GSE212008
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.