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Viswanatha, Raghuvir

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Viswanatha

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Raghuvir

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Viswanatha, Raghuvir

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2016 - 20183

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Author's Publications: search.filters.isAuthorOfPublication.5a97c617-171c-45e3-bdc1-a8e480018e13

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    Pooled Genome-Wide CRISPR Screening for Basal and Context-Specific Fitness Gene Essentiality in Drosophila Cells
    (eLife Sciences Publications, Ltd, 2018-07-27) Viswanatha, Raghuvir; Li, Zhongchi; Hu, Yanhui; Perrimon, Norbert
    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.
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    Ezrin activation by LOK phosphorylation involves a PIP2-dependent wedge mechanism
    (eLife Sciences Publications, Ltd, 2017) Pelaseyed, Thaher; Viswanatha, Raghuvir; Sauvanet, Cécile; Filter, Joshua J; Goldberg, Michael L; Bretscher, Anthony
    How cells specify morphologically distinct plasma membrane domains is poorly understood. Prior work has shown that restriction of microvilli to the apical aspect of epithelial cells requires the localized activation of the membrane-F-actin linking protein ezrin. Using an in vitro system, we now define a multi-step process whereby the kinase LOK specifically phosphorylates ezrin to activate it. Binding of PIP2 to ezrin induces a conformational change permitting the insertion of the LOK C-terminal domain to wedge apart the membrane and F-actin-binding domains of ezrin. The N-terminal LOK kinase domain can then access a site 40 residues distal from the consensus sequence that collectively direct phosphorylation of the appropriate threonine residue. We suggest that this elaborate mechanism ensures that ezrin is only phosphorylated at the plasma membrane, and with high specificity by the apically localized kinase LOK. DOI: http://dx.doi.org/10.7554/eLife.22759.001
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    CRISPR Guide RNA Design for Research Applications
    (Wiley-Blackwell, 2016-09) Mohr, Stephanie; Hu, Yanhui; Ewen-Campen, Benjamin; Housden, Benjamin; Viswanatha, Raghuvir; Perrimon, Norbert
    The rapid rise of CRISPR as a technology for genome engineering and related research applications has created a need for algorithms and associated online tools that facilitate design of on‐target and effective guide RNAs (gRNAs). Here, we review the state of the art in CRISPR gRNA design for research applications of the CRISPR‐Cas9 system, including knockout, activation, and inhibition. Notably, achieving good gRNA design is not solely dependent on innovations in CRISPR technology. Good design and design tools also rely on availability of high‐quality genome sequence and gene annotations, as well as on availability of accumulated data regarding off‐targets and effectiveness metrics.