Generation of eco-friendly channel catfish,Ictalurus punctatus, harboring alligator cathelicidin gene with robust disease resistance by harnessing different CRISPR/Cas9-mediated systems

Abstract

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.

Enhancing aquaculture disease resistance: Antimicrobial peptides and gene editing

“…Therefore, fish‐, AMG‐ and pathogen‐moderator analyses were performed mainly for these three parameters. Recently, Wang et al 34 demonstrated that different loci for transgene integration significantly affect survival after bacterial infection in cathelicidin‐ and cecropin‐transgenic channel catfish. As a result, we also conducted the locus‐moderator analysis to determine the effect of AMG integration loci on disease resistance enhancement.…”

“…From the first AMG‐transgenic catfish, Dunham et al 25 concluded that cecropin can enhance CSR from 27% to 96% when channel catfish were infected with F. columnare , and a similarly positive result was also observed in medaka 39 . Additionally, survival improvements of over 30% were achieved in lactoferrin‐transgenic grass carp, lysozyme‐/hepcidin‐transgenic zebrafish, cecropin‐transgenic rainbow trout and cathelicidin‐transgenic channel catfish against various bacteria, respectively 26,27,29,34,53 . Regarding viruses, GCRV and IHNV were investigated in lactoferrin‐transgenic grass carp and cecropin‐/ CF‐17 ‐transgenic rainbow trout, respectively.…”

“…Publications have already shown that single or multiple transgenes can be integrated into the fish genome by single sgRNA‐mediated genome editing (ssGE) or multi‐sgRNA‐mediated multiple genome editing assisted using plasmid donors 92,93 . Wang et al 34 demonstrated that the cathelicidin transgene can be knocked into the LH locus of channel catfish by double‐stranded DNA‐mediated ssGE. In addition, a double‐cut plasmid containing a foreign AMG transgene as a donor is beneficial to improve integration rates using CRISPR/Cas9‐mediated ssGE 58,92 .…”

“…In addition, the expression of granulin peptide (GRN‐41) was induced in the spleen of GRN‐41‐transgenic zebrafish at 6 h post V. vulnificus infection 36 . Interestingly, cathelicidin was significantly overexpressed in a variety of tissues in cathelicidin‐integrated channel catfish, even in the absence of pathogenic infections 33,34 . Furthermore, a considerable number of innate IRGEs can be induced or activated in AMG‐integrated fish after pathogen infections.…”

“…[26][27][28][29][30] Although the most intuitive advantage of AMG-integrated fish is a significantly higher survival rate when they are infected by pathogens, an increasing number of studies indicated that decreased numbers of colony-forming units (CFUs) of bacteria, enhanced enzyme activity and increased expression of AMGs and immune-related genes (IRGEs) were contributing to improved survival rate in individuals harbouring foreign AMGs. 24,[31][32][33][34] For example, the tail tissue of hepcidin-transgenic zebrafish (Danio rerio) and convict cichlid (Archocentrus nigrofasciatus) exhibited significant reduction in CFUs for Vibrio vulnificus compared to WT fish. 28 In addition, the caudal peduncle from transgenic zebrafish carrying epinecidin inhibited bacterial growth when fish were challenged with V. vulnificus.…”