Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Engineering APOBEC3A deaminase for highly accurate and efficient base editing

Abstract

Cytosine base editors (CBEs) are effective tools for introducing C-to-T base conversions, but their clinical applications are limited by off-target and bystander effects. Through structure-guided engineering of human APOBEC3A (A3A) deaminase, we developed highly accurate A3A-CBE (haA3A-CBE) variants that efficiently generate C-to-T conversion with a narrow editing window and near-background level of DNA and RNA off-target activity, irrespective of methylation status and sequence context. The engineered deaminase domains are compatible with PAM-relaxed SpCas9-NG variant, enabling accurate correction of pathogenic mutations in homopolymeric cytosine sites through flexible positioning of the single-guide RNAs. Dual adeno-associated virus delivery of one haA3A-CBE variant to a mouse model of tyrosinemia induced up to 58.1% editing in liver tissues with minimal bystander editing, which was further reduced through single dose of lipid nanoparticle-based messenger RNA delivery of haA3A-CBEs. These results highlight the tremendous promise of haA3A-CBEs for precise genome editing to treat human diseases.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Structure-based engineering of A3A.
Fig. 2: Further engineering of A3A to develop haA3A-CBE.
Fig. 3: Efficient base editing in methylated regions and GC contexts by haA3A-CBEs.
Fig. 4: Further characterization of haA3A-CBEs.
Fig. 5: Precise editing of pathogenic SNVs by haA3A-CBEs in HEK293T cell lines.
Fig. 6: Efficient and precise C-to-T base editing in FahNS/NS mice.

Similar content being viewed by others

Data availability

HTS data have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database under the accession codes PRJNA938162, PRJNA938284, PRJNA938523, PRJNA938572, PRJNA938580 and PRJNA1032216. RNA-seq data and HTS data of A3A hotspots have been deposited in the SRA database under the accession code PRJNA938578. WGS data have been deposited in the SRA database under the accession code PRJNA1042830. Data for mouse liver treated with CBE have already been posted in the SRA database under the accession code PRJNA937584. The hg38 reference genome (Homo sapiens genome assembly GRCh38.p14, NCBI, NLM (nih.gov)) was used for alignment in the analysis of RNA-seq and WGS data from HEK293T cells. The mm10 reference genome (Mus musculus genome assembly GRCm38.p6, NCBI, NLM (nih.gov)) was used for the analysis of RNA-seq data from mice. ClinVar database (ClinVar (nih.gov)) was used to identify pathogenic SNVs that can be correct by cytosine base editing. The published structure of A3A (PDB ID 5SWW) can be accessed at the RCSB Protein Data Bank (PDB) 5SWW: Crystal Structure of Human APOBEC3A complexed with ssDNA. There are no restrictions on data availability. Source data are provided with this paper.

References

  1. Anzalone, A. V., Koblan, L. W. & Liu, D. R. Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors. Nat. Biotechnol. 38, 824–844 (2020).

  2. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Zhao, D. D. et al. New base editors change C to A in bacteria and C to G in mammalian cells. Nat. Biotechnol. 39, 35–40 (2021).

    Article  PubMed  CAS  Google Scholar 

  5. Kurt, I. C. et al. CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nat. Biotechnol. 39, 41–46 (2021).

    Article  PubMed  CAS  Google Scholar 

  6. Kim, K. et al. Highly efficient RNA-guided base editing in mouse embryos. Nat. Biotechnol. 35, 435–437 (2017).

    Article  PubMed  CAS  Google Scholar 

  7. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  8. 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).

    Article  CAS  Google Scholar 

  9. Shimatani, Z. et al. Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nat. Biotechnol. 35, 441–443 (2017).

    Article  PubMed  CAS  Google Scholar 

  10. Villiger, L. et al. Treatment of a metabolic liver disease by in vivo genome base editing in adult mice. Nat. Med. 24, 1519–1525 (2018).

    Article  PubMed  CAS  Google Scholar 

  11. Rossidis, A. C. et al. In utero CRISPR-mediated therapeutic editing of metabolic genes. Nat. Med. 24, 1513–1518 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Li, G. L. et al. Gene editing and its applications in biomedicine. Sci. China Life Sci. 65, 660–700 (2022).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Zuo, E. et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364, 289–292 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Jin, S. et al. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science 364, 292–295 (2019).

    Article  PubMed  CAS  Google Scholar 

  15. Grunewald, J. et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 569, 433–437 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Zhou, C. et al. Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis. Nature 571, 275–278 (2019).

