Pooled genome-wide CRISPR screening for basal and context-specific fitness gene essentiality in<i>Drosophila cells</i>

Viswanatha, Raghuvir; Li, Zhongchi; Hu, Yanhui; Perrimon, Norbert

doi:10.1101/274464

Cited by 21 publications

(51 citation statements)

References 58 publications

(54 reference statements)

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“…We first tested the efficacy of ssDNA drop-in constructs as a substrate for homology-directed repair in Drosophila S2R+ cells stably transfected with Cas9 (S2R+-MT::Cas9; Viswanatha et al, 2018). Specifically, we were interested in generating a collection of Drosophila S2R+ cells in which different organelles are marked by a protein tagged with a superfolder GFP (sfGFP) in each cell line (Pédelacq et al, 2006).…”

Section: Resultsmentioning

confidence: 99%

“…Drosophila cells stably expressing Cas9 (S2R+-MT::Cas9; Drosophila Genomics Resource Center cell stock #268; Viswanatha et al 2018) were cultured in Schneider's Drosophila Medium 1X (ThermoFisher Scientific #21720024) with 10% FBS and 1% penicillin/streptomycin (referred to as regular media). We note that this cell line is a derivative of S2R+ NPT005 (DGRC #229) and thus contains an mCherry tag in the Clic locus (Neumüller et al, 2012).…”

Section: Cell Culture and Regular Mediamentioning

confidence: 99%

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An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms

Kanca

Zirin

García-Marqués

et al. 2019

Preprint

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We previously reported a CRISPR-mediated knock-in strategy into introns of Drosophila genes, generating an attP-FRT-SA-T2A-GAL4-polyA-3XP3-EGFP-FRT-attP transgenic library for multiple uses (Lee et al., 2018b). The method relied on double stranded DNA (dsDNA) homology donors with ~1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely in vivo. The approach is fast, cheap, and scalable.

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Section: Resultsmentioning

confidence: 99%

Section: Cell Culture and Regular Mediamentioning

confidence: 99%

An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms

Kanca

Zirin

García-Marqués

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

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“…Therefore, whether a gene or set of genes is essential can be context specific, varying upon different growth conditions or treatments etc. (32,33). In our screen, we identified 61 genes with distinct essentiality in macrophages compared to a database collection of ~421 cell lines CRISPR screens ( Figure 1E, S1B).…”

Section: Macrophage-specific Genes Involved In Viabilitymentioning

confidence: 99%

High throughput CRISPR screening identifies genes involved in macrophage viability and inflammatory pathways

Covarrubias

Vollmers

Capili

et al. 2019

Preprint

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Macrophages are critical cells of the innate immune system involved in the recognition and destruction of invading microbes in addition to the resolution of inflammation and maintenance of homeostasis. Understanding the genes involved in all aspects of macrophage biology is essential to gaining new insights into immune system dysregulation during diseases that range from autoinflammatory to cancer. Here we utilize high throughput clustered regularly interspaced short palindromic repeats (CRISPR) screening to generate a resource that identifies genes required for macrophage viability and function. First, we employ a pooled based CRISPR/Cas nuclease active screening approach to identify essential genes required for macrophage viability by targeting genes within coding exons. In addition, we also target 3'UTRs to gain insights into new cis-regulatory regions that control expression of these essential genes. Second, using our recently generated NF-κB reporter macrophage line, we perform a fluorescence-activated cell sorting (FACS)-based high-throughput genetic screen to identify regulators of inflammation. We identify a number of novel positive and negative regulators of the NF-κB pathway as well as unraveling complexities of the TNF signaling cascade showing it can function in an autocrine manner to negatively regulate the pathway. Utilizing a single complex library design we are capable of interrogating various aspects of macrophage biology, generating a resource for future studies. SignificanceExcess inflammation is associated with a variety of autoimmune diseases and cancers. Macrophages are important mediators of this inflammatory response. Defining the genes involved in their viability and effector function is needed to completely understand these two important aspects of macrophage biology. Here we screened over 21,000 genes and generated a resource guide of genes required for macrophage viability as well as novel positive and negative regulators of NF-κB signaling. We reveal important regulatory aspects of TNF signaling and showing that membrane-bound TNF primarily functions in an autocrine fashion to negatively regulate inflammation. /body

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“…C16orf72 loss is broadly deleterious to the fitness of cancer cell lines particularly those derived from kidney tumors ( Figure 5B). Evolutionarily, C16orf72 is highly conserved through C. elegans ( Figure 5C), with CRISPR-Cas9 fitness screening data in Drosophila cells (Viswanatha et al, 2018) indicating that its importance for cellular viability is also conserved ( Figure 5D).…”

Section: C16orf72 Is a Stress-inducible Modulator Of The Cellular Adamentioning

confidence: 99%

Coessential Genetic Networks Reveal the Organization and Constituents of a Dynamic Cellular Stress Response

Amici¹,

Jackson²,

Metz³

et al. 2019

Preprint

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The interrelated programs essential for cellular fitness in the face of stress are critical to understanding tumorigenesis, neurodegeneration, and aging. However, modelling the combinatorial landscape of stresses experienced by diseased cells is challenging, leaving functional relationships within the global stress response network incompletely understood. Here, we leverage genome-scale fitness screening data from 625 cancer cell lines, each representing a unique biological context, to build a network of "coessential" gene relationships centered around master regulators of the response to proteotoxic, oxidative, hypoxic, and genotoxic stress. This approach organizes the stress response into functional modules, identifies genes connecting distinct modules, and reveals mechanisms underlying cellular dependence on individual modules. As an example of the power of this approach, we discover that the previously unannotated HAPSTR (C16orf72) promotes resilience to diverse stressors as a stress-inducible regulator of the E3 ligase HUWE1. Altogether, we present a broadly applicable framework and interactive tool (http://fireworks.mendillolab.org/) to interrogate biological networks using unbiased genetic screens.

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Pooled genome-wide CRISPR screening for basal and context-specific fitness gene essentiality inDrosophila cells

Cited by 21 publications

References 58 publications

An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms

An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms

High throughput CRISPR screening identifies genes involved in macrophage viability and inflammatory pathways

Coessential Genetic Networks Reveal the Organization and Constituents of a Dynamic Cellular Stress Response

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