Disrupting pegRNA intramolecular complementarity via PBS and spacer sequence alterations can enhance prime editing...

Area of Science:

• Molecular Biology
• Genetic Engineering

Background:

• Prime editing is a powerful gene editing technology.
• The primer binding site (PBS) and spacer regions of prime editing guide RNAs (pegRNAs) are critical for efficiency.
• Intramolecular complementarity between the PBS and spacer can impede prime editing.

Purpose of the Study:

• To investigate the impact of PBS length and sequence on prime editing efficiency.
• To explore strategies for weakening PBS-spacer complementarity to enhance editing.
• To develop a streamlined prime editing approach (SPELL) for improved efficiency.

Main Methods:

• Literature analysis of over 3000 pegRNAs.
• Experimental testing of over 300 modified pegRNAs with altered PBS-spacer interactions.
• Development and application of the SPELL method utilizing a fixed-length PBS with deletions.

Main Results:

• Optimal PBS length did not significantly shift for enhanced pegRNAs (epegRNAs) compared to conventional pegRNAs.
• Introducing mismatches in the spacer or PBS sequence improved editing efficiency up to seven-fold.
• Combining spacer mismatches and PBS deletions further enhanced editing, though optimization is complex.
• The SPELL approach achieved near-optimal editing efficiency without extensive pegRNA optimization.

Conclusions:

• Weakening PBS-spacer complementarity is a key strategy for enhancing prime editing.
• SPELL offers a streamlined and efficient method for prime editing.
• Further optimization of spacer mismatches and PBS deletions can yield significant improvements in editing efficiency.