Establishment Of TALENs And CRISPR/Cas9in Tilapia And Their Applications On Sex Determination And Differentiation

Clafrication the sex determination and differentiation of organisms with sexual reproducation is one of the most basic biological issues. Growth rates of many economic animals, such as Nile tilapia Oreochromis niloticus, showed significant sexual dimorphism, with the male grow faster than the female by50%. The molecular mechanism of tilapia sex determination and sex control has become the hot issue of aquaculture because of results from tilapia are useful for both basic research and aquaculture. With the development of genomic library and high-throughput DNA sequencing technology, the genome sequence of tilapia are available. The main challenge is to interpret the function of these sequences. For a long time, the lack of effective methodology of gene knockout seriously hampered the studies of tilapia sex determination. Recently, genome editing nucleases (TALEN) and the CRISPR/Cas9system have provided two powerful approaches to explore any gene related to sex determination. In the present study, TALEN and CRISPR/Cas9were established in tilapia and subsequently dmrtl, foxl2, the newly discovered igf3and nanos were mutated these technologies so as to elucidate the function of these genes in tilapia sex determination and differentiation. The main results are listed as follows:1) A high-quality Fosmid library of the XY Nile tilapia was successfully constructed, microarrayed and characterized using the vector pCC2FOS. It encompasses115200non-redundant clones with an average insert size of40kb and3-fold genome coverage. All clones were arrayed in300plates of384-well plate. For each plate, column-, row-and plate-pools were constructed, and therefore, totally4800column pools,7200row pools and300plate pools were constructed and arrayed. Finally,25super pools were made with each covering twelve384-well plates. Stability of the library was tested by100generation passage experiment. No loss or rearrangement of the inserted DNA fragment was found during the continuous passage. Any targeted gene can be fast screened only by minimally77colony PCR due to the array of super-pools, plate-pools, row-and column-pools. Twenty genes related to sex differentiation and growth were successfully isolated from this library, with each having2-5positive clones. The Fosmid library will be helpful to isolate sex linked marker, sex determining gene, genome editing and genetic and breeding.2) Transcription activator-like effector nucleases (TALENs) have been proved to be effective in several organisms genome editing. In this study, we reported that TALENs can induce somatic mutations in Nile tilapia, an important species for worldwide aquaculture, with reliably high efficiency. Two pairs of TALENs were constructed to target genes related to sex differentiation, including dmrtl,foxl2, and both resulted in indel mutations with maximum efficiencies of up to81%at the targeted loci. Effects of dmrtl and foxl2mutation on gonadal phenotype, sex differentiation and related gene expression were analyzed by histology, immunohistochemistry and real-time PCR. In Dmrtl deficiency testes, phenotypes of significant testicular regression, including deformed efferent duct, degenerated spermatogonia or even a complete loss of germ cells, and proliferated steroidogenic cells, were observed. In addition, disruption of Dmrtl in XY fish resulted in increased foxl2, cyp19ala expression, serum estradiol-17β and11-ketotestosterone levels. On the contrary, deficiency of Foxl2in XX fish exhibited varying degrees of oocyte degeneration, significantly decreased aromatase gene expression and serum estradiol-17β levels. Some Foxl2deficiency fish even exhibited complete sex reversal with high expression level of Dmrtl and Cyp11b2. Taken together, our data demonstrated that TALENs are an effective tool for targeted gene editing in tilapia genome. Foxl2and Dmrtl play antagonistic roles in sex differentiation in Nile tilapia via regulating cyp19a1a expression and estrogen production.3) CRISPR/Cas9system has recently become powerful tool for targeted genome editing in model animals. Here, we report that disruption of selected genes, including nanos2, nanos3, dmrtl and foxl2, in tilapia through CRISPR/Cas9system was achieved with high efficiency (up to95%). Furthermore, obvious phenotypes were observed in GO generation after mutation of germ cell or somatic cell specific genes. For example, loss of Nanos2and Nanos3in XY and XX fish resulted in germ cell-deficient gonads as demonstrated by GFP labeling and Vasa staining, respectively, and masculinization of somatic cells in both XY and XX gonads as