Current base editors (BEs) catalyze only base transitions (C to T and A to G) and cannot produce base transversions. Here we present BEs that cause C-to-A transversions in Escherichia coli and C-to-G transversions in mammalian cells. These glycosylase base editors (GBEs) consist of a Cas9 nickase, a cytidine deaminase and a uracil-DNA glycosylase (Ung). Ung excises the U base created by the deaminase, forming an apurinic/apyrimidinic (AP) site that initiates the DNA repair process. In E. coli, we used activation-induced cytidine deaminase (AID) to construct AID-nCas9-Ung and found that it converts C to A with an average editing specificity of 93.8% ± 4.8% and editing efficiency of 87.2% ± 6.9%. For use in mammalian cells, we replaced AID with rat APOBEC1 (APOBEC-nCas9-Ung). We tested APOBEC-nCas9-Ung at 30 endogenous sites, and we observed C-to-G conversions with a high editing specificity at the sixth position of the protospacer between 29.7% and 92.2% and an editing efficiency between 5.3% and 53.0%. APOBEC-nCas9-Ung supplements the current adenine and cytidine BEs (ABE and CBE, respectively) and could be used to target G/C disease-causing mutations.
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This research was financially supported by the National Key Research and Development Program of China (2019YFA0904900), the Key Research Program of the Chinese Academy of Science (KFZD-SW-215), the National Natural Science Foundation of China (31861143019), a Newton Fund PhD placement program grant (ID 352639434) under the UK–China Joint Research and Innovation Partnership Fund and the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (TSBICIP-PTJS-003). We gratefully thank F. Gu (Wenzhou Medical University) for providing the plasmid pAPOBEC-nCas9-UGI and Y. Li (Tianjin Institute of Industrial Biotechnology) for assisting with the data analysis.
A provisional patent has been submitted in part entailing the reported approach.
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a, Results obtained following treatment with nCas9-AID in E. coli MG1655 Δung. b, Results obtained following treatment with dCas9-AID in wild type E. coli MG1655. For all plots, dots represent individual biological replicates, bars represent mean values, and error bars represent the s.d. of three independent biological replicates. Source data
Supplementary Tables 1, 2, 4 and 7; Supplementary Figs. 1 and 2; and DNA sequences for vector components.
Mutations of APOBEC-nCas9-UGI and APOBEC-nCas9-Ung at RP11-177B4-3, PSMB2-1 and EMX1-site5 using mismatched sgRNAs.
The main primers used for this work.
The main plasmids used for this work.
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Zhao, D., Li, J., Li, S. et al. Glycosylase base editors enable C-to-A and C-to-G base changes. Nat Biotechnol 39, 35–40 (2021). https://doi.org/10.1038/s41587-020-0592-2
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