A Highly Efficient CRISPR-Cas9-Based Genome Engineering Platform in Acinetobacter baumannii to Understand the H2O2-Sensing Mechanism of OxyR

Yu Wang; Zhipeng Wang; Yan Chen; Xiaoting Hua; Yunsong Yu; Quanjiang Ji

doi:10.1016/j.chembiol.2019.09.003

A Highly Efficient CRISPR-Cas9-Based Genome Engineering Platform in Acinetobacter baumannii to Understand the H₂O₂-Sensing Mechanism of OxyR

Cell Chem Biol. 2019 Dec 19;26(12):1732-1742.e5. doi: 10.1016/j.chembiol.2019.09.003. Epub 2019 Sep 20.

Authors

Yu Wang¹, Zhipeng Wang¹, Yan Chen², Xiaoting Hua², Yunsong Yu², Quanjiang Ji³

Affiliations

¹ School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, China.
² Department of Infectious Diseases, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou 310016, China; Key Laboratory of Microbial Technology and Bioinformatics of Zhejiang Province, Hangzhou 310016, China.
³ School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, China. Electronic address: quanjiangji@shanghaitech.edu.cn.

PMID: 31548010
DOI: 10.1016/j.chembiol.2019.09.003

Abstract

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.

Keywords: Acinetobacter baumannii; CRISPR-Cas9; base editing; cytidine deaminase; genome editing.

MeSH terms

Acinetobacter baumannii / drug effects
Acinetobacter baumannii / genetics*
Anti-Bacterial Agents / pharmacology
Bacterial Proteins / genetics
Bacterial Proteins / metabolism*
CRISPR-Cas Systems / genetics*
Drug Resistance, Bacterial / genetics
Gene Editing / methods*
Hydrogen Peroxide / analysis*
Microbial Sensitivity Tests
Point Mutation
Repressor Proteins / genetics
Repressor Proteins / metabolism*

Substances

Anti-Bacterial Agents
Bacterial Proteins
Repressor Proteins
Hydrogen Peroxide