Frontiers in genome editing最新文献

{"title":"CRISPR/Cas9-mediated knockout of <i>DFR</i> alters pigmentation and shifts flavonoid accumulation in red leaf lettuce without detectable growth penalties.","authors":"Ai Nagamine, Masaki Ono, Osuke Sato, Eiji Goto, Hiroshi Ezura","doi":"10.3389/fgeed.2026.1755922","DOIUrl":"10.3389/fgeed.2026.1755922","url":null,"abstract":"<p><p>Red leaf lettuce (<i>Lactuca sativa</i> L. cv. 'Red Fire') is a preferred crop in plant factories with artificial light (PFALs) due to its short cultivation cycle and high anthocyanin content, which increases both its nutritional value and visual appeal. However, anthocyanins strongly influence leaf coloration and antioxidant profiles, and their levels are highly responsive to the light environment. Therefore, targeted editing of flavonoid biosynthesis may provide a breeding strategy to diversify pigment composition and associated functional traits under PFAL conditions. In this study, we used CRISPR/Cas9 to knock out <i>DFR</i> (dihydroflavonol 4-reductase), a key enzyme in the anthocyanin pathway. Genome-edited lines were generated via a dual-guide RNA system, resulting in a successfully edited red leaf genotype. The <i>DFR</i>-knockout lines displayed a complete loss of red pigmentation and a visibly distinct green phenotype. Metabolite profiling revealed a significant decrease in anthocyanin levels, accompanied by an increase in total flavonoid levels in some lines. Growth traits, including shoot dry weight and leaf number, were not significantly affected, suggesting that <i>DFR</i> knockout does not compromise growth under PFAL conditions. These findings highlight <i>DFR</i> as a promising target for creating pigment-altered lettuce lines for controlled-environment cultivation, including PFAL systems.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"8 ","pages":"1755922"},"PeriodicalIF":5.9,"publicationDate":"2026-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12996048/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147488619","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":"Evaluation of genetic variation in tumor suppressor miRNA encoding and their target genes in breast cancer; focus on miRNA interaction and expression analysis.","authors":"Yogita Chhichholiya, Sandeep Singh, Rajesh Vashistha, Manjit Kaur Rana, Anjana Munshi","doi":"10.3389/fgeed.2026.1705463","DOIUrl":"10.3389/fgeed.2026.1705463","url":null,"abstract":"<p><strong>Background: </strong>Genetic variations in tumor suppressor miRNAs and the 3'UTR of their target genes influence tumor biology and breast cancer (BC) risk.</p><p><strong>Objective: </strong>This study investigated genetic variations in tumor suppressor miRNAs (hsa-let-7c, hsa-miR-34a, hsa-miR-145a) and their target genes (KRAS, IGFBP6, IGF1R), and their functional significance in BC patients.</p><p><strong>Methods: </strong>The miRNA encoding regions and 3'UTRs of the selected target genes were sequenced in 208 BC patients. Functional analyses were performed using luciferase assay, RT-PCR, IHC, and Western blotting. RNAfold, TNM plot, Kaplan-Meier Plotter, and ROC Plotter were used for structural predictions, survival, and therapy response analysis.</p><p><strong>Results: </strong>Two variants, rs712 and rs9266, were found in the 3'UTR of KRAS. Luciferase assay confirmed that rs9266 disrupts the binding of hsa-let-7c and hsa-miR-181c, leading to increased KRAS expression. KRAS expression was highest in heterozygous, followed by homozygous mutant, and lowest in wild-type genotypes. Higher hsa-let-7c and hsa-miR-181c expression correlated with better survival. ROC analysis identified KRAS as a potential predictive biomarker for chemotherapy response.</p><p><strong>Conclusion: </strong>Variants rs712 and rs9266 in the KRAS 3'UTR impair miRNA binding, enhancing KRAS expression and tumorigenesis, while elevated hsa-let-7c and hsa-miR-181c levels predict favourable survival outcomes in BC patients.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"8 ","pages":"1705463"},"PeriodicalIF":5.9,"publicationDate":"2026-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12982462/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147470437","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":"Correction: Diversity of transgene integration and gene-editing events in wheat (<i>Triticum aestivum</i> L.) transgenic plants generated using <i>Agrobacterium</i>-mediated transformation.","authors":"Louie Cris Lopos, Natalia V Bykova, Janeen Robinson, Susan Brown, Kerry Ward, Andriy Bilichak","doi":"10.3389/fgeed.2026.1787940","DOIUrl":"10.3389/fgeed.2026.1787940","url":null,"abstract":"<p><p>[This corrects the article DOI: 10.3389/fgeed.2023.1265103.].