CRISPRtest™ Essential-Gene Cas9 Activity Kit is an easy-to-use, highly sensitive kit for measuring functional Cas9 activity in mouse cell lines. The kit contains pseudoviral-packaged, lentiviral constructs with green fluorescent protein (GFP), and an sgRNA that targets an essential gene. After transduction, knockout of the target gene kills off the cells that picked up the GFP marker. Measurement of the depletion of these fluorescently labeled cells, normalized against the negative control BFP cells, provides a quantitative estimate of Cas9 activity.
This Assay is similar to the CRISPR-Test Cas9 Fluorescent Reporter Assay in that both use the disappearance of a fluorescent marker to assess Cas9 activity. However, the mechanism of the Essential-Gene Cas9 and Fluorescent Reporter Assays is a bit different. The Fluorescent Report Assay directly disrupts the GFP reporter, whereas the Essential-Gene Cas9 Assay relies on cell death induced by disruption of a gene required for viability. The Essential-Gene Cas9 Assay take a few more days to complete, but more closely mimics the phenotypic change that occurs in a “dropout” viability screen with a pooled lentiviral CRISPR library.
The kit provides enough reagents to measure this activity in 5-10 cell lines.