Protocol of CRISPR-Cas9 knockout screens for identifying ferroptosis regulators

Summary: Ferroptosis, an iron-dependent programmed cell death triggered by excessive lipid peroxidation, has shown promising therapeutic potentials in human diseases. Here, we describe a protocol of a CRISPR-Cas9 loss-of-function screen to identify regulators in response to different inducers of ferroptosis. We emphasize the steps of library amplification, drug treatment, high-throughput sequencing preparation, and bioinformatics analysis using model-based analysis of genome-wide CRISPR-Cas9 knockout (MAGeCK). We also present a method to discover the regulators of ferroptosis and verify the potential targets efficiently.For complete details on use and execution of this protocol, please refer to Yang et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.