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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