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. 2017 Jun 20;16(1):137–150. doi: 10.1111/pbi.12755

Figure 4.

Figure 4

Mutation at GhCLA1 target sites with T7E1 digestion assay and Sanger sequencing. (a) GhCLA1 and Cas9 positivity check of the wild type (WT), control (CK) and mutants. Mutants m3 and m9 are in chimeric and green phenotype. Mutants m6, m4, m3 and m9 were targeted at sgRNA7 and sgRNA8 sites. Mutants m24 and m26 were targeted at sgRNA9 and sgRNA10 sites. (b) and (c) T7E1 digestion assay of the mutants at the target sites. M, marker. A large fragment deletion was occurred in a representative albino mutant m4. Its Sanger sequencing results (d) and the detailed chromatograms (e) at the target sites are illustrated. In (d), the SNP is highlighted in black. In (e), the top chart illustrates the large fragment deletion of one GhCLA1 copy. The charts at middle and bottom represent mutations at separate target sites of the same gene locus. The PAM regions and mutated target sites are underlined with black and red lines, respectively.