Construction Of African Swine Fever Double Gene Deletion Virus By CRISPR/Cas9 And Cre/Loxp

African swine fever（ASF）is an acute,febrile and highly contact infectious disease of swine caused by the African swine fever virus（ASFV）.It is also called African swine fever or verrucous swine disease.It is characterized by high fever,loss of appetite,bleeding of the skin and internal organs and high mortality.The disease belongs to the animal epidemic disease which is required to be reported legally by the world organization for animal health（OIE）.Since the first report o f ASFV cases in August 2018,the current epidemic has spread to 31provinces and regions in China,bringing huge economic losses to China’s pig breeding industry and great challenges to China’s prevention and control of ASFV.As there is no specific drug for African swine fever,killing and eradicating it will take a long time and pay a huge price.In contrast,the vaccine is the most economical and effective way to prevent and control African swine fever.However,the traditional inactivated vaccine,subunit vaccine and so on have poor or no protection ability to swine,so live attenuated vaccine caused by gene deletion has become the main research direction of ASFV vaccine.The main purpose of this project is to study whether CRISPR/cas9 and Cre/loxp gene editing system can effectively construct African classical swine fever gene deletion strain and study the performance of the recombinant strain,in order to provide a reference for the preparation of African classical swine fever gene deletion vaccine.In this study,CRISPR/cas9 and Cre/loxp gene-editing systems were used to construct the candidate strains of African swine fever live vaccine.Firstly,several transfection methods of ASFV were screened to determine the optimal transfection method.Then,the CD2v single gene deletion strain（CHINA 2018/1/△CD2v/e GFP）of African swine fever was constructed by CRISPR/cas9 system,and the obtained strain was purified,passed on and identified.O n the basis of CHINA 2018/1/△CD2v/e GFP,a CD2v/UK double gene deletion strain（CHIN A 2018/1/△CD2v/△UK）was constructed by CRISPR/Cas9system.The purified CHINA 2018/1/△CD2v/△UK was titrated,proliferative performance-tested,exogenous factor tested and stability studied.At the same time,in order to reduce the impact of foreign genes on the recombinant virus,the e GFP gene in CHINA2018/1/△CD2v/e GFP was knocked out by Cre/loxp system,and the recombinant virus（CHINA 2018/1/△CD2v）was identified by PCR and sequencing.The results showed that liposome transfection was the most suitable method for ASFV transfection.Compared with sg RN A+Cas9 protein transfection in vitro and electroporation,the virus strain obtained by liposome transfection could be stably passaged.In addition,the CD2v single gene deletion strain（CHINA 2018/1/△CD2v/e GFP）of African swine fever was successfully constructed,and the CD2v/UK double gene deletion strain（CHINA 2018/1/△CD2v/△UK）was constructed on this basis;meanwhile,the fluorescent gene e GFP in CHINA 2018/1/△CD2v/e GFP was knocked out by Cre/loxp system,and the gene deletion strain（CHINA 2018/1/△CD2v）was obtained.For CHINA 2018/1/△CD2v/△UK,the virus titer was 10⁷TCID₅₀/m L.compared with the parent strain（CHINA 2018/1）,the replication ability of the recombinant strain was higher than that of the parent strain.In this study,CRISPR/Cas9 and Cre/loxp gene-editing technology were used to construct African swine fever double gene deletion strain.The recombinant strain not only has high recombination efficiency but also has better replication ability compared with the parent strain,which is very important for the current research and development of the African swine fever gene deletion vaccine.