4. Protocol Overview

Both the CaT-Active (CaT-A) and CaT-Background (CaT-B) reagents contain lentiviruses that express RFP and GFP from separate transcripts, each driven by its own promoter. The RFP transcript is driven by the strong CMV or EF1L promoter, while GFP is driven by the weak UbiC promoter (see Figure below).

CaT-A and CaT-B with CMV lentiviral vector

CaT-A and CaT-B with EF1L lentiviral vector

The CaT-A lentivirus expresses a sgRNA targeting the UbiC promoter. When transduced into cells expressing a functional dCas9-TA fusion protein (such as Cellecta’s dCas9-VPH), GFP expression increases in response to the sgRNA/dCas9-TA mediated transactivation of the UbiC promoter.

The CaT-B lentivirus expresses a control non-targeting sgRNA which does not recruit the dCas9-transactivator onto the UbiC promoter, so GFP expression is unaffected.

To assay cells for dCas9-TA activity, transduce two separate samples of the same dCas9-TA expressing cells with the CaT-A and CaT-B viruses. GFP fluorescence in the cells transduced to the CaT-A virus will be significantly increased as compared to the CaT-B cells. RFP fluorescence in both samples will be unaffected. The difference in the mean GFP fluorescence between the transduced cells of the two samples (normalized against the mean RFP fluorescence) provides a quantitative measure of the activity of the dCas9-TA transactivator in the dCas9-TA cells.