Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

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

Subjects

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

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.

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.

References

  1. Goldberg, A. D., Allis, C. D. & Bernstein, E. Epigenetics: a landscape takes shape. Cell 128, 635–638 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Hoernes, T. P. & Erlacher, M. D. Translating the epitranscriptome. Wiley Interdiscip. Rev. RNA 8, 1–18 https://doi.org/10.1002/wrna.1375 (2017).

  3. Zhao, B. S., Roundtree, I. A. & He, C. Post-transcriptional gene regulation by mRNA modifications. Nat. Rev. Mol. Cell Biol. 18, 31–42 (2017).

    Article  CAS  PubMed  Google Scholar 

  4. Meyer, K. D. & Jaffrey, S. R. The dynamic epitranscriptome: N6-methyladenosine and gene expression control. Nat. Rev. Mol. Cell Biol. 15, 313–326 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Liu, J. et al. N6-Methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcription. Science 367, 580–586 (2020).

  6. Xiao, W. et al. Nuclear m6A reader YTHDC1 regulates mRNA splicing. Molecular Cell 61, 507–519 (2016).

    Article  CAS  PubMed  Google Scholar 

  7. Roundtree, I. A. et al. YTHDC1 mediates nuclear export of N6-methyladenosine methylated mRNAs. eLife 6, e31311 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Shi, H. et al. YTHDF3 facilitates translation and decay of N6-methyladenosine-modified RNA. Cell Res. 27, 315–328 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Wang, X. et al. N6-Methyladenosine-dependent regulation of messenger RNA stability. Nature 505, 117–120 (2014).

    Article  PubMed  CAS  Google Scholar 

  10. Wang, X. et al. N6-Methyladenosine modulates messenger RNA translation efficiency. Cell 161, 1388–1399 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Dominissini, D. et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485, 201–206 (2012).

    Article  CAS  PubMed  Google Scholar 

  12. Meyer, KateD. et al. Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRs and near stop codons. Cell 149, 1635–1646 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Batista, P. J. et al. m6A RNA modification controls cell fate transition in mammalian embryonic stem cells. Cell Stem Cell 15, 707–719 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Zhou, J. et al. Dynamic m6A mRNA methylation directs translational control of heat shock response. Nature 526, 591–594 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Xu, K. et al. Mettl3-mediated m6A regulates spermatogonial differentiation and meiosis initiation. Cell Res. 27, 1100–1114 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Zhao, B. S. et al. m6A-dependent maternal mRNA clearance facilitates zebrafish maternal-to-zygotic transition. Nature 542, 475–478 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Patil, D. P. et al. m6A RNA methylation promotes XIST-mediated transcriptional repression. Nature 537, 369–373 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Xiang, Y. et al. RNA m6A methylation regulates the ultraviolet-induced DNA damage response. Nature 543, 573–576 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhong, X. et al. Circadian clock regulation of hepatic lipid metabolism by modulation of m6A mRNA methylation. Cell Rep. 25, 1816–1828 (2018). e1814.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Jaffrey, S. R. & Kharas, M. G. Emerging links between m6A and misregulated mRNA methylation in cancer. Genome Med. 9, 2 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Zhang, Z. et al. METTL3-mediated N6-methyladenosine mRNA modification enhances long-term memory consolidation. Cell Res. 28, 1050–1061 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Han, D. et al. Anti-tumour immunity controlled through mRNA m6A methylation and YTHDF1 in dendritic cells. Nature 566, 270–274 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Engel, M. & Chen, A. The emerging role of mRNA methylation in normal and pathological behavior. Genes Brain Behav. 17, e12428 (2018).

    Article  CAS  PubMed  Google Scholar 

  24. Jaffrey, S. R. & Kharas, M. G. Emerging links between m6A and misregulated mRNA methylation in cancer. Genome Med. 9, 2 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Cui, Q. et al. m6A RNA methylation regulates the self-renewal and tumorigenesis of glioblastoma stem cells. Cell Rep. 18, 2622–2634 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Liu, J. et al. A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat. Chem. Biol. 10, 93–95 (2014).

    Article  CAS  PubMed  Google Scholar 

  27. Sledz, P. & Jinek, M. Structural insights into the molecular mechanism of the m6A writer complex. eLife 5, e18434 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Wang, X. et al. Structural basis of N6-adenosine methylation by the METTL3–METTL14 complex. Nature 534, 575–578 (2016).

    Article  CAS  PubMed  Google Scholar 

  29. Wang, P., Doxtader, K. A. & Nam, Y. Structural basis for cooperative function of Mettl3 and Mettl14 methyltransferases. Mol. Cell 63, 306–317 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Scholler, E. et al. Interactions, localization, and phosphorylation of the m6A generating METTL3–METTL14–WTAP complex. RNA 24, 499–512 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Zheng, G. et al. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol. Cell 49, 18–29 (2013).

