Enhancing CRISPR prime editing by reducing misfolded pegRNA interactions

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biochemistry

Background:

• CRISPR prime editing (PE) utilizes a Cas9 nickase-reverse transcriptase fusion protein (PE2) and a prime editing guide RNA (pegRNA).
• pegRNAs are engineered to guide PE to target genomic sequences and encode desired edits.
• Potential limitations in pegRNA structure can impede Cas9 complexation and reduce PE efficiency.

Purpose of the Study:

• To investigate the impact of internal sequence complementarity within pegRNAs on PE efficiency.
• To develop and validate methods for improving pegRNA structure and function.
• To enhance the efficiency of CRISPR prime editing technology.

Main Methods:

• Analysis of sequence complementarity within pegRNAs.
• Development of a pegRNA refolding procedure.
• Introduction of point mutations to disrupt internal pegRNA interactions.
• Assessment of PE efficiency in zebrafish embryos using ribonucleoprotein complexes.

Main Results:

• Sequence complementarity between the 5' and 3' regions of pegRNAs negatively affects Cas9 complexation.
• A simple pegRNA refolding procedure increased PE efficiencies by up to 25-fold in zebrafish embryos.
• Introducing point mutations to disrupt internal pegRNA interactions further improved PE efficiencies by up to sixfold.

Conclusions:

• Internal sequence complementarity is a critical factor limiting pegRNA efficacy.
• PegRNA refolding and targeted mutagenesis are effective strategies to enhance CRISPR prime editing efficiency.
• These findings provide practical methods for optimizing PE for broader research and therapeutic applications.