Establishment Of A CRISPR/Cas9-based Screening Platform For Functional Receptors Mediating Classical Swine Fever Virus Invasion

Classical swine fever(CSF)is a fatal disease caused by classical swine fever virus(CSFV),which results in a huge economic loss to pig husbandry in the world.CSFV does not have their own metabolic and metabolic systems and has to rely on host cells to complete replication cycles,which begins with specific interactions of viral particles with cellular receptors.The interaction between CSFV surface proteins and cellular receptors determines its tissue tropism and host range.More importantly,virus entry can be prevented by blocking virus-host interactions as an antiviral strategy.Therefore,the study of cellular receptors will be benefit for understanding the entry mechanism of viruses and providing accurate targets for developing novel antiviral preparation.Previous studies have shown that during CSFV infections,laminin receptor(LamR)acts as adsorption receptor;CD46 has been shown to be involved in the adsorption process;heparan sulfate(HS)acts as adsorption receptors for cellular adaptation strains of CSFV.Nevertheless,there are also LamR,CD46 and HS expressions in some CSFV non-susceptible cells,suggesting that functional receptor(s)that mediate CSFV invade into the non-permissive cells have not been identified.Hence,a technology platform is needed to screen functional receptors that mediate CSFV invasion.It is possible that the improvement of receptor screening methods and their wide application for the researchers to find cellular receptors for more viruses.The previous screening methods are mainly protein interaction,such as virus overlay protein blot assay,but the emerging RNA interference(RNAi),haploid cell genetic screening,and CRISPR/Cas9 screening technology have been the important strategies in field of biotechnology screening virus cellular receptor(s).Compared with the above two technologies,the screening conditions of CRISPR/Cas9 technology are extremely strict,since Cas9 nuclease targeting the genome DNA causes permanent damage,which greatly reduces false-positive results.Thus,the selected host factors are the key host factors that regulate viral infection.In this study,the sgRNAs library of porcine-derived cell membrane proteins was conducted to construct and evaluate a screening platform based on CRISPR/Cas9 technology to lay the foundation for screening functional receptors that mediate CSFV invasion.In this study,first,we constructed the recombinant CSFV that stably expresses the Venus fluorescent protein(rCSFV-Venus)using the reverse genetic operating system,and optimized the growth condition of the recombinant virus.In addition,we constructed the PK-15 cell lines that stably express the Cas9 protein using lentivirus system,and screened for 6#-PK-Cas9 monoclonal cell lines with high expression of Cas9 protein and strong cleavage activity using Western blotting and flow cytometry assays.Furthermore,we analyzed and screened 3500 porcine cellular membrane proteins from the transmembrane regions of porcine and human membrane protein genes,and designed 3 sgRNAs for each membrane protein gene,cloned the sgRNAs into a lentiviral vector and packaged them into lentivirus to construct the lentiviral sgRNAs library of porcine cellular membrane,transducing the lentiviral library into PK-15 cells to test the infection efficiency of the sgRNAs lentiviral library.The results showed that we have gained 10 500 sgRNAs lentivirus and each sgRNA lentivirus can infect cells,indicating the availability and effectiveness of the sgRNAs lentiviral library for screening functional receptors mediating CSFV invasion.Finally,sgRNAs lentiviral library was transduced into PK-Cas9 monoclonal cell lines at an MOI of 0.3 dose,and infected with rCSFV-Venus at high-dose after pressure screening,then the uninfected cells were sorted by flow sorting system,the genomic DNA of the cells was extracted,and the sgRNAs sequences were amplified by PCR,and high-throughput sequencing was performed to assess the screening effects of CRISPR/Cas9 screening platform.The results showed that we have screened 12 high-scoring candidate genes that were located on the cell membrane.This study shows that the CRISPR/Cas9 screening platform constructed in this study can be used to screen functional receptors that mediate the invasion of classical swine fever virus.In summary,CRISPR/Cas9 screening platform was constructed using PK-15 cells stably expressing Cas9 protein,porcine cellular membrane proteins sgRNAs lentiviral library and recombinant CSFV stably expressing Venus fluorescent protein,which can be used to screen and identify functional receptors mediating CSFV invasion,it is great significance to elucidate CSFV pathogenic mechanisms,discover novel antiviral targets,develop new vaccines and provide novel targets for antiviral strategies.Moreover,the screening platform can be used to screen the functional receptors for other porcine viruses.