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

Abstract

N⁶-Methyladenosine (m⁶A) is the most widespread internal messenger RNA 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 elucidate causal relationships between the presence of a specific m⁶A and phenotypic outcomes. In the present study, 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 that can reveal the effect of individual m⁶A modifications and dissect their functional roles.