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Programmable m6A modification of cellular RNAs with a Cas13-directed methyltransferase

Nature Biotechnology volume 38, pages 1431–1440 (2020)Cite this article

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Abstract

N6-Methyladenosine (m6A) is the most widespread internal messenger RNA modification in humans. Despite recent progress in understanding the biological roles of m6A, the inability to install m6A site specifically in individual transcripts has hampered efforts to elucidate causal relationships between the presence of a specific m6A and phenotypic outcomes. In the present study, we demonstrate that nucleus-localized dCas13 fusions with a truncated METTL3 methyltransferase domain and cytoplasm-localized fusions with a modified METTL3:METTL14 methyltransferase complex can direct site-specific m6A incorporation in distinct cellular compartments, with the former fusion protein having particularly low off-target activity. Independent cellular assays across multiple sites confirm that this targeted RNA methylation (TRM) system mediates efficient m6A installation in endogenous RNA transcripts with high specificity. Finally, we show that TRM can induce m6A-mediated changes to transcript abundance and alternative splicing. These findings establish TRM as a tool for targeted epitranscriptome engineering that can reveal the effect of individual m6A modifications and dissect their functional roles.

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Fig. 1: Overview of cellular modification of adenine to m6A in mRNA, and design of a targeted RNA methylation system.
Fig. 2: Validation of TRM in E. coli.
Fig. 3: Methylation of reporter transcripts in human cells.
Fig. 4: Cellular localization of TRM editors and targeted methylation of endogenous transcripts in human cells.
Fig. 5: Specificity and off-target methylation of TRM editors.
Fig. 6: TRM effect on RNA abundance and alternative splicing.

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Data availability

Key plasmids from this work are available from Addgene (depositor: D. R. Liu) and other plasmids are available upon request. All unmodified reads for sequencing-based data in the manuscript are available on the NCBI Sequence Read Archive, accession number PRJNA559201. Amino acid sequences of TRM editors reported in the present study are provided in the Supplementary Sequences.

Code availability

All scripts used in this study are available at https://github.com/CwilsonBroad.

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Acknowledgements

We thank the Harvard Bauer Core facility staff for assistance with automatic liquid handling and NGS library preparation; J. Nelson, A. Anzalone, and R. Chen for helpful discussions; the Pattern team at the Broad Institute for data visualization assistance; and A. Vieira for assistance editing the manuscript. This work was supported by the Ono Pharma Foundation, US NIH (grant nos. RM1 HG009490, U01 AI142756 and R35 GM118062) and HHMI. C.W. is the Marion Abbe Fellow of the Damon Runyon Cancer Research Foundation (DRG-2343–18). P.J.C. is supported by an NSF Graduate Research Fellowship.

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Author notes

  1. These authors contributed equally: Christopher Wilson, Peter J. Chen.

Authors and Affiliations

  1. Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA

    Christopher Wilson, Peter J. Chen, Zhuang Miao & David R. Liu

  2. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    Christopher Wilson, Peter J. Chen, Zhuang Miao & David R. Liu

  3. Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA

    Christopher Wilson, Peter J. Chen, Zhuang Miao & David R. Liu

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Contributions

C.W. and P.J.C. designed the research and performed experiments. Z.M. performed immunofluorescence experiments and assisted with plasmid cloning and RNA purification. D.R.L. designed and supervised the research. C.W., P.J.C. and D.R.L. wrote the manuscript. All authors contributed to editing the manuscript.

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Correspondence to David R. Liu.

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Competing interests

D.R.L. is a consultant and co-founder of Beam Therapeutics, Prime Medicine, Editas Medicine and Pairwise Plants, companies that use genome editing. D.R.L., P.J.C. and C.W. have filed patent applications on aspects of this work.

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Supplementary Figs. 1–21, Tables 1–6, Sequences 1–2 and References.

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Wilson, C., Chen, P.J., Miao, Z. et al. Programmable m6A modification of cellular RNAs with a Cas13-directed methyltransferase. Nat Biotechnol 38, 1431–1440 (2020). https://doi.org/10.1038/s41587-020-0572-6

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