Effects of CRISPR/Cas9 targeting of the myo-inositol biosynthesis pathway on hyper-osmotic tolerance of tilapia cells.

ABSTRACT

Myo-inositol is an important compatible osmolyte in vertebrates. This osmolyte is produced by the myo-inositol biosynthesis (MIB) pathway composed of myo-inositol phosphate synthase and inositol monophosphatase. These enzymes are among the highest upregulated proteins in tissues and cell cultures from teleost fish exposed to hyperosmotic conditions indicating high importance of this pathway for tolerating this type of stress. CRISPR/Cas9 gene editing of tilapia cells produced knockout lines of MIB enzymes and control genes. Metabolic activity decreased significantly for MIB KO lines in hyperosmotic media. Trends of faster growth of the MIB knockout lines in isosmotic media and faster decline of MIB knockout lines in hyperosmotic media were also observed. These results indicate a decline in metabolic fitness but only moderate effects on cell survival when tilapia cells with disrupted MIB genes are exposed to hyperosmolality. Therefore MIB genes are required for full osmotolerance of tilapia cells.