Allele-specific silencing of the gain-of-function mutation in Huntington’s disease using CRISPR/Cas9
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
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
View: Text | PDF
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

×

Figure 3

Molecular consequences of mutant HTT-specific CRISPR/Cas9.

Options: View larger image (or click on image) Download as PowerPoint
Molecular consequences of mutant HTT-specific CRISPR/Cas9. (A) Our mutan...
(A) Our mutant-specific CRISPR/Cas9 strategy using the NGG PAM site generated by rs363099 is illustrated. The NGG PAM site occurs on the minus strand of the mutant HTT (red underline). The PAS (rs363099, red) and the crRNA hybridization site (black underline) are highlighted. (B) An HD iPSC with a juvenile-onset CAG repeat was transfected with a plasmid vector expressing either SpCas9 only (empty vector [EV]) or SpCas9/gRNA to establish targeted clonal lines. Three non-HD single-cell clones were also developed after EV treatment. Three independent targeted single-cell clones with inactivated mutant HTT were confirmed by Sanger sequencing. All 3 clonal lines showed out-of-frame deletion at the 29th exon. Dashed lines represent deletions. (C–F) Subsequently, 3 independent clones for non-HD control (left; Normal + EV), EV-treated HD clones (middle; HD + EV), and CRISPR/Cas9 treated HD clones (right; HD + CRISPR) were analyzed to characterize HTT DNA, RNA, and protein (n = 3 for each group). (C) The presence of an expanded CAG repeat in the DNA of the targeted clones (upper band) was confirmed by PCR assay of genomic DNA. (D) The expression levels of HTT mRNA were measured by RT-PCR analysis designed to detect the CAG repeat region. (E and F) Total and mutant HTT protein levels were determined by immunoblot analysis using a panhuntingtin antibody (MAB2166) and mutant huntingtin-specific antibody (1F8), respectively. Images of the full gels of immunoblot analysis are provided in Supplemental Figure 7.