Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA, USA.
Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA, USA; W.M. Keck Center for Noncoding RNAs, University of California, San Francisco, San Francisco, CA, USA; Institute for Molecular Medicine, Martin Luther University Halle-Wittenberg, Halle, Germany.
Cell Rep. 2020 Dec 29;33(13):108541. doi: 10.1016/j.celrep.2020.108541.
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.
巨噬细胞是免疫系统的关键效应细胞，了解其存活和功能相关的基因对于深入了解疾病期间免疫系统失调至关重要。我们使用高通量、基于池的 CRISPR-Cas 筛选方法来鉴定巨噬细胞存活所需的必需基因。此外，我们靶向 3'UTR，以深入了解以前未被识别的控制这些必需基因的顺式调控区域。接下来，我们使用最近生成的核因子 κB (NF-κB)报告基因系，进行基于荧光激活细胞分选 (FACS)的高通量遗传筛选，发现了许多以前未被识别的 NF-κB 途径的正调控因子和负调控因子。我们揭示了 TNF 信号级联的复杂性，表明它可以在巨噬细胞中以自分泌的方式发挥作用，从而负调控该途径。利用单个复杂文库设计，我们能够研究巨噬细胞生物学的各个方面，从而为未来的研究生成资源。
