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

5-methylcytosine, 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 m5C, the inability to introduce m5C at specific sites within transcripts has hindered efforts to elucidate direct links between specific m5C and phenotypic outcomes. Here we developed a CRISPR-Cas13d-based tool, named reengineered m5C modification systems (termed ‘RCMS’), for targeted m5C methylation and demethylation in specific transcripts. The RCMS editors consist of a nuclear-localised dCasRx conjugated to either to a methyltransferase, NSUN2/NSUN6 or a demethylase, the catalytic domain of mouse Tet2 (Tet2 CD), enabling the manipulation of methylation events at precise m5C sites. We demonstrate that the RCMS editors can direct site-specific m5C incorporation and demethylation. Furthermore, we confirm their effectiveness in modulating m5C levels within tRNAs and their ability to induce changes in transcript abundance and cell proliferation through m5C-mediated mechanisms. These findings collectively establish RCMS editors as a focused epitranscriptome engineering tool, facilitating the identification of individual m5C alterations and their consequential effects. To investigate whether the RCMS activity leads to substantial non-specific methylation, we assessed the impact of the RCMS dCasRx-NSUN6 editor, guided by gRNA -150 nt targeting RPSA m5C234, on the overall m5C content using BSP-seq.We then performed the m5C methylation levels in mRNA by the dCasRx-NSUN6 and dCasRx-dNSUN6 with the RPSA gRNA −150 nt or nontarget gRNA.Comparative m5C methylation level profiling analysis of BSP-RNAseq in HEK293T cells( NT,NSUN6,dNSUN6）