Protein-Level Verification Without The Need For Antibodies Using SWATH^® Acquisition. Fast, Comprehensive, and Highly Reproducible

Although humans have been able to influence the traits of plants and animals for thousands of years through domestication and selective breeding, it wasn’t until the early 1970s and the development of genetic engineering techniques that man could directly manipulate the genetic outcome of another organism.¹ These early genetic engineering experiments sparked a revolution of discovery and innovation that has led to the development of cutting-edge biotechnology, synthetic biology, and genome editing techniques.

Today, modern gene editing techniques allow the insertion, deletion, alteration, or replacement of DNA at precise locations within the genome.² Because of its ease-of-use and versatility, the CRISPR-Cas9 system³ (which stands for Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR associated protein 9) is now the most widely used of these techniques and has been embraced around the world for many applications.⁴ For example, in food production, researchers are using CRISPR-Cas9 to manipulate plant genomes to increase crop yield, consume less water, intensify flavor, and improve disease resistance. ⁴ In livestock research, scientists are developing heat-resistant cows that can thrive in warmer climates to help deal with global warming. ⁴ In medical diagnostics, the CRISPR system is being used as the basis of a consumer kit that could be used at home for early detection of cancer and infectious diseases. ⁴ To eradicate malaria, researchers are manipulating the genome of the host mosquito to be resistant to the malaria parasite. ⁴ And of course, it has found wide-ranging uses in biomedical research to understand gene function in cell cultures and model organisms such as a mouse. CRISPR-Cas9 is being investigated for gene therapy to replace a mutated gene with a functional therapeutic gene.

So how does it work? CRISPR-Cas9 consists of two components, a guide RNA that is specific to the target DNA sequence, and an endonuclease protein, in this case, Cas9, that acts as molecular scissors to cut double-stranded DNA. After cutting, the cell naturally repairs the cut. This process can be hijacked to remove whole sections of DNA or add new or modified portions of DNA as the repair piece. After the gene is edited, assays are typically performed for DNA-level and RNA-level verification. However, sometimes these assays can be inconclusive. For example, when attempting to knockout a gene, often nature will splice around the knockout point in an attempt to create some version of the gene. These mRNA levels may not correlate well with what’s actually happening at the protein level. Because the entire reason for gene editing is often to study or cause some change within an organism’s protein expression or observable traits, protein-level verification becomes very important.

The most common assays for protein-level verification involve immunoblotting techniques such as western blot and ELISA. These techniques require antibodies. However, often antibodies aren’t available, are low quality, are too costly to produce, or there is a large time lag for their production.

Alternatively, proteomics using mass spectrometry is universally applicable without the need for antibodies. In particular, SWATH Acquisition using a TripleTOF^® system can provide comprehensive protein-level quantitation across the entire proteome. This means that not only can the affected gene product of the gene editing event be verified, but also corresponding changes throughout the entire proteome can be quantified. Additionally, SWATH Acquisition is highly sensitive and reproducible, capable of consistently detecting and quantifying thousands of proteins from cell lines across multiple instruments, operators, and laboratories.⁵

Recently researchers in Singapore used SWATH Acquisition for protein-level verification after CRISPR-Cas9 gene editing of the Major Vault Protein (MVP) in zebrafish.⁶ Initial verification studies using mRNA assays were inconclusive showing truncated forms of the gene still being transcribed in the mutant lines. No zebrafish antibodies were available for protein-level verification. Using SWATH Acquisition, the researchers were not only able to confirm complete knock-out of the gene, but also quantify concomitant proteome responses. In total, about 3800 proteins were detected and quantified from each sample in a single run, and of those, a number of proteins were significantly up- or down-regulated. While MVP proteins have no known function, the proteins that were significantly changed belonged to several different groups of related processes helping researchers further unravel the mysteries of this protein.

Advantages of MS Analysis for protein level confirmation after CRISPR-Cas9 gene editing:

• mRNA level verification can be inconclusive. Thus protein-level confirmation is often critical
• SWATH Acquisition provides confirmation at the protein-level without the need for antibodies, so research can progress faster
  • Antibodies are costly and can require a long time to produce
  • For some organisms and protein types, antibodies may not be available
  • Because there is no need to wait for antibody production, research can move faster
• Additional protein expression changes that result from the gene editing are readily observed because SWATH Analysis is global and comprehensive in its application
• No special method creation or reagents are required
• A comprehensive digital archive is created for every sample
  • Future investigations only require examining the data not re-running the sample
• SWATH Acquisition is amenable to high throughput profiling for screening arrays of knockouts

SWATH Acquisition is ideally suited for verification at the protein level of any gene editing event. Additionally, SWATH Acquisition provides comprehensive information regarding farther reaching biological implications of the experiment. For more information about SWATH Acquisition including videos, testimonials, and application notes, please visit our SWATH technology web site.

References

1. Timeline of Genetic Engineering: https://www.brighthub.com/science/genetics/articles/21983.aspx
2. What is Genome Editing?: https://www.yourgenome.org/facts/what-is-genome-editing
3. CRISPR 101: Your Guide To Understanding CRISPR: https://app.hubspot.com/documents/2418554/view/38573039?accessId=08e364
4. 8 Amazing CRISPR projects that could change life as we know it: https://www.digitaltrends.com/cool-tech/8-example-crispr-projects-changing-world/
5. Collins, B. C.*, Hunter, C.*, Liu, Y.*, Stefani, T., Chan, D., Zhang, H., Bader, S., Moritz, R. L., Schilling, B., Gibson, B. W., Krisp, C., Molloy, M. P., Hou, G., Lin, L., Liu, S., Hirayama, M., Ohtsuki, S., Selevsek, N., Schlapback, R., Tzeng, S.-C., Held, J. M., Larsen, B., Gingras, A.-C., and Aebersold, R. “Multi-laboratory assessment of reproducibility, qualitative and quantitative performance of SWATH-mass spectrometry”, Nature Communications (2017), 8.  https://www.nature.com/articles/s41467-017-00249-5
6. Technical Note: “Confirming Gene Mutation by CRISPR-Cas9 at the Protein Level and Identifying Proteome-Wide Changes,” Q Lin, LWL Low, S Tate, J Gunaratne, and TJ Carney.