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.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Sequence Read Archive
Krokan, H. E., Drabløs, F. & Slupphaug, G. Uracil in DNA—occurrence, consequences and repair. Oncogene 21, 8935–8948 (2002)
Lewis, C. A. Jr, Crayle, J., Zhou, S., Swanstrom, R. & Wolfenden, R. Cytosine deamination and the precipitous decline of spontaneous mutation during Earth’s history. Proc. Natl Acad. Sci. USA 113, 8194–8199 (2016)
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)
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)
Komor, A. C., Badran, A. H. & Liu, D. R. Editing the genome without double-stranded DNA breaks. ACS Chem. Biol. (2017)
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)
Rees, H. A. et al. Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat. Commun. 8, 15790 (2017)
Satomura, A. et al. Precise genome-wide base editing by the CRISPR Nickase system in yeast. Sci. Rep. 7, 2095 (2017)
Lu, Y. & Zhu, J.-K. Precise editing of a target base in the rice genome using a modified CRISPR/Cas9 system. Mol. Plant 10, 523–525 (2017)
Zong, Y. et al. Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nat. Biotechnol. 35, 438–440 (2017)
Zhang, Y. et al. Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system. Nat. Commun. 8, 118 (2017)
Billon, P. et al. CRISPR-mediated base editing enables efficient disruption of eukaryotic genes through induction of STOP codons. Mol. Cell 67, 1068–1079 (2017)
Kuscu, C. et al. CRISPR-STOP: gene silencing through base-editing-induced nonsense mutations. Nat. Methods 14, 710–712 (2017)
Kim, K. et al. Highly efficient RNA-guided base editing in mouse embryos. Nat. Biotechnol. 35, 435–437 (2017)
Chadwick, A. C., Wang, X. & Musunuru, K. In vivo base editing of PCSK9 (proprotein convertase subtilisin/kexin Type 9) as a therapeutic alternative to genome editing. Arterioscler. Thromb. Vasc. Biol. 37, 1741–1747 (2017)
Liang, P. et al. Correction of β-thalassemia mutant by base editor in human embryos. Protein Cell https://doi.org/10.1007/s13238-017-0475-6 (2017)
Li, G. et al. Highly efficient and precise base editing in discarded human tripronuclear embryos. Protein Cell 8, 776–779 (2017)
Tang, W., Hu, J. H. & Liu, D. R. Aptazyme-embedded guide RNAs enable ligand-responsive genome editing and transcriptional activation. Nat. Commun. 8, 15939 (2017)
Yasui, M. et al. Miscoding properties of 2′-deoxyinosine, a nitric oxide-derived DNA Adduct, during translesion synthesis catalyzed by human DNA polymerases. J. Mol. Biol. 377, 1015–1023 (2008)
Zheng, Y., Lorenzo, C. & Beal, P. A. DNA editing in DNA/RNA hybrids by adenosine deaminases that act on RNA. Nucleic Acids Res. 45, 3369–3377 (2017)
Kim, J. et al. Structural and kinetic characterization of Escherichia coli TadA, the wobble-specific tRNA deaminase. Biochemistry 45, 6407–6416 (2006)
Wolf, J., Gerber, A. P. & Keller, W. tadA, an essential tRNA-specific adenosine deaminase from Escherichia coli. EMBO J. 21, 3841–3851 (2002)
Matthews, M. M. et al. Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity. Nat. Struct. Mol. Biol. 23, 426–433 (2016)
Grunebaum, E., Cohen, A. & Roifman, C. M. Recent advances in understanding and managing adenosine deaminase and purine nucleoside phosphorylase deficiencies. Curr. Opin. Allergy Clin. Immunol. 13, 630–638 (2013)
Gerber, A. P. & Keller, W. An adenosine deaminase that generates inosine at the wobble position of tRNAs. Science 286, 1146–1149 (1999)
Fukui, K. DNA mismatch repair in eukaryotes and bacteria. J. Nucleic Acids 2010, 260512 (2010)
Shi, K. et al. Structural basis for targeted DNA cytosine deamination and mutagenesis by APOBEC3A and APOBEC3B. Nat. Struct. Mol. Biol. 24, 131–139 (2017)
Macbeth, M. R. et al. Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing. Science 309, 1534–1539 (2005)
Losey, H. C., Ruthenburg, A. J. & Verdine, G. L. Crystal structure of Staphylococcus aureus tRNA adenosine deaminase TadA in complex with RNA. Nat. Struct. Mol. Biol. 13, 153–159 (2006)
Lau, A. Y., Wyatt, M. D., Glassner, B. J., Samson, L. D. & Ellenberger, T. Molecular basis for discriminating between normal and damaged bases by the human alkyladenine glycosylase, AAG. Proc. Natl Acad. Sci. USA 97, 13573–13578 (2000)
Vik, E. S. et al. Endonuclease V cleaves at inosines in RNA. Nat. Commun. 4, 2271 (2013)
Paquet, D. et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533, 125–129 (2016)
Kim, D. et al. Genome-wide target specificities of CRISPR RNA-guided programmable deaminases. Nat. Biotechnol. 35, 475–480 (2017)
Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015)
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, B. et al. KLF1 drives the expression of fetal hemoglobin in British HPFH. Blood 130, 803–807 (2017)
Alexander, J. & Kowdley, K. V. HFE-associated hereditary hemochromatosis. Genet. Med. 11, 307–313 (2009)
Landrum, M. J. et al. ClinVar: public archive of interpretations of clinically relevant variants. Nucleic Acids Res. 44, D862–D868 (2016)
Badran, A. H. et al. Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance. Nature 533, 58–63 (2016)
Müller, K. M. et al. Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution. Nucleic Acids Res. 33, e117 (2005)
Esvelt, K. M., Carlson, J. C. & Liu, D. R. A system for the continuous directed evolution of biomolecules. Nature 472, 499–503 (2011)
Kwart, D., Paquet, D., Teo, S. & Tessier-Lavigne, M. Precise and efficient scarless genome editing in stem cells using CORRECT. Nat. Protocols 12, 329–354 (2017)
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.
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.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Mutations are coloured according to the round of evolution in which they were identified.
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 ml−1 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.
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.
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.
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.
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.
About this article
Cite this article
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
BMC Plant Biology (2020)
Pharmacology & Therapeutics (2020)
Cardiovascular Research (2020)
Nature Communications (2020)