Multi-species genome-wide CRISPR screens identify conserved suppressors of cold-induced cell death

Abstract

During hibernation of Syrian hamsters, the core body temperature shows a remarkable decrease, going from 37°C to 4°C. Although this ability to survive at low temperatures could in principle be due to systemic factors that occur during hibernation, we and others have seen that cells from hibernating rodents cultured in vitro maintain this ability. Although others have studied characteristics of cells from hibernating and non-hibernating organisms, the genes and pathways that are involved in cold-induced cell death have not been systematically explored. In this thesis, we conduct two genome-wide CRISPR-Cas9 screens in both a cold-sensitive (K562) and cold-resistant (BHK-21) cell line, and uncover GPX4 and related selenocysteine incorporation genes as important for protection against cold-induced cell death. Using genetic knockdowns, along with overexpression of GPX4, we confirm our findings and demonstrate that levels of GPX4 may be limiting in K562 cells, contributing to their cold sensitivity. Additionally, pharmacological validation using inhibitors of GPX4 reveal that the catalytic activity of GPX4 is dependent on the selenocysteine in the active site. Our findings are extended across multiple cell lines and cell types across six species. Taken together, our results suggest that GPX4 may be a powerful and conserved suppressor of cold-induced cell death. Building on our initial findings, we go on to show that cold exposure leads to increases in membrane permeability. This membrane permeability is transient, as rewarming of the cells reduces permeability to baseline levels. We also test the role of lipid peroxidation in contributing to membrane permeability and find that although it contributes in some cell lines, it is not the sole contributor as ferroptosis inhibitors do not fully mitigate membrane permeability. We go on to test different membrane channels and do not see decreases in membrane permeability, potentially indicating pathway-independent effects of temperature on membrane permeability. Altogether, this work provides a foundation for understanding how cold exposure influences mammalian cells.