A programmable system to methylate and demethylate N(6)-methyladenosine (m(6)A) on specific RNA transcripts in mammalian cells

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.