Genome editing technology not only enables in-depth study of gene function in model organism, such as zebrafish, medaka to reveal relevant biological mechanisms, but also plays a part in improving important economic characteristics of aquaculture fish species, such as improving meat quality, the number of intramuscular spines and enhancing the growth speed (Table 1).
Species Gene Methods Phenotypes of mutation Generation References Zebrafish (Danio rerio) stat3 TALEN Spine malformation and dysregulated immune response F2 Xiong et al. (2017a) stat5.1 CRISPR/Cas9 Reduction of body length and weight F2 Xiong et al. (2017b) stat5.2 TALEN No significant difference between mutant and wild type F2 and F3 greb1 TALEN Convergence and extension (CE) movement defect during zebrafish gastrulation F2 Li et al. (2017) Common carp (Cyprinus carpio) mstnba sp7a TALEN; CRISPR/Cas9 Increased muscle cells F1 Zhong et al. (2016) TALEN; CRISPR/Cas9 Severe bone defect F1 Zhong et al. (2016) Channel catfish (Ictalurus punctatus) mstn CRISPR/Cas9 Increased muscle mass and weight F2 Dong et al. (2014) Olive flounder (Paralichthys olivaceus) mstn CRISPR/Cas9 Increased muscle mass and weight F2 Kim et al. (2019) Yellow catfish (Pelteobagrus fulvidraco) Mstna ZFN Not reported F1 Dong et al. (2011) CRISPR/Cas9 Increased muscle mass and weight F2 Zhang et al.(2020a, b) mstnb TALEN No significant difference F1 Dong et al. (2014)
Table 1. Phenotypes of growth-related gene mutation induced by genome editing
Myostatin (MSTN) is an important gene related to muscle growth, which negatively regulates skeletal muscle development. In 2011, ZFN was applied to mstna knockout in yellow catfish (Pelteobagrus fulvidraco) by Qingshun Zhao's team, Nanjing University in China, gene knockout in aquaculture fish was achieved for the first time (Dong et al. 2011). In 2014, they took the lead in using TALEN technology to knock out mstnb in yellow catfish and built a gene editing platform of TALEN for non-model fish (Dong et al. 2014). These achievements suggested the effectiveness of using ZFN and TALEN technologies in fish. Later, Khalil et al. (2017) and Kim et al. (2019) successively knocked out mstn in Ictalurus punctatus and Paralichthys olivaceus, respectively, using CRISPR/Cas9, which leads to muscle thickening of the mutants and significant increase of the body weight, meanwhile modulating the expression of major muscle-derived regulatory factors. Another growth-related trait is the number of intermuscular spines, which affects the taste of fish. Researchers endeavored to select new varieties without intermuscular spines to meet the consumers' needs. Common carp (Cyprinus carpio) is one of the important aquaculture species containing many intermuscular spines. Seven genes involved in bone and muscle formation were knocked out, respectively, in carp to construct single-gene knockout mutants using TALEN and CRISPR/Cas9, whereas knockout efficiency of CRISPR/Cas9 was found higher than TALEN generally (Zhong et al. 2016). Phenotype of mutant carps demonstrated that the muscle cells in mstnba mutant increased, and severe bone defects appeared in sp7a mutant. Also, the team constructed sp7a and mstnba double-knockout carps successfully with high-mutation efficiency verifying the feasibility of multiple-knockout in polyploid fish by CRISPR/Cas9 technology. Growth hormone (GH) is essential for individual development and metabolism. Knockout of Stat5.1, a downstream regulator of GH signaling, in zebrafish by CRISPR/Cas9 led to significant reduction of body length and weight of mutants, and gh1 and ghra were confirmed to be potential regulatory targets of Stat5 protein through chromatin immunoprecipitation combined with deep sequencing (ChIP-seq) and related experiments (Xiong et al. 2017b). Previously, they had knocked out stat3, another member of STAT family, in zebrafish by TALEN technology, and found that STAT3 was related to spine development and the immune response of zebrafish (Xiong et al. 2017a). Furthermore, greb1 is a potential target gene regulated by nodal signal in zebrafish. Li et al. (2017) used TALEN technology to knock out greb1 gene resulting in convergence and extension (CE) movement defect during zebrafish gastrulation, and the growth and development of adult zebrafish were hindered.
