TITLE: LucifeRace: A luminescence-based competition assay
AUTHORS: Andrew Giacomelli, Hans R. Widlund, Joseph Rosenbluh, William C. Hahn

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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. </div><div class = "text-block"><span>Firefly (</span><span style = "font-style:italic;">Photinus pyralis</span><span>) and Renilla (</span><span style = "font-style:italic;">Renilla reniformis</span><span>) 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).</span></div><div class = "text-block">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:</div><div class = "text-block"><a href="https://www.addgene.org/118016" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.addgene.org/118016/</span></a></div><div class = "text-block"><a href="https://www.addgene.org/118017" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.addgene.org/118017/</span></a></div><div class = "text-block">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:</div><div class = "text-block"><a href="https://www.addgene.org/118018" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.addgene.org/118018/</span></a></div><div class = "text-block"><a href="https://www.addgene.org/117731" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.addgene.org/117731/</span></a></div><div class = "text-block">This protocol is designed for the use of sgRNA-expressing vectors that contain a puromycin resistance cassette (i.e. lentiGuide-Puro):</div><div class = "text-block"><a href="https://www.addgene.org/52963" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.addgene.org/52963/</span></a></div><div class = "text-block">Lentivirus packaging plasmids developed by the Weinberg lab can also be obtained from Addgene (Stewart et al. RNA. 2003):</div><div class = "text-block"><a href="https://www.addgene.org/8455" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.addgene.org/8455/</span></a></div><div class = "text-block"><a href="https://www.addgene.org/8454" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.addgene.org/8454/</span></a></div><div class = "text-block">(Protocol Image adapted from Giacomelli et al. Nat. Genet. 2018)</div><div class = "text-block"><span style = "font-weight:bold;">References</span></div><div class = "text-block"><span>Cheung, H.W.</span><span style = "font-style:italic;">et al. </span><span>Systematic investigation of genetic vulnerabilities across cancer cell lines reveals lineage-specific dependencies in ovarian cancer. </span><span style = "font-style:italic;">Proc Natl Acad Sci USA </span><span style = "font-weight:bold;">108</span><span>, 12372-7 (2011).</span></div><div class = "text-block"><span>Cowley, G.S.</span><span style = "font-style:italic;">et al. </span><span>Parallel genome-scale loss of function screens in 216 cancer cell lines for the identification of context-specific genetic dependencies. </span><span style = "font-style:italic;">Scientific Data </span><span style = "font-weight:bold;">1 </span><span>(2014).</span></div><div class = "text-block"><span>Giacomelli, A.O. </span><span style = "font-style:italic;">et al. </span><span>Mutational processes shape the landscape of TP53 mutations in human cancer. </span><span style = "font-style:italic;">Nat Genet </span><span style = "font-weight:bold;">50</span><span style = "font-style:italic;font-weight:bold;"> </span><span>(2018).</span></div><div class = "text-block"><span>Takeda, D.S. </span><span style = "font-style:italic;">et al.</span><span> A Somatically Acquired Enhancer of the Androgen Receptor Is a Noncoding Driver in Advanced Prostate Cancer. </span><span style = "font-style:italic;">Cell </span><span style = "font-weight:bold;">174</span><span> (2018).</span></div><div class = "text-block"><span>Chan, E.M. </span><span style = "font-style:italic;">et al. </span><span>WRN helicase is a synthetic lethal target in microsatellite unstable cancers. </span><span style = "font-style:italic;">Nature</span><span style = "font-style:italic;font-weight:bold;"> </span><span style = "font-weight:bold;">568</span><span> (2019).</span></div><div class = "text-block"><span>Price, C.P. </span><span style = "font-style:italic;">et al.</span><span> Genome-Wide Interrogation of Human Cancers Identifies EGLN1 Dependency in Clear Cell Ovarian Cancers. </span><span style = "font-style:italic;">Cancer Res </span><span style = "font-weight:bold;">79 </span><span>(2019).</span></div><div class = "text-block"><span>Shauer, N.J. </span><span style = "font-style:italic;">et al.</span><span> Selective USP7 inhibition elicits cancer cell killing through a p53-dependent mechanism. </span><span style = "font-style:italic;">Sci Rep </span><span style = "font-weight:bold;">10 </span><span>(2020).</span></div><div class = "text-block"><span>Stewart, S.A. </span><span style = "font-style:italic;">et al.</span><span> Lentivirus-delivered stable gene silencing by RNAi in primary cells. </span><span style = "font-style:italic;">RNA</span><span style = "font-weight:bold;"> 9 </span><span>(2003).</span></div><div class = "text-block"><span>Dyer, B.W., Ferrer, F.A., Klinedinst, D.K. & Rodriguez, R. A noncommercial dual luciferase enzyme assay system for reporter gene analysis. </span><span style = "font-style:italic;">Anal Biochem </span><span style = "font-weight:bold;">282 </span><span>(2000).</span></div><div class = "text-block"><span>Hampf, M. and Gossen, M. A protocol for combined Photinus and Renilla luciferase quantification compatible with protein assays. </span><span style = "font-style:italic;">Anal Biochem</span><span> 356 (2006).</span></div></div>

[STEPS]
?. [Generate stable cell lines]
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.
