Programmable RNA N -Methyladenosine Demethylation by a Cas13d-Directed Demethylase.

N -methyladenosine (m A) is a prevalent and reversible RNA modification, which plays a crucial role in the regulation of RNA fate and gene expression. However, the lack of tools to precisely manipulate m A sites in specific transcripts has hindered efforts to clarify the association between a specific m A-modified transcript and its phenotypic outcomes. Here we develop a CRISPR-Cas13d-based tool called reengineered m A modification valid eraser (termed "REMOVER") for targeted m A demethylation of a specific transcript. The catalytically inactive RfxCas13d (dCasRx) is fused to the m A demethylase ALKBH3, and the dCasRx-ALKBH3 fusion protein can mediate potent demethylation of m A-modified RNAs. We further find that REMOVER can specifically demethylate m A of MALAT1 and PRUNE1 RNAs, thereby significantly increasing their stability. Our study establishes REMOVER as a tool for targeted RNA demethylation of specific m A-modified transcripts, which enables further elucidation of the relationship between m A modification of specific transcripts and their phenotypic outcomes.