Cas9-mediated myostatin disruption enhances muscle mass in the olive flounder Paralichthys olivaceus

Aquaculture 512 (2019) 734336

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CRISPR/Cas9-mediated myostatin disruption enhances muscle mass in the olive flounder Paralichthys olivaceus

T

Julan Kima, Ja Young Choa, Ju-Won Kima, Hyun-Chul Kimb, Jae Koo Noha, Young-Ok Kima, Hyung-Kyu Hwangb, Woo-Jin Kimb, Sang-Yeob Yeoc, Cheul Min Ana, Jung Youn Parka, ⁎ Hee Jeong Konga, a

Biotechnology Research Division, National Institute of Fisheries Science, Busan 46083, Republic of Korea Genetics and Breeding Research Center, National Institute of Fisheries Science, Geoje 53334, Republic of Korea c Division of Applied Chemistry and Biotechnology, Hanbat National University, Daejeon 34158, Republic of Korea b

A R T I C LE I N FO

A B S T R A C T

Keywords: Paralichthys olivaceus Microinjection Genome editing CRISPR/Cas9 Myostatin Muscle mass

Myostatin (MSTN), a negative regulator of skeletal muscle development, is the major gene of interest for improving animal breeding productivity, including for aquaculture. We introduced a CRISPR/Cas9 system to disrupt Paralichthys olivaceus MSTN (PoMSTN) using microinjection. Cas9 mRNA and sgRNAs targeting the first exon of the PoMSTN gene were co-injected into embryos of P. olivaceus. On-target mutations on the PoMSTN locus were generated in somatic tissues with 75.6% efficiency, and founders harboring germline mutations were produced in the F0 generation. In the F1 generation, restriction fragment length polymorphism analysis was applied to select biallelic mutants, and heterozygous biallelic mutants with PoMSTN disruption were obtained. PoMSTN-disrupted P. olivaceus exhibited greater body thickness and increased condition factor, indicating the enhancement of muscle mass with muscle hyperplasia. Expression of PoMSTN mRNA and protein was significantly reduced in the muscle of PoMSTN-disrupted P. olivaceus, and the mRNA expression of major myogenic regulatory factors (myogenin, MyoD, and Myf5) was differentially affected in PoMSTN-disrupted mutants. These results suggest that the establishment of a CRISPR/Cas9 system in P. olivaceus provides powerful tools for related genetic studies and breeding.

1. Introduction

previous studies have indicated that inhibiting MSTN function enhances muscle growth, even in fish (Lee et al., 2009; Chisada et al., 2011). These findings have suggested strategies for increasing fish production in aquaculture through increasing growth performance by disrupting the MSTN gene in fish. Paralichthys olivaceus is one of the most commercially important fish species in Korea, China, and Japan (Fujiwara et al., 2007). More than 37,000 tons were produced in 2018, worth approximately 422 million USD, or 14.9% of the total aquaculture value of Korea (Korean Statistical Information Service, 2019). To improve the productivity of P. olivaceus, selective breeding technology has been applied to control economically important traits such as growth rate, disease resistance, and body shape (Ogata et al., 2002; Fuji et al., 2006; Fuji et al., 2007; Kim et al., 2011). Because the whole genome and transcriptome of P. olivaceus have been published, the next step will be to understand the information encoded by the genome sequence (Shao et al., 2017). Transgenic and non-transgenic genetic engineering technology based

Myostatin (MSTN, also known as GDF-8), a member of the transforming growth factor β (TGF-β) superfamily, functions as a negative regulator of skeletal muscle development and growth (McPherron et al., 1997; Kambadur et al., 1997). Mammalian MSTN is primarily expressed in skeletal muscle, and MSTN deficient animals exhibit visibly distinct muscular hypertrophy or hyperplasia, commonly known as doublemuscled phenotypes, in humans, cattle, pigs, and sheep (Kambadur et al., 1997; Clop et al., 2006; Schuelke et al., 2004; Wang et al., 2017). In fish, however, MSTN has been shown to be expressed in a wide range of tissues, in addition to skeletal muscle, and was suggested to play more diverse roles than mammalian MSTN (Rodgers and Weber, 2001). Recently, MSTN was reported to be involved in the response to viral infection in fish, orange-spotted grouper (Epinephelus coioides), but the study of the diverse function of MSTN is still lacking in fish (Chen et al., 2017). Despite the contrast between mammalian MSTN and fish MSTN,

⁎ Corresponding author at: Biotechnology Research Division, National Institute of Fisheries Science (NIFS), 216 Gijanghaean-ro, Gijang-eup, Gijang-gun, Busan 46083, Republic of Korea. E-mail address: [email protected] (H.J. Kong).

https://doi.org/10.1016/j.aquaculture.2019.734336 Received 18 April 2019; Received in revised form 20 July 2019; Accepted 22 July 2019 Available online 23 July 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.

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mMESSAGE mMACHINE kit (Thermo Fisher Scientific, Waltham, MA, USA).

on the delivery of DNA, RNA, or proteins have been prompted to speed up the process of basic and applied research in fish (Gong et al., 2003; Pohajdak et al., 2004; Kishimoto et al., 2018). However, the hard chorion of the P. olivaceus embryo makes gene delivery complicated (Goto et al., 2019). This issue must be overcome to enable the exploration of developing transgenic or genome-edited P. olivaceus. Targeted genome editing can generate mutations at specific genomic loci via either error-prone non-homologous end joining (NHEJ) or high fidelity homologous recombination (HR) DNA repair pathways following the induction of double strand breaks (DSB) (Chandrasegaran and Carroll, 2016). Recently, the clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/ Cas9) system has emerged as a powerful tool for genome editing with both high precision and high efficiency (Wang et al., 2016). The CRISPR/Cas9 system uses single-guide RNA (sgRNA) complementary to the target DNA sequence, and Cas9 nuclease is programmed to bind to the DNA when the protospacer adjacent motif (PAM) sequence is on the forward strand to cleave the desired genomic site (Kleinstiver et al., 2015). Compared to the traditional knockdown strategy, CRISPR/Cas9induced genome editing is significantly more efficient in disrupting target genes in several aquaculture species, including channel catfish (Ictalurus punctatus), Atlantic salmon (Salmo salar), common carp (Cyprinus carpio), and red sea bream (Pagrus major) (Kishimoto et al., 2018; Khalil et al., 2017; Edvardsen et al., 2014; Zhong et al., 2016). In the case of Atlantic salmon, the efficiency was high enough to induce biallelic mutations in the first (F0) generation (Edvardsen et al., 2014). Even though most F0 individuals exhibit genetic mosaics, homozygous mutants can be obtained in the F1 or F2 generations. Genome editing technology is regarded as a powerful tool to speed up trait development and fish breeding (Ye et al., 2015). The P. olivaceus MSTN (PoMSTN) gene has been isolated and characterized previously (Zhong et al., 2008). Genomic organization of the PoMSTN gene and its expression pattern were identified. However, the role of PoMSTN has been suggested to involve in varied physiological processes including skeletal muscle growth inhibition, and remains to be elucidated. In this study, we edited the PoMSTN gene using the CRISPR/Cas9 system based on the microinjection into 1-cell or 2cell stage embryos of P. olivaceus. PoMSTN gene disruption was demonstrated in both somatic and germ cells of the F0 generation and a mutant allele of F0 was successfully transmitted to F1 offspring. We evaluated the growth performance and analyzed the expression of PoMSTN and transcript levels of three myogenic regulatory factors (MRFs; myogenin, MyoD, and Myf5) in the muscle tissues of the WT and PoMSTN-disrupted mutants. We established a PoMSTN-disrupted P. olivaceus breed in the F1 generation using a CRISPR/Cas9 system for genome editing.

