Figure 4.

Allele-specific CRISPR-Cas9 targeting of HTT SNPs in the adult striatum of a mouse model of HD. (A) Cartoon depicting the AAV vector used to deliver the Exon50Tg1 and control sgRNAs and the delivery of AAV to mouse brains. The HD model mice have two alleles at the exon50 SNP—the BAC97 transgene with the T allele and the YAC18 transgene with the C allele. The Ex50Tg1 sgRNA targets the T allele of the SNP heterozygosity. The gRNA and a turboRFP reporter gene are delivered by AAV injection into the adult striatum of 8-week-old mice. Cas9 activity is provided as a mouse transgene. (B) Analysis at multiple time points. At 2, 4 and 6 weeks after AAV treatment, the frequency of the BAC97 allele is reduced, and InDel mutations are induced by the Ex50T-programmed nuclease. (C) Analysis of flanking exons. Heterozygous SNPs were examined by Illumina sequencing at two flanking exons in the 4 weeks samples. There is no significant change in the ratio of BAC97 to YAC18 alleles in exons 48 or 57, indicating that cleavage and repair at exon 50 do not induce a high frequency of deletions large enough to remove these SNPs. n = 3 mice and error bars represent the SEM. p-values <0.05 were considered significant. Exact p-values are reported in Supplementary File 3. AAV, adeno-associated virus. Color images are available online.