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RNA modification in the form of N 6 -methyladenosine (m 6 A) regulates nearly all the post-transcriptional processes. The asymmetric m 6 A deposition suggests that regional methylation may have distinct functional consequences. However, current RNA biology tools do not distinguish the contribution of individual m 6 A modifications. Here we report the development of 'm 6 A editing', a powerful approach that enables m 6 A installation and erasure from cellular RNAs without changing the primary sequence. We engineered fusions of CRISPR-Cas9 and a single-chain m 6 A methyltransferase that can be programmed with a guide RNA. The resultant m 6 A 'writers' allow functional comparison of single site methylation in different messenger RNA regions. We further engineered m 6 A 'erasers' by fusing CRISPR-Cas9 with ALKBH5 or FTO to achieve site-specific demethylation of RNAs. The development of programmable m 6 A editing not only expands the scope of RNA engineering, but also facilitates mechanistic understanding of epitranscriptome.

Programmable RNA N6-methyladenosine editing by CRISPR-Cas9 conjugates