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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.

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

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