Preliminary Study On Screening Of Host Factors Related To JEV Replication By CRISPR Screening

Japanese encephalitis virus(JEV),a member of the flaviridae family,is a single positive strand RNA virus that causes viral encephalitis mainly in southeast Asia and the western Pacific,JEV is mainly transmited by mosquitoes biting people or animals,there is currently no effective drug,the disease has had a huge impact on stockbreeding and human health.JEV must rely on host cell components for infection,replication,and proliferation.Host proteins are an important class.Some proteins are used by the virus to promote its replication,while others inhibit it.Screening host functional proteins is crucial to the analysis of the life cycle and pathogenesis of viruses.Using CRISPR system can cut double-strand DNA which cause loss of gene function.CRISPR screening method is a high-throughput method developed in recent years to quickly identify functional genes.The basic process is as follows,researchers designed a sg RNA library that can target the entire genome,the mixed library is packaged as lentivirus,and then cells are infected by the library lentivirus to obtain a mixed cell line,imposing screening pressures,and finally,high-throughput sequencing was used to compare sg RNA abundances of different phenotypes to identify functional genes.In this study,a two-vector system in the CRISPR screening system was selected.It consists of a Lenti Cas9-Blast plasmid,which contains the blasticidin screening marker and expresses Cas9 protein,and a Lenti Guide-Puro plasmid which contains a puromycin screening marker and transcribes sg RNA.First,a plasmid stably expressing Cas9 protein was packaged as a lentivirus,and A549 cell were infected.Cas9-A549 cell line was obtained by screening for blasticidin,then,a sg RNA library containing 123411 sg RNAs and targeting 19050 human-derived genes was packaged as a lentivirus.Cas9-A549 cell lines were infected at a 0.3 MOI dose,and a mixed cell line of the library was obtained by puromycin selection,then,infecting the mixed cells with JEV at a dose of 30 MOI and the surviving cells were expanded,related software performs related analysis on gene enrichment,gene function,and gene enrichment pathway.In this study,CRISPR screening technology was used for the first time to screen the host factors related to replication and proliferation of JEV.Using the sg RNA library,under the premise of p <0.01,a total of 285 host factors that may be related to JEV replication in human cells were identified.Genes with higher scores analyzed by software are mainly including IFIT2,ANKS3,PHIP,SMIM20,DHX57,NF2,etc.The function of these genes are mainly focused on natural immunity,vesicle transport,ER stress,transcriptional regulation and other related signaling pathways and biological processes.The research results provide effective data for the study of dependent host factors for replication of JEV,and provide scientific data for the selection of antiviral drug targets and the design of new vaccines.