Engineering Compact Base Editors by AlphaFold‐Guided Mutation Scan and Escherichia coli ‐Based Tri‐Selection

ABSTRACT APOBEC1‐based cytosine base editors such as BE4max enable base conversion, but many alternative deaminases show low activity and cytotoxicity, especially when miniaturized for delivery. SsdA tox , a DNA deaminase toxin from Pseudomonas syringae that is two‐thirds the size of APOBEC1, is attractive for compact base editors but, in native form, shows low C‐to‐T editing efficiency and high cytotoxicity. Guided by an AlphaFold‐ and CASTpFold‐based alanine scan, we identified K31 as a gatekeeping residue whose substitution enlarges the modeled DNA binding pocket. Site‐saturation mutagenesis at K31 produced variants with ten‐fold higher activity but increased indel formation. To further enhance activity while reducing indels and cytotoxicity, we developed Trinity‐Screen, an Escherichia coli ( E. coli) ‐based three‐in‐one directed evolution platform that selects for high activity and reduced double‐strand break‐associated indels. Trinity‐Screen revealed four additional DNA‐binding positions; combinatorial mutagenesis at these sites generated four‐ and five‐site SsdA tox variants that retained high activity yet showed lower indel rates and rescued bacterial viability. To standardize comparisons, we defined the Base Editor Performance Index (BEPI), which integrates C‐to‐T conversion and indel frequency. Optimized SsdA tox variants achieved up to 31‐fold improvement relative to wild type, outperforming BE4max at multiple endogenous targets and displaying ten‐fold lower cytotoxicity in E. coli .