Reprogramming Mycobacterium tuberculosis CRISPR System for Gene Editing and Genome-wide RNA Interference Screening

Khaista Rahman; Muhammad Jamal; Xi Chen; Wei Zhou; Bin Yang; Yanyan Zou; Weize Xu; Yingying Lei; Chengchao Wu; Xiaojian Cao; Rohit Tyagi; Muhammad Ahsan Naeem; Da Lin; Zeshan Habib; Nan Peng; Zhen F. Fu; Gang Cao

Genomics, Proteomics & Bioinformatics (Dec 2022)

Reprogramming Mycobacterium tuberculosis CRISPR System for Gene Editing and Genome-wide RNA Interference Screening

Khaista Rahman,
Muhammad Jamal,
Xi Chen,
Wei Zhou,
Bin Yang,
Yanyan Zou,
Weize Xu,
Yingying Lei,
Chengchao Wu,
Xiaojian Cao,
Rohit Tyagi,
Muhammad Ahsan Naeem,
Da Lin,
Zeshan Habib,
Nan Peng,
Zhen F. Fu,
Gang Cao

Affiliations

Khaista Rahman: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China
Muhammad Jamal: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China
Xi Chen: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China
Wei Zhou: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China
Bin Yang: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China
Yanyan Zou: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China
Weize Xu: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China
Yingying Lei: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China
Chengchao Wu: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China
Xiaojian Cao: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China
Rohit Tyagi: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China
Muhammad Ahsan Naeem: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China
Da Lin: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China
Zeshan Habib: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China
Nan Peng: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
Zhen F. Fu: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China; Corresponding authors.
Gang Cao: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China; Bio-Medical Center, Huazhong Agricultural University, Wuhan 430070, China; Key Laboratory of Preventive Veterinary Medicine in Hubei Province, the Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China; Corresponding authors.

Journal volume & issue: Vol. 20, no. 6
pp. 1180 – 1196

Abstract

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Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), which is still the leading cause of mortality from a single infectious disease worldwide. The development of novel anti-TB drugs and vaccines is severely hampered by the complicated and time-consuming genetic manipulation techniques for M. tuberculosis. Here, we harnessed an endogenous type III-A CRISPR/Cas10 system of M. tuberculosis for efficient gene editing and RNA interference (RNAi). This simple and easy method only needs to transform a single mini-CRISPR array plasmid, thus avoiding the introduction of exogenous protein and minimizing proteotoxicity. We demonstrated that M. tuberculosis genes can be efficiently and specifically knocked in/out by this system as confirmed by DNA high-throughput sequencing. This system was further applied to single- and multiple-gene RNAi. Moreover, we successfully performed genome-wide RNAi screening to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be extensively used for exploring the functional genomics of M. tuberculosis and facilitate the development of novel anti-TB drugs and vaccines.

Published in Genomics, Proteomics & Bioinformatics

ISSN: 1672-0229 (Print); 2210-3244 (Online)
Publisher: Oxford University Press
Country of publisher: United Kingdom
LCC subjects: Science: Biology (General)
Website: https://academic.oup.com/gpb

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