A Genome-scale Pooled CRISPR Knockout Screen to Identify Modulators of TREM2 Hong Sun1, Zuo Zhang1, Sidney Hsieh2, David Alvarado1, Uwe Mueller3, Alexander A Szewczak3, Rebecca S. Mathew1, Chris Mirescu2, Mathew Kennedy2, Joel A Klappenbach1 1Genetics and Pharmacogenomics, 2Neuroscience Discovery, 3Pharmacology, Merck & Co., Inc.
Triggering receptor expressed on myeloid cells 2 (TREM2) is a surface expressed receptor with strong genetic association of loss-of-function variants with increased risk for Alzheimer's disease (AD) and potentially other neurodegenerative diseases. Gene expression profiling of single-cells and sorted cell populations has demonstrated that in the brain, TREM2 is expressed nearly exclusively in microglial cells. Microglia are resident macrophages of the CNS that play key roles in both neuro-inflammation and phagocytic clearance of extracellular protein aggregates, neurotoxic debris and synapses. It is hypothesized that activation of TREM2 may dampen neuro-inflammation, promote clearance of amyloid beta (Aβ) plaques and ameliorate neuronal damage associated with neurodegeneration, which presents a unique opportunity to develop novel therapeutic strategies for the treatment of AD. We have performed a genome-wide pooled CRISPR-Cas9 screen to identify genes that when knocked out in the human monocytic cell line, THP-1, upregulate endogenous surface TREM2 and thus potentiate TREM2 signal transduction. Using a pooled, flow cytometry based surface TREM2 detection, we positively enriched high and low surface TREM2 populations in THP-1 under both suspension and adherent conditions. Our pooled CRISPR screen identified TYROBP (DAP12) and a DAP12 binding protein, SIRPB1, as hits positively associated with TREM2 surface expression, consistent with their functions as signaling adaptors with TREM2 interaction. Interestingly, in addition to positive modulators of TREM2, we identified 210 putative negative modulators of TREM2 not previously associated with TREM2 expression. Pathway enrichment analysis indicates the association of infection and phagocytosis with positive modulation of TREM2, which is consistent with published work. In summary, we have completed a genome scale CRISPR KO screen in human THP1 cells and discovered novel positive and negative modulators of TREM2 expression.These primary hits will provideinsights into the mechanistic role of TREM2 in AD biology and help identify novel therapeutic targets.
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