Jul 23, 2020
Guide RNA Library Titration of Cas9 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 Titration of Cas9 Cell Lines. protocols.io https://dx.doi.org/10.17504/protocols.io.bgxujxnw
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 29, 2020
Last Modified: July 23, 2020
Protocol Integer ID: 37588
Abstract
This protocol is for the gRNA library titration of cas9 expressing cancer cell lines using the Kusuke Yusa v1.1 whole genome gRNA library. gRNA library titration is performed on all the cas9 cancer cell lines prior to gRNA library screening. The library titration allows us to determine the volume of library virus required to transduce cas9 cancer cells at 30% transduction efficiency which is calculated using FACS analysis.
Process diagram:
Guidelines
This protocol is for the titration of 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 aim of this protocol is to identify how much virus to dose each cell line for transduction to avoid host cells taking up more than one gRNA, therefore an MOI of 30% should be aimed for.
The volumes of library titrated may differ depending on the batch of virus, it may require diluting prior to titrating. It may also differ for different libraries. An increased number of titration points may also be required. This may require some R&D to determine optimal volumes to titrate.
This protocol takes 4 days.
1.25x106 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
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
- P200 pipette and tips
- P20 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 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
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 1.25x106 cells in 8 mL complete media to make a final concentration of 1.56x106 cells per ml.
Add 6.4 µL of polybrene (a concentration of 8µg/ml) to the cell suspension and mix well using a 10ml stripette and pipetboy.
Transfer 1.6 mL of the cell suspension into 4 wells of a 6 well plate (this equates to 2.5x105 cells per well) and add the relevant media volume, see table 1, column 4.
| Titration Volume (µl) | Cell + polybrene suspension (ml) | Volume of media (ml) | |
| 0 | 1.6 | 400 | |
| 15 | 1.6 | 385 | |
| 50 | 1.6 | 350 | |
| 150 | 1.6 | 250 |
Table 1. Library titration volumes, per well of a 6 well plate
FIgure 1. Library titration plate layout
Dilute the gRNA library in complete media if required. The defrosted gRNA 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.
Add the diluted gRNA library volumes in Table 1 to the 6 well plate according to the layout in Figure 1. Mix well by rocking the plate gently.
Place the plate in the incubator.
Day 2
Day 2
Carefully replace media with 2 mL fresh media per well, without dislodging the cells.
Day 4: Fixing
Day 4: Fixing
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.
Note
Day of harvest may be adjusted if cell lines take longer to express BFP. Some optimisation may be required.
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 . Carefully aspirate the supernatent without dislodging the pellet.
Note
Centrifuge using a minifuge inside the MSC.
Follow "Fixing Cell Pellets" protocol found here: https://protocols.io/view/fixing-cell-pellets-bg2fjybn.html
Flow Cytometry and analysis
Flow Cytometry and analysis
Set up an experiment ensuring parameters: FSC-A, FSC-W, SSC-A, 450/50-A (BFP) are selected.
Note
The following steps are for use on the BD LSRFortessa flow cytometer. It can be adapted for use on other flow cytometers.
Run the mock sample (0) 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 these cells (see Figure 2, top panel). If required, adjust FSC and SSC voltages so that the main population lies within the plot.
Note
FSC will need to be reduced for very large cell lines.
Figure 2. Setting up plots and gates for the mock sample
Check that the mock cells are negative for BFP using 450/50 (405)-A vs SSC-A and insert a polygon gate with the negative population positioned to the left of the gate, see Figure 2, bottom left panel. Run and record 10,000 events per sample.
Next run the titrated samples, starting with the lowest volume of virus (15µl). The BFP positive cells should appear in the BFP+ gate, shown in Figure 2, bottom right panel. Run and record 10,000 events per sample.
Note
Library infectivity is defined as the percentage of single cells that are BFP positive.
Export FCS files. Carry out analysis using appropriate analysis software, such as FlowJo. Analysis and gating protocol are the same used to set up the flow cytometer as shown in Figure 2.
Export data as the percentage of single cells that are BFP positive as an excel file for each titration.
Plot a titration curve in Microsoft Excel by plotting the percentage of BFP% cells at each virus concentration, to calculate the amount of virus required for 35% transduction efficiency based on the linear portion of the curve. See Figure 3.
Note
Although the transduction efficiency of 30% is ideal, we aim for 35% when scaling up to account for errors when increasing the surface area from a 6 well plate to a flask.
For example, in Figure 3, the equation to determine the amount of virus that infects 35% of cells in one well of a 6 well plate is (35-11.324)/0.5057= 48.8µl. This value can then be scaled up to the desired surface area for transduction.
Figure 3. Example of transduction efficiency titration curve