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Learn how gesicles are made and used, and the problems they were designed to overcome.
Gesicles gene editing webinar
Watch Dr. Baz Smith discuss gesicle technology and how it's used to perform CRISPR/Cas9-mediated gene editing.
While CRISPR/Cas9 is a powerful technique for genome manipulation, two significant challenges remain: obtaining efficient delivery of the Cas9-sgRNA complex to all cell types, and leaving no additional footprint (i.e., persistent and elevated expression of Cas9 in target cells) that could lead to off-target effects. To address these challenges, we have developed a system of cell-derived nanovesicles called gesicles. Gesicles contain active Cas9 protein complexed with an sgRNA specific to a gene of interest. Thus, there is no persistent expression of Cas9, because no coding gene is present. Additionally, they are engineered with glycoproteins on their surface that mediate binding and fusion with the membrane of a wide range of target cells. These features enable gesicles to knock out genes with high efficiency and in a broader range of cell types than plasmid-based delivery methods. Finally, use of this method allows for control of the dose and duration of the Cas9-sgRNA complex in the cell, further reducing the chance of off-target effects.
Mechanism for producing gesicles to deliver a Cas9-sgRNA ribonucleoprotein complex
Gesicles are made by transfecting a 293T-based producer cell line with a vector containing an sgRNA for your target locus and our gesicle packaging mix, which contains everything you need in an optimized format. Loaded gesicles can then be collected from the supernatant and added to target cells for efficient editing with no footprint.
293T producer cells that have been transfected with our gesicle packaging mix and an sgRNA expression plasmid begin to form gesicles, stimulated by a nanovesicle-inducing glycoprotein
The producer cells express active Cas9 endonuclease which forms a ribonucleoprotein (RNP) complex with your expressed sgRNA. Gesicle loading utilizes the iDimerize system, which enables inducible protein-protein interactions: The membrane-bound CherryPicker red fluorescent protein on the surface of the gesicle is tagged with one dimerization domain, and the Cas9 endonuclease contains another dimerization domain. A small heterodimerizer ligand is added to link these two domains together, thereby enriching the Cas9-sgRNA RNP complex into the gesicle.
The red fluorescent protein-labeled gesicles are now loaded with active Cas9 complexed with your target sgRNA. Loaded gesicles pinch off from the producer cells and saturate the surrounding medium. Gesicles can be collected from the supernatant, yielding a concentrated stock of your Cas9-sgRNA gesicles. The gesicles can be used immediately on your target cells, or stored for over one year at –70°C.
Harvested gesicles can be applied to a broad range of target cell types. In the presence of protamine sulfate, gesicles fuse to the membrane of the target cell via cell-surface glycoproteins and release the Cas9-sgRNA RNP complex into the cell. The membranes of cells that have been successfully targeted with gesicles are transiently labeled with red fluorescent protein. Critically, the absence of dimerizer ligand in the target-cell culture medium and the presence of a nuclear localization signal on the Cas9 endonuclease ensures that the complex dissociates from the red fluorescent protein and is transported to the nucleus. Once there, efficient targeted gene editing takes place. After editing occurs, the Cas9-sgRNA RNP complex is degraded over time by endogenous cell processes, leaving no remaining footprint that would be capable of producing off-target effects.
Gesicle production system components
The Guide-it CRISPR/Cas9 Gesicle Production System contains everything you need to easily produce gesicles that efficiently target your gene of interest. The kit includes lyophilized Xfect Transfection Reagent and gesicle packaging mixes which contain coding sequences for the nanovesicle-inducing glycoprotein, Cas9 endonuclease, and CherryPicker red fluorescent protein. A pre-linearized vector for cloning your target sgRNA and A/C Heterodimerizer for loading gesicles are also provided. Gesicle production can be performed in any 293T-based cell line, but we recommend the Gesicle Producer 293T Cell Line for optimal production results. Older or non-validated cell lines may produce gesicles less efficiently, thereby decreasing the gesicle concentration and subsequent editing efficiency.
Gesicle production system workflow
The Guide-it CRISPR/Cas9 Gesicle Production System workflow is simple and straightforward. Your sgRNA for a gene of interest is cloned into the provided linearized expression plasmid, pGuide-it-sgRNA1. Next, this cloned plasmid is mixed with dH2O and the Guide-It CRISPR/Cas9 Gesicle Packaging Mixes 1 and 2. Following a 10-minute incubation, the complete nanoparticle mix is applied to 293T-based producer cells in the presence of the provided A/C Heterodimerizer ligand. After 48–72 hours, gesicles containing active Cas9 protein complexed with your sgRNA can be collected and concentrated via centrifugation. Gesicles can be used immediately, or stored at –70°C for more than one year.
The Guide-it CRISPR/Cas9 Gesicle Production System is a complete kit that enables highly efficient gene editing without any additional footprint. This kit contains all of the reagents necessary to prepare custom gesicles to deliver Cas9 and a user-defined, gene-specific guide sequence. Gesicle-based delivery of Cas9 protein and sgRNA using this system results in successful genome editing in a broader range of cell types compared to delivery by plasmid transfection. Critically, gesicles do not result in persistence of the Cas9 endonuclease, which in turn reduces the chances of off-target effects and provides precise control of the dose and timing of gene editing.