Programmable RNA N¹ -Methyladenosine Demethylation by a Cas13d-Directed Demethylase

N¹ -methyladenosine (m¹ 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 m¹ A sites in specific transcripts has hindered efforts to clarify the association between a specific m¹ A-modified transcript and its phenotypic outcomes. Here we develop a CRISPR-Cas13d-based tool called reengineered m¹ A modification valid eraser (termed "REMOVER") for targeted m¹ A demethylation of a specific transcript. The catalytically inactive RfxCas13d (dCasRx) is fused to the m¹ A demethylase ALKBH3, and the dCasRx-ALKBH3 fusion protein can mediate potent demethylation of m¹ A-modified RNAs. We further find that REMOVER can specifically demethylate m¹ A of MALAT1 and PRUNE1 RNAs, thereby significantly increasing their stability. Our study establishes REMOVER as a tool for targeted RNA demethylation of specific m¹ A-modified transcripts, which enables further elucidation of the relationship between m¹ A modification of specific transcripts and their phenotypic outcomes.