12a and G-quadruplex-ThT-based biosensors, enabling sensitive and specific detection of ATP, a crucial biomarker in cardiovascular diseases, at concentrations as low as 23 nM. Our method demonstrates substantial improvements over traditional ATP detection techniques, such as high-performance liquid chromatography and enzymatic assays, which often require complex sample preparation methods and costly equipment. The feasibility of the biosensor was further demonstrated in various models, including heart failure in mice and hypoxic conditions in cardiomyocytes. This successfully showcased its ability to function as a practical tool for diagnosing and monitoring diseases characterized by ATP dysregulation, highlighting its effectiveness in real-world clinical scenarios. This biosensor is notable for its rapid response, ease of use, and potential for integration into point-of-care diagnostics. These features offer significant advantages for the early diagnosis and management of ischemic heart disease and other conditions where ATP serves as a key metabolic biomarker. This technology also offers significant potential for early diagnosis and monitoring of myocardial ischemia and cardiovascular diagnostics. These findings underscore the biosensor's capacity for real-time ATP monitoring, offering crucial insights into mitochondrial function and disease progression, particularly in cardiovascular and inflammatory diseases.}, }
@article {pmid39577177, year = {2025}, author = {Zhuang, T and Gao, C and Zhao, W and Yu, H and Liu, Y and Zhang, N and Li, N and Ji, M}, title = {A minimal transcription template-based amplification-free CRISPR-Cas13a strategy for DNA detection.}, journal = {Biosensors &amp; bioelectronics}, volume = {270}, number = {}, pages = {116918}, doi = {10.1016/j.bios.2024.116918}, pmid = {39577177}, issn = {1873-4235}, mesh = {*CRISPR-Cas Systems ; Humans ; *Biosensing Techniques/methods ; DNA, Viral/genetics/analysis/isolation &amp; purification ; Adenoviruses, Human/genetics/isolation &amp; purification ; }, abstract = {CRISPR-Cas13a has shown great potential for the rapid and accurate detection of pathogen nucleic acids. However, conventional CRISPR-Cas13a-based assays typically require pre-amplification, which can introduce aerosol contamination and operational complexities. In this study, we developed a Minimalist transcription template-based Amplification-free CRISPR-Cas13a strategy for DNA detection (MAD). This strategy facilitates the release of pathogen DNA and its annealing with primers from nasopharyngeal swab samples in a straightforward manner, followed by T7 transcription and CRISPR-Cas13a detection, completing the entire process within 40 min. MAD eliminates the need for DNA extraction and pre-amplification while maintaining high sensitivity after optimization, allowing for result visualization via lateral flow strips. Furthermore, evaluation of 167 clinical pediatric samples identified 18 positive cases of human adenovirus, demonstrating a 99.4% concordance in detection compared to standard qPCR. We believe that MAD offers new insights into CRISPR-Cas diagnostics and, due to its simplicity, rapidity, and safety, is poised for widespread application in clinical practice.}, }
@article {pmid39576839, year = {2024}, author = {Harrison, EN and Jay, AN and Kent, MR and Sukienik, TP and LaVigne, CA and Kendall, GC}, title = {Engineering an fgfr4 knockout zebrafish to study its role in development and disease.}, journal = {PloS one}, volume = {19}, number = {11}, pages = {e0310100}, pmid = {39576839}, issn = {1932-6203}, support = {R01 CA272872/CA/NCI NIH HHS/United States ; T32 CA269052/CA/NCI NIH HHS/United States ; }, mesh = {Animals ; *Zebrafish/genetics ; *Receptor, Fibroblast Growth Factor, Type 4/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Knockout Techniques ; Zebrafish Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals, Genetically Modified ; }, abstract = {Fibroblast growth factor receptor 4 (FGFR4) has a role in many biological processes, including lipid metabolism, tissue repair, and vertebrate development. In recent years, FGFR4 overexpression and activating mutations have been associated with numerous adult and pediatric cancers. As such, FGFR4 presents an opportunity for therapeutic targeting which is being pursued in clinical trials. To understand the role of FGFR4 signaling in disease and development, we generated and characterized three alleles of fgfr4 knockout zebrafish strains using CRISPR/Cas9. To generate fgfr4 knockout crispants, we injected single-cell wildtype zebrafish embryos with fgfr4 targeting guide RNA and Cas9 proteins, identified adult founders, and outcrossed to wildtype zebrafish to create an F1 generation. The generated mutations introduce a stop codon within the second Ig-like domain of Fgfr4, resulting in a truncated 215, 223, or 228 amino acid Fgfr4 protein compared to 922 amino acids in the full-length protein. All mutant strains exhibited significantly decreased fgfr4 mRNA expression during development, providing evidence for successful knockout of fgfr4 in mutant zebrafish. We found that, consistent with other Fgfr4 knockout animal models, the fgfr4 mutant fish developed normally; however, homozygous fgfr4 mutant zebrafish were significantly smaller than wildtype fish at three months post fertilization. These fgfr4 knockout zebrafish lines are a valuable tool to study the role of FGFR4 in vertebrate development and its viability as a potential therapeutic target in pediatric and adult cancers, as well as other diseases.}, }
@article {pmid39576609, year = {2025}, author = {Fal, K and Carles, CC}, title = {dCas-Based Tools to Visualize Chromatin or Modify Epigenetic Marks at Specific Plant Genomic Loci.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2873}, number = {}, pages = {305-332}, pmid = {39576609}, issn = {1940-6029}, mesh = {*Chromatin/genetics/metabolism ; *Epigenesis, Genetic ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genome, Plant ; Gene Expression Regulation, Plant ; Plants/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genetic Loci ; Plants, Genetically Modified/genetics ; }, abstract = {Development of locus-specific approaches targeting precise regions on chromatin, for locus/transcription visualization or transcription/epigenetic marks editing, is a critical challenge in functional genetics and epigenetics. Systems engineered from the clustered regularly interspaced short palindromic repeats (CRISPR) and its associated endonuclease (Cas) operate through DNA sequence-specific recognition by so-called guide RNAs, which provides high flexibility and modularity for precise chromatin visualization or edition. Here, we provide an overview of the CRISPR/Cas-derived tools developed for visualization of chromatin loci in live imaging or for effective modification of gene expression. These tools make use of effector modules that combine activators, repressors, and epigenetic modifiers with a deactivated Cas protein (dCas). We present how their use in plants brought advances in visualizing or manipulating the expression of loci involved in agronomically interesting traits such as flowering time and response to drought or heat. We also discuss the limitations and future improvements of the dCas-related technologies, such as more compact and combinatorial systems, spatiotemporal targeting for fine-tuning of gene expression, and live visualization of chromatin dynamics.}, }
@article {pmid39576313, year = {2025}, author = {Teng, P and Gao, Z and Quan, Q and He, G and Song, Q and Zhang, X and Xiao, W and Zhao, J and Cao, D and Liang, J and Tang, Y}, title = {SERS-based CRISPR/Cas12a assays for protein biomarker prostate-specific antigen detection.}, journal = {Analytical and bioanalytical chemistry}, volume = {417}, number = {3}, pages = {573-582}, pmid = {39576313}, issn = {1618-2650}, support = {No. 2022B1111020003//Key-Area Research and Development Program of Guangdong Province/ ; No. 32301266//the National Natural Science Foundation of China/ ; 82372099//the National Natural Science Foundation of China/ ; No. 202201011351//the Technology Research Program of Guangzhou City/ ; 2023A03J0283//the Technology Research Program of Guangzhou City/ ; 2024A03J1076//the Technology Research Program of Guangzhou City/ ; No. 2023CXB014//Outstanding Innovative Talents Cultivation Funded Programs for Doctoral Students of Jinan University/ ; No. 2022A1515110772//GuangDong Basic and Applied Basic Research Foundation/ ; 2021A1515220067//GuangDong Basic and Applied Basic Research Foundation/ ; 2021A1515110//GuangDong Basic and Applied Basic Research Foundation/ ; }, mesh = {*Prostate-Specific Antigen/blood/analysis ; *Spectrum Analysis, Raman/methods ; Humans ; *Limit of Detection ; Male ; CRISPR-Cas Systems ; Enzyme-Linked Immunosorbent Assay/methods ; Biomarkers/blood/analysis ; Biosensing Techniques/methods ; Biomarkers, Tumor/blood/analysis ; }, abstract = {Sensitive and accurate detection of protein biomarkers is crucial for disease diagnosis, especially for early diagnosis. Here, we describe surface-enhanced Raman scattering (SERS)-based CRISPR/Cas12a assays (S-CRISPR) for protein biomarker detection. Firstly, an S-CRISPR-driven enzyme-linked immunosorbent assay (S-CasLISA) was developed utilizing a capture antibody coated on a microplate to recognize the target and a detection antibody labeled with active DNA to trigger the activity of CRISPR/Cas12a. With this assay, we achieved detection of prostate-specific antigen (PSA) as models at the picogram level. The limit of detection (LoD) of S-CasLISA was 0.17 pg mL[-1] and in the range of 0.1 pg mL[-1] to 10 ng mL[-1]. Further, we applied aptamer to S-CRISPR (S-Apt-CRISPR), combining the high sensitivity of SERS with the high selectivity of aptamers, while simplifying the operation process of CRISPR detection of protein biomarkers. The proposed S-Apt-CRISPR also could detect picogram-level PSA and without repeated washing steps. The LoD of S-Apt-CRISPR was 0.35 pg mL[-1] and in the range of 0.1 pg mL[-1] to 10 ng mL[-1]. Both SERS-based CRISPR/Cas12a assays were validated with clinical samples and demonstrated accuracy consistent with the chemiluminescence immunoassay. The introduction of the CRISPR/Cas12a system with SERS has the effect of improving the analytical capabilities of the system, thereby broadening and facilitating its application in the analysis of sensitive and accurate protein biomarkers.}, }
@article {pmid39576177, year = {2024}, author = {Samudra, SP and Park, S and Esser, EA and McDonald, TP and Borges, AM and Eggenschwiler, J and Menke, DB}, title = {A new cell culture resource for investigations of reptilian gene function.}, journal = {Development (Cambridge, England)}, volume = {151}, number = {22}, pages = {}, pmid = {39576177}, issn = {1477-9129}, support = {R01 HG013006/HG/NHGRI NIH HHS/United States ; 1827647//National Science Foundation/ ; R01HG013006/NH/NIH HHS/United States ; //University of Georgia/ ; }, mesh = {Animals ; *Hedgehog Proteins/metabolism/genetics ; *Lizards/genetics ; Mice ; *Cilia/metabolism/genetics ; *Signal Transduction/genetics ; CRISPR-Cas Systems/genetics ; Cell Line ; Fibroblasts/metabolism/cytology ; Limb Buds/metabolism/cytology/embryology ; Cell Culture Techniques/methods ; Zinc Finger Protein GLI1/metabolism/genetics ; Gene Editing/methods ; }, abstract = {The establishment of CRISPR/Cas9 gene editing in Anolis sagrei has positioned this species as a powerful model for studies of reptilian gene function. To enhance this model, we developed an immortalized lizard fibroblast cell line (ASEC-1) for the exploration of reptilian gene function in cellular processes. We demonstrate the use of this cell line by scrutinizing the role of primary cilia in lizard Hedgehog (Hh) signaling. Using CRISPR/Cas9 mutagenesis, we disrupted the ift88 gene, which is required for ciliogenesis in diverse organisms. We determined that loss of itf88 from lizard cells leads to an absence of primary cilia, a partial derepression of gli1 transcription, and an inability of the cells to respond to the Smoothened agonist, SAG. Through a cross-species analysis of SAG-induced transcriptional responses in cultured limb bud cells, we further determined that ∼46% of genes induced as a response to Hh pathway activation in A. sagrei are also SAG responsive in Mus musculus limb bud cells. Our results highlight conserved and diverged aspects of Hh signaling in anoles and establish a new resource for investigations of reptilian gene function.}, }
@article {pmid39576014, year = {2024}, author = {Saito, S and Nakamura, Y and Miyashita, S and Sato, T and Hoshina, K and Okada, M and Hasegawa, H and Oishi, M and Fujii, Y and Körbelin, J and Kubota, Y and Tainaka, K and Natsumeda, M and Ueno, M}, title = {CRISPR/CasRx suppresses KRAS-induced brain arteriovenous malformation developed in postnatal brain endothelial cells in mice.}, journal = {JCI insight}, volume = {9}, number = {22}, pages = {}, pmid = {39576014}, issn = {2379-3708}, mesh = {Animals ; Mice ; *Proto-Oncogene Proteins p21(ras)/genetics/metabolism ; *Endothelial Cells/metabolism ; *CRISPR-Cas Systems ; *Intracranial Arteriovenous Malformations/genetics/pathology/metabolism ; *Disease Models, Animal ; Brain/blood supply/pathology/metabolism ; Humans ; Mutation ; }, abstract = {Brain arteriovenous malformations (bAVMs) are anomalies forming vascular tangles connecting the arteries and veins, which cause hemorrhagic stroke in young adults. Current surgical approaches are highly invasive, and alternative therapeutic methods are warranted. Recent genetic studies identified KRAS mutations in endothelial cells of bAVMs; however, the underlying process leading to malformation in the postnatal stage remains unknown. Here we established a mouse model of bAVM developing during the early postnatal stage. Among 4 methods tested, mutant KRAS specifically introduced in brain endothelial cells by brain endothelial cell-directed adeno-associated virus (AAV) and endothelial cell-specific Cdh5-CreERT2 mice successfully induced bAVMs in the postnatal period. Mutant KRAS led to the development of multiple vascular tangles and hemorrhage in the brain with increased MAPK/ERK signaling and growth in endothelial cells. Three-dimensional analyses in cleared tissue revealed dilated vascular networks connecting arteries and veins, similar to human bAVMs. Single-cell RNA-Seq revealed dysregulated gene expressions in endothelial cells and multiple cell types involved in the pathological process. Finally, we employed CRISPR/CasRx to knock down mutant KRAS expression, which efficiently suppressed bAVM development. The present model reveals pathological processes that lead to postnatal bAVMs and demonstrates the efficacy of therapeutic strategies with CRISPR/CasRx.}, }
@article {pmid39575850, year = {2024}, author = {Zhong, J and Wu, X and Guo, C and Liu, C and Zhang, Q and Chen, Y and Liu, Y}, title = {A Single-Tube, Single-Enzyme Clustered Regularly Interspaced Short Palindromic Repeats System (UNISON) with Internal Controls for Accurate Nucleic Acid Detection.}, journal = {Analytical chemistry}, volume = {96}, number = {49}, pages = {19348-19353}, doi = {10.1021/acs.analchem.4c03403}, pmid = {39575850}, issn = {1520-6882}, mesh = {*CRISPR-Cas Systems/genetics ; *Hepatitis B virus/genetics ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins have been widely applied in molecular diagnostics. Unlike the Ct value quantification method of PCR, the CRISPR system mainly relies on the rise of the rate of the fluorescence signal to indicate the concentration of the target nucleic acid, which is susceptible to system errors caused by various factors, such as reaction conditions and instrument performance. Therefore, establishing internal controls is essential to improve the accuracy, reliability, and commercial feasibility of the CRISPR system. However, the nonspecific trans-cleavage activity of Cas proteins presents a challenge in establishing internal controls. In this study, we developed unified nucleic acid detection with a single-tube, one-enzyme system (UNISON) for accurate nucleic acid detection with internal controls. By extending the crRNA and modifying it with different fluorophores and quenchers, we achieved that the specific target can only specifically cleave the corresponding folded crRNA and generate a corresponding fluorescence signal. With this design, we established an internal control, achieving accurate and reliable detection of clinical samples of the hepatitis B virus. Integrating internal controls into the CRISPR/Cas system demonstrates significant potential in medical diagnostics and virus monitoring.}, }
@article {pmid39575683, year = {2024}, author = {Siniscalco, AM and Perera, RP and Greenslade, JE and Veeravenkatasubramanian, H and Masters, A and Doll, HM and Raj, B}, title = {Barcoding Notch signaling in the developing brain.}, journal = {Development (Cambridge, England)}, volume = {151}, number = {24}, pages = {}, pmid = {39575683}, issn = {1477-9129}, support = {R00 HD098298/HD/NICHD NIH HHS/United States ; DGE-2236662//National Science Foundation/ ; //University of Pennsylvania/ ; DP2 NS131787/NS/NINDS NIH HHS/United States ; R00HD098298/NH/NIH HHS/United States ; }, mesh = {Animals ; *Zebrafish/genetics/embryology/metabolism ; *Brain/metabolism/embryology/growth &amp; development ; *Signal Transduction/genetics ; *Receptors, Notch/metabolism/genetics ; *Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Neurons/metabolism/cytology ; Gene Expression Regulation, Developmental ; Single-Cell Analysis ; }, abstract = {Developmental signaling inputs are fundamental for shaping cell fates and behavior. However, traditional fluorescent-based signaling reporters have limitations in scalability and molecular resolution of cell types. We present SABER-seq, a CRISPR-Cas molecular recorder that stores transient developmental signaling cues as permanent mutations in cellular genomes for deconstruction at later stages via single-cell transcriptomics. We applied SABER-seq to record Notch signaling in developing zebrafish brains. SABER-seq has two components: a signaling sensor and a barcode recorder. The sensor activates Cas9 in a Notch-dependent manner with inducible control, while the recorder obtains mutations in ancestral cells where Notch is active. We combine SABER-seq with an expanded juvenile brain atlas to identify cell types derived from Notch-active founders. Our data reveal rare examples where differential Notch activities in ancestral progenitors are detected in terminally differentiated neuronal subtypes. SABER-seq is a novel platform for rapid, scalable and high-resolution mapping of signaling activity during development.}, }
@article {pmid39574587, year = {2024}, author = {Kushawah, G and Amaral, DB and Hassan, H and Gogol, M and Nowotarski, SH and Bazzini, AA}, title = {Critical role of Spatio-Temporally Regulated Maternal RNAs in Zebrafish Embryogenesis.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, pmid = {39574587}, issn = {2692-8205}, support = {R01 GM136849/GM/NIGMS NIH HHS/United States ; R21 OD034161/OD/NIH HHS/United States ; }, abstract = {The maternal-to-zygotic transition shifts regulatory control from maternal to zygotic messenger RNAs (mRNA) through maternal mRNA degradation. While temporal aspects of maternal mRNA decay are known, spatial mechanisms remain underexplored. Using CRISPR-Cas9 and CRISPR-Cas13d systems, we functionally dissected the contribution of maternal versus zygotic fractions and overcame challenges of studying embryonic lethal genes. We identified differentially distributed maternal mRNAs in specific cells and evidenced the critical role of five maternal mRNAs, cth1, arl4d, abi1b, foxa and lhx1a, in embryogenesis. Further, we focused on the functionally uncharacterized cth1 gene, revealing its essential role in gametogenesis and embryogenesis. Cth1 acts as a spatio-temporal RNA decay factor regulating mRNA stability and accumulation of its targets in a spatio-temporal manner through 3'UTR recognition during early development. Furthermore, Cth1 3'UTR drives its spatio-temporal RNA localization. Our findings provide new insights into spatio-temporal RNA decay mechanisms and highlight dual CRISPR-Cas strategies in studying embryonic development.}, }
@article {pmid39574297, year = {2025}, author = {Zhang, Y and Chen, J and Wu, Z and Zhao, C and Wang, R and Li, Z and Wang, J and Wang, D}, title = {CRISPR/Cas Enzyme Catalysis in Liquid-Liquid Phase-Separated Systems.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {12}, number = {3}, pages = {e2407194}, pmid = {39574297}, issn = {2198-3844}, support = {20230101356JC//Outstanding Youth Foundation of Jilin Province, China/ ; 2024A1515011281//Guangdong Basic and Applied Basic Research/ ; JJKH20241346KJ//Scientific Research project of Education Department of Jilin Province/ ; }, mesh = {*CRISPR-Cas Systems/genetics ; Catalysis ; *CRISPR-Associated Proteins/metabolism/genetics ; Gene Editing/methods ; }, abstract = {The clustered regularly interspaced palindromic repeats (CRISPR) /CRISPR-associated proteins (Cas) system is the immune system in bacteria and archaea and has been extensively applied as a critical tool in bioengineering. Investigation of the mechanisms of catalysis of CRISPR/Cas systems in intracellular environments is essential for understanding the underlying catalytic mechanisms and advancing CRISPR-based technologies. Here, the catalysis mechanisms of CRISPR/Cas systems are investigated in an aqueous two-phase system (ATPS) comprising PEG and dextran, which simulated the intracellular environment. The findings revealed that nucleic acids and proteins tended to be distributed in the dextran-rich phase. The results demonstrated that the cis-cleavage activity of Cas12a is enhanced in the ATPS, while its trans-cleavage activity is suppressed, and this finding is further validated using Cas13a. Further analysis by increasing the concentration of the DNA reporter revealed that this phenomenon is not attributed to the slow diffusion of the reporter, and explained why Cas12a and Cas13a do not randomly cleave nucleic acids in the intracellular compartment. The study provides novel insights into the catalytic mechanisms of CRISPR/Cas systems under physiological conditions and may contribute to the development of CRISPR-based molecular biological tools.}, }
@article {pmid39572140, year = {2024}, author = {Chen, X and Stafford, DW and Tie, JK}, title = {Assessment of gamma-glutamyl carboxylase activity in its native milieu.}, journal = {Methods in enzymology}, volume = {708}, number = {}, pages = {207-236}, doi = {10.1016/bs.mie.2024.10.011}, pmid = {39572140}, issn = {1557-7988}, mesh = {Humans ; *Carbon-Carbon Ligases/metabolism/genetics ; *Vitamin K/metabolism ; Animals ; CRISPR-Cas Systems ; Enzyme Assays/methods ; HEK293 Cells ; Gene Knockout Techniques/methods ; }, abstract = {Gamma-glutamyl carboxylase (GGCX), a polytopic membrane protein found in the endoplasmic reticulum, catalyzes the posttranslational modification of a variety of vitamin K-dependent (VKD) proteins to their functional forms. GGCX uses the free energy from the oxygenation of reduced vitamin K to remove the proton from the glutamate residue to drive VKD carboxylation. During the process of carboxylation, reduced vitamin K is oxidized to vitamin K epoxide. Therefore, GGCX is a dual-function enzyme that possesses both glutamate carboxylation and vitamin K epoxidation activities. Genetic variations in GGCX are mainly associated with bleeding disorders referred to as combined VKD coagulation factors deficiency. Comorbid non-bleeding phenotypes are also observed in patients carrying GGCX mutations. Our current knowledge concerning GGCX's function has been obtained mainly from in vitro experimentation under artificial conditions, which limits its use in interpreting the clinical phenotypes associated with GGCX genotypes. In this chapter, we describe the background, establishment, and application of mammalian cell-based assays for both the carboxylation and epoxidation activities of GGCX. We provide detailed procedures for making the reporter cell lines, creating CRISPR-Cas9-mediated gene-knockout reporter cell lines, and using these cell lines for functional studies of GGCX and its naturally occurring mutations. Combined with different reporter proteins, this cell-based strategy has been successfully used for the functional study of vitamin K-related enzymes, high-throughput screening of VKD carboxylation inhibitors, and genome-wide CRISPR-Cas9 knockout library screening of the unknown enzymes associated with vitamin K reduction.}, }
