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Reverse genetics approaches are critical for uncovering complex biological processes and genetic engineering. Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) system (CRISPR/Cas9) has been widely used for generating mutants in a large number of species. The Nile tilapia (Oreochromis niloticus), a gonochoristic teleost with XX/XY sex determination system, is a good model for studying fish sex determination and reproduction. Here, we describe a detailed procedure for gene mutation in tilapia by CRISPR/Cas9. This protocol covers selection of target sites, in vitro RNA transcription, artificial insemination and microinjection of one cell stage embryos. We also provide details for detection of somatic and germline transmitted mutations, fast establishment of mutant lines and discuss some practical advices. This protocol will facilitate broader applications of CRISPR/Cas9 in studies of tilapia as well as other aquaculture fishes. [ABSTRACT FROM AUTHOR]

A novel insulin-like growth factor (igf3), which is exclusively expressed in the gonads, has been widely identified in fish species. Recent studies have indicated that Igf3 regulates spermatogonia proliferation and differentiation in zebrafish; however, detailed information on the role of this Igf needs further in vivo investigation. Here, using Nile tilapia (Oreochromis niloticus) as an animal model, we report that igf3 is required for spermatogenesis and reproduction. Knockout of igf3 by CRISPR/Cas9 severely inhibited spermatogonial proliferation and differentiation at 90 days after hatching, the time critical for meiosis initiation, and resulted in less spermatocytes in the mutants. Although spermatogenesis continued to occur later, more spermatocytes and less spermatids were observed in the igf3−/− testes when compared with wild type of testes at adults, indicating that Igf3 regulates spermatocyte to spermatid transition. Importantly, a significantly increased occurrence of apoptosis in spermatids was observed after loss of Igf3. Therefore, igf3−/− males were subfertile with drastically reduced semen volume and sperm count. Conversely, the overexpression of Igf3 in XY tilapia enhanced spermatogenesis leading to more spermatids and sperm count. Transcriptomic analysis revealed that the absence of Igf3 resulted in dysregulation of many genes involved in cell cycle, meiosis and pluripotency regulators that are critical for spermatogenesis. In addition, in vitro gonadal culture with 17α-methyltetosterone (MT) and 11-ketotestosterone (11-KT) administration and in vivo knockout of cyp11c1 demonstrated that igf3 expression is regulated by androgens, suggesting that Igf3 acts downstream of androgens in fish spermatogenesis. Notably, the igf3 knockout did not affect body growth, indicating that this Igf specifically functions in reproduction. Taken together, our data provide genetic evidence for fish igf3 in the regulation of reproductive capacity by controlling spermatogenesis. [ABSTRACT FROM AUTHOR]

TSP1 was reported to be involved in multiple biological processes including the activation of TGF-β signaling pathways and the regulation of angiogenesis during wound repair and tumor growth, while its role in ovarian folliculogenesis remains to be elucidated. In the present study, Tsp1a was found to be expressed in the oogonia and granulosa cells of phase I to phase IV follicles in the ovaries of Nile tilapia by immunofluorescence. tsp1a homozygous mutants were generated by CRISPR/Cas9. Mutation of tsp1a resulted in increased oogonia, reduced secondary growth follicles and delayed ovary development. Expression of the cell proliferation marker PCNA was significantly up-regulated in the oogonia of the mutant ovaries. Furthermore, transcriptomic analysis revealed that expressions of DNA replication related genes were significantly up-regulated, while cAMP and MAPK signaling pathway genes which inhibit cell proliferation and promote cell differentiation were significantly down-regulated. In addition, aromatase (Cyp19a1a) expression and serum 17β-estradiol (E2) concentration were significantly decreased in the mutants. These results indicated that lacking tsp1a resulted in increased proliferation and inhibited differentiation of oogonia, which in turn, resulted in increased oogonia, reduced secondary growth follicles and decreased E2. Taken together, our results indicated that tsp1a was essential for ovarian folliculogenesis in Nile tilapia. [ABSTRACT FROM AUTHOR]

Wilms tumor 1 (Wt1) is an essential factor for urogenital system development. Teleosts have two wt1s , named as wt1a and wt1b . In this study, the expression pattern of wt1a and wt1b and their functions on the urogenital system were analyzed by in situ hybridization and CRISPR/Cas9. wt1a was found to be expressed in the glomerulus at 3 dah (days after hatching), earlier than wt1b. wt1a and wt1b were simultaneously expressed in the somatic cells of gonads at 3 dah, while their cell locations were similar, but not identical in adult fish gonads. The wt1a -/- fish displayed pericardial edema and yolk sac edema at 3 dah and subsequently expanded as general body edema at 6 dah, failed to develop glomerulus and died during 6–10 dah, whereas the wt1b -/- fish were phenotypically normal. Immunohistochemical analyses revealed that the germ cell marker Vasa was expressed, while somatic cell genes Cyp19a1a, Amh, Gsdf and Dmrt1 were not expressed in the wt1a -/- gonads at 6 dah. The sex phenotypes of XX and XY in the wt1b -/- fish were not affected. Real-time PCR revealed that the ovarian cyp19a1a expression was up-regulated in XX wt1b -/- fish, compared with XX control at 90 dah. Serum estradiol-17β level was also up-regulated in XX wt1b -/- fish at 90 and 180 dah. The XY wt1b -/- fish had normal serum estradiol-17β and 11-ketotestosterone levels and remained fertile. These results suggest that Wt1a and Wt1b have different functions in the kidneys and gonads of tilapia. [ABSTRACT FROM AUTHOR]