N6-methyladenosine (m6A) is rapidly being studied and uncovered to play a significant role in various biological processes as well as in RNA fate and functions, while the effects of defined m6A sites are rarely characterized for the lack of convenient tools. To provide an applicable method to remove m6A modification at specific loci, an m6A editing system called "targeted RNA demethylation by SunTag system (TRADES)" is engineered. In this system, the targeting element dCas13b is fused to ten copies of GCN4 peptides which can recruit multiple scFv-fusion RNA demethylase to demethylate the target m6A site. Owing to this design, TRADES is more flexible to the indistinct m6A sites for its wide editing window. By site-specific demethylation of messenger RNA (mRNA) SON A2699, the lifetime of SON RNA is successfully prolonged in HeLa cells. Meanwhile, TRADES negligibly influences the lifetime of other non-targeted transcripts. TRADES also can regulate the gene expression of target transcript in an m6A-dependent manner. Moreover, the interference occuring for HBV and HIV replications demonstrates that the TRADES system holds potential in viral life cycle regulation and clinical applications.
Keywords: N6‐methyladenosine; RNA modification; gene expression; nucleic acids; site‐specific demethylation.
© 2020 The Authors. Published by Wiley‐VCH GmbH.