Alfalfa (Medicago sativa) is the most widely cultivated forage crop worldwide, owing to its high protein content, balanced amino acid profile, and abundance of vitamins and minerals. However, weed infestation severely threatens alfalfa yield and quality. The lack of herbicide‑resistant cultivars, combined with the diversity of weed species, has made herbicide tolerance a key breeding objective. To address this challenge, we developed a highly optimized prime editing system and, for the first time, achieved prime editing in alfalfa by enhancing the expression efficiency of its components. Specifically, we introduced mutations in three target genes MsALS1, MsALS2 and MsACC1 to confer herbicide resistance: mutations in MsALS1 and MsALS2 were designed to confer resistance to ALS‑inhibiting herbicides (based on evidence from their rice homologs), while those in MsACC1 were targeted to confer resistance to aryloxyphenoxypropionate (APP) herbicides. Using the Csy4 nuclease system, we performed simultaneous prime editing of all three genes, ultimately generating alfalfa lines resistant to nicosulfuron and haloxyfop‑P‑methyl. This study represents the first successful multi‑gene prime editing in alfalfa, providing a novel tool and a valuable approach for alfalfa molecular breeding.
