Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein
Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein
复制标题
使用工程化 dCas13b-ALKBH5 融合蛋白进行靶向 mRNA 去甲基化
DOI：
10.1093/nar/gkaa269
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发表时间：
2020-06-04
影响因子：
14.9
通讯作者：
Wang, Hongsheng
中科院分区：
文献类型：
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作者：
Li, Jiexin;Chen, Zhuojia;Wang, Hongsheng
近年来，关于N6-甲基腺苷(M6A)修饰mRNA生物学功能的研究如雨后春笋般涌现。在这里，我们首次构建并鉴定了一种基于CRISPR-Cas13b的工具，该工具通过将Prevotea sp.P5-125的VI-B型Cas13催化死亡酶(DPspCas13b)与m6A去甲基酶ALKBH5(命名为dm6ACRISPR)融合，靶向m6A甲基化。随后，这种系统被证明特异性地去甲基化靶mRNA的m6A，如CyB5A，以增加其mRNA的稳定性。此外，dm6ACRISPR系统似乎提供了目标基因的有效去甲基化，具有微弱的脱靶效应。总之，我们提供了一种可编程的体内操作工具，用于研究特定基因的mRNA修饰及其潜在的生物学功能。
Studies on biological functions of N6-methyladenosine (m6A) modification in mRNA have sprung up in recent years. Here we construct and characterize a CRISPR-Cas13b-based tool for the first time that targeted m6A methylation of mRNA by fusing the catalytically dead Type VI-B Cas13 enzyme from Prevotella sp.P5-125 (dPspCas13b) with the m6A demethylase ALKBH5, which is named as dm6ACRISPR. Subsequently, such system is shown to specific demethylase the m6A of target mRNA such as CYB5A to increase its mRNA stability. In addition, the dm6ACRISPR system appeared to afford efficient demethylation of the target genes with tenuous off-target effects. Together, we provide a programmable and in vivo manipulation tool to study mRNA modification and its potential biological functions of specific gene.