Abstract
Adenine base editors (ABEs) catalyze A-to-G transitions showing broad applications, but their bystander mutations and off-target editing effects raise safety concerns. Through structure-guided engineering, we found ABE8e with an N108Q mutation reduced both adenine and cytosine bystander editing, and introduction of an additional L145T mutation (ABE9), further refined the editing window to 1–2 nucleotides with eliminated cytosine editing. Importantly, ABE9 induced very minimal RNA and undetectable Cas9-independent DNA off-target effects, which mainly installed desired single A-to-G conversion in mouse and rat embryos to efficiently generate disease models. Moreover, ABE9 accurately edited the A5 position of the protospacer sequence in pathogenic homopolymeric adenosine sites (up to 342.5-fold precision over ABE8e) and was further confirmed through a library of guide RNA–target sequence pairs. Owing to the minimized editing window, ABE9 could further broaden the targeting scope for precise correction of pathogenic single-nucleotide variants when fused to Cas9 variants with expanded protospacer adjacent motif compatibility. bpNLS, bipartite nuclear localization signals.
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Data availability
HTS data have been uploaded to the NCBI Sequence Read Archive (SRA) database under accession codes PRJNA812697, PRJNA812700 and PRJNA862289. RNA-seq raw data have been uploaded into the SRA database under accession code PRJNA811343. Data for rat embryos treated with ABE7.10 have already been posted in the SRA database under accession code PRJNA471163. There are no restrictions on data availability. Source data are provided with this paper.
Change history
22 April 2024
A Correction to this paper has been published: https://doi.org/10.1038/s41589-024-01622-4
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Acknowledgements
We are grateful to S. Siwko (Texas A&M University Health Science Center) for proofreading the manuscript and support from East China Normal University Public Platform for innovation (011). We thank Y. Zhang from the Flow Cytometry Core Facility of School of Life Sciences in ECNU and Haixia Jiang from the Core Facility and Technical Service Center for SLSB of School of Life Sciences and Biotechnology in SJTU. We thank L. Ji (MedSci) for drawing schematic diagrams. 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 (No.32025023 to D.L., No.32230064 to D.L. and No.31971366 to L.W.); grants from the Shanghai Municipal Commission for Science and Technology (20MC1920400 and 21JC1402200 to D.L.), the Innovation Program of Shanghai Municipal Education Commission (2019-01-07-00-05-E00054 to D.L.), the Fundamental Research Funds for the Central Universities; and support from East China Normal University Outstanding Doctoral Students Academic Innovation Ability Improvement Project (YBNLTS2021-026 to L.C.).
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Authors and Affiliations
Contributions
L.C. and D.L. designed the project. L.C., S.Z., N.X., M.H., X.Z., J.Y., S.B., Y.H., C.L., B.Z. and L.W. performed ABE8e variant screening experiments. L.C., S.Z., N.X., M.H., J.Y., S.B. and Y.H. performed characterization of ABE9. L.C., S.Z. and N.X. assessed off-target effects of ABE9. L.C., M.H., S.Z. and Me.L. performed animal studies. M.H., G.R. and H.G. generated stable cell lines and other assays. S.Z., N.X. and D.Z. performed target library analysis. L.C., S.Z., N.X., M.H., J.Y., S.B., Y.H., H.M., H.W., C.Y., Mi.L., L.Z., Y.C. and D.L. analyzed the sequencing data, D.L., L.C., Y.Z. and G.S. wrote the manuscript with input from all the authors. D.L. supervised the research.
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L.C., D.L., S.Z., N.X. and M.L. have submitted patent applications on the basis the results reported in this study. The remaining authors declare no competing interests.
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Nature Chemical Biology thanks Hui Yang, Wen Xue and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 ABE8e induces severe bystander mutations and global random off-target editing.
a, Comparison of base editing efficiency of ABEmax or ABE8e at 6 endogenous genomic loci in HEK293T cells. Partial data are derived from Fig. 1b, c. b, The C-to-T/G/A editing efficiency of ABEmax or ABE8e was examined at 5 endogenous genomic loci in HEK293T cells. Partial data are derived from Fig. 1c. c, The schematic diagram of orthogonal R-loop assay-based nickase SaCas9 (nSaCas9) (left panel); Cas9-independent DNA off-target analysis of ABE8e using the modified orthogonal R-loop assay at each R-loop site with nSaCas9-sgRNA plasmid (right panel). Data are mean ± s.d. (n = 3 independent experiments). Data are derived from Fig. 4b. In a and b, the heatmap represents average editing percentage derived from three independent experiments and editing efficiency was determined by HTS. Statistical source data are provided online.
Extended Data Fig. 2 Comparison of the editing window between ABE8e and ABE8e-N108Q.
a, Comparison of A-to-G base editing window of ABE8e or ABE8e-N108Q at 12 target sites in HEK293T cells. b, Comparison of C-to-T/G/A base editing window of ABE8e or ABE8e-N108Q at 9 target sites in HEK293T cells. In a and b, data are from Fig. 2a (a) and Fig. 2b (b), and each point represents mean from three independent experiments. Statistical source data are provided online.
