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

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

Content Provider	Europe PMC
Author	Rau, Kristina Rösner, Lukas Rentmeister, Andrea
Copyright Year	2019
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 down-regulation 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 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 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 studying the function of distinct m6A sites.
Page Count	13
ISSN	13558382
Journal	RNA
Volume Number	25
PubMed Central reference number	PMC6800472
Issue Number	10
PubMed reference number	31263003
e-ISSN	14699001
DOI	10.1261/rna.070706.119
Language	English
Publisher	Cold Spring Harbor Laboratory Press
Publisher Date	2019-07-01
Access Restriction	Open
Rights License	This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/. © 2019 Rau et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society
Subject Keyword	RNA modification N6-methyladenosine (m6A) Cas9 FTO demethylation sgRNA PAMmer
Content Type	Text
Resource Type	Article
Subject	Molecular Biology

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