164 CRISPR/Cas9-mediated gene repair in the COL7A1 gene

Abstract

The CRISPR/Cas9 system turned out to be a powerful tool for genome editing and is therefore a promising option for the specific repair of gene mutations causing the blistering skin disease epidermolysis bullosa (EB). We have exploited the CRISPR/Cas9-mediated homology-directed repair (HDR) approach for the correction of a homozygous mutation in COL7A1 exon 80, leading to a complete loss of type VII collagen within the basement membrane zone of the skin. We have predicted a guide RNA (gRNA) specific for intron 80 of COL7A1, which was then cloned either into a wild-type Cas9 dual vector system, inducing double strand breaks, or a D10A Cas9 dual vector system, causing single strand breaks within the target intron. Homology COL7A1 arms for HDR were cloned into a donor vector, including a selection cassette. Transfected patient keratinocytes were selected either via antibiotic selection or fluorescent-activated cell sorting (FACS). RT-PCR on genomic DNA of treated cells and subsequent restriction enzyme digest analysis of the resulting PCR products showed the genetic correction of the COL7A1 mutation. The mutation-specific enzymatic digest revealed the presence of 26% reverted alleles. Additionally, type VII collagen restoration was confirmed via Western blot analysis and immunofluorescence staining. Our data indicate that genome editing using the CRISPR/Cas9 system can be an elegant tool for the repair of genes involved in the severe skin disease epidermolysis bullosa.