Genome editing in fishes and their applications

Accepted Manuscript Genome Editing in Fishes and Their Applications Bo ZHU, Wei GE PII: DOI: Reference:

S0016-6480(17)30644-5 http://dx.doi.org/10.1016/j.ygcen.2017.09.011 YGCEN 12755

To appear in:

General and Comparative Endocrinology

Received Date: Revised Date: Accepted Date:

15 January 2017 15 August 2017 13 September 2017

Please cite this article as: ZHU, B., GE, W., Genome Editing in Fishes and Their Applications, General and Comparative Endocrinology (2017), doi: http://dx.doi.org/10.1016/j.ygcen.2017.09.011

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Genome Editing in Fishes and Their Applications

Bo ZHU and Wei GE

Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Taipa, Macau, China

KEY WORDS: genome editing; ZFN; TALEN; CRISPR/Cas9; fish; teleosts RUNNING TITLE: Genome editing in fishes

*Correspondence: Wei Ge, Faculty of Health Sciences, The University of Macau, Taipa, Macau, China. Tel: +853-8822-4996; Email: [email protected];

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Abstract There have been revolutionary progresses in genome engineering in the past few years. The newly-emerged genome editing technologies including zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats associated with Cas9 (CRISPR/Cas9) have enabled biological scientists to perform efficient and precise targeted genome editing in different species. Fish represent the largest group of vertebrates with many species having values for both scientific research and aquaculture industry. Genome editing technologies have found extensive applications in different fish species for basic functional studies as well as applied research in such fields as disease modeling and aquaculture. This mini-review focuses on recent advancements and applications of the new generation of genome editing technologies in fish species, with particular emphasis on their applications in understanding reproductive functions because the reproductive axis has been most systematically and best studied among others and its function has been difficult to address with reverse genetic approach. Introduction Genome editing is a powerful tool for reverse genetics study of gene functions. The first generation of genome editing method or gene targeting/knockout was established three decades ago in the mouse model (Doetschman et al., 1987; Thomas and Capecchi, 1987; Thompson et al., 1989). This technology, however, has not been applicable to non-rodent models including fish species for functional studies, in particular for gene functions in adults. The most commonly used reverse genetics approach in fish, especially the model species such as the zebrafish (Bill et al., 2009) and medaka (Aller et al., 2013), is gene knockdown by antisense morpholino oligomers (MOs) (Bedell et al., 2011). Despite its convenience and efficiency, this approach can only be used to study gene functions in the first 5 days of development, and the phenotypes induced by MOs are not heritable (Eisen and Smith, 2008). Another similar reverse genetics approach is RNAi-induced gene silencing; however, this technology has not been well established in fish species despite a few successful reports (De Rienzo et al., 2012; Shinya et al., 2013; Zhao et al., 2001). Although gene knockdown has been the dominant reverse genetic approach in model fish species, it often suffers the problems of low specificity and high toxicity. TILLING (Targeting Induced Local Lesions IN Genomes) is also a reverse genetics approach that identifies individuals carrying mutations in genes of interest from populations treated with mutagens (Till et al.,

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2007). Since TILLING involves screening large mutant libraries with DNA sequencing, it is often beyond the capacity of most laboratories (Huang et al., 2012). In recent years, we have witnessed revolutionary progresses in the field of reverse genetics with the development of novel genome editing technologies, including ZFN, TALEN, and CRISPR/Cas9, which all adopt an engineered sequence-specific nuclease for DNA cleavage. These newly emerged genome editing technologies for the first time allow for gene targeting or editing in a wide range of species including fishes with great efficiency (Gaj et al., 2013). So far, more than 40 fish species have had their genomes sequenced, including model species such as the zebrafish (Howe et al., 2013) and medaka (Takeda, 2008). These sequenced genomes and the availability of genome editing technologies are now providing tremendous potential for functional studies of genes in a variety of fish species for both science and application. The knockout approach also allows scientists to revisit many of the genes that have been studied with MO-based gene knockdown. An increasing number of studies has shown discrepancy between MO-induced knockdown and nuclease-induced knockout (Lebedeva et al., 2017; Novodvorsky et al., 2015), further illustrating the power of gene knockout. In this mini-review, we will summarize recent advances and applications of ZFN, TALEN, and CRISPR/Cas9 in fish species, with particular emphasis on their application for studying gene functions in reproduction because the reproductive axis is so far the best studied system with genome editing. ZFN, TALEN and CRISPR/Cas9 First established in 1996 (Kim et al., 1996), ZFN (Zinc finger nuclease) is an artificial restriction enzyme consisting of a zinc finger DNA-binding domain and a FokI DNA cleavage nuclease (Urnov et al., 2010). Each ZFN is typically comprised of three to six zinc finger motifs, and each motif specifically recognizes three nucleotides in DNA; therefore, each ZFN is able to recognize or target 9 to 18 base pairs (Carlson et al., 2012) . The cleavage of target DNA requires dimerization of two ZFNs for the FokI enzyme to cleave the DNA sequence, resulting in double strand break (DSB) at the target locus (Durai et al., 2005). Theoretically, ZFN is an ideal tool for inducing mutations at target DNA sites in any organisms (Gupta and Musunuru, 2014). However, its application has been constrained by limitations in zinc finger domain design and construction as well as low efficiency (Urnov et al., 2010). The recently-emerged TALEN provides us a more advanced approach for genome editing. Similar to ZFN, the cleavage of target DNA sequence requires dimerized TALENs. 3

