Programmable m(6)A modification of cellular RNAs with a Cas13-directed methyltransferase

N(6)-methyladenosine (m(6)A) is the most widespread internal mRNA modification in humans. Despite recent progress in understanding the biological roles of m(6)A, the inability to install m(6)A site-specifically in individual transcripts has hampered efforts to elucidate causal relationships between the presence of a specific m(6)A 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 m(6)A 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 m(6)A installation in endogenous RNA transcripts with high specificity. Finally, we show that TRM can induce m(6)A-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 m(6)A modifications and dissect their functional roles.