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 crRNA and tracrRNA for a completely DNA-free genome engineering system

Cas9 nuclease in the CRISPR-Cas9 system

In type II CRISPR-Cas systems, 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 mRNA, in conjunction with our synthetic crRNA:tracrRNA, enables researchers to perform genome engineering experiments in a completely DNA-free manner. The Cas9 Nuclease mRNA is a highly pure, stable molecule with a 5' cap and a 3' poly A tail. The benefits to this approach are:

  • No external DNA added to system
  • Ensure against the possibility of incorporating plasmid DNA into the host cell lines genome (see supporting data)
  • No issues with incompatabilities between promoter and cell line
  • Transient expression of Cas9 nuclease, which 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.
  
HazardousNo
Shelf Life12 Months
Shipping ConditionDry Ice
Storage Condition-80 C
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).


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Cas9 Nuclease Selection Guide

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Select the Cas9 Nuclease option that best suits your experimental needs and conditions


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

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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.


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

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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 RNA Configurator (http://dharmacon.gelifesciences.com/gene-editing/crispr-rna-configurator/). UT = untransfected sample.


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

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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 RNA Configurator (http://dharmacon.gelifesciences.com/gene-editing/crispr-rna-configurator/). UT = untransfected sample.


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Sequential electroporations with Edit-R Cas9 Nuclease mRNA and synthetic crRNA:tracrRNA

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K562 cells were plated at 10 M cells in a 150 mm cell culture plate. The next day, 2 M cells were collected and centrifuged at ~500 x g for 5 minutes. After centrifuging, cell pellets were resuspended in electroporation buffer and mixed with Cas9 mRNA (5 µg), then electroporated. Cells were transferred to one well of a 6-well plate for 6 hours, then collected and centrifuged at ~500 x g for 5 minutes at room temperature and electroporated with crRNA:tracrRNA (5.4 µM) synthetic positive control targeting PPIB (Cat #U-007000-xx) or Non Targeting Control #3 (Cat# U-007503-xx). 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.


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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.