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Volume 3 Issue 3
Aug.  2021
Article Contents

Citation:

The application of genome editing technology in fish

  • Corresponding author: Jianguo Lu, lujianguo@mail.sysu.edu.cn
  • Received Date: 2020-06-17
    Accepted Date: 2021-01-11
    Published online: 2021-05-27
  • Edited by Xin Yu.
  • The advent and development of genome editing technology has opened up the possibility of directly targeting and modifying genomic sequences in the field of life sciences with rapid developments occurring in the last decade. As a powerful tool to decipher genome data at the molecular biology level, genome editing technology has made important contributions to elucidating many biological problems. Currently, the three most widely used genome editing technologies include: zinc finger nucleases (ZFN), transcription activator like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR). Researchers are still striving to create simpler, more efficient, and accurate techniques, such as engineered base editors and new CRISPR/Cas systems, to improve editing efficiency and reduce off-target rate, as well as a near-PAMless SpCas9 variants to expand the scope of genome editing. As one of the important animal protein sources, fish has significant economic value in aquaculture. In addition, fish is indispensable for research as it serves as the evolutionary link between invertebrates and higher vertebrates. Consequently, genome editing technologies were applied extensively in various fish species for basic functional studies as well as applied research in aquaculture. In this review, we focus on the application of genome editing technologies in fish species detailing growth, gender, and pigmentation traits. In addition, we have focused on the construction of a zebrafish (Danio rerio) disease model and high-throughput screening of functional genes. Finally, we provide some of the future perspectives of this technology.
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The application of genome editing technology in fish

    Corresponding author: Jianguo Lu, lujianguo@mail.sysu.edu.cn
  • 1. School of Marine Sciences, Sun Yat-Sen University, Zhuhai 519082, China
  • 2. Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Zhuhai 519080, China

Abstract: The advent and development of genome editing technology has opened up the possibility of directly targeting and modifying genomic sequences in the field of life sciences with rapid developments occurring in the last decade. As a powerful tool to decipher genome data at the molecular biology level, genome editing technology has made important contributions to elucidating many biological problems. Currently, the three most widely used genome editing technologies include: zinc finger nucleases (ZFN), transcription activator like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR). Researchers are still striving to create simpler, more efficient, and accurate techniques, such as engineered base editors and new CRISPR/Cas systems, to improve editing efficiency and reduce off-target rate, as well as a near-PAMless SpCas9 variants to expand the scope of genome editing. As one of the important animal protein sources, fish has significant economic value in aquaculture. In addition, fish is indispensable for research as it serves as the evolutionary link between invertebrates and higher vertebrates. Consequently, genome editing technologies were applied extensively in various fish species for basic functional studies as well as applied research in aquaculture. In this review, we focus on the application of genome editing technologies in fish species detailing growth, gender, and pigmentation traits. In addition, we have focused on the construction of a zebrafish (Danio rerio) disease model and high-throughput screening of functional genes. Finally, we provide some of the future perspectives of this technology.

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Introduction
  • Genome editing technology, which is a reverse genetic technology, is an epoch-making method that enables us to manipulate the genetic material of a living organism precisely. This is compared to "molecular scissors" cutting the targeted gene sequence directionally. In pace with the development of high-throughput sequencing technology, fish genome data have increased exponentially. So far, genomes of nearly 100 of fish species have been sequenced, including the genomes of Cynoglossus semilaevis (Chen et al. 2014), Cyprinus carpio (Xu et al. 2014), Larimichthys crocea (Ao et al. 2015), Hippocampus comes (Lin et al. 2016) and Ctenopharyngodon idellus (Wang et al. 2015). These extensive genome data sets and the availability of genome editing technologies make it possible to decipher the functional genes in many fish species for both science and applications.

    Genome editing technology can cleave targeted genes on the genome to cause DNA double-strand breaks. Damaged DNA is repaired by activating the endogenous DNA repair mechanisms, including non-homologous end joining (NHEJ) and homology directed repair (HDR). In the presence of homologous donor DNA, activated HDR uses homologous donor DNA as the template to repair damaged DNA, whereas NHEJ makes repairs by ligating DNA ends together without a homologous template. When all alleles in the cell are broken, gene knock-in may be realized by providing damaged DNA with an artificially designed homologous template using HDR. Undoubtedly, there would be high-frequency indels or replacements resulting in frameshift mutations using NHEJ, thus changing the gene function and achieving the purpose of gene knockout. Compared with RNA interference (RNAi) technology, genome editing technology can permanently change the genetic code at the DNA level. As a new technology emerging in the last decade, its rapid innovation will be conducive to the utilization of fish genome resources. Specifically, genome editing in fish makes it easier for us to identify gene functions, illuminate molecular mechanisms of biological processes, obtain desired economic traits, and construct zebrafish (Danio rerio) disease models.

