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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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.
References
Collins, F. S., Brooks, L. D. & Chakravarti, A. A DNA polymorphism discovery resource for research on human genetic variation. Genome Res. 8, 1229–1231 (1998).
Gaudelli, N. M. et al. Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).
Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Ran, F. A. et al. Genome engineering using the CRISPR–Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).
Li, X. et al. Base editing with a Cpf1–cytidine deaminase fusion. Nat. Biotechnol. 36, 324–327 (2018).
Rees, H. A. & Liu, D. R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770–788 (2018).
Ma, Y. et al. Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells. Nat. Methods 13, 1029–1035 (2016).
Wang, X. et al. Efficient base editing in methylated regions with a human APOBEC3A–Cas9 fusion. Nat. Biotechnol. 36, 946–949 (2018).
Gehrke, J. M. et al. An APOBEC3A–Cas9 base editor with minimized bystander and off-target activities. Nat. Biotechnol. 36, 977–982 (2018).
Zong, Y. et al. Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A. Nat. Biotechnol. 36, 950–953 (2018).
Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, aaf8729 (2016).
Komor, A. C. et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci. Adv 3, eaao4774 (2017).
Cheng, T.-L. et al. Expanding C–T base editing toolkit with diversified cytidine deaminases. Nat. Commun. 10, 3612 (2019).
Wang, L. et al. Enhanced base editing by co-expression of free uracil DNA glycosylase inhibitor. Cell Res. 27, 1289–1292 (2017).
Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat. Biotechnol. 36, 843–846 (2018).
Kim, Y. B. et al. Increasing the genome-targeting scope and precision of base editing with engineered Cas9–cytidine deaminase fusions. Nat. Biotechnol. 35, 371–376 (2017).
Huang, T. P. et al. Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors. Nat. Biotechnol. 37, 626–631 (2019).
Zafra, M. P. et al. Optimized base editors enable efficient editing in cells, organoids and mice. Nat. Biotechnol. 36, 888–893 (2018).
Thuronyi, B. W. et al. Continuous evolution of base editors with expanded target compatibility and improved activity. Nat. Biotechnol. 37, 1070–1079 (2019).
Hess, G. T. et al. Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nat. Mehods 13, 1036–1042 (2016).
Jiang, W. et al. BE-PLUS: a new base editing tool with broadened editing window and enhanced fidelity. Cell Res. 28, 855–861 (2018).
Dickey, T. H., Altschuler, S. E. & Wuttke, D. S. Single-stranded DNA-binding proteins: multiple domains for multiple functions. Structure 21, 1074–1084 (2013).
Bae, S., Park, J. & Kim, J.-S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).
Kim, D. et al. Genome-wide target specificities of CRISPR RNA-guided programmable deaminases. Nat. Biotechnol. 35, 475–480 (2017).
Grünewald, J. et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 569, 433–437 (2019).
Zhou, C. et al. Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis. Nature 571, 275–278 (2019).
Grünewald, J. et al. CRISPR DNA base editors with reduced RNA off-target and self-editing activities. Nat. Biotechnol. 37, 1041–1048 (2019).
Kim, K. et al. Highly efficient RNA-guided base editing in mouse embryos. Nat. Biotechnol. 35, 435–437 (2017).
Traxler, E. A. et al. A genome-editing strategy to treat β-hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition. Nat. Med. 22, 987–990 (2016).
Wienert et al. Wake-up sleepy gene: reactivating fetal globin for β-hemoglobinopathies. Trends Genet. 34, 927–940 (2018).
Ye, L. et al. Genome editing using CRISPR–Cas9 to create the HPFH genotype in HSPCs: an approach for treating sickle cell disease and β-thalassemia. Blood 113, 10661–10665 (2016).
Antoniani, C. et al. Induction of fetal hemoglobin synthesis by CRISPR/Cas9-mediated editing of the human β-globin locus. Proc. Natl Acad. Sci. USA 131, 1960–1973 (2018).
Wang, L. et al. Reactivation of γ-globin expression through Cas9 or base editor to treat β-hemoglobinopathies. Cell Res. 30, 276–278 (2020).
Dedoussis, G., Sinopoulou, K., Gyparaki, M. & Loutradis, A. Fetal hemoglobin expression in the compound heterozygous state for −117 (G → A) Aγ HPFH and IVS‐1 nt 110 (G → A) β+ thalassemia: a case study. Eur. J. Haematol. 65, 93–96 (2000).
Martyn, G. E. et al. Natural regulatory mutations elevate the fetal globin gene via disruption of BCL11A or ZBTB7A binding. Nat. Genet. 50, 498–503 (2018).
Wienert, B. et al. KLF1 drives the expression of fetal hemoglobin in British HPFH. Blood 130, 803–807 (2017).
Guilinger, J. P., Thompson, D. B. & Liu, D. R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 32, 577–582 (2014).
Tsai, S. Q. et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat. Biotechnol. 32, 569–576 (2014).
Kim, D. et al. Digenome-seq: genome-wide profiling of CRISPR–Cas9 off-target effects in human cells. Nat. Methods 12, 237–243 (2015).
Zuo, E. et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364, 289–292 (2019).
Doman, J. L., Raguram, A., Newby, G. A. & Liu, D. R. Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors. Nat. Biotechnol. https://doi.org/10.1038/s41587-020-0414-6 (2020).
Hu, J. H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63 (2018).
Nishimasu, H. et al. Engineered CRISPR–Cas9 nuclease with expanded targeting space. Science 361, 1259–1262 (2018).
Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).
Kleinstiver, B. P. et al. Engineered CRISPR–Cas9 nucleases with altered PAM specificities. Nature 523, 481–484 (2015).
Zhu, B., Chen, L., Zhang, X. & Li, D. Efficient generation of site-specific point mutations in cell lines by hyper active CBEs (hyCBEs). Protoc. Exch. https://doi.org/10.21203/rs.3.pex-841/v1 (2020).
Yang, L. et al. Increasing targeting scope of adenosine base editors in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants. Protein Cell 9, 814–819 (2018).
Li, D. et al. Heritable gene targeting in the mouse and rat using a CRISPR–Cas system. Nat. Biotechnol. 31, 681–683 (2013).
Liu, H. et al. Nuclear cGAS suppresses DNA repair and promotes tumorigenesis. Nature 563, 131–136 (2018).
Hwang, G.-H. et al. Web-based design and analysis tools for CRISPR base editing. BMC Bioinformatics 19, 542 (2018).
Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).
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).
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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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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.
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.
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.
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.
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.
Supplementary information
Supplementary Information
Supplementary Notes 1 and 2 and Supplementary Sequences 1 and 2.
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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DOI: https://doi.org/10.1038/s41556-020-0518-8
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