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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 P1 individuals harboring the transgene, and high transgene expression was observed in the blood of some P1 fish. 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.

One of the major goals in aquaculture is to protect fish against infectious diseases as disease outbreaks could lead to economic losses if not controlled. Antimicrobial peptides (AMPs), a class of highly conserved peptides known to possess direct antimicrobial activities against invading pathogens, were evaluated for their ability to protect Channel Catfish Ictalurus punctatus and hybrid catfish (female Channel Catfish × male Blue Catfish I. furcatus) against infection caused by the fish pathogen Aeromonas hydrophila ML09‐119. To identify effective peptides, the minimum inhibitory concentrations against bacterial pathogens Edwardsiella ictaluri S97‐773, Edwardsiella piscicida E22‐10, A. hydrophila ML09‐119, Aeromonas veronii 03X03876, and Flavobacterium columnare GL‐001 were determined in vitro. In general and overall, cathelicidins derived from alligator and sea snake exhibited more potent and rapid antimicrobial activities against the tested catfish pathogens as compared to cecropin and pleurocidin AMPs and ampicillin, the antibiotic control. When the peptides (2.5 µg of peptide/g of fish) were injected into fish and simultaneously challenged with A. hydrophila through immersion, increased survival rates in Channel Catfish and hybrid catfish were observed in both cathelicidin (alligator and sea snake) treatments as compared to other peptides and the infected control (P < 0.001) with alligator cathelicidin being the overall best treatment. Bacterial numbers in the kidney and liver of Channel Catfish and hybrid catfish also decreased (P < 0.05) for cathelicidin‐injected groups at 24 and 48 h after challenge infection. These results show the potential of cathelicidin to protect catfish against bacterial infections and suggest that an approach overexpressing the peptide in transgenic fish, which is the long‐term goal of this research program, may provide a method of decreasing bacterial disease problems in catfish as delivering the peptides via individual injection or feeding would not be economically feasible.

Flying fish is considered a low-value fish in the Philippines but is a good source of inexpensive animal protein. The challenge is to effectively utilize this vulnerable raw material to increase its market value. The study evaluated the physicochemical changes and sensory attributes of flying fish (Cheilopogon intermedius) fillets marinated in three different marinating solutions (Marinade 1: “ham flavor”; Marinade 2: “salty-sour flavor”; Marinade 3: “spiced flavor”) and control (un-marinated) followed by vacuum-packaging then stored at 4 °C for 20 d. Chemical analyses revealed a significant reduction (p < 0.05) in the total volatile base nitrogen (TVB-N), trimethylamine nitrogen (TMA-N), histamine level, and thiobarbituric acid (TBA) value in the marinated fillets in comparison with the control. After 20 d of storage at 4 °C, mesophilic and psychrophilic bacterial counts reached 107–109 and 106–108 CFU/g, respectively. The panelists considered samples unfit for human consumption on Day 12 when the mesophilic and psychrophilic bacterial counts exceeded 106 CFU/g. No significant differences (p > 0.05) were detected for the overall sensory acceptability of the marinated samples; however, Marinade 1 was found to be most acceptable, followed by Marinades 2 and 3. The shelf-life of the marinated flying fish based on microbiological and sensory analyses at refrigerated storage (4 °C) was 12 d.

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The CRISPR/Cas9 platform holds promise for modifying fish traits of interest as a precise and versatile tool for genome manipulation. To reduce introgression of transgene and control reproduction, catfish species have been studied for upscaled disease resistance and intervening of reproduction to lower the potential environmental risks of introgression of escapees' as transgenic animals. Taking advantage of the CRISPR/Cas9-mediated system, we succeeded in integrating the cathelicidin gene from an alligator (Alligator sinensis; As-Cath) into the target luteinizing hormone (LH) locus of channel catfish (Ictalurus punctatus) using two delivery systems assisted by double-stranded DNA (dsDNA) and single-stranded oligodeoxynucleotides (ssODNs), respectively. In this study, high knock-in (KI) efficiency (22.38%, 64/286) but low on-target was achieved using the ssODN strategy, whereas adopting a dsDNA as the donor template led to an efficient on-target KI (10.80%, 23/213). On-target KI of As-Cath was instrumental in establishing the LH knockout (LH--As-Cath+) catfish line, which displayed heightened disease resistance and reduced fecundity compared to the wild-type sibling fish. Furthermore, implanting with HCG and LHRHa can restore the fecundity, spawnability and hatchability of the new transgenic fish line. Overall, we replaced the LH gene with an alligator cathelicidin transgene and then administered hormone therapy to gain complete reproductive control of disease-resistant transgenic catfish in an environmentally sound manner. This strategy not only effectively improves the consumer-valued traits, but also guards against genetic contamination. This is a breakthrough in aquaculture genetics to confine fish reproduction and prevent the establishment of transgenic or domestic genotypes in the natural environment.

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