Developing a CRISPR-assisted base-editing system for genome engineering of Pseudomonas chlororaphis

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Pseudomonas chlororaphis is a plant growth-promoting bacterium known for antibiotic production.
• Existing genome editing in P. chlororaphis relies on inefficient homologous recombination (HR).
• HR methods are laborious, time-consuming, and require specific DNA repair proteins and templates.

Purpose of the Study:

• To establish a novel and efficient CRISPR-assisted base-editing (CBE) system for P. chlororaphis.
• To demonstrate the system's ability to introduce targeted mutations without DNA strand breaks.
• To assess the applicability of CBE in other Pseudomonas species.

Main Methods:

• Developed a CBE system by fusing rat cytidine deaminase (rAPOBEC1), enhanced-specificity Cas9 nickase (eSpCas9ppD10A), and uracil DNA glycosylase inhibitor (UGI).
• Engineered premature STOP codons in target genes (hmgA and phzO) using the CBE system.
• Validated the CBE system in P. chlororaphis strains and extended its use to P. fluorescens 10586.

Main Results:

• Successfully interrupted hmgA and phzO genes in P. chlororaphis with high efficiency using CBE.
• The phzO-inactivated strain generated by CBE showed identical phenotypes to one created by HR.
• Achieved high editing efficiency across different P. chlororaphis subspecies and in P. fluorescens.

Conclusions:

• The developed CBE system provides an efficient, DNA break-free method for genome editing in P. chlororaphis.
• This technology bypasses the limitations of traditional HR-based methods.
• The wide applicability of this CBE system will advance research in bacterial physiology and metabolic engineering.