Jun 18, 2020
LucifeRace: A luminescence-based competition assay
This protocol is a draft, published without a DOI.
- Andrew O Giacomelli1,
- Hans R. Widlund2,
- Joseph Rosenbluh3,
- William C. Hahn4
- 1Princess Margaret Cancer Centre;
- 2Brigham and Women's Hospital, Harvard Medical School;
- 3Monash University;
- 4Dana-Farber Cancer Institute, Broad Institute, Brigham and Women's Hospital, Harvard Medical School
- Cancer Dependency Map Target ValidationTech. support email: depmap-tda@broadinstitute.org
Protocol Citation: Andrew O Giacomelli, Hans R. Widlund, Joseph Rosenbluh, William C. Hahn 2020. LucifeRace: A luminescence-based competition assay. protocols.io https://protocols.io/view/luciferace-a-luminescence-based-competition-assay-bhm2j48e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 18, 2020
Last Modified: June 18, 2020
Protocol Integer ID: 38298
Keywords: genetic dependency, small molecule sensitivity, high-throughput competition assay, luminescence,
Abstract
Fluorescence-based cell competition assays have been used effectively to validate genetic dependencies predicted by the Cancer Dependency Map (Cheung et al. PNAS. 2011, Cowley et al. Scientific Data. 2014). However, these can be labor-intensive and are dependent upon the availability of flow cytometry equipment. To overcome these limitations we developed LucifeRace, a luminescence-based competition assay. It offers several advantages over the traditional competition assay in terms of ease of use, scalability, and application in a wider array of cell biology labs.
Firefly (Photinus pyralis) and Renilla (Renilla reniformis) luciferases are evolutionarily distinct enzymes, and have different substrate, cofactor, and pH requirements. When present in a mixture, they can be measured independently using a multiplexed sequential dual luminescence assay. In the LucifeRace assay, two cell populations, one labeled with firefly luciferase and the other with Renilla luciferase, are mixed together and after some incubation period in the presence of a chemical or genetic perturbagen are subjected to a dual luminescence assay to measure the contribution of each cell type to the mixture. Variations of this protocol have been used to assess the effects of small molecules and genetic perturbations in human cancer cell lines (Giacomelli et al. Nat. Genetics. 2018, Takeda et al. Cell. 2018, Chan et al. Nature. 2019, Price et al. Cancer Research. 2019, Shauer et al. Sci. Rep. 2020).
To efficieintly label cell lines with firefly or Renilla luciferase, we created a pair of lentiviral vectors built on the pLX313 backbone. These vectors promote constitutive expression of the luciferases under control of the human EF1α promoter, and a contain a hygromycin resistance cassette to allow for the selection of transduced cells. pLX313-firefly and -Renilla luciferase expression vectors can be obtained from Addgene:
pLX311-Cas9 and pLX311-LacZ allow for constitutive expression of Cas9 or LacZ under control of the human EF1α promoter, and a contain a blasticidin resistance cassette to allow for the selection of transduced cells:
This protocol is designed for the use of sgRNA-expressing vectors that contain a puromycin resistance cassette (i.e. lentiGuide-Puro):
Lentivirus packaging plasmids developed by the Weinberg lab can also be obtained from Addgene (Stewart et al. RNA. 2003):
(Protocol Image adapted from Giacomelli et al. Nat. Genet. 2018)
References
Cheung, H.W.et al. Systematic investigation of genetic vulnerabilities across cancer cell lines reveals lineage-specific dependencies in ovarian cancer. Proc Natl Acad Sci USA 108, 12372-7 (2011).
Cowley, G.S.et al. Parallel genome-scale loss of function screens in 216 cancer cell lines for the identification of context-specific genetic dependencies. Scientific Data 1 (2014).
Giacomelli, A.O. et al. Mutational processes shape the landscape of TP53 mutations in human cancer. Nat Genet 50 (2018).
Takeda, D.S. et al. A Somatically Acquired Enhancer of the Androgen Receptor Is a Noncoding Driver in Advanced Prostate Cancer. Cell 174 (2018).
Chan, E.M. et al. WRN helicase is a synthetic lethal target in microsatellite unstable cancers. Nature 568 (2019).
Price, C.P. et al. Genome-Wide Interrogation of Human Cancers Identifies EGLN1 Dependency in Clear Cell Ovarian Cancers. Cancer Res 79 (2019).
Shauer, N.J. et al. Selective USP7 inhibition elicits cancer cell killing through a p53-dependent mechanism. Sci Rep 10 (2020).
Stewart, S.A. et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA 9 (2003).
