CRISPR/Cas9 mediated editing of phytoene desaturase gene in squash

Gene editing using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9 (CRISPR/Cas9) system has become an important biotechnological tool for studying gene function and improving crops. In the present study, the potential of the system was assessed for squash ( Cucurbita pepo subspecies pepo ) by targeting phytoene desaturase ( PDS ) gene using the particle bombardment method. The recombinant pHSE401 vector, carrying two sgRNAs ( gRNA1 and gRNA2 ) specific to the PDS homolog ( Cp4.1LG08g06310, CpPDS ) under the control of Arabidopsis U6 promoter and the Cas9 protein was developed and bombarded into cotyledonary node explants of squash cv. Black Beauty. The transformation efficiency of 4.5% was observed and all the transformants exhibited albino/bleached phenotype. The CpPDS knockout system generated easily detectable bleached/albino explants within 6–8 weeks. The albino phenotype was confirmed through Sanger sequencing which detected several deletion mutations (single, two and three bp deletion) within the CpPDS-gRNA1 target. However, no mutations were found within the CpPDS-gRNA2 target. This study demonstrated CRISPR/Cas9 as a viable tool for gene editing in squash and provides a platform for the modification of economically important traits in the crop.