Development of genome editing technologies
  • Since the discovery of the DNA double-helix structure in the last century, researchers have been exploring methods of direct genetic modification. In 1979, Scherer and Davis (1979) used homologous recombination to replace his3 on chromosome 15 of Saccharomyces cerevisiae. From the late 1980s to the early 1990s, researchers discovered that cells are capable of repairing double-strand breaks (DSB) spontaneously via two endogenous repair mechanisms: NHEJ and HDR (Rouet et al. 1994; Rudin et al. 1989). Therefore, targeted DNA damage repair has become the focus of gene editing technology. Meanwhile, it has been reported that zinc-finger protein (ZFP) recognizes 3–4 bp nucleotide sequences specifically (Chevalier et al. 2002; Miller et al. 1985; Pavletich and Pabo 1991). Researchers fused ZFPs to the catalytic domain of Fok Ⅰ endonuclease to form ZFNs, and implemented it in a variety of plants and animals, including the fruit fly (Drosophila melanogaster) (Bibikova et al. 2002, 2003), rat (Rattus norvegicus) (Geurts et al. 2009), zebrafish (Doyon et al. 2008; Meng et al. 2008), and corn (Zea mays) (Shukla et al. 2009). Compared with homologous recombination, zinc finger nucleases (ZFNs) achieve a high editing efficiency of ~ 30%. However, the ZF domain exhibits a context-dependent effect between adjacent modules, and it is impossible to design a specific and efficient ZFN for any sequence. Plus, due to the toxicity induced by high off-target effects and the high cost of modular assembly, ZFN technology has been gradually replaced by the second-generation nuclease technology, which is transcription activator-like effector nucleases (TALEN) technology. TAL effectors (TALEs) were first discovered in the plant pathogenic bacterial genus Xanthomonas as a toxic factor (Bonas et al. 1989), which is secreted by the bacterial type Ⅲ secretion system. TALEs would result in plant disease as it binds to specific promoter elements of the susceptible gene, and activates the gene transcription in the nucleus. In 2009, the code for nucleotide sequence corresponding to the TALE repeat variable di-residue (RVD) was deciphered (Boch et al. 2009; Moscou and Bogdanove 2009). TALEs with characteristic of DNA binding specificity were officially applied to gene editing technology. Similar to ZFN, TALENs were formed by the fusion of TALEs to the catalytic domain of Fok Ⅰ endonuclease, a complex which could be targeted to specific sites cutting double-stranded DNA (Christian et al. 2010). The emergence of TALEN technology has once again expedited the development of gene editing technology, and it was selected as among the top ten scientific breakthroughs in 2012 by Science. TALEN technology was then quickly applied to research on gene function studies of various animals and plants, such as the rat (Rattus nrvegicus) (Tesson et al. 2011), pig (Sus scrofa) (Carlson et al. 2012), Xenopus laevis (Lei et al. 2012), fruit fly (Drosophila) (Liu et al. 2012), zebrafish (Huang et al. 2011), and rice (Oryza sativa) (Li et al. 2012).

    Early in 1987, a peculiar sequence located at the 3′ end flanking region of the iap gene was discovered in Escherichia coli, and had a repeat sequence containing a dyad symmetry of 14 base pairs (Ishino et al. 1987). Later, this sequence was found in ~ 90% archaea and 40% bacteria (Groenen et al. 1993; Hoe et al. 1999; Mojica et al. 1995, 2000). It was named clustered regularly interspaced short palindromic repeats (CRISPR) according to its characteristics that the repeat sequence was interspaced by the spacer sequence (Jansen et al. 2002a), most of which in CRISPR are derived from viruses and plasmids (Bolotin et al. 2005; Mojica et al. 2005; Pourcel et al. 2005). Besides CRISPR arrays, the other crucial tool is Cas protein encoded by CRISPR-associated (cas) genes that is located adjacent to the CRISPR locus (Jansen et al. 2002b). A hypothesis was proposed that the CRISPR/Cas system could defend against exogenous DNA sequence (viruses and plasmids) invasion (Makarova et al. 2006), which was then proved through infecting Streptococcus thermophilus with lytic phages, suggesting that the CRISPR/Cas system is the adaptive immune system in bacteria (Barrangou et al. 2007). In 2008, CRISPR RNAs (crRNA) was shown to direct Cas proteins to target DNA (Brouns et al. 2008).