Sex determination and differentiation in fish is complex and involves many aspects such as genetics, embryogenesis, and endocrinology. Many genes (e.g., steroid biosynthesis genes, hormone receptor genes, TGFB pathway genes, encoded sex-related DNA binding protein genes) are found to co-regulate these processes. The molecular mechanism under sex determination and differentiation is also a major research topic in the field of life science. Thus, a series of studies on the function of key sex-related genes was carried out using genome editing technology in model fish species due to their biological properties. Furthermore, in the long-term farming practice, significant sexual dimorphism in size between male and females has been observed in at least 20 farmed species (Mei and Gui 2015). Growth speed or individual size has marked difference between male and females. Therefore, sex becomes an important research topic for farmed fish with sexual dimorphism, whereas yield and economic benefits could be increased by cultivating monosex fish population. At present, researchers have performed genome editing on sex-related genes in a variety of fish, such as yellow catfish, tilapia (Oreochromis niloticus) and Chinese tongue sole (Cynoglossus semilaevis) (Table 2).
Species Gene Methods SD gene (yes/no) Phenotypes of mutation Generation References Zebrafish (Danio rerio) Gsdf TALEN No Extended bipotential gonad state; sterile female and accumulating immature follicles; fertile male F2 Yan et al. (2017) cyp19a1a TALEN; CRISPR/Cas9 No Female to male sex reversal; delayed male sex differentiation F2 Lau et al. (2016) and Yin et al. (2017) Amh CRISPR/Cas9 No Hypertrophic testes and ovaries; impaired differentiation of germ cells F2 Lin et al. (2017) and Zhang et al.(2020a, b) dmrt1 TALEN; CRISPR/Cas9 No Regressed testis lobes with a mass of adipose tissue; impaired male germ cell development F2 Lin et al. (2017) and Webster et al. (2017) bmp15 TALEN No Reduced or no estrogen-producing granulosa cells and female to male sex reversal F2 Dranow et al. (2016) fshb; lhb TALEN No Delay of ovary and testis development in fshb mutant; female sterility due to a failure of oocyte maturation in lhb mutant F2 Zhang et al. (2015b) fshr; lhcgr TALEN No Delayed initiation of spermatogenesis in male fshr mutant and retarded ovarian growth and failed follicle activation in female fshr mutant; normal phenotype in lhcgr mutant; male fshr and lhcgr double mutants are sterile F2 Zhang et al. (2015a) gnrh3 TALEN No Normal gametogenesis and reproductive performance F3 Spicer et al. (2016) esr2a; esr2b; esr1 CRISPR/Cas9 No Enlarged and deformed chorion in esr2a mutants; female esr2a; esr2b double mutants and esr2a; esr2b; esr1 tri-mutants to male sex reversal F2 Lu et al. (2017) pgr TALEN No Females are infertile due to ovulation defects F2 Zhu et al. (2015) sox3 CRISPR/Cas9 No Follicle development retardation and a reduced fecundity F2 Hong et al. (2019) wnt4a CRISPR/Cas9 No Reproductive duct defects result in both male and female sterility; mutants develop predominantly as males F2 Kossack et al. (2019) mettl3 TALEN No Disrupted oocyte maturation and blocked sperm maturation F3 Xia et al. (2018) Ar TALEN; CRISPR/Cas9 No male infertility; immature oocytes in female F2 Crowder et al. (2018), Yu et al. (2018) and Tang et al. (2018) cyp17a1 TALEN No Insufficient androgen levels and all male offspring F3; F7 Zhai et al. (2017) and Shu et al. (2020) cyp11c1 CRISPR/Cas9 No Defective natural mating but possessed mature gametes; reduced egg spawning and failed germinal vesicle breakdown in females; delayed and prolonged juvenile ovary-to-testis transition in males F2 Zhang et al.