羊口疮又名羊传染性脓疱，是由羊口疮病毒(Orf virus,ORFV)引起的接触性传染病，严重危害养羊业的发展。前人研究发现ORFV的dUTPase蛋白具有生物学活性，但其生物学功能尚不清楚。有人根据病毒基因同源性提出其可能是ORFV毒力基因的假说，但缺乏实验依据，尚存争议。本项目拟利用CRISPR/Cas9系统，设计sgRNA oligos并克隆至PX459载体，将其与含同源臂及RFP表达盒的重组质粒，以及ORFV DNA共转染MDBK细胞。经荧光筛选、噬斑纯化、PCR、Western blot及IFA鉴定获得dUTPase基因缺失株。再从细胞水平和动物实验对基因缺失株的生长特性、毒力及免疫原性进行全面的研究。本项目有望通过实验明确dUTPase基因是否为ORFV的毒力基因，阐明dUTPase基因缺失对其生物学特性及免疫原性的影响。为揭示ORFV致病机理及基因缺失活疫苗研究奠定基础。

Orf, also known as contagious ecthyma, is an contagious disease caused by orf virus (ORFV). The disease has an economic impact on sheep industry due to decreases in production. The previous study has found that dUTPase gene of ORFV encoded a protein with biological activity. However, the biological function is unclear.There is a hypothesis that the dUTPase may be ORFV's virulence gene based on gene homologous compared with other viruses. However, the opinion lacks experimental basis and remains disputes. In the present project, CRISPR/Cas9 system will be employed for the dUTPase gene deletion. The sgRNA oligos were designed and cloned to PX459 vector. Then, the homology arms of dUTPase gene and RFP expression cassette were cloned to the pcDNA3.1 plasmid. Moreover, the two recombinant plasmids above will be co-transfected into MDBK cells with ORFV genomic DNA. After been fluorescent screening, plaque purification and PCR, Western blot and IFA identification, the dUTPase gene deletion mutant was obtained. Furthermore, the growth properties, pathogenicity and immunogenicity of the dUTPase gene deletion mutant was comprehensively studied at the cellular level and animal experiments. The project will clarify whether the dUTPase gene is a virulence gene of ORFV and the influence of the gene deletion on the biological feature and immunogenicity. In conclusion, this project will lay the foundation for revealing the pathogenesis of ORFV and vaccine development of the gene deletion mutant .