A modular toolbox for the optogenetic deactivation of transcription

Abstract

Light-controlled transcriptional activation is a commonly used optogenetic strategy that allows researchers to regulate gene expression with high spatiotemporal precision. The vast majority of existing tools are, however, limited to light-triggered induction of gene expression. Here, we inverted this mode of action and created optogenetic systems capable of efficiently terminating transcriptional activation in response to blue light. First, we designed highly compact regulators by photo-controlling the VP16 (pcVP16) transactivation peptide. Then, applying a two-hybrid strategy, we engineered LOOMINA (light off-operated modular inductor of transcriptional activation), a versatile transcriptional control platform for mammalian cells that is compatible with various effector proteins. Leveraging the flexibility of CRISPR systems, we combined LOOMINA with dCas9 to control transcription with blue light from endogenous promoters with exceptionally high dynamic ranges in multiple cell lines. Functionally and mechanistically, the versatile LOOMINA platform and the exceptionally compact pcVP16 transactivator represent valuable additions to the optogenetic repertoire for transcriptional regulation.

Graphical Abstract

Figure 1.

Development of potent light-inhibited transcriptional regulators. (A) Amino acid sequences of the pcVP16 lead candidates. The sequences corresponding to the C-terminus of the AsLOV2 Jα helix and the VP16 transactivation peptide are depicted for comparison. (B) Schematic of transcriptional control with a photocontrolled Gal4-VP16. GOI: gene of interest. (C) Flowchart of the luciferase screening assay. (D) HEK293T cells were transfected with plasmids encoding (i) a Renilla luciferase, (ii) a Gal4-inducible firefly luciferase and (iii) the indicated Gal4-TAD fusions, followed by incubation of samples under blue light exposure or in the dark. The luciferase activity was measured 48 h after transfection. Gal4-VP16-dom.: Gal4 fused to the complete VP16 transactivator domain. (E) Mechanism of action of the LOOMINA system. GOI: gene of interest. (F) Schematic of the constructs tested. (G–I) HEK293T cells were transfected with plasmids encoding (i) a firefly luciferase downstream of 13x TetO sites, (ii) a Renilla luciferase, (iii) dCas9 and (iv) VP64 (G), p65 (H) or VPR (I) as indicated by the circled numbers. As positive control a plasmid expressing a genetic fusion between dCas9 and the transactivator was used instead of the split constructs. The samples were either illuminated or kept in darkness, followed by measurements of luciferase activity after 48 h. (D and G–I) Bars represent the mean of three biological replicates. Data points indicate the individual biological replicates, which are the mean of three technical replicates. Error bars indicate S.D. Fold changes between illuminated and dark samples are indicated. Vector mass ratios between the dCas9-expressing construct and the transactivator construct are indicated for each sample. Experiments for (G–I) were performed together on a single plate and the same reporter only control was used as reference. Rep. only: reporter-only control.

Figure 2.

LOOMINA facilitates spatially confined gene expression and mediates robust transcriptional control in various cell lines. (A) HEK293T cells were transfected with plasmids encoding (i) a tet-inducible mCherry reporter, (ii) a TetO-targeting sgRNA and (iii/iv) LOOMINA in the NLS-dCAs9-3xLOV/Zdk3-VPR configuration. Cells were illuminated through a photomask covering half of the plate for 48 h. Fluorescence microscopy images are shown along with a quantification of the fluorescent signal across the x-axis of the image. (B) Overview of the constructs and cell lines used. (C) U2OS, HEPG2 or HeLa cells were transfected with plasmids encoding (i) a firefly luciferase downstream of 13x TetO sites, (ii) a Renilla luciferase and (iii) the LOOMINA constructs as indicated by the circled numbers. As positive control a plasmid expressing a genetic fusion between dCas9 and the transactivator was used instead of the split constructs. Samples were either illuminated or kept in the dark, followed by measurements of luciferase activity after 48 h. Bars represent means of three biological replicates. Data points indicate individual biological replicates, which are the mean of three technical replicates. Error bars indicate S.D. Fold changes between illuminated and dark samples are indicated. Rep. only: reporter-only control.

Figure 3.

LOOMINA enables endogenous transcriptional control with high dynamic range. (A) Overview of the experimental workflow for activating transcription at endogenous loci. (B) Schematics of the vectors that were used for the control of genomic promoters. (C–G) HEK293T or HeLa cells were co-transfected with (i) a cocktail of plasmids targeting different sites either in the promoter regions of IL1RN (C–E), MYOD (F) or OCT4 (G) and (ii) plasmids encoding for the individual LOOMINA components. As a positive control, a plasmid expressing a fusion between dCas9 and either VP64 or VPR was used. The circled numbers refer to the combination of Cas9 and TAD used and correspond to the vectors outlined in (B). The vector mass ratios between the dCas9-expressing construct and the transactivator construct are indicated. Samples were either illuminated or kept in the dark, followed by RT-qPCR analysis of gene expression after 72 h. Bars represent the mean of three biological replicates. Data points indicate individual biological replicates, which are the mean of three technical replicates. Error bars indicate S.D. Fold changes between illuminated and dark samples are indicated. high c., 700 ng of the combined LOOMINA components were transfected instead of 400 ng.

Figure 4.

LOOMINA enables light-controlled expression of endogenous genes in a neuronal-like cell line. (A) Schematic of the LOOMINA configurations used in N2a cells. Overview of the experimental workflow for the activation of endogenous transcription in N2a cells. The number of multiplexed sgRNAs for each target gene is indicated. (B) Cells were co-transfected with plasmids encoding (i) the respective sgRNAs and (ii) the indicated LOOMINA constructs, followed by incubation under light exposure or in darkness for 48 h. Expression of dCas9 (pink), mCherry (red) and GFP (green) was assessed by combined immunocytochemistry. Arc was visualized using sequence-specific RNAscope probes. Representative confocal RNAscope images are shown. (C) Quantification of the mean fluorescence intensity of the Arc-probe from (B). Bars represent the mean of individual values from single cells. The number of cells analyzed per condition is shown in Supplementary Figure S17. (D) N2a cells were transfected with the lead LOOMINA constructs alongside sgRNA expressing plasmids targeting either the Kir2.1 (left) or Neurog2 (right). A direct dCas9-VPR fusion was used as a positive control. Samples were either illuminated or kept in darkness for 48 h, followed by RT-qPCR analysis of gene expression. Bars represent the mean of two biological replicates. Data points represent the individual biological replicates, which are the mean of three technical replicates. (B–D) Vector mass ratios between the dCas9-expressing construct and the transactivator construct are indicated. Circled numbers refer to the vectors outlined in panel (A). Error bars indicate S.D. Fold changes between illuminated and dark samples are indicated. Statistical significance was assessed by two-way ANOVA with Sidak correction for multiple comparisons; n.s.: not significant; *P-value < 0.05; **P-value < 0.01, ****P-value < 0.0001. nt sgRNA: non-targeting sgRNA control.

Figure 5.

LOOMINA—a modular toolbox for the tunable optogenetic control of transcriptional induction. The combination of the LOVTRAP dissociation system with diverse DBDs and TADs enables the customized optogenetic transcriptional control of reporter expression or endogenous genes in various human cell lines.