Development of an Efficient Genome Editing Method in Salmonid Species

CRISPR/Cas9 system has been used widely in animals and plants for an efficient genome editing, but still due to the biology of salmonids, it has been proved difficult to implement in practice. To this purpose, in our lab we are addressing key issues regarding the implementation of this method in order to understand disease resistance. We analyze the rates of transfection, viability and cell growth in different cell lines, optimizing the delivery system of DNA transfection (calfectin and lipofectamin) using a novel plasmid containing both, Cas9 and sgRNA in fish cell lines (SK, CHSE-214 and SHK-1).

An eukaryotic expression plasmid encoding the protein spCas9 which included the sgRNA-GFP, was constructed. This was done by subcloning the U6 zebrafish promoter reported by (Shinya et al. 2003) into LCmCherryV2. Additionally, a stable fish line cell (CHSE-214) was genetically engineered to overexpress a citosolyc eGFP:Puromycin driven by the mammalian CMV promoter. In order to determine the efficiency of selection media and toxicity, fish cell culture was treated for two weeks with Puromicyn antibiotic in three different concentrations (0,2/0,5 and 1 mg/ml). The results show that the mammalian CMV promoter is one of the best candidates driving the gene expression in fish line cell (not toxic). Moreover, we concluded that the CSH-214 (Salmon embryo derived from a Chinook salmon) and lipofectamin presented the best transfection efficiency rates at 72 hours post transfection.

In summary, we demonstrate improved transfection of cell lines onto salmonid cell lines using nucleofection; we also propose a method for selection of transfected cells in selective medium containing puromicyn. This is the first approach for studies involving genome editing, which in turn may be a powerful biotechnology tool in the salmon industry.

Funding: FONDECYT POSTDOC N°3160370