Mutant Allele-Specific CRISPR Disruption in DYT1 Dystonia Fibroblasts Restores Cell Function

Abstract Most individuals affected with DYT1 dystonia have a heterozygous three base-pair deletion in the TOR1A gene (c.907_909delGAG). The mutation appears to act through a dominant negative mechanism compromising normal torsinA function, and it is proposed that reducing mutant torsinA may normalize torsinA activity. Here we used an engineered Cas9 variant from Streptococcus pyogenes (SpCas9-VRQR) to target the mutation in TOR1A gene in order to disrupt mutant torsinA in DYT1 patient fibroblasts. Selective targeting of the DYT1 allele was highly efficient with most common non-homologous end joining (NHEJ) edits leading to a predicted premature stop codon with loss of the torsinA C-terminus [delta 302-332 amino acid (aa)]. Structural analysis predicted a functionally inactive status of this truncated torsinA due to the loss of residues associated with ATPase activity and binding to LULL1. Immunoblotting showed a reduction on the torsinA protein level in Cas9-edited DYT1 fibroblasts, and a functional assay using HSV infection indicated a phenotypic recovery towards that observed in control fibroblasts. These findings suggest that the selective disruption of the mutant TOR1A allele using CRISPR-Cas9 inactivates mutant torsinA allowing the remaining wild-type torsinA to exert normal function.