@article {pmid39571855, year = {2024}, author = {Wan, Y and Huang, C and Feng, D and Wang, L and Lin, X and Zhao, X and Han, L and Zhu, Y and Hao, L and Du, H and Huang, L}, title = {Characterizing the collateral activity of CRISPR/Cas13 in mammalian cells: Implications for RNA editing and therapeutic applications.}, journal = {International journal of biological macromolecules}, volume = {283}, number = {Pt 4}, pages = {137861}, doi = {10.1016/j.ijbiomac.2024.137861}, pmid = {39571855}, issn = {1879-0003}, mesh = {Humans ; *RNA Editing/genetics ; *CRISPR-Cas Systems ; Animals ; Swine ; Cell Proliferation ; HEK293 Cells ; Cell Survival ; Embryonic Development/genetics ; }, abstract = {The CRISPR/Cas13 system has garnered attention as a potential tool for RNA editing. However, the degree of collateral activity among various Cas13 orthologs and their cytotoxic effects in mammalian cells remain contentious, potentially impacting their applications. In this study, we observed differential collateral activities for LwaCas13a and RfxCas13d in 293T and U87 cells by applying both sensitive dual-fluorescence (mRuby/GFP) reporter and quantifiable dual-luciferase (Fluc/Rluc) reporter, with LwaCas13a displaying notable activity contrary to previous reports. However, significant collateral RNA cleavage exerted only a modest impact on cell viability. Furthermore, collateral activity of LwaCas13a mildly impeded, but did not arrest, porcine embryo development. Our findings reveal that distinct collateral RNA cleavage by Cas13 slightly suppresses mammalian cell proliferation and embryo development. This could account for the lack of reported collateral effects in numerous prior studies and offers new insights into the implications of the collateral activity of Cas13 for clinical application.}, }
@article {pmid39571575, year = {2024}, author = {Wang, Y and Zhang, Y and Kim, K and Han, J and Okin, D and Jiang, Z and Yang, L and Subramaniam, A and Means, TK and Nestlé, FO and Fitzgerald, KA and Randolph, GJ and Lesser, CF and Kagan, JC and Mathis, D and Benoist, C}, title = {A pan-family screen of nuclear receptors in immunocytes reveals ligand-dependent inflammasome control.}, journal = {Immunity}, volume = {57}, number = {12}, pages = {2737-2754.e12}, pmid = {39571575}, issn = {1097-4180}, support = {R01 AI150686/AI/NIAID NIH HHS/United States ; T32 AI007061/AI/NIAID NIH HHS/United States ; R37 AI049653/AI/NIAID NIH HHS/United States ; R01 AI169795/AI/NIAID NIH HHS/United States ; R37 AI116550/AI/NIAID NIH HHS/United States ; P30 DK034854/DK/NIDDK NIH HHS/United States ; T32 HL116275/HL/NHLBI NIH HHS/United States ; K00 CA264434/CA/NCI NIH HHS/United States ; R01 AI116550/AI/NIAID NIH HHS/United States ; R01 AI167993/AI/NIAID NIH HHS/United States ; }, mesh = {Animals ; *Inflammasomes/metabolism/immunology ; Mice ; *Receptors, Cytoplasmic and Nuclear/metabolism ; Cell Differentiation ; Mice, Inbred C57BL ; Ligands ; Retinoic Acid Receptor gamma ; Macrophages, Peritoneal/immunology/metabolism ; Mice, Knockout ; Pyroptosis/immunology ; Receptors, Retinoic Acid/metabolism ; CARD Signaling Adaptor Proteins/metabolism ; GATA6 Transcription Factor/metabolism ; Retinoid X Receptor alpha/metabolism ; CRISPR-Cas Systems ; }, abstract = {Ligand-dependent transcription factors of the nuclear receptor (NR) family regulate diverse aspects of metazoan biology, enabling communications between distant organs via small lipophilic molecules. Here, we examined the impact of each of 35 NRs on differentiation and homeostatic maintenance of all major immunological cell types in vivo through a "Rainbow-CRISPR" screen. Receptors for retinoic acid exerted the most frequent cell-specific roles. NR requirements varied for resident macrophages of different tissues. Deletion of either Rxra or Rarg reduced frequencies of GATA6[+] large peritoneal macrophages (LPMs). Retinoid X receptor alpha (RXRα) functioned conventionally by orchestrating LPM differentiation through chromatin and transcriptional regulation, whereas retinoic acid receptor gamma (RARγ) controlled LPM survival by regulating pyroptosis via association with the inflammasome adaptor ASC. RARγ antagonists activated caspases, and RARγ agonists inhibited cell death induced by several inflammasome activators. Our findings provide a broad view of NR function in the immune system and reveal a noncanonical role for a retinoid receptor in modulating inflammasome pathways.}, }
@article {pmid39571094, year = {2025}, author = {Waite, EL and Tigue, M and Yu, M and Lahori, D and Kelly, K and May, CL and Naji, A and Roman, J and Doliba, N and Avrahami, D and Nguyen-Ngoc, KV and Sander, M and Glaser, B and Kaestner, KH}, title = {The IsletTester Mouse: An Immunodeficient Model With Stable Hyperglycemia for the Study of Human Islets.}, journal = {Diabetes}, volume = {74}, number = {3}, pages = {332-342}, pmid = {39571094}, issn = {1939-327X}, support = {UM1 DK126194/DK/NIDDK NIH HHS/United States ; UG3 DK122639/DK/NIDDK NIH HHS/United States ; 1782/18//Israel Science Foundation/ ; 5UG3DK122639/DK/NIDDK NIH HHS/United States ; U01 DK123594/DK/NIDDK NIH HHS/United States ; U01 DK134995/DK/NIDDK NIH HHS/United States ; P30 DK050306/DK/NIDDK NIH HHS/United States ; 2019314//United States-Israel Binational Science Foundation/ ; }, mesh = {Animals ; Humans ; *Hyperglycemia/metabolism/genetics ; Mice ; *Islets of Langerhans Transplantation ; *Islets of Langerhans/metabolism ; Disease Models, Animal ; Glucokinase/genetics/metabolism ; CRISPR-Cas Systems ; *Insulin-Secreting Cells/metabolism ; Gene Editing ; Mice, Inbred NOD ; Female ; }, abstract = {UNLABELLED: The gold standard for assessing the function of human islets or β-like cells derived from stem cells involves their engraftment under the kidney capsule of hyperglycemic, immunodeficient mice. Current models, such as streptozotocin treatment of severely immunodeficient mice or the NRG-Akita strain, are limited due to unstable and variable hyperglycemia and/or high morbidity. To address these limitations, we developed the IsletTester mouse via CRISPR/Cas9-mediated gene editing of glucokinase (Gck), the glucose sensor of the β-cells, directly in NSG zygotes. IsletTester mice are heterozygous for an Arg345→stop mutation in Gck and present with stable random hyperglycemia (∼250 mg/dL [14 mmol/L]), normal lifespan, and fertility. We demonstrate the utility of this model through functional engraftment of both human islets and human embryonic stem cell-derived β-like cells. The IsletTester mouse will enable the study of human islet biology over time and under different physiological conditions and can provide a useful preclinical platform to determine the functionality of stem cell-derived islet products.<br><br>ARTICLE HIGHLIGHTS: Current mouse models for assessing islet function in&#xa0;vivo are limited due to unstable and variable hyperglycemia and/or high morbidity. We derived the IsletTester mouse to address these limitations. Leveraging a previously characterized glucokinase mutation and CRISPR/Cas9 technology, we successfully developed a moderately hyperglycemic and immunodeficient mouse model for the in&#xa0;vivo assessment of islet function. Our IsletTester mouse has stable, moderate hyperglycemia that can be corrected with primary human islets or stem cell-derived insulin-producing cells. The IsletTester mouse provides a reliable, easy-to-use platform for the preclinical assessment of stem cell-derived islet products or islet-targeted drugs.}, }
@article {pmid39570677, year = {2024}, author = {Raddaoui, A and Chebbi, Y and Frigui, S and Ammeri, RW and Ben Abdejlil, N and Abbassi, MS and Achour, W}, title = {Deciphering the Resistome and Mobiolme of an Avian-Associated Enterococus faecalis ST249 Clone that Acquired Vancomycin Resistance Isolated from Neutropenic Patient in Tunisia.}, journal = {Microbial drug resistance (Larchmont, N.Y.)}, volume = {30}, number = {12}, pages = {481-488}, doi = {10.1089/mdr.2024.0144}, pmid = {39570677}, issn = {1931-8448}, mesh = {Humans ; *Enterococcus faecalis/drug effects/genetics/isolation &amp; purification ; Tunisia ; *Anti-Bacterial Agents/pharmacology ; *Microbial Sensitivity Tests ; *Neutropenia/microbiology ; Animals ; Child ; *Vancomycin/pharmacology/therapeutic use ; Whole Genome Sequencing ; Vancomycin Resistance/genetics ; Plasmids/genetics ; Gram-Positive Bacterial Infections/drug therapy/microbiology ; DNA Transposable Elements ; Bacterial Proteins/genetics ; Vancomycin-Resistant Enterococci/genetics/drug effects/isolation &amp; purification ; }, abstract = {This study aimed to characterize the first vancomycin-resistant Enterococcus faecalis (VREfs) isolate from patient with neutropenic in Tunisia by whole-genome sequencing (WGS). This strain was detected from routine rectal swab from an 8-year-old child with bone marrow aplasia, residing in a rural area, on September 20, 2021. The strain was isolated after 12 days of hospitalization at the National Bone Marrow Transplant Center. Minimum Inhibitory Concentrations of vancomycin and teicoplanin were >256 and 16 mg/L, respectively. WGS revealed that the strain belonged to the ST249 clone, exclusively reported in avian (poultry and ducks) vancomycin-susceptible E. faecalis isolates in six studies from four countries, primarily Denmark. The vanA gene was carried by the Tn1546 transposon mobilized by a pTW9-like plasmid. The ardA gene, a CRISPR-Cas system neutralization factor, was detected in this strain. In summary, this is the first report of avian-associated E. faecalis ST249 in clinical samples. Initially vancomycin susceptible, the strain acquired a pTW9-like plasmid carrying the classical vanA-Tn1546 transposon. This acquisition was facilitated by the sex pheromone-response mechanisms and the ardA gene and CRISPR-Cas system neutralization.}, }
@article {pmid39570312, year = {2024}, author = {Iturralde, AB and Weller, CA and Giovanetti, SM and Sadhu, MJ}, title = {Comprehensive deletion scan of anti-CRISPR AcrIIA4 reveals essential and dispensable domains for Cas9 inhibition.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {121}, number = {48}, pages = {e2413743121}, pmid = {39570312}, issn = {1091-6490}, support = {ZIA HG200401/ImNIH/Intramural NIH HHS/United States ; 1ZIAHG200401//HHS | NIH | National Human Genome Research Institute (NHGRI)/ ; }, mesh = {*CRISPR-Associated Protein 9/metabolism/genetics ; *CRISPR-Cas Systems ; Sequence Deletion ; Protein Domains ; Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; }, abstract = {Delineating a protein's essential and dispensable domains provides critical insight into how it carries out its function. Here, we developed a high-throughput method to synthesize and test the functionality of all possible in-frame and continuous deletions in a gene of interest, enabling rapid and unbiased determination of protein domain importance. Our approach generates precise deletions using a CRISPR library framework that is free from constraints of gRNA target site availability and efficacy. We applied our method to AcrIIA4, a phage-encoded anti-CRISPR protein that robustly inhibits SpCas9. Extensive structural characterization has shown that AcrIIA4 physically occupies the DNA-binding interfaces of several SpCas9 domains; nonetheless, the importance of each AcrIIA4 interaction for SpCas9 inhibition is unknown. We used our approach to determine the essential and dispensable regions of AcrIIA4. Surprisingly, not all contacts with SpCas9 were required, and in particular, we found that the AcrIIA4 loop that inserts into SpCas9's RuvC catalytic domain can be deleted. Our results show that AcrIIA4 inhibits SpCas9 primarily by blocking PAM binding and that its interaction with the SpCas9 catalytic domain is inessential.}, }
@article {pmid39570287, year = {2025}, author = {Su, KC and Radul, E and Maier, NK and Tsang, MJ and Goul, C and Moodie, B and Marescal, O and Keys, HR and Cheeseman, IM}, title = {Functional genetics reveals modulators of antimicrotubule drug sensitivity.}, journal = {The Journal of cell biology}, volume = {224}, number = {2}, pages = {}, pmid = {39570287}, issn = {1540-8140}, support = {R35 GM126930/GM/NIGMS NIH HHS/United States ; //Massachusetts Institute of Technology Office of Graduate Education/ ; HFCR-18-03-02//Hope Funds for Cancer Research/ ; //G. Harold &amp; Leila Y. Mathers Charitable Foundation/ ; /NH/NIH HHS/United States ; R35GM126930/GM/NIGMS NIH HHS/United States ; }, mesh = {Humans ; *Microtubules/metabolism/drug effects/genetics ; *Paclitaxel/pharmacology ; *Tubulin Modulators/pharmacology ; *Nocodazole/pharmacology ; *CRISPR-Cas Systems ; *Kinesins/genetics/metabolism ; Microtubule-Associated Proteins/metabolism/genetics ; }, abstract = {Microtubules play essential roles in diverse cellular processes and are important pharmacological targets for treating human disease. Here, we sought to identify cellular factors that modulate the sensitivity of cells to antimicrotubule drugs. We conducted a genome-wide CRISPR/Cas9-based functional genetics screen in human cells treated with the microtubule-destabilizing drug nocodazole or the microtubule-stabilizing drug paclitaxel. We further conducted a focused secondary screen to test drug sensitivity for ∼1,400 gene targets across two distinct human cell lines and to additionally test sensitivity to the KIF11 inhibitor, STLC. These screens defined gene targets whose loss enhances or suppresses sensitivity to antimicrotubule drugs. In addition to gene targets whose loss sensitized cells to multiple compounds, we observed cases of differential sensitivity to specific compounds and differing requirements between cell lines. Our downstream molecular analysis further revealed additional roles for established microtubule-associated proteins and identified new players in microtubule function.}, }
@article {pmid39570020, year = {2025}, author = {Yin, L and Zhao, Z and Wang, C and Zhou, C and Wu, X and Gao, B and Wang, L and Man, S and Cheng, X and Wu, Q and Hu, S and Fan, H and Ma, L and Xing, H and Shen, L}, title = {Development and evaluation of a CRISPR/Cas12a-based diagnostic test for rapid detection and genotyping of HR-HPV in clinical specimens.}, journal = {Microbiology spectrum}, volume = {13}, number = {1}, pages = {e0225324}, pmid = {39570020}, issn = {2165-0497}, support = {32302218//MOST | National Natural Science Foundation of China (NSFC)/ ; (2022YL05B,2022YL12A,2022YL07B//Science and Technology Plan (in the field of Medical and health care) of Xiangyang/ ; 82002192//MOST | National Natural Science Foundation of China (NSFC)/ ; 24YDTPJC00800//Tianjin Science and technology planning project/ ; 21JCQNJC01410//Tianjin Municipal Science and Technology Committee (Tianjin Municipal Science and Technology Commission)/ ; 2021M702460//China Postdoctoral Science Foundation (China Postdoctoral Foundation Project)/ ; 2022KF0203//Liquor Making Biological Technology and Application of Key Laboratory of Sichuan Province Microbiology/ ; 2022CFB539,2022CFD107//General Project of Natural Science Foundation of Hubei Province/ ; Q20222605//Young and middle-aged Talents Project of Hubei Provincial Education Department/ ; 2021kpgj06//Scientific Research Ability Cultivation Fund of Hubei University of Arts and Science/ ; }, mesh = {Humans ; *Papillomavirus Infections/diagnosis/virology ; *CRISPR-Cas Systems ; Female ; *Sensitivity and Specificity ; *Genotype ; *Papillomaviridae/genetics/classification/isolation &amp; purification ; *Nucleic Acid Amplification Techniques/methods ; Uterine Cervical Neoplasms/diagnosis/virology ; Molecular Diagnostic Techniques/methods ; Genotyping Techniques/methods ; DNA, Viral/genetics ; Diagnostic Tests, Routine/methods ; }, abstract = {Persistent infection with high-risk human papillomavirus (HR-HPV) is the principal etiological factor of cervical cancer. Considering the gradual progression of cervical cancer, the early, rapid, sensitive, and specific identification of HPV, particularly HR-HPV types, is crucial in halting the advancement of the illness. Here, we established a rapid, highly sensitive, and specific HR-HPV detection platform, leveraging the CRISPR/Cas12a assay in conjunction with multienzyme isothermal rapid amplification. Our platform enables the detection and genotyping of 14 types of HR-HPV by using type-specific crRNAs. The outcomes of the detection can be interpreted either through a fluorescence reader or visually. Furthermore, we achieved one-tube multiplex detection of 14 HR-HPV types through the use of multiple amplifications and a crRNA pool. The detection sensitivity of this method is 2 copies/μL with no cross-reactivity, and the results can be obtained within 30 minutes. This method exhibited 100% clinical sensitivity and 100% clinical specificity when applied to 258 clinical specimens. Based on these findings, our CRISPR/Cas-based HR-HPV detection platform holds promise as a novel clinical detection tool, offering a visually intuitive and expedited alternative to existing HPV infection diagnostics and providing fresh perspectives for clinical cervical cancer screening.IMPORTANCEThis study developed a novel high-risk human papillomavirus (HR-HPV) detection platform based on CRISPR/Cas12a technology. This platform not only enables the rapid, highly sensitive, and specific detection and genotyping of 14 types of HR-HPV but also achieves single-tube multiplex detection of 14 HR-HPV types through ingenious design. The outcomes of the detection can be interpreted either through a fluorescence reader or visually. To the best of our knowledge, this is the first paper to utilize CRISPR/Cas diagnostic technology for the simultaneous detection of 14 types of HPV and to evaluate its feasibility in clinical sample detection using a large number of clinical samples. We hope that this work will facilitate the rapid and accurate detection of HPV and promote the broader application of CRISPR/Cas diagnostic technology.}, }
@article {pmid39569987, year = {2024}, author = {Yamamoto, S and Afifi, OA and Lam, LPY and Takeda-Kimura, Y and Osakabe, Y and Osakabe, K and Bartley, LE and Umezawa, T and Tobimatsu, Y}, title = {Disruption of aldehyde dehydrogenase decreases cell wall-bound p-hydroxycinnamates and improves cell wall digestibility in rice.}, journal = {The Plant journal : for cell and molecular biology}, volume = {120}, number = {6}, pages = {2828-2845}, doi = {10.1111/tpj.17148}, pmid = {39569987}, issn = {1365-313X}, support = {Exploratory Research on Humanosphere Science 2021//Research Institute for Sustainable Humanosphere, Kyoto University/ ; Exploratory Research on Humanosphere Science 2023//Research Institute for Sustainable Humanosphere, Kyoto University/ ; DE-SC0021126//Office of Science/ ; #20H03044//Japan Society for the Promotion of Science/ ; #22J13457//Japan Society for the Promotion of Science/ ; #24K01827//Japan Society for the Promotion of Science/ ; #1015621//National Institute of Food and Agriculture/ ; }, mesh = {*Cell Wall/metabolism ; *Oryza/genetics/metabolism/enzymology ; *Coumaric Acids/metabolism ; *Lignin/metabolism ; *Plant Proteins/genetics/metabolism ; Aldehyde Dehydrogenase/metabolism/genetics ; Xylans/metabolism ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; }, abstract = {In grass cell walls, ferulic acid (FA) serves as an important cross-linker between cell wall polymers, such as arabinoxylan (AX) and lignin, affecting the physicochemical properties of the cell walls as well as the utilization properties of grass lignocellulose for biorefinering. Here, we demonstrate that hydroxycinnamaldehyde dehydrogenase (HCALDH) plays a crucial role in the biosynthesis of the FA used for cell wall feruloylation in rice (Oryza sativa). Bioinformatic and gene expression analyses of aldehyde dehydrogenases (ALDHs) identified two rice ALDH subfamily 2C members, OsHCALDH2 (OsALDH2C2) and OsHCALDH3 (OsALDH2C3), potentially involved in cell wall feruloylation in major vegetative tissues of rice. CRISPR-Cas9 genome editing of OsHCALDH2 and OsHCALDH3 revealed that the contents of AX-bound ferulate were reduced by up to ~45% in the cell walls of the HCALDH-edited mutants, demonstrating their roles in cell wall feruloylation. The abundance of hemicellulosic sugars including arabinosyl units on AX was notably reduced in the cell walls of the HCALDH-edited mutants, whereas cellulose and lignin contents remained unaffected. In addition to reducing cell wall-bound ferulate, the loss of OsHCALDH2 and/or OsHCALDH3 also partially reduced cell wall-bound p-coumarate and sinapate in the vegetative tissues of rice, whereas it did not cause detectable changes in the amount of γ-oryzanol (feruloyl sterols) in rice seeds. Furthermore, the HCALDH-edited mutants exhibited improved cell wall saccharification efficiency, both with and without alkaline pretreatment, plausibly due to the reduction in cell wall cross-linking FA. Overall, HCALDH appears to present a potent bioengineering target for enhancing utilization properties of grass lignocellulose.}, }
@article {pmid39569586, year = {2024}, author = {Kanke, KL and Rayner, RE and Bozik, J and Abel, E and Venugopalan, A and Suu, M and Nouri, R and Stack, JT and Guo, G and Vetter, TA and Cormet-Boyaka, E and Hester, ME and Vaidyanathan, S}, title = {Single-stranded DNA with internal base modifications mediates highly efficient knock-in in primary cells using CRISPR-Cas9.}, journal = {Nucleic acids research}, volume = {52}, number = {22}, pages = {13561-13576}, pmid = {39569586}, issn = {1362-4962}, support = {//Comprehensive Transplant Center Human Tissue Biorepository,&#xa0;OSU&#xa0;Wexner Medical Center/ ; R00 HL151900/HL/NHLBI NIH HHS/United States ; HL151900-03/HL/NHLBI NIH HHS/United States ; DK106829//National Institute of Diabetes, Digestive and Kidney Diseases/ ; UL1TR002733//Center for Clinical and Translational Science/ ; //Ohio State University/ ; MCCOY19Ro//Nationwide Children's Hospital/ ; VAIDYA22A0-KB//Cystic Fibrosis Foundation/ ; UL1 TR002733/TR/NCATS NIH HHS/United States ; U54 DK106829/DK/NIDDK NIH HHS/United States ; K99 HL151900/HL/NHLBI NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems ; Humans ; *DNA, Single-Stranded/genetics/metabolism ; *Gene Knock-In Techniques ; *Induced Pluripotent Stem Cells/metabolism ; Receptors, CCR5/genetics/metabolism ; Gene Editing/methods ; Cells, Cultured ; Hematopoietic Stem Cells/metabolism ; }, abstract = {Single-stranded DNA (ssDNA) templates along with Cas9 have been used for knocking-in exogenous sequences in the genome but suffer from low efficiency. Here, we show that ssDNA with chemical modifications in 12-19% of internal bases, which we denote as enhanced ssDNA (esDNA), improve knock-in (KI) by 2-3-fold compared to end-modified ssDNA in airway basal stem cells (ABCs), CD34 + hematopoietic cells (CD34 + cells), T-cells and endothelial cells. Over 50% of alleles showed KI in three clinically relevant loci (CFTR, HBB and CCR5) in ABCs using esDNA and up to 70% of alleles showed KI in the HBB locus in CD34 + cells in the presence of a DNA-PKcs inhibitor. This level of correction is therapeutically relevant and is comparable to adeno-associated virus-based templates. The esDNA templates did not improve KI in induced pluripotent stem cells (iPSCs). This may be due to the absence of the nuclease TREX1 in iPSCs. Indeed, knocking out TREX1 in other cells improved KI using unmodified ssDNA. esDNA can be used to modify 20-30 bp regions in primary cells for therapeutic applications and biological modeling. The use of this approach for gene length insertions will require new methods to produce long chemically modified ssDNA in scalable quantities.}, }
