Efficient Gene Reframing Therapy for Recessive Dystrophic Epidermolysis Bullosa with CRISPR/Cas9.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system induces site-specific double-strand breaks, which stimulate cellular DNA repair through either the homologous recombination or non-homologous end-joining pathways. The non-homologous end-joining pathway, which is activated more frequently than homologous recombination, is prone to introducing small insertions and/or deletions at the double-strand break site, leading to changes in the reading frame. We hypothesized that the non-homologous end-joining pathway is applicable to genetic diseases caused by a frameshift mutation through restoration of the reading frame. Recessive dystrophic epidermolysis bullosa is a hereditary skin disorder caused by mutations in COL7A1. In this study, we applied gene reframing therapy to a recurrent frameshift mutation, c.5819delC, in COL7A1, which results in a premature termination codon. CRISPR/Cas9 targeting this specific mutation site was delivered to recessive dystrophic epidermolysis bullosa patient fibroblasts. After genotyping a large collection of gene-edited fibroblast clones, we identified a significant number (17/50) of clones in which the frameshift in COL7A1 was restored. The reframed COL7 was functional, as shown by triple-helix formation assay in vitro, and was correctly distributed in the basement membrane zone in mice. Our data suggest that mutation site-specific non-homologous end-joining might be a highly efficient gene therapy for inherited disorders caused by frameshift mutations.