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

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.