@article {pmid39569582, year = {2024}, author = {Arantes, PR and Chen, X and Sinha, S and Saha, A and Patel, AC and Sample, M and Nierzwicki, &#x141; and Lapinaite, A and Palermo, G}, title = {Dimerization of the deaminase domain and locking interactions with Cas9 boost base editing efficiency in ABE8e.}, journal = {Nucleic acids research}, volume = {52}, number = {22}, pages = {13931-13944}, pmid = {39569582}, issn = {1362-4962}, support = {R01 GM141329/GM/NIGMS NIH HHS/United States ; //Advanced Cyberinfrastructure Coordination Ecosystem: Services &amp; Support (ACCESS) program/ ; CHE-2144823//National Science Foundation/ ; R01GM141329/NH/NIH HHS/United States ; BIO230007//Pittsburgh Supercomputer Center/ ; DP2 GM149550/GM/NIGMS NIH HHS/United States ; TC-24-063//Camille and Henry Dreyfus Foundation/ ; MCB160059//San Diego Supercomputing Center/ ; FG-2023-20431//Sloan Foundation/ ; 2138259//National Science Foundation/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism/chemistry ; *DNA/metabolism/genetics/chemistry ; Protein Multimerization ; Humans ; Adenosine Deaminase/metabolism/genetics/chemistry ; Protein Domains ; Deamination ; Molecular Dynamics Simulation ; Adenine/metabolism/chemistry ; }, abstract = {CRISPR-based DNA adenine base editors (ABEs) hold remarkable promises to address human genetic diseases caused by point mutations. ABEs were developed by combining CRISPR-Cas9 with a transfer RNA (tRNA) adenosine deaminase enzyme and through directed evolution, conferring the ability to deaminate DNA. However, the molecular mechanisms driving the efficient DNA deamination in the evolved ABEs remain unresolved. Here, extensive molecular simulations and biochemical experiments reveal the biophysical basis behind the astonishing base editing efficiency of ABE8e, the most efficient ABE to date. We demonstrate that the ABE8e's DNA deaminase domain, TadA8e, forms remarkably stable dimers compared to its tRNA-deaminating progenitor and that the strength of TadA dimerization is crucial for DNA deamination. The TadA8e dimer forms robust interactions involving its R98 and R129 residues, the RuvC domain of Cas9 and the DNA. These locking interactions are exclusive to ABE8e, distinguishing it from its predecessor, ABE7.10, and are indispensable to boost DNA deamination. Additionally, we identify three critical residues that drive the evolution of ABE8e toward improved base editing by balancing the enzyme's activity and stability, reinforcing the TadA8e dimer and improving the ABE8e's functionality. These insights offer new directions to engineer superior ABEs, advancing the design of safer precision genome editing tools.}, }
@article {pmid39568260, year = {2025}, author = {Wang, B and Fan, A and Liu, M and Zhou, Y and Zhang, W and Yan, J}, title = {An Integrated Rapid Detection of Botryosphaeriaceae Species in Grapevine Based on Recombinase Polymerase Amplification, CRISPR/Cas12a, and Lateral Flow Dipstick.}, journal = {Plant disease}, volume = {109}, number = {5}, pages = {1102-1110}, doi = {10.1094/PDIS-08-24-1615-RE}, pmid = {39568260}, issn = {0191-2917}, mesh = {*Vitis/microbiology ; *Ascomycota/genetics/isolation &amp; purification ; *Plant Diseases/microbiology ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Recombinases/genetics/metabolism ; Sensitivity and Specificity ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Grapevine Botryosphaeria dieback (GBD), caused by Botryosphaeriaceae species, is an important grapevine trunk disease that poses a threat to grape yield and quality in global viticultural regions. Pathogen diagnosis at the species level using morphological methods is difficult and time-consuming. Therefore, this study aimed to develop a rapid and accurate detection method for the pathogens causing GBD. Recombinase polymerase amplification (RPA) with CRISPR/Cas12a cleavage was combined for detecting pathogens associated with GBD, and lateral flow dipsticks were employed to monitor the outcomes. Based on the β-tubulin sequences of Botryosphaeriaceae and their related species, specific RPA primers and CRISPR/Cas12a CrRNA were designed and subsequently selected for specifically detecting pathogens associated with GBD. Under optimized reaction conditions and systems, the developed RPA/CRISPR-Cas12a detection system specifically detected Botryosphaeriaceae species within 30 min of RPA and 25 min of CRISPR/Cas12a reactions at 37°C. Specificity tests showed that specific fragments were amplified with the RPA primers in the DNA of six Botryosphaeriaceae species found in China, while none of the fragments were amplified in the other 22 nontarget fungal pathogen species of grapevine. The detection sensitivity of this method was 1 pg/μl, which is equal to that of real-time PCR. In summary, our method is simple to perform, produces visual results, does not rely on expensive equipment, and therefore possesses high practical value, providing an efficient and robust detection platform to accelerate the field detection of pathogens associated with GBD.}, }
@article {pmid39567138, year = {2025}, author = {Lu, X and Wang, Y and Zhen, X and Che, Y and Yu, H and Ge, Y and Wang, X and Li, R and Geng, M and Zhou, B and Liu, J and Guo, J and Yao, Y}, title = {Editing of the soluble starch synthase gene MeSSIII-1 enhanced the amylose and resistant starch contents in cassava.}, journal = {Carbohydrate polymers}, volume = {348}, number = {Pt B}, pages = {122903}, doi = {10.1016/j.carbpol.2024.122903}, pmid = {39567138}, issn = {1879-1344}, mesh = {*Manihot/genetics/chemistry/metabolism ; *Starch Synthase/genetics/metabolism ; *Amylose/metabolism/chemistry ; *Resistant Starch/metabolism ; Gene Editing/methods ; Mutation ; CRISPR-Cas Systems/genetics ; Starch/metabolism/chemistry ; Plant Proteins/genetics/metabolism/chemistry ; Amylopectin/chemistry/metabolism ; Viscosity ; }, abstract = {Foods with high amylose and resistant starch (RS) contents have great potential to enhance human health. In this study, cassava soluble starch synthase MeSSIII-1 gene mutants were generated using CRISPR/Cas9 system. The results showed that the storage roots of messiii-1 mutants had higher contents of amylose, RS, and total starch than those in CK. The rates of small and large-sized starch granules were increased. Additionally, amylopectin starch in messiii-1 mutants had a higher proportion of medium- and long- chains, and a lower proportion of short-chains than those in CK. The onset, peak, and conclusion temperatures of starch gelatinization in messiii-1 mutants were significantly lower than those in CK, and the peak viscosity, trough viscosity and final viscosity all increased. MeSSIII-1 mutation could increase the contents of sucrose, glucose, and fructose in cassava storage roots. We hypothesize that these soluble sugars serve a dual role: they provide the necessary carbon source for starch synthesis and act as sugar signals to trigger the transcriptional reprogramming of genes involved in starch biosynthesis. This process results in a collective enhancement of amylose, RS, and total starch contents, accompanied by changes in starch granule morphology, fine structure, and physicochemical properties.}, }
@article {pmid39566816, year = {2025}, author = {Baek, M and Kim, CL and Kim, SH and la Cour Karottki, KJ and Hefzi, H and Grav, LM and Pedersen, LE and Lewis, NE and Lee, JS and Lee, GM}, title = {Unraveling productivity-enhancing genes in Chinese hamster ovary cells via CRISPR activation screening using recombinase-mediated cassette exchange system.}, journal = {Metabolic engineering}, volume = {87}, number = {}, pages = {11-20}, doi = {10.1016/j.ymben.2024.11.009}, pmid = {39566816}, issn = {1096-7184}, mesh = {CHO Cells ; *Cricetulus ; Animals ; *CRISPR-Cas Systems ; Antibodies, Bispecific/genetics/metabolism ; Recombinases/genetics/metabolism ; Cricetinae ; }, abstract = {Chinese hamster ovary (CHO) cells, which are widely used for therapeutic protein production, have been genetically manipulated to enhance productivity. Nearly half of the genes in CHO cells are silenced, which are promising targets for CHO cell engineering. To identify novel gene targets among the silenced genes that can enhance productivity, we established a genome-wide clustered regularly interspaced short palindromic repeats activation (CRISPRa) screening platform for bispecific antibody (bsAb)-producing CHO (CHO-bsAb) cells with 110,979 guide RNAs (gRNAs) targeting 13,812 silenced genes using a virus-free recombinase-mediated cassette exchange-based gRNA integration method. Using this platform, we performed a fluorescence-activated cell sorting-based cold-capture assay to isolate cells with high fluorescence intensity, which is indicative of high specific bsAb productivity (qbsAb), and identified 90 significantly enriched genes. To verify the screening results, 14 high-scoring candidate genes were individually activated in CHO-bsAb cells via CRISPRa. Among these, 10 genes demonstrated enhanced fluorescence intensity of CHO-bsAb cells in the cold-capture assay when activated. Furthermore, the overexpression of the identified novel gene target Syce3 in CHO-bsAb cells resulted in a 1.4- to 1.9-fold increase in the maximum bsAb concentration, owing to improved qbsAb and specific growth rate. Thus, this virus-free CRISPRa screening platform is a potent tool for identifying novel engineering targets in CHO cells to improve bsAb production.}, }
@article {pmid39566812, year = {2025}, author = {Delshad, M and Davoodi-Moghaddam, Z and Khademi, M and Pourbagheri-Sigaroodi, A and Zali, MR and Bashash, D}, title = {Advancements in gene therapy for human diseases: Trend of current clinical trials.}, journal = {European journal of pharmacology}, volume = {986}, number = {}, pages = {177143}, doi = {10.1016/j.ejphar.2024.177143}, pmid = {39566812}, issn = {1879-0712}, mesh = {Humans ; *Genetic Therapy/methods ; *Clinical Trials as Topic ; Genetic Vectors ; }, abstract = {In an era of rapid scientific advancement, gene therapy has emerged as a groundbreaking approach with the potential to revolutionize the treatment of a myriad of diseases and medical conditions. The trend of current clinical trials suggests that there is growing interest and investment in exploring gene therapy as a viable treatment option. In 2023, a significant milestone was achieved with the approval of seven gene therapies by the Food and Drug Administration (FDA). Projections indicate that between 10 and 20 gene therapies could receive annual FDA approval by 2025. In this review, we conducted a comprehensive analysis of registered clinical trials on Clinicaltrials.gov to determine the progression status of gene therapies. Upon extraction of the data, we conducted a comprehensive analysis of the 2809 included studies. This involved a systematic approach, commencing with an overview, followed by a detailed examination of gene therapy strategies employed in various malignant and non-malignant disorders. Additionally, the study will cover the types of vectors utilized in current trials. Lastly, a meticulous review of 105 phase III-IV clinical trials was conducted to identify potential therapies demonstrating promise. We trust that the comprehensive overview provided will serve as a solid foundation for forthcoming research and study designs, ultimately contributing to the progression of gene therapy and its practical application within healthcare settings. Also, we anticipate that such inquiries will bolster the formulation of practical policies and guidelines for pharmaceutical companies engaged in gene therapy research and development.}, }
@article {pmid39566771, year = {2024}, author = {Panda, G and Ray, A}, title = {Deciphering Cas9 specificity: Role of domain dynamics and RNA:DNA hybrid interactions revealed through machine learning and accelerated molecular simulations.}, journal = {International journal of biological macromolecules}, volume = {283}, number = {Pt 4}, pages = {137835}, doi = {10.1016/j.ijbiomac.2024.137835}, pmid = {39566771}, issn = {1879-0003}, mesh = {*Molecular Dynamics Simulation ; *CRISPR-Associated Protein 9/chemistry/metabolism/genetics ; *Machine Learning ; *DNA/chemistry/metabolism ; *RNA/chemistry/metabolism ; Francisella/enzymology ; Protein Domains ; CRISPR-Cas Systems ; Substrate Specificity ; Bacterial Proteins/chemistry/metabolism/genetics ; }, abstract = {CRISPR/Cas9 technology is widely used for gene editing, but off-targeting still remains a major concern in therapeutic applications. Although Cas9 variants with better mismatch discrimination have been developed, they have significantly lower rates of on-target DNA cleavage. This study compares the dynamics of the highly specific Cas9 from Francisella novicida (FnCas9) to the commonly used SpCas9. Using long-scale atomistic Gaussian accelerated molecular dynamic simulations and machine learning techniques, we deciphered the structural factors behind FnCas9's higher specificity in native and off-target forms. Our analysis revealed that Cas9's cleavage specificity relies more on its domain rearrangement than on RNA:DNA heteroduplex shape, with significant conformational variations in Cas9 domains among off-target forms, while the RNA:DNA hybrid showed minimal changes, especially in FnCas9 compared to SpCas9. REC1-REC3 domains contacts with the RNA:DNA hybrid in FnCas9 acted as critical discriminator of off-target effects playing a pivotal role in influencing specificity. In FnCas9, allosteric signal transmission involves the REC3 and HNH domain, bypassing REC2, leading to a superior efficiency in information transmission. This study offers a quantitative framework for understanding the structural basis of elevated specificity, paving the way for the rational design of Cas9 variants with improved precision and specificity in genome editing applications.}, }
@article {pmid39566123, year = {2024}, author = {Dara, M and Dianatpour, M and Azarpira, N and Tanideh, N and Tanideh, R}, title = {Integrating CRISPR technology with exosomes: Revolutionizing gene delivery systems.}, journal = {Biochemical and biophysical research communications}, volume = {740}, number = {}, pages = {151002}, doi = {10.1016/j.bbrc.2024.151002}, pmid = {39566123}, issn = {1090-2104}, mesh = {*Exosomes/genetics ; Humans ; *Gene Transfer Techniques ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genetic Therapy/methods ; }, abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) serves as an adaptive immune system in bacteria and archaea, offering a defense mechanism against invading genetic elements such as viruses (bacteriophages) and plasmids. Today, CRISPR has evolved into a powerful gene-editing technology that enables highly specific and rapid modifications of DNA within a genome. It has a broad range of applications across various fields, including medicine, agriculture, and fundamental research. One of the significant challenges facing this technology is the efficient transfer of CRISPR constructs into target cells for gene editing. There are several methods to deliver this system into target cells, which can be classified as viral and non-viral methods. Each of these approaches has its own advantages and disadvantages. Recently, the use of extracellular vesicles for delivery has garnered particular attention. Exosomes are nano-sized extracellular vesicles that have emerged as promising carriers for drug delivery due to their unique properties. These naturally occurring vesicles, typically ranging from 30 to 150 nm in diameter, facilitate intercellular communication by transferring bioactive molecules such as proteins, lipids, and nucleic acids between cells. Exosome therapy has surfaced as a promising strategy in regenerative medicine, utilizing small extracellular vesicles to deliver therapeutic molecules to target cells. One of the emerging options for transferring the CRISPR system is exosomes. The integration of these two advanced technologies holds significant potential for developing efficient and targeted gene editing and advancing precision medicine. In contemporary medicine, there is an increasing focus on personalized and targeted treatments that cater to the distinct genetic and molecular profiles of individual patients. The synergy of CRISPR technology and exosome therapy presents a remarkable opportunity to develop highly targeted and effective therapeutic strategies customized to individual patient requirements. This review article examines the potential of incorporating CRISPR technology within exosomes for precision therapeutic applications.}, }
@article {pmid39565688, year = {2024}, author = {Asano, K and Yoshimi, K and Takeshita, K and Mitsuhashi, S and Kochi, Y and Hirano, R and Tingyu, Z and Ishida, S and Mashimo, T}, title = {CRISPR Diagnostics for Quantification and Rapid Diagnosis of Myotonic Dystrophy Type 1 Repeat Expansion Disorders.}, journal = {ACS synthetic biology}, volume = {13}, number = {12}, pages = {3926-3935}, pmid = {39565688}, issn = {2161-5063}, mesh = {*Myotonic Dystrophy/genetics/diagnosis ; Humans ; *CRISPR-Cas Systems/genetics ; *Trinucleotide Repeat Expansion/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Sensitivity and Specificity ; }, abstract = {Repeat expansion disorders, exemplified by myotonic dystrophy type 1 (DM1), present challenges in diagnostic quantification because of the variability and complexity of repeat lengths. Traditional diagnostic methods, including PCR and Southern blotting, exhibit limitations in sensitivity and specificity, necessitating the development of innovative approaches for precise and rapid diagnosis. Here, we introduce a CRISPR-based diagnostic method, REPLICA (repeat-primed locating of inherited disease by Cas3), for the quantification and rapid diagnosis of DM1. This method, using in vitro-assembled CRISPR-Cas3, demonstrates superior sensitivity and specificity in quantifying CTG repeat expansion lengths, correlated with disease severity. We also validate the robustness and accuracy of CRISPR diagnostics in quantitatively diagnosing DM1 using patient genomes. Furthermore, we optimize a REPLICA-based assay for point-of-care-testing using lateral flow test strips, facilitating rapid screening and detection. In summary, REPLICA-based CRISPR diagnostics offer precise and rapid detection of repeat expansion disorders, promising personalized treatment strategies.}, }
@article {pmid39565578, year = {2025}, author = {Hwang, I and Nikoli, H and Paik, J}, title = {Functional Motif Discovery in FOXO1 Through CRISPR/Cas9 Exon Tiling Scan.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2871}, number = {}, pages = {57-68}, pmid = {39565578}, issn = {1940-6029}, mesh = {*Forkhead Box Protein O1/metabolism/genetics ; *CRISPR-Cas Systems ; *Exons/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {The study of FOXO1, a pivotal transcription factor, has garnered significant attention due to its critical role in diverse cellular processes, including lineage differentiation, apoptosis, cell cycle regulation, and metabolism. To comprehensively understand the functional intricacies of FOXO1, an innovative approach is essential. This chapter highlights employing CRISPR exon scanning as a strategic tool to dissect the functional domains of FOXO1 and unravel its diverse regulatory functions. CRISPR exon scan allows for the identification of functionally important domains based on the levels of sgRNA depletion or enrichment within the FOXO1 gene, providing a unique opportunity to investigate the domain function under relevant biological contexts. This approach enables the systematic exploration of FOXO1's structural domains, shedding light on how distinct regions contribute to its overall function. The comprehensive exon scan analysis using CRISPR technology allows gaining a nuanced understanding of FOXO1's functional diversity and regulatory mechanisms.}, }
@article {pmid39565132, year = {2025}, author = {Khongthongdam, M and Phetruen, T and Chanarat, S}, title = {Development of ptxD/Phi as a new dominant selection system for genetic manipulation in Cryptococcus neoformans.}, journal = {Microbiology spectrum}, volume = {13}, number = {1}, pages = {e0161824}, pmid = {39565132}, issn = {2165-0497}, mesh = {*Cryptococcus neoformans/genetics/pathogenicity ; *Virulence Factors/genetics ; CRISPR-Cas Systems ; Fungal Proteins/genetics/metabolism ; Cryptococcosis/microbiology ; Oxidoreductases/genetics/metabolism ; }, abstract = {Cryptococcus neoformans is a globally distributed pathogenic fungus posing a significant threat to immunocompromised individuals, particularly those with HIV/AIDS. Effective genetic manipulation tools are essential for understanding its biology and developing new therapies. However, current genetic tools, including the variation of versatile selectable markers, are limited. This study develops and validates the phosphite dehydrogenase gene (ptxD)/phosphite (Phi) selection system as a non-antibiotic selectable marker for genetic manipulation in C. neoformans. A codon-optimized ptxD gene from Pseudomonas stutzeri was cloned under the TEF promoter. Using the transient CRISPR-Cas9 coupled with electroporation system, we integrated the ptxD gene into the C. neoformans genome and assessed the impact of ptxD integration on cell growth and virulence factors. The ptxD/Phi system effectively selected transformed cells on Phi-containing media. Growth assays showed that ptxD integration did not adversely affect cell growth or key virulence factors, including pleomorphism, capsule size, and melanin production. Additionally, we successfully disrupted the ADE2 gene using this system, confirming its applicability for gene deletion. Taken together, the ptxD/Phi system provides a robust and versatile tool for genetic manipulation in C. neoformans, facilitating further research into its biology and pathogenicity.IMPORTANCECryptococcus neoformans is a type of fungus that can cause serious illnesses in people who have weakened immune systems, like those with HIV/AIDS. To better study this fungus and find new treatments, scientists need tools to change its genes in precise ways. However, the current tools available for this are somewhat limited. This research introduces a new tool called the phosphite dehydrogenase gene/phosphite system, which does not rely on antibiotics to work. It uses a gene from a different bacterium that helps select and grow only the fungus cells that have successfully incorporated new genetic information. This is particularly useful because it does not interfere with the normal growth of the fungus or the features that make it harmful (like its ability to change shape or produce protective coatings). By making it easier and more effective to manipulate the genetics of C. neoformans, this tool opens up new possibilities for understanding how this fungus operates and for developing therapies to combat its infections. This is crucial for improving the treatment of infections in vulnerable populations.}, }
@article {pmid39563182, year = {2024}, author = {Wang, Y and Chen, P and Wen, H and Gui, Y and Yan, D and Huang, D and Wang, D and Tang, BZ and Tan, H}, title = {Advanced Nanoplatform Mediated by CRISPR-Cas9 and Aggregation-Induced Emission Photosensitizers to Boost Cancer Theranostics.}, journal = {ACS nano}, volume = {18}, number = {48}, pages = {33168-33180}, doi = {10.1021/acsnano.4c11757}, pmid = {39563182}, issn = {1936-086X}, mesh = {*Photosensitizing Agents/chemistry/pharmacology ; *Theranostic Nanomedicine ; Humans ; *CRISPR-Cas Systems/genetics ; Mice ; Animals ; *Reactive Oxygen Species/metabolism ; Nanoparticles/chemistry ; Neoplasms/therapy/genetics/diagnostic imaging/pathology ; Cell Line, Tumor ; Antineoplastic Agents/pharmacology/chemistry ; Immunotherapy ; Mice, Inbred BALB C ; Cell Proliferation/drug effects ; Drug Screening Assays, Antitumor ; }, abstract = {Immunotherapy combined with phototherapy is emerging as a promising strategy to treat omnipotent cancers. In this study, a clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system, aggregation-induced emission (AIE) photosensitizer (PS) and surface coating of polyethylene imine/hyaluronic acid were combined to construct a multifunctional nanoplatform, denoted as TCPH nanoparticles (NPs), for comprehensive cancer theranostics. TCPH NPs are featured by intrinsic functions including efficient reactive oxygen species (ROS) production, good photothermal conversion, programmed death-ligand 1 (PD-L1)-eliminating capability, and effective intracellular transport. The generated ROS and hyperthermia do not only achieve primary tumor elimination but also regulate the tumor immune microenvironment. Genomic disruption of PD-L1 conspicuously augments its therapeutic efficacy, especially in tumor metastasis and recurrence. Exceptional multimodal imaging navigation has also been developed. Excellent theranostics performance was substantiated in diverse tumor models, implying that this synergistic strategy of phototheranostics and immunotherapy provides a paradigm shift in emerging CRISPR-mediated nanomedicines.}, }
