CRISPRpi: Inducing and Curing Prophage Using the CRISPR Interference.

Area of Science:

• Microbiology
• Molecular Biology
• Bacteriophage Research

Background:

• Bacteriophages, viruses that infect bacteria, can integrate into the host genome as prophages.
• Prophage induction is crucial for studying phage biology and for phage therapy, but many prophages are non-inducible.
• Existing methods for prophage curing are often inefficient or limited to specific phage types.

Purpose of the Study:

• To develop a novel CRISPR-interference (CRISPRi) based protocol for efficient prophage induction.
• To demonstrate the utility of this CRISPRi system for curing bacterial hosts of prophages, including non-inducible ones.
• To establish a rapid and high-efficiency method for prophage management in bacterial systems.

Main Methods:

• Utilized CRISPR interference (CRISPRi) technology to target and silence the transcription of prophage repressor genes.
• Engineered a plasmid carrying specific CRISPRi components and guide RNAs (spacers) targeting the prophage repressor.
• Introduced the CRISPRi system into bacterial hosts via plasmid electroporation.

Main Results:

• Successfully triggered prophage induction in bacterial hosts, even for previously non-inducible prophages.
• Demonstrated efficient curing of bacterial hosts from integrated prophages using the CRISPRi system.
• Achieved high efficiency of both prophage induction and curing within one to two weeks.

Conclusions:

• CRISPR interference provides a versatile and effective tool for manipulating prophage states in bacteria.
• This protocol offers a significant advancement for bacteriophage research and potential therapeutic applications.
• The developed method is rapid, efficient, and applicable to a broad range of prophages.