Programmable RNA 5-methylcytosine (m⁵C) modification of cellular RNAs by dCasRx conjugated methyltransferase and demethylase

5-Methylcytosine (m(5)C), an abundant RNA modification, plays a crucial role in regulating RNA fate and gene expression. While recent progress has been made in understanding the biological roles of m(5)C, the inability to introduce m(5)C at specific sites within transcripts has hindered efforts to elucidate direct links between specific m(5)C and phenotypic outcomes. Here, we developed a CRISPR-Cas13d-based tool, named reengineered m(5)C modification system (termed 'RCMS'), for targeted m(5)C methylation and demethylation in specific transcripts. The RCMS editors consist of a nuclear-localized dCasRx conjugated to either a methyltransferase, NSUN2/NSUN6, or a demethylase, the catalytic domain of mouse Tet2 (ten-eleven translocation 2), enabling the manipulation of methylation events at precise m(5)C sites. We demonstrate that the RCMS editors can direct site-specific m(5)C incorporation and demethylation. Furthermore, we confirm their effectiveness in modulating m(5)C levels within transfer RNAs and their ability to induce changes in transcript abundance and cell proliferation through m(5)C-mediated mechanisms. These findings collectively establish RCMS editors as a focused epitranscriptome engineering tool, facilitating the identification of individual m(5)C alterations and their consequential effects.