{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T10:19:43Z","timestamp":1785406783681,"version":"3.56.0"},"reference-count":44,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,4,22]],"date-time":"2026-04-22T00:00:00Z","timestamp":1776816000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Defeat Duchenne Foundation","award":["53320215"],"award-info":[{"award-number":["53320215"]}]},{"DOI":"10.13039\/501100000024","name":"Canadian Institutes of Health Research","doi-asserted-by":"crossref","award":["492510"],"award-info":[{"award-number":["492510"]}],"id":[{"id":"10.13039\/501100000024","id-type":"DOI","asserted-by":"crossref"}]},{"award":["492510"],"award-info":[{"award-number":["492510"]}],"id":[{"id":"https:\/\/ror.org\/01gavpb45","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Duchenne muscular dystrophy (DMD) is a fatal X-linked neuromuscular disorder caused by mutations in the dystrophin gene. Prime editing is a versatile genome editing technology capable of introducing precise nucleotide changes without generating double-strand DNA breaks, making it a promising approach for correcting pathogenic point mutations. In this study, we applied prime editing to modify mdx-4cv and mdx-5cv mutation-equivalent sites in mouse C2C12 myoblasts in vitro. Initial editing efficiencies were unexpectedly low and were associated with the presence of a 5\u2032-TTCT-3\u2032 motif within engineered prime editing guide RNAs (epegRNAs). epegRNA designs containing this motif exhibited reduced prime editing efficiency, whereas silent substitution eliminating the motif significantly improved editing outcomes, indicating that specific sequence features within epegRNAs can influence editing performance. Rational redesign of epegRNAs to remove this motif substantially enhanced editing efficiency, achieving up to 20% modification at the 4cv target site using an NGG PAM and 21% editing at the 5cv locus using an NGAG PAM. These findings highlight an important sequence-dependent constraint in epegRNA design and provide practical guidance for optimizing prime editing strategies targeting Dmd mutations in vitro.<\/jats:p>","DOI":"10.3390\/cells15090740","type":"journal-article","created":{"date-parts":[[2026,4,22]],"date-time":"2026-04-22T10:38:14Z","timestamp":1776854294000},"page":"740","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Successful In Vitro Modification of the Dmd Gene Using Prime Editing"],"prefix":"10.3390","volume":"15","author":[{"given":"Ayesha","family":"Siddika","sequence":"first","affiliation":[{"name":"D\u00e9partement de M\u00e9decine Mol\u00e9culaire, Universit\u00e9 Laval, Quebec City, QC G1V 0A6, Canada"},{"name":"Centre de Recherche du Centre Hospitalier Universitaire de Qu\u00e9bec, Quebec City, QC G1E 6W2, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fatima El","family":"Husseiny","sequence":"additional","affiliation":[{"name":"D\u00e9partement de M\u00e9decine Mol\u00e9culaire, Universit\u00e9 Laval, Quebec City, QC G1V 0A6, Canada"},{"name":"Centre de Recherche du Centre Hospitalier Universitaire de Qu\u00e9bec, Quebec City, QC G1E 6W2, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jo\u00ebl","family":"Rousseau","sequence":"additional","affiliation":[{"name":"Centre de Recherche du Centre Hospitalier Universitaire de Qu\u00e9bec, Quebec City, QC G1E 6W2, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9404-9195","authenticated-orcid":false,"given":"Jacques P.","family":"Tremblay","sequence":"additional","affiliation":[{"name":"D\u00e9partement de M\u00e9decine Mol\u00e9culaire, Universit\u00e9 Laval, Quebec City, QC G1V 0A6, Canada"},{"name":"Centre de Recherche du Centre Hospitalier Universitaire de Qu\u00e9bec, Quebec City, QC G1E 6W2, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e3015","DOI":"10.1002\/jgm.3015","article-title":"Gene therapy clinical trials worldwide to 2017: An update","volume":"20","author":"Ginn","year":"2018","journal-title":"J. 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