Understand disease mechanisms and validate potential therapeutic targets with knock-out (KO) cell lines.
Knock-out (KO) cell lines help overcome the challenge of accessing the correct controls during assay development and enable confident interrogation of the relationship between genotype and phenotype.
However, generating CRISPR KO cell lines is time-consuming, with an average of three to four repeats of the CRISPR workflow before they obtain the specific CRISPR edits required (Synthego. CRISPR Benchmark Report, 2019).
To give you this time back, we have established a large and expanding range of ready-made and made-to-order knock-outs in commonly used cell lines that are ready to go, making CRISPR knock-out cell lines as easy to buy as reagents.
Our CRISPR knock-out cell line catalog is designed to cut more than four months from your preparation time, taking you from months of experimental work to a simple click to achieve the CRISPR knock-out cell line you need – meaning you can get more data and publish faster.
It's the quickest and most cost-effective option to accelerate your program.
Last edited Fri 17 Mar 2023Gene-edited cell lines play an important role in many stages of the drug discovery pipeline, including validating reagents and targets. We offer an extensive catalog of high-quality ready-made and made-to-order KO cell lines so you can save time and move your project forward.
Benefits of our gene editing services:
1. Cell line selection
Every knock-out cell line starts with the selection of the most appropriate target gene and background cell line. Our selections are made in close collaboration with the research community to ensure that we’re always supporting our customers with the cell lines that are most relevant to their research and application needs.
We obtain our background cell lines from standard repositories, such as the American Type Culture Collection (ATTC), which are fully validated and certified mycoplasma-free.
If we haven’t used a line before, we conduct a thorough evaluation to check suitability, including confirming that the protein of interest is expressed in the wild-type cells and ensuring that the cells will grow and remain viable after the target is knocked out.
In addition to using workhorse cell lines such as HEK and HeLa, we produce knock-outs in various physiologically relevant and disease-relevant backgrounds. For example, CD74 is highly expressed in lymphoma, so we generate our knock-out line for human CD74 in a Raji lymphoma cell line.
2. CRISPR guide design
Our high-throughput platform allows us to automate the production of our CRISPR knock-out cell lines at scale. This reduces the time typically required to produce gene-edited monoclonal cell lines, so you can gain back valuable lab time, have confidence in scalable and long-term supply, and achieve consistent results over time.
Creating a successful CRISPR knock-out cell line depends on selecting suitable guide RNAs; our CRISPR guide RNAs are carefully designed and optimized using comprehensive web-based gRNA design tools – including alignment-based scoring and hypothesis-driven methods – to increase the accuracy and efficiency of targeting.
Crucially, we employ a dual RNA system, using two guides to recognize flanking sites around the target sequence rather than a single site. This generates a small fragment deletion, guaranteeing a complete protein knock-out and increasing our typical knock-out efficiency to over 95% compared with a single guide efficiency of around 55%. This reduces our screening time 2,3 and translates into cost efficiency and reliability for our customers.
3. Transfection
Once we’ve designed our RNA guides, the CRISPR components need to be transfected into cells to begin the editing process.
Conventionally, the components of the CRISPR system are delivered into cells using DNA plasmids, relying on the cell to express the Cas enzyme and guide RNAs needed for editing. But this technique can introduce uncertainty because it relies on efficient transcription of all the CRISPR system components by the cell. It also allows the CRISPR system to be expressed continuously, increasing the potential for off-target edits, and leaves external plasmid DNA in the cells, which may cause problems down the line.
We take a different approach. By delivering pre-assembled CRISPR ribonucleoprotein complexes directly into cells, we benefit from a transient editing system that dissipates, so there is no continuous CRISPR expression, and no extra genetic material left behind. As well as enhancing accuracy, this system allows us to chemically modify guide RNAs before transfection to increase specificity even further4.
4. Enrichment and single-cell expansion
Once the editing process is complete, we use a clonal selection workflow (followed by an extensive validation process) to create a homologous knock-out cell line.