2.2. Artificial fertilization of P. olivaceus P. olivaceus were raised at the Genetics and Breeding Research Center, National Institute of Fisheries Science (NIFS; Geoje, Republic of Korea) and maintained in 10-ton flow-through tanks at 20 ± 0.3 °C under a natural photoperiod. During the spawning season, mature eggs were obtained by gently squeezing the abdomens of ovulated females and were stored at room temperature. Semen was collected by gently massaging the abdomens of males with particular attention to prevent exposure to seawater, which causes sperm activation, and stored at 4 °C until artificial fertilization. Artificial fertilization was accomplished by adding 500 μL sperm to 10 mL eggs and gently swirling on a petri dish. After 1 min, seawater was added to the eggs and mixed together to activate the sperm. Eggs were rinsed carefully with seawater, and floating eggs were collected. Animal experiments were conducted in accordance with the Animal Protection Act of the Ministry of Agriculture, Food and Rural Affairs, Republic of Korea, and were approved by the Institutional Animal Care and Use Committee, NIFS. 2.3. Microinjection The fertilized embryos were injected on a microinjection plate. The embryos were aligned and held in square-shaped troughs for a width of 0.9 ± 0.05 mm and a depth of 0.9 ± 0.05 mm made with a plastic mold in 2% agarose. A borosilicate glass capillary tube (World Precision Instruments, Inc., Sarasota, FL, USA) was pulled with a micropipette puller device (Narishige, Tokyo, Japan) to produce a glass microinjection needle. The end of the tip was melted slightly in a loop of platinum wire to form a brake to prevent backflow of cytoplasm from the embryo and ground with a micropipette grinder (Narishige) to permit smooth penetration into the cytoplasm. sgRNAs were microinjected at a final concentration of 1 μg/μL together with 1–1.5 μg/μL Cas9 mRNA, 200 mM KCl, and phenol red solution (Sigma-Aldrich, St. Louis, MO, USA) into 1-cell or 2-cell stage embryos using an air pressure microinjector (PicoPump; World Precision Instruments, Inc.). After microinjection, embryos were incubated and reared in seawater tanks with continuous aeration at 18 °C until hatching. 2.4. Fish breeding Hatched larvae were firstly fed enriched rotifers, Brachionus plicatilis, at a density of 3–8 individuals/mL from 3 to 18 days post-hatching (dph). Brine shrimp, Artemia salina, nauplii were fed to larvae at a density of 0.3–1.5 individuals/mL from 13 to 32 dph. The feed density was maintained twice a day. The fish were gradually switched to pelleted feed (Love Larva; Hayashikane Sangyo, Shimonoseki, Yamaguchi, Japan) of the appropriate size at around 18 dph, then hand-fed to satiation with a commercial extruded diet (Jeil Feed, Daejeon, Korea) multiple times per day to ensure sufficient food supply. Filtered seawater was supplied with constant aeration in the rearing tank and the water temperature was maintained at 20 ± 0.3 °C using temperature control equipment (Aquatron; Yuwon Engineering Co., Korea) from hatching (0 dph). The photoperiod was controlled to be as close as possible to natural daylight (or the natural aquatic environment) using fluorescent lighting on an electronic timer (Yuwon Engineering Co., Korea) in the indoor rearing tanks.

2. Materials and methods 2.1. Preparation of sgRNA and Cas9 mRNA sgRNAs (single guide RNAs) targeting the P. olivaceus MSTN gene (GenBank Accession No. DQ997779) were designed using the online CRISPR RGEN Tools (www.rgenome.net/cas-designer). To identify the efficiency of sgRNAs, in vitro activity of these sgRNAs was analyzed with Cas9 nuclease. Two PoMSTN sgRNAs (PoMSTN-sgRNA-#1, 5′-GCT GCT GAT GGT GCG TCT CTT GG-3′; PoMSTN-sgRNA-#3, 5′-TCT GAG CAA ACT GCG AAT GAA GG-3′) were synthesized and a plasmid encoding Cas9, pRGEN-Cas9-CMV, was purchased from ToolGen (Seoul, Korea). A PCR amplicon containing two PoMSTN sgRNA target sites was synthesized using specific primers (PoMSTN-Amp-F, 5′-CTC ACA GTC TGC GTC CCT TT-3′; PoMSTN-Amp-R, 5′-CAC GTC GTA CTG GTC GAG AA-3′) and was treated with recombinant Cas9 nucleases and each PoMSTN sgRNA, or untreated. Each reaction was separated using agarose gel electrophoresis. After linearization with XhoI (Roche, Basel, Switzerland), the Cas9 template was in vitro transcribed using the T7

2.5. Mutation Detection (T7 endonuclease I (T7E1) assay, restriction fragment length polymorphism (RFLP), and Sanger sequencing of PCR amplicons) Genomic DNA was extracted from individual fin-clip samples at 185 dph or embryos at 1 day post-fertilization (dpf) using DNeasy Blood & 2

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Aldrich), and anti-DAPI (Calbiochem, San Diego, CA, USA). The secondary antibodies used were Alexa Fluor 488-conjugated goat antimouse, Alexa Fluor 488-conjugated goat anti-rabbit IgG, Alexa Fluor 405-conjugated goat anti-mouse IgG, and Alexa Fluor 568-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR, USA). Digital photos were taken using a confocal laser scanning microscope (LSM510; Carl Zeiss, Oberkochen, Germany).