</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"8 ","pages":"1787940"},"PeriodicalIF":4.4,"publicationDate":"2026-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12961196/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147379964","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-based programmable genome editing in chickens: concepts, applications and regulatory issues.","authors":"Gautham Kolluri, Adnan Naim, Shiva Kumar Kurva, Jagbir Singh Tyagi, Mohd Matin Ansari, Simmi Tomar, Ashok Kumar Tiwari, Laxmi Chouhan","doi":"10.3389/fgeed.2025.1729535","DOIUrl":"10.3389/fgeed.2025.1729535","url":null,"abstract":"<p><p>The advent of genetics, molecular biology, and genome sequencing has rapidly accelerated the development of elite genetic lines across various species, including poultry. It is now possible to introduce intra- or inter-species single nucleotide polymorphisms into chicken lines to enhance productivity. This advancement may mark the beginning of a new agricultural revolution, dramatically reducing the time required to improve poultry lines for commercial production environments. Transgenic technologies, including lentiviral vectors and piggyBac transposition, have enabled the generation of animals expressing exogenous genes. The emergence of new genome-editing tools is transforming avian biotechnology, allowing the creation of customized organisms for specific traits. Genome editing has become the most efficient method for studying gene function. First and second generation tools, such as zinc finger nucleases and transcription activator-like effector nucleases (TALENs), are limited by complex design and off-target effects. In contrast, the third generation Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9), represents a significant breakthrough. It encompasses guided RNA (gRNA) and the Cas9 endonuclease which together target specific DNA sequences and induces double-strand breaks that are repaired <i>via</i> error-prone non-homologous end joining, frequently causing insertions or deletions that disrupt gene function. Targeting specificity is achieved through gRNA-DNA base pairing and recognition of a protospacer adjacent motif by Cas9. Beyond gene knockout, CRISPR/Cas9 enables functional analysis of non-coding elements such as enhancers and insulators. Delivered <i>via</i> plasmid systems, Cas9 and gRNA are transiently expressed and degrade within 48-72 h, leaving no permanent genetic footprint. Since no exogenous DNA is integrated, this approach is generally considered less contentious than traditional transgenic methods in the context of genetically modified organism regulation. CRISPR/Cas9 has diverse applications in poultry, including enhancing disease resistance to avian influenza and Marek's disease, improving productivity traits such as growth, feed efficiency, and egg-laying, and enabling early in-ovo sexing to address ethical concerns around male chick culling. It also allows control of reproductive traits for breeding management, supports bio-pharming by producing therapeutic proteins or vaccines in eggs, and facilitates functional genomics through precise gene knockouts to study development, immunity, and metabolism.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"7 ","pages":"1729535"},"PeriodicalIF":4.4,"publicationDate":"2026-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12827552/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146055014","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":"Therapeutic applications of CRISPR-Cas9 gene editing.","authors":"Aditya Bharti, Joann Mudge","doi":"10.3389/fgeed.2025.1724291","DOIUrl":"10.3389/fgeed.2025.1724291","url":null,"abstract":"<p><p>CRISPR-Cas9 is a gene editing tool used extensively in biological research that is now making its way into clinical therapies. With the first CRISPR therapy obtaining approval by the United States' Food and Drug Administration (FDA) in late 2023, we look at clinical trials of emerging therapies involving CRISPR-Cas9, currently the most prevalent CRISPR-based tool in these trials. A CRISPR-based therapy is currently approved for treatment of both sickle-cell anemia and transfusion-dependent β-thalassemia but clinical trials for CRISPR-based therapeutics include a much broader range of targets. CRISPR-Cas9 is being explored to treat cancer, infectious disease, and more. This review highlights CRISPR-Cas9 clinical trials registered at clinicaltrials.gov as of 12/31/2024.