Genome-wide pooled CRISPR knockout screen for novel regulators of macrophage efferocytosis.. Genome-wide pooled CRISPR knockout screen for novel regulators of macrophage efferocytosis.

We performed a FACS-based genome-wide CRISPR knockout screen in primary murine macrophages to identify regulators of efferocytosis, the phagocytic clearance of dying cells. The screen identified known and novel regulators of macrophage efferocytosis. More broadly, the screen approach can be applied to interrogate complex functional phenotypes in primary macrophages. Overall design: This is a pooled FACS-based genome-wide CRISPR knockout screening for efferocytosis regulators. Bone marrow (BM) cells were isolated from Rosa26-Cas9 knock-in mice constitutively expressing Cas9 endonuclease (JAX #026179), transduced with lentiviral Brie gRNA libraries and then differentiated to BMDMs for efferocytosis and screening. The success of the screening relies on the effective enrichment of macrophages with high vs. low efferocytosis capacity. Since efferocytosis is a binary event, to facilitate an effective separation and enrichment, we performed two rounds of efferocytosis sequentially. Specifically, human Jurkat cells, an acute T cell leukemia cell line routinely used for in vitro efferocytosis assays, were treated with staurosporine to induce apoptosis, then labeled with fluorescent linkers, PKH67 (Ex/Em: 490/502 nm) or PKH26 (Ex/Em: 551/567 nm). BMDMs were first incubated with PKH67-labeled apoptotic cells (ACs) for efferocytosis, followed by the second round of incubating with PKH26-labeled ACs. BMDMs were then collected for flow cytometry sorting, which separated the BMDMs that engulfed both PKH67+ and PKH26+ ACs, i.e., the efficient eaters (~5%), and BMDMs that did not engulf any ACs, i.e., the non-eaters. We have also collected BMDMs without performing efferocytosis, i.e. the input samples. The non-eaters are expected to enrich for gRNAs targeting positive regulators essential for efferocytosis, i.e., knockout would impair efferocytosis. The efficient eaters are expected to enrich for gRNAs targeting negative regulators, i.e. knockout would enhance efferocytosis. We sequenced the sorted non-eaters, efficient eaters, and the input samples for each of the two replicates and performed MAGeCK analysis to identify the top hits.

我们在原代小鼠巨噬细胞中开展了基于荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）的全基因组CRISPR敲除筛选，以鉴定胞葬作用（efferocytosis，即死亡细胞的吞噬清除过程）的调控因子。本次筛选既发现了已知的巨噬细胞胞葬作用调控因子，也鉴定出了全新的调控靶点。从更广泛的应用场景来看，该筛选策略可用于探究原代巨噬细胞的复杂功能表型。
总体设计：本研究采用基于FACS的混合全基因组CRISPR敲除筛选体系，用于筛选胞葬作用调控因子。我们从组成型表达Cas9核酸酶的Rosa26-Cas9敲入小鼠（杰克逊实验室品系编号JAX #026179）中分离骨髓细胞，通过慢病毒Brie向导RNA（gRNA）文库进行转导，随后将其分化为骨髓来源巨噬细胞（BMDMs）以开展胞葬作用实验与筛选。本筛选的成功与否，取决于能否有效富集胞葬能力高低不同的巨噬细胞。由于胞葬作用属于二元事件（即要么发生要么不发生），为实现高效的细胞分离与富集，我们依次开展了两轮胞葬作用实验。
具体而言，我们选用体外胞葬作用实验中常用的人急性T细胞白血病细胞系Jurkat，经星形孢菌素处理诱导其凋亡后，分别使用荧光染料PKH67（激发/发射波长：490/502 nm）与PKH26（激发/发射波长：551/567 nm）进行标记，得到凋亡细胞（ACs）。首先将BMDMs与PKH67标记的凋亡细胞共孵育以启动胞葬作用，随后再与PKH26标记的凋亡细胞进行第二轮共孵育。之后收集BMDMs进行流式细胞术分选，将同时吞噬了PKH67阳性与PKH26阳性凋亡细胞的高效吞噬细胞（约占总细胞的5%），与未吞噬任何凋亡细胞的非吞噬细胞分离开来。此外我们还收集了未开展胞葬作用实验的BMDMs作为初始样本。
预期非吞噬细胞中会富集靶向胞葬作用必需正向调控因子的gRNA，即敲除此类因子会削弱胞葬作用；而高效吞噬细胞中则会富集靶向负向调控因子的gRNA，即敲除此类因子会增强胞葬作用。我们对两个生物学重复的分选后非吞噬细胞、高效吞噬细胞以及初始样本进行了测序，并通过MAGeCK分析鉴定出了显著富集的命中靶点。

搜集汇总

背景与挑战

背景概述

该数据集是一个全基因组CRISPR敲除筛选项目，旨在识别小鼠原代巨噬细胞中胞葬作用（即凋亡细胞清除）的新调节因子。研究采用基于FACS的筛选方法，通过两轮吞噬实验和流式分选，结合MAGeCK数据分析，发现了已知和未知的胞葬作用调节基因，为巨噬细胞功能研究提供了新工具和见解。