CAS9D10AGFPP
CMV-CAS9D10A-2A-GFP Plasmid
Synonym(s):
CAS9D10A Plasmid
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About This Item
UNSPSC Code:
12352200
NACRES:
NA.51
Recommended Products
recombinant
expressed in E. coli
Quality Level
form
liquid
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
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General description
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.
Application
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:
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.
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
Other Notes
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).
Legal Information
Storage Class Code
10 - Combustible liquids
WGK
nwg
Flash Point(F)
Not applicable
Flash Point(C)
Not applicable
Certificates of Analysis (COA)
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A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone
Vazquez N, et al.
BMC Molecular Biology, 19(1), 3-3 (2018)
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