Programmable m6A modification of cellular RNAs with a Cas13-directed methyltransferase

Abstract

N6-methyladenosine (m6A) is the most widespread internal mRNA modification in humans. Despite recent progress in understanding the biological roles of m6A, the inability to install m6A site-specifically in individual transcripts has hampered efforts to establish causal relationships between the presence of a specific m6A and phenotypic outcomes. Here we demonstrate that nucleus-localized dCas13 fusions with a truncated METTL3 methyltransferase domain and cytoplasm-localized fusions with a modified METTL3:METTL14 methyltransferase complex can direct site-specific m6A incorporation in distinct cellular compartments, with the former fusion protein having particularly low off-target activity. Independent cellular assays across multiple sites confirm that this targeted RNA methylation (TRM) system mediates efficient m6A installation in endogenous RNA transcripts with high specificity. Finally, we show that TRM can induce m6A-mediated changes to transcript abundance and alternative splicing. These findings establish TRM as a tool for targeted epitranscriptome engineering to help reveal the effect of individual m6A modifications and dissect their functional roles.