Fig. 3

Characterization of activity of synthetic pATF promoters. A Circuit design of dCas9 based artificial transcription factor-controlled activation of synthetic promoters (pATFs). Specific gRNAs are produced by U6 promoter while the expression of the dCas9-VP64 is under the control of the 35S promoter. Reporter genes are under the control of the synthetic promoter (3 repeats of the gRNA followed by minimal 35S promoter to the artificial promoter (gRNA binding site) upstream of a specific fluorescence reporter. The distance between gRNA binding sites and between the minimal 35S promoter are indicated B Fluorescence microscope image showing Agrobacterium mediated transient expression of YFP, BFP and RFP into Nicotiana benthamiana leaves with dCas9-VP64 (bottom panels) and without dCas9-VP64 (upper panels) using three different gRNAs. Images were captured using the appropriate filter (Materials and Methods) at same exposure. C. Relative integrated density of each fluorescence signal (shown in panel B). Integrated density was measured using image J software and normalized to that of the control (con;—dCAS9-VP64). Error bars: S.D. (n = 3, independent replicates). Asterisks indicate statistical significance in a student t-test (P < 0.05)