Studies of gene function in non-model animals have been limited by the approaches available for eliminating gene function. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated) system has recently become a powerful tool for targeted genome editing. Here, we report the use of the CRISPR/Cas9 system to disrupt selected genes, including nanos2, nanos3, dmrt1, and foxl2, with efficiencies as high as 95%. In addition, mutations in dmrt1 and foxl2 induced by CRISPR/Cas9 were efficiently transmitted through the germline to F 1 . Obvious phenotypes were observed in the G0 generation after mutation of germ cell or somatic cell-specific genes. For example, loss of Nanos2 and Nanos3 in XY and XX fish resulted in germ cell-deficient gonads as demonstrated by GFP labeling and Vasa staining, respectively, while masculinization of somatic cells in both XY and XX gonads was demonstrated by Dmrt1 and Cyp11b2 immunohistochemistry and by up-regulation of serum androgen levels. Our data demonstrate that targeted, heritable gene editing can be achieved in tilapia, providing a convenient and effective approach for generating loss-of-function mutants. Furthermore, our study shows the utility of the CRISPR/Cas9 system for genetic engineering in non-model species like tilapia and potentially in many other teleost species. R ECENTLY, a simple and efficient genome editing technology, type II CRISPR/Cas9, has been developed based on the Streptococcus pyogenes clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas9) adaptive immune system. It requires three components for effective DNA cleavage: the nuclease Cas9, a targeting CRISPR RNA (crRNA), and an additional transactivating crRNA (tracrRNA) (Gasiunas et al. 2012;Jinek et al. 2012;Cho et al. 2013;Cong et al. 2013;Hwang et al. 2013;Mali et al. 2013). Further improvement of the system was achieved by fusing the crRNA and tracrRNA to form a single guide RNA (gRNA) that is sufficient to direct Cas9-mediated target cleavage (Hwang et al. 2013). Importantly, previous studies performed in vitro (Jinek et al. 2012), in bacteria , and in human cells (Cong et al. 2013) have shown that Cas9-mediated cleavage can be abolished by single mismatch at the gRNA-target site interface, particularly in the last 10-12 nucleotides located in the 39 end of the 20-nt gRNA targeting region. Compared to the other two engineered nuclease genome-editing technologies, zinc-finger nucleases (ZFNs) (Urnov et al. 2005;Doyon et al. 2008) and transcription activator-like effector nucleases (TALENs) (Huang et al. 2011;Sander et al. 2011;Tesson et al. 2011), the CRISPR/Cas9 system is substantially less expensive and much easier to program for editing new target sites. This new approach has been widely used for genome engineering in model animals, including Caenorhabditis elegans (Dickinson et al. 2013;Friedland et al. 2013;Tzur et al. 2013), Drosophila (Bassett et al. 2013;Ren et al. 2013;Yu et al. 2013), zebrafish (Chang et al. 2013Hrusc...

Gonadal soma-derived factor (gsdf) is critical for testicular differentiation in teleosts, yet detailed analysis of Gsdf on testicular differentiation is lacking. In the present study, we knocked out tilapia gsdf using CRISPR/Cas9. F0 gsdf-deficient XY fish with high mutation rate (≥58%) developed as intersex, with ovotestes 90 days after hatching (dah), and become completely sex-reversed with ovaries at 180 and 240 dah. Those individuals with a low mutation rate (<58%) and XY gsdf(+/-) fish developed as males with normal testes. In F2 XY gsdf(-/-) fish, the gonads first expressed Dmrt1, which initiated the male pathway at 10 dah, then both male and female pathways were activated, as reflected by the simultaneous expression of Dmrt1 and Cyp19a1a in different cell populations at 18 dah, shifted to the female pathway expressing only Cyp19a1a at 36 dah, and finally developed into functional ovaries as adults. The male pathway and Dmrt1 expression was initiated, but failed to be maintained, in the absence of Gsdf. Aromatase-inhibitor treatment from 10 to 35 dah, however, rescued the phenotype, resulting in XY gsdf(-/-) with normal testes that expressed Dmrt1 and Cyp11b2. In vitro promoter analyses demonstrated that Dmrt1 activated gsdf expression in a dose-dependent manner in the presence of Sf1, even though Dmrt1 alone could not. Taken together, our results demonstrated that gsdf is a downstream gene of dmrt1. Gsdf probably inhibits estrogen production to trigger testicular differentiation. Mol. Reprod. Dev. 83: 497-508, 2016. © 2016 Wiley Periodicals, Inc.

