The CRISPRaTest™ Functional dCas9-Activator Assay Kit measures functional dCas9 transactivator (e.g., dCas-VPH) activity in any mammalian cell system. The CRISPRaTest Kit provides reagents to measure the activator strength of any Streptococcus pyogenes dCas9 transactivator (dCas9-TA) hybrid protein in cells designed to be used for CRISPRa experiments using a FACS-based assay. 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 promoter while the GFP gene is driven by the weak UbiC promoter (see Figure below).
The CaT-A lentivirus expresses an sgRNA targeting the UbiC promoter of the GFP gene. When transduced into cells, also expressing a functional dCas9-TA fusion protein (such as Cellecta’s dCas9-VPH), GFP expression increases in response to the sgRNA mediated dCas9-transactivation. The CaT-B lentivirus also expresses an sgRNA but it is non-targeting so it cannot recruit the dCas9-transactivator and GFP expression is unaffected.
The kit provides enough reagents to measure this activity in 5-15 cell lines.