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A Highly Efficient CRISPR-Cas9-Based Genome Engineering Platform in Acinetobacter baumannii to Understand the H2O2-Sensing Mechanism of OxyR.
Cell Chemical Biology ( IF 6.6 ) Pub Date : 2019-09-20 , DOI: 10.1016/j.chembiol.2019.09.003
Yu Wang 1 , Zhipeng Wang 1 , Yan Chen 2 , Xiaoting Hua 2 , Yunsong Yu 2 , Quanjiang Ji 1
Affiliation  

The rapid emergence of extensively drug-resistant A. baumannii has posed a major threat to global public health, emphasizing the desperate need for novel therapeutic strategies. We report the development of a highly efficient genome-engineering platform in A. baumannii by coupling a Cas9 nuclease-mediated genome cleavage system with the RecAb recombination system. We applied the CRISPR-Cas9/RecAb system to dissect the oxidative stress-sensing mechanism of OxyR by performing alanine scanning mutagenesis of 13 residues residing in the H2O2-sensing pocket, pinpointing new vital factors for H2O2 sensing. Moreover, we developed a cytidine base-editing system, enabling programmed C to T conversions. Exploiting this powerful technique, we systematically investigated the drug-resistant mechanisms in a clinically isolated multidrug-resistant A. baumannii strain by generating premature stop codons in the possible resistance genes, unveiling distinct roles of these genes in drug resistance. The development of these genome-engineering methods will facilitate new therapeutic-means development in A. baumannii and related organisms.

中文翻译:

鲍曼不动杆菌中一个基于CRISPR-Cas9的高效基因组工程平台,以了解OxyR的H2O2传感机制。

广泛耐药的鲍曼不动杆菌的迅速出现对全球公共卫生构成了重大威胁,强调了对新型治疗策略的迫切需求。我们通过结合Cas9核酸酶介导的基因组裂解系统与RecAb重组系统,报告了鲍氏不动杆菌中高效基因组工程平台的发展。我们应用CRISPR-Cas9 / RecAb系统通过对H2O2感应袋中的13个残基进行丙氨酸扫描诱变来剖析OxyR的氧化应激感应机制,为H2O2感应找到新的重要因素。此外,我们开发了一个胞苷碱基编辑系统,可实现从C到T的程序化转换。利用这一强大的技术,我们系统地研究了临床分离的多药耐药性A中的耐药机制。通过在可能的抗性基因中产生过早的终止密码子,鲍曼氏菌菌株揭示了这些基因在耐药性中的独特作用。这些基因组工程方法的发展将促进鲍曼不动杆菌和相关生物中新的治疗手段的发展。
更新日期:2019-11-09
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