Guide-itTM CRISPR/Cas9 Gesicle Packaging Set

Description

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 generated using the Guide-it CRISPR/Cas9 Gesicle Packaging Set 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 and mediates binding and fusion with the cellular membrane of target cells. Simultaneous overexpression of additional macromolecular cargoes, in this case the Cas9 protein fromStreptococcus pyogenesand 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. The Guide-it CRISPR/Cas9 Gesicles Packaging Mixes 1 and 2 are each supplied prealiquoted and lyophilized in single vials containing Xfect Transfection Reagent and an optimized combination of gesicle-producing components. Gesicles are produced by sequentially reconstituting the lyophilized mixtures with your guide RNA vector of choice in sterile water and transfecting the sample into Gesicle Producer 293T cells according to the protocol in the Guide-it CRISPR/Cas9 Gesicle Production System User Manual. It is essential to use the pGuide-itsgRNA1 Vector for expression/packaging of your guide RNA, since using other commonly used guide RNA vectors will not result in effective Cas9/sgRNA complexes. Our products are to be used forResearch Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval. Back 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 Back Successful knockout ofCD81in hiPS cells.Gesicles containing Cas9-sgRNA complexes designed to target human CD81 were harvested and added to Cellartis Human iPS Cell Line 18 hiPSC ChiPSC18 , cultured in Cellartis DEF-CS Culture System for 6 and 24 hr, and then cultured in gesicle-free DEF-CS culture media for an additional 7 days. The surface expression of CD81 on gesicle-treated cells and untreated control cells was determined via flow cytometry analysis using FITC-labeled antibodies against CD81. Panel A. CD81 negative left and positive right labeling controls with hiPSC ChiPSC18 cells. Panel B. DEF-hiPSC ChiPSC18 cells after 6 hr left and 24 hr right of gesicle treatment, labeled with anti-CD81 FITC antibodies. Back