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An expanded CRISPR–Cas9-assisted recombineering toolkit for engineering genetically intractable Pseudomonas aeruginosa isolates

Nature Protocols volume 18, pages 3253–3288 (2023)Cite this article

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Abstract

Much of our current understanding of microbiology is based on the application of genetic engineering procedures. Since their inception (more than 30 years ago), methods based largely on allelic exchange and two-step selection processes have become a cornerstone of contemporary bacterial genetics. While these tools are established for adapted laboratory strains, they have limited applicability in clinical or environmental isolates displaying a large and unknown genetic repertoire that are recalcitrant to genetic modifications. Hence, new tools allowing genetic engineering of intractable bacteria must be developed to gain a comprehensive understanding of them in the context of their biological niche. Herein, we present a method for precise, efficient and rapid engineering of the opportunistic pathogen Pseudomonas aeruginosa. This procedure relies on recombination of short single-stranded DNA facilitated by targeted double-strand DNA breaks mediated by a synthetic Cas9 coupled with the efficient Ssr recombinase. Possible applications include introducing single-nucleotide polymorphisms, short or long deletions, and short DNA insertions using synthetic single-stranded DNA templates, drastically reducing the need of PCR and cloning steps. Our toolkit is encoded on two plasmids, harboring an array of different antibiotic resistance cassettes; hence, this approach can be successfully applied to isolates displaying natural antibiotic resistances. Overall, this toolkit substantially reduces the time required to introduce a range of genetic manipulations to a minimum of five experimental days, and enables a variety of research and biotechnological applications in both laboratory strains and difficult-to-manipulate P. aeruginosa isolates.

Key points

  • This protocol provides an expanded toolkit for engineering genetically intractable Pseudomonas aeruginosa isolates, utilizing two plasmids to enable Ssr-mediated recombineering and CRISPR–Cas9 counterselection of mutated bacterial colonies in a single-step procedure.

  • The toolkit markedly reduces the time required to introduce a range of genetic manipulations compared with previous allelic exchange methods, facilitating a variety of research and biotechnological applications in both laboratory strains and difficult-to-manipulate P. aeruginosa isolates.

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Fig. 1: The CRISPR–Cas9-recombineering system for genomic manipulation in P. aeruginosa.
Fig. 2: Design of spacer DNA sequences and recombineering oligonucleotides.
Fig. 3: Overview of the protocol workflow.
Fig. 4: Expected results for each of the described applications.

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Data availability

The authors declare that no new data were generated during the development of this protocol. The original publication describing the method expanded herein is available in the primary research paper (https://doi.org/10.1038/s41522-022-00268-1). All plasmids described in this protocol are available at Addgene (Table 1). Source data are provided with this paper.

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Acknowledgements

We thank V. de Lorenzo and E. Martinez-García for their inspiring work in recombineering methods and for sharing valuable materials. This work was supported by grants from the European Union (ERC Consolidator grant COMBAT 724290) and the excellence cluster RESIST (Resolving Infection Susceptibility; EXC 2155—project number 390874280). Furthermore, S.H. received funding from the German Research Foundation (Deutsche Forschungsgemeinschaft grant SPP 1879), the Lower Saxony Ministry for Science and Culture (BacData grant ZN3428) and the Novo Nordisk Foundation (grant NNF18OC0033946). P.I.N. received financial support from the Novo Nordisk Foundation through grants NNF20CC0035580, LiFe (NNF18OC0034818) and TARGET (NNF21OC0067996), the Danish Council for Independent Research (SWEET, DFF–research project 8021-00039B) and the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement no. 814418 (SinFonia). Biorender (Biorender.com) was used to create the figures.

Author information

Author notes

  1. These authors contributed equally: Debbie Pankratz, Nicolas Oswaldo Gomez.

  2. These authors jointly supervised this work: Susanne Häussler, Alejandro Arce-Rodriguez.

Authors and Affiliations

  1. Department of Molecular Bacteriology, Helmholtz Centre for Infection Research, Braunschweig, Germany

    Debbie Pankratz, Nicolas Oswaldo Gomez, Agnes Nielsen, Fabián Arce-Rodriguez, Susanne Häussler & Alejandro Arce-Rodriguez

  2. Institute for Molecular Bacteriology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Germany

    Debbie Pankratz, Melisa Gür & Susanne Häussler

  3. Institute of Microbiology, Technische Universität Braunschweig, Braunschweig, Germany

    Ayten Mustafayeva & Alejandro Arce-Rodriguez

  4. Department of Clinical Microbiology, Copenhagen University Hospital—Rigshospitalet, Copenhagen, Denmark

    Melisa Gür & Susanne Häussler

  5. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark

    Pablo Ivan Nikel

  6. Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Hannover, Germany

    Susanne Häussler

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  1. Debbie Pankratz
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Contributions

A.A.-R., S.H. and P.I.N. designed the protocol. P.I.N. developed the original CRISPR–Cas9-bearing plasmid. D.P., N.O.G., A.A.-R., A.N. and A.M. constructed the new vector set for the toolkit. D.P., A.A.-R., N.O.G., A.N., A.M. and F.A.-R. performed the experiments and validated the protocol in laboratory strains. M.G. and D.P. performed the experiments in clinical isolates. A.A.-R., S.H. and P.I.N. supervised the project. A.A.-R., D.P, N.O.G. and S.H. wrote the protocol.

Corresponding authors

Correspondence to Susanne Häussler or Alejandro Arce-Rodriguez.

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The authors declare no competing interests.

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Nature Protocols thanks Andrea Smania and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Jeske, A. et al. NPJ Biofilms Microbiomes 8, 6 (2022): https://doi.org/10.1038/s41522-022-00268-1

Borgert, S. R. et al. Nat. Commun. 13, 7402 (2022): https://doi.org/10.1038/s41467-022-35030-w

Supplementary information

Supplementary Table 1

Oligonucleotides used in this study.

Source data

Source Data Fig. 4

Unprocessed agarose gels for Fig. c–e.

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Pankratz, D., Gomez, N.O., Nielsen, A. et al. An expanded CRISPR–Cas9-assisted recombineering toolkit for engineering genetically intractable Pseudomonas aeruginosa isolates. Nat Protoc 18, 3253–3288 (2023). https://doi.org/10.1038/s41596-023-00882-z

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