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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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Updated: Oct 21, 2025

A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization
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A versatile genetic engineering toolkit for E. coli based on CRISPR-prime editing.

Yaojun Tong1,2, Tue S Jørgensen3, Christopher M Whitford3

  • 1The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark. yaojun.tong@sjtu.edu.cn.

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Summary

This study introduces a new CRISPR-Prime Editing toolkit for bacterial genome engineering. It enables precise DNA insertions, deletions, and substitutions in E. coli, expanding beyond single-nucleotide changes.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Biotechnology

Background:

  • CRISPR base editing facilitates bacterial genome engineering but is limited to single-nucleotide substitutions.
  • A need exists for versatile genetic manipulation tools in prokaryotes that allow for larger edits.

Purpose of the Study:

  • To adapt a CRISPR-Prime Editing system for versatile genetic manipulation in prokaryotes.
  • To develop a DNA-free, single-nucleotide resolution toolkit for introducing substitutions, deletions, and insertions in E. coli.

Main Methods:

  • Adaptation of a CRISPR-Prime Editing system for prokaryotic use.
  • Application of the toolkit for genetic modifications in both plasmids and the E. coli chromosome.
  • Testing of editing efficiencies for various types of genetic modifications, including deletions and insertions of different sizes.

Main Results:

  • The developed CRISPR-Prime Editing toolkit successfully introduced substitutions, deletions, and insertions in E. coli with high fidelity.
  • High efficiency (up to 40%) was achieved for 1-bp deletions under optimal conditions.
  • Successful deletions of up to 97 bp and insertions of up to 33 bp were demonstrated, though efficiency decreased with larger fragment sizes.

Conclusions:

  • This CRISPR-Prime Editing toolkit represents a significant advancement for E. coli genome engineering, offering versatility beyond base editing.
  • The toolkit provides a foundation for developing similar advanced genetic manipulation systems for other bacterial species.