?. [Generate stable cell lines]
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:https://portals.broadinstitute.org/gpp/public/resources/protocols*Always follow appropriate biosafety protocols for working with human cell lines and lentivirus.
?. [Day 0 - Mix and seed luciferase-labeled cells]
Trypsinize exponentially-growing stable cell lines (pLX311-Cas9/pLX313-Firefly and pLX311-LacZ/pLX313-Renilla) to generate two single-cell suspensions.
?. [Day 0 - Mix and seed luciferase-labeled cells]
Determine the density of each cell suspension using a hemocytometer or other cell counting device.
?. [Day 0 - Mix and seed luciferase-labeled cells]
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.
?. [Day 0 - Mix and seed luciferase-labeled cells]
Pipette or carefully decant the cell mixture into a multichannel reservoir.
?. [Day 0 - Mix and seed luciferase-labeled cells]
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 puromycin5) 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.
?. [Day 0 - Mix and seed luciferase-labeled cells]
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.
?. [Day 0 - Mix and seed luciferase-labeled cells]
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.
?. [Day 1 - Infect cells with sgRNA-expressing lentivirus]
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.
?. [Day 1 - Infect cells with sgRNA-expressing lentivirus]
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).
?. [Day 1 - Infect cells with sgRNA-expressing lentivirus]
Optional: A spinfection protocol may be used to enhance infection efficiency.
?. [Day 1 - Infect cells with sgRNA-expressing lentivirus]
Incubate infected cells in a 37°C tissue-culture incubator overnight.
?. [Day 2 - Select for infected cells using puromycin]
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.
?. [Day 2 - Select for infected cells using puromycin]
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.
?. [Day 2 - Select for infected cells using puromycin]
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.
?. [Day 4 - Replate cells]
Using a multichannel pipette, carefully aspirate all ~200 μL of the supernatant from the 96-well plate and dispose appropriately.
?. [Day 4 - Replate cells]
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).
?. [Day 4 - Replate cells]
Add 150 μL of fresh (serum-containing) media and mix to resuspend cells.
?. [Day 4 - Replate cells]
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.
?. [Day 4 - Replate cells]
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.
?. [Day 5 - Perform dual luciferase assay and replate cells]
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.
?. [Day 5 - Perform dual luciferase assay and replate cells]
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.
?. [Day 5 - Perform dual luciferase assay and replate cells]
Obtain raw firefly luminescence readings using a luminometer that is compatible with 96-well plates.
?. [Day 5 - Perform dual luciferase assay and replate cells]
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.
?. [Day 5 - Perform dual luciferase assay and replate cells]
Obtain raw Renilla luminescence readings using a luminometer that is compatible with 96-well plates.
?. [Day 5 - Perform dual luciferase assay and replate cells]
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.
?. [Day 5 - Perform dual luciferase assay and replate cells]
Add 50 μL of trypsin-EDTA to each well and incubate cells at 37ºC until all cells lift from plate (~1-10 min).
?. [Day 5 - Perform dual luciferase assay and replate cells]
Add 150 μL of fresh (serum-containing) media and mix to resuspend cells.
?. [Day 5 - Perform dual luciferase assay and replate cells]
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.
?. [Day 5 - Perform dual luciferase assay and replate cells]
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).
?. [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]
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.