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A fast and robust iterative genome-editing method based on a Rock-Paper-Scissors strategy.

Jichao Wang1, Xinyue Sui1,2, Yamei Ding3

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This study introduces a novel Rock-Paper-Scissors genome-editing method for bacteria. This efficient technique accelerates strain optimization by reducing cultivation steps and eliminating the need for separate plasmid curing.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Synthetic Biology

Background:

  • Iterative genome editing is crucial for developing optimized bacterial strains with specific phenotypes.
  • Traditional methods often involve time-consuming steps for marker elimination and plasmid curing, leading to potential errors and delays.

Purpose of the Study:

  • To develop a faster and more robust iterative genome-editing method for bacterial strain engineering.
  • To streamline the genome-editing process by minimizing cultivation steps and eliminating the need for separate plasmid curing procedures.

Main Methods:

  • A novel Rock-Paper-Scissors (RPS) genome-editing strategy utilizing three interdependent sgRNA plasmids.
  • Each plasmid in the RPS system can cure or be cured by the other two, enabling simultaneous editing and curing.
  • Demonstrated application in *Escherichia coli* and *Klebsiella pneumoniae* for gene deletions and replacements.

Main Results:

  • Achieved enhanced curing efficiency and an embedded double-check mechanism, negating the need for separate plasmid curing or confirmation steps.
  • Reduced cultivation requirements to only two steps per genome-editing round, significantly accelerating the process.
  • Successfully performed gene deletions and replacements in *E. coli* and *K. pneumoniae*.

Conclusions:

  • The RPS method represents the fastest and most robust iterative genome-editing technique to date.
  • The minimized cultivation steps decrease the likelihood of spontaneous genome mutations during strain optimization.
  • This method offers a significant advancement for bacterial strain engineering and synthetic biology applications.