Abstract: The prime editing (PE) system is a precise genome editing technology that works efficiently in monocots; however, its application is limited by low editing efficiency in dicots, particularly Cucurbitaceae and Solanaceae plants. Here, we first significantly improved the transformation efficiency by introducing spectinomycin in cucurbits, then used the tomato elongation factor 1‐alpha (SlEF1α) promoter to enhance PE protein expression, and incorporated the Csy4 ribonuclease to process pegRNAs, collectively addressing multiple constraints limiting PE efficiency in cucurbits. The optimized PE systems, particularly Csy4‐PE6d, achieved an average desired editing frequency of 80.83% at targeted loci in cucumber via stable genetic transformation, with frequencies reaching up to 100% at certain sites. Moreover, Csy4‐PE6d generated homozygous edits in 36.43% of transgenic lines and demonstrated robust editing activity in melon, pumpkin, and potato. Using the Csy4‐PE6d tool, we generated heritable edited cucumber lines with dual resistance to bacterial angular leaf spot and downy mildew by targeting the CsSGR gene. Collectively, this optimized system substantially enhances PE efficiency in Cucurbit crops, providing an effective solution to common challenges such as low editing efficiency and limited heritability in these species.

Key words: cucurbits, genetic transformation, prime editing, resistance