This protocol should be used ONLY if you do not have the 561 nm (or 530 nm) laser to detect the RFP protein. The CRISPRtest and CRISPRtest_blue kits can be used even if you do not have access to a flow cytometer that can detect TagRFP (Ex.: 561 nm / Em.: ~600 nm or Ex.: 530 nm / Em.: ~600 nm).
Day 0 – Transduction
Start the experiment with actively growing cells. Maintain cells in log phase throughout the
- Suspend parental cells and Cas9 cells in growth media supplemented with 1X CRISPRtest Transduction Reagent (dilute supplied Transduction Reagent 1,000-fold in growth media), at a density of 100,000 cells/ml.
- 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 (depending on the version of the kit) in the 4 different wells, mix, and incubate for 24 hours at 37°C, 5% CO2.
Day 1 – Media Change
24 hours post-transduction, replace cell growth medium with fresh growth medium. Continue incubation in the CO2 incubator for an additional 48 hours.
Day 3 – CRISPRtest_Blue Dilution Selection
- Collect an aliquot of cells from each well and analyze by flow cytometry (FACS analysis) using the following settings:
Channel 1: Excitation 488 nm, Emission ~530 nm (for tagGFP2 — CRISPRtest with GFP)
Channel 1: Excitation 400 nm, Emission ~450 nm (TagBFP2 — for CRISPR_blue)
- For each sample, determine the percentage of GFP+ (or BFP+) cells.
- For each cell line, choose the CRISPRtest Virus dilution (from Step 3) where the GFP (or %BFP) is in the range of 10%-20 (it should be the same dilution for both cell lines), then discard the other dilutions.
- Grow the selected CRISPRtest dilutions for an additional 7 days. As cells must be in log phase for the whole duration of the experiment, passage the cells accordingly.
Day 10 – Read
- Analyze cells by flow cytometry, using the same settings as on Day 3.
- Calculate the %GFP (or %BFP) in both cell lines.
- Assess Cas9 activity using the following formula:
%KO = 1 – [%GFP (or BFP) in Cas9 cells / GFP (or BFP) in Parental cells]
The closer the KO value is to 100, the more active Cas9 is in the cells.
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