Multiplex CRISPR/Cas9-mediated genome editing of the FAD2 gene in rice: a model genome editing system for oil palm

• Published in: Journal of Genetic Engineering and Biotechnology Vol. 19; no. 1; pp. 86 - 13
• Main Authors: Bahariah, Bohari, Masani, Mat Yunus Abdul, Rasid, Omar Abd, Parveez, Ghulam Kadir Ahmad
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 11.06.2021; Springer; Springer Nature B.V; Elsevier
• Subjects: biolistics; Biomedical Engineering and Bioengineering; biotechnology; callus; CRISPR; CRISPR-Cas systems; Crop improvement; Deoxyribonucleic acid; DNA; DNA sequencing; Editing; Efficiency; Elaeis guineensis; Engineering; FAD2; FAD2 gene; Fatty acids; Gene sequencing; Genes; genetic engineering; Genetic modification; Genetic transformation; Genetically altered foods; Genetically modified organisms; Genome editing; Genomes; Genomics; High oleic acid; Laboratories; leaves; loci; Model monocot; Multiplex CRISPR/Cas9; Multiplexing; Mutagenesis; Mutation; nucleotide sequences; Nucleotide sequencing; Oryza sativa; Plant genetics; protoplasts; Rice; RNA polymerase; Seeds; Transfection; China; United States > US; Model monocot; FAD2; Genome editing; High oleic acid; Multiplex CRISPR/Cas9; Rice
• ISSN: 1687-157X; 2090-5920
• DOI: 10.1186/s43141-021-00185-4

Background Genome editing employing the CRISPR/Cas9 system has been widely used and has become a promising tool for plant gene functional studies and crop improvement. However, most of the applied CRISPR/Cas9 systems targeting one locus using a sgRNA resulted in low genome editing efficiency. 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. Conclusion The results suggested that the application of the multiplex sgRNA-CRISPR/Cas9 genome editing system may be useful for crop improvement in monocot species that are recalcitrant to genetic modification, such as oil palm.