TITLE: Single-gene long-term CRISPRi knockdown viability assay
AUTHORS: Nolan Bick, Benjamin Gaeta, Tsukasa Shibue, Francisca Vazquez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>As an alternative to '</span><span style = "font-weight:bold;">Single-gene short-term CRISPR ko viability assay</span><span> (</span><a href="https://www.protocols.io/edit/single-gene-short-term-crispr-ko-viability-assay-bc6jizcn/description" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/edit/single-gene-short-term-crispr-ko-viability-assay-bc6jizcn/description</span></a><span>)', we have also developed a method for assessing the </span><span style = "font-weight:bold;">long-term viability effect </span><span>upon suppression of a gene of interest. Using CRISPRko (CRISPR/Cas9-mediated gene knockout) is not compatible with evaluating long-term viability effects due to incomplete penetrance of gene inactivation. Instead, we use </span><span style = "font-weight:bold;">CRISPRi</span><span> with catalytically dead Cas9 (dCas9) fused to Krüppel associated box (KRAB) domain to suppress gene expression with higher penetrance. Following perturbation with CRISPRi, cells are seeded at a clonogenic density and their growth is monitored for up to 2 weeks. </span></div><div class = "text-block">We provide sequences of CRISPRi negative and positive sgRNAs controls, which are critical for evaluating the strength of the dependency, as well as a series of templates to record and normalize the data produced throughout this assay. In addition to permitting evaluation of long-term viability effects, this CRISPRi-based assay might also enable the assessment of the effects of partial suppression of gene expression if appropriate gRNAs are available. </div></div>

[STEPS]
?. [Virus Titration]
Viral Titration
?. [Virus Titration]
Mix following in a 12-well plate,  AB1cells500,000 cells2virus(see below; with differing amount of virus)38 mg/mL polybrene1 uL410% FBS, RPMIup to 2 mLPlate map (volume of virus)  ABCD10 uL (0 uL)0 uL (0 uL)225 uL (200 uL)50 uL (400 uL)3100 uL (800 uL)200 uL (1600 uL) Centrifuge at 900xg, 1.5h, 37ºC
AB1cells500,000 cells2virus(see below; with differing amount of virus)38 mg/mL polybrene1 uL410% FBS, RPMIup to 2 mL
ABCD10 uL (0 uL)0 uL (0 uL)225 uL (200 uL)50 uL (400 uL)3100 uL (800 uL)200 uL (1600 uL)
?. [Virus Titration]
Titrate the virus (see titration protocol above STEP5.1-5.3) to determine the amount of virus giving you maximal infection and minimal toxicity. Archiving >90% infection efficiency (post-antibiotic selection) is optimal.If titrating the virus is not possible, select 3 concentrations to use during the experiment. We found that for most cell lines we can use 100 uL of concentrated virus or 800 uL on non-concentrated virus
?. [Virus Titration]
Add optimal virus to cells and spin plate at 900g (RCF) @ 37°C for 90 minutes. Aspirate media and wash with PBS and add selection antibiotic
?. [Seeding: Day 3]
Check to see cells are selecting out from selection (Ideally >10% dead cells). When testing 3 different MOIs, pick the one with minimal viral toxicity and maximal survival against selection.
