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Gene editing constitutes a novel approach for precisely correcting disease-causing gene mutations. Frameshift mutations inCOL7A1 causing recessive dystrophic epidermolysis bullosaare amenable to open reading frame restoration by non-homologous end joining repair-based approaches. Efficient targeteddeletion of faulty COL7A1 exons in polyclonal patient keratinocytes would enable the translation of this therapeutic strategy to the clinic. In this study, using a dual single-guide RNA(sgRNA)-guided Cas9 nuclease delivered as a ribonucleoprotein complex through electroporation, we have achieved veryefficient targeted deletion of COL7A1 exon 80 in recessivedystrophic epidermolysis bullosa (RDEB) patient keratinocytescarrying a highly prevalent frameshift mutation. This ex vivonon-viral approach rendered a large proportion of correctedcells producing a functional collagen VII variant. The effectivetargeting of the epidermal stem cell population enabled longterm regeneration of a properly adhesive skin upon graftingonto immunodeficient mice. A safety assessment by next-generation sequencing (NGS) analysis of potential off-target sitesdid not reveal any unintended nuclease activity. Our strategycould potentially be extended to a large number of COL7A1mutation-bearing exons within the long collagenous domainof this gene, opening the way to precision medicine for RDEB.