Method Article

Rapid Optimization of a Light-Inducible System to Control Mammalian Gene Expression

DOI:

10.3791/68779

⸱

November 4th, 2025

 ,  ,  , 
1Department of Chemical Engineering, University of California, Davis, 2Department of Microbiology and Molecular Genetics, University of California, Davis

In This Article

Summary

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This study describes a method for high-throughput experiments using a 3D-printed LED array to optimize light-inducible gene expression in HEK293T cells.

Abstract

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Inducible gene expression tools can open novel applications in human health and biotechnology, but current options are often expensive, difficult to reverse, and have undesirable off-target effects. Optogenetic systems use light-responsive proteins to control the activity of regulators such that expression is controlled with the "flip of a switch". This study optimizes a simplified light activated CRISPR effector (2pLACE) system, which provides tunable, reversible, and precise control of mammalian gene expression. The OptoPlate-96 enables high-throughput screening via flow cytometry for single-cell analysis and rapid optimization of 2pLACE. This study demonstrates how to use the 2pLACE system with the OptoPlate-96 in HEK293T cells to identify the optimal component ratios for maximizing dynamic range and to find the blue light intensity response curve. Similar workflows can be developed for other mammalian cells and for other optogenetic systems and wavelengths of light. These advancements enhance the precision, scalability, and adaptability of optogenetic tools for biomanufacturing applications.

Introduction

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Synthetic biology tools such as inducible gene expression systems have provided significant contributions to biological research. Their ability to regulate gene expression in a tunable, reversible, and precise manner can improve control of protein production1,2,3,4,5,6, cell morphology7,8,9,10,11,12, metabolic pathways13,14,15,16,17,18, and other targets for biomanufacturing and therapeutic applications. Chemical inducible systems can have residual19,20 or off-target effects. Chemical additives can also be very expensive and difficult to scale up industrially due to additional downstream purification processes21,22,23. Light-inducible gene expression systems can offer a scalable and precise method for biomanufacturing practices24,25,26,27.

Many light-inducible gene expression systems have been developed as useful synthetic biology tools in a variety of applications28,29,30,31,32,33,34,35,36 and wavelengths of blue35,37,38, red/far-red12,39,40, or green41 light. In the case of CRISPR-based optogenetic gene regulation, modifying the amount of individual guide RNAs (gRNAs) delivered can affect the mRNA expression of endogenous genes42, and will need to be optimized for different cell lines. Transactivation proteins such as VP6437,42,43,44, VP1639,40,44,45,46, p6544, or fusions of them47 can also be used, increasing the modularity of optogenetic systems. In order to investigate complex and intertwined biological pathways12,40,48,49,50,51,52,53, different combinations of these known components can be tested. Additionally, different cell lines can show differences in induction with the same system52. Different induction levels can lead to insufficient gene expression or sensitivity in the precise control of gene expression, requiring rigorous testing to find an optimal optogenetic system in novel or more biologically relevant cell lines. With a growing pace and widening applicability of optogenetic gene regulation, it is imperative to rapidly characterize these different systems.

Current methods in characterizing optogenetic tools either use stable or transient expression of genes12,54. Generating stably expressing cell lines and optimizing system dynamics for maximum expression can be time-consuming and therefore costly. Transient expression enables quick and valuable insights, especially when testing multicomponent optogenetic systems. Here, we used the blue light activated CRISPR-dCas9 effector (LACE)37 system, composed of cryptochrome 2 (CRY2) and cryptochrome-interacting basic-helix-loop-helix N-terminal (CIBN). It has been used to control gene expression in mammalian cells such as HEK293T (human embryonic kidney cells)37,38, CHO-DG44 (Chinese hamster ovary cells)55, and C2C12 (mouse myoblast cells)38. Its modular nature is ideal for quickly characterizing system performance through transient expression and high-throughput methods. For example, multicomponent systems like LACE may need optimization of mass ratios to improve induction, a step not typically employed in optogenetic system characterization.

