Fig. 3

Reporter gene activation by chemically inducible pol-II expressed guide RNAs in combination with dCasEV2.1. a Schematic representation of cloning strategy and post-transcriptional processing of guide RNA (gRNA). The tRNA and scaffold-tRNA sequences are excised from a Level -1 plasmid and assembled together with the protospacer in a Level 0 plasmid. The tRNA-protospacer-scaffold-tRNA unit is transferred to a Level 1 vector to generate the gRNA expression cassette. b Reporter gene activation with constitutive dCasEV2.1 and differently expressed gRNAs. The pDFR:FLuc reporter was activated by a constitutive dCasEV2.1 under two 35S promoter (Cs/Cs dCasEV2.1) combined with differently expressed gRNAs targeting the DFR promoter of the FLuc reporter. U6-26 is a constitutive promoter for polymerase III, and pNOS and p35S are constitutive promoters for polymerase II. CBS is the operator for copper induction and AlcA is the operator for ethanol induction. Both CBS and AlcA are assembled next to a minimal DFR promoter. c Triple induction of dCasEV2.1 components to activate gene expression. The pDFR:FLuc reporter was activated by a dually induced dCasEV2.1 using a minDFR promoter (Cu/Cu dCasEV2.1) in combination with differently expressed gRNAs targeting the DFR promoter of the FLuc reporter. Activation is expressed as Normalized FLuc/RLuc ratios obtained for the N. benthamiana leaves transiently expressing the genetic constructs. All FLuc/RLuc ratios were normalized using the FLuc/RLuc ratios of N. benthamiana leaves expressing a constitutive pNOS:Fluc reporter. Error bars indicate SD (n = 3). Statistical analyses were performed using one-way ANOVA (Tukey’s multiple comparisons test, P-Value ≤ 0.05). Variables within the same statistical groups are marked with the same letters. The figure includes images from Biorender (biorender.com)