A programmable system to methylate and demethylate N6-methyladenosine (m6A) on specific RNA transcripts in mammalian cells

Abstract

RNA N 6 -methyladenosine (m 6 A) is the most abundant internal mRNA modification and forms part of an epitranscriptomic system that modulates RNA function. m 6 A is reversibly catalyzed by specific enzymes, and those modifications can be recognized by RNA-binding proteins that in turn regulate biological processes. Although there are many reports demonstrating m 6 A participation in critical biological functions, this exploration has mainly been conducted through the global KO or knockdown of the writers, erasers, or readers of m 6 A. Consequently, there is a lack of information about the role of m 6 A on single transcripts in biological processes, posing a challenge in understanding the biological functions of m 6 A. Here, we demonstrate a CRISPR/dCas13a-based RNA m 6 A editors, which can target RNAs using a single or multiple CRISPR RNA array to methylate or demethylate m 6 A in human 293T cells and mouse embryonic stem cells. We systematically assay its capabilities to enable the targeted rewriting of m 6 A dynamics, including modulation of circular RNA translation and transcript half-life. Finally, we use the system to specifically modulate m 6 A levels on the noncoding XIST (X-inactive specific transcript) to modulate X chromosome silencing and activation. The editors described here can be used to explore the roles of m 6 A in biological processes.