This protocol details the rapid optimization and characterization of two plasmid LACE (2pLACE)38 system. In this setup, 2pLACE controls expression of a fluorescent reporter, eGFP (enhanced green fluorescent protein), in HEK293T cells. Activation is performed in black glass-bottom 96-well plates using an OptoPlate-96, a 3D printed LED array designed and constructed by Lukasz Bugaj and colleagues56,57,58,59. This protocol specifically optimizes maximal expression with different mass ratios of the two-plasmid system. It also includes user-friendly code to control different light intensities to investigate the tunability of the system and its full dynamic range. Flow cytometry is used for data collection and analysis. Protocols for generating plasmid constructs and 3D printing materials for the LED array can be found in previous publications54,56. These methods highlight a quick, high-throughput pipeline for characterizing light-inducible gene expression systems.

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Protocol

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1. Plating HEK293T cells in a 96-well format

  1. In a biosafety cabinet, collect a bleach waste container, serological pipettes, pipette-aid, Dulbecco's Phosphate Buffered Saline (DPBS), Dulbecco-modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS), and a T-75 cell culture flask with confluent HEK293T cells. Prewarm the media to 37 °C.
  2. In a T-75 flask, check that the confluency is about 80%-100% confluent through an inverted microscope.
  3. In the biosafety cabinet, use a serological pipette to aspirate the spent media from the cell culture flask. Avoid touching the surface or plastic of the flask. Dispose of the spent media and aspirator in the waste container.
  4. Gently wash the cells with about 10 mL of DPBS by aspirating it into a corner of the flask and gently swirling it around the surface. Aspirate the DPBS from the flask and dispose of the wash and the aspirator in the waste container.
  5. Add 1.5 mL of 0.05% Trypsin-EDTA to cover the surface of the flask and incubate at room temperature for 1 min. Gently tap the flask to loosen the cells from the surface.
  6. Add 8.5 mL of fresh DMEM into the flask. Resuspend and mix cells with a serological pipette until all aggregates have been broken up by aspirating up and down.
  7. Collect 9 mL of cell suspension into a 15 mL conical tube.
    1. Add 9 mL of fresh DMEM into the flask and place it into an incubator at 37 °C and 5% CO2
  8. Using a hemocytometer, mix 10 µL of suspended cells with 10 µL of Trypan blue dye at a 1:1 ratio to calculate the concentration of cells. Use this value to prepare enough cells to seed on the plate.
  9. Seed ~35,000 cells in 100 µL for each well of a high-performance #1.5 black 96-well glass bottom plate and place in an incubator at 37 °C and 5% CO2 for 24 h.

2. 2pLACE transfection optimization

  1. For one well and 10% excess, aliquot 11 µL of warm serum-free DMEM (SFM) into a 1.5 mL microcentrifuge tube.
  2. Using a CRY2-eGFP: CIBN-gRNA plasmid mass ratios equal to 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7: 3, 8:2, and 9:1, aliquot 110 ng for each well of total DNA to the aliquots of SFM (Table 1).
  3. As a positive control, aliquot the same mass of CMV-eGFP plasmid to different aliquots of SFM.
  4. For each well, aliquot 11 µL of SFM for transfection reagent dilution.
  5. Prepare the transfection reagent at a 1:3 mass (µg) to volume (µL) ratio of DNA and transfection reagent.
  6. Add diluted transfection reagent to the diluted DNA and incubate for 12 min at room temperature to create the transfection complexes.
  7. Add ~20 µL of the transfection complex to each well. Ensure that it does not touch the walls of the well.
  8. Wrap the plate in aluminum foil and place it in an incubator at 37 °C and 5% CO2 for 24 h.

3. 2pLACE activation

  1. Modify the microcontroller input code to illuminate the desired wells using these settings (Supplementary Coding File 1, Lines 147 - 158).
    1. LED Intensity: 9.27 mW/cm2 (Supplementary Coding File 1, Lines 200 - 215).
    2. Pulse Length: 1 s (Supplementary Coding File 1, Lines 280 - 290).
    3. Pulse Frequency: 0.067 Hz (every 15 s) (Supplementary Coding File 1, Lines 264 - 278).
      ​NOTE: Wavelength settings are determined by the types of LEDs installed.
  2. Spray the 3D printed lid of the OptoPlate with 70% ethanol and let it dry in a biosafety cabinet.
  3. Turn on a darkroom red light lamp and place the 96-well plate into a biosafety cabinet, and replace the lid with the dried 3D printed lid.
    NOTE: One can optionally view the CMV-eGFP cells using fluorescence microscopy to estimate transfection efficiency.
  4. Take the 96-well plate and place it onto the LED array to construct the LED array apparatus. Ensure that the plate snaps into place.
  5. Connect the microcontroller, LED, and fan ports to a power source.
  6. Carefully spray wires down with 70% ethanol and wipe dry.
  7. Place the LED array apparatus into an incubator at 37 °C and 5% CO2 for 24 h. Ensure that the LEDs turn on as intended and that the wires are not taut when the incubator is sealed.

