Genome editing of WSSV CRISPR/Cas9 and immune activation extends the survival of infected Penaeus vannamei.

White spot syndrome virus (WSSV) is an exceptionally harmful virus that generally causes high levels of mortality in cultured shrimp. Attempts at viral suppression have been made to control the disease and have achieved limited efficiency. Recent advances in genome editing technology using CRISPR/Cas9 have led to potential innovations to prevent or treat many viral diseases. In this study, a CRISPR/Cas9 system was applied to WSSV genome cleavage to suppress WSSV infection in shrimp. The U6 promoter sequence was identified. A chimeric DNA vector consisting of the shrimp U6 promoter with gRNA expression sequences specific to two sites of the WSSV genome and the WSSV ribonucleotide reductase promoter with the Cas9 DNA sequence in pAC-sgRNA-Cas9 was constructed. The expression of gRNAs specific to the WSSV genome and Cas9 was determined in primary cultured hemocyte cells and in shrimp tissue via RT‒PCR. The efficacy of CRISPR/Cas9-WSSV for WSSV genome cleavage was determined in vitro and against WSSV-infected Penaeus vannamei. The reaction of synthetic gRNAs and recombinant Cas9 was able to cleave WSSV DNA amplicons, and shrimp that received CRISPR/Cas9-WSSV presented significantly lower WSSV DNA. In addition to interfering with viral DNA propagation, CRISPR/Cas9-WSSV encapsulated with IHHNV-VLP also stimulated an immune-related gene response. Treatment with CRISPR/Cas9-WSSV against WSSV challenge resulted in a significantly longer survival period. This finding has led to the development and application of a CRISPR/Cas9 system for WSSV infectious disease control, which could be used for managing shrimp aquaculture in the future.