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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
Summary
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.
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.
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