4.4. Alternative Protocol when Unable to Detect RFP (561 nm)

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
entire experiment.

1. 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.

2. For each cell line, aliquot 1 ml of cell suspension per well in 4 wells of a 12-well plate.

3. 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

1. 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)
   

2. For each sample, determine the percentage of GFP+ (or BFP+) cells.

3. 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.

4. 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

1. Analyze cells by flow cytometry, using the same settings as on Day 3.

2. Calculate the %GFP (or %BFP) in both cell lines.

3. 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.