CRISPR/Cas9-enhanced ssDNA recombineering for Pseudomonas putida

Area of Science:

• Microbiology
• Molecular Biology
• Synthetic Biology

Background:

• Single-stranded DNA (ssDNA) recombineering offers versatile genetic modification in Pseudomonas putida.
• Low efficiency of ssDNA recombineering hinders the identification of desired mutant clones.
• CRISPR/Cas9 can be employed for counterselection of wild-type sequences to overcome efficiency limitations.

Purpose of the Study:

• To develop and optimize a combined ssDNA recombineering and CRISPR/Cas9 system for efficient genomic editing in P. putida.
• To demonstrate the utility of this method for precise gene deletions and sequential modifications.
• To enhance the genetic tractability of P. putida for biotechnological applications.

Main Methods:

• Integration of ssDNA recombineering with CRISPR/Cas9-mediated counterselection.
• Design of CRISPR plasmids to target wild-type sequences for Cas9 cleavage.
• Optimization of the workflow for P. putida, including gene deletion examples (edd and pyrF).
• Utilization of incompatible CRISPR plasmids with distinct antibiotic markers for sequential edits.

Main Results:

• Successful implementation of a streamlined protocol for genomic editing in P. putida.
• Demonstration of efficient deletion of the edd gene involved in glucose metabolism.
• Achieved sequential deletion of the pyrF gene in an edd mutant strain using incompatible CRISPR plasmids.
• Validated the applicability of the method for multiple, consecutive genomic modifications.

Conclusions:

• The combined ssDNA recombineering and CRISPR/Cas9 system significantly enhances the efficiency of genetic manipulation in P. putida.
• This approach simplifies the process of generating complex mutant strains.
• The optimized protocol expands the genetic toolkit for P. putida, reinforcing its role as a valuable chassis for biotechnology.