Frontiers in genome editing最新文献

{"title":"Ethical dimensions and societal implications: ensuring the social responsibility of CRISPR technology.","authors":"Irfan Biswas","doi":"10.3389/fgeed.2025.1593172","DOIUrl":"10.3389/fgeed.2025.1593172","url":null,"abstract":"<p><p>CRISPR-Cas9 is a breakthrough genome-editing platform that can cut chosen DNA sequences with unprecedented speed, accuracy, and affordability. By reprogramming a single guide RNA, researchers now alter gene function, correct pathogenic variants, or introduce novel traits. Earlier tools such as zinc-finger nucleases and TALENs performed similar tasks but were significantly more complex and costly. Yet CRISPR's very power raises urgent ethical concerns: Who controls its use, and how can society prevent germ-line enhancement, eugenic selection, or unequal access that favors wealthy nations and patients? A well-publicized case of embryo editing already showed how premature, unregulated experiments can erode public trust. This perspective therefore frames CRISPR's scientific promise alongside its social responsibilities, arguing that proactive, globally coordinated governance is essential to unlock benefits while preventing new forms of genetic inequality.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"7 ","pages":"1593172"},"PeriodicalIF":4.4,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12405698/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145002078","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Editorial: Gene editing to achieve Zero Hunger.","authors":"Shakeel Ahmad, Iqrar Ahmad Rana, Kevin M Folta, Christian Damian Lorenzo, Sultan Habibullah Khan","doi":"10.3389/fgeed.2025.1632120","DOIUrl":"10.3389/fgeed.2025.1632120","url":null,"abstract":"","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"7 ","pages":"1632120"},"PeriodicalIF":4.4,"publicationDate":"2025-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12350413/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144877050","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CATS: a bioinformatic tool for automated Cas9 nucleases activity comparison in clinically relevant contexts.","authors":"Ettore Rocchi, Federico Magnani, Gastone Castellani, Antonio Carusillo, Martina Tarozzi","doi":"10.3389/fgeed.2025.1571023","DOIUrl":"10.3389/fgeed.2025.1571023","url":null,"abstract":"<p><strong>Introduction: </strong>With the growing number of Cas9 nucleases available to genetic engineers, selecting the most suitable one for a given application can be challenging. A major complication arises from the differing protospacer adjacent motif (PAM) sequence requirements of each Cas9 variant, which makes direct comparisons difficult. To ensure a fair comparison, it is essential to identify common target sites that are not biased by the natural genetic landscape of the chosen target.</p><p><strong>Methods: </strong>To address this challenge, we developed CATS (Comparing Cas9 Activities by Target Superimposition), a novel bioinformatic tool. CATS automates the detection of overlapping PAM sequences across different Cas9 nucleases and identifies allele-specific targets, particularly those arising from pathogenic mutations. One of the key parameters in CATS is the proximity of PAM sites, which helps minimize sequence composition bias. The tool integrates data from continuously updated sources and includes ClinVar information to facilitate the targeting of disease-causing mutations.</p><p><strong>Results: </strong>CATS significantly reduces the time and effort required for CRISPR/Cas9 experimental design. It streamlines the comparison of Cas9 nucleases with different PAM requirements, enabling researchers to select the most appropriate nuclease for their specific target. The tool's automation, speed, and user-friendly interface make it accessible to researchers regardless of their computational expertise.</p><p><strong>Discussion: </strong>By enabling the identification of overlapping PAMs and allele-specific targets, CATS supports the implementation of Cas9-based applications in both research and clinical settings. Its ability to incorporate genetic variants makes it particularly useful for designing therapeutic approaches that selectively target mutated alleles while sparing healthy ones. Ultimately, CATS contributes to the development of more effective and precise genetic therapies.