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Genome Editing


Products >  Genome_Editing >  CRISPR_Cas9 >  AAV_Systems

Helper Virus-Free Preparation of AAV particles to deliver Cas9 and sgRNA

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 SaCas9. 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. No. 632608) and the AAVpro CRISPR/SaCas9 Helper Free System (Cat. No. 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. Note that SpCas9 and SaCas9 can have different targeting efficiencies even when used on the same region of the genome – see Images & Data for details.

  At-A-Glance   Documents   Images & Data   Resources

Features

  • Viral delivery of the Cas9 gene and a 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 the human microRNA miR-342 from the packaging plasmid significantly increases AAV titer

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 AAVpro CRISPR/Cas9 Systems User Manual for details
  • 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 – 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 AAVpro CRISPR SaCas9 Systems User Manual for details
  • 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. No. 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. No. 632609)

  • AAVpro CRISPR/Cas9 Vector Set
  • Guide-it Ligation Components
  • Stellar Competent Cells

AAVpro CRISPR/SaCas9 Helper Free System (AAV2) (Cat. No. 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. No. 632618)

  • AAVpro CRISPR/SaCas9 Vector Set
  • Guide-it Ligation Components
  • Stellar Competent Cells

Additional 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.

 
 
Products
Cat. # Product Contents Size Price License Units Select
632608 AAVpro® CRISPR/Cas9 Helper Free System (AAV2) 1 System $894.00 License Statements
632609 AAVpro® CRISPR/Cas9 Vector System 1 System $502.00 License Statements
632618 AAVpro® CRISPR/SaCas9 Vector System 1 System $492.00 License Statements
632619 AAVpro® CRISPR/SaCas9 Helper Free System (AAV2) 1 System $876.00 License Statements
 

 

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