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Adv."],"published-print":{"date-parts":[[2026,6,5]]},"abstract":"<jats:p>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\u2013driven 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.<\/jats:p>","DOI":"10.1126\/sciadv.ady7359","type":"journal-article","created":{"date-parts":[[2026,6,5]],"date-time":"2026-06-05T17:59:36Z","timestamp":1780682376000},"update-policy":"https:\/\/doi.org\/10.34133\/aaas_crossmark","source":"Crossref","is-referenced-by-count":0,"title":["Uncovering functional variants using a high-efficiency PE3-based screening platform"],"prefix":"10.1126","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7869-0043","authenticated-orcid":true,"given":"Shisheng","family":"Huang","sequence":"first","affiliation":[{"name":"The Second People\u2019s Hospital of Changzhou, The Third Affiliated Hospital of Nanjing Medical University, Changzhou Medical Center, Nanjing Medical University, Changzhou, Jiangsu 213003, China."},{"name":"School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, 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