The EpiQuik™ CRISPR/Cas9 Assay ELISA Kit (Colorimetric) is suitable for specifically measuring CRISPR-associated protein 9 (Cas9 and dCas9: S. pyogenes) amounts quantitatively with use of purified Cas9 nuclease and whole cell extracts isolated from tissues and cultured cells of various species. The kit has the following advantages:
- Quick and efficient procedure, which can be finished within 3 hours.
- Innovative colorimetric assay in high throughput format to quantitatively measure Cas9 protein amount without the need for electrophoresis, especially suitable for screening Cas9-transfected clones.
- High sensitivity and specificity. The detection limit is as low as 0.1 ng/well with dynamic range of 0.5-20 ng/well within the indicated amount range of the whole cell extracts. Only recognizes Cas9 (S. pyogenes).
- The control is conveniently included for the quantification of Cas9 amount.
- Strip microplate format makes the assay flexible: manual or high throughput.
- Simple, reliable, and consistent assay conditions.
The discovery of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas9 (CRISPR associated system or CRISPR associated protein 9 nuclease) found in bacteria to work as a defense mechanism against foreign DNA has proven to be an invaluable tool to target and modify a genetic sequence in gene editing and genome engineering applications. The system, known as CRISPR/Cas9, allows for sequence-specific cleavage of a targeted genomic locus by delivering the RNA-guided Cas9 nuclease and appropriate guide RNAs (gRNA) into a cell. In addition, Protospacer Adjacent Motif (PAM) sequence immediately following the specificity sequence is necessary for successful binding of the Cas9 nuclease. It is important and critical to monitor the level of Cas9 editing protein or track the Cas9 editing protein in transfected cells, as it will tell transfection efficiency and optimize the editing process in the total cell population.
Principle & Procedure
This kit is designed for measuring Cas9 level in whole cell extracts or purified Cas9 proteins from various cells and tissues. In an assay with this kit, the Cas9 proteins in samples are tightly and stably spotted on the strip wells. The bound Cas9 proteins can then be recognized with detection antibody followed by a color development reagent. The ratio of Cas9 is proportional to the intensity of absorbance. The absolute amount of Cas9 can be quantitated by comparing to the Cas9 control.
Input materials should be purified Cas9 nuclease or whole cell extract. The amount of whole cell extract for each assay can be between 0.5 µg and 5 µg with an optimal range of 1 to 2 µg. Total volume of the input material should not be more than 4 µl.
WB (10X Wash Buffer)
CBB (Cas9 Binding Buffer)
DS (Developer Solution)
SS (Stop Solution)
8-Well Assay Strips (With Frame)
Adhesive Covering Film
DAb (Detection Antibody, 1000X)*
SI (Signal Indicator, 1000X)*
ES (Enhancer Solution, 1000X)*
Cas9 Standard Control (100 µg/ml)
*For maximum recovery of the products, centrifuge the original vial after thawing prior to opening the cap.