Genome editing of candidate genes related to disease resistance to Piscirickettsia salmonis in Atlantic salmon (Salmo salar)


Salmon Rickettsial Syndrome (SRS), caused by the bacterium Piscirickettsia salmonis, is one of the most severe infectious diseases threatening the Chilean Atlantic salmon industry. Among the leading causes of mortality and morbidity, SRS significantly affect the seawater production stage, where biomass losses account for a major economic impact. One potential avenue to tackle SRS is the improvement of host resistance using selective breeding. To accomplish this, insight into the genetic basis of host response, identifying specific genes and pathways involved in this response, and comprehending the potential function these genes have in infection overcome, is valuable. Consequently, this study aims to identify functional genes and pathways that contribute to genetic host resistance to SRS and investigate the effect of CRISPR/Cas9 knockout on these genes during P.salmonis infection. Candidate genes were identified from a previous in vivo large-scale infection study of 2,265 Atlantic salmon smolts injected with P.salmonis and genotyped. These data were used to estimate SRS resistance breeding values. Head-kidney and liver samples for RNA-Seq were obtained from 48 individuals at pre-infection, 3 and 9 days post-infection, and tests of differential expression between pre- and post-infection, and between high and low resistance breeding values were performed. From the thousands of differentially expressed genes, enrichment of several KEGG pathways related to immune response such as bacterial internalisation, intracellular trafficking, apoptosis, and inflammasome was observed in both tissues in fish relatively more resistant to infection. A literature review of the biological function of genes in these pathways highlighted the most suitable candidates for functional studies. Subsequently, five genes related to SRS resistance were successfully edited using a CRISPR/Cas9 Ribonucleoprotein (RNP) transfection to knockout these genes in an Atlantic salmon cell line (SHK-1). An in vitro infection challenge model of the knockout and control cell lines with P.salmonis was performed to elucidate the impact on cytopathic damage, cell viability and bacterial load during infection. These findings suggest a promising avenue of research into the genetic architecture of host resistance to SRS

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