Abstract

A novel brain cell line, CIB, derived from grass carp, was established and characterized. • CIB was sensitive to Grass Carp Reovirus Type I (GCRV-I) and elicited a robust innate immune response upon viral infection. • CIB demonstrated high plasmid transfection efficiency, facilitating the expression of exogenous genes. • A CIB clonal cell line with a large-fragment gene knockout was established using CRISPR/Cas9 technology. Fish cell lines serve as valuable tools in aquaculture research, particularly in immunology, pathology, and toxicology. In this study, we successfully established a novel cell line derived from brain tissue of grass carp, designated CIB. This cell line has been subcultured over 100 times and exhibits a fibroblast-like morphology. Chromosomal analysis revealed that the diploid chromosome number of CIB cells is 2n = 48, while sequencing of the 18S rRNA gene confirmed the cell line's origin. Notably, CIB cells demonstrated a transfection efficiency of 62.4% with pEGFP-N3, highlighting their potential for studies involving exogenous gene expression. Furthermore, CIB cells were susceptible to grass carp reovirus genotype I (GCRV-I), as evidenced by cytopathic effects (CPE), increased synthesis of viral proteins, and accumulation of viral particles within the cells. Both viral infection and poly(I:C) stimulation significantly increased the expression of intracellular interferon-related signaling molecules. Additionally, electroporation of a gRNA-Cas9 ribonucleoprotein (RNP) complex into CIB cells achieved the first successful large-fragment gene knockout in cultured grass carp cells and generated a homozygous clonal line, thereby enhancing the antiviral response. In summary, this novel cell line represents a significant advancement for studying gene functions, host-virus interactions, and genetic engineering in teleost fish.