Integration of alligator cathelicidin gene via two CRISPR/Cas9-assisted systems enhances bacterial resistance in blue catfish, Ictalurus furcatus

Abstract

CRISPR/Cas9-mediated genome editing has paved new avenues for improving production-valued traits in aquaculture by knocking out or disrupting functional genes. However, utilizing CRISPR/Cas9-based knock-in (KI) of exogenous genes can expedite genetic improvement of traits such as disease resistance, which remains problematic in farmed fish. In this study, we successfully generated transgenic blue catfish (Ictalurus furcatus) of primarily Rio Grande strain ancestry with site-specific KI of the alligator (Alligator sinensis) cathelicidin (As-Cath) gene into the luteinizing hormone (lh) locus via two CRISPR/Cas9-mediated KI systems, assisted by the linear double-stranded DNA (dsDNA) and double-cut plasmid, respectively. High integration rates were observed with linear dsDNA (16.67%, [13/78]) and double-cut plasmid strategies (24.53%, [26/106]). In addition, the on-target KI efficiency of the double-cut plasmid strategy (16.04%, [17/106]) was 1.67 times higher than that of the linear dsDNA strategy (10.26%, [8/78]) based on the odds ratio. The relative expression of the As-Cath transgene of P1 founders was detected in nine tissues, dominated by the kidney, skin, and muscle (14.30-, 7.71- and 6.92-fold change, P < 0.05). Moreover, the As-Cath transgenic blue catfish showed a higher cumulative survival rate than that of wild-type controls (80% vs. 30%, P < 0.05) following Flavobacterium covae infection. Survival during culture supports the challenge data as survival of As-Cath transgenic individuals was 97.1% while that of pooled non-transgenic individuals was observed to be less 87.0% (P = 0.15). The growth rates and external morphology of the transgenic and wild-type siblings were not different (P > 0.05), indicating no pleiotropic effects of the As-Cath transgene integration at the lh locus in the P1 founders for this trait. Taken together, our findings demonstrate that CRISPR/Cas9-assisted KI of an antimicrobial peptide gene can be achieved in blue catfish with high integration efficiency, and As-Cath transgenic blue catfish have improved disease resistance, which is a promising strategy for disease reduction in aquaculture.