Cellecta CRISPR-Test Assay Kits contain a mix of lentiviral particles with both green fluorescent and red fluorescent protein (GFP and RFP). The GFP fluorescent marker responds to the Cas9 or dCas9-variant activity measured by the Kit, and the RFP fluorescence provides a normalization control.
Would you like to accurately measure the activity of CRISPR Cas9 nuclease (for CRISPR KO), dCas9-KRAB (for CRISPRi), or dCas9-activator such as dCas9-VPH (for CRISPRa) before starting a CRISPR screen or other CRISPR study? Cellecta’s CRISPR-Test™ Assay Kits provide a convenient and quantitative assay for this purpose. Each Assay Kit contains a mix of ready-to-transduce viral particles with one construct that is responsive to the Cas9 variant being measured and the other a negative control. Each of the constructs have different fluorescent protein markers (i.e., GFP or RFP) so the change in green fluorescent cells containing the responsive construct relative to the red cells with the negative control can be assessed by flow analysis.
Additionally, a set of four Cas9 expression constructs is available at a reduced price when purchased in conjunction with one of the CRISPR-Test Assay Kits.
(1) Transduce your target population of putative Cas9, dCas9-activator, or dCas9-repressor cells with the ready-to-transduce lentiviral mix which contains GFP-sensitive and RFP-control constructs.
(2) Determine the baseline GFP:RFP ratio by assessing the number of green to red fluorescent cells by flow cytometry.
(3) After 4-6 days, again measure the ratio of green to red fluorescent cells by flow.
(4) Assess Cas9 or dCas9-variant activity based on the difference in the initial vs. end-point green cell vs. red cell ratios.
Conveniently and accurately measure the activity levels of CRISPR Cas9, dCas9-repressors (e.g., dCas9-KRAB), or dCas9-activators (e.g., dCas9-VP64, dCas-VPR, or Cellecta’s dCas9-VPH). The assay works with any S. pyogenes Cas9 or dCas9 variant that complexes with a standard sgRNA design.
These Kits are useful for assessing Cas9 activity in cells before running CRISPR screens with pooled sgRNA libraries and for studies using CRISPRa or CRISPRi gene modulation where the dCas9-activators must be consistently expressed for the transactivation or repression of the target gene to remain in effect.