"a. Objective Type 1 myotonic dystrophy (DM1) originates from an amplification of CTG microsatellites in the DMPK gene. The pathology is primarily explained by a toxic gain of function where the expanded-CUG DMPK transcripts induce, among other mechanisms, the loss of function of the MBNL proteins, triggering a wide spliceopathy. Several therapeutic strategies have been tested to neutralize the toxic DMPK transcripts or their consequences. Nevertheless, they all have at least one drawback such as lack of specificity limiting clinical applicability. Here, we investigated a new therapeutic strategy consisting of the inhibition of the DMPK promoter by a CRISPRi system in patient-derived myotubes. b. Methodology Our DMPK silencing strategy by CRISPRi was tested in immortalized myoblasts from a DM1 patient or a healthy donor. Stable cell lines expressing a deactivated Cas9 conjugated to an inhibitory KRAB domain in addition to their own sgRNAs were produced by lentiviral transduction. The efficacy and specificity of our therapeutic strategy were then assessed in differentiated myotubes. c. Results Our DMPK promoter inhibition strategy is highly efficient to reduce toxic DMPK transcript quantities up to 80%. This level of inhibition allows to correct the DM1 hallmark defects by reducing the presence of foci, improving the spliceopathy and normalizing an electrophysiological parameter in DM1 myotubes. Furthermore, this approach displays unprecedented high specificity as evidenced by a complete lack of off-target effects on the transcriptome from unaffected myotubes. d. Conclusions We conclude that DMPK promoter inhibition is a promising strategy to be developed for DM1 treatment."