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

Abstract N6-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.