?. [Seeding: Day 3]
Trypsinize cells and count cells (SELECTION-FREE MEDIA)
?. [Seeding: Day 3]
Seed cells into colony formation plate, seeding density varies based on cell doubling time (make dilutions if needed)
?. [Seeding: Day 3]
24-well: Total volume to 500ulFast growing (cell doubling time 24-72 hours): 500 cells per well at lowest densityMedium growing (cell doubling time 72-120 hours): 1000 cells per well at lowest densitySlow growing (cell doubling time >120+ hours): 2500 cells per well at lowest density
?. [Seeding: Day 3]
Plate layout (24-well):  ABCDEFG1(seeding density)Ch2-2 (neg control)POLR2D (pos control)SF3B1 (pos control)Target- sgRNA-1Target- gRNA-2Target-sgRNA-321x32x44x58x
ABCDEFG1(seeding density)Ch2-2 (neg control)POLR2D (pos control)SF3B1 (pos control)Target- sgRNA-1Target- gRNA-2Target-sgRNA-321x32x44x58x
?. [Seeding: Day 3]
DO NOT add antibiotics at this point (antibiotic treatment will be restarted on Day 6)
?. [Seeding: Day 3]
Plate rest of negative control and target cells for Western blot if desired
?. [Culturing: Day 6]
Working quickly, aspirate media (collect and spin if suspension cells), and re-add media with antibiotic selection
?. [Culturing: Day 6]
Do not wash with PBSAvoid drying in center of wellsDon’t touch bottom of wellCarefully add media to wells (avoid detachment of cells)
?. [Culturing: Day 9]
Working quickly, aspirate media (collect and spin if suspension cells), and re-add media with antibiotic selection
?. [Culturing: Day 12]
Working quickly, aspirate media (collect and spin if suspension cells), and re-add media with antibiotic selection
?. [Staining with CV: Day 14]
Staining with Crystal Violet (Adherent Cells)
?. [Staining with CV: Day 14]
Aspirate Media and gently wash with PBS
?. [Staining with CV: Day 14]
Add 4% PFA or 10% Neutral-buffered formalin (under fume hood) to wells and put on shaker for 15 minutesFor 24-well, 250ul
?. [Staining with CV: Day 14]
Remove PFA or Formalin (under fume hood) and dispose of in appropriate hazardous waste container
?. [Staining with CV: Day 14]
Wash wells with DI water For 24-well, 250ul
?. [Staining with CV: Day 14]
Remove water and dispose of in PFA/Formalin waste container
?. [Staining with CV: Day 14]
Add crystal violet to each well (under fume hood) and put on shaker for 20 minutes For 24-well, 250ul
?. [Staining with CV: Day 14]
Remove crystal violet (under fume hood) and dispose of in appropriate hazardous container
?. [Staining with CV: Day 14]
Wash wells with DI water For 24-well, 250ul
?. [Staining with CV: Day 14]
Remove water and dispose of in crystal violet waste container
?. [Staining with CV: Day 14]
Fill ice bucket with DI water and gently dunk plates to rinse, repeat for a total of 2 dunks
?. [Staining with CV: Day 14]
Let plates air dry (overnight is usually best)
?. [Quantification with CTG: Day 14]
Quantifying with Cell Titer-Glo (Suspension Cells)
?. [Quantification with CTG: Day 14]
Without removing media, add 150 uL/well of CTG reagent (24-well plates) (undiluted)
?. [Quantification with CTG: Day 14]
Shake the plate on the shaker, 30 min @ Room Temp.
?. [Quantification with CTG: Day 14]
Aliquot 100 uL x 3 wells into opaque (white/black) 96-well plate
?. [Quantification with CTG: Day 14]
Read luminescenceUse the same Excel template as above (see STEP 17.1) for the analysis of the result.The intensity of crystal violet staining (or luminescence) of experimental samples (with knockdown) relative to those of negative and positive control samples will indicate how strong the viability effect is.
?. [Quantification of CV: After day 14]
After scanning pictures of plate(s), add 10% acetic acid to each well and put on shaker for at least 20 minutesFor 24-well, 325ul
?. [Quantification of CV: After day 14]
Mix well, add 100ul in triplicates into clear bottom 96-well plate
?. [Quantification of CV: After day 14]
Add 10% acetic acid as account for background to empty well(s)
?. [Quantification of CV: After day 14]
Read absorbance at 595 nm
?. [Quantification of CV: After day 14]
Dispose of acetic acid + crystal violet in appropriate hazardous waste container
?. [Reagent Preparation/Set-Up]
Select cell lines that are putatively dependent (dep) or nondependent (nondep) on a gene of interest using depmap.org. Usually, 3 or more cell lines from both dep and nondep classes are selected.
?. [Reagent Preparation/Set-Up]
OPTIONAL: If the original medium for the cell line is not RPMI, consider adapting the cells with RPMI + 10% fetal bovine serum (FBS) so that all the cell lines are grown in the same media.
?. [Reagent Preparation/Set-Up]
Plasmids/CloningWe created the pXPR_BRD023-D lentivector to express dCas9 + KRAB alongside sgRNAs targeting our gene of interest in addition to positive and negative controls (sequences included in materials). Step 3.1: describes our method to create the pXPR_BRD023-D vectorSteps 3.2 to 3.7: describes insertion of specific sgRNA sequences for the gene of interest. and controls.
