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Adeno-associated virus delivery-CRISRP/Cas9 system (Two vector)

AAV CRISPR/Cas9 Vector System

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Á¦Á¶»ç Á¦Ç°ÄÚµå Á¦Ç°¸í ¿ë·® °¡°Ý
(ºÎ°¡¼¼º°µµ)
ºñ°í »ç¿ëÀڸŴº¾ó
Clontech
632608
AAVpro¢ç CRISPR/Cas9 Helper Free System (AAV2)
°ü·ÃÇмú ±¸¸ÅÇϱâ LMOÁ¦Ç°  ¶óÀ̼±½º 
1 System
(Package)
1,707,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x32374, x33215
Clontech
632609
AAVpro¢ç CRISPR/Cas9 Vector System
°ü·ÃÇмú ±¸¸ÅÇϱâ LMOÁ¦Ç°  ¶óÀ̼±½º 
1 System
(Package)
822,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x32374, x33215

¡Ø SaCas9 (from S. aureus) ¸¦ ÀÌ¿ëÇÑ AAV meditated CRISPR/Cas9 Genome EditingÀº AAVpro¢ç CRISPR/SaCas9 Helper Free System (AAV2) (Code 632619)À» È®ÀÎÇϼ¼¿ä.
  • Adeno-Associated Virus (AAV)¸¦ ÀÌ¿ëÇÑ ¼¼Æ÷ ³» Cas9, sgRNA À¯ÀüÀÚ µµÀÔ
  • TransfectionÀÌ ¾î·Á¿î Mammalian cell (ºÐ¿­, ºñºÐ¿­ ¼¼Æ÷)¿¡¼­ Genome Editing
  • AAV¿¡ ÀÇÇÑ Cas9 µµÀÔÀº genome integrationÀ» ¾ïÁ¦ÇÏ°í Cas9ÀÇ ¹ßÇöÀ» Áö¼Ó½Ãų »Ó¸¸ ¾Æ´Ï¶ó, off-target effect¸¦ ¾ïÁ¦
Á¦Ç°¼³¸í
AAVpro¢ç CRISPR/Cas9 Helper free systemÀº CRISPR/Cas9 genome editing¿¡ »ç¿ëµÇ´Â single guide RNA (sgRNA)¿Í Cas9 À¯ÀüÀÚ¸¦ ¹ßÇöÇÏ´Â AAV2 (Adeno-Associated virus)¸¦ Helper virus ¾øÀÌ Á¦ÀÛÇÒ ¼ö ÀÖ´Ù. º» Á¦Ç°Àº 4.1kbÀÇ Cas9 À¯ÀüÀÚ¸¦ 2Á¾·ùÀÇ AAV º¤ÅÍ (pAAV-Guide-it-Up, pAAV-Guide-it-Down vector)¿¡ ³ª´©¾î Á¦ÀÛÇÔÀ¸·Î½á AAVÀÇ »çÀÌÁî Á¦ÇÑÀ» ±Øº¹ÇÏ¿´À» »Ó¸¸ ¾Æ´Ï¶ó ´Ù¾çÇÑ mammalin cell in vitro ±¸Ãà ¹× in vivo¿¡¼­ÀÇ È¿À²ÀûÀÎ Genome EditingÀ» °¡´ÉÄÉ ÇÏ¿´´Ù. º» Á¦Ç°Àº ÃÑ 10ȸºÐÀÇ sgRNA ¹ßÇöÀ» À§ÇÑ linearized vector, °íÈ¿À²ÀÇ Ligation mix¿Í Stellar competent cellÀÌ Æ÷ÇԵǾîÀÖÀ¸¸ç, modified AAV2 Á¦ÀÛÀ» À§ÇÑ pRC2-mi342 Vector¿Í pHelper vector, AAV2 ÀÔÀÚ ÃßÃâÀ» À§ÇÑ AAV Extraction Solutionµµ Æ÷ÇÔÇÏ°í ÀÖ´Ù.


Figure 1. 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.


Figure 2. AAV vector recombination to produce full-length Cas9 and sgRNA. 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.


Figure 3. 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, 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 pre-linearized vector.
Applications
AAV-based delivery of a user-defined sgRNA and Cas9 for mammalian genome editing using CRISPR/Cas9 technology
±¸¼ºÇ° (ÀÚ¼¼ÇÑ ³»¿ëÀº CoA¸¦ ÂüÁ¶Çϼ¼¿ä)
AAVpro¢ç CRISPR/Cas9 Helper Free System (AAV2) (Cat. No. 632608)
  • AAVpro¢ç CRISPR/Cas9 Vector Set
  • Guide-it Ligation Components
  • Stellar Competent Cells
  • pRC2-mi342 Vector
  • pHelper Vector
  • AAVpro¢ç Extraction Solution

AAVpro¢ç CRISPR/Cas9 Vector System (Cat. No. 632609)
  • AAVpro¢ç CRISPR/Cas9 Vector Set
  • Guide-it Ligation Components
  • Stellar Competent Cells




Keyword : AAV,AAV CRISPR,in vivo,µµÀÔÈ¿À²,spCas9,CRISPR,CRISPR/Cas9,Cas9,sgRNA,Å©¸®½ºÆÛ,gene editing,À¯ÀüÀÚÆíÁý

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