期刊
NUCLEIC ACIDS RESEARCH
卷 48, 期 10, 页码 5684-5694出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkaa269
关键词
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资金
- National Natural Science Foundation of China [81973343, 81673454, 81672608, 31801197]
- Fundamental Research Funds for the Central Universities (Sun Yat-sen University) [19ykpy130, 19ykzd24]
- China Postdoctoral Science Foundation [2018M643354]
- Guangdong Provincial Key Laboratory of Construction Foundation [2017B030314030]
- Guangdong Provincial Key Laboratory of Chiral Molecule and Drug Discovery [2019B030301005]
- US NIH [1RO1CA251698-01]
- CPRIT [RP180349, RP190077]
- Welch Foundation [I-1805]
Studies on biological functions of N-6-methyladenosine (m(6)A) 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 dm(6)ACRISPR, was created by linking a catalytically inactive Type VI-B Cas13 enzyme from Prevotella sp. P5-125 (dPspCas13b) to m(6)A demethylase AlkB homolog 5 (ALKBH5). dm(6)ACRISPR specifically demethylates m(6)A of targeted mRNA such as cytochrome b5 form A (CYB5A) to increase its mRNA stability. It can also demethylate beta-catenin-encoding CTNNB1 mRNA that contains multiple m(6)A sites to trigger its translation. In addition, the dm(6)ACRISPR 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.
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