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

️ 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/

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