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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

Sinónimos:

eSpCas9-EGFP, eSpCas9-GFP

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About This Item

Código UNSPSC:
12352202
NACRES:
NA.51

recombinante

expressed in E. coli

Ensayo

≥90% (SDS-PAGE)

Formulario

lyophilized powder

envase

pkg of 1 kit (3 components)

gen reportero

GFP

Condiciones de envío

wet ice

temp. de almacenamiento

−20°C

Descripción general

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.

Aplicación

  • Functional Genomics
  • Target Validation
  • Genome Editing
  • Fluorescence Microscopy
  • Flow Cytometry

Características y beneficios

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

Envase

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

Componentes

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

Principio

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.

Reconstitución

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.

Otras notas

Use our CRISPR Selection Tool to order gRNA

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

Información legal

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

Código de clase de almacenamiento

11 - Combustible Solids

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Certificados de análisis (COA)

Lot/Batch Number
0000200483
0000308665
0000200483
0000308665
0000142408

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Visite la Librería de documentos

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
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
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
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

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Validate CRISPR gene editing experiments easily with Sigma-Aldrich® T7E1 mismatch detection kit, ensuring successful editing.

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Validate CRISPR gene editing experiments easily with Sigma-Aldrich® T7E1 mismatch detection kit, ensuring successful editing.

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