(2020a, b) Medaka (Oryzias latipes) Dmy TALEN Yes Male to female sex reversal F3 Luo et al. (2015) foxl3 TALEN No XX developed functional sperm in ovaries F2 Nishimura et al. (2015) gsdf TALEN/ZFN No XY developed into ovaries in the early stage, but two-thirds of them developed into testes in adult F2 Imai et al. (2015) and Zhang et al. (2016) esr2a CRISPR/Cas9 No Oviduct atresia resulted in female sterility F2 Kayo et al. (2019) fshb TALEN No Folliculogenesis was arrested at the yolk accumulation stage F2 Takahashi et al. (2016) gnrh1 TALEN No Ovulation failure F2 Takahashi et al. (2016) lhb TALEN No Ovulation failure F2 Takahashi et al. (2016) Medaka (O. dancena) sox3Y ZFN Yes Male to female sex reversal F2 Takehana et al. (2014) Tilapia (Oreochromis niloticus) foxl2 TALEN No Partial sex reversal F0 Li et al. (2013) cyp19a1a TALEN No Partial female to male sex reversal F0 Li et al. (2013) sf-1 CRISPR/Cas9 No Part of female to male sex reversal F0 Xie et al. (2016) R-spondin1 TALEN No Delay in ovarian differentiation and sperm formation F0 Wu et al. (2016a) β-catenin1/2 TALEN No Delay in ovarian differentiation and masculinization F0 Wu et al. (2016b) esr2a CRISPR/Cas9 No Delay in ovarian development and abnormal development of testis F2 Yan et al. (2019) esr2b CRISPR/Cas9 No Reproductive duct defects result in male and female sterility F2 Yan et al. (2019) dmrt1 TALEN No Regression of testis F2 Li et al. (2013) gsdf CRISPR/Cas9 No Male to female sex reversal F2 Jiang et al. (2016) amhy CRISPR/Cas9 Yes Male to female sex reversal F2 Li et al. (2015) igf3 CRISPR/Cas9 No Male sterility; reduced semen volume and sperm count F2 Li et al. (2020) pgr CRISPR/Cas9 No Decline of sperm count and sperm motility and fertility F2 Fang et al. (2018b) rln3a CRISPR/Cas9 No Disrupted spermatogenesis; decline of sperm motility F2 Yang et al. (2020) Yellow catfish (Pelteobagrus fulvidraco) pfpdz1 CRISPR/Cas9 No XY ovary differentiates into testis-like tissue F2 Dan et al. (2018) Chinese tongue sole (Cynoglossus semilaevis) dmrt1 TALEN Yes Ovary-like testis and faster growth rates in ZZ; intersex gonad in ZW (a testis on one side and an ovary on the other side) F2 Cui et al. (2017) Rainbow trout (Oncorhynchus mykiss) sdY ZFN Yes Male to female sex reversal F1 Yano et al. (2012) Sterlet (Acipenser ruthenus) dnd1 CRISPR/Cas9 No Mutant sterility F2 Baloch et al. (2019) SD sex-determining gene
Table 2. Phenotypes of sex-related gene mutation induced by genome editing
Steroidogenesis is a key process of hormonal synthesis that lead to sexual differentiation and maintenance, gonad development and maturation, reproduction and fertility. Sex hormones are steroid hormones that are produced mainly by gonads. Cyp19a1a, which is a gene encoding aromatase that catalyzes the conversion of androgen to estrogen, was targeted by TALEN and CRISPR/Cas9 in zebrafish. All cyp19a1a−/− female mutants reversed to males and male sex differentiation was delayed (Lau et al. 2016; Wu et al. 2020; Yin et al. 2017). Conversely, Wu et al. (2020) used CRISPR/Cas9 to knock out dmrt1, rescuing all male phenotype of cyp19a1a homozygous mutants. In tilapia, cyp19a1a-deficient F0 fish displayed partial female to male sex reversal in XX fish using TALEN (Li et al. 2013). Another important gene in steroid biosynthesis, cyp17a1, cyp17a1−/− zebrafish were all male and lost mating behavior, but mutants showed insufficient androgen levels. Moreover, the expression of amh was downregulated, whereas that of sox9a was upregulated. Gene knockout in this study provides a model of cyp17a1−/− mutant to elucidate that AR interacts with SOX9A to regulate amh transcription (Shu et al. 2020; Zhai et al. 2017). 11-ketotestosterone (11-KT), which is a male-specific androgen, is vital for testis development, spermatogenesis, and reproduction. The conversion of testosterone to 11-KT is catalyzed by 11β-hydroxylase encoded by cyp11c1. Targeted disruption of cyp11c1 in zebrafish using CRISPR/Cas9 caused reduced 11-KT, defective mating behaviors, but produced mature gametes. Delayed and prolonged juvenile ovary-to-testis transition was observed in cyp11c1−/− males during testis development. Blocked egg spawning and germinal vesicle breakdown were observed in cyp11c1−/− females (Zhang et al. 2020a, b).
