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CRISPR–Cas9, CRISPRi and CRISPR-BEST-mediated genetic manipulation in streptomycetes

Nature Protocols volume 15, pages 2470–2502 (2020)Cite this article

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Abstract

Streptomycetes are prominent sources of bioactive natural products, but metabolic engineering of the natural products of these organisms is greatly hindered by relatively inefficient genetic manipulation approaches. New advances in genome editing techniques, particularly CRISPR-based tools, have revolutionized genetic manipulation of many organisms, including actinomycetes. We have developed a comprehensive CRISPR toolkit that includes several variations of ‘classic’ CRISPR–Cas9 systems, along with CRISPRi and CRISPR-base editing systems (CRISPR-BEST) for streptomycetes. Here, we provide step-by-step protocols for designing and constructing the CRISPR plasmids, transferring these plasmids to the target streptomycetes, and identifying correctly edited clones. Our CRISPR toolkit can be used to generate random-sized deletion libraries, introduce small indels, generate in-frame deletions of specific target genes, reversibly suppress gene transcription, and substitute single base pairs in streptomycete genomes. Furthermore, the toolkit includes a Csy4-based multiplexing option to introduce multiple edits in a single experiment. The toolkit can be easily extended to other actinomycetes. With our protocol, it takes <10 d to inactivate a target gene, which is much faster than alternative protocols.

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Fig. 1: A mechanistic overview of the all-in-one pot ssDNA bridging method for sgRNA cloning.
Fig. 2: An overview of the CRISPR-based genetic manipulation system included in this protocol.
Fig. 3: A three-sgRNA multiplexed example.
Fig. 4: An overview of the entire protocol.
Fig. 5: An overview of CRISPR–Cas9 plasmid construction for in-frame deletion or insertion of foreign DNA.
Fig. 6: Experimental setup for collecting spores.
Fig. 7: Anticipated results.

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Data availability

No new data were generated or analyzed with this protocol; all presented data were previously published9,15.

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Acknowledgements

We thank S. Shaw for proofreading the manuscript. This work was supported by grants from the Novo Nordisk Foundation (NNF10CC1016517, NNF15OC0016226, NNF16OC0021746). S.Y.L. was also supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557) from the Ministry of Science and ICT through the National Research Foundation (NRF) of Korea.

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Authors and Affiliations

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

    Yaojun Tong, Christopher M. Whitford, Kai Blin, Tue S. Jørgensen, Tilmann Weber & Sang Yup Lee

  2. Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea

    Sang Yup Lee

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  1. Yaojun Tong
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Contributions

Y.T. designed and developed the protocol; K.B. designed the spacer identification software CRISPy-web; T.S.J. established the genome-wide off-target evaluation pipeline. Y.T., and C.M.W. performed the experiments; T.W. and S.Y.L. supervised and steered the project; and Y.T., T.W., and S.Y.L. wrote the protocol.

Corresponding authors

Correspondence to Tilmann Weber or Sang Yup Lee.

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Competing interests

Y.T., T.W., and S.Y.L. are co-inventors on a patent application for an actinomycete CRISPR application (WO2016150855A1) filed by Technical University of Denmark.

Additional information

Peer review information Nature Protocols thanks Christophe Corre, Ioannis Mougiakos, and Fabrizio Alberti for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Key references using this protocol

Tong, Y., Charusanti, P., Zhang, L., Weber, T. & Lee, S. Y. ACS Synth. Biol. 4, 1020–1029 (2015): https://doi.org/10.1021/acssynbio.5b00038

Tong, Y. et al. Proc. Natl. Acad. Sci. USA 116, 20366–20375 (2019): https://doi.org/10.1073/pnas.1913493116

Blin, K., Pedersen, L. E., Weber, T. & Lee, S. Y. Synth. Syst. Biotechnol. 1, 118–121 (2016): https://doi.org/10.1016/j.synbio.2016.01.003

Blin, K., Shaw, S., Tong, Y. & Weber, T. Synth. Syst. Biotechnol. 5, 99–102 (2020): https://doi.org/10.1016/j.synbio.2020.05.005

Supplementary information

Reporting Summary

Supplementary Video 1

A step-by-step protocol of interspecific E. coli–Streptomyces conjugation. The video demonstrates how to carry out Streptomyces spore–E.coli conjugation. It includes Streptomyces spore collection (Fig. 6), mixing Streptomyces spores with the target plasmid carrying ET12567/pUZ8002 E. coli culture, and overlaying the plate surface with 1 mg apramycin and 1 mg nalidixic acid containing sterilized ddH2O.

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Tong, Y., Whitford, C.M., Blin, K. et al. CRISPR–Cas9, CRISPRi and CRISPR-BEST-mediated genetic manipulation in streptomycetes. Nat Protoc 15, 2470–2502 (2020). https://doi.org/10.1038/s41596-020-0339-z

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