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.

中文翻译：

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用于定点 A 至 I RNA 编辑的短链向导 RNA


定点RNA编辑是一种很有前景的基因修饰技术，可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-gRNA) 的腺苷脱氨酶，将天然人 ADAR2 的 A 到 I RNA 编辑活性引导到可编程靶位点。在这项研究中，我们开发了短链 AD-gRNA (shAD-gRNA) 作为实用 RNA 编辑寡核苷酸的潜在基本框架。基于之前 AD-gRNA 的知识，shAD-gRNA 被设计为具有尽可能短的序列来诱导编辑活动。在体外，与原始AD-gRNA相比，shAD-gRNA表现出相似或优越的编辑诱导活性，具体取决于目标RNA序列，并且在目标位点周围具有较低的脱靶编辑活性，预计将成为热点用于脱靶编辑。此外，shAD-gRNA 在培养细胞中实现了外源性和内源性人类 ADAR 的目标 RNA 编辑。我们的结果将 shAD-gRNA 作为一个简短的基本框架，可用于进一步开发实用的 RNA 编辑寡核苷酸。