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Pooled genome-wide CRISPR screening for basal and context-specific fitness gene essentiality in Drosophila cells.

Raghuvir Viswanatha | Zhongchi Li | Yanhui Hu | Norbert Perrimon
eLife | 2018

Genome-wide screens in Drosophila cells have offered numerous insights into gene function, yet a major limitation has been the inability to stably deliver large multiplexed DNA libraries to cultured cells allowing barcoded pooled screens. Here, we developed a site-specific integration strategy for library delivery and performed a genome-wide CRISPR knockout screen in Drosophila S2R+ cells. Under basal growth conditions, 1235 genes were essential for cell fitness at a false-discovery rate of 5%, representing the highest-resolution fitness gene set yet assembled for Drosophila, including 407 genes which likely duplicated along the vertebrate lineage and whose orthologs were underrepresented in human CRISPR screens. We additionally performed context-specific fitness screens for resistance to or synergy with trametinib, a Ras/ERK/ETS inhibitor, or rapamycin, an mTOR inhibitor, and identified key regulators of each pathway. The results present a novel, scalable, and versatile platform for functional genomic screens in invertebrate cells.

Pubmed ID: 30051818

Associated grants

  • Agency: NIGMS NIH HHS, United States
    Id: R01GM067761
  • Agency: NIGMS NIH HHS, United States
    Id: P41 GM132087
  • Agency: NIGMS NIH HHS, United States
    Id: R01 GM067761
  • Agency: LAM Foundation, International
    Id: LAM0122P01-17
  • Agency: NIGMS NIH HHS, United States
    Id: T32 GM007748
  • Agency: NIGMS NIH HHS, United States
    Id: 5T32GM007748-39
  • Agency: Howard Hughes Medical Institute, United States
  • Agency: U.S. Department of Defense, International
    Id: W81XWH-16-1-0127

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This is a list of tools and resources that we have found mentioned in this publication.


Drosophila RNAi Screening Center (tool)

RRID:SCR_000733

Database that provides free online tools to users to allow the retrieval of information related to the Drosophila genome and allows access to genome-wide and related cell-based screening of Drosophila at Harvard Medical School (for a fee) . Tools available include SnapDragon, and RNAi designer, a heat map tool for viewing screen data, and gene and amplicon search and download tools. The DRSC mainly exists to provide Drosophila genome screening services, including help with assay development and optimization, data and image analysis, and planning of follow-up assays.

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GenomeNet (tool)

RRID:SCR_004165

GenomeNet is a Japanese network of database and computational services for genome research and related research areas in biomedical sciences, operated by the Kyoto University Bioinformatics Center. GenomeNet was established in September 1991 under the Human Genome Program of the then Ministry of Education, Science and Culture (Monbusho). The GenomeNet service has been developed by the Kanehisa Laboratory in Kyoto University as part of the research projects. GenomeNet is operated using the Supercomputer System of the Institute for Chemical Research, Kyoto University. LinkDB is supported by the National Bioscience Database Center of the Japan Science and Technology Agency.

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Thermo Fisher Scientific (tool)

RRID:SCR_008452

Commercial vendor and service provider of laboratory reagents and antibodies. Supplier of scientific instrumentation, reagents and consumables, and software services.

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KEGG (tool)

RRID:SCR_012773

Integrated database resource consisting of 16 main databases, broadly categorized into systems information, genomic information, and chemical information. In particular, gene catalogs in completely sequenced genomes are linked to higher-level systemic functions of cell, organism, and ecosystem. Analysis tools are also available. KEGG may be used as reference knowledge base for biological interpretation of large-scale datasets generated by sequencing and other high-throughput experimental technologies.

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New England Biolabs (tool)

RRID:SCR_013517

An Antibody supplier

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GLAD (tool)

RRID:SCR_001284

Software for analysis of array CGH data: detection of breakpoints in genomic profiles and assignment of a status (gain, normal or loss) to each chromosomal regions identified.

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TagDust (tool)

RRID:SCR_004175

A program to eliminate artifactual reads from next-generation sequencing data sets.

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FlyBase (tool)

RRID:SCR_006549

Database of Drosophila genetic and genomic information with information about stock collections and fly genetic tools. Gene Ontology (GO) terms are used to describe three attributes of wild-type gene products: their molecular function, the biological processes in which they play a role, and their subcellular location. Additionally, FlyBase accepts data submissions. FlyBase can be searched for genes, alleles, aberrations and other genetic objects, phenotypes, sequences, stocks, images and movies, controlled terms, and Drosophila researchers using the tools available from the "Tools" drop-down menu in the Navigation bar.

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