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"7 ","pages":"1571023"},"PeriodicalIF":4.4,"publicationDate":"2025-07-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12328375/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144801116","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CRISPR-Cas9 editing of <i>TNFAIP3</i> variants in salivary gland epithelial cells to study Sjögren's disease pathogenesis.","authors":"Subhashis Ghosh, Qisheng Tu, Zoe Xiaofang Zhu, Sreelakshmi Panginikkod, Jake Jinkun Chen","doi":"10.3389/fgeed.2025.1625393","DOIUrl":"10.3389/fgeed.2025.1625393","url":null,"abstract":"<p><p>Sjögren's disease (SD) is a systemic autoimmune disease that particularly affects the salivary and lacrimal glands, causing sicca symptoms. Genetic polymorphism in the <i>TNFAIP3</i> gene has been implicated in the pathogenesis of SD. In this study, we aimed to functionally determine the impact of two specific single-nucleotide polymorphisms (SNPs) in <i>TNFAIP3</i>, rs6920220 (G/A) and rs2230926 (T/C/G), on the pathogenesis of SD. Using CRISPR-Cas9, we edited human salivary gland epithelial cells (SGECs) to incorporate <i>TNFAIP3</i> SNPs rs6920220 (G/A) and rs2230926 (T/C/G) and co-cultured them with Jurkat cells. We performed assays to examine gene expression, inflammatory cytokine levels, and related signaling pathways to investigate the effects of these genetic variants on <i>TNFAIP3</i> function and cellular response. Our results demonstrated that these SNPs reduced <i>TNFAIP3</i> expression, increased NF-κB activation, and elevated pro-inflammatory cytokine production. These findings provide strong evidence for the functional significance of these genetic variants in the pathogenesis of SD and underscore the utility of CRISPR-Cas9 technology in elucidating genetic contributions to autoimmune disorders.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"7 ","pages":"1625393"},"PeriodicalIF":4.4,"publicationDate":"2025-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12326134/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144796308","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Simply cut out - Combining CRISPR/Cas9 RNPs and transiently selected telomere vectors for marker free-gene deletion in <i>Trichoderma atroviride</i>.","authors":"Mario Gründlinger, Chiara Ellensohn, Leo Drechsel, Ulrike Schreiner, Siebe Pierson, Clara Baldin, Susanne Zeilinger","doi":"10.3389/fgeed.2025.1623963","DOIUrl":"10.3389/fgeed.2025.1623963","url":null,"abstract":"<p><p><i>Trichoderma atroviride</i> is a well-known mycoparasitic fungus widely used for the biological control of fungal plant pathogens. However, genetic manipulation in this organism remains challenging due to the limited availability of versatile and efficient molecular tools. Here, we present a CRISPR/Cas9-based method for targeted gene manipulation using ribonucleoprotein (RNP) complexes combined with a transiently stable telomere vector. We successfully inactivated three genes-<i>pks4</i> (spore pigment production), <i>pyr4</i> (pyrimidine biosynthesis), and <i>pex5</i> (peroxisomal matrix protein import receptor)-to demonstrate the system's utility. Although double-strand breaks induced by Cas9 can be repaired via homology-directed repair (HDR), using donor templates, the most effective gene inactivations in our case were achieved via non-homologous end joining (NHEJ), by co-transforming the transiently stable telomere vector carrying the hygromycin-resistance gene (<i>hph</i>), which was rapidly lost under non-selective conditions. This strategy enables marker-free genetic manipulation, supports vector recycling, and simplifies successive transformations. Overall, our method expands the genetic toolbox for <i>T. atroviride</i>, offering a fast and reliable approach for reverse genetics in this agriculturally important fungus.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"7 ","pages":"1623963"},"PeriodicalIF":4.9,"publicationDate":"2025-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12263576/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144651405","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comprehensive approaches to design efficient gRNA for SDN1-CRISPR/Cas9 genome editing in wheat.","authors":"Tushadri Singh, H M Mamrutha, Rajender Singh, J P Jaiswal, Zeenat Wadhwa, Rakesh Kumar, Omvir Singh, O P Ahlawat, Ratan Tiwari","doi":"10.3389/fgeed.2025.1579165","DOIUrl":"10.3389/fgeed.2025.1579165","url":null,"abstract":"<p><strong>Background: </strong>CRISPR/Cas9 technology has gained popularity due to its efficient, widely applicable, and relatively easy genome editing. Furthermore, the removal of regulation on site-directed nuclease1- (SDN1) and SDN2-developed products in many countries has made it a more revolutionary technology for adoption in crop improvement. Designing accurate guide RNA (gRNA) is the initial and most crucial step that decides the success of the editing. Although the gene editing technique is widely used in crops, a detailed and comprehensive method for designing efficient gRNA in wheat is still lacking. By virtue of wheat being a hexaploid crop and having a large genome size with repetitive DNA, a tailor-made strategy for designing the gRNA is crucial.