Skip to main content

Thank you for visiting 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 RNA N6-methyladenosine editing by CRISPR-Cas9 conjugates


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.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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).


  1. Roundtree, I. A., Evans, M. E., Pan, T. & He, C. Dynamic RNA modifications in gene expression regulation. Cell 169, 1187–1200 (2017).

    CAS  Article  Google Scholar 

  2. Lewis, C. J., Pan, T. & Kalsotra, A. RNA modifications and structures cooperate to guide RNA-protein interactions. Nat. Rev. Mol. Cell Biol. 18, 202–210 (2017).

    CAS  Article  Google Scholar 

  3. Bokar, J. A., Shambaugh, M. E., Polayes, D., Matera, A. G. & Rottman, F. M. Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase. RNA 3, 1233–1247 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  Article  Google Scholar 

  5. Ping, X. L. et al. Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase. Cell Res. 24, 177–189 (2014).

    CAS  Article  Google Scholar 

  6. 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).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  8. Fu, Y., Dominissini, D., Rechavi, G. & He, C. Gene expression regulation mediated through reversible m(6)A RNA methylation. Nat. Rev. Genet. 15, 293–306 (2014).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  10. Edupuganti, R. R. et al. N(6)-methyladenosine (m(6)A) recruits and repels proteins to regulate mRNA homeostasis. Nat. Struct. Mol. Biol. 24, 870–878 (2017).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  13. Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

    CAS  Article  Google Scholar 

  14. Doudna, J. A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014).

    Article  Google Scholar 

  15. Komor, A. C., Badran, A. H. & Liu, D. R. CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell 169, 559 (2017).

    CAS  Article  Google Scholar 

  16. Stricker, S. H., Koferle, A. & Beck, S. From profiles to function in epigenomics. Nat. Rev. Genet. 18, 51–66 (2017).

    CAS  Article  Google Scholar 

  17. Allis, C. D. & Jenuwein, T. The molecular hallmarks of epigenetic control. Nat. Rev. Genet. 17, 487–500 (2016).

    CAS  Article  Google Scholar 

  18. Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247 e217 (2016).

    CAS  Article  Google Scholar 

  19. Morita, S. et al. Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions. Nat. Biotechnol. 34, 1060–1065 (2016).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    Article  Google Scholar 

  22. Wang, X. et al. Structural basis of N(6)-adenosine methylation by the METTL3-METTL14 complex. Nature 534, 575–578 (2016).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  24. Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    CAS  Article  Google Scholar 

  25. Ke, S. et al. m6A mRNA modifications are deposited in nascent pre-mRNA and are not required for splicing but do specify cytoplasmic turnover. Genes Dev. 31, 990–1006 (2017).

    CAS  Article  Google Scholar 

  26. Linder, B. et al. Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome. Nat. Methods 12, 767–772 (2015).

    CAS  Article  Google Scholar 

  27. Zhou, J. et al. N(6)-methyladenosine guides mRNA alternative translation during integrated stress response. Mol. Cell 69, 636–647 e637 (2018).

    CAS  Article  Google Scholar 

  28. Xiao, Y. et al. An elongation- and ligation-based qPCR amplification method for the radiolabeling-free detection of locus-specific N(6)-methyladenosine modification. Angew. Chem. Int. Ed. Engl. 57, 15995–16000 (2018).

    CAS  Article  Google Scholar 

  29. Liu, Y. et al. Targeting cellular mRNAs translation by CRISPR-Cas9. Sci. Rep. 6, 29652 (2016).

    CAS  Article  Google Scholar 

  30. Meyer, K. D. et al. 5′ UTR m(6)A promotes cap-independent translation. Cell 163, 999–1010 (2015).

    CAS  Article  Google Scholar 

  31. Zhou, J. et al. Dynamic m(6)A mRNA methylation directs translational control of heat shock response. Nature 526, 591–594 (2015).

    CAS  Article  Google Scholar 

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

    Article  Google Scholar 

  33. 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).

    CAS  Article  Google Scholar 

  34. Zou, S. et al. N(6)-Methyladenosine: a conformational marker that regulates the substrate specificity of human demethylases FTO and ALKBH5. Sci. Rep. 6, 25677 (2016).

    CAS  Article  Google Scholar 

  35. Mauer, J. et al. Reversible methylation of m6Am in the 5′ cap controls mRNA stability. Nature 541, 371–375 (2017).

    CAS  Article  Google Scholar 

  36. Wei, J. et al. Differential m(6)A, m(6)Am, and m(1)A demethylation mediated by FTO in the cell nucleus and cytoplasm. Mol. Cell 71, 973–985 e975 (2018).

    CAS  Article  Google Scholar 

  37. Liu, N. et al. N(6)-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions. Nature 518, 560–564 (2015).

    CAS  Article  Google Scholar 

Download references


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).

Author information

Authors and Affiliations



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.

Corresponding author

Correspondence to Shu-Bing Qian.

Ethics declarations

Competing interests

The authors declare no competing interests.

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 Table 1–3, Supplementary Figs. 1–11

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Liu, XM., Zhou, J., Mao, Y. et al. Programmable RNA N6-methyladenosine editing by CRISPR-Cas9 conjugates. Nat Chem Biol 15, 865–871 (2019).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing