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Glycosylase-based base editors for efficient T-to-G and C-to-G editing in mammalian cells

Nature Biotechnology (2024)Cite this article

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Abstract

Base editors show promise for treating human genetic diseases, but most current systems use deaminases, which cause off-target effects and are limited in editing type. In this study, we constructed deaminase-free base editors for cytosine (DAF-CBE) and thymine (DAF-TBE), which contain only a cytosine-DNA or a thymine-DNA glycosylase (CDG/TDG) variant, respectively, tethered to a Cas9 nickase. Multiple rounds of mutagenesis by directed evolution in Escherichia coli generated two variants with enhanced base-converting activity—CDG-nCas9 and TDG-nCas9—with efficiencies of up to 58.7% for C-to-A and 54.3% for T-to-A. DAF-BEs achieve C-to-G/T-to-G editing in mammalian cells with minimal Cas9-dependent and Cas9-independent off-target effects as well as minimal RNA off-target effects. Additional engineering resulted in DAF-CBE2/DAF-TBE2, which exhibit altered editing windows from the 5′ end to the middle of the protospacer and increased C-to-G/T-to-G editing efficiency of 3.5-fold and 1.2-fold, respectively. Compared to prime editing or CGBEs, DAF-BEs expand conversion types of base editors with similar efficiencies, smaller sizes and lower off-target effects.

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Fig. 1: Design and evolution of CDG-nCas9/TDG-nCas9 in E. coli.
Fig. 2: Evaluation of the most efficient CDG-nCas9s/TDG-nCas9s in E. coli.
Fig. 3: Characterization of DAF-CBE and TBE editing activities in HEK293T cells.
Fig. 4: Comparison of DAF-CBE with CGBEs and DAF-TBE with PEs.
Fig. 5: Construction of pathogenic SNVs by DAF-BEs in hiPSCs.
Fig. 6: Engineering DAF-BEs by redesigning the fusion of glycosylase variants and nCas9.

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Data availability

The high-throughput sequencing data have been deposited in the National Center for Biotechnology Information database with accession codes PRJNA900229 and PRJNA1001163 (refs. 42,43). Source data for Figs. 2–4 and 6 and Supplementary Figs. 4 and 5 are presented with the paper. There are no restrictions on data availability. Source data are provided with this paper.

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Acknowledgements

This research was financially supported by the National Key Research and Development Program of China (2019YFA0904900); the National Natural Science Foundation of China (32225031, 32171449, 32271483, 32001041 and 81903776); the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (TSBICIP-KJGG-017); the Youth Innovation Promotion Association, Chinese Academy of Sciences (2022177); and the Tianjin Natural Science Foundation (20JCYBJC00310). The authors thank Zibing Jin (Capital Medical University, China) for providing hiPSCs.

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

Authors and Affiliations

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

    Lijun Ye, Dongdong Zhao, Yiran Wang, Bo Li, Yuanzhao Yang, Xueting Hou, Huibin Wang, Zhandong Wei, Xiaoqi Liu, Yaqiu Li, Siwei Li, Yajing Liu, Xueli Zhang & Changhao Bi

  2. University of Chinese Academy of Sciences, Beijing, China

    Lijun Ye, Dongdong Zhao, Bo Li, Xueli Zhang & Changhao Bi

  3. National Technology Innovation Center of Synthetic Biology, Tianjin, China

    Lijun Ye, Dongdong Zhao, Yiran Wang, Bo Li, Yuanzhao Yang, Xueting Hou, Huibin Wang, Zhandong Wei, Xiaoqi Liu, Yaqiu Li, Siwei Li, Yajing Liu, Xueli Zhang & Changhao Bi

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

    Ju Li & Yiran Wang

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

    Yuanzhao Yang & Huibin Wang

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Contributions

X.Z. and C.B. designed the research, analyzed data and wrote the manuscript. L.Y. and D.Z. designed the research, performed experiments, analyzed data and wrote the manuscript. J.L. and S.L. designed the research. Y.W., B.L., X.H., H.W., Z.W., X.L. and Y.L. performed experiments. Y.Y. and Y.L. analyzed data.

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Correspondence to Xueli Zhang or Changhao Bi.

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X.Z., C.B., L.Y. and D.Z. jointly filed patent applications on DAF-BEs.

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Ye, L., Zhao, D., Li, J. et al. Glycosylase-based base editors for efficient T-to-G and C-to-G editing in mammalian cells. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-023-02050-w

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