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Single vector를 이용한 효율적인 sgRNA, Cas9 발현

CRISPR Plasmid System

제조사	제품코드	제품명	용량	가격 (부가세별도)	비고	사용자매뉴얼
Clontech	632601	Guide-it™ CRISPR/Cas9 System (Green)	1 System (Package)	728,000원		,
Clontech	632602	Guide-it™ CRISPR/Cas9 System (Red)	1 System (Package)	728,000원		,

Product index

Overivew
유전자 편집 전 단계
편집 유전자 도입
유전자 편집 후 확인
CRE recombinase
- CRE Recombinase Gesicl..
- AAV Helper Free System..

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 ZsGreen1
또는 tdTomato 사용
Simple Plasmid construction: vector가 미리 선형화(Pre-linearized vector)되어있어 원하는 유전자의 합성 sgRNA를 간단히
ligation & clone
Complete system: sgRNA/Cas9 system에 필요한 제품이 모두 포함

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

Cloning and expression of a target single guide RNA (sgRNA) for mammalian genome editing using CRISPR/Cas9 technology

구성품 (자세한 내용은 CoA를 참조하세요)

Guide-it CRISPR/Cas9 System (Green) (Cat. No. 632601)

pGuide-it-ZsGreen1 Vector (Linear)
Guide-it Ligation Components
Stellar Competent Cells

Guide-it CRISPR/Cas9 System (Red) (Cat. No. 632602)

pGuide-it-tdTomato Vector (Linear)
Guide-it Ligation Components
Stellar Competent Cells

보존

Stellar Competent Cells:-70℃
기타:-20℃

Keyword : CRISPR vector,Cas9 vector,CRISPR,CRISPR/Cas9,Cas9,sgRNA,크리스퍼,gene editing,유전자편집

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