Research towards the effective disruption of reproductive competence in Nile tilapia Oreochromis niloticus

Abstract

Reproductive containment in farmed fish is highly desired for sustainable aquaculture to prevent genetic introgression with wild conspecifics and enhance productivity by suppressing sexual maturation. A number of strategies have already been implemented or have been tested in commercially important fish (e.g. triploidy, monosexing, hormonal therapies); however, they either do not result in 100% containment, or they cannot be applied to all species. One promising new approach consists in disrupting primordial germ cells (PGCs), at the origin of germline cells, to induce sterility. The work carried out in this doctoral thesis aimed to investigate the genes involved in the survival of germ cells and subsequently conduct a functional analysis of candidate genes using CRISPR/Cas9 gene editing system to ultimately provide the basis for the development of a novel sterilisation technique. Nile tilapia was chosen as the experimental animal as it is a major aquaculture species worldwide and the control of reproduction plays a critical role in the farming productivity in this species. In addition, the species has clear advantages as its whole genome sequence is accessible, the generation time is relatively short and zygotes can be available all year round. Initially, a panel of 11 candidate genes with reported roles in survival of PGCs was investigated during the ontogenic development which led to the selection of piwi-like (piwil) gene as a target for genome editing. Then, high temperature was tested as a means to induce germ cell loss to better understand the mechanism underlying germ cell survival and apoptosis, and this study confirmed the functional importance of piwil genes in relation to germ cell loss and proliferation. In addition, the study suggested potential subfunctionalisation within the Bcl-2 gene family which requires further investigation. The next step aimed to optimise the CRISPR/Cas9 gene editing method by improving the microinjection system and testing different concentrations of sgRNAs. Over 95% of injected embryos showed on-target mutation in piwil2 via zygote injection of CRISPR/Cas9 reagents and complete KO larvae were shown in half of the mutants, producing putative sterile fish. However, there was no clear association between the phenotypes in PGCs and the mutation rate. Further comparative studies of mutant screening methods including T7E1, RGEN, HRMA, fragment analysis and NGS revealed that the genotypes of F0 are highly mosaic, suggesting that deep sequencing is recommended for accurate and high throughput F0 screening and further improvement for predictable genome editing is required for a reliable gene functional analysis in F0. In summary, the current thesis provided new scientific knowledge and supporting evidence for the use of the CRISPR/Cas9 gene editing platform to study gene function associated with sterility, with the ultimate goal to develop an alternative sterilisation method in fish.