Figure 2.

Allele-specific targeting of mHTT in BAC97/YAC18 primary neurons. (A) Cartoon depicting the generation and infection of primary cortical neurons from mice heterozygous for Yac18 (wt) and Bac97 (mutant) human transgenes as well as a Cas9 transgene. (B) An allele-specific sgRNA targeting the T allele of Htt Exon 50 (Ex50T) was introduced into BAC97/YAC18/Cas9 mouse primary neurons by lentiviral transduction using the LentiCRISPRcmvGFP vector. An sgRNA targeting the Rosa26 locus was used as a control. Genomic DNA was prepared 7 days after infection. PCR amplicons containing the targeted SNPs were barcoded and analyzed by Illumina sequencing. InDel allele frequencies were determined using the CRISPResso software package. The control sample has a higher frequency of the BAC97 allele than YAC18 due to a higher copy number of the BAC97 transgene. In the treated sample, 40% of reads are induced InDel mutations. 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. (C) The distribution of InDel sizes shows a strong bias toward frameshift mutations (open bars) rather than in frame mutations (filled bars). The insertion of a single base is the dominant allele type. InDel, insertion/deletion; PCR, polymerase chain reaction; SEM, standard error of the mean; sgRNA, single guide RNA; wt, wild type. Color images are available online.