Among hormone genes, single, double, and triple mutation of esr2a, esr2b and esr1 (three nuclear estrogen receptors, nERs) in zebrafish were constructed by CRISPR/Cas9. Esr2a mutants appeared enlarged with deformed chorion. Esr2a; esr2b double mutants and esr2a; esr2b; esr1 tri-mutants blocked folliculogenesis that resulted in sex reversal to males. Other mutants were normal compared with the wild type. Moreover, CRISPR/Cas9-induced esr2a mutants medaka caused female infertility due to oviduct atresia. Similarly, esr1, esr2a, esr2b mutants were generated by CRISPR/Cas9 in Nile tilapia. Consistent with esr1 mutant zebrafish, esr1 mutants were normal in oogenesis and spermatogenesis, whereas ers2a mutants occurred retarded follicle growth in female and subfertility in male and ers2b mutant displayed malformation of reproductive ducts in both male and female causing infertile, which were different from zebrafish mutants. The hypothalamus-pituitary-gonad axis controls reproduction in fish. Gonadotropins secreted from pituitary and their receptors (e.g., FSH, LH, FSH receptor, and LH receptor) can also regulate the gonadal growth and development. Using TALEN, fshb zebrafish mutants exhibited a delay in ovary and testis development but caught up afterward, and were fertile finally. On the contrary, lhb zebrafish mutants presented normal gonadal development in both male and females but were infertile in the females due to a failure of oocyte maturation (Zhang et al. 2015b). In addition, delayed initiation of spermatogenesis in fshr-deficient males and retarded ovarian growth and failed follicle activation in fshr-deficient females by TALEN. And then females reversed to males. However, lhcgr-deficient zebrafish showed normal phenotype. Significantly, fshr–/– and lhcgr–/– double mutants developed into sterile males (Zhang et al. 2015a). Gonadotropin-releasing hormone (GNRH), a major neuropeptide regulator, promoted the synthesis and release of LH and FSH to regulate gonad development and reproduction. Interestingly, TALEN-mediated zebrafish gnrh3 mutants had normal gametogenesis and reproductive performance, and all gnrh3−/− were fertile (Spicer et al. 2016). Contrary to zebrafish, medaka gnrh1 mutants exhibited blocked ovulation leading to infertility (Takahashi et al. 2016). Ovulation may be controlled by the binding of progestin to nuclear progestin receptor (Pgr or nPR). In zebrafish, TALEN-induced pgr−/− female mutants failed to ovulate due to lack of functional Pgr-mediated genomic progestin signaling in the follicular cells adjacent to the oocytes. Thus, the establishment of knockout pgr zebrafish model in this study was able to assist in researching the effect of non-genomic steroid receptors on physiological processes (Zhu et al. 2015). In tilapia, pgr deficiency caused a decrease in sperm count, reduced sperm motility, and subfertility by CRISPR/Cas9 (Fang et al. 2018a, b). Androgens act by binding to androgen receptors (AR), members of the nuclear hormone receptor superfamily, which are necessary for gonad differentiation and development in vertebrates, such as spermatogenesis and oocyte maturation. Ar−/− male zebrafish by CRISPR/Cas9 exhibited female secondary sex characteristics and were unable to release sperm due to abnormal cyst formation with no central pool of spermatozoa and lack of seminiferous tubule (Crowder et al. 2018). Also, research has found that male infertility on account of defective spermatogenesis by TALEN or CRISPR/Cas9. Moreover, ar−/− female zebrafish had reduced fecundity and more immature oocytes (Tang et al. 2018; Yu et al. 2018).