Tissue Kits (Qiagen, Venlo, The Netherlands) or PrimePrep Direct PCR Reagent (GenetBio, Daejeon, Korea) following the manufacturer's instructions. The genomic regions surrounding the CRISPR target site were amplified using PCR with the following primers: PoMSTN-T7E1-F (5′-GCA TCT GTC TCA CAT TGT GC-3′), PoMSTN-T7E1-R (5′-TTT GGG CAG GAG CTG CTT CA-3′), PoMSTN-seq-F (5′-CCT CCC ACC AGA GAA AAT GC-3′), and PoMSTN-seq-R (5′-TGA CCA CAT CCC TGT TGT CAT C-3′). The PCR amplification procedure was as follows: initial denaturation for 5 min at 95 °C, followed by 25 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s; and a final elongation at 72 °C for 5 min. To detect mutations in PCR products using a T7E1 assay, the PCR products were denatured and slowly re-annealed to facilitate heteroduplex formation. The procedure includes a 5-min denaturing step at 95 °C, followed by cooling to 85 °C at −2 °C/s and further to 25 °C at −0.1 °C/s. The re-annealed products were digested with T7EI (New England BioLabs, Beverly, MA, USA) at 37 °C for 30 min and separated on 3% agarose gel. For RFLP analysis, the PCR products were digested with Esp3I (Thermo Fisher Scientific) for 2 h and the cleavages of fragments were visualized on 3% agarose gel. To identify mutation sequences in each fish, purified PCR products were cloned into the pGEM T easy vector (Promega, Madison, WI, USA). For each sample, 8–11 random colonies were sequenced using universal primer T7 promoter, SP6, and an ABI3730xl automatic sequencer (Applied Biosystems, Inc., Foster City, CA, USA).

2.9. Whole-mount in situ hybridization (WISH) WISH was carried out as described by Yeo et al., 2007. Antisense riboprobes were synthesized from cDNA for EGFP (Clontech Laboratories, Inc., Mountain View, CA, USA). Images of embryos were obtained using a differential interference contrast microscope (Axioplan2, Carl Zeiss).

2.10. H&E staining For each fish, a muscle tissue sample was removed from the left dorsal muscle and the samples were placed in cryostat to be held at thermal equilibrium. Each muscle cryosection (8 μm) was stained with hematoxylin and eosin (H&E) solution. Tissue sections were dehydrated with ethyl alcohol, and then cleared in xylene and mounted with Permount (Thermo Fisher Scientific). H&E-stained images were captured with a differential interference contrast microscope (Axioplan2; Carl Zeiss).

2.6. mRNA extraction and reverse transcription PCR (RT-PCR) Total RNA was extracted from tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA samples were treated with RQ1 RNase-free Dnase I (Qiagen) and subjected to reverse transcription (RT) for cDNA synthesis. RT-PCR was performed using Ex-Taq (Takara Bio Inc., Shiga, Japan) and specific primers: PoMSTN-RT-F (5′-CAA GAG ACG CAC CAT CAG CA-3′), PoMSTN-RT-R (5′-GGA GCT GCT TCA CAA TGT CTC-3′), PoMyogenin-RT-F (5′-AAG AGG AGC ACC CTG ATG AA-3′), PoMyogenin-RT-R (5′-GGA GAT CCT CGC TGC TGT AG-3′), PoMyoDRT-F (5′-ACG GCA TGA CGG ATT TTA AC-3′), PoMyoD-RT-R (5′-TAG ATC AGG TTG GGG TCC TG-3′), PoMyf5-RT-F (5′-AGG CTG AAG AAG GTC AAC CA-3′), PoMyf5-RT-F (5′-ACA CTG GAC TGT TGC TGT CG-3′), Po18S-RT-F (5′-ATG GCC GTT CTT AGT TGG TG-3′), and Po18S-RT-R (5′-CAC ACG CTG ATC CAG TCA GT-3′). The PCR reaction conditions were a denaturation for 5 min at 95 °C, followed by 30 or 35 cycles of 95 °C for 30 s, 60 °C for 30 s, 95 °C for 30 s, and a final extension step at 72 °C for 7 min.

2.11. Statistical analysis The results were expressed as the mean ± standard deviation (SD) (n = 3). Statistical analyses of the total length, body weight, and condition factor were conducted using Tukey's honestly significant difference (HSD) test (P ≤ .05). We analyzed differences in the H&E staining using t-tests (P ≤ .05). Statistical analyses were conducted using SPSS software version 18 (SPSS Inc., Chicago, IL, USA).

3. Results 3.1. Development of microinjection system for P. olivaceus embryos We optimized a microinjection needle for microinjecting P. olivaceus embryos (Fig. 1A). The microinjection needle was generated using a vertical pipette puller and the end of the tip was slightly ground to a 30° angle to penetrate the chorion of P. olivaceus embryos with minimal damage. A constriction brake was constructed in the taper to prevent the backflow of cytoplasm. To optimize the microinjection site in P. olivaceus embryos, rhodamine B-labeled dextran was injected into the yolk, yolk syncytial layer (YSL), and cytosol (Fig. 1B). Injection into the YSL and cytosol caused cytoplasmic diffusion of fluorescent substances, whereas injection into yolk led to immediate embryonic death. In addition to the nonspecific diffusion of rhodamine B labeleddextran, the expression of EGFP was demonstrated in embryos microinjected with a mixture of EGFP mRNA and rhodamine B-labeled dextran (Fig. 1C). Furthermore, gene expression and cellular localization of EGFP in embryos injected with NLS-Myc tag-EGFP mRNA at the 2-cell stage was analyzed at the blastula stage by immunostaining (Fig. 1D). EGFP was observed in the nuclei with a mosaic expression pattern, but not in all DAPI-positive cells, due to the non-uniform distribution of injected mRNA from one of the cells at the 2-cell stage or later. At the 50% epiboly stage, EGFP mRNA was detected using whole mount in situ hybridization (WISH) (Fig. 1E). These results revealed that our microinjection system was efficient for delivering genetic material and analyzing gene expression in P. olivaceus embryos.

