CRISPR/Cas9 Plasmid Systems
CRISPR/Cas9 Plasmid Systems
Brand: Takara Bio.
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CRISPR/Cas9 Plasmid Systems
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.
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
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
Genomic DNA modifications introduced using the pGuide-it-ZsGreen1 plasmid
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; 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, 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).
The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells
The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells. The CRISPR/Cas9 system relies on a single guide RNA (sgRNA) directing the Cas9 endonuclease to induce a double strand break at a specific target sequence three base-pairs upstream of a PAM sequence in genomic DNA. This DNA cleavage can be repaired in one of two ways: 1) nonhomologous end joining, (NHEJ) resulting in gene knockout due to error-prone repair (orange), or 2) homology-directed repair (HDR), resulting in gene knockin due to the presence of a homologous repair template (purple).
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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
Genomic DNA modifications introduced using the pGuide-it-ZsGreen1 plasmid
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; 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, 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).
The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells
The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells. The CRISPR/Cas9 system relies on a single guide RNA (sgRNA) directing the Cas9 endonuclease to induce a double strand break at a specific target sequence three base-pairs upstream of a PAM sequence in genomic DNA. This DNA cleavage can be repaired in one of two ways: 1) nonhomologous end joining, (NHEJ) resulting in gene knockout due to error-prone repair (orange), or 2) homology-directed repair (HDR), resulting in gene knockin due to the presence of a homologous repair template (purple).
