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

By Zhen Xia, Min Tang, Hongyan Zhang, Ryan Gimple, Briana Prager, Hongzhen Tang, Chongran Sun, Fuyi Liu, Peng Lin, Yutang Mei, Ruoxin Du, Jeremy Rich, Qi Xie

Posted 27 Oct 2020
bioRxiv DOI: 10.1101/2020.10.27.356436

N6-methyladenosine (m6A) is the most common modification on endogenous RNA transcripts in mammalian cells. Currently, the lack of precise single-nucleotide RNA modification tools makes it challenging to understand the relationship between site-specific RNA methylation and the corresponding phenotypic outcomes. 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 specifically and efficiently edited m6A modifications at targeted sites, 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 the YTH paralogs act together to mediate RNA 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. ### Competing Interest Statement The authors have declared no competing interest.

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