Each TALE protein is comprised of a tandem array of highly conserved 33 to 35 amino acid repeat modules with the 12th and 13th amino acids being variable, named Repeat Variable Diresidue (RVD). Different RVD allows each module to specifically recognize one individual nucleotide instead of three nucleotides as in ZFN (Moscou and Bogdanove, 2009). The dimerized FokI randomly cleaves the DNA sequence between the left and right TALEN target sites (Christian et al., 2010). Compared with ZFN, it is much easier to construct plasmids for expressing TALE proteins, making this technology easily available to most molecular biology laboratories. Because of this and its high specificity and efficiency, TALEN has quickly replaced ZFN as a dominant platform for genome editing since its establishment in 2011 (Miller et al., 2011). While TALEN was gaining popularity in various organisms in early 2010s, another powerful genome editing technology, CRISPR/Cas9, emerged, and it has been sweeping across life sciences since 2013 due to its superior advantages (Cong et al., 2013). Compared with TALEN, the assembly of the components for performing CRISPR/Cas9 is even more cost effective, convenient and efficient, which allows for large-scale and high-throughput genome modification in many species. In particular, the CRIRSP/Cas9 system allows for simultaneous multiplex targeting (Cong et al., 2013). Unlike ZFN and TALEN, the nuclease Cas9 is guided towards the target DNA site by a small guide RNA followed by random cleavage of the DNA. Similar to ZFN and TALEN, Cas9 cleavage also generates DSB that triggers the DNA repairing system including non-homologous end joining (NHEJ) and homology-induced DNA repair (HDR) (Durai et al., 2005). The NHEJ often induces insertion or deletion (indel) mutations at the target site, leading to the loss of the functional gene, whereas the HDR is used for precise targeted editing or knock-in of an exogenous DNA fragment (Gratz et al., 2014). One advantage of CRISPR/Cas9 method is its high efficiency, which allows for biallelic disruption of alleles (Jao et al., 2013). This is particularly useful for non-model species with long generation time because mutant phenotypes can be seen immediately in the founder generation. The major drawback of the CRISPR/Cas9 system is its potential off-targeting effects (Cho et al., 2014; Schaefer et al., 2017). Because of this, TALEN will continue to be an alternative choice for genome editing. Genome editing in fish species In recent years, fish species, especially the model species such as the zebrafish, have played important roles in testing new protocols of genome editing because of the biological

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advantages of fish models. A large number of genes have been disrupted or modified in fish species for functional studies, especially those involved in reproduction. Functional analysis of genes in fish hypothalamic-pituitary-gonadal axis Compared with other organ systems, the reproductive axis is so far the major function in adults that has been systematically studied with genome editing technologies, and most studies have been performed in the zebrafish (Liu and Lin, 2017), medaka (Luo et al., 2015a; Nishimura et al., 2015) and tilapia (Li et al., 2014; Li and Wang, 2017). Gene targeting in the hypothalamus Hypothalamic-pituitary-gonadal (HPG) axis is the neuroendocrine and endocrine system that controls reproduction (Sower et al., 2009). The hypothalamus is the highest commander in the HPG axis, and it controls the biosynthesis and secretion of pituitary gonadotropins, namely follicle-stimulating hormone (FSH) and luteinizing hormone (LH), by releasing stimulatory and inhibitory neurohormones, in particular gonadotropin-releasing hormone (GnRH). Although GnRH has been extensively studied in all groups of vertebrates including fish (Roch et al., 2010; Zohar et al., 2010), there has been no genetic data on its functional importance in fish reproduction because of the lack of reverse genetics tools. This situation has changed recently with the development of the new generation of genome editing technologies. The zebrafish Gnrh3/gnrh3 gene, which is believed to be hypophysiotropic, was deleted recently with TALEN. Surprisingly, the loss of gnrh3 gene had no effect on sexual maturation and gametogenesis in both sexes, and gnrh3 mutants were all fertile (Spicer et al., 2016). In contrast, a recent study in medaka reported that TALEN-induced disruption of gnrh1 (hypophysiotropic form in medaka) led to female infertility due to anovulation (Takahashi et al., 2016). With this discrepancy, it would be interesting to see more studies in different species. As the master hormone in the HPG axis, GnRH is subject to regulation by various inputs from different levels of the axis, and one of the key regulators is kisspeptin(s) (Kiss1) (Dungan et al., 2006). In recent years, kisspeptin and its receptor Gpr54 (Lee et al., 1999) have been extensively studied in various vertebrate models including fish for their regulation of GnRH (Biran et al., 2008). Disruption of Kiss1 and its receptor Gpr54 in the mouse induced infertility (Lapatto et al., 2007). Recently, both kisspeptins (kiss1/2) and their receptors (kissr1/2) were knocked out with TALEN in the zebrafish (Tang et al., 2015). Surprisingly but similar to the phenotype of gnrh3 mutation, the loss of the kiss/kissr system 5

