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Dryad

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

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Aug 29, 2022 version files 3.59 MB

Abstract

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 mutant HTT protein indicate that our allele-specific CRISPR/Cas9 strategy completely inactivates mutant HTT through NMD. RNAseq analysis also supported high levels of on-target gene specificity because no other genes except HTT were altered in clonal lines developed through our NMD-CRISPR/Cas9 strategy. Together, our data demonstrating significant target population, selective inactivation of mutant HTT, good targeting efficiency, and lack of recurrent off-targeting establish its therapeutic value of novel rs363099-based mutant HTT-specific NMD-CRISPR/Cas9 strategy in HD.