Researchers at ETH Zurich in Basel report they have used CRISPR-Cas technology to decipher how mutations in a cell’s genome affect its function. With their new approach, the researchers can generate thousands of cells with different gene variants in Petri dishes and determine which of the variants contribute to cancer development.
The findings are published in Nature Biotechnology in an article titled, “Multimodal scanning of genetic variants with base and prime editing,” and led by ETH professor Randall Platt, PhD.
“Mutational scanning connects genetic variants to phenotype, enabling the interrogation of protein functions, interactions, and variant pathogenicity,” the researchers wrote. “However, current methodologies cannot efficiently engineer customizable sets of diverse genetic variants in endogenous loci across cellular contexts in high throughput. Here, we combine cytosine and adenine base editors and a prime editor to assess the pathogenicity of a broad spectrum of variants in the epithelial growth factor receptor gene (EGFR). Using pooled base editing and prime editing guide RNA libraries, we install tens of thousands of variants spanning the full coding sequence of EGFR in multiple cell lines and assess the role of these variants in tumorigenesis and resistance to tyrosine kinase inhibitors.”
Scientists already had the ability to make individual changes to the genome of cells. However, the ETH researchers modified one gene in two human cell lines in over 50,000 different ways, thereby 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 various types of cancer, including lung, brain, and breast cancer.
Platt and his team combined two CRISPR-Cas methods, base editing and prime editing, to produce many variants of the EGFR gene.
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 regions in which mutations either cause a healthy cell to transform into a cancer cell or make a cancer cell become resistant or, conversely, sensitive to a drug. Because it is not possible to create all of these gene variants using base editing, the researchers tied in the other method, prime editing.
The researchers finally 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 it: some of these variants may play a role in the onset of cancer, while others may make it resistant to certain drugs. During this study, the ETH researchers also discovered a potentially new mechanism by which a mutation in the EGFR gene can cause cancer. They also found six gene variants that appear to play a role in cancer but had never been described.
“In conclusion, we established a multimodal precision base editing and prime editing mutational scanning framework to interrogate genetic elements at high and single-nucleotide resolution,” the researchers wrote. “Our approach yielded fundamentally new insights into EGFR activation and resistance to clinically approved TKIs, opening avenues for further work that may one day guide clinical decision-making. We anticipate that multimodal mutational scanning using precision editing can be expanded in the future to fully saturate genetic variant diversity in seemingly any genetic element, helping us to interpret the importance of yet-to-be-discovered genetic variants in a clinical context.”
