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CAS9GEMP Sigma-Aldrich

Cas9 Geminin plasmid

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Properties

recombinant   expressed in E. coli
packaging   vial of 50 μL
concentration   20 ng/μL in TE buffer; DNA (1μg of plasmid DNA)
application(s)   CRISPR: suitable
Promoter   Promoter name: EF1-alpha
reporter gene   GFP
selection   ampicillin
shipped in   dry ice
storage temp.   −20°C

Description

General description

This product is an expression plasmid that utilizes the EF1a promoter for strong transient expression of a Cas9-GFP-Geminin fusion (EF1a-Cas9-GFP-Geminin) allowing for easy visualization of successful transfection. The Cas9-Geminin expression plasmid is one part of a two part CRISPR system with individual Cas9-Geminin and gRNA expression vectors.

To order gRNA in any format click here

Application

Functional Genomics/Target Validation
• Performing HDR mediated targeted integration in multiple cell lines
• Creation of knock-in cell lines with promoters, fusion tags or reporters integrated into endogenous genes

Features and Benefits

• Highly specific and highly active
• Sequence verified
• Ready to use purified plasmid DNA

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. While HDR is absent in G1, NHEJ is active throughout the cell cycle and is largely favored over HDR. Consequently HDR can be increased by directly synchronizing the expression of Cas9 with cell-cycle progression by fusing Cas9 to human Geminin (a protein expressed in S and G2 phases). The Cas9-geminin fusion protein then regulates gene editing by promoting repair during S and G2 phases when homology directed repair (HDR) occurs. The result is creation of double strand breaks in cells at times that are more able to incorporate donor sequences.

Legal Information

CRISPR Label License

Safety & Documentation

Safety Information

RIDADR 
NONH for all modes of transport
WGK Germany 
WGK 3
Flash Point(F) 
Not applicable
Flash Point(C) 
Not applicable

Documents

Certificate of Analysis (COA)

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Protocols & Articles

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