Catalog #: CRTEST
CRISPRtest™ Essential-Gene Cas9 Activity Kit is an easy-to-use, highly sensitive kit for measuring functional Cas9 activity in human or mouse cell lines. The kit contains two pseudoviral-packaged, lentiviral constructs--(1) that expresses an sgRNA that targets an essential gene and one fluorescent protein (e.g., GFP, BFP) as well as a second construct with a non-target sgRNA and a second fluorescent marker (usually RFP or BFP). After transduction, knockout of the target gene kills off the cells that picked up sgRNA with the one fluorescent marker. Measurement of the depletion of cells with the lethal sgRNA fluorescent protein as compared to ones with the other fluorescent marker and non-target sgRNA 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 is a bit different from Cas9 Fluorescent Reporter Assays. 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 takes 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.
Each kit provides enough reagents to measure this activity in 5-10 cell lines.