High-precision cytosine base editors by evolving nucleic-acid-recognition hotspots in deaminase.
basic_science · Level V
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- Record sourced from PubMed, PMID 40624252.
- Also identified by DOI 10.1038/s41587-025-02678-w and PMC identifier 12240478.
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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-KRAS<sup>G12D</sup> (ACC) and TP53<sup>R248Q</sup> (CCG)-in vitro. Our approach offers a general strategy to access precise base editors for potential clinical applications.