AAVpro CRISPR/Cas9 Vector System

Description

The AAVpro CRISPR/Cas9 Vector System is a complete system for cloning a target single guide RNA sgRNA for CRISPR/Cas9-mediated genome editing in mammalian cells. The included AAVpro CRISPR/Cas9 Vector Set consists of two plasmids pAAV-Guide-it-Up and pAAV-Guide-it-Down that each contain a truncated, upstream or downstream portion of the Cas9 gene with a 1.6-kb region of homology. Due to the nature of the AAV genome, when target cells are transduced with both viruses Up and Down , recombination occurs at the region of homology to create a full-length Cas9 gene with an upstream CMV promoter. pAAV-Guide-it-Down is supplied pre-linearized for simple cloning of a target-specific sgRNA sequence under the control of the human U6 promoter. This kit also contains all of the components needed for cloning, including ligation mix, necessary controls, and Stellar Competent Cells. 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 The AAVpro CRISPR/Cas9 system results in more mutations than transfection, especially in hard-to-transfect cell lines.The indicated cell types 1.0 x 105cells were seeded in 12-well plates one day before transduction. Cells were transduced with 1.0 x 105MOI genomic titer each of AAV2-Up and AAV2-Down viral particles targeting theCCR5gene. After 72 hours, cells were harvested and analyzed using the Guide-it Mutation Detection Kit. As a control, cells were transfected with a plasmid P: 2.5 mug encoding Cas9 and a guide sequence targetingCCR5, using Xfect Transfection Reagent. Back Production of full-length Cas9 and sgRNA.The pAAV-Guide-it-Up and pAAV-Guide-it-Down vectors encode truncated upstream and downstream portions of the Cas9 gene respectively, with a 1.6-kb overlapping region of homology. A user-defined single guide RNA sgRNA can be cloned into pAAV-Guide-it-Down see Vector Maps . Separate AAV2-Up and AAV2-Down particles are generated by transfecting HEK 293T cells with either pAAV-Guide-it-Up or pAAV-Guide-it-Down along with pRC2-mi342 and pHelper vectors. The resulting viral particles are isolated using AAV Extraction Solution. Target cells are cotransduced with both AAV2-Up and AAV2-Down particles. The 1.6-kb homologous overlapping region results in recombination during AAV genome processing giving rise to a full-length Cas9 gene that is transcribed and translated into protein. Back AAV vector recombination to produce full-length Cas9 and sgRNA.The large size of the Cas9 gene precludes its packaging into a single AAV particle. pAAV-Guide-it-Up and pAAV-Guide-it-Down vectors contain truncated upstream and downstream portions of Cas9, respectively, with a shared 1.6-kb overlapping region of homology. Separate viruses are generated with each plasmid. Transduction of target cells with both viruses results in recombination to generate the full-length Cas9 4.1 kb gene. Subsequent expression in target cells results in functional Cas9 protein guided to the appropriate genomic site by the sgRNA.. Back Back Vector map of pAAV-Guide-it-Up.The structure of the pAAV-Guide-it-Up vector. This vector is part of the AAVpro CRISPR/Cas9 Helper Free System, a kit that produces AAV particles for cell delivery of Cas9 and sgRNA genes, the components necessary for CRISPR/Cas9 genome editing. The large size of the Cas9 gene precludes its packaging into a single AAV particle. To allow AAV-mediated delivery of the Cas9 gene, this system uses two separate Cas9 plasmids; the pAAV-Guide-it-Up vector contains a truncated portion of the gene 3,106 bp that encodes the N-terminal 1,035 amino acids of Cas9 under the control of the CMV promoter.. Back Vector map of pAAV-Guide-it-Down.The structure of the pre-linearized pAAV-Guide-iT-Down vector. This vector is part of the AAVpro CRISPR/Cas9 Helper Free system, a kit that produces AAV particles for cell delivery of the Cas9 and sgRNA genes, the components necessary for CRISPR/Cas9 genome editing. The large size of the Cas9 gene precludes its packaging into a single AAV virus. To allow AAV-mediated delivery of the Cas9 gene, this system uses two separate Cas9 plasmids. The pAAV-Guide-it-Down vector contains a truncated downstream portion of the gene 2,616 bp that encodes the C-terminal 872 amino acids of Cas9. A user-defined single guide RNA sgRNA can be cloned into the pAAV-Guide-it-Down vector downstream of the human U6 promoter. To construct the vector, a pair of oligos corresponding to the genomic target guide sequence are annealed to form a duplex. The duplexed DNA is then cloned into the prelinearized vector. Back