Objective  Porcine epidemic diarrhea, caused by porcine epidemic diarrhea virus (PEDV), is a highly contagious viral disease and results in high mortality of pigs and huge lost of pig industry. The CRISPR/Cas13b system can mediate a highly efficient cleavage or editing to target RNA, thereby offering a novel strategy for interfering the infection of RNA viruses. We here tried to use the CRISPR/Cas13b system to cleave the PEDV RNA genome, in order to explore a novel strategy to inhibit PEDV infection.

Method  We designed four CRISPR RNA (crRNA) sequences which respectively recognize four regions in the PEDV genome. The CRISPR/Cas13b targeting vectors were constructed and transfected into Vero cells. The transfected cells were infected by PEDV, and then we analyzed the viral load of PEDV in cultured cells.

Result  The CRISPR/Cas13b system significantly inhibited PEDV propagation in Vero cells. In the viral immunofluorescence assay, the transfected cells with targeting vectors U6-crRNA3 and U6-crRNA4 had obviously fewer fluorophores compared with normal cells. The quantitative PCR results showed that CRISPR/Cas13b decreased PEDV load in cultured cells by above 50%.

Conclusion  The constructed CRISPR/Cas13b system can effectively interfere the propagation of PEDV. This study provides an alternative approach for effective RNA virus prevention and control, and creation of disease-resistant pig models.