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There are no current therapeutic approaches with the ability to excise large multi-exon duplications, leaving this patient cohort without mutation-specific treatment. Using CRISPR\/Cas9 could provide a valid alternative to achieve targeted excision of genomic duplications of any size. Here we show that the expression of a single CRISPR\/Cas9 nuclease targeting a genomic region within a <jats:italic>DMD<\/jats:italic> duplication can restore the production of wild-type dystrophin in vitro. We assessed the extent of dystrophin repair following both constitutive and transient nuclease expression by either transducing DMD patient-derived myoblasts with integrating lentiviral vectors or electroporating them with CRISPR\/Cas9 expressing plasmids. Comparing genomic, transcript and protein data, we observed that both continuous and transient nuclease expression resulted in approximately 50% dystrophin protein restoration in treated myoblasts. Our data demonstrate that a high transient expression profile of Cas9 circumvents its requirement of continuous expression within the cell for targeting <jats:italic>DMD<\/jats:italic> duplications. This proof-of-concept study therefore helps progress towards a clinically relevant gene editing strategy for in vivo dystrophin restoration<jats:italic>,<\/jats:italic> by highlighting important considerations for optimizing future therapeutic approaches.<\/jats:p>","DOI":"10.1038\/s41598-022-07671-w","type":"journal-article","created":{"date-parts":[[2022,3,8]],"date-time":"2022-03-08T11:06:27Z","timestamp":1646737587000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Transiently expressed CRISPR\/Cas9 induces wild-type dystrophin in vitro in DMD patient myoblasts carrying duplications"],"prefix":"10.1038","volume":"12","author":[{"given":"Veronica","family":"Pini","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Virginie","family":"Mariot","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julie","family":"Dumonceaux","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John","family":"Counsell","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Helen C.","family":"O\u2019Neill","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah","family":"Farmer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francesco","family":"Conti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francesco","family":"Muntoni","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2022,3,8]]},"reference":[{"key":"7671_CR1","doi-asserted-by":"publisher","first-page":"395","DOI":"10.1002\/humu.22758","volume":"36","author":"CL Bladen","year":"2015","unstructured":"Bladen, C. 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The remaining authors declare no competing interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"3756"}}