Abstract 637: Genome-wide Crispr Screen in Primary Macrophages for Identification of a Regulatory Network for Macrophage Efferocytosis

Abstract

Defective macrophage efferocytosis drives important diseases including atherosclerosis, whereas enhancing efferocytosis has potential therapeutic benefits. A systematic approach to identify regulators of efferocytosis in an unbiased manner holds promise for novel molecular mechanisms and targets. This study establishes an experimental framework using a novel CRISPR knockout screen to systematically map the regulatory networks of macrophage efferocytosis. We infected bone marrow-derived macrophages (BMDM) isolated from Cas9 transgenic mice with a pooled, genome-wide lentiviral library with 78,637 gRNAs, and induced efferocytosis by fluorescently-labeled apoptotic Jurkat cells. We used FACS to isolate BMDM that engulfed multiple apoptotic cells (enriched in gRNAs targeting negative regulators) or did not engulf apoptotic cells (enriched in gRNAs targeting positive regulators), and determined gRNA abundance by deep sequencing. The top-ranked 1,000 genes analyzed by two published methods MAGeCK and PBNPA showed substantial overlap (53% for positive regulators, 36% for negative regulators). We recovered many of the known regulators. e.g., Actr2 , Actr3 and Arpc3 , key components of the Arp2/3 complex regulating actin polymerization, were ranked 2, 8 and 4; Atp6v0b and Atp6v0c , subunits of vacuolar ATPase responsible for acidification of intracellular organelles, were ranked 22 and 27. Cd36 , a well-known efferocytosis receptor, was ranked 5. The top ranked positive regulators were enriched in GO terms “positive regulation of response to stimulus”, “Fcγ receptor signaling pathway” and “phagocytosis” (FDR-adjusted P = 2.19E-14, 1.10E-12 and 4.08E-11, respectively). Individual gRNAs verified a number of top hits from the pooled screen. In summary, we have developed a functional screen that has defined number of genes essential for macrophage efferocytosis. By focusing on a functional phenotype beyond cell viability and growth, we illustrate the versatility of CRISPR screens and provide a general strategy for systematically identifying gene of interest and uncovering novel regulators of complex macrophage functions.