Fig. 5

DSB activity and expression of sgRNAs. A Mutation frequency in GT-negative EMB-GFP KI transgenic plants. Mutation frequencies at EMB2410 target sites were analyzed in different GT-negative EMB-GFP T1 transgenic plants. B Mutation frequency in GT-negative ROS1-GFP KI transgenic plants. Mutation frequencies at ROS1 target sites were analyzed in the GT-negative ROS1-GFP T1 transgenic plants. C, D Mutation patterns in GT-negative EMB-GFP (C) and ROS1-GFP (D) KI transgenic plants. The relationship between average mutation pattern (bp) (Additional file 3: Table S3) and GT efficiency is shown. E Expression analysis of ROS1 sgRNA. Expression levels of sgRNAs targeting ROS1 were determined by qRT-PCR in ROS1-GFP T1 transgenic plants. ROS1-sgRNA2 expression was determined in single-sgRNA ROS1-GFP-sg2, triple-sgRNA ROS1-GFP-sg222, ROS1-GFP-sg234. ROS1-sgRNA3 and ROS1-sgRNA4 were analyzed in different triple sgRNAs ROS1-GFP-sg234 T1 transgenic plants. Actin7 gene (At5g09810) was used as an internal reference gene. The numbers in parenthesis represent the number of individual samples analyzed. ns; not significant. One way ANOVA and Tukey test were used for difference analysis (P < 0.05)