Integration of antimicrobial peptide genes via CRISPR/Cas9 for disease resistance enhancement and reversible sterility in catfish

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 transgenes and control reproduction, catfish were studied for upscaled disease resistance coupled with intervention of reproduction to lower the potential environmental risks of introgression of transgenic escapees.

To generate disease resistance and sterility in channel catfish (Ictalurus punctatus), CRISPR/Cas9 systems were utilized to integrate the cathelicidin gene from an alligator (Alligator sinensis; As-Cath) into the target luteinizing hormone (lh) locus of channel catfish using two delivery systems assisted by double-stranded DNA (dsDNA) and single-stranded oligodeoxynucleotides (ssODNs), respectively. 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, implantion with HCG and LHRHa restored the fecundity, spawnability and hatchability of the new transgenic fish line.

To establish disease resistance and sterility in blue catfish (I. furcatus), transgenic blue catfish of primarily Rio Grande strain ancestry were generated with site-specific KI of the As-Cath transgene into the lh locus via two CRISPR/Cas9-mediated KI systems, assisted by the linear dsDNA and double-cut plasmid (dcPlasmid), respectively. High integration rates were observed with linear dsDNA (16.67%, [13/78]) and dcPlasmid strategies (24.53%, [26/106]). In addition, the on-target KI efficiency of the dcPlasmid 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 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 for growth of the As-Cath transgene integration at the lh locus was observed in the P1 founders.

To generate transgenic channel catfish carrying two exogenous antimicrobial peptide genes (AMGs), CRISPR/Cas9-assisted microinjection of cecropin (Cec) and As-Cath was employed to create dual-AMG integrated (*_Cec+/*_Cath+) transgenic embryos with high integration rates. Additionally, a univariate-multiple logit regression model was fitted to determine the synergistic expression of transgenes and endogenous AMGs in the head kidney post-bacterial infection. Transgenic-embryo-based genome editing significantly increased the efficiency of dual-AMG integration from 17.6% to 37.3%. The survival rate of single-AMG (50% vs. 20%, P = 0.023) and dual-AMG (70% vs. 20%, P = 0.005) integrated fish was dramatically higher than that of wild-type fish (20%) following Edwardsiella ictaluri challenge. More dual-AMG fry survived than expected based on integration and inheritance rates of single-AMG transgenics compared to other genotypes. Logistic regression analysis indicated that individual body weight and gender did not affect survival, while the transgenes Cec and As-Cath contributed directly to the survival during the bacterial infection. Furthermore, transgenes enhanced fish disease resistance by regulating the expression of TCP and NK-lysin genes.

To establish transgenic sterile channel catfish lines with elevated disease resistance and fast growth rate, single-sgRNA-based genome editing (ssGE) and multi-sgRNA-based MGE (msMGE) were used to replace the lh and melanocortin-4 receptor (mc4r) genes with the As-Cath transgene and the myostatin (two target sites: mstn1, mstn2) gene with the Cec transgene, respectively. A total of 9,000 embryos were microinjected from three families, and 1,004 live fingerlings were generated and analyzed. There was no significant difference in hatchability (all P > 0.05) and fry survival (all P > 0.05) between ssGE and msMGE. Compared to ssGE, CRISPR/Cas9-mediated msMGE assisted by the mixture of dsDNA and dcPlasmid donors yielded a higher KI efficiency of As-Cath (19.93%, [59/296] vs. 12.96%, [45/347]; P = 0.018) and Cec (22.97%, [68/296] vs. 10.80%, [39/361]; P = 0.003) transgenes, respectively. The msMGE strategy can be used to generate transgenic fish carrying two transgenes at multiple loci. In addition, double and quadruple mutant individuals can be produced with high efficiency (36.3% ~ 71.1%) in one-step microinjection.

Overall, the lh gene was replaced with the As-Cath transgene and then hormone therapy was administered to gain complete reproductive control of disease-resistant transgenic catfish in an environmentally sound manner. In addition, potential sterile catfish with enhanced disease resistance carrying two AMGs at multiple loci using transgenic-embryo-based genome editing or msMGE strategy was achieved. This strategy not only effectively improves the consumer-valued traits, but also guards against genetic contamination of wild populations. This is a breakthrough in aquaculture genetics to confine fish reproduction and prevent the establishment of transgenic or domestic genotypes in the natural environment.