Transgene Insertion of Cathelicidin Gene in Channel Catfish Ictalurus punctatus using CRISPR/Cas9 Knock-in Technology and Cathelicidin Activity Against Catfish Pathogens
Type of DegreePhD Dissertation
Fisheries and Allied Aquacultures
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Protection of fish against infectious diseases is a major challenge in aquaculture, and economic losses due to these diseases limit profitability. 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 (I. punctatus ♀ x blue catfish, I. furcatus ♂) against infection caused by the catfish pathogens, Aeromonas hydrophila ML09-119 and Edwardsiella ictaluri S97-773. To identify effective peptides, the minimum inhibitory concentrations (MICs) against pathogens namely Edwardsiella ictaluri S97-773, E. piscicida E22-10, A. hydrophila ML09-119, A. veronii 03X03876 and Flavobacterium columnare GL-001 were determined in vitro. Cathelicidins derived from alligator and sea snake exhibited more potent and rapid antimicrobial activities against the tested catfish pathogens when compared to cecropin and pleurocidin AMPs, and ampicillin, the antibiotic control. When the peptides (50 µg/ml) were injected into fish and simultaneously challenged with each pathogen through immersion, increased survival rates in channel and hybrid catfish were observed for both cathelicidins (alligator and sea snake) as compared to other peptides and the infected control. Bacterial numbers were also reduced in the liver and kidney of channel catfish and hybrid catfish in the cathelicidin treatments 24 h post-infection. After 8 days of E. ictaluri challenge, serum was collected to determine immune-related parameters such as bactericidal activity, lysozyme, serum protein, albumin and globulin. These immune-related parameters were significantly and consistently elevated in fish injected with the two cathelicidins as compared to other peptides and the infected control. These results show the potential of cathelicidin to protect catfish against bacterial infections and suggests an approach that overexpressing the peptide in transgenic fish may provide a method of decreasing bacterial disease problems in catfish. To produce disease-resistant lines of channel catfish carrying cathelicidin gene, targeted gene insertion using CRISPR/Cas9 knock-in system was used. Two types of donor vectors, dsDNA and plasmid DNA, were designed, which were driven by two different promoters, zebrafish ubiquitin promoter and common carp β-actin promoter, harboring a 250-bp homologous sequences flanking both sides of the genomic target locus in a non-coding region of channel catfish chromosome 1. High integration rates were observed using dsDNA and plasmid DNA construct driven by zebrafish ubiquitin promoter. However, upon analysis, integration rates were higher in dead fry than in live fingerlings, indicating either off-target effects or pleiotropic effects. Additionally, we may be targeting a sensitive area of the genome. 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 localization of cathelicidin in neutrophils and macrophage granules as also observed in most antimicrobial peptides. Overall, these findings prove a successful targeted exogenous gene insertion in a non-coding chromosomal region in non-model fish using CRISPR/Cas9 knock-in system.