🎙️ Episode 21: High-Throughput Variant Discovery — Pooled Prime Editing in Human Cells

🧬 In this episode of Base by Base, we explore a cutting-edge platform for scalable functional screening of human genetic variants reported by Herger et al. (2025) in Cell Genomics. The study introduces a pooled prime editing workflow in haploid HAP1 cells that optimizes pegRNA scaffold design, employs co-selection via an ATP1A1 resistance mutation, and integrates surrogate targets to enrich and filter highly active edits—enabling interrogation of both coding and non-coding variants in their native genomic context .

🔍 Study highlights:
The platform achieves improved editing efficiencies by incorporating stabilized pegRNA scaffolds, optimized prime editor expression, and co-selection for an ATP1A1 resistance mutation in HAP1 cells .
Deep sequencing of pegRNA–surrogate target cassettes enables negative selection screening in SMARCB1 exons 8–9, accurately identifying loss-of-function single-nucleotide and multinucleotide variants that impair protein function .
Positive selection using 6-thioguanine in MLH1 screens yields precise function scores for SNVs in exon 10, with AUCs reaching 1.00 under stringent surrogate editing thresholds, demonstrating robust discrimination of pathogenic variants .
A comprehensive library covering 874 non-coding ClinVar variants across 60 kb of MLH1 reveals pathogenic splice and regulatory variants at scale, with surrogate target filtering ensuring high data quality .
Validation assays confirm that surrogate target editing correlates strongly with endogenous variant installation, supporting accurate function scoring across large genomic regions .

🧠 Conclusion:
This pooled prime editing platform offers a versatile and scalable tool for functional assessment of genetic variants across both coding and non-coding regions, paving the way for accelerated discovery and classification of disease-associated alleles .

📖 Reference:
Herger, M., Kajba, C. M., Buckley, M., Cunha, A., Strom, M., & Findlay, G. M. (2025). High-throughput screening of human genetic variants by pooled prime editing. Cell Genomics, 5, 100814. https://doi.org/10.1016/j.xgen.2025.100814

📜 License:
This episode is based on an open access article published under the Creative Commons Attribution 4.0 International license (CC BY 4.0) – https://creativecommons.org/licenses/by/4.0/