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Fig. 5 | Plant Methods

Fig. 5

From: Orthogonal control of gene expression in plants using synthetic promoters and CRISPR-based transcription factors

Fig. 5

Design and characterization of gRNA expression modules under the control of Pol II promoters. A OCS1-1 circuit generates RNA using U6 (Pol III) promoter while OCS1-5 circuit generates gRNA using 35S (Pol II) promoter flanked by self-cleaving ribozymes – HammerHead (HHR) and Hepatitis Delta Virus (HDV). B Fluorescence microscope images showing Agrobacterium mediated transient expression of OCS constructs with two modalities of gRNA expression (OCS1-1 and OCS1-5). Control images were taken without dCAS9-VP64 expression. Scale bars: 200 μm C and D. Quantification of the gRNA-1 expression in OCS constructs (OCS 1–1 (C) and OCS 1–5 (D)) using qPCR relative to 5S rRNA. Error bars: S.D. (n = 3, independent replicates) E. 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). NS: not significant

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Plant Methods

ISSN: 1746-4811