    The widespread CRISPR/Cas systems contain mainly the type Ⅱ Cas9 system, type Ⅴ Cas12 system, and type Ⅵ Cas13 system. Cleavage mechanisms and identification of nucleic acid types are distinct in different CRISPR/Cas systems, among which the CRISPR/Cas9 system has been extensively explored. In 2011, Emmanuelle Charpentier's team found RNase Ⅲ and Cas9 are required for crRNA maturation. Cas9, which is an endonuclease, targets the gene with the assistance of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) recognizing the spacer and the protospacer adjacent motif (PAM), and then cleaves DNA to produce blunt ends (Deltcheva et al. 2011). Subsequently, this is repaired through NHEJ and HDR endogenous repair mechanisms (Deltcheva et al. 2011; Deveau et al. 2008; Garneau et al. 2010; Gasiunas et al. 2012; Jinek et al. 2012; Mojica et al. 2009). CRISPR/Cas9 technology launched the revolution of the third generation of gene editing technology, which is widely used owing to its simple operation and precise gene targeting. In 2013, Feng Zhang's team demonstrated for the first time that CRISPR/Cas9 technology could be applied to mammalian cells (Cong et al. 2013; Ran et al. 2013). Other CRISPR/Cas systems have been developed, and include Cas12 and Cas13. The Cas12 system targets DNA, which is consistent with Cas9, whereas the Cas13 system targets RNA. The new variants based on CRISPR/Cas9 were engineered, especially using base editing technologies that enabled the conversion of the specific base into another. Recently, a variant of Streptococcus pyogenes Cas9 (SpCas9) have been developed, a near-PAMless SpCas9 variant called SpRY, which has overcome the limitations of specific PAM (e.g., NGG) and expanded the range of target sites (Walton et al. 2020). The developments in gene editing have been greatly accelerated by the advent of programmable nucleases from concept to practice, and enabled scientists to maneuver literally any gene in a wide variety of cell types and species. In short, the era of gene editing technology has arrived (Fig. 1).

    Figure 1.  Schematic chart of genome editing technology. REPAIR RNA Editing for Programmable A to I Replacement, RESCUE RNA Editing for Specific C to U Exchange

    Aquatic products are the world's third largest source of animal protein. Fish provides an economical and high-quality animal protein. Therefore, the application of gene editing technology in fish genetics so as to obtain more and more high-quality fishery resources has a positive effect on the development of fisheries. In addition, fish are evolutionarily connected, and the differentiation of gene pool and gene function is complicated after two rounds of genome duplication in vertebrate ancestors and a third round of genome duplication in bony fish. Gene editing technology can dig deep into the information behind genome data and verify gene function and evolutionary mechanism from the molecular level. From 2008 to 2013, ZFN, TALEN, and CRISPR/Cas9 were used to model zebrafish as experimental subjects to implement gene editing successfully in fish for the first time. Then for the first time, the application of ZFN and TALEN in Pelteobrus fulvidraco achieved gene editing of cultured fish. In 2013, CRISPR/Cas9 was applied successfully in tilapia (Oreochromis niloticus) and Atlantic salmon (Salmo salar L.). So far, gene editing technology has been successfully applied to economically valuable fish, such as tilapia, carp (Cyprinus carpio), grass carp (Ctenopharyngodon idellus), Atlantic salmon, and tongue sole (Cynoglossus semilaevis). Moreover, mutations in germline cells are transmitted to offspring. From effective gene knockout to theoretical research on sex, growth, development, and immunity, to genetic breeding for disease resistance, stress tolerance, rapid growth or sterility traits, the application of gene editing technology in fish has gradually matured, and provides help for promoting the breeding of excellent aquatic varieties.