@article {pmid39563029, year = {2025}, author = {Broksø, AD and Bendixen, L and Fammé, S and Mikkelsen, K and Jensen, TI and Bak, RO}, title = {Orthogonal transcriptional modulation and gene editing using multiple CRISPR-Cas systems.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {33}, number = {1}, pages = {71-89}, pmid = {39563029}, issn = {1525-0024}, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Streptococcus pyogenes/genetics ; Staphylococcus aureus/genetics ; Gene Knockout Techniques ; CRISPR-Associated Protein 9/metabolism/genetics ; T-Lymphocytes/metabolism ; Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation ; Transcriptional Activation ; Endonucleases/metabolism/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {CRISPR-Cas-based transcriptional activation (CRISPRa) and interference (CRISPRi) enable transient programmable gene regulation by recruitment or fusion of transcriptional regulators to nuclease-deficient Cas (dCas). Here, we expand on the emerging area of transcriptional engineering and RNA delivery by benchmarking combinations of RNA-delivered dCas and transcriptional modulators. We utilize dCas9 from Staphylococcus aureus and Streptococcus pyogenes for orthogonal transcriptional modulation to upregulate one set of genes while downregulating another. We also establish trimodal genetic engineering by combining orthogonal transcriptional regulation with gene knockout by Cas12a (Acidaminococcus; AsCas12a) ribonucleoprotein delivery. To simplify trimodal engineering, we explore optimal parameters for implementing truncated single guide RNAs (sgRNAs) to make use of SpCas9 for knockout and CRISPRa. We find the Cas9 protein/sgRNA ratio to be crucial for avoiding sgRNA cross-complexation and for balancing knockout and activation efficiencies. We demonstrate high efficiencies of trimodal genetic engineering in primary human T cells while preserving basic T cell health and functionality. This study highlights the versatility and potential of complex genetic engineering using multiple CRISPR-Cas systems in a simple one-step process yielding transient transcriptome modulation and permanent DNA changes. We believe such elaborate engineering can be implemented in regenerative medicine and therapies to facilitate more sophisticated treatments.}, }
@article {pmid39563028, year = {2025}, author = {Nieland, L and Vrijmoet, AB and Jetten, IW and Rufino-Ramos, D and de Reus, AJEM and Breyne, K and Kleinstiver, BP and Maguire, CA and Broekman, MLD and Breakefield, XO and Abels, ER}, title = {CRISPR targeting of mmu-miR-21a through a single adeno-associated virus vector prolongs survival of glioblastoma-bearing mice.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {33}, number = {1}, pages = {133-151}, pmid = {39563028}, issn = {1525-0024}, support = {P01 CA069246/CA/NCI NIH HHS/United States ; U19 CA179563/CA/NCI NIH HHS/United States ; }, mesh = {Animals ; *Glioblastoma/therapy/genetics/pathology ; *Dependovirus/genetics ; *MicroRNAs/genetics ; Mice ; *Genetic Vectors/genetics/administration &amp; dosage ; Humans ; *CRISPR-Cas Systems ; Gene Editing/methods ; Cell Line, Tumor ; Genetic Therapy/methods ; Disease Models, Animal ; Brain Neoplasms/therapy/genetics/pathology ; RNA, Guide, CRISPR-Cas Systems/genetics ; Xenograft Model Antitumor Assays ; Gene Targeting ; }, abstract = {Glioblastoma (GB), the most aggressive tumor of the central nervous system (CNS), has poor patient outcomes with limited effective treatments available. MicroRNA-21 (miR-21(a)) is a known oncogene, abundantly expressed in many cancer types. miR-21(a) promotes GB progression, and lack of miR-21(a) reduces the tumorigenic potential. Here, we propose a single adeno-associated virus (AAV) vector strategy targeting mmu-miR-21a using the Staphylococcus aureus Cas9 ortholog (SaCas9) guided by a single-guide RNA (sgRNA). Our results demonstrate that AAV8 is a well-suited AAV serotype to express SaCas9-KKH/sgRNA at the tumor site in an orthotopic GB model. The SaCas9-KKH induced a genomic deletion, resulting in lowered mmu-miR-21a levels in the brain, leading to reduced tumor growth and improved overall survival. In this study, we demonstrated that disruption of genomic mmu-miR-21a with a single AAV vector influenced glioma development, resulting in beneficial anti-tumor outcomes in GB-bearing mice.}, }
@article {pmid39562753, year = {2025}, author = {Cirincione, A and Simpson, D and Yan, W and McNulty, R and Ravisankar, P and Solley, SC and Yan, J and Lim, F and Farley, EK and Singh, M and Adamson, B}, title = {A benchmarked, high-efficiency prime editing platform for multiplexed dropout screening.}, journal = {Nature methods}, volume = {22}, number = {1}, pages = {92-101}, pmid = {39562753}, issn = {1548-7105}, support = {RM1HG009490//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R35 GM138167/GM/NIGMS NIH HHS/United States ; T32HG003284//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; P30 CA072720/CA/NCI NIH HHS/United States ; F31 HD113443/HD/NICHD NIH HHS/United States ; R35GM138167//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; T32 GM007388/GM/NIGMS NIH HHS/United States ; R01 GM076275/GM/NIGMS NIH HHS/United States ; T32 HG003284/HG/NHGRI NIH HHS/United States ; DGE-2039656//National Science Foundation (NSF)/ ; P30CA072720//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; F31HD113443//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; 2239957//National Science Foundation (NSF)/ ; T32GM007388//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01-GM076275//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; }, mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {Prime editing installs precise edits into the genome with minimal unwanted byproducts, but low and variable editing efficiencies have complicated application of the approach to high-throughput functional genomics. Here we assembled a prime editing platform capable of high-efficiency substitution editing suitable for functional interrogation of small genetic variants. We benchmarked this platform for pooled, loss-of-function screening using a library of ~240,000 engineered prime editing guide RNAs (epegRNAs) targeting ~17,000 codons with 1-3 bp substitutions. Comparing the abundance of these epegRNAs across screen samples identified negative selection phenotypes for 7,996 nonsense mutations targeted to 1,149 essential genes and for synonymous mutations that disrupted splice site motifs at 3' exon boundaries. Rigorous evaluation of codon-matched controls demonstrated that these phenotypes were highly specific to the intended edit. Altogether, we established a prime editing approach for multiplexed, functional characterization of genetic variants with simple readouts.}, }
@article {pmid39562573, year = {2024}, author = {Kunitake, K and Mizuno, T and Hattori, K and Oneyama, C and Kamiya, M and Ota, S and Urano, Y and Kojima, R}, title = {Barcoding of small extracellular vesicles with CRISPR-gRNA enables comprehensive, subpopulation-specific analysis of their biogenesis and release regulators.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {9777}, pmid = {39562573}, issn = {2041-1723}, support = {24H00868//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JPMJPR17H5//MEXT | JST | Precursory Research for Embryonic Science and Technology (PRESTO)/ ; CDA-00008/2019-C//Human Frontier Science Program (HFSP)/ ; K05 DA000008/DA/NIDA NIH HHS/United States ; JPMJCR19H1, JPMJCR23B7//MEXT | JST | Core Research for Evolutional Science and Technology (CREST)/ ; }, mesh = {*Extracellular Vesicles/metabolism/genetics ; Humans ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Tetraspanin 29/metabolism/genetics ; *Tetraspanin 30/metabolism/genetics ; HEK293 Cells ; Exosomes/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Small extracellular vesicles (sEVs) are important intercellular information transmitters in various biological contexts, but their release processes remain poorly understood. Herein, we describe a high-throughput assay platform, CRISPR-assisted individually barcoded sEV-based release regulator (CIBER) screening, for identifying key players in sEV release. CIBER screening employs sEVs barcoded with CRISPR-gRNA through the interaction of gRNA and dead Cas9 fused with an sEV marker. Barcode quantification enables the estimation of the sEV amount released from each cell in a massively parallel manner. Barcoding sEVs with different sEV markers in a CRISPR pooled-screening format allows genome-wide exploration of sEV release regulators in a subpopulation-specific manner, successfully identifying previously unknown sEV release regulators and uncovering the exosomal/ectosomal nature of CD63[+]/CD9[+] sEVs, respectively, as well as the synchronization of CD9[+] sEV release with the cell cycle. CIBER should be a valuable tool for detailed studies on the biogenesis, release, and heterogeneity of sEVs.}, }
@article {pmid39562558, year = {2024}, author = {Li, W and Jiang, X and Wang, W and Hou, L and Cai, R and Li, Y and Gu, Q and Chen, Q and Ma, P and Tang, J and Guo, M and Chuai, G and Huang, X and Zhang, J and Liu, Q}, title = {Discovering CRISPR-Cas system with self-processing pre-crRNA capability by foundation models.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10024}, pmid = {39562558}, issn = {2041-1723}, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Cleavage ; CRISPR-Associated Proteins/metabolism/genetics ; }, abstract = {The discovery of CRISPR-Cas systems has paved the way for advanced gene editing tools. However, traditional Cas discovery methods relying on sequence similarity may miss distant homologs and aren't suitable for functional recognition. With protein large language models (LLMs) evolving, there is potential for Cas system modeling without extensive training data. Here, we introduce CHOOSER (Cas HOmlog Observing and SElf-processing scReening), an AI framework for alignment-free discovery of CRISPR-Cas systems with self-processing pre-crRNA capability using protein foundation models. By using CHOOSER, we identify 11 Casλ homologs, nearly doubling the known catalog. Notably, one homolog, EphcCasλ, is experimentally validated for self-processing pre-crRNA, DNA cleavage, and trans-cleavage, showing promise for CRISPR-based pathogen detection. This study highlights an innovative approach for discovering CRISPR-Cas systems with specific functions, emphasizing their potential in gene editing.}, }
@article {pmid39561776, year = {2025}, author = {Xu, Y and Morrow, CA and Laksir, Y and Holt, OM and Taylor, K and Tsiappourdhi, C and Collins, P and Jia, S and Andreadis, C and Whitby, MC}, title = {DNA nicks in both leading and lagging strand templates can trigger break-induced replication.}, journal = {Molecular cell}, volume = {85}, number = {1}, pages = {91-106.e5}, pmid = {39561776}, issn = {1097-4164}, mesh = {*Schizosaccharomyces/genetics/metabolism ; *DNA Replication ; *Schizosaccharomyces pombe Proteins/metabolism/genetics ; *DNA Breaks, Double-Stranded ; *DNA Breaks, Single-Stranded ; *DNA, Fungal/metabolism/genetics ; CRISPR-Cas Systems ; DNA End-Joining Repair ; DNA-Binding Proteins/metabolism/genetics ; DNA Repair ; Recombinational DNA Repair ; DNA Helicases/metabolism/genetics ; Homologous Recombination ; }, abstract = {Encounters between replication forks and unrepaired DNA single-strand breaks (SSBs) can generate both single-ended and double-ended double-strand breaks (seDSBs and deDSBs). seDSBs can be repaired by break-induced replication (BIR), which is a highly mutagenic pathway that is thought to be responsible for many of the mutations and genome rearrangements that drive cancer development. However, the frequency of BIR's deployment and its ability to be triggered by both leading and lagging template strand SSBs were unclear. Using site- and strand-specific SSBs generated by nicking enzymes, including CRISPR-Cas9 nickase (Cas9n), we demonstrate that leading and lagging template strand SSBs in fission yeast are typically converted into deDSBs that are repaired by homologous recombination. However, both types of SSBs can also trigger BIR, and the frequency of these events increases when fork convergence is delayed and the non-homologous end joining protein Ku70 is deleted.}, }
@article {pmid39561528, year = {2024}, author = {Zhang, K and Wang, P and Li, S and Xie, X and Wang, Z and Li, Y and Jiao, X and Li, Q}, title = {Type I-E CRISPR-Cas system regulates fimZY and T3SS1 genes expression in Salmonella enterica serovar Pullorum.}, journal = {Veterinary microbiology}, volume = {299}, number = {}, pages = {110301}, doi = {10.1016/j.vetmic.2024.110301}, pmid = {39561528}, issn = {1873-2542}, mesh = {*CRISPR-Cas Systems ; *Salmonella enterica/genetics ; Animals ; *Chickens ; *Gene Expression Regulation, Bacterial ; Bacterial Proteins/genetics/metabolism ; Salmonella Infections, Animal/microbiology ; Poultry Diseases/microbiology ; Virulence/genetics ; Type III Secretion Systems/genetics ; Plasmids/genetics ; Cell Line, Tumor ; Serogroup ; }, abstract = {Clustered regularly interspaced short palindromic repeats and associated Cas proteins (CRISPR-Cas) provide prokaryotes with adaptive immunity against invasion by plasmids or phages. In Salmonella, the type I-E CRISPR-Cas system is typically considered silent in immunity against foreign genetic elements. To elucidate the role of the CRISPR-Cas system, we chose Salmonella enterica serovar Pullorum S06004 as a model organism due to its four spacers and well-defined biological characteristics observed in previous studies. Western blot analysis revealed expression of Cas3 in S06004 cultured in vitro, but plasmid transformation assays demonstrated that both wild-type (WT) and S06004 strains overexpressing LeuO (a positive regulator of CRISPR-Cas) showed no immunity against the target plasmid. RNA-Seq analysis detected significant downregulation of the fim cluster, encoding type I fimbriae, and T3SS1-related genes in the cas cluster mutant compared to the WT. This downregulation was further confirmed in mutants of CR1 and individual cas genes by qRT-PCR. Consequently, mutants of CR1 and cas clusters exhibited decreased invasion of chicken hepatocellular carcinoma cells. The consistent regulation of T3SS1 genes by the CRISPR-Cas system in S. Pullorum, S. Enteritidis, and S. Typhimurium indicates a common role for the type I-E CRISPR-Cas system in promoting bacterial virulence. However, the specific molecular mechanisms underlying this regulation require further investigation.}, }
@article {pmid39561249, year = {2025}, author = {Sakagami, H and Shiroshima, T and Nemoto, N and Niimura, T and Sugawara, T and Hara, Y and Saito, K and Okubo, T and Fukaya, M}, title = {Disruption of Iqsec1 in mice leads to embryonic lethality with reduced large apical vacuoles in the visceral endoderm.}, journal = {FEBS letters}, volume = {599}, number = {4}, pages = {581-591}, doi = {10.1002/1873-3468.15058}, pmid = {39561249}, issn = {1873-3468}, support = {23K06322//Japan Society for the Promotion of Science/ ; }, mesh = {Animals ; *Endoderm/metabolism/ultrastructure/pathology ; Mice ; *Vacuoles/metabolism/ultrastructure/pathology/genetics ; Mice, Knockout ; *Guanine Nucleotide Exchange Factors/genetics/metabolism/deficiency ; Embryonic Development/genetics ; Yolk Sac/metabolism/ultrastructure ; *Embryo Loss/genetics/pathology ; Embryo, Mammalian/metabolism ; CRISPR-Cas Systems ; Female ; }, abstract = {Iqsec1 (IQ motif and Sec7 domain-containing protein 1), also known as BRAG2 (Brefeldin A-resistant Arf-GEF 2), is a guanine nucleotide exchange factor that regulates membrane trafficking, cytoskeletal organization, and signal transduction by activating class II and III ADP-ribosylation factors. To investigate the physiological role of Iqsec1 at the whole animal level, we generated Iqsec1-deficient mice using CRISPR/Cas9-mediated gene editing. Nearly all Iqsec1[-/-] mice (99%) exhibited embryonic lethality with severe growth retardation. Electron microscopy revealed that Iqsec1[-/-] embryos at embryonic day 8.5 lacked large apical vacuoles in visceral endoderm cells of the yolk sac, compared with controls. These findings suggest that Iqsec1 plays a critical role in embryogenesis, likely through regulation of membrane trafficking in visceral endoderm cells.}, }
@article {pmid39561207, year = {2024}, author = {Osia, B and Merkell, A and Lopezcolorado, FW and Ping, X and Stark, JM}, title = {RAD52 and ERCC6L/PICH have a compensatory relationship for genome stability in mitosis.}, journal = {PLoS genetics}, volume = {20}, number = {11}, pages = {e1011479}, pmid = {39561207}, issn = {1553-7404}, support = {R01 CA256989/CA/NCI NIH HHS/United States ; R01 CA240392/CA/NCI NIH HHS/United States ; T32 CA186895/CA/NCI NIH HHS/United States ; F32 CA275207/CA/NCI NIH HHS/United States ; P30 CA033572/CA/NCI NIH HHS/United States ; }, mesh = {*Rad52 DNA Repair and Recombination Protein/genetics/metabolism ; *Genomic Instability ; *Mitosis/genetics ; Humans ; *DNA Helicases/genetics/metabolism ; Poly-ADP-Ribose Binding Proteins/genetics/metabolism ; DNA Repair/genetics ; Anaphase/genetics ; CRISPR-Cas Systems ; HeLa Cells ; Synthetic Lethal Mutations/genetics ; DNA Damage/genetics ; }, abstract = {Mammalian RAD52 is a DNA repair factor with strand annealing and recombination mediator activities that appear important in both interphase and mitotic cells. Nonetheless, RAD52 is dispensable for cell viability. To query RAD52 synthetic lethal relationships, we performed genome-wide CRISPR knock-out screens and identified hundreds of candidate synthetic lethal interactions. We then performed secondary screening and identified genes for which depletion causes reduced viability and elevated genome instability (increased 53BP1 nuclear foci) in RAD52-deficient cells. One such factor was ERCC6L, which marks DNA bridges during anaphase, and hence is important for genome stability in mitosis. Thus, we investigated the functional interrelationship between RAD52 and ERCC6L. We found that RAD52 deficiency increases ERCC6L-coated anaphase ultrafine bridges, and that ERCC6L depletion causes elevated RAD52 foci in prometaphase and interphase cells. These effects were enhanced with replication stress (i.e. hydroxyurea) and topoisomerase IIα inhibition (ICRF-193), where post-treatment effect timings were consistent with defects in addressing stress in mitosis. Altogether, we suggest that RAD52 and ERCC6L co-compensate to protect genome stability in mitosis.}, }
@article {pmid39560750, year = {2025}, author = {Noruzi, S and Mohammadi, R and Jamialahmadi, K}, title = {CRISPR/Cas9 system: a novel approach to overcome chemotherapy and radiotherapy resistance in cancer.}, journal = {Naunyn-Schmiedeberg's archives of pharmacology}, volume = {398}, number = {4}, pages = {3373-3408}, pmid = {39560750}, issn = {1432-1912}, mesh = {*CRISPR-Cas Systems ; Humans ; *Neoplasms/genetics/therapy/radiotherapy/drug therapy ; *Drug Resistance, Neoplasm/genetics ; Animals ; Gene Editing/methods ; *Radiation Tolerance/genetics ; Antineoplastic Agents/therapeutic use/pharmacology ; }, abstract = {Cancer presents a global health challenge with rising incidence and mortality. Despite treatment advances in cancer therapy, radiotherapy and chemotherapy remained the most common treatments for all types of cancers. However, resistance phenotype in cancer cells leads to unsatisfactory results in the efficiency of therapeutic strategies. Therefore, researchers strive to propose effective solutions to overcome treatment failure, which requires a deep knowledge of treatment-resistant mechanisms. The progression and occurrence of tumors can be attributed to gene mutation. Over the past decade, the emergence of clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) genome editing has revolutionized cancer research. This versatile technology enables cancer modeling, manipulation of specific DNA sequences, and genome-wide screening. CRISPR/Cas9 is an effective tool for identifying radio- and chemoresistance genes and offering potential adjunctive treatments to overcome tumor recurrence after chemo- and radiotherapy. This article aims to explain the potential of the CRISPR/Cas9 system in improving the effectiveness of chemo- and radiotherapy and ultimately overcoming treatment failure.}, }
@article {pmid39560384, year = {2024}, author = {Steiner, S and Roy, CR}, title = {CRISPR-Cas9-based approaches for genetic analysis and epistatic interaction studies in Coxiella burnetii.}, journal = {mSphere}, volume = {9}, number = {12}, pages = {e0052324}, pmid = {39560384}, issn = {2379-5042}, support = {R21 AI171333/AI/NIAID NIH HHS/United States ; R56 AI182383/AI/NIAID NIH HHS/United States ; S10 OD030363/OD/NIH HHS/United States ; R21AI171333,R56AI182383//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; }, mesh = {*Coxiella burnetii/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Epistasis, Genetic ; Bacterial Proteins/genetics ; Humans ; Animals ; Phenotype ; }, abstract = {UNLABELLED: Coxiella burnetii is an obligate intracellular bacterial pathogen that replicates to high numbers in an acidified lysosome-derived vacuole. Intracellular replication requires the Dot/Icm type IVB secretion system, which translocates over 100 different effector proteins into the host cell. Screens employing random transposon mutagenesis have identified several C. burnetii effectors that play an important role in intracellular replication; however, the difficulty in conducting directed mutagenesis has been a barrier to the systematic analysis of effector mutants and to the construction of double mutants to assess epistatic interactions between effectors. Here, two CRISPR-Cas9 technology-based approaches were developed to study C. burnetii phenotypes resulting from targeted gene disruptions. CRISPRi was used to silence gene expression and demonstrated that silencing of effectors or Dot/Icm system components resulted in phenotypes similar to those of transposon insertion mutants. A CRISPR-Cas9-mediated cytosine base editing protocol was developed to generate targeted loss-of-function mutants through the introduction of premature stop codons into C. burnetii genes. Cytosine base editing successfully generated double mutants in a single step. A double mutant deficient in both cig57 and cig2 had a robust and additive intracellular replication defect when compared to either single mutant, which is consistent with Cig57 and Cig2 functioning in independent pathways that both contribute to a vacuole that supports C. burnetii replication. Thus, CRISPR-Cas9-based technologies expand the genetic toolbox for C. burnetii and will facilitate genetic studies aimed at investigating the mechanisms this pathogen uses to replicate inside host cells.<br><br>IMPORTANCE: Understanding the genetic mechanisms that enable C. burnetii to replicate in mammalian host cells has been hampered by the difficulty in making directed mutations. Here, a reliable and efficient system for generating targeted loss-of-function mutations in C. burnetii using a CRISPR-Cas9-assisted base editing approach is described. This technology was applied to make double mutants in C. burnetii that enabled the genetic analysis of two genes that play independent roles in promoting the formation of vacuoles that support intracellular replication. This advance will accelerate the discovery of mechanisms important for C. burnetii host infection and disease.}, }
