CRISPR-Cas9 and CRISPR-Assisted Cytidine Deaminase Enable Precise and Efficient Genome Editing in Klebsiella pneumoniae

被引:153
作者
Wang, Yu [1 ]
Wang, Shanshan [2 ]
Chen, Weizhong [1 ]
Song, Liqiang [1 ]
Zhang, Yifei [1 ]
Shen, Zhen [3 ]
Yu, Fangyou [4 ]
Li, Min [3 ]
Ji, Quanjiang [1 ]
机构
[1] ShanghaiTech Univ, Sch Phys Sci & Technol, Shanghai, Peoples R China
[2] Wenzhou Med Univ, Dept Lab Med, Wenzhou, Peoples R China
[3] Shanghai Jiao Tong Univ, Sch Med, Ren Ji Hosp, Dept Lab Med, Shanghai, Peoples R China
[4] Tongji Univ, Sch Med, Shanghai Pulm Hosp, Dept Clin Lab, Shanghai, Peoples R China
基金
中国国家自然科学基金; 国家重点研发计划; 中国博士后科学基金;
关键词
CRISPR; Cas9; Klebsiella pneumoniae; genetic engineering; genome editing; base editing; ESCHERICHIA-COLI; STAPHYLOCOCCUS-AUREUS; GENE REPLACEMENT; DNA; BASE; 1,3-PROPANEDIOL; EPIDEMIOLOGY; RESISTANCE; SYSTEM;
D O I
10.1128/AEM.01834-18
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Klebsiella pneumoniae is a promising industrial microorganism as well as a major human pathogen. The recent emergence of carbapenem-resistant K. pneumoniae has posed a serious threat to public health worldwide, emphasizing a dire need for novel therapeutic means against drug-resistant K. pneumoniae. Despite the critical importance of genetics in bioengineering, physiology studies, and therapeutic-means development, genome editing, in particular, the highly desirable scarless genetic manipulation in K. pneumoniae, is often time-consuming and laborious. Here, we report a two-plasmid system, pCasKP-pSGKP, used for precise and iterative genome editing in K. pneumoniae. By harnessing the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome cleavage system and the lambda Red recombination system, pCasKP-pSGKP enabled highly efficient genome editing in K. pneumoniae using a short repair template. Moreover, we developed a cytidine base-editing system, pBECKP, for precise C -> T conversion in both the chromosomal and plasmid-borne genes by engineering the fusion of the cytidine deaminase APOBEC1 and a Cas9 nickase. By using both the pCasKP-pSGKP and the pBECKP tools, the bla(KPC-2) gene was confirmed to be the major factor that contributed to the carbapenem resistance of a hypermucoviscous carbapenem-resistant K. pneumoniae strain. The development of the two editing tools will significantly facilitate the genetic engineering of K. pneumoniae. IMPORTANCE Genetics is a key means to study bacterial physiology. However, the highly desirable scarless genetic manipulation is often time-consuming and laborious for the major human pathogen K. pneumoniae. We developed a CRISPR-Cas9-mediated genome-editing method and a cytidine base-editing system, enabling rapid, highly efficient, and iterative genome editing in both industrial and clinically isolated K. pneumoniae strains. We applied both tools in dissecting the drug resistance mechanism of a hypermucoviscous carbapenem-resistant K. pneumoniae strain, elucidating that the bla(KPC-2), gene was the major factor that contributed to the carbapenem resistance of the hypermucoviscous carbapenem-resistant K. pneumoniae strain. Utilization of the two tools will dramatically accelerate a wide variety of investigations in diverse K. pneumoniae strains and relevant Enterobacteriaceae species, such as gene characterization, drug discovery, and metabolic engineering.
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页数:15
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