Functional benefit of CRISPR-Cas9-induced allele deletion for dominant mutation.

More than 700 pathogenic or probably pathogenic variations have been identified in the gene causing various myopathies collectively known as "-related myopathies." There is no treatment for these myopathies, and gene therapy stands out as one of the most promising approaches. In the context of a dominant form of central core disease due to a mutation, we aimed at showing the functional benefit of inactivating specifically the mutated allele by guiding CRISPR-Cas9 cleavages onto frequent single-nucleotide polymorphisms (SNPs) segregating on the same chromosome. Whole-genome sequencing was used to pinpoint SNPs localized on the mutant allele and identified specific CRISPR-Cas9 guide RNAs. Lentiviruses encoding these guide RNAs and the nuclease were used to transduce immortalized patient myoblasts, inducing the specific deletion of the mutant allele. The efficiency of the deletion was assessed at DNA and RNA levels, and at the functional level after monitoring calcium release induced by the stimulation of the RyR1-channel. This study provides proof of concept regarding the benefits of mutant allele deletion, in the case of a dominant mutation, from both a molecular and functional perspective, and could apply potentially to 20% of all patients with a mutation.