Enhancing prime editor flexibility with coiled-coil heterodimers

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• Prime editing (PE) allows precise DNA modifications without double-strand breaks but faces delivery challenges due to large size.
• Adeno-associated virus (AAV) packaging limits hinder in vivo delivery of large prime editors.
• Existing split PE systems require complex engineering and may reduce editing efficiency.

Purpose of the Study:

• To develop a simplified and efficiently deliverable prime editing system.
• To enhance the in vivo applicability of prime editing technology.
• To engineer prime editors with expanded editing scopes.

Main Methods:

• Developed CC-PE using non-covalent recruitment of reverse transcriptase to Cas9 nickase via coiled-coil heterodimers.
• Engineered SpCas9-NG-PE and SpRY-PE variants using coiled-coil heterodimers for broader editing scope.
• Delivered dual AAV vectors carrying CC-PE into mice to target the Pcsk9 gene in the liver.

Main Results:

• CC-PE demonstrated maintained or enhanced editing efficiency compared to unsplit PE across multiple cell types.
• Engineered NG-CC-PE and SpRY-CC-PE achieved efficient precise editing with expanded scopes.
• In vivo delivery of CC-PE in mice resulted in significant Pcsk9 gene editing and reduced cholesterol levels.

Conclusions:

• The modular CC-PE system offers enhanced flexibility and overcomes in vivo delivery limitations.
• This simplified split prime editor facilitates precise gene editing in vivo.
• CC-PE technology holds promise for advancing therapeutic gene editing strategies.