Dyer, B.W., Ferrer, F.A., Klinedinst, D.K. & Rodriguez, R. A noncommercial dual luciferase enzyme assay system for reporter gene analysis. Anal Biochem 282 (2000).
Hampf, M. and Gossen, M. A protocol for combined Photinus and Renilla luciferase quantification compatible with protein assays. Anal Biochem 356 (2006).
Materials
Promega™ offers a pre-made, easy-to-use dual luminescence assay reagent called Dual-Glo®. Alternatively, the following recipe can be used to produce homemade FLR and RLR reagents (Dyer et al. Analytical Biochemistry. 2000. and Hampf and Gossen. Analytical Biochemistry. 2006).
Refer to MSDS for each of the components and take all appropriate safety precautions.
Several components need to be dissolved to create the stock solutions, which need to be combined to create the Firefly Luciferase Reagent (FLR) and the Renilla Luciferase Reagent (RLR).
Reagents should be made in batches, aliquoted, and frozen at -20 to -80°C. The long-term stability of the reagents has not yet been thoroughly tested, and multiple rounds of freeze-thaw should be avoided.
If using the homemade reagents to perform the dual luminescence assay, 5X Passive Lysis Buffer (PLB from Promega™) and Phosphate Buffered Saline (PBS) must also be purchased and mixed with the FLR at a 25:15:10 ratio (FLR:PBS:PLB) prior to performing the assay.
Table 1. Use the following recipes to make high concentration stocks for each component of the firefly luciferase reagent (FLR):
| Reagent | Final Conc. | Dissolution | |
| glycylglycine | 1 M | Dissolve 1.32 g in 10 mL ddH2O | |
| KxPO4, pH = 8 | 1 M | Dissolve 13.73 g of KH2PO4 and 156.6 g of K2HPO4 in 1 liter ddH2O | |
| EGTA | 0.5 M | Dissolve 1.9 g in 10 mL ddH2O | |
| ATP | 200 mM | Dissolve 0.55 g in 5 mL ddH2O | |
| DTT | 1 M | Dissolve 1.54 g in 10 mL ddH2O | |
| MgSO4 | 1 M | Dissolve 1.2 g in 10 mL ddH2O | |
| CoA | 10 mM | Dissolve 7.6 mg in 1 mL ddH2O | |
| D-luciferin | 10 mM | Dissolve 28 mg in 10 mL ddH2O |
Table 2. Combine the stock components together according to the following table to make 50 mL of FLR.
| Reagent | Stock | Final Conc. | Volume (in 50 mL total) | |
| glycylglycine | 1 M | 25 mM | 1.25 mL | |
| KxPO4, pH = 8 | 1 M | 15 mM | 750 μL | |
| EGTA | 0.5 M | 4 mM | 400 μL | |
| ATP | 200 mM | 2 mM | 500 μL | |
| DTT | 1 M | 1 mM | 50 μL | |
| MgSO4 | 1 M | 15 mM | 750 μL | |
| CoA | 10 mM | 0.1 mM | 500 μL | |
| D-luciferin | 10 mM | 75 μM | 375 μL | |
| ddH2O | to 50 mL |
Table 3. Use the following recipes to make high concentration stocks for each component of the renilla luciferase reagent (RLR):
| Reagent | Final Conc. | Dissolution | |
| NaCl | 5 M | ||
| EDTA | 0.5 M | ||
| KxPO4, pH = 5.1 | 1 M | Dissolve 13.73 g of KH2PO4 and 156.6 g of K2HPO4 in 1 liter ddH2O | |
| BSA | 10 mg/mL | ||
| NaN3 | 0.5 M | Dissolve 325 mg in 10 mL ddH2O | |
| coelenterazine | 0.5 mM | Dissolve 1 mg in 2.36 mL of methanol and 2.36 mL of ethanol |
Table 4. Combine the stock components together using the following table to make 50 mL of RLR.
| Reagent | Stock | Final Conc. | Volume (in 50 mL total) | |
| NaCl | 5M | 1.1 M | 11 mL | |
| EDTA | 0.5 M | 2.2 mM | 200 μL | |
| KxPO4, pH = 5.1 | 1M | 220 mM | 11 mL | |
| BSA | 100 mg/ml | 0.44 mg/mL | 220 μL | |
| NaN3 | 0.5 M | 1.3 mM | 130 μL | |
| coelenterazine | 0.5 mM | 1.43 μM | 143 μL | |
| ddH2O | to 50 mL |
Safety warnings
Always follow appropriate biosafety protocols for working with human cell lines and lentivirus.
