biostudies-literatureWang ZHubei Key Research ProjectHubei Province to support the high-quality development of the seed industry1782https://www.ebi.ac.uk/biostudies/studies/S-EPMC11544962biostudies-literatureUnknown13(21)CRISPR/Cas9 is a powerful genome editing tool for trait improvement in various crops; however, enhancing mutation efficiency using CRISPR/Cas9 in watermelon and melon remains challenging. We designed four CRISPR systems with different sgRNA expression cassettes to target the phytoene desaturase (<i>PDS</i>) gene in melon. The constructed vectors were delivered to host plants using <i>Agrobacterium</i>-mediated transformation. Phenotypic and genotypic analyses of the edited melon seedlings revealed that the CRISPR systems with tRNA and Csy4 spacers driven by the Pol II-type promoter significantly improved mutation efficiency, reaching 25.20% and 42.82%, respectively. Notably, 78.95% of the mutations generated by the Csy4 system involved large-fragment deletions (LDs) between the two target sites. In watermelon, the Csy4 system achieved a <i>PDS</i> editing efficiency of 41.48%, with 71.43% of the edited seedlings showing LD between the two target sites. Sequencing analysis indicated that the edited melon seedlings exhibited heterozygous, three-allele mutation and chimeric events; the edited watermelon seedlings included 2/14 homozygous mutations. Compared to the commonly used Pol III promoter, using the Pol II promoter to drive sgRNA expression cassettes containing Csy4 showed the best improvement in CRISPR/Cas9 editing efficiency in melon; this system was also effective in watermelon.CellsImproving the Genome Editing Efficiency of CRISPR/Cas9 in Melon and Watermelon.PMC115449622022BBA00622021BBA101HBZY2023B004-5Tang MRen JWan LZhang NZeng HWei JWang ZfalseImproving the Genome Editing Efficiency of CRISPR/Cas9 in Melon and Watermelon.CRISPR/Cas9 is a powerful genome editing tool for trait improvement in various crops; however, enhancing mutation efficiency using CRISPR/Cas9 in watermelon and melon remains challenging. We designed four CRISPR systems with different sgRNA expression cassettes to target the phytoene desaturase (<i>PDS</i>) gene in melon. The constructed vectors were delivered to host plants using <i>Agrobacterium</i>-mediated transformation. Phenotypic and genotypic analyses of the edited melon seedlings revealed that the CRISPR systems with tRNA and Csy4 spacers driven by the Pol II-type promoter significantly improved mutation efficiency, reaching 25.20% and 42.82%, respectively. Notably, 78.95% of the mutations generated by the Csy4 system involved large-fragment deletions (LDs) between the two target sites. In watermelon, the Csy4 system achieved a <i>PDS</i> editing efficiency of 41.48%, with 71.43% of the edited seedlings showing LD between the two target sites. Sequencing analysis indicated that the edited melon seedlings exhibited heterozygous, three-allele mutation and chimeric events; the edited watermelon seedlings included 2/14 homozygous mutations. Compared to the commonly used Pol III promoter, using the Pol II promoter to drive sgRNA expression cassettes containing Csy4 showed the best improvement in CRISPR/Cas9 editing efficiency in melon; this system was also effective in watermelon.2024-01-01T00:00:00Z2024 Oct2025-04-22T19:42:01.247Z2025-04-06T02:51:59.249ZS-EPMC115449623951388910.3390/cells13211782