CRISPR/Cas9-induced (CTG⋅CAG)n repeat instability in the myotonic dystrophy type 1 locus: implications for therapeutic genome editing

Myotonic dystrophy type 1 (DM1) is caused by (CTG⋅CAG)n-repeat expansion within the DMPK gene and thought to be mediated by a toxic RNA gain of function. Current attempts to develop therapy for this disease mainly aim at destroying or blocking abnormal properties of mutant DMPK (CUG)n RNA. Here, we explored a DNA-directed strategy and demonstrate that single clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-cleavage in either its 5′ or 3′ unique flank promotes uncontrollable deletion of large segments from the expanded trinucleotide repeat, rather than formation of short indels usually seen after double-strand break repair. Complete and precise excision of the repeat tract from normal and large expanded DMPK alleles in myoblasts from unaffected individuals, DM1 patients, and a DM1 mouse model could be achieved at high frequency by dual CRISPR/Cas9-cleavage at either side of the (CTG⋅CAG)n sequence. Importantly, removal of the repeat appeared to have no detrimental effects on the expression of genes in the DM1 locus. Moreover, myogenic capacity, nucleocytoplasmic distribution, and abnormal RNP-binding behavior of transcripts from the edited DMPK gene were normalized. Dual sgRNA-guided excision of the (CTG⋅CAG)n tract by CRISPR/Cas9 technology is applicable for developing isogenic cell lines for research and may provide new therapeutic opportunities for patients with DM1

Recovery in the myogenic program of congenital myotonic dystrophy myoblasts after excision of the expanded (CTG)n repeat

The congenital form of myotonic dystrophy type 1 (cDM) is caused by the large-scale expansion of a (CTG•CAG)n repeat in DMPK and DM1-AS. The production of toxic transcripts with long trinucleotide tracts from these genes results in impairment of the myogenic differentiation capacity as cDM’s most prominent morpho-phenotypic hallmark. In the current in vitro study, we compared the early differentiation programs of isogenic cDM myoblasts with and without a (CTG)2600 repeat obtained by gene editing. We found that excision of the repeat restored the ability of cDM myoblasts to engage in myogenic fusion, preventing the ensuing myotubes from remaining immature. Although the cDM-typical epigenetic status of the DM1 locus and the expression of genes therein were not altered upon removal of the repeat, analyses at the transcriptome and proteome level revealed that early abnormalities in the temporal expression of differentiation regulators, myogenic progression markers, and alternative splicing patterns before and immediately after the onset of differentiation became normalized. Our observation that molecular and cellular features of cDM are reversible in vitro and can be corrected by repeat-directed genome editing in muscle progenitors, when already committed and poised for myogenic differentiation, is important information for the future development of gene therapy for different forms of myotonic dystrophy type 1 (DM1)