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ECAS9GFPPR

Sigma-Aldrich

eSpCas9-GFP Protein

from Streptococcus pyogenes with mutations conferring enhanced specificity, fused with enhanced GFP, recombinant, expressed in E. coli, 3X NLS

Synonyme(s) :

eSpCas9-EGFP, eSpCas9-GFP

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About This Item

Code UNSPSC :
12352202
Nomenclature NACRES :
NA.51

Produit recombinant

expressed in E. coli

Pureté

≥90% (SDS-PAGE)

Forme

lyophilized powder

Conditionnement

pkg of 1 kit (3 components)

Gène reporter

GFP

Conditions d'expédition

wet ice

Température de stockage

−20°C

Description générale

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

Caractéristiques et avantages

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

Conditionnement

pkg of 50 μg ( ≥260 pmol )
pkg of 250 μg ( ≥1300 pmol)

Composants

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

Principe

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.

Autres remarques

Use our CRISPR Selection Tool to order gRNA

Check out our other MISSION® Cas9 Proteins at SigmaAldrich.com/CRISPRproteins

Informations légales

CRISPR Use License Agreement
MISSION is a registered trademark of Merck KGaA, Darmstadt, Germany

Code de la classe de stockage

11 - Combustible Solids

Certificats d'analyse (COA)

Recherchez un Certificats d'analyse (COA) en saisissant le numéro de lot du produit. Les numéros de lot figurent sur l'étiquette du produit après les mots "Lot" ou "Batch".

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Retrouvez la documentation relative aux produits que vous avez récemment achetés dans la Bibliothèque de documents.

Consulter la Bibliothèque de documents

Ian M Slaymaker et al.
Science (New York, N.Y.), 351(6268), 84-88 (2015-12-03)
The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can

Articles

Validating CRISPR/Cas9-mediated Gene Editing

After you have performed a CRISPR experiment it is important to determine which gRNAs performed successfully editing. There are many ways to validate CRISPR gene editing experiments. A quick and easy way to check for cutting is by using the Sigma-Aldrich® T7E1 mismatch detection kit.

Protocoles

Ribonucleoprotein (RNP) Protocols Using Synthetic sgRNAs and Cas9 proteins

Combine guaranteed sgRNAs with our comprehensive range of CRISPR products and tools, including Cas9 and delivery reagents, for efficient genome modification with higher specificity.

Contenu apparenté

CRISPR Cas9 Ribonucleoprotein (RNP) Application Note

Combine guaranteed sgRNAs with our comprehensive range of CRISPR products and tools, including Cas9 and delivery reagents, for efficient genome modification with higher specificity.

Visualize your CRISPR Experiment with MISSION

Watch Cas9-GFP proteins work inside your cells and deliver the powerful nuclease editing you have come to trust and expect. MISSION™ SpCas9-GFP and eSpCas9-GFP proteins are multi-functional enzymes with high on-target cleavage efficiency fused to Enhanced Green Fluorescent Protein (EGFP) for real-time observation of Cas9 delivery and clearance from cells.

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