Application

Functional Genomics Target Validation Genome Editing Fluorescence Microscopy Flow Cytometry

Components

Each kit consists of: one vial of lyophilized eSpCas9-GFP recombinant protein one vial containing 1 mL of 1x dilution buffer one vial containing 1 mL of nuclease-free water with glycerol

Features and Benefits

•Enhanced specificity compared to wild type Cas9•Highly active•Ready-to-inject/transfect

General description

Recombinant enhanced specificity Cas9-GFP protein from Streptococcus pyogenes (~192 KD) is a ready-to-use reagent for genome engineering experiments. When combined with target-specific guide RNAs, eSpCas9-GFP 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. An N-terminally fused enhanced green fluorescent protein with an excitation peak at 488 nm and emission peak at 509 nm allows for visualization of transfected RNP complex in addition to utility in flow cytometry applications.  The protein also contains three varied nuclear localization sequences postioned for optimal activity.

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 ( ≥260 pmol )pkg of 250 µg ( ≥1300 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 single 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. Newly engineered eSpCas9 (1.1) enables the efficient targeted gene editing of established CRISPR systems with the benefit of reduced off-target effects. Point mutations (K848A/K1003A/R1060A)  in the chromosome-binding motif of SpCas9, as described by Slaymaker, et al., provide higher on-target fidelity without loss of cleavage efficiency.

Reconstitution

Lyophilized enhanced specificity S. pyogenes eSpCas9-GFP 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.