Development of a versatile nuclease prime editor with upgraded precision

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biochemistry

Background:

• Prime editors (PEs) offer precise genome editing but are limited by complex editing outcomes and off-target effects.
• Chemical inhibition of DNA-PK enhances PE insertions but poses specificity and toxicity concerns.
• Developing alternative strategies to improve PE precision and safety is crucial for advancing gene editing applications.

Purpose of the Study:

• To develop a novel prime editor (PE) platform, termed uPEn, that enhances precise DNA editing efficiency and specificity.
• To mitigate unintended edits caused by prime editors (PEs) through a non-chemical approach.
• To establish a high-activity editing platform with broad applications in genetic engineering.

Main Methods:

• Co-introduction of prime editor (PEn) with a 53BP1-inhibitory ubiquitin variant to restrain nonhomologous end joining (NHEJ).
• Exploration of homologous region-containing pegRNA (HR-pegRNA) configurations for optimized editing.
• Assessment of uPEn efficiency for insertions, deletions, and base substitutions in HEK293T cells.

Main Results:

• The uPEn platform significantly enhances precise DNA editing, achieving high efficiencies for insertions (38%), deletions (43%), and replacements (52%).
• uPEn demonstrates superior activity compared to PE3/5max for challenging base substitutions and small-block edits.
• The 53BP1-inhibitory ubiquitin variant effectively mitigates unintended edits, improving overall editing precision.

Conclusions:

• uPEn represents a highly efficient and precise prime editor platform, overcoming limitations of previous PE systems.
• This novel approach broadens the application potential of prime editing for complex genetic modifications.
• uPEn offers a promising alternative to chemical inhibitors for improving gene editing outcomes.