in the zebrafish (kiss1-/-, kiss2-/-, kissr1-/-, kissr2-/-, kiss1-/-;kiss2-/-, and kissr1-/;kissr2-/-) produced no phenotype in either females or males, and the fish were normal and fertile in both sexes. This is confirmed by a triple knockout of gnrh3 and kiss1/2 genes (Liu et al., 2017a). These results in the zebrafish are in sharp contrast to that reported in the mouse (Lapatto et al., 2007), and contradictory to the current view on the role of the kiss/kissr system in controlling fish reproduction (Mechaly et al., 2013). Although the lack of phenotypes in the gnrh3 and kiss/kissr mutant zebrafish could be explained by the compensatory roles played by other stimulatory circuits in the HPG (Liu et al., 2017a; Trudeau, 2015), the discoveries are undoubtedly challenging the dogma regarding the role of GnRH in neuroendocrine regulation of fish reproduction. More studies are urgently needed in different fish species to confirm the results in the zebrafish before making any general conclusions. Gene targeting in the pituitary Pituitary FSH and LH are among the best studied hormones in fish (Aizen et al., 2007; Chen and Ge, 2012; Hildahl et al., 2012). However, due to the lack of genetic tools for studying reproduction in fish models, the functional importance of these hormones had remained elusive until recently. Using TALEN, we and others have recently knocked out FSH (fshb) and LHlhb) subunits in the zebrafish. As expected, the loss of fshb caused a severe delay in puberty onset and follicle activation to enter the vitellogenic growth in females, while the loss of lhb gene had no effect on follicle growth but the females were infertile due to failed oocyte maturation and ovulation (Chu et al., 2015; Chu et al., 2014; Zhang et al., 2015b). The anovulatory phenotype of lhb mutant is likely due to reduced igf3 expression (Li et al., 2015a). Interestingly, the fshb-/-;lhb-/- double mutant zebrafish were all males due to sex reversal (Zhang et al., 2015b). The functionality of lhb and fshb has also been assessed by TALEN in medaka. Similar to that in the zebrafish, female lhb mutant medaka was infertile due to anovulation. Also similar to the zebrafish, female fshb mutant medaka showed severe blockade of folliculogenesis at pre-vitellogenic stage; however, unlike the zebrafish mutant, the medaka fshb mutant was infertile (Akiko Takahashi, 2016). It would be interesting to compare different model species with the loss-of-function approach for a better understanding of gonadotropins and their receptors in controlling reproduction in fish. Recently, the lhb gene was mutated by ZFN in the channel catfish, resulting in sterility in mutant founders. However, no homozygous mutants were reported (Qin et al., 2016a).

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In addition to gonadotropins, other pituitary hormones are also being assessed by genome editing. Prolactin (Prl/prl), with important function in osmoregulation and potential role in reproduction, was recently disrupted in the zebrafish with TALEN. The results showed that Prl was dispensable for reproduction since PRL deficient zebrafish were fertile; however, the prl-deficient mutants could not survive beyond 16 dpf (days post-fertilization) in freshwater but exhibited no phenotype in brackish water (Shu et al., 2016), confirming its ancient physiological role in osmoregulation as a freshwater-adapting hormone. Gene targeting in the gonads Gonads (ovary and testis) are central players of the reproductive system, and they are responsible for production of gametes (gametogenesis) and sex steroids (steroidogenesis) after sexual maturation. A large number of genes involved in gonadal differentiation and function have been characterized in a variety of vertebrates including fish (Devlin and Nagahama, 2002; Kobayashi and Nagahama, 2009). However, most studies on these genes have focused on expression analysis. With genome editing technologies available, people are now revisiting these genes with the aim of providing new genetic insights into the regulatory networks in the gonads that control sex determination and differentiation as well as gonadal gametogenesis and steroidogenesis in mature adults. Genes in primordial germ cell development In both ovary and testis, the germ cells (eggs and sperm) are derived from the primordial germ cells (PGCs) in the embryos. A few genes have been implicated in PGC formation and maintenance, including dead end (dnd), nanos2, and nanos3. As an RNAbinding protein and germ plasm component, Dnd is essential for PGC formation and migration in the zebrafish (Weidinger et al., 2003). Recently, the dnd gene was disrupted in the Atlantic salmon by CRISPR/Cas9, and the F0 mutant fish with complete loss of pigmentation displayed a loss of germ cells in the gonads, confirming an important role for dnd in germline determination. Interestingly, dnd knockout F0 fish could still form ovary and testis, but without oocytes or spermatogonia, indicating germ cell-independent gonadal differentiation in the Atlantic salmon (Wargelius et al., 2016). This is different from the situation in the zebrafish and medaka (Kurokawa et al., 2007; Siegfried and NussleinVolhard, 2008). In addition to dnd, other genes such as the nanos family have also been implicated in germ cell development. In the zebrafish, nanos1 was reported to be essential for PGC migration and survival (Koprunner et al., 2001). The mutations of nanos2 and nanos3