@article {pmid39560197, year = {2024}, author = {Wang, Y and He, D and Li, W and Dong, Y and Fang, L and Liu, D and Tang, Y and Xiao, S}, title = {Field-deployable porcine epidemic diarrhea virus diagnostics utilizing CRISPR-Cas13a.}, journal = {Virulence}, volume = {15}, number = {1}, pages = {2429022}, pmid = {39560197}, issn = {2150-5608}, mesh = {*Porcine epidemic diarrhea virus/genetics/isolation &amp; purification ; Animals ; Swine ; *CRISPR-Cas Systems ; *Swine Diseases/diagnosis/virology ; *Coronavirus Infections/diagnosis/veterinary/virology ; Sensitivity and Specificity ; CRISPR-Associated Proteins/genetics ; }, abstract = {Porcine epidemic diarrhoea virus (PEDV), a pathogenic microorganism that induces epidemic diarrhoea in swine, causes substantial economic damage to swine-farming nations. To prevent and control PEDV infections, the availability of upgraded and rapid virus detection techniques is crucial. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas)13a system, namely, programmability of CRISPR RNA (crRNA) and "collateral" promiscuous RNase activity of Cas13a after target RNA identification. In this study, we aimed to develop a recombinase polymerase amplification (RPA)-based CRISPR-Cas13a approach for PEDV diagnosis for the first time. The results showed that up to 10 copies of the target PEDV DNA standard/µL were detected after 40 min at 37 °C. PEDV detection exhibited remarkable specificity compared to that of other selected pathogens. Additionally, this RPA-based CRISPR-Cas13a approach could be used to clinical samples, with similar performance to that of reverse transcription-quantitative polymerase chain reaction (RT - qPCR). The results of our proposed approach were visualized using either lateral flow strips or fluorescence for field-deployable viral diagnostics, thereby facilitating its use in endemic regions. Overall, our proposed approach showed good reliability, sensitivity, and specificity, suggesting that it is applicable for detecting other viruses in diagnosing diseases and inspecting food safety.}, }
@article {pmid39558904, year = {2024}, author = {Chen, Y and Kang, R and Jiang, Y and Zheng, Q and Yang, Y and Liu, J and Wu, G and Zhao, W and Li, Z and Peng, C and Zhang, P and Peng, F and Liu, Q and Hu, S and Luo, X and Wu, G and Cui, K and Huang, J and Wang, Y and Songyang, Z and Liang, P}, title = {Rationally designed Campylobacter jejuni Cas9 enables efficient gene activation and base editing.}, journal = {Molecular therapy. Nucleic acids}, volume = {35}, number = {4}, pages = {102366}, pmid = {39558904}, issn = {2162-2531}, abstract = {Compact and adaptable CRISPR-Cas systems enable genome engineering applications in various contexts via high-efficiency delivery. The adeno-associated virus (AAV) is a widely used delivery system. One of the most compact type II-C Cas9 orthologs-CjCas9, derived from Campylobacter jejuni, is particularly appealing for AAV delivery. However, the editing efficiency of CjCas9 limits its applications. In this study, we used structure-guided protein engineering to improve the editing efficiency of CjCas9. Subsequently, we developed a miniature transcriptional activator (LDE-CjCas9-VPR) and base editors engineered from CjCas9 (LDE-CjABE and LDE-CjCBE). LDE-CjABE effectively induced genome editing in human and mouse cells. Through AAV delivery, LDE-CjABE enhanced the on-target editing efficiency, and off-target editing was not detected in the mouse retina. Therefore, the compact size and high editing efficiency of LDE-CjCas9 broadens the target scope of transcription activation and base editing toolsets for therapeutic applications.}, }
@article {pmid39558417, year = {2024}, author = {Santos, IFM and Moreira, DS and Costa, KF and Ribeiro, JM and Murta, SMF and Santi, AMM}, title = {Ascorbate peroxidase modulation confirms key role in Leishmania infantum oxidative defence.}, journal = {Parasites &amp; vectors}, volume = {17}, number = {1}, pages = {472}, pmid = {39558417}, issn = {1756-3305}, support = {Finance Code 001//Coordena&#xe7;&#xe3;o de Aperfei&#xe7;oamento de Pessoal de N&#xed;vel Superior/ ; CNPq 309994-2023-3//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; APQ-02816-21; BDP-00657; RED-00104-22//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de Minas Gerais/ ; Chamada de Redes Colaborativas de Pesquisa do Instituto Ren&#xe9; Rachou-Fiocruz Minas (Rede FarVac)//Funda&#xe7;&#xe3;o Oswaldo Cruz/ ; }, mesh = {*Leishmania infantum/genetics/drug effects/enzymology ; *Ascorbate Peroxidases/genetics/metabolism ; *Oxidative Stress ; *Macrophages/parasitology ; Hydrogen Peroxide/pharmacology ; Animals ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Protozoan Proteins/genetics/metabolism ; Antiprotozoal Agents/pharmacology ; Mice ; Drug Resistance/genetics ; }, abstract = {BACKGROUND: Ascorbate peroxidase (APX) has emerged as a promising target for chemotherapy because of its absence in humans and crucial role in the antioxidant defence of trypanosomatids. APXs, which are class I haeme-containing enzymes, reduces hydrogen peroxide using ascorbate to produce water and monodehydroascorbate, thereby preventing cell damage caused by H2O2.<br><br>METHODS: We aimed to create an APX-knockout L. infantum line using CRISPR/Cas9. Despite unsuccessful attempts at full knockouts, we achieved deletion of chromosomal copies post-APX episomal insertion, yielding Li&#x394;chrAPX::LbAPX parasites. We performed phenotypic characterization to assess the impact of these genetic modifications, which included the determination of APX transcript expression levels using quantitative PCR, drug sensitivity, infectivity, and parasite survival in macrophages.<br><br>RESULTS: Quantitative polymerase chain reaction (PCR) analysis revealed a 10- to 13-fold reduction in APX transcript expression in Li&#x394;chrAPX::LbAPX compared with wild-type (LiWT) and APX-overexpressing (Li::Cas9::LbAPX) parasites, respectively. The episomes in those knockdown parasites remained stable even after 20 drug-free passages in vitro. Li::Cas9::LbAPX parasites showed increased resistance to trivalent antimony (Sb[III]) and isoniazid, reduced tolerance to H2O2, and unchanged macrophage infectivity compared with LiWT. In contrast, Li&#x394;chrAPX::LbAPX parasites were more sensitive to Sb[III] and isoniazid, exhibited greater susceptibility to H2O2-induced oxidative stress, and 72&#xa0;h post-infection, showed fewer infected macrophages and intracellular amastigotes compared with LiWT parasites.<br><br>CONCLUSIONS: Our findings hint at the indispensability of APX in L. infantum and raise the possibility of its potential as a therapeutic target for leishmaniasis.}, }
@article {pmid39558180, year = {2025}, author = {Wang, K and Liu, S and Zhou, S and Qileng, A and Wang, D and Liu, Y and Chen, C and Lei, C and Nie, Z}, title = {Ligand-Responsive Artificial Protein-Protein Communication for Field-Deployable Cell-Free Biosensing.}, journal = {Angewandte Chemie (International ed. in English)}, volume = {64}, number = {4}, pages = {e202416671}, doi = {10.1002/anie.202416671}, pmid = {39558180}, issn = {1521-3773}, support = {2020YFA0907500//Chongqing Municipal Key Research and Development Program of China/ ; 22034002//National Natural Science Foundation of China/ ; 22374036//National Natural Science Foundation of China/ ; 92253304//National Natural Science Foundation of China/ ; 22061160466//National Natural Science Foundation of China/ ; 22277063//National Natural Science Foundation of China/ ; 2022JJ20004//Natural Science Foundation of Hunan Province/ ; }, mesh = {*Biosensing Techniques/methods ; Ligands ; Cell-Free System ; *Transcription Factors/metabolism/chemistry ; CRISPR-Cas Systems ; }, abstract = {Natural protein-protein communications, such as those between transcription factors (TFs) and RNA polymerases/ribosomes, underpin cell-free biosensing systems operating on the transcription/translation (TXTL) paradigm. However, their deployment in field analysis is hampered by the delayed response (hour-level) and the complex composition of in vitro TXTL systems. For this purpose, we present a de novo-designed ligand-responsive artificial protein-protein communication (LIRAC) by redefining the connection between TFs and non-interacting CRISPR/Cas enzymes. By rationally designing a chimeric DNA adaptor and precisely regulating its binding affinities to both proteins, LIRAC immediately transduces target-induced TF allostery into rapid CRISPR/Cas enzyme activation within a homogeneous system. Consequently, LIRAC obviates the need for RNA/protein biosynthesis inherent to conventional TXTL-based cell-free systems, substantially reducing reaction complexity and time (from hours to 10 minutes) with improved sensitivity and tunable dynamic range. Moreover, LIRAC exhibits excellent versatility and programmability for rapidly and sensitively detecting diverse contaminants, including antibiotics, heavy metal ions, and preservatives. It also enables the creation of a multi-protein communication-based tristate logic for the intelligent detection of multiple contaminants. Integrated with portable devices, LIRAC has been proven effective in the field analysis of environmental samples and personal care products, showcasing its potential for environmental and health monitoring.}, }
@article {pmid39558174, year = {2024}, author = {Stadelmann, T and Heid, D and Jendrusch, M and Mathony, J and Aschenbrenner, S and Rosset, S and Correia, BE and Niopek, D}, title = {A deep mutational scanning platform to characterize the fitness landscape of anti-CRISPR proteins.}, journal = {Nucleic acids research}, volume = {52}, number = {22}, pages = {e103}, pmid = {39558174}, issn = {1362-4962}, support = {453202693//German Research Foundation/ ; //Aventis Foundation/ ; //German Academic Scholarship Foundation/ ; //Biltema Foundation/ ; //National Center of Competence in Research Chemical Biology/ ; 716058/ERC_/European Research Council/International ; /SNSF_/Swiss National Science Foundation/Switzerland ; }, mesh = {*CRISPR-Cas Systems ; *Escherichia coli/genetics ; *Gene Editing/methods ; Mutation ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Associated Proteins/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Deep mutational scanning is a powerful method for exploring the mutational fitness landscape of proteins. Its adaptation to anti-CRISPR proteins, which are natural CRISPR-Cas inhibitors and key players in the co-evolution of microbes and phages, facilitates their characterization and optimization. Here, we developed a robust anti-CRISPR deep mutational scanning pipeline in Escherichia coli that combines synthetic gene circuits based on CRISPR interference with flow cytometry coupled sequencing and mathematical modeling. Using this pipeline, we characterized comprehensive single point mutation libraries for AcrIIA4 and AcrIIA5, two potent inhibitors of CRISPR-Cas9. The resulting mutational fitness landscapes revealed considerable mutational tolerance for both Acrs, suggesting an intrinsic redundancy with respect to Cas9 inhibitory features, and - for AcrIIA5 - indicated mutations that boost Cas9 inhibition. Subsequent in vitro characterization suggested that the observed differences in inhibitory potency between mutant inhibitors were mostly due to changes in binding affinity rather than protein expression levels. Finally, to demonstrate that our pipeline can inform Acrs-based genome editing applications, we employed a selected subset of mutant inhibitors to increase CRISPR-Cas9 target specificity by modulating Cas9 activity. Taken together, our work establishes deep mutational scanning as a powerful method for anti-CRISPR protein characterization and optimization.}, }
@article {pmid39557836, year = {2024}, author = {Tsui, CK and Twells, N and Durieux, J and Doan, E and Woo, J and Khosrojerdi, N and Brooks, J and Kulepa, A and Webster, B and Mahal, LK and Dillin, A}, title = {CRISPR screens and lectin microarrays identify high mannose N-glycan regulators.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {9970}, pmid = {39557836}, issn = {2041-1723}, support = {F32 AG069388/AG/NIA NIH HHS/United States ; 5F32AG069388-03//U.S. Department of Health &amp; Human Services | NIH | National Institute on Aging (U.S. National Institute on Aging)/ ; }, mesh = {*Polysaccharides/metabolism ; *Mannose/metabolism/chemistry ; Humans ; Glycosylation ; *Golgi Apparatus/metabolism ; *Lectins/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; HEK293 Cells ; }, abstract = {Glycans play critical roles in cellular signaling and function. Unlike proteins, glycan structures are not templated from genetic sequences but synthesized by the concerted activity of many genes, making them historically challenging to study. Here, we present a strategy that utilizes CRISPR screens and lectin microarrays to uncover and characterize regulators of glycosylation. We applied this approach to study the regulation of high mannose glycans - the starting structure of all asparagine(N)-linked-glycans. We used CRISPR screens to uncover the expanded network of genes controlling high mannose levels, followed by lectin microarrays to fully measure the complex effect of select regulators on glycosylation globally. Through this, we elucidated how two high mannose regulators - TM9SF3 and the CCC complex - control complex N-glycosylation via regulating Golgi morphology and function. Notably, this allows us to interrogate Golgi function in-depth and reveals that similar disruption to Golgi morphology can lead to drastically different glycosylation outcomes. Collectively, this work demonstrates a generalizable approach for systematically dissecting the regulatory network underlying glycosylation.}, }
@article {pmid39557480, year = {2024}, author = {Shi, Y and Yu, Y and Lü, YL and Lü, H}, title = {Design and practice of educational experiments on genetic epistasis.}, journal = {Yi chuan = Hereditas}, volume = {46}, number = {11}, pages = {958-970}, doi = {10.16288/j.yczz.24-248}, pmid = {39557480}, issn = {0253-9772}, mesh = {*Epistasis, Genetic ; *Saccharomyces cerevisiae/genetics ; CRISPR-Cas Systems ; Mutation ; Phenotype ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; Adenine/metabolism ; }, abstract = {Genetic epistasis is a fundamental concept in genetics that describes how interactions between genes determine phenotypic traits. To enhance students' understanding and practical application of genetic epistasis, this experiment is designed and conducted using gene mutations in the adenine biosynthesis pathway of Saccharomyces cerevisiae (baker's yeast). S. cerevisiae is a classic model organism for genetic teaching experiments. In its adenine biosynthesis pathway, a mutation in the ADE2 gene leads to the accumulation of the intermediate 5'-phosphoribosylaminoimidazole (AIR), causing the cells to appear red. However, if a gene upstream of ADE2 in the adenine biosynthesis pathway (such as ADE8) is defective, the red phenotype of yeast will disappear. Conversely, a defect in a gene downstream of ADE2 (such as ADE1) does not alter the red phenotype. Therefore, ADE8 is epistatic to ADE2. In this experiment, the CRISPR-Cas9 genome editing technology is employed, allowing students to perform single knockout of ade2Δ, as well as double knockouts of ade2Δade8Δ and ade2Δade1Δ in S. cerevisiae. By observing the phenotypic changes in yeast mutants from white to red and back to white, students gain a profound understanding of the basic genetic theory of how genes determine phenotypes and the concept of epistasis in gene interactions. This experiment also enables students to master fundamental yeast genetic techniques, significantly enhancing their ability to design and conduct experiments in real research environments. This is of great significance for their future research work and academic development.}, }
@article {pmid39557225, year = {2025}, author = {Zuo, M and Du, J and Liu, Y and Chen, M and Liu, B and Li, G and Li, M and Huang, S and Yu, G}, title = {Deletion of the gsk-3β (Glycogen synthase kinase-3β) in zebrafish results in decreased susceptibility to Aeromonas hydrophila.}, journal = {Microbial pathogenesis}, volume = {198}, number = {}, pages = {107129}, doi = {10.1016/j.micpath.2024.107129}, pmid = {39557225}, issn = {1096-1208}, mesh = {Animals ; *Aeromonas hydrophila/pathogenicity ; *Zebrafish/microbiology/immunology ; *Glycogen Synthase Kinase 3 beta/genetics/metabolism ; *Gram-Negative Bacterial Infections/immunology/microbiology ; *Fish Diseases/microbiology/immunology ; *Immunity, Innate/genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Gene Knockout Techniques ; Autophagy/genetics ; Disease Models, Animal ; Gene Deletion ; Aquaculture ; Zebrafish Proteins/genetics/metabolism ; Disease Susceptibility ; }, abstract = {Aeromonas hydrophila is a significant pathogen in the field of fish farming, resulting in substantial financial losses for the aquaculture industry. As the pathogen's resistance to commercially available antibiotics continues to rise, the identification of novel antimicrobial strategies becomes increasingly crucial. This study aims to explore the modulatory impact of gsk-3β (Glycogen synthase kinase-3β) on the intrinsic immunity against Aeromonas hydrophila in zebrafish, with the objective of uncovering a new avenue for enhancing fish antimicrobial activity through gene editing. Our investigation involved an analysis of the evolutionary patterns and protein sequence of gsk-3β, elucidating its conserved characteristics in zebrafish and fish species of economic importance. In this research, CRISPR-Cas9 technology was employed to generate a zebrafish model with a knockout of gsk-3β, resulting in a decreased resistance of zebrafish to Aeromonas hydrophila (ATCC 7966) infection. Furthermore, we conducted preliminary investigations into the potential mechanisms through which gsk-3β governs antimicrobial immunity. Our findings revealed that knockout of gsk-3β resulted in diminished activation of innate immunity, antioxidant capacity, and autophagy. Hence, the findings of this study are highly significant in improving the economic benefits of aquaculture and in effectively preventing and controlling infection caused by Aeromonas hydrophila.}, }
@article {pmid39556729, year = {2024}, author = {Du, SW and Newby, GA and Salom, D and Gao, F and Menezes, CR and Suh, S and Choi, EH and Chen, PZ and Liu, DR and Palczewski, K}, title = {In vivo photoreceptor base editing ameliorates rhodopsin-E150K autosomal-recessive retinitis pigmentosa in mice.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {121}, number = {48}, pages = {e2416827121}, pmid = {39556729}, issn = {1091-6490}, support = {UG3 AI150551/AI/NIAID NIH HHS/United States ; F30 EY033642/EY/NEI NIH HHS/United States ; F30EY029136 T32GM007250 T32AR080622//HHS | NIH (NIH)/ ; F30 EY029136/EY/NEI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; UG3AI150551 U01AI142756 R35GM118062 RM1HG009490//HHS | NIH (NIH)/ ; R35 GM118062/GM/NIGMS NIH HHS/United States ; R00HL163805//HHS | NIH (NIH)/ ; U01 AI142756/AI/NIAID NIH HHS/United States ; R01 EY034501/EY/NEI NIH HHS/United States ; T32 GM007250/GM/NIGMS NIH HHS/United States ; R01 EY009339/EY/NEI NIH HHS/United States ; P30 EY034070/EY/NEI NIH HHS/United States ; T32 GM008620/GM/NIGMS NIH HHS/United States ; R00 HL163805/HL/NHLBI NIH HHS/United States ; R01EY009339 R01EY034501 P30EY034070//HHS | NIH (NIH)/ ; T32 AR080622/AR/NIAMS NIH HHS/United States ; T32GM008620 F30EY033642//HHS | NIH (NIH)/ ; }, mesh = {Animals ; *Rhodopsin/genetics/metabolism ; *Retinitis Pigmentosa/genetics/therapy/metabolism/pathology ; Mice ; *Gene Editing/methods ; Disease Models, Animal ; CRISPR-Cas Systems ; Retinal Rod Photoreceptor Cells/metabolism/pathology ; Mutation ; }, abstract = {Rhodopsin, the prototypical class-A G-protein coupled receptor, is a highly sensitive receptor for light that enables phototransduction in rod photoreceptors. Rhodopsin plays not only a sensory role but also a structural role as a major component of the rod outer segment disc, comprising over 90% of the protein content of the disc membrane. Mutations in RHO which lead to structural or functional abnormalities, including the autosomal recessive E150K mutation, result in rod dysfunction and death. Therefore, correction of deleterious rhodopsin mutations could rescue inherited retinal degeneration, as demonstrated for other visual genes such as RPE65 and PDE6B. In this study, we describe a CRISPR/Cas9 adenine base editing strategy to correct the E150K mutation and demonstrate precise in vivo editing in a Rho-E150K mouse model of autosomal recessive retinitis pigmentosa (RP). Using ultraviolet-visible spectroscopy, mass spectrometry, and the G-protein activation assay, we characterized wild-type rhodopsin and rhodopsin variants containing bystander base edits. Subretinal injection of dual-adeno-associated viruses delivering our base editing strategy yielded up to 44% Rho correction in homozygous Rho-E150K mice. Injection at postnatal day 15, but not later time points, restored rhodopsin expression, partially rescued retinal function, and partially preserved retinal structure. These findings demonstrate that in vivo base editing can restore the function of mutated structural and functional proteins in animal models of disease, including rhodopsin-associated RP and suggest that the timing of gene-editing is a crucial determinant of successful treatment outcomes for degenerative genetic diseases.}, }
@article {pmid39556419, year = {2024}, author = {Ipoutcha, T and Tsarmpopoulos, I and Gourgues, G and Baby, V and Dubos, P and Hill, GE and Arfi, Y and Lartigue, C and Thébault, P and Bonneaud, C and Sirand-Pugnet, P}, title = {Evolution of the CRISPR-Cas9 defence system in Mycoplasma gallisepticum following colonization of a novel bird host.}, journal = {Microbial genomics}, volume = {10}, number = {11}, pages = {}, pmid = {39556419}, issn = {2057-5858}, mesh = {*CRISPR-Cas Systems ; *Mycoplasma gallisepticum/genetics/pathogenicity ; Animals ; *Evolution, Molecular ; Bacteriophages/genetics ; Phylogeny ; }, abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are bacterial defences that target bacteriophages and mobile genetic elements. How these defences evolve in novel host environments remains largely unknown. We studied the evolution of the CRISPR-Cas system in Mycoplasma gallisepticum (also named Mycoplasmoides gallisepticum), a bacterial pathogen of poultry that jumped into a passerine host ~30 years ago. Over the decade following the host shift, all isolates displaying a functional CRISPR-Cas system were found not only to harbour completely new sets of spacers, but the DNA protospacer adjacent motif recognized by the main effector M. gallisepticum Cas9 (MgCas9) was also different. These changes in CRISPR-Cas diversity and specificity are consistent with a change in the community of phages and mobile elements infecting M. gallisepticum as it colonized the novel host. In the years following the host shift, we also detected a gradual rise in isolates displaying non-functional MgCas9. After 12 years, all circulating isolates harboured inactive forms only. This loss of CRISPR-Cas function comes at a time when the passerine host is known to have evolved widespread resistance, which in turn drove the evolution of increasing M. gallisepticum virulence through antagonistic coevolution. Such striking concordance in the rise of inactivated forms of CRISPR-Cas and the evolution of host resistance suggests that the inactivation of the CRISPR-Cas system was necessary for enabling adaptive bacterial responses to host-driven selection. We highlight the need to consider both host and pathogen selection pressures on bacteria for understanding the evolution of CRISPR-Cas systems and the key factors driving the emergence of a pathogenic bacterium in a novel host.}, }
