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Programmable RNA N6-methyladenosine editing by CRISPR-Cas9 conjugates

Abstract

RNA modification in the form of N6-methyladenosine (m6A) regulates nearly all the post-transcriptional processes. The asymmetric m6A deposition suggests that regional methylation may have distinct functional consequences. However, current RNA biology tools do not distinguish the contribution of individual m6A modifications. Here we report the development of ‘m6A editing’, a powerful approach that enables m6A installation and erasure from cellular RNAs without changing the primary sequence. We engineered fusions of CRISPR-Cas9 and a single-chain m6A methyltransferase that can be programmed with a guide RNA. The resultant m6A ‘writers’ allow functional comparison of single site methylation in different messenger RNA regions. We further engineered m6A ‘erasers’ by fusing CRISPR-Cas9 with ALKBH5 or FTO to achieve site-specific demethylation of RNAs. The development of programmable m6A editing not only expands the scope of RNA engineering, but also facilitates mechanistic understanding of epitranscriptome.

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Fig. 1: Targeted 5′ UTR methylation by engineered m6A ‘writers’
Fig. 2: Physiological effects of targeted methylation by engineered m6A ‘writers’
Fig. 3: Targeted RNA demethylation by engineered m6A ‘erasers’
Fig. 4: Physiological effects of targeted demethylation by engineered m6A ‘erasers’

Data availability

All sequencing raw data and processed files have been deposited in the Gene Expression Omnibus (GSE132051).

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Acknowledgements

We would like to thank Qian laboratory members for helpful discussion. We are grateful to Cornell University Life Sciences Core Laboratory Center for sequencing support. This work was supported by grants to S.-B.Q. from US National Institutes of Health (R01GM122814 and R21CA227917) and HHMI Faculty Scholar (55108556).

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S.-B.Q. conceived the project, designed the experiments and wrote the manuscript. X.-M.L. performed most of the experiments and wrote the manuscript. Y.M. conducted the sequencing data analysis. J.Z. contributed to the single nucleotide m6A printing assay and Q.J. helped with data interpretation. All authors discussed the results and edited the manuscript.

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Correspondence to Shu-Bing Qian.

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The authors declare no competing interests.

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Supplementary Table 1–3, Supplementary Figs. 1–11

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Liu, XM., Zhou, J., Mao, Y. et al. Programmable RNA N6-methyladenosine editing by CRISPR-Cas9 conjugates. Nat Chem Biol 15, 865–871 (2019). https://doi.org/10.1038/s41589-019-0327-1

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  • DOI: https://doi.org/10.1038/s41589-019-0327-1

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