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induced by CRISPR/Cas9 in the F0 founder tilapia could not be transmitted to the F1 generation due to the loss of germ cells (Li et al., 2014). Genes in sex determination Unlike the situation in mammals, the mechanisms underlying gender commitment of PGCs or sex determination are only unknown in a few teleosts. Several potential sexdetermining genes have been proposed in teleosts, and the functionality of these genes has been evaluated recently by genome editing including ZFN, TALEN or CRISPR/Cas9. The dmy identified in medaka is functionally equivalent to SRY gene in mammals (Matsuda et al., 2002). Disruption of dmy by TALEN resulted in sex reversal of XY males to females, while dmy-deficient XX female mutants were fertile (Luo et al., 2015b). Similarly, a different sexdetermining gene, sdy, has been identified in the rainbow trout. The F0 mosaic mutation of sdy induced by ZFN technology could be transmitted to the F1 progeny, and the heterozygous F1 XY fish showed obvious ovarian structure resulting from male-to-female sex reversal, supporting a role for sdy in male determination and differentiation in the rainbow trout (Yano et al., 2014). Recently, a third sex-determining gene was characterized in tilapia, which was a Y-linked duplication of amh (amhy) that is nearly identical to the X-linked amh. Knockout of amhy with CRISPR/Cas9 in XY fish resulted in male-to-female sex reversal (Li et al., 2015b). In the Chinese tongue sole, a recent study using CRISPR/Cas9 provided evidence that dmrt1 might likely function as the sex-determining gene in this species to initiate male development (Cui et al., 2017). Genes in sex differentiation Although the number of sex-determining genes identified in fish is limited, a large of genes have been characterized for their roles in fish gonadal differentiation. DMRT1 (Doublesex and mab-3 related transcription factor 1) is critical for testis development in mice (Raymond et al., 2000). This has been confirmed recently in fish by gene knockout. In tilapia, Dmrt1-deficient F0 fish generated by TALEN showed significant testicular regression, including spermatogonial degeneration or even loss of germ cells (Li et al., 2014; Li et al., 2013). Similarly, TALEN-induced loss of dmrt1 gene in the zebrafish caused male-to-female sex reversal and the males were infertile with testis dysgenesis (Webster et al., 2017). In Chinese tongue sole, deletion of dmrt1 by TALEN led to development of ovary-like testis with increased expression of female-related genes including foxl2 and cyp19a1a but decreased expression of male-related genes such as sox9a and amh, which resulted in 8

disrupted spermatogenesis (Cui et al., 2017). In addition to transcription factors like Dmrt1, gonadal differentiation is also influenced by secreted growth factors. Gsdf (gonadal somatic cell derived factor) is a fish-specific TGF-βsuperfamily member expressed predominantly in the Sertoli cells in the testis, and its role in gonadal differentiation has gained increasing attention. Recently, gsdf was deleted in medaka by ZFN, and the homozygous mutant fish showed all-female phenotype and decreased dmrt1 expression (Zhang et al., 2016). Similar result has been obtained in tilapia. Disruption of tilapia gsdf by CRISPR/Cas9 resulted in male-to-female sex reversal with ovotestis in the F0 mosaic XY fish, and homozygous gsdf mutants were all females with significantly increased estradiol (E2) levels compared to the controls (XY gsdf +/+ and XY gsdf +/-), suggesting that Gsdf may suppress estrogen production in the gonads (Jiang et al., 2016). Genes in spermatogenesis Spermatogenesis in mature testis is a complicated process that involves numerous regulatory factors, including gonadotropins, growth factors, androgens and various transcription factors (Schulz et al., 2010). Similar to that in females, loss of pituitary FSH but not LH in the zebrafish caused a significant delay of spermatogenesis at puberty (Zhang et al., 2015b) and similar phenotype was observed in TALEN-induced fshr knockout (Zhang et al., 2015a). Interestingly, although fshr and lhcgr mutant males were fertile with normal spermatogenesis after sexual maturation, double knockout of fshr and lhcgr genes resulted in male sterility with testis dysgenesis, demonstrating the importance of gonadotropin signaling in controlling spermatogenesis (Zhang et al., 2015a). A recent study using double mutants (fshb;fshr and lhb;lhcgr) in the zebrafish showed that the canonical FSH-Fshr and LH-Lhcgr pathways were functionally redundant in promoting spermatogenesis and the loss of either pathway had no effect male reproduction (Xie et al., 2017). Also recently in the zebrafish, a study using TALEN showed that the deletion of cyp17a1 caused a dramatic decrease in serum androgen levels; however, all fish turned out to be males (Zhai et al., 2017). This study raises an interesting question about the role of androgens in spermatogenesis. It is well known that the initiation and progression of spermatogenesis involve various transcription factors. In addition to Dmrt1 discussed above, similar studies have also been carried out on other transcription factors. Deletion of steroidogenic factor-1 (SF-1, nr5a1), one of the most important factors in controlling steroidogenesis, with CRISPR/Cas9 in Nile tilapia caused increased expression of foxl2 and cyp19a1a and decreased expression of dmrt1, amh and cyp11b2 in mutant XY males of F0 generation with testis dysgenesis. The phenotype could

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be recused by 17-methyltestosterone (MT) (Xie et al., 2016). Evidence has emerged recently that disruption of meiosis caused abnormality in spermatogenesis. A recent study in tilapia showed that R-spondin 1 (rspo1) significantly increased its expression right before meiotic onset in both ovary and testis. Interestingly, although rspo1 is considered a female-related gene favoring ovarian development, its deletion with TALEN also caused a delay in spermatogenesis (Wu et al., 2016). In agreement with this, the knockout of Dmc1, a recombinase involved in meiosis (Fukushima et al., 2000), by TALEN in medaka increased apoptosis of spermatocytes and production of malfunctional sperm with abnormal morphology such as multiple tails or heads. As expected, the mutant males were infertile (Chen et al., 2016). Genes in folliculogenesis In mature female vertebrates, folliculogenesis refers to the process of follicle growth and maturation in the ovary (van den Hurk and Zhao, 2005). In teleosts, the folliculogenesis can be divided into several distinct stages: primary growth (PG), pre-vitellogenic (PV), early vitellogenic (EV), mid-vitellogenic (MV), late vitellogenic (LV), full-grown (FG) and maturation (Ge, 2005b). Although fish folliculogenesis has been extensively studied for its regulation, the functional significance of various regulatory factors has not been well studied using genetic approach. The new generation of genome editing technologies promises to provide critical insight into the regulatory networks that govern folliculogenesis in fish. As the major hormones that control folliculogenesis, gonadotropins (FSH and LH) act in the ovary through their cognate receptors Fshr and Lhcgr (Ge, 2005a; Swanson et al., 2003). Using TALEN technology, we and others have recently deleted both fshr and lhcgr genes in the zebrafish. Intriguingly, the disruption of Fshr/fshr and Lhcgr/lhcgr could not phenocopy the mutants of fshb and lhb (Chu et al., 2015; Chu et al., 2014; Zhang et al., 2015a; Zhang et al., 2015b), presumably because of the cross-reaction of Fshr by LH in the zebrafish (So et al., 2005). The loss of fshr led to a complete arrest of folliculogenesis at early PG stage, and all fshr-deficient females eventually changed sex to become males at different times of development. Surprisingly, the disruption of lhcgr produced no visible phenotype in folliculogenesis (Zhang et al., 2015a). The phenotype of fshr knockout in the zebrafish is similar to that reported in medaka fshr mutant, which was all-male due to sex reversal presumably induced by reduced expression of aromatase (cyp19a1a) (Murozumi et al., 2014).