TGF-β family contains significant factors related to gonadal development, sex determination, and differentiation. Gsdf (Gonadal somatic cell derived factor), a sex-determining gene in the allied species O. luzonensis Y chromosome (Myosho et al. 2012), was knocked out on the autosomal chromosome of O. latipes using ZFN and TALEN, respectively. All homozygous mutant XY gonads developed into ovaries in the early stage, but two-thirds of them developed into testes in adults, which demonstrated that gsdf gene is essential for early male development (Imai et al. 2015). Similarly, all gsdf homozygous mutant XYs constructed by TALEN were found to be female in O. latipes, and follow-up experiments testified that gsdf was the downstream gene of dmy (Zhang et al. 2016). Yan et al. (2017) used TALEN to knock out gsdf in zebrafish, and gsdf−/− female mutants accumulated a large number of immature follicles without yolk resulting in sterility. In tilapia, mutations of gsdf were generated by CRISPR/Cas9. All XY gsdf−/− mutants reversed as females, suggesting that gsdf might be downstream to dmrt1 and induced testis differentiation by inhibiting estrogen secretion (Jiang et al. 2016). Bmp15, another TGFB family member, is an oocyte-produced signal system that regulates granulosa cell to maintain female phenotype. Bmp15 mutants via TALEN appeared as an arrest of oogenesis and degenerated oocytes and granulosa cells without cyp19a1a expression produced no or reduced estrogen, which led to sex-reversion (Dranow et al. 2016). Anti-Mullerian hormone (AMH) can induce Müllerian duct regression in mammals, and is also crucial for Leydig cell differentiation and function and follicular development in adult females. Zebrafish amh mutants displayed hypertrophic gonads in both male and females, and impaired differentiation of germ cells due to their excessive proliferation (Lin et al. 2017; Zhang et al. 2020a, b). In Nile tilapia, Wang's team from Southwest University targeted amhy, a tandem duplicate of anti-Mullerian hormone with a missense SNP on the Y chromosome, by CRISPR/Cas9. Amhy XY mutants sex reversed to female fish, indicating that amhy may be a sex-determining gene (Li et al. 2015).
Sex determination and differentiation are associated also with gene encoded sex-related DNA-binding protein. It is widely acknowledged that dmrt1 is pivotal for testis development, sex determination, and differentiation in males. The first sex-determining gene found in teleost fish is dmy that is a duplication of dmrt1 on the Y chromosome of Oryzias latipes (Matsuda et al. 2002). Lu et al. (2015) used the TALEN technology to knock out dmy in O. latipes resulting in XYDMY− sex reversal female medaka. Transcriptome sequencing of the mutants revealed the mechanism of sex reversal caused by mutation of dmy in O. latipes. However, in O. dancena, loss of sox3, a gene that encodes transcription factor containing HMG-box motif which can bind to DNA, on the Y chromosome caused sex reversal in all XY, and sox3Y was finally determined to be the sex-determining gene in O. dancena (Takehana et al. 2014). Forkhead box transcriptional factor, such as foxl2, foxl3 (a duplicated copy of foxl2), is crucial for sex determination and differentiation in female. Disruption of foxl3 in medaka caused XX female ovaries filled with functional sperm using TALEN (Nishimura et al. 2015).
Many studies have been carried out in another model species zebrafish. Zebrafish mutants of the male sex-related gene dmrt1 that were constructed using CRISPR/Cas9 occurred with regressed testis lobes (Lin et al. 2017). However, loss-of-function of sox3 by CRISPR/Cas9 in zebrafish exhibited follicle development retardation and a reduced fecundity in females, which led to the downregulated expression of cyp19a1a (Hong et al. 2019). This is similar to bmp15 mutant zebrafish (Dranow et al. 2016).
