Multimodal scanning of genetic variants with base and prime editing

Mutational scanning connects genetic variants to phenotype, enabling the interrogation of protein functions, interactions and variant pathogenicity. 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. Our EGFR variant scan identifies important hits, supporting the robustness of the approach and revealing underappreciated routes to EGFR activation and drug response. We anticipate that multimodal precision mutational scanning can be applied broadly to characterize genetic variation in any genetic element of interest at high and single-nucleotide resolution.