Epitranscriptomic editing of the RNA N6-methyladenosine modification by dCasRx conjugated methyltransferase and demethylase

N6-methyladenosine (m6A) is a common modification on endogenous RNA transcripts in mammalian cells. The precise modification of the RNA m6A level at a specific site helps to better understand its biological function. Here, we developed a bidirectional dCasRx epitranscriptome editing platform composed of a nucleus-localized dCasRx conjugated with either a methyltransferase, METTL3, or a demethylase, ALKBH5, to manipulate methylation events at targeted m6A sites in HEK293T and glioma stem cells. This platform edited m6A modifications at targeted sites with high specificity and efficiency, modulating both gene expression and cell proliferation. We then employed the dCasRx epitranscriptomic editor to further elucidate the molecular function of m6A-binding proteins YTH (DF1, DF2, DF3) family and found that all three of YTHDFs promoted m6A-mediated mRNA degradation. These findings collectively demonstrate that the dCasRx epitranscriptome perturbation platform reported in this study can be employed as site-specific m6A editors for delineating the functional roles of individual m6A modifications in the mammalian epitranscriptome.