Design of single guide RNA for CRISPR/Cas9 mediated genome engineering in rice – for semi-dwarfism and thermo-sensitive genic male sterility

Abstract

CRISPR/Cas9 causes high precision genome editing in an efficient manner. By designing an 18 to 20 nucleotide ‘spacer’ which is immediately followed by the protospacer adjacent motif, the Cas9 can be directed to cause double stranded breaks in a genome. The CRISPR plasmid pRGEB32 has been optimized to edit plant genomes. We designed sgRNAs to target the TMS5 and SD1 gene of rice using CRISPR-PLANT, an online platform for designing guide RNAs. The efficiency of the sgRNAs were assessed for various parameters such as GC content, potential off-target activity and specificity. The sgRNAs were cloned into the pRGEB32 plasmid background, transformed to competent E. coli and finally mobilized to AGL-1 strain of Agrobacterium. This protocol could be utilized to develop transformation ready CRISPR constructs in a short-time.