{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T08:48:09Z","timestamp":1785746889222,"version":"3.56.0"},"reference-count":29,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2026,8,1]],"date-time":"2026-08-01T00:00:00Z","timestamp":1785542400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science (JSPS) KAKENHI","doi-asserted-by":"publisher","award":["25K02195"],"award-info":[{"award-number":["25K02195"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science (JSPS) KAKENHI","doi-asserted-by":"publisher","award":["23H03860"],"award-info":[{"award-number":["23H03860"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["JP223fa627008"],"award-info":[{"award-number":["JP223fa627008"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"award":["JP223fa627008"],"award-info":[{"award-number":["JP223fa627008"]}],"id":[{"id":"https:\/\/ror.org\/004rtk039","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["J. Dev. Biol.","JDB"],"abstract":"<jats:p>Background\/Objectives: CRISPR\/Cas9-mediated genome editing enables efficient generation of knockout mouse models; however, frameshift mutations do not always result in complete loss of function. The factors influencing functional inactivation following frameshift mutations remain incompletely understood. Here, we tested whether frameshift-dominant targeting of exon 1 is sufficient to generate a null allele of Fgf10, a gene essential for limb formation. Methods: Guide RNAs (gRNAs) were selected using a machine learning-based pipeline to favor microhomology-mediated end joining (MMEJ)-dominant repair. Editing efficiency and indel profiles were assessed via amplicon sequencing in mouse embryonic stem cells (mESCs) and preimplantation embryos. Edited embryos were transferred to surrogate females and analyzed at embryonic day 15.5 (E15.5). Results: Amplicon sequencing confirmed &gt;97% editing efficiency and &gt;80% frameshift alleles in both mESCs and preimplantation embryos, with a predominant 7 bp deletion. Despite highly efficient frameshift-dominant editing, most of the E15.5 embryos were morphologically normal, indicating that exon 1 targeting did not reliably produce null phenotypes. In silico analysis suggested the possible presence of alternative downstream translation initiation sites, and our secretion assay supported this possibility. Initiation from a downstream ATG in a +2 reading frame (e.g., 7 bp deletion) may restore the downstream coding sequence and partially preserve protein function. Conclusions: Frameshift mutations in exon 1 of Fgf10 do not consistently result in functional knockout. The functional outcome depends on the specific reading frame and may be influenced by alternative translation initiation and protein domain architecture. These findings highlight important considerations for designing genome editing strategies to achieve complete gene inactivation.<\/jats:p>","DOI":"10.3390\/jdb14030033","type":"journal-article","created":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T08:04:59Z","timestamp":1785744299000},"page":"33","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Frameshift Mutations in Exon 1 of Fgf10 Frequently Yield Morphologically Normal Embryos"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8532-7402","authenticated-orcid":false,"given":"Khanui","family":"Lkhagvadorj","sequence":"first","affiliation":[{"name":"Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Otsu 520-2192, Shiga, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eiichi","family":"Okamura","sequence":"additional","affiliation":[{"name":"Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Otsu 520-2192, Shiga, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Toshifumi","family":"Morimura","sequence":"additional","affiliation":[{"name":"Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Otsu 520-2192, Shiga, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seiya","family":"Mizuno","sequence":"additional","affiliation":[{"name":"Laboratory Animal Resource Center, Trans-Border Medical Research Center, University of Tsukuba, Tsukuba 305-8575, Ibaraki, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0645-6183","authenticated-orcid":false,"given":"Masatsugu","family":"Ema","sequence":"additional","affiliation":[{"name":"Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Otsu 520-2192, Shiga, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"eadd8643","DOI":"10.1126\/science.add8643","article-title":"CRISPR technology: A decade of genome editing is only the beginning","volume":"379","author":"Wang","year":"2023","journal-title":"Science"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1038\/nprot.2017.153","article-title":"Easi-CRISPR protocol for creating knock-in and conditional knockout mouse models using long ssDNA donors","volume":"13","author":"Miura","year":"2018","journal-title":"Nat. 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