CRISPR screening of porcine sgRNA library identified host factors essential for Japanese encephalitis virus replication

ABSTRACT Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus that causes encephalitis and reproductive disorders in mammalian species. However, key host genes involved in the JEV life cycle and cell death are poorly understood. Here, we designed 85,674 single guide RNAs (sgRNAs) targeting 17,743 protein-coding genes, 11,053 long ncRNAs (lncRNAs), and 551 microRNAs (miRNAs) in the porcine genome, and subsequently developed a porcine sgRNA library and genome-scale CRISPR/Cas9 knockout (PigGeCKO) system. These sgRNAs were delivered into porcine kidney-15 (PK-15) cells that constitutively express Cas9, positive selection screening of the resulting PigGeCKO cell collection for resistance to JEV-induced cell death led to the identification of several previously unreported genes required for JEV infection. We conducted follow-up studies to verify the dependency of JEV on these genes, and identified functional contributions for six of the many candidate JEV-related host genes, including an endoplasmic reticulum membrane protein complex subunit 3 (EMC3) and calreticulin (CALR). Additionally, we identified that four genes associated with heparan sulfate proteoglycans (HSPGs) metabolism, specifically those responsible for HSPG sulfurylation, facilitated JEV entry into PK-15 cells. Thus, beyond our development of the largest CRISPR-based functional genomic screening platform for pig research to date, this work deepens our basic understanding of flavivirus infection and identifies multiple potentially vulnerable targets for the development of medical and breeding technologies to prevent and treat diseases caused by Japanese encephalitis virus.