</p><p><strong>Result: </strong>The manuscript explains the comprehensive strategies and methods for efficient gRNA designing by considering the physical and structural expression of the target gene in the genome and explains the on-target and off-target effects of gRNA for its precise editing through the CRISPR/Cas9-mediated SDN1 method of genome editing in wheat.</p><p><strong>Conclusion: </strong>The present manuscript is first of its kind to address the holistic approach, starting from efficient gene selection, gRNA designing, and post-gRNA designing issues like gRNA stability, binding efficiency, and functionality for SDN1-CRISPR/Cas9 genome editing in wheat. This manuscript will be a ready reference for wheat researchers designing effective gRNA for wheat improvement to meet future food demand.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"7 ","pages":"1579165"},"PeriodicalIF":4.9,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12259558/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144644291","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"DNA-free CRISPR genome editing in raspberry (<i>Rubus idaeus</i>) protoplast through RNP-mediated transfection.","authors":"Ryan Creeth, Andrew Thompson, Zoltan Kevei","doi":"10.3389/fgeed.2025.1589431","DOIUrl":"10.3389/fgeed.2025.1589431","url":null,"abstract":"<p><p>Protoplast-based systems have been utilised in a wide variety of plant species to enable genome editing without chromosomal introgression of foreign DNA into plant genomes. DNA-free genome editing followed by protoplast regeneration allows elite cultivars to be edited without further genetic segregation, preserving their unique genetic composition and their regulatory status as non-transgenic. However, protoplast isolation presents a barrier to the development of advanced breeding technologies in raspberry and no protocol has been published for DNA-free genome editing in the species. Pre-assembled ribonucleoprotein complexes (RNPs) do not require cellular processing and the commercial availability of Cas9 proteins and synthetic guide RNAs has streamlined genome editing protocols. This study presents a novel high-yielding protoplast isolation protocol from raspberry stem cultures and RNP-mediated transfection of protoplast with CRISPR-Cas9. Targeted mutagenesis of the phytoene desaturase gene at two intragenic loci resulted in an editing efficiency of 19%, though estimated efficiency varied depending on the indel analysis technique. Only amplicon sequencing was sensitive enough to confirm genome editing in a low efficiency sample. To our knowledge, this study constitutes the first use of DNA-free genome editing in raspberry protoplast. This protocol provides a valuable platform for understanding gene function and facilitates the future development of precision breeding in this important soft fruit crop.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"7 ","pages":"1589431"},"PeriodicalIF":5.9,"publicationDate":"2025-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12256493/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144638866","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genome editing for sustainable agriculture in Peru: advances, potential applications and regulation.","authors":"Marilu Mestanza, Angel David Hernández-Amasifuen, Alexandra Jherina Pineda-Lázaro, Dennis Eriksson, Juan Carlos Guerrero-Abad","doi":"10.3389/fgeed.2025.1611040","DOIUrl":"10.3389/fgeed.2025.1611040","url":null,"abstract":"<p><p>Peruvian agriculture is characterize by crops such as potato, maize, rice, asparagus, mango, banana, avocado, cassava, onion, oil palm, chili, papikra, blueberry, coffee, cacao, grapes, quinoa, olive, citrus and others. All of them have challenges in production in their specific agroecosystems under stress due to pests, diseases, salinity, drought, cold among others. Gene editing through CRISPR/Cas is a key tool for addressing critical challenges in agriculture by improving resilience to biotic and abiotic stress, increasing yield and enhancing the nutritional value of the crops. This approach allows precise mutation on site-specific gene at the DNA level, obtaining desirable traits when its function is altered. The CRISPR/Cas system could be used as a transgene-free genome editing tool when the ribonucleoprotein (RNP) acts as a carrier to delivered the CRISPR/Cas components into the plant cell protoplasts, or when the tRNA-like sequence (TLS) motifs are fused to single-guide RNA (sgRNA) and Cas mRNA sequence and expressed in transgenic plants rootstock to produce \"mobile\" CRISPR/Cas components to upper tissue (scion). Those innovations could be a potential approach to strengthen the Peruvian agriculture, food security and gricultural economy, especially in the tropical, Andean and coastal regions. This review article examines the advances and strategies of gene editing, focusing on transgene-free methodologies that could be adopted for research, development and use, and also identifies potential applications in key crops for Peru and analyzes their impact in the productivity and reduction of agrochemicals dependence. Finally, this review highlights the need to establish regulatory policies that strengthen the use of biotechnological precise innovations, ensuring the conservation and valorization of agrobiodiversity for the benefit of Peruvian farmers.