CRISPR/cas9  

All-in-one vector system for CRISPR-based genome editing
Transfect, enrich, screen, and publish—using our GeneArt® CRISPR Nuclease Vector Kit. The clustered regulatory interspaced short palindromic repeats (CRISPR) Nuclease system offers a simple, ready-to-use, all-in-one expression vector system consisting of both a Cas9 nuclease expression cassette and a guide RNA cloning cassette for rapid and efficient cloning of a target-specific crRNA. This system allows you to edit and engineer the genomic locus of your choice in a sequence-specific manner from a single plasmid. After relevant targets have been identified with GeneArt® CRISPRs, the biologically-relevant mutations can be validated with GeneArt® Precision TALs to reduce potential off-targeting.

Now available in two formats:

  1. Build-it-yourself ready to use kit
  2. Custom made to order

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.

CRISPR double strand break

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.

CRISPR nuclease vectors

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).

 

CRISPR/cas9 mediated cleavage efficiency

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.

CRISPR/cas9 enrichment of gRNA cell populations

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.

Ordering Information

Sku Name Size Price Qty
A21174 GeneArt® CRISPR Nuclease Vector with OFP Reporter Kit 10 reactions USD 495.00
A21175 GeneArt® CRISPR Nuclease Vector with CD4 Enrichment Kit 10 reactions USD 495.00
A21177 GeneArt® CRISPR Nuclease Vector with CD4 Enrichment Kit (with competent cells) 10 reactions USD 625.00
A24372 GeneArt® Genomic Cleavage Detection Kit 20 reactions USD 220.00
A21178 GeneArt® CRISPR Nuclease Vector with OFP Reporter Kit (with competent cells) 10 reactions USD 625.00

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.

Maximize the efficiency of genetic modifications

Use Lipofectamine 3000 to improve the cleavage efficiency with GeneArt Precision TALs or CRISPR. Download the Application Note here.

Technical resources

Free webinar on CRISPR/Cas9

If you haven’t heard of CRISPR, you must view this recorded webinar.  In this webinar you’ll get an overview of CRISPR/Cas9–mediated genome editing and learn about the GeneArt® CRISPR Nuclease System.

For Research Use Only. Not for use in diagnostic procedures.