4.1. Assay Procedure

Two populations of putative Cas9-positive cells are transduced with the CRISPRuTest™ CT-A and CT-B viral mix reagents, respectively. After 4 days, each population is analyzed by flow by cytometry. The disappearance of GFP in the CT-A population as compared with the CT-B population provides a quantitative measure of the Cas9 activity in the target cells.

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 (see Additional Required Materials). 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.

*Start the experiment with actively growing cells. Maintain cells in log phase throughout the entire experiment.

Day 0

1. Quickly thaw the CRISPRuTest™ CT-A and CT-B lentiviral particles 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.

2. Suspend Cas9+ cells in growth medium supplemented with 1X Transduction Reagent, at a density of ca. 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.

3. Aliquot 1 ml of cell suspension (100,000 cells) into each of 2 wells of a 12-well plate.

4. Add 20 µl of CT-A virus into one well and 20 µl of CT-B virus into the other well, and then mix and return cells to incubator.

Note: For most cell lines, 20 µl of CT viral reagents will suffice to obtain at least 50% RFP+ cells (the recommended minimum percentage of transduced cells for optimal assay sensitivity). For hard-to-transduce cell lines, more virus might be needed. If in doubt, it is recommended to set up two sets of transductions with 20 µl of CT-A and CT-B as described above, and 50 µl of each in the second transductions. For the final calculation, use the samples that have 50%-80% RFP+ cells on Day 4. A control lentiviral vector may be used before running the assay to test the transduction efficiency of your cells (see Additional Required Materials).

Days 1-6

Exchange medium with fresh growth medium and grow cells under standard conditions for 3 days. Passage cells as needed. Cells should not become confluent.

Day 7

1. Analyze cells by flow cytometry, using settings below:

Channel 1: excitation 488nM, emission 530/20nm (GFP)
Channel 2: excitation 561nM, emission 590/20nm (RFP)

2. For both CT-B and CT-A samples, gate in RFP+ cells as shown below:

#_Plot gated cells of CT-B sample GFP vs. RFP as shown below, adjusting Channel 1 and 2 intensity so that GFP and RFP signal falls into a diagonal. Then, gate all cells below diagonal (“GFP_low” cells):

3. Do the same for CT-A sample, applying the same “GFP_low” gate:

*While some Cas9 lines can show a significant loss of GFP activity as soon as Day 7, depending on the cell line readings may have to be taken at multiple time points until the loss of GFP activity plateaus. Some cell lines may take 10, 14, or even 21 days to reach maximum activity.

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