Modified pegRNAs mitigate scaffold-derived prime editing by-products.

Area of Science:

• Molecular Biology
• Gene Editing Technologies

Background:

• Prime editing (PE) enables precise genomic alterations using reverse transcriptase (RT) and a prime editing guide RNA (pegRNA).
• A limitation of prime editing is the potential for RT to extend beyond the pegRNA template, incorporating scaffold sequences and reducing editing precision.

Purpose of the Study:

• To develop and validate a method to prevent RT extension into the pegRNA scaffold.
• To enhance the precision of prime editing by mitigating scaffold-derived by-products.

Main Methods:

• Incorporation of an internal abasic spacer or 2'-O-methylation within the pegRNA sequence.
• Utilizing modified pegRNAs with processive prime editors (PEs) like PE6d or PE** in various cell types.
• Benchmarking the efficiency and precision of modified pegRNAs compared to standard pegRNAs.

Main Results:

• Modified pegRNAs successfully terminated RT at the end of the template sequence.
• Scaffold-derived by-products were significantly mitigated.
• High prime editing precision was maintained across different cell types and with processive PEs.

Conclusions:

• Internal abasic spacers or 2'-O-methylation in pegRNAs offer a simple and effective strategy to prevent RT extension.
• This approach substantially improves prime editing precision by eliminating common by-products.
• The developed method is compatible with existing prime editing systems and various cell types.