Coordinating structure-guided design and cis-regulatory optimization to engineer elite herbicide-tolerance alleles in crops.

Genome editing facilitates the rapid creation of elite alleles, yet identifying editable sites for durable trait improvement without functional trade-offs remains challenging. Most current strategies modify a single genetic axis, limiting their effectiveness and stability. Here we developed a dual-layer allele engineering framework integrating structure-guided protein design with precise cis-regulatory tuning to overcome such constraints. Using rice 4-hydroxyphenylpyruvate dioxygenase (OsHPPD) as a model, structural analysis enabled the rational design of coding variants that retain enzymatic activity while conferring herbicide resistance. To further enhance resistance, we introduced a highly efficient 22-bp expression-modulating element into the engineered alleles, generating combinatorial coding and regulatory variants with significantly improved resistance, up to four times the recommended field application rate. Importantly, the dual-layer strategy was successfully applied in polyploid wheat, demonstrating its robustness and cross-species applicability. Our work establishes a versatile framework for functional allele engineering, offering a precise and broadly applicable route for crop improvement.