Science advancesJun 5, 2026

Finding functional gene variants using an efficient PE3-based screening method

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jun 27, 2026

Abstract

Essence

EvoPRIME is a PE3-based prime-editing screening platform for finding functional genetic variants, including mutations linked to osimertinib resistance.

Evidence

This platform experiment validated EvoPRIME in loss-of-function dropout, EGFR gain-of-function saturation mutagenesis, and phosphorylation-mutation screens.

Caveat

The evidence is limited to screening performance and functional assays rather than clinical validation of the identified resistance mutations.

Simplified

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.

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Finding functional gene variants using an efficient PE3-based screening method

Abstract

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