Jul 23, 2020
Assessment of Cas9 Activity in Cas9 Transduced 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. Assessment of Cas9 Activity in Cas9 Transduced Cancer Cell Lines. protocols.io https://dx.doi.org/10.17504/protocols.io.bgvtjw6n
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: May 27, 2020
Last Modified: July 23, 2020
Protocol Integer ID: 37523
Abstract
Cas9 expressing cancer cell lines need to be assessed for cas9 activity to ensure they are capable of efficiently knocking out genes. The activity is assessed by transducing the cells with a BFP-GFP vector containing an anti-GFP guide RNA. If the cas9 is active, the GFP will be knocked down so these cells will express BFP but not GFP as detected by flow cytometry.
Process diagram:
Guidelines
Cas9 cancer cell lines should be produced following the protocol: Cas9 Transduction of Cancer Cell Lines. Cas9 lines should be over 50% confluent in a T150 flask within 4 weeks of transduction to be assessed for cas9 activity.
This protocol takes 4 days.
1x106 cells are required for this protocol.
Materials
MATERIALS
DPBSInvitrogen - Thermo FisherCatalog #14190
TrypLE™ Express Enzyme (1X), no phenol redThermo FisherCatalog #12604021
6 Well Clear TC-Treated Multiple Well PlatesCorningCatalog #3516
Polybrene Infection / Transfection ReagentEmd MilliporeCatalog #TR-1003-G
Falcon Round bottomed 5ml tube with cell strainer lidVWR international LtdCatalog #734-0001
Microcentrifuge tube Safe-Lock write-on 1.5mL Eppendorf TubeEppendorfCatalog # 0030 120.086
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.
BFP/GFP/gGFP Vector (Reporter Vector): lentiviral reporter pKLV2-U6gRNA(gGFP)-PGKBFP2AGFP-W
BFP/GFP Vector (Control Vector): pKLV2-U6gRNA(Empty)-PGKBFP2AGFP-W
- Further information regarding these vectors can be found in Figure S1:
Tzelepis K, Koike-Yusa H, De Braekeleer E, et al. A CRISPR Dropout Screen Identifies Genetic Vulnerabilities and Therapeutic Targets in Acute Myeloid Leukemia.Cell Rep. 2016;17(4):1193-1205. doi:10.1016/j.celrep.2016.09.079
Equipment
- Microbiological Safety Cabinet (MSC)
- Centrifuge
- Microfuge
- 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
Pre-warm cell culture media to room temperature
Thaw 10mg/mL polybrene to room temperature
Thaw BFP/GFP Vector (Control Vector) to room temperature
Thaw BFP/GFP/gGFP Vector (Reporter Vector) to room temperature
Day 1
Day 1
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
Prepare a cell suspension containing 1x106 cells in 7.2 mL media (this equates to 2.5x105 cells per well in 1.8 mL ).
Add 6.4 µL polybrene (8µg/ml concentration) to the cell suspension and mix using a 10ml stripette and pipetboy.
Transfer 1.8 mL of the cell suspension into 3 wells of a 6 well plate to include a mock, control, and reporter transduction with the final cell number and reagent volumes shown in table 1. Ensure the plate is clearly labelled as per Figure 1.
| Well | Transfection | Number of Cells | Cell + polybrene suspension (ml) | Vector | |
| 1 | Mock | 2.5 x10^5 | 1.8ml | 200µl media | |
| 2 | Control | 2.5 x10^5 | 1.8ml | 200µl BFP/GFP | |
| 3 | Reporter | 2.5 x10^5 | 1.8ml | 200µl BFP/GFP/gGFP |
Table 1. Reagents per well of a 6 well plate for transduction set up.
Figure 1. 6 well plate layout
Add 200 µL of media to the mock well, and 200 µL of the designated control or reporter virus to the appropriate well (see table 1). Mix well by rocking the plate gently.
Place the plate in the incubator.
