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Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing

Abstract

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.

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Fig. 1: Engineering of TadA-8e-derived CGBEs.
Fig. 2: Evolution and characterization of Td-CBEs in mammalian cells.
Fig. 3: Off-target assessment of Td-CGBE and Td-CBEs.
Fig. 4: Examination of mouse embryos with Td-CGBE.
Fig. 5: Precise editing of pathogenic SNVs by TadA-derived base editors and target library analysis for eTd-CBEs.

Data availability

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.

Code availability

The relevant codes of analysis for Detect-seq data were deposited in GitHub (https://github.com/menghaowei/Detect-seq)55.

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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 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.).

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L.C. and D.L. designed the experiments. L.C., B.Z., G.R., H.M., Y.Y., M.H., C.L., S.Z., H.G., S.B., C.L., R.D. and N.X. performed the experiments. L.C., B.Z., G.R., H.M., Y.Y., M.H., D.Z., C.L., H.W., C.L., Z.L., Y.C., Y.G., S.S., C.Y., G.S., L.W., C.Y., M.L. and D.L. analyzed the data. D.L., L.C., B.Z., G.R., H.M., C.Y. and G.S. wrote the manuscript with inputs from all the authors. D.L. supervised the research.

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Correspondence to Chengqi Yi, Mingyao Liu or Dali Li.

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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.

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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

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