Precision genome editing screens to study genetic variants of uncertain significance

Abstract

Genetic tests play an increasingly pivotal role in predicting disease risks, prognosis and treatment response in the clinics. However, their interpretation is hindered by the prevalence of variants of uncertain significance (VUS) whose phenotypic effects remain elusive. Addressing this issue, multiplexed assays of variant effect (MAVEs) have been developed that enable the interrogation of protein functions and the assessment of variant pathogenicity. However, current workflows are limited in their ability to replicate variant diversity, genomic background, expression levels and cellular context. Here, we leveraged and combined multiple precision genome editing technologies, including cytosine and adenine base editors along with a prime editor, to assess the pathogenicity of a broad spectrum of variants in the genome of cells. We applied our multimodal approach to interrogate EGFR, uncovering pathogenic genetic variants driving oncogenesis and drug resistance. We identified known and novel hits, thus supporting the performance of the screening approach and providing new insights into underappreciated routes to EGFR activation and drug response. In the future, we envision that multimodal precision mutational scanning screens can be widely employed to comprehensively characterize genetic variation down to single nucleotide resolution across diverse genomic backgrounds, thereby contributing to the development of personalized treatment protocols.