Unless you’ve been hiding under a rock for the past decade, you have probably heard something about CRISPR or the CRISPR-Cas9 system. Basically, scientists have figured out how to exploit the immune system of bacteria to edit genes in other organisms. This amazingly powerful tool has been used to edit genes in plants, animals and even humans. Not only is the CRISPR system easy to use, it is also fast, has high accuracy and is inexpensive compared to other methods.
After a gene has been edited, the next step is to confirm that the mutation knocked down the targeted gene. Typically, genomic PCR and genetic sequencing tests are performed to confirm successful deletions or insertions, however, screening directly for protein expression is a more definitive measure of gene knockdown, avoiding signal from in-frame deletions/mutations1. Therefore, it makes sense to implement a Western/dot blot as a post-editing checkpoint.
If your world revolves around the Western blot, maybe you haven’t ventured out to try the CRISPR technology yet. There is one application for this powerful technology that may be very useful for Western blotters, antibody validation. If you have ever struggled with the lack of reproducibility of Western blots due to non-specific antibodies or spent a lot of money on commercial antibodies that just don’t work, listen up. The CRISPR technology is a game changer that may very well be the tool that ends the reproducibility crisis that plagues Western blotting. Using knock out validation ensures that primary antibodies are highly specific, which will produce robust results. With the overabundance of antibodies that lack selectivity, each antibody should be validated against a positive and negative control. The CRISPR technology offers a fast and convenient means of generating that negative control. Validating your primary antibodies with a wild type and knock out control will produce the most reliable results, returning confidence to your Western blot data.
- Estep, JA et al. Immunoblot screening of CRISPR/Cas9-mediated gene knockouts without selection. BMC molecular biology 17.1 (2016): 9
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