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.

  • Letter
  • Published:

Glycosylase base editors enable C-to-A and C-to-G base changes

Nature Biotechnology volume 39, pages 35–40 (2021)Cite this article

Subjects

A Publisher Correction to this article was published on 29 July 2020

This article has been updated

Abstract

Current base editors (BEs) catalyze only base transitions (C to T and A to G) and cannot produce base transversions. Here we present BEs that cause C-to-A transversions in Escherichia coli and C-to-G transversions in mammalian cells. These glycosylase base editors (GBEs) consist of a Cas9 nickase, a cytidine deaminase and a uracil-DNA glycosylase (Ung). Ung excises the U base created by the deaminase, forming an apurinic/apyrimidinic (AP) site that initiates the DNA repair process. In E. coli, we used activation-induced cytidine deaminase (AID) to construct AID-nCas9-Ung and found that it converts C to A with an average editing specificity of 93.8% ± 4.8% and editing efficiency of 87.2% ± 6.9%. For use in mammalian cells, we replaced AID with rat APOBEC1 (APOBEC-nCas9-Ung). We tested APOBEC-nCas9-Ung at 30 endogenous sites, and we observed C-to-G conversions with a high editing specificity at the sixth position of the protospacer between 29.7% and 92.2% and an editing efficiency between 5.3% and 53.0%. APOBEC-nCas9-Ung supplements the current adenine and cytidine BEs (ABE and CBE, respectively) and could be used to target G/C disease-causing mutations.

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Fig. 1: Base editing in E. coli using nCas9-AID and Ung-nCas9-AID fusion.
Fig. 2: Base editing in HEK293T cells using the GBE APOBEC-nCas9-Ung and control APOBEC-nCas9-UGI.
Fig. 3: Base-editing efficiency, indel frequency and the fraction of C substitutions at C6 from APOBEC-nCas9-Ung and APOBEC-nCas9-UGI.
Fig. 4: NBE in E. coli.

Similar content being viewed by others

Data availability

There is no restriction on data associated with this study. The raw data for Figs. 1 and 4 and Extended Data Fig. 1 have been submitted with the manuscript. High-throughput sequencing data have been deposited in the NCBI database (accession code SRP265375). Source data are provided with this paper.

Change history

  • 29 July 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

References

  1. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    Article  CAS  Google Scholar 

  2. Fujii, W., Kawasaki, K., Sugiura, K. & Naito, K. Efficient generation of large-scale genome-modified mice using gRNA and Cas9 endonuclease. Nucleic Acids Res. 41, e187 (2013).

    Article  CAS  Google Scholar 

  3. Hwang, W. Y. et al. Efficient genome editing in zebrafish using a CRISPR–Cas9 system. Nat. Biotechnol. 31, 227–229 (2013).

    Article  CAS  Google Scholar 

  4. Shalem, O. et al. Genome-scale CRISPR–Cas9 knockout screening in human cells. Science 343, 84–87 (2014).

    Article  CAS  Google Scholar 

  5. Jiang, Y. et al. Multigene editing in the Escherichia coli genome via the CRISPR–Cas9 system. Appl. Environ. Microbiol. 81, 2506–2514 (2015).

    Article  CAS  Google Scholar 

  6. 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).

    Article  CAS  Google Scholar 

  7. Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, aaf8729 (2016).

  8. 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  Google Scholar 

  9. Ma, Y. et al. Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells. Nat. Methods 13, 1029–1035 (2016).

    Article  CAS  Google Scholar 

  10. Zhang, Y. et al. Programmable base editing of zebrafish genome using a modified CRISPR–Cas9 system. Nat. Commun. 8, 118 (2017).

    Article  CAS  Google Scholar 

  11. Banno, S., Nishida, K., Arazoe, T., Mitsunobu, H. & Kondo, A. Deaminase-mediated multiplex genome editing in Escherichia coli. Nat. Microbiol. 3, 423–429 (2018).

    Article  CAS  Google Scholar 

  12. Li, C. et al. Expanded base editing in rice and wheat using a Cas9–adenosine deaminase fusion. Genome Biol. 19, 59 (2018).

    Article  Google Scholar 

  13. Ryu, S.-M. et al. Adenine base editing in mouse embryos and an adult mouse model of Duchenne muscular dystrophy. Nat. Biotechnol. 36, 536–539 (2018).

    Article  CAS  Google Scholar 

  14. Shimatani, Z. et al. Inheritance of co-edited genes by CRISPR-based targeted nucleotide substitutions in rice. Plant Physiol. Biochem. 131, 78–83 (2018).

  15. Wang, Y. et al. MACBETH: multiplex automated Corynebacterium glutamicum base editing method. Metab. Eng. 47, 200–210 (2018).

    Article  CAS  Google Scholar 

  16. Hess, G. T. et al. Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nat. Methods 13, 1036–1042 (2016).

    Article  CAS  Google Scholar 

  17. Komor, A. C., Badran, A. H. & Liu, D. R. CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell 168, 20–36 (2016).

