CRISPR screens for SARS-CoV-2 Host Factors

CRISPR 筛选 SARS-CoV-2 宿主因子

• 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
• 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

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.

项目概要 我们将与 Wilen 博士（耶鲁大学）和 Goujon 博士（法国国家科学研究中心）合作，建立细胞系 模型并进行全基因组 CRISPR 筛选，以确定 SARS-CoV-2 所需的宿主基因 感染。我们将首先使用来自非洲绿猴的 Vero 细胞，因为它们是一个成熟的模型 许多病原体，我们之前已经为该物种生成了一个全基因组文库。初步数据 表明我们还生成了可以作为有效模型系统的人类细胞系，并且这些 一旦模型得到充分验证，将立即进行筛选。通过这些屏幕，我们期望 找到感染所需的宿主因子，例如病毒的表面结合靶标、ACE2 和 预计筛选将识别出额外的基因，当这些基因被敲除时，可以预防病毒细胞病变 影响。此外，我们可以调节选择压力来识别一些因素，当这些因素丢失时，会使 SARS-CoV-2 细胞，这将为宿主细胞生物学提供补充观点。同样，我们也会 进行 CRISPR 激活筛选以识别宿主基因，这些基因在过度表达时可提供保护 对抗感染，这可能会确定病毒必须克服的限制因素。主屏幕将是 通过二级库进行验证，这也将允许在更多条件和细胞类型中测试基因， 建立结果的普遍性。最后，将进行组合筛选以生成 命中基因的无偏见遗传相互作用图谱，可以识别部分被掩盖的冗余 主要筛选，以及跨途径的意想不到的协同作用。有针对性、机械化的基因追踪 这些屏幕所识别的内容超出了本提案的范围，但对 Wilen 和 古容群体。

项目成果

Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs.

双向全基因组 CRISPR 筛选揭示了调节 SARS-CoV-2、MERS-CoV 和季节性 HCoV 的宿主因子。

• 发表时间: 2022-08
• 影响因子: 30.8
• 作者: Rebendenne, Antoine;Roy, Priyanka;Bonaventure, Boris;Chaves Valadão, Ana Luiza;Desmarets, Lowiese;Arnaud;Rouillé, Yves;Tauziet, Marine;Giovannini, Donatella;Touhami, Jawida;Lee, Yenarae;DeWeirdt, Peter;Hegde, Mudra;Urbach, Serge
• 通讯作者: Urbach, Serge

Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal coronaviruses.

双向全基因组 CRISPR 筛选揭示了调节 SARS-CoV-2、MERS-CoV 和季节性冠状病毒的宿主因子。

• 发表时间: 2021-05-21
• 作者: Rebendenne, Antoine;Roy, Priyanka;Bonaventure, Boris;Chaves Valadão, Ana Luiza;Desmarets, Lowiese;Rouillé, Yves;Tauziet, Marine;Arnaud;Giovannini, Donatella;Lee, Yenarae;DeWeirdt, Peter;Hegde, Mudra;Garcia de Gracia, Francisco;M
• 通讯作者: M

Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs.

双向全基因组 CRISPR 筛选揭示了调节 SARS-CoV-2、MERS-CoV 和季节性 HCoV 的宿主因子。

• 发表时间: 2021-05-27
• 作者: Rebendenne, Antoine;Roy, Priyanka;Bonaventure, Boris;Chaves, Valadão Ana Luiza;Desmarets, Lowiese;Rouillé, Yves;Tauziet, Marine;Arnaud;Giovannini, Donatella;Lee, Yenarae;DeWeirdt, Peter;Hegde, Mudra;Garcia de, Gracia Francisco;M
• 通讯作者: M