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

Abstract

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. While editing the HPPD protein or its expression alone falls short, doing both at once gives strong herbicide tolerance in rice and wheat, pointing to a general way to engineer dosage-sensitive crop traits.