Aptamer‐Mediated Reversible Transactivation of Gene Expression by Light

Abstract The investigation and manipulation of cellular processes with subcellular resolution requires non‐invasive tools with spatiotemporal precision and reversibility. Building on the interaction of the photoreceptor PAL with an RNA aptamer, we describe a variation of the CRISPR/dCAS9 system for light‐controlled activation of gene expression. This platform significantly reduces the coding space required for genetic manipulation and provides a strong on‐switch with almost no residual activity in the dark. It adds to the current set of modular building blocks for synthetic biological circuit design and is broadly applicable.

dCas9 constructions: As PCR template for the final pdCas9_eGFP, the plasmid dCAS9-VP64_GFP was used. dCAS9-VP64_GFP was a gift from Feng Zhang (Addgene plasmid # 61422; http://n2t.net/addgene:61422; RRID: Addgene_61422). The backbone was amplified by PCR, excluding the VP64 sequence. For the plasmid amplification, Phusion Flash High-Fidelity PCR Master Mix was used. The primers for the insert were designed to have an overlap with the target backbone. The amplicon was phosphorylated and ligated to gain pdCas9_T2A_eGFP. sgRNA constructions: The sgRNAs containing PAL aptamers were designed using the sgRNA 2.0 scaffold as a template. The stem regions of the tetraloop and stem-loop 2 were preserved and artificial stem sequences were added. The sequences were tested for correct folding using mFold software. As host plasmid pENTR.hU6 was used. The plasmid backbone was amplified using Phusion Flash High-Fidelity PCR Master Mix. The sgRNA inserts were ordered from Ella Biotech (Martinsried, Germany) and cloned into the pENTR.hU6 backbone using AQUA cloning (Table S2, Supporting Information). New seed sequences were introduced by PCR, where the forward primer contains the new seed sequence as 5' extension.
Optogenetic gene activation assays: HeLa cells were seeded at 70.000 cells per well in 24-well plates and cultured for 24h at 37 °C and 5% CO 2 . HeLa cells were transfected using Lipofectamine 2000. For the experiments four plasmids were mixed in a specific ratio and a final amount of 200 fmol. The ratio used was 2:1:1:1 (dCas9:Effector:sgRNA:Reporter). For MS2 dependent activation MPH was used. For PAL dependent activation the plasmid PHP or HPP was used. For all experiments the pdCas9-T2A-eGFP plasmid was used for dCas9 expression. For the expression of the sgRNAs, pENTR plasmids that contains a hU6 promoter were used. The transfection was incubated for 4 hours at 37ºC and 5% CO 2 in the presence of blue light of 465 nm or in the absence of light. After the replacement of medium the cells were incubated for 24 hours in the light or in the dark. For flow cytometry analysis, the cells were transfected with the pGL3-Basic-8xgRNA-eBFP reporter plasmid. In short, cells were first washed with 500 µl warm DPBS (Gibco) and then detached using 200 µl Accutase (Gibco). The volume was filled up to 500 µl using cultivation medium and the resuspended cells were transferred into FACS tubes (BD). The cells were centrifuged for 5 min at 200 g. Then the supernatant was removed and the cells were washed with 1 ml warm DPBS. The cells were measured using a BD FACS Canto II. The voltage for FSC was set to 50 V, SSC to 300 V and Pacific Blue Laser (405 nm) to 187 V. The FSC threshold was set to 5.000. HeLa cells were gated for single cells with an auto fluorescence of 1% ( Figure S2e, Supporting Information). The activation was normalized using the feature scaling method. For Metridia luciferase reporter assays, 50.000 HeLa cells were seeded. As reporter plasmid pGL3-Basic-8xgRNA-metLuc was used. For measurement, 50 µl of supernatant was collected from each sample and transferred to a LUMITRAC 200 96-well plate. Luciferase reagents were prepared according to the manufacturers manual (Clontech). For the reversible Metridia luciferase reporter assays, after transfection 50 µl of supernatant was collected for measurement while the rest was replaced by fresh cell culture medium. The replacement of supernatant was done before every switching illumination setting from light to dark or dark to light.
Optogenetic activation of endogenous ASCL1: HeLa cells were seeded at 70.000 cells per well in 24-well plates and cultured for 24h at 37 °C and 5% CO 2 . HeLa cells were transfected using Lipofectamine 2000. For the experiments three plasmids were mixed in a specific ratio and a final amount of 200 fmol. The ratio used was 2:1:1 (dCas9:Effector:sgRNA). For MS2 dependent activation MPH was used. For PAL dependent activation the plasmid PHP was used. For all experiments the pdCas9-T2A-eGFP plasmid was used for dCas9 expression. For the expression of the sgRNAs, pENTR plasmids that contains a hU6 promoter were used. The transfection was incubated for 4 hours at 37ºC and 5% CO 2 in the presence of blue light of 465 nm or in the absence of light. After the replacement of medium the cells were incubated for 24 hours in the light or in the dark. For qPCR analysis, cellular RNA isolation and reversetranscription were done with the Cells-to-Ct kit (Thermo Fisher Scientific) using TaqMan gene expression master mix (Thermo Fisher Scientific). TaqMan probes were used for ASCL1 and GAPDH detection (Life Technologies; TaqMan gene expression assay IDs were Hs04187546_g1 (ASCL1) and Hs99999905_m1 (GAPDH)). The fold induction of mRNA was calculated by first calculating the Δc t value for each biological sample. The Δc t values were then normalized to control sgRNA to obtain ΔΔc t values. For the fold induction of mRNA, 2^ΔΔc t was calculated.
Cas9 cleavage assays: First, a 1 µM Cas9 solution was prepared from a 20 µM Cas9 stock solution (NEB, Ipswich) in 1x Cas9 buffer (NEB, Ipswich). The DNA cleavage substrate was amplified from pGL3-Basic-8xgRNA-eBFP. Per assay 150 ng of substrate was used. In the assay molar ratios of 8:1 (Cas9:substrate) and 1:1 (Cas9:gRNA) were used in a final volume of 20 µl. First ddH2O, Cas9 buffer and sgRNA were mixed in a single reaction tube. For sgRNA refolding, the mixture was heated for 2 min at 95 °C and then cooled down to 20 °C with 6 °C/min. For complex formation Cas9 was added and incubated for 5 min at 20 °C. The substrate was added to start the reaction. The cleavage reaction was incubated for 35 min at 37 °C. Subsequently 1 µl of 20 mg/ml Proteinase K (Roth) was added and incubated for 35 min at 55 °C to stop the reaction. The reaction was loaded on a 1% agarose gel. The samples were run for 30 min at 130 V and then stained with ethidium bromide. The cleavage products were visualized using UV irradiation and images were made. The cleavage fractions were analyzed using ImageJ 1.50i software.
In vitro transcription of gRNAs: sgRNA templates with a 5' attached T7 promoter were ordered from Ella Biotech (Martinsried, Germany). The in vitro transcription was done overnight and the RNA was isolated and purified by PAGE and electro elution.

