Jul 23, 2020
Guide RNA Library Transduction of Cas9 Cancer Cell Lines
- Verity Goodwin1,
- Emily Souster1,
- Charlotte Beaver1,
- Adam Jackson1,
- Rizwan Ansari1,
- Mathew Garnett1,
- Fiona Behan1
- 1Wellcome Sanger Institute
- Cellular Generation and Phenotyping
Protocol Citation: Verity Goodwin, Emily Souster, Charlotte Beaver, Adam Jackson, Rizwan Ansari, Mathew Garnett, Fiona Behan 2020. Guide RNA Library Transduction of Cas9 Cancer Cell Lines. protocols.io https://dx.doi.org/10.17504/protocols.io.bg2njyde
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 01, 2020
Last Modified: July 23, 2020
Protocol Integer ID: 37678
Abstract
This protocol is for the whole-genome CRISPR screening of stably expressing Cas9 cancer cell lines in triplicate using the commercially available Kusuke Yusa v1.1 whole genome gRNA library. It can be adapted for other gRNA libraries, under the assumption that there is a BFP reporter in the gRNA library.
The protocol can be followed assuming the following is known:
- The number of cells to be transduced
- The number of days required for screening
- How many cells to maintain throughout the screen
- The number of cells required per pellet
- The required coverage of the library
This protocol takes 16 days.
Process diagram:
Guidelines
This library transduction is carried out in 33 x106 cells per replicate. These 33 x106 cells should occupy ~80% of the available surface area. Determine the surface area for each cell line as follows: Using previously collected data, estimate the surface area required for 33 x106 cells and select the smallest flask size that will accommodate 33x106 cells at 80% confluency.
Unless otherwise noted, all steps should be performed under sterile conditions in a biological safety cabinet.
Materials
MATERIALS
DPBSInvitrogen - Thermo FisherCatalog #14190
TrypLE™ Express Enzyme (1X), no phenol redThermo FisherCatalog #12604021
6 Well Clear TC-Treated Multiple Well PlatesCorningCatalog #3516
Cell Culture Treated T150 FlasksCorningCatalog #430825
Falcon Tissue Culture Treated 3 Layer Multi-Flask 525 Cm2BD BiosciencesCatalog #353143
Falcon Tissue Culture Treated 5 Layer Multi-Flask 875 Cm2 BD BiosciencesCatalog #353144
Polybrene Infection / Transfection ReagentEmd MilliporeCatalog #TR-1003-G
DNA LoBind 2.0ml PCR Clean Eppendorf TubesEppendorfCatalog #0030 108.078
Puromycin (10mg/ml)InvivoGenCatalog #ant-pr-1
250ml Storage Bottle - Disposable With Plug Seal Cap Sterile 45mm neckCorningCatalog #430281
Microcentrifuge tube Safe-Lock write-on 1.5mL Eppendorf TubeEppendorfCatalog # 0030 120.086
Human Improved Genome-wide Knockout CRISPR LibraryaddgeneCatalog #67989
Select an appropriate culture media for your cell line. Common culture medias used for cancer cell lines are serum supplemented Advanced DMEM D-12 or RPMI in the presence of pen-strep.
Equipment
- Microbiological Safety Cabinet (MSC)
- Centrifuge
- Microfuge
- Media dispensing pump (recommended)
- Pipetboy
- Stripettes
- P1000 pipette and tips
- 37 °C waterbath
- 37 °C humidified incubator (5% CO2)
- Light microscope
Safety warnings
Chemical safety warnings:
Biological safety warnings:
- Cell lines may contain adventitious agents, including viruses. No attempt will be made to culture these agents deliberately. Correct use of PPE will drastically reduce the risks.
- Lentiviruses used in this protocol can infect human cells but are non-replicating and therefore the pathogenicity of these viruses is negligible. Correst use of PPE will drastically reduce the risks.
