Programmable RNA N6-methyladenosine editing by CRISPR-Cas9 conjugates

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.