CRISPR/Cas13d mediated inhibition of Siniperca chuatsi rhabdovirus infection in fish cells

Abstract

Siniperca chuatsi rhabdovirus (SCRV) is an RNA virus causing lethal disease in Mandarin fish (Siniperca chuatsi). In the present study, the CRISPR/Cas13d system was successfully established and validated in fish cells to suppress SCRV infection. A total of 25 sgRNA spacers were designed to target the genome or mRNA of SCRV, from which five highly effective sgRNAs were selected that consistently inhibited SCRV replication at 12, 24, and 48 h post-infection (hpi). Then, the five sgRNA spacers were combined into a CRISPR array and stable CRISPR/Cas13d-expressing cells were developed to evaluate their inhibitory effect to SCRV infection. The results showed that the Cas13d effector combined with the CRISPR array efficiently suppressed viral amplification within 48 hpi. The CRISPR array also showed a stronger inhibitory effect than that of single sgRNAs. These results demonstrated that the established CRISPR/Cas13d system can effectively target and inhibit SCRV replication and gene expression in fish cells and identified efficient target sequences that could be served as potential antiviral-specific sites. These findings provided new insights into the development of novel strategies for controlling RNA viruses infection including SCRV.