Both the CT-Active (CT-A) and CT-Background (CT-B) reagents contain a lentivirus expressing RFP and GFP from one transcript, separated by a T2A peptide linker sequence (see Figure below). Transduction of either of these reagents into cells will initially produce cells expressing both GFP and RFP fluorescence.
The difference between the two viral reagents is that the CT-A expresses a GFP-targeting sgRNA, whereas the CT-B viral reagent expresses a non-targeting sgRNA. Therefore, within a few days after transduction, CT-A transduced cells will lose GFP and only have RFP fluorescence, whereas CT-B transduced cells will continue to exhibit both RFP and GFP fluorescence. The decline in GFP fluorescence in the CT-A cell population, then, can be used to assess Cas9 activity and forms the basis of the CRISPRuTest assay.
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