Uncovering functional variants using a high-efficiency PE3-based screening platform
Shisheng Huang1,2, Wanyu Tao2,3, Xiangyang Li4
1The Second People's Hospital of Changzhou, The Third Affiliated Hospital of Nanjing Medical University, Changzhou Medical Center, Nanjing Medical University, Changzhou, Jiangsu 213003, China.
Abstract:
Prime editing (PE) enables precise nucleotide changes but has limited utility in high-throughput functional screening due to low editing efficiency. Here, we developed EvoPRIME, a PE3-based screening platform that integrates Csy4-mediated processing of Pol II-driven guide RNAs with a fluorescence-based enrichment strategy. EvoPRIME achieves comparable efficiency to state-of-the-art prime editing systems without requiring mismatch repair (MMR) suppression. We validated the performance of EvoPRIME through a loss-of-function (LOF) dropout screen targeting essential genes, which showed improved sensitivity and reproducibility, and a gain-of-function (GOF) saturation mutagenesis screen of the EGFR tyrosine kinase domain under osimertinib selection, which identified both known and uncharacterized resistance-conferring mutations. Leveraging EvoPRIME, we further conducted a high-throughput screen that functionally assesses both gain- and loss-of-phosphorylation (GOP and LOP) mutations. Among the hits, GAB1 S419E emerged as a phosphomimetic mutation that activates AKT signaling and confers osimertinib resistance. These studies establish EvoPRIME as a versatile platform for uncovering functional variants.
