CRISPR/Cas9 system is one of the most advance technologies for efficient genome editing in plants. The guide RNAs (gRNAs) 20 bp long were designed using the online platform CRISPR-P v2.0 for the rice transcription factor OsMADS26. Best three gRNAs were selected after evaluating their features such as on-score value, GC content, potential off-target sites, and location in genome. The binary vector, pRGEB32, with rice codon optimized Cas9 under the control of rice ubiqutin promoter has been utilized to clone the gRNAs. The gRNAs were annealed and ligated to the pRGEB32 vector, cloned into E. coli strain DH5α, and then mobilized into Agrobacterium tumefaciens strain EHA105 for their further mobilization into rice.
Key words: CRISPR/Cas9, drought tolerance, gRNA, OsMADS26, pRGEB32
|
