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Prime Editing of Mouse Primary Neurons
Colin D Robertson1, Ryan R Richardson1, Marilyn Steyert1,2
1Department of Pharmacology and Physiology, University of Maryland School of Medicine, Baltimore, MD, USA.
Methods in Molecular Biology (Clifton, N.J.)
|April 12, 2025
Summary
Prime editing precisely modifies genomes using a hybrid technology. This method enables accurate DNA synthesis from RNA, minimizing unwanted genetic alterations in neuronal studies.
Area of Science:
- Molecular Biology
- Genetics
- Neuroscience
Background:
- Prime editing is an advanced genome editing technology.
- It precisely introduces small edits into the genome.
- This technology combines nickase Cas9 with reverse transcriptase.
Purpose of the Study:
- To describe a protocol for prime editing in mouse primary neurons.
- To enable precise genome manipulation in a native neuronal context.
- To facilitate the study of protein structure/function and pathogenic variants.
Main Methods:
- Design of prime editing guide RNAs (pegRNAs) and PE3b gRNAs using PegAssist.
- Cloning of plasmids for prime editing.
- Nucleofection of mouse primary neurons.
- Preparation for next-generation sequencing.
Main Results:
- The protocol provides directions for pegRNA and PE3b gRNA design and construction.
- Successful implementation of prime editing in mouse primary neurons.
- Maintained endogenous gene expression during genome manipulation.
Conclusions:
- Prime editing offers a precise method for studying gene function and disease in neurons.
- This technique is valuable for investigating protein structure/function relationships.
- It allows for the study of pathogenic variants within their native cellular environment.
