CGSIV Infected 293T Cell Line AND Inhibiting CGSIV Replication And Proliferation With CRISPR-Cas9 System

Chinese giant salamander iridovirus(CGSIV)is an emerging virus that were extracted and isolated from the diseased giant salamanders in China.CGSIV is a member of the amphibian-like ranavirus(ALRV)in the genus Ranavirus of Iridoviridea family.It can infected a variety of vertebrates,such as osteichthyes,amphibians,and reptils,resulting in high morbidity and mortality.At present,the molecular biology and gene function research on CGSIV is still in its early stages of development.Selecting a suitable cell line as a model is an important foundation for virus research,but traditional fish cell lines has the disadvantages of low transfection efficiency and immature technical means.Therefore,based on the extensive host characteristics of the iridescent virus and the applicability of mammalian cell lines in FV3 and TFV studies,we chose the 293 T cell line as a potential host cell line for CGSIV.The infectivity of CGSIV on 293 T cell line at low temperrature were confirmed by PCR,Western Blot,and Transmission Electron Microscope.We demonstrated that CGSIV can successfully infected the 293 T cell line at 26°C and completed its life cycle in the cell to produced infectious progeny virus.This provided a theoretical basis and realistic basis for the development of the 293 T as a new cell line model for CGSIV virus research;Clustered regularly interspaced short palindromic repeats /CRISPR-associated protein 9 system(CRISPR-Cas9)is a powerful and multiplexable genome editing tool,enabling researchers to accurately manipulate and modify target genes.CRISPR-Cas9 system was widely used in animal,plant,yeast and virus research.To determined the applicability of CRISPR-Cas9 technology in the study of iridovirus,we designed six single-guide RNAs targeting the CGSIV PCNA and 18 K genes,to verify the effectiveness of CRISPR-Cas9 system in iridovirus.In our study,we demonstrated that expression of Cas9 endonuclease and single-guide RNA specific for CGSIV PCNA and 18 K,was able to induced cleavage of the CGSIV genome,resulted in the introduction of long deletion mutations and short insertions into the PCNA and 18 K gene.We also found significant inhibition of virus replication and proliferation in 293 T cells expressed Cas9 and CGSIV-specific single-guide RNA.Our study may provided a new theoretical basis and strategy for the reduction of virus particles in host cells and the treatment of iridovirus diseases.