Developing a Base Editing System for Marine Roseobacter Clade Bacteria

Area of Science:

• Marine Microbiology
• Synthetic Biology
• Genome Engineering

Background:

• Roseobacter clade bacteria are crucial for marine ecosystems and biogeochemical cycles.
• Their metabolic versatility makes them promising candidates for marine synthetic biology.
• Efficient genome editing tools are needed for this important bacterial group.

Purpose of the Study:

• To adapt and apply a CRISPR-Cas-based base editing system for precise genome modification in Roseobacter clade bacteria.
• To demonstrate the system's efficacy in the model organism Roseovarius nubinhibens.
• To investigate the beta-ketoadipate pathway and identify novel regulatory elements.

Main Methods:

• Adaptation of a CRISPR-Cas9 system with nuclease-deactivated Cas9 and deaminase for base editing.
• Application of the system in Roseovarius nubinhibens for single-nucleotide resolution genome editing.
• Introduction of premature STOP codons to interrogate gene essentiality in the beta-ketoadipate pathway.

Main Results:

• Achieved precise and efficient genome editing without double-strand breaks or donor DNA.
• Demonstrated the essentiality of genes within the beta-ketoadipate pathway.
• Experimentally identified PcaQ as a transcription activator for the first time.

Conclusions:

• This work represents the first CRISPR-Cas-based genome editing in the Roseobacter clade.
• The developed base editing system provides a powerful tool for studying marine microbial ecology and biogeochemistry.
• It opens new possibilities for the synthetic biology of Roseobacter bacteria.