Engineering Adenine Deaminase TadA for Precise and PAM‐Flexible Point Mutagenesis and Gradient‐Tuning Endogenous Protein Design

Abstract Base editing enables precise nucleotide substitutions within a relatively broad editing window (5–6 nucleotides). However, considerable bystander editing significantly compromise its accuracy. Point mutagenesis, a powerful approach for gradient‐tuning protein function, facilitates the generation of diverse plant phenotypes to meet the demands of complex environments and consumer preferences. Here, a series of plant base editors is engineered by fusing three optimized TadA8e variants, TadA9, TadA‐LM, and TadA‐dual, with a PAM‐flexible SpRY nickase (SpRYn, with 5′‐NNN PAM recognition). These editors enable A‐to‐G, C‐to‐T, and dual‐base (simultaneous A‐to‐G and C‐to‐T) conversions within a highly condensed active window (1–3 nucleotides). Performance evaluations reveal that the TadDBE (TadA Dual‐Base Editor) achieves the most robust outcomes, delivering dual‐base editing efficiencies ranging from 2.3% to 61.4%, while maintaining minimal off‐target activity. Utilizing TadDBE, targeted point mutagenesis is performed on OsBadh2 , a gene encoding betaine aldehyde dehydrogenase that plays a critical role in the biosynthesis of 2‐acetyl‐1‐pyrroline (2‐AP), a key aromatic compound. This approach yields rice lines exhibiting gradient‐tuned aromatic profiles and optimized levels of 2‐AP and γ‐aminobutyric acid (GABA). These evolved TadA‐derived editors provide a precise, PAM‐flexible platform for base editing and represent a versatile strategy for generating genome‐edited plants with gradient‐tuned agronomic traits.