Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein

Jiexin Li, Zhuojia Chen, Feng Chen, Guoyou Xie, Yuyi Ling, Yanxi Peng, Yu Lin, Nan Luo, Cheng Ming Chiang, Hongsheng Wang

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

Studies on biological functions of N6-methyladenosine (m6A) modification in mRNA have drawn significant attention in recent years. Here we describe the construction and characterization of a CRISPR-Cas13b-based tool for targeted demethylation of specific mRNA. A fusion protein, named dm6ACRISPR, was created by linking a catalytically inactive Type VI-B Cas13 enzyme from Prevotella sp. P5-125 (dPspCas13b) to m6A demethylase AlkB homolog 5 (ALKBH5). dm6ACRISPR specifically demethylates m6A of targeted mRNA such as cytochrome b5 form A (CYB5A) to increase its mRNA stability. It can also demethylate β-catenin-encoding CTNNB1 mRNA that contains multiple m6A sites to trigger its translation. In addition, the dm6ACRISPR system incurs efficient demethylation of targeted epitranscriptome transcripts with limited off-target effects. Targeted demethylation of transcripts coding for oncoproteins such as epidermal growth factor receptor (EGFR) and MYC can suppress proliferation of cancer cells. Together, we provide a programmable and in vivo manipulation tool to study mRNA modification of specific genes and their related biological functions.

Original languageEnglish (US)
Pages (from-to)5684-5694
Number of pages11
JournalNucleic acids research
Volume48
Issue number10
DOIs
StatePublished - Jun 4 2020

ASJC Scopus subject areas

  • Genetics

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