CRISPR-Test Cas9 and dCas9 Activity Test Kits

Measure the activity of the Cas9 variant expressed by your cells

Cellecta CRISPR-Test Assay Kits provide a convenient flow assay to accurately measure the activity of the Cas9 variant expressed by your cells. Each kit contains a mix of lentiviral particles with two different fluorescent markers. One fluorescent marker responds to the Cas9 or dCas9-variant activity measured by the kit, and the other marker provides a normalization control.

• Measure functional activity of Cas9 nuclease, dCas9-repressor, or dCas9-activator in any cell line
• Quantitative, flow-based, read-outs
• Convenient, complete, easy-to-use kits with ready-to-transduce lentiviral particle

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) in your engineered cells 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 the level of one of the fluorescent markers relative to the other be assessed by flow analysis and provides an accurate measurement of the activity of the Cas9 variant expressed in the cells.

How It Works

Fluorescent-Reporter CRISPR-Test™ Assays directly knockout, knockdown, or induce a GFP reporter gene. Whereas, Essential-Gene CRISPRtest™ Cas9 Assays knockout of an essential endogenous gene required for viability, which leads to loss of cells with the fluorescent marker. Both assays have a similar protocol:

(1) Transduce your target population of putative Cas9, dCas9-activator, or dCas9-repressor cells with the ready-to-transduce lentiviral mix which contains fluorescently labeled lentiviral constructs, and sgRNA compatible with the Cas9 variant being measured.

(2) Determine the baseline fluorescence ratio of two fluorescent markers by flow cytometry.

(3) After several days, again measure the fluorescent cells by flow.

(4) Assess Cas9 or dCas9-variant activity based on the difference in the initial vs. end-point fluorescence ratios.

Applications

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.

The Fluorescent-Reporter CRISPR-Test™ Assays for Cas9, dCas9-activator, and dCas9-Repressor directly knockout, knockdown, or induce expression of a GFP reporter gene, which leads to loss (or activation for the dCas9-activator assay) of fluorescence. These assays work in any mammalian cell and typically can be run in ~4 days.

The Essential-Gene CRISPRtest™ Cas9 Activity Assays knockout an essential gene required for cell viability. The disruption of this gene leads to loss of cells with the fluorescent marker. A construct with a non-targeting sgRNA has a second fluorescent marker, which is used for normalization. These assays are specific for a target organism--human or mouse cells. Since loss of fluorescence is dependent on cell death, the assay depends on the cell growth rate and usually take 6-10 days to perform. These assays are offered with both GFP and BFP readouts.