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PE-STOP: A versatile tool for installing nonsense substitutions amenable for precise reversion.

Ziguo Song1, Guiquan Zhang2, Shuhong Huang1

  • 1International Joint Agriculture Research Center for Animal Bio-Breeding of Ministry of Agriculture and Rural Affairs, College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China.

The Journal of Biological Chemistry
|June 21, 2023
PubMed
Summary

We developed PE-STOP, a prime editing-based tool for gene inactivation. This method precisely installs stop codons, offering improved versatility and specificity for gene function studies.

Keywords:
PE-STOPbase editinggene inactivationnonsense mutationoff-targetprime editingstop codon

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Genome editing technologies accelerate gene function studies in model systems.
  • Current stop/start codon-targeting tools have limitations in versatility, precision, and specificity.
  • Prime editing (PE) offers versatile, precise edits templated by a guide RNA.

Purpose of the Study:

  • To establish a prime editing-empowered method (PE-STOP) for precise gene inactivation via nonsense mutations.
  • To complement existing gene-targeting tools with enhanced versatility and specificity.
  • To demonstrate precise genetic rescue of PE-STOP-induced mutations using the PE platform.

Main Methods:

  • Utilized a novel prime editor (PE) with reverse transcriptase activity.
  • Developed the PE-STOP method for installing nonsense substitutions using specialized prime editing guide RNA.
  • Validated PE-STOP in human embryonic kidney 293T and N2a cells.
  • Assessed on-target precision and off-target effects.

Main Results:

  • PE-STOP efficiently introduced stop codons in human cell lines (29% in HEK293T, 25% in N2a).
  • The method demonstrated high on-target precision and minimal off-target effects.
  • A strategy for precise genetic rescue of PE-STOP-induced mutations was successfully implemented.

Conclusions:

  • PE-STOP provides a versatile and specific tool for gene inactivation.
  • This method enables functional interrogation of nonsense mutations.
  • The PE platform facilitates both gene inactivation and precise genetic rescue.