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High-Throughput CRISPR Screening Identifies Genes Involved in Macrophage Viability and Inflammatory Pathways.

  • Author(s): Covarrubias, Sergio
  • Vollmers, Apple Cortez
  • Capili, Allyson
  • Boettcher, Michael
  • Shulkin, Aaron
  • Correa, Michele Ramos
  • Halasz, Haley
  • Robinson, Elektra K
  • O'Briain, Laura
  • Vollmers, Christopher
  • Blau, James
  • Katzman, Sol
  • McManus, Michael T
  • Carpenter, Susan
  • et al.
Abstract

Macrophages are critical effector cells of the immune system, and understanding genes involved in their viability and function is essential for gaining insights into immune system dysregulation during disease. We use a high-throughput, pooled-based CRISPR-Cas screening approach to identify essential genes required for macrophage viability. In addition, we target 3' UTRs to gain insights into previously unidentified cis-regulatory regions that control these essential genes. Next, using our recently generated nuclear factor κB (NF-κB) reporter line, we perform a fluorescence-activated cell sorting (FACS)-based high-throughput genetic screen and discover a number of previously unidentified positive and negative regulators of the NF-κB pathway. We unravel complexities of the TNF signaling cascade, showing that it can function in an autocrine manner in macrophages to negatively regulate the pathway. Utilizing a single complex library design, we are capable of interrogating various aspects of macrophage biology, thus generating a resource for future studies.

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