Haplotyping SNPs for allele-specific gene editing of the expanded huntingtin allele using long-read sequencing

HGG Adv. 2022 Sep 26;4(1):100146. doi: 10.1016/j.xhgg.2022.100146. eCollection 2023 Jan 12.


Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by CAG trinucleotide repeat expansions in exon-1 of huntingtin (HTT). Currently, there is no cure for HD, and the clinical care of individuals with HD is focused on symptom management. Previously, we showed allele-specific deletion of the expanded HTT allele (mHTT) using CRISPR-Cas9 by targeting nearby (<10 kb) SNPs that created or eliminated a protospacer adjacent motif (PAM) near exon-1. Here, we comprehensively analyzed all potential PAM sites within a 10.4-kb genomic region flanking exon-1 of HTT in 983 individuals with HD using a multiplex targeted long-read sequencing approach on the Oxford Nanopore platform. We developed computational tools (NanoBinner and NanoRepeat) to de-multiplex the data, detect repeats, and phase the reads on the expanded or the wild-type HTT allele. One SNP common to 30% of individuals with HD of European ancestry emerged through this analysis, which was confirmed as a strong candidate for allele-specific deletion of the mHTT in human HD cell lines. In addition, up to 57% HD individuals may be candidates for allele-specific editing through combinatorial SNP targeting. Cumulatively, we provide a haplotype map of the region surrounding exon-1 of HTT in individuals affected with HD. Our workflow can be applied to other repeat expansion diseases to facilitate the design of guide RNAs for allele-specific gene editing.

Keywords: CRISPR; Huntington’s disease; SNP; long-read sequencing; repeat detection.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Gene Editing*
  • Haplotypes / genetics
  • Humans
  • Neurodegenerative Diseases*
  • Polymorphism, Single Nucleotide / genetics