TITLE: Assessment of Cas9 Activity in Cas9 Transduced Cancer Cell Lines
AUTHORS: Verity Goodwin, Emily Souster, Charlotte Beaver, Adam Jackson, Rizwan Ansari, Mathew Garnett, Fiona Behan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div><div class = "text-block"><span style = "font-weight:bold;">Process diagram:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/11f7e279594fdb8947a627a79a011af77912ade8923993cae1dcb9e618960e5f/ccmqbdtbp.png" /></div></div>

[STEPS]
?. [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
?. [Day 1]
Prepare a cell suspension containing 1x10^6 cells in  media (this equates to 2.5x10^6 cells per well in  .
7.2 mL
1.8 mL
?. [Day 1]
?. [Day 1]
Prepare a cell suspension containing 1x106 cells in  media (this equates to 2.5x105 cells per well in  ).
7.2 mL
1.8 mL
?. [Day 1]
Add   polybrene (8µg/ml concentration) to the cell suspension and mix using a 10ml stripette and pipetboy.
6.4 µl
?. [Day 1]
Transfer   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.  ABCDE1WellTransfectionNumber of CellsCell + polybrene suspension (ml)Vector21Mock2.5 x10^51.8ml200µl media32Control2.5 x10^51.8ml200µl BFP/GFP43Reporter2.5 x10^51.8ml200µl BFP/GFP/gGFP
1.8 mL
ABCDE1WellTransfectionNumber of CellsCell + polybrene suspension (ml)Vector21Mock2.5 x10^51.8ml200µl media32Control2.5 x10^51.8ml200µl BFP/GFP43Reporter2.5 x10^51.8ml200µl BFP/GFP/gGFP
?. [Day 1]
Add   of media to the mock well, and   of the designated control or reporter virus to the appropriate well (see table 1). Mix well by rocking the plate gently.
200 µl
200 µl
?. [Day 1]
Place the plate in the incubator.
?. [Day 2]
if cells are   fresh media per well, without dislodging the cells.
2 mL
?. [Day 2]
If cells are >90% confluent, passage each well:
?. [Day 2]
Aspirate culture media from each well, and carefully wash each well with   PBS by angling the pipette at the side of the wells. Aspirate PBS.
2 mL
?. [Day 2]
Add   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.
500 µl
?. [Day 2]
When cells have detached from the base of the well, add  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.
500 µl
?. [Day 2]
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]
If cells are >90% confluent, passage at a 1:2 split ratio, following steps 7.1 - 7.5.
?. [Day 4: Fixing ]
Detach and collect cells following steps 7.1 - 7.4. Carefully aspirate supernatent without disturbing the pellet.
?. [Day 2]
Collect the 1ml cell suspension in a labelled 1.5ml centrifuge tube and centrifuge
Centrifuge: 300 34, 3 min
Centrifuge using a minifuge inside the MSC.
?. [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.
?. [Flow Cytometry]
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.
?. [Flow Cytometry]
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.
?. [Flow Cytometry]
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.
?. [Flow Cytometry]
Finally, run and record 10,000 events for the reporter sample.
?. [Flow Cytometry]
Export the .fcs files. Carry out analysis using appropriate analysis software.
?. [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.
The following steps are for the use of FlowJo analysis software but can be adapted for use on other analysis software.
?. [FACS Analysis]
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.
?. [FACS Analysis]
Double-click inside the gate to open a new pane containing single cells.
?. [FACS Analysis]
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.
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.
?. [FACS Analysis]
Copy these gates across to the control sample and check that it identifies BFP+ cells as shown in Figure 3, right panel.
?. [FACS Analysis]
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.
Pass criteria: 75% cas9 positivity.
?. [FACS Analysis]
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.
?. [Day 4: Fixing ]
Follow "Fixing cell pellets" protocol found here: https://protocols.io/view/fixing-cell-pellets-bg2fjybn.html