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

Abstract

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 emerging roles of N6-methyladenosine (m6A) deregulation in liver carcinogenesis

“…It has been shown that removal of the m6A modification in lncRNA MALAT1 at A2577 resulted in structural change and altered the interaction with the RNA binding protein hnRNPC [87]. A similar dCas9-FTO system has also been reported by another group [88]. In another study, Rauch et al made use of the newly identified RNA-guide RNA targeting CRISPR/ Cas13 system to interrogate the functional consequence of the binding of different m6A "reader" proteins to the targeted RNA.…”

TRADES: Targeted RNA Demethylation by SunTag System

“…So we still lack a flexible approach to remove m 6 A with spatial and temporal specificity. Recently, some effective RNA m 6 A editing methods were reported by CRISPR-dCas9 conjugates and other programmable RNA binding proteins, [29][30][31] but with sufficient space for improvement.…”

“…[29] Rcas9 was also chosen as the RNA-targeting protein to realize sequence-specific m 6 A demethylation. [30] However, these technologies still need to be improved for two reasons. First, additional PAMer DNA with multiple 2′-OMe modifications should be co-transfected via two kinds of transfection reagents that increase the cost and complexity.…”

Function and evolution of RNA N6-methyladenosine modification

“…Albeit SCARLET with low-throughput and fancy features, the feasibility and accuracy allow SCARLET to be a frequently-used strategy for examining the accuracy of high-throughput detection of m 6 A modification. And SCARLET can also be allowed to screen other types of epigenetic modifications in RNA, such as m 5 C and Ψ modifications [25]. Golovina et al reported a strategy based on high-resolution melting analysis for monitoring m 6 A sites in a specific RNA position [26].…”