PAM-altering SNP-based allele-specific CRISPR-Cas9 therapeutic strategies for Huntington's disease

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.