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Cas9 protein
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Cas9 protein, in complex with guide RNA, offers benefit for CRISPR/Cas9 technology over plasmid or mRNA transfection. The biggest benefit is that you can make RNP complex that is ready to work in vivo. Cas9 protein is short lived (about 24 hours of half life in cells), thus minimize the off-target effects and mosaicism. You can also easily test the activity of guide RNA in vitro by a simple cleavage assay. In addition, Cas9 protein is pretty stable, and activity confirmation is much easier compared to mRNA.

Cas9 forms a sequence-specific endonuclease when complexed with two RNAs, one of which guides target selection (crRNA) and and the other is a constant component (tracrRNA). RGENs recognize a target specific sequence that is 23bp in length, ending with two guanines (GG). This minimal requirement for target site selection means that RGENs can be designed to cleave any region of the genome (every 8bp in theory). We provide custom CRISPR/Cas9 (RGEN) synthesis service with or without validation. IVT sgRNA Synthesis Service (aRGEN).

CRISPR/Cas systems are employed by bacteria and archaea as a defense against invading viruses and plasmids. Recently, the type II CRISPR/Cas system from the bacterium Streptococcus pyogenes has been engineered to function in eukaryotic systems using two molecular components: a single Cas9 protein and a non-coding guide RNA (gRNA). The Cas9 endonuclease can be programmed with a gRNA, directing a DNA double-strand break (DSB) at a desired genomic location. CAS9 contains 1388 amino acids. This protein is predicted to contain a RuvC/ ribonuclease (RNase) H domain involved in crRNA maturation and McrA/HNH signature domain involved in the DNA degradation step. Recombinant Cas9 protein from Streptococcus pyogenes (~160 KD) is a ready-to-use reagent for genome engineering experiments.

Take the power of CRISPR/Cas9 technology to the next level. In addition, the more transient nature of Cas9 protein compared to plasmid or mRNA delivery further reduces off-target activity without decreasing on-target efficiency1,2. In vitro transfection of cells. In vitro cleavage assays for functional gRNA screens. With oporatable Cas9 protein, you can: Increase mutation efficiency. Reduce off-target events.

Several meth-ods for delivery of Cas9 RNPs have been reported.

OriGene Cas9 Nuclease can be used for the pre-screening of highly efficient gRNA sequence through in-vitro cleavage assays. Cas9 protein is an RNA-guided endonuclease that can be used for the site-specific cleavage of double stranded DNA. OriGene offers a complete set of Cas9 products from DNA clone to purified protein and specific antibody. All of them are created based on wild type Streptococcus pyogenes Cas9 protein. Why Choose OriGene Cas9 Protein? Sterile protein with NLS signals. High RNA-guided endonuclease activity. High protein concentration & purity. Featured products: SKU.

Figure 1. Cas9 protein cleavage of HUWE DNA fragments in vitro. Three different sgRNA targeting the HUWE gene were designed and pre-incubated with Cas9 proteins respectively for the cleavage assay. Cleaved DNA bands are indicated by red marks. Ct, uncut HUWE DNA fragment control. Figure 2. Cas9 performance comparison. HUWE DNA fragments were used as template. The sgRNA targeting the HUWE gene was pre-incubated with Cas9 protein for the cleavage assay. The GeneHero™ Cas9 Nuclease (lane 3-5) and IDT Alt-R® Cas9 nuclease (lane 6-8) were serial diluted from 250 ng, 100 ng to 50 ng per reaction

Cas9 (CRISPR associated protein 9) is a protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses, and which is heavily utilized in genetic engineering applications. Its main function is to cut DNA and therefore it can alter a cell's genome. More technically, Cas9 is an RNA-guided DNA endonuclease enzyme associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immunity system in Streptococcus pyogenes.