GeneArt® CRISPR Nuclease Vectors

GeneArt® CRISPR Nuclease Vector Kits are reporter vector systems for expression of the functional components needed for CRISPR-Cas genome editing. The kits facilitate the generation of constructs to express noncoding guide RNA (including crRNA and tracrRNA), using a plasmid vector that also expresses Cas9 endonuclease.

GeneArt® CRISPR Nuclease Vectors with OFP (orange fluorescent protein) allow for flow cytometry–based sorting of crRNA-expressing cell populations, whereas GeneArt® CRISPR Nuclease Vectors with CD4 enable bead-based enrichment of crRNA-expressing cells.

GeneArt® CRISPR Nuclease Vectors.(A) GeneArt® CRISPR Nuclease: OFP Reporter Plasmid map and features of GeneArt® CRISPR Nuclease: OFP Reporter. The vector is supplied linearized between nucleotides 6,732 and 6,752, with 5 bp 5´ overhangs on each strand as indicated. (B) GeneArt® CRISPR Nuclease: CD4 Enrichment Plasmid map and features of GeneArt® CRISPR Nuclease: CD4 Enrichment. The vector is supplied linearized between nucleotides 7,336 and 7,355, with 5 bp 5´ overhangs on each strand as indicated. 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.

To use either GeneArt® CRISPR Nuclease Vector Kit, you will first need to design two single-stranded DNA oligonucleotides; one encoding the target-specific crRNA (forward-strand oligonucleotide) and the other its complement (reverse-strand oligonucleotide). You can generate a double-stranded oligonucleotide suitable for cloning into the linearized vector provided in the kit by simply annealing the complementary oligonucleotides.

The design of the single-stranded oligonucleotides is critical to the success of the cloning procedure and to the effectiveness of the construct as a genome editing tool. General guidelines are provided here to help you choose the target sequence and to guide your design of the single-stranded oligonucleotides.

Design strategy and target specificity

To use the GeneArt® CRISPR Nuclease Vector Kit, you first design two single-stranded DNA oligonucleotides (24–25 bp), one encoding the target-specific crRNA (forward-strand oligonucleotide) and the other its complement (reverse-strand oligonucleotide). The choice of genomic target sequence can significantly affect the degree of cleavage observed. Therefore, we recommend that you test more than one target-specific crRNA sequence per locus of interest. Guidelines are provided below for choosing your target sequence, and an example is provided in the figure. Note that these are general recommendations only and exceptions may occur.

Order CRISPR oligos

Length—Choose a target sequence ranging from 19 to 20 nucleotides in length that is adjacent to an NGG proto-spacer adjacent motif (PAM) sequence on the 3´ end of the target sequence. The 5´ G required for transcription initiation from the U6 Pol III promoter is already included in the vector overhangs and does not need to be included in the target sequence.

Homology—Make sure that the target sequence does not contain significant homology to other genes, as this can increase off-target effects. Recently published work has shown that gRNA-Cas9 complexes can potentially tolerate 1–3 or more mismatches, depending on their location in the gRNA. Refer to published articles for more insights into choosing a target sequence.

Orientation—You may choose a target sequence encoding the sense sequence of the target locus or the antisense sequence. Thus, you can generate CRISPR RNA in two possible orientations, provided that it meets the PAM requirements on the 3´ end.

3. Transfect CRISPR plasmids for cell culture

a. Delivery method: The method of transfections varies based on cell type. Consult original references or the supplier of your cell line for the optimal method of transfection. Pay particular attention to medium requirements, when to passage the cells, and at what dilution to split the cells. For high-efficiency transfection in a broad range of mammalian cell lines, we recommend using the cationic lipid–based Lipofectamine^® 3000 Reagent

b. DNA purity: For transfection into eukaryotic cells, DNA must be pure and free of contamination with phenol and sodium chloride. We recommend using high-quality DNA prepared with the PureLink^® HiPure Plasmid Midiprep Kit. Store plasmid DNA stocks at –20°C.

c. Amount of DNA: Depending on the transfection reagent and cell line, the dosage of DNA that yields the best transfection efficiency will vary. It is advisable to do a dose–response study to determine optimal transfection conditions. In our experience with 293FT cells in a 6-well transfection format, 3 μg of CRISPR-Cas9 expression plasmid gives optimal transfection efficiency when cells are at 70% confluency.

