Construction And Identification Of A Marek’s Disease Virus Mutant With MiR-M11 Deletion Utilizing CRISPR/Cas9-based Gene Editing

ObjectiveAs an important member of the alpha-herpesvirus family,serotype 1 Marek’s disease virus(MDV-1)is tumorigenic,and it could induce rapid-onset T-cell lymphoma and Marek’s disease(MD)in the host,which is considered a good biomedical model for study the biology,genetics,and immunology of virus-induced tumorigenesis.Some MDV-1-encoded micro RNAs(mi RNAs)have been shown to play important roles in the biological process of viruses,among which mi R-M11 may be involved in regulating the replication of MDV-1,but its function remains unclear In this study,we chose the domestic MDV-1 isolated virus strain HN302 as the research object and used CRISPR/Cas9 gene-editing technology to construct the mi R-M11 gene deletion virus strain of HN302 and identify its biological characteristics.In this way,the effect of mi R-M11 on the replication ability of MDV-1 in vitro was explored,and it provided important clues for further elucidating the regulatory function and molecular mechanism of MDV-1-encoded mi RNA.Besides,it provided important biological materials for subsequent animal challenge experiments and vaccine development.Methods1.Using a double knockout strategy,two g RNAs were designed on the upstream and downstream of the mi R-M11 precursor gene using the g RNA online design website and then connected to the p X459 vector to construct a gene-editing plasmid: p X459-g RNA.The effect of different g RNA combinations on the gene-editing effect was tested by transfection of p X459-g RNA plasmid and HN302 virus infection.Then,the combination with higher gene editing efficiency was screened out.2.The short DNA fragment after gene deletion was identified by sequencing,which confirmed that the mi R-M11 precursor gene was edited and deleted at the expected site.After multiple rounds of monoclonal virus plaque purification,the purified virus was passaged to test its stability.Finally,a stable mi R-M11 gene deletion virus strain HN302ΔM11 was obtained.3.RT-q PCR was used to detect the relative expression levels of some genes and mi RNAs of HN302ΔM11 virus;IFA was used to identify the expression of some protein-coding genes of MDV-1;finally,a standard curve was constructed,and absolute quantitative PCR was used to draw the in vitro proliferation curve of the virus.Results1.The g RNA combinations that can efficiently edit the HN302 mi R-M11 precursor gene were screened: g R2 and g R4.2.After three rounds of virus monoclonal plaque purification and 15 consecutive passages,a stable mi R-M11 precursor gene deletion strain HN302ΔM11 was obtained,indicating that CRISPR/Cas9 gene-editing technology can efficiently and stably transform the MDV-1 genome.3.The results of the virus in the vitro proliferation curve showed that after the deletion of the mi R-M11 precursor gene-editing,the in vitro proliferation rate of the HN302ΔM11 virus was significantly faster than that of the parental virus strain,and the viral gene copy number of HN302ΔM11 was significantly higher than that of the parental virus strain at 96 h and 120 h(P<0.05),indicating that the editing deletion of mi R-M11 can significantly enhance the replication ability of MDV-1 in vitro.4.The expression of mi R-M11-5p and mi R-M11-3p was not detected after the editing deletion of mi R-M11 encoded by HN302,and the expressions of other mi RNAs and genes could be detected.However,compared with the parental virus strain,the expression levels of mi R-M31-3p and mi R-M4-5p encoded by the gene deletion virus strain were significantly increased(P<0.05).Conclusions1.Using CRISPR/Cas9 gene editing technology,a stable MDV-1-encoded mi RM11 gene deletion strain HN302ΔM11 was successfully constructed.2.mi R-M11 is not a necessary gene for MDV-1 replication in vitro,but its deletion can enhance the in vitro replication ability of MDV-1 to a certain extent.3.mi R-M11 may participate in the regulation of MD tumor formation together with other mi RNAs.