2.7. Western blot analysis Protein was extracted from muscle tissue and the protein concentration was evaluated using a BCA protein assay. Equal amounts of protein were subjected to SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA). Immunoblotting was carried out using a 1:1000 dilution of antibodies anti-GDF-8 (Abcam, Cambridge, UK) and anti-β-actin (Abcam) according to the manufacturer's instructions. Primary antibodies were visualized using the enhanced chemiluminescent development reagent (Amersham Pharmacia Biotech Ltd., Little Chalfont, UK) following the peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). 2.8. Fluorescence image and immunohistochemistry Enhanced green fluorescent protein (EGFP) fluorescence of transgenic embryos was detected under a DM5000B microscope (Leica Microsystems, Wetzlar, Germany). Immunohistochemistry was performed as described by Yeo and Chitnis, 2007. The primary antibodies used were anti-Myc (Sigma-Aldrich), anti-phalloidin-TRITC (Sigma3

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Fig. 1. Development of a microinjection system for Paralichthys olivaceus. (A) Microinjection needle optimized for P. olivaceus embryos. Upper panel, end of the tip of the microinjection needle; lower panel, constriction brake in the microinjection needle. (B) Optimization of the microinjection site in P. olivaceus embryos. At the 2-cell stage, rhodamine B-labeled dextran was introduced into the yolk (I), yolk syncytial layer (YSL) (II), and cytosol (III), respectively. Fluorescence was visualized at the 4-cell stage. Scale bars, 500 μm. (C) Fluorescence expression of EGFP mRNA and rhodamine B-labeled dextran-injected embryos. Embryos were microinjected at the 2-cell stage and fluorescence was visualized at the 16-cell and blastula stages. Scale bars, 500 μm. (D) Immunostaining analysis of an embryo that was injected with NLS-Myc tag-EGFP mRNA. Embryos were microinjected at the 2-cell stage and stained with anti-Myc antibody, phalloidin-TRITC, and DAPI at the blastula stage. Scale bars at 50× magnification, 200 μm; Scale bars at 400× magnification, 50 μm. (E) Whole mount in situ hybridization of embryo injected with NLS-Myc tag-EGFP mRNA. Embryos were microinjected at the 2-cell stage and analyzed using a GFP riboprobe at the 50% epiboly stage.

high in vitro cleavage efficiencies, and were selected for further genome editing of PoMSTN (Fig. 2B). Based on the efficiency of the two sgRNAs in vitro, both sgRNAs were microinjected into approximately 3100 fertilized eggs at the 1- or 2-cell stage with Cas9 mRNA. A total of 284 injected eggs survived until 185 dph, and genomic DNA was extracted from fin clips of 160 fishes and subjected to PCR amplification around

3.2. PoMSTN modification in P. olivaceus using CRISPR/Cas9 system To induce PoMSTN disruption in P. olivaceus, two sgRNAs (PoMSTN sgRNA-#1, PoMSTN sgRNA-#3) targeting the first exon of PoMSTN were designed for complete disruption of the C-terminal domain of PoMSTN (Fig. 2A). Two sgRNAs with recombinant Cas9 nuclease had 4

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Fig. 2. CRISPR/Cas9 system-mediated modifications of PoMSTN in P. olivaceus. (A) Schematic diagram of sgRNAs targeting the PoMSTN loci. Exons of PoMSTN are shown in the light green box. The PoMSTN-sgRNA target sequences are in bold and underlined. PAM sequences are indicated in bold and red. (B) In vitro cleavage assay of two PoMSTN sgRNAs. A PCR amplicon containing two PoMSTN sgRNA target sites was synthesized from genomic DNA. The PCR amplicon was treated with recombinant Cas9 nucleases and each PoMSTN sgRNA, or untreated. Each reaction was separated using agarose gel electrophoresis. (C) TA-sequencing results at the target site of the PoMSTN sgRNA-#1 detected in the fin of P. olivaceus F0. (D) TA-sequencing results at the target site of the PoMSTN sgRNA-#3 detected in the fin of P. olivaceus F0. Target sequences complementary to each PoMSTN sgRNA are underlined; the PAM sequence is in red; (−) indicates deletion; blue denotes insertion; insertions (+) and deletions (−) are shown to the right of each allele. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

population of gene-disrupted individuals. To investigate the germline transmission of the mutant alleles to the F1 generation, we crossed the PoMSTN-modified F0 with the WT counterparts. Maturation and spawning of P. olivaceus took at least 2 years for females and 1 year for males, thus sexually mature F0 fishes were selected from the pool of F0 candidates and subsequently examined. Four out of five females undergoing sexual maturity were analyzed for germline transmission. We used a T7E1 assay to analyze the somite stage embryos (n = 78) from the mating group of female F-#1 and a WT male and detected on-target cleavage of PoMSTN, thereby indicating heterozygous monoallelic mutants (PoMSTN+/−) (Fig. 3A). The germline transmission rates were 14.10% (n = 11/78) in female F-#1. The mutation type of all embryos examined was an 8-base deletion (Fig. 3B). Meanwhile, 20 males releasing sperm were investigated for germline transmission among all 284 fish. Eight somite stage embryos from each mating group of males

the sgRNAs target site of PoMSTN exon 1. Subsequently, indels induced by the CRISPR/Cas9 system were identified in the target region of two sgRNAs using TA-cloning sequencing. A representative mutant sequence is shown in Fig. 2C and D. Diverse types of mutations were generated in the target region of the PoMSTN sequence, and most of the mutation types resulted in frameshift or nonsense mutations, leading to truncated PoMSTN proteins. Out of 160 fish, the PoMSTN sgRNA-#1 target region was mutated in 121 fish (75.6%), whereas the PoMSTN sgRNA-#3 target region was mutated in 59 fish (36.9%) (supplementary data).

3.3. Germline transmission of CRISPR/Cas9-induced mutation to the F1 generation Germline transmission is a critical milestone for expanding the 5

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Fig. 3. Germline transmission of PoMSTN mutation to F1 generation. (A) Detection of modified PoMSTN loci by T7E1 assay. A female F-#1 was crossed with a WT male. PCR products from 16 embryos were subjected to a T7E1 assay. Red arrows indicate embryos with mutations. (B) Sequences of modified PoMSTN loci of embryos obtained from mating a female F-#1 and a WT male. Target sequences complementary to PoMSTN sgRNA-#1 are underlined; the PAM sequence is in red. (C) Detection of modified PoMSTN loci by T7E1 assay. Each of the eight males (M-#1, #2, #3, #4, #5, #6, #7, and #8) was crossed with a WT female. PCR products from eight embryos were subjected to T7E1 assay. Red arrows indicate the embryos with mutations. (D) Sequences of modified PoMSTN loci of embryos from mating male M-#1 and a WT female. The target sequence complementary to PoMSTN sgRNA-#1 is underlined; the PAM sequence is in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

mutations were obtained in the F0 generation, and targeted germline mutations induced by the CRISPR/Cas9 system were successfully transmitted to subsequent generations.

