Sequence-specific m6A demethylation in RNA by FTO fused to RCas9

Abstract

N6-methyladenosine (m6A) is the most common internal modification in eukaryotic mRNA and associated with numerous cellular processes in health and disease. Up- and downregulation of its “writer” or “eraser” proteins alter the global m6A level, however, modifying distinct m6A sites has remained elusive. We genetically fused the dioxygenase FTO responsible for m6A demethylation to RCas9 as RNA-targeting module. The resulting RCas9 FTO retained demethylation activity and bound to RNA in a sequence-specific manner depending on the sgRNA and PAMmer. Using SCARLET analysis, we quantified the m6A level at a specific site and analyzed the effect of the PAM-to-m6A distance on activity. Sequence-specific demethylation by RCas9-FTO was tested on different RNA combinations and showed up to 15-fold sequence preference for target RNA compared to off-target RNA. Taken together, RCas9-FTO represents a new tool for sequence-specific demethylation of m6A in RNA that can be readily adapted to any given RNA sequence and opens the door to study the function of distinct m6A sites.