Gene knockout workflow using Edit-R Cas9 Protein Hybrid NLS & Edit-R synthetic guide RNA

Gene editing with Edit-R Cas9 Protein Hybrid NLS and Edit-R synthetic guide RNA is performed by co-electroporating or co-transfecting all components with DharmaFECT transfection reagents. One may then observe phenotypes directly. A DNA mismatch detection assay, TIDE, or NGS analysis workflow can be used to estimate gene editing efficiency.

Efficient protein knockout is achieved after nucleofection of CD4+ primary human T-cells with Edit-R Cas9 Protein Hybrid NLS RNPs

CD4+ cells were stimulated with CD3/CD28 T-cell activation beads (BioLegend) at a 1:1 bead:T-cell ratio and grown for 8 days with 30 U/mL of IL-2. The Lonza 4D shuttle system was used to nucleofect RNPs composed of Edit-R Cas9 Protein Hybrid NLS and Edit-R synthetic guide RNA individual and target pools for CXCR3 (Gene ID: 2833) and CD3D (Gene ID: 915). Functional knockout was measured by flow cytometry analysis of target prevalence for CXCR3 (Clone ID: G025H7) and CD3D (Clone ID: OKT3) (BioLegend). Representative flow cytometry analysis plots of CD4+ T-cells shown after target knockdown by RNP nucleofection.

Efficient indel formation following nucleofection of U2OS cells with Edit-R Cas9 Protein Hybrid NLS RNPs

U2OS cells were seeded at a density of 7,500 cells/well in a 96-well and grown for 18 hours at standard cell culture conditions.  The Lonza 4D shuttle system was used to nucleofect RNPs composed of either Cas9 nuclease protein NLS (V1) or Edit-R Cas9 Protein Hybrid NLS (V2) and Edit-R synthetic guide RNA target pools. Cells were incubated for 72 hours and subjected to direct lysis and target amplicon PCR from gDNA.  Resulting amplicon PCR material was used for T7E1 and TIDE editing analyses.  Data represents an average of three biological replicates.

Efficient indel formation following lipid transfection of U2OS cells with Edit-R Cas9 Protein Hybrid NLS and Edit-R synthetic sgRNA

U2OS cells were seeded at 10,000 cells/well in 96-well plates and co-transfected using DharmaFECT 1 transfection reagent (0.4 mL/well) with Edit-R Cas9 Protein Hybrid NLS and Edit-R synthetic single guide RNAs targeting CD3D and TFRC or a validated control Edit-R Synthetic sgRNA T-cell Receptor Targets sgRNA targeting TRAC.  Gene editing was assessed by T7E1 mismatch repair assay and TIDE analysis.

Increased activity of Edit-R Cas9 Protein Hybrid NLS assessed by an in vitro Cas9 cleavage assay

RNPs were assembled at a 1.2:1 ratio of sgRNA:Cas9 protein and allowed to complex for 10 minutes at room temperature.  200 ng of substrate (1.5 kb DNA duplex with two, 20-nt sgRNA target sequences starting at position 715 and separated by a 55-nt linker) was added to each reaction and incubated for 30 minutes at 37°C.  To halt the reaction, 0.6 U of proteinase K was added to each reaction and incubated for 15 minutes at 37°C. Cleavage reactions were separated by gel electrophoresis on a 1.5% agarose gel.