Application

Functional Genomics/Target Validation/Genome Editing

Biochem/physiol Actions

CAS9 plays a vital role in plasmid DNA interference. It is the only cas protein needed to deliver resistance against foreign DNA. CAS9 stimulates both RNA-guided genome editing and gene regulation in various organisms, but it can facilitate only one activity at a time within any given cell.

Components

Each kit consists of: one vial of Cas9 recombinant protein one vial containing 1 mL of 1× dilution buffer one vial containing 1 mL of nuclease-free water with glycerol

Features and Benefits

Highly specific Highly active Ready-to-inject/transfect

General description

Recombinant Cas9 protein from Streptococcus pyogenes (~160 KD) is a ready-to-use reagent for genome engineering experiments. When combined with target-specific guide RNAs, wild type Streptococcus pyogenes Cas9 protein will act as a targeted nuclease suitable for transfection of cell cultures and for the accelerated development of genetically-modified animals via one-cell embryo injection.

CAS9, also known as cas5 and csn1, is the signature gene of the type II clustered regularly interspaced short palindromic repeats (CRISPR)-RuvC (RNase H-like fold) cas system. 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.

Legal Information

MISSION is a registered trademark of Sigma-Aldrich Co. LLC

CRISPR Use License Agreement

Other Notes

Use our CRISPR Selection Tool to order gRNACheck out our other MISSION^® Cas9 Proteins at SigmaAldrich.com/CRISPRproteins

Packaging

pkg of 50 µg (≥ 300 pmol)pkg of 250 µg (≥ 1500 pmol)

Principle

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. Similar to DSBs induced by zinc finger nucleases (ZFNs), the cell then activates endogenous DNA repair processes, either non-homologous end joining (NHEJ) or homology-directed repair (HDR), to heal the targeted DSB.

Reconstitution

Lyophilized S. pyogenes Cas9 protein should be resuspended in the Reconstitution solution provided to desired concentration. Gently tap tube to completely dissolve lyophilized powder, incubate for 10 minutes on ice, and spin tube to bring material to bottom of tube.