4.1. Assay Procedure

Parental and dCas9-Rep cells are transduced with the CRISPRiTest™ Virus (CiT Viral Mix). After 4 days, cells are analyzed by flow cytometry to compare the mean RFP and GFP fluorescence levels. The mean GFP and RFP fluorescent values are then used to calculate dCas9-Rep activity in dCas9-Repressor cells.

The assay was optimized using MDA-MB-231 cells. Parental and dCas9-KRAB MDA-MB-231 cells are available from Cellecta to 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.

Day 0

1. Quickly thaw the CiT Viral Mix 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 -70°C, and used again for subsequent experiments.

2. Suspend both parental and dCas9-Rep cells in growth medium supplemented with 1X CRISPRTest Transduction Reagent, at a density of ca. 100,000 cells/ml.

3. For each cell line, aliquot 1ml of cell suspension/well in 1 well of a 12-well plate.

4. Add 40µl of CiT Viral Mix to each well, mix and return cells to incubator.

Note: For most cell lines, 40µl of CiT Viral Mix would suffice to obtain at least 70% RFP+ cells (the recommended minimum % of transduced cells for optimal assay sensitivity). For hard to transduce cells, more virus (and/or spinoculation) might be needed. When in doubt, it is recommended to use 40µl, 80µl, and 160µl of CiT Viral Mix in separate wells. For the final calculation, use the samples that have 70%-85% 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).

Day 1

Replace medium with fresh growth medium, grow cells under standard conditions for 3 days. Passage cells as needed. Cell should not become confluent.

Day 4

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. Calculate the mean GFP and mean RFP intensity for both parental and dCas9-Rep transduced cells, then calculate the relative GFP/RFP expression for each sample (normalized GFP intensity).

Transcriptional repression efficiency in the dCas9-Rep cell line is calculated as the ratio between the normalized GFP intensity of Parental Cells and the normalized GFP intensity of dCas9-Rep Cells.

Note: The Calculated Transcriptional repression efficiency is an underestimation of the actual transcriptional repression, due to % of non-transduced cells.