and WT females were evaluated using a T7E1 assay, and the inheritance of mutations by offspring was observed in 9 out of 20 F0 males (45%) (Fig. 3C). In male M-#1, four mutation types (−1 bp, −2 bp, −4 bp, and −12 bp) were identified in germ cells, in contrast with germinal mutation of female F-#1 with only one mutation type (−8 bp) (Fig. 3B, D). The frequency and type of induced mutation varied between the somatic tissue and germ cells in 20 F0 males. Germline mutations were not detected in 11 out of 20 F0 males, even though they harbored targeted mutations on the PoMSTN locus of genomic DNA extracted from fins, suggesting mosaicism of CRISPR/Cas9-induced PoMSTN mutations in various tissues of each individual (data not shown). Taken together, these results indicate that P. olivaceus harboring germline

3.4. Generation of biallelic mutant F1s with complete PoMSTN disruption Due to the previous validation of germline transmission, heterozygous monoallelic mutant parents (female, F-#1; male, M-#1) were crossed to obtain biallelic mutant F1 fish with complete PoMSTN gene disruption. In the F1 generation, five out of nine individuals exhibited cleavage products according to the T7E1 assay, indicating monoallelic mutants (PoMSTN+/−) or heterozygous biallelic mutants (described as 6

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PoMSTN−/−). On the other hand, we used restriction enzyme Esp3I in RFLP for rapid mutation detection because all previously identified mutation types in the target site of PoMSTN sgRNA-#1 destroyed the restriction recognition site of Esp3I, whereas the WT sequence had an intact restriction recognition site. As a result, F1 #1 and #2 had

restriction digest bands by Esp3I, suggesting that they carry at least one intact allele. F1 #1 was demonstrated as WT (PoMSTN+/+). Fig. 4A shows a comparison between the T7E1 assay and RFLP analysis of F1 s. We confirmed the mutation types of both alleles of F1 #2, #3, and #4 based on sequencing results, and they were classified as PoMSTN+/− 7

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Fig. 4. Generation of biallelic mutants in the F1 generation. (A) T7E1 assay and restriction fragment length polymorphism (RFLP) analysis of F1 from heterozygous parents (female, F-#1; male, M-#1). Identical sets of PCR amplicons of modified PoMSTN loci from F1 individuals were cleaved with T7E1 and restriction enzyme Esp3I for 30 min. Red dots reveal a cleaved band from the T7E1 assay and yellow dots denote restriction digestion. (B) Sequence of modified PoMSTN loci identified in F1 mutants (#2, #3, and #4). The PAM sequence is in red; the recognition site of the Esp3I restriction enzyme is in green. (C) RFLP analysis to determine the genotypes of the mutations. PCR amplicons from WT (+/+), heterozygous monoallelic mutants (+/−), and heterozygous biallelic mutants (−/−) were digested with Esp3I for different times, as indicated (10 min, 17 h). The yellow dots denote restriction digestion. (D) Representative data of RFLP analysis of F1 generation from heterozygous parents (female, F-#1; male, M-#1, #2, #3, #4, #5, #6, #7, and #8). PCR amplicons were digested with Esp3I for 2 h. Red arrows indicate heterozygous biallelic mutants (PoMSTN−/−). Yellow arrows denote WT (PoMSTN+/+). (E) Sequences of both alleles of modified PoMSTN loci identified in 20 PoMSTN−/− mutants in the F1 generation from heterozygous parents (female, F-#1; male, M-#1, #2, #3, #4, #5, #6, #7, and #8). Red indicates PAM and green indicates the recognition site of Esp3I. Recognition sequences were destroyed in all mutant alleles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

−2

(F1 #2) and PoMSTN−/− (F1 #3, PoMSTN−8/−2; #4, PoMSTN−8/−1) (Fig. 4B). Notably, the T7E1 assay did not distinguish between heterozygous monoallelic mutations and heterozygous biallelic mutations, but RFLP analysis to detect indel mutations of each allele did. RFLP analysis was also used to identify the genotype between WT and monoallelic mutation when the PCR products of the target region were digested overnight (Fig. 4C). To expand the pool of the F1 generation, including biallelic mutants, F1 was generated by artificial fertilization of germline edited eggs from one female F-#1 and sperm from eight males (M-#1, #2, #3, #4, #5, #6, #7, and #8). Based on the sensitivity and reliability of the RFLP analysis, the genotypes of 240 F1 individuals were determined using Esp3I. As a result, 20 (20/240 = 8.33%), 204 (204/240 = 85%), and 16 (16/240 = 6.67%) F1 s were categorized as PoMSTN−/−, PoMSTN+/−, and WT, respectively (Fig. 4D). The mutation types of 20 PoMSTN−/− mutants were analyzed by sequencing (Fig. 4E). These results indicate that biallelic mutants (PoMSTN−/−) with complete PoMSTN disruption were produced in the F1 generation.

and WT at 251 dph. PoMSTN primers for RT-PCR were designed to span the deletion junction of mutant alleles to detect intact mRNA of PoMSTN. PoMSTN mRNA was detected in all tissues examined, but expressed almost exclusively in the skeletal muscle tissue of the WT specimens (Fig. 6A). However, intact PoMSTN mRNA was expressed at a low level in all tissues of PoMSTN−8/−2 (Fig. 6A). As MSTN is known to be a negative regulator of myogenesis by deactivating downstream MRFs such as myogenin, MyoD, and Myf5, the transcript levels of the three MRFs in muscle tissue were subsequently compared among two PoMSTN−/− individuals (PoMSTN−/−-#1, PoMSTN−8/−2; #2, PoMSTN−8/−1) and WT (Fig. 6B). PoMSTN mRNA levels in muscle tissue were reduced in both PoMSTN−/− mutants. In the muscle tissue of PoMSTN−/−-#1, constant myogenin mRNA, constant MyoD mRNA, and decreased Myf5 mRNA expression were demonstrated, whereas constant myogenin mRNA, decreased MyoD mRNA, and decreased Myf5 mRNA expression were confirmed in the muscle tissue of PoMSTN−/−-#2. The mRNA expression patterns of myogenin, MyoD, and Myf5 varied between the PoMSTN−/− mutants, suggesting that the expression levels of the three MRFs were not directly regulated by the PoMSTN level. The expression of PoMSTN protein in muscle tissue among PoMSTN−/−-#1, PoMSTN−/−-#2, and WT was compared by western blotting using antibodies for the C-terminal domain of MSTN (Fig. 6C). The expression of PoMSTN protein was lower in PoMSTN−8/ −2 and PoMSTN−8/−1, showing disruption of mature MSTN protein in both PoMSTN−8/−2 and PoMSTN−8/−1. Because deletion on the PoMSTN locus, in which the number of deleted base pairs is not divisible by three, was predicted to induce frameshift and nonsense mutations, we expected the PoMSTN−8/−2 and PoMSTN−8/−1 to express the truncated form of PoMSTN protein. These data suggest that PoMSTN mRNA and protein were disrupted by mutations on the PoMSTN locus, and PoMSTN disruption did not appear to be critical for regulating the expression of MRFs in PoMSTN-disrupted P. olivaceus.

