Targeting N-Terminal Huntingtin with a Dual-sgRNA Strategy by CRISPR/Cas9
Targeting N-Terminal Huntingtin with a Dual-sgRNA Strategy by CRISPR/Cas9
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通过 CRISPR/Cas9 采用双 sgRNA 策略靶向 N 末端亨廷顿蛋白
DOI：
10.1155/2019/1039623
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发表时间：
2019-11-16
影响因子：
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通讯作者：
Zhang，Chun-Li
中科院分区：
文献类型：
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作者：
Wu，Junjiao;Tang，Yu;Zhang，Chun-Li
亨廷顿病 (HD) 是一种常染色体显性遗传性进行性神经退行性疾病，由亨廷顿 (HTT) 基因中的 CAG/聚谷氨酰胺 (polyQ) 重复扩增引起。 PolyQ 束位于外显子 1 的 N 末端 HTT 中并由其转录。HTT 是一种大型多面蛋白，参与一系列细胞功能。先前的研究表明，缺乏 N 末端的截短 HTT 保留了可以产生神经保护作用的特定功能。它表明可以通过使用 CRISPR/Cas9 去除有害的 N 端 HTT 来修复 HD，而不损害剩余 HTT 肽的功能。为了成功生成功能性截短 HTT 蛋白，需要能够启动截短 HTT 表达的替代下游 ATG 起始密码子。在这项研究中，我们搜索了外显子7之前所有可能的框内ATG，并证明其中一个确实可以启动质粒中下游的GFP表达。然后我们尝试通过 CRISPR/Cas9 采用优化的双 sgRNA 策略去除内源性 N 端 HTT；然而，我们无法检测到截短的 HTT 表达的明显特征。我们的结果表明，N 端 HTT 的非规范 ATG 在基因组环境中可能无效，就像在构建体环境中一样。尽管如此，我们的研究检验了下游非经典 ATG 对蛋白质翻译的治疗功效，并为 HTT 基因的进一步基因组操作提供了优化的双 sgRNA 策略。
Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder, caused by a CAG/polyglutamine (polyQ) repeat expansion in the Huntingtin (HTT) gene. The polyQ tract is located in and transcribed from N-terminal HTT of exon 1. HTT is a large multifaceted protein, which participates in a range of cellular functions. Previous studies have shown that truncated HTT, which lacks N-terminus, retains specific functions that can produce neuroprotective benefits. It gives an insight that it is possible to repair HD by removing deleterious N-terminal HTT with CRISPR/Cas9, without compromising functions of remaining HTT peptides. To successfully generate functional truncated HTT proteins, an alternative downstream ATG start codon that is capable of initiating truncated HTT expression is required. In this study, we searched all possible in-frame ATGs before exon 7 and demonstrated that one of them can indeed initiate the downstream GFP expression in plasmids. We then tried to remove endogenous N-terminal HTT with an optimized dual-sgRNA strategy by CRISPR/Cas9; however, we cannot detect obvious traits of truncated HTT expression. Our results suggest that noncanonical ATGs of N-terminal HTT may not be effective in the genomic context, as in the construct context. Nevertheless, our study examined the therapeutic efficacy of downstream noncanonical ATGs for protein translation and also provided an optimized dual-sgRNA strategy for further genome manipulation of the HTT gene.