    Article  PubMed  CAS  Google Scholar 

  17. Rothgangl, T. et al. In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels. Nat. Biotechnol. 39, 949–957 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Tan, J. J., Zhang, F., Karcher, D. & Bock, R. Engineering of high-precision base editors for site-specific single nucleotide replacement. Nat. Commun. 10, 439 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Gehrke, J. M. et al. An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities. Nat. Biotechnol. 36, 977–982 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Lee, S. et al. Single C-to-T substitution using engineered APOBEC3G-nCas9 base editors with minimum genome- and transcriptome-wide off-target effects. Sci. Adv. 6, eaba1773 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Zuo, E. et al. A rationally engineered cytosine base editor retains high on-target activity while reducing both DNA and RNA off-target effects. Nat. Methods 17, 600–604 (2020).

    Article  PubMed  CAS  Google Scholar 

  23. Wang, L. et al. Eliminating base-editor-induced genome-wide and transcriptome-wide off-target mutations. Nat. Cell Biol. 23, 552–563 (2021).

    Article  PubMed  Google Scholar 

  24. Wang, X. et al. Efficient base editing in methylated regions with a human APOBEC3A-Cas9 fusion. Nat. Biotechnol. 36, 946–949 (2018).

    Article  PubMed  CAS  Google Scholar 

  25. 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. 38, 620–628 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. 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).

    Article  PubMed  CAS  Google Scholar 

  27. Kouno, T. et al. Crystal structure of APOBEC3A bound to single-stranded DNA reveals structural basis for cytidine deamination and specificity. Nat. Commun. 8, 15024 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat. Biotechnol. 36, 843–846 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Nishimasu, H. et al. Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science 361, 1259–1262 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. 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).

    Article  PubMed  CAS  Google Scholar 

  31. Sharma, S. et al. APOBEC3A cytidine deaminase induces RNA editing in monocytes and macrophages. Nat. Commun. 6, 6881 (2015).

    Article  PubMed  CAS  Google Scholar 

  32. Andreucci, E. et al. TRPV4 related skeletal dysplasias: a phenotypic spectrum highlighted byclinical, radiographic, and molecular studies in 21 new families. Orphanet J. Rare Dis. 6, 37 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Dorval, I. et al. Analysis of 160 CF chromosomes: detection of a novel mutation in exon 20. Hum. Genet 91, 254–256 (1993).

    Article  PubMed  CAS  Google Scholar 

  34. Higuchi, M. et al. Molecular characterization of severe hemophilia A suggests that about half the mutations are not within the coding regions and splice junctions of the Factor VIII gene. Proc. Natl Acad. Sci. USA 88, 7405–7409 (1991).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Cuesta-Munoz, A. L. et al. Severe persistent hyperinsulinemic hypoglycemia due to a de novo glucokinase mutation. Diabetes 53, 2164–2168 (2004).

    Article  PubMed  CAS  Google Scholar 

  36. Hermans, M. M. et al. Twenty-two novel mutations in the lysosomal alpha-glucosidase gene (GAA) underscore the genotype-phenotype correlation in glycogen storage disease type II. Hum. Mutat. 23, 47–56 (2004).

    Article  PubMed  CAS  Google Scholar 

  37. Guldberg, P. et al. A novel missense mutation in the phenylalanine hydroxylase gene of a homozygous Pakistani patient with non-PKU hyperphenylalaninemia. Hum. Mol. Genet 2, 1061–1062 (1993).

    Article  PubMed  CAS  Google Scholar 

  38. Janecke, A. R. et al. Mutations in RDH12 encoding a photoreceptor cell retinol dehydrogenase cause childhood-onset severe retinal dystrophy. Nat. Genet. 36, 850–854 (2004).

    Article  PubMed  CAS  Google Scholar 

  39. Liu, X. Z. et al. Digenic inheritance of non-syndromic deafness caused by mutations at the gap junction proteins Cx26 and Cx31. Hum. Genet 125, 53–62 (2009).

    Article  PubMed  CAS  Google Scholar 

  40. Machado, R. D. et al. Pulmonary arterial hypertension: a current perspective on established and emerging molecular genetic defects. Hum. Mutat. 36, 1113–1127 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Yang, L. et al. Amelioration of an inherited metabolic liver disease through creation of a de novo start codon by cytidine base editing. Mol. Ther. 28, 1673–1683 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Yin, S. et al. Enhanced genome editing to ameliorate a genetic metabolic liver disease through co-delivery of adeno-associated virus receptor. Sci. China Life Sci. 65, 718–730 (2022).

