Rapid manipulation of the porcine epidemic diarrhea virus genome by CRISPR/Cas9 technology.

• A reverse genetics system for highly virulent PEDV strain AJ1102 was established. • A recombinant PEDV was generated using CRISPR/Cas9 technology. • Recombinant PEDV can be created within one week using this strategy. • Provides an efficient platform for PEDV genome manipulation and vaccine development. Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus causing lethal watery diarrhea in suckling piglets. Reverse genetics is a valuable tool to study the functions of viral genes and to generate vaccine candidates. In this study, a full-length infectious cDNA clone of the highly virulent PEDV strain AJ1102 was assembled in a bacterial artificial chromosome (BAC). The rescued virus (rAJ1102) exhibited similar proliferation characteristics in vitro to the wildtype AJ1102. Using CRISPR/Cas9 technology, a recombinant virus rAJ1102-ΔORF3-EGFP in which the ORF3 gene was replaced with an EGFP gene, was successfully generated, and its proliferation characteristics were compared with the parental rAJ1102. Importantly, it just took one week to construct the recombinant PEDV rAJ1102-ΔORF3-EGFP using this method, providing a more efficient platform for PEDV genome manipulation, which could also be applied to other RNA viruses. [ABSTRACT FROM AUTHOR]