Coupling CRISPR/Cas9 and Lambda Red Recombineering System for Genome Editing of Gallinarum and the Effect of Knock-Out Mutant on the Virulence of Bacteria.

The poultry industry in developing countries still faces a significant threat from fowl typhoid, a disease caused by Gallinarum that has been well contained in more economically developed countries. In addition to the virulence exhibited by large virulence plasmid (85 kb), Salmonella Pathogenicity Island 2 in . Gallinarum plays a key role in mediating disease through its type III secretion systems (TTSS). The TTSS secrete effector protein across the containing vacuoles and mediate the internalization of bacteria by modulating vesicular passage. In this study, candidate virulent gene (~1 kb) encoding type III secretion system was successfully deleted from indigenously isolated . Gallinarum genome through homology-directed repair using CRISPR/Cas9 and lambda recombination systems. CRISPR/Cas9-based genome editing of poultry-derived Gallinarum has not been previously reported, which might be linked to a lack of efficiency in its genetic tools. This is the first study which demonstrates a complete CRISPR/Cas9-based gene deletion from this bacterial genome. More importantly, a poultry experimental model was employed to assess the virulence potential of this mutant strain (ΔssaU_G18) which was unable to produce any mortality in the experimentally challenged birds as compared to the wild type strain. No effect on weight gain was observed whereas bacteria were unable to colonize the intestine and liver in our challenge model. This in vivo loss of virulence in mutant strain provides an excellent functionality of this system to be useful in live vaccine development against this resistant and patho genic bacteria.