CRISPR/Cas9 mediated gene editing in Edwardsiella piscicida

Abstract

Edwardsiella piscicida is a fish pathogenic bacteria that induces Edwardsiellosis in various host species causing severe loss. Especially, in olive flounder (Paralichthys olivaceus) farms, heavy mortarlities have been caused by E. piscicida. CRISPR/Cas9 is considered as a kind of adaptive immune system of prokaryotes and is currently used for gene editing in various species. Bacteria can cleave genomes of infecting phages by this system, which characteristics are used to cut double stranded (ds) DNA. In this study, we developed a CRISPR/Cas9 system that can edit genome of E. piscicida. To construct efficient gene editing platforms, we optimized CRISPR/Cas9 system in E. piscicida including the most efficient combination of vectors. The group comprised vectors containing Cas9, λ-Red system, single guide RNA scaffold and homologous arms. Furthermore, editing of several genes was performed based on the established system. Auxotrophic E. piscicida mutants by the knockout of alanine racemase (alr) and aspartate-semialdehyde dehydrogenase (asd) were generated respectively by partial removal of each gene. When targeting the alr gene, gene substitution was also carried out to change 3 bp. As a result, gene editing was successful and precise gene modification to remove only 1 bp was conducted well. In the present work, we conducted to edit not only one gene but also multi-genes. When two genes were targeted, although the editing efficiency was significantly reduced, we generated two-gene edited mutant at one trial, indicating that this system is much more convenient compared to the other gene editing technologies. In addition, in the present study, a foreign gene cassette expressing green fluorescence protein (GFP) was inserted into the alr gene of E. piscicida chromosome using the CRISPR/Cas9 system, and verified the expression of GFP in the alr gene knockout E. piscicida mutant. The auxotrophic mutant of E.piscicida produced by CRISPR/Cas9 system was used for live-attenuated vaccines. In serum agglutination test and vaccine efficacy test in goldfish and olive flounder, the live-attenuated vaccine showed higher protective efficacy and serum agglutination titer than a formalin-killed vaccine.