Functional benefit of CRISPR/Cas9-induced allele deletion for RYR1 dominant mutation

More than 700 pathogenic or probably pathogenic variations have been identified in the RYR1 gene 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 a RYR1 mutation, we aimed at showing the functional benefit of inactivating specifically the mutated RYR1 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 RYR1 allele and identified specific CRISPR/Cas9 guide-RNAs. Lentiviruses encoding these guide-RNAs and the SpCas9 nuclease were used to transduce immortalized patient muscle cells, inducing the specific deletion of the mutant RYR1 allele. 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 provides in-cellulo proof of concept regarding the benefits of mutant RYR1 allele deletion, in the case of a dominant RYR1 mutation, from both a molecular and functional perspective. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/576997v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@484a3borg.highwire.dtl.DTLVardef@1a842org.highwire.dtl.DTLVardef@cda3d5org.highwire.dtl.DTLVardef@befd91_HPS_FORMAT_FIGEXP M_FIG C_FIG eTOC synopsisMutations in the RYR1 gene, 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 mutant RYR1 allele using CRISPR/Cas9, in the case of a dominant mutation, leaving the wild type allele alone.