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Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing
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Generation of an Avian Myeloblastosis Virus (AMV) Reverse Transcriptase Prime Editor
Yi-Ting Tsai1, Bruna Lopes da Costa1,2,3, Salvatore Marco Caruso1,2,3,4
1Department of Biomedical Engineering, Columbia University, New York, NY, USA.
Advances in Experimental Medicine and Biology
|July 13, 2023
Summary
Prime editing offers a new gene editing approach for inherited retinal diseases. This study developed a novel prime editor using avian myeloblastosis virus reverse-transcriptase for precise genetic correction.
Area of Science:
- Molecular Biology
- Gene Editing Technologies
- Ophthalmology
Background:
- Inherited retinal diseases (IRDs) involve over 280 genes, presenting significant challenges for gene editing therapies.
- Traditional genome editing methods often cause complications due to their reliance on double-strand breaks.
- Prime editing (PE) is a promising double-strand break-independent gene editing technology with potential for treating IRDs.
Purpose of the Study:
- To develop a novel prime editor system utilizing avian myeloblastosis virus (AMV)-reverse transcriptase (RT).
- To demonstrate the efficacy of the developed prime editor for precise genetic modifications relevant to IRDs.
Main Methods:
- Construction of a prime editor system by fusing a SpCas9 nickase mutant with an AMV-RT.
- Design and utilization of a prime editing guide RNA (pegRNA) for target site recognition and editing.
- Application of the developed prime editor in HEK293 cells to introduce a specific mutation (PRPH2 c.828+1G>A).
Main Results:
- Successful development of a functional prime editor based on AMV-RT.
- Demonstrated capability of the prime editor to install the PRPH2 c.828+1G>A mutation in HEK293 cells.
- The new system shows potential for precise correction of mutations associated with IRDs.
Conclusions:
- The AMV-RT-based prime editor is a viable alternative for gene editing applications.
- This technology holds promise for developing targeted therapies for a wide range of inherited retinal diseases.
- Further research can explore the application of this system for diverse genetic mutations causing IRDs.
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