Functional benefit of CRISPR/Cas9-induced allele deletion forRYR1dominant mutation

Abstract

AbstractMore than 700 pathogenic or probably pathogenic variations have been identified in theRYR1gene causing various myopathies collectively known as “RYR1-related myopathies”. Currently, 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 aRYR1mutation, we aimed at showing the functional benefit of inactivating specifically the mutatedRYR1allele 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 mutantRYR1allele and identified specific CRISPR/Cas9 guide-RNAs. Lentiviruses encoding these guide-RNAs and theSpCas9nuclease were used to transduce immortalized patient muscle cells, inducing the specific deletion of the mutantRYR1allele. The efficiency of the deletion was assessed at both DNA and RNA levels and at the functional level after monitoring calcium release induced by the stimulation of the RyR1-channel. This study providesin-celluloproof of concept regarding the benefits of mutantRYR1allele deletion, in the case of a dominantRYR1mutation, from both a molecular and functional perspective.Graphical abstracteTOC synopsisMutations in theRYR1gene, encoding a calcium channel required for muscle contraction, cause severe myopathies. In this study, Marty and colleagues demonstrate the functional benefit of suppression of a mutantRYR1allele using CRISPR/Cas9, in the case of a dominant mutation, leaving the wild type allele alone.