Frontiers in genome editing最新文献

{"title":"Development of a homeolog-specific gene editing system in an evolutionary model for the study of polyploidy in nature.","authors":"Shengchen Shan, Michael T Pisias, Evgeny V Mavrodiev, Jonathan P Spoelhof, Bernard A Hauser, W Brad Barbazuk, Pamela S Soltis, Douglas E Soltis, Bing Yang","doi":"10.3389/fgeed.2025.1645542","DOIUrl":"10.3389/fgeed.2025.1645542","url":null,"abstract":"<p><p>Polyploidy, or whole-genome duplication (WGD), is a significant evolutionary force. Following allopolyploidy, duplicate gene copies (homeologs) have divergent evolutionary trajectories: some genes are preferentially retained in duplicate, while others tend to revert to single-copy status. Examining the effect of homeolog loss (i.e., changes in gene dosage) on associated phenotypes is essential for unraveling the genetic mechanisms underlying polyploid genome evolution. However, homeolog-specific editing has been demonstrated in only a few crop species and remains unexplored beyond agricultural applications. <i>Tragopogon</i> (Asteraceae) includes an evolutionary model system for studying the immediate consequences of polyploidy in nature. In this study, we developed a CRISPR-mediated homeolog-specific editing platform in allotetraploid <i>T. mirus</i>. Using the <i>MYB10</i> and <i>DFR</i> genes as examples, we successfully knocked out the targeted homeolog in <i>T. mirus</i> (4<i>x</i>) without editing the other homeolog (i.e., no off-target events). The editing efficiencies, defined as the percentage of plants with at least one allele of the targeted homeolog modified, were 35.7% and 45.5% for <i>MYB10</i> and <i>DFR</i>, respectively. Biallelic modification of the targeted homeolog occurred in the T₀ generation. These results demonstrate the robustness of homeolog-specific editing in polyploid <i>Tragopogon</i>, laying the foundation for future studies of genome evolution following WGD in nature.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"7 ","pages":"1645542"},"PeriodicalIF":4.4,"publicationDate":"2025-08-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12426060/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145066218","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":"Prime editing using paired pegRNAs targeting NG- or NGG-PAM in rice.","authors":"Ayako Nishizawa-Yokoi, Keiko Iida, Akiko Mori, Seiichi Toki","doi":"10.3389/fgeed.2025.1550308","DOIUrl":"10.3389/fgeed.2025.1550308","url":null,"abstract":"<p><p>Prime editing (PE) enables precise genome modification, i.e., all 12 types of base substitution, as well as designed insertion and deletion. Previously, we developed an efficient PE system using a pair of engineered pegRNAs (epegRNAs), appending an RNA pseudoknot sequence to the 3'ends of pegRNAs to enhance stability and prevent degradation of the 3'extension. Using a wild-type nSpCas9-based PE system (PE-wt) recognizing an NGG-protospacer adjacent motif (PAM) in this approach, two NGG-PAMs (NGG and CCN) adjacent to the target site are required for targeting by paired pegRNAs; however, this is not the PAM configuration available at most target sites. Using an nSpCas9-NG variant recognizing NG-PAM in PE (PE-NG) can expand applicability. Here, we compare the PE efficiency of PE-wt with paired epegRNAs targeting a distal NGG-PAM <i>versus</i> PE-NG with paired epegRNAs targeting NG-PAMs adjacent to the target site. By introducing substitution and designated deletion mutations into target genes via PE-wt and PE-NG with paired epegRNAs, we demonstrated that PE-wt could edit the target site efficiently despite targeting the distal PAM site when either of the paired epegRNAs for PE-NG targets PGC-PAM. If epegRNAs for PE-NG are designed to recognize NGA and NGT-PAM, there is no significant difference in frequency between PE-NG and PE-wt. These findings indicate that PE efficiency via PE-NG is particularly low at the NGC-PAM in rice.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"7 ","pages":"1550308"},"PeriodicalIF":4.4,"publicationDate":"2025-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12426525/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145066257","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":"Stem cell and CRISPR/Cas9 gene editing technology in Alzheimer's disease therapy: from basic research to clinical innovation.","authors":"Cong He, Baojiang Chen, Ciai Yan, Xiaoqing Zhou","doi":"10.3389/fgeed.2025.1612868","DOIUrl":"10.3389/fgeed.2025.1612868","url":null,"abstract":"<p><p>Alzheimer's disease (AD), a progressive neurodegenerative disorder characterized by Aβ plaques, tau protein neuronal fiber tangles, and neuroinflammation, poses a significant global health problem, and current therapies focus on the symptoms rather than the cause. This paper gives a new multidimensional therapeutic form to AD treatment by exploring the integrated application of stem cell therapy and CRISPR/Cas9 gene editing technology. The study comprehensively dissected the roles of neural stem cells (NSCs), induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) in neural replacement, neuroinflammation modulation and neuroplasticity enhancement, and also explored the application of CRISPR/Cas9 in modifying the pathogenic variants of AD-related genes (APP, PSEN1 and PSEN2). The key findings suggest that gene-edited iPSCs can reduce abnormal Aβ and tau protein accumulation in AD models, improve cognitive function, and provide a platform for disease modeling and drug screening. Stem cell transplantation promotes neurogenesis and synaptic plasticity by secreting neurotrophic factors to improve the brain microenvironment. Despite the challenges of off-target effects, immune rejection, and long-term safety, the synergistic application of these two technologies offers a breakthrough solution for AD treatment. This paper highlights the translational potential of combining stem cells with gene editing technology, which is expected to drive clinical applications in the next 5-10 years. The integration of these advanced technologies not only addresses the limitations of current AD treatments, but also paves the way for a personalized medical approach that is expected to revolutionize the AD treatment landscape and bring new hope to patients worldwide.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"7 ","pages":"1612868"},"PeriodicalIF":4.4,"publicationDate":"2025-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12417523/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145042584","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":"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":4.9,"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}