Encoded sex-related DNA-binding protein genes were also widely studied in cultured fish. Wang's team have performed studies with the loss of sex-related genes dmrt1 and foxl2 in tilapia, respectively, by TALEN. Dmrt1 mutant males of F0 founders exhibited testicular regression including degenerated spermatogonia, loss of germ cells, and abnormal efferent ducts. However, sex reversal did not happen. In contrast, foxl2 mutant females of F0 founders showed varying degrees of oocyte degeneration. Moreover, the expression levels of aromatase gene and serum estradiol-17 were significantly reduced, while some foxl2 mutant females even showed complete sex reversal (Li et al. 2013). Using CRISPR/Cas9 technology, this team have created knockout mutants for sex-related genes nanos2, nanos3, dmrt1, and foxl2 in Nile tilapia with mutation rates as high as 95%. Gene mutation was effectively inherited to the F1 generation, and thereby the platform for CRISPR/Cas9 gene knockout of non-model fish was successfully constructed (Li et al. 2014). Cui et al. (2017) knocked out dmrt1 on the Z chromosome in tongue sole by TALEN technology, and found that gonads of ZZ mutants developed into intersex gonads. Here, the growth rate of dmrt1-deficient ZZ mutants was significantly faster than that of normal ZZ males.
In rainbow trout, sdY, a truncated, divergent form of interferon regulatory factor 9, is a sex-determining gene. Using ZFN, targeted sdY gene caused ovarian structure in F1 males but did not lead to a male-to-female sex reversal (Yano et al. 2012, 2014). Using CRISPR/Cas9 technology, Dan et al. (2018) knocked out pfpdz1 in the Y chromosome resulting in the differentiation of male gonads into ovaries, which demonstrated the important role of pfpdz1 in male differentiation and maintenance. Contrary to yellow catfish and Nile tilapia, female Chinese tongue sole (Cynoglossus semilaevis) grows faster than males. Mutants had male-biased sex ratios and presented no sex reversal and no female phenotype during the early juvenile stage. Recently, a study targeted mettl3, which is a gene-encoded Mettl3 methyltransferase that catalyzed m6A, using TALEN. It was demonstrated that gamete maturation was disrupted and fertility declined in F3 generation mettl3 mutants, which were obtained by the self-crossing of F2 heterozygotes with the same mutation (Xia et al. 2018). Moreover, some genes, such as wnt4a, related to reproductive duct formation. Loss-of-function of wnt4a caused reproductive duct malformation in both males and females by CRISPR/Cas9, and thus mature gametes faired to release leading to sterility. Gene knockout may contribute to the construction of sterile individuals. Germ cell transplantation techniques may be used to transplant germ cells from the endangered Chinese sturgeon (Acipenser sinensis) into sterlet (Acipenser ruthenus) with short sexual maturity, thus helping endangered species to achieve the goal of "surrogate pregnancy". The infertile sterlet, an ideal recipient in germ cell transplantation experiments, is unable to produce germ cells by itself, but only provides an environment suitable for germ cell development, and thus improving the efficiency of transplantation. Using CRISPR/Cas9 technology, sterile sterlet was successfully obtained through a knockout of dnd1 in 2019 (Baloch et al. 2019).
Most teleost fish own colorful and diverse pigment patterns. Eight different types of pigment cell have been identified in the skin of teleosts so far. Conversely, only one type has been found in mammals (Schartl et al. 2016). The pigment cells of teleosts originate from the neural crest (Sato and Yamamoto 2001) and the multipotency of neural crest cells and the diversity of pigment cells in fish make them an ideal model for studying the formation, differentiation, and migration of different types of pigment cells. The evolutionary mechanism behind the complex and diverse pigment patterns in fish is also very popular in evolutionary biology research. In addition, ornamental fish (for example Koi carp) and farmed fish with gorgeous or healthy body color are worth higher market values. Thus, genome editing technologies have been applied to pigmentation-related studies, elucidating functions, and interactions of pigment-related genes by constructing single-gene mutants or multi-gene mutants (Table 3).
