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Modular (de)construction of complex bacterial phenotypes by CRISPR/nCas9-assisted, multiplex cytidine base-editing

Author

Listed:
  • Daniel C. Volke

    (Technical University of Denmark)

  • Román A. Martino

    (Universidad Nacional de Córdoba
    Universidad Nacional de Córdoba)

  • Ekaterina Kozaeva

    (Technical University of Denmark)

  • Andrea M. Smania

    (Universidad Nacional de Córdoba
    Universidad Nacional de Córdoba)

  • Pablo I. Nikel

    (Technical University of Denmark)

Abstract

CRISPR/Cas technologies constitute a powerful tool for genome engineering, yet their use in non-traditional bacteria depends on host factors or exogenous recombinases, which limits both efficiency and throughput. Here we mitigate these practical constraints by developing a widely-applicable genome engineering toolset for Gram-negative bacteria. The challenge is addressed by tailoring a CRISPR base editor that enables single-nucleotide resolution manipulations (C·G → T·A) with >90% efficiency. Furthermore, incorporating Cas6-mediated processing of guide RNAs in a streamlined protocol for plasmid assembly supports multiplex base editing with >85% efficiency. The toolset is adopted to construct and deconstruct complex phenotypes in the soil bacterium Pseudomonas putida. Single-step engineering of an aromatic-compound production phenotype and multi-step deconstruction of the intricate redox metabolism illustrate the versatility of multiplex base editing afforded by our toolbox. Hence, this approach overcomes typical limitations of previous technologies and empowers engineering programs in Gram-negative bacteria that were out of reach thus far.

Suggested Citation

  • Daniel C. Volke & Román A. Martino & Ekaterina Kozaeva & Andrea M. Smania & Pablo I. Nikel, 2022. "Modular (de)construction of complex bacterial phenotypes by CRISPR/nCas9-assisted, multiplex cytidine base-editing," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
  • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-30780-z
    DOI: 10.1038/s41467-022-30780-z
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    References listed on IDEAS

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    Cited by:

    1. Javier M. Hernández-Sancho & Arnaud Boudigou & Maria V. G. Alván-Vargas & Dekel Freund & Jenny Arnling Bååth & Peter Westh & Kenneth Jensen & Lianet Noda-García & Daniel C. Volke & Pablo I. Nikel, 2024. "A versatile microbial platform as a tunable whole-cell chemical sensor," Nature Communications, Nature, vol. 15(1), pages 1-18, December.
    2. Lan Hu & Jing Han & Hao-Da Wang & Zhou-Hua Cheng & Chang-Ce Lv & Dong-Feng Liu & Han-Qing Yu, 2025. "A universal and wide-range cytosine base editor via domain-inlaid and fidelity-optimized CRISPR-FrCas9," Nature Communications, Nature, vol. 16(1), pages 1-17, December.

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