Wild-type (AB line) and mutant zebrafish were maintained in the Zebrafish Facility of the International Institute of Molecular and Cell Biology in Warsaw (license no. PL14656251) according to standard procedures and ethical regulations. All experiments involving the zebrafish embryos/larvae were carried out in accordance with the Polish Laboratory Animal Science Association and Local Ethics Committee for Animal Experimentation in Warsaw (permission no. WAW2/181/2019).
To perform CRISPR-Cas9 knock-out, 2 nl of a solution containing two gRNAs (0.04 mg/ml of each) and Cas9 protein (0.2 mg/ml, see below) were injected into the cytoplasm of one-cell stage wild-type AB zebrafish embryos. Fifty embryos of each, gdap1 and ghitm, as well as two variants of kcng4b (variants #1 and #2) knock-outs were grown up after injection to 4 months to be in-crossed as the F0 founders. The phenotype of the F1 off-spring was analyzed at 1–5 dpf under a Leica M165 FC microscope. All F0 founders were fin-clipped for DNA sampling and HRM analysis. The mutation-positive ones were analyzed by DNA sequencing.
Standard single gRNA mutagenesis was performed as a control. For this, the 5′-site gRNAs of each set (all gRNAs-1 and Kcng4b-gRNA-3) were injected into 50 embryos as described above. The fish were analyzed by HRM at 5 days post fertilization (dpf) stage.
DNA oligonucleotides encoding gRNAs with invariant adapter sequence were used, four for kcng4b and two for gdap1 and ghitm: Kcng4b-gRNA-1, TAATACGACTCACTATATGAAGAGAGACTCCTTTTCTGTTTTAGAGCTAGAA; Kcng4b-gRNA-2, TAATACGACTCACTATAGTTAGCAATGGCCCAAGAAAGTTTTAGAGCTAGAA; Kcng4b-gRNA-3, TAATACGACTCACTATAAGCAGTGAGGGTTGGCTGAAGTTTTAGAGCTAGAA; Kcng4b-gRNA-4, TAATACGACTCACTATACATGCAGCAGCAGTGAGGGTGTTTTAGAGCTAGAA; Gdap1-gRNA-1, TAATACGACTCACTATAGGGAGTCTACGGTGATCTCTGTTTTAGAGCTAGAA; Gdap1-gRNA-2, TAATACGACTCACTATAGGCTGGTATGTGGGAGTCTAGTTTTAGAGCTAGAA; Ghitm-gRNA-1, TAATACGACTCACTATACGCGGGCAGTGTGGGCCTGAGTTTTAGAGCTAGAA; Ghitm-gRNA-2, TAATACGACTCACTATATGTGGGCCTGACGGCGCTCTGTTTTAGAGCTAGAA (gRNA sequences are underlined, all sequences here and below have the 5′–3′ direction).
For gRNA synthesis each of them was mixed with the constant oligonucleotide, containing a complementary adapter part and a T7 promoter sequence, Const-gRNA: AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC, both 10 μM, and a double-stranded product was synthesized according to the following protocol: 95 °C—3 min and 35 cycles of 95 °C—15 s, 20 °C—15 s, 72 °C—15 s using PCR Mix Plus mixture (A&A Biotechnology, Poland). The resulting DNA was purified by Clean-Up Concentrator kit (A&A Biotechnology, Poland) and verified by 2.5% agarose gel-electrophoresis. 0.2 μg of DNA was used for RNA in vitro transcription by T7 RNA-Polymerase (A&A Biotechnology, Poland) according to the manufacturer’s protocol.
Cas9 protein production
DNA encoding Streptococcus pyogenes Cas9 protein with N- and C-terminal nuclear localization signals (NLS) was PCR amplified using PCR Mix Plus mixture (A&A Biotechnology, Poland) and pCS2-nCas9n plasmid  as a template and cloned into pETM-60 vector (Novagen) using NcoI and XhoI restriction endonuclease sites. The final construct encoded an N-terminal hexa-histidine-NusA tag followed by peptide cleavage site for tobacco etch virus (TEV) protease and C-terminal hexa-histidine (His) tag. The protein was expressed in BL21 Rosetta Escherichia coli strain (Novagen) in simplified Studier’s autoinduction media  at 37 °C for 4 h followed by 20 h at 20 °C. Cells were harvested by centrifugation at 4000 × g for 30 min and stored at −20 °C.
