Programmable RNA N⁶,2´-O-Dimethyladenosine Editing

N⁶,2'-O-dimethyladenosine (m⁶Am) is a prevalent RNA modification located at the first transcribed nucleotide adjacent to the 5' cap of mRNAs, where it has been implicated in gene regulation. However, the lack of methods for precise, transcript-specific manipulation of m⁶Am has limited its functional dissection. Here, we develop a programmable RNA-editing platform, termed Targeted m⁶Am Methylation (TAmM), that enables site-specific installation of m⁶Am on selected cellular transcripts. TAmM is engineered by fusing the catalytically inactive RfxCas13d (dCasRx) with the m⁶Am methyltransferase PCIF1, allowing guided deposition of m⁶Am at cap-proximal nucleotides. Using TAmM, we achieve efficient and specific m⁶Am installation at single-nucleotide resolution, as validated by LC-MS/MS. Targeted m⁶Am editing does not alter steady-state mRNA abundance but modulates protein output in a transcript-dependent manner. Mechanistically, m⁶Am installation enhances polysome association, indicating a direct role in translational regulation. Functional interrogation demonstrates that cap-proximal m⁶Am deposition on CTNNB1 increases β-catenin protein expression, promoting cell proliferation, clonogenicity, and migration in cancer cell models. Importantly, TAmM exhibits high fidelity, with negligible effects on the global m⁶Am landscape, transcriptome, or proteome. Our study establishes TAmM as a precise and versatile platform for programmable m⁶Am manipulation and reveals transcript-specific roles of m⁶Am in gene regulation.