Edit-R Cas9 Nuclease mRNA

A DNA-free option for Cas9 Nuclease expression


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Purified Cas9 Nuclease mRNA for co-transfection with synthetic guide RNA for a completely DNA-free genome engineering system

Edit-R Cas9 nuclease mRNA expresses a human codon-optimized version of the S. pyogenes Cas9 gene with two nuclear localization signals (NLS). The Cas9 endonuclease complexes with a guide RNA for genomic DNA target recognition and cleavage.

Edit-R Cas9 Nuclease mRNA, in conjunction with Edit-R synthetic sgRNA or crRNA:tracrRNA, enables researchers to perform genome engineering experiments in a completely DNA-free manner.

Highlights

  • A DNA-free system eliminates the possibility of incorporating external DNA into the host cell line genome
  • Transient expression of Cas9 nuclease may reduce off-targeting for more specific results
  • Easily co-transfect Edit-R synthetic guide RNA and Cas9 mRNA using DharmaFECT Duo transfection reagent or co-electroporate into cells that are difficult to transfect.
  
HazardousNo
Shipping ConditionDry Ice
Stability at Recommended Storage ConditionsAt least 12 months
Storage Condition-80 C
Cas9 Nuclease Selection Guide

Cas9 Nuclease Selection Guide

Cas9 Nuclease Selection Guide

Cas9 nuclease is not one-size-fits-all. Determining the most appropriate Cas9 nuclease reagent for your experiment is dependent on the particular application or cell type. Take a look at the features in this table to get started on selecting the best product for you.


Workflow using Edit-R Cas9 Nuclease mRNA and synthetic crRNA:tracrRNA

Workflow using Edit-R Cas9 Nuclease mRNA and synthetic crRNA:tracrRNA

Workflow using Edit-R Cas9 Nuclease mRNA and synthetic crRNA:tracrRNA

Gene editing with Edit-R Cas9 Nuclease mRNA 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.


Efficiency of gene editing with 2x MS modified or unmodified crRNA:tracrRNA

Efficiency of gene editing with 2x MS modified or unmodified crRNA:tracrRNA

Efficiency of gene editing with 2x MS modified or unmodified crRNA:tracrRNA

A. Modifications to block degradation by nucleases are required for successful co-electroporation of Cas9 mRNA and crRNA:tracrRNA. B. Co-electroporation of Cas9 protein and crRNA:tracrRNA does not require stabilizing modifications, but results may be improved with their addition.


Editing of VEGFA gene in HEK293T cells using Edit-R Cas9 Nuclease mRNA delivered by DharmaFECT transfection reagents

Editing of VEGFA gene in HEK293T cells using Edit-R Cas9 Nuclease mRNA delivered by DharmaFECT transfection reagents

Editing of VEGFA gene in HEK293T cells using Edit-R Cas9 Nuclease mRNA delivered by DharmaFECT transfection reagents

HEK293T cells were plated at 20,000 cells/well in 96-well plates and co-transfected using DharmaFECT transfection reagents with 200 ng of Edit-R-Cas9 mRNA and synthetic crRNA:tracrRNA targeting VEGFA. 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. DF1 = DharmaFECT 1, Duo = DharmaFECT Duo, UT = untreated sample, MW = FastRuler Low Range DNA Ladder (Thermo Scientific).


Plasmid DNA may be introduced into the genome from CRISPR-Cas9 gene editing with plasmid delivery

Plasmid DNA may be introduced into the genome from CRISPR-Cas9 gene editing with plasmid delivery

Plasmid DNA may be introduced into the genome from CRISPR-Cas9 gene editing with plasmid delivery

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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Edit-R Cas9 Nuclease mRNA gene editing in three cell lines

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HEK293T, A549 and U2OS cells were plated at 20,000 cells/well in 96-well plates and co-transfected using DharmFECT Duo Transfection Reagent with Cas9 mRNA (200 or 100 ng) and synthetic crRNA:tracrRNA (50 nM) targeting VEGFA. 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. The synthetic crRNA was designed using the Dharmacon CRISPR Design Tool. UT = untransfected sample.


Edit-R Cas9 Nuclease mRNA dose curve for efficient gene editing

Edit-R Cas9 Nuclease mRNA dose curve for efficient gene editing

Edit-R Cas9 Nuclease mRNA dose curve for efficient gene editing

HEK293T and U2OS cells were plated at 20,000 cells/well in 96-well plates and co-transfected using DharmFECT Duo Transfection Reagent with Cas9 mRNA (200, 100, 50, 25 or 10 ng) and synthetic crRNA:tracrRNA (50 nM) targeting VEGFA. 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. It is recommended to optimize the amount of Cas9 mRNA (50-200 ng/well in 96-well format) in your cells of interest and for your application. The synthetic crRNA targeting VEGFA was designed using the Dharmacon CRISPR Design Tool. UT = untransfected sample.


Synthetic crRNA:tracrRNA is compatible with all Cas9 Nuclease formats

Synthetic crRNA:tracrRNA is compatible with all Cas9 Nuclease formats

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.