Both Cas9-GFP and eSpCas9-GFP exhibit similar transfection rates in terms of efficiency. However, in terms of editing activity, Cas9-GFP has greater activity than eSpCas9-GFP. The eSpCas9-GFP variant, while more specific, compromises on activity, particularly noticeable with difficult-to-edit targets. The eSpCas9 variant is suitable when high specificity is a priority, the target is relatively easy to modify, and the model system can effectively recover after transfection to enable the isolation of a decreased population of edited cells.
ECAS9GFPPR
eSpCas9-GFP Protein
from Streptococcus pyogenes with mutations conferring enhanced specificity, fused with enhanced GFP, recombinant, expressed in E. coli, 3X NLS
Sinónimos:
eSpCas9-EGFP, eSpCas9-GFP
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50 μG
MXP 6,054.00
250 μG
MXP 12,108.00
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50 μG
MXP 6,054.00
250 μG
MXP 12,108.00
About This Item
UNSPSC Code:
12352202
NACRES:
NA.51
Productos recomendados
recombinant
expressed in E. coli
assay
≥90% (SDS-PAGE)
form
lyophilized powder
packaging
pkg of 1 kit (3 components)
reporter gene
GFP
shipped in
wet ice
storage temp.
−20°C
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.
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
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)
pkg of 250 μg ( ≥1300 pmol)
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
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.
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.
Other Notes
Use our CRISPR Selection Tool to order gRNA
Check out our other MISSION® Cas9 Proteins at SigmaAldrich.com/CRISPRproteins
Check out our other MISSION® Cas9 Proteins at SigmaAldrich.com/CRISPRproteins
Legal Information
MISSION is a registered trademark of Merck KGaA, Darmstadt, Germany
Storage Class
11 - Combustible Solids
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Artículos
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.
Validate CRISPR gene editing experiments easily with Sigma-Aldrich® T7E1 mismatch detection kit, ensuring successful editing.
Protocolos
Combine guaranteed sgRNAs with our comprehensive range of CRISPR products and tools, including Cas9 and delivery reagents, for efficient genome modification with higher specificity.
Contenido relacionado
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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What are the differences between CAS9GFPPRO and ECAS9GFPPR in terms of transfection efficiency and applicability in various cell types?
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