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CAS9D10AGFPP

Sigma-Aldrich

CMV-CAS9D10A-2A-GFP Plasmid

Synonym(s):
CAS9D10A Plasmid
NACRES:
NA.51

Quality Level

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

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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:

  • 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

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).

Storage Class Code

12 - Non Combustible Liquids

WGK

nwg

Flash Point(F)

Not applicable

Flash Point(C)

Not applicable

Certificate of Analysis

Certificate of Origin

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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