A thermostable type I-B CRISPR-Cas system for orthogonal and multiplexed genetic engineering

Zhiheng Yang, Zilong Li, Bixiao Li, Ruihong Bu, Gao-Yi Tan, Zhengduo Wang, Hao Yan, Zhenguo Xin, Guojian Zhang, Ming Li, Hua Xiang, Lixin Zhang () and Weishan Wang ()
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Zhiheng Yang: East China University of Science and Technology (ECUST)
Zilong Li: Chinese Academy of Sciences
Bixiao Li: Chinese Academy of Sciences
Ruihong Bu: Chinese Academy of Sciences
Gao-Yi Tan: East China University of Science and Technology (ECUST)
Zhengduo Wang: East China University of Science and Technology (ECUST)
Hao Yan: Chinese Academy of Sciences
Zhenguo Xin: Chinese Academy of Sciences
Guojian Zhang: Ocean University of China
Ming Li: Chinese Academy of Sciences
Hua Xiang: Chinese Academy of Sciences
Lixin Zhang: East China University of Science and Technology (ECUST)
Weishan Wang: Chinese Academy of Sciences

Nature Communications, 2023, vol. 14, issue 1, 1-14

Abstract: Abstract Thermophilic cell factories have remarkably broad potential for industrial applications, but are limited by a lack of genetic manipulation tools and recalcitrance to transformation. Here, we identify a thermophilic type I-B CRISPR-Cas system from Parageobacillus thermoglucosidasius and find it displays highly efficient transcriptional repression or DNA cleavage activity that can be switched by adjusting crRNA length to less than or greater than 26 bp, respectively, without ablating Cas3 nuclease. We then develop an orthogonal tool for genome editing and transcriptional repression using this type I-B system in both thermophile and mesophile hosts. Empowered by this tool, we design a strategy to screen the genome-scale targets involved in transformation efficiency and established dynamically controlled supercompetent P. thermoglucosidasius cells with high efficiency ( ~ 108 CFU/μg DNA) by temporal multiplexed repression. We also demonstrate the construction of thermophilic riboflavin cell factory with hitherto highest titers in high temperature fermentation by genome-scale identification and combinatorial manipulation of multiple targets. This work enables diverse high-efficiency genetic manipulation in P. thermoglucosidasius and facilitates the engineering of thermophilic cell factories.

Date: 2023
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DOI: 10.1038/s41467-023-41973-5

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