Uncovering functional variants using a high-efficiency PE3-based screening platform
- Shisheng Huang
- Wanyu Tao
- Xiangyang Li
- Junfan Guo
- Yu Zhang
- Guanglei Li
- Pengfei Zhang
- Guiquan Zhang
- Gaoyang Li
- Gaofeng Fan
- Shang-Min Zhang
- Xingxu Huang
2026-06-05
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.