Cytosine base editors (CBEs) efficiently generate precise C·G-to-T·A base conversions, but the activation-induced cytidine deaminase/apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (AID/APOBEC) protein family deaminase component induces considerable off-target effects and indels. To explore unnatural cytosine deaminases, we repurpose the adenine deaminase TadA-8e for cytosine conversion. The introduction of an N46L variant in TadA-8e eliminates its adenine deaminase activity and results in a TadA-8e-derived C-to-G base editor (Td-CGBE) capable of highly efficient and precise C·G-to-G·C editing. Through fusion with uracil glycosylase inhibitors and further introduction of additional variants, a series of Td-CBEs was obtained either with a high activity similar to that of BE4max or with higher precision compared to other reported accurate CBEs. Td-CGBE/Td-CBEs show very low indel effects and a background level of Cas9-dependent or Cas9-independent DNA/RNA off-target editing. Moreover, Td-CGBE/Td-CBEs are more efficient in generating accurate edits in homopolymeric cytosine sites in cells or mouse embryos, suggesting their accuracy and safety for gene therapy and other applications.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Nature Communications Open Access 26 January 2023
Nature Communications Open Access 13 January 2023
Subscribe to Nature+
Get immediate online access to Nature and 55 other Nature journal
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
HTS data have been deposited in the NCBI Sequence Read Archive database under accession codes PRJNA822038, PRJNA871961, PRJNA855334, PRJNA835691, PRJNA835701 and PRJNA882574 (refs. 47,48,49,50,51,52). RNA-seq data have been deposited in the NCBI Sequence Read Archive database under accession codes PRJNA871962 and PRJNA830998 (refs. 53,54). There are no restrictions on data availability. Source data are provided with this paper.
Rees, H. A. & Liu, D. R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770–788 (2018).
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).
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).
Wang, L. et al. Enhanced base editing by co-expression of free uracil DNA glycosylase inhibitor. Cell Res. 27, 1289–1292 (2017).
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).
Zhao, D. et al. Glycosylase base editors enable C-to-A and C-to-G base changes. Nat. Biotechnol. 39, 35–40 (2021).
Koblan, L. W. et al. Efficient C·G-to-G·C base editors developed using CRISPRi screens, target-library analysis, and machine learning. Nat. Biotechnol. 39, 1414–1425 (2021).
Chen, L. et al. Programmable C:G to G:C genome editing with CRISPR-Cas9-directed base excision repair proteins. Nat. Commun. 12, 1384 (2021).
Zuo, E. et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364, 289–292 (2019).
Jin, S. et al. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science 364, 292–295 (2019).
Zhou, C. et al. Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis. Nature 571, 275–278 (2019).
Grunewald, J. et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 569, 433–437 (2019).
Gehrke, J. M. et al. An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities. Nat. Biotechnol. 36, 977–982 (2018).
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).
Kim, H. S., Jeong, Y. K., Hur, J. K., Kim, J. S. & Bae, S. Adenine base editors catalyze cytosine conversions in human cells. Nat. Biotechnol. 37, 1145–1148 (2019).
Grunewald, J. et al. CRISPR DNA base editors with reduced RNA off-target and self-editing activities. Nat. Biotechnol. 37, 1041–1048 (2019).
Chen, L. et al. Engineering precise adenine base editor with infinitesimal rates of bystander mutations and off-target editing. Nat. Chem. Biol. https://doi.org/10.1038/s41589-022-01163-8 (2022).
Lapinaite, A. et al. DNA capture by a CRISPR-Cas9-guided adenine base editor. Science 369, 566–571 (2020).
Jeong, Y. K. et al. Adenine base editor engineering reduces editing of bystander cytosines. Nat. Biotechnol. 39, 1426–1433 (2021).
Tan, J., Zhang, F., Karcher, D. & Bock, R. Engineering of high-precision base editors for site-specific single nucleotide replacement. Nat. Commun. 10, 439 (2019).
Thuronyi, B. W. et al. Continuous evolution of base editors with expanded target compatibility and improved activity. Nat. Biotechnol. 37, 1070–1079 (2019).
Nishimasu, H. et al. Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science 361, 1259–1262 (2018).
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).
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).
Bae, S., Park, J. & Kim, J. S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).
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).
Wang, L. et al. Eliminating base-editor-induced genome-wide and transcriptome-wide off-target mutations. Nat. Cell Biol. 23, 552–563 (2021).
Lei, Z. et al. Detect-seq reveals out-of-protospacer editing and target-strand editing by cytosine base editors. Nat. Methods 18, 643–651 (2021).
Lei, Z. et al. Mitochondrial base editor induces substantial nuclear off-target mutations. Nature 606, 804–811 (2022).
Yuan, T. et al. Optimization of C-to-G base editors with sequence context preference predictable by machine learning methods. Nat. Commun. 12, 4902 (2021).
Auer-Grumbach, M., Strasser-Fuchs, S., Robl, T., Windpassinger, C. & Wagner, K. Late onset Charcot-Marie-Tooth 2 syndrome caused by two novel mutations in the MPZ gene. Neurology 61, 1435–1437 (2003).
Rodriguez-Escudero, I. et al. A comprehensive functional analysis of PTEN mutations: implications in tumor- and autism-related syndromes. Hum. Mol. Genet. 20, 4132–4142 (2011).
