EvoPRIME Boosts Prime-Editing Screens and Identifies EGFR Resistance Mutations

The platform, called EvoPRIME, combines a PE3-based editing architecture with Csy4-mediated guide RNA processing and a fluorescence-based enrichment strategy that selectively isolates successfully edited cells. In benchmark experiments, EvoPRIME achieved editing efficiencies exceeding 50% at multiple endogenous loci and outperformed several existing prime-editing configurations, including PE3-based systems enhanced with mismatch repair suppression.

To demonstrate the platform’s utility, the researchers performed a saturation mutagenesis screen across exons 18–21 of EGFR, which encode the receptor’s tyrosine kinase domain and harbour many clinically relevant resistance mutations in non-small cell lung cancer (NSCLC). Following treatment with the third-generation EGFR inhibitor osimertinib, the screen identified several known resistance-associated mutations, including alterations at C797 and L792, validating the approach. The screen also uncovered additional candidate resistance variants, including mutations affecting residues D800, P733, I780, L828 and T847, as well as an in-frame deletion spanning amino acids 733–736.

The study further expanded prime-editing screening into phosphosite biology. Using phosphoproteomic data from osimertinib-resistant NSCLC models, the researchers designed a library targeting more than 2,000 phosphorylation sites and introduced phosphomimetic or phospho-deficient substitutions directly into endogenous genes. The screen identified dozens of phosphorylation sites associated with resistance.

Among the strongest hits was GAB1 S419E, a phosphomimetic mutation that increased AKT signalling, reduced apoptosis and promoted resistance to osimertinib. Pharmacological inhibition of AKT partially restored drug sensitivity, suggesting that the pathway may represent a potential therapeutic vulnerability.

Although EvoPRIME is primarily a research tool rather than a therapeutic platform, the authors argue that its improved efficiency could facilitate large-scale functional studies aimed at identifying clinically relevant variants, resistance mechanisms and candidate drug targets.

The study was led by Shisheng Huang, Shang-Min Zhang and Xingxu Huang at ShanghaiTech Uni-
versity, China. It was published on 5 June in Science Advances.