Multiplex CRISPR/Cas9-mediated genome editing of the FAD2 gene in rice: a model genome editing system for oil palm
Autor: | Mat Yunus Abdul Masani, Omar Abd Rasid, Ghulam Kadir Ahmad Parveez, Bohari Bahariah |
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Jazyk: | angličtina |
Rok vydání: | 2021 |
Předmět: |
0106 biological sciences
0301 basic medicine FAD2 Locus (genetics) Computational biology Biology QH426-470 01 natural sciences DNA sequencing 03 medical and health sciences Genome editing Multiplex CRISPR/Cas9 Genetics CRISPR Multiplex Gene High oleic acid Cas9 Research fungi food and beverages Model monocot Transformation (genetics) 030104 developmental biology Rice TP248.13-248.65 010606 plant biology & botany Biotechnology |
Zdroj: | Journal of Genetic Engineering and Biotechnology, Vol 19, Iss 1, Pp 1-13 (2021) Journal of Genetic Engineering & Biotechnology |
ISSN: | 2090-5920 |
Popis: | 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. |
Databáze: | OpenAIRE |
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