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

View ORCID ProfileSergio Covarrubias, View ORCID ProfileApple Vollmers, Allyson Capili, View ORCID ProfileMichael Boettcher, Elektra K. Robinson, Laura O’Briain, View ORCID ProfileChristopher Vollmers, James Blau, View ORCID ProfileMichael McManus, View ORCID ProfileSusan Carpenter
doi: https://doi.org/10.1101/807164
Sergio Covarrubias
Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, CA, USA
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Apple Vollmers
Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, CA, USA
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  • ORCID record for Apple Vollmers
Allyson Capili
Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, CA, USA
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Michael Boettcher
Department of Microbiology and Immunology, University of California, San Francisco, CA 94143, USADiabetes Center, University of California, San Francisco, CA, USAWM Keck Center for Noncoding RNAs, University of California, San Francisco, CA, USAInstitute for Molecular Medicine, Martin Luther University Halle-Wittenberg, Halle, Germany
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Elektra K. Robinson
Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, CA, USA
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Laura O’Briain
Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, CA, USA
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Christopher Vollmers
Department of Biomolecular Engineering, University of California, Santa Cruz, CA, USA
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James Blau
Department of Microbiology and Immunology, University of California, San Francisco, CA 94143, USADiabetes Center, University of California, San Francisco, CA, USAWM Keck Center for Noncoding RNAs, University of California, San Francisco, CA, USA
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Michael McManus
Department of Microbiology and Immunology, University of California, San Francisco, CA 94143, USADiabetes Center, University of California, San Francisco, CA, USAWM Keck Center for Noncoding RNAs, University of California, San Francisco, CA, USA
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Susan Carpenter
Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, CA, USA
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  • ORCID record for Susan Carpenter
  • For correspondence: sucarpen@ucsc.edu
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Abstract

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.

Significance Excess 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.

Footnotes

  • Author Contributions SeC and SuC conceptualized and designed research study. SeC and AV performed the screen. MB, JB, MM provided the custom whole-genome sgRNA library. AC, EKR and LO performed candidate validation experiments. CV developed data analysis tools. SeC analyzed and interpreted data and wrote paper.

  • Figures were re-sized to fit standard size sheet of paper and leave a one-inch margin from the top of the page.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted October 17, 2019.
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High throughput CRISPR screening identifies genes involved in macrophage viability and inflammatory pathways
Sergio Covarrubias, Apple Vollmers, Allyson Capili, Michael Boettcher, Elektra K. Robinson, Laura O’Briain, Christopher Vollmers, James Blau, Michael McManus, Susan Carpenter
bioRxiv 807164; doi: https://doi.org/10.1101/807164
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High throughput CRISPR screening identifies genes involved in macrophage viability and inflammatory pathways
Sergio Covarrubias, Apple Vollmers, Allyson Capili, Michael Boettcher, Elektra K. Robinson, Laura O’Briain, Christopher Vollmers, James Blau, Michael McManus, Susan Carpenter
bioRxiv 807164; doi: https://doi.org/10.1101/807164

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