Designing of CRISPR/Cas9 Construct to Mediate Editing of Transcription Factor TFIIAƔ5 Gene Towards Increasing Resistance Against Bacterial Leaf Blight Disease in Rice

CRISPR/Cas9 system is an advanced genome editing technology to efficiently edit precisely the plant genomes. The 20bp long guide RNA (gRNAs) sequences are challenged to be designed in order to obtain optimal gRNAs that can efficiently edit on-target editing with minimal editing at unintended locations. The best gRNAs generated by online gRNA designing tool CRISPR direct were chosen by assessing their on-score value, GC content, off-target sites, and location in the genome. A binary vector, pRGEB32 has been utilized to clone the gRNA. The vector is already inserted with codon-optimized Cas9 and the gRNA was ligated into the vector at the BsaI site adjacent to the gRNA scaffold that was already cloned in the vector. Both components are under the control of the rice ubiquitin promoter. The cloned vector was mobilized into E.coli strain TOP10 and then Agrobacterium tumefacient strain EHA105 prior to further mobilization into rice callus for gene editing study.