Huntington's disease (HD) is caused by an expanded CAG repeat in huntingtin (HTT). Since HD is dominant, and loss of HTT leads to neurological abnormalities, safe therapeutic strategies require selective inactivation of mutant HTT. Previously, we proposed a concept of CRISPR-Cas9 using mutant-specific PAM sites generated by SNPs to selectively inactivate mutant HTT. Aiming at revealing suitable targets for clinical development, we analyzed the largest HD genotype dataset to reveal target PAM-altering SNPs (PAS) and subsequently evaluated their allele specificities. The gRNAs based on the PAM sites generated by rs2857935, rs16843804, and rs16843836 showed high levels of allele specificity in patient-derived cells. Simultaneous use of two gRNAs based on rs2857935-rs16843804 or rs2857935-rs16843836 produced selective genomic deletions in mutant HTT and prevented the transcription of mutant HTT mRNA without impacting the expression of normal counterpart or re-integration of the excised fragment elsewhere in the genome. RNAseq and off-target analysis confirmed high levels of allele specificity and the lack of recurrent off-targeting. Approximately 60% of HD subjects are eligible for mutant-specific CRISPR-Cas9 strategies of targeting one of these 3 PAS in conjunction with one non-allele-specific site, supporting high applicability of PAS-based allele-specific CRISPR approaches in the HD patient population.

To fully characterize the molecular consequences of our mutant-specific CRISPR-Cas9 strategy, we performed RNAseq analysis. We transfected two HD iPSC lines (iPSC-A and iPSC-B) carrying adult onset CAG repeats with mutant-specific CRISPR-Cas9 (PX551 EFS vector expression SpCas9 and PX552 EFS plasmid expressing our test gRNA; experimental group; L4-R4 or L4-R6) or empty vector (PX551 EFS vector for SpCas9 and empty PX552 vector without gRNA; control group). Single cell clones were subsequently developed by limited dilution. Finally, 12 and 20 clonal lines were developed for the empty vector (EV) and TP-CRISPR group respectively. Clonal lines were further validated by Sanger sequencing and MiSeq analysis of genomic DNA. Then, genome-wide RNAseq analysis was performed by the Broad Institute. Sequence data were processed by STAR aligner as part of the Broad Institute's standard RNAseq analysis pipeline. For differential gene expression (DGE) analysis, we used transcripts per million (TPM) data computed by the TPMCalculator (https://github.com/ncbi/TPMCalculator). Expression levels in 20,260 protein-coding genes based on Ensembl (ftp://ftp.ensembl.org/pub/release-75/gtf/homo_sapiens/) were normalized; 3,420 genes were excluded due to zero TPM values in at least one sample.