Short-Chain Guide RNA for Site-Directed A-to-I RNA Editing
Short-Chain Guide RNA for Site-Directed A-to-I RNA Editing
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DOI：
10.1089/nat.2020.0866
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发表时间：
2020-11-10
影响因子：
4
通讯作者：
Fukuda, Masatora
中科院分区：
文献类型：
--
作者：
Nose, Kanako;Hidaka, Kota;Fukuda, Masatora
定点RNA编辑是一种很有前途的基因修饰技术，用于治疗和制药应用。我们之前构建了作用于RNA （ADAR）引导RNA （AD-gRNAs）的腺苷脱氨酶，将天然人类ADAR2的a -to- i RNA编辑活性引导到可编程的目标位点。在这项研究中，我们开发了短链AD-gRNA （shade - grna）作为实际rna编辑寡核苷酸的潜在基本框架。基于对以往AD-gRNA的了解，我们设计了最短序列的shade - grna来诱导编辑活性。在体外实验中，与原始AD-gRNA相比，根据靶RNA序列的不同，shade - grna表现出类似或更强的编辑诱导活性，而靶位点周围的脱靶编辑活性较低，预计将成为脱靶编辑的热点。此外，在培养的细胞中，shade - grnas用外源性和内源性人类ADARs都实现了靶RNA编辑。我们的研究结果表明，shade - grna作为一个简短的基本框架，将适用于进一步开发实用的rna编辑寡核苷酸。
Site-directed RNA editing is a promising genetic modification technology for therapeutic and pharmaceutical applications. We previously constructed adenosine deaminases acting on RNA (ADAR)-guiding RNAs (AD-gRNAs) that direct A-to-I RNA editing activity of native human ADAR2 into a programmable target site. In this study, we developed the short-chain AD-gRNA (shAD-gRNA) as a potential basic framework for practical RNA-editing oligonucleotides. Based on knowledge of previous AD-gRNA, shAD-gRNAs were designed to have the shortest possible sequence for the induction of editing activity. In vitro, compared to the original AD-gRNA, the shAD-gRNAs showed similar or superior editing induction activity, depending on the target RNA sequence, and had lower off-target editing activity around the target site, which is predicted to be a hotspot for off-target editing. Moreover, shAD-gRNAs achieved target RNA editing with both exogenous and endogenous human ADARs in cultured cells. Our results present shAD-gRNA as a short basic framework that would be applicable to further development for practical RNA-editing oligonucleotides.