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Increasing the efficiency and targeting range of cytidine base editors through fusion of a single-stranded DNA-binding protein domain

Nature Cell Biology volume 22, pages 740–750 (2020)Cite this article

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Abstract

Cytidine base editors are powerful genetic tools that catalyse cytidine to thymidine conversion at specific genomic loci, and further improvement of the editing range and efficiency is critical for their broader applications. Through insertion of a non-sequence-specific single-stranded DNA-binding domain from Rad51 protein between Cas9 nickase and the deaminases, serial hyper cytidine base editors were generated with substantially increased activity and an expanded editing window towards the protospacer adjacent motif in both cell lines and mouse embryos. Additionally, hyeA3A-BE4max selectively catalysed cytidine conversion in TC motifs with a broader editing range and much higher activity (up to 257-fold) compared with eA3A-BE4max. Moreover, hyeA3A-BE4max specifically generated a C-to-T conversion without inducing bystander mutations in the haemoglobin gamma gene promoter to mimic a naturally occurring genetic variant for amelioration of β-haemoglobinopathy, suggesting the therapeutic potential of the improved base editors.

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Fig. 1: Design and optimization of BE4max through fusion of an ssDBD.
Fig. 2: Characterization of hyA3A-BE4max in HEK293T cells.
Fig. 3: Highly efficient base editing by hyA3A-BE4max in mouse embryos.
Fig. 4: Characterization of hyeA3A-BE4max in HEK293T cells.
Fig. 5: Investigation of the off-target editing activity and potential toxicity of hyCBEs.
Fig. 6: Highly efficient base editing by hyeA3A-BE4max in mouse embryos.
Fig. 7: hyeA3A-BE4max generates accurate mutation in the HBG promoter in HUDEP-2 cells.

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Data availability

HTS data have been deposited in the NCBI Sequence Read Archive database under accession codes PRJNA566262, PRJNA566253 and PRJNA602779. RNA-Seq data have been deposited in the NCBI Sequence Read Archive database under accession code PRJNA599328. WGS data have been deposited in the NCBI Sequence Read Archive database under accession code PRJNA610447. Source data for Figs. 1–7 and Extended Data Figs. 2–6 are presented with the paper. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank M. Crossley from the School of Biotechnology and Biomolecular Sciences, University of New South Wales, Australia, who kindly provided the HUDEP-2(ΔGγ) cells and edited the manuscript, and X. Huang from ShanghaiTech University for discussions and suggestions in relation to this study. We thank H. Jiang from the Core Facility and Technical Service Center for SLSB, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University for technical support with FACS analysis. This work was partially supported by grants from the National Key Research and Development Program of China (2019YFA0110802 and 2019YFA0802800), National Natural Science Foundation of China (81670470 and 81873685) and Shanghai Municipal Science and Technology Commission (18411953500), and from the Innovation program of the Shanghai Municipal Education Commission (2019-01-07-00-05-E00054). This work was also supported by Fundamental Research Funds for the Central Universities, and the ECNU Public Platform for innovation (011).

Author information

Author notes

  1. These authors contributed equally: Xiaohui Zhang, Liang Chen, Biyun Zhu.

Authors and Affiliations

  1. Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China

    Xiaohui Zhang, Liang Chen, Biyun Zhu, Liren Wang, Caiyu Chen, Mengjia Hong, Yifan Huang, Huiying Li, Weishi Yu, Shuming Yin, Lei Yang, Meizhen Liu, Ying Zhang, Yuxuan Wu, Mingyao Liu & Dali Li

  2. Bioray Laboratories, Shanghai, China

    Honghui Han

  3. Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, School of Medicine, Tongji University, Shanghai, China

    Bailian Cai & Zhiyong Mao

  4. Cipher Gene, Beijing, China

    Weishi Yu & Zuozhen Yang

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Contributions

X.Z. and D.L. designed the experiments and analysed the data. X.Z., L.C., B.Z., L.W., C,C., M.H., Y.H., H.H., B.C., W.Y., S.Y., L.Y., Z.Y., Meizhen Liu, Y.Z., H.L. Z.M. and Y.W. performed the experiments. D.L., X.Z., L.C. and B.Z. analysed the data and wrote the manuscript with the input from all of the authors, Mingyao Liu and D.L. supervised the research.

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Correspondence to Dali Li.

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The authors have submitted a patent application (application numbers 2019113109698, 2019113125440 and 2019113125370) based on the results reported in this study.

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Extended data

Extended Data Fig. 1 Highly efficient base editing by A3A-BE4max or hyA3A-BE4max in mouse embryos.

(a, b) Genotyping of F0 generation pups by A3A-BE4max and hyA3A-BE4max. The frequencies of WT and mutant alleles were determined by analyzing HTS using BE-analyzer. The percentage on the right represents the frequency of the indicated mutant allele with the corresponding mutation-induced amino acid conversion shown in parentheses. The frequency of the wild-type allele was omitted. Wt, wild-type.

Extended Data Fig. 2 Off-target analysis and germline transmission of the founders derived from hyA3A-BE4max injection.

(a) HTS was performed with mouse tails to determine editing efficiencies at 15 potential off-target sites in three Dmd mutant F0 mice (#BD03, #BD04 and #BD07). Mismatched nucleotide letters are indicated in lowercase. Data are means ± SD (n = 3 mice).(b) HTS alignments of mutant sequences from F1 generated by mating founder #BD12(♀) with Wt (♂). The column on the right indicates frequencies of mutant alleles. Wt, wild-type.Statistical source data are provided in Source Data Extended Data Fig. 2.

Source data

Extended Data Fig. 3 Comparison of base editing efficiency and protein levels by CBEs and hyCBEs in HEK293T cells.

