Genome editing in Streptococcus mutans through self-targeting CRISPR arrays.

Area of Science:

• Microbiology
• Genetics
• Biotechnology

Background:

• Streptococcus mutans is the primary cause of dental caries.
• Glucosyltransferases (Gtfs) are key virulence factors, synthesizing extracellular polysaccharides (EPS) essential for dental plaque biofilm formation.
• Existing methods for controlling S. mutans biofilms are limited.

Purpose of the Study:

• To develop a novel self-targeting gene editing technology to inhibit S. mutans biofilm formation.
• To investigate the essential components of the CRISPR-Cas9 system in S. mutans UA159 for defense against foreign DNA.
• To engineer S. mutans to reduce EPS synthesis and biofilm development.

Main Methods:

• Validated the necessity of PAM sites, tracrRNA, Cas9, and RNase III for S. mutans CRISPR-Cas9 functionality.
• Designed self-targeting CRISPR arrays targeting glucosyltransferase genes (gtfB).
• Constructed and transformed plasmids containing CRISPR arrays and editing templates into S. mutans UA159.

Main Results:

• Confirmed the essential components for the S. mutans CRISPR-Cas9 system.
• Successfully edited gtfB or gtfBgtfC genes in S. mutans using the self-targeting technology.
• Achieved significant reduction in EPS synthesis and breakdown of biofilm formation.

Conclusions:

• The developed self-targeting CRISPR-Cas9 technology is effective for gene editing in S. mutans.
• This approach significantly reduces EPS production and inhibits biofilm formation.
• This technology holds promise as a future tool for preventing S. mutans biofilms in dental clinics.