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.
Keywords: CRISPR‐Cas13; N6,2´‐O‐dimethyladenosine; RNA editing; RNA modification.
© 2026 The Author(s). Advanced Science published by Wiley‐VCH GmbH.