Allele-specific silencing of the gain-of-function mutation in Huntington’s disease using CRISPR/Cas9
Jun Wan Shin, … , Richard Z. Chen, Jong-Min Lee
Jun Wan Shin, … , Richard Z. Chen, Jong-Min Lee
Published August 30, 2022
Citation Information: JCI Insight. 2022;7(19):e141042. https://doi.org/10.1172/jci.insight.141042.
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Research Article Genetics

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 that selective silencing of mutant HTT produces robust therapeutic benefits. Here, capitalizing on exonic protospacer adjacent motif–altering (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 inactivates mutant HTT through NMD. In addition, GUIDE-Seq analysis and subsequent validation experiments support high levels of on-target gene specificity. Our data demonstrate 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.

Authors

Jun Wan Shin, Eun Pyo Hong, Seri S. Park, Doo Eun Choi, Ihn Sik Seong, Madelynn N. Whittaker, Benjamin P. Kleinstiver, Richard Z. Chen, Jong-Min Lee

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Figure 4

Allele specificity and on-target gene selectivity of mutant HTT-specific CRISPR/Cas9.

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Allele specificity and on-target gene selectivity of mutant HTT-specific...
(A) Two iPSC lines (iPSC-A and iPSC-B) carrying expanded CAG repeats on hap.01 (mutant HTT) and normal CAG repeats on hap.08 haplotype (normal HTT) were analyzed by RNA-Seq. This most frequent diplotype carries 10 heterozygous SNPs on exons of HTT (including our target PAS rs363099; red), permitting ASE analysis of RNA-Seq data. (B) For each SNP site, we counted the RNA-Seq sequence reads carrying hap.08 alleles (normal HTT) in EV-treated clonal lines (blue; n = 12) and those in CRISPR/Cas9-treated clonal lines (red; n = 12 samples/group). Each box plot shows the maximum, upper quarter, median, lower quarter, and minimum based on n = 12 independent clones/group. Circles represent outliers defined by a standard interquartile outlier detection method. Student t test (2 tailed) was performed separately to determine the statistical significance of each site. None of them were significant by a Bonferroni multiple-test–corrected P value. (C) Similarly, we compared sequence read counts of alleles of hap.01 at 10 heterozygous exonic SNPs (representing mutant HTT) in EV- and CRISPR/Cas9-treated clones (n = 12 samples/group) and performed Student t test (2 tailed). Asterisks denote statistical significance after Bonferroni multiple-test correction (corrected P < 0.05). (D) DGE analysis was performed to identify genes whose expression levels were altered by our mutant-specific CRISPR/Cas9. A volcano plot was based on 16,840 expressed genes in our clonal lines. The y axis and x axis represent the levels of significance (–log10[FDR]) and effect size (log2[fold change]), respectively. A dashed horizontal line represents the significance threshold (FDR = 0.05). Red circles represent genes containing or flanking the predicted off-target sites. (E) We also tested whether a gene set comprising all predicted off-targets was significantly enriched by comparing the true gene set score (sum of significance values) of predicted off-targets (red triangle) to a null distribution of gene set scores obtained from 1,000,000 random sampling of 52 genes.