Cas9 Geminin plasmid CAS9GEMP-1UG

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.
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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.
CRISPR Label License,

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

RIDADR	NONH for all modes of transport
WGK Germany	WGK 2
Flash Point F	Not applicable
Flash Point C	Not applicable