School of Physical Science and Technology, ShanghaiTech University, Shanghai, China.
Department of Laboratory Medicine, Wenzhou Medical University, Wenzhou, China.
Appl Environ Microbiol. 2018 Nov 15;84(23). doi: 10.1128/AEM.01834-18. Print 2018 Dec 1.
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.
是一种有前途的工业微生物，也是一种主要的人类病原体。最近出现的耐碳青霉烯 对全球公共健康构成了严重威胁，强调迫切需要新的治疗手段来对抗耐药性 尽管遗传学在生物工程、生理学研究和治疗手段开发中至关重要，但基因组编辑，特别是在 中，理想的无痕遗传操作，往往既费时又费力。在这里，我们报告了一个双质粒系统，pCasKP-pSGKP，用于 在利用成簇规律间隔短回文重复 (CRISPR)-Cas9 基因组切割系统和 lambda Red 重组系统的情况下，pCasKP-pSGKP 使用短修复模板在 中实现了高效的基因组编辑。此外，我们开发了一种胞嘧啶碱基编辑系统 pBECKP，通过工程融合胞嘧啶脱氨酶 APOBEC1 和 Cas9 切口酶，用于在染色体和质粒携带基因中精确的 C→T 转换。通过使用 pCasKP-pSGKP 和 pBECKP 工具， 基因被确认为导致 hypermucoviscous 耐碳青霉烯的耐碳青霉烯 菌株产生碳青霉烯耐药性的主要因素。这两种编辑工具的开发将极大地促进 基因的遗传工程。遗传学是研究细菌生理学的关键手段。然而，对于主要的人类病原体 ，理想的无痕遗传操作往往既费时又费力。我们开发了一种 CRISPR-Cas9 介导的基因组编辑方法和一种胞嘧啶碱基编辑系统，能够在工业和临床分离的 菌株中快速、高效、迭代地进行基因组编辑。我们将这两种工具应用于剖析 hypermucoviscous 耐碳青霉烯的耐碳青霉烯 菌株的耐药机制，阐明 基因是导致该菌株对碳青霉烯类药物耐药的主要因素。这两种工具的利用将极大地加速对不同 菌株和相关 物种的各种研究，如基因特征描述、药物发现和代谢工程。
