CRISPR Functional Cas9 Activity Test Kits

 The CRISPR test assays allow for measurement of functionalactivity of Cas9 nuclease, dCas9-repressor, or dCas9-activator in any mammalian cell line. The results are obtained in a quantitative FACS-based read out. All of the kits are complete with read-to-transduce lentiviral particles. They are useful for assessing Cas9 activity in cells before the starting CRISPR screens or CRISPRa or CRISPRi gene modulation.  There are two different mechanisms to measure the different Cas9 variants.

• The Fluorescent-Reporter CRISPR-Test™ Functional Assays directly knock out, knockdown, or induce a GFP reporter gene

• The Essential-Gene CRISPRtest™ Cas9 Activity Assays knock out an endogenous gene required for viability, leading to loss of cells with fluorescent marker.

Both assay mechanisms follow a similar protocol. The GFP fluorescent markers respond to specific Cas9 activity, while the RFP fluorescent markers provide normalization control. After transducing the target population with fluorescently labeled lentiviral constructs, determine the baseline GFP:RFP fluorescence ratio using flow cytometry. Then after several days, measure the fluorescence ratio by flow and compare the difference in initial vs end-point fluorescence. 

These tests measure the Cas9 nuclease activity in any vertebrate cell line. Using a FACS-based assay, the kit contains reagents to measure the nuclease activity in cells expressing Streptococcus pyogenes Cas9. It contains pseudoviral lentiviral constructs with GFP whose fluorescence is disrupted with active Cas9. The activity is measured by loss of GFP in the cell line.