4. Flow cytometry preparation

  1. While in a red-light environment, take out the OptoPlate and place the plate into a biosafety cabinet
    NOTE: One can optionally view the cells by fluorescence microscopy to visually confirm light induction.
  2. Carefully aspirate all media from each well.
  3. Detach the cells using 30 µL of 0.05% Trypsin-EDTA and incubate for 1 min at room temperature.
  4. Mix each well with 100 µL cold FACS buffer (1% FBS in PBS) until single-celled.
  5. Transfer each sample into a V-bottom 96-well plate.
  6. Centrifuge the V-bottom 96-well plate at 164 x g for 10 min at room temperature.
  7. Aspirate 80 µL of supernatant without disturbing the pellet.
  8. Add 200 µL of cold FACS buffer to resuspend the pellet.
  9. Wrap the V-bottom 96-well plate in aluminum foil for light insulation.
  10. Place the plate into a styrofoam box with ice for transportation.

5. Flow cytometry gating and data collection

  1. Turn on the flow cytometer using the switch in the back, and open the respective flow cytometry software on the computer.
  2. Run the system startup program and load 2 mL of DI water in the sample loader (Supplementary Figure 1A).
  3. Run quality control (QC) using the provided QC beads (Supplementary Figure 1B).
  4. Ensure the flow cytometer is in plate sampling mode (Supplementary Figure 1C).
  5. Set up and specify sample wells (Supplementary Figure 1D).
  6. Create the following scatter plots and tables (Supplementary Figure 2A).
    1. Side Scatter (SSC-A) vs Forward Scatter (FSC-A).
    2. SSC-H vs SSC-A.
    3. SSC-A vs FITC-A.
    4. FITC-A statistics table (Supplementary Figure 2B).
  7. Snap the V-bottom plate into the plate loader of the cytometer.
  8. Select an untransfected well and click on Initialize, and then click on Run.Ensure volume sample rate is 10 µL/min (Supplementary Figure 2C).
  9. Adjust SSC and FITC voltages to center the population of interest on the SSC-A vs FSC-A plot (Supplementary Figure 2C).
    1. Create a polygon to gate the healthy cell population of interest (Supplementary Figure 2D).
    2. Create a polygon to gate for doublet discrimination on the SSC-H vs SSC-A plot.
    3. Adjust FITC voltage to place untransfected cells towards the left of the SSC-A vs FITC-A plot (Supplementary Figure 2D).
  10. Repeat for the remaining untransfected wells and record Mean FITC-A data.
  11. Select a CMV-eGFP transfected well and click on Run.
  12. Adjust FITC voltage to capture both autofluorescing and fluorescing cells in the SSC-A vs FITC-A plot.
    1. Create a polygon to gate the fluorescing cells against the non-fluorescing cells while referencing the initial gate in untransfected cells (Supplementary Figure 2E).
  13. Auto-record samples at 60 µL/min until 200 s have been reached and/or events have reached 10,000 (Supplementary Figure 2F).
  14. Export Mean FITC as a CSV file and analyze the data.
  15. Clean the flow cytometer through the Daily Clean option (Supplementary Figure 2G).

6. LED intensity optimization

  1. Edit the microcontroller script to test desired LED intensities (Supplementary Coding File 1, Lines 200-215).
    NOTE: Equivalent LED intensities with respect to the values in the microcontroller script are reported elsewhere38. Self-calibration may be necessary when using different LEDs.
  2. Ensure the script contains the following values in (Supplementary Coding File 1, Lines 200-215) as designed in Table 2, and upload the code to the microcontroller.
  3. Repeat steps 1-5.

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Results

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Adopting workflow elements from previous literature37,38,55, we added high-throughput simultaneous illumination from the OptoPlate-96 and demonstrated a pipeline to rapidly optimize a light-inducible gene expression system in mammalian cells (Figure 1).

For inducible gene expression systems, high dynamic ranges are a critical performance marker, in addition to absolute on...