Day 2
Day 2
if cells are <90% confluent, carefully replace media with 2 mL fresh media per well, without dislodging the cells.
If cells are >90% confluent, passage each well:
Aspirate culture media from each well, and carefully wash each well with 2 mL PBS by angling the pipette at the side of the wells. Aspirate PBS.
Add 500 µL TrypLE to each well and place in the incubator for 3-5 minutes. Use the microscope if necessary to check that all the adherent cells have been dislodged. If not, return them to the incubator for a few more minutes until detached.
When cells have detached from the base of the well, add 500 µL complete media to each well. Mix by pietting up and down using a P1000, and wash the base of the well ensuring cells are fully detached.
Collect the 1ml cell suspension in a labelled 1.5ml centrifuge tube and centrifuge 300 x g, 00:03:00
Note
Centrifuge using a minifuge inside the MSC.
Carefully aspirate the supernatent without disturbing the cell pellet. Resuspend each pellet in an appropriate volume of media to carry out either a 1:2 or 1:3 split ratio, dependent on growth rate. Discard extra cells.
Day 3
Day 3
If cells are >90% confluent, passage at a 1:2 split ratio, following steps 7.1 - 7.5.
Day 4: Fixing
Day 4: Fixing
Detach and collect cells following steps 7.1 - 7.4. Carefully aspirate supernatent without disturbing the pellet.
Follow "Fixing cell pellets" protocol found here: https://protocols.io/view/fixing-cell-pellets-bg2fjybn.html
Flow Cytometry
Flow Cytometry
Set up an experiment ensuring that parameters FSC-A, FSC-W, SSC-A, 450/50-A (BFP) and 530/30-A (GFP) are selected.
Run the mock sample first and use this to separate debris (FSC-A vs SSC-A) and identify single cells (FSC-A vs FSC-W). Use the polygon gate to select for this cells (see Figure 2). If required, adjust FSC and SSC voltages to ensure the main population lies within the plot.
Figure 2. Setting up plots and gates for the mock sample.
Check that the mock cells are negative for BFP and GFP using the filters: BFP 450/50 (405)-A and GFP 530/30 (488)-A and insert a polygon gate with the negative population positioned on the left side (Figure 2, bottom left panel). Run and record 10,000 events per sample.
Next, run the control sample. The control sample should contain cells that are positive for both BFP and GFP. Run and record 10,000 events.
Finally, run and record 10,000 events for the reporter sample.
Export the .fcs files. Carry out analysis using appropriate analysis software.
FACS Analysis
FACS Analysis
Using the analysis software, start by analysing the mock sample. Firstly, identify single cells by drawing a gate around the main population in FSC-A vs SSC-A, as shown in Figure 2, top left panel.
Note
The following steps are for the use of FlowJo analysis software but can be adapted for use on other analysis software.
Double-click inside the gate to open a new pane containing the selected cells. Identify single cells by drawing a gate around the population shown in Figure 2, top right panel.
Double-click inside the gate to open a new pane containing single cells.
Change the plot axis to show BFP vs SSC-A and draw a BFP+ gate based on the negative cells, see Figure 3, left panel.
Note
Cas9 activity is defined as the percentage of cells that have taken up the vector that have silenced GFP expression, so we need to identify BFP positive cells that are GFP negative.
Figure 3. Setting BFP+ gates for the mock and control cells
Copy these gates across to the control sample and check that it identifies BFP+ cells as shown in Figure 3, right panel.
Using the control cells, double-click on the BFP+ gate population and change the plot axis to BFP vs GFP as shown in Figure 4, left panel. Gate the region of BFP+ cells that are not GFP+. Name this gate Cas9.
Figure 4. Identifying Cas9 positive cells
Copy all the gates over to the reporter sample. Check that the Cas9 gate does not include any GFP+ cells. The majority of cells should have shifted to BFP+ GFP-. This will provide the percentage of cas9 positive cells in the cas9 cell line.
Note
Pass criteria: 75% cas9 positivity.