Dataset Information

Highly efficient base editing in Staphylococcus aureus using an engineered CRISPR RNA-guided cytidine deaminase.

ABSTRACT: Novel therapeutic means against Staphylococcus aureus infections are urgently needed due to the emergence of drug-resistant S. aureus. We report the development of a CRISPR RNA-guided cytidine deaminase (pnCasSA-BEC), enabling highly efficient gene inactivation and point mutations in S. aureus. We engineered a fusion of a Cas9 nickase (Cas9D10A) and a cytidine deaminase (APOBEC1) that can be guided to a target genomic locus for gene inactivation via generating a premature stop codon. The pnCasSA-BEC system nicks the non-edited strand of the genomic DNA, directly catalyzes the conversion of cytidine (C) to uridine (U), and relies on DNA replication to achieve C ? T (G ? A) conversion without using donor repair templates. The development of the base-editing system will dramatically accelerate drug-target exploration in S. aureus and provides critical insights into the development of base-editing tools in other microbes.

SUBMITTER: Gu T

PROVIDER: S-EPMC5932532 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Highly efficient base editing in Staphylococcus aureus using an engineered CRISPR RNA-guided cytidine deaminase.

Gu Tongnian T Zhao Siqi S Pi Yishuang Y Chen Weizhong W Chen Chuanyuan C Liu Qian Q Li Min M Han Dali D Ji Quanjiang Q

Chemical science 20180222 12

Novel therapeutic means against Staphylococcus aureus infections are urgently needed due to the emergence of drug-resistant S. aureus. We report the development of a CRISPR RNA-guided cytidine deaminase (pnCasSA-BEC), enabling highly efficient gene inactivation and point mutations in S. aureus. We engineered a fusion of a Cas9 nickase (Cas9D10A) and a cytidine deaminase (APOBEC1) that can be guided to a target genomic locus for gene inactivation via generating a prema ...[more]

PMID: 29780457

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Project description: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.

Dataset Information

Highly efficient base editing in Staphylococcus aureus using an engineered CRISPR RNA-guided cytidine deaminase.

Publications

Highly efficient base editing in <i>Staphylococcus aureus</i> using an engineered CRISPR RNA-guided cytidine deaminase.

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