Materials and reagents. A Sylgard 184 poly(dimethylsiloxane) (PDMS) kit was purchased from Dow Corning Co. (Midland, MI, USA). Glass plates coated with chromium and photoresist for chip fabrication were obtained from Shaoguang Microelectronics Corp. (Hunan, China). All the other chemicals were of analytical grade and used without further purification. Milli-Q grade water (Millipore Inc., Bedford, MA, USA) was used for preparing all solutions and cleaning microchannels. 10 mM phosphate-buffered saline (PBS, pH 7.4) containing 137 mmol/L NaCl, 2.7 mmol/L KCl, 8.72 mmol/L Na 2 HPO 4 , and 1.41 mmol/L KH 2 PO 4 . Glass capillary (1.0 mm id, 10 cm length) was purchased from Sichuan University Inc. (Chengdu, China). Free hemoglobin test kit was acquired from Jiancheng Bioengineering Institute (Nanjing, China). Silicone tube was from Nuoyawei Inc. (Shenzhen, China). Theophylline was from National Institutes for Food and Drug Control (Beijing, China). Other chemicals were of analytical grade and used without purification.

It is well accepted that Forkhead box protein L2 (Foxl2) and aromatase (Cyp19a1; the enzyme responsible for estrogen synthesis) are critical for ovarian development in vertebrates. Knockouts of Foxl2 and Cyp19a1 in goat, mouse, and zebrafish have revealed similar but not identical functions across species. Functional analyses of these two genes in other animals are needed to elucidate their conserved roles in vertebrate sexual development. In this study, we established foxl2 and cyp19a1a mutant lines in Nile tilapia. Both foxl2-/- and cyp19a1a-/- XX fish displayed female-to-male sex reversal. Sf1, Dmrt1, and Gsdf were upregulated in the foxl2-/- and the cyp19a1a-/- XX gonads. Downregulation of Cyp19a1a and serum estradiol-17β level, and upregulation of Cyp11b2 and serum 11-ketotestosterone level were observed in foxl2-/- XX fish. The mutant phenotype of foxl2-/- XX individuals could be rescued by 17β-estradiol treatment from 5 to 30 days after hatching (dah). Upregulation of Star1, the enzyme involved in androgen production in tilapia, was also observed in the foxl2-/- XX gonad at 30 and 90 dah. In vitro promoter analyses consistently demonstrated that Foxl2 could suppress the transcription of star1 in a dose-dependent manner. In addition, compared with the control XX gonad, fewer germ cells were detected in the foxl2-/- XX, cyp19a1a-/- XX, and control XY gonads 10 dah. These results demonstrate that Foxl2 promotes ovarian development by upregulating Cyp19a1a expression and repressing male pathway gene expression. These results extend the study of Foxl2 and Cyp19a1a loss of function to a commercially important fish species.

Multidrug resistance (MDR) has become a major impediment to a successful treatment for liver cancer patients, and one of the common reasons for MDR is the activation of ABCB1 gene, leading to the over-expression of P-glycoprotein (P-gp), which conferred cancer cells be resistant to a broad range of anticancer drugs. MicroRNAs (miRNAs) are a class of short, non-coding RNA moleculars that can regulate gene expression at the post-transcriptional level. In the current study, the aim is to explore whether miRNA participates in the regulation of MDR mediated by ABCB1. We found that the expression of ABCB1 was correlated with the doxorubicin IC50 dose in eight hepatocellular carcinoma (HCC) cell lines: Hep3B, HCC3, LM-6, SMMC7721, Huh-7, SK-Hep-1, HepG2 and BEL-7402. Using the bioinformatics, we discovered that there were several miRNAs that can bind to the 3'UTR of ABCB1 gene. Among these candidate miRNAs, miR-223 was chosen for further study. Then, EGFP reporter assay, real-time PCR and Western blot were performed to verify that miR-223 targeted ABCB1 3'UTR directly, and miR-223 downregulated ABCB1 at both mRNA and protein levels. Finally, we found that the over-expression of miR-223 increased the HCC cell sensitivity to anticancer drugs, and the inhibition of miR-223 had the opposite effect. Importantly, the over-expression or silencing of ABCB1 can rescue the cell response to the anticancer drugs mediated by miR-223 over-expression or inhibition, respectively. In conclusion, our findings indicated that miR-223 played an important role in the regulation of MDR mediated by ABCB1, and it suggests that miR-223 may be considered as a therapeutic biomarker for HCC patients who had MDR problems induced by high expression of ABCB1.

