Development of CRISPR/Cas9 plasmid for multiple sites genome editing in oil palm (Elaeis guineensis Jacq.)

Genome editing technology via CRISPR/Cas9 system is a versatile technique with numerous potential applications, particularly in agriculture. In this study, we attempted to develop a CRISPR/Cas9 plasmid containing four sgRNA to allow multiple gene editing in the oil palm genome. In the first step, we used an in silico approach to finding the optimum 20-nt guides from four gene regions across oil palm genome. These guides were later joined with a promoter and tracr-RNA fragment to construct a 472 bp module, and together with three tetranucleotide linkers and restriction sites at both terminals gave an insert of length 1 918 bp. This insert was then incorporated into CRISPR/Cas9 vector, and the final plasmid was sequence validated.