Long-read sequencing for the assessment of therapeutic CRISPR/Cas9 strategy for the removal of aberrant DM1-causing CTG repeats in DMPK gene locus

The genetic cause of Myotonic Dystrophy Type 1 (DM1) is the expansion of unstable CTG-repeats in the 3´ untranslated region of the DMPK gene. Therapeutic gene editing for treatment of monogenic diseases could in principle remove definitively the disease-causing genetic defect. Here we describe the application of the CRISPR/Cas9 strategy to remove the CTG-expansion in the DMPK gene in myogenic cells derived from a DM1 patient that is heterozygous for the expansion. The efficacy of on-target genomic events was assessed by performing long-read DNA sequencing (Oxford Nanopore Technologies) after applying Cas9-mediated target enrichment of the Regions Of Interest (ROI), enabling the simultaneous analysis (a) of cuts by both sgRNAs resulting in the effective excision of the CTG expansion, (b) of cuts induced only by a single sgRNA, (c) of the potential occurrence of aberrant events such as inversions, and (d) the existence of non-edited sequences, within the same sample. Overall design: New guide RNA pairs were designed and assessed by PCR. The most effective pair (sgC3/sg384) was utilized to perform editing in proliferating (Growth Medium, GM) and differentiated (Differentiation Medium, DM) myogenic cells. For target enrichment, CRISPR RNAs (crRNAs) were designed to cut the ROI four times, i.e., twice upstream on the plus strand and twice downstream on the minus strand, to avoid incomplete cleavage. crRNA pools against two ROI were used: one targeting the full-length DMPK gene (~14kbp, ROI1) to screen for potential long deletions, the other flanking the sgC3/sg384 editing sites within the 3'UTR region (~4kbp, ROI2). Five libraries were prepared and sequenced from edited DM1 myogenic cell populations in both DM (1 ROI1 and 2 ROI2 samples) and GM (2 ROI2 samples), yielding an overall sequencing coverage = 140X for both cell states.