    Article  Google Scholar 

  18. Kim, K. et al. Highly efficient RNA-guided base editing in mouse embryos. Nat. Biotechnol. 35, 435–437 (2017).

    Article  CAS  Google Scholar 

  19. Lindahl, T. An N-glycosidase from Escherichia coli that releases free uracil from DNA containing deaminated cytosine residues. Proc. Natl Acad. Sci. USA 71, 3649–3653 (1974).

    Article  CAS  Google Scholar 

  20. Zheng, K. et al. Highly efficient base editing in bacteria using a Cas9–cytidine deaminase fusion. Commun. Biol. 1, 32 (2018).

    Article  Google Scholar 

  21. Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).

    Article  CAS  Google Scholar 

  22. Kleinstiver, B. P. et al. Engineered CRISPR–Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015).

    Article  Google Scholar 

  23. Hu, J. H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63 (2018).

    Article  CAS  Google Scholar 

  24. Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).

    Article  CAS  Google Scholar 

  25. Engler, C., Kandzia, R. & Marillonnet, S. A one pot, one step, precision cloning method with high- throughput capability. PLoS ONE 3, e3647 (2008).

    Article  Google Scholar 

  26. Hillson, N. J., Rosengarten, R. D. & Keasling, J. D. j5 DNA assembly design automation software. ACS Synth. Biol. 1, 14–21 (2011).

    Article  Google Scholar 

  27. Zhao, J. et al. Engineering central metabolic modules of Escherichia coli for improving β-carotene production. Metab. Eng. 17, 42–50 (2013).

    Article  CAS  Google Scholar 

  28. Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

    Article  CAS  Google Scholar 

  29. Feng, X., Zhao, D., Zhang, X., Ding, X. & Bi, C. CRISPR/Cas9 assisted multiplex genome editing technique in Escherichia coli. Biotechnol. J. 13, e1700604 (2018).

  30. Bae, S., Park, J. & Kim, J. S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This research was financially supported by the National Key Research and Development Program of China (2019YFA0904900), the Key Research Program of the Chinese Academy of Science (KFZD-SW-215), the National Natural Science Foundation of China (31861143019), a Newton Fund PhD placement program grant (ID 352639434) under the UK–China Joint Research and Innovation Partnership Fund and the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (TSBICIP-PTJS-003). We gratefully thank F. Gu (Wenzhou Medical University) for providing the plasmid pAPOBEC-nCas9-UGI and Y. Li (Tianjin Institute of Industrial Biotechnology) for assisting with the data analysis.

Author information

Author notes

  1. These authors contributed equally: Dongdong Zhao, Ju Li, Siwei Li.

Authors and Affiliations

  1. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China

    Dongdong Zhao, Siwei Li, Xiuqing Xin, Muzi Hu, Changhao Bi & Xueli Zhang

  2. College of Life Science, Tianjin Normal University, Tianjin, China

    Ju Li

  3. College of Biotechnology, Tianjin University of Science and Technology, Tianjin, China

    Xiuqing Xin

  4. School of Biological Engineering, Dalian Polytechnic University, Dalian, China

    Muzi Hu

  5. Centre for Synthetic and Systems Biology and UK Centre for Mammalian Synthetic Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK

    Marcus A. Price & Susan J. Rosser

Authors
  1. Dongdong Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ju Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Siwei Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xiuqing Xin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Muzi Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Marcus A. Price
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Susan J. Rosser
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Changhao Bi
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Xueli Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

X.Z., C.B. and J.L. designed the research, analyzed data and wrote the manuscript. D.Z. designed the research, performed experiments, analyzed data and wrote the manuscript. S.L. performed experiments and analyzed data. M.A.P. performed experiments and wrote the manuscript. S.J.R. wrote the manuscript. X.X. and M.H. performed experiments.

Corresponding authors

Correspondence to Changhao Bi or Xueli Zhang.

Ethics declarations

Competing interests

A provisional patent has been submitted in part entailing the reported approach.

Additional information

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

Extended data

Extended Data Fig. 1 Sequencing data results following CBE.

a, Results obtained following treatment with nCas9-AID in E. coli MG1655 Δung. b, Results obtained following treatment with dCas9-AID in wild type E. coli MG1655. For all plots, dots represent individual biological replicates, bars represent mean values, and error bars represent the s.d. of three independent biological replicates.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1, 2, 4 and 7; Supplementary Figs. 1 and 2; and DNA sequences for vector components.

Reporting Summary

Supplementary Table 3

Mutations of APOBEC-nCas9-UGI and APOBEC-nCas9-Ung at RP11-177B4-3, PSMB2-1 and EMX1-site5 using mismatched sgRNAs.

Supplementary Table 5

The main primers used for this work.

Supplementary Table 6

The main plasmids used for this work.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 1

Statistical source data.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhao, D., Li, J., Li, S. et al. Glycosylase base editors enable C-to-A and C-to-G base changes. Nat Biotechnol 39, 35–40 (2021). https://doi.org/10.1038/s41587-020-0592-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41587-020-0592-2

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