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Published 01 October, 2021
The evolution of CRISPR technology has enabled scientists worldwide to push their programs forward to ensure therapeutics reach the market and the patients who need them as quickly as possible.
Knock-out cell lines are becoming increasingly popular for applications in drug discovery, including interrogating the relationships between genotype and phenotype and validating drug targets.
Although CRISPR is conceptually simple, it can be challenging to get right, and many labs don’t have the time, expertise, or facilities to generate gene-edited cell lines. A poorly designed or conducted process can result in low editing efficiency, leading to unintended, off-target edits and multiple repeats to achieve the desired outcome1. Extensive validation of the cell line is also critical to ensure accurate and reproducible results are achieved, but this uses valuable time and resources.
Ready-made cell lines are the easiest and most cost-effective way to accelerate your workflow if the cells you use are high-quality and provide reliable, reproducible results. At Abcam, we have optimized our CRISPR editing process to ensure quality at every stage, generating robust, well-characterized cell lines.
Here we explain our process for making and validating our gene-edited cell lines so that you can have confidence in our cell lines and, subsequently, your results.
Figure 1: how Abcam generates CRISPR-edited knock-out cell lines
1. Cell line selection
Our extensive range of over 2,500 ready-made KO cell lines covers targets across many fields, from cell adhesion molecules to cytokine receptors, transcription factors, and kinases. But every new knock-out cell line starts with deciding the target gene and appropriate background cell line.
We obtain our background cell lines from standard repositories, such as the American Type Culture Collection (ATTC), which are validated and certified mycoplasma-free. If we haven’t used the 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.
Where possible, we produce our knock-outs in physiologically relevant cell lines. Therefore, in addition to providing knock-outs in workhorse cell lines such as HEK and HeLa, we also create knock-outs in 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
CRISPR is a two-component gene-editing system made from enzyme ‘scissors’ (Cas nuclease) that precisely cut DNA and a guide RNA. Guide RNAs are critical for the specificity of gene editing as these small oligonucleotides form complexes with the Cas nucleases and direct them to the desired location by binding to a complementary sequence within the genome.
Making a CRISPR knock-out cell line depends on picking suitable guide RNAs. We carefully design and optimize all our CRISPR guide RNAs using comprehensive web-based gRNA design tools, including alignment-based scoring and hypothesis-driven methods, to increase the accuracy and efficiency of targeting.
We also 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%, which reduces our screening time2,3.
Once we’ve designed our RNA guides, the CRISPR components must be transfected into cells to begin the editing process.
Conventionally, scientists deliver the components of the CRISPR system into cells using DNA plasmids, relying on the cell to express the Cas enzyme and guide RNAs needed for editing.
However, this system introduces uncertainty as it depends on the efficient transcription of all components of the CRISPR system 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 line4.
Instead, we deliver CRISPR ribonucleoprotein complexes directly into cells, which creates a transient editing system that dissipates, so there is no continuous CRISPR expression and no extra genetic material left behind. This system also allows us to chemically modify guide RNAs before transfection to increase specificity even further4.
Once the editing process is complete, we use a clonal selection workflow combined with an extensive validation process to create a homologous knock-out cell line.
CRISPR is carried out on bulk transfected cell ‘pools,’ which are then screened using next-generation sequencing to confirm the editing efficiency. Pools containing successfully edited cells are then single-cell cloned and expanded to create clonal cell populations.
We screen these 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 go forward for proteomic validation.
We use western blotting as our standard technique for proteomic validation, often using our highly specific recombinant monoclonal antibodies, and compare protein expression in the parent cell with the knock-out cell to check that it’s entirely knocked out.
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 rely on ELISA for validation.
You can see examples of this throughout our range of ready-made knock-out 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.
Creating gene-edited cell lines can be expensive and time-consuming, involving months of work and costing up to $8,000. In contrast, ready-made KO cell lines can be delivered within five days, making the process quicker, easier, and cheaper than doing it yourself.
With over a decade of experience with CRISPR, we’ve optimized our workflow to ensure all our gene-edited cell lines are reliable and well-characterized. Each cell line in our extensive range of over 2,500 ready-made KO cell lines has been precisely generated to ensure the generation of robust cell lines to accelerate the transition from discovery through to manufacturing.
Gene-edited cell lines have high utility in many stages of the drug discovery pipeline. Extensively validated KO cell lines aid the generation of reproducible and accurate results, allowing you to progress through your workflow and get to market faster.