CRISPR-Cas9 Screen for SARS-CoV-2 Host Dependency Factors

CRISPR-Cas9 筛选 SARS-CoV-2 宿主依赖性因素

• 批准号: 10487066
• 负责人: Alex Compton
• 金额: $ 21.62万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10487066
• 关键词: 2019-nCoV; Antiviral Therapy; Biological Assay; COVID-19 pandemic; COVID-19 screening; CRISPR library; CRISPR screen; Cell Survival; Cells; Collaborations; Communicable Diseases; Dependence; Development; Gene Silencing; Genes; Genetic Screening; Guide RNA; Hela Cells; Human; Infection; Integration Host Factors; Lentivirus Vector; Medical Research; Modality; National Institute of Diabetes and Digestive and Kidney Diseases; Puromycin; RNA Sequences; Research; Research Institute; Resistance; Resources; Role; SARS-CoV-2 infection; Screening Result; Severe Acute Respiratory Syndrome; Time; Virus; Work; cell type; novel; response; transcriptome sequencing

This project was initiated in spring of 2020 and is now ready for the virus challenge stage. We have obtained CRISPR-Cas9 libraries from Juan Bonifacino that can be delivered to diverse cell types by lentiviral vectors. Scarlett Shi, a Research Fellow in the lab, is leading the work on this project. She has applied lentiviral vectors carrying CRISPR-Cas9 libraries to HeLa, Vero, and HEK293T-hACE2 cells and has selected cells with diverse gene inactivations using puromycin. These cells were given to Rajini Mudhasani of USAMRIID for infection with wild-type SARS-CoV-2. Cells that are missing a crucial factor needed for infection will survive the otherwise cytopathic effects of productive SARS infection, resulting in the progressive enrichment of virus-resistant cells in culture over time. These cells will be subjected to bulk RNA sequencing in order to identify the guide RNA sequences associated with cell survival, thereby identifying the genes encoding host dependency factors. The initial results of the screen will be followed with targeted assays to confirm the functional role of a given gene during SARS-CoV-2 infection.

该项目于2020年春季启动，现已进入病毒挑战阶段。我们已经从Juan Bonifacino获得了CRISPR-Cas9文库，可以通过慢病毒载体递送到不同的细胞类型。Scarlett Shi是实验室的研究员，负责该项目的工作。她将携带CRISPR-Cas9文库的慢病毒载体应用于HeLa、Vero和HEK 293 T-hACE 2细胞，并选择了使用嘌呤霉素进行不同基因失活的细胞。这些细胞被给予USAMRIID的Rajini Mudhasani，用于感染野生型SARS-CoV-2。缺少感染所需的关键因子的细胞将在生产性SARS感染的细胞病变效应中存活下来，导致随着时间的推移，培养物中的病毒抗性细胞逐渐富集。将对这些细胞进行批量RNA测序，以鉴定与细胞存活相关的指导RNA序列，从而鉴定编码宿主依赖性因子的基因。筛选的初步结果将随后进行靶向测定，以确认SARS-CoV-2感染期间给定基因的功能作用。