Abstract: 【Objective】 This study was aimed to establish a rapid and accurate method for targeted insertion of exogenous genes into the Duck enteritis virus (DEV) genome using the HDR-CRISPR/Cas9 gene editing technology under optimized conditions,so as to lay the foundation for the development of recombinant vaccines utilizing DEV as a vector. 【Method】 The DEV vaccine strain was isolated,propagated,and the viral titer was determined.The gRNA was designed and synthesized based on the non-coding sequence between UL26 and UL27 genes,then inserted into PX459-V2.0 to form the gRNA-Cas9 plasmid.Simultaneously,the enhanced green fluorescent protein (EGFP) reporter gene was inserted into the PVAX-1 vector via conventional gene cloning techniques to construct a recombinant expression plasmid containing the eukaryotic expression cassette P_CMV-EGFP-BGH pA.Using the DEV genome as a template,upstream and downstream homologous arm sequences of the gRNA target site were amplified,fused with P_CMV-EGFP-BGH pA,and cloned into PUC19 to obtain donor plasmid PUC19-UP-EGFP-DOWN. Recombinant virus rDEV-EGFP was constructed by transfecting plasmid and infecting parent DEV.The experimental conditions for the construction of the recombinant virus were optimized by controlling single variable method,and the optimal conditions were determined based on the number of green fluorescent plaques formed under different conditions.The plaques expressing green fluorescence were screened and purified using the limited dilution method to obtain the recombinant virus rDEV-EGFP.Subsequently,the genetic stability and in vitro replication ability of the recombinant virus rDEV-EGFP were evaluated. 【Result】 PCR amplification and sequencing results showed that the gRNA-Cas9 plasmid targeting the non-coding region between UL27 and UL26 genes of DEV genome and the donor plasmid containing EGFP eukaryotic expression box were successfully constructed.The chick embryo fibroblast (CEF) was infected with DEV after being transfected with gRNA-Cas9 and donor plasmid,and green fluorescence plaque could be observed under fluorescence microscope.The results showed that the EGFP reporter gene was successfully inserted into DEV genome using HDR-CRISPR/Cas9 gene editing technology.The optimal experimental conditions were as follows:Multiplicity of infection (MOI) of DEV was 0.2,the transfection ratio of gRNA-Cas9 plasmid to donor DNA was 1∶2,the transfection form of donor DNA was DNA fragment,the interval between transfection and infection was 6 h,and the collection time of recombinant virus was 48 h after DEV infection.After optimization,the knockin efficiency of EGFP reporter gene was significantly increased (P ＜ 0.05).Recombinant virus rDEV-EGFP was passed in CEF for 15 consecutive passages,and the EGFP eukaryotic expression box was still stably integrated in the non-coding region between UL26 and UL27 genes,showing good genetic stability.The results of growth curve showed that the replication ability of recombinant virus rDEV-EGFP in CEF was not significantly different from that of the DEV vaccine strain,and the growth trend was consistent,indicating good in vitro replication ability. 【Conclusion】 In this study,a method for rapidly constructing recombinant DEV based on HDR-CRISPR/Cas9 gene editing technology was established.Recombinant virus rDEV-EGFP with good genetic stability and in vitro replication ability was successfully constructed,which provided a theoretical basis and technical platform for the development of recombinant DEV vector vaccine candidate strains.

Key words: Duck enteritis virus; CRISPR/Cas9 gene editing; recombinant vector