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).
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.
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 × 10^6 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).
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
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.
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.