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

Abstract

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 nonsensemediated 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 mutantspecific 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.

Personalized allele-specific CRISPR-Cas9 strategies for myofibrillar myopathy 6

“…Since our allele-specific NMD-CRISPR was predicted to produce small indels, determining the editing efficiency was more feasible compared to TP-CRISPR. As previously observed [27, 28, 32-34], editing efficiencies of CRISPR-Cas9 strategies in iPSC were modest. Nevertheless, our allele-specific NMD-CRISPR strategy using a PPS showed complete allele specificities (Table 2).…”

“…Taking advantage of the strong requirement of NGG PAM sequence for SpCas9 [30], we previously developed allelespecific CRISPR-Cas9 approaches, capitalizing on genetic variations that generate or eliminate the NGG PAM sequence, taking Huntington's disease as an example [29]. As expected, our PAM-altering SNP-based CRISPR-cas9 strategies showed high levels of allele specificity [27][28][29], supporting its utility in other dominant disorders. In this study, we developed personalized allele-specific CRISPR-Cas9 strategies for MFM6 using PAM-altering SNPs, and showed their complete allele specificities.…”

“…We previously developed allele-specific CRISPR-Cas9 strategies using DNA variations that generate or eliminate the CRISPR-CAS9 PAM sequence (i.e., PAM-altering SNP; S. Fig. 1A), and demonstrated their high levels of allele specificities [27][28][29]. In addition, genetic variations that introduce mismatches near the PAM site can be used to edit the gene in an allele-selective manner (i.e., PAM-proximal SNP; S. Fig.…”

“…Subsequently, we performed a series of sequencing analyses for each trio to identify genetic variations that permit mutant-specific TP-CRISPR and NMD-CRISPR. Since PAS approaches demonstrated high levels of allele specificity, we focused on DNA variants that alter the NGG PAM sequence [27][28][29]. For TP-CRISPR strategies for subject #3, we could not identify allelespecific PAS upstream of the TSS due to many homopolymers around the candidate sites.…”

Allele-specific CRISPR/Cas9 editing inactivates a single nucleotide variant associated with collagen VI muscular dystrophy

“…Allele-specific CRISPR/Cas9-induced gene inactivation has been tested in various cellular and animal models of dominant diseases, with variable degrees of Cas9 on-target activity and allele discrimination. [23][24][25][26][27][28][29] Here, we applied this precision medicine to a recurrent glycine substitution in COL6A1 (G290R). 9,10 We show that the indel profiles favor gene inactivation, and that the gRNA design can be optimized for enhanced allele selectivity with the addition of base mismatches.…”