Screening Of Essential Host Genes Required To Support Swine Influenza Virus Replication Utilizing Pig Genome-scale CRISPR/Cas9 Knockout Libraries

Swine influenza(SI)is a respiratory infectious disease caused by Swine influenza virus(SIV),of which the morbidity can reach up to 100%.SI is often secondary to porcine reproductive and respiratory syndrome,mycoplasma pneumonia and porcine circovirus diseases,later causes immune damage,and then prolongs the course of respiratory diseases and increase the mortality rate.Thus,as a prevalent and serious disease in large-scale pig farms,SI poses a serious threat to pig breeding and public health.Influenza A virus is an important zoonotic disease,and pigs are the common susceptible hosts of avian,swine and human influenza viruses,which therefore are considered as the "mixer" for influenza virus genes and the "incubator" for new epidemic influenza strains generation.So it is getting easier for influenza viruses to spread across species after infecting pigs.The prevention and control of SIV epidemic is of great significance for the huge threat of SI to the healthy and efficient pig breeding,public health in China and worldwide.In this study,we utilized an acquired CRISPR/Cas9 genome-wide knockout library in pig kidney cells(PK-15-GeCKO)to screen the host genes required to support the replication of currently popular Eurasian avian like SIV.Otherwise,we knocked the screened genes down in PK-15-Cas9 to preliminarily verify the effect of the gene knockdown against SIV.In addition,we attempted to construct a GeCKO in newborn pig trachea(NPTr)cell,which is the better cell model of more closely nature infectious state.In this study,we constructed an NPTr cell line stably expressing Cas9 protein(NPTr-Cas9-Blast),which laid a foundation for screening host factors necessary for SIV replication and provided theoretical basis for breeding gene editing pigs resistant to SIV.The main research contents are as follows:1 Essential host genes screening for SIV replication in PK-15-GeCKOAll cells in the control group(PK-15-Cas9)died 96 hpi with SIV.Hemagglutination units of the control group and the experimental group(PK-15-GeCKO)were determined.The surviving cells were expanded to bear the next round of challenge.After four rounds of challenge,almost all the cells in the experimental group survived,and the virus almost ceased to proliferate.Then high-throughput sequencing was performed in cells survived from the second,third and fourth rounds of challenge.The candidate genes were analyzed by bioinformatic analysis including GO and KEGG pathway.The results showed that the candidate genes of essential host factors for SIV infection were screened within the whole pig genome,and the proteins expressed by these genes involve in multiple biological processes,cell components and molecular functions.2 Preliminarily verification of the effect of candidate genes on virus proliferationAccording to the results of screening,we chose sgRNAs of the top 5 enriched genes and respectively integrated into PK-15-Cas9 through lentivirus infection.Then we preliminarily verified the antiviral effects on these polyclonal knockout cell lines.Polyclonal knockout cell lines were inoculated with HuB virus,and supernatant was collected at 12,24 and 36 hpi for the virus titers measure.In order to determine whether the knockdown of these genes specifically affected the proliferation of swine influenza virus,these knockout cell lines also were infected with human influenza viruses for the virus titer test.The results showed SLC,R6,GG and AD gene knockdown inhibited the proliferation of both swine and human influenza virus.3 Construction of Cas9 stably expressed NPTr cell lineConsidering that influenza virus firstly attacks the respiratory organs,we selected NPTr cells as the experiment model for the second screening.In this study,we constructed the NPTr cell line stably expressing Cas9(NPTr-Cas9-Blast).First we explored the optimal lentivirus packaging and infecting condition,and packaged lentivirus expressing Cas9 in 293 T cells and infected wide-type NPTr cells,then we purified cell lines using the limited dilution method.After that,an sgRNA with the knockout activity targeting endogenous gene ANPEP was integrated into the candidate cell line by lentivirus infection.Basing on T7E1 enzyme digestion assay and sequencing analysis,a cell line with the highest Cas9 knockout activity was indentified as the NPTr-Cas9-Blast cell lines,which was used to construct NPTr-GeCKO.