Takara Helper virus-free preparation of AAV particles to deliver Cas9 and sgRNA

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

More Information

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

Applications

• AAV-based delivery of a user-defined sgRNA and Cas9 for mammalian genome editing using CRISPR/Cas9 technology

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

Additional product information

Please see the product`s Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.