CRISPR/Cas9-mediated knock-in of alligator cathelicidin gene in a non-coding region of channel catfish genome

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.

Single nucleotide replacement in the Atlantic salmon genome using CRISPR/Cas9 and asymmetrical oligonucleotide donors

“…To make precise genome alterations, it is a necessity to induce homology directed repair (HDR) by supplying a repair template homologous to the CRISPR target site, thereby allowing to change SNPs, insert affinity tags for protein detection and modify regulatory elements to alter expression of target genes. An example is channel catfish, where HDR mediated editing was used to increase disease resistance [25]. A single nucleotide replacement (SNR) can be used to introduce favorable wild type alleles and could be a promising and time saving solution compared to traditional breeding with backcrossing and selection.…”

Effectiveness of Cathelicidin Antimicrobial Peptide against Ictalurid Catfish Bacterial Pathogens

“…In this regard, we have transferred the cathelicidin gene to Channel Catfish (Simora et al. 2020) and have future plans to expand the population to allow the testing of disease resistance. Cecropin transgenic Channel Catfish exhibited enhanced disease resistance compared to non‐transgenic controls (Dunham et al.…”

Genome editing on finfish: Current status and implications for sustainability

“…13,14,25,49,51,53,57,59,60,61,63,69,72,73,83 Some studies suspect embryo mortality 35,79 and embryo malformation followed by death 12 to be related to off-target effects. Simora et al 77 experienced that increased mutation rate implied increased embryo mortality after inserting an alligator (Alligator mississippiensis) cathelicidin gene for pathogen resistance in Channel catfish (Ictalurus punctatus Rafinesque), suspecting this to be either offtarget effects or pleiotropic effects. Elaswad et al 79 argue that the specificity of the CRISPR/Cas9 depends on the protospacer adjacent motif (PAM) and the gRNA.…”