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Clipos™ CAS 9-RNA Lipid Nanoparticles-HA

Catalog:CDPM05

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StorageStore at 4°C
DescriptionCRISPR-Cas9 is a relatively new technology that has enabled scientists and medical researchers to edit parts of a genome by removing, adding, and altering sections of a DNA sequence. It is currently the simplest, most versatile, and precise method of genetic manipulation. As a CRISPRassociated enzyme, Cas-9 is utilized as an RNA-guided endonuclease that requires a guide RNA product for genome DNA target recognition and ultimately, the generation of DNA double-strand breaks. Cas-9 mRNA encodes a specific protein made of Nitrogen and Carbon terminal nuclear localization signals (NLS). The incorporation of two NLS signals within the mRNA increases the frequency of delivery to the nucleus, thus increasing the rate of DNA cleavage. A Carbon terminal HA epitope tag is also utilized to aid detection, isolation, and purification of the Cas-9 protein. mRNA encapsulated in lipid nanoparticle, primarily in PBS (-ca, -mg). Dilute levels of: sodium acetate, ethanol, free mRNA & free lipids.
Technical NotesWork with mRNA-LNP on ice. It is important to minimize the time that the product spends at room temperature; after handling product during experiments, return immediately to ice. Upon receiving product, briefly pulse spin before opening to ensure product is at bottom of container; it is important not to spin for too long as this may rupture mRNA-LNP’s. Do not vortex. mRNA-LNP products should only be handled with certified RNAase-free reagents and consumables; use of filtered pipette tips is highly recommended.
Usage1. Thaw & rinse PBMC in CAR-T medium, then suspend the cells at 1 x 10^6 cells per mL. 2. Pipet 500 uL of PBMC into 1 well of a 24-well plate containing CD3/CD28 antibodies - either bound to beads or bound to the plate itself. 3. When the T cells are growing well (4-7 days later), pipet 1 million cells into 3 wells of a 12-well plate and add medium to 1 mL per well. 4. Add the Cas9 RNA nanoparticles to well #1. 5. The next day, electroporate the cells with the sgRNA of choice. 1. LNP-treated cells (well #1): electroporate only with the sgRNA 2. Non-treated cells (well #2): electroporate only with the sgRNA 3.Non-treated cells (well #3): electroporate with a mixture of the sgRNA and Cas9 protein. 6. Culture the cells for 3-7 days, during which time the levels of the gene product will steadily decrease in the cells with both alleles knocked out. 7. Analyze the cells by flow cytometry with an antibody against the gene product .
Encoding mRNACas9
In Vitro QC (Cell Types)HEK293S
Technology ApplicationsControl
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