demonstrated by Dmrtl and Cyp11b2immunohistochemistry and by up-regulation of serum androgen levels. In addition, mutations in dmrtl and foxl2induced by CRISPR/Cas9were efficiently transmitted through the germline to F1. Mutations were found to be transmitted to their F1progeny with a rate of22.2%(4of18, dmrtl) and58.3%(10of24, foxl2), respectively. The F1foxl2larvae carried deletion mutations including in-frame and frame-shift deletions as their GO founders. In contrast, the F1dmrtl larvae only carried3or21bp in-frame deletions, the same as found in the sperms used for fertilization but different from the GO founders which carried both in-frame and frame-shift deletions. Therefore, frame-shift deletions in Dmrtl in tilapia germ cells probably affect their development, meiosis, and maturation in tilapia. Taken together, our data demonstrate that targeted, heritable gene editing can be achieved in tilapia, providing an effective approach for generating loss-of-function mutants.4) Previous studies from our lab have reported that a novel igf3, which is distinct from the conventional igfl and igf2, is only expressed in fish gonads, thus implying its special roles in gonad sex differentiation. In this study, igf3mRNA expression in the tilapia ovary was found to be higher than in the testis from5to40days after hatching (dah), but was lower than the testis from50to70dah. Consistently, Igf3protein signal was detected in the somatic cells of the XX and XY gonads from10dah till adulthood by immunohistochemistry using a specific Igf3polyclonal antibody. Incubation of ovarian and testicular cells in primary culture with recombinant Igf3significantly increased nr5a1,foxl2, dmrtl, cyp19a1a, cyp11a1, cyp11b2, and hsd3b2and cyp17a1expression in time-and dose-dependent manners. Igf3was successfully mutated by CRISPR/Cas9in XX and XY tilapia with high efficiency reach at95%. Histology examination showed Igf3deficiency testis have almost no spermatocyte and sperm, but with spermatogonia, and weak Cyp11b2expression detected. Spermatogonia, spermatocyte and sperm existed in the3month control XY testis, which expressed strong Cyp11b2. Real-time PCR results showed that expression level of dmrtl, nr5al and cypllb2were decreased in Igf3deficiency testis, compared with the control. Consistent with these results, knockout of Igf3in the XY fish resulted in decreased level of serum11-KT compared with the control fish. On the other hand, a large number of oogonia, little phage I and II oocyte was observed in the3months Igf3deficiency ovary, which expressed low Cypl9ala. In control XX ovary, little oogonia, different stage of I, II, III oocyte was observed, and Cypl9ala was highly expressed in the interstital cells of3months control ovary. Real-time PCR results showed that expression level of foxl2, nr5a1and cypl9ala were down regulated after loss of Igf3, compared with the control. Consistent with these results, knockout of Igf3in the XX fish resulted in decreased level of E2compared with the control fish. These results implied the gonad specific Igf3take part in teleost meiosis through regulating transcription factors and steroids. On the other hand, the igf3promoter sequence was obtained from Fosmid library. Promoter analysis using luciferase assays in HEK293cells revealed that igf3promoter activity was directly activated by Nr5a1(Sf1), and further enhanced by Foxl2, NrObla (Dax1) and NrOblb (Dax2) but repressed by Dmrtl and estrogen receptor along with17P-estradiol treatment. In addition, igf3promoter activity was increased slightly by Forskolin treatment alone, but synergistically up-regulated by transfection with nr5a1. Consistent with the results from promoter analysis, igf3expression was up-regulated after Dmrtl and Foxl2deficiency. These in vitro results correlated well with the expression profile of igf3during early gonad differentiation. Our results indicated that igf3is involved in fish gonad steroidogenesis because of its ability to regulate the expression of foxl2, dmrtl, nr5a1and steroidogenic enzymes. The expression of igf3is in turn regulated by the transcription factors Foxl2, Dmrtl and Nr5al as well as by17β-estradiol treatment.In summary, microarrayed Fosmid library, TALEN and CRISPR/Cas9were successfully established in a worldwide cultured fish, Nile tilapia. The function of Dmrtl, Foxl2, Nanos2, Nanos3and Igf3in teleost sex determination and differentiation was studied by TALEN and CRISPR/Cas9, which showing the advantages of these two technologies to elucidate the molecular mechanisms of teleost sex determination and differentiation. The establishment of these platforms promotes the studies of functional genomics research and sex control in aquaculture.