Uncovering functional variants using a high-efficiency PE3-based screening platform.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Prime editing (PE) offers precise genome editing but faces limitations in high-throughput functional screening due to low efficiency.
• Developing efficient and scalable screening platforms is crucial for understanding gene function and identifying therapeutic targets.

Purpose of the Study:

• To develop and validate EvoPRIME, a novel prime editing-based screening platform.
• To enhance the efficiency and applicability of prime editing for high-throughput functional genetic screens.

Main Methods:

• EvoPRIME integrates Csy4-mediated processing of RNA polymerase II-driven guide RNAs with a fluorescence-based enrichment strategy.
• The platform was validated using loss-of-function (LOF) dropout screens and gain-of-function (GOF) saturation mutagenesis screens.
• Functional assessment of gain- and loss-of-phosphorylation (GOP and LOP) mutations was performed.

Main Results:

• EvoPRIME demonstrates comparable editing efficiency to existing prime editing systems without needing mismatch repair suppression.
• The platform showed improved sensitivity and reproducibility in LOF screens targeting essential genes.
• GOF screens identified known and novel osimertinib resistance mutations in the EGFR tyrosine kinase domain.
• A GOP screen identified a GAB1 S419E phosphomimetic mutation activating AKT signaling and conferring osimertinib resistance.

Conclusions:

• EvoPRIME is a versatile and efficient platform for high-throughput functional genetic screening.
• The platform facilitates the discovery of functional variants, including those involved in drug resistance and signaling pathways.
• EvoPRIME advances the utility of prime editing for large-scale genetic studies.