Cas9-sgRNA Gesicle Production
Cas9-sgRNA Gesicle Production
Brand: Takara Bio.
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Cas9-sgRNA Gesicle Production
The Guide-it CRISPR/Cas9 Gesicle Production System is a system for producing high yields of target-specific CRISPR/Cas9 gesicles for gene editing. Gesicles are cell-derived nanovesicles used to deliver macromolecular cargoes to a broad range of target cells, including cells that are difficult to transfect with plasmids. The nanovesicles are produced in a Gesicle Producer 293T Cell Line (Cat. No. 632617) via co-overexpression of packaging mix components, which include a nanovesicle-inducing glycoprotein and a protein that is displayed on the cell surface that mediates binding and fusion with the cellular membrane of target cells. Simultaneous overexpression of additional macromolecular cargoes, in this case the Cas9 protein from Streptococcus pyogenes and a target-specific guide RNA (sgRNA), results in incorporation of the Cas9/sgRNA complex within the gesicles. After the resulting Cas9/sgRNA gesicles are harvested and applied to your target cells in the presence of protamine sulfate, they will efficiently enter the cells and mediate gene editing. This system provides the components needed to clone and express your target-specific guide RNA, and packaging reagents to produce CRISPR/Cas9 gesicles. It is essential to use the pGuide-it-sgRNA1 Vector for expression and packaging of your guide RNA, since using other commonly used guide RNA vectors will not result in effective Cas9/sgRNA complexes.
Overview
- Broad tropism: Efficient delivery of active Cas9 protein and target-specific sgRNA to a broad range of cell types
- Reduced off-target effects: Delivery of Cas9 protein eliminates genomic integration and reduces off-target effects
- Genome editing only when you want it: Tight control over dose and timing of delivery and editing
- Notes:
- The traditional guide RNA scaffold provided with most commonly used vectors will not work with Cas9 delivered using gesicle technology. It is essential to use the pGuide-it-sgRNA1 Vector supplied in Cat. # 631613 or Cat. # 631612 when making gesicles, since this vector contains an optimized scaffold sequence for increased assembly and stability.
- The Gesicle Producer 293T Cell Line is not provided with the system and may be purchased separately (Cat. # 632617)
- Stem cell applications: We have demonstrated high-efficiency editing of endogenous genes in human iPS cells using a combination of CRISPR/Cas9 gesicles and our DEF-CS culture system.
- Note regarding primary CD34+ cells and primary T cells: While CRISPR/Cas9 gesicles have worked well in editing hard-to-transfect cell lines such as Jurkat cells, we have so far been unable to demonstrate high editing efficiency in primary CD34+ cells and primary T cells.
Applications
- CRISPR/Cas9-mediated gene editing
- Stem cell research
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.
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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.
