Allele-Specific Knockdown of Mutant Huntingtin Protein via Editing at Coding Region Single Nucleotide Polymorphism Heterozygosities
Sarah Oikemus, Edith L. Pfister, Ellen Sapp, Kathryn Chase, Lori Kennington, Edward Hudgens, Rachael Miller, Lihua Julie Zhu, Akanksh Chaudhary, Eric Mick, Miguel Sena‐Esteves, Scot A. Wolfe, Marian DiFiglia, Neil Aronin, Michael H. Brodsky
Abstract
Huntington's disease (HD) is a devastating, autosomal dominant neurodegenerative disease caused by a trinucleotide repeat expansion in the huntingtin (HTT) gene. Inactivation of the mutant allele by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 based gene editing offers a possible therapeutic approach for this disease, but permanent disruption of normal HTT function might compromise adult neuronal function. Here, we use a novel HD mouse model to examine allele-specific editing of mutant HTT (mHTT), with a BAC97 transgene expressing mHTT and a YAC18 transgene expressing normal HTT. We achieve allele-specific inactivation of HTT by targeting a protein coding sequence containing a common, heterozygous single nucleotide polymorphism (SNP). The outcome is a marked and allele-selective reduction of mHTT protein in a mouse model of HD. Expression of a single CRISPR-Cas9 nuclease in neurons generated a high frequency of mutations in the targeted HD allele that included both small insertion/deletion (InDel) mutations and viral vector insertions. Thus, allele-specific targeting of InDel and insertion mutations to heterozygous coding region SNPs provides a feasible approach to inactivate autosomal dominant mutations that cause genetic disease.