构建人源肺类器官模型，发现调控新型冠状病毒RNA合成的宿主基因

当前爆发的新型冠状病毒疫情严重威胁全球人类健康。冠状病毒复制涉及独特的病毒RNA“连续转录”和“非连续转录”的合成方式，虽然明确报道宿主因子是病毒RNA合成不可或缺的组分，但是这些宿主因子的身份和调控分子机制还不清楚。由于安全限制，目前只有P3实验室可研究病毒RNA合成，而且往往在人源或者其他物种的永生化细胞甚至肿瘤细胞中进行，不能全面有效模拟病人体内感染特征。在此，我们拟构建更接近人体生理的人源肺类器官模型来发现调控新型冠状病毒RNA合成的宿主基因。为此，我们拟基于新冠病毒RNA骨架，设计、测试并优化病毒RNA合成双组份报告系统，基因敲入该报告系统至人类胚胎干细胞中，分化干细胞为肺类器官，利用CRISPR微小文库和全基因组文库，功能性筛选并验证调控病毒RNA合成的关键宿主基因。此模型更贴近体内感染，并且可在常规实验室使用，将是研究新冠病毒RNA合成机制以及寻找药物新靶点的一种重要工具。

The current outbreak of SARS-CoV-2 seriously threatens human health worldwide. Coronavirus replication involves a unique synthesis method of "continuous transcription" and "discontinuous transcription" of viral RNA. Although it is clearly reported that host factors are indispensable components of viral RNA synthesis, the identity and regulatory molecular mechanisms of these host factors remain unknown. Due to safety restrictions, currently only P3 laboratories can study viral RNA synthesis, which is generally explored in immortalized cells or tumor cells of human origin or other species, which cannot fully and effectively simulate the characteristics of infection in patients. Here, we plan to develop a human lung organoid model that is closer to human physiology to discover host genes that regulate SARS-CoV-2 RNA synthesis. To this end, we plan to design, test, and optimize a two-component reporting system for viral RNA synthesis based on SARS-CoV-2 RNA backbone. This reporting system is then inserted into the genome of human embryonic stem cells via a knock-in method. The host genes regulating viral RNA synthesis are functionally screened in stem cell-derived lung organoids via a mini-library and a genomic library, which are based on CRSIPR/Cas9 technology. Key host genes are further verified. This model is closer to infection in vivo and can be used in routine laboratories. It will be an important tool for studying the RNA synthesis mechanism of SARS-CoV-2 and finding potential drug targets.