Using CRISPR to decipher whether gene variants lead to cancer

In recent years, scientists have developed a series of new methods based on CRISPR-Cas technology to precisely edit the genetic material of living organisms. One application is in cell therapy: a patient’s immune cells can be specifically reprogrammed to fight cancer more effectively.

Researchers from the Department of Biosystems Science and Engineering at ETH Zurich in Basel have now found a further application for these new CRISPR-Cas methods: led by ETH Professor Randall Platt, the researchers use them to decipher how mutations in the genome of influence a cell’s functioning. function. For example, the order of the DNA building blocks in tumor cells differs from that in healthy cells. With the new approach, the researchers can generate tens of thousands of cells with different gene variants in petri dishes. They can then decipher which of the variants contribute to the development of cancer and which make the cancer cells resistant to standard drugs.

Combine two methods

Scientists already had the ability to make individual changes to the genome of cells. But the ETH researchers’ project plans were much more complex: They modified one gene in two human cell lines in more than 50,000 different ways, creating a correspondingly large number of different cell variants, and then tested the function of those cells. For their proof of concept, they worked with the EGFR gene, which is central to the development of several types of cancer, including lung, brain and breast cancer.

Platt and his team combined two CRISPR-Cas methods to produce a large number of variants of this gene. Both methods have been developed in recent years by researchers at MIT and Harvard University in the United States, and both have advantages and disadvantages. One of these methods, base editing, makes it possible to modify individual building blocks of DNA, also called bases, very easily and reliably. However, the possibilities of base editing are limited: it can generally swap DNA base C with base T, or A with G.

Several tens of thousands of cells changed

The second method the researchers used is prime editing. Theoretically, this method is very powerful: similar to the “search and replace” function of a word processing program, it can modify individual sequences of genetic code in a targeted manner. “We can use it to swap any DNA base for another. Or we can, for example, insert three or 10 bases into the genome or delete the same number,” says Platt. “You can basically do whatever you want with it.”

However, Prime editing does not work reliably. This has made it difficult to use it to create an entire collection of several tens of thousands of differently modified cells that could then be subjected to screening. Platt and his team have now achieved this.

Important for oncology

Cell pools with different gene variants are crucial for research. This is because oncologists are increasingly analyzing the genetic information in patients’ tumor cells on a basis-by-basis. This information often gives them clues about which medications might work for an individual patient.

In recent years, scientists have built up databases containing thousands of different genetic variants found in patients. The databases also contain a thorough description of their effects for about half of these variants. The other half is only known to occur in patients; it is not clear what impact they have on the development or treatment of cancer. Scientists call these ‘variants of uncertain significance’. If a doctor finds such a variant in a patient, the information is of little use to him.

Researchers are convinced that oncology would benefit enormously from more information about these variants. That is why they try to produce cells with these gene variants in the laboratory, so that they can then analyze the function of those cells. In recent years, researchers have done a lot of work to prepare for this possibility. They already had the option to use basic editing as a method, but the problem was that basic editing alone is not enough. “You can only produce about a tenth of these variants,” explains Olivier Belli, a PhD student in Platt’s group and, together with master’s student Kyriaki Karava, first author of the study.

New relevant variants found

To systematically generate cells with virtually all possible relevant variants of the EGFR gene, Platt and his team first identified the cancer-relevant regions in this gene. These are areas where mutations cause a healthy cell to turn into a cancer cell, or a cancer cell to become resistant or, conversely, sensitive to a drug. Since it is not possible to create all these gene variants using base editing, the studies paired the other method, prime editing.

Finally, the researchers analyzed these cells. For ten EGFR gene variants whose effect on cancer progression was previously uncertain, they have now been able to provide evidence that they are significant and describe this: some of these variants may play a role in the development of cancer, while others can cause cancer to develop. resistant to certain drugs. In the course of this study, the ETH researchers also discovered a potential new mechanism by which a mutation in the EGFR gene can cause cancer. In addition, they found six gene variants that appear to play a role in cancer, but have never been described: completely new, relevant gene variants.

The EGFR gene is just one of hundreds of human genes associated with cancer. This new research approach is now ready to also decode variants of uncertain significance in all other genes.