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.
Keywords: CRISPR-Cas13; N1-methyladenosine; RNA; mRNA; targeted demethylation.
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