PARTIAL INACTIVATION OF MATERNAL GENE (PIMG) TECHNOLOGY FOR THE GENETIC CONTAINMENT OF GE FISH

The major goalof thisproject is to engineer commercial farmfish to be sterile.Objective 1. Identify 3'UTR motifs essential for maternal delivery of mRNA into the germ plasm/PGCs.In this objective we will use reporter mRNA injection experiments to identify 3'UTR motifs necessary to mediate PGC specific expression. Working in iterative cycles, we will design, synthesize, and test the effect of disrupting either one or a combination of conserved 3'UTR cis-acting-regulatory motifs, to identify those required and sufficient to mediate PGC specific expression. These efforts will yield information on 3'UTR regulatory networks controlled by miRs and RNA Binding Proteins that modulate mRNA stability and will deliver insights into the mechanisms that are functionally important during PGC development.Objective 2. Generation of F0 founder carrying 'de novo' alleles for 3 germ line genes.In objective 2, we will perform homology-directed repair (HDR) facilitated by phenotypic selectionto cut out the WT (wild-type)-3'UTR sequences and paste the new 3'UTR alleles with altered cis-regulatory elements selected as part of objective1. Three DNA variant with 3'UTR mutations flanked by CRISPR target sites will be generated synthetically and cloned into a donor plasmid. One-cell stage tilapia embryos will be injected with a solution containing Cas9mRNA, sgRNAs with the donor plasmid DNA. To test for efficient HDR, fin DNA samples from selected fish at 2-3 months of age will be extracted and analyzed by junction fragment PCR. Based on previous work at CAT, we expect that injection of 600 embryos will produce new mutant lines with defective PGC development pathway. Batches of these F0 treated embryos will be raised for further analysis. We expect mosaic mutant females to produce embryos, some of which with reduced PGC counts.Objective 3. Establish Tilapia lines, characterize their phenotypes, and demonstrate maternal effect PGC ablation. In objective 3 we will first outcross F0 mutated males with WT females carrying the GFP transgene driving a PGC expression patternto establish lines. F1 progeny will then be screened to locate individuals carrying the precise allelic replacement event. Finally, F1 progeny carrying the same 3'UTR mutation will be incrossed to produce F2 progeny. This F2 progeny will be raised to adulthood, sexed, and genotyped to identify homozygous mutant. Homozygous mutant females carrying the GFP transgene should produce PGC depleted progeny that will grow into sterile or sub-fertile adult. Sterility will be studied at the morphological cellular and molecular level in 3 and 5-month-old F3 progenies. In contrast to females, homozygous mutant males should produce progeny with a normal PGC count.