@article {pmid39556313, year = {2024}, author = {Liu, S and Zhao, Y and Mo, Q and Sun, Y and Ma, H}, title = {Engineering CjCas9 for Efficient Base Editing and Prime Editing.}, journal = {The CRISPR journal}, volume = {7}, number = {6}, pages = {395-405}, doi = {10.1089/crispr.2024.0018}, pmid = {39556313}, issn = {2573-1602}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism ; Humans ; Campylobacter jejuni/genetics ; Cytosine/metabolism ; Genetic Therapy/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Adenine/metabolism ; Bacterial Proteins/genetics ; Streptococcus pyogenes/genetics/enzymology ; }, abstract = {The CRISPR-Cas9 system has been applied for clinical applications of gene therapy. Most CRISPR-based gene therapies are derived from Streptococcus pyogenes Cas9, which is challenging to package into a single adeno-associated virus vector and limits its clinical applications. Campylobacter jejuni Cas9 (CjCas9) is one of the smallest Cas9 proteins. CjCas9-mediated base editing (CjBE) efficiency varies across genomic sites, while CjCas9-mediated prime editing (CjPE) efficiency is less than 5% on average. Here we developed enhanced cytosine base editors (enCjCBEs) and adenine base editors (enCjABEs) by engineered CjCas9[P47K]. We demonstrated the robust C-to-T conversion (70% on average) by enCjCBE or A-to-G conversion (76% on average) by enCjABE. Meanwhile, we applied the CjCas9[P47K] variant to generate enhanced CjPE (enCjPE), which increases the editing efficiency 17-fold at the PRNP site over wild-type CjPE. Fusing nonspecific DNA binding protein Sso7d to enCjCas9 and MS2 stem-loop RNA aptamer to the 3-terminal of cognate pegRNA resulted in 12% editing efficiency on average with a 24-fold increase over wild-type CjPE, and we termed it SsenCjPE. The SsenCjPE can also be combined with hMLH1dn to further increase the editing efficiency and MMLV RTaseΔRnH to reduce size. Finally, we introduced an additional mutation D829R into SsenCjPE and generated SsenCjPE-M2 with a 61-fold increase of PE efficiency over wild-type at the PRNP site. In summary, enCjBEs, SsenCjPEs, or SsenCjPE-M2 are compact Cas9-derived BE or prime editors in biological research or biomedical applications.}, }
@article {pmid39556245, year = {2024}, author = {Poth, T and Schirmacher, P}, title = {[Centers for experimental animal pathology still occupy a small but valuable niche in translational medicine-time for change?].}, journal = {Pathologie (Heidelberg, Germany)}, volume = {45}, number = {Suppl 1}, pages = {31-36}, pmid = {39556245}, issn = {2731-7196}, mesh = {Animals ; *Translational Research, Biomedical/methods ; Humans ; Disease Models, Animal ; Mice ; Pathology/methods ; Germany ; }, abstract = {Since CRISPR/Cas systems can be easily used to establish new genetically engineered mouse models, application of these models in the translational research field to explore predictive and therapeutic approaches for human diseases is rising.Integrative centers for experimental animal pathology, such as the CMCP in Heidelberg and the CEP in Munich, link the veterinary and human pathology discipline and contribute substantially to meaningful study results by combining the technical, research, and diagnostic expertise for phenotyping, evaluating, and interpretating complex animal models in the context of human pathology. They provide high-quality tissue processing in a broad spectrum of standard and specialized tissue-based technologies. The histopathology platform enables animal model evaluation including phenotyping, scoring, and detecting species-specific "background lesions." By providing essential prerequisites for high-quality, reproducible, and sustainable research in translational medicine, facilities for experimental animal pathology optimally fulfill the requirements for publications in top-class professional journals, contribute to the implementation of the 3R principle for animal welfare, and help to reduce costs in the preclinical research.The importance of animal models reflecting human diseases will increase in the future but the requirements for high-quality tissue processing and comparative pathology/morphological phenotyping cannot be met by single facilities in a niche position. To cover the existing gap and expected rising demand in the German and European translational research landscape, further facilities for experimental animal pathology that are integrated in the human pathology environment should be established.}, }
@article {pmid39555930, year = {2025}, author = {Krausfeldt, LE and Samuel, PS and Smith, RP and Urakawa, H and Rosen, BH and Colwell, RR and Lopez, JV}, title = {Transcriptional profiles of Microcystis reveal gene expression shifts that promote bloom persistence in in situ mesocosms.}, journal = {Microbiology spectrum}, volume = {13}, number = {1}, pages = {e0136924}, pmid = {39555930}, issn = {2165-0497}, support = {W912HZ-19-SOI-0022//DOD | USA | U.S. Army Corps of Engineers (USACE)/ ; }, mesh = {*Microcystis/genetics/metabolism/growth &amp; development ; *Harmful Algal Bloom ; *Nitrogen/metabolism ; Gene Expression Regulation, Bacterial ; Ecosystem ; Transcriptome ; Microcystins/genetics/metabolism ; Urea/metabolism ; Photosynthesis ; Gene Expression Profiling ; }, abstract = {Harmful algal blooms caused by cyanobacteria threaten aquatic ecosystems, the economy, and human health. Previous work has tried to identify the mechanisms that allow blooms to form, focusing on the role of nutrients. However, little is known about how introduced nutrients influence gene expression in situ. To address this knowledge gap, we used in situ mesocosms initiated with water experiencing a Microcystis bloom. We added pulses of nutrients that are commonly associated with anthropogenic sources to the mesocosms for 72 hours and collected samples for metatranscriptomics to examine how the physiological function of Microcystis and bloom status changed. The addition of nitrogen (N) as urea, but not the addition of PO4, resulted in conspicuous bloom persistence for at least 9 days after the final introduction of nutrients. The addition of urea initially resulted in the upregulation of photosynthesis machinery, as well as phosphate, carbon, and N transport and metabolism. Once Microcystis presumably became N-replete, upregulation of amino acid metabolism, microcystin biosynthesis, and other processes associated with biomass generation occurred. These capacities coincided with the upregulation of toxin-antitoxin systems, CRISPR-cas genes, and transposases suggesting that phage defense and genome rearrangement are critical in bloom persistence. Overall, our results show the stepwise transcriptional response of a Microcystis bloom to the introduction of nutrients, specifically urea, as it is sustained in a natural setting. The transcriptomic shifts observed herein may serve as markers of the longevity of blooms while providing insight into why Microcystis blooms over other cyanobacteria.IMPORTANCEHarmful algal blooms represent a threat to human health and ecosystems. Understanding why blooms persist may help us develop warning indicators of bloom persistence and create novel mitigation strategies. Using mesocosm experiments initiated with water with an active bloom, we measured the stepwise transcription changes of the toxin-producing cyanobacterium Microcystis in response to the addition of nutrients that are important in causing blooms. We found that nitrogen (N), but not phosphorus, promoted bloom longevity. The initial introduction of N resulted in the upregulation of genes involved in photosynthesis and N import. At later times in the bloom, upregulation of genes involved in biomass generation, phage protection, genomic rearrangement, and toxin production was observed. Our results suggest that Microcystis first fulfills nutritional requirements before investing energy in pathways associated with growth and protection against competitors, which allowed bloom persistence more than a week after the final addition of nutrients.}, }
@article {pmid39555828, year = {2024}, author = {Fontana, M and Solomon, SD and Kachadourian, J and Walsh, L and Rocha, R and Lebwohl, D and Smith, D and Täubel, J and Gane, EJ and Pilebro, B and Adams, D and Razvi, Y and Olbertz, J and Haagensen, A and Zhu, P and Xu, Y and Leung, A and Sonderfan, A and Gutstein, DE and Gillmore, JD}, title = {CRISPR-Cas9 Gene Editing with Nexiguran Ziclumeran for ATTR Cardiomyopathy.}, journal = {The New England journal of medicine}, volume = {391}, number = {23}, pages = {2231-2241}, doi = {10.1056/NEJMoa2412309}, pmid = {39555828}, issn = {1533-4406}, mesh = {Aged ; Aged, 80 and over ; Female ; Humans ; Male ; Middle Aged ; *Amyloid Neuropathies, Familial/blood/complications/genetics/therapy ; *Cardiomyopathies/blood/genetics/therapy ; CRISPR-Cas Systems ; Gene Editing/methods ; *Genetic Therapy/adverse effects/methods ; Infusions, Intravenous/adverse effects ; Natriuretic Peptide, Brain/blood ; Peptide Fragments/blood ; *Prealbumin/genetics ; Troponin T/blood ; *Liposomes/administration &amp; dosage/adverse effects ; *RNA, Guide, Kinetoplastida/administration &amp; dosage/adverse effects/genetics ; *Nanoparticles/administration &amp; dosage/adverse effects ; }, abstract = {BACKGROUND: Transthyretin amyloidosis with cardiomyopathy (ATTR-CM) is a progressive, often fatal disease. Nexiguran ziclumeran (nex-z) is an investigational therapy based on CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease) targeting the gene encoding transthyretin (TTR).<br><br>METHODS: In this phase 1, open-label trial, we administered a single intravenous infusion of nex-z to patients with ATTR-CM. Primary objectives included assessment of the effect of nex-z on safety and pharmacodynamics, including the serum TTR level. Secondary end points included changes in N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels, high-sensitivity cardiac troponin T levels, the 6-minute walk distance, and the New York Heart Association (NYHA) class.<br><br>RESULTS: A total of 36 patients received nex-z and completed at least 12 months of follow-up. Of these patients, 50% were in NYHA class III and 31% had variant ATTR-CM. The mean percent change from baseline in the serum TTR level was -89% (95% confidence interval [CI], -92 to -87) at 28 days and -90% (95% CI, -93 to -87) at 12 months. Adverse events were reported in 34 patients. Five had transient infusion-related reactions, and two had transient liver-enzyme elevations that were assessed as treatment-related. Serious adverse events, most of which were consistent with ATTR-CM, were reported in 14 patients. The geometric mean factor change from baseline to month 12 was 1.02 (95% CI, 0.88 to 1.17) in the NT-proBNP level and 0.95 (95% CI, 0.89 to 1.01) in the high-sensitivity cardiac troponin T level. The median change from baseline to month 12 in the 6-minute walk distance was 5 m (interquartile range, -33 to 49). A total of 92% of the patients had either improvement or no change in their NYHA class.<br><br>CONCLUSIONS: In this phase 1 study involving patients with ATTR-CM, treatment with a single dose of nex-z was associated with transient infusion-related reactions and consistent, rapid, and durable reductions in serum TTR levels. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.).}, }
@article {pmid39555799, year = {2024}, author = {Inen, J and Han, CM and Farrel, DM and Bilousova, G and Kogut, I}, title = {CIRCLE-Seq for Interrogation of Off-Target Gene Editing.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {213}, pages = {}, pmid = {39555799}, issn = {1940-087X}, support = {R01 AR078551/AR/NIAMS NIH HHS/United States ; T32 AR007411/AR/NIAMS NIH HHS/United States ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; DNA/genetics ; Sequence Analysis, DNA/methods ; }, abstract = {Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of unintended cleavage sites of CRISPR-Cas9 through targeted sequencing of CRISPR-Cas9 cleaved DNA. The protocol involves circularizing genomic DNA (gDNA), which is subsequently treated with the Cas9 protein and a guide RNA (gRNA) of interest. Following treatment, the cleaved DNA is purified and prepared as a library for Illumina sequencing. The sequencing process generates paired-end reads, offering comprehensive data on each cleavage site. CIRCLE-seq provides several advantages over other in vitro methods, including minimal sequencing depth requirements, low background, and high enrichment for Cas9-cleaved gDNA. These advantages enhance sensitivity in identifying both intended and unintended cleavage events. This study provides a comprehensive, step-by-step procedure for examining the off-target activity of CRISPR-Cas9 using CIRCLE-seq. As an example, this protocol is validated by mapping genome-wide unintended cleavage sites of CRISPR-Cas9 during the modification of the AAVS1 locus. The entire CIRCLE-seq process can be completed in two weeks, allowing sufficient time for cell growth, DNA purification, library preparation, and Illumina sequencing. The input of sequencing data into the CIRCLE-seq pipeline facilitates streamlined interpretation and analysis of cleavage sites.}, }
@article {pmid39555789, year = {2024}, author = {Sheikh, MA and Afandi, FH and Iannello, G and Corneo, B and Emerald, BS and Ansari, SA}, title = {CRISPR-Cas9 Mediated Gene Deletion in Human Pluripotent Stem Cells Cultured Under Feeder-Free Conditions.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {213}, pages = {}, doi = {10.3791/67296}, pmid = {39555789}, issn = {1940-087X}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Pluripotent Stem Cells/cytology/metabolism ; Gene Deletion ; Gene Knockout Techniques/methods ; Human Embryonic Stem Cells/cytology/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Lentivirus/genetics ; Gene Editing/methods ; }, abstract = {The CRISPR-Cas9 system for genome editing has revolutionized gene function studies in mammalian cells, including stem cells. However, the practical application of this technique, particularly in pluripotent stem cells, presents certain challenges, such as being time- and labor-intensive and having low editing efficiency. Here, we describe the generation of a CRISPR-mediated gene knockout in a human embryonic stem cell (hESC) line stably expressing sgRNAs for the L2HGDH gene, using a highly efficient and stable lentiviral-mediated gene delivery system. The sgRNAs targeting exon 1 of the L2HGDH gene were chemically synthesized and cloned into the lentiCRISPR v2-puro vector, which combines the constitutive expression of sgRNAs with Cas9 in a highly efficient single-vector system to achieve higher lentiviral titers for hESC infection and stable selection using puromycin. Puromycin-selected cells were further expanded, and single-cell clones were obtained using the limited dilution method. The single clones were expanded, and several homozygous knockout clones for the L2HGDH gene were obtained, as confirmed by a 100% reduction in L2HGDH expression using Western blot analysis. Furthermore, using MSBSP-PCR, the CRISPR mutation site was mapped upstream of the PAM recognition sequence of Cas9 in the selected homozygous clones. Sanger sequencing was performed to analyze the exact insertions/deletions, and functional characterization of the clones was conducted. This method produced a significantly higher percentage of homozygous deletions compared to previously reported non-viral gene delivery methods. Although this report focuses on the L2HGDH gene, this robust and cost-effective approach can be used to create homozygous knockouts for other genes in pluripotent stem cells for gene function studies.}, }
@article {pmid39555173, year = {2024}, author = {Chuksina, TA and Fatkulin, AA and Sorokina, NP and Smykov, IT and Kuraeva, EV and Masagnaya, ES and Smagina, KA and Shkurnikov, MY}, title = {Genome Characterization of Two Novel Lactococcus lactis Phages vL_296 and vL_20A.}, journal = {Acta naturae}, volume = {16}, number = {3}, pages = {102-109}, pmid = {39555173}, issn = {2075-8251}, abstract = {Fermented dairy products are produced using starter cultures. They ferment milk to create products with a certain texture, aroma, and taste. However, the lactic acid bacteria used in this production are prone to bacteriophage infection. We examined the genomes of two newly discovered bacteriophage species that were isolated from cheese whey during the cheesemaking process. We have determined the species and the lytic spectrum of these bacteriophages. Phages vL_20A and vL_296 were isolated using lactococcal indicator cultures. They have unique lytic spectra: of the 21 possible identified host bacteria, only four are shared amongst them. The vL_20A and vL_296 genomes comprise linear double-stranded DNA lengths with 21,909 and 22,667 nucleotide pairs, respectively. Lactococcus phage bIL67 (ANI 93.3 and 92.6, respectively) is the closest to the phages vL_20A and vL_296. The analysis of the CRISPR spacers in the genomes of starter cultures did not reveal any phage-specific vL_20A or vL_296 among them. This study highlights the biodiversity of L. lactis phages, their widespread presence in dairy products, and their virulence. However, the virulence of phages is balanced by the presence of a significant number of bacterial strains with different sensitivities to phages in the starter cultures due to the bacterial immune system.}, }
@article {pmid39554814, year = {2024}, author = {Zhou, Y and Yan, Z and Zhou, S and Li, W and Yang, H and Chen, H and Deng, Z and Zeng, Q and Sun, P and Wu, Y}, title = {ERA-CRISPR/Cas12a-based, fast and specific diagnostic detection for Chlamydia pneumoniae.}, journal = {Frontiers in cellular and infection microbiology}, volume = {14}, number = {}, pages = {1477422}, pmid = {39554814}, issn = {2235-2988}, mesh = {*Chlamydophila pneumoniae/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; Humans ; *Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; Molecular Diagnostic Techniques/methods ; CRISPR-Associated Proteins/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Chlamydophila Infections/diagnosis ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; }, abstract = {Chlamydia pneumoniae (C. pneumoniae) is a specialized intracellular parasitic pathogen capable of causing pneumonia, sinusitis, bronchitis, and other respiratory diseases, which pose significant public health challenges. Therefore, rapid, accurate, and sensitive diagnosis is crucial for the prevention and treatment of respiratory diseases caused by C. pneumoniae. In this study, we combined enzymatic recombination amplification (ERA) with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 12a system (CRISPR/Cas12a) to develop a dual detection platform termed the Cpn-ERA-CRISPR/Cas12a dual system. This system integrates both the ERA-CRISPR/Cas12a fluorescence system and the ERA-CRISPR/Cas12a lateral flow system. Detection results can be measured using a fluorescence detector or observed with the naked eye on lateral flow strips. The fluorescence system and the lateral flow system detect C. pneumoniae in 30 minutes and 15 minutes, respectively. This dual system exhibits no cross-reactivity with the other seven pathogens, demonstrating high specificity, and achieves a sensitivity of 10[0] copies/µL. Additionally, the Cpn-ERA-CRISPR/Cas12a dual system was employed to analyze 39 clinical samples, comprising 19 positive and 20 negative samples. The detection rate for positive samples was 100%, with no positive results in the negative samples, indicating a high level of concordance with qPCR results. In summary, the Cpn-ERA-CRISPR/Cas12a dual system represents a novel tool for diagnosing C. pneumoniae and holds promising application potential in grassroots community hospitals.}, }
@article {pmid39554198, year = {2024}, author = {Bamidele, N and Ansodaria, A and Chen, Z and Cheng, H and Panwala, R and Jazbec, E and Sontheimer, EJ}, title = {Rational Design of Enhanced Nme2Cas9 and Nme2[Smu]Cas9 Nucleases and Base Editors.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, pmid = {39554198}, issn = {2692-8205}, support = {F31 GM143879/GM/NIGMS NIH HHS/United States ; R01 GM150273/GM/NIGMS NIH HHS/United States ; R25 GM113686/GM/NIGMS NIH HHS/United States ; }, abstract = {CRISPR-Cas genome editing tools enable precise, RNA-guided modification of genomes within living cells. The most clinically advanced genome editors are Cas9 nucleases, but many nuclease technologies provide only limited control over genome editing outcomes. Adenine base editors (ABEs) and cytosine base editors (CBEs) enable precise and efficient nucleotide conversions of A:T-to-G:C and C:G-to-T:A base pairs, respectively. Therapeutic use of base editors (BEs) provides an avenue to correct approximately 30% of human pathogenic variants. Nonetheless, factors such as protospacer adjacent motif (PAM) availability, accuracy, product purity, and delivery limit the full therapeutic potential of BEs. We previously developed Nme2Cas9 and its BE derivatives, including ABEs compatible with single adeno-associated virus (AAV) vector delivery, in part to enable editing near N4CC PAMs. Further engineering yielded domain-inlaid BEs with enhanced activity, as well as Nme2Cas9/SmuCas9 chimeras that target single-cytidine (N4C) PAMs. Here we further enhance Nme2Cas9 and Nme2[Smu]Cas9 editing effectors for improved efficiency and vector compatibility through site-directed mutagenesis and deaminase linker optimization. Finally, we define the editing and specificity profiles of the resulting variants by using paired guide-target libraries.}, }
@article {pmid39552996, year = {2024}, author = {Shetty, A and Kwas, H and Rajhi, H and Rangareddy, H and Fryer, J}, title = {Revolutionizing Tuberculosis Management With Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas Technology: A Comprehensive Literature Review.}, journal = {Cureus}, volume = {16}, number = {10}, pages = {e71697}, pmid = {39552996}, issn = {2168-8184}, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have gained attention for their revolutionary potential in tuberculosis (TB) management, providing a novel approach to both diagnostics and treatment. This technology, renowned for its ability to accurately target and modify genetic material, offers a promising solution to the limitations of current TB diagnostic methods, which often rely on time-consuming culture techniques or polymerase chain reaction (PCR)-based assays. One of the key advantages of CRISPR-Cas systems is their high specificity and sensitivity, making them well-suited for detecting Mycobacterium tuberculosis, even in low-bacterial-load samples. Techniques such as CRISPR-Cas12 and Cas13 have been employed for rapid detection, utilizing their trans-cleavage activity to produce a fluorescent signal upon recognition of the TB genome. Furthermore, these methods often use isothermal amplification techniques like recombinase polymerase amplification (RPA) or loop-mediated isothermal amplification (LAMP), which require less equipment compared to traditional PCR. Beyond diagnostics, CRISPR-Cas technologies show promise in studying TB resistance mechanisms and potentially treating drug-resistant strains. Genome-editing capabilities enable researchers to manipulate the M. tuberculosis genome, investigating genes linked to virulence or antibiotic resistance. Although challenges such as the development of multiplexed CRISPR assays for detecting multiple mutations simultaneously remain, advancements continue to improve the technology's practicality for clinical use. Incorporating CRISPR into TB management could enhance early detection, inform personalized treatment, and potentially contribute to developing more effective therapies, especially in regions where TB remains a significant public health threat.}, }
@article {pmid39552005, year = {2024}, author = {Gu, X and Zhang, J and Liang, J and Liu, X and He, X and Jin, X and Yan, C and Wang, L and Song, C}, title = {CRISPR/Cas13a Trans-Cleavage and Catalytic Hairpin Assembly Cascaded Signal Amplification Powered SERS Aptasensor for Ultrasensitive Detection of Gastric Cancer-Derived Exosomes.}, journal = {Analytical chemistry}, volume = {96}, number = {47}, pages = {18681-18689}, doi = {10.1021/acs.analchem.4c03063}, pmid = {39552005}, issn = {1520-6882}, mesh = {Humans ; *Stomach Neoplasms/diagnosis ; *Aptamers, Nucleotide/chemistry ; *Spectrum Analysis, Raman ; *Exosomes/chemistry ; *Silver/chemistry ; CRISPR-Cas Systems ; Mucin-1/blood/metabolism ; Biosensing Techniques ; Nanotubes/chemistry ; Limit of Detection ; Cell Line, Tumor ; }, abstract = {Cancer-derived exosomes carry a large number of specific molecular profiles from cancer cells and have emerged as ideal biomarkers for early cancer diagnosis. Accurate detection of ultralow-abundance exosomes in complex biological samples remains a great challenge. Herein, a novel SERS aptasensor powered by cascaded signal amplification of CRISPR/Cas13a trans-cleavage and catalytic hairpin assembly (CHA) was proposed for ultrasensitive detection of gastric cancer-derived exosomes, which included hairpin-structured recognition aptamers (MUC1-apt), cascaded signal amplification (i.e., CRISPR/Cas13a trans-cleavage and CHA), SERS tags, and silver nanorods (AgNRs) sensing chip. In the presence of SGC-7901 cell-derived exosomes, MUC1-apt specifically bound to MUC1 proteins highly expressed on exosomes via its contained MUC1 aptamer with its exposed RNA fragments activating the CRISPR/Cas13a trans-cleavage to cleave the uracil-modified hairpin reporter, and the cleavage products further triggered the downstream CHA reaction to form numerous duplexes, which can, in turn, capture a large number of SERS tags onto the AgNRs sensing chip to generate a significantly enhanced Raman signal. The proposed SERS aptasensor exhibits good performance on analysis of exosomes, i.e., rapid response within 60 min, single-particle sensitive detection from a 2 μL biological sample, good specificity in distinguishing SGC-7901 cell-derived exosomes against other exosomes, good uniformity, excellent repeatability, and satisfactory recoveries in human serum, and good universality to expand the detection of multiplex exosomes, which indicates that the SERS aptasensor provides a valuable reference for clinical diagnosis of early cancer.}, }
