Abstract:[Background] Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas9) has been proven to be an efficient and powerful third-generation gene editing tool and has achieved great progress in the discovery of functional genes. However, few studies have reported about using this method to screen the host genes associated with porcine epidemic diarrhea virus (PEDV). [Objective] To screen out the genes related to PEDV replication in the whole genome by using the CRISPR/Cas9 system, and to perform preliminary verification of candidate genes, so as to provide scientific reference for breeding PEDV-resistant pigs. [Methods] A genome-wide knockout library of human hepatoma cell line (Huh-7) was constructed via CRISPR/Cas9 technology, and the Huh-7 library cells were infected with PEDV. Then, the key host factors affecting PEDV replication were screened by high-throughput sequencing. The interference and detection experiments of virus replication were performed to preliminarily verify the candidate genes that affected PEDV replication. [Results] A CRISPR/Cas9 system was successfully constructed to screen the genes related to PEDV replication in the whole genome. The genes with top enrichment degree, including those encoding integrin α11 (ITGA11), replication protein A2 (RPA2), kinesin family member 2A (KIF2A), induced myeloid leukemia cell differentiation protein 1 (MCL1), poly (ADP-ribose) polymerase 1 (PARP1), and solute carrier family 18 member A1 (vesicular monoamine transporter, SLC18A1), were verified. Compared with the control group, the interference of ITGA11 via siRNA significantly down-regulated the mRNA and protein levels of PEDV-N and reduced the virus titer in the IPEC-J2 cells infected with PEDV. [Conclusion] The genome-wide knockout library based on the CRISPR/Cas9 system can be used as an effective tool for screening PEDV replication-related functional genes. ITGA11 may be a potential target gene for the breeding of PEDV-resistant pigs.
