4.2. Cas9 Activity Calculation

The difference between the percentage of “GFP_low” cells in the CT-A population and percentage of “GFP_low” cells in the CT-B population indicates the percentage of cells which underwent Cas9-mediated GFP knockout in the CT-A sample. The percentage of “GFP_low” cells in the CT-B population should be close to 0% (i.e., no sgRNA targeting GFP), where as in the CT-A population it should be near 100% (sgRNA targeting GFP).

The flow cytometry data from the the previous section using the positive control cell line MDA-MB-231-Cas9-Blast (see Additional Required Materials to obtain this cell line) can be used to calculate the percentage of GFP knockout, which is a direct measurement of the Cas9 activity, as follows:

Percentage GFP Knockout in Target Cas9 cells = 93.8% – 1.62% = 92.2%

Note: If the GFP Knockout in the target cells is less than 80%, you should continue to incubate the cells for a few more days. For some cell lines, it may take 10, 14, or even 21 days to reach maximum activity.

Note: For a Cas9-negative cell line, the percentage of “GFP_low” cells in both CT-A and CT-B should be similarly close to 0%.