@article {pmid39551936, year = {2024}, author = {Chou-Zheng, L and Howell, O and Boyle, TA and Hossain, M and Walker, FC and Sheriff, EK and Aslan, B and Hatoum-Aslan, A}, title = {AcrIIIA1 is a protein-RNA anti-CRISPR complex that targets core Cas and accessory nucleases.}, journal = {Nucleic acids research}, volume = {52}, number = {22}, pages = {13490-13514}, pmid = {39551936}, issn = {1362-4962}, support = {R01 AI173022/AI/NIAID NIH HHS/United States ; 1020298//Burroughs Wellcome Fund/ ; 1R01AI173022/NH/NIH HHS/United States ; 2054755//U.S. National Science Foundation/ ; }, mesh = {*CRISPR-Associated Proteins/metabolism/genetics ; *CRISPR-Cas Systems ; Bacterial Proteins/metabolism/genetics ; Bacteriophages/genetics ; Viral Proteins/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Escherichia coli/genetics/metabolism ; }, abstract = {Clustered regularly-interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins protect bacteria and archaea from their viruses, and anti-CRISPRs (Acrs) are small virus-encoded proteins that inhibit CRISPR-Cas immunity. Over 80 families of Acrs have been described to date; however, only three of these subvert Type III CRISPR-Cas immunity. Type III systems employ a complex network of Cas and accessory nucleases to degrade viral nucleic acids. Here, we discover and characterize AcrIIIA1, the first Type III-A specific anti-CRISPR protein. We demonstrate that AcrIIIA1 binds to Csm2 within the Cas10-Csm effector complex and attenuates Cas10's DNase activity and second messenger production. Additionally, AcrIIIA1 associates with fragmented t(m)RNAs (acrIIIA1-RNAs), and we show that they co-purify with the Cas10-Csm complex during phage infection. Although the precise role(s) of acrIIIA1-RNAs remain unclear, we found that they bind stably to RNase R, a host-encoded nuclease known to bolster immunity, and RNase R has the capacity to degrade them. Altogether, our results support a model in which AcrIIIA1 and its associated RNAs target both core Cas and accessory nucleases to provide robust protection against Type III CRISPR-Cas immunity.}, }
@article {pmid39551873, year = {2025}, author = {Krishnamoorthy, V and Daly, J and Kim, J and Piatnitca, L and Yuen, KA and Kumar, B and Taherzadeh Ghahfarrokhi, M and Bui, TQT and Azadi, P and Vu, LP and Wisnovsky, S}, title = {The glycosyltransferase ST3GAL4 drives immune evasion in acute myeloid leukemia by synthesizing ligands for the glyco-immune checkpoint receptor Siglec-9.}, journal = {Leukemia}, volume = {39}, number = {2}, pages = {346-359}, pmid = {39551873}, issn = {1476-5551}, mesh = {Humans ; *Leukemia, Myeloid, Acute/immunology/pathology/metabolism/genetics ; *Sialyltransferases/metabolism/genetics ; *Sialic Acid Binding Immunoglobulin-like Lectins/metabolism ; Ligands ; *Immune Evasion ; *Antigens, CD/metabolism ; beta-Galactoside alpha-2,3-Sialyltransferase ; Glycosylation ; Cell Line, Tumor ; CRISPR-Cas Systems ; }, abstract = {Immunotherapy has demonstrated promise as a treatment for acute myeloid leukemia (AML). However, there is still an urgent need to identify new molecules that inhibit the immune response to AML. Most prior research in this area has focused on protein-protein interaction interfaces. While carbohydrates also regulate immune recognition, the role of cell-surface glycans in driving AML immune evasion is comparatively understudied. The Siglecs, for example, are an important family of inhibitory, glycan-binding signaling receptors that have emerged as prime targets for cancer immunotherapy in recent years. In this study, we find that AML cells express ligands for the receptor Siglec-9 at high levels. Integrated CRISPR genomic screening and clinical bioinformatic analysis identified ST3GAL4 as a potential driver of Siglec-9 ligand expression in AML. Depletion of ST3GAL4 by CRISPR-Cas9 knockout (KO) dramatically reduced the expression of Siglec-9 ligands in AML cells. Mass spectrometry analysis of cell-surface glycosylation in ST3GAL4 KO cells revealed that Siglec-9 primarily binds N-linked sialoglycans on these cell types. Finally, we found that ST3GAL4 KO enhanced the sensitivity of AML cells to phagocytosis by Siglec-9-expressing macrophages. This work reveals a novel axis of immune evasion and implicates ST3GAL4 as a possible target for immunotherapy in AML.}, }
@article {pmid39551783, year = {2024}, author = {Auradkar, A and Corder, RM and Marshall, JM and Bier, E}, title = {A self-eliminating allelic-drive reverses insecticide resistance in Drosophila leaving no transgene in the population.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {9961}, pmid = {39551783}, issn = {2041-1723}, support = {R01 AI162911/AI/NIAID NIH HHS/United States ; R01 GM117321/GM/NIGMS NIH HHS/United States ; R01 GM144608/GM/NIGMS NIH HHS/United States ; }, mesh = {Animals ; *Insecticide Resistance/genetics ; *Alleles ; *Drosophila melanogaster/genetics ; *Transgenes ; Male ; Female ; CRISPR-Cas Systems ; Drosophila Proteins/genetics/metabolism ; Animals, Genetically Modified ; Voltage-Gated Sodium Channels/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Drive Technology/methods ; }, abstract = {Insecticide resistance (IR) poses a significant global challenge to public health and welfare. Here, we develop a locally-acting unitary self-eliminating allelic-drive system, inserted into the Drosophila melanogaster yellow (y) locus. The drive cassette encodes both Cas9 and a single gRNA to bias inheritance of the favored wild-type (1014 L) allele over the IR (1014 F) variant of the voltage-gated sodium ion channel (vgsc) target locus. When enduring a fitness cost, this transiently-acting drive can increase the frequency of the wild-type allele to 100%, depending on its seeding ratio, before being eliminated from the population. However, in a fitness-neutral "hover" mode, the drive maintains a constant frequency in the population, completely converting IR alleles to wild-type, even at low initial seeding ratios.}, }
@article {pmid39551276, year = {2025}, author = {Xu, L and Lu, X and Li, Y and Butaye, P and Qin, S and Wang, Z and Li, R}, title = {Eliminating the tigecycline resistance RND efflux pump gene cluster tmexCD-toprJ in bacteria using CRISPR/Cas9.}, journal = {International journal of antimicrobial agents}, volume = {65}, number = {1}, pages = {107390}, doi = {10.1016/j.ijantimicag.2024.107390}, pmid = {39551276}, issn = {1872-7913}, mesh = {*Tigecycline/pharmacology ; *CRISPR-Cas Systems ; *Plasmids/genetics ; *Multigene Family/genetics ; *Anti-Bacterial Agents/pharmacology ; *Microbial Sensitivity Tests ; Drug Resistance, Bacterial/genetics ; Drug Resistance, Multiple, Bacterial/genetics ; Membrane Transport Proteins/genetics ; Bacterial Proteins/genetics ; Humans ; Electroporation ; Conjugation, Genetic ; }, abstract = {OBJECTIVES: Tigecycline, a last-resort antibiotic in the tetracycline class, has been effective in treating infections caused by multidrug-resistant bacteria. However, the emergence of the tigecycline resistance gene cluster tmexCD-toprJ, which encodes a resistance-nodulation-division efflux pump, has significantly limited its therapeutic effectiveness. This study aims to explore the potential of CRISPR/Cas9-based plasmids to target and cleave tmexCD-toprJ gene cluster from bacterial plasmids and chromosomal integrative conjugative elements (ICEs), respectively.<br><br>METHODS: We developed two CRISPR/Cas9-based plasmids, pCas9Kill and pCas9KillTS. The pCas9Kill plasmid designed to eliminate tmexCD-toprJ from plasmids through electroporation, while the pCas9KillTS plasmid, delivered through conjugation, targeted tmexCD-toprJ within ICEs on the bacterial chromosome. The plasmid modifications were assessed using nanopore long-read sequencing.<br><br>RESULTS: Electroporation with the pCas9Kill plasmid resulted in the removal of tmexCD-toprJ from plasmids, restoring bacterial susceptibility to tigecycline. Nanopore sequencing revealed that the plasmids were repaired by insertion sequences after tmexCD-toprJ removal. In contrast, the pCas9KillTS plasmid introduced via conjugation to target tmexCD-toprJ gene cluster on ICEs within the chromosome. This approach led to chromosomal cleavage and subsequent bacterial cell death.<br><br>CONCLUSION: Our results demonstrate that both plasmids effectively inactivated tmexCD-toprJ, with pCas9Kill restoring tigecycline susceptibility in plasmid-bearing strains and pCas9KillTS causing targeted cell death in chromosomal ICE-harbouring bacteria. This study highlights the potential of CRISPR/Cas9 systems in addressing antibiotic resistance, providing a promising strategy to combat tigecycline-resistant pathogens.}, }
@article {pmid39549700, year = {2024}, author = {Siozios, S and Nadal-Jimenez, P and Azagi, T and Sprong, H and Frost, CL and Parratt, SR and Taylor, G and Brettell, L and Liew, KC and Croft, L and King, KC and Brockhurst, MA and Hypša, V and Novakova, E and Darby, AC and Hurst, GDD}, title = {Genome dynamics across the evolutionary transition to endosymbiosis.}, journal = {Current biology : CB}, volume = {34}, number = {24}, pages = {5659-5670.e7}, doi = {10.1016/j.cub.2024.10.044}, pmid = {39549700}, issn = {1879-0445}, mesh = {*Symbiosis/genetics ; *Genome, Bacterial ; Biological Evolution ; Gene Transfer, Horizontal ; Evolution, Molecular ; Enterobacteriaceae/genetics/physiology ; }, abstract = {Endosymbiosis-where a microbe lives and replicates within a host-is an important contributor to organismal function that has accelerated evolutionary innovations and catalyzed the evolution of complex life. The evolutionary processes associated with transitions to endosymbiosis, however, are poorly understood. Here, we leverage the wide diversity of host-associated lifestyles of the genus Arsenophonus to reveal the complex evolutionary processes that occur during the transition to a vertically transmitted endosymbiotic lifestyle from strains maintained solely by horizontal (infectious) transmission. We compared the genomes of 38 strains spanning diverse lifestyles from horizontally transmitted pathogens to obligate interdependent endosymbionts. Among culturable strains, we observed those with vertical transmission had larger genome sizes than closely related horizontally transmitting counterparts, consistent with evolutionary innovation and the rapid gain of new functions. Increased genome size was a consequence of prophage and plasmid acquisition, including a cargo of type III effectors, alongside the concomitant loss of CRISPR-Cas genome defense systems, enabling mobile genetic element expansion. Persistent endosymbiosis was also associated with loss of type VI secretion, which we hypothesize to be a consequence of reduced microbe-microbe competition. Thereafter, the transition to endosymbiosis with strict vertical inheritance was associated with the expected relaxation of purifying selection, gene pseudogenization, metabolic degradation, and genome reduction. We argue that reduced phage predation in endosymbiotic niches drives the loss of genome defense systems driving rapid genome expansion upon the adoption of endosymbiosis and vertical transmission. This remodeling enables rapid horizontal gene transfer-mediated evolutionary innovation and precedes the reductive evolution traditionally associated with adaptation to endosymbiosis.}, }
@article {pmid39549144, year = {2024}, author = {Javaid, A and Hameed, S and Li, L and Zhang, Z and Zhang, B and -Rahman, MU}, title = {Can nanotechnology and genomics innovations trigger agricultural revolution and sustainable development?.}, journal = {Functional &amp; integrative genomics}, volume = {24}, number = {6}, pages = {216}, pmid = {39549144}, issn = {1438-7948}, mesh = {*Agriculture/methods ; CRISPR-Cas Systems ; *Crops, Agricultural/genetics ; Gene Editing/methods ; *Genomics/methods ; *Nanotechnology/methods ; Sustainable Development ; }, abstract = {At the dawn of new millennium, policy makers and researchers focused on sustainable agricultural growth, aiming for food security and enhanced food quality. Several emerging scientific innovations hold the promise to meet the future challenges. Nanotechnology presents a promising avenue to tackle the diverse challenges in agriculture. By leveraging nanomaterials, including nano fertilizers, pesticides, and sensors, it provides targeted delivery methods, enhancing efficacy in both crop production and protection. This integration of nanotechnology with agriculture introduces innovations like disease diagnostics, improved nutrient uptake in plants, and advanced delivery systems for agrochemicals. These precision-based approaches not only optimize resource utilization but also reduce environmental impact, aligning well with sustainability objectives. Concurrently, genetic innovations, including genome editing and advanced breeding techniques, enable the development of crops with improved yield, resilience, and nutritional content. The emergence of precision gene-editing technologies, exemplified by CRISPR/Cas9, can transform the realm of genetic modification and enabled precise manipulation of plant genomes while avoiding the incorporation of external DNAs. Integration of nanotechnology and genetic innovations in agriculture presents a transformative approach. Leveraging nanoparticles for targeted genetic modifications, nanosensors for early plant health monitoring, and precision nanomaterials for controlled delivery of inputs offers a sustainable pathway towards enhanced crop productivity, resource efficiency, and food safety throughout the agricultural lifecycle. This comprehensive review outlines the pivotal role of nanotechnology in precision agriculture, emphasizing soil health improvement, stress resilience against biotic and abiotic factors, environmental sustainability, and genetic engineering.}, }
@article {pmid39549122, year = {2025}, author = {Yoshimura, M and Ishida, T}, title = {Generation of viable hypomorphic and null mutant plants via CRISPR-Cas9 targeting mRNA splicing sites.}, journal = {Journal of plant research}, volume = {138}, number = {1}, pages = {189-196}, pmid = {39549122}, issn = {1618-0860}, support = {JP23K05803//Japan Society for the Promotion of Science London/ ; }, mesh = {*CRISPR-Cas Systems ; *Arabidopsis/genetics/growth &amp; development ; *Arabidopsis Proteins/genetics/metabolism ; Gene Editing ; RNA Splice Sites/genetics ; RNA Splicing/genetics ; Mutation ; RNA, Messenger/genetics/metabolism ; }, abstract = {Genetic analysis is important for modern plant molecular biology, and in this regard, the existence of specific mutants is crucial. While genome editing technologies, particularly CRISPR-Cas9, have revolutionized plant molecular biology by enabling precise gene disruption, knockout methods are ineffective for lethal genes, necessitating alternatives like gene knockdown. This study demonstrates the practical generation of a hypomorphic mutant allele, alongside severe null mutant alleles, via the targeting of mRNA splicing sites using CRISPR-Cas9. The Arabidopsis HIGH PLOIDY 2 (HPY2) encodes a yeast NSE2 ortholog, part of the conserved eukaryotic SMC5/6 complex, with SUMO E3 ligase activity essential for cell cycle progression and plant development. Loss-of-function HPY2 mutants exhibit severe dwarfism and seedling lethality, making functional analysis challenging. To overcome these limitations, we created HPY2 knockdown mutants as novel tools to investigate gene function. Of the three mutant alleles, the hpy2-cr1 and hpy2-cr2 mutants resembled the existing severe hpy2-1 allele, both harboring a single base pair insertion in one exon, causing significant root shortening and seedling lethality. In contrast, the hypomorphic mutant hpy2-cr3, which has a five bp deletion at an intron-exon junction, showed relatively longer root growth and survived until the reproductive stage. RT-PCR analysis of hpy2-cr3 revealed atypical mRNAs producing truncated polypeptides that retained some HPY2 function, explaining the milder phenotype. These results establish the successful generation of novel hypomorphic mutant alleles critical for studying the lethal gene HPY2, and demonstrate the usefulness of CRISPR-Cas9 for producing viable hypomorphic mutants for investigating complex genetic interactions.}, }
@article {pmid39548711, year = {2024}, author = {Derollez, E and Lesterlin, C and Bigot, S}, title = {Design, potential and limitations of conjugation-based antibacterial strategies.}, journal = {Microbial biotechnology}, volume = {17}, number = {11}, pages = {e70050}, pmid = {39548711}, issn = {1751-7915}, support = {ANR-21-AAMR-0007-01//Joint Programming Initiative on Antimicrobial Resistance/ ; EQU202103012587//Fondation pour la Recherche M&#xe9;dicale/ ; ANR-22-CE35-0017//Agence Nationale de la Recherche/ ; }, mesh = {*Anti-Bacterial Agents/pharmacology/chemistry ; *Conjugation, Genetic ; *Bacteria/drug effects/genetics ; CRISPR-Cas Systems ; Drug Resistance, Bacterial ; Humans ; }, abstract = {Over the past few decades, the global spread of antimicrobial resistance has underscored the urgent need to develop innovative non-antibiotic antibacterial strategies and to reduce antibiotic use worldwide. In response to this challenge, several methods have been developed that rely on gene transfer by conjugation to deliver toxic compounds or CRISPR systems specifically designed to kill or resensitize target bacterial strains to antibiotics. This review explores the design, potential, and limitations of these conjugation-based antibacterial strategies, focusing on the recent advances in the delivery of CRISPR systems as antibacterial effectors.}, }
@article {pmid39548497, year = {2024}, author = {Piaggio, AJ and Gierus, L and Taylor, DR and Holmes, ND and Will, DJ and Gemmell, NJ and Thomas, PQ}, title = {Building an eDNA surveillance toolkit for invasive rodents on islands: can we detect wild-type and gene drive Mus musculus?.}, journal = {BMC biology}, volume = {22}, number = {1}, pages = {261}, pmid = {39548497}, issn = {1741-7007}, mesh = {Animals ; *Introduced Species ; Mice ; *Gene Drive Technology/methods ; *DNA, Environmental/analysis/genetics ; Islands ; }, abstract = {BACKGROUND: Invasive management strategies range from preventing new invasive species incursions to eliminating established populations, with all requiring effective monitoring to guide action. The use of DNA sampled from the environment (eDNA) is one such tool that provides the ability to surveille and monitor target invasive species through passive sampling. Technology being developed to eliminate invasive species includes genetic biocontrol in the form of gene drive. This approach would drive a trait through a population and could be used to eliminate or modify a target population. Once a gene drive organism is released into a population then monitoring changes in density of the target species and the spread of the drive in the population would be critical.<br><br>RESULTS: In this paper, we use invasive Mus musculus as a model for development of an eDNA assay that detects wild-type M. musculus and gene drive M. musculus. We demonstrate successful development of an assay where environmental samples could be used to detect wild-type invasive M. musculus and the relative density of wild-type to gene drive M. musculus.<br><br>CONCLUSIONS: The development of a method that detects both wild-type M. musculus and a gene drive M. musculus (tCRISPR) from environmental samples expands the utility of environmental DNA. This method provides a tool that can immediately be deployed for invasive wild M. musculus management across the world. This is a proof-of-concept that a genetic biocontrol construct could be monitored using environmental samples.}, }
@article {pmid39547516, year = {2025}, author = {Goolab, S and Terburgh, K and du Plessis, C and Scholefield, J and Louw, R}, title = {CRISPR-Cas9 mediated knockout of NDUFS4 in human iPSCs: A model for mitochondrial complex I deficiency.}, journal = {Biochimica et biophysica acta. Molecular basis of disease}, volume = {1871}, number = {2}, pages = {167569}, doi = {10.1016/j.bbadis.2024.167569}, pmid = {39547516}, issn = {1879-260X}, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Electron Transport Complex I/deficiency/metabolism/genetics ; *CRISPR-Cas Systems ; *Mitochondrial Diseases/genetics/metabolism/pathology ; Gene Knockout Techniques ; Mitochondria/metabolism/genetics ; }, abstract = {Mitochondrial diseases, often caused by defects in complex I (CI) of the oxidative phosphorylation system, currently lack curative treatments. Human-relevant, high-throughput drug screening platforms are crucial for the discovery of effective therapeutics, with induced pluripotent stem cells (iPSCs) emerging as a valuable technology for this purpose. Here, we present a novel iPSC model of NDUFS4-related CI deficiency that displays a strong metabolic phenotype in the pluripotent state. Human iPSCs were edited using CRISPR-Cas9 to target the NDUFS4 gene, generating isogenic NDUFS4 knockout (KO) cell lines. Sanger sequencing detected heterozygous biallelic deletions, whereas no indel mutations were found in isogenic control cells. Western blotting confirmed the absence of NDUFS4 protein in KO iPSCs and CI enzyme kinetics showed a ~56 % reduction in activity compared to isogenic controls. Comprehensive metabolomic profiling revealed a distinct metabolic phenotype in NDUFS4 KO iPSCs, predominantly associated with an elevated NADH/NAD[+] ratio, consistent with alterations observed in other models of mitochondrial dysfunction. Additionally, β-lapachone, a recognized NAD[+] modulator, alleviated reductive stress in KO iPSCs by modifying the redox state in both the cytosol and mitochondria. Although undifferentiated iPSCs cannot fully replicate the complex cellular dynamics of the disease seen in vivo, these findings highlight the utility of iPSCs in providing a relevant metabolic milieu that can facilitate early-stage, high-throughput exploration of therapeutic strategies for mitochondrial dysfunction.}, }
@article {pmid39547360, year = {2025}, author = {Xie, L and Fan, N and Ding, X and Zhang, T and Wang, W and Ji, P and Wu, H}, title = {Comparative transcriptomic and metabolomic analysis of FTO knockout and wild-type porcine iliac artery endothelial cells.}, journal = {Gene}, volume = {936}, number = {}, pages = {149094}, doi = {10.1016/j.gene.2024.149094}, pmid = {39547360}, issn = {1879-0038}, mesh = {Animals ; *Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics/metabolism ; *Endothelial Cells/metabolism ; Swine ; *Gene Knockout Techniques ; *Transcriptome ; *Iliac Artery/metabolism ; Gene Expression Profiling/methods ; Metabolomics/methods ; Metabolome ; Cell Line ; CRISPR-Cas Systems ; }, abstract = {The fat mass and obesity associated (FTO) gene, previously identified as a pivotal genetic locus associated with adiposity, has recently been linked to various cancers. In this study, we established an FTO knockout (KO) cell line in porcine iliac artery endothelial cells (PIECs) utilizing CRISPR/Cas9 technology to systematically investigate the gene's function and effect through transcriptomic and metabolomic analysis. Our results revealed significant gene expression and metabolic profiles differences between the FTO KO and wild-type (WT) cells. Furthermore, enrichment analysis highlighted the involvement of differentially expressed genes in metabolic processes, cellular components, and molecular functions, as well as in complement and coagulation cascades, mineral absorption, glutathione metabolism, insulin signaling, fluid shear stress, and atherosclerosis pathways. The metabolomic profiling revealed clear distinctions between the FTO KO and WT cells, indicating profound modifications in cellular metabolism. Correlation analysis of transcriptomic and metabolomic data revealed a significant association between six metabolites and twenty genes, with melatonin showing specific correlations with the expression of several genes, indicating a complex regulatory network between gene expression and metabolic changes. This study provides a foundation for further research on the FTO gene's role in cellular processes and molecular mechanisms underlying physiological and pathological conditions.}, }
