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TECHNOLOGY OVERVIEW
Gesicles enable CRISPR/Cas9­mediated gene editing with
high efficiency and no additional footprint
The Guide­it CRISPR/Cas9 Gesicle Production System contains everything you need to easily produce gesicles
that will efficiently target your gene of interest.
Gesicle production mechanism >>
Gesicle production system components >>
Gesicle production workflow >>
Overview
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 off­target effects. To address these challenges,
we have developed a system of cell­derived 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 plasmid­based delivery methods. Finally, use
of this method allows for control of the dose and duration of the Cas9­sgRNA complex in the cell, further reducing
the chance of off­target effects.
Mechanism for producing gesicles to deliver a Cas9­sgRNA ribonucleoprotein complex
Gesicles are made by transfecting a 293T­based 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 nanovesicle­inducing 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 protein­protein interactions: The membrane­bound 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 Cas9­sgRNA RNP complex into the gesicle.
The red fluorescent protein­labeled 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
Cas9­sgRNA 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 cell­surface glycoproteins
and release the Cas9­sgRNA 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 Cas9­sgRNA 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 Guide­it 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 nanovesicle­inducing glycoprotein, Cas9 endonuclease,
and CherryPicker red fluorescent protein. A pre­linearized vector for cloning your target sgRNA and A/C
Heterodimerizer for loading gesicles are also provided. Gesicle production can be performed in any 293T­based
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 Guide­it 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, pGuide­it­sgRNA1. Next, this cloned
plasmid is mixed with dH2O and the Guide­It CRISPR/Cas9 Gesicle Packaging Mixes 1 and 2. Following a 10­
minute incubation, the complete nanoparticle mix is applied to 293T­based 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.
Conclusions
The Guide­it 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 user­defined, gene­specific guide sequence. Gesicle­based 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 off­target effects and provides precise control of the dose and timing of gene editing.
http://www.clontech.com/US/Products/Genome_Editing/CRISPR_Cas9/Resources/CRISPR_Cas9_Gesicles_Overview