文献类型：期刊文献

英文题名：A thermostable type I-B CRISPR-Cas system for orthogonal and multiplexed genetic engineering

作者：Yang, Zhiheng[1,2,3];Li, Zilong[3];Li, Bixiao[3];Bu, Ruihong[3,4];Tan, Gao-Yi[1,2];Wang, Zhengduo[1,2];Yan, Hao[3];Xin, Zhenguo[3];Zhang, Guojian[4];Li, Ming[3,5];Xiang, Hua[3,5];Zhang, Lixin[1,2];Wang, Weishan[3,5]

机构：[1]China Univ Sci & Technol ECUST, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]East China Univ Sci & Technol ECUST, Sch Biotechnol, Shanghai 200237, Peoples R China;[3]Chinese Acad Sci, State Key Lab Microbial Resources, Inst Microbiol, Beijing 100101, Peoples R China;[4]Ocean Univ China, Sch Med & Pharm, Qingdao 266003, Peoples R China;[5]Univ Chinese Acad Sci, Beijing 100049, Peoples R China

年份：2023

卷号：14

期号：1

外文期刊名：NATURE COMMUNICATIONS

收录：;WOS:【SCI-EXPANDED(收录号:WOS:001097145300013)】；

基金：This work was supported by the National Key Research and Development Program of China (2020YFA0906800 to H.X. and Z.L.); the National Natural Science Foundation of China (grants 32150020 to M.L., 32170095 to W.W., and 32100066 to H.Y.); the Youth Innovation Promotion Association CAS (Y202027 to W.W.); and 2023 Double World-class Project-Key Program-Intelligent Biomanufacturing to L.Z.

语种：英文

摘要：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 ( similar to 10(8) CFU/mu g 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.