Allele-specific CRISPR/Cas9-induced NHEJ repair disrupts a single nucleotide variant associated with collagen VI muscular dystrophy

Development of allele-specific gene editing tools can expand the therapeutic options for dominant genetic conditions, either via allelic gene inactivation or via gene correction. Here, we applied allelic gene inactivation using allele-specific CRISPR/Cas9 guide RNAs (gRNAs) to introduce frameshifting indels to a dominant-negative allele. The target is a single nucleotide variant in the COL6A1 gene (c.868G>A; G290R) that acts as dominant negative and is associated with a severe form of congenital muscular dystrophy. We expressed spCas9 along with allele-specific gRNAs, without providing a repair template, in primary fibroblasts derived from four patients and one control subject. Amplicon deep-sequencing showed that single nucleotide deletions accounted for the majority of indels introduced, for two gRNAs tested. While activity of the two gRNAs was greater at the G290R allele, both gRNAs were also active at the wild-type allele. To enhance allele selectivity, we introduced deliberate mismatches to one gRNA. One of these optimized gRNAs showed minimal activity at the WT allele, while generating productive edits and improving collagen VI matrix in cultured patient fibroblasts. This study strengthens the potential of gene editing to treat dominant-negative disorders, but also underscores the challenge in achieving allele selectivity with gRNAs.