@article {pmid39547205, year = {2024}, author = {Maniatis, T}, title = {From bacterial operons to gene therapy: 50 years of the journal Cell.}, journal = {Cell}, volume = {187}, number = {23}, pages = {6417-6420}, doi = {10.1016/j.cell.2024.10.037}, pmid = {39547205}, issn = {1097-4172}, mesh = {Animals ; Humans ; *Anemia, Sickle Cell/therapy/genetics ; Bacteria/genetics/metabolism ; beta-Thalassemia/therapy/genetics ; CRISPR-Cas Systems/genetics ; DNA, Recombinant/genetics ; *Genetic Therapy/methods ; History, 20th Century ; History, 21st Century ; *Operon ; }, abstract = {Recombinant DNA technology has profoundly advanced virtually every aspect of biological and medical sciences, from basic research to biotechnology. Here, I discuss conceptual connections linking fundamental discoveries that were enabled by the technology, advances in the understanding of gene regulation in both prokaryotes and eukaryotes, and the recent FDA-approved CRISPR-based gene therapy for sickle cell anemia and β-thalassemia based on transcriptional derepression.}, }
@article {pmid39547118, year = {2024}, author = {Jiang, C and Li, Y and Wang, R and Sun, X and Zhang, Y and Zhang, Q}, title = {Development and optimization of base editors and its application in crops.}, journal = {Biochemical and biophysical research communications}, volume = {739}, number = {}, pages = {150942}, doi = {10.1016/j.bbrc.2024.150942}, pmid = {39547118}, issn = {1090-2104}, mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics ; *CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; Genome, Plant/genetics ; }, abstract = {Genome editing technologies hold significant potential for targeted mutagenesis in crop development, aligning with evolving agricultural needs. Point mutations, or single nucleotide polymorphisms (SNPs), define key agronomic traits in various crop species and play a pivotal role. The implementation of single nucleotide variations through genome editing-based base editing offers substantial promise in expediting crop improvement by inducing advantageous trait variations. Among many genome editing techniques, base editing stands out as an advanced next-generation technology, evolved from the CRISPR/Cas9 system.Base editing, a recent advancement in genome editing, enables precise DNA modification without the risks associated with double-strand breaks. Base editors, designed as precise genome editing tools, enable the direct and irreversible conversion of specific target bases. Base editors consist of catalytically active CRISPR-Cas9 domains, including Cas9 variants, fused with domains like cytidine deaminase, adenine deaminase, or reverse transcriptase. These fusion proteins enable the introduction of specific point mutations in target genomic regions. Currently developed are cytidine base editors (CBEs), mutating C to T; adenine base editors (ABEs), changing A to G; and prime editors (PEs), enabling arbitrary base conversions, precise insertions, and deletions. In this review, the research, development, and progress of various base editing systems, along with their potential applications in crop improvement, were intended to be summarized. The limitations of this technology will also be discussed. Finally, an outlook on the future of base editors will be provided.}, }
@article {pmid39546944, year = {2024}, author = {Haferkamp, U and Telugu, N and Krieg, K and Schaefer, W and Lam, D and Binkle-Ladisch, L and Friese, MA and Diecke, S and Pless, O}, title = {Generation of two isogenic human iPSC lines (ZIPi013-B-1, ZIPi013-B-2) carrying a CRISPR/Cas9-mediated deletion of TRPM4.}, journal = {Stem cell research}, volume = {81}, number = {}, pages = {103609}, doi = {10.1016/j.scr.2024.103609}, pmid = {39546944}, issn = {1876-7753}, mesh = {Humans ; *TRPM Cation Channels/metabolism/genetics ; *CRISPR-Cas Systems ; *Induced Pluripotent Stem Cells/metabolism ; Cell Line ; Gene Deletion ; }, abstract = {Two isogenic hiPSC lines, ZIPi013-B-1 and ZIPi013-B-2, were generated by CRISPR/Cas9-mediated indels in the TRPM4 gene of the previously published ZIPi013-B. TRPM4 belongs to the evolutionarily conserved family of transient receptor potential (TRP) channels. It is expressed ubiquitously and its activity is regulated by intracellular calcium binding, changes in membrane potential, phosphoinositide lipids in the plasma membrane and the local concentration of cytoplasmic ATP and ADP. TRPM4 has been implicated in various diseases, including neurological and immune system disorders, cardiac diseases and cancer. Both new cell lines offer the opportunity to model human diseases and test therapeutic modalities addressing these.}, }
@article {pmid39546609, year = {2024}, author = {Reding, K and Chung, M and Heath, A and Hotopp, JD and Pick, L}, title = {Same rule, different genes: Blimp1 is a pair-rule gene in the milkweed bug Oncopeltus fasciatus.}, journal = {Science advances}, volume = {10}, number = {46}, pages = {eadq9045}, pmid = {39546609}, issn = {2375-2548}, support = {R01 GM113230/GM/NIGMS NIH HHS/United States ; }, mesh = {Animals ; *Gene Expression Regulation, Developmental ; *Heteroptera/genetics ; Transcription Factors/genetics/metabolism ; Body Patterning/genetics ; CRISPR-Cas Systems ; Insect Proteins/genetics/metabolism ; RNA Interference ; Mutation ; }, abstract = {Morphological features of organismal body plans are often highly conserved within large taxa. For example, segmentation is a shared and defining feature of all insects. Screens in Drosophila identified genes responsible for the development of body segments, including the "pair-rule" genes (PRGs), which subdivide embryos into double-segment units in a previously unexpected pre-patterning step. Here we show that the milkweed bug Oncopeltus fasciatus also uses a pair rule for embryo subdivision but Oncopeltus employs different genes for this process. We identified the gene Blimp1 as an Oncopeltus PRG based on its expression pattern, tested its function with RNA interference and CRISPR-Cas9, and generated the first PR mutant in this species. Although it does not have PR function in Drosophila, like Drosophila PRGs, Blimp1 encodes a transcription factor required for embryonic viability. Thus, pair-rule subdivision of the insect body plan is more highly conserved than the factors mediating this process, suggesting a developmental constraint on this pre-patterning step.}, }
@article {pmid39546384, year = {2024}, author = {Birchler, JA and Kelly, J and Singh, J and Liu, H and Zhang, Z and Char, SN and Sharma, M and Yang, H and Albert, PS and Yang, B}, title = {Synthetic minichromosomes in plants: past, present, and promise.}, journal = {The Plant journal : for cell and molecular biology}, volume = {120}, number = {6}, pages = {2356-2366}, doi = {10.1111/tpj.17142}, pmid = {39546384}, issn = {1365-313X}, support = {PGRP 2221891//National Science Foundation/ ; }, mesh = {*Chromosomes, Plant/genetics ; *Chromosomes, Artificial/genetics ; *Centromere/genetics ; *CRISPR-Cas Systems ; Gene Editing/methods ; Genetic Engineering/methods ; Plants/genetics ; Telomere/genetics ; Plants, Genetically Modified ; }, abstract = {The status of engineered mini-chromosomes/artificial chromosomes/synthetic chromosomes in plants is summarized. Their promise is that they provide a means to accumulate foreign genes on an independent entity other than the normal chromosomes, which would facilitate stacking of novel traits in a way that would not be linked to endogenous genes and that would facilitate transfer between lines. Centromeres in plants are epigenetic, and therefore the isolation of DNA underlying centromeres and reintroduction into plant cells will not establish a functional kinetochore, which obviates this approach for in vitro assembly of plant artificial chromosomes. This issue was bypassed by using telomere-mediated chromosomal truncation to produce mini-chromosomes with little more than an endogenous centromere that could in turn be used as a foundation to build synthetic chromosomes. Site-specific recombinases and various iterations of CRISPR-Cas9 editing provide many tools for the development and re-engineering of synthetic chromosomes.}, }
@article {pmid39545734, year = {2025}, author = {Xu, T and Ni, Y and Li, H and Wu, S and Yan, S and Chen, L and Yu, Y and Wang, Y}, title = {Discovery and characterization of complete genomes of 38 head-tailed proviruses in four predominant phyla of archaea.}, journal = {Microbiology spectrum}, volume = {13}, number = {1}, pages = {e0049224}, pmid = {39545734}, issn = {2165-0497}, support = {32370151//MOST | National Natural Science Foundation of China (NSFC)/ ; }, mesh = {*Archaea/genetics/virology/classification ; *Archaeal Viruses/genetics/classification ; *Phylogeny ; *Genome, Archaeal ; *Proviruses/genetics ; Genome, Viral/genetics ; CRISPR-Cas Systems ; }, abstract = {Archaea play a significant role in natural ecosystems and the human body. Archaeal viruses exert a considerable influence on the structure and composition of archaeal communities and their associated ecological environments. The present study revealed the complete genomes of 38 archaeal head-tailed proviruses through comprehensive data mining. The hosts of these proviruses were identified as belonging to the following four dominant phyla: Halobacteriota, Thermoplasmatota, Thermoproteota, and Nanoarchaeota. In addition to the 14 proviruses of halophilic archaea related to the Graaviviridae family, the remaining proviruses exhibited limited genetic similarities to known (pro)viruses, suggesting the existence of 14 potential novel families. Of the 38 archaeal proviruses, 30 have the potential to lyse host cells. Eleven proviruses contain genes linked to antiviral defense mechanisms, including those involved in restriction modification (RM), clustered regularly interspaced short palindromic repeat (CRISPR)-associated (CRISPR-Cas) nucleases, defense island system associated with restriction-modification (DISARM), and DNA degradation (Dnd). Moreover, auxiliary metabolic genes were identified in the proviruses of Bathyarchaeia and Halobacteriota archaea, including those involved in carbohydrate and amino acid metabolism. Our findings indicate the diversity of archaeal viruses, their interactions with archaeal hosts, and their roles in the adaptation of the host.IMPORTANCEThe field of archaeal virology has seen a rapid expansion through the use of metagenomics, yet the diversity of these viruses remains largely uncharted. In this study, the complete genomes of 38 novel archaeal proviruses were identified for the following four dominant phyla: Halobacteriota, Thermoplasmatota, Thermoproteota, and Nanoarchaeota. Two families and six genera of Archaea were the first to be identified as hosts for viruses. The proviruses were found to contain diverse genes that were involved in distinct adaptation strategies of viruses to hosts. Our findings contribute to the expansion of the lineages of archaeal viruses and highlight their intricate interactions and essential roles in enabling host survival and adaptation to diverse environmental conditions.}, }
@article {pmid39544011, year = {2025}, author = {Fang, CG and James, B and Williams, M and Bachler, A and Tay, WT and Walsh, T and Frese, M}, title = {Cry1 resistance in a CRISPR/Cas9-mediated HaCad1 gene knockout strain of the Australian cotton bollworm Helicoverpa armigera conferta (Lepidoptera: Noctuidae).}, journal = {Pest management science}, volume = {81}, number = {2}, pages = {959-965}, pmid = {39544011}, issn = {1526-4998}, support = {//Commonwealth Scientific and Industrial Research Organisation/ ; //University of Canberra/ ; }, mesh = {Animals ; *Moths/genetics/growth &amp; development ; *CRISPR-Cas Systems ; *Bacillus thuringiensis Toxins ; *Endotoxins/pharmacology/genetics ; *Hemolysin Proteins/pharmacology/genetics ; *Bacterial Proteins/genetics ; *Insecticide Resistance/genetics ; *Insect Proteins/genetics/metabolism ; Gene Knockout Techniques ; Larva/genetics/growth &amp; development ; Cadherins/genetics ; Australia ; Insecticides/pharmacology ; Helicoverpa armigera ; }, abstract = {BACKGROUND: Helicoverpa armigera is a highly polyphagous species that causes huge losses to agricultural and horticultural crops worldwide. In the cotton industry, H. armigera, including the Australian subspecies Helicoverpa armigera conferta, is largely managed using genetically modified crops that express insecticidal toxins, such as Cry1Ac. Resistance to Cry1 proteins occurs and, in some cases, is mediated by changes to HaCad1, a gene that encodes the midgut protein cadherin. Around the world, numerous resistance-associated polymorphisms have been identified in the HaCad1 gene of H. armigera, but Cry1Ac resistance is rare in the Australian subspecies. We used CRISPR/Cas9 to disrupt the cadherin gene in H. armigera conferta and characterised the resulting phenotype with bioassays and transcriptomics.<br><br>RESULTS: Compared to the parental strain, the newly generated HaCad1 knockout strain is 44-fold and 16-fold more resistant to Cry1Ac and Cry1A.105, respectively, while wild-type and knockout insects were equally insensitive to Cry1F.<br><br>CONCLUSION: The disruption of the HaCad1 gene causes Cry1Ac resistance in Australian H. armigera conferta. However, Cry1Ac resistance remains rare in Australian field populations suggesting that Australia's approach to pest management in cotton has prevented widespread Cry1Ac resistance. &#xa9; 2024 CSIRO. Pest Management Science published by John Wiley &amp; Sons Ltd on behalf of Society of Chemical Industry.}, }
@article {pmid39543704, year = {2024}, author = {Gelsomino, L and Caruso, A and Tasan, E and Leonetti, AE and Malivindi, R and Naimo, GD and Giordano, F and Panza, S and Gu, G and Perrone, B and Giordano, C and Mauro, L and Nardo, B and Filippelli, G and Bonofiglio, D and Barone, I and Fuqua, SAW and Catalano, S and Andò, S}, title = {Evidence that CRISPR-Cas9 Y537S-mutant expressing breast cancer cells activate Yes-associated protein 1 to driving the conversion of normal fibroblasts into cancer-associated fibroblasts.}, journal = {Cell communication and signaling : CCS}, volume = {22}, number = {1}, pages = {545}, pmid = {39543704}, issn = {1478-811X}, mesh = {Humans ; *Breast Neoplasms/pathology/genetics/metabolism ; *YAP-Signaling Proteins ; *Cancer-Associated Fibroblasts/metabolism/pathology ; Animals ; *CRISPR-Cas Systems/genetics ; Female ; *Mutation/genetics ; *Adaptor Proteins, Signal Transducing/metabolism/genetics ; Mice ; Mice, Nude ; MCF-7 Cells ; Fibroblasts/metabolism ; Cell Proliferation ; Transcription Factors/metabolism/genetics ; Tumor Microenvironment/genetics ; Estrogen Receptor alpha/metabolism/genetics ; Cell Movement/genetics ; Receptor, IGF Type 1/metabolism/genetics ; }, abstract = {BACKGROUND: Endocrine therapy (ET) has improved the clinical outcomes of Estrogen receptor alpha-positive (ERɑ +) breast cancer (BC) patients, even though resistance to ET remains a clinical issue. Mutations in the hormone-binding domain of ERɑ represent an acquired intrinsic mechanism of ET resistance. However, the latter also depends on the multiple functional interactions between BC cells and the tumor microenvironment (TME). Here, we investigated how the most common Y537S-ERɑ mutation may influence the behavior of fibroblasts, the most prominent component of the TME.<br><br>METHODS: We conducted coculture experiments with normal human foreskin fibroblasts BJ1-hTERT (NFs), cancer-associated fibroblasts (CAFs), isolated from human BC specimens, and Y537S CRISPR-expressing MCF-7 BC cells (MCF-7YS). Mass spectrometry (MS) and Metacore analyses were performed to investigate how the functional interactions between BC cells/fibroblasts may affect their proteomic profile. The impact of fibroblasts on BC tumor growth and metastatic potential was evaluated in nude mice.<br><br>RESULTS: Mutant BC conditioned medium (CM) affected the morphology/proliferation/migration of both NFs and CAFs. 198 deregulated proteins signed the proteomic similarity profile of NFs exposed to the YS-CM and CAFs. Among the upregulated proteins, Yes-associated protein 1 (YAP1) was the main central hub in the direct interaction network. Increased YAP1 protein expression and activity were confirmed in NFs treated with MCF-7YS-CM. However, YAP1 activation appears to crosstalk with the insulin growth factor-1 receptor (IGF-1R). Higher amount of IGF-1 were noticed in the MCF-7YS-CM cells compared to the MCF-7P, and IGF-1 immunodepletion reversed the enhanced YAP1 expression and activity. Mutant cells upon exposure to the NF- and CAF-CM exhibited an enhanced proliferation/growth/migration/invasion compared to the MCF-7P. MCF-7YS cells when implanted with CAFs showed an early relative increased tumor volume compared to YS alone. No changes were observed when MCF-7P cells were co-implanted with CAFs. Compared with that in MCF-7P cells, the metastatic burden of MCF-7YS cells was intrinsically greater, and this effect was augmented upon treatment with NF-CM and further increased with CAF-CM.<br><br>CONCLUSIONS: YS mutant BC cells induced the conversion of fibroblasts into CAFs, via YAP, which represent a potential therapeutic target which interrupt the functional interactions between mutant cells/TME and to be implemented in the novel therapeutic strategy of a subset of metastatic BC patients carrying the frequent Y537S mutations.}, }
@article {pmid39543397, year = {2025}, author = {Loveless, TB and Carlson, CK and Dentzel Helmy, CA and Hu, VJ and Ross, SK and Demelo, MC and Murtaza, A and Liang, G and Ficht, M and Singhai, A and Pajoh-Casco, MJ and Liu, CC}, title = {Open-ended molecular recording of sequential cellular events into DNA.}, journal = {Nature chemical biology}, volume = {21}, number = {4}, pages = {512-521}, pmid = {39543397}, issn = {1552-4469}, support = {Predoctoral Fellowship//American Heart Association (American Heart Association, Inc.)/ ; R00GM140254//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; DP2 GM119163/GM/NIGMS NIH HHS/United States ; DP2GM119163//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; GRFP//National Science Foundation (NSF)/ ; R35 GM136297/GM/NIGMS NIH HHS/United States ; K99GM140254//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R21GM126287//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R21 GM126287/GM/NIGMS NIH HHS/United States ; 1763272//National Science Foundation (NSF)/ ; }, mesh = {*DNA/genetics ; Humans ; CRISPR-Cas Systems ; Gene Editing/methods ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; }, abstract = {Genetically encoded DNA recorders noninvasively convert transient biological events into durable mutations in a cell's genome, allowing for the later reconstruction of cellular experiences by DNA sequencing. We present a DNA recorder, peCHYRON, that achieves high-information, durable, and temporally resolved multiplexed recording of multiple cellular signals in mammalian cells. In each step of recording, prime editor, a Cas9-reverse transcriptase fusion protein, inserts a variable triplet DNA sequence alongside a constant propagator sequence that deactivates the previous and activates the next step of insertion. Insertions accumulate sequentially in a unidirectional order, editing can continue indefinitely, and high information is achieved by coexpressing a variety of prime editing guide RNAs (pegRNAs), each harboring unique triplet DNA sequences. We demonstrate that the constitutive expression of pegRNA collections generates insertion patterns for the straightforward reconstruction of cell lineage relationships and that the inducible expression of specific pegRNAs results in the accurate recording of exposures to biological stimuli.}, }
@article {pmid39543038, year = {2025}, author = {Chu, HY and Peng, J and Mou, Y and Wong, ASL}, title = {Quantifying Protein-Nucleic Acid Interactions for Engineering Useful CRISPR-Cas9 Genome-Editing Variants.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2870}, number = {}, pages = {227-243}, pmid = {39543038}, issn = {1940-6029}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics ; *Mutation ; *Streptococcus pyogenes/genetics/metabolism/enzymology ; DNA/genetics/metabolism ; Protein Binding ; }, abstract = {Numerous high-specificity Cas9 variants have been engineered for precision genome editing. These variants typically harbor multiple mutations designed to alter the Cas9-single guide RNA (sgRNA)-DNA complex interactions for reduced off-target cleavage. By dissecting the contributions of individual mutations, we attempt to derive principles for designing high-specificity Cas9 variants. Here, we computationally modeled the specificity harnessing mutations of the widely used Cas9 isolated from Streptococcus pyogenes (SpCas9) and investigated their individual mutational effects. We quantified the mutational effects in terms of energy and contact changes by comparing the wild-type and mutant structures. We found that these mutations disrupt the protein-protein or protein-DNA contacts within the Cas9-sgRNA-DNA complex. We also identified additional impacted amino acid sites via energy changes that constitute the structural microenvironment encompassing the focal mutation, giving insights into how the mutations contribute to the high-specificity phenotype of SpCas9. Our method outlines a strategy to evaluate mutational effects that can facilitate rational design for Cas9 optimization.}, }
@article {pmid39542433, year = {2024}, author = {Capelli, L and Pedrini, F and Di Pede, AC and Chamorro-Garcia, A and Bagheri, N and Fortunati, S and Giannetto, M and Mattarozzi, M and Corradini, R and Porchetta, A and Bertucci, A}, title = {Synthetic Protein-to-DNA Input Exchange for Protease Activity Detection Using CRISPR-Cas12a.}, journal = {Analytical chemistry}, volume = {96}, number = {47}, pages = {18645-18654}, pmid = {39542433}, issn = {1520-6882}, mesh = {*CRISPR-Cas Systems/genetics ; *Matrix Metalloproteinase 2/metabolism/genetics ; Humans ; DNA/chemistry/metabolism ; Peptide Nucleic Acids/chemistry/metabolism ; CRISPR-Associated Proteins/metabolism/chemistry ; Peptides/chemistry/metabolism ; DNA, Single-Stranded/chemistry/metabolism ; Biosensing Techniques/methods ; Fluorescence Resonance Energy Transfer ; }, abstract = {We present a novel activity-based detection strategy for matrix metalloproteinase 2 (MMP2), a critical cancer protease biomarker, leveraging a mechanism responsive to the proteolytic activity of MMP2 and its integration with CRISPR-Cas12a-assisted signal amplification. We designed a chemical translator comprising two functional units─a peptide and a peptide nucleic acid (PNA), fused together. The peptide presents the substrate of MMP2, while the PNA serves as a nucleic acid output for subsequent processing. This chemical translator was immobilized on micrometer magnetic beads as a physical support for an activity-based assay. We incorporated into our design a single-stranded DNA partially hybridized with the PNA sequence and bearing a region complementary to the RNA guide of CRISPR-Cas12a. The target-induced nuclease activity of Cas12a results in the degradation of FRET-labeled DNA reporters and amplified fluorescence signal, enabling the detection of MMP2 in the low picomolar range, showing a limit of detection of 72 pg/mL. This study provides new design principles