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A versatile one-step CRISPR-Cas9 based approach to plasmid-curing.

Ida Lauritsen1, Andreas Porse1, Morten O A Sommer1

  • 1Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800, Kongens Lyngby, Denmark.

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Summary

A new universal plasmid-curing system (pFREE) efficiently removes plasmids from bacterial cells using CRISPR-Cas9 technology. This rapid method aids genetic engineering and eliminates the need for specific plasmid removal techniques.

Keywords:
CRISPR-Cas9Genome engineeringPlasmid-curingPseudomonas putidaReplicon analysispFREE

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Plasmids are crucial for molecular biology but impose metabolic burdens and are often transiently used.
  • Current methods for genetic manipulation lack a universal tool for rapid plasmid removal from bacterial cells.

Purpose of the Study:

  • To develop a universal and rapid plasmid-curing system for bacterial cells.
  • To create a method for efficient removal of common cloning and expression vectors.

Main Methods:

  • Analysis of replicon abundance and sequence conservation in bacterial vectors.
  • Construction of a CRISPR-Cas9 based plasmid-curing system (pFREE).
  • Development of a one-step protocol and PCR for identifying plasmid-free clones.

Main Results:

  • The pFREE system demonstrates broad applicability across common bacterial cloning and expression vectors due to shared sequence similarities.
  • Curing efficiencies ranged from 40% to 100% for widely used plasmids.
  • Successful application in Escherichia coli and Pseudomonas putida highlights its broad host context expandability.

Conclusions:

  • The pFREE system offers a fast, freely available, and universal solution for plasmid removal, potentially replacing individualized vector suicide methods.
  • This system is particularly valuable for complex genetic engineering applications involving multiple plasmids, such as genome editing and combinatorial pathway engineering.