AAV CRISPR/Cas9 Systems
AAV CRISPR/Cas9 Systems
Brand: Takara Bio.
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AAV CRISPR Cas9 Systems
AAV2 CRISPR/Cas9 system
We offer two kits for helper virus-free preparation of AAV2 particles for genome editing with Cas9: the AAVpro CRISPR/Cas9 Helper Free System and the AAVpro CRISPR/SaCas9 Helper Free System. These kits simultaneously deliver expression cassettes for Cas9 and a user-defined single guide RNA (sgRNA) to mammalian cells. Cas9 sequences are derived from either Streptococcus pyogenes (the CRISPR/Cas9 systems) or Staphylococcus aureus (the CRISPR/SaCas9 systems).
The use of an AAV system allows for efficient genome modification in a wide variety of mammalian cells in vitro, with the CRISPR/Cas9 system using two AAV vectors to deliver the larger S. pyogenes Cas9 (SpCas9), while the CRISPR/SaCas9 system delivers everything in a single vector due to the smaller size of the SaCas9 gene. sgRNAs can be cloned directly into the prelinearized pAAV-Guide-it-Down or pAAV-Guide-it-1 plasmids in a single step. High-titer AAV2 particles can be prepared without a helper virus by simply transfecting HEK 293T cells with the included plasmids. Both the AAVpro CRISPR/Cas9 Helper Free System (Cat. # 632608) and the AAVpro CRISPR/SaCas9 Helper Free System (Cat. # 632619) are complete systems, containing all the reagents necessary to clone sgRNAs and prepare AAV particles. AAV Extraction Solution is also included for efficient AAV2 viral particle isolation. Please note that SpCas9 and SaCas9 can have different targeting efficiencies even when used on the same region of the genome—refer to Image Data (via the Details arrow in the product table below) for more information.
Overview
- Viral delivery of the Cas9 gene and an sgRNA allows for genome editing in hard-to-transfect mammalian cells, including proliferating and non-proliferating cells
- AAV delivery of Cas9 precludes genomic integration and persistent expression of Cas9, reducing off-target effects
- Expression of human microRNA miR-342 from the packaging plasmid significantly increases AAV titer
- AAV Extraction Solution is also included for efficient AAV2 viral particle isolation
Applications
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AAV-based delivery of a user-defined sgRNA and Cas9 for mammalian genome editing using CRISPR/Cas9 technology
AAVpro CRISPR/Cas9 Helper Free Systems (with S. pyogenes Cas9)
- The SpCas9 gene is split between pAAV-Guide-it-Up and pAAV-Guide-it-Down plasmids with a 1.6-kb region of homology; the homologous region results in recombination in target cells, producing a full-length SpCas9 gene
- SpCas9 uses the Protospacer Adjacent Motif (PAM) sequence NGG where N is any base—see the AAVpro CRISPR/Cas9 Systems User Manual for details
- The pAAV-Guide-it-Down plasmid is provided prelinearized, facilitating single-step cloning of sgRNAs with reduced background due to plasmid re-ligation
AAVpro CRISPR/SaCas9 Helper Free Systems (with S. aureus Cas9)
- The SaCas9 gene fits onto a single plasmid, pAAV-Guide-it-1, which that also contains the sgRNA expression cassette
- SaCas9 (from Staphylococcus aureus) uses the PAM sequence NNGRR(T) where N is any base, R is an A or G base, and the (T) is strongly preferred—see the AAVpro CRISPR SaCas9 Systems User Manual for details
- The pAAV-Guide-it-1 plasmid is provided prelinearized, facilitating single-step cloning of sgRNAs with reduced background due to plasmid re-ligation
Components
- AAVpro CRISPR/Cas9 Helper Free System (AAV2) (Cat. # 632608)
- AAVpro CRISPR/Cas9 Vector Set
- Guide-it Ligation Components
- Stellar Competent Cells
- pRC2-mi342 Vector
- pHelper Vector
- AAVpro Extraction Solution
- AAVpro CRISPR/Cas9 Vector System (Cat. # 632609)
- AAVpro CRISPR/Cas9 Vector Set
- Guide-it Ligation Components
- Stellar Competent Cells
- AAVpro CRISPR/SaCas9 Helper Free System (AAV2) (Cat. # 632619)
- AAVpro CRISPR/SaCas9 Vector Set
- Guide-it Ligation Components
- Stellar Competent Cells
- pRC2-mi342 Vector
- pHelper Vector
- AAVpro Extraction Solution
- AAVpro CRISPR/SaCas9 Vector System (Cat. # 632618)
- AAVpro CRISPR/SaCas9 Vector Set
- Guide-it Ligation Components
- Stellar Competent Cells
The AAVpro CRISPR/Cas9 system results in more mutations than transfection, especially in hard-to-transfect cell lines
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 105 cells) were seeded in 12-well plates one day before transduction. Cells were transduced with 1.0 x 105 MOI (genomic titer) each of AAV2-Up and AAV2-Down viral particles targeting the CCR5 gene. 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 µg) encoding Cas9 and a guide sequence targeting CCR5, using Xfect Transfection Reagent.