Species Gene Methods Related genes in biological processes Generation References Zebrafish (Danio rerio) pcdh10a; pcdh10b TALEN; CRISPR/Cas9 Melanophore migration (pcdh10a) F2 Williams et al. (2018) thraa; thrab; thrb CRISPR/Cas9 Regulation of maturation in pigment cell lineages F2 Saunders et al. (2019) scarb1 CRISPR/Cas9 Uptake of carotenoid F2 Saunders et al. (2019) plin6 TALEN Storage and accumulation of carotenoid F2 Granneman et al. (2017) edn3a; edn3b; ednrb1a CRISPR/Cas9 Iridophore proliferation (edn3b) F2 Spiewak et al. (2018) alk; ltk; aug-α1; aug-α2; aug-β CRISPR/Cas9 Iridophore development F2 Mo et al. (2017) mc1r CRISPR/Cas9 Dorso-ventral countershading F2 Cal et al. (2019a) asip1 CRISPR/Cas9 Dorso-ventral countershading F2 Cal et al. (2019b) sox10, sox5 CRISPR/Cas9 Chromatophore differentiation F2 Nagao et al. (2018) Medaka (Oryzias latipes) sox10a; sox10b; sox5 TALEN; TILLING Chromatophore differentiation F2 Cal et al. (2019b) kitlga CRISPR/Cas9 Regulation of melanogenesis
Melanophore proliferation and migration
F2 Otsuki et al. (2020) tyr CRISPR/Cas9 Melanin biosynthesis F2 Fang et al. (2018a) Atlantic Salmon (Salmo salar L.) tyr CRISPR/Cas9 Melanin biosynthesis F0 Edvardsen et al. (2014) slc45a2 CRISPR/Cas9 Regulation of tyrosinase activity F0 Edvardsen et al. (2014) Oujiang color common carp (Cyprinus carpio var. color) mc1r CRISPR/Cas9 Regulation of melanogenesis F2 Mandal et al. (2020) White crucian carp (Carassius auratus cuvieri) tyr CRISPR/Cas9 Melanin biosynthesis F2 Liu et al. (2019b) Astyanax mexicanus oca2 CRISPR/Cas9 Melanin biosynthesis F2 Klaassen et al. (2018) Pundamilia nyererei agrp2 CRISPR/Cas9 Black stripe patterns F2 Kratochwil et al. (2018) TILLING Targeting Induced Local Lesions IN Genomes
Table 3. Phenotypes of pigment gene mutation induced by genome editing
Single-gene knockout contributes to the study of the functions of key genes involved in fish body color formation. In 2014, Edvardsen et al. (2014) knocked out pigment-related genes tyrosinase (tyr) and solute carrier family 45, member 2 (slc45a2) in Atlantic salmon (Salmo salar L.), respectively. This resulted in pigment lost to varying degrees in the F0 generation, and was achieved with the application of CRISPR/Cas9 technology to cold water marine species for the first time. Knockout of tyr introduced by CRISPR/Cas9 technology in O. latipes resulted in a red-eyed albino mutant medaka with colorless melanophores, which demonstrated the important role of tyr in melanin synthesis (Fang et al. 2018a). The evolution of fish body color is closely related to the living environment. For example, Astyanax mexicanus comes in two distinct forms: a normal pigment form with eyes living on the surface, and an albino form without eyes living in caves. Here, eye degeneration is caused by the dark environment. Both oca2-mutant surface fish and hybrids of cavefish and oca2-mutant surface generated by CRISPR/Cas9 showed a phenotype similar to that of cavefish. This proves that the loss of oca2 was the main reason for the albinism of the cave fish in the evolutionary process (Klaassen et al. 2018). Various pigmentation and pattern changes of cichlid fishes are the result of radiation adaptation to different environments. The black horizontal stripes disappeared and reappeared many times during evolution. Knockout of agrp2 by CRISPR/Cas9 in Pundamilia nyererei, which is a family of cichlids with black vertical stripes, led to the appearance of black horizontal stripes. This confirms a vital role of agrp2 in modulating the appearance of black stripes in cichlids (Kratochwil et al. 2018).
