Cas9-sgRNA Gesicle Production

Cas9 protein delivery in culture cells

Cas9 protein delivery in culture cells. Immunohistochemistry was performed on RPE cells stably expressing ZsGreen1 and treated with Cas9 gesicles. Cells were stained 12 hr after addition of gesicles. Cas9 was detected using the Guide-it Cas9 Polyclonal Antibody (Cat. # 632607) together with the Alexa 350-conjugated anti-rabbit IgG secondary antibody. Red fluorescence from the CherryPicker fluorescent protein could also be detected in the cells

Knockout efficiency of fluorescent reporter by Cas9-sgRNA protein complexes delivered to various cell types using gesicles

Knockout efficiency of fluorescent reporter by Cas9-sgRNA protein complexes delivered to various cell types using gesicles. Cell lines were created that contained an integrated ZsGreen1 fluorescent protein expression cassette. In this system, successful Cas9-mediated cleavage can be measured by loss of ZsGreen1 expression. These cell lines were treated with gesicles loaded with Cas9-sgRNA protein complexes (with the sgRNA generated against ZsGreen1), and then analyzed by flow cytometry. Cas9-sgRNA protein complex delivery and ZsGreen1 knockout via gesicles was efficient and comparable to plasmid-based delivery in easier-to-transfect cell types (left graph) and surpassed the results achieved via plasmid-based delivery in harder-to-transfect cell types (right graph).

Editing efficiency is increased by an improved sgRNA scaffold design in gesicles

Editing efficiency is increased by an improved sgRNA scaffold design in gesicles. HT1080 cells containing an integrated fluorescent protein expression cassette were transfected with a plasmid encoding for Cas9 and AcGFP1-specific sgRNA or treated with gesicles. Both delivery methods were tested using either the traditional or an optimized, sgRNA scaffold targeting AcGFP1. The optimized scaffold has an extension of the Cas9-binding hairpin and removes four consecutive uracils. The knockout efficiency was measured six days later by flow cytometry analysis. The optimized sgRNA scaffold had no effect on editing efficiency for plasmid-based delivery. However, the optimized sgRNA scaffold increased knockout efficiency by 36.4 for gesicles. Thus, only the pGuide-it-sgRNA1 vector containing the optimized scaffold is recommended for gesicle production.

Using an improved sgRNA scaffold design in gesicles enables a dose-dependent increase in knockout effect

Using an improved sgRNA scaffold design in gesicles enables a dose-dependent increase in knockout effect. HT1080 cells containing an integrated fluorescent protein expression cassette were treated with 5 µl, 10 µl, 20 µl, or 30 µl of gesicles produced with either the traditional or an optimized, sgRNA scaffold targeting AcGFP1. The optimized scaffold has an extension of the Cas9-binding hairpin and removes four consecutive uracils. The knockout efficiency was measured six days later by flow cytometry analysis. Editing efficiency using the traditional scaffold was low, regardless of gesicle dose. However, gesicles using the optimized scaffold demonstrated a dose-dependent increase in knockout up to 59. Thus, only the pGuide-it-sgRNA1 vector containing the optimized scaffold is recommended for gesicle production.

Efficient knockout of an endogenous protein (CD81) using gesicles containing Cas9-sgRNA complexes

Efficient knockout of an endogenous protein (CD81) using gesicles containing Cas9-sgRNA complexes. The cell-surface protein receptor CD81 was knocked out in Jurkat cells using either plasmid cotransfection of Cas9 DNA and sgRNA or gesicles preloaded with a Cas9-sgRNA ribonucleoprotein complex. The knockout efficiency was measured six days later via antibody labeling of the membrane receptor followed by flow cytometry analysis. Results for delivery via gesicles were significantly greater than results achieved with plasmid transfection.

Gesicles utilize an optimized sgRNA scaffold

Gesicles utilize an optimized sgRNA scaffold. The traditional sgRNA scaffold was modified by extending the Cas9-binding hairpin and removing four consecutive uracils. This optimized scaffold is critical for successful gesicle-based editing due to improved knockout efficiency.