Description
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.
Application
Functional Genomics
Target Validation
Genome Editing
Fluorescence Microscopy
Flow Cytometry
Biochem/physiol Actions
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.
Features and Benefits
Enhanced specificity compared to wild type Cas9
Highly active
Ready-to-inject/transfect
Packaging
pkg of 50 μg ( ≥260 pmol )
pkg of 250 μg ( ≥1300 pmol)
Preparation Note
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.
Other Notes
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
Use our CRISPR Selection Tool to order gRNA
Check out our other MISSION® Cas9 Proteins at SigmaAldrich.com/CRISPRproteins
Legal Information
CRISPR Use License Agreement
MISSION is a registered trademark of Merck KGaA, Darmstadt, Germany
