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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	700,000원		,
Clontech	632602	Guide-it™ CRISPR/Cas9 System (Red)	1 System	700,000원		,

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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℃

Citation

Capturing human trophoblast development with naive pluripotent stem cells in vitro.
Cell stem cell | 2021 Jun 24 | PubMed ID: 33831365 | Read Article

Resolving Cell Fate Decisions during Somatic Cell Reprogramming by Single-Cell RNA-Seq
Molecular Cell | 2019 Feb 01 | PubMed ID: 30772174 | Read Article

The Dual Function of KDM5C in Both Gene Transcriptional Activation and Repression Promotes Breast Cancer Cell Growth and Tumorigenesis
Advanced Science | 2021 Feb 18 | PubMed ID: 33977073 | Read Article

Pro-death signaling of cytoprotective heat shock factor 1: upregulation of NOXA leading to apoptosis in heat-sensitive cells
Cell Death and Differentiation | 2020 Jan 29 | PubMed ID: 31996779 | Read Article

GCN2 kinase Activation by ATP-competitive Kinase Inhibitors
Nature chemical biology | 2021 Dec 23 | PubMed ID: 34949839 | Read Article

Selective degradation of splicing factor CAPERα by anticancer sulfonamides
Nature Chemical Biology | 2017 Apr 24 | PubMed ID: 28437394 | Read Article

Improved secretion of glycoproteins using an N-glycan-restricted passport sequence tag recognized by cargo receptor
Nature Communications | 2020 Mar 13 | PubMed ID: 32170195 | Read Article

Frequency and mechanisms of LINE-1 retrotransposon insertions at CRISPR/Cas9 sites
Nature Communications | 2022 Jun 27 | PubMed ID: 35760782 | Read Article

TRAF3IP3 negatively regulates cytosolic RNA induced anti-viral signaling by promoting TBK1 K48 ubiquitination
Nature Communications | 2020 May 04 | PubMed ID: 32366851 | Read Article

Classical non-homologous end-joining pathway utilizes nascent RNA for error-free double-strand break repair of transcribed genes
Nature Communications | 2016 Oct 05 | PubMed ID: 27703167 | Read Article

Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3
Nature Communications | 2022 Aug 30 | PubMed ID: 36042215 | Read Article

Overcoming resistance to αPD-1 of MMR-deficient tumors with high tumor-induced neutrophils levels by combination of αCTLA-4 and αPD-1 blockers
Journal for Immunotherapy of Cancer | 2022 Jul 27 | PubMed ID: 35896284 | Read Article

Serine racemase enhances growth of colorectal cancer by producing pyruvate from serine
Nature Metabolism | 2020 Jan 01 | PubMed ID: 32694681 | Read Article

E74-Like Factor 3 Is a Key Regulator of Epithelial Integrity and Immune Response Genes in Biliary Tract Cancer
Cancer Research | 2020 Dec 08 | Read Article

ASGCT Annual Meeting Abstracts
Molecular Therapy | 2017 May 01 | Read Article

A genetically linked pair of NLR immune receptors shows contrasting patterns of evolution
Proceedings of the National Academy of Sc… | 2022 Jun 30 | PubMed ID: 35771942 | Read Article

Pluripotent stem cell models of Blau syndrome reveal an IFN-γ–dependent inflammatory response in macrophages
Journal of Allergy and Clinical Immunology | 2018 Jan 01 | PubMed ID: 28587749 | Read Article

Tissue-specific enhancement of OsRNS1 with root-preferred expression is required for the increase of crop yield
Journal of Advanced Research | 2022 May 01 | PubMed ID: 35609869 | Read Article

Tumor cell-derived ANGPTL2 promotes β-catenin-driven intestinal tumorigenesis
Oncogene | 2022 Jul 13 | PubMed ID: 35831580 | Read Article

CTP synthase is essential for early endosperm development by regulating nuclei spacing
Plant Biotechnology Journal | 2021 Jun 19 | Read Article

ENTREP/FAM189A2 encodes a new ITCH ubiquitin ligase activator that is downregulated in breast cancer
EMBO Reports | 2021 Dec 20 | PubMed ID: 34927784 | Read Article

Critical Role of ATP-P2X7 Axis in UV-Induced Melanogenesis
Journal of Investigative Dermatology | 2019 Jul 01 | PubMed ID: 30926287 | Read Article

Highly efficient generation of isogenic pluripotent stem cell models using prime editing
eLife | 2022 Sep 07 | PubMed ID: 36069759 | Read Article

Highly efficient generation of isogenic pluripotent stem cell models using prime editing
eLife | 2022 Sep 07 | PubMed ID: 36069759 | Read Article

Ratiometric sensing of BiP-client versus BiP levels by the unfolded protein response determines its signaling amplitude
eLife | 2017 Dec 18 | PubMed ID: 29251598 | Read Article

Post-transcriptional repression of circadian component CLOCK regulates cancer-stemness in murine breast cancer cells
eLife | 2021 Apr 23 | PubMed ID: 33890571 | Read Article

Scribble sub-cellular localization modulates recruitment of YES1 to regulate YAP1 phosphorylation
Cell Chemical Biology | 2021 Aug 01 | PubMed ID: 33730553 | Read Article

The Plasmodium falciparum Nuclear Protein Phosphatase NIF4 Is Required for Efficient Merozoite Invasion and Regulates Artemisinin Sensitivity
mBio | 2022 Aug 08 | PubMed ID: 35938722 | Read Article

Hepatocyte-Derived Lipotoxic Extracellular Vesicle Sphingosine 1-Phosphate Induces Macrophage Chemotaxis
Frontiers in Immunology | 2018 Dec 19 | PubMed ID: 30619336 | Read Article

GPRC5A facilitates cell proliferation through cell cycle regulation and correlates with bone metastasis in prostate cancer.
International journal of cancer | 2020 Feb 07 | PubMed ID: 31276604 | Read Article

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

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