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

Abstract

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.

Gene Editing–Mediated Disruption of Epidermolytic Ichthyosis–Associated KRT10 Alleles Restores Filament Stability in Keratinocytes

“…TALEN_6.21 demonstrated high on-target activity in immortalized KCs from a patient with EI (56.8%), compared with a recent geneediting approach targeting KRT5 in KCs derived from a patient with epidermolysis bullosa simplex (25%) (Aushev et al, 2017). These high efficiencies were achieved without the use of selection methods commonly employed in gene editing of KCs (Hainzl et al, 2017;Kocher et al, 2017).…”

“…Transfection efficiency was evaluated as previously described (Hainzl et al, 2017;Kocher et al, 2017).…”

Anex vivoRNAtrans‐splicing strategy to correct human generalized severe epidermolysis bullosa simplex

“…Treatment of medically intractable hereditary diseases requires replacement or correction of the affected gene or RNA in order to facilitate expression of a functional wild‐type protein. Whereas cDNA therapy was previously shown to be an option for many recessively inherited genetic diseases in clinical trials, therapy of dominant negative mutations requires different approaches, which will either repair or knock down the disease‐causing allele or gene product . Such genome editing approaches might involve CRISPR (clustered regularly interspaced short palindromic repeats) or TALEN (transcription activator‐like effector nuclease) technologies.…”

Epidermolysis bullosa: Advances in research and treatment

“…Dominant‐negative mutations such as those common in EBS can be corrected in different ways. In addition to a mutation‐specific repair approach for a KRT14 mutation, we developed another treatment approach based on deletion of a mutant keratin 5 allele while leaving the wild‐type allele intact and taking over the function . The latter study demonstrated that one gene‐specific designer nuclease could be employed to inactivate any EBS‐causing mutant KRT5 allele by inducing insertions or deletions (indels) via the error‐prone non‐homologous end‐joining (NHEJ) repair pathway causing a frameshift at a site within the target gene that is independent on the location of the disease‐causing mutation.…”

“…Due to the straightforward use and rapid advancement of the technology, CRISPR is today dominating the gene editing field. Whereas most CRISPR approaches harnessed the DNA double‐strand break inducing Streptococcus pyogenes Cas9 (SpCas9) nuclease variants, we have recently developed double nicking approaches based on single‐strand DNA breaks (nicks) at the targeting site with the intention to reduce off‐target site effects . Compared to the extremely high numbers of off‐target site modifications inherent to replacement gene therapies resulting from multiple random integrations in the treated cells, off‐target mutations are rare or non‐existent upon designer nuclease treatment …”