?. [Reagent Preparation/Set-Up]
The Cas9 sequence was removed from all-in-one CRISPR KO plasmid, pXPR_BRD023, and replaced with the dCas9-KRAB sequence from pXPR_BRD121. Restriction enzymes Nhel (BRD023),  XbaI(BRD121), and BamHI(both) were used for digestion. The dCas9-KRAB insert and BRD023 backbone were subsequently ligated, transformed, and plasmid DNA extracted.
?. [Staining with CV: Day 14]
Take images of plates once dry using scannerPlace plates face up (bottom of well is closer to scanner)Resolution: 600 dpi
?. [Reagent Preparation/Set-Up]
·Ligate digested BRD023-D backbone to each pair of annealed sgciRNA oligos. ·Immediately transform ligation mix to Stbl3 competent cells from ThermoFisher (Cat: C737303). ·Leave on ice for 10 minutes, heat shock at 42C for 30 seconds, recover by adding 200ul of SOC media per transformation, and incubate at 37C for 1 hour.·Plate 50ul of each transformation mixture on LB agar + Carbenicillin plates and incubate at 37C overnight (16 hours).
?. [Reagent Preparation/Set-Up]
Order oligos for sgRNA sequences:3 sgRNAs targeting the gene to be tested2 positive controls and 1 negative control:  AB1Negative control (intergenic sequence) Chr2-2GGTGTGCGTATGAAGCAGTG2Positive control 1 POLR2DCGGCGGGTGGCAGCGATCCG3Positive control 2 SF3B1AAGATCGCCAAGACTCACGA
AB1Negative control (intergenic sequence) Chr2-2GGTGTGCGTATGAAGCAGTG2Positive control 1 POLR2DCGGCGGGTGGCAGCGATCCG3Positive control 2 SF3B1AAGATCGCCAAGACTCACGA
?. [Reagent Preparation/Set-Up]
Anneal oligo sgciRNA oligo pairs. For each pair, add the following in a PCR tube.·1.5 μL of forward oligo (100uM)·1.5 μL of reverse oligo (100uM)·5 μL of 10x NEB buffer 3.1·42 μL ddH20----------------------50ul total·Incubate for 4 min at 95°C, 10 minutes at 70C and then let cool to room temperature.
?. [Reagent Preparation/Set-Up]
Digest the generic pXPR_BRD023-D vector with NEB enzymes, EcoRI and BsmBI (visit NEB website for digestion procedures consistent with enzyme parameters). Purify backbone and insert via agarose gel.
?. [Reagent Preparation/Set-Up]
Pick 6 bacterial colonies per plate and amplify each colony in 4ml of LB + 50ug/ml Carbenicillin media for 16 hours at 37C in a bacterial shaker.
?. [Reagent Preparation/Set-Up]
Perform miniprep (per manufacturer's instructions) on all samples to extract plasmid DNA.
?. [Reagent Preparation/Set-Up]
Prepare lentivirus for each CRISPRi all-in-one construct using 15 cm-plates (protocol for generic virus prodocution is included in attached files).
?. [Reagent Preparation/Set-Up]
Determine the number of 15 cm plates for virus production per type of virus based on the need and resource available (we typically need 5 or more 15 cm plates per virus type per target).
?. [Reagent Preparation/Set-Up]
Filter media containing virus via 0.45 um filter.
?. [Reagent Preparation/Set-Up]
Concentrate the virus to approximately 2 mL per 15 cm plate, using Amicon Ultra-15 Centrifugal FIlter Unit (30K NMWL, MIlipore SIgma, C7715).
?. [Reagent Preparation/Set-Up]
To concentrate virus,  spin ~15mLs of virus-containing media at 2500g's for 30 minutes at 4°C. We use 30kDa or 100kDa 15-mL filter centrifuge tubes. This approach typically yields  ~ 1mL of concentrated virus.
?. [Virus Titration]
A no infection well with antibiotic selection is helpful to check on the selection progress of cells
?. [Quantification of CV: After day 14]
Use an Excel template attached here to analyze the result
?. [Virus titration/infection: Day 0 ]
24 hours after seeding/infecting change medium  ABCD1Purono Puro2PuroPuro3PuroPuroPuro: 10% FBS RPMI + 2 ug/mL puromycin, 1 mLno Puro:  10% FBS RPMI, 1 mL
ABCD1Purono Puro2PuroPuro3PuroPuro
?. [Virus titration/infection: Day 0 ]
48 hours after starting Puro selection, pick the well withNo apparent death by PuromycinNo apparent virus toxicity