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A strategy for Cas13 miniaturization based on the structure and AlphaFold.
Feiyu Zhao1, Tao Zhang1, Xiaodi Sun1
1State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin University, 130062, Changchun, China.
Nature Communications
|September 8, 2023
Summary
Researchers developed a new protein miniaturization strategy, creating smaller Cas13 variants for RNA editing and degradation. This advance enables efficient CRISPR/Cas13 systems for research and therapeutics.
Area of Science:
- Molecular Biology
- Biotechnology
- Genetics
Background:
- The small size of Cas nucleases fused with effector domains is crucial for diverse functions.
- Efficient and generalizable methods for Cas nuclease complex miniaturization are lacking.
Purpose of the Study:
- To establish a protein miniaturization strategy for creating compact Cas13 variants.
- To develop downsized CRISPR/Cas13 systems for RNA targeting applications.
Main Methods:
- Development of the Interaction, Dynamics and Conservation (IDC) strategy for protein miniaturization.
- Utilizing AlphaFold2 for structure prediction to guide the design of compact Cas13 variants.
- Construction of an RNA base editor (mini-Vx) and a single adeno-associated virus (AAV) delivery system.
Main Results:
- Generation of five compact Cas13 variants retaining full RNA binding and cleavage activity.
- Demonstration of efficient RNA editing by mini-Vx.
- Successful RNA knockdown using the single AAV-delivered mini-RfxCas13d and crRNA cassette.
Conclusions:
- The IDC strategy combined with AlphaFold2 provides a feasible approach for generating miniaturized CRISPR/Cas13 systems.
- Downsized CRISPR/Cas13 systems are effective for targeted RNA degradation and RNA editing.
- These findings support applications in basic research and therapeutic development.
