AAV CRISPR/Cas9 Systems

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.