The CRISPRuTest™ Functional Cas9 Activity Assay Kit can be used to measure the Cas9 nuclease activity in any mammalian cell system. The CRISPRuTest Kit provides a FACS-based assay to quantitatively assess the Cas9 nuclease activity in cells expressing Streptococcus pyogenes Cas9.
The kit contains two vials of pseudoviral packaged lentiviral constructs, both expressing RFP and GFP from one transcript, separated by T2A. 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.
The kit provides enough reagents to measure Cas9 activity in 5-15 cell lines.