Department of Medical Informatics, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China; Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Sun Yat-sen University, Guangzhou 510080, China.
Center for Biosystems Dynamics Research, RIKEN, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan; Graduate School of Biostudies, Kyoto University, Yoshida hon-machi, Kyoto 606-8501, Japan; Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang 110016, China.
Methods. 2022 Jul;203:56-61. doi: 10.1016/j.ymeth.2022.03.006. Epub 2022 Mar 17.
N-methyladenosine (mA) is a reversible and prevalent internal modification in RNAs and can be dynamically modulated by methyltransferase and demethylase. Targeted manipulation of mA RNA modification is critical in studying the functions of specific mA sites as well as developing molecular therapies through targeting mA. The CRISPR-Cas systems including CRISPR-Cas9 and CRISPR-Cas13 have been widely used to edit and modify specific nucleotides on DNA and RNA through fusing effective proteins such as enzymes with Cas9/13. Through taking advantage of the mA methyltransferase and demethylase, a series of CRISPR-Cas-based methods have also been developed to manipulate the mA methylation at specific RNA sites. This review summarizes the latest CRISPR-Cas13 and Cas9 toolkits for mA site-specific manipulation, including fundamental components, on-target efficiency, editing window, PAM/PFS requirement, and subcellularly localized targeting as well as potential limitations. We thus aim to provide an overview to assist researchers to choose an optimal tool to manipulate mA for different purposes and also point out possible optimization strategies.
N6-甲基腺苷（m6A）是 RNA 中一种可逆且普遍存在的内部修饰，可被甲基转移酶和去甲基酶动态调节。靶向操纵 m6A RNA 修饰对于研究特定 m6A 位点的功能以及通过靶向 m6A 开发分子治疗至关重要。CRISPR-Cas 系统包括 CRISPR-Cas9 和 CRISPR-Cas13，已被广泛用于通过融合 Cas9/13 等有效蛋白来编辑和修饰 DNA 和 RNA 上的特定核苷酸。通过利用 m6A 甲基转移酶和去甲基酶，还开发了一系列基于 CRISPR-Cas 的方法来操纵特定 RNA 位点的 m6A 甲基化。本综述总结了最新的 CRISPR-Cas13 和 Cas9 工具包用于 m6A 位点特异性操作，包括基本组成、靶效率、编辑窗口、PAM/PFS 要求以及亚细胞定位靶向以及潜在的局限性。因此，我们旨在提供一个概述，以帮助研究人员为不同目的选择最佳工具来操纵 m6A，并指出可能的优化策略。
