Cut and Paste: Efficient Homology-Directed Repair of a Dominant Negative KRT14 Mutation via CRISPR/Cas9 Nickases

• Published in: Molecular therapy Vol. 25; no. 11; pp. 2585 - 2598
• Main Authors: Kocher, Thomas, Peking, Patricia, Klausegger, Alfred, Murauer, Eva Maria, Hofbauer, Josefina Piñón, Wally, Verena, Lettner, Thomas, Hainzl, Stefan, Ablinger, Michael, Bauer, Johann Wolfgang, Reichelt, Julia, Koller, Ulrich
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.11.2017; Elsevier Limited; American Society of Gene & Cell Therapy
• Subjects: Base Sequence; Cells, Cultured; CRISPR; CRISPR-Cas Systems; CRISPR/Cas9; Cytoplasm; Deoxyribonuclease I - genetics; Deoxyribonuclease I - metabolism; Deoxyribonucleic acid; DNA; Epidermolysis bullosa; epidermolysis bullosa simplex; Epidermolysis Bullosa Simplex - genetics; Epidermolysis Bullosa Simplex - metabolism; Epidermolysis Bullosa Simplex - pathology; Epidermolysis Bullosa Simplex - therapy; Exons; Gene Editing - methods; Gene Expression; Gene therapy; Genes; Genome editing; Genomes; High-Throughput Nucleotide Sequencing; Homology; homology-directed repair double nicking; Humans; Immunofluorescence; Introns; Keratin; keratin 14; Keratin-14 - genetics; Keratin-14 - metabolism; Keratinocytes; Keratinocytes - metabolism; Keratinocytes - pathology; Keratinocytes - transplantation; Molecular Targeted Therapy; Mutation; Mutation hot spots; Next-generation sequencing; Nicking endonuclease; Original; Plasmids - chemistry; Plasmids - metabolism; Recombination; Recombinational DNA Repair; Ribonucleic acid; RNA; RNA, Guide, CRISPR-Cas Systems - genetics; United States > US; Germany; homology-directed repair double nicking; keratin 14; CRISPR/Cas9; epidermolysis bullosa simplex
• ISSN: 1525-0016; 1525-0024; 1525-0024
• DOI: 10.1016/j.ymthe.2017.08.015

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.