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