CRISPR-Cas9-mediated cleavage efficiency

Cleavage efficiency can be detected using the GeneArt® Genomic Cleavage Detection Assay, which is a technique that leverages mismatch detection endonucleases to detect insertions and deletions (indels) generated during cellular NHEJ repair.

CRISPR-Cas9-mediated cleavage efficiency. Gel analysis of a cleavage assay using the GeneArt® Genomic Cleavage Detection Assay for the HPRT locus. (A) Results obtained 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.

Transfection efficiency monitored using FACS

Since Cas9 is linked to either OFP or CD4 in the expression vector, transfection efficiency can be monitored using fluorescence microscopy or FACs

Transfection efficiency. (A) Transfection efficiency in 293T cells using the GeneArt® CRISPR Nuclease OFP Vector encoding crRNA specific for the RelA locus. Data show >90% OFP-positive cells in transfected samples. (B) CD4 functionality for the GeneArt® CRISPR Nuclease CD4 Vector. 293FT 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 to measure transfection efficiency. A portion of the stained cells was also seeded on a plate for analysis by fluorescence microscopy.

Cells that express Cas9 and gRNA can be enriched by magnetic bead–based enrichment using the Dynabeads® CD4 magnetic beads CD4 reporter.

Maximize the efficiency of genetic modifications

Use Lipofectamine® 3000 reagent to improve cleavage efficiency with GeneArt® Precision TALs or CRISPRs.

Download the application note

Q: How do you compare the CRISPR-Cas9 system with TALENs in terms of specificity for the target?
A: It is generally believed that TALENs have higher specific cleavage of the target than CRISPRs. Carefully designing the CRISPR target typically results in lower off-target effects. The cleavage efficiency of CRISPR or TALEN for the target locus can be measured using the GeneArt® Genomic Cleavage Detection Kit.

Q: How do I avoid off-target effects by inducing NHEJ repair when targeting efficiency is not significantly affected?
A: Careful design helps ensure minimal off-target effects. BLAST search the genome to make sure that no more than 14 bp of sequence identical to the 3' of CRISPR target sequence are present in genome, other than at the target locus. Download our Technical Product Bulletin for optimal CRISPR design tips.

Q: Can you simultaneously target different genes with the same construct?
A: The vector we currently offer allows users to clone one target only. You can co-transfect two constructs to simultaneously target two loci.

Q: Do you have any tools to assist with the design of the target sequence?
A: Yes, download our Technical Product Bulletin for tips and recommendations for optimal CRISPR design.

Q: Can you elaborate on your suggestion to use CRISPR as a screening tool before going to TALs?
A: Since cleavage efficiency at a particular locus depends on the accessibility of the locus, chromatin state, and sequence, it is advisable to test multiple different loci/regions within a gene of interest. With CRISPR-Cas9–mediated genome editing, for each target of interest the user needs only to change the 19–20 bp target-specific oligo. After targets have been screened and the sequence/locus with best cleavage efficiency has been identified with the fast and easy-to-use GeneArt® CRISPR system, the biologically relevant mutations can be precisely created with high-specificity GeneArt® Precision TALs.

Q: How is percent indel calculated?
A: The formula used is: % Indel = 1 – ((1 – fraction cleaved) 1/2)

Q: Will you offer a service to generate custom cell lines using CRISPR?
A: Yes, we are currently offering this service.

Q: What are the advantages/disadvantages of OFP vs. CD4?
A: One could perform FACS-based sorting with OFP (orange fluorescent protein); for CD4, either FACS or bead-based enrichment can be used. Find more details in the user manual.

Q: Will you also provide custom designed, precloned CRISPR vectors for my target?
A: Yes, we do have a custom CRISPR vector service option.