DOI: 10.5061/dryad.1g1jwstsb
title: Complete allele-specific silencing of the gain-of-function mutation of Huntington's disease

description: Dominant gain-of-function mechanism in Huntington's disease (HD)
 suggests selective inactivation of mutant HTT produces the biggest
 therapeutic benefit. Here, we developed a complete allele-specific
 CRISPR/Cas9 strategy to permanently silence mutant HTT through
 nonsense-mediated decay (NMD), capitalizing on an exonic PAM (protospacer
 adjacent motif)-Altering SNP (PAS). Comprehensive sequence/haplotype
 analysis identified PAS-generated NGG PAM sites on exons of common HTT
 haplotypes in HD patients, revealing a single clinically meaningful
 PAS-based mutant-specific NMD-CRISPR/Cas9 strategy. The alternative allele
 of rs363099 eliminates NGG PAM site on the most frequent normal HTT
 haplotype in HD, permitting mutant HTT-specific CRISPR/Cas9 therapeutics
 in ~20% of HD patients with European ancestry. Our rs363099-based
 CRISPR/Cas9 showed perfect allele specificity and good targeting
 efficiencies in cells derived from HD patients. Dramatically reduced
 mutant HTT mRNA and complete loss of mutant HTT protein indicate that our
 allele-specific CRISPR/Cas9 strategy completely inactivates mutant HTT
 through NMD. RNAseq analysis also supported high levels of on-target gene
 specificity because no other genes except HTT were altered in clonal lines
 developed through our NMD-CRISPR/Cas9 strategy. Together, our data
 demonstrating significant target population, selective inactivation of
 mutant HTT, good targeting efficiency, and lack of recurrent off-targeting
 establish its therapeutic value of novel rs363099-based mutant
 HTT-specific NMD-CRISPR/Cas9 strategy in HD.
description: We transfected two HD iPSC
 lines carrying adult onset CAG repeats (42 and 46; both carrying hap.01
 and hap.08 diplotype) for mutant-specific NMD-CRISPR/Cas (PX551 vector for
 <em>sp</em>Cas9 and PX552 for our test gRNA;  experimental
 group) or empty vector (PX551 vector <em>sp</em>Cas9 and empty
 PX552 vector without gRNA; control group) and subsequently developed
 single cell clones by limited dilution. 12 clonal lines were developed for
 each group and 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. Expression
 levels of genes were based on 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, and subsequently, 3,420 genes were excluded because of zero TPM values in at least one sample. Therefore, we provide 16,840 genes expressed in all 24 samples (HD.NMD-CRISPR.RNAseq.24.Sample.200601.txt). Three files are associated with this data set: HD.NMD-CRISPR.RNAseq.24.Sample.200601.txt : RNAseq expression data NMD-CRISPR.RNAseq.Meta.Data.csv : Sample metadata describing sample characteristics and covariates README.txt : Description of columns in the metadata file