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Dual base editor catalyzes both cytosine and adenine base conversions in human cells

Abstract

Although base editors are useful tools for precise genome editing, current base editors can only convert either adenines or cytosines. We developed a dual adenine and cytosine base editor (A&C-BEmax) by fusing both deaminases with a Cas9 nickase to achieve C-to-T and A-to-G conversions at the same target site. Compared to single base editors, A&C-BEmax’s activity on adenines is slightly reduced, whereas activity on cytosines is higher and RNA off-target activity is substantially decreased.

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Fig. 1: Design and optimization of A&C-BE.
Fig. 2: Efficient editing of HBG promoter by A&C-BEmax in HUDEP-2 cells.

Data availability

Targeted amplicon sequencing data have been deposited in the NCBI Sequence Read Archive database under accession codes PRJNA558944, PRJNA559260, PRJNA559237, PRJNA559051, PRJNA592341 and PRJNA592333. The RNA-sequencing data used in this study have been deposited in the NCBI Sequence Read Archive database under accession code PRJNA592597. Plasmids encoding A&C-BEmax, AID-BE4max, Lenti ABE7.10-N-AIDmax and Lenti AID-BEmax are available from Addgene.

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Acknowledgements

We thank M. Crossley at the University of New South Wales, Australia, for providing the HUDEP-2(ΔGγ) cell line and H. Yang and C. Zhou at the Institute of Neuroscience, Chinese Academy of Sciences, for the suggestions of RNA off-targeting analysis. This work was partially supported by grants from the National Key R&D Program of China (2019YFA0110802 to D.L. and 2019YFA0802800 to M.L.), the National Natural Science Foundation of China (81670470 and 81873685 to D.L., and 31925011 and 91940306 to L.Y.), grants from the Shanghai Municipal Commission for Science and Technology (18411953500 to D.L.), the Innovation Program of the Shanghai Municipal Education Commission (2019-01-07-00-05-E00054 to D.L.), the support of the ECNU Public Platform for Innovation (011) and Fundamental Research Funds for the Central Universities.

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Authors

Contributions

X.Z., D.L. and M.L. designed the experiments and analyzed the data. X.Z., B.Z., L.C., L.X., L.L., W.H., S.Y., L.Y., Ha.H., Ho.H., Y.L., L.W., X.M., X.P. and Y.W. performed experiments. X.Z., W.Y., Z.Y. and G.C. analyzed the transcriptome data. R.K. and Y.N. generated HUDEP-2 cells. Y.W. and Li.Y. performed bioinformatic analysis to profile A&C-BEmax-editable variants and SNPs. D.L. and M.L. supervised the research. D.L., X.Z., S.S., H.G. and B.Z. analyzed the data and wrote the manuscript with input from all the authors.

Corresponding author

Correspondence to Dali Li.

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Competing interests

The authors have submitted patent applications (nos. 201810929391.3, 201810930979.0 and 201810930980.3) based on the results reported in this study.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–17, Supplementary Note, Supplementary Tables 3–6 and Supplementary Sequences 1–3.

Reporting Summary

Supplementary Table 1

Number of pathogenic sites or SNPs correctable by A&C-BEmax.

Supplementary Table 2

Analysis of potential codon and amino acid conversion by A&C-BEmax.

Unprocessed blots for Supplementary Fig. 4b.

Source data

Source Data Fig. 1

Statistical source data.

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Zhang, X., Zhu, B., Chen, L. et al. Dual base editor catalyzes both cytosine and adenine base conversions in human cells. Nat Biotechnol 38, 856–860 (2020). https://doi.org/10.1038/s41587-020-0527-y

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