The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells
The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells. The CRISPR/Cas9 system relies on a single guide RNA (sgRNA) directing the Cas9 endonuclease to induce a double strand break at a specific target sequence three base-pairs upstream of a PAM sequence in genomic DNA. This DNA cleavage can be repaired in one of two ways: 1) nonhomologous end joining, (NHEJ) resulting in gene knockout due to error-prone repair (orange), or 2) homology-directed repair (HDR), resulting in gene knockin due to the presence of a homologous repair template (purple).
AAV vector recombination to produce full-length Cas9 and sgRNA
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.
The AAVpro CRISPR/SaCas9 systems express SaCas9 and sgRNA from a single vector
The AAVpro CRISPR/SaCas9 systems express SaCas9 and sgRNA from a single vector. pAAV-Guide-it-1 is supplied pre-linearized and ready for insertion of the sgRNA target sequence (sgRNA shown in orange). When target cells are transduced with a virus packaged with the vector, the cells will express both SaCas9 and the sgRNA in order to achieve genome editing.
Guide-it Mutation Detection Kit
Guide-it Mutation Detection Kit for side-by-side genomic DNA modification comparison using the AAVpro CRISPR/Cas9 System (SpCas9) and the AAVpro CRISPR/SaCas9 System. Two different genomic regions in CYP2 Exon 1 were targeted and the results are shown in wells 1 and 2 for each enzyme, and the target regions are shown below the gel. The indel percentage is indicated below each well for each cell type. To carry out the modification, 1 x 105 cells of three different cell lines were seeded in each well of 12-well plates one day before transduction. Cells were transduced with either the two-vector AAVpro CRISPR/Cas9 System (SpCas9) or the one-vector AAVpro CRISPR/SaCas9 System at 1 x 105 MOI for each vector as determined by genomic titer. After 72 hr, cells were harvested and analyzed using the Guide-it Mutation Detection Kit.
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Components
- AAVpro CRISPR/Cas9 Helper Free System (AAV2) (Cat. # 632608)
- AAVpro CRISPR/Cas9 Vector Set
- Guide-it Ligation Components
- Stellar Competent Cells
- pRC2-mi342 Vector
- pHelper Vector
- AAVpro Extraction Solution
- AAVpro CRISPR/Cas9 Vector System (Cat. # 632609)
- AAVpro CRISPR/Cas9 Vector Set
- Guide-it Ligation Components
- Stellar Competent Cells
- AAVpro CRISPR/SaCas9 Helper Free System (AAV2) (Cat. # 632619)
- AAVpro CRISPR/SaCas9 Vector Set
- Guide-it Ligation Components
- Stellar Competent Cells
- pRC2-mi342 Vector
- pHelper Vector
- AAVpro Extraction Solution
- AAVpro CRISPR/SaCas9 Vector System (Cat. # 632618)
- AAVpro CRISPR/SaCas9 Vector Set
- Guide-it Ligation Components
- Stellar Competent Cells
The AAVpro CRISPR/Cas9 system results in more mutations than transfection, especially in hard-to-transfect cell lines
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 105 cells) were seeded in 12-well plates one day before transduction. Cells were transduced with 1.0 x 105 MOI (genomic titer) each of AAV2-Up and AAV2-Down viral particles targeting the CCR5 gene. 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 µg) encoding Cas9 and a guide sequence targeting CCR5, using Xfect Transfection Reagent.
The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells
The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells. The CRISPR/Cas9 system relies on a single guide RNA (sgRNA) directing the Cas9 endonuclease to induce a double strand break at a specific target sequence three base-pairs upstream of a PAM sequence in genomic DNA. This DNA cleavage can be repaired in one of two ways: 1) nonhomologous end joining, (NHEJ) resulting in gene knockout due to error-prone repair (orange), or 2) homology-directed repair (HDR), resulting in gene knockin due to the presence of a homologous repair template (purple).
AAV vector recombination to produce full-length Cas9 and sgRNA
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.
The AAVpro CRISPR/SaCas9 systems express SaCas9 and sgRNA from a single vector
The AAVpro CRISPR/SaCas9 systems express SaCas9 and sgRNA from a single vector. pAAV-Guide-it-1 is supplied pre-linearized and ready for insertion of the sgRNA target sequence (sgRNA shown in orange). When target cells are transduced with a virus packaged with the vector, the cells will express both SaCas9 and the sgRNA in order to achieve genome editing.
Guide-it Mutation Detection Kit
Guide-it Mutation Detection Kit for side-by-side genomic DNA modification comparison using the AAVpro CRISPR/Cas9 System (SpCas9) and the AAVpro CRISPR/SaCas9 System. Two different genomic regions in CYP2 Exon 1 were targeted and the results are shown in wells 1 and 2 for each enzyme, and the target regions are shown below the gel. The indel percentage is indicated below each well for each cell type. To carry out the modification, 1 x 105 cells of three different cell lines were seeded in each well of 12-well plates one day before transduction. Cells were transduced with either the two-vector AAVpro CRISPR/Cas9 System (SpCas9) or the one-vector AAVpro CRISPR/SaCas9 System at 1 x 105 MOI for each vector as determined by genomic titer. After 72 hr, cells were harvested and analyzed using the Guide-it Mutation Detection Kit.
