Ethics
All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals from Tokyo University of Marine Science and Technology, Japan.
Fish
Nibe croaker broodstock (approximate body weight (BW) of 300 g and total length of 250 mm) were maintained in a 0.5-m3 circular fiber-reinforced plastic (FRP) tank at Tateyama Station (Banda), Field Science Center of Tokyo University of Marine Science and Technology (Chiba, Japan). Water temperature was maintained at 23°C–26°C using aquarium heaters, and the photoperiod was set at 16–18 h day length for the first 3 months, following the natural photoperiod. Fertilized eggs were collected by water flow into an egg collection net.
Preparation of genome‑editing tools and their microinjection into fertilized eggs of nibe croaker
A guide RNA (gRNA) targeting the dnd gene (GenBank Accession No. LC317114) of nibe croaker was designed on the basis of a report of dnd KO in Atlantic salmon (Wargelius et al., 2016). First, in the area of the nibe croaker dnd gene, “CHOP CHOP” (http://chopchop.cbu.uib.no/index.php) was used to search for candidate sequences. Then, microhomologies at both ends of the cleavage sites of the obtained target sequences were searched using the “search for the CRISPR target site with microhomology sequences” (http://viewer.shigen.info/cgi-bin/crispr/crispr.cgi) and candidate sequences that prevent in-frame mutations caused by microhomology within the sequence. Using the abovementioned process, a gRNA target site (5′-GCCCCACCGAGCTGAACAGGGGG-3′, Fig. 1) was designed. CRISPR RNA (crRNA) complementary to the target site sequence and trans-activating crRNA (tracrRNA) were chemically synthesized and then purified using high-performance liquid chromatography by Fasmac Co., Ltd. (Kanagawa, Japan). Fertilized eggs of nibe croaker were microinjected following the procedure of Kawamura et al. (2022) with a slight modification. The glass needles for microinjection were constructed using a puller (PC-10; Narishige Co., Ltd., Tokyo, Japan) and a microgrinder (EG-400; Narishige Co., Ltd.) to produce a needle with a 7-µm tip. Fertilized eggs were aligned in a groove of a 2% agar plate filled with sterilized seawater at 25℃. A total of 1 nL of CRISPR/Cas9 solution containing 40 ng/µL of crRNA, 40 ng/µL of tracrRNA, and 100 ng/µL of Alt-R S.p. Cas9 Nuclease V3 (Integrated DNA Technologies, Coralville, IA, USA) was injected into the cytoplasm of a one-cell-stage embryo using a micromanipulator (MP-2R; Narishige Co., Ltd.) and microinjector (IM-9B; Narishige Co., Ltd.) attached to a stereoscopic microscope (SZX-10; Nikon, Tokyo, Japan). The injected eggs were incubated in a 1-L glass beaker filled with sterilized seawater at 25°C until the embryo-formation stage (20 h postfertilization). The hatching rate of the injected eggs was calculated by dividing the number of successfully hatched larvae by the number of injected eggs and then multiplying the result by 100. Larval rearing of nibe croaker was performed as previously described (Takeuchi et al., 2009). In brief, injected eggs were transferred to a 100-L polycarbonate tank supplied with flow-through seawater and maintained at 23°C–26°C. The feeding of larvae began 3 dph. The rotifer Brachionus rotundiformis, fed with freshwater Chlorella (fresh Chlorella V12; Cholera Industry, Tokyo, Japan), was added to the tank two times a day. The densities of rotifers and Nannochloropsis sp. (Marine fresh; ISC, Fukuoka, Japan) in the tank were maintained at 30–40 individuals/mL and 5 × 105 cells/mL, respectively. Artemia nauplii were provided from 14 dph. The rotifers and Artemia nauplii were incubated with Hyper Gloss (Marine Tech Co., Ltd., Aichi, Japan) for 6 to 12 h before feeding to increase the n-3 fatty acid concentration in live food. Starting at 20 dph, the larvae were fed an artificial diet (Otohime; Marubeni Nisshin Feed, Tokyo, Japan). Founders were reared in 100-L tanks for approximately 60 days and then transferred into 0.5-m3 tanks with flow-through seawater maintained under a natural photoperiod and water temperature.
