Ferroptosis induction via genetic approaches - CRISPR/Cas9-based disruption on key anti-ferroptotic genes

Unlike apoptosis, necroptosis, or pyroptosis which are executed by dedicated proteins, ferroptosis is a distinct form of regulated cell death driven by lipid peroxidation downstream of metabolic dysfunction. In most physiological settings, the cyst(e)ine/glutathione/glutathione peroxidase 4 (GPX4) axis constitutes the central anti-ferroptotic machinery, and disruption of this axis is usually sufficient to trigger ferroptosis. For in vitro studies, commonly employed ferroptosis inducers include erastin, which blocks cystine uptake by targeting system x_c^-, and (1S,3R)-RSL3, which inhibits GPX4 activity. However, both compounds exhibit off-target effects - erastin can activate voltage-dependent anion channels in mitochondria, whereas (1S,3R)-RSL3 affects other selenoproteins in addition to GPX4. Thus, genetic approaches to induce ferroptosis provide a valuable complement to chemical inducers by excluding off-target concerns. Here, we describe an efficient CRISPR/Cas9-based strategy to generate SLC7A11- and GPX4-knockout HT1080 cells. These knockout lines require routine culture in medium supplemented with β-mercaptoethanol or liproxstatin-1, while withdrawal of these supplements readily induces ferroptosis.