CRISPR/Cas9-based Genome Editing in Pseudomonas aeruginosa and Cytidine Deaminase-Mediated Base Editing in Pseudomonas Species

Weizhong Chen; Ya Zhang; Yifei Zhang; Yishuang Pi; Tongnian Gu; Liqiang Song; Yu Wang; Quanjiang Ji

doi:10.1016/j.isci.2018.07.024

CRISPR/Cas9-based Genome Editing in Pseudomonas aeruginosa and Cytidine Deaminase-Mediated Base Editing in Pseudomonas Species

iScience. 2018 Aug 31:6:222-231. doi: 10.1016/j.isci.2018.07.024. Epub 2018 Aug 1.

Authors

Weizhong Chen¹, Ya Zhang¹, Yifei Zhang¹, Yishuang Pi¹, Tongnian Gu¹, Liqiang Song¹, Yu Wang¹, Quanjiang Ji²

Affiliations

¹ School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, China.
² School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, China. Electronic address: quanjiangji@shanghaitech.edu.cn.

Abstract

Pseudomonas species are a large class of gram-negative bacteria that exhibit significant biomedical, ecological, and industrial importance. Despite the extensive research and wide applications, genetic manipulation in Pseudomonas species, in particular in the major human pathogen Pseudomonas aeruginosa, remains a laborious endeavor. Here we report the development of a genome editing method pCasPA/pACRISPR by harnessing the CRISPR/Cas9 and the phage λ-Red recombination systems. The method allows for efficient and scarless genetic manipulation in P. aeruginosa. By engineering the fusion of the cytidine deaminase APOBEC1 and the Cas9 nickase, we further develop a base editing system pnCasPA-BEC, which enables highly efficient gene inactivation and point mutations in a variety of Pseudomonas species, such as P. aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, and Pseudomonas syringae. Application of the two genome editing methods will dramatically accelerate a wide variety of investigations, such as bacterial physiology study, drug target exploration, and metabolic engineering.

Keywords: Biotechnology; Genetic Engineering; Genetics; Microbial Genetics.