Application

Functional Genomics/Target Validation/Genome Editing/Molecular Biology/Synthetic Biology/DNA detection/isolation/DNA imaging

Components

Each kit consists of: one vial of dCas9 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•Suitable for genomic DNA detection/isolation•Ready-to-inject/transfect

General description

Recombinant dCas9-3X FLAG-Biotin protein from Streptococcus pyogenes (~165 KD) is a ready-to-use reagent for genome engineering and genomic DNA detection, isolation and imaging experiments. When combined with target-specific guide RNAs, dead Streptococcus pyogenes Cas9 protein acts as a DNA targeting protein suitable for transfection of cell cultures, microinjection, DNA pull-down, chromatin enrichment, microbial identification and in situ DNA imaging. At the C terminus, the protein is tagged with a SV40 large T antigen nuclear localization signal (NLS) for nuclear import and both a 3XFLAG epitope and biotin for anti-FLAG^®-based or streptavidin-based experiments.

Legal Information

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

CRISPR Use License Agreement

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

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 (≥ 1500pmol)

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 rendered inactive (dCas9) with mutations to the two protein domains, RuvC and HnH (D10A and H840A respectively), which are responsible for nuclease activity. The protein can then be programmed with a gRNA and directed to bind at a desired sequence of DNA.

Reconstitution

Lyophilized dCas9 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.