Statistics: Statistical analysis for pairwise comparisons between light/dark experiments was performed using Welch's two-tailed t-test
and confidence intervals of 95% in Prism 6.   Table S3. *p <0.05, **p <0.01, ***p<0.005 versus the sample in the dark. (e) Gating strategy for optogenetic eBFP activation assays. HeLa cells were analyzed by flow cytometry and 30.000 events were recorded.
From all events, first the main single-cell population was isolated using SSC-A to SSC-H plot. The isolated population was then plotted to visualize the signal intensity of the Pacific Blue-A channel. A quadrant gate was applied, which separates the population from cells with more (Q2) and less (Q1) than 1% fluorescence.
Events that have a Pacific Blue-A intensity, which exceeds 1% (Q2 gate) are considered as activated cells.  Table S3. *p <0.05, **p <0.01, ***p<0.005 versus the sample in the dark. (g) Light-dependent upregulation of eBFP using sgRNAs with mutants and varying aptamer stem-lengths showing normalized data. The data is presented as mean values ± s.d and was normalized to the sgRNA 2.0 control using min-max scaling (n = 6 from 3 individual experiments with two cell culture replicates. Welch's two-tailed t-test was performed for the fold induction in the light versus the sample in the dark. The resulting p-values of the samples are given in the Supporting Information in Table S3. *p <0.05, **p <0.01, ***p<0.005 versus the sample in the dark. (h) Light-dependent upregulation of eBFP using sgRNAs with mutants and varying aptamer stem-lengths. The number of eBFP positive cells gained from (g) is presented as a function of eBFP intensity threshold (THD). The data is presented as mean values ± s.d (n = 6 from 3 individual experiments with two cell culture replicates). (i) Light-dependent upregulation of Metridia Luciferase using sgRNAs with varying aptamer stem-lengths and point mutants showing normalized data. The data is presented as mean values ± s.d and was normalized to the sgRNA 2.0 control using min-max scaling (n = 6 from 3 individual experiments with two cell culture replicates. Welch's two-tailed t-test was performed for the fold induction in the light versus the sample in the dark. The resulting pvalues of the samples are given in the Supporting Information in Table S3. *p <0.05, **p <0.01, ***p<0.005 versus the sample in the dark. (j) Light-dependent activation of Metridia luciferase expression over time. The data is presented as mean values ± s.d (n = 6 from 3 individual experiments with two cell culture replicates).
Welch's two-tailed t-test was performed for the fold induction in the light versus the sample in the dark. The resulting p-values of the samples are given in the Supporting Information in Table S3. *p <0.05, **p <0.01, ***p<0.005 versus the sample in the dark. Table S1. Target sequences of sgRNAs.