Compound delivery of eVLPs enhances prime editing for targeted genome engineering and high-throughput screening

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Engineered virus-like particles (eVLPs) offer transgene-free delivery for genome editing.
• Prime editing provides precise genomic modifications but faces efficiency limitations.
• High-throughput applications for eVLP-mediated genome editing require optimized strategies.

Purpose of the Study:

• To develop an optimized strategy for high-throughput prime editing using eVLPs.
• To enhance prime editing efficiency and expand its applications in functional genomics.
• To present PRIME-VLP, a novel method leveraging eVLP kinetics for improved genome editing.

Main Methods:

• Developed PRIME-VLP (Progressive Repeated Infections for Maximized Editing via Virus-Like Particles).
• Utilized sequential sub-saturating eVLP transductions at optimal intervals.
• Employed pegRNA-free eVLPs to decouple pegRNA and editor delivery.

Main Results:

• Achieved 1.5- to 2.9-fold improvements in prime editing efficiency across various targets and cell types.
• Demonstrated no increase in off-target editing, cellular toxicity, or transcriptional perturbations.
• Enabled pooled prime editing screens and overcame transgene silencing limitations.
• Identified TP53 loss-of-function variants conferring Nutlin-3 resistance using a 6,000-pegRNA library.

Conclusions:

• PRIME-VLP significantly enhances prime editing efficiency and expands the utility of eVLPs.
• The method is suitable for high-throughput functional genomics screens, including pooled screens.
• PRIME-VLP offers a promising alternative to conventional delivery methods for genome editing applications.