Abstract
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.
Highlights
clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based gene knockout in animal cells, 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
We developed three transgene constructs carrying the cathelicidin gene: two double-stranded DNA constructs each carrying different promoters namely zebrafish (Danio rerio) ubiquitin promoter and common carp (Cyprinus carpio) β-actin promoter and a plasmid DNA construct driven by zebrafish ubiquitin promoter
We demonstrated that targeted gene integration can be performed using CRISPR/Cas[9] knock-in system in channel catfish
Summary
CRISPR/Cas9-based gene knockout in animal cells, 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. 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). This study marks the first use of CRISPR/Cas[9] 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. Expression of genes encoding peptides with in vitro antimicrobial activity can result in enhanced resistance to bacterial pathogens in transgenic fish[25,26]. Inserting alligator cathelicidin gene in channel catfish using CRISPR/Cas[9] system might enhance its resistance to various pathogens. An innately disease-resistant fish would not require specific vaccination for certain pathogens, and will provide an economical solution to bacterial disease p roblems[25]
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