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Mini-PE, a prime editor with compact Cas9 and truncated reverse transcriptase
Ting Lan1,2, Huangyao Chen1,3,2, Chengcheng Tang1
1Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Wuyi University, Jiangmen 529020, China.
Molecular Therapy. Nucleic Acids
|September 8, 2023
Summary
Researchers developed a mini-prime editor (PE) by optimizing the Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). This smaller PE tool enables precise gene editing in cells and mouse retinas, paving the way for potential genetic disease therapies.
Area of Science:
- Molecular Biology
- Gene Editing Technologies
- Biotechnology
Background:
- Prime editing (PE) is a powerful genome editing technology that allows for precise DNA modifications without requiring DNA donors or inducing double-strand breaks.
- The large size of current prime editor systems poses a significant challenge for efficient in vivo delivery and application.
- Developing smaller, more manageable prime editor variants is crucial for advancing in vivo gene editing therapies.
Purpose of the Study:
- To engineer a miniaturized prime editor (mini-PE) system with enhanced delivery capabilities for in vivo applications.
- To identify and optimize key components of the prime editor, specifically the reverse transcriptase (RT) domain.
- To evaluate the efficiency and precision of the developed mini-PE in cellular and animal models.
Main Methods:
- Screening of truncated Moloney murine leukemia virus (MMLV) reverse transcriptase (RT) variants, including those with the F155Y mutation, to identify smaller, efficient components.
- Optimization of pegRNAs and incorporation of nicking sgRNAs to enhance the editing activity of the mini-PE system.
- Delivery of the mini-PE system using adeno-associated viruses (AAVs) for in vivo gene editing in mouse models.
Main Results:
- Identified the smallest functional truncated MMLV RT variants that retain high gene editing efficiency.
- The optimized mini-PE system achieved up to 10% precise editing at target sites in human and mouse cells.
- Demonstrated successful in vivo editing of the mouse Hsf1 gene in the retina using AAV delivery, albeit with efficiencies below 1%.
Conclusions:
- A miniaturized prime editor (mini-PE) has been successfully developed by optimizing MMLV RT, enabling efficient gene editing in vitro.
- The mini-PE system shows promise for in vivo gene editing applications, as demonstrated by editing in the mouse retina.
- Further improvements in editing efficiency are needed to fully exploit the therapeutic potential of mini-PE for human genetic diseases.
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