Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage

Abstract

The spontaneous deamination of cytosine is a major source of transitions from C•G to T•A base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently convert targeted A•T base pairs to G•C could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR–Cas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted A•T base pairs efficiently to G•C (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.

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Figure 1: Scope and overview of base editing by an A•T to G•C base editor.
Figure 2: Protein evolution and engineering of ABEs.
Figure 3: Evolved ABEs mediate A•T to G•C base editing at human genomic DNA sites.
Figure 4: Product purity of late-stage ABEs.
Figure 5: Comparison of ABE7.10-mediated base editing and Cas9-mediated HDR, and application of ABE7.10 to two disease-relevant SNPs.

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Change history

  • 02 May 2018

    Please see accompanying Publisher correction (https://doi.org/10.1038/s41586-018-0070-x). In this Article, owing to an error during the production process, in Fig. 1a, the dark blue and light blue wedges were incorrectly labelled as 'G•C → T•A' and 'G•C → A•T', instead of 'C•G → T•A' and 'C•G → A•T', respectively. This error has been corrected online.

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Acknowledgements

This work was supported by DARPA HR0011-17-2-0049, US NIH RM1 HG009490, R01 EB022376, and R35 GM118062, and HHMI. A.C.K. and D.I.B. were Ruth L. Kirchstein National Research Service Awards Postdoctoral Fellows (F32 GM 112366 and F32 GM106621, respectively). M.S.P. was an NSF Graduate Research Fellow and was supported by training grant T32 GM008313. We thank Z. Niziolek for technical assistance. N.M.G. thanks A. E. Martin for his encouragement.

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Authors

Contributions

N.M.G designed the research, performed all evolution experiments, conducted human cell experiments, analysed data, and wrote the manuscript. A.C.K assisted with experimental design and human cell experiments and analysed data. H.A.R. performed HDR and off-target experiments. M.S.P. performed computational data analyses and developed HTS processing scripts. A.H.B contributed to selection design and evolution strategy. D.I.B. assisted with cloning of late-stage ABEs. D.R.L designed and supervised the research and wrote the manuscript. All of the authors contributed to editing the manuscript.

Corresponding author

Correspondence to David R. Liu.

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Competing interests

N.M.G., A.C.K., and D.R.L. have filed patent applications on this work. D.R.L. is a consultant and co-founder of Editas Medicine, Beam Therapeutics, and Pairwise Plants, companies that use genome editing technologies.

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Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 Genotypes of 57 ABEs described in this work.

Mutations are coloured according to the round of evolution in which they were identified.

Extended Data Figure 2 Base editing efficiencies of additional early-stage ABE variants.

a, Table of 19 human genomic DNA test sites (left) with corresponding locations on human chromosomes (right). The sequence context (target motif) of the edited A in red is shown for each site. PAM sequences are shown in blue. b, A•T to G•C base editing efficiencies in HEK293T cells of various wild-type RNA adenine deaminases fused to Cas9 nickase at six human genomic target DNA sites. Values reflect the mean and s.d. of three biological replicates performed on different days. c, A•T to G•C base editing efficiencies in HEK293T cells of ABE2 editors with altered fusion orientations and linker lengths at six human genomic target DNA sites. d, A•T to G•C base editing efficiencies in HEK293T cells at six human genomic target DNA sites of ABE2 editors fused to catalytically inactivated alkyl-adenosine glycosylase (AAG) or endonuclease V (EndoV), two proteins that bind inosine in DNA. e, A•T to G•C base editing efficiencies of ABE2.1 in HAP1 cells at site 1 with or without AAG. Values and error bars in b and c are the mean and s.d. of three independent biological replicates performed on different days.

Extended Data Figure 3 High-throughput DNA sequencing analysis of HEK293T cells treated with ABE2.1 and sgRNAs targeting each of six human genomic sites.

One representative replicate is shown. Data from untreated HEK293T cells are shown for comparison.

