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.

N ⁶ -甲基腺苷 (m ⁶ A) 是人类最普遍的内部信使 RNA 修饰。尽管最近在理解 m ⁶ A 的生物学作用方面取得了进展，但无法在单个转录本中专门安装 m ⁶ A 位点阻碍了阐明特定 m ⁶ A 的存在与表型结果之间的因果关系的努力。在本研究中，我们证明了具有截短的 METTL3 甲基转移酶结构域的核定位 dCas13 融合和具有修饰的 METTL3:METTL14 甲基转移酶复合物的细胞质定位融合可以指导位点特异性 m ⁶在不同的细胞区室中掺入，前一种融合蛋白具有特别低的脱靶活性。跨多个位点的独立细胞测定证实，这种靶向 RNA 甲基化 (TRM) 系统可在具有高特异性的内源性 RNA 转录物中介导有效的 m ⁶ A 安装。最后，我们表明 TRM 可以诱导 m ⁶ A 介导的转录本丰度和可变剪接变化。这些发现将 TRM 确立为靶向外转录组工程的工具，可以揭示单个 m ⁶ A 修饰的影响并剖析它们的功能作用。