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With this approach problematic wounds could be grafted with gene edited, patient\u2010specific\u00a0skin equivalents. Precise gene editing using homology\u2010directed repair (HDR) is the ultimate goal, however low efficiencies have hindered progress. Reframing strategies based on highly efficient non\u2010homologous end joining (NHEJ) repair aimed at excising dispensable, mutation\u2010harboring exons offer a promising alternative approach for restoring the\n                    <jats:italic>COL7A1<\/jats:italic>\n                    open reading frame. To this end, we employed an exon skipping strategy using dual single guide RNA (sgRNA)\/Cas9 ribonucleoproteins (RNPs) targeted at three novel\n                    <jats:italic>COL7A1<\/jats:italic>\n                    exons (31, 68, and 109) containing pathogenic heterozygous mutations, and achieved exon deletion rates of up to 95%. Deletion of exon 31 in both primary human RDEB keratinocytes and fibroblasts resulted in the restoration of type VII collagen (C7), leading to increased cellular adhesion in vitro and accurate C7 deposition at the dermal\u2010epidermal junction in a 3D skin model. Taken together, we extend the list of\n                    <jats:italic>COL7A1<\/jats:italic>\n                    exons amenable to therapeutic deletion. As an incidental finding, we find that long\u2010read Nanopore sequencing detected large on\u2010target structural variants comprised of deletions up to &gt;5\u2009kb at a frequency of ~10%. Although this frequency may be acceptable given the high rates of intended editing outcomes, our data demonstrate that standard short\u2010read sequencing may underestimate the full range of unexpected Cas9\u2010mediated editing events.\n                  <\/jats:p>","DOI":"10.1002\/btm2.10640","type":"journal-article","created":{"date-parts":[[2024,1,18]],"date-time":"2024-01-18T00:00:36Z","timestamp":1705536036000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Highly efficient\n                    <scp>CRISPR<\/scp>\n                    \/Cas9\u2010mediated exon skipping for recessive dystrophic epidermolysis bullosa"],"prefix":"10.1002","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9481-4149","authenticated-orcid":false,"given":"Alex","family":"du Rand","sequence":"first","affiliation":[{"name":"School of Biological Sciences The University of Auckland  Auckland New 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