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A thermostable type I-B CRISPR-Cas system for orthogonal and multiplexed genetic engineering
Zhiheng Yang1,2, Zilong Li2, Bixiao Li2
1State Key Laboratory of Bioreactor Engineering, and School of Biotechnology, East China University of Science and Technology (ECUST), 200237, Shanghai, China.
Nature Communications
|October 4, 2023
Summary
Researchers developed a new CRISPR-Cas tool for genetic engineering in thermophilic bacteria. This system enhances transformation efficiency and enables the creation of improved cell factories for industrial applications.
Area of Science:
- Synthetic Biology
- Microbial Engineering
- CRISPR-Cas Systems
Background:
- Thermophilic microorganisms offer significant potential for industrial biotechnology but are hindered by limited genetic tools.
- Efficient genetic manipulation is crucial for optimizing these organisms as cell factories.
Purpose of the Study:
- To identify and characterize a novel thermophilic type I-B CRISPR-Cas system for genetic engineering.
- To develop a versatile tool for genome editing and transcriptional repression in thermophilic and mesophilic hosts.
- To engineer enhanced thermophilic cell factories for high-value product synthesis.
Main Methods:
- Identification and characterization of a type I-B CRISPR-Cas system from Parageobacillus thermoglucosidasius.
- Development of a switchable CRISPR-Cas tool for transcriptional repression and DNA cleavage by adjusting crRNA length.
- Application of the tool for genome-wide screening of transformation efficiency targets.
- Engineering of dynamically controlled supercompetent cells and a thermophilic riboflavin cell factory.
Main Results:
- The identified CRISPR-Cas system exhibits tunable transcriptional repression (<26 bp crRNA) and DNA cleavage (>26 bp crRNA) activities.
- An orthogonal tool was established for both thermophilic and mesophilic hosts.
- High transformation efficiency (~10^8 CFU/μg DNA) was achieved in P. thermoglucosidasius through multiplexed repression.
- A thermophilic riboflavin cell factory with record titers was constructed via genome-scale engineering.
Conclusions:
- This work provides a powerful and versatile genetic manipulation toolkit for P. thermoglucosidasius.
- The developed system significantly advances the engineering of thermophilic cell factories for industrial biotechnology.
- Enables diverse high-efficiency genetic manipulation in thermophiles, facilitating the development of robust industrial microbial platforms.
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