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Updated: Aug 13, 2026
Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
Published on: June 16, 2017
Protocol of CRISPR-Cas9 knockout screens for identifying ferroptosis regulators
Xin Yang1, Shoufu Duan1, Zhiming Li1
1Institute for Cancer Genetics, and Herbert Irving Comprehensive Cancer Center, Vagelos College of Physicians & Surgeons, Columbia University, New York, NY, USA.
Abstract:
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.
Insights
This study details a CRISPR-Cas9 screening protocol to find regulators of ferroptosis, an iron-dependent cell death. The method efficiently identifies key genes controlling ferroptosis, aiding therapeutic development.
Area of Science:
- Biochemistry
- Genetics
- Cell Biology
Background:
- Ferroptosis is an iron-dependent programmed cell death involving lipid peroxidation.
- It holds therapeutic potential for various human diseases.
- Identifying ferroptosis regulators is crucial for developing targeted therapies.
Purpose of the Study:
- To establish a CRISPR-Cas9 loss-of-function screening protocol.
- To identify novel regulators of ferroptosis induced by different agents.
- To provide an efficient method for target discovery and validation.
Main Methods:
- CRISPR-Cas9 loss-of-function screen.
- Library amplification and drug treatment.
- High-throughput sequencing and bioinformatics analysis using MAGeCK.
Main Results:
- A robust protocol for identifying ferroptosis regulators was developed.
- The screen enables discovery of genes modulating ferroptosis.
- The method allows for efficient verification of potential therapeutic targets.
Conclusions:
- The described protocol facilitates systematic identification of ferroptosis regulators.
- This approach can accelerate the discovery of new therapeutic strategies targeting ferroptosis.
- Efficient target validation is a key feature of this screening method.
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