Methyltransferases modify a wide range of biomolecules using S-adenosyl-l-methionine (AdoMet) as cosubstrate. Enzymatic generation of nucleoside-modified AdoMet analogues and conversion by different methyltransferases is shown.

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. Sequencespecific 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.

The transcriptome of each individual cell contains numerous RNA species, each of which can be controlled by multiple mechanisms during their lifetime. The standard transcriptome analysis focuses on the expression levels of the genes of interest. To gain additional insights into spatiotemporal RNA distribution and the underlying trafficking processes, RNA labeling and imaging are necessary—ideally in living cells. This perspective will summarize state-of-the-art RNA imaging methods including their strengths and weaknesses.

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Cas9 can be implemented as an RNA-targeting system to track mRNA in living cells. Nuclear export is enabled by efficient targeting of GFP-fused Cas9 to an endogenous mRNA. The approach provides a new and versatile platform for RNA-targeting with applications in RNA imaging and beyond.