Programmable RNA N 1 ‐Methyladenosine Demethylation by a Cas13d‐Directed Demethylase

N 1 ‐methyladenosine (m 1 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 1 A sites in specific transcripts has hindered efforts to clarify the association between a specific m 1 A‐modified transcript and its phenotypic outcomes. Here we develop a CRISPR‐Cas13d‐based tool called re engineered m 1 A mo dification v alid er aser (termed “REMOVER”) for targeted m 1 A demethylation of a specific transcript. The catalytically inactive RfxCas13d (dCasRx) is fused to the m 1 A demethylase ALKBH3, and the dCasRx‐ALKBH3 fusion protein can mediate potent demethylation of m 1 A‐modified RNAs. We further find that REMOVER can specifically demethylate m 1 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 1 A‐modified transcripts, which enables further elucidation of the relationship between m 1 A modification of specific transcripts and their phenotypic outcomes.