Edit-R Cas9 Nuclease protein NLS

A DNA-free option for Cas9 Nuclease expression


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Purified Cas9 Nuclease protein NLS for co-transfection with synthetic crRNA and tracrRNA for a completely DNA-free genome engineering system

Cas9 nuclease in the CRISPR-Cas9 system

In the S. pyogenes CRISPR-Cas system, the CRISPR-associated enzyme Cas9 is an RNA-guided endonuclease that requires a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA) for genomic DNA target recognition and cleavage using two active sites that together generate DNA double-strand breaks (DSB).

Cas9 Nuclease protein, in conjunction with Edit-R synthetic crRNA:tracrRNA, enables researchers to perform genome engineering experiments in a completely DNA-free manner. The Cas9 Nuclease protein is a highly pure, stable molecule and contains a nuclear localization signal (NLS) for targeted delivery in your cells of interest.

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 incompatabilities between promoter and cell line
  • Transient expression of Cas9 nuclease may 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 Edit-R Cas9 Nuclease protein NLS reagents

 
Catalog # Concentration Volume Quantity
µM µg/µl µl µg pMol
CAS11728 20 µM 3.2 12.5 40 250
CAS11200 20 µM 3.2 25 80 500
CAS11201 20 µM 3.2 50 160 1000
CAS11729 40 µM 6.4 12.5 80 500
CAS11730 40 µM 6.4 25 160 2 x 500

 

  
HazardousNo
Shipping ConditionDry Ice
Stability at Recommended Storage ConditionsAt least 12 months
Storage Condition-20 C
Editing of PSMD7 gene in U2OS-(Ubi)EGFP cells using Cas9 protein delivered by DharmaFECT transfection reagents

Editing of PSMD7 gene in U2OS-(Ubi)EGFP cells using Cas9 protein delivered by DharmaFECT transfection reagents

Editing of PSMD7 gene in U2OS-(Ubi)EGFP cells using Cas9 protein delivered by DharmaFECT transfection reagents

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).


Editing of PPIB gene in U2OS cell line using Edit-R Cas9 Nuclease protein NLS in electroporation

Editing of PPIB gene in U2OS cell line using Edit-R Cas9 Nuclease protein NLS with electroporation

Editing of PPIB gene in U2OS cell line using Edit-R Cas9 Nuclease protein NLS in electroporation

U2OS cells were plated at 2.5 × 10^6 cells into a 150 mm dish. The next day, Edit-R Cas9 Nuclease protein NLS (150 pmol) was complexed with crRNA:tracrRNA (300 pmol) for 10 minutes at room temperature before electroporation. U2OS cells were trypsinized and 1 × 106 cells were collected and centrifuged at ~500 × g for 5 minutes. After centrifuging, cell pellets were resuspended in electroporation buffer and mixed with complexed Cas9 protein and crRNA:tracrRNA. The cell-complex mix was electroporated using the Lonza Nucleofector™ IIb, program X-001. Electroporated cells were then transferred into one well of a 6-well dish containing pre-warmed/equilibrated medium. Cells were harvested 72 hours post-electroporation and the relative frequency of gene editing was calculated based on a DNA mismatch detection assay with T7 Endonuclease I. The synthetic crRNA targeting PPIB is Edit-R PPIB Synthetic crRNA Control (Cat #U-007000-xx).


Undesirable plasmid DNA insertions can be avoided with DNA-free Cas9 nuclease reagents

Undesirable plasmid DNA insertions can be avoided with DNA-free Cas9 nuclease reagents

Undesirable plasmid DNA insertions can be avoided with DNA-free Cas9 nuclease reagents

HEK293T cells were plated in 6-well plates and transfected with hCMV-mKate2-Cas9 expression plasmid and crRNA:tracrRNA complex targeting the human PPIB gene in exon 2. Cells were harvested 72 hours post-transfection and sorted using the mKate2 fluorescent reporter. Fluorescent cells were plated at two, four or six individual cells per well in 96-well plates and further grown for clonal isolation. To precisely determine the genotype, Sanger sequencing was performed on PCR products amplified from gDNA spanning the crRNA target site and analyzed for indels. Various indels were observed, but notably, several of the clonal lines contained insertions homologous to components of the Cas9 expression plasmid. Similar observations were also reported in Hendel et al. (2014). See Application Note for more experimental details: http://dharmacon.gelifesciences.com/uploadedFiles/Resources/edit-r-experimental-workflow-appnote.pdf


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Cas9 Nuclease Selection Guide

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Choose which Cas9 option is ideal for your experiment


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Cas9 Protein Workflow

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Gene editing with Edit-R Cas9 Nuclease protein NLS and crRNA:tracrRNA 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.


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Synthetic crRNA:tracrRNA is compatible with all Cas9 Nuclease formats

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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.