CRISPR/Cas9-based gene knockout in animal cells, particularly in teleosts, has proven to be very efficient with regards to mutation rates, but the precise insertion of exogenous DNA or gene knock-in via the homology-directed repair (HDR) pathway has seldom been achieved outside of the model organisms. Here, we succeeded in integrating with high efficiency an exogenous alligator cathelicidin gene into a targeted non-coding region of channel catfish (Ictalurus punctatus) chromosome 1 using two different donor templates (synthesized linear dsDNA and cloned plasmid DNA constructs). We also tested two different promoters for driving the gene, zebrafish ubiquitin promoter and common carp β-actin promoter, harboring a 250-bp homologous region flanking both sides of the genomic target locus. Integration rates were found higher in dead fry than in live fingerlings, indicating either off-target effects or pleiotropic effects. Furthermore, low levels of mosaicism were detected in the tissues of P₁individuals harboring the transgene, and high transgene expression was observed in the blood of some P₁fish. This can be an indication of the localization of cathelicidin in neutrophils and macrophage granules as also observed in most antimicrobial peptides. This study marks the first use of CRISPR/Cas9 HDR for gene integration in channel catfish and may contribute to the generation of a more efficient system for precise gene integration in catfish and other aquaculture species, and the development of gene-edited, disease-resistant fish.

Transcription activator-like effector nuclease (TALEN) plasmids targeting the channel catfish gonadotropin-releasing hormone (cfGnRH) gene were delivered into fertilized eggs with double electroporation to sterilize channel catfish (Ictalurus punctatus). Targeted cfGnRH fish were sequenced and base deletion, substitution, and insertion were detected. The gene mutagenesis was achieved in 52.9% of P₁ fish. P₁ mutants (individuals with human-induced sequence changes at the cfGnRH locus) had lower spawning rates (20.0–50.0%) when there was no hormone therapy compared to the control pairs (66.7%) as well as having lower average egg hatch rates (2.0% versus 32.3–74.3%) except for one cfGnRH mutated female that had a 66.0% hatch rate. After low fertility was observed in 2016, application of luteinizing hormone-releasing hormone analog (LHRHa) hormone therapy resulted in good spawning and hatch rates for mutants in 2017, which were not significantly different from the controls (p > 0.05). No exogenous DNA fragments were detected in the genome of mutant P₁ fish, indicating no integration of the plasmids. No obvious effects on other economically important traits were observed after the knockout of the reproductive gene in the P₁ fish. Growth rates, survival, and appearance between mutant and control individuals were not different. While complete knock-out of reproductive output was not achieved, as these were mosaic P₁ brood stock, gene editing of channel catfish for the reproductive confinement of gene-engineered, domestic, and invasive fish to prevent gene flow into the natural environment appears promising.

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.