Reducing the inherent auto-inhibitory interaction within the pegRNA enhances prime editing efficiency

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Genomics

Background:

• Prime editing enables precise genome modification without double-strand breaks.
• Previous studies suggested an optimal primer binding site (PBS) length of ~13 nucleotides for prime editing guide RNAs (pegRNAs).
• Optimal PBS length characterization was previously limited to plasmid or lentiviral expression systems.

Purpose of the Study:

• To investigate the impact of PBS-spacer complementarity on prime editor (PE) ribonucleoprotein (RNP) complex efficiency.
• To determine optimal pegRNA design parameters for enhanced prime editing outcomes using PE-RNP systems.
• To validate refined PE-pegRNA designs in cellular models and in vivo.

Main Methods:

• Characterization of auto-inhibitory interactions between PBS and spacer sequences in pegRNAs.
• Systematic variation of PBS length and complementarity to optimize pegRNA binding and target recognition.
• Assessment of prime editing efficiency in mammalian cells using end-protected pegRNAs.
• Application of transient cold shock treatment to enhance prime editing outcomes.
• Validation of optimized PE-RNP complexes in patient-derived fibroblasts, primary human T cells, and zebrafish.

Main Results:

• Reduced complementarity between the PBS-spacer region enhances prime editing efficiency by destabilizing auto-inhibitory interactions.
• For end-protected pegRNAs, shorter PBS lengths with a melting temperature near 37°C are optimal in mammalian cells.
• Transient cold shock treatment post-delivery further improves prime editing efficiency.
• Optimized PE-RNP complexes successfully corrected disease-related mutations and installed precise edits in various cell types and organisms.

Conclusions:

• Refined pegRNA design parameters, including optimized PBS length and reduced complementarity, significantly enhance prime editing efficiency with PE-RNPs.
• The findings provide a more robust and efficient prime editing system for therapeutic applications and genetic research.
• This study advances the precision and applicability of prime editing technology for correcting genetic defects and engineering genomes.