UC Davis

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 are produced transiently by a plasmid vector with hygromycin resistance. Hygromycin selected Cas9-OmB1 cells transfected with this vector displayed high gene editing frequency(up to 81%). Substitution of the previously used U6 promoters into the new system yielded negligible mutation frequency, thereby identifying heterologous U6 promoters as the primary cause for lack of gene edits in initial attempts. This CRISPR/Cas9 system was then used to generate three knock-out (KO) clonal cell lines for MIPS, IMPA1.1, NFAT5 and a non-essential gene group as controls for any deleterious side effects of the gene editing and cell selection process. Measurement of metabolic activity and growth/survival of MIB KO and control cell lines exposed to both HO and basal iso-osmotic (IO) conditions were performed over a 288 hour period. The results show increased growth rates of both MIPS and IMPA1.1 KO lines over the control cells in basal IO conditions but decreased survival (although not significant) in HO conditions. HO challenge of the NFAT5 KO and control lines followed by qRT-PCR targeting MIB enzyme transcripts showed a notable reduction of HO induced transcriptional upregulation in both IMPA1.1 (37-49%) and MIPS (6-37%). In wild-type (WT) OmB cells, co-transfection with vectors expressing dominant negative (DN) and WT versions of tilapia NFAT5 and a GFP reporter vector driven by the IMPA1.1 promoter caused statistically significant changes of reporter activity. Reporter activity was increased 5.1 fold by WT NFAT5 in basal iso-osmotic (IO) conditions and reduced ~45% by DN NFAT5 in HO conditions. These results provide evidence for an interaction between this transcription factor and the IMPA1.1 promoter regulatory elements. In conclusion, a novel DNA vector-based CRISPR/Cas9 delivery platform can achieve high mutation rates when component expression is driven by tilapia endogenous promoters allowing proficient development of target gene KO cell lines. Inactivating the MIB pathway has moderate impact on survival of HO challenged OmB cells but has a growth promoting regulatory effect in IO conditions. We conclude that NFAT5 is an osmotic transcriptional regulator of the tilapia MIB pathway that is responsible for approximately 50% of IMPA1.1 and <37% of MIPS induction under the hyperosmotic stress conditions applied in this study.