Gao Minsong, Su Shichen, Cao Jie, Xiang Siying, Huang Ye, Shu Xiao, Ma Jinbiao, Liu Jianzhao
MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Zheda Road 38, Hangzhou 310027, China.
State Key Laboratory of Genetic Engineering, Collaborative Innovation Centre of Genetics and Development, Department of Biochemistry, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200438, China.
ACS Chem Biol. 2022 Apr 15;17(4):854-863. doi: 10.1021/acschembio.1c00895. Epub 2022 Mar 16.
Development of tools for precise manipulation of cellular mRNA mA methylation at the base level is highly required. Here, we report an RNA-guided RNA modification strategy using a fusion protein containing deactivated nuclease Cas13b and mA methyltransferase METTL14, namely, dCas13b-M14, which is designedly positioned in the cytoplasm. dCas13b-M14 naturally heterodimerizes with endogenous METTL3 to form a catalytic complex to methylate specific cytoplasmic mRNA under a guide RNA (gRNA). We developed assays to screen and validate the guiding specificity of varied gRNAs at single-base resolution. With an optimum combination of dCas13b-M14 and gRNAs inside cells, we have successfully tuned methylation levels of several selected mRNA mA sites. The off-target effect was evaluated by whole transcriptome mA sequencing, and a very minor perturbation on the methylome was revealed. Finally, we successfully utilized the editing tool to achieve methylations on five selected mRNA sites. Together, this study paves the way for studying position-dependent roles of mA methylation in a particular transcript.
目前非常需要开发能够在碱基水平上精确操纵细胞mRNA m⁶A甲基化的工具。在此，我们报道了一种RNA引导的RNA修饰策略，该策略使用一种融合蛋白，其包含失活的核酸酶Cas13b和m⁶A甲基转移酶METTL14，即dCas13b-M14，特意将其定位在细胞质中。dCas13b-M14与内源性METTL3自然异源二聚化，形成催化复合物，在引导RNA（gRNA）的作用下对特定的细胞质mRNA进行甲基化。我们开发了检测方法，以单碱基分辨率筛选和验证不同gRNA的引导特异性。通过在细胞内优化组合dCas13b-M14和gRNA，我们成功地调节了几个选定的mRNA m⁶A位点的甲基化水平。通过全转录组m⁶A测序评估脱靶效应，结果显示对甲基化组的干扰非常小。最后，我们成功利用该编辑工具在五个选定的mRNA位点实现了甲基化。总之，本研究为研究m⁶A甲基化在特定转录本中的位置依赖性作用铺平了道路。