The dmrt6 gene has been isolated from tetrapods and recently from a coelacanth, Latimeria chalumnae. Its evolutionary history and exact function remain unclear. In the present study, dmrt6 was isolated from Perciformes (five cichlids and stickleback), Siluriformes (southern catfish), and Lepisosteiformes (spotted gar). Syntenic and phylogenetic analyses indicated that dmrt6 experienced gene transposition after the divergence of teleosts from other bony fish as gene loci surrounding dmrt6 were conserved among teleosts (but was completely different from gene loci surrounding dmrt6 in tetrapods and spotted gar), while these gene loci were conserved among nonteleost species. Real-time PCR and in situ hybridization revealed that dmrt6 was highly expressed in the XY gonads from 90 days after hatching (dah) onward and was observed exclusively in spermatocytes of the testes in tilapia. Dmrt6 knockout by CRISPR/Cas9 resulted in fewer spermatocytes, down-regulated Cyp11b2 in testes, and consequently produced a lower level of serum 11-ketotestosterone (11-KT) in Dmrt6-deficient XY fish compared with the XY control at 120 dah. From 150 to 180 dah, spermatogenesis gradually recovered, and cyp11b2 expression and serum 11-KT level were restored to the same levels as those of the XY control fish. In addition, a Dmrt6 mutation was observed in genomic DNA of sperm of G0 mutant fish and F1 fish. Taken together, our data suggest that dmrt6 also exists in bony fish. Its absence in most fish genomes was probably due to incomplete sequencing and/or secondary loss. The dmrt6 gene is highly expressed in spermatocytes and is involved in spermatogenesis in tilapia.

The mitotic kinesin superfamily protein KIF14 is essential for cytokinesis and chromosome segregation and increased KIF14 expression is related to a variety of human cancers. In this study, we investigate KIF14 expression in association with clinical variables and the role of KIF14 during tumorigenesis. We found that KIF14 is overexpressed in most primary hepatocellular carcinoma (HCC) tissues compared with the adjacent normal liver tissues and KIF14 overexpression is associated with tumor grade (P = 0.002), stage (P = 0.013) and poor survival (P < 0.001). Downregulation of KIF14 decreased the capacity of proliferation both in vitro and in vivo. Furthermore, suppression of KIF14 not only decreases cancer cell migration but also induces apoptosis of cells with inactivation of the phosphatidylinositol 3-kinase-Akt signaling pathway. Therefore, our current study indicates that KIF14 promotes HCC carcinogenesis and may serve as a potential therapeutic target for human HCC.

We report an approach for fabricating a tunable wettability surface by electroless gold plating on poly(dimethylsiloxane) (PDMS). A two-layer structured gold film with a tight layer and a loose layer can be obtained on the surface of a PDMS chip when the PDMS chip is immersed in a gold plating solution at 30 degrees C for 4 h. Its wettability can be rapidly switched between superhydrophilicity and superhydrophobicity by plasma and heat treatments without any self-assembled monolayer, and the superhydrophobicity can be even changed from the gecko-foot-hair-like character to the lotus-leaf-like character. Benefiting from the various wettabilities of the prepared gold/PDMS composites, protein patterning is successfully achieved on a patterned superhydrophobic/superhydrophilic gold/PDMS composite; a superhydrophobic needle for transferring supersmall water droplets (1 microL) to a superhydrophobic surface is successfully fabricated.