제품설명
Guide-it CRISPR/Cas9 system 제품은 CRISPR/Cas9 기술의 mammalian genome modification (or genome editing)을 위해 target single guide RNAs(sgRNAs)의 클로닝 및 발현을 할 수 있도록 제작된 제품이다. 본 제품에 포함되어있는 벡터는 Cas9 nuclease, target-specific sgRNA, 형광단백질을 발현할 수 있으며, 발현된 형광단백질은 transfection 효율을 모니터링 하거나 Flow cytometry를 이용하여 transfected cell을 isolating/enriching 에 사용할 수 있다.



그림 1. The structure of the pGuide-it vector. This vector is part of the Guide-it CRISPR/Cas9 Systems, kits for the cloning of a target single guide RNA (sgRNA) for CRISPR/Cas9 genome editing. The pGuide-it vector is used to simultaneously express Cas9 nuclease, a target-specific sgRNA, and an exceptionally bright fluorescent protein (either ZsGreen1 or tdTomato). Cas9 and the fluorescent protein are co-expressed from a bidirectional CMV promoter, and a user-defined sgRNA is cloned downstream of a human U6 promoter. To construct the vector, a pair of oligos corresponding to the genomic target (your guide sequence) are annealed to form a duplex. Then, the duplexed DNA is cloned into the pre-linearized vector using the included high-efficiency ligation mix and competent cells.



그림 2. Genomic DNA modifications introduced using the pGuide-it-ZsGreen1 plasmid. 1 x 105 cells (HEK 293, HeLa, U2OS, or HT1080 cell lines) were seeded in 12-well plates 16 hr prior to transfection. Cells were transfected with 2.5 μg of pGuide-it-ZsGreen1 plasmid using the Xfect Transfection Reagent (Code 631317); the plasmid expressed Cas9, ZsGreen1, and an sgRNA targeting the either CCR5, C4BPB, or EMX1. After 48 hr, fluorescence was analyzed by microscopy (A), and by FACS (B, Transfection efficiency). The efficiency of gene modification was also evaluated using the Guide-it Mutation Detection Kit (Code 631443), a PCR-based method for testing for the presence of indels (B). The target sequence was amplified directly from cells, the amplicon was melted and hybridized to form mismatched targets that were cleaved with Guide-it Resolvase. Cleavage products were resolved on an agarose gel and quantified by densitometry to estimate the frequency of indels (B, Indel).
적용
구성품 (자세한 내용은 CoA를 참조하세요)
Guide-it CRISPR/Cas9 System (Green) (Cat. No. 632601)
Guide-it CRISPR/Cas9 System (Red) (Cat. No. 632602)
보존
Stellar Competent Cells:-70℃
기타:-20℃