A programmable system to methylate and demethylate m6A on specific mRNAs

Abstract

RNA N6-Methyladenosine (m6A) is the most abundant mRNA modification, and forms part of an epitranscriptomic system that modulates RNA function. RNA modifications can be reversibly catalyzed by several specific enzymes, and those modifications can be recognized by RNA binding proteins that in turn regulate biological processes. Although there are many reports demonstrating m6A participation in critical biological functions, this exploration has mainly been conducted through the global knockout or knockdown of the writers, erasers, or readers of m6A. Consequently, there is a lack of information about the role of m6A on single transcripts in biological processes, posing a challenge in understanding the biological functions of m6A. Here, we demonstrate a CRISPR/dCas13a-based RNA m6A-editor which can target mRNAs using single crRNA or multiple crRNAs array to methylate or demethylate m6A. We systematically assay its capabilities to enable the targeted rewriting of m6A dynamics, including modulation of circular RNA translation and transcript half-life. Finally, we demonstrate the utility of the system by specifically modulating XIST m6A levels, which can control X chromosome silencing and activation. Based on our editors, m6A on single and multiple transcripts can be modified to allow the exploration of the role of m6A on in biological processes.