CRISPR/Cas9 mediated editing of phytoene desaturase gene in squash

Abstract

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.