A Genome-wide CRISPR Screen Identifies EIF2A as a Host Factor Required for Porcine Epidemic Diarrhea Virus (PRJCA019306)
In this study, we performed a porcine genome-wide CRISPR screen to identify potential host factors required for PEDV infection. Especially, we founded that eukaryotic translation initiation factor 2A (EIF2A) is a critical host factor for the PEDV. Knocking out EIF2A gene can inhibit on PEDV infection. Notably, RNASeq results showed that anti-PEDV capability of EIF2A-KO cell is not achieved by enhancing expression of antiviral genes. We designed a lentiviral sgRNA library of 115,738 sgRNAs that targeted 19,745 protein-coding pig genes. About 964 million IPEC-J2-Cas9 cells (~500 cells per sgRNA) were transduced with the lentiviral sgRNA library at low MOI (MOI = 0.3) and puromycin was used to select the sgRNA-transduced cells. cells were collected after 5 rounds of PEDV challenge, and control group cells without PEDV challenged were also collected. The genomic DNA of each sample was extracted using a Blood & Cell Culture DNA Midi Kit (QIAGEN). The sgRNA-coding region was amplified by PCR using Q5 Hot Start High-Fidelity DNA Polymerase (NEB) in a reaction volume of 50 L. PCR products were mixed and purified with a MinElute PCR purification Kit (QIAGEN). All PCR products were pooled and purified with a MinElute PCR purification Kit (QIAGEN), followed by Illumina HiSeq 3000 Next-generation sequencing. EIF2A-KO cell line and wild-type IPEC-J2 cell line were infected by PEDV., mock group and infected group cells were collected for RNA sequencing at 36hpi. four biological repeats for each group were performed to prepare RNA-Seq library. The 32 libraries were sequenced on Illumina HiSeq 2500.