CRISPR screening of porcine sgRNA library identifies host factors associated with Japanese encephalitis virus replication

Changzhi Zhao,Bo Zuo,Xinyun Li,Yi-Liang Miao,Xiaosong Han,Shuhong Zhao,Mengjin Zhu,Ping Qian,Shengsong Xie,Hailong Liu,Liuxing Qin,Tianhe Xiao,Xiongwei Nie,Zichang Wang,Jinxue Ruan,Jinfu Zhang,Kui Yang

doi:10.1038/s41467-020-18936-1

Abstract

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus that causes encephalitis and reproductive disorders in mammalian species. However, the host factors critical for its entry, replication, and assembly are poorly understood. Here, we design a porcine genome-scale CRISPR/Cas9 knockout (PigGeCKO) library containing 85,674 single guide RNAs targeting 17,743 protein-coding genes, 11,053 long ncRNAs, and 551 microRNAs. Subsequently, we use the PigGeCKO library to identify key host factors facilitating JEV infection in porcine cells. Several previously unreported genes required for JEV infection are highly enriched post-JEV selection. We conduct follow-up studies to verify the dependency of JEV on these genes, and identify functional contributions for six of the many candidate JEV-related host genes, including EMC3 and CALR. Additionally, we identify that four genes associated with heparan sulfate proteoglycans (HSPGs) metabolism, specifically those responsible for HSPGs sulfurylation, facilitate JEV entry into porcine cells. Thus, beyond our development of the largest CRISPR-based functional genomic screening platform for pig research to date, this study identifies multiple potentially vulnerable targets for the development of medical and breeding technologies to treat and prevent diseases caused by JEV.

Highlights

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus that causes encephalitis and reproductive disorders in mammalian species
To minimize the chance of inserting multiple single guide RNAs (sgRNAs) into the same porcine kidney-15 (PK-15)-Cas[9] cells, we employed a low multiplicity of infection (MOI) to obtain a transduction rate of around 30% according to a previous study[27]
Our results highlight the power of CRISPR/Cas9-based screening for functional analyses in pigs, and we present in Fig. 7 a preliminary proposed model for JEV entry and replication in porcine cells based on our findings

Summary

Introduction

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus that causes encephalitis and reproductive disorders in mammalian species. Work in human cell lines based on CRISPR-based screening strategies (with virus-induced cell death readout phenotypes) have successfully identified required host genes for infection by DENV, ZIKV, WNV, YFV, and HCV20–24 These studies have repeatedly illustrated that genome-scale CRISPR screening represents a powerful tool for both basic biology and medical research. We generate gene knockout (KO) and knockdown cell lines for six of the candidate genes, and successfully confirm their requirement for JEV infection in porcine cells These newly discovered host genes are potential targets for the development of therapies for the treatment of Japanese encephalitis and porcine diseases caused by JEV, and can be used in the construction of genetically edited disease-resistant animal models. We anticipate that our benchmark-setting CRISPR/Cas[9] screening resources will greatly facilitate basic and applied functional genomics research in pigs

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