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.

中文翻译：

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通过编码区编辑对突变亨廷顿蛋白进行等位基因特异性敲除 单核苷酸多态性 杂合性


亨廷顿病 (HD) 是一种毁灭性的常染色体显性神经退行性疾病，由亨廷顿 (HTT) 基因中的三核苷酸重复扩增引起。通过基于成簇规则间隔短回文重复序列 (CRISPR)-Cas9 的基因编辑使突变等位基因失活，为这种疾病提供了一种可能的治疗方法，但正常 HTT 功能的永久性破坏可能会损害成人神经元功能。在这里，我们使用一种新型 HD 小鼠模型来检查突变 HTT (mHTT) 的等位基因特异性编辑，其中 BAC97 转基因表达 mHTT，YAC18 转基因表达正常 HTT。我们通过靶向含有常见杂合单核苷酸多态性 (SNP) 的蛋白质编码序列来实现 HTT 的等位基因特异性失活。结果是 HD 小鼠模型中 mHTT 蛋白显着且等位基因选择性减少。单个 CRISPR-Cas9 核酸酶在神经元中的表达在目标 HD 等位基因中产生高频突变，其中包括小插入/缺失 (InDel) 突变和病毒载体插入。因此，等位基因特异性靶向 InDel 和杂合编码区 SNP 的插入突变提供了一种灭活导致遗传病的常染色体显性突变的可行方法。