基于CRISPR/Cas9的铜绿假单胞菌基因组编辑及假单胞菌属中胞苷脱氨酶介导的碱基编辑

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

作者信息

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

机构信息

School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, China.

School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, China.

出版信息

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

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.

摘要

假单胞菌属是一大类革兰氏阴性细菌,在生物医学、生态和工业方面具有重要意义。尽管进行了广泛的研究并得到了广泛应用,但对假单胞菌属进行基因操作,尤其是对主要人类病原体铜绿假单胞菌进行基因操作,仍然是一项艰巨的任务。在此,我们报告了一种利用CRISPR/Cas9和噬菌体λ-Red重组系统开发的基因组编辑方法pCasPA/pACRISPR。该方法可在铜绿假单胞菌中进行高效且无痕的基因操作。通过设计胞苷脱氨酶APOBEC1与Cas9切口酶的融合蛋白,我们进一步开发了一种碱基编辑系统pnCasPA-BEC,它能够在多种假单胞菌属细菌中实现高效的基因失活和点突变,如铜绿假单胞菌、恶臭假单胞菌、荧光假单胞菌和丁香假单胞菌。这两种基因组编辑方法的应用将极大地加速各种研究,如细菌生理学研究、药物靶点探索和代谢工程。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2c33/6137401/059a6558d75a/fx1.jpg

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