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Advancing Fast-Track Genome Engineering in Bacillus subtilis Phages.

Arthur Loubat1, Cédric Wolfender1, Magali Calabre1

  • 1Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78350 Jouy-en-Josas, France.

ACS Synthetic Biology
|January 15, 2026
PubMed
Summary
This summary is machine-generated.

QuickPhage enables rapid and precise engineering of Bacillus subtilis phages using CRISPR-Cas9. This method facilitates gene deletion, reporter gene insertion, and controlled protein production, advancing phage synthetic biology.

Keywords:
Bacillus subtilisCRISPRQuickPhageSPP1genome engineeringphage

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Area of Science:

  • Synthetic Biology
  • Microbial Genetics
  • Bacteriophage Biology

Background:

  • Phage genome engineering is crucial for understanding phage biology and developing genetic tools.
  • Existing methods can be time-consuming and technically demanding.

Purpose of the Study:

  • To develop a rapid, accessible, precise, and cost-effective method for engineering Bacillus subtilis phages.
  • To demonstrate the utility of this method for gene manipulation and protein production.

Main Methods:

  • Utilized CRISPR-Cas9 as a counter-selection system for isolating SPP1 phage mutants.
  • Employed short homologous repair patches (40 nucleotides) for efficient genome editing.
  • Applied the method for gene deletion and reporter gene insertion (e.g., GFP).

Main Results:

  • Achieved highly accurate genome edits within a single day.
  • Demonstrated synthetic regulation of protein production (GFP) with up to 400-fold induction.
  • Identified an efficient superinfection arrest mechanism active within 13 minutes.

Conclusions:

  • QuickPhage offers a streamlined approach for Bacillus subtilis phage genome engineering.
  • Phages show potential for protein production and metabolic engineering applications.
  • This work expands the synthetic biology toolbox for B. subtilis, enabling genome refactoring and DNA delivery tools.