Fig. 2

Design of a GUUS reporter system to evaluate the gene editing activity of CRISPR/Cas9 in Arabidopsis transgenic plants. a Schematics of the GUUS reporter construct and the CRISPR/Cas9 construct used for plant transformation. CRISPR/Cas9 induces cleavage of the GUUS reporter gene at the intergenic region (IR). The double-stranded break (DSB) could be subsequently repaired by the homology-dependent recombination pathway (HdR) or the error-prone non-homologous end joining pathway (NHEJ). In both cases, the sgRNA binding site in the IR would be mutated hampering binding of the FP1 primer. In this way, the PCR amplification efficiency of the “U” fragment would be reduced compared to the non-targeted “S” fragment. FP1/RP1, PCR primers designed to amplify the “U” fragment of the GUUS gene. FP0/RP0, PCR primers designed to amplify the “S” fragment of GUUS gene. b A single copy CRISPR/Cas9 transgenic line was identified with the T-DNA inserted into the sixth intron of the At1g75730 gene. Cas9, the Cas9 expression module as shown in a; sgR, sgRNA expression module as shown in a; HYG, hygromycin resistance module derived from pCAMBIA1300; LB, T-DNA left border; RB, T-DNA right border. c A single copy GUUS reporter transgenic line was identified with the T-DNA inserted into the second intron of the At2g16970 gene. GUUS, GUUS expression module as shown in a; NPT, Neomycin resistance module derived from pCAMBIA2300