Application

Functional Genomics/Target Validation/Genome Editing

Components

Each kit consists of: one vial of Cas9-D10A Nickase 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

Cas9 (CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein-9 nuclease) nickase creates double-stranded breaks through two nuclease domains, named RuvC and HNH.

Recombinant Cas9-D10A Nickase protein from Streptococcus pyogenes (~160 KD) is a ready-to-use reagent for genome engineering experiments. When combined with target-specific paired guide RNAs, S. pyogenes Cas9-D10A nickase 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.

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/CRISPRproteinsSee the product in our recent article on bioRxiv Highly efficient and specific genome editing in human cells with paired CRISPR-Cas9 nickase ribonucleoproteins

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-D10A Nickase 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.