Before start
- Know library volume from library titration
- Pre-warm culture media to room temperature
- Thaw polybrene (10mg/ml)
- Thaw virus (the virus should only be thawed twice)
- All lentiviral waste should be deactivated with 1% Virkon solution for a minimum of 1 hour.
Day 1: Transduction
Day 1: Transduction
Detach and collect cas9 cells as per protocol "Passaging adherent cancer cell lines" steps 1-8 found here: https://protocols.io/view/passaging-adherent-cancer-cell-lines-bgtbjwin.html
Dilute the gRNA library if required, in complete media. The defrosted library should be stored on ice and used within 1 hour.
Safety information
Lentivirus: Lentiviruses used in this protocol can infect human cells but are non-replicating and therefore the pathogenicity of these viruses is negligible. Correst use of PPE will drastically reduce the risks.
Carry out each library transduction in triplicate. Prepare each replicate in a 250ml storage bottle as per Table 1 to transduce the cells at 100x coverage. Mix well, and seed into the appropriate labware. Clearly label each replicate (R1, R2, and R3).
Safety information
It is recommended to use a media dispensing pump to dispense volumes greater than 50ml.
Note
It is recommended to select the flask size for 33 million cells to comfortably fit for each cell line (see guidelines for more information).
| Components | T150 | T525 | T875 | 2x T875s | |
| Cells | 33 x10^6 | 33 x10^6 | 33 x10^6 | 33 x10^6 | |
| Polybrene (µL) | 8 µg/mL = 26.7 | 8 µg/mL = 93.33 | 8 µg/mL = 156.66 | 8 µg/mL = 313.32 | |
| Volume of virus library (mL) | (LT result x 15.6*) /1000 | (LT result x 54.7*) /1000 | (LT result x 91.1*) /1000 | (LT result x 182.3*) /1000 | |
| Media (mL) | 33.33 – (virus volume + cell volume + polybrene) | 116.67 – (virus volume + cell volume + polybrene) | 194.4 – (virus volume + cell volume + polybrene) | 388.8 – (virus volume + cell volume + polybrene) | |
| Total volume per flask (mL) | 33.33 | 116.67 | 194.4 | 388.8 |
*Scaling Factor: This is the calculated based on the surface area of a 6 well plate (9.6cm2) as this is the labware for the library titration, and the labware for the transduction.
Table 1. Table of components per replicate for transduction at 100x coverage for the gRNA library
Remove 1 mL of stock cell suspension and seed into two wells of a 6 well plate (500 µL per well). Add 1.5 mL media to each well. Place plate in the incubator. One well will be fixed on day 4 with a sample of transduced cells to act as a negative control to identify the transduction efficiency.
Note
Maintain the second well for the remainder of the screen, passaging when required. This well will be fixed when puromycin selection efficiency is assessed to act as the negative control.
Day 2
Day 2
Media change the flasks. Passage cells if required, keeping all cells (see protocol "Passaging adherent cancer cell lines", hyperlink in step 1).
Safety information
It is recommended to use a media pump to dispense volumes greater than 50ml.
Day 3
Day 3
Passage cells if required, keeping all cells (see protocol "Passaging adherent cancer cell lines", hyperlink in step 1).
Day 4: Puromycin Selection
Day 4: Puromycin Selection
Detach and collect cas9 cells as per protocol "Passaging adherent cancer cell lines" steps 1-8 (hyperlink in step 1).
Take an aliquot of ~200,000 cells in a 1.5ml tube to test transduction efficiency using FACS.
Note
The transduction efficiency for the library needs to be >15% to ensure a sufficient level of library representation. It should be ~30% for each replicate.
Detach and collect the negative control cells from the 6 well plate following steps 8-11 in the protocol "Library Titration of Cas9 Cancer Cell Lines" found here: https://protocols.io/view/guide-rna-library-titration-of-cas9-cell-lines-bgxujxnw.html
Fix the 4 cell suspensions following protocol "Fixing cell pellets" (R1, R2, R3, negative control) found here: https://protocols.io/view/fixing-cell-pellets-bg2fjybn.html
Carry out flow cytometry and FACS analysis following steps 16-20 in the protocol "Library Titration of Cas9 Cancer Cell Lines" (hyperlink in step 8.1).
