Development of CRISPR/Cas9 Construct in Rice (Oryza sativa subsp. indica) Using Golden Gate Cloning Method Towards Drought Tolerance

Rice (Oryza sativa) is a staple food consumed by the majority of world’s population. Climate change, however, has created significant threat to our security as it posed severe effects on rice production. The emergence genome editing technology opened new era in crop improvement. Hence, this study aims develop CRISPR/Cas9 construct drought tolerance for O. sativa subsp. indica cv. IR64 using Golden Gate cloning method. For purpose, generation constructs involved several stages, i.e., characterization SUMO E2-Conjugating Enzyme (OsSCE1) gene, single-guide RNA (sgRNA) design and vector construction. FGENESH, GeneMarkS, InterProScan, Blast2GO programmes – were used OsSCE1 gene characterisation. putative isolated from was then verified sequencing, 585 bp long showed 99% identity with chromosome 10. In silico analysis concluded abiotic stress regulation. 20 sgRNA designed manually aid gRNA prediction including CCTop, Benchling. virtual validated Cloning approach later confirmed through sequencing. assembly separate vectors containing sequence, plant selectable marker, Cas9 cassette standardised elements hierarchical modular (MoClo). This demonstrated that format, insertion are achievable, resulting speedier more efficient process which may contribute improve rice. Further Agrobacterium-mediated transformation developed be conducted determine efficacy knocking out candidate genes improving ability