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CRISPR screens for SARS-CoV-2 Host Factors

CRISPR 筛选 SARS-CoV-2 宿主因子

基本信息

批准号：
10163544
负责人：
John Doench
金额：
$ 44万
依托单位：
BROAD INSTITUTE, INC.
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-15 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10163544
关键词：
2019-nCoV African Green Monkey Binding Bioinformatics Biological Assay Biological Models Biology CRISPR library CRISPR screen CRISPR/Cas technology Cell Line Cell model Cells Cellular biology Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Complement Data Foundations France Genes Genetic Genetic Screening Genome Genomic DNA Human Human Cell Line Infection Infectious Agent Institutes Integration Host Factors Knock-out Laboratories Libraries Life Cycle Stages Mammalian Cell Maps Marshal Masks Modeling Monkey Cell Line Pathogenesis Pathway interactions Phenotype Positioning Attribute Process Production Resources Sampling Surface Techniques Technology Testing Therapeutic Intervention Time Universities Validation Vero Cells Viral Viral Cytopathogenic Effect Virus Virus Diseases Virus Receptors biosafety level 3 facility cell type combinatorial design established cell line experience experimental study follow-up functional genomics gene function genome-wide in vivo insight interest loss of function overexpression pathogen pressure prevent response screening synergism vector

项目摘要

PROJECT SUMMARY In collaboration with Dr. Wilen (Yale University) and Dr. Goujon (CNRS, France), we will establish cell line models and conduct genome-wide CRISPR screens to identify host genes that are necessary for SARS-CoV-2 infection. We will first use Vero cells from the African Green Monkey, as they are a well-established model for many pathogens, and we had previously generated a genome-wide library for this species. Preliminary data suggest that we have also generated human cell lines that can serve as effective model systems, and these screens will be conducted as soon as the models are sufficiently validated. Across these screens, we expect to find host factors that are necessary for infection, such as the surface binding target for the virus, ACE2, and anticipate that the screens will identify additional genes that, when knocked out, prevent viral cytopathic effects. Additionally, we can modulate the selective pressure to identify factors that, when lost, sensitize the cells to SARS-CoV-2, which will provide a complementary view into host cell biology. Likewise, we will also conduct CRISPR activation screens to identify host genes that, when overexpressed, provide protection against infection, which may identify restriction factors that the virus must overcome. Primary screens will be validated with secondary pools, which will also allow for testing of genes across more conditions and cell types, establishing the generalizability of the results. Finally, combinatorial screens will be conducted to generate unbiased genetic interaction maps of hit genes, which can identify redundancies that are partially masked in a primary screen, as well as unexpected synergies across pathways. Focused, mechanistic follow-up of genes identified by these screens is outside the scope of this proposal, but is of immediate interest to the Wilen and Goujon groups.

项目总结