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Discussion

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This protocol demonstrates the optogenetic control of a multicomponent transient gene expression in mammalian cells, allowing other researchers to rapidly and simultaneously characterize modular optogenetic systems with a fluorescent reporter.

In this case, a two-plasmid light-inducible system was optimized for the expression of eGFP in HEK293T cells. For successful co-transfection, it is essential to optimize seeding densities and mass ratios for the highest transfection efficiency. This is e...

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Disclosures

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The authors declare no competing interests.

Acknowledgements

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This work is supported by the Translational Research Institute through NASA Cooperative Agreement NNX16AO69A and by the Good Foods Institute. This project was supported by the UC Davis Flow Cytometry Shared Resource Laboratory with technical assistance from Bridget McLaughlin, Jonathan Van Dyke, and Ashley Karajeh, with funding from the NCI P30 CA093373 (Comprehensive Cancer Center) and S10OD018223 (Beckman Coulter “Cytoflex” cytometer).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1.5 mL microcentrifuge tubesVWR10025-724
10 mL Reagent ReservoirsVWR77395-252
10 mL serological pipettesVWR75816-100
1000 μL filter tipsVWR76322-154
15 mL  High-Performance Centrifuge Tubes, flat capVWR89039-664
1931-C Optical Power MeterNewport1931-C
2 mL serological pipettesVWR75816-104
20 μL filter tipsVWR76322-134
200 μL filter tipsVWR76322-150
50 mL High-Performance Centrifuge Tubes, flat capVWR89039-656
96-well v-bottom platesBRANDplates781661
A21, LED Red light bulbBluex BulbsN/Ared light source
Arduino IDE SoftwareArduinoN/A
Biosafety Cabinet, Class IINuAireN/A
Bright-Line HemocytometerHausser Scientific3110
Cell counting slidesBioRad1450016
Cell Culture Plate 96-well, #1.5H glass bottom plateCellvisP96-1.5H-N
CytExpert SoftwareBeckman CoulterN/A
CytoFLEX Ready to Use Daily QC FluorospheresBeckman CoulterC657194 °C
CytoFLEX-S Flow Cytometer (4 violet, 2 blue, 4 Yellow Green, 3 Red channels)Beckman CoulterC09766
Dulbecco's Phosphate Buffered Saline powder, no calicum, no magnesiumFisher Scientific21600069Room Temperature
Eppendorf Centrifuge 5804Eppendorf 022622501v-bottom plate centrifuge step
Eppendorf Centrifuge 5810REppendorf22625501
Eppendorf Research plus, 8-channel, variable volume, 30 - 300 μLEppendorf3125000052
Eppendorf Research plus, single-channel, variable volume, 100 - 1000 μLEppendorf3123000063
Eppendorf Research plus, single-channel, variable volume, 2 - 20 μLEppendorf3123000039
Eppendorf Research plus, single-channel, variable volume, 20 - 200 μLEppendorf3123000055
General Purpose Laboratory Labeling TapeVWR89097-920
Gibco DMEM, powder, high glucoseFisher Scientific121000614 °C
Gibco Trypan Blue Solution, 0.4%Fisher Scientific15-250-061Room Temperature
Gibco Value Heat Inactivated FBSFisher ScientificA5256901-20 °C
Gibco, Trypsin-EDTA (0.05%), with EDTA, Animal Origin, 1X, Phenol Red, 500 mLGibco25300062-20 °C
HEK293T CellsATCCCRL-11268-80 °C, Liquid Nitrogen
Lab markers, MicronovaVWR89205-944
Metal Desk LampSimple DesignsN/ALamp for red light
OptoPlate-96LABmakerN/A
Pipet-aid XPDrummond4-000-101
Plasmid: CIBN-gRNAN/AN/A-20 °C
Plasmid: CMV-eGFPN/AN/A-20 °C
Plasmid: CRY2-eGFPN/AN/A-20 °C
PolyJet DNA In Vitro Transfection ReagentFisher ScientificNC15361174 °C
T-75 Cell Culture FlaskVWR10062-860
Water Jacketed CO2 IncubatorVWR10810-884

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Optogenetic SystemsInducible Gene ExpressionCRISPR EffectorFlow CytometryHigh Throughput ScreeningBlue Light ActivationHEK293T CellsFluorescence Imaging

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