合成病毒基因组在基础病毒学研究以及疫苗和抗病毒药物的开发中发挥着重要作用。 1 型单纯疱疹病毒 (HSV-1) 是一种大型 DNA 病毒，广泛用于溶瘤病毒疗法。尽管此前已有HSV-1基因组从头合成的报道，但合成过程仍远未达到高效，而且合成的基因组中含有可能影响其复制和应用的载体序列。在本研究中，我们开发了一种有效的无载体策略来合成和拯救合成 HSV-1。与用包含载体的完全合成的基因组转染哺乳动物细胞的传统方法相反，通过酵母中的转化相关重组(TAR)合成的重叠HSV-1片段被线性化并共转染到哺乳动物细胞中以拯救合成病毒。利用这一策略，生成了包含 HSV-1 F 株完整基因组的合成病毒 F-Syn。 F-Syn的生长曲线和电镜证实其复制动力学和形态发生与亲本病毒相似。此外，通过将TAR与体外CRISPR/Cas9编辑相结合，生成了删除病毒基因ICP6、ICP34.5和ICP47的溶瘤病毒F-Syn-O。 F-Syn-O的抗肿瘤作用在体外进行了测试。 F-Syn-O 在多种人类肿瘤细胞系中成功感染并诱导剂量依赖性细胞毒性作用。这些策略将促进 HSV-1 基因组的便捷和系统操作，并可进一步应用于溶瘤疱疹病毒的设计和构建。

Synthesizing viral genomes plays an important role in fundamental virology research and in the development of vaccines and antiviral drugs. Herpes simplex virus type 1 (HSV-1) is a large DNA virus widely used in oncolytic virotherapy. Although de novo synthesis of the HSV-1 genome has been previously reported, the synthetic procedure is still far from efficient, and the synthesized genome contains a vector sequence that may affect its replication and application. In the present study, we developed an efficient vector-free strategy for synthesis and rescue of synthetic HSV-1. In contrast to the conventional method of transfecting mammalian cells with a completely synthesized genome containing a vector, overlapping HSV-1 fragments synthesized by transformation-associated recombination (TAR) in yeast were linearized and cotransfected into mammalian cells to rescue the synthetic virus. Using this strategy, a synthetic virus, F-Syn, comprising the complete genome of the HSV-1 F strain, was generated. The growth curve and electron microscopy of F-Syn confirmed that its replication dynamics and morphogenesis are similar to those of the parental virus. In addition, by combining TAR with in vitro CRISPR/Cas9 editing, an oncolytic virus, F-Syn-O, with deleted viral genes ICP6, ICP34.5, and ICP47 was generated. The antitumor effect of F-Syn-O was tested in vitro. F-Syn-O established a successful infection and induced dose-dependent cytotoxic effects in various human tumor cell lines. These strategies will facilitate convenient and systemic manipulation of HSV-1 genomes and could be further applied to the design and construction of oncolytic herpesviruses.