The Myo-inositol Biosynthesis Pathway and its Regulation by NFAT5 in Tilapia Salinity Tolerance Assessed by Genetic Manipulation of Cultured Cells

The constantly changing physical world subjects its inhabitants to variable abiotic conditions. Organisms function optimally within a range of abiotic parameters. Extreme divergence form that range due to conditions such as salinity fluctuations in some aquatic environments, imposes strain on their physiological systems such as hyper-osmotic (HO) stress. Persistence in such an environment is restricted to species capable of the necessary adaptive responses. The economically important Mozambique tilapia (Oreochromis mossambicus) is such a species, making it an ideal model for studying salinity stress tolerance. Previous work showed increased abundances of the myo-inositol biosynthesis (MIB) pathway enzymes myo-inositol phosphate synthase (MIPS) and inositol monophosphatase 1 (IMPA1.1) in tissues of O. mossambicus subjected to elevated salinity. Previous work also suggested an involvement of the transcription factor NFAT5 (nuclear factor of activated T-cells 5) in the osmotic regulation of the MIB pathway. However, the causality of these relationships with salinity tolerance phenotypes had yet to be established. The objective of this research was to test the overall hypothesis that upregulation of the MIB pathway enzymes contributes to salinity tolerance and is mediated by NFAT5 using CRISPR/Cas9 gene editing and synthetic biology approaches in a simplified but representative tilapia cell line model (OmB) to manipulate the genetic loci encoding these proteins. Initial CRISPR/Cas9 editing attempts utilizing gRNA/Cas9 protein complexes and expression vectors optimized for zebrafish or mammalian cells failed to yield detectable gene edits. Therefore, a system customized for tilapia cells was developed using endogenous O. mossambicus EF1 alpha and U6 promoters to drive Cas9 and gRNA expression respectively. A version of the OmB cell line (Cas9-OmB1) was engineered to express the Cas9 protein by transposon mediated genomic integration of the Cas9 gene while different gRNAs