※ SaCas9 (from S. aureus) 를 이용한 AAV meditated CRISPR/Cas9 Genome Editing은 AAVpro® CRISPR/SaCas9 Helper Free System (AAV2) (Code 632619)을 확인하세요.
제품설명
AAVpro® CRISPR/Cas9 Helper free system은 CRISPR/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 제한을 극복하였을 뿐만 아니라 다양한 mammalin cell in vitro 구축 및 in vivo에서의 효율적인 Genome Editing을 가능케 하였다. 본 제품은 총 10회분의 sgRNA 발현을 위한 linearized vector, 고효율의 Ligation mix와 Stellar competent cell이 포함되어있으며, modified AAV2 제작을 위한 pRC2-mi342 Vector와 pHelper vector, AAV2 입자 추출을 위한 AAV Extraction Solution도 포함하고 있다.


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
구성품 (자세한 내용은 CoA를 참조하세요)
AAVpro® CRISPR/Cas9 Helper Free System (AAV2) (Cat. No. 632608)
AAVpro® CRISPR/Cas9 Vector System (Cat. No. 632609)