Fig. 1.

Outline of the sequential transformation strategy for gene targeting and screening procedure. HDR donor constructs containing a selection marker and sgRNAs targeting genes of interest were transformed into parental lines, which were already transgenic for an incidental GL2-targeting sgRNA and, importantly, for Cas9 driven by a specific promoter. T1 transgenic lines were selected with Basta, and T2 seedlings were obtained and analyzed in bulk. The positive lines were used for further experiments with individual T2 plants. Portions of the images were obtained from the Microsoft PowerPoint clip art data base