Glycosylase base editors enable C-to-A and C-to-G base changes (2024)

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Subjects Abstract Access options Additional access options: Similar content being viewed by others Linking CRISPR–Cas9 double-strand break profiles to gene editing precision with BreakTag Plasmid targeting and destruction by the DdmDE bacterial defence system Improving prime editing with an endogenous small RNA-binding protein Data availability Change history References Acknowledgements Author information Authors and Affiliations Contributions Corresponding authors Ethics declarations Competing interests Additional information Extended data Extended Data Fig. 1 Sequencing data results following CBE. Supplementary information Supplementary Information Reporting Summary Supplementary Table 3 Supplementary Table 5 Supplementary Table 6 Source data Source Data Fig. 1 Source Data Fig. 4 Source Data Extended Data Fig. 1 Rights and permissions About this article Cite this article This article is cited by Targeted C•G-to-T•A base editing with TALE-cytosine deaminases in plants Adenine transversion editors enable precise, efficient A•T-to-C•G base editing in mammalian cells and embryos CRISPR/Cas9-mediated base editors and their prospects for mitochondrial genome engineering Recent advances in CRISPR-based functional genomics for the study of disease-associated genetic variants Precise genome-editing in human diseases: mechanisms, strategies and applications References
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Nature Biotechnology volume39,pages 35–40 (2021)Cite this article

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  • Biotechnology
  • Molecular biology

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

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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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Glycosylase base editors enable C-to-A and C-to-G base changes (1)
Glycosylase base editors enable C-to-A and C-to-G base changes (2)
Glycosylase base editors enable C-to-A and C-to-G base changes (3)
Glycosylase base editors enable C-to-A and C-to-G base changes (4)

Similar content being viewed by others

Glycosylase base editors enable C-to-A and C-to-G base changes (6)

Plasmid targeting and destruction by the DdmDE bacterial defence system

Article 13 May 2024

Glycosylase base editors enable C-to-A and C-to-G base changes (7)

Improving prime editing with an endogenous small RNA-binding protein

Article Open access 03 April 2024

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

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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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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

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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.

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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.

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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Glycosylase base editors enable C-to-A and C-to-G base changes (8)

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

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Glycosylase base editors enable C-to-A and C-to-G base changes (2024)

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