<p>Recent studies claim that <i>N</i>⁴-acetylcytidine (ac⁴C) modification of RNA confers crucial regulatory roles, such as increasing translation efficiency and prolonging its half-life. However, the absence of methods for selectively acetylating specific RNA molecules hampers linking ac⁴C to cell physiology. Here, we developed an efficient molecular tool that incorporates ac⁴C on a specific transcript of interest. Through protein engineering, we developed a hyperactive variant of <i>N</i>-acetyltransferase 10 (NAT10), designated enhanced NAT10 (eNAT10). When fused to the programmable RNA-targeting protein dCas13, eNAT10 enables robust acetylation of various target RNAs in multiple contexts. RNA acetylation by dCas13–eNAT10 was highly dependent on co-transfected guide RNA, highlighting its specificity. We also describe the programmable RNA chemical modification in vivo using dual-adeno-associated virus. Using our system, we found that acetylation of RNA may modulate the subcellular localization of modified transcripts. We anticipate that our tool will facilitate numerous studies on ac⁴C functions across different cellular and disease contexts.</p><p></p>