A SEVA-based, CRISPR-Cas3-assisted genome engineering approach for Pseudomonas with efficient vector curing

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• CRISPR-Cas systems offer precise genome engineering capabilities.
• Developing adaptable tools for Gram-negative bacteria like Pseudomonas is crucial for synthetic biology and biotechnology.
• Efficient vector removal is essential for streamlined genetic manipulation workflows.

Purpose of the Study:

• To present a versatile CRISPR-Cas3 editing system for genomic alterations in Pseudomonas putida and Pseudomonas aeruginosa.
• To demonstrate the system's utility for universal vector curing by targeting the origin of transfer.
• To establish a flexible and easily transferable genome engineering platform for diverse Gram-negative hosts.

Main Methods:

• Utilized a CRISPR-Cas3 system provided as Golden Gate-compatible vectors with antibiotic markers.
• Employed the Standard European Vector Architecture (SEVA) vector set for homology repair templates.
• Designed a spacer targeting the origin-of-transfer for vector curing applications.

Main Results:

• Successfully created genomic alterations in Pseudomonas species using the CRISPR-Cas3 system.
• Demonstrated efficient removal of up to three SEVA vectors within days by targeting the origin of transfer.
• Showcased the system's flexibility and portability across multiple Gram-negative hosts.

Conclusions:

• The presented CRISPR-Cas3 system provides a straightforward method for genome editing in Pseudomonas.
• The system serves as a universal and effective tool for vector curing, simplifying bacterial genetic engineering.
• This approach has broad implications for advancing genomic engineering and managing plasmids in bacteria.