For designing all-in-one CRISPR-Cas9 vectors, use Invitrogen GeneArt CRISPR Nuclease Vector Kits, which incorporate both a Cas9 nuclease cassette and a targeted guide RNA (gRNA) cloning cassette, allowing you to edit a genomic locus of choice from a single Cas9 plasmid.
The kits include tools for CRISPR-Cas9 vector design, construction, and propagation, along with reporters that enable sorting or enrichment of transfected populations.
GeneArt CRISPR Nuclease Vector Kits make it easy to express a guide RNA (including crRNA and tracrRNA) using a plasmid vector that also expresses Cas9 endonuclease. Choose the GeneArt CRISPR Nuclease Vector with OFP (orange fluorescent protein) for flow cytometry-based sorting of crRNA-expressing cell populations, or the GeneArt CRISPR Nuclease Vector with CD4 for bead-based enrichment of crRNA-expressing cells.
TIP: If you’re new to CRISPR editing, we recommend using TrueCut Cas9 Protein with a TrueGuide Synthetic gRNA rather than a Cas9 DNA plasmid. The ribonuceoprotein complex becomes active immediately upon transfection and is cleared from the nucleus faster than the CRISPR plasmid, reducing the opportunity for off-target effects or DNA integration.
Figure 1. GeneArt CRISPR nuclease vector maps. The vector is supplied prelinearized with 5-bp 5’ overhangs as shown for easy cloning of your double-stranded 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).
Figure 2 summarizes the three-step workflow to create a CRISPR vector and express it in cells.
Figure 2. Workflow for utilization of GeneArt CRISPR Nuclease Vector Kit. Following annealing and cloning of DNA oligos coding for target-specific crRNA into a Cas9 nuclease reporter vector, the vector is transformed into E. coli cells. E. coli are screened for the desired CRISPR clone, which is then transfected into your cells for gene editing and analysis.
In addition to using our GeneArt CRISPR Nuclease Vector Kits for an all-in-one CRISPR plasmid system, you may choose to use separate Cas9 plasmids and gRNA plasmids.
Plasmids for Cas nuclease expression and gRNA cloning vectors can be obtained from open-access resources (e.g., Addgene, the nonprofit global plasmid repository). For constructing your own plasmid for gRNA expression, you may need oligonucleotides containing your gRNA sequence, as well as FastDigest restriction and cloning enzymes.
We offer a variety of competent E. coli cells for propagation of your Cas9 plasmid, including One Shot MAX Efficiency DH5a T1R Chemically Competent Cells as well as the PureLink Expi Endotoxin-Free Maxi Plasmid Purification Kit for plasmid isolation and purification.
For Research Use Only. Not for use in diagnostic procedures.