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

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.

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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.

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.

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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.

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Authors

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.

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

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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.

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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

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