CRISPR/Cas9-mediated knockout of tnf-1 in zebrafish reduces disease resistance after Edwardsiella piscicida bacterial infection

Tumor necrosis factor (TNF) is an important cytokine involved in immune responses to bacterial infections in vertebrates, including fish. Although Tnf-alpha is a well-studied cytokine, there are contradictory findings about Tnf-alpha function following bacterial infection. In this study, we analyzed the expression and function of the Tnf-alpha-type I isoform (Tnf-alpha 1) in zebrafish by knockout experiments using the CRISPR/Cas9 gene-editing tool. The open reading frame of tnf-alpha 1 encodes a 25.82 kDa protein with 234 amino acids (aa). The expression of tnf-alpha 1 in the early stages of zebrafish was observed from the 2-cell stage. Adult zebrafish spleens showed the highest expression of tnf-alpha 1. To evaluate the function of Tnf-alpha 1, an 8 bp deletion in the target region, resulting in a short truncated protein of 55 aa, was used to create the tnf-alpha 1 knockout mutant. The pattern of downstream gene expression in 7-day larvae in wild-type (WT) and tnf-alpha 1 knockout fish was examined. We also verified the fish mortality rate after Edwardsiella piscicida challenge and found that it was much higher in tnf-alpha 1 knockout fish than in WT fish. Additionally, downstream gene expression analyses after E. piscicida exposure revealed a distinct expression pattern in tnf-alpha 1 knockout fish compared to that in WT fish. Overall, our study using tnf-alpha 1 deletion in zebrafish confirmed that Tnf-alpha 1 is critical for immune regulation during bacterial infection.