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bioRxiv · 10.1101/2025.08.11.669692

Compound Delivery of eVLPs Enhances Prime Editing for Targeted Genome Engineering and High-Throughput Screening

Langley, J.·Baudrier, L.·Curry, J.·Narta, K.·Todesco, H. M.·Potts, K.·Morrissy, S.·Mahoney, D. J.·Billon, P.
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Abstract

Engineered virus-like particles (eVLPs) enable transgene-free ribonucleoprotein delivery for genome editing applications, yet optimized delivery strategies for high-throughput applications remain unexplored. Prime editing enables precise genomic modifications but suffers from limited efficiency that constrains its widespread adoption. Here, we present PRIME-VLP (Progressive Repeated Infections for Maximized Editing via Virus-Like Particles), a delivery strategy that enhances prime editing efficiency for both targeted genome engineering and high-throughput prime editing screening. PRIME-VLP leverages the temporal dynamics of eVLP-mediated editing through multiple sequential transductions with sub-saturating eVLP doses delivered at optimal intervals. This approach achieves 1.5 to 2.8-fold improvements in editing efficiency across diverse genomic targets and cell types. PRIME-VLP maintains high specificity without increasing off-target effects, compromising cellular viability or causing transcriptional perturbations. By decoupling pegRNA and editor delivery through pegRNA-free eVLPs, PRIME-VLP enables pooled prime editing screens, circumventing transgene silencing limitations of conventional lentiviral-based screens. Using a 6,000-pegRNA library targeting TP53, PRIME-VLP achieved 2.8-fold higher editing efficiency and improved reproducibility compared to conventional lentiviral delivery. An eVLP-based screen identified functional TP53 loss-of-function variants that confer resistance to MDM2 inhibition by Nutlin-3. This work expands the versatility of eVLPs beyond their current in vivo therapeutic applications, demonstrating their promise for high-throughput functional genomics by overcoming the delivery limitations of lentiviral systems.

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Langley, J., Baudrier, L., Curry, J., Narta, K., Todesco, H. M., Potts, K., Morrissy, S., Mahoney, D. J., Billon, P.. 2025-08-11. Compound Delivery of eVLPs Enhances Prime Editing for Targeted Genome Engineering and High-Throughput Screening. https://doi.org/10.1101/2025.08.11.669692

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