Principles and classification of gene editing technology

    ZFN technology

  • ZFN is composed of two parts: DNA-cleavage domain of Fok Ⅰ endonuclease and DNA-binding zinc finger domain. The zinc finger protein consists of tandem repeating Cys2/His2 zinc fingers. Each zinc finger (ZF) contains about 30 amino acids, forming two anti-parallel β sheets and an α helix structure (Beerli and Barbas 2002). Four amino acid residues (− 1, + 2, + 3, + 6) on the surface of α helix. − 1, + 3, + 6 positions bind to the continuous 3 bp on the coding strand, respectively, and + 2 position recognizes a neighboring nucleotide on the non-coding strand (Klug 2010). Tandem array of ZF modules recognizes the longer DNA sequence of 9–18 bp in length (Liu et al. 1997). The cleavage domain of Fok Ⅰ endonuclease can only be activated after dimerization (Schornack et al. 2006). Thus, it is necessary to design three or four ZF tandem modules to form two zinc finger arrays (ZFAs) according to the target sequences on the sense strand and antisense strand, respectively. Moreover, the distance of two target sites on the double-strand DNA should be suitable for cleavage (6–8 bp), so as to guide Fok Ⅰ to form dimers at the target sites. Subsequently, the dimerized Fok Ⅰ cleaves DNA and causes DSB, thereby enabling targeted editing. However, if natural modules or modified ZF module are assembled to form ZFAs, it cannot entirely comply with the activity and specificity of single ZF to recognize DNA. The interaction between adjacent zinc fingers affected the binding activity of the recombinant ZFA, which is called context-dependent interactions between neighboring fingers (Fig. 2a). Therefore, various construction strategies, e.g., modular assembly (MA), Oligomerized Pool Engineering (OPEN), context-dependent assembly (CoDA) and two-finger archive have been developed to obtain highly active ZFAs (Bhakta and Segal 2010; Gupta et al. 2012; Maeder et al. 2008; Sander et al. 2011).

    Figure 2.  The schematic diagram of gene editing mechanism. a ZFN. b TALEN. c CRISPR locus. d CRISPR/Cas9

  • TALEN technology

  • TALEN is created by fusion of the DNA recognition domain of TALE protein and the cleavage domain of Fok Ⅰ nuclease. The structure of TALE protein includes three parts: N-terminal region, which contains type Ⅲ translocation system required for secretion; C-terminal region which contains nuclear localization signal (NLS) and activation domain (AD); and central repeat domain which can specifically bind to DNA. The central region is composed mostly of 12–27 highly conserved repeats, each of which includes 33–35 amino acids. They are basically the same, besides of the hypervariable residues at the 12th and the 13th position, also known as repeat variable di-residue (RVD). Repeats with different RVDs recognize different bases, and there is a one-to-one correspondence between RVDs and nucleotides in the target DNA sequence, forming a simple code (Boch et al. 2009; Cermak et al. 2011). NN, NI, HD, and NG recognize guanine (G), adenine (A), cytosine (C), and thymine (T), respectively. Also, NH, NK, and HN recognize guanine (G). Most TALEs identify target sites that begin with a T, which is required for TALEs activity (Cermak et al. 2011; Cong et al 2012). Similar with ZFN, two TALEs target DNA sequence, respectively, and the dimerized Fok Ⅰ cleaves DNA sequence (Fig. 2b). Because it is tedious to clone the repeat arrays, "Golden Gate" molecular cloning (Cermak et al. 2011), high-throughput solid-phase assembly (Briggs et al. 2012; Reyon et al. 2012) and ligation-independent cloning techniques (Schmid-Burgk et al. 2013) have been developed to simplify the construction steps and take a shorter period of time. "Golden Gate" is currently the mainstream strategy of TALE repeat arrays assembly based on the property of Bsa I.