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"7 ","pages":"1724291"},"PeriodicalIF":4.4,"publicationDate":"2025-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12748150/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145879606","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":"Development of efficient targeted insertion mediated by CRISPR-Cas12a and homology-directed repair in maize.","authors":"Brenden Barco, Shujie Dong, Yuki Matsuba, Ashley Crook, Ruiji Xu, Yingxiao Zhang, Chengjin Zhang, Ryan Carlin, Kevin Potter, Stephen B Rigoulot, Jeongmoo Park, Erin M Seaberry, Allison Parrish, Sivamani Elumalai, Sam Nalapalli, Craig Schuller, Anna Prairie, Anna Mangum, Kangfeng Mei, Hao Wu, Melissa Murray, Kristin Setliff, Francine Johnson, Dawn McNamara, Ling Zhu, Mark Rose, Weining Gu, Hao Hu, Yuanji Zhang, Yaping Jiang, Wenling Wang, Guozhu Tang, Lizhao Geng, Jianping Xu, Wan Shi, Jason Nichols, Tim Kelliher, Liang Shi, Ian Jepson, Qiudeng Que","doi":"10.3389/fgeed.2025.1713347","DOIUrl":"10.3389/fgeed.2025.1713347","url":null,"abstract":"<p><p>Targeted insertion (TIN) of transgenic trait cassettes has the potential to reduce timeline and cost for GM product development and commercialization. Precise genome engineering has made remarkable progress over the last several decades, particularly with the use of site-directed nucleases as core editing machinery. However, there are still many critical factors that can impact TIN efficiency including insertion site selection, nuclease optimization and expression, donor vector design, gene delivery, and stable event regeneration. Here, we develop workflows for target site sequence identification and gRNA screening for CRISPR-Cas12a system and demonstrate its successful application for TIN in maize with donor sequences up to 10 kilobase pairs (kb) in size. We first prioritize genomic regions for inserting transgenes <i>in silico</i> using bioinformatics tools and then test gRNA performance using a leaf protoplast transient assay. Despite its known low efficiency, we identify homology-directed repair (HDR) as the preferential pathway for directing targeted insertions of large sequences in immature embryos and demonstrate double-junction integrations at a rate of up to 4%. We further apply a molecular analysis workflow using large amplicon TaqMan assays and nanopore sequencing for streamlined identification and characterization of high-quality insertion events with intact large inserts. Analysis of TIN events across generations suggests that efficiency bottlenecks are not limited to donor targeted integration; attrition in efficiency also results from partial or additional donor insertion, chimerism, and close linkage with undesired sequence insertions such as those encoding the editing machinery. This work represents a major step forward in realizing the potential of precise genome engineering in maize for basic research and biotech trait development applications.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"7 ","pages":"1713347"},"PeriodicalIF":4.4,"publicationDate":"2025-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12711819/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145806231","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":"Emerging tools in plant genome editing.","authors":"Shilpi Sharma, Naveen Kumar Saroha, Abhilasha Sehrawat, Guiliang Tang, Deepali Singh, Sachin Teotia","doi":"10.3389/fgeed.2025.1588089","DOIUrl":"10.3389/fgeed.2025.1588089","url":null,"abstract":"<p><p>Plant genome editing has undergone a transformative shift with the advent of advanced molecular tools, offering unprecedented levels of precision, flexibility and efficiency in modifying genetic material. While classical site-directed nucleases such as ZFNs, TALENs and CRISPR-Cas9 have revolutionized genome engineering by enabling targeted mutagenesis and gene knockouts, the landscape is now rapidly evolving with the emergence of novel systems that go beyond the conventional double strand break (DSB)-mediated approaches. Advanced and recent tools include LEAPER, SATI, RESTORE, RESCUE, ARCUT, SPARDA, helicase-based