Directed evolution of an m(6)A eraser for site-selective epitranscriptome editing.

N (6)-methyladenosine (m(6)A) is a critical RNA modification, but tools to manipulate it in cells remain limited. Current m(6)A demethylases (FTO and ALKBH5) exhibit low catalytic efficiency, hindering their applications in epitranscriptomic research and biotechnology. Here, we develop a fluorescence-based directed evolution platform that enables rapid selection of m(6)A demethylases with enhanced activity. Using this platform, we generated FTO-818, an evolved FTO variant with 13-fold increased catalytic efficiency against m(6)A compared to wild-type FTO. FTO-818 demonstrates superior demethylation performance both on purified biological RNAs in vitro and on endogenous m(6)A when overexpressed in human cells. Furthermore, we constructed a targeted m(6)A editor by fusing FTO-818 to catalytically-dead Cas13b (dCas13b-FTO-818), achieving efficient, site-specific removal of m(6)A from individual mRNA and lncRNA. This directed evolution platform and the resulting FTO-818 enzyme provide powerful new tools for investigating m(6)A biology and advancing applications in epitranscriptomic research and biotechnology.