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Related Experiment Video

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A flexible split prime editor using truncated reverse transcriptase improves dual-AAV delivery in mouse liver.

Chunwei Zheng1, Shun-Qing Liang1, Bin Liu1

  • 1RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA.

Molecular Therapy : the Journal of the American Society of Gene Therapy
|January 9, 2022
PubMed
Summary
This summary is machine-generated.

We developed a compact prime editor (PE) without the RNase H domain, enabling efficient in vivo gene editing. This novel PE, delivered via dual adeno-associated viruses, advances prime editing applications for gene therapy.

Keywords:
AAVmouse liverprime editingreverse transcriptasesplit prime editor

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Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • Biotechnology

Background:

  • Prime editing (PE) offers significant potential for gene therapy but faces delivery challenges due to its large size (>6.3 kb).
  • Current methods using dual adeno-associated viruses (AAVs) have limitations in selecting Cas9 split sites, impacting editing efficiency.
  • Overexpression of reverse transcriptase in full-length PE may cause unintended translation termination due to its RNase H domain.

Purpose of the Study:

  • To develop a compact prime editor (cPE) lacking the RNase H domain for improved in vivo delivery and function.
  • To identify an optimal Cas9 split site for efficient dual-AAV delivery of the cPE.
  • To evaluate the editing efficiency and safety profile of the cPE in cellular and in vivo models.

Main Methods:

  • Engineered a compact prime editor (cPE) by removing the RNase H domain.
  • Utilized a specific Cas9 split site (Glu 573) for dual-AAV delivery of the cPE.
  • Assessed editing efficiency in cell lines and mouse liver models.
  • Investigated the interaction of cPE with peptidyl release factor 1 (eRF1) and stop codon readthrough effects.

Main Results:

  • The cPE demonstrated editing efficiency comparable to full-length PE.
  • The selected Cas9 split site (Glu 573) enabled robust editing (up to 93% of full-length PE) in cells and mouse liver.
  • Dual-AAV8 delivery of split-cPE573 successfully mediated a 3-bp TGA insertion in the Pcsk9 gene in mouse liver.
  • The cPE lacking the RNase H domain showed reduced binding to eRF1, mitigating stop codon readthrough.

Conclusions:

  • A compact prime editor (cPE) without the RNase H domain was successfully developed.
  • The flexible split design, exemplified by cPE573, enhances the in vivo delivery and utility of prime editing.
  • This optimized PE system holds promise for advancing gene therapy applications by overcoming delivery hurdles and improving safety.