Syrbe, S. et al. De novo loss- or gain-of-function mutations in KCNA2 cause epileptic encephalopathy. Nat. Genet. 47, 393–399 (2015).
Esteghamat, F. et al. CELA2A mutations predispose to early-onset atherosclerosis and metabolic syndrome and affect plasma insulin and platelet activation. Nat. Genet. 51, 1233–1243 (2019).
Ropero, P. et al. Hb Johnstown [β 109 (G11) Val–>Leu]: second case described and associated for the first time with β0-thalassemia in two Spanish families. Am. J. Hematol. 65, 298–301 (2000).
Fazeli, W. et al. A TUBB6 mutation is associated with autosomal dominant non-progressive congenital facial palsy, bilateral ptosis and velopharyngeal dysfunction. Hum. Mol. Genet. 26, 4055–4066 (2017).
Wu, C. H. et al. Mutations in the profilin 1 gene cause familial amyotrophic lateral sclerosis. Nature 488, 499–503 (2012).
Arbab, M. et al. Determinants of base editing outcomes from target library analysis and machine learning. Cell 182, 463–480 (2020).
Walton, R. T., Christie, K. A., Whittaker, M. N. & Kleinstiver, B. P. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science 368, 290–296 (2020).
Li, J. et al. Structure-guided engineering of adenine base editor with minimized RNA off-targeting activity. Nat. Commun. 12, 2287 (2021).
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).
Zhang, X. et al. Dual base editor catalyzes both cytosine and adenine base conversions in human cells. Nat. Biotechnol. 38, 856–860 (2020).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Kruger, S. et al. Sensorimotor polyneuropathy and systemic amyloidosis as paraneoplastic symptoms of a carcinoid-like well differentiated carcinoma of the breast. Dtsch. Med. Wochenschr. 123, 179–184 (1998).
Li, D. et al. Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat. Biotechnol. 31, 681–683 (2013).
Hwang, G. H. et al. Web-based design and analysis tools for CRISPR base editing. BMC Bioinformatics 19, 542 (2018).
Chen, L. et al. Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing. NCBI SRA, BioProject PRJNA822038 https://www.ncbi.nlm.nih.gov/bioproject/PRJNA822038 (2022).
Chen, L. et al. Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing. NCBI SRA, BioProject PRJNA871961 https://www.ncbi.nlm.nih.gov/bioproject/PRJNA871961 (2022).
Chen, L. et al. Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing. NCBI SRA, BioProject PRJNA855334 https://www.ncbi.nlm.nih.gov/bioproject/PRJNA855334 (2022).
Chen, L. et al. Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing. NCBI SRA, BioProject PRJNA835691 https://www.ncbi.nlm.nih.gov/bioproject/PRJNA835691 (2022).
Chen, L. et al. Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing. NCBI SRA, BioProject PRJNA835701 https://www.ncbi.nlm.nih.gov/bioproject/PRJNA835701 (2022).
Chen, L. et al. Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing. NCBI SRA, BioProject PRJNA882574 https://www.ncbi.nlm.nih.gov/bioproject/PRJNA882574 (2022).
Chen, L. et al. Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing. NCBI SRA, BioProject PRJNA871962 https://www.ncbi.nlm.nih.gov/bioproject/PRJNA871962 (2022).
Chen, L. et al. Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing. NCBI SRA, BioProject PRJNA830998 https://www.ncbi.nlm.nih.gov/bioproject/PRJNA830998 (2022).
Chen, L. et al. Td-BE-Detect-seq analysis. GitHub https://github.com/menghaowei/Detect-seq (2022).
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 the School of Life Sciences in ECNU and H. Jiang from the Core Facility and Technical Service Center for the SLSB of the School of Life Sciences and Biotechnology in SJTU. We thank L. Ji (MedSci) for designing schematic diagrams. This work is partially supported by grants from the National Key R&D Program of China (2019YFA0802800 to M.L., 2019YFA0110802 to D.L., 2019YFA0802200 to C.Y., and 2019YFA0110900 to C.Y.), the National Natural Science Foundation of China (32025023 to D.L., 32230064 to D.L., 31971366 to L.W., 82230002 to M.L., 21825701 to C.Y., 91953201 to C.Y. and 92153303 to C.Y.), the Shanghai Municipal Commission for Science and Technology (21CJ1402200 to D.L. and 20140900200 to D.L.), and the Innovation Program of the Shanghai Municipal Education Commission (2019-01-07-00-05-E00054 to D.L.), the Fundamental Research Funds for the Central Universities (NK2022010207 to D.L.), the State Key Laboratory of Drug Research (SIMM2205KF-01 to C.Y.) and support from the East China Normal University Outstanding Doctoral Students Academic Innovation Ability Improvement Project (YBNLTS2021-026 to L.C.).
The authors have submitted patent applications based on the results reported in this study (L.C., D.L., G.R., C.L., H.G., B.Z., J.Y., S.B., R.D. and M.L.). The remaining authors declare no competing interests.
Peer review information
Nature Biotechnology thanks Francisco Sanchez-Rivera and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Chen, L., Zhu, B., Ru, G. et al. Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing. Nat Biotechnol (2022). https://doi.org/10.1038/s41587-022-01532-7
This article is cited by
Nature Communications (2023)
Nature Communications (2023)