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

Jiashuo Liu1, Jingjing Zhang2, Tingting Zhao3

  • 1Department of Obstetrics and Gynecology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China.

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A new SunTag-Prime Editing (PE) system efficiently delivers gene editing tools using adeno-associated virus (AAV) vectors. This breakthrough overcomes size limitations for treating genetic diseases, matching traditional prime editing efficiency.

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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.