Construction And Preliminary Identification Of RPRV Carrying SgRNA/ASFV P30-CRISPR/Cas9

African Swine Fever(ASF)is an acute,febrile and hemorrhagic infectious disease affecting swine with a 100% mortality rate in domestic pigs and wild boar,while most infected African wild pigs(warthogs and bush pigs)only show subclinical symptoms.An outbreak of African Swine Fever was first reported in Kenya in 1921,before it spread to many other regions,including Western and Eastern Europe,and South America.It was first introduced to China in August 2018,with the first ASF outbreak occurring in Shenbei district,Shenyang City,where 47 pigs were infected and later died.OIE reported that 28,783 pigs were culled,which had a severe effect on the pig industry.At present,there are no effective treatments or vaccines for African Swine Fever.The subunit vaccines,inactivated vaccines,and attenuated vaccines that have been developed against African Swine Fever cannot provide complete protection for the immunized animals.The development of new methods for the prevention and treatment of this disease is of crucial importance.In this study,a CRISPR/Cas9 gene editing system for p30(an essential gene for ASFV replication)was constructed.Through targeted cutting of the p30 gene,the eukaryotic expression of the p30 fusion plasmid was inhibited,which provided the possibility of effectively inhibiting virus replication.In this experiment,the following experiments were undertaken for the preliminary identification and construction of the CRISPR/Cas9 gene editing system targeting the ASFV p30 gene:1.Cloned ASFV p30 gene and constructed p30 fusion eukaryotic expression plasmid containing mCherry red fluorescent reporter gene.Designed 5 pairs of sgRNAs targeting the p30 gene through the website and constructed CRISPR-Cas9 gene editing plasmids PX458-1,PX458-2,PX458-3,PX458-4,PX458-5 targeting the p30 gene.Co-transfected PX458 plasmids targeting the p30 gene and the p30-mCherry plasmids into HEK 293 T cells,and identified the cutting efficiency of these Cas9 plasmids by using fluorescence observation,real-time quantitative PCR(qPCR)and Western Blot methods to detect p30 expression.The results show that PX458-3 and PX458-4 effectively cut the p30 gene and significantly reduced the expression of p30(p<0.05).Therefore,PX458-3 and PX458-4 were selected as two effective targets for subsequent experiments.2.Selected the PX458-3 plasmid,and inserted its CRISPR/Cas9 gene editing system sequence into the PRV transfer plasmid pc DNA3.1-LR to construct a PRV eukaryotic transfer plasmid 3.1LR-Cas9.Constructed recombinant virus K61-Cas9 targeting ASFV p30 gene and expressing Cas9 protein with3.1LR-Cas9 and PRV Bartha K61 strain virus by homologous recombination.The K61-Cas9 was first infected with Vero E6 cells,and transfected p30-mCherry plasmids after 4 hours.After 48 hours,the expression of p30 was detected by the use of fluorescence observation,q PCR and Western Blot methods.The results show that the fluorescence expression of p30 was significantly weakened.Results of qPCR indicate that the CRISPR/Cas9 gene editing system has an inhibitory effect on the expression of p30,which provides new ideas for further research into preventing the replication of ASFV and eventual eradication of this epidemic disease.