Precision Genome Engineering in Streptococcus suis Based on a Broad-Host-Range Vector and CRISPR-Cas9 Technology

Area of Science:

• Microbiology
• Genetics
• Molecular Biology

Background:

• Streptococcus suis is a significant zoonotic pathogen causing severe invasive diseases in both pigs and humans.
• Existing genome engineering methods for S. suis are inefficient, relying on antibiotic resistance markers and lacking precision for small genomic mutations.

Purpose of the Study:

• To develop a novel CRISPR-based genome editing system for S. suis.
• To enable rapid, precise genetic modifications, including multiple gene deletions and nucleotide changes.

Main Methods:

• Utilized linear DNA fragments for homologous recombination (HR) in S. suis.
• Implemented a plasmid-based negative selection system to eliminate unedited bacteria.
• Engineered a broad-host-range replicon for the CRISPR plasmid to enhance applicability.
• Characterized a Cas9-sgRNA escape mechanism involving slow-growing persister-like colonies.

Main Results:

• Successfully demonstrated rapid introduction of multiple gene deletions in successive editing rounds.
• Achieved precise nucleotide changes in essential genes.
• Identified a novel S. suis escape mechanism from CRISPR-Cas9 targeting, characterized by slow-growing colonies that do not impede editing utility.
• The developed system accelerates mutant construction and simplifies antibiotic marker removal.

Conclusions:

• The developed CRISPR-based system significantly enhances the genetic engineering toolbox for S. suis.
• This method allows for faster and more precise genome editing compared to traditional approaches.
• The system's broad-host-range capability and efficient marker removal streamline genetic studies of S. suis and potentially other bacteria.