NCM 2022: CRISPRoff as potential treatment strategy for collagen VI congenital muscular dystrophy

Collagen VI congenital muscular dystrophy (ColVI-CMD) is a rare spectrum disorder for which there is still no treatment. One group of mutations that is often observed is heterozygous dominant-negative glycine substitutions in the triple helix of one of the three ColVI genes. Therapeutic strategies currently under investigation in cell culture aim to knockdown the pathogenic allele, and since many pathogenic variants are very rare, we aim to develop a therapeutic approach that is allele specific but mutation independent. Using Cas9 enrichment, we sequenced a 60-kb region including COL6A2 and its known regulatory elements to identify and phase common variants in this region. The next step was to design allele-specific guide RNAs and test those with CRISPRoff in patient-derived primary fibroblasts. Since the treatment aims to methylate the target, we used Cas9 enrichment again to sequence this region and Remora, Clair3, and methylartist to analyse allele-specific methylation.