Applied Microbiology and Biotechnology

, Volume 103, Issue 6, pp 2783–2795 | Cite as

Combination of ssDNA recombineering and CRISPR-Cas9 for Pseudomonas putida KT2440 genome editing

  • Zhixin Wu
  • Zhongqiu Chen
  • Xinyue Gao
  • Jing Li
  • Guangdong ShangEmail author
Methods and protocols


Pseudomonas putida KT2440 is a Gram-negative, biosafety strain that plays important roles in environmental and biotechnological applications. Highly efficient genome editing strategy is essential to the elucidation of gene function and construction of metabolic engineered strains. Building on our previously established recombineering-mediated markerless and scarless P. putida KT2440 chromosomal gene deletion methods, herein we combined single-stranded DNA (ssDNA) recombineering and CRISPR-Cas9 technologies for P. putida KT2440 genome editing. Firstly, an inactive kanamycin resistance gene was knocked into the P. putida KT2440 chromosome. Then, based on kanamycin selection, recombinase gene selection, ssDNA recombineering condition optimization, and gRNA expression promoter selection were performed. A two-plasmid genome editing system was established; the first is a broad host range, RK2 replicon–based plasmid cloned with the tightly regulated redβ and cas9 genes; the second is a broad host range, pBBR1 replicon–based, sgRNA expression plasmid. Gene point mutations and gene deletions were carried out; the genome editing efficiency is as high as 100%. The method will expedite the P. putida KT2440 metabolic engineering and synthetic biology studies.


Pseudomonas putida KT2440 ssDNA recombineering CRISPR-Cas9 Genome editing 



We thank Dr. Josef Altenbuchner, Dr. Svein Valla, and Dr. Barry Wanner for providing the plasmids used in this study.

Funding information

Funding was provided by National Natural Science Foundation of China (NSFC 81273412) and National High Technology Research and Development Program of China (2012AA02A702).

Compliance with ethical standards

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the author.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

253_2019_9654_MOESM1_ESM.pdf (859 kb)
ESM 1 (PDF 859 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life SciencesNanjing Normal UniversityNanjingChina

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