Production of homozygous KO mutants
When the injected individuals reached 4 months of age, mature male specimens were confirmed by gently squeezing their abdomen to check spermiation. The sperm from these males were artificially inseminated with eggs derived from wild-type females to produce the F1 generation. The resulting F1-generation larvae were sampled and used for mutation detection by the T7 endonuclease I (T7EI) assay. This assay was performed following a procedure modified from Kawamura et al. (2022). In brief, genomic DNA was extracted from whole hatched larvae. A 244-bp fragment flanking the target site was amplified using the primers nc_dnd_Fw (5′-CCCACTGGATGCCTACGAG-3′) and nc_dnd_Rv (5′-TGGCAGGTCTTCGATACAGAG-3′). PCR was conducted in a 10-µL reaction volume containing 1× PCR Buffer II, 200 µM of dNTPs, 1.5 mM MgCl2, 1.25 U of AmpliTaq Gold DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), 10 ng of template DNA, and 1 µM of each primer. The thermal cycling conditions were as follows: 1 cycle of 95°C for 10 min, then 40 cycles of 95°C for 15 s, 52°C for 30 s, and 72°C for 30 s, followed by a final elongation step at 72°C for 5 min. The PCR products were purified using the ISOSPIN PCR Product (Nippon Gene, Tokyo, Japan). In addition, the purified PCR products were digested with T7E1 (New England Biolabs, Beverly, MA, USA) in accordance with the manufacturer’s instructions, and the digestion products were separated on a 2% agarose gel. The PCR products of F1 individuals, which have a mutation in the dnd gene, were sequenced using the dideoxy method (Sanger et al. 1977).
F1 populations containing mutants were raised until males and females reached maturation at 1 year old. Then, they were subjected to the abovementioned T7EI assay to identify and select heterozygous dnd mutants. The resulting F1 heterozygous mutant females and males were mated to produce the F2 generation. Of the resulting F2 population, 50 individuals were sampled for genotyping: wild-type dnd (+/+), heterozygous KO dnd (+/−), and homozygous KO dnd (−/−), each with specific forward primers for the wild-type allele; nc_dnd_Fw_WTp (5′-AGGATCTGTTGATCCCCCTG-3′) and a specific forward primer for the mutant allele; nc_dnd_Fw_KOp (5′-TGGCAGGTCTTCGATACAGAG-3′) and PCR with common reverse primers; nc_dnd_Rv (5′-TGGCAGGTCTTCGATACAGAG-3′). PCR amplification with the primer sets was conducted using HiDi DNA polymerase (myPOLSBiotec, Konstanz, Germany) that efficiently amplifies from primers that are completely matched at the 3′-end and discriminates templated DNA containing single-nucleotide variations (Drum et al. 2014). In addition, PCR amplification was conducted in a 10-µL reaction volume tube containing 1× HiDi Buffer, 200 µM dNTPs, 1 U HiDi DNA polymerase, 25 ng of template DNA, and 0.4 µM of each primer. Then, the PCR mixture was cycled under the following conditions: 1 cycle of 95°C for 3 min, then 30 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 1 min, followed by a final elongation step at 72°C for 3 min. Afterward, the PCR products were verified under UV light after electrophoresis on a 2.0% agarose gel.
Histological analysis of the gonads of dnd KO fish
In investigating the gonadal development of dnd KO nibe croaker in the F2 population, wild-type and homozygous dnd KO individuals were selected by PCR for genotyping, and histological and immunohistochemical analyses of the gonads obtained from 10-dph, 20-dph, and 6-months-old nibe croaker were performed. The gonads were paraffin embedded and sliced into 4-µm-thick sections in accordance with the conventional method. Immunohistochemistry was performed in accordance with the method of Yazawa et al. (2021), and germ cells were detected using an anti-nibe croaker Vasa polyclonal antibody (Yoshikawa et al., 2018). Primary antibodies, anti-nibe croaker Vasa polyclonal antibody, were diluted to 1:200 in Can Get Signal Immunostain (Toyobo). Secondary antibodies, goat anti-rabbit Alexa Fluor 488 (Invitrogen Life Technologies, San Diego, CA, USA), were used in accordance with the manufacturer’s instructions. The total number of PGCs was counted by observing the anti-Vasa staining on serial histological sections.
