While the risk of human-to-human transmission is still low, the pervasive reservoirs and appearance of new, concerning mutations or those identified as a threat science.org/doi/10.1126/...
demand a very high level of vigilance and preparedness

I am also very proud to share this authorship with my former master’s student Kyriaki Karava who began working with me on this project two years ago. Her sharp scientific reasoning, hard work and amazing team spirit made this work possible but also a truly enjoyable collaboration!

This marks the end of my PhD journey at ETH Zürich and I cannot finish without thanking my supervisor Randall Platt for his continuous support throughout this journey, as well as Rick Farouni for sharing his code magic with us and all reviewers for their insightful comments.

We believe that this work will facilitate the accurate interpretation of VUS in cancer patients and the optimization of treatment regimens. These complementary screening approaches could also be expanded to investigate the function of many more genes of interest in the future.

This new approach identified known and novel hits, including short insertions that could not be introduced by base editing. We also identified key parameters for prime editing screen design and demonstrated the use of synonymous edits to differentiate true and false positives.

While base editing scanning screens proved useful in identifying novel pathogenic mutations, they can only replicate 17% of known EGFR variants. We thus designed a prime editing library introducing almost 3000 patient-derived mutations and conducted a new EGFR activation screen.

Previous experiments were conducted in an EGFR WT cell line but drug resistance often emerges in patients due to secondary mutations. We thus repeated our screens in PC-9 cells harboring an hyperactive EGFR variant and identified both overlapping and context-dependent hits.

We then leveraged the same approach to identify variants impacting cell sensitivity to two clinically approved tyrosine kinase inhibitors. We found hits resistant to one or both molecules, suggesting that these data could be used to help prioritize drug treatments in the clinics.

After controlling for constructs impacting cell viability, we identified 19 hits that led to EGF-independent proliferation. 8 of these likely oncogenic variants were listed as VUS while 6 others were absent from databases, including unexpected C-terminal truncating mutations.

First, we delivered an sgRNA library tiling all EGFR exons along with a C-to-T or an A-to-G base editor to an EGFR WT cell line. We then selected for cells with constitutive EGFR activity by removing EGF from the culture medium before quantifying relative library distributions.

To address this, we used base and prime editing pooled screens to assess the pathogenicity and drug sensitivities of tens of thousands of variants in the Epithelial Growth Factor Receptor (EGFR), an oncogene frequently mutated in lung, brain and breast cancers.

Each human genome contains about 4 million variants, including 10,000 non-synonymous ones that can influence disease risk and treatment response. Yet, nearly half of the 3 million mutations listed in ClinVar are variants of uncertain significance (VUS) whose impact is unknown.