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"7 ","pages":"1611040"},"PeriodicalIF":4.9,"publicationDate":"2025-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12258049/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144638867","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A long shelf-life melon created via CRISPR/Cas9 RNP-based <i>in planta</i> genome editing.","authors":"Kentaro Sasaki, Kaoru Urano, Naozumi Mimida, Satoko Nonaka, Hiroshi Ezura, Ryozo Imai","doi":"10.3389/fgeed.2025.1623097","DOIUrl":"10.3389/fgeed.2025.1623097","url":null,"abstract":"<p><p>Genome editing in melon (<i>Cucumis melo</i> L.) remains a significant challenge due to the inefficiencies associated with conventional cell culture-based transformation methods. In the present study, a novel <i>in planta</i> Particle Bombardment (iPB) approach was developed to enable DNA-free genome editing in melon without the need for cell culture. CRISPR/Cas9 ribonucleoproteins (RNPs) were coated onto gold particles and delivered directly into shoot apical meristem tissue, which harbors potential germline cells, via particle bombardment. This method was applied to enhance fruit shelf-life by targeting an ethylene biosynthesis gene (<i>CmACO1</i>). The resulting <i>cmaco1</i> mutant demonstrated a significantly extended shelf-life, attributable to reduced ethylene production during fruit ripening. This delayed ripening phenotype was reversed upon treatment with exogenous ethylene, confirming the functional impact of <i>CmACO1</i> disruption. Because this strategy bypasses cell culture, the iPB-RNP method offers a solution to common limitations in genome editing, such as genotype dependence and somaclonal variation. Consequently, this technique holds substantial promise for advancing commercial melon breeding efforts and may be broadly applicable to other species within the Cucurbitaceae family.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"7 ","pages":"1623097"},"PeriodicalIF":4.9,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12213888/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144556081","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Long read sequencing reveals transgene concatemerization and vector sequences integration following AAV-driven electroporation of CRISPR RNP complexes in mouse zygotes.","authors":"Muhammad W Luqman, Piroon Jenjaroenpun, Jessica Spathos, Nikhil Shingte, Mitchell Cummins, Pattaraporn Nimsamer, Lars M Ittner, Thidathip Wongsurawat, Fabien Delerue","doi":"10.3389/fgeed.2025.1582097","DOIUrl":"10.3389/fgeed.2025.1582097","url":null,"abstract":"<p><p>Over the last decade CRISPR gene editing has been successfully used to streamline the generation of animal models for biomedical research purposes. However, one limitation to its use is the potential occurrence of on-target mutations that may be detrimental or otherwise unintended. These bystander mutations are often undetected using conventional genotyping methods. The use of Adeno-Associated Viruses (AAVs) to bring donor templates in zygotes is currently being deployed by transgenic cores around the world to generate knock-ins with large transgenes (i.e., 1-4 kb payloads). Thanks to a high level of efficiency and the relative ease to establish this technique, it recently became a method of choice for transgenic laboratories. However, a thorough analysis of the editing outcomes following this method is yet to be developed. To this end, we generated three different types of integration using AAVs in two different murine genes (i.e., <i>Ace2</i> and <i>Foxg1</i>) and employed Oxford Nanopore Technologies long read sequencing to analyze the outcomes. Using a workflow that includes Cas9 enrichment and adaptive sampling, we showed that unintended on-target mutations, including duplication events and integration of viral sequences (sometimes reported using other workflows) can occur when using AAVs. This work highlights the importance of in-depth validation of the mutant lines generated and informs the uptake of this new method.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"7 ","pages":"1582097"},"PeriodicalIF":5.9,"publicationDate":"2025-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12174137/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144327947","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}