□ 특징

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● Single vector system : 하나의 벡터에서 target-specific sgRNA, Cas9 nuclease, 형광단백질(ZsGreen1 또는 tdTomato)

발현

● Exceptionally bright fluorescent protein : trasnfection 효율을 확인할 수 있는 리포터로써 EGFP보다 2.5배~6배 더 밝은

ZsGreen1또는 tdTomato 사용

● Simple Plasmid construction : vector가미리 선형화(Pre-linearized vector)되어있어 원하는 유전자의 합성 sgRNA를

간단히 ligation & clone

● Complete system : sgRNA/Cas9 system에필요한 제품이 모두 포함

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□ 제품설명

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Guide-it CRISPR/Cas9system 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 cytometry를이용하여 transfected cell을isolating/enriching 에 사용할 수 있다.그림1. The structure of the pGuide-it vector. Thisvector is part of the Guide-it CRISPR/Cas9 Systems, kits for the cloning of atarget single guide RNA (sgRNA) for CRISPR/Cas9 genome editing. The pGuide-itvector is used tosimultaneously express Cas9 nuclease, a target-specificsgRNA, and an exceptionally bright fluorescent protein (either ZsGreen1 ortdTomato). Cas9 and the fluorescent protein are co-expressed from abidirectional CMV promoter, and a user-defined sgRNA is cloned downstream of ahuman U6 promoter. To construct the vector, a pair of oligos corresponding tothe genomic target (your guide sequence) are annealed to form a duplex. Then,the duplexed DNA is cloned into the pre-linearized vector using the includedhigh-efficiency ligation mix and competent cells.그림2. Genomic DNA modifications introduced using the pGuide-it-ZsGreen1 plasmid. 1 x 10⁵ cells (HEK 293, HeLa, U2OS, or HT1080 celllines) were seeded in 12-well plates 16 hr prior to transfection. Cells weretransfected with 2.5 μg of pGuide-it-ZsGreen1 plasmid using the XfectTransfection 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, Transfectionefficiency). The efficiency of gene modification was also evaluated using the Guide-itMutation Detection Kit (Code 631443), a PCR-based method for testing forthe presence of indels (B). The target sequence was amplified directly fromcells, the amplicon was melted and hybridized to form mismatched targets thatwere cleaved with Guide-it Resolvase. Cleavage products were resolved on anagarose gel and quantified by densitometry to estimate the frequency of indels(B, Indel).

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□ 적용

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​□ 구성품

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□ 보존

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Stellar Competent Cells : -70℃기타 : -20℃