3.5. Growth performance of PoMSTN-disrupted P. olivaceus To investigate the phenotypic effect of PoMSTN disruption in P. olivaceus, we evaluated the morphological differences between PoMSTN−/− and WT. WT and biallelic mutant, which were similar in total length (27 ± 0.4 cm), were selected for a representative comparison, and the body of PoMSTN−/− was considerably thicker (28 mm) than that of WT (24 mm) at 251 dph (Fig. 5A). The thicker bodies of PoMSTN-disrupted P. olivaceus indicates that they carried more muscle mass. To monitor the growth performance of PoMSTN−/ − , we assessed the increase in the total length and body weight of each group (n = 3) of PoMSTN−/− (two PoMSTN−8/−1 and one PoMSTN−8/ −2 ) and WT from 159 dph to 251 dph (Fig. 5B, C). PoMSTN−/− tended to be slightly shorter in total length and weighed more than the WT, but no significant differences were observed. On the other hand, the condition factor, which is an index of the weight-length relationship in fish, of PoMSTN−/− was higher than that of WT from 159 dph to 251 dph (Fig. 5D). Considering the body thickness of PoMSTN−8/−2, the higher relative weight suggests more muscle growth in the PoMSTN-disrupted mutants. To evaluate the muscle histology of PoMSTN-disrupted muscle, H&E staining was conducted in skeletal muscle tissue of both WT and PoMSTN−8/−2 specimens (Fig. 5E). The number of muscle fibers per unit area in PoMSTN−8/−2 was not significantly different from that in WT, indicating that PoMSTN disruption induced muscle growth by increasing the number of muscle fibers. There is no difference in the sizes of the muscle fibers between PoMSTN−8/−2 and WT. Taken together, PoMSTN disruption facilitated muscle growth in P. olivaceus through muscle hyperplasia but not hypertrophy.

4. Discussion and conclusion MSTN, a negative regulator of skeletal muscle development, is the major gene of interest in animal breeding to improve productivity, because mutations in MSTN increase skeletal muscle growth in some animals (Kambadur et al., 1997; Clop et al., 2006; Schuelke et al., 2004; Wang et al., 2017). In fish, RNA interference-mediated MSTN suppression was shown to be associated with the double-muscle effect (Lee et al., 2009; Terova et al., 2013). Genome editing technology has been applied recently to knock out MSTN in several fish species, including model and marine fish (Lee et al., 2009; Chisada et al., 2011; Kishimoto et al., 2018). These studies inspired us to disrupt the PoMSTN gene using the CRISPR/Cas9 system to investigate the function of PoMSTN as a reverse genetics approach and to enhance the growth performance of P. olivaceus. To introduce targeted mutation using the CRISPR/Cas9 system, we developed a microinjection system for P. olivaceus embryos (Fig. 1). The microinjection technique was firstly applied to model fish, zebrafish and medaka, and is now widely applied in experiments with marine fish, such as Atlantic salmon (Salmo salar L.), red sea bream (Pagrus major), Pacific bluefin tuna (Thunnus orientalis) and barfin flounder

3.6. Gene expression of PoMSTN and MRFs in PoMSTN-disrupted P. olivaceus As the PoMSTN target region was successfully edited in P. olivaceus, the gene expression levels of PoMSTN and other MRFs were evaluated to investigate the molecular effects of PoMSTN disruption. The tissue distribution of PoMSTN mRNA was demonstrated in both PoMSTN−8/ 8

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Fig. 5. Evaluation of growth performance of PoMSTN-disrupted P. olivaceus. (A) Cross-sections of the WT (left) and PoMSTN−8/−2 (right) at 251 dph. The red line indicates the body thickness of PoMSTN−/− (right). (B) Total length changes in WT and PoMSTN−8/−2 from 159 dph to 251 dph (n = 3 per group). (C) Body weight changes in WT and PoMSTN−/− from 159 dph to 251 dph (n = 3 per group). (D) Condition factor changes in WT and PoMSTN−/− from 159 dph to 251 dph. The condition factor (CF) was calculated as CF = (body weight in g)/(total length in cm)3 × 1000 (n = 3 per group). Means sharing the same superscript letter were not significantly different from each other (Tukey's HSD, P ≤ .05). (E) H&E staining of cross-sectional areas of muscle from WT and PoMSTN−8/−2 at 251 dph. Scale bars, 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(Verasper moseri) (Kishimoto et al., 2018; Edvardsen et al., 2014; Rosen et al., 2009; Porazinski et al., 2010; Otani et al., 2008; Goto et al., 2015). To optimize the microinjection system for the P. olivaceus

embryo, the size and form of the microinjection glass needle had to be considered carefully to ensure that it would penetrate the hard chorion of marine fish eggs (Goto et al., 2019). We also examined optimal 9

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Fig. 6. Gene expression analysis of PoMSTN-disrupted P. olivaceus. (A) Tissue distribution of PoMSTN mRNA in WT and PoMSTN−8/−2 at 251 dph. (B) RT-PCR analysis of PoMSTN and MRFs (myogenin, MyoD, and Myf5) mRNA in the muscle of PoMSTN−/−-#1 (PoMSTN−8/−2), PoMSTN−/−-#2 (PoMSTN−8/−1), and WT at 251 dph. (C) Western blotting analysis of PoMSTN protein in the muscles of PoMSTN−/−-#1 (PoMSTN−8/−2), PoMSTN−/ − -#2 (PoMSTN−8/−1), and WT at 251 dph.

