Prime Editing of

5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the primary target of the broad-spectrum herbicide glyphosate, and several well-characterized amino acid substitutions in EPSPS are known to confer glyphosate resistance. Here, we introduced the P106S substitution into wheat TaEPSPS via prime editing, resulting in resistance to the field-recommended dosage of glyphosate. Either homozygous or heterozygous P106S in a single TaEPSPS homeolog was sufficient to confer resistance, with tolerance levels of 461 g a.e. ha-1 at 4 °C and 1383-1845 g a.e. ha-1 at 28 °C. The P106S substitution reduced shikimate accumulation by 60-77% under glyphosate treatment, did not alter the native expression patterns of TaEPSPS, and incurred negligible fitness costs under nonglyphosate conditions. Although P106S-edited lines exhibited growth inhibition and an approximate 10-day growth delay following glyphosate application, no significant yield penalty was observed at maturity. These glyphosate-resistant P106S alleles hold promising potential for integration into breeding programs to enhance weed control strategies.