Takara Cloning and expression systems for sgRNAs

The Guide-it CRISPR/Cas9 systems are kits for the cloning and expression of single guide RNAs (sgRNAs) for mammalian genome editing using CRISPR/Cas9 technology. The vectors in these systems simultaneously express Cas9 nuclease, a target-specific sgRNA, and an exceptionally bright fluorescent protein for monitoring transfection efficiency and/or for further enriching/isolating transfected cells by flow cytometry (ZsGreen1 and tdTomato versions are available).

The Guide-it CRISPR/Cas9 systems are kits for the cloning and expression of single guide RNAs (sgRNAs) for mammalian genome editing using CRISPR/Cas9 technology. The vectors in these systems simultaneously express Cas9 nuclease, a target-specific sgRNA, and an exceptionally bright fluorescent protein for monitoring transfection efficiency and/or for further enriching/isolating transfected cells by flow cytometry (ZsGreen1 and tdTomato versions are available). Generating a plasmid that expresses a sequence-specific sgRNA with this system is simple: a pair of user-provided oligos corresponding to the target genomic sequence of interest are annealed to form a duplex, and the duplexed oligos are inserted into the pre-linearized vector using the included high-efficiency ligation mix. The kit also includes Stellar Competent Cells to ensure high-efficiency transformation.

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Overview

• A single vector expresses your target-specific sgRNA from the human U6 promoter; Cas9 nuclease and a bright fluorescent marker (either ZsGreen1 or tdTomato) are expressed from a bidirectional CMV promoter
• tdTomato and ZsGreen1 are exceptionally bright fluorescent proteins, 6x and 2.5x brighter than EGFP, respectively; these markers can be used to monitor transfection efficiency and/or to enrich/isolate transfected cells by flow cytometry
• Simple plasmid construction—design oligos specific to your target sequence and clone into the pre-linearized plasmid using the included ligation reagents and high-efficiency competent cells
• Includes sufficient reagents for construction of 10 different target (sgRNA) expression plasmids

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Applications

• Cloning and expression of a target sgRNA for mammalian genome editing using CRISPR/Cas9 technology

Components

• Guide-it CRISPR/Cas9 System (Green) (Cat. # 632601)

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

• Guide-it CRISPR/Cas9 System (Red) (Cat. # 632602)

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

Additional product information

Please see the product`s Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.