We perform CRISPR on bulk transfected cell ‘pools,’ which are then screened using next-generation and Sanger sequencing to confirm editing efficiency. Pools containing successfully edited cells are then single-cell-cloned and expanded to create clonal cell populations.
5. Validation
We screen clones by sequencing the target site and checking that we have created complete biallelic knock-outs at the site of interest. Based on this genomic validation, we select three knock-out clones to take forward.
We then use western blotting as our standard technique for proteomic validation, often using our highly specific recombinant monoclonal antibodies to ensure accuracy. We compare protein expression in the parent cell to check that the gene has been completely ablated.
Where additional or alternative validation is needed, we choose another appropriate technique. For example, if denaturing the target is problematic, we might use immunocytochemistry for validation. Or, if the target is a secreted protein, such as a cytokine, we will often employ a highly specific ELISA for validation.
You can see examples of this throughout our range of ready-made knock-out Abcam cell lines. For instance, our human CXCL10 knock-out A549 and IL1B knock-out THP-1 cell lines are validated with western blot and ELISA, while our human ANPEP (CD13) knock-out THP-1 cell line has been validated with western blot, immunocytochemistry, and flow cytometry.
Please review the individual cell lines datasheet for specific validation for that product.
How we generate our gene edited cell lines broken down into 6 steps. 1 - target selection and dual RNA guide design 2 - Transfection with CRISPR/CAS complexes 3 - Pooled DNA sequencing to confirm editing efficiency 4- Sublconing, expansion and confirmatory DNA sequencing 5 - Proteomic validation with western blot or alternative methods (ELISA, ICC etc.) where necessary 6 - CRISPR knockout cell line
Our KO cell lines were generated using CRISPR/Cas9 technology. The KO cell lines are single-clonal and validated using Sanger sequencing. Please refer to the datasheet for the specific mutation description.
Do you test the KO cell lines for mycoplasma?
Yes, the cell line banks are tested for mycoplasma using an independent third party, Bionique, using broth and agar method. We also spot-check our stocks regularly.
Can I obtain a copy of the mycoplasma-free certificate?
Yes. Please contact our scientific support team and they can provide this to you.
How should I store the cells once I receive them?
If you do not plan to resuscitate immediately, we recommend that you store the vial in liquid nitrogen immediately upon receipt.
Can I use my own WT cell line, or do I have to purchase it from you?
We recommend you use our WT, provided free of charge with each knock-out cell line order (not available for individual purchase). Please refer to the datasheet to determine which WT to add to your KO cell line order. Alternatively, you can contact us if you have any questions.
Which wild type (WT) cell line should I purchase to use as a control with my KO cell line?
When you buy a KO cell line the WT is available free of charge, however you will need to add it separately to your order. Please refer to the General Notes section within the KO cell line's datasheet for our recommended WT and how to order. If you have questions you can contact our scientific support team.
What is the biological safety level (BSL) of your cells?
Our KO cell lines are classified as BSL1 and BSL2 depending on the cell line. Cell lines such as A549, HCT116, HEPG2, and MCF7 are BSL1. EK-293T and HeLa cell lines are BSL2.
What guarantees are in place for your KO cell lines?
Our product promise™ guarantee covers our KO cell lines. This means we guarantee the cell lines are mycoplasma free and validated at the genomic level by Sanger sequencing.
What is the passage number of the cell lines?
The cells are all less than passage number 20. Please contact us if you would like the exact passage number of your lot.
Are there antibiotics in the cells?
The clones are selected using puromycin resistance, however the cells are not grown in the presence of puromycin or other antibiotics.
How should I culture my cells?
Please refer to the handling procedure on the KO cell line's datasheet and our cell culture protocol.
What is the standard media formulation that your cells are in?
The cells are in Cell Freezing Medium-DMSO serum-free media. Please refer to the KO cell line's datasheet for more information.