All experiments were conducted at the Fish Genetics Research Unit, E.W. Shell Fisheries Research Center, Auburn University, AL, USA. All experimental protocols used in this experiment were approved by the Auburn University Institutional Animal Care and Use Committee (IACUC) before the experiment was initiated and followed the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.
Design and preparation of sgRNA and CRISPR/Cas9 System
A common carp β-actin promoter (Accession ID: M24113.1) was used to drive the expression of masu salmon elongase gene (Accesion ID: KC847063.1). An antifreeze polyA terminator from ocean pout, Zoarces americanus (Accession ID: S65567.1) was used to terminate transcription. This synthetic construct (ccβA-msElovl2), totaling 9,267bp, was built by GenScript (USA Inc., Piscataway, NJ 08854, USA). Two sets of two CRISPR short guide RNAs (sgRNA) plasmids were designed, targeting the coding region in exon one of the mc4r gene in channel catfish (MC4R-A and MC4R-D) and the non-coding region adjacent to the protospacer adjacent motif (PAM) sequence of the transgene (PUC57) (Table 1). The corresponding ccβA-msElovl2 transgene was designed to contain two arms, upstream and downstream, for homologous recombination, matching the cuts created by the sgRNA. sgRNAs and ssODNs were constructed for 2H2OP insertion of ccβA-msElovl2 into mc4r locus (Table 1; Figure 1). Each of the oligos and plasmid was reconstituted using DNase/RNase Free water to 10 mM. The sgRNA templates were generated by synthesizing double stranded DNA through T7 run-off as described by Varshney et al. (2015) with modifications from Khalil et al. (2017). The two oligos were annealed using EconoTaqÒ Plus 2x Master Mix (Lucigen, Middleton, WI). The sgRNAs were synthesized using the Maxiscript T7 kit (Thermo Fisher Scientific), following the manufacture guidelines. The obtained sgRNAs were purified using Zymo RNA Clean and Concentrator kit (Zymo Research). The sgRNAs were stored in -80°C freezer. The Cas9 protein was acquired from PNA Bio (3541 Old Conejo Rd, Newbury Park, CA 91320) and reconstituted in dH20 to a concentration of 1 mg/mL. The ccβA-msElovl2 plasmid was reconstituted to 250 ng/µL. Each of the ssODNs were reconstituted to 100 ng/µL. Twenty minutes prior to fertilizing the eggs, two sets of injection solutions were prepared by mixing 2µL (200 ng) of each pair of ssODN, 1 µL ccβA-msElovl2 plasmid (125 ng), 1 µL ccβA-msElovl2 sgRNA (PUC57) (300 ng), 1 µL MC4R-(A or D) sgRNA (300 ng), 2 µL Cas9 and 2 µL phenol red (10%) to a total volume of 11 µL.
Brood stock selection, husbandry, selection, and spawning
The Kansas strain of channel catfish was chosen as brood stock due to their superior growth and fry output when induced by injection of luteinizing hormone releasing hormone analogs (LHRHa). Brood stock were cultured in 0.04-ha earthen ponds averaging 1-meter in depth at a density of 3500 fish/ha. They were fed a 32% protein catfish pellet at 1-2% of their body weight five days per week. Dissolved oxygen was maintained above 3 mg/L using a ½ horsepower surface aerator (Air-O-Lator). Brood stock spawning followed the procedures described by Qin et al. (2016).
Transgenic fish production
Approximately 200-300 g of eggs were transferred to a greased pan for fertilization. Approximately 3 mL of sperm solution was added to the eggs and mixed gently with fingertips. Freshwater was added to barely cover the eggs to activate the sperm and eggs and the water was swirled to form a single layer and prevent sticking. After 2 min, the eggs should be fertilized, and three more cm of water was added, and the eggs were left to harden for 15 min. While the embryos were hardening, 5-10 µL of the injection mixture was loaded into 1.0 mm OD borosilicate glass capillary microinjection needles using a microloader. After 15 minutes 100-200 embryos were transferred in a single layer to a greased 100 mm petri dish and covered with Holtfreter’s solution (59 mM NaCl, 0.67 mM KCl, 2.4 mM NaHCO3, 0.76 mM CaCl2, 1.67 mM MgSO4) for microinjection. Microinjection procedure followed that described by Khalil et al. (2017) and Elaswad et al. (2018). Each embryo was injected with 5 nL of solution at the one cell stage. Control embryos were injected with 5 nL of 12% phenol red solution.
After microinjection, embryos were placed in 4 L tubs of Holtfreter’s solution (Bart and Dunham 1996) with 10 mg/L doxycycline kept at 27°C with continuous aeration. The solution was changed, and dead embryos were removed daily. After about 5 days, or when the embryos were moving rapidly within the egg membrane and close to hatch, doxycycline treatment was discontinued. Fry were kept at 20 fry/L and fed Purina® AquaMax® powdered starter feed (50% crude protein, 17% crude fat, 3% crude fiber and 12% ash) (Purina Animal Nutrition LLC, Shoreview, MN) four times a day for two months. At 20 days post hatch, fry were moved to 60 L aquaria in recirculating aquaculture systems (RAS).
