P.278 - CRISPR/Cas9-mediated genome editing corrects splicing alterations in myotonic dystrophy type 1

Abstract

Myotonic dystrophy type 1 (DM1) belongs to the group of nucleotide repeat disorders. More specifically this autosomal form of muscular dystrophy is caused by the expansion of the CTG trinucleotide repeat located at the 3' untranslated region (3′-UTR) of the DMPK gene. Elongated CUG repeats of the mutated DMPK mRNAs become sequestration sites for splicing factors, and induce the formation of stable ribonucleoprotein complexes visualized as foci. As a consequence, the alternative splicing of numerous transcripts is dysregulated, which leads to the DM1 pathological alterations affecting various tissues. We have developed a strategy to delete the CTG repeat expansion in the human DMPK locus by using the CRISPR/Cas9 system. For that purpose, we constructed different expression platforms for small size Cas9 nucleases under either a ubiquitous or a muscle-specific promoter and guide RNAs (sgRNAs) targeting the 3'-UTR of the DMPK gene. Co-transfection of these constructs in DM1 cells resulted in the deletion of the CTG repeat. In order to optimize the delivery of Cas9 and sgRNAs in myoblast cell lines, these constructs were cloned into lentiviral vectors. Cells transduced with Cas9-sgRNA lentiviral vectors were tested for the deletion of DMPK CTG repeats and for the presence of nuclear foci by FISH. Furthermore, upon muscle cell differentiation, we investigated the mis-splicing of transcripts that are dysregulated in the DM1 disease. Our data overall demonstrate that the deletion of the CTG repeats at the 3′-UTR of the DMPK gene reverts the DM1 phenotype at molecular and cellular levels.