This Cas9 (S. pyogenes) BioAssay(TM) ELISA Kit is a sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of endogenous levels of Cas9 (S. pyogenes). The CRISPR associated protein 9 (Cas9) is an RNA-guided DNA nuclease and part of the Streptococcus pyogenes CRISPR antiviral immunity system that provides adaptive immunity against extrachromosomal genetic material (1). The CRISPR antiviral mechanism of action involves three steps: (i), acquisition of foreign DNA by host bacterium, (ii), synthesis and maturation of CRISPR RNA (crRNA) followed by the formation of RNA-Cas nuclease protein complexes, and (iii), target interference through recognition of foreign DNA by the complex and its cleavage by Cas nuclease activity (2). The type II CRISPR/Cas antiviral immunity system provides a powerful tool for precise genome editing and has potential for specific gene regulation and therapeutic applications (3). The Cas9 protein and a guide RNA consisting of a fusion between a crRNA and a trans-activating crRNA (tracrRNA) must be introduced or expressed in a cell. A 20-nucleotide sequence at the 5 end of the guide RNA directs Cas9 to a specific DNA target site. As a result, Cas9 can be programmed to cut various DNA sites both in vitro and in cells and organisms. CRISPR/Cas9 genome editing tools have been used in many organisms, including mouse and human cells (4,5). Research studies demonstrate that CRISPR can be used to generate mutant alleles or reporter genes in rodents and primate embryonic stem cells (6-8). Assay Principle: To perform the assay, sample is incubated with a capture antibody conjugated with a proprietary tag and a second detection antibody linked to HRP, forming a sandwich with Cas9 (S. pyogenes) in solution. This entire complex is immobilized to the plate via an anti-tag antibody. The wells are then washed to remove unbound material. TMB is then added. The magnitude of observed signal is proportional to the quantity of Cas9 (S. pyogenes). Kit Components: ELISA Microwell Strip Plate, 1x96wells Cas9 (S. pyogenes) Rabbit Capture mAb, 1xvial (Lyophilized) Cas9 Mouse HRP-linked mAb, 1xvial (Lyophilized) ELISA Capture Antibody Diluent, 1x3ml ELISA HRP Antibody Diluent, 1x3ml TMB Substrate, 1x1ml STOP Solution, 1x1ml ELISA Wash Buffer (20X), 1x25ml Cell Extraction Buffer (5X), 1x10ml Cell Extraction Enhancer Solution (50X), 1x1ml Positive Control Type 2, 1xvial Storage and Stability: Store all components at RT. Stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
