Allele-specific silencing of the gain-of-function mutation in Huntington's disease using CRISPR-Cas9.

Dominant gain-of-function mechanisms in Huntington's disease (HD) suggest selective silencing of mutant HTT produces robust therapeutic benefits. Here, capitalizing on exonic PAM-Altering SNP (PAS), we developed an allele-specific CRISPR-Cas9 strategy to permanently inactivate mutant HTT through nonsense- mediated decay (NMD). Comprehensive sequence/haplotype analysis identified SNP-generated NGG PAM sites on exons of common HTT haplotypes in HD subjects, revealing a clinically relevant PAS-based mutant- specific CRISPR-Cas9 strategy. Alternative allele of rs363099 (29th exon) eliminates the NGG PAM site on the most frequent normal HTT haplotype in HD, permitting mutant-specific CRISPR-Cas9 therapeutics in a predicted ~20% of HD subjects with European ancestry. Our rs363099-based CRISPR-Cas9 showed perfect allele specificity and good targeting efficiencies in patient-derived cells. Dramatically reduced mutant HTT mRNA and complete loss of mutant protein suggest that our allele-specific CRISPR-Cas9 strategy inactivate mutant HTT through NMD. In addition, GUIDE-seq analysis and subsequent validation experiments supported high levels of on-target gene specificity. Together, our data demonstrated a significant target population, complete mutant specificity, decent targeting efficiency in patient-derived cells, and minimal off-target effects on protein-coding genes, proving the concept of PAS-based allele-specific NMD-CRISPR-Cas9 and supporting its therapeutic potential in HD.