4. Protocol Overview

Both the CT-Active (CT-A/CT-A EF1L) and CT-Background (CT-B/CT-B EF1L) 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.

CRISPRuTest Vector Diagrams CT-A and CT-B

CRISPRuTest EF1L Vector Diagram CT-A EF1L and CT-B EF1L

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/saCas9 activity and forms the basis of the CRISPRuTest assay.