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We report the generation of the first large animal model of <jats:italic>OTOF<\/jats:italic> mutations using the CRISPR system associated with different Cas9 components (mRNA or protein) assisted by single strand oligodeoxynucleotides (ssODN) to induce homology-directed repair (HDR). Zygote microinjection was performed with two sgRNA targeting exon 5 and 6 associated to Cas9 mRNA or protein (RNP) at different concentrations in a mix with an ssODN template targeting HDR in exon 5 containing two STOP sequences. A total of 73 lambs were born, 13 showing indel mutations (17.8%), 8 of which (61.5%) had knock-in mutations by HDR. Higher concentrations of Cas9-RNP induced targeted mutations more effectively, but negatively affected embryo survival and pregnancy rate. This study reports by the first time the generation of <jats:italic>OTOF<\/jats:italic> disrupted sheep, which may allow better understanding and development of new therapies for human deafness related to genetic disorders. These results support the use of CRISPR\/Cas system assisted by ssODN as an effective tool for gene editing in livestock.<\/jats:p>","DOI":"10.1038\/s41598-020-62879-y","type":"journal-article","created":{"date-parts":[[2020,4,7]],"date-time":"2020-04-07T10:02:45Z","timestamp":1586253765000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":33,"title":["Otoferlin gene editing in sheep via CRISPR-assisted ssODN-mediated Homology Directed Repair"],"prefix":"10.1038","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2494-9574","authenticated-orcid":false,"given":"A.","family":"Menchaca","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"P. 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