Before start
1) Prior to performing genetic perturbation experiments with a new cell line, you should determine:
a) The maximum density of these cells in 96-well plates at confluence
b) The doubling time of these cells in 96-well plates
c) The linearity of the luminescence signal with respect to cell number
This information will help you identify the optimal seeding density, duration between passages, and dilution ratio at each passage. For example, a cell line with a maximum density of 20,000 cells/well and a doubling time of 1 day can be seeded at ~2500 cells/well (1250 expressing firefly luciferase + 1250 expressing Renilla luciferase) and passaged every 4 days at a 1/16 dilution.
Generate stable cell lines
Generate stable cell lines
2w
2w
Select an appropriate cell line using the Cancer Dependency Map portal. This protocol has been optimized for adherent cell lines that are propagated in serum-containing medium and are resilient when treated with trypsin.
2w
Using lentiviral transduction, engineer 2 stable derivative cell lines from the selected parental cell line. One of these derivatives should express Cas9 and firefly luciferase (i.e. pLX311-Cas9/pLX313-Firefly), and the other should express a control ORF (or empty vector) and Renilla luciferase (i.e. pLX311-LacZ/pLX313-Renilla). When generating these lines, aim for a multiplicity of infection (MOI) of ~0.3 to ensure that most cells contain a single integrant of each expression vector.
*For production of lentivirus and stable cell lines please see:
*Always follow appropriate biosafety protocols for working with human cell lines and lentivirus.
Day 0 - Mix and seed luciferase-labeled cells
Day 0 - Mix and seed luciferase-labeled cells
10m
10m
Trypsinize exponentially-growing stable cell lines (pLX311-Cas9/pLX313-Firefly and pLX311-LacZ/pLX313-Renilla) to generate two single-cell suspensions.
10m
Determine the density of each cell suspension using a hemocytometer or other cell counting device.
2m
Dilute each cell suspension to an appropriate density (see Before Starting note) and thoroughly mix the two cell suspensions together at a 1:1 (cell:cell) ratio in a 50 mL conical vial.
10m
Pipette or carefully decant the cell mixture into a multichannel reservoir.
1m
Transfer 200 μL of the cell mixture into mutliple wells of a 96-well plate using a multichannel pipette.
As you plan the scale of your experiments, be sure to account for the inclusion of technical replicates as well as the following controls:
1) Negative control sgRNAs that target:
• non-essential genes
• non-coding regions
• non-human genes
*These help control for non-specific effects associated with lentiviral transduction, selection in puromycin, and cell fitness effects associated with DNA cleavage by Cas9.
2) sgRNAs that target firefly luciferase
*These act as internal controls for Cas9 activity.
3) sgRNAs that target essential genes
*These allow you to determine the expected maximal killing effect of lethal sgRNAs.
4) Uninfected cells selected in puromycin
5) Uninfected cells not selected in puromycin
*Controls 4 and 5 are used to determine the extent of puromycin-mediated killing.
6) Wells containing media only or PBS
*These help reduce evaporation from experimental wells, and can be used to determine the level of background luminescence.
5m
Allow cells to settle to the bottom of the 96-well plate at room temperature on a level surface (~15 minutes) and then incubate the plate in a 37°C tissue-culture incubator overnight.
15m
Optional: You may wish to seed the cell mixture into a few wells of an additional 96-well plate. This plate can be subjected to a dual luminescence assay at an early time point to determine the relative proportions of the two labeled populations prior to infection. Note that raw Renilla luminescence values are typically 10-fold greater than raw firefly luminescence values.
*See Step 22-26 below for details on how to perform the dual luminescence assay.
5m
Day 1 - Infect cells with sgRNA-expressing lentivirus
Day 1 - Infect cells with sgRNA-expressing lentivirus
30m
30m
Using a multichannel pipette, add Polybrene infection reagent to each well of the 96-well plate to acheive a final concentration of 5 μg/mL. Pre-dilute the Polybrene in fresh media, if necessary.
5m
Add high-titer lentiviruses that encode the chosen experimental or control sgRNAs (e.g. lentiGuide-Puro, pXPR003 backbones) (5-10 μL of virus per well, aim for 80-100% infection efficiency).
5m
Optional: A spinfection protocol may be used to enhance infection efficiency.
2h
Incubate infected cells in a 37°C tissue-culture incubator overnight.
18h
Day 2 - Select for infected cells using puromycin
Day 2 - Select for infected cells using puromycin
5m
5m
Using a multichannel pipette, carefully aspirate all ~200 μL of the supernatant from the 96-well plate and safely dispose of waste.
*Remember, the supernatant contains active lentivirus.
5m
Add 200 μL of fresh media containing puromycin (1 μg/mL) to all infected wells and a portion of the uninfected control wells.