Allogenic testicular cell transplantation into homozygous dnd KO recipients
The testes from two male nibe croakers, individual “donor-A” (15.0 cm in standard length (SL), 59.3 g BW, and gonad somatic index (GSI) of 0.58%) and individual “donor-B” (14.5 cm in SL, 53.7 g BW, and GSI of 1.42%) caught using hook and line in Tateyama Bay, Chiba Prefecture, Japan, were isolated and cryopreserved following the procedure of Lee et al. (2013) with a slight modification. The isolated testes were minced and equilibrated in a cryotube containing 1 mL of cryomedium (1.3 M DMSO, 10% egg yolk, 0.1 M trehalose in Leibo-vitz’s L15 medium (Thermo Fischer Scientific, Waltham, MA)) on ice for 60 min and then frozen at − 1°C/min for 90 min using the Bicell freezing container (Nihon Freezer, Tokyo, Japan) in a deep freezer (− 80°C) before preservation in liquid nitrogen. After 6 months of cryopreservation, the cryotubes were thawed in a 25°C water bath for 1–2 min, and the cells were dehydrated in an L-15 medium (pH7.5) supplemented with 1% (vol/vol) fetal bovine serum and 25 mM Hepes. Subsequently, the thawed testes were enzymatically dissociated and used as donor testicular cells for intraperitoneal transplantation in accordance with the procedure reported by Takeuchi et al. (2009). Approximately 20,000 cells were transplanted into the peritoneal cavity of 12-dph F2 larvae resulting from mating between heterozygous dnd KO (300 recipients for each donor individual) using the method described by Takeuchi et al. (2009). Recipients were reared until 6 months, and homozygous dnd KO recipients were identified by genotyping PCR. The resulting homozygous dnd KO recipients were then used for the progeny test. In 12-month-old individuals, ten individuals among the homozygous dnd KO recipients identified from 50 transplanted recipients were subjected to histological observations of the gonads according to the aforementioned method.
Progeny tests using dnd KO recipients
Group mating experiments were conducted in each of the two strains using 6-month-old mature dnd KO recipients in a 0.5-m3 FRP tank to confirm whether donor-derived offspring can be produced through the natural spawning of dnd KO recipients. In inducing spawning, the water temperature was increased from approximately 20°C to 26°C, and the photoperiod was fixed at 16 h. Floating eggs were collected using an egg collection net attached to the drain of the fish tank. The total number of spawned eggs, floating eggs, and hatched larvae obtained from each spawning event was recorded, and 1-dph larvae were sampled and used for DNA analysis. Microsatellite analysis was performed to confirm whether or not each resultant larva carried donor-derived nDNA. Two loci (501 and 1001) of microsatellite DNA markers designed on the basis of microsatellite regions on the genome of large yellow croaker (GenBank Accession No. GCA_900246015.1) were used for parentage assignment following a procedure described by Morishima et al. (2009) with a slight modification. The microsatellite marker loci were amplified using the primer set 501 (Fwd; 5′-GTGATGACAGACTGTGATAAGCT-3′, Rev; 5′-GTCACGACGTTGTAAGAGCATTTGATGTAGCAGTGT-3′) and 1001 (Fwd; 5′- ACTCAAACCCACACCTGACA-3′, Rev; 5′-GTCACGACGTTGTAAGGCCATTGGTGACTGATGA-3′). A 10-µL reaction volume comprising 1× ExTaq reaction buffer (TakaraBio, Tokyo, Japan), 200-µM dNTPs, 0.25 U of ExTaq DNA polymerase (TakaraBio), 20 ng of template DNA, 0.1 µM of forward primer, and a mixture of 0.01 µM of reverse primer and 0.1 µM of fluorescently labeled primer (Fam-GTCACGACGTTGTA for 501 and Vic-GTCACGACGTTGTA for 1001) was used to perform PCR amplification. The thermal cycling conditions were as follows: 95°C for 3 min, followed by 30 cycles of 94°C for 30 s, 62°C for 30 s, and 72°C for 1 min, and a final elongation step at 72°C for 5 min. The resultant samples were electrophoresed on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems by Life Technology, USA), and the fluorescently labeled PCR products were analyzed using GeneScan (Applied Biosystems by Life Technology).