The cell pellet was resuspended in 40 mM Tris–HCl buffer (pH 7.5) containing 5% of glycerol, 0.5 M NaCl, 40 mM imidazole, 1.0 mM phenylmethylsulfonyl fluoride (PMSF) and 2 mM 2-mercaptoethanol, then ultrasonicated and centrifugated at 10,000 × g 4 °C for 20 min. The supernatant was loaded on a HisTrap FF Crude® column (GE Healthcare) equilibrated by the same buffer. After washing with 20 mM Tris–HCl buffer (pH 7.5) containing 5% of glycerol, 0.5 mM NaCl and 80 mM imidazole, the protein was eluted by the same buffer containing 0.3 M imidazole and the N-terminal His-NusA tag was cleaved off by TEV protease (1 mg per 100 mg of Cas9 protein) during overnight dialysis against 20 mM Tris–HCl buffer (pH 7.5) containing 5% of glycerol, 0.15 mM NaCl and 2 mM 2-mercaptoethanol. The protein solution was then clarified by centrifugation (10,000 × g, 10 min) and loaded on a Q Sepharose® Fast Flow column (GE Healthcare) equilibrated by the same buffer. The flow-through was collected and dialyzed overnight against 20 mM HEPES buffer (pH 7.5) containing 0.2 mM KCl and 20% of glycerol. As a final step the protein sample was concentrated on a Vivaspin 500 centrifugal concentrator (Merk) and stored at −80 °C.
All Cas9 purification steps were controlled by spectrophotometry and by SDS-polyacrylamide gel electrophoresis.
Fish mutation analysis
The clipped fins of adult fish or 5-day-old embryos chilled on ice were placed into 30 μl of 96% ethanol, heated at 80 °C until ethanol evaporates (~10 min) and 50 μl of TE buffer (pH 8.0) were added. After 10 min of heating (80 °C) samples were chilled and incubated 3 h at 55 °C with 0.5 mg/ml Proteinase K (Merck). After another heating (10 min, 95 °C) and chilling, 1 μl of the solution was used as a template for PCR and HRM analysis.
The HRM analysis was performed according to the manufacturers’ protocols (Roche and BioRad). PCR was performed in 10 μl volume with a 0.4 μM of each primer using Light Cycler 480 High-Resolution Melting Master Mix, 2 mM MgCl2 (Roche) or using Precision Melt Supermix (BioRad). Melting curves were taken by Light Cycler 96 (Roche) or by CFX Connect Real-Time PCR System (BioRad). The following oligonucleotides were used as the primers:
For kcng4b #1: Kg4-HRM-1f, GATGCCACTGCTAAAGAGG, and Kg4-HRM-1r, GGTTGGCTGAATGGCAGC; and for kcng4b #2: Kg4-HRM-2f, TGCTAACAGAAATAGCCTGC, and Kg4-HRM-2r, CTCATTAAAACATACAGAAATCC.
For gdap1: GDAP1-HRM-f, GGAAGCACGTATTATCATCG; and GDAP1-HRM-r, TGCGAATGTGTGTAGTGGC.
For ghitm: GHITM-HRM-f, CGATCTGGCCGCAGTACG; and GHITM-HRM-r, TACCAGCCAGGAGTTGCTC.
The following PCR conditions were used: pre-incubation: 95 °C—10 min; 3 step amplification (45 cycles): 95 °C—10 s, 60 °C—15 s, 72 °C—15 s; high resolution melting: 95 °C—60 s, 40 °C—60 s, 65 °C—1 s, 97 °C—1 s.
For the PCR and following sequencing kcng4b-specific primers were used: Kcng4b-seq-fvd, CGTTCATATCACGAACTGAAG, and Kcng4b-seq-rev, GGTAGGTCAAATCTTTGAAAAC; while the gdap1 and ghitm fragments were amplified using the HRM primers. The following PCR conditions were used: pre-incubation: 95 °C—3 min; 3 step amplification (35 cycles): 95 °C—30 s, 58 °C—30 s, 68 °C—15 s. PCR-products were got using PCR Mix Plus mixture from A&A Biotechnology (Poland) and sequenced from the same primers. Heterozygous sequences were analyzed using the TIDE web tool .