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As the key enzyme involved in estrogen biosynthesis, the ovarian aromatase (cyp19a1a) is essential for female differentiation and function in teleosts (Nakamura et al., 2003). Using both TALEN and CRISPR/Cas9, we have recently created several knockout mutants for cyp19a1a in the zebrafish. As expected, the mutant showed an all-male phenotype, which was due to failed conversion of the “juvenile ovary” to true ovary during sex differentiation (Lau et al., 2016). This is in sharp contrast to the situation in the mouse in which the loss of aromatase led to female infertility but not ovarian formation (Fisher et al., 1998). In tilapia, the disruption of cyp19a1a with CRISPR/Cas9 in XX female fish induced female-to-male sex reversal in mosaic F0 mutant with increased expression of dmrt1 and cyp11b2 and decreased estrogen production (Li et al., 2013). These results show that the ovarian aromatase is crucial for both sex differentiation and maintenance of female status in fish. The aromatase is well known to be controlled by transcription factor FOXL2 in both mammals and fish (Georges et al., 2014; Wang et al., 2007). In teleosts, two paralogs of foxl2, named foxl2a (foxl2) and foxl2b (foxl3), have been described in some species including medaka (Nishimura et al., 2015). The functional importance of Foxl2 (foxl2a) has recently been evaluated by TALEN-induced mutagenesis in tilapia, which revealed a female-to-male sex reversal in the mosaic F0 mutant due to decreased expression of cyp19a1a (Li et al., 2013) . This result supports a functional role for Foxl2 in regulating cyp19a1a expression and therefore sex differentiation and maintenance. In the medaka, foxl3 is abundantly expressed in XX females compared to XY males. Interestingly, disruption of foxl3 with TALEN caused production of sperm in the ovary of XX females without affecting cyp19a1a expression, suggesting an inhibitory role for foxl3 in blocking spermatogenesis (Nishimura et al., 2015). In addition to foxl2, the expression of cyp19a1a may also be regulated by other transcription factors including SF-1 (nr5a). The function of SF-1 in mammals remains unclear since NR5A1 deficient mice suffer from postnatal embryonic lethality that limits further functional analysis. A recent loss-of-function study with CRISPR/Cas9 in tilapia has provided insights into the function of sf-1 in teleosts. It was discovered that some sf-1deficient F0 XX females and most sf-1 heterozygous F1 XX females underwent female-tomale sex reversal. Treatment with E2 could partially rescue the phenotype of sf-1-deficient XX females, suggesting a role for Sf-1 in regulating cyp19a1a expression and estrogen production in females (Xie et al., 2016).

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The expression of cyp19a1a is also subject to regulation by growth factors that act in the ovary in a paracrine manner. As a member of TGF-β superfamily, bone morphogenetic protein 15 (BMP15) is specifically expressed in the oocyte in mammals (Persani et al., 2014). Recently the bmp15 gene was disrupted in the zebrafish with TALEN, which caused an arrest of folliculogenesis at PV stage or stage II, partly due to reduced cyp19a1a expression (Dranow et al., 2016). Like the ovary in mammals, the fish ovary also produces two major types of female steroids, i.e., estrogens and progestogens. The major functional form of progestogens in fish is 17, 20-dihydroxy-4-pregnen-3-one (DHP), which signals partly via nuclear progestin receptor (pgr). The function of Pgr was recently evaluated in the zebrafish by TALENinduced mutagenesis. The female mutant was infertile due to anovulation (Tang et al., 2016; Zhu et al., 2015), and the defect could partly be due to lower expression of fshb and lhb in the pituitary (Wang et al., 2016). Similar to progestogens, estrogens also signal mostly via nuclear receptors. In teleosts, there exist three nuclear estrogen receptors (nERs), namely esr1, esr2a and esr2b. We have recently evaluated the functions of these nERs in zebrafish reproduction by using CRISPR/Cas9-mediated gene knockout. Surprisingly, none of the single nER mutant produced significant phenotype in reproduction of either males or females. However, further analysis with double and triple knockouts (esr1-/-;esr2a-/-;esr2b-/-) showed that nERs were essential for female reproduction, especially Esr2a and Esr2b. This study further demonstrates the power of the zebrafish model, which allows for generation of mutants with multiple mutations. The loss of Esr2a and Esr2b caused an arrest of folliculogenesis at PV stage, and these females eventually changed to males through sex reversal, resulting in all-male adults (Lu et al., 2017). Interestingly, even the triple knockout of nERs could not fully mimic the phenotype of aromatase mutant (Lau et al., 2016), suggesting alternative signalling mechanisms for estrogens in the zebrafish. Functional analysis of genes in non-reproductive functions of fish In addition to the genes involved in the reproductive axis, the genome editing technologies have also been used to study functions of non-reproductive genes, in particular those involved in pigmentation, development and growth. Some of the genes targeted are exogenous genes such as EGFP and mCherry in transgenic fishes mainly for testing the efficacy of the technologies (Ansai et al., 2012; Jao et al., 2013). Since development and