Add 200 μL of fresh media lacking puromycin to the remaining uninfected control wells and empty wells.
5m
Incubate plates in a 37°C tissue-culture incubator for 48 hours. This will allow the puromycin to kill all cells that did not become infected with an sgRNA-expressing lentivirus.
2d
Day 4 - Replate cells
Day 4 - Replate cells
5m
5m
Using a multichannel pipette, carefully aspirate all ~200 μL of the supernatant from the 96-well plate and dispose appropriately.
5m
Add 50 μL of 0.25% trypsin-EDTA to each well and incubate cells at 37ºC until all cells lift from plate (~1-10 min).
15m
Add 150 μL of fresh (serum-containing) media and mix to resuspend cells.
5m
Split the entire mixture into two replica plates:
• a Dual Luciferase Assay plate and
• a Trypsinize and Reseed plate.
*It is easiest to first load 100 μL of fresh media into each replica plate and then aliquot 100 μL of the cell mixture into those plates.
5m
Allow cells to settle to the bottom of the 96-well plates at room temperature on a level surface (~15 minutes) and then incubate the plates in a 37°C tissue-culture incubator overnight.
15m
Day 5 - Perform dual luciferase assay and replate cells
Day 5 - Perform dual luciferase assay and replate cells
5m
5m
For the Dual Luciferase Assay plate:
Using a multichannel pipette, carefully aspirate all ~200 μL of the supernatant from the 96-well plate and dispose appropriately.
5m
Add 50 μL of the firefly luciferase reagent (FLR) containing passive cell lysis buffer and PBS (see Materials), or Dual-Glo® Luciferase Reagent from Promega™, which already contains lysis buffer.
Protect from light, and incubate plate for 20 minutes at room temperature.
25m
Obtain raw firefly luminescence readings using a luminometer that is compatible with 96-well plates.
1m 36s
Add 25 μL of the Renilla luciferase reagent (RLR) or Promega™ Stop & Glo® reagent.
Protect from light, and incubate plate for 20 minutes at room temperature.
25m
Obtain raw Renilla luminescence readings using a luminometer that is compatible with 96-well plates.
1m 36s
For the Trypsinize and Reseed plate:
Using a multichannel pipette, carefully aspirate all ~200 μL of the supernatant from the 96-well plate and dispose appropriately.
Add 50 μL of trypsin-EDTA to each well and incubate cells at 37ºC until all cells lift from plate (~1-10 min).
15m
Add 150 μL of fresh (serum-containing) media and mix to resuspend cells.
5m
Split the mixture into two new replica plates at an appropriate dilution (see Before Starting note):
• a Dual Luciferase Assay plate and
• a Trypsinize and Reseed plate.
*It is easiest to first load a defined amount of fresh media into each replica plate and then aliquot the appropriate dilution of the cell suspension into those plates. For example, for a 1/16 dilution, first load 187.5 μL of fresh media into each new replica plate, and then add 12.5 μL of the mixed cell suspension.
5m
Allow cells to settle to the bottom of the 96-well plate at room temperature on a level surface (~15 minutes) and then incubate the plate in a 37°C tissue-culture incubator for the desired amount of time (typically 2-4 days).
15m
Day 9 (and onwards) - Perform dual luciferase assay and replate cells
Day 9 (and onwards) - Perform dual luciferase assay and replate cells
Repeat all steps from Day 5. Continue the process of reading and replating for as long as replicates remain consistent (tested up to 17 days).
Data Analysis
Data Analysis
We recommend performing the following calculations to arrive at an approximation of the log-ratio of edited:unedited cells:
1) Divide the raw firefly luminescence reading of each experimental well by the mean raw firefly luminescence readings of wells infected with the chosen set of negative control sgRNAs (from Step 7). This is the fractional firefly luminescence reading (i.e. the % viability of the edited cells).
2) Divide the raw Renilla luminescence reading of each experimental well by the mean raw Renilla luminescence readings of wells infected with the same set of negative control sgRNAs. This is the fractional Renilla luminescence reading (i.e. the % viability of the unedited cells).
3) Divide the fractional firefly luminescence reading by the fractional Renilla luminescence reading. This is the edited to unedited ratio.
4) Calculate the logarithm of the edited to unedited ratio (or plot the ratio on a log-scale axis).
*Because Cas9 is expressed by cells that express firefly luciferase:
- Log ratios near 0 suggest that the edited and unedited cells had equal fitness.
- Log ratios less than 0 suggest that edited cells had a competitive fitness disadvantage relative to unedited cells.
- Log ratios greater than 0 suggest that edited cells had a competitive fitness advantage relative to unedited cells.