approaches like HACE and Type IV-A CRISPR system, and transposon-based techniques like TATSI and piggyBac. These tools unlock previously inaccessible avenues of genome and transcriptome modulation. Some of these technologies allow DSB-free editing of DNA, precise base substitutions and RNA editing without altering the genomic DNA, a significant advancement for regulatory approval and for species with complex genomes or limited regeneration capacity. While LEAPER, RESCUE and RESTORE are the new advents in the RNA editing tool, SATI allows DSB-free approach for DNA editing, ARCUT offers less off-target and cleaner DNA repairs and Type IV-A CRISPR system induces gene silencing rather than editing. The transposon-based approaches include TATSI, piggyBac and TnpB, and helicases are used in HACE and Type IV-A CRISPR system. The prokaryotic Argonaute protein is used in SPARDA tool as an endonuclease to edit DNA. The transient and reversible nature of RNA editing tools such as RESTORE and LEAPER introduces a new layer of epigenetics-like control in plant systems, which could be harnessed for tissue-specific and environmentally-responsive trait expression. Simultaneously, innovations like ARCUT and SPARDA utilize chemically-guided editing, minimizing reliance on biological nucleases and reducing off-target risks. Their modularity and programmability are enabling gene function studies, synthetic pathway designs and targeted trait stacking. These advances represent a novel synthesis of genome engineering and systems biology, positioning plant genome editing not just as a tool of modification but as a platform for designing adaptive and intelligent crops, tailored to future environmental and nutritional challenges. Although, many of these recent tools remain to be applied on plant systems, they are proven to be effective elsewhere and hold a great potential to be effective in creating climate-resilient crops.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"7 ","pages":"1588089"},"PeriodicalIF":4.4,"publicationDate":"2025-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12713537/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145806348","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 mediated editing of <i>starch branching enzyme</i>, <i>SBE2</i> gene in potato for enhanced resistant starch for health benefits.","authors":"Sudha Batta, Sundaresha Siddappa, Neha Sharma, Rajender Singh, Reena Gupta, Dinesh Kumar, Brajesh Singh, Ajay Kumar Thakur","doi":"10.3389/fgeed.2025.1686412","DOIUrl":"10.3389/fgeed.2025.1686412","url":null,"abstract":"<p><p>Potato is an important vegetatively propagated, starch-rich tuber crop. High amylose potatoes containing more resistant starch offer healthier food alternatives. However, the resistant starch content is low in most cultivated potato varieties. In this study, targeted mutation of the <i>starch branching enzyme2</i> (<i>SBE2.1</i> & <i>SBE2.2</i> isoforms) had been done in the commercially significant potato cultivar, Kufri Chipsona-I using Clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9 system) to develop high-amylose potato lines. <i>SBE2</i> is one of the key enzymes involved in amylopectin biosynthesis, a starch component. Two isoforms, <i>SBE2.1 & SBE2.2,</i> were mutated using CRISPR-Cas9-mediated genome editing. After <i>Agrobacterium-mediated</i> genetic transformation, fifty transformed lines were generated on herbicide Basta selection medium, out of which 70% were found positive for <i>bar</i> and <i>Cas9</i> genes. Overall, six mutant lines, <i>viz.</i> K301, K302, K303, K304, K305, K306, derived from distinct events, exhibited deletions and substitutions in the target exons. The CRISPR-Cas9 edited K304 potato line exhibited both insertion-deletion (indel) and substitution mutations in three out of the four selected targets across both genes, and was therefore identified as the most efficiently edited line. The harvested tubers from <i>SBE2.1 & SBE2.2</i> mutant K304 line showed the highest amylose (95.91%) and resistant starch content (8.69 g/100 g). Evaluation of starch using X-ray crystallography (XRD) illustrated an altered crystallinity index (CI%) in all six mutant events in comparison to the wild study. Furthermore, ¹H-NMR study demonstrated a substantial decline in branch chain elongation in amylopectin, and thus a low degree of branching in a range of 1.15%-3.66% was reported in mutant lines, relative to the wild type (5.46%). The present study demonstrated the efficacy of CRISPR-Cas9-mediated mutagenesis of starch biosynthetic genes to develop high-amylose potato lines with elevated resistant starch content for improved health benefits.