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growth is a vast area with tremendous information, this review only focuses on some selected literature to illustrate the power of genome editing technologies. Targeting of genes in pigmentation Pigmentation involves melanin production in melanocytes (Cichorek et al., 2013). In recent years, the genes involved in pigmentation have been repeatedly targeted in fish species by ZFN, TALEN, and CRISPR/Cas9 mostly for testing different protocols because of the convenience for phenotype analysis. The golden gene (slc24a5) is essential for pigmentation, and it is highly conserved across vertebrates (Lamason et al., 2005). In the zebrafish, the golden gene has been disrupted by all three major platforms of genome engineering including ZFN, TALEN and CRISPR/Cas9 (Dahlem et al., 2012; Doyon et al., 2008; Jao et al., 2013), resulting in lighter-colored eyes in F0 mosaic larvae, and the mutations could be transmitted to the F1 progeny. In addition to slc24a5, other genes in fish pigmentation have also been targeted by CRISPR/Cas9 including tyrosinase (tyr) and slc45a2/alb. In the zebrafish, slc45a2-deficient F0 larvae produced by CRISPR/Cas9 displayed varying degrees of pigment loss (Irion et al., 2014). In Atlantic salmon, tyr and slc45a2 were successfully mutated by CRISRP/Cas9, and the F0 mosaic founders also showed varying degrees of pigment loss (Edvardsen et al., 2014). Similar study has also been performed in the lamprey, the most primitive type of vertebrates. In the Northeast Chinese lamprey, disruption of the slc24a5 by CRISPR/Cas9 caused loss of pigmentation in the skin and retina to different levels in the F0 founders (Zu et al., 2016). The use of genome editing (TALEN) has also helped identify a gene (oculocutaneous albinism 2; oca2) in the cavefish that is responsible for reduced pigmentation (Ma et al., 2015). These studies demonstrated the efficiency of genome editing in a wide range of fish species from model organisms (e.g., zebrafish) to large species with economic importance ( e.g., Atlantic salmon), and from evolutionarily primitive species (e.g., lamprey) to ones with special adaptations (e.g., cavefish). Targeting of genes in development and growth A variety of genes in development and growth have been functionally evaluated in teleosts by using ZFN, TALEN, and/or CRISPR/Cas9. No tail (ntl) in the zebrafish is the ortholog of brachyury in the mouse (Schultemerker et al., 1994). As expected, the ZFNinduced mutation of ntl in F0 zebrafish caused a phenotype of defective tails (Doyon et al., 2008). With the development of TALEN and CRISPR/Cas9, a large number of genes in development and growth have been disrupted in fish for functional studies, including four

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Mesp genes (mespaa, mespab, mespba and mespbb) involved in anteroposterior specification (Yabe et al., 2016) and stat3 in spine development and immune function (Xiong et al., 2017) by TALEN in the zebrafish, the genes involved in the development of bone (sp7a/b, runx2, bmp2a, spp1, and opg) by both TALEN and CRISPR/Cas9 in the common carp, which showed significant phenotypes in F0 mosaic mutants (Zhong et al., 2016), and the genes involved in zebrafish survival and growth (akt2) (Zhang et al., 2017a) and development of the cranial vasculature (gspt1l) (Wang et al., 2017) by CRISPR/Cas9. In the field of developmental biology, the MO-mediated gene knockdown has been a dominant approach for reverse genetics in fish models. With the availability of efficient genome editing technologies, it is anticipated that the nuclease-induced gene knockout will quickly out-perform MO- or RNAi-mediated gene knockdown, and a large number of genes that have been studied with knockdown will be re-evaluated by gene knockout. Targeting of genes in other functions In addition to the genes in reproduction, development and growth, a variety of genes involved in the functions of other organ systems have been studied in teleosts by genome editing and the list of genes investigated is increasing quickly. Most of the genes have been studied in the zebrafish, including kdrl (Doyon et al., 2008) by ZFN; gria3a and hey2 (Sander et al., 2011), ryr3, ryr1a and tbx6 (Dahlem et al., 2012), rnf213a (Wen et al., 2016), six7 (Sotolongo-Lopez et al., 2016), zap70 (Moore et al., 2016), nr1d1 (Huang et al., 2016), and leg1a (Hu et al., 2016) by TALEN; and mitfa and ddx19 (Jao et al., 2013), Seta/b (Serifi et al., 2016), nrg1-I (Samsa et al., 2016), and fus (Lebedeva et al., 2016) by CRISPR/Cas9. In the rohu (Labeo rohita), an important formed carp in India, the TLR22 gene involved in innate immunity was silenced by CRISPR/Cas9 through knock-in of a large exogenous fragment of about 3.2 kb (Chakrapani et al., 2016). In Atlantic killifish, AHR2 proteins (ahr2a and ahr2b) seem to play major roles in mediating the effects induced by aryl hydrocarbon pollutants (AHPs). The homozygous mutants (ahr2a-/- and ahr2b-/-) created by CRISPR/Cas9 and double homozygous mutants (ahr2a-/-;ahr2b-/-) may be useful tools for monitoring AHPs in marine environments (Aluru et al., 2015). Recently, the CRISPR/Cas9-based genome editing technology has been successfully established in the short-lived African turquoise killifish, a rising model for aging in vertebrates (Harel et al., 2015; Harel et al., 2016). Genome editing for generation of disease models in fish

