Reverse genetic systems are a critical tool for studying viruses and identifying countermeasures. In response to the ongoing COVID-19 pandemic, we recently developed an infectious complementary DNA (cDNA) clone for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The reverse genetic system can be used to rapidly engineer viruses with desired mutations to study the virus in vitro and in vivo. Viruses can also be designed for live-attenuated vaccine development and engineered with reporter genes to facilitate serodiagnosis, vaccine evaluation and antiviral screening. Thus, the reverse genetic system of SARS-CoV-2 will be widely used for both basic and translational research. However, due to the large size of the coronavirus genome (~30,000 nucleotides long) and several toxic genomic elements, manipulation of the reverse genetic system of SARS-COV-2 is not a trivial task and requires sophisticated methods. Here, we describe the technical details of how to engineer recombinant SARS-CoV-2. Overall, the process includes six steps: (i) prepare seven plasmids containing SARS-CoV-2 cDNA fragment(s), (ii) prepare high-quality DNA fragments through restriction enzyme digestion of the seven plasmids, (iii) assemble the seven cDNA fragments into a genome-length cDNA, (iv) in vitro transcribe RNA from the genome-length cDNA, (iv) electroporate the genome-length RNA into cells to recover recombinant viruses and (vi) characterize the rescued viruses. This protocol will enable researchers from different research backgrounds to master the use of the reverse genetic system and, consequently, accelerate COVID-19 research.

反向遗传系统是研究病毒和确定对策的关键工具。为应对持续的 COVID-19 大流行，我们最近开发了一种用于严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 的传染性互补 DNA (cDNA) 克隆。反向遗传系统可用于快速设计具有所需突变的病毒，以在体外和体内研究病毒。病毒还可以设计用于减毒活疫苗开发，并使用报告基因进行工程改造，以促进血清诊断、疫苗评估和抗病毒筛选。因此，SARS-CoV-2 的反向遗传系统将广泛用于基础研究和转化研究。然而，由于冠状病毒基因组的大小（约 30,000 个核苷酸长）和一些有毒的基因组元素，操纵 SARS-COV-2 的反向遗传系统并非易事，需要复杂的方法。在这里，我们描述了如何设计重组 SARS-CoV-2 的技术细节。总体而言，该过程包括六个步骤：（i）制备包含 SARS-CoV-2 cDNA 片段的七个质粒，（ii）通过七个质粒的限制酶消化制备高质量的 DNA 片段，（iii）组装七个将 cDNA 片段化为基因组长度的 cDNA，(iv) 从基因组长度的 cDNA 体外转录 RNA，(iv) 将基因组长度的 RNA 电穿孔到细胞中以回收重组病毒和 (vi) 表征拯救的病毒。该协议将使来自不同研究背景的研究人员能够掌握反向遗传系统的使用，从而加速 COVID-19 研究。