For this assay, you should transduce the CRISPRtest Virus into the Cas9-expressing cells and the parental cells which were used to generated the Cas9 cell line.
The assay was optimized using MDA-MB-231 cells. These Cas9-expressing MDA-MB-231 with blasticidin selection marker cells are available from Cellecta for use in the assay as a positive control. Some optimization may be needed based on the growth characteristics of your target cells. If a negative control is also desired, include a cell line not expressing Cas9 (ideally, the parental cells for the Cas9-positive target cells) in a parallel run of the assay.
Day 0 – Transduction
- Quickly thaw the CRISPRtest Virus in a water bath at 37°C. Transfer the thawed particles to a laminar flow hood, gently mix by rotation, inversion, or gentle vortexing, and keep on ice. Unused reagent can be aliquoted, refrozen at -80°C, and used again for subsequent experiments.
- Suspend parental cells and Cas9 cells in growth media supplemented with 1X CRISPRtest Transduction Reagent (dilute supplied Transduction Reagent 1000-fold in growth media), at a density of 100,000 cells/ml.
Note: This cell density was calculated for MDA-MB-231 cells. Depending on cell size and growth, you may need to use a different concentration and correspondingly-sized plate. As a rule of thumb, cells should be transduced at a density such that they would become confluent in ~48 hours. For the assay, you should plate at least 100,000 cells.
- For each cell line, aliquot 1 ml of cell suspension per well in 4 wells of a 12-well plate.
- Add 0 µl, 3 µl, 10 µl, and 30 µl of CRISPRtest (or CRISPRtest_Blue) Virus in the 4 different wells, mix, and incubate for 24 hours at 37°C, 5% CO2.
Day 1 – Change Medium
24 hours post-transduction, change cell growth medium with fresh growth medium. Continue incubation in the CO2 incubator for an additional 48 hours.
Day 3 – CRISPRtest Dilution Selection
- Collect an aliquot of cells from each well and analyze by flow cytometry (FACS analysis) using the following settings:
Flow Cytometry Settings for TagRFP Excitation: 561nm (yellow laser) [530nm laser is still acceptable] Emission: 590/20nm band-pass filter, or similar Flow Cytometry Settings for TagGFP2 Excitation: 488nm (blue laser) Emission: 530/20nm band-pass filter, or similar Flow Cytometry Settings for TagBFP Excitation: 405nm (violet laser) Emission: 470/20nm band-pass filter, or similar
- For each sample, calculate the percentage of GFP- RFP+ cells (i.e., %RFP) and the percentage of GFP+ RFP- cells (i.e., %GFP) or, if using CRISPRtest_blue, calculate BFP- RFP+ cells (i.e., %RFP) and BFP+ RFP- cells (i.e., %BFP), accordingly.
- For each cell line, choose the CRISPRtest Virus dilution (from Step 3) where the RFP is between 10%-20 (it should be the same dilution for both cell lines), then discard the other dilutions.
Note CRISPRtest Virus dilutions should give similar %RFP in both parental and Cas9 cell lines. At Day 3, the GFP:RFP (or BFP:RFP) ratio should be ~1 in both cell lines.
| Calculations | Parental Cells Day 3 | Cas9 Cells Day 3 |
|---|---|---|
| %RFP | 13% | 13% |
| %GFP (or BFP) | 18% | 17% |
| GFP (BFP) : RFP Ratio | = 18 / 13 = 1.38 | = 17 / 10 = 1.3 |
- Grow the selected CRISPRtest dilutions for an additional 7 days. Cells must be in log phase for the whole duration of the experiment so passage the cells as needed.
Day 10 – Read
- Analyze cells by flow cytometry, using the same settings as on Day 3.
- Calculate the fluorescent ratio in both cell lines and assess Cas9 activity
- Use the following formula to determine Cas9 activity:
%KO = 1 – [(GFP:RFP)Cas9 / (GFP:RFP)Parental] (for GFP/RFP kits)
%KO = 1 – [(BFP:RFP)Cas9 / (BFP:RFP)Parental] (for BFP/RFP kits)
The calculated KO value is the percentage of cells in the Cas9 cell line where the PCNA gene was knocked out (i.e., where CRISPR knockout was effective for both alleles). The closer this value is to 100, the more active Cas9 is in the cells.
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