  • CRISPR/Cas technology

  • The emergence of CRISPR/Cas systems originated from adaptive immunity in prokaryotes. The CRISPR locus in bacteria and archaea that plays a key role in defense is composed of the cas gene operon, the leader sequence and CRISPR array, whereas the CRISPR array consists of short and conserved DNA repeats (repeats) and highly variable sequences (spacers) interspacing repeats. The leader sequence included transcription initiation sites of CRISPR array, and is located between the cas gene operon and CRISPR array. Otherwise, the tracrRNA gene that is in the vicinity of CRISPR array and the cas gene (Fig. 2c). Type Ⅱ Cas9 system is currently the most widely used CRISPR/Cas system. As foreign DNA (such as bacteriophage) invades the bacterial cell, the Cas1 and Cas2 protein, encoded within the cas gene operon, will form Cas1-Cas2 complex that intercept the foreign DNA fragment, which is referred to as the protospacer. Acquisition of this protospacer is based on recognition of the protospacer adjacent motif (PAM) on foreign DNA by the Cas1-Cas2 complex. Then, the Cas1-Cas2 complex is loaded with the protospacer to dock leader-proximal repeat (the proximal end of the first repeat) with the assistance of the leader-anchoring site (LAS) of recognition. Finally, this complex cleaves the first repeat sequence 3'end of two strands to allow the protospacer insert into the cleavage site to form a new spacer (Anders et al. 2014; Nishimasu et al. 2014; Nunez et al. 2014). When foreign DNA invades the bacterial cell again, the promoter at the leader initiates transcription that CRISPR transcribes into pre-CRISPR RNA (pre-crRNA), and tracrRNA gene transcribes into tracrRNA which is a small trans-encoded RNA and can be partly complementary to the repeats followed by the forming of pre-crRNA and tracrRNA complex via base pairing. The pre-crRNA is processed into crRNA by RNase Ⅲ in the presence of Cas9 (Deltcheva et al. 2011). Each crRNA corresponds to a spacer. The tracrRNA-crRNA complex directs cas9 to the foreign DNA by the recognition of Cas9 Pam-interacting (PI) domain to PAM sequences and the formation of a 20 bp crRNA-target DNA heteroduplex. The active Cas9 has HNH and RuvC nuclease domains and these two domains can cleave a single strand complementary to crRNA and non-complementary strand, respectively, so as to achieve the purpose of gene knockout (Gasiunas et al. 2012; Hale et al. 2009; Horvath and Barrangou 2010; Jackson et al. 2017; Nishimasu et al. 2014) (Fig. 2d). By the combination of crRNA and tracrRNA into single guide RNA (sgRNA), the CRISPR/Cas9 system may be further simplified to include only sgRNA and Cas9 artificially making the application of Cas9 system easier and faster.

    Subsequently, researchers further optimized and modified Cas9 protein to engineer-derived Cas9 nickase (Cas9n) (Ran et al. 2013) and nuclease dead Cas9 (dCas9) (Qi et al. 2013). Cas9n that can only cleave single-stranded DNA has two mutant structures, one is Cas9 (D10A) nickase formed by replacing aspartic acid with alanine at the RuvC domain, and the other is Cas9 (H840A) nickase formed by replacing histidine with alanine at HNH domain. Only when Cas9n mediated by a pair of sgRNA recognize the target site correctly can DSB form, and therefore off-target rate will decrease. The dCas9 that was inactivated in both domains by mutations can only recognize but not cleave double-stranded DNA. Similarly, researchers fused dCas9 with Fok Ⅰ. When both fusion proteins recognize the target site correctly that causes the dimerization of Fok Ⅰ, dimerized Fok Ⅰ endonuclease can exercise its function of cleaving double-stranded DNA. Due to dual recognition of two Cas9n molecules or two dCas9 molecules with Fok Ⅰ to the target site than single recognition of one Cas9 molecule, the off-target effects are reduced. In addition, dCas9 binds to transcriptional activators or inhibitors to regulate gene transcription levels (Gilbert et al. 2014). Since 2015, researchers have successively developed enhanced SpCas9 (eSpCas9) (Slaymaker et al. 2016), SpCas9-HF1 with low off-target efficiency (Joung et al. 2016), HypaCas9 formed by changing the REC3 domain (Chen et al. 2017), highly specific xCas9 (Hu et al. 2018) and other Cas9 proteins with low off-target rate.

    Cas genes and CRISPR array have evolved quickly. CRISPR/Cas systems were initially divided into three major types (Makarova et al. 2011; Makarova and Koonin 2015). Then, Makarova et al. (2015) suggested an updated two-class classification according to Cas protein composition differences and sequence divergence according to Cas protein composition differences and sequence divergence. Class 1 CRISPR/Cas systems included types Ⅰ, Ⅲ, and Ⅳ, which possess multiple effector Cas proteins. Class 2 CRISPR/Cas systems included Ⅱ, Ⅴ and Ⅵ, which possessed a single, large effector Cas protein. In 2019, new type of Cas protein was involved in the class 2 CRISPR/Cas system. As of now, the new classification includes two classes, six types, and 33 subtypes (Makarova et al. 2019). Cas12a is class 2 V-A type CRISPR effector Cas protein (Zetsche et al. 2015). Contrary to Cas9, which cuts DNA to produce blunt ends, Cas12a cut DNA or RNA to produce sticky ends, which contribute to the insertion of designed DNA fragment. Cas12a required only crRNA in length of 42–44 nt but no tracrRNA, thus making the experiment operation more convenient. The effector protein of the V-B CRISPR system is Cas12b, which can maintain optimal nuclease activity in a wide temperature range (31–59 ℃) (Teng et al. 2018). The smaller V-A Cas12f1 (Cas14a) can target single-stranded DNA (Harrington et al. 2018), and HEPN domains of Cas13a and Cas13d in type Ⅵ can target RNA cleavage (Abudayyeh et al. 2016; Konermann et al. 2018).

