Construction Of MDV Meq Gene Deletion Strain By CRISPR/Cas9 System And Establishment Of Its Detection Method

Marek's disease(MD)is a lymphoproliferative disease of chickens caused by Marek's disease virus(MDV),which causes huge losses in the poultry industry in countries around the world.The use of MD vaccine controls the disease well,but its pathogen MDV still shows a trend of continuous evolution,making it difficult to prevent and control the disease.The gene-deleted vaccine is the most promising MD vaccine because it removes the disease-causing gene and retains its immunogenicity to the greatest extent.The vaccine has good safety and higher immunity,But it can not prevent infection of MDV wild strain as the MD vaccine can not establish an immune barrier,which makes it difficult to detect and monitor MDV.Epidemiology,pathogenic research and development of new vaccines for MDV strains are the main measures to control the disease.It is necessary to develop better genetic editing methods and relevant detection methods.A strain of MDV wild strain BS/15 isolated from our laboratory showed that it has unique mutations in Chinese strains,and it has strong ability to replicate in vivo,with stronger cytolytic ability and late virulence.The construction of a gene-deleted vaccine for the parental virus has the potential to be a highly effective MD vaccine.This study utilised CRISPR/Cas9 system to delete the meq gene of BS/15 isolates.According to the meq gene sequence of Chinese MDV isolate BS/15 strain,eight small guide RNAs(sgRNAs)targeting meq gene were designed and cloned into pX330 plasmid,and the high efficiency of sgRNA was initially screened by sgRNA transfection and sequencing analysis.Two sgRNAs with the highest efficiency were selected and transfected into chicken embryo fibroblast(CEF).The meq gene-deleted strain was screened by PCR detection.Finally,a meq gene-deleted strain was successfully cloned.Sequence analysis revealed that the obtained MDV recombinant strain had been deleted the meq gene at the expected cleavage site.It had been proved that the meq-deleted strain and the parent strain had similar replication ability on CEF.This study provides a simple and rapid method for editing MDV genes,providing technical support for the study of MDV functional genes,molecular evolution and novel genetic engineering vaccines.In order to effectively detect and monitor MDV gene-modified strains and MDV isolates,this study established a quantitative quantification of MDV isolates and meq gene-deficient strains based on the meq gene-deficient strain rBS?Meq strain and another strain of rMS?Meq strain.It has been proved that the methods can accurately quantify meq gene deletion strain and MDV isolate respectively with high detection sensitivity,good specificity and high stability.This is of great significance for the diagnosis of MD in chickens and immune surveillance of vaccine.In summary,this study established a method for editing MDV isolates using CRISPR/Cas9 system,and using this method to construct a MDV meq gene deletion rBS?Meq strain,which provided technical support for studying the gene function and molecular evolution of MDV.At the same time,a q-PCR method for distinguishing MDV isolates from meq gene deletion strain was established,which is of great significance and application value for studying the infection process of MDV,immunesurveillance of vaccines and diagnosis of MD.