单向导 RNA 酶实现可编程 RNA 碱基编辑。

Programmable RNA base editing with a single gRNA-free enzyme.

机构信息

Bio-Med Big Data Center, CAS Key Laboratory of Computational Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China.

University of Chinese Academy of Sciences, Beijing 100049, China.

出版信息

Nucleic Acids Res. 2022 Sep 9;50(16):9580-9595. doi: 10.1093/nar/gkac713.

DOI:10.1093/nar/gkac713

PMID:36029126

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9458445/

Abstract

Programmable RNA editing enables rewriting gene expression without changing genome sequences. Current tools for specific RNA editing dependent on the assembly of guide RNA into an RNA/protein complex, causing delivery barrier and low editing efficiency. We report a new gRNA-free system, RNA editing with individual RNA-binding enzyme (REWIRE), to perform precise base editing with a single engineered protein. This artificial enzyme contains a human-originated programmable PUF domain to specifically recognize RNAs and different deaminase domains to achieve efficient A-to-I or C-to-U editing, which achieved 60-80% editing rate in human cells, with a few non-specific editing sites in the targeted region and a low level off-target effect globally. The RNA-binding domain in REWIREs was further optimized to improve editing efficiency and minimize off-target effects. We applied the REWIREs to correct disease-associated mutations and achieve both types of base editing in mice. As a single-component system originated from human proteins, REWIRE presents a precise and efficient RNA editing platform with broad applicability.

摘要

可编程 RNA 编辑可在不改变基因组序列的情况下重写基因表达。目前特定的 RNA 编辑工具依赖于将向导 RNA 组装成 RNA/蛋白质复合物，这导致了递送障碍和低编辑效率。我们报告了一种新的无 gRNA 系统，即单个 RNA 结合酶的 RNA 编辑（REWIRE），该系统可使用单个工程化蛋白进行精确的碱基编辑。这种人工酶包含一个源自人类的可编程 PUF 结构域，可特异性识别 RNA，并具有不同的脱氨酶结构域，可实现高效的 A 到 I 或 C 到 U 编辑，在人类细胞中实现了 60-80%的编辑率，在靶向区域有几个非特异性编辑位点，整体脱靶效应较低。REWIRE 中的 RNA 结合结构域进一步得到优化，以提高编辑效率并最小化脱靶效应。我们将 REWIRE 应用于纠正与疾病相关的突变，并在小鼠中实现了两种类型的碱基编辑。作为源自人类蛋白的单一组分系统，REWIRE 提供了一种精确、高效的 RNA 编辑平台，具有广泛的适用性。