    Article  PubMed  CAS  Google Scholar 

  43. Nathwani, A. C. et al. Self-complementary adeno-associated virus vectors containing a novel liver-specific human Factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver. Blood 107, 2653–2661 (2006).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Song, C. Q. et al. Adenine base editing in an adult mouse model of tyrosinaemia. Nat. Biomed. Eng. 4, 125–130 (2020).

    Article  PubMed  CAS  Google Scholar 

  45. Bulliard, Y. et al. Structure-function analyses point to a polynucleotide-accommodating groove essential for APOBEC3A restriction activities. J. Virol. 85, 1765–1776 (2011).

    Article  PubMed  CAS  Google Scholar 

  46. Thuronyi, B. W. et al. Continuous evolution of base editors with expanded target compatibility and improved activity. Nat. Biotechnol. 37, 1070–1079 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Chen, L. et al. Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing. Nat. Biotechnol. 41, 663–672 (2023).

    Article  PubMed  CAS  Google Scholar 

  48. Neugebauer, M. E. et al. Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity. Nat. Biotechnol. 41, 673–685 (2023).

    Article  PubMed  CAS  Google Scholar 

  49. Lam, D. K. et al. Improved cytosine base editors generated from TadA variants. Nat. Biotechnol. 41, 686–697 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  50. Greig, J. A. et al. Integrated vector genomes may contribute to long-term expression in primate liver after AAV administration. Nat. Biotechnol. https://doi.org/10.1038/s41587-023-01974-7 (2023).

  51. Hwang, G. H. et al. Web-based design and analysis tools for CRISPR base editing. BMC Bioinf. 19, 542 (2018).

    Article  CAS  Google Scholar 

  52. Chen, L. et al. Engineering a precise adenine base editor with minimal bystander editing. Nat. Chem. Biol. 19, 101–110 (2023).

    Article  PubMed  CAS  Google Scholar 

  53. Zhang, X. 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).

    Article  PubMed  CAS  Google Scholar 

  54. McGrath, E. et al. Targeting specificity of APOBEC-based cytosine base editor in human iPSCs determined by whole genome sequencing. Nat. Commun. 10, 5353 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to the 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 the East China Normal University. This work was partially supported by grants from National Key R&D Program of China (grants 2022YFC3400203 to Y.G., 2023YFC3403400 to D.L., 2023YFE0209200 to D.L., 2019YFA0110802 to D.L., 2019YFA0802800 to Mingyao Liu, 2019YFA0110902 to C.Y. and 2019YFA0801402 to F.Z.), the National Natural Science Foundation of China (grants 32025023 to D.L., 32230064 to D.L., 31971366 to L.W., 82230002 to Mingyao Liu, 82100773 to Y.G., 82271890 to F.Z., 21825701 to C.Y., 91953201 to C.Y. and 92153303 to C.Y.), grants from the Shanghai Municipal Commission for Science and Technology (grant 21JC1402200 to D.L.), the Innovation program of Shanghai Municipal Education Commission (grant 2019-01-07-00-05-E00054 to D.L.), the China Postdoctoral Science Foundation (grant 2022M721161 to L.Y.) and Innovative Research Team of High-Level Local Universities in Shanghai (grant SHSMU-ZDCX20212200 to D.L. and F.Z.).

Author information

Authors and Affiliations

Authors

Contributions

L.Y. and D.L. conceived the rational engineering of A3A. L.Y., Y.H., M.W. performed the experiments of plasmid construction, cell culture, cell transfection, genomic DNA preparation, AAV virus production, lentiviral vector production and the creation of stable cell lines. L.Y., Y.H., M.W. and X.R. performed the experiments of PCR, with reverse transcription and qPCR and prepared the HTS libraries. L.Y., Y.H., M.W., T.Z. and Y.G. performed the western blot, IHC and HE staining experiments. L.Y., Y.H., M.W., T.Z., S.Y., Dexin Zhang and J.M. performed the experiments of cell sorting. L.Y. and T.Z. performed the experiments of bisulfite sequencing. L.Y., Y.H., M.W. and Meizhen Liu performed the animal experiments. L.Y., Y.H., M.W., Dan Zhang, L.W. and D.L. analyzed the HTS data. G.S. and L.Y. analyzed the structure of A3A. Dan Zhang, H.W. and H.M. analyzed the RNA-seq and WGS data. X.C. and J.L. performed the experiments for production of CBE mRNAs and LNP encapsulation, respectively. D.L., L.Y., L.W., F.Z., Mingyao Liu, C.Y., B.F., Y.C. and G.S. designed the experiments and wrote the manuscript with the input from all the authors. D.L. supervised the research.

