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

• Published in: Applied and environmental microbiology 2018-12, Vol.84 (23)
• Main Authors: Wang, Yu, Wang, Shanshan, Chen, Weizhong, Song, Liqiang, Zhang, Yifei, Shen, Zhen, Yu, Fangyou, Li, Min, Ji, Quanjiang
• Format: Article
• Language: English
• Subjects: Bacteria; Bioengineering; CRISPR; Cytidine deaminase; Drug resistance; Editing; Genes; Genetic engineering; Genetics; Genome editing; Genomes; Health risks; Klebsiella; Klebsiella pneumoniae; Methods; Pathogens; Public health; Recombination
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.01834-18

is a promising industrial microorganism as well as a major human pathogen. The recent emergence of carbapenem-resistant has posed a serious threat to public health worldwide, emphasizing a dire need for novel therapeutic means against drug-resistant 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 , is often time-consuming and laborious. Here, we report a two-plasmid system, pCasKP-pSGKP, used for precise and iterative genome editing in 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 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 gene was confirmed to be the major factor that contributed to the carbapenem resistance of a hypermucoviscous carbapenem-resistant strain. The development of the two editing tools will significantly facilitate the genetic engineering of 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 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 strains. We applied both tools in dissecting the drug resistance mechanism of a hypermucoviscous carbapenem-resistant strain, elucidating that the gene was the major factor that contributed to the carbapenem resistance of the hypermucoviscous carbapenem-resistant strain. Utilization of the two tools will dramatically accelerate a wide variety of investigations in diverse strains and relevant species, such as gene characterization, drug discovery, and metabolic engineering.