RAPID: Comparative CRISPR Genetic Screens to Understand Zoonotic Transmission of SARS-CoV-2

迄今为止，新冠肺炎大流行已经感染了300多万人，并导致全球约20万人死亡。病毒依靠它们的目标细胞为它们的生命周期提供适当的机制。鉴于导致新冠肺炎的冠状病毒(SARS-CoV-2)可以感染蝙蝠和人类，该项目旨在确定在这两种生物中感染所需的共同和独特的分子相互作用。作为对美国国家科学基金会“亲爱的同事来信”的回应，这项提案使用了CRISPR/Cas9等尖端遗传工具来获取有关病毒复制所需的细胞因子的知识。在整个研究过程中，将创造基因工具来识别蝙蝠细胞中的这些相互作用，为任何希望进一步研究这些相互作用的研究人员提供资源。这些信息还将使研究人员了解蝙蝠和人类细胞之间常见的冠状病毒复制所需的特定相互作用，提供一个框架，以充分了解这种SARS-CoV-2，以及未来未发现的病原体。蝙蝠和人类细胞系特有的分子相互作用使我们能够深入了解在多个物种中为了生存需要克服哪些相互作用，也可能有助于深入了解感染如何在一个物种(蝙蝠)中是良性的，而在另一个物种(人类)中却是良性的。该项目将发现冠状病毒生物学所必需的新知识，为今后的深入研究提供基础。除了增加关于SARS-CoV-2生物学的知识外，这项提案还支持培训一名博士后研究员，扩大对STEM的参与。SARS-CoV-2从马蹄蝠向人类的人畜传播是冠状病毒生物学中的一个大问题。蝙蝠和人类之间的宿主-病毒相互作用和共同的细胞途径是什么，从而允许两个物种的有效复制和病毒生存？为了回答这个问题，CRISPR/Cas9将被用来有效地敲除蝙蝠和人类细胞系中的每一个基因，以确定冠状病毒在每个细胞系中复制所需的基因。虽然CRISPR/Cas9是一个强大的遗传工具，但目前这些工具仅限于对人类和小鼠模型的研究，这对BAT细胞系中病毒复制的研究构成了巨大的挑战。将使用一种名为“MerryCRISPR”的新型计算管道来设计BAT CRISPR试剂，并随后建立这项工作所需的工具。结合在BAT和人类细胞系中平行研究的整个宿主基因组-病毒相互作用研究，除了确定特定物种的相互作用外，还将提供必要的信息，以建立功能和全面的宿主-冠状病毒相互作用图。总而言之，这些研究将为未来询问特定和独特的宿主-冠状病毒相互作用提供基础，为研究人员提供冠状病毒基本生物学的信息。这一快速奖项是由综合组织系统生物部的生理和结构系统集群利用冠状病毒援助、救济和经济安全(CARE)法案的资金颁发的。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

To date the COVID-19 pandemic has infected over 3,000,000 people and resulted in approximately 200,000 deaths worldwide. Viruses rely upon their target cells to provide the proper machinery for their life cycle. Seeing as the coronavirus (SARS-CoV-2) responsible for COVID-19 can infect both bats and humans, this project aims to identify common and unique molecular interactions required for infection in both organisms. In response to the NSF’s “Dear Colleague Letter” this proposal uses cutting-edge genetic tools such as CRISPR/Cas9 to gain knowledge about cellular factors needed for virus replication. Throughout the course of this research, genetic tools to identify these interactions in bat cells will be created, providing a resource for any researcher who wishes to study these interactions further. This information will also inform researchers on the specific interactions necessary for coronavirus replication that are common between bat and human cells, providing a framework to fully understand this SARS-CoV-2, in addition to future, undiscovered pathogens. Molecular interactions that are unique to bat and human cell lines provide insight into what interactions are overcome for survival in multiple species and also may provide insight into how an infection can be benign in one species (bats) and cause disease in another (humans). This project will discover new knowledge essential to coronavirus biology, providing the basis for future in depth studies. In addition to increasing knowledge about SARS-CoV-2 biology, this proposal also supports the training of a post-doctoral fellow, broadening participation in STEM. The zoonotic transmission of SARS-CoV-2 from the horseshoe bat to humans represents a large question in coronavirus biology. What are the host-virus interactions and cellular pathways in common between bats and humans that allow for efficient replication and virus survival in both species? To answer this question, CRISPR/Cas9 will be used to effectively knockout every gene in both bat and human cell lines to identify genes are required for coronavirus replication in each cell line. While CRISPR/Cas9 is a powerful genetic tool, currently these tools are limited to the study of human and murine models, representing a significant challenge for the study of viral replication in bat cell lines. A novel computational pipeline named “MerryCRISPR” will be used to design bat CRISPR reagents and subsequently build the tools necessary for this work. The combination of whole host genome-viral interactions studies studied in parallel in both bat and human cell lines will provide the necessary information to build functional and comprehensive host-coronavirus interaction maps in addition to identifying species-specific interactions. Collectively, these studies will provide the basis for future interrogation of specific and unique host-coronavirus interactions, informing researchers on the essential biology of coronaviruses. This RAPID award is made by the Physiological and Structural Systems Cluster in the BIO Division of Integrative Organismal Systems, using funds from the Coronavirus Aid, Relief, and Economic Security (CARES) Act.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.