Corresponding authors

Correspondence to Fanyi Zeng, Liren Wang or Dali Li.

Ethics declarations

Competing interests

A patent application (application number CN202210791520.3) based on the A3A mutants such as Y130A, VA and Y130G reported in this study has been submitted, not yet authorized. The patent applicant is East China Normal University, and D.L., L.Y., Y.H. and M.W. are the inventors. The remaining authors declare no competing interests.

Peer review

Peer review information

Nature Chemical Biology thanks the anonymous reviewer(s) for their contribution to the peer review of this work.

Additional information

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

Extended data

Extended Data Fig. 1 Optimizing human cell orthogonal R-loop assay to evaluate the Cas9-independent off-target activity of SpCas9-based CBEs in HEK293T cells.

a, Architecture of A3A-BE4max for all CBE constructs used in this study. b, Schematic of Cas9-independent deamination of cytosines within nSaCas9(D10A)-induced R-loops by SpCas9-based CBEs. c, Cas9-independent off-target editing frequencies for A3A-BE4max at six SaCas9 loci using dSaCas9 or nSaCas9. d, On-target editing frequencies for A3A-BE4max at ABL1 site 1 when either dSaCas9 or nSaCas9 were used to target six SaCas9 loci. e, Cas9-independent off-target C-to-G editing frequencies for A3A-BE4max at SaCas9 site 5 and SaCas9 site 6 using dSaCas9 or nSaCas9. f, Frequencies of indels induced by A3A-BE4max’s Cas9-independent off-target deamination at six SaCas9 loci using dSaCas9 or nSaCas9. g, Frequencies of indels induced by A3A-BE4max at ABL1 site 1 using dSaCas9 or nSaCas9. h, Frequencies of indels induced by A3A-BE4max variants at ABL1 site 1 and Sa site 5 in HEK293T cells. Dots represent individual values, and bars represent mean ± s.d. of three independent biological replicates.

Extended Data Fig. 2 Further characterize the on-target activity of haA3A-CBEs.

a, Editing frequencies for haA3A-CBEs at additional 15 genomic sites in HEK293T cells from three independent biological replicates, editing frequencies higher than 10% are labeled in cells. b, Performance of CBE variants in editing favored cytosines in TC context (n = 16, 14, 16, 9, 10, and 10 cytosines for A3A-BE4max, YE1 –BE4max, eA3A-BE4max, haA3A-CBE-A, haA3A-CBE-VA, and haA3A-CBE-G, respectively.), CC context (n = 10, 7, 5, 9, 8, and 8 cytosines for A3A-BE4max, YE1 –BE4max, eA3A-BE4max, haA3A-CBE-A, haA3A-CBE-VA, and haA3A-CBE-G, respectively.), GC context (n = 8, 7, 8, 8, 8, and 8 cytosines for A3A-BE4max, YE1 –BE4max, eA3A-BE4max, haA3A-CBE-A, haA3A-CBE-VA, and haA3A-CBE-G, respectively.), and AC context (n = 7, 11, 10, 13, 13, and 13 cytosines for A3A-BE4max, YE1 –BE4max, eA3A-BE4max, haA3A-CBE-A, haA3A-CBE-VA, and haA3A-CBE-G, respectively.) from 39 sites with NGG PAM shown in a, Fig. 1c, Fig. 2a,b and Fig. 3a. Each dot represents the average editing frequency of the most highly edited cytosine in each site calculated from three independent experiments, and bars represent mean ± s.d. P values were calculated using the two-tailed Student’s t-test.

Extended Data Fig. 3 Editing window plots (a) and editing precision (b) for CBE variants.

In a, the lower and upper dotted lines in each plot denote the editing window cutoff and central editing window cutoff, respectively. The editing window and central editing window are defined as the protospacer positions for which the average editing efficiency is ≥30% and ≥70%, respectively, of the average editing at the maximally edited position. In b, each dot indicates the editing precision for each CBE variant at each tested site shown in Fig. 1c, Fig. 2a,b, Fig. 3a, and Extended Data Fig. 2a (n = 39 tested sites). Editing precision is defined as the ratio of the editing efficiency of the most highly edited base to that of the second highly edited base in each target site. Bars represent mean ± s.d.