were edited by the CRISPR/Cas9 system. According to the difference between the numbers of mutant alleles and mutation rate of germ cells of F-#1 and M-#1, it is possible to infer the timing of the DSB generation during genome editing. Although Cas9 nuclease protein can trigger earlier genomic cuts than mRNA (Albadri et al., 2017), we used Cas9 mRNA because of the aggregation of Cas9 nuclease protein in the microinjection needle developed for P. olivaceus embryos. In the CRISPR/Cas9 methodology, screening and identification of mutants is one of the most time-consuming, laborious, and cost intensive. T7E1 mismatch detection assays is a quick means of detecting indels and therefore it is the most widely used method for detecting mutations induced by genome editing. However, homozygous mutations cannot be detected by T7E1 assays. Sequencing analysis reveals both the type of mutation and its frequency at the target locus, but it is time-consuming, expensive, and labor-intensive. We carried out T7E1 assays, sequencing, and RFLP analysis to detect mutations induced by the CRISPR/Cas9 system. RFLP analysis is used to detect site-directed mutations from two independent alleles per locus when a target region contains a suitable restriction site (Zischewski et al., 2017). RFLP analysis was suggested as a rapid and reliable method for detecting PoMSTN disruption in the F1 generation (Fig. 4). As all mutations induced by Cas9/PoMSTN sgRNA-#1 destroyed the Esp3I restriction recognition site, whereas the WT sequence had an intact restriction recognition site. RFLP analysis by Esp3I restriction enzyme was particularly useful for distinguishing WT (PoMSTN+/+) and heterozygous monoallelic mutations (PoMSTN+/−) from heterozygous biallelic mutations (PoMSTN−/−) in the F1 generation in our study. Sensitive and convenient RFLP analysis can be combined with other methods for detecting on-target mutations and improving the entire CRISPR/Cas9 methodology. We produced a PoMSTN-disrupted breed of P. olivaceus in the F1 generation by mating germline-edited founders (F0). This procedure greatly reduced the period for establishing homozygotes, which generally requires two generations of breeding.

microinjection sites in embryos to induce cytoplasmic diffusion of microinjected substances. The microinjection system optimized for P. olivaceus embryos potentially has versatile research applications, including gene function analysis, transgene techniques, and genome editing. We developed a CRISPR/Cas9 system for genome editing in P. olivaceus, and the PoMSTN locus was disrupted in both somatic cells and germ cells. Two sgRNAs (PoMSTN sgRNA-#1, PoMSTN sgRNA-#3) in the CRISPR/Cas9 system induced mutations in each target locus (Fig. 2C, D). This suggests the possibility of multiple gene knockout in studying interactions among various genes. The in vitro cleavage assay was reported not to correlate with the in vivo efficiency (Albadri et al., 2017). To select high-quality sgRNA for efficient genome editing, in vivo tests can be used for pre-screening sgRNAs, rather than in vitro assays, even though this is a laborious process. Meanwhile, PoMSTN sgRNA-#1 in the CRISPR/Cas9 system achieved 75.6% editing efficiency in P. olivaceus, and this mutation rate was higher than observed in other aquaculture species in fish, such as Atlantic salmon (22–40%) and tilapia (24–50%) (Edvardsen et al., 2014; Li et al., 2014). According to these results, the designed CRISPR/Cas9 system was effective in inducing indels at specific sites of PoMSTN and generating PoMSTN-modified P. olivaceus in the F0 generation. The type and frequency of germline mutations induced by the CRISPR/Cas9 system in PoMSTN varied among individuals of the F0 generation (Fig. 3). Embryos from the mating group of female F-#1 and WT male were shown to be WT or heterozygous monoallelic mutants harboring one mutation type, an 8-base deletion (PoMSTN ± 8), indicating that germ cells of female F-#1 harbored WT alleles or 8-basedeleted alleles. The germ cells of male M-#1 were identified to harbor one of five types of alleles: WT, −1 bp, −2 bp, −4 bp, and − 12 bp. The type of germinal mutation transmitted to the F1 generation was maintained constantly in the separate artificial fertilization of the F0 generation. These results suggest that primordial germ cells (PGCs) 10

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MSTN were characterized in flatfish species, including Verasper variegatus, Paralichthys adspersus and P. olivaceus, and has been shown to be involved in muscle growth in flatfish (Li et al., 2012; Delgado et al., 2008; Zhong et al., 2008; Kim et al., 2019). However, the unique function of MSTN compared to other teleosts remains to be elucidated. Growth performance were evaluated to monitor muscle gain of PoMSTN-disrupted P. olivaceus. Biallelic mutants, PoMSTN−/−, exhibited increased muscle mass through muscle hyperplasia with greater body thickness and increased condition factor from 159 dph to 251 dph. This suggests that PoMSTN plays a role in the regulation of muscle growth of P. olivaceus. In the fish industry, skeletal muscle constitutes the main edible part of the fish. The PoMSTN-disrupted breed of P. olivaceus may therefore be more productive and profitable in aquaculture because its muscle mass is larger than that of the WT. There were no clear differences in body shape between PoMSTN-8/−2 and the WT, even though several reports have shown that MSTN deficiency is related to unusual body shapes, bone structures, and body fat mass in mice and fish (Kishimoto et al., 2018; Elkasrawy and Hamrick, 2010; Braga et al., 2013). Significant differences of growth performance were observed between MSTN-disrupted mutants and WT in a marine fish, P. major, as the fish grew older than 300 days (Kishimoto et al., 2018). Considering that P. olivaceus continues to grow over 5 years, and fish at least 2 years in age (over 1.1 kg) are at a marketable age, the growth performance of PoMSTN-disrupted mutants remains to be elucidated under additional monitoring for several years. Moreover, the morphological and genetic effects of PoMSTN disruption should be analyzed in detail with a larger pool of PoMSTN-disrupted mutants in further studies. PoMSTN-knockout P. olivaceus can be utilized as a useful model for studying the characteristics of the PoMSTN gene. The tissue distribution of intact PoMSTN mRNA in WT and PoMSTN−8/−2 demonstrated that PoMSTN mRNA in muscle was significantly reduced, showing the effects of genomic PoMSTN modification in PoMSTN−8/−2 (Fig. 6A). Modified PoMSTN mRNA would be translated into the truncated form of PoMSTN protein, and the protein levels of PoMSTN were reduced in the muscle tissue of PoMSTN−8/−2 and PoMSTN−8/−1-, as predicted (Fig. 6C). The faint band detected in PoMSTN−8/−2 and PoMSTN−8/−1 may be due to the cross-reactivity of antibodies with GDF11, which shares a highly conserved C-terminal region with MSTN (Zhong et al., 2008). PoMSTN disruption was not correlated with the expression of MRFs in PoMSTN-disrupted P. olivaceus, even though MSTN is known to be a negative regulator of relevant MRFs (Fig. 6B). The transcript levels of three major MRFs, myogenin, MyoD, and Myf5, showed inter-individual variation in PoMSTN−/− mutants, suggesting that PoMSTN does not directly regulate the expression of the MRFs and the expression of the MRFs does not determine muscle growth performance in P. olivaceus. Previously, MSTN genotype (MSTN+/+, MSTN+/−, and MSTN−/−) was reported to have no significant effect on the mRNA expression of MyoD and myogenin in mice (Rachagani et al., 2010). In IGF-1 KO mice, the mRNA expression of myogenin was downregulated, whereas the MSTN expression was not affected by IGF-1 deletion, indicating that MSTN and MRFs may work indirectly (Miyake et al., 2007). Most fishes possess two or more paralogs for MSTN or relevant MRFs, and their paralogs showed different expression patterns during development in brown trout Salmo trutta L (Churova et al., 2017). In particular, P. olivaceus is one species of the order Pleuronectiformes, also known as flatfish, which exhibit the most extreme asymmetric body morphology among vertebrates (Shao et al., 2017). Flatfish develop eye-sidedness as they grow from larval to juvenile stage (positional asymmetry), and have much greater fast skeletal muscle mass on the eyed side of adults compared with the blind side (volumetric asymmetry) (Schreiber, 2013). In one species of flatfish, Atlantic halibut (Hippoglossus hippoglossus L.), MyoD mRNA was transiently expressed in a novel bilaterally asymmetric pattern during early somitogenesis (Galloway et al., 2006). This suggests that muscle growth factors including PoMSTN and relevant MRFs are involved in

