GeneArt® CRISPR Nuclease Vector
All-in-one vector system for CRISPR-based genome editing
Now available in two formats:
Three-component CRISPR-based editing
Genome editing uses engineered nucleases in conjunction with endogenous repair mechanisms to alter the DNA in a cell. The CRISPR/Cas system takes advantage of a short guide RNA (gRNA) to target the bacterial Cas9 endonuclease to specific genomic loci. Because the gRNA supplies the specificity, changing the target only requires a change in the design of the sequence encoding the gRNA.
The CRISPR/Cas system used in gene editing consists of three components: the Cas nuclease Cas9 (a double-stranded DNA endonuclease), a target complementary crRNA, and an auxiliary transactivating crRNA (Figure 1). The crRNA and tracrRNA of the GeneArt® CRISPR Nuclease Vector are expressed together as a guide RNA that mimics the natural crRNA-tracrRNA chimera in bacterial systems. All that's needed is to introduce a double-stranded oligonucleotide encoding a desired sequence to express the crRNA portion of the chimera.
Figure 1. CRISPR/Cas9 targeted double-strand break. Cleavage occurs on both strands, 3 base pairs upstream of the NGG protospacer adjacent motif (PAM) sequence on the 3’ end of the target sequence.
GeneArt® CRISPR Nuclease Vector Kits
GeneArt® CRISPR Nuclease Vector Kits are reporter vector systems for expression of the functional components needed for CRISPR/Cas genome editing in mammalian cells. These kits are available with two different reporters: GeneArt® CRISPR Nuclease Vectors with OFP (orange fluorescent protein) allow for flow cytometry–based sorting (FACS) of Cas9 and CRISPR RNA-expressing cell populations (Figure 2A), and GeneArt® CRISPR Nuclease Vectors with CD4 enable bead-based enrichment of Cas9 and CRISPR RNA expressing cells (Figure 2B).
The linearized GeneArt® CRISPR Nuclease Vectors provide a rapid and efficient way to clone double-stranded oligonucleotides encoding a crRNA representing a desired target into an expression cassette that allows sequence-specific targeting of the Cas9 nuclease.
Figure 2. GeneArt® CRISPR nuclease vector maps. The vector is prelinearized with 5 base pair overhangs for easy cloning of your doublestranded DNA oligo that encodes a target-specific crRNA. Maps are shown of the vectors with (A) OFP reporter and (B) CD4 reporter. The gRNA, Cas9, and reporter are expressed from the same vector. Cas9 is directed to the nucleus by nuclear localization signals (NLS1 and NLS2).
Measure gene modification efficiency using a GeneArt® Genomic Cleavage Detection Assay
Locus specific cleavage efficiency can be detected using GeneArt® Genomic Cleavage Detection Kit, a technique that leverages mismatch detection endonucleases to detect indels generated by insertions and/or deletions incorporated during cellular NHEJ repair (Figure 3).
Figure 3. CRISPR/Cas9-mediated cleavage efficiency. Gel image of a cleavage assay using the GeneArt® Genomic Cleavage Detection Assay (set to launch in January 2014) for the HPRT locus. (A) Results using the GeneArt® CRISPR Nuclease OFP Vector expressing HPRT-specific CRISPR RNA. (B) Results obtained using the GeneArt® CRISPR Nuclease CD4 Vector expressing HPRT-specific CRISPR RNA. Following transfection into HeLa cells, triplicate cleavage assays were performed and the percentage of indels were calculated.
Enrichment of the Cas9- and gRNA-expressing cell population
Since the Cas9 gene is linked to either OFP or CD4, the transfection efficiency using GeneArt® CRISPR Nuclease all-in-one vector system can be monitored by fluorescence microscopy or FACS in the case of the OFP-containing plasmid or by using anti-CD4 fluorescent antibodies in the case of the CD4 plasmid.
Figure 7. Enrichment of the Cas9- and gRNA-expressing cell population. (A) Transfection efficiency in 293T cells using the GeneArt® CRISPR Nuclease OFP Vector encoding crRNA specific for the RelA locus. Data shows >90% OFP-positive cells in transfected samples. (B) CD4 functionality for the GeneArt® CRISPR Nuclease CD4 Vector. 293 FT cells were transfected with AAVS1-specific GeneArt ® CRISPR Nuclease CD4 Vector. Cells were harvested and stained with anti-CD4 FITC antibody and analyzed by flow cytometry for measuring transfection efficiency. A portion of the stained cells were also seeded on a plate for analysis by fluorescence microscopy.
Want us to design your target oligo and clone it for you?
Let us know at CRISPR@lifetech.com, we’ll design and provide you with 100 µg of transfection-quality DNA.
For Research Use Only. Not for use in diagnostic procedures.