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).
Purified Cas9 nuclease NLS protein offers a ready-to-use option, enabling researchers to accelerate genome engineering experiments in a completely DNA-free manner.
Cas9 protein highlights
- Straightforward co-transfection or co-electroporation with synthetic guide RNA
- No external DNA added to system to ensure against the possibility of incorporating plasmid DNA into the host cell's genome
- No issues with incompatibilities between promoter and cell line
- Transient expression of Cas9 nuclease to reduce off targeting
DharmaFECT transfection reagents are highly recommended for use with Edit-R™ gene editing reagents and should be purchased separately. Refer to the DharmaFECT Cell Type Guide to find the appropriate formulation for your cell type.
Quantity & concentration of Cas9 nuclease protein NLS reagents
|CAS11730||40 µM||6.4||25||160||2 x 500|
Gene knockout workflow using Cas9 nuclease protein & Edit-R synthetic guide RNA
Gene editing with Cas9 Nuclease protein NLS and guide RNA is performed by co-transfecting all components with DharmaFECT Duo Transfection Reagent (or other DharmaFECT transfection reagent suitable to your specific cells of interest). One may then observe phenotypes directly. A DNA mismatch detection assay can be used to estimate gene editing efficiency prior to clonal cell line generation and characterization.
Cas9 nuclease protein NLS data
Synthetic guide RNAs co-delivered with Cas9 nuclease mRNA or protein
Guide RNAs were co-electroporated in K-562 cells with Cas9 mRNA (A) or Cas9 protein (B) and gene editing was assessed by DNA mismatch detection assay. In U2OS, guide RNAs were co-transfected with Cas9 mRNA (C) or Cas9 protein (D) and phenotypic gene knockout was assessed by EGFP fluorescence.
Synthetic crRNA:tracrRNA is compatible with all Cas9 nuclease formats
U2OS cells were plated at 10,000 cells/well one day prior to transfection. Cells were transfected with either Edit-R Cas9 Nuclease plasmid (200 ng), Edit-R Cas9 Nuclease mRNA(200 ng) or Cas9 nuclease protein (25 nM) and crRNA:tracrRNA (25 nM) targeting PPIB using DharmaFECT Duo transfection reagent (0.4 L/well) in biological triplicates.
DharmaFECT dose curve transfection optimization for highly-efficient gene editing using Cas9 nuclease protein
Efficient gene editing with Cas9 nuclease protein NLS demonstrated by DNA mismatch assay using T7 Endonuclease I. U2OS-(Ubi)EGFP cells were plated at 10,000 cells/well in 96-well plates and co-transfected using DharmaFECT Duo or DharmFECT 1 transfection reagents with Edit-R Cas9 Nuclease protein NLS and synthetic crRNA:tracrRNA at the following ratios of Cas9:RNA nM (2:1 = 25:12.5, 1:1 = 25:25, 1:2 = 25:50, 1:4 = 25:100) targeting PSMD7. Cells were harvested 72 hours post-transfection and the relative frequency of gene editing was calculated based on a DNA mismatch detection assay with T7 Endonuclease I. UT = untreated sample, Ladder = FastRuler Low Range DNA Ladder (Thermo Scientific).
|Shipping Condition||Dry Ice|
|Storage Conditions||-20 C|
|Stability at Recommended Storage Conditions||At least 12 months|