Extended Data Fig. 4 Editing activities of haA3A-CBEs in Hela cells.

In heatmaps, editing efficiencies shown represent the mean of three biologically independent replicates, editing frequencies higher than 10% are labeled in cells.

Extended Data Fig. 5 Protein expression levels of CBE variants.

Western blot showing the protein expression of the indicated CBE variants in HEK293T cells.

Source data

Extended Data Fig. 6 Correction of human pathogenic SNVs.

a, Information of nine human pathogenic SNVs caused by T-to-C or A-to-G mutations from ClinVar database. b, Sequences represent the protospacers and PAMs (blue), pathogenic SNVs are labeled in red, and bystander Cs edited by A3A-BE4amx are labeled in green. For SNV 7, SNV8 and SNV9, upper sequences represent the protospacers with NGG PAMs, and lower sequences represent the protospacers with NG PAMs.

Extended Data Fig. 7 Efficient and precise correction of the disease-causing mutation in FahNS/NS mice.

a, Diagram of two sgRNAs (sg1 and sg2) designed to target the exon 1 of FahNS/NS allele. PAMs are indicated by blue lines and protospacers by red lines. Blue numbers denote the positions of the individual bases within the protospacers, while the subscript numbers denote the base positions relative to the first base of the CDS. The disease-causing mutation1A>G (p.M1 > V) is shown in red. The C1-to-T1 (shown in green) conversion in the non-coding strand leads to the desired G1-to-A1 (shown in green) conversion in the coding strand, which restores the start codon for methionine (shown in green). b, The editing frequencies and correction rates of the FahNS/NS allele by CBE variants in HEK293T reporter cells. Heatmaps represent the average editing frequencies of individual cytosines from three biologically independent replicates. Histograms represent the frequencies of perfect edits (desired C1-to-T1 edits without any other edits) of the FahNS/NS allele by CBE variants. The numbers beside bars display fold changes for accurate CBE variants in creating desired G1-to-A1 (without bystander edits) edits compared with A3A-BE4max. c, Schematic view of intein-mediated split haA3A-CBE-G AAV constructs (haA3A-CBE-G-N and haA3A-CBE-G-C). ITR, inverted terminal repeat; NLS, nuclear localization signal; bGH, bovine growth hormone poly (A) signal; U6, RNA polymerase III promoter for human U6 snRNA; P2A and T2A, 2 A peptide from porcine teschovirus-1 polyprotein and Thosea asigna virus capsid protein, respectively. d, The experimental scheme for in vivo base editing. e, Editing frequencies of computationally predicted off-target loci of sg1. f, The editing frequencies of the FahNS/NS allele in different tissues from untreated and treated mice 9 weeks postinjection. Dots represent individual values, and bars represent mean ± s.d. of three independent biological replicates (b) or three mice (e and f).

Extended Data Fig. 8 Transcriptomic changes following AAV delivery of haA3A-CBE-G in FahNS/NS mice.

a, Heatmap of 273 differentially expressed genes amongst untreated and treated FahNS/NS mice. b, Volcano plot showing fold-change and p-value of genes up-regulated (red) and down-regulated (blue) in treated compared to untreated FahNS/NS mice. P values were obtained using DESeq2 (Methods).

Extended Data Fig. 9 Editing profiles of CBE variants delivered by plasmid or mRNA in HEK293T reporter cells.

a, Editing frequencies for CBE variants delivered by plasmid or mRNA in HEK293T reporter cells. In heatmaps, editing efficiencies shown represent the mean of three biologically independent replicates. b, Editing precision of each CBE variant delivered by plasmid or mRNA. Frequencies of perfect edits (c), invalid edits (d) and indels (e) of FahNS/NS allele induced by CBE variants in HEK293T reporter cells. Dots represent individual values, and bars represent mean ± s.d. of three independent biological replicates.

Supplementary information

Supplementary Information

Supplementary Figs. 1–8, Sequences 1 and 2, Tables 1–7 and Note.

Reporting Summary

Source data

Source Data Fig. 6

Unprocessed scan of blot for Fig. 6e.

Source Data Extended Data Fig. 5

Unprocessed scan of blot for Extended Data Fig. 5.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yang, L., Huo, Y., Wang, M. et al. Engineering APOBEC3A deaminase for highly accurate and efficient base editing. Nat Chem Biol 20, 1176–1187 (2024). https://doi.org/10.1038/s41589-024-01595-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-024-01595-4

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing