Cascade-Cas3 facilitates high-accuracy genome engineering in Pseudomonas using phage-encoded homologous recombination

Abstract

Phage-encoded homologous recombination (PEHR) is an efficient tool for bacterial genome editing. We previously developed and utilized a Pseudomonas-specific PEHR system. However, when using the PEHR system for Pseudomonas genome editing, false positives can be a problem. In this study, we combined a compact Cascade-Cas3 system from P. aeruginosa (PaeCas3c) with a Pseudomonas-specific PEHR system, and the results of our recombineering assay showed that this compact Cascade-Cas3 system can significantly improve PEHR recombineering accuracy.

Keywords

• Phage-encoded homologous recombination
• Pseudomonas
• Cascade-Cas3 system
• Genome engineering