Continuous multiplexed phage genome editing using recombitrons.

Area of Science:

• Molecular Biology
• Microbiology
• Synthetic Biology

Background:

• Bacteriophage genome editing is crucial for improving phage therapy efficacy against pathogenic bacteria.
• Current phage genome editing methods are inefficient, requiring extensive screening and counterselection.
• There is a need for scalable and efficient methods for bacteriophage genome modification.

Purpose of the Study:

• To develop a scalable and efficient method for bacteriophage genome editing.
• To overcome the limitations of current laborious phage genome editing techniques.
• To enable rapid generation of modified phages for therapeutic applications.

Main Methods:

• Utilized modified bacterial retrons (recombitrons) to generate recombineering donor DNA.
• Employed single-stranded binding and annealing proteins for efficient DNA integration into phage genomes.
• Demonstrated the system's ability to perform continuous, multiplexable genome modifications.

Main Results:

• Achieved efficient genome modifications in multiple phages without counterselection.
• Successfully performed large insertions and deletions with >99% efficiency when combined with counterselection.
• Installed up to five distinct mutations on a single lambda phage genome in hours.
• Identified a residue-level epistatic interaction in the T7 gp17 tail fiber.

Conclusions:

• The recombitron-based system offers a scalable and efficient approach for bacteriophage genome editing.
• This method significantly reduces the labor and time required for phage modification.
• The developed system facilitates the rapid engineering of phages for enhanced therapeutic potential.