With the ability to induce rapid and efficient repair of disease-causing mutations, CRISPR/Cas9 technology is ideally suited for gene therapy approaches for recessively and dominantly inherited monogenic disorders. In this study, we have corrected a causal hotspot mutation in exon 6 of the keratin 14 gene (KRT14) that results in generalized severe epidermolysis bullosa simplex (EBS-gen sev), using a double-nicking strategy targeting intron 7, followed by homology-directed repair (HDR). Co-delivery into EBS keratinocytes of a Cas9 D10A nickase (Cas9n), a predicted single guide RNA pair specific for intron 7, and a minicircle donor vector harboring the homology donor template resulted in a recombination efficiency of >30% and correction of the mutant KRT14 allele. Phenotypic correction of EBS-gen sev keratinocytes was demonstrated by immunofluorescence analysis, revealing the absence of disease-associated K14 aggregates within the cytoplasm. We achieved a promising safety profile for the CRISPR/Cas9 double-nicking approach, with no detectable off-target activity for a set of predicted off-target genes as confirmed by next generation sequencing. In conclusion, we demonstrate a highly efficient and specific gene-editing approach for KRT14, offering a causal treatment option for EBS. Kocher et al. established a CRISPR/Cas9-based gene-editing strategy with the aim to correct a recurrent dominantly inherited KRT14 hotspot mutation causal for the phenotypic manifestation in epidermolysis bullosa simplex (EBS). The homology-directed repair (HDR) was induced via Cas9 nickases in a double-nicking configuration showing an improved safety profile compared with the commonly used wild-type Cas9 nuclease. Treatment of patient keratinocytes and correction of the mutation at genomic level resulted in a full reversion of the disease phenotype.
Kocher, T., Peking, P., Klausegger, A., Murauer, E. M., Hofbauer, J. P., Wally, V., … Koller, U. (2017). Cut and Paste: Efficient Homology-Directed Repair of a Dominant Negative KRT14 Mutation via CRISPR/Cas9 Nickases. Molecular Therapy, 25(11), 2585–2598. https://doi.org/10.1016/j.ymthe.2017.08.015