All-in-one plasmid system 

Transfect, enrich, screen, and publish—using our GeneArt CRISPR Nuclease Vector Kit. The CRISPR Nuclease system offers a ready-to-use, all-in-one expression vector system with a Cas9 nuclease expression cassette and a guide RNA cloning cassette for fast 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 TALs to reduce potential off-targeting.

Buy now  Request Resource Guide

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. The kits make it easy to express noncoding guide RNA (including crRNA and tracrRNA), using a plasmid vector that also expresses Cas9 endonuclease.

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

 

GeneArt® CRISPR nuclease vectors

GeneArt® CRISPR nuclease vectors

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.


Optimal CRISPR design at the touch of a finger

Quickly search and design optimal CRISPR gRNAs with minimal off-target affects on your desktop.

Start designing today

Tips for designing CRISPR oligos

Choose genomic DNA target sequence

Choose a 19–20 bp target sequence upstream of the NGG PAM site. You can choose a target site either in the sense or antisense strand of the genomic DNA provided it meets the PAM requirements.

    CRISPR oligos  
 

Step 1

 

Step 2

 

Step 3

 

Step 4

Top strand oligo design

Add a 5-base, GTTTT 3' overhang needed for cloning to the selected 19–20 bp target sequence to generate the top strand oligo. Note that the PAM site is not included in the oligo.

    CRISPR oligos  
 

Step 1

 

Step 2

 

Step 3

 

Step 4

Bottom strand oligo design

Generate reverse complementary sequence specific to the 19–20 bp target sequence and add a 5-base, CGGTG 3' overhang to generate the bottom strand oligo.

    CRISPR oligos  
 

Step 1

 

Step 2

 

Step 3

 

Step 4

Anneal oligos

Anneal top and a bottom strand oligos to generate a double-stranded (ds) oligo with compatible ends for cloning into GeneArt® CRISPR Nuclease Vector.

    CRISPR oligos  
 

Step 1

 

Step 2

 

Step 3

 

Step 4


CRISPR cloning workflow

1. Anneal and clone DNA oligos

For cloning, simply anneal DNA oligos that code for your target-specific crRNA and ligate into the pre-linearized vector. Sufficient reagents are provided for 10 reactions, including:

  • Linearized cloning vector, 10X annealing buffer, T4 DNA ligase, 5X DNA ligase buffer, and DNase/RNase-free water.
  • A control double-stranded DNA oligo for monitoring cloning efficiency.
  • U6 forward sequencing primer for checking the orientation and sequence of the crRNA-specific double-stranded oligo insert.

2. Transform

Once you have completed the ligation reaction, transform One Shot® TOP10 Chemically Competent E. coli with the resulting CRISPR nuclease construct. One Shot® TOP10 Chemically Competent E. coli are ideal for high-efficiency cloning and plasmid propagation. They allow stable replication of high copy number plasmids. The genotype of TOP10 cells is similar to that of the DH10B™ strain. One tube of One Shot® TOP10 E. coli is required for each ligation reaction.

3. Transfect CRISPR plasmids for cell culture

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.

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.

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.


Monitoring CRISPR genome editing success

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

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

CRISPR-Cas9 transfection efficiency

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.

Enrichment of the Cas9- and gRNA-expressing cell population using Dynabeads® magnetic beads

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

Bead-based enrichment using the GeneArt® CRISPR Nuclease Vector with CD4  

 


Bead-based enrichment using the GeneArt CRISPR Nuclease Vector with CD4
. The sample had very low CD4-expressing cell populations and hence lower cleavage efficiency (1.4%). Following transfection, CD4-expressing cells were enriched from the rest of the cell population using anti-CD4 antibody–coated magnetic beads. A GeneArt Genomic Cleavage Detection Assay was performed on both pre- and post-enriched cell pellets. Cleavage efficiency increased to 48.1% after enrichment.

 

Maximize the efficiency of genetic modifications

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

CRISPR FAQs

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.

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.

A. The vector we currently offer allows users to clone one target only. You can co-transfect two constructs to simultaneously target two loci.

Yes, download our Technical Product Bulletin for tips and recommendations for optimal CRISPR design.

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® TALs.

A. The formula used is: % Indel = 1 – ((1 – fraction cleaved) 1/2)

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.

Ordering information

Custom CRISPR for every gene; we'll design your target & clone it for you. Ready-to-transfect. 100 μg   Contact us