Genome-wide CRISPR-Cas9 viability screen reveals genes involved in TNF-α-induced apoptosis of human umbilical vein endothelial cells.

Tumor necrosis factor α (TNF-α), a pivotal cytokine in sepsis, protects the host against pathogens by promoting an inflammatory response while simultaneously inducing apoptosis of the vascular endothelium. Unfortunately, inhibitors targeting certain components of the TNF-α signaling pathway to reduce cellular apoptosis have failed to translate into clinical applications, partly due to the adverse effects of excessive immunosuppression. In an attempt to discover potential targets in the TNF-α signaling pathway to modulate moderate inflammation and apoptosis during the development of sepsis, we performed a pooled genome-wide CRISPR/Cas9 knockout screen in human umbilical vein endothelial cells (HUVECs). Tumor necrosis factor receptor superfamily member 1A (TNFRSF1A), B-cell lymphoma 2 (BCL2), Bcl2-associated death promoter (BAD), and NLR family member X1 (NLRX1) deficiencies were identified as the effective genetic suppressors of TNF-α cytotoxicity on a list of candidate regulators. CRISPR-mediated NLRX1 knockout conferred cellular resistance to challenge with TNF-α, and NLRX1 could be induced to colocalize with mitochondria following TNF-α stimulation. Thus, our work demonstrates the advantage of genome-scale screening with Cas9 and validates NLRX1 as a potential modulator of TNF-α-induced vascular endothelial apoptosis during sepsis.