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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
PubMed
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.

Keywords:
CRISPRGenome editingNeuronsNucleofectionPegAssistPrime editingpegRNA

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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.