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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

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Synonym(s):
eSpCas9-EGFP, eSpCas9-GFP

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

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CAS9GFPPROESPCAS9PROPECAS9
Cas9-GFP Protein from Streptococcus pyogenes, fused with enhanced GFP, recombinant, expressed in E. coli, 3X NLS

Sigma-Aldrich

CAS9GFPPRO

Cas9-GFP Protein

eSpCas9 Protein from Streptococcus pyogenes with mutations conferring enhanced specificity, recombinant, expressed in E. coli, 1X NLS

Sigma-Aldrich

ESPCAS9PRO

eSpCas9 Protein

assay

≥90% (SDS-PAGE)

assay

≥90% (SDS-PAGE)

assay

≥95% (SDS-PAGE)

assay

≥95% (SDS-PAGE)

form

lyophilized powder

form

lyophilized powder

form

lyophilized powder

form

lyophilized powder

packaging

pkg of 1 kit (3 components)

packaging

pkg of 1 kit (3 components)

packaging

pkg of 1 kit (3 components)

packaging

pkg of 1 kit (4 components)

shipped in

wet ice

shipped in

wet ice

shipped in

ambient

shipped in

wet ice

storage temp.

−20°C

storage temp.

−20°C

storage temp.

−20°C

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.

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)

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.

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

Legal Information

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

Storage Class Code

11 - Combustible Solids

WGK

WGK 3


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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
Patryk Janus et al.
Cell death and differentiation, 27(7), 2280-2292 (2020-01-31)
Heat shock can induce either cytoprotective mechanisms or cell death. We found that in certain human and mouse cells, including spermatocytes, activated heat shock factor 1 (HSF1) binds to sequences located in the intron(s) of the PMAIP1 (NOXA) gene and
Kristina Seiler et al.
Cell death & disease, 13(5), 448-448 (2022-05-11)
The family of hexokinases (HKs) catalyzes the first step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. While HK1 and HK2 are ubiquitously expressed, the less well-studied HK3 is primarily expressed in hematopoietic cells and tissues and is highly
Natalia Vydra et al.
eLife, 10 (2021-11-17)
Heat shock factor 1 (HSF1), a key regulator of transcriptional responses to proteotoxic stress, was linked to estrogen (E2) signaling through estrogen receptor α (ERα). We found that an HSF1 deficiency may decrease ERα level, attenuate the mitogenic action of

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.

Protocols

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.

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