  • Prime editing technology

  • In 2019, David R. Liu's team developed Prime Editing technology, which can accurately insert, delete or replace the desired modified base at the target site. Prime Editing technology is a further improvement of Base Editing technology, which was reported by the same team previously (Gaudelli et al. 2017; Komor et al. 2016). Substitution types of Base Editing have limitations, whereas Prime Editing implements all 12 types of base substitutions (8 base transversions, 4 base conversions), and a maximum range of 44 base insertions and 80 base deletions. In this process, only single-strand breaks occur without DSB, and the repair mechanism no longer depends on HDR that requires a donor template. Prime Editors (PEs) is improved on the basis of CRISPR/Cas9 system. Prime Editor 1 is to transform sgRNA by adding a RNA sequence that consists of a primer complementary to the target sequence and a reverse transcription template (RT template) carrying the desired sequence at its 3' end. This RNA sequence is called prime editing guide RNA (pegRNA). Cas9 (H840A) nickase was fused with reverse transcriptase and targeted to DNA under the guidance of pegRNA. Cas9 nickase cleaves only the single strand containing PAM and the reverse transcription template is reverse transcribed into DNA that inserted into the cleaved single strand sequence. At this time, unlike DSB-activated repair mechanism, the broken single strand repairs gap and replaces the original sequence through equilibration between the edited 3' flap and the unedited 5' flap and complementary strand is also replaced through the Mismatch repair (MMR) mechanism, thus completing specific site gene editing (Fig. 3). Prime Editor 2 and Prime Editor 3 have raised editing efficiency compared to Prime Editor 1 (Anzalone et al. 2019). However, its off-target rate needs to be studied further.

    Figure 3.  The schematic diagram of Prime Editing mechanism. A prime editing complex is composed of a PE protein and a pegRNA. The PE protein contains a Cas9(H840A) nickase, and an RT domain. The pegRNA consists of a primer and an RT template. a The PE-pegRNA complex binds the antisense strand and nicks the PAM on the sense strand. b The primers on the pegRNA binds to the sense chain by base pairing. c Under the effect of reverse transcriptase, the reverse transcription inserts the designed edited sequence into the sense strand. d Flap endonuclease 1 (FEN1) resects specific 5' flap. According to equilibration between the edited 3' flap and the unedited 5' flap, 3' flap replaces the original sequence to repair the gap, and the antisense strand is also replaced

Application of genome editing technology in fish species

    Application in growth traits

  • 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.

  • Application in sex traits

  • 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).

  • Hormone and hormone receptor genes

  • 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 pathway genes

  • 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).

  • Encode sex-related DNA-binding protein genes

  • 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.

  • Other genes

  • 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).

  • Application in pigment traits

  • 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).

    SpeciesGeneMethodsRelated genes in biological processesGenerationReferences
    Zebrafish (Danio rerio)pcdh10a; pcdh10bTALEN; CRISPR/Cas9Melanophore migration (pcdh10a)F2Williams et al. (2018)
    thraa; thrab; thrbCRISPR/Cas9Regulation of maturation in pigment cell lineagesF2Saunders et al. (2019)
    scarb1CRISPR/Cas9Uptake of carotenoidF2Saunders et al. (2019)
    plin6TALENStorage and accumulation of carotenoidF2Granneman et al. (2017)
    edn3a; edn3b; ednrb1aCRISPR/Cas9Iridophore proliferation (edn3b)F2Spiewak et al. (2018)
    alk; ltk; aug-α1; aug-α2; aug-βCRISPR/Cas9Iridophore developmentF2Mo et al. (2017)
    mc1rCRISPR/Cas9Dorso-ventral countershadingF2Cal et al. (2019a)
    asip1CRISPR/Cas9Dorso-ventral countershadingF2Cal et al. (2019b)
    sox10, sox5CRISPR/Cas9Chromatophore differentiationF2Nagao et al. (2018)
    Medaka (Oryzias latipes)sox10a; sox10b; sox5TALEN; TILLINGChromatophore differentiationF2Cal et al. (2019b)
    kitlgaCRISPR/Cas9Regulation of melanogenesis
    Melanophore proliferation and migration
    F2Otsuki et al. (2020)
    tyrCRISPR/Cas9Melanin biosynthesisF2Fang et al. (2018a)
    Atlantic Salmon (Salmo salar L.)tyrCRISPR/Cas9Melanin biosynthesisF0Edvardsen et al. (2014)
    slc45a2CRISPR/Cas9Regulation of tyrosinase activityF0Edvardsen et al. (2014)
    Oujiang color common carp (Cyprinus carpio var. color)mc1rCRISPR/Cas9Regulation of melanogenesisF2Mandal et al. (2020)
    White crucian carp (Carassius auratus cuvieri)tyrCRISPR/Cas9Melanin biosynthesisF2Liu et al. (2019b)
    Astyanax mexicanusoca2CRISPR/Cas9Melanin biosynthesisF2Klaassen et al. (2018)
    Pundamilia nyerereiagrp2CRISPR/Cas9Black stripe patternsF2Kratochwil 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.