Extended Data Fig. 3 Evaluation of ABE8e, ABE9 and individual saturation variants.
a, Base editing efficiency of N108- or L145-saturated variants at 2 endogenous genomic loci in HEK293T cells. The heatmap represents an average editing percentage derived from two or three independent experiments with editing efficiency determined by HTS. b, The normalized precision (ABE8e is used for standardization) is defined as the highest / all other A-to-G base editing of ABE8e-N108Q or ABE9 at the 12 target sites in Fig. 3b. Data represent mean ± s.d. from three independent experiments. P values above each group indicated the comparison of ABE8e-N108Q and ABE9. c, Comparison of A-to-G base editing window of ABE7.10, ABE8e, ABE8e-N108Q or ABE9 at 12 target sites from Fig. 3b. in HEK293T cells. Data points represent mean from three independent experiments. d, Comparison of indels induced by ABE7.10, ABE8e, ABE8e-N108Q or ABE9 at 12 target sites from Fig. 3b. Each data point represents the average indel frequency at each target site calculated from 3 independent experiments. Error bar and P value are derived from these 12 data points. Data are mean ± s.d. In b and d, P value was determined by two-tailed Student’s t test. Statistical source data are provided online.
Extended Data Fig. 4 Editing activities comparison of ABE8e-N108Q and ABE9 in Hela cells.
The A-to-G editing efficiency of ABE8e-N108Q or ABE9 was examined at 3 endogenous genomic loci containing multiple As. The heatmap represents an average editing percentage derived from three independent experiments with editing efficiency determined by HTS. Statistical source data are provided online.
Extended Data Fig. 5 Cas9-dependent off-target assessment of ABE9.
a, Cas9-dependent DNA on- and off-target analysis of the indicated targets (CCR5-sg1p and ABE site 16) by ABE8e, ABE8e-N108Q and ABE9 in HEK293T cells. Data are mean ± s.d. (n = 3 independent experiments). On-target data are derived from Fig. 3b. b, Comparison of indels induced by ABE8e, ABE8e-N108Q or ABE9 at 44 Cas9-dependent DNA off-target target sites from a and Fig. 4a. Each data point represents the average indel frequency at each target site calculated from 2 independent experiments merely in HEK site 2-GUIDE-seq-OT1, 2 treated with ABE8e-N108Q and from 3 independent experiments in the rest of biological samples. Error bar and P value are derived from these 44 data points. Data are mean ± s.d. P value was determined by two-tailed Student’s t test. Statistical source data are provided online.
Extended Data Fig. 6 Cas9-independent off-target assessment in the R-loop assay.
a, On-target base editing induced by ABE8e, ABE8e-N108Q or ABE9 using the modified orthogonal R-loop assay at each R-loop site with nSaCas9-sgRNA plasmid. Data are mean ± s.d. (n = 3 independent experiments). b, Comparison of indels induced by ABE8e, ABE8e-N108Q or ABE9 at six R-loop sites from Fig. 4b. Each data point represents the average indel frequency at each target site calculated from 3 independent experiments. Error bar and P value are derived from these 6 data points. Data are mean ± s.d. P value was determined by two-tailed Student’s t test. Statistical source data are provided online.
Extended Data Fig. 7 Highly efficient and precise editing by ABE9 in rodent embryos.
a, Genotyping of F0 generation pups treated by ABE8e or ABE9. b, Comparison of indels induced ABE8e (n = 19) or ABE9 (n = 16) in the target sequence of the splicing acceptor site in intron 3 of the mouse Tyr gene. Data are mean ± s.d. c, Summary of the numbers of embryos used and the pups generated after microinjection of ABE8e/sgRNA or ABE9/sgRNA. d, e, Genotyping of F0 rats induced by ABE7.10 (d) and ABE9 (e) (desired editing in blue or undesired in red). f, Comparison of indels induced by ABE7.10 (n = 28) or ABE9 (n = 8) in the target sequence of exon 13 in the rat Gaa gene. Data are mean ± s.d. g, Ratio of desired reads to total reads in F0 rats induced by ABE7.10 or ABE9. In a, d and e, the percentage on the right represents the frequency determined by the rate of indicated mutant alleles to total alleles counts. Percentiles of each allele reads <1% are omitted. Statistical source data are provided online.
Extended Data Fig. 8 Allele tables for ABE9 in four stable HEK293T cell lines.
a-d, Allele tables for ABE8e, ABE8e-N108Q and ABE9 in four stable HEK293T cell lines: COL1A2 c.1136 G > A (a), CARD14 c.424 G > A (b), BVES c.602 C > T (c) and KCNA5 c.1828G > A (d). The percentile and sequencing reads of each allele from two or three independent experiments are listed on the right. Desired A5-to-G percentiles of alleles are exhibited, while percentiles of top ten invalid allele types are presented and percentiles of invalid allele types less than 1% are omitted.
Extended Data Fig. 9 Unbiased analysis of target library of ABE9.
Analysis of absolute mean editing efficiency of ABE8e, ABE8e-N108Q and ABE9. Positions of the protospacer are shown at the bottom of the heatmap, counting the protospacer adjacent motif (PAM) as positions 21–23.
Supplementary information
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Supplementary Figure 1, Supplementary Tables 1–4 and Supplementary Sequence 1–3.
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Chen, L., Zhang, S., Xue, N. et al. Engineering a precise adenine base editor with minimal bystander editing. Nat Chem Biol 19, 101–110 (2023). https://doi.org/10.1038/s41589-022-01163-8
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DOI: https://doi.org/10.1038/s41589-022-01163-8
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