Directed evolution of an m⁶A eraser for site-selective epitranscriptome editing

N ⁶-methyladenosine (m⁶A) is a critical RNA modification, but tools to manipulate it in cells remain limited. Current m⁶A 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 m⁶A demethylases with enhanced activity. Using this platform, we generated FTO-818, an evolved FTO variant with 13-fold increased catalytic efficiency against m⁶A compared to wild-type FTO. FTO-818 demonstrates superior demethylation performance both on purified biological RNAs in vitro and on endogenous m⁶A when overexpressed in human cells. Furthermore, we constructed a targeted m⁶A editor by fusing FTO-818 to catalytically-dead Cas13b (dCas13b-FTO-818), achieving efficient, site-specific removal of m⁶A from individual mRNA and lncRNA. This directed evolution platform and the resulting FTO-818 enzyme provide powerful new tools for investigating m⁶A biology and advancing applications in epitranscriptomic research and biotechnology.