CRISPR/Cas9-Mediated Allele-Specific Disruption of a Dominant COL6A1 Pathogenic Variant Improves Collagen VI Network in Patient Fibroblasts.

Collagen VI-related disorders are the second most common congenital muscular dystrophies for which no treatments are presently available. They are mostly caused by dominant-negative pathogenic variants in the genes encoding α chains of collagen VI, a heteromeric network forming collagen; for example, the c.877G>A; p.Gly293Arg COL6A1 variant, which alters the proper association of the tetramers to form microfibrils. We tested the potential of CRISPR/Cas9-based genome editing to silence or correct (using a donor template) a mutant allele in the dermal fibroblasts of four individuals bearing the c.877G>A pathogenic variant. Evaluation of gene-edited cells by next-generation sequencing revealed that correction of the mutant allele by homologous-directed repair occurred at a frequency lower than 1%. However, the presence of frameshift variants and others that provoked the silencing of the mutant allele were found in >40% of reads, with no effects on the wild-type allele. This was confirmed by droplet digital PCR with allele-specific probes, which revealed a reduction in the expression of the mutant allele. Finally, immunofluorescence analyses revealed a recovery in the collagen VI extracellular matrix. In summary, we demonstrate that CRISPR/Cas9 gene-edition can specifically reverse the pathogenic effects of a dominant negative variant in COL6A1.