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As members of vertebrates, fish models such as the zebrafish and medaka are now gaining increasing popularity in biomedical sciences for understanding human genetic diseases and drug screening because of their amenability to genetics (Hsu et al., 2007; Lin et al., 2016a; Sullivan et al., 2017; Witten et al., 2017). With 75% gene similarity with humans and more than 84% genes causing human genetic diseases (Howe et al., 2013), zebrafish is now and will be one of the top animal models for studying human genetic diseases. The biological advantages of the zebrafish and the availability of the genome editing technologies will make it a top choice for disease modeling and drug screening. Here are some examples for the power of disease modeling in the zebrafish involving genome editing. Human congenital amegakaryocytic thrombocytopenia (CAMT) is known to be caused by mutations in the myeloproliferative leukemia (MPL) protein gene. Recently, a zebrafish mpl mutant line was created by TALEN. The mutant exhibited reduced number of thrombocytes and high chance of bleeding, mimicking the symptoms of human CAMT. This mutant zebrafish line provides a valuable model for understanding human CAMT and a potential platform for screening drugs to treat the disease (Lin et al., 2016b). Human epilepsies in childhood are linked to mutations in syntaxin-binding protein 1 (STXBP1). Zebrafish has two forms of the protein (stxbp1a and stxbp1b). These two genes were recently mutated in the zebrafish by using CRISPR/Cas9, and the two mutants displayed typical symptoms of human epilepsies, making them an excellent animal model for studying disease mechanisms and developing therapy (Grone et al., 2016). As a popular model for cancer research, zebrafish carries most of human oncogenes and tumor suppressors, making it an excellent model for studying the functions of these genes and screening for target drugs. For example, a recent study using TALEN created a mosaic zebrafish mutant with the tumor suppressor retinoblastoma1 (rb1) knocked out in some somatic cells, which led to tumor development as early as 3.5 months and mostly in the brain (Solin et al., 2015). Osteoporosis is a major health problem in the elderly. Genome-wide association studies (GWAS) have identified large number of potential genetic factors for osteoporosis, including ATP6V1H. Mutation of zebrafish ATP6V1H homologue (atp6v1h) by CRISPR/Cas9 generated a mutant that exhibited phenotypes similar to human osteoporosis. Further analysis of the mutant identified two genes (mmp9 and mmp13) that mediated atp6v1h deficiency. Treatment of the mutant fish with small molecule inhibitors of MMP9 and MMP13 successfully rescued the phenotypes of the atp6v1h mutant (Zhang et al., 2017b). These studies demonstrate the great potential of fish species and genome editing in human disease modeling and therapy development.

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Potential application of genome editing in aquaculture Although it is still in its infancy stage, genome editing will find tremendous applications in the field of aquaculture. This, together with transgenesis, makes it possible to precisely change genome composition according to the needs in aquaculture, therefore allowing for precision breeding. Many traits of great significance in aquaculture could be targets for improvement by genome editing, including growth and reproductive performance, disease resistance, feed conversion efficiency, and tolerance to environmental stressors (temperature, salinity and oxygen) (Abdelrahman et al., 2017). Sterility is often desired in aquaculture for controlling escape of domestic and transgenic fish. This has been achieved in the channel catfish by disrupting the  subunit gene of pituitary LH hormone with ZFN (Qin et al., 2016b). Recently, a gene coding for myostatin (mstnba) (an inhibitor of skeletal muscle growth) was mutated by CRISPR/Cas9 in the common carp, which showed increased muscles in F0 founders (Zhong et al., 2016). In contrast, disruption of mstna with ZFN (Dong et al., 2011) and mstnb with TALEN (Dong et al., 2014) in the yellow catfish displayed no significant phenotype in muscle growth in either F0 or F1 generation. The discrepancy between common carp and yellow catfish suggests functional variation among different species. It would be interesting to see the phenotypes in homozygous mutants in both species. The studies in the yellow catfish represents the first attempt to apply genome editing technologies in a farmed fish species (Dong et al., 2011; Dong et al., 2014). These studies show great potential for the application of genome editing in aquaculture for breeding new varieties with valuable traits. Compared with traditional transgenesis, genome editing normally does not introduce foreign DNA into the genome of target species, therefore alleviating public concerns on the safety of genetically modified organisms (GMOs). Technological innovations in fish models With the fast development of genome editing technologies, fish models especially the zebrafish are often the top choice to test new protocols. With the methods for global gene knockout becoming mature, the demand for advanced versions of genome editing is increasing, such as gene knock-in or insertion of DNA fragments at specific sites and cellspecific conditional knockout. Recently, a CRISPR/Cas9 and Tol2-based transgenic vector system was designed for efficient cell-specific disruption of target genes in the zebrafish. The vector contained three expression cassettes, namely a U6 promoter-driven gRNA (ruod), a cell-specific promoter (gata1)-driven Cas9 and heart-specific promoter (cmlc2)-driven GFP for selection of transgene integration (Ablain et al., 2015). This system is efficient and

