Gene Editing of the Follicle-Stimulating Hormone Gene to Sterilize Channel Catfish, Ictalurus punctatus, Using a Modified Transcription Activator-like Effector Nuclease Technology with Electroporation

Abstract

Follicle-stimulating hormone (fsh) plays an important role in sexual maturation in catfish. Knocking out the fsh gene in the fish zygote should suppress the reproduction of channel catfish (Ictalurus punctatus). In this study, transcription activator-like effector nuclease (TALEN) plasmids targeting the fsh gene were electroporated into fertilized eggs with the standard double electroporation technique. Targeted fsh cleavage efficiency was 63.2% in P₁fsh-knockout catfish. Ten of fifteen (66.7%) control pairs spawned, and their eggs had 32.3-74.3% average hatch rates in 2016 and 2017. Without hormone therapy, the spawning rates of P₁ mutants ranged from 33.3 to 40.0%, with an average egg hatching rate of 0.75%. After confirmation of the low fertility of P₁ mutants in 2016, human chorionic gonadotropin (HCG) hormone therapy improved the spawning rates by 80% for female mutants and 88.9% for male mutants, and the mean hatch rate was 35.0% for F₁ embryos, similar to that of the controls (p > 0.05). Polymerase chain reaction (PCR) identification showed no potential TALEN plasmid integration into the P₁ channel catfish genome. Neither the P₁ nor the F₁ mutant fish showed any noticeable changes in in body weight, survival rate, and hatching rate when the reproductive gene was knocked out. F₁ families had a mean inheritance rate of 50.3%. The results brought us one step closer to allowing implementation of certain genetic techniques to aquaculture and fisheries management, while essentially eliminating the potential environment risk posed by transgenic, hybrid, and exotic fish as well as domestic fish.

Keywords: channel catfish; follicle-stimulating hormone; hormone therapy; transcription activator-like effector nucleases.

Figure 1

Surveyor mutation detection test results of P₁ channel catfish (Ictalurus punctatus). The mutation was targeted at the follicle-stimulating hormone (fsh) gene. The “-” on the left of the molecular marker indicates the control, which is wild-type fish DNA. The right “-” denotes the negative control using water as the DNA template; “m” indicates 1 kb molecular marker. Mutated channel catfish are shown in lanes 1, 3, 4, and 5, and wild-type channel catfish are displayed in lanes 2 and 6. Figure 1 was derived from the full-length gel shown in Supplementary Figure S2.

Figure 2

Sequence analysis of the mutated follicle-stimulating hormone (fsh) gene in channel catfish (Ictalurus punctatus). (A) Sequence analysis of the nucleic acid in the mutated fsh gene. The channel catfish fsh nucleotide sequence of the wild-type is displayed at the top. Orange highlighted sequences represent the target binding sites of the transcription activator-like effector nucleases (TALENs). Blue colored sequences represent the predicted cleavage sites of the nucleases. The mutation types (deletion/insertion/substitution) of nucleotides are shown by red dashes and letters. Numbers of nucleotides deleted (-) or inserted (+) in the edited fsh gene are denoted on the right side of the sequences. (B) Sequence analysis of corresponding predicted amino acid in the fsh mutants. Frameshift reading resulted in a premature stop codon (red colored). Single amino acid deletions or substitutions were caused by changes in one nucleotide mutations or three nucleotide deletions that are highlighted in red. (C) Bright-green shaded areas are the Cys residues, and N-linked glycosylation sites are underlined. The asterisk “*” indicated the same amino acid between normal human and wild-type catfish.

Figure 3

Analysis of transcription activator-like effector nuclease (TALEN) plasmid integration into the genome of channel catfish (Ictalurus punctatus) using polymerase chain reaction (PCR) genome. In (A,B), from the left to the right lanes of “-” denote the negative controls. The left one indicates using water as a template, and the right one indicates the wild-type channel fish sample; the two “+” denote the positives. The left one indicates the TALEN plasmids containing the left DNA binding site, and the right one indicates TALEN plasmids containing the right DNA binding site; “m” indicates 1 kb molecular weight standards. The same six fsh-mutated channel catfish individuals (numbered 1–6) were checked for plasmid DNA element integration. The cytomegalovirus (CMV) promoter region was resolved in (A). The transcription activator-like (TAL) repeats region was resolved in (B). The original full gel image of Figure 3 can be found in Supplementary Figure S3.

Figure 4

Mutation analysis of follicle-stimulating hormone (fsh) gene edited F₁ channel catfish (Ictalurus punctatus) using the Surveyor mutation detection test. “-” represents the wild-type DNA as negative control; “m” represents 1 kb DNA molecular weight standards; numbers (2, 3, 4, 5 and 6) show the mutated channel catfish individuals; lanes numbered 1 and 7 show progeny without anticipated mutation. The full gel picture of Figure 4 can be found in Supplementary Figure S4.