An auto-excision system for rapid and efficient genetic manipulation in Glaesserella parasuis

Site-specific recombination systems are widely used in bacterial gene editing due to their precision and efficiency. However, traditional gene editing methods often require labor-intensive plasmid construction and multiple transformation steps, which can be time-consuming and inefficient. In this study, we developed an Auto-Excision (AE) system that overcomes these limitations by optimizing the entire process—from the preparation of targeting sequences to the screening of marker-free mutants. The AE system simplifies the knockout process by eliminating the need to construct targeting plasmids for each target gene, requiring only a single transformation, and allowing for the direct selection of markerless mutants in the presence of antibiotics. We validated the AE system's ability to enable rapid and efficient gene knockout in Glaesserella parasuis (G. parasuis), demonstrating its potential as a rapid and labor-efficient gene manipulation tool. This method reduces the overall timeline to as little as one day, with a hands-on time of less than one hour, while achieving a knockout efficiency greater than 90 %. Additionally, the system successfully performed multi-gene knockouts, targeting five genes in succession. This approach offers substantial time and labor savings, with the entire process achievable within a single bacterial colony growth cycle. This positions the AE system as a rapid bacterial genetic manipulation method currently known, with broad potential applications across diverse bacterial species.