  • Genome editing in constructing zebrafish disease models

  • 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.

Other applications of genome editing in fish

    Genome editing technology in high-throughput screening of functional genes

  • With the development of high-throughput sequencing technology, massive amounts of gene sequence data have been produced. To identify the more functional genes quickly and accurately, genome editing technology may be used to generate mutations in zebrafish. With a short reproduction cycle, large number of offspring may be produced allowing the construction of a variety of zebrafish mutants. Shah et al. (2015) constructed sgRNA pools of 48 electrical synapse-related genes in eight rows and six columns, as each row or column were mixed into an sgRNA pool of 100 pg in total and injected into zebrafish embryos to specifically generate knockout mutants. The rows and columns corresponding to zebrafish with obvious electrical synapse loss were marked through phenotype, and the intersections of these rows and columns were located. In the end, two new genes related to electrical synapse were identified successfully. In the same year, Varshney et al. (2015) established a high-throughput mutagenesis pipeline (multiplexed gene targeting) based on CRISPR/Cas9 technology in zebrafish to screen functional genes. Specifically, 162 loci of 83 genes (designing multiple target sites for one gene) were targeted, and sequencing of mutation sites was using ABI or Illumina Miseq technology to screen for mutants. Then, large-scale phenotypic analysis was used to identify genes related to human hearing and vestibular function. Hearing impairment in humans is related to the loss of function of hair cells which is considered to be the mechanoreceptor in the inner ear. Zebrafish hair cells have regenerative ability, which exists not only in the inner ear, but also in the lateral line, and the hair cells (known as neuromast hair cells) are located in the of body surface. Thus, it is convenient to use them in pathological research. Pei et al. (2018) achieved a total of 254 gene mutations in zebrafish induced by retroviral insertional mutation technology and CRISPR/Cas9 technology. This approach established a high-throughput screening pipeline for mining genes related to hair cell development and regeneration; seven genes that affect hair cell regeneration were identified. Also, large-scale screening has been applied to research on retinal diseases. Eroglul et al. (2018) employed CRISPR/Cas9 technology to target > 300 genes related to retinal regeneration or degeneration, and adopted strategies of either targeting the same gene with multiple sgRNAs, or targeting multiplexed gene at the same time to screen for key genes in zebrafish retinal diseases model.

  • Application of efficient and precise genome editing technology in fish

  • Improving editing efficiency and minimizing off-target efficiency are the main goals for genome editing technology. By optimizing the type and structure of Cas protein and the number of sgRNA, researchers have developed a variety of new optimized versions of genome editing technologies, which have been applied in the model organism, zebrafish. The structure of Cas protein affects its efficiency of binding DNA. Slaymaker et al. (2016) replaced the three amino acid residues of Cas9 protein with alanine, weakening its interaction with non-target sequences, thereby reducing the off-target rate, and the modified Cas9 protein was then called eSpCas9. In the same year, Joung et al. (2016) engineered SpCas9-HF1 by changing the four sites of Cas9 protein that binds to DNA backbone for transforming the long amino acid side chain into the short chain, which could not bind to the DNA backbone; thus the off-target rate was also reduced. The two modification strategies mentioned above achieved off-target effect reduction, but also reduced the binding ability of Cas9 to the target sequence to a certain extent. Then, Chen et al. (2017) developed HypaCas9 through replacing four amino acid residues with alanine, which altered the Cas9 protein conformation and achieved the purpose of reducing the binding ability with non-target sequences without affecting the binding ability with target sequences. The evoCas9 and HiFi Cas9 (Cas9 point mutation R691A) had even lower off-target rate without changing the target efficiency (Casini et al. 2018; Vakulskas et al. 2018). Most recently, the protocol for HypaCas9 and HiFi Cas9 gene knockout in zebrafish has been published (Prykhozhij et al. 2020). In addition, Liu et al. (2019a) fused the nuclear localization signal SV40 NLS with Cas12a and applied it to zebrafish. These workers found that the improved Cas12a had high-knockout efficiency, and the off-target rate and toxicity were reduced. In terms of the amount of sgRNA, Wu et al. (2018) provided a four-guide RNA lookup table of 21, 386 genes in zebrafish, using four sgRNA and Cas9 protein complex (four-guided Cas9 RNP) to perform zebrafish embryo injection. The result was that the knockout efficiency in G0 exceeded 90%, whereas the embryo malformation rate (toxicity) was < 17%. The key gene of heart development, zbtb16a, was finally identified.

