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 reengineered m(1)A modification valid eraser (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.