Multiple gene knockouts facilitate research on the similarities and differences of homologous gene regulation mechanism in pigment cells. Iridophores are of great significance to the formation of striped patterns in zebrafish. Spiewak et al. (2018) used CRISPR/Cas9 technology to construct single-gene mutant or multi-gene mutants of endothelin genes edn3a and edn3b and their receptor ednrb1a in zebrafish. The edn3b mutants were mostly close to the phenotype of Danio nigrofasciatus, i.e., broken stripes and lack of iridophores and melanophores. The construction of knockout mutants was the foundation for further study of the relationship between the variation of edn3b cis-regulatory elements and the phenotype of D. nigrofasciatus. Single-gene mutant and multi-gene mutants of anaplastic lymphoma kinase (Alk) and leukocyte tyrosine kinase (Ltk) and their ligands augmentor-α and augmentor-β were constructed via CRISPR/Cas9. Comparison of the different mutants revealed that different ligands and receptors had specific spatiotemporal expression during the formation of iridophores in zebrafish (Mo et al. 2017). Pigment pattern is closely related to the correct migration and differentiation of pigment cells. Williams et al. (2018) constructed single-gene and double-gene knockout mutants of zebrafish pcdh10a and pcdh10b through TALEN and CRISPR/Cas9 technologies. It is demonstrated that deletion of pcdh10a resulted in abnormal migration of melanophore precursors; deletion of pcdh10b led to somatic defects. Double knockout of pcdh10a and pcdh10b resulted in an increasing abnormal migration of melanophore precursors and embryonic lethality. Nagao et al. (2018) used TALEN, CRISPR/Cas9, and Targeting Induced Local Lesions IN Genomes (TILLING) to construct single-gene mutant and multi-gene mutants of sox5 and sox10 in zebrafish and medaka. Through comparative analysis of the phenotypes, this study showed that different interaction patterns of sox5 and sox10 in zebrafish and medaka played crucial roles in the specification and fate determination of the pigment progenitor cells differentiating into different pigment cells.
Zebrafish is one of the model organisms with plenty of advantages, such as transparent embryo, short sexual maturity cycle, strong reproductive capacity, small size, and ease with feeding. Therefore, it is an ideal experimental system for drug screening, environmental monitoring, and research on the function of various human disease-related genes. The establishment of zebrafish disease models by genome editing technology has great potential to clarify the pathogenesis of nervous system, cardiovascular, and bone diseases.
Alzheimer's disease (AD), which is a neurodegenerative disease, is the result of excessive aggregation of amyloid protein in the brain caused by beta-site APP cleaving enzyme (BACE1) hydrolysis of amyloid precursor protein (APP). Van Bebber et al. (2013) used ZFN technology to knock out bace1 to construct a zebrafish AD disease model resulting in hypomyelination in the peripheral (PNS) but not the central nervous system (CNS) of the mutants. This model provides a way to characterize the effect of BACE1 inhibitors in vivo. In terms of the research related to human polycystic ovary syndrome (PCOS) caused by endocrine and metabolic abnormalities, Yan et al. (2017) employed TALEN technology to knock out gsdf in zebrafish. Here, the mutants showed similarities to the PCOS phenotypes, such as the accumulation of immature antral follicles, ovulation disorders, excessive androgen secretion, and obesity. Also, the gene knockout zebrafish model has been applied to human bone disease research. Thus, Gao et al. (2017) used the CRISPR/Cas9 technology to knock out the mapk7 gene for a zebrafish disease model of adolescent idiopathic scoliosis (AIS). Here, the mutants displayed scoliosis, indicating that mapk7 may be the causative gene of AIS. Zhang et al. (2017) constructed the zebrafish disease model of atp6v1h mutant by CRISPR/Cas9 mediated gene knockout, which led to lethality in homozygous mutants. In contrast, the heterozygous mutants showed reductions in the number of mature calcified bone cells, bone mineral density and bone mass leading to curved vertebra. This was basically consistent with the phenotype of human osteoporosis, indicating that the atp6v1h mutation might be one of the causes of osteoporosis. Many diseases are induced by single-base pair mutations. For example, the rare human genetic syndrome, i.e., Cantú syndrome (CS, ) which leads to cardiovascular disorders, is caused by the missense mutations of the kcnj8 or abcc9 genes. Tessadori et al. (2018) constructed the knock-in lines of kcnj8 and abcc9 gene missense mutation in zebrafish by CRISPR/cas9 technology with specific oligonucleotide sequence as template (F0 generation editing efficiency of all lines was as high as 75—100%). The zebrafish model with the same CS phenotype was successfully obtained further achieved the purpose of single base precise editing.