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"7 ","pages":"1686412"},"PeriodicalIF":5.9,"publicationDate":"2025-11-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12689564/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145745898","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: Social aspects of crop genome editing.","authors":"Srividhya Venkataraman, Kathleen Hefferon","doi":"10.3389/fgeed.2025.1740380","DOIUrl":"https://doi.org/10.3389/fgeed.2025.1740380","url":null,"abstract":"","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"7 ","pages":"1740380"},"PeriodicalIF":4.4,"publicationDate":"2025-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12687441/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145727637","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":"Construction of EpCAM overexpression and knockdown vectors and their implications in colorectal cancer research.","authors":"Bingping Wang, Jinkai Duan, Jie Zhou, Hulin Ma, Meng Ren, Liangquan Chen, Rina Su, Hao Zhang, Shuang Zhang, Yanwei Gao","doi":"10.3389/fgeed.2025.1679698","DOIUrl":"10.3389/fgeed.2025.1679698","url":null,"abstract":"<p><strong>Background: </strong>The functional characterization of Epithelial Cell Adhesion Molecule (EpCAM) in colorectal cancer (CRC) progression has been constrained by methodological limitations, particularly the potential for truncated protein isoforms to confound traditional genetic knockout approaches. This study aimed to develop a novel CRISPR/Cas9 strategy to overcome this challenge and systematically elucidate the context-dependent oncogenic roles of EpCAM across diverse CRC models.</p><p><strong>Methods: </strong>We engineered EpCAM overexpression (pCDH-EpCAM) and CRISPR/Cas9 knockdown (pGMC-KO-EpCAM) vectors using restriction digestion and T4 DNA ligation. A strategic dual-exon targeting approach (exons 1 and 3) was employed to minimize the risk of functional escape variants. Selected CRC cell lines (HT-29, HT-115, HRT-18) were genetically modified using optimized Lipofectamine 2000 transfection. Functional impacts were quantitatively assessed through: (i) flow cytometry for EpCAM surface expression (CD326-PE); (ii) daily cell counting over 8 days for proliferation kinetics; and (iii) scratch wound healing (0/24/48 h) and Transwell migration assays (8-μm pores, 18 h) to evaluate metastatic potential.</p><p><strong>Results: </strong>Successful genetic modulation was achieved and validated: HT-29-OE-EpCAM-2 exhibited an 89% EpCAM-positive rate versus 12% in wild-type (WT) (*p*<0.001), while HRT-18-KD-EpCAM-3 showed a significant reduction to 4% EpCAM-positive cells (vs. 15% in WT, *p*<0.001). EpCAM overexpression accelerated proliferation, with HT-29-OE cells showing a 20.1% increase in peak density on day 5 (30.76 ± 0.15 × 10⁴ vs. WT 25.62 ± 0.25 × 10⁴; *p*<0.001). Conversely, EpCAM knockdown in HRT-18 cells prolonged the doubling time by 8.8% (30.8 h vs. WT 28.3 h; *p*<0.05). Migration capacity was profoundly altered: HT-115-OE cells achieved complete scratch closure (100% vs. 74.05% in WT, *p*<0.001), whereas HRT-18-KD cells showed an 80.5% reduction (*p*<0.001). Transwell migration hierarchy confirmed the pro-metastatic role of EpCAM (HT-29-OE > HT-115-OE > HRT-18-KD; ANOVA *p* = 0.0024).</p><p><strong>Conclusion: </strong>This study establishes a robust dual-vector toolkit for reliable EpCAM manipulation, highlighting a novel exon-targeting strategy that mitigates the limitations of previous approaches. Our findings demonstrate that EpCAM is a master regulator of CRC aggressiveness, dictating proliferative and metastatic phenotypes in a cell context-dependent manner. The genetically defined models provide a validated platform for therapeutic screening and safety assessment, forming a foundational resource for advancing EpCAM-targeted therapies and diagnostic applications.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"7 ","pages":"1679698"},"PeriodicalIF":4.4,"publicationDate":"2025-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12675343/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145703022","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}