Complete allele-specific silencing of the gain-of-function mutation of Huntington's disease

Dominant gain-of-function mechanism in Huntington's disease (HD) suggests selective inactivation of mutant HTT produces the biggest therapeutic benefit. Here, we developed a complete allele-specific CRISPR/Cas9 strategy to permanently silence mutant HTT through nonsense-mediated decay (NMD), capitalizing on an exonic PAM (protospacer adjacent motif)-Altering SNP (PAS). Comprehensive sequence/haplotype analysis identified PAS-generated NGG PAM sites on exons of common HTT haplotypes in HD patients, revealing a single clinically meaningful PAS-based mutant-specific NMD-CRISPR/Cas9 strategy. The alternative allele of rs363099 eliminates NGG PAM site on the most frequent normal HTT haplotype in HD, permitting mutant HTT-specific CRISPR/Cas9 therapeutics in ~20% of HD patients with European ancestry. Our rs363099-based CRISPR/Cas9 showed perfect allele specificity and good targeting efficiencies in cells derived from HD patients. Dramatically reduced mutant HTT mRNA and complete loss of muta...