    Article  CAS  PubMed  Google Scholar 

  32. Jia, G. et al. N6-Methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat. Chem. Biol. 7, 885–887 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Wang, X. & He, C. Reading RNA methylation codes through methyl-specific binding proteins. RNA Biol. 11, 669–672 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Alarcon, C. R. et al. HNRNPA2B1 Is a mediator of m6A-dependent nuclear RNA processing events. Cell 162, 1299–1308 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Liu, N. et al. N6-Methyladenosine-dependent RNA structural switches regulate RNA–protein interactions. Nature 518, 560–564 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Shi, H., Wei, J. & He, C. Where, when, and how: context-dependent functions of RNA methylation writers, readers, and erasers. Mol. Cell 74, 640–650 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Yang, Y., Hsu, P. J., Chen, Y. S. & Yang, Y. G. Dynamic transcriptomic m6A decoration: writers, erasers, readers and functions in RNA metabolism. Cell Res. 28, 616–624 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Meyer, K. D. & Jaffrey, S. R. Rethinking m6A readers, writers, and erasers. Annu. Rev. Cell Dev. Biol. 33, 319–342 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Liu, X.-M., Zhou, J., Mao, Y., Ji, Q. & Qian, S.-B. Programmable RNA N6-methyladenosine editing by CRISPR-Cas9 conjugates. Nat. Chem. Biol. 15, 865–871 (2019).

  40. Abudayyeh, O. O. et al. RNA targeting with CRISPR–Cas13. Nature 550, 280–284 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. East-Seletsky, A. et al. Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection. Nature 538, 270–273 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Abudayyeh, O. O. et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353, aaf5573 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Li, F. et al. A radioactivity-based assay for screening human m6A-RNA methyltransferase, METTL3–METTL14 complex, and demethylase ALKBH5. J. Biomol. Screen. 21, 290–297 (2016).

    Article  PubMed  CAS  Google Scholar 

  44. Cox, D. B. T. et al. RNA editing with CRISPR-Cas13. Science 358, 1019–1027 (2017).

  45. Abudayyeh, O. O. et al. A cytosine deaminase for programmable single-base RNA editing. Science 365, 382 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Slaymaker, I. M. et al. High-resolution structure of Cas13b and biochemical characterization of RNA targeting and cleavage. Cell Rep. 26, 3741–3751.e3745 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Schellenberger, V. et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat. Biotechnol. 27, 1186–1190 (2009).

    Article  CAS  PubMed  Google Scholar 

  48. Meyer, K. D. et al. Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRs and near stop codons. Cell 149, 1635–1646 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Nelles, D. A. et al. Programmable RNA tracking in live cells with CRISPR/Cas9. Cell 165, 488–496 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Liu, N. et al. Probing N6-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA. RNA 19, 1848–1856 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Cui, Q. et al. m6A RNA methylation regulates the self-renewal and tumorigenesis of glioblastoma stem cells. Cell Rep 18, 2622–2634 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Zhang, S. et al. m6A demethylase ALKBH5 maintains tumorigenicity of glioblastoma stem-like cells by sustaining FOXM1 expression and cell proliferation program. Cancer Cell 31, 591–606 (2017). e596.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Geula, S. et al. m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation. Science 347, 1002–1006 (2015).

    Article  CAS  PubMed  Google Scholar 

  54. Garcia-Campos, M. A. et al. Deciphering the ‘m6A code’ via antibody-independent quantitative profiling. Cell 178, 731–747.e716 (2019).

    Article  CAS  PubMed  Google Scholar 

  55. Zhang, Z. et al. Single-base mapping of m6A by an antibody-independent method. Sci. Adv. 5, eaax0250 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Imanishi, M., Tsuji, S., Suda, A. & Futaki, S. Detection of N6-methyladenosine based on the methyl-sensitivity of MazF RNA endonuclease. Chem. Commun. 53, 12930–12933 (2017).

    Article  CAS  Google Scholar 

  57. Bartosovic, M. et al. N6-Methyladenosine demethylase FTO targets pre-mRNAs and regulates alternative splicing and 3′-end processing. Nucleic Acids Res. 45, 11356–11370 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Xiao, W. et al. Nuclear m6A reader YTHDC1 regulates mRNA splicing. Mol. Cell 61, 507–519 (2016).

    Article  CAS  PubMed  Google Scholar 

  59. O’Connell, M. R. et al. Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature 516, 263–266 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  60. Alarcón, C. R., Lee, H., Goodarzi, H., Halberg, N. & Tavazoie, S. F. N6-Methyladenosine marks primary microRNAs for processing. Nature 519, 482 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  61. Gaudelli, N. M. et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Dominissini, D., Moshitch-Moshkovitz, S., Salmon-Divon, M., Amariglio, N. & Rechavi, G. Transcriptome-wide mapping of N6-methyladenosine by m6A-seq based on immunocapturing and massively parallel sequencing. Nat. Protoc. 8, 176–189 (2013).

    Article  CAS  PubMed  Google Scholar 

  63. Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta CT) method. Methods 25, 402–408 (2001).

    Article  CAS  PubMed  Google Scholar 

  64. Cui, X. et al. MeTDiff: a novel differential RNA methylation analysis for MeRIP-Seq data. IEEE/ACM Trans. Comput. Biol. Bioinform. 15, 526–534 (2015).

    Article  Google Scholar 

  65. Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Law, C. W., Chen, Y., Shi, W. & Smyth, G. K. voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol. 15, R29 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  67. Ritchie, M. E. et al. Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47–e47 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

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.

Author information

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

Authors
  1. Christopher Wilson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Peter J. Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhuang Miao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David R. Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding author

Correspondence to David R. Liu.

Ethics declarations

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.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

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

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41587-020-0572-6

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research