CRISPR/Cas9 microinjection of transgenic embryos enhances the dual-gene integration efficiency of antimicrobial peptide genes for bacterial resistance in channel catfish, Ictalurus punctatus

Abstract

Integrating a vector-engineered antimicrobial peptide gene (AMG) into the fish genome effectively modulated the innate immune system and increased resistance to infectious disease in channel catfish (Ictalurus punctatus). CRISPR/Cas9-assisted microinjection of cecropin (Cec) and cathelicidin (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 (LR) analysis indicated that individual body weight and gender did not affect survival, while the transgenes Cec and 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. This study demonstrates that it is promising to generate dual-gene integrated genetic lines with a high integration efficiency by adopting transgenic-embryo-based CRISPR/Cas9-mediated genome editing, and an LR model is feasible for assessing the synergistic effects of gene expression.