Overcoming the pitfalls of validating knockout cell lines by western blot

When making a knockout cell line using CRISPR, the first step is to isolate edited clones and sequence them to check for the presence of a frameshift mutation. Next, it is important to verify that the function of the protein of interest has been disrupted.

Using western blotting for verification can be a helpful tool, but correct interpretation of the results is crucial to avoid misidentification of cell lines with functional proteins as knockouts (and vice versa). In this blog post we will look at the possible fates of a protein after gene knockout using CRISPR and explore how best to validate a knockout cell line.

What happens to the protein after a frameshift mutation?

A frameshift mutation can result in a premature stop codon which is often presumed to lead to a knockout. Depending on the region targeted by the guide RNA, how the DNA is repaired by the cell, and the location of the stop codon¹, there are multiple possible outcomes at the protein level:

- mRNA degradation by nonsense-mediated decay (NMD) before translation, so no protein is produced (Fig. 1b).

- translation to make a truncated protein (Fig. 1c).

- translation beginning from an alternative start codon after the premature stop codon producing a truncated protein (Fig. 1d).

- exon skipping, excluding the exon containing the indel, to produce a splice variant (Fig. 1e)

- translation of a truncated portion of the wildtype protein followed by a novel amino acid sequence (Fig. 1f).

Some of these fates can co-occur in the same cell line. For example, mRNA where the targeted exon is skipped may be present alongside mRNA including the targeted exon.²

Figure 1. DNA, mRNA, and protein in a wildtype cell line (a) and possible outcomes after a frameshift mutation (b-f).

A common way to check clones for knockout is western blotting. Western blot results will depend on where the antibody binds, and the protein expressed by the cell line; figures 2 and 3 show examples of how a western blot could look.

Why are bands visible in a western blot using a knockout cell line?

The western blot in Figure 2 uses an antibody recognizing an epitope in the N-terminus of the wildtype protein. If the mRNA is destroyed by NMD, or if a splice variant without the N-terminus is generated, bands will not be observed on the blot. However, if the protein is truncated at the C-terminus, or if the wildtype N-terminus is followed by a novel amino acid sequence, a band may be observed. Such bands may lead to misinterpretation of the results.

Figure 2. Schematic representation of a western blot showing protein bands detected by an antibody against the wildtype protein’s N-terminus, for each of the proteins outlined in Figure 1.

Using a second antibody, for example one binding an epitope in the center of the protein (Figure 3), will provide more information. Analyzing the bands observed in both blots gives additional insight into whether the wildtype protein is still present, but it cannot confirm whether the protein detected is functional.

Figure 3. Schematic representation of a western blot showing protein bands detected by an antibody against the middle of the wildtype protein, for each of the proteins outlined in Figure 1.

Therefore, we recommend using a functional assay specific to the protein of interest to assess a function which is important for downstream experiments. Here is a list of functional assays researchers have used with Horizon’s HAP1 knockout cell lines; this list of assays could be applied to check for protein function in any knockout cell line.

How can a knockout cell line be validated?

Whether the cell line in question is a HAP1 knockout cell line, a cancer-related knockout cell line, or a knockout cell line you have made yourself, here are some tips for validating the knockout:

- If possible, use a functional assay to investigate an important function of the targeted protein.

- In all assays, use the parental cell line as the control to account for cell line-specific effects.

- When using antibodies, ensure that they have been validated using positive and negative controls to ensure that they are binding the correct target.

- For all experiments, consider appropriate controls, including assay controls.

References

Popp, M. W. and Maquat, L. E., Leveraging Rules of Nonsense-Mediated mRNA Decay for Genome Engineering and Personalized Medicine. Cell 165 (2016). https://doi.org/10.1016/j.cell.2016.05.053
Tuladhar, R., Yeu, Y., Tyler Piazza, J. et al. CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation. Nat Commun 10, 4056 (2019). https://doi.org/10.1038/s41467-019-12028-5

Written by Janine Ostick, Ph.D, Scientific Support Specialist

Janine is a Scientific Support Specialist in the Scientific Support team. During her PhD, she gained practical experience using CRISPR for gene editing, as well as culturing and differentiating iPSCs. She enjoys helping customers with their scientific questions about Dharmacon and Horizon products.

Written by Ivonne Rubio, Ph.D, Scientific Support Specialist

Ivonne has been part of the scientific support team since August 2013. With several additional years of hands on experience in the lab and a broad scientific background. She loves assisting customers with their scientific questions, especially about different strategies in gene modulation or CRISPR based studies.