Culture and growth
Fry from each genetic type were stocked into 3-replicate 50 L aquaria in RAS for growth experiments. Fish were kept at a density of 2 fish/L. Feed size was adjusted as the fish grew. Fry were fed Aquaxcel WW Fish Starter 4512 (45% crude protein, 12% crude fat, 3% crude fiber and 1% phosphorus) (Cargill Animal Nutrition, Minneapolis, MN) twice a day for four months. Juvenile fish were fed with WW 4010 Transition (40% crude protein, 10% crude fat, 4% crude fiber and 1% phosphorus) (Cargill Animal Nutrition, Minneapolis, MN) once a day. All fish were fed every day to satiation.
At 6 months post-hatch, pelvic fin-clip samples (10-20 mg) were collected in sterile 1.5 mL Eppendorf tubes and kept in a -80°C freezer until DNA extraction. Genomic DNA was extracted using proteinase K digestion followed by protein precipitation and iso-propanol precipitation of DNA, as described by Kurita et al. (2004). DNA concentration and purity was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific) and concentration was adjusted to 500 ng/µL.
To determine the presence of the transgene, the primer set Elovl2-F, Elovl2-R (Table 1) was designed using Primer3plus to amplify a partial sequence of the transgene. The Expand High FidelityPLUS PCR System (Roche) was used with 500 ng of genomic DNA. A Bio-Rad T100 Thermal Cycler was used to run PCR amplification with the following procedure: initial denaturing at 95°C for 3 min, 34 cycles of denaturation at 95°C for 30 s, annealing at 62°C for 40 s with a ramp speed of -0.2°C/s, extension at 72°C for 40 s; and final elongation at 72°C for 10 min. The PCR product was confirmed with gel electrophoresis on a 1% TAE tris base, acetic acid, and EDTA agarose gel. The knock-in mutation was preliminarily identified by presence of the amplified DNA fragment.
The primer set MC4R-F and MC4R-R (Table 1) was designed using Primer3plus to encapsulate all possible mutation sites in the mc4r gene. The PCR amplification procedure was as follows: initial denaturing at 95°C for 3 min; 34 cycles of denaturation at 95°C for 30 s, annealing at 62°C for 40 s with a ramp speed of -0.2°C/s, extension at 72°C for 40 s; and final elongation at 72°C for 10 min. The PCR product was confirmed on a 1% TAE tris base, acetic acid, and EDTA agarose gel. The Surveyor® mutation detection kit (Integrated DNA Technologies, Coralville, IA) was used to detect mutations. The PCR product from the treatment fish was mixed with PCR product from a wild-type control of the same family at a 1:1 ratio. The combined product was then hybridized in a BioRad Thermocycler using the following procedure: Initial denaturing at 95°C for 3 min; 95 to 85°C at -0.2°C/s, 85 to 25°C at -0.2°C/s. Hybridized PCR products were mixed with Nuclease S, Enhancer S, MgCl2 and Reaction Buffer (2) according to kit instructions and incubated at 42°C for 1 h. The digested products were separated on a 1.5% TBE (tris borate EDTA) agarose gel and compared with that of control samples.
To confirm and identify the mutations, positive samples were sequenced, and the DNA cloned, using the TA cloning method. First, genomic DNA from mutants was amplified with PCR using Expand High FidelityPLUS PCR System (Roche) using the above protocol. The PCR product was verified using a 1% TAE agarose gel and cloned into the TOPO® TA Cloning® Kit (Invitrogen) with 20 clones per sample and sent to MCLabs for sequencing. Finally, the resulting sequences were interpreted using the MAFFT sequence alignment tool.
Fatty Acid Analysis
Muscle was sampled from 3 fish containing the Elovl2 transgene and mutated at the mc4r gene, 3 fish containing the mc4r mutation and not the Elovl2 transgene, and 3 control fish with no mutation. Muscle samples were taken immediately after euthanizing the individual by pithing. They were then prepared for lipid extraction by first grinding into a slurry with a coffee grinder. Ground tissue (2g) was homogenized per extraction using a handheld tissue homogenizer until the tissue was sufficiently homogenized. Lipid extraction was performed using the chloroform-methanol protocol from Folch et al. (1957) and performed in triplicate for each sample. Extracts in hexane were kept in 2 mL borosilicate glass vials with PTFE caps and stored at -20°C until they could be injected into a gas chromatograph (GC-MS7890A). The concentration of the fatty acid was measured using gas chromatography-mass spectrometry (GC-MS, Agilent Technologies 7890A GC with 5975C MS) equipped with a DB-1701 GC column. Helium was used as the carrier gas. The initial temperature of the oven was set at 100°C for 5 min, followed with a ramp of 4°C/min to reach 250°C and then held for 10 min. The detector was kept at 225°C. Peaks were identified and quality check of the resulting spectra, and calculation of concentrations and was done by comparing sample retention times to a standard mix (Supelco® C4-24 Fatty Acid Methyl Ester (FAME) Mix; Lot: LRAC7954).
To calculate differences in body weight and fatty acid levels between ccβA-msElovl2 knockout/MC4R knock in mutants, mc4r knockout mutants and controls, a one-way ANOVA and Tukey’s multiple comparisons test were performed using RStudio version 1.1.463 (R Core Team, Vienna, Austria). Logistic regression was used to determine significant difference in mutation rates between MC4R-D and MC4R-A using RStudio. Principal component analysis (PCA) was plotted in RStudio using the ggplot2 3.1.0 package. PCA was based on a correlation matrix.