A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana

Abstract

Background: CRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana. In the presence of copper, the copper-responsive factor CUP2 undergoes a conformational change and binds a DNA motif named copper-binding site (CBS).

Results: In this study, we tested several activation domains fused to CUP2 and found that the non-viral Gal4 domain results in strong activation of a reporter gene equipped with a minimal promoter, offering advantages over previous designs. To connect copper regulation with downstream programmable elements, several copper-dependent configurations of the strong dCasEV2.1 PTA were assayed, aiming at maximizing activation range, while minimizing undesired background expression. The best configuration involved a dual copper regulation of the two protein components of the PTA, namely dCas9:EDLL and MS2:VPR, and a constitutive RNA pol III-driven expression of the third component, a guide RNA with anchoring sites for the MS2 RNA-binding domain. With these optimizations, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold and 245-fold for the endogenous N. benthamiana DFR and PAL2 genes, respectively, with negligible expression in the absence of the trigger.

Conclusions: The tight regulation of copper over CI/dCasEV2.1 makes this system ideal for the conditional production of plant-derived metabolites and recombinant proteins in the field.

Keywords: CRISPR/Cas9-based programmable transcriptional activator; Copper switch; Inducible expression; Minimal promoter; Nicotiana benthamiana; Plant synthetic biology.

Fig. 1

Copper-inducible reporter gene activation in transient expression in Nicotiana benthamiana. a Schematic representation of the copper-inducible gene expression system. The CUP2 fused to an activation domain such as Gal4 changes its conformation upon copper binding and binds a Copper Binding Site (CBS) operator allowing transcription of the downstream gene (green arrow). b Copper-mediated activation of Firefly Luciferase (FLuc) reporter gene by different activation domains (Gal4, VP16, VPR, and TV) fused to CUP2 after 5 mM copper sulfate application. c Copper-mediated activation of FLuc by different concentrations of copper sulfate. The construct CUP2:Gal4 was used as the copper-dependent transcriptional factor. d Pictures of leaves 2 days after treatment with different concentrations of copper sulfate. For b and c, activation is expressed as Normalized FLuc/RLuc ratios of 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)

Fig. 2

Copper-inducible reporter gene activation by dCasEV2.1 in N. benthamiana. a Schematic representation of dCasEV2.1 components. b Genetic constructs for dual constitutive (Cs/Cs) transcription (35S promoter for both dCas9:EDLL and MS2:VPR), single copper-regulated (Cu/Cs) transcription (CBS:minimal DFR promoter for dCas9:EDLL and 35S promoter for MS2:VPR), and dual copper-regulated (Cu/Cu) transcription (CBS:minimal DFR promoter for both dCas9:EDLL and MS2:VPR). c Efficiency of single vs. dual copper-regulated transcription of dCasEV2.1 under a minimal DFR promoter (minDFR) to activate a pDFR:FLuc reporter. d Efficiency of dual copper-regulated transcription of dCasEV2.1 under a minimal 35S (min35S) promoter to activate a pDFR:FLuc reporter. Activation is expressed as Normalized FLuc/RLuc ratios of 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)

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)

Fig. 4

Application of copper-inducible dCasEV2.1 for activating endogenous genes in N. benthamiana. a Schematic representation of the strategy of copper-mediated dCasEV2.1 induction for targeted gene activation in planta. b Activation of the N. benthamiana DFR (NbDFR) and PAL2 (NbPAL2) genes by the dually copper-regulated dCasEV2.1 under minDFR promoter (Cu/Cu dCasEV2.1) and the constitutive dCasEV2.1 under 35S promoter (Cs/Cs dCasEV2.1). Transcripts were quantified by RT-qPCR at 5 days post-infiltration of dCasEV2.1 in combination with a constitutive guide RNA targeting either NbDFR (gNbDFR), NbPAL2 (gNbPAL2), or the S. lycopersicum MTB gene (unspecific gRNA) and a constitutive CUP2:Gal4. Leaves were treated either with 5 mM copper sulfate or water at 3 days post infiltration. Error bars indicate SD (n = 3). The asterisks represent a significant difference between treatments. Statistical analysis was performed using an unpaired two-tailed t-test for two samples comparisons (P-value ≤ 0.05). The figure includes images from Biorender (biorender..com)