SunTag-PE: a modular prime editing system enables versatile and efficient genome editing

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• Prime editing (PE) offers precise genetic modification for treating inherited disorders.
• The large size of PE effectors limits delivery via adeno-associated virus (AAV) vectors, hindering clinical applications.
• Existing split-PE strategies often compromise editing efficiency.

Purpose of the Study:

• To develop a novel, modular, and efficient split-prime editing system for improved AAV-mediated delivery.
• To overcome the packaging capacity limitations of AAV for prime editing applications.
• To enhance the clinical translatability of prime editing technology.

Main Methods:

• Developed the SunTag-PE system, splitting PE effectors into GCN4-nCas9 and scFv-tethered reverse transcriptase (RT).
• Optimized SunTag-PE configurations, identifying 1×GCN4 at the N terminus of nCas9 as most effective.
• Validated SunTag-PE efficiency and specificity in PE2 and PE3 formats, comparing it to canonical fused-PE and other split-PE strategies (sPE, MS2-PE).
• Assessed the modularity of SunTag-PE with different Cas9 orthologs (SauCas9, FrCas9).
• Utilized dual AAVs for delivering SunTag-ePE3 to correct a pathogenic mutation in an HBB mutant cell line.

Main Results:

• The SunTag-PE system achieved editing efficiencies comparable to canonical fused-PE and superior to other split-PE methods.
• The optimized 1×GCN4 SunTag-PE configuration demonstrated high efficiency without increasing indel byproducts.
• The SunTag-PE system's modularity was successfully validated across different Cas9 orthologs.
• Efficient correction of a pathogenic HBB mutation was achieved in a cellular model using dual AAV-delivered SunTag-ePE3.

Conclusions:

• The SunTag-PE system represents an efficient and modular strategy for splitting prime editing effectors.
• This approach overcomes AAV packaging limitations, facilitating the clinical translation of prime editing.
• SunTag-PE enhances gene editing delivery and efficiency, paving the way for new genetic disease therapies.