CRISPR-Based Functional Characterization of Immune Genes Associated Pathogen Resistance in Aquaculture Species

Abstract

Streptococcosis caused by Streptococcus agalactiae constitutes a serious challenge to Nile tilapia (Oreochromis niloticus) aquaculture with increased mortality and heavy economic burden. Elucidation of the underlying molecular mechanism in the host immune system is important for designing effective disease intervention strategies. In this experiment, the functions of three immune-related genes tlr2, myd88, and il1b on Nile tilapia defense response to S. agalactiae were analyzed using CRISPR-Cas9 technology. The in-silico screening of candidate guide RNA revealed a promising on-target efficiency (79.2-88.0%) and off-target specificity (88.5-95.0%) with il1b being the most favorable. The genome editing verified that all the selected target sites were successfully modified in 83.3% of fish per gene group. After infection, the expression of all three genes was significantly increased in the unedited fish while gene-edited groups exhibited transcriptional inhibition of their target genes. The gene-modified fish displayed lower immune downstream signaling, lower inflammatory markers, higher bacterial burden (6.50–7.00 log10 CFU/g), and lower survival rate (50.0-66.7%) than that of the controls (75.0%). From this integration, the functions of tlr2, myd88, and il1b were determined to be important and interconnected for innate immunity and pathogen defense mechanisms. This information confirms the suggested signaling model of TLR2-MyD88-IL-1β, where the disruption of these genes reduces resistance to S. agalactiae in Nile tilapia.