asymmetric skeletal muscle growth during myogenesis in P. olivaceus. The molecular role of PoMSTN in the muscle growth remains unresolved, and PoMSTN-disrupted P. olivaceus can provide further information for understanding the mechanism causing asymmetry in P. olivaceus. In conclusion, we successfully modified the PoMSTN locus using CRISPR/Cas9 technology and produced PoMSTN-disrupted P. olivaceus in the F1 generation. Biallelic mutants (PoMSTN−/−) exhibited more muscle mass, with thicker bodies and higher condition factor compared to the WT. The establishment of a CRISPR/Cas9 system in P. olivaceus provides fundamental tools for genetic studies and breeding, and further analysis of PoMSTN-disrupted mutants might provide useful information for understanding the role of PoMSTN. Acknowledgements This research was supported by a grant from the National Institute of Fisheries Science (NIFS) of the Republic of Korea (R2019018). Declaration of Competing Interest None. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.aquaculture.2019.734336. References Albadri, S., Del Bene, F., Revenu, C., 2017. Genome editing using CRISPR/Cas9-based knock-in approaches in zebrafish. Methods 121–122, 77–85. Braga, M., Pervin, S., Norris, K., Bhasin, S., Singh, R., 2013. Inhibition of in vitro and in vivo brown fat differentiation program by myostatin. Obesity (Silver Spring) 21, 1180–1188. Chandrasegaran, S., Carroll, D., 2016. Origins of programmable nucleases for genome engineering. J. Mol. Biol. 428, 963–989. Chen, Y.T., Lin, C.F., Chen, Y.M., Lo, C.E., Chen, W.E., Chen, T.Y., 2017. Viral infection upregulates myostatin promoter activity in orange-spotted grouper (Epinephelus coioides). PLoS One 12, e0186506. Chisada, S.I., Okamoto, H., Taniguchi, Y., Kimori, Y., Toyoda, A., Sakaki, Y., Takeda, S., Yoshiura, Y., 2011. Myostatin-deficient medaka exhibit a double-muscling phenotype with hyperplasia and hypertrophy, which occur sequentially during post-hatch development. Dev. Biol. 359, 82–94. Churova, M.V., Meshcheryakova, O.V., Ruchev, M., Nemova, N.N., 2017. Age- and stagedependent variations of muscle-specific gene expression in brown trout Salmo trutta L. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 211, 16–21. Clop, A., Marcq, F., Takeda, H., Pirottin, D., Tordoir, X., Bibé, B., Bouix, J., Caiment, F., Elsen, J.M., Eychenne, F., Larzul, C., Laville, E., Meish, F., Milenkovic, D., Tobin, J., Charlier, C., Georges, M., 2006. A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep. Nat. Genet. 38, 813–818. Delgado, I., Fuentes, E., Escobar, S., Navarro, C., Corbeaux, T., Reyes, A.E., Vera, M.I., Alvarez, M., Molina, A., 2008. Temporal and spatial expression pattern of the myostatin gene during larval and juvenile stages of the Chilean flounder (Paralichthys adspersus). Comp. Biochem. Physiol. B Biochem. Mol. Biol. 151, 197–202. Edvardsen, R.B., Leininger, S., Kleppe, L., Skaftnesmo, K.O., Wargelius, A., 2014. Targeted mutagenesis in Atlantic salmon (Salmo salar L.) using the CRISPR/Cas9 system induces complete knockout individuals in the F0 generation. PLoS One 9, e108622. Elkasrawy, M.N., Hamrick, M.W., 2010. Myostatin (GDF-8) as a key factor linking muscle mass and bone structure. J. Musculoskelet. Neuronal Interact. 10, 56–63. Fuji, K., Kobayashi, K., Hasegawa, O., Coimbra, M.R., Sakamoto, T., Okamoto, N., 2006. Identification of a single major genetic locus controlling the resistance to lymphocystis disease in Japanese flounder (Paralichthys olivaceus). Aquaculture. 254, 203–210. Fuji, K., Hasegawa, O., Honda, K., Kumasaka, K., Sakamoto, T., Okamoto, N., 2007. Marker-assisted breeding of a lymphocystis disease-resistant Japanese flounder (Paralichthys olivaceus). Aquaculture. 272, 291–295. Fujiwara, A., Fujiwara, M., Nishida-Umehara, C., Abe, S., Masaoka, T., 2007. Characterization of Japanese flounder karyotype by chromosome bandings and fluorescence in situ hybridization with DNA markers. Genetica. 131, 267–274. Galloway, T.F., Bardal, T., Kvam, S.N., Dahle, S.W., Nesse, G., Randøl, M., Kjørsvik, E., Andersen, O., 2006. Somite formation and expression of MyoD, myogenin and myosin in Atlantic halibut (Hippoglossus hippoglossus L.) embryos incubated at different temperatures: transient asymmetric expression of MyoD. J. Exp. Biol. 209,

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