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abfrontier/AAVpro CRISPR/Cas9载体系统/AAVpro CRISPR/Cas9载体系统,1系统/632608
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선택 | Cat.No. | 제품명 | 가격(VAT별도) | 수량 |
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제품특징
□ 특징
● Adeno-Associated Virus (AAV)를 이용한 세포 내 Cas9, sgRNA 유전자 도입
● Transfection이 어려운 Mammalian cell (분열, 비분열 세포)에서 Genome Editing
● AAV에 의한 Cas9 도입은 genome integration을 억제하고 Cas9의 발현을 지속시킬 뿐만 아니라, off-target effect를 억제
□ 제품설명
AAVpro® CRISPR/Cas9 Helper free system은CRISPR/Cas9 genome editing에 사용되는 single guide RNA(sgRNA)와 Cas9 유전자를 발현하는 AAV2(Adeno-Associated virus)를 Helper virus 없이 제작할 수 있다. 본 제품은 4.1kb의 Cas9 유전자를 2종류의 AAV 벡터 (pAAV-Guide-it-Up,pAAV-Guide-it-Down vector)에 나누어 제작함으로써 AAV의 사이즈 제한을극복하였을 뿐만 아니라 다양한 mammalin cell in vitro 구축 및 in vivo에서의 효율적인 Genome Editing을 가능케 하였다. 본 제품은 총 10회분의sgRNA 발현을 위한 linearized vector, 고효율의 Ligation mix와 Stellar competent cell이포함되어있으며, modified AAV2 제작을 위한pRC2-mi342 Vector와 pHelper vector, AAV2 입자 추출을 위한 AAV Extraction Solution도 포함하고 있다.Figure1. Production of full-length Cas9 and sgRNA. ThepAAV-Guide-it-Up and pAAV-Guide-it-Down vectors encode truncated upstream anddownstream portions of the Cas9 gene respectively, with a 1.6-kb overlappingregion of homology. Figure2. AAV vector recombination to produce full-length Cas9 and sgRNA. Separate virusesare generated with each plasmid. Transduction oftarget cells with both viruses results in recombination to generate the fulllength Cas9 (4.1 kb) gene. Subsequent expression in target cells results infunctional Cas9 protein guided to the appropriate genomic site by the sgRNA.Figure3. Vector Map of pAAV-Guide-it-Down. The structureof the pre-linearized pAAV-Guide-iT-Down vector. This vector is part of theAAVpro CRISPR/Cas9 Helper Free system, a kit that produces AAV particles forcell delivery of the Cas9 and sgRNA genes, components necessary for CRISPR/Cas9genome editing. The large size of the Cas9 gene precludes its packaging into asingle AAV virus. To allow AAV-mediated delivery of the Cas9 gene, this systemuses two separate Cas9 plasmids. The pAAV-Guide-it-Down vector contains atruncated downstream portion of the gene (2,616 bp) that encodes the C-terminal872 amino acids of Cas9. A user-defined single guide RNA (sgRNA) can be clonedinto the pAAV-Guide-it-Down vector downstream of the human U6 promoter. Toconstruct the vector, a pair of oligos corresponding to the genomic target(guide sequence) are annealed to form a duplex.The duplexed DNA is then clonedinto the pre-linearized vector.
□ Applications
AAV-based delivery of a user-defined sgRNA and Cas9 for mammalian genome editing using CRISPR/Cas9 technology
□ 구성품
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