All experimental animal care was performed in accordance with institutional and national guidelines and regulations. The study protocol was approved by the Institutional Animal Care and Use Committee of the respective universities (Gunma University Permit # 17–051; Osaka University, RIMD Permit # R02-04). The study is reported in accordance with ARRIVE guidelines.
Maintenance of fish lines
The GRZ (GRZ-AD) strain of N. furzeri3 was gifted by prof. Adam Antebi. Fish were maintained at 26.5 ° C, 0.7 conductivity on 12/12 h light / dark cycle in the fish breeding system (MEITO, Nagoya, Japan) at a density of 1 fish per 1.4 L tank for adult fish. Fish were fed freshly hatched brine shrimp twice a day from Monday to Saturday, and once a day on Sunday, fish that were over 2 weeks old were also fed bloodworms (Kyorin, Himeji, Japan). For mating, one adult male and 3–4 female fish were kept in 4 L tanks, and females spawned on a sand substrate in the plastic cases. Embryos were collected and incubated in the egg water (0.03% sea salt containing methylene blue). Ten days later, the embryos were plated on sterile dry peat moss until they were ready to hatch. Usually, one month after egg collection, embryos were ready to hatch and incubated in the 0.07% ice-cold humic acid solution (53680, Sigma-Aldrich, St. Louis, MO, USA) for 30–60 min, and transferred into the 4 L hatching tank with air supply. Hatched fry were kept in a hatching tank, and half of the solution was changed with the fish breeding system water every day. After 2 weeks, by which time they had grown to be around 1–1.5 cm, they were transferred to 1.4 L tanks similar to the adult fish.
The sgRNA targeting sites are listed in Supplementary table 1 and the gene sequences were retrieved using NCBI Gene IDs (tyrosinase: 107380455, tcf7l1: 107393717, tbx16: 107393822). We selected three target sites on the protein coding sequences for each gene that did not overlap with other genomic sequences and were close to the translational initiation sequence, aiming to induce frameshift mutation, using the chop-chop program. The protocol for sgRNA synthesis was based on a previously reported method24. The oligonucleotides containing a T7 promoter sequence, target sequence, and the sgRNA templates were PCR-amplified from pDR274 vectors25 using the oligonucleotides and primer sgRNA-RV with PrimeSTAR Max (TaKaRa, Kusatsu, Japan) and purified using the NucleoSpin Gel and PCR Clean-up Kit (MACHEREY – NAGEL, Düren, Germany). sgRNAs were synthesized using the CUGA7 gRNA Synthesis Kit (Nippon Gene, Tokyo, Japan), and their concentrations were measured using a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The injection solutions consisted of three sgRNAs (20 pg each), Cas9 protein (1 nM, M0646, New England Biolabs, MA, USA), and phenol red (P0290, Sigma-Aldrich) in RNase-free water. The solution was injected into the cell body of 1-cell stage fertilized eggs or 4-cells stage embryos to generate genetic mosaics, and then the embryos with the red-colored cell body were selected. The DsRed2-transgenic line was created by injecting the plasmid (Ola.Actb: LOXP-DsRed2-LOXP-EGFP)26 with 15 pg tol2 mRNA. The efficiency of DNA digestion was analyzed by PCR, including the targeting by following the primer (listed in Supplementary table 1) and T7E1 reaction (313-08801, NIPPONGENE), and then PCR fragments were analyzed by a microchip electrophoresis system (MCE-202 MultiNA, Shimadzu, Kyoto, Japan) according to the manufacturer’s instructions using a DNA-1000 reagent kit (Shimadzu).
CRISPR / Cas9-mediated reporter knock-in
A donor DNA plasmid (pBS-Tbait-olhs-GFP) and sgRNA for the bait sequence were prepared according to a previously described method15.16. Capped mRNA encoding Cas9 nuclease was synthesized using the pCS2 + hSpCas9 vector as a template using the mMessage mMachine SP6 Kit (Thermo Fisher Scientific). Targeting sites of sgRNA (listed in Supplementary table 1) were selected from the 5 ‘UTR region using the chop-chop program, and genes sequences were retrieved from the NCBI Gene database (Gene ID of note: 107391787, tbx16: 107393822, hba: 107378369, entpd5a: 107392375).
The injection plates were generated by melting 2% agarose with egg water in a 100 mm petri-dish, and the injection mold was floated with 0.9 mm width 1.0 mm height (AM6540-0904-1, IPN-Factory, Hekinan, Japan). For injection needle preparation, a glass capillary (G-1, Narishige, Tokyo, Japan) was pulled by one-step pulling using a PC-10 puller (Narishige), and the tip of the capillary was slightly broken off. RNA and DNA solutions were introduced into the needle using a microloader (5242956003, Eppendorf) and injected using an IM 300 microinjector (Narishige).
In situ hybridization
Embryos were fixed with 4% paraformaldehyde. Whole-mount in situ hybridization was performed according to a standard protocol. Probes for the note and tbx16 genes were generated from N. furzeri embryonic cDNA by PCR using the sequences retrieved using the gene IDs from the NCBI Gene database (note: 107391787, tbx16:107393822).
Bright-field images of embryos and fry were captured using a stereomicroscope (Leica, Wetzlar, Germany). Fluorescent images of the embryos were captured using FV3000 (Olympus, Tokyo, Japan).