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versatile as it can be easily applied to any genes in any tissues. A similar but different approach was recently reported in the zebrafish to achieve cell-specific knockout. Instead of introducing gRNA and Cas9 in the same vector under different promoters, this approach involved generating two separate transgenic lines expressing gRNA globally and Cas9 in specific cells respectively. By crossing the two transgenic lines, the gRNA and Cas9 were co-expressed in the same cells, achieving cell-specific knockout (Yin et al., 2015). Cellspecific knockout has also been reported in the zebrafish by using bacterial artificial chromosome-rescue-based knockout (BACK) in combination with Cre/loxp system (Liu et al., 2017b). Recently, a novel optogenetic gene expression system has been attempted in the zebrafish to achieve site- and time-specific expression of Cas9, which, together with ubiquitously expressed sgRNAs, would induce gene mutation in specific spatiotemporal manners (Reade et al., 2017). In addition to gene knockout, the genome editing methods have also been used to introduce DNA fragments at specific sites (gene knock-in) (Kawahara et al., 2016). Bedell et al. first demonstrated HDR-mediated knock-in of a short EcoRV site and a modified loxP sequence into the zebrafish genome by microinjection of a donor ssOligo with TALEN mRNAs (Bedell et al., 2012). Also using TALEN, Zu et al. subsequently achieved sitespecific knock-in of a large DNA fragment encoding EGFP by using a dsDNA donor with two long arms (Zu et al., 2013). The method is quickly becoming mature and reliable, and a variety of exogenous DNA sequences of various sizes, such as NotI and EcoRI sequences, EGFP and mCherry, FRT and loxP, have been introduced at specific DNA sites in the genome by TALEN (Hoshijima et al., 2016). Recently, large exogenous DNA fragments have been introduced at specific sites in a homology-independent manner by CRISPR/Cas9 (Auer et al., 2014). The CRISPR/Cas9 method has recently found a new and interesting application, i.e., cell lineage tracing. Briefly, an array of 10 CRISPR/Cas9 target sites (barcode) is synthesized and introduced into the genome of zebrafish by transgenesis. This will be followed by co-injection of Cas9 and 10 sgRNAs that recognize the target sites in the barcode. The random editing by these sgRNAs in vivo will generate different combinations of mutations, which are shared by cells of different lineages. This provides a powerful tool to trace the ancestry and lineage of different cells in multi-cellular organisms like the zebrafish (McKenna et al., 2016). Concluding remarks 17

With the high efficiency of genome editing in both model fish species and those with economic importance, we are now entering an era that will witness an explosive adoption of these powerful technologies in a wide range of fish species to study gene functions or create new genetically modified stains with special characteristics. The increasing popularity of genome editing in fish models is partly due to the biological and technical advantages of fish species. At present, most genome editing experiments have been performed on three popular fish species, i.e., zebrafish, medaka, and tilapia, and many genes studied in detail are concerned about reproductive functions. Although reproduction will continue to be a main area for the application of genome editing because this function had been difficult to study with reverse genetics, there will be increasing use of these technologies for studying gene functions in other organ systems. Furthermore, with the technologies becoming mature in various fish models, it is anticipated that they will find more applications in aquaculture for improving traits of economical values such as growth and disease resistance, in environmental science for monitoring pollutants that interfere with specific physiological events and pathways, and in biomedical science for disease modeling and drug screening. As the largest group of vertebrates with the greatest biodiversity, the availability and application of genome editing technologies in fish species will provide tremendous information and insights into gene functions and their evolution across vertebrates. Acknowledgement The work related to genome editing in our laboratory has been supported by grants from the University of Macau (MYRG2014-00062-FHS, MYRG2015-00227-FHS, and CPG2014-00014-FHS) and The Macau Fund for Development of Science and Technology (FDCT114/2013/A3 and FDCT/089/2014/A2) to W.G. References Abdelrahman, H., ElHady, M., Alcivar-Warren, A., Allen, S., Al-Tobasei, R., Bao, L., Beck, B., Blackburn, H., Bosworth, B., Buchanan, J., Chappell, J., Daniels, W., Dong, S., Dunham, R., Durland, E., Elaswad, A., Gomez-Chiarri, M., Gosh, K., Guo, X., Hackett, P., Hanson, T., Hedgecock, D., Howard, T., Holland, L., Jackson, M., Jin, Y., Kahlil, K., Kocher, T., Leeds, T., Li, N., Lindsey, L., Liu, S., Liu, Z., Martin, K., Novriadi, R., Odin, R., Palti, Y., Peatman, E., Proestou, D., Qin, G., Reading, B., Rexroad, C., Roberts, S., Salem, M., Severin, A., Shi, H., Shoemaker, C., Stiles, S., Tan, S., Tang, K.F., Thongda, W., Tiersch, T., Tomasso, J., Prabowo, W.T., Vallejo, R., van der Steen, H., Vo, K., Waldbieser, G., Wang, H., Wang, X., Xiang, J., Yang, Y., Yant, R., Yuan, Z., Zeng, Q., Zhou, T., 2017. Aquaculture genomics, genetics and breeding in the United States: current status, challenges, and priorities for future research. BMC Genomics 18, 191. Ablain, J., Durand, E.M., Yang, S., Zhou, Y., Zon, L.I., 2015. A CRISPR/Cas9 vector system for tissue-specific gene disruption in zebrafish. Dev Cell 32, 756-764.

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Highlights 1. The new generation of genome editing technologies are applicable to fish species. 2. A large of number of genes have been investigated by genome editing in fish. 3. There is a great potential for genome editing in fish for both science and application.

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