technology overview gesicles enable …technology overview gesicles enable crispr/cas9mediated gene...
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TECHNOLOGY OVERVIEWGesicles enable CRISPR/Cas9mediated gene editing withhigh efficiency and no additional footprintThe Guideit CRISPR/Cas9 Gesicle Production System contains everything you need to easily produce gesiclesthat will efficiently target your gene of interest.
Gesicle production mechanism >>Gesicle production system components >>Gesicle production workflow >>
OverviewWhile CRISPR/Cas9 is a powerful technique for genome manipulation, two significant challenges remain: obtainingefficient delivery of the Cas9/sgRNA complex to all cell types, and leaving no additional footprint (i.e., persistentand 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 proteincomplexed with an sgRNA specific to a gene of interest. Thus, there is no persistent expression of Cas9, becauseno coding gene is present. Additionally, they are engineered with glycoproteins on their surface that mediatebinding and fusion with the membrane of a wide range of target cells. These features enable gesicles to knock outgenes with high efficiency and in a broader range of cell types than plasmidbased delivery methods. Finally, useof this method allows for control of the dose and duration of the Cas9sgRNA complex in the cell, further reducingthe chance of offtarget effects.
Mechanism for producing gesicles to deliver a Cas9sgRNA ribonucleoprotein complexGesicles are made by transfecting a 293Tbased producer cell line with a vector containing an sgRNA for yourtarget locus and our gesicle packaging mix, which contains everything you need in an optimized format. Loadedgesicles 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 sgRNAexpression 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 enablesinducible proteinprotein interactions: The membranebound CherryPicker red fluorescent proteinon the surface of the gesicle is tagged with one dimerization domain, and the Cas9 endonucleasecontains another dimerization domain. A small heterodimerizer ligand is added to link these twodomains together, thereby enriching the Cas9sgRNA RNP complex into the gesicle.
The red fluorescent proteinlabeled gesicles are now loaded with active Cas9 complexed with yourtarget sgRNA. Loaded gesicles pinch off from the producer cells and saturate the surroundingmedium. Gesicles can be collected from the supernatant, yielding a concentrated stock of yourCas9sgRNA gesicles. The gesicles can be used immediately on your target cells, or stored forover one year at –70°C.
Harvested gesicles can be applied to a broad range of target cell types. In the presence ofprotamine sulfate, gesicles fuse to the membrane of the target cell via cellsurface glycoproteinsand release the Cas9sgRNA RNP complex into the cell. The membranes of cells that have beensuccessfully 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 nuclearlocalization signal on the Cas9 endonuclease ensures that the complex dissociates from the redfluorescent protein and is transported to the nucleus. Once there, efficient targeted gene editingtakes place. After editing occurs, the Cas9sgRNA RNP complex is degraded over time byendogenous cell processes, leaving no remaining footprint that would be capable of producing offtarget effects.
Gesicle production system components
The Guideit CRISPR/Cas9 Gesicle Production System contains everything you need to easily produce gesiclesthat efficiently target your gene of interest. The kit includes lyophilized Xfect Transfection Reagent and gesiclepackaging 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/CHeterodimerizer for loading gesicles are also provided. Gesicle production can be performed in any 293Tbasedcell line, but we recommend the Gesicle Producer 293T Cell Line for optimal production results. Older or nonvalidated cell lines may produce gesicles less efficiently, thereby decreasing the gesicle concentration andsubsequent editing efficiency.
Gesicle production system workflow
The Guideit CRISPR/Cas9 Gesicle Production System workflow is simple and straightforward. Your sgRNA for agene of interest is cloned into the provided linearized expression plasmid, pGuideitsgRNA1. Next, this clonedplasmid is mixed with dH2O and the GuideIt CRISPR/Cas9 Gesicle Packaging Mixes 1 and 2. Following a 10minute incubation, the complete nanoparticle mix is applied to 293Tbased producer cells in the presence of theprovided A/C Heterodimerizer ligand. After 48–72 hours, gesicles containing active Cas9 protein complexed withyour sgRNA can be collected and concentrated via centrifugation. Gesicles can be used immediately, or stored at–70°C for more than one year.
ConclusionsThe 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 inturn reduces the chances of offtarget effects and provides precise control of the dose and timing of gene editing.
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