Extended Data Figure 4 Base editing efficiencies of additional ABE2 and ABE3 variants, and the effect of adding A142N to TadA*–dCas9 on antibiotic selection survival in E. coli.

a, A•T to G•C base editing efficiencies in HEK293T cells at six human genomic target DNA sites of ABE2 variants with different engineered dimeric states. A control ABE variant containing two wild-type TadA domains and no evolved TadA* domains (ABE0.2) did not result in A•T to G•C editing at the six genomic sites tested, confirming that dimerization alone is insufficient to mediate ABE activity. b, A•T to G•C base editing efficiencies in HEK293T cells at six human genomic target DNA sites of ABE3.1 variants differing in their dimeric state (homodimer of TadA*–TadA*–Cas9 nickase, or heterodimer of wild-type TadA–TadA*–Cas9 nickase), in the length of the TadA–TadA linker, and in the length of the TadA–Cas9 nickase linker. See Extended Data Fig. 1 for ABE genotypes and architectures. c, Colony-forming units on 2×YT agar with 256 μg ml1 of spectinomycin of E. coli cells expressing an sgRNA targeting the I89T defect in the spectinomycin resistance gene and a TadA*-dCas9 editor lacking or containing the A142N mutation identified in evolution round 4. Successful A•T to G•C base editing at the target site restores spectinomycin resistance. Values and error bars in a and b show the mean and s.d. of three independent biological replicates performed on different days.

Extended Data Figure 5 Base editing efficiencies of additional ABE5 variants.

a, A•T to G•C base editing efficiencies in HEK293T cells at six human genomic target DNA sites of two ABE3.1 variants with two pairs of mutations isolated from spectinomycin selection of the round 5 library. b, A•T to G•C base editing efficiencies in HEK293T cells at six human genomic target DNA sites of ABE5 variants with different linker lengths. See Extended Data Fig. 1 for ABE genotypes and architectures. Values and error bars show the mean and s.d. of three independent biological replicates performed on different days.

Extended Data Figure 6 Base editing efficiencies of ABE7 variants at 17 genomic sites.

a, b, A•T to G•C base editing efficiencies in HEK293T cells at 17 human genomic target DNA sites of ABE7.1–ABE7.5 (a), and ABE7.6-7.10 (b). See Extended Data Fig. 1 for ABE genotypes and architectures. c, A•T to G•C base editing efficiencies in U2OS cells at six human genomic target DNA sites of ABE7.8–ABE7.10. The lower editing efficiencies observed in U2OS cells compared with HEK293T cells are consistent with differences in transfection efficiency between the two cell lines; we observed transfection efficiencies of about 40–55% in U2OS cells under the conditions used in this study, compared to about 65–80% in HEK293T cells. Values and error bars show the mean and s.d. of three independent biological replicates performed on different days.

Extended Data Figure 7 Activity window of late-stage ABEs.

a, Relative A•T to G•C base editing efficiencies in HEK293T cells of late-stage ABEs at protospacer positions 1–9 in two human genomic DNA sites that together place an adenine at each of these positions. Values are normalized so that the maximum observed efficiency at each of the two sites for each ABE is 1. b, Relative A•T to G•C base editing efficiencies in HEK293T cells of late-stage ABEs at protospacer positions 1–18 and 20 across all 19 human genomic DNA sites tested. Values are normalized so that the maximum observed efficiency at each of the 19 sites for each ABE is 1. Values and error bars show the mean and s.d. across 19 sites, each with three independent biological replicates performed on different days.

Extended Data Figure 8 Rounds of evolution and engineering increased ABE processivity.

The calculated mean normalized linkage disequilibrium between nearby target adenines at 6–17 human genomic target DNA sites for the most active ABEs emerging from each round of evolution and engineering. Higher linkage disequilibrium values indicate that an ABE is more likely to edit an adenine if a nearby adenine in the same DNA strand (the same sequencing read) is also edited. Linkage disequilibrium values are normalized from 0 to 1 in order to be independent of editing efficiency. Values and error bars show the mean and s.d. of normalized linkage disequilibrium values across 6–17 sites, each with three independent biological replicates performed on different days.

Extended Data Figure 9 High-throughput DNA sequencing analysis of HEK293T cells treated with five late-stage ABE variants and an sgRNA targeting -198T in the promoter of HBG1 and HBG2.

One representative replicate is shown of DNA sequences at the HBG1 (a) and HBG2 (b) promoter targets. ABE-mediated base editing installs a −198T→C mutation on the strand complementary to the one shown in the sequencing data tables. Data from untreated HEK293T cells are shown for comparison.

Extended Data Table 1 Analysis of cellular RNAs in ABE7.10-treated cells compared with untreated cells

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This file contains Supplementary Tables 1-10, Supplementary Sequences 1-3, Supplementary Notes 1-4 and Supplementary References. (PDF 1680 kb)

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Gaudelli, N., Komor, A., Rees, H. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017). https://doi.org/10.1038/nature24644

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