CMV-CAS9D10A-2A-GFP Plasmid CAS9D10AGFPP-1EA

NACRES:

NA.51

The Cas9-D10A nickase plasmid co-expressing GFP uses the CMV promoter for strong transient expression of Cas9-D10A. Alternate promoters can be substituted by replacement of CMV using MluI and NheI. Also, the Cas9D10A expression plasmids can be linearized using XbaI for T7-based mRNA production.
CMV-CAS9D10A-2A-GFP Plasmid has been used in CRISPR-Cas9 mutagenesis to truncate the forkhead box O3 (FOXO3) gene in the mammalian cell.
Functional Genomics
Use of Paired Cas9 Nickase + GFP for:

• 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 1ug of Cas9-D10A Nickase-GFP plasmid.
Please note, this product does not contain any guide RNA sequence. A pair of gRNA plasmids must be purchased separately through the Custom CRISPR paired nickase product tab.
Physical form
1 ug of Sigma Cas9-D10A nickase-GFP plasmid
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. GFP is co-expressed from the same mRNA as the Cas9 nickase protein via a 2A peptide linkage, enabling tracking of transfection efficiency and enrichment of genome editing activity in cell populations via fluorescence activated cell sorting (FACS).
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	GFP
selection	kanamycin
shipped in	dry ice
storage temp.	20°C

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