Abstract
Base editors (BEs), covalent fusions of a cytosine or adenine deaminase with a nuclease-impaired CRISPR protein, mediate site-specific conversion of C:G to T:A (CBEs) or A:T to G:C (ABEs) in the genome. Existing BEs modify all cytosines or adenines within the editing window, which limits their precision. Here we engineer nucleotide and context specificity of the Escherichia coli transfer RNA-specific adenosine deaminase (TadA) to pinpoint cytosine editing. Strategically sampling multiple nucleic-acid-recognition hotspots through directed evolution, we develop 16 TadA-derived NCN-specific deaminases that cover every possible −1 and +1 context for a target cytosine, providing on-demand deaminase choices for editor customization. We apply these variants to (1) correct disease-associated T:A-to-C:G transitions documented by ClinVar, achieving greater accuracy than conventional CBEs in 81.5% of cases, and (2) model two cancer-driver mutations—KRASG12D (ACC) and TP53R248Q (CCG)—in vitro. Our approach offers a general strategy to access precise base editors for potential clinical applications.
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Data availability
All NGS data were deposited to the National Center for Biotechnology Information Sequence Read Archive under BioProject PRJNA976736.
Code availability
Codes for analyzing the paired sgRNA–target library are available from GitHub (https://github.com/yuanwuCB/PairedLib_analysis).
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Acknowledgements
We thank all W.T. lab members for discussion and K. M. Watters for scientific editing of the paper. This work was completed in part with computing resources provided by the University of Chicago Research Computing Center. We thank the Single-Cell Immunophenotyping Core Facility at the University of Chicago for sequencing support. This work was supported in part by the National Institutes of Health (R21GM141670 to W.T.). W.T. is supported by the Searle Scholars Program (SSP-2021-113), the Cancer Research Foundation Young Investigator Program, the American Cancer Society (RSG-22-043-01-ET) and the David and Lucile Packard Foundation (2022-74685).
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Y.W. and W.T. conceptualized and designed the study. Y.W. carried out the first and second rounds of directed evolution. Y.L.X. carried out the third and fourth rounds of directed evolution. Y.W. and Y.L.X. performed 15 parallel evolutions of TadAC(NCN) and purified TadAC(NCN). Y.W. characterized TadAC1.1 and TadAC(NCN) in vitro. Y.W. and Y.L.X. prepared HEK293T cells carrying the paired sgRNA–target library and characterized Tad-CBEs using these cells. Y.W. prepared mRNA, evaluated Tad-CBEs at endogenous loci and conducted genome-wide off-target analysis. Y.L.X. conducted transcriptome-wide off-target analysis. Y.W. performed all NGS and analyzed the data. W.T. supervised the study. Y.W. and W.T. wrote the paper with input from Y.L.X.
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The University of Chicago has filed a provisional patent on TadAC proteins and their applications in gene editing. All authors are listed as inventors.
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Supplementary Notes 1 and 2, Figs. 1–38 and Tables 1 and 3–13.
Supplementary Table 2
Genotypes of wildtype TadA, TadA8.20, TadA8e, TadA8r, and TadA-derived cytosine deaminases.
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Wu, Y., Xiao, YL. & Tang, W. High-precision cytosine base editors by evolving nucleic-acid-recognition hotspots in deaminase. Nat Biotechnol (2025). https://doi.org/10.1038/s41587-025-02678-w
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DOI: https://doi.org/10.1038/s41587-025-02678-w
