Figure 5

Validation of the CRISPR/Cas toolkit in Arabidopsis . (A) Physical maps of the T-DNAs of two pGreen-derived CRISPR/Cas9 binary vectors, each carrying two-gRNAs targeting three Arabidopsis genes (TRY, CPC and ETC2). The alignment of gRNA with its target gene is shown. Only aligned regions of interest are displayed. -rc, reverse complement. (B) Representative phenotypes of p2gR-TRI-A T1 transgenic lines. S, strong phenotypes similar to that of try cpc etc2 triple mutant, with highly clustered trichomes on leaf blades and petioles; M, moderate phenotypes with parts of leaf blades or a partial leaf blade displaying the phenotypes of the try cpc double mutant or the triple mutant; W, plants with weak or no mutant phenotypes. The total number of T1 transgenic plants, the number of T1 transgenic plants displaying strong, moderate, and weak phenotypes, and the percentage (in parentheses) of the total number are shown. The T0 seeds were screened on hygromycin MS plates for 13 days and grown in soil for 10 days before photographing. (C) Magnified image of a detached leaf displaying highly clustered trichomes on petioles, which is similar to the phenotype of the try cpc etc2 triple mutant. (D) Sequencing analysis of target gene mutations of a representative p2gR-TRI-A line. Dots, deleted bases. Highlighting denotes the degree of homology of the aligned fragments. The type of indel and the number of indels of the same type are indicated.