RESULTS: Here, we demonstrate the modification of the FAD2 gene in rice using a multiplex sgRNA-CRISPR/Cas9 genome editing system. To test the system's efficiency for targeting multiple loci in rice, we designed two sgRNAs based on FAD2 gene sequence of the Oryza sativa Japonica rice. We then inserted the validated sgRNAs into a CRISPR/Cas9 basic vector to construct pYLCRISPRCas9PUbi-H:OsFAD2. The vector was then transformed into protoplast cells isolated from rice leaf tissue via PEG-mediated transfection, and rice calli using biolistic transformation. Direct DNA sequencing of PCR products revealed mutations consisting of deletions of the DNA region between the two target sgRNAs.

RESULTS: A total of four single-guide RNAs (sgRNAs) were designed in silico based on the genomic sequences of EgFAD2 and EgPAT. Using robust plant CRISPR/Cas9 vector technology, multiple sgRNA expression cassettes were efficiently constructed into a single-binary CRISPR/Cas9 vector to edit the EgFAD2 and EgPAT genes. Each of the constructed transformation vectors was then delivered into oil palm embryogenic calli using the biolistic, Agrobacterium-mediated, and PEG-mediated protoplast transformation methods. Sequence analysis of PCR products from 15 samples confirmed that mutations were introduced at four target sites of the oil palm EgFAD2 and EgPAT genes. Single- and double-knockout mutants of both genes were generated, with large and small deletions within the targeted regions. Mutations found at EgFAD2 and EgPAT target sites indicate that the Cas9/sgRNA genome-editing system effectively knocked out both genes in oil palm.