(a)Comparison of base editing efficiency induced by A3A-BE4max or hyeA3A-BE4max in HEK293T cells. The average mutation percentage derived from three independent experiments of A3A-BE4max and hyeA3A-BE4max at the same site is listed. Some of the data (hyeA3A-BE4max) are the same as presented in Fig. 4a. Statistical source data are provided in Source Extended Data Fig. 3. (b) The protein levels of BE4max, hyBE4max, A3A-BE4max, hyA3A-BE4max, eA3A-BE4max and hyeA3A-BE4max were determined by Western blotting in HEK293T cells 3 days after transfection of similar amounts of plasmid DNA. Specific antibodies against Cas9 (top) or GAPDH (bottom) were used. Western blotting images are representative of three independent experiments. Unprocessed blots are shown in Source Data Extended Data Fig. 3.

Source data

Extended Data Fig. 4 Comparison of base editing product purity induced by variant base editors in HEK293T cells.

(a) Comparison of base editing products induced by BE4max vs hyBE4max. HTS data were analyzed and the ratio of each type of nucleotides was listed on each target position. Data are means ± SD (n = 3 independent experiments). (b) Comparison of base editing products induced by A3A-BE4max vs hyA3A-BE4max. HTS data were analyzed and the ratio of each type of nucleotides was listed on each target position. Data are means ± SD (n = 3 independent experiments) (c) Comparison of base editing product induced by eA3A-BE4max vs hyeA3A-BE4max. HTS data were analyzed and the ratio of each type of nucleotides was listed on each target position. The individual data points are shown as black (C > T), light green (C > A) and light red (C > G) dots. Data are means ± SD (n = 3 independent experiments). Statistical source data are provided in Source Data Extended Data Fig. 4.

Source data

Extended Data Fig. 5 Whole genome sequencing of Dmd F0 (#DD11) and wild-type (Wt) mice.

(a) Summary of genome sequencing analysis. WGS for a Dmd mutant mouse (#DD11) and a wild type mouse (Wt) were performed. A total of 82,573 and 62,359 SNPs were identified for #DD11 and Wt, respectively. After filtering out dbSNP (naturally occurring variants in the SNP database), 20,387 SNPs were obtained in the #DD11 genome. Then the sequences at the remaining SNP sites were compared with all on-/off-target sequences (20 bp). (b) Summary of on-/off-target site information. A total of 175,058 sites, including 1 on-target site and 20; 374; 2,869; 22,335; and 148,569 off-target sites with 3, 4, 5, 6, or 7 mismatch/es, respectively, were analyzed. (c) Summary of the whole-genome sequencing. (d) Summary of off-target analysis. After comparing the sequences at the remaining SNP sites with the 175,058 on-/off-target sequences (20 bp), the C-to-T substitution was only detected within the on-target sequencing in #DD11. (e) Validation the off-target candidate site determined in (d) using targeted deep sequencing of genomic DNA isolated from various #DD11 organs (heart, liver, lung and tail). Mismatched nucleotides and PAM sequences are shown in red and in blue, respectively. Data represent mean from two independent experiments. Statistical source data are provided in Source Data Extended Data Fig. 5.

Source data

Extended Data Fig. 6 Indels and differentiation stage evaluation of HUDEP-2 (ΔGγ) cells after viral infection.

(a) Schematic representation of lentivirus constructs for HUDEP-2 infection.Psi+, Psi packaging signal; RRE, Rev response element; cPPT, central polypurine tract; EFS, elongation factor 1a short promoter; Bp-NLS, bipartite nuclear localization signals; A3A, derived from human Apobec3A; Rad51DBD, derived human rad51 single strand DNA binding protein domain; spCas9n, Cas9 D10A; P2A, 2 A self-cleaving peptide; WPRE, post-transcriptional regulatory element; UGI, Uracil DNA glycosylase inhibitor; EGFP, a maker for FACS. (b) Comparison of indels generated by lenti-hyA3A-BE4max or lenti-hyeA3A-BE4max treated HUDEP-2(ΔGγ) cells. Data are means ± SD (n = 3 independent experiments). P value was determined by two-tailed Student’s t test. (c) Erythroid differentiation validation of HUDEP-2 (ΔGγ) cells evaluated by anti α4-integrin(APC) and anti CD235a(FITC) surface markers, 8 days after differentiation. FACS data analysis are representative of three independent experiments. Statistical source data are provided in Source Data Extended Data Fig. 6.

Source data

Supplementary information

Supplementary Information

Supplementary Notes 1 and 2 and Supplementary Sequences 1 and 2.

Reporting Summary

Supplementary Tables 1–5

Supplementary Table 1: Highly efficient base editing by A3A-BE4max and hyA3A-BE4max in embryos. Summary of the numbers of embryos used and the pups generated after microinjection of CBE/sgRNA. Supplementary Table 2: Highly efficient base editing by eA3A-BE4max and hyeA3A-BE4max in embryos. Summary of the numbers of embryos used and the pups generated after microinjection of CBE/sgRNA. Supplementary Table 3: Target protospacer sequences analysed in this study.Target Cs are shown in green with a subscripted number denoting spacer position, and the PAM sequence is in blue. Supplementary Table 4: PCR primers used in this study. Supplementary Table 5: Off-target sites and off-target site PCR primers used in this study.

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Zhang, X., Chen, L., Zhu, B. et al. Increasing the efficiency and targeting range of cytidine base editors through fusion of a single-stranded DNA-binding protein domain. Nat Cell Biol 22, 740–750 (2020). https://doi.org/10.1038/s41556-020-0518-8

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