Seed all remaining cells at a 1:3 split ratio (slow growing lines can be split 1:2) in appropriately sized labware (e.g.: if the cells were in 1x T875, re-seed all cells into 3x T875s) in the presence of puromycin at the concentration determined by the puromycin titration, see Table 2 for volumes.
Safety information
It is recommended to use a media pump to dispense volumes greater than 50ml.
Puromycin is toxic if swallowed and harmful in contact with skin.
| Size of Flask | Volume of Media (ml) | 1µg/ml Puromycin (µl) | 2µg/ml Puromycin (µl) | 3µg/ml Puromycin (µl) | 4µg/ml Puromycin (µl) | |
| T525 | 50 | 60 | 120 | 180 | 240 | |
| T875 | 100 | 100 | 200 | 300 | 400 |
Table 2. Volume of 1mg/ml puromycin stock to add to each flask
Day 7-11
Day 7-11
Note
It is important that at least 50 million cells are retained at every passage going forward to maintain the integrity of the library as this maintains 5 times the original 100x coverage.
Inspect cells under a microscope. If cells are <90% confluent, media change each flask with fresh media plus puromycin.
If cells are >90% confluent, passage each replicate, and carry out FACS analysis to determine if puromycin selection has been successful:
Detach and collect cas9 cells as per protocol "Passaging adherent cancer cell lines" steps 1-8 (hyperlink in step 1).
Take an aliquot of ~200,000 cells in a 1.5ml tube per replicate.
Detach and collect the negative control cells from the 6 well plate following steps 8-11 in the protocol "Library Titration of Cas9 Cancer Cell Lines" (hyperlink in step 8.1).
Fix the 4 cell suspensions following protocol "Fixing cell pellets" (R1, R2, R3, negative control) found here: https://protocols.io/view/fixing-cell-pellets-bg2fjybn.html
Carry out flow cytometry and FACS analysis following steps 16-20 in the protocol "Library Titration of Cas9 Cancer Cell Lines" (hyperlink in step 8.1).
Note
If puromycin selection has been successful, the proportion of BFP positive cells should be >80%
If puromycin selection was successful, re-seed a minimum of 50 million cells per replicate into an appropriate number of flasks, following protocol "Passaging adherent cancer cell lines" (hyperlink in step 1) without puromycin. The screen can be continued in the absence of puromycin.
If puromycin selection was unsuccessful, re-seed all cells into an appropriate number of flasks, following protocol "Passaging adherent cancer cell lines" (hyperlink in step 1) in the presence of puromycin.
Note
7 days post puromycin selection, media change the cells without puromycin. The remainder of the screen can be continued in the absence of puromycin.
Following puromycin selection, passage cells when >80% confluent, keeping a minimum of 50 million cells. If the cells are slow growing, keep a minimum of 100 million cells.
Note
If by day 10, cells have not been passaged, media change the flasks without puromycin.
If the cells do not require passaging until the day of pelleting, follow steps 11.2 - 11.4 to determine the puromycin selection efficiency on the day of pellet.
Day 14/15/16: Pellet
Day 14/15/16: Pellet
Pellet cells when confluent, between day 14 and day 16.
Detach cells, collect, and count each replicate following protocol "Passaging adherent cancer cell lines" steps 1-8 (hyperlink in step 1).
Note
Collect the cell suspensions in PBS at step 7.
Aliquot 25 x106 cells into up to 6x 2ml PCR clean/DNA LoBind tubes and centrifuge at 300 x g for 5 minutes using a microcentrifuge in the MSC.
Carefully remove the supernatent using a P1000 pipette, ensuring not to dislodge the cell pellet. These cell pellets can be stored in a -80 °C freezer until DNA extraction is required.