Fig. 2

Schematic representation of CRISPR/Cas9-mediated targeted mutagenesis in the rice Os8N3 gene. a Schematic diagram of Os8N3 gene and xa13m targeting sequence. Rice Os8N3 contains five exons, represented by black rectangles, and the untranslated region portion, represented by white rectangles. The enlarged area indicated by the black broken line shows the coding sequence and position of the first exon of Os8N3. The 20-bp sgRNA targeting sequence (xa13m) and protospacer adjacent motif (PAM) sequence are shown in red and in underlined lower-case letters, respectively. The vertical arrowhead indicates an expected cleavage site. The underlined bold ATG indicates a translation initiation codon. b T-DNA region of the recombinant OsU6a::xa13m-sgRNA/pHAtC vector carrying xa13m-sgRNA under the control of the OsU6a promoter. Expression of Cas9 is driven by the Cauliflower mosaic virus 35S (CaMV35S) promoter; expression of the xa13m-sgRNA is driven by the OsU6a promoter; expression of hygromycin (HPT) is driven by the nopalin synthase (NOS) promoter; NLS: nuclear localization signal of Simian virus 40 (SV40) large T antigen; nos-t: gene terminator; LB and RB: left and right border, respectively. Primers used in the PCR are indicated by black arrows