The specificity of the N-dCas9/dCas9-N tools in Arabidopsis and cucumber. A) to C) RT-qPCR validation of the relative expression levels of AtFLS2 when N-dCas9 is targeted to T1 (A) and T3 (B) sites in Arabidopsis protoplasts and the expression level of Csa2G238880 when dCas9-N is targeted to T1 site (C) in cucumber hairy roots. Data are shown as means ± Sd (n = 2). Differences between 2 groups were tested using the Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). D) to F) Expression level of AtFLS2 when N-dCas9 is targeted to T1 (D) and T3 (E) sites in Arabidopsis protoplasts and expression level of Csa2G238880 when dCas9-N is targeted to T1 site (F) in cucumber calculated using RNA-seq CPM values. Data are shown as means ± Sd (n = 2). Differences between 2 groups were tested using the Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). G) and H) Pearson's correlation analysis with RNA-seq data reveals the specificity when N-dCas9 is targeted to the T1 (D) and T3 (E) sites of AtFLS2 in Arabidopsis protoplasts. I) Pearson's correlation analysis with RNA-seq data reveals the specificity when dCas9-N is targeted to the T1 site of Csa2G238880 in cucumber hairy roots. On-target and potential off-target genes are marked in each plot respectively, in G to I.