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Zheng, J. ; Zhang, W. ; Conrad, M.

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

In:. Elsevier, 2026. DOI: 10.1016/bs.mcb.2026.05.002 (Methods Cell Biol.)
DOI
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 xc−, 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.
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Publication type Article: Periodical or book chapter
Keywords Crispr/cas9 ; Ferroptosis ; Gpx4 ; Ht1080 Cells ; Knockout ; Slc7a11
ISSN (print) / ISBN 0091-679X
Journal Methods in Cell Biology
Publisher Elsevier
Reviewing status Peer reviewed
Institute(s) Institute of Metabolism and Cell Death (MCD)