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

N-6-methyladenosine (m(6)A) is the most common internal modification in eukaryotic mRNA and associated with numerous cellular processes in health and disease. Up- and down-regulation of its writer or eraser proteins alter the global m(6)A level; however, modifying distinct m(6)A sites has remained elusive. We genetically fused the dioxygenase FTO responsible for m(6)A demethylation to RCas9 as an 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 m(6)A level at a specific site and analyzed the effect of the PAM-to-m(6)A 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 m(6)A in RNA that can be readily adapted to any given RNA sequence and opens the door to studying the function of distinct m(6)A sites.