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

RAPID：比较 CRISPR 基因筛选以了解 SARS-CoV-2 的人畜共患传播

• 批准号: 2032072
• 负责人: WILLIAM MCDOUGALL
• 金额: $ 20万
• 依托单位: University of Massachusetts Medical School
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2032072&HistoricalAwards=false
• 关键词: RAPID; Comparative; CRISPR; Genetic; Screens

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.

迄今为止，COVID-19大流行已在全球感染了300多万人，造成约20万人死亡。病毒依靠它们的靶细胞为它们的生命周期提供适当的机制。鉴于导致COVID-19的冠状病毒（SARS-CoV-2）可以感染蝙蝠和人类，该项目旨在确定两种生物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“亲爱的同事信”的回应，该提案使用尖端的遗传工具，如CRISPR/Cas9，来获取病毒复制所需的细胞因子的知识。在整个研究过程中，识别蝙蝠细胞中这些相互作用的遗传工具将被创建，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，为充分了解这种SARS-CoV-2以及未来未发现的病原体提供一个框架。蝙蝠和人类细胞系所特有的分子相互作用，可以让我们了解为了在多个物种中生存而克服哪些相互作用，也可以让我们了解一种感染在一个物种（蝙蝠）中是良性的，而在另一个物种（人类）中是如何引起疾病的。该项目将发现对冠状病毒生物学至关重要的新知识，为未来的深入研究提供基础。除了增加对SARS-CoV-2生物学的了解外，该提案还支持培养博士后，扩大STEM的参与。SARS-CoV-2从马蹄蝠到人类的人畜共患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-病毒相互作用和细胞途径有哪些共同之处，从而允许病毒在这两个物种中有效复制和存活？为了回答这个问题，CRISPR/Cas9将用于有效敲除蝙蝠和人类细胞系中的每个基因，以确定每个细胞系中冠状病毒复制所需的基因。虽然CRISPR/Cas9是一种强大的遗传工具，但目前这些工具仅限于人类和小鼠模型的研究，这对研究蝙蝠细胞系中的病毒复制构成了重大挑战。一种名为“MerryCRISPR”的新型计算管道将用于设计CRISPR试剂并随后构建这项工作所需的工具。在蝙蝠和人类细胞系中并行进行的全宿主基因组-病毒相互作用研究的结合，除了确定物种特异性相互作用外，还将为构建功能和全面的宿主-冠状病毒相互作用图谱提供必要的信息。总的来说，这些研究将为未来研究特定和独特的宿主-冠状病毒相互作用提供基础，为研究人员提供有关冠状病毒基本生物学的信息。该RAPID奖由综合有机体系统生物学部的生理和结构系统集群获得，资金来自《冠状病毒援助、救济和经济安全（CARES）法案》。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。