Saturation profiling of drug-resistant genetic variants using prime editing

Methods to characterize the functional effects of genetic variants of uncertain significance (VUSs) have been limited by incomplete coverage of the mutational space. In clinical oncology, drug resistance arising from VUSs can prevent optimal treatment. Here we introduce PEER-seq, a high-throughput method based on prime editing that can evaluate the functional effects of single-nucleotide variants (SNVs). PEER-seq introduces both intended SNVs and synonymous marker mutations using prime editing and deep sequences the endogenous target regions to identify the introduced SNVs. We generate and functionally evaluate 2,476 SNVs in the epidermal growth factor receptor gene (EGFR), including 99% of all possible variants in the canonical tyrosine kinase domain. We determined resistance profiles of 95% of all possible EGFR protein variants encoded in the whole tyrosine kinase domain against the common tyrosine kinase inhibitors afatinib, osimertinib and osimertinib in the presence of the co-occurring substitution T790M, in PC-9 cells. Our study has the potential to substantially improve the precision of therapeutic choices in clinical settings.