Conclusions and future perspectives
  • Genome editing technology has been widely used in fish, but most of them are limited to ZFN, TALEN and traditional CRISPR/Cas9 technology. With the development of various improved genome editing and new CRISPR/Cas systems, we are able to utilize precise genome editing technology to study gene function. However the application of these improved technologies is still relatively rare in fish. The barriers include the success rate of microinjection in different fish embryos are still low, the genome editing efficiency needs to be improved and calling for reduced the off-target effect.

    The efficiency of microinjection in fish embryos is one of the key factors affecting the success of gene editing technology. It is difficult to microinject the fish because the eggs are sticky and the hard chorion hindered the insertion of the microinjection needles. Mucus is also easily attached to the tip of the needles and causing mechanical damage of the eggs or broken needles, and thereby the injection efficiency and embryo survival rate were not satisfactory. 0.25% trypsin was found capable of debonding the viscous eggs that can significantly improve microinjection efficiency (Zhi et al. 2016). The chorion of marine fish eggs gradually hardens as development proceeds. Additionally, high internal pressure of eggs causes back flow of egg material. The loss of internal material may reduce embryo survival rate. A modified needle can prevent back flow and the end of the tip is ground to the certain angle to penetrate the chorion easily (Goto et al. 2019; Kim et al. 2019). Experimental conditions of debonding treatment of different fish mucus on eggs together with the promotion of experimental instrument accuracy leading to improved microinjection success rates need to be further explored.

    In addition, a variety of new genome editing technologies could be used in fish. For example, to reduce the off-target rate, dCas9, Cas9n dual target sequences or modified Cas9 proteins and other efficient CRISPR/Cas systems can be employed in fish. Plus, the base editing technique reported by David R. Liu's team could be used to study the effect of SNP sites on fish traits (Anzalone et al. 2019; Komor et al. 2016). However, high-quality, monosex and stress-resistant varieties obtained through gene knockout have yet to be gradually accepted by consumers. In terms of fish diseases, the CRISPR/Cas13 mediated genome editing and RNA viruses detecting shed the lights for studying viral diseases in fish. Freije et al. (2019) performed this CRISPR/Cas13 in human cell lines to successfully suppress human RNA viruses. Feng Zhang' team established the SHERLOCK platform based on Cas13, which may be used to detect RNA viruses (Gootenberg et al. 2017).

    Various life activities of organisms are closely related to precise regulation of gene expression. Gene knockout provides a simple and efficient method for studying different spatiotemporal gene expression and visually identifying gene function. However, the sole use of genome editing technology to solve biological problems has certain limitations. The combination of genome editing technology and multi-omics could pave the way for better understanding gene functions. Gene knockout may verify the functional genes identified by multi-omics analysis, and in return, multi-omics could reflect the changes of mutants at different molecular levels (such as transcription level or protein level) and metabolic level after gene knockout. Moreover, the combination of high-throughput sequencing and gene editing technology (mainly CRISPR/Cas9) exploited good strategies for the rapid screening of functional genes on a large scale, and has been applied to model organism such as mice, zebrafish, and their cells lines (Datlinger et al. 2017).

    With deeper exploration into this technology, it is reasonable to believe that genome editing technology has the potential to be applied to fish in the future to ultimately promote theoretical research and genetic breeding of excellent traits in fish species.

Acknowledgements
  • This work was supported by Guangzhou Science and Technology Project [No. 201803020017], National Natural Science Foundation of China [No. 31902427] and China Postdoctoral Science Foundation [No. 2018M631016].

Author contributions
  • JGL, WYF, JRH, and SZL reviewed literature and wrote the manuscript, improved and corrected the manuscript. The manuscript has been approved by all authors.

Compliance with ethical standards

    Conflict of interest

  • The authors declare no conflicts of interests.

  • Animal and human rights statement

  • No animal and human rights are involved in this article.

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