Cas9 nuclease in the CRISPR-Cas9 system
The CRISPR-associated enzyme Cas9 is an RNA-guided endonuclease that requires a guide RNA for genomic DNA target recognition and generation of DNA double-strand breaks (DSB).
Cas9 nuclease expression plasmids encode a human codon-optimized version of the S. pyogenes Cas9 gene under the control of an RNA pol II promoter. Cas9 nuclease expression plasmids are non-lentiviral vectors provided as endotoxin-free purified DNA for direct co-transfection with Edit-R synthetic guide RNA.
Cas9 nuclease expression plasmid highlights
- Choose from three plasmid options, based on preference of enrichment method and cell type
- mKate2 fluorescent reporter
- Puromycin-resistance marker
- hCMV promoter and Blasticidin resistance marker
- Choose from 6 SMARTchoice promoter options
Not all RNA pol II promoters are equally active in different cellular environments
The activity of any given promoter controlling the transcription of Cas9 nuclease can differ greatly from one biological context to another, resulting in variable Cas9 expression levels and thus varying levels of DNA cleavage. Choosing an optimal promoter for your cell line or type will therefore affect the degree of gene editing in your experimentation. The Cas9 nuclease expression plasmids are offered with six different, well-characterized cellular promoters from which you can choose. For help determining the optimal promoter for your experiment, see the SMARTchoice promoter selection plate.
|hCMV||human cytomegalovirus immediate early promoter|
|mCMV||mouse cytomegalovirus immediate early promoter|
|hEF1α||human elongation factor 1 alpha promoter|
|mEF1α||mouse elongation factor 1 alpha promoter|
|PGK||mouse phosphoglycerate kinase promoter|
|CAG||chicken beta actin hybrid promoter|
Gene knockout workflow using Cas9 nuclease expression plasmid & Edit-R synthetic guide RNA
An illustration of the Edit-R CRISPR-Cas9 components required for gene editing:plasmid expressing Cas9 nuclease, tracrRNA, and crRNA designed to the target site of interest. All three components are co-transfected into the mammalian cell of choice using DharmaFECT Duo Transfection Reagent to perform gene disruption. Enrichment of transfected cells can then be carried out by fluorescent cell sorting or selection for Puromycin resistance.
Less time preparing, more time experimenting
The Edit-R Gene Engineering system utilizes high-quality synthetic tracrRNA and crRNA to program Cas9 nuclease, thereby eliminating the need to clone individual sgRNAs thus saving time and labor.
Cas9 nuclease expression plasmid data
Enrichment of Cas9-expressing U2OS cells using SMARTCas9-mKate2 expression plasmid by FACS results in increased target gene editing
Enrichment of Cas9-expressing U2OS cells using SMARTCas9-mKate2 expression plasmid by FACS results in increased % editing of human PPIB. U2OS cells were transfected with SMARTCas9-mKate2 (with human CMV promoter) expression plasmid and tracrRNA:crRNA targeting the human PPIB gene. Cells were sorted at 72 hours on a MoFlo XDP 100 instrument into three bins corresponding to negative, low, and high mKate2 fluorescence. SURVEYOR™ DNA mismatch assay was performed on sorted U2OS cells and % gene editing was compared with the unsorted (US) and control untransfected (UT) cells. The level of editing was calculated using densitometry (% editing). An increase in % gene editing is observed in the sorted cells, correlating with the increased mKate2 expression.
Vector elements and promoter options of Cas9-mKate2 expression plasmid
The Cas9-mKate2 plasmid expresses the monomeric red fluorescent protein mKate2 and the human codon-optimized Cas9 nuclease from S. pyogenes, driven by one of six choices of RNA pol II promoters. By linking expression of mKate2 to Cas9 nuclease using the self-cleaving peptide T2A, sorting mKate2-positive cells by FACS will enrich for Cas9-expressing cells and increase the percentage of cells which have undergone the gene editing event.
Vector elements and promoter options of Cas9-PuroR expression plasmid
The Cas9-PuroR plasmid expresses the Puromycin-resistance selection marker and the human codon-optimized Cas9 nuclease from S. pyogenes, driven by one of six choices of RNA pol II promoters. By linking expression of the Puromycin-resistance marker to Cas9 nuclease using the self-cleaving peptide T2A, selecting cells by treatment with puromycin will enrich for Cas9-expressing cells and thus increase the percentage of cells which have undergone the gene editing event.
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|Storage Conditions||-20 C|
|Stability at Recommended Storage Conditions||At least 12 months|