CMV-CAS9-2A-RFP Plasmid CAS9RFPP-1EA

NACRES:

NA.51

The Cas9 expression plasmids use the CMV promoter for strong transient expression of Cas9. Alternate promoters can be substituted by replacement of CMV using MluI and NheI. Also, the Cas9 expression plasmids can be linearized using XbaI for T7-based mRNA production. The addition of a fluorophore that is translationally co-expressed with the Cas9 nuclease allows for easy visualization of successful transfection.
Functional Genomics/Target Validation

• Creation of gene knockouts in multiple cell lines
• Complete knockout of genes not amenable to RNAi
• Creation of knock-in cell lines with promoters, fusion tags, or reporters integrated into endogenous genes

Components
1 vial containing 1 µg of Cas9-2A-RFP plasmid.
Please note, product does not contain guideRNA sequence. This must be purchased separately through the Custom CRISPR product tab.
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.
Physical form
Sigma Cas9-2A-RFP plasmid DNA is supplied at concentrations of 50ng/μl in 20μl.
Must be used in conjunction with a U6-gRNA plasmid in order to mediate a double strand break in the DNA.
Typical transfection concentrations used in literature are in the ranges of >= 1.0 µg/µL and <= 5 µL of Cas9 plasmid combined with >= 1.0 µg/µL and <= 5 µL of U6-gRNA plasmids. (All dosages above assume 0.5 to 1 million cells nucleofected).
CRISPR Use License Agreement,

Quality Level	200,
recombinant	expressed in E. coli
packaging	vial of 50 µL
concentration	20 ng/µL in TE buffer; DNA (1µg of plasmid DNA)
Promoter	Promoter name: CMV
reporter gene	RFP
shipped in	dry ice
storage temp.	20°C

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