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A Simple Method to Generate Super-sensitive AID (ssAID)-based Conditional Knockouts using CRISPR-based Gene Knockout
Kohei Nishimura1,2, Tatsuo Fukagawa1
1Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan.
Bio-Protocol
|August 16, 2021
Summary
Researchers developed a super-sensitive auxin-inducible degron (ssAID) system for conditional knockout of essential genes. This method significantly reduces auxin concentration, minimizing cytotoxicity and enabling efficient gene function studies.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Gene knockout is crucial for analyzing protein function.
- CRISPR/Cas-9 is widely used but not suitable for essential genes.
- Conditional knockout systems are needed for essential genes.
Purpose of the Study:
- To introduce an improved method for conditional knockout of essential genes.
- To present a super-sensitive auxin-inducible degron (ssAID) system.
- To enable efficient investigation of essential gene roles.
Main Methods:
- Established a super-sensitive auxin-inducible degron (ssAID) system.
- Developed a single-step method for generating AID-based conditional knockout cells.
- Utilized ssAID system in various cell lines.
Main Results:
- Achieved over 1,000-fold reduction in required auxin concentration.
- Demonstrated efficient protein depletion with minimal cytotoxicity.
- Successfully generated conditional knockout cells using the ssAID system.
Conclusions:
- The improved ssAID system is a powerful tool for studying essential genes.
- Reduced auxin concentrations mitigate cytotoxicity associated with AID systems.
- This method facilitates conditional knockout in diverse eukaryotic cell lines.
