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To identify the origin of recruited osteoblasts, cell lineage tracing using Cre\/loxP recombination is commonly used. However, a confounding factor is the use of transgenic Cre drivers that do not accurately recapitulate endogenous gene expression or the use of knock-in Cre drivers that alter endogenous protein activity or levels. Here, we describe a CRISPR\/Cas9 homology-directed repair knock-in approach that allows efficient generation of Cre drivers controlled by the endogenous gene promoter. In addition, a self-cleaving peptide preserves the reading frame of the endogenous protein. Using this approach, we generated col10a1p2a-CreERT2 knock-in medaka and show that tamoxifen-inducible CreERT2 efficiently recombined loxP sites in col10a1 cells. Similar knock-in efficiencies were obtained when two unrelated loci (osr1 and col2a1a) were targeted. Using live imaging, we traced the fate of col10a1 osteoblast progenitors during bone lesion repair in the medaka vertebral column. We show that col10a1 cells at neural arches represent a mobilizable cellular source for bone repair. Together, our study describes a previously unreported strategy for precise cell lineage tracing via efficient and non-disruptive knock-in of Cre.<\/jats:p>","DOI":"10.1242\/dev.200238","type":"journal-article","created":{"date-parts":[[2022,5,20]],"date-time":"2022-05-20T11:20:14Z","timestamp":1653045614000},"source":"Crossref","is-referenced-by-count":4,"title":["A non-disruptive and efficient knock-in approach allows fate tracing of resident osteoblast progenitors during repair of vertebral lesions in medaka"],"prefix":"10.1242","volume":"149","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7576-3198","authenticated-orcid":false,"given":"Wen Hui","family":"Tan","sequence":"first","affiliation":[{"name":"National University of Singapore Department of Biological Sciences and Centre for Bioimaging Sciences , , Singapore 117543 , Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4688-6241","authenticated-orcid":false,"given":"Christoph","family":"Winkler","sequence":"additional","affiliation":[{"name":"National University of Singapore Department of Biological Sciences and Centre for Bioimaging Sciences , , Singapore 117543 , Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"237","published-online":{"date-parts":[[2022,6,21]]},"reference":[{"key":"2022062110585448800_DEV200238C1","doi-asserted-by":"publisher","first-page":"77","DOI":"10.1016\/j.ymeth.2017.03.005","article-title":"Genome editing using CRISPR\/Cas9-based knock-in approaches in zebrafish","volume":"121-122","author":"Albadri","year":"2017","journal-title":"Methods"},{"key":"2022062110585448800_DEV200238C2","doi-asserted-by":"publisher","first-page":"1732","DOI":"10.1038\/s41598-021-81239-y","article-title":"Endogenous zebrafish proneural Cre drivers generated by CRISPR\/Cas9 short homology directed targeted integration","volume":"11","author":"Almeida","year":"2021","journal-title":"Sci. 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