Abstract

Piscirickettsia salmonis is a gram-negative intracelular bacterium (non-motile), that severely threatens the sustainability of the salmon industry in Chile. This bacterium is known to be the etiological agent of Salmonid Rickettsial Septicaemia (SRS) or Piscirickettsiosis, a disease that produces a systemic aggressive infection that involves several organs and tissues including kidney, spleen, liver, intestine, brain, skin, ovary and gills. Despite the high impact that P. salmonis provoke on the farm industry, crucial aspects of its biology, pathogenesis and virulence are completely unknown. In this context, using deep sequencing of the transcriptome, our laboratory has demonstrated that certain immune related genes present a strong correlation in resistant families of Salmo salar to P. salmonis infection. Moreover, the results showed that the bacterial load was significantly lower in this family. The present study aimed to understand the biological function which are underlying the resistant family. To this purpose a targeted mutagenesis in Atlantic salmon using the CRISPR/Cas9 system will be conducted. This system has been demostrated to be and efficient and reliable genome editing technique in vertebrates. In order to cloning the target sites for gRNAs numerous over-expressed genes were selected and evaluated an online tool which excluded unwanted off-targeted sequences (chop chop Harvard tool). In this context, a set of candidates genes and suitable oligonucleotides were selected and listed. The present work suggests the functional study by double-allelic mutations including the fusion of lysosomes with phagasomes, actin regulators and cytolytic proteins genes. Additionally, we present the preliminary studies of cell culture.