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

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.

Data availability

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

References

  1. 1.

    Watve, M. G., Tickoo, R., Jog, M. M. & Bhole, B. D. How many antibiotics are produced by the genus Streptomyces? Arch. Microbiol. 176, 386–390 (2001).

    CAS  PubMed  Google Scholar 

  2. 2.

    Berdy, J. Bioactive microbial metabolites. J. Antibiot. (Tokyo) 58, 1–26 (2005).

    CAS  Google Scholar 

  3. 3.

    Newman, D. J. & Cragg, G. M. Natural products as sources of new drugs from 1981 to 2014. J. Nat. Prod. 79, 629–661 (2016).

    CAS  PubMed  Google Scholar 

  4. 4.

    Blin, K. et al. antiSMASH 4.0-improvements in chemistry prediction and gene cluster boundary identification. Nucleic Acids Res. 45, W36–W41 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Weber, T. et al. Metabolic engineering of antibiotic factories: new tools for antibiotic production in actinomycetes. Trends Biotechnol. 33, 15–26 (2015).

    CAS  PubMed  Google Scholar 

  6. 6.

    Hwang, K. S., Kim, H. U., Charusanti, P., Palsson, B. O. & Lee, S. Y. Systems biology and biotechnology of Streptomyces species for the production of secondary metabolites. Biotechnol. Adv. 32, 255–268 (2014).

    CAS  PubMed  Google Scholar 

  7. 7.

    Sander, J. D. & Joung, J. K. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Wang, H., La Russa, M. & Qi, L. S. CRISPR/Cas9 in genome editing and beyond. Annu. Rev. Biochem. 85, 227–264 (2016).

    CAS  PubMed  Google Scholar 

  9. 9.

    Tong, Y., Charusanti, P., Zhang, L., Weber, T. & Lee, S. Y. CRISPR-Cas9 based engineering of actinomycetal genomes. ACS Synth. Biol. 4, 1020–1029 (2015).

    CAS  PubMed  Google Scholar 

  10. 10.

    Cobb, R. E., Wang, Y. J. & Zhao, H. M. High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system. ACS Synth. Biol. 4, 723–728 (2015).

    CAS  PubMed  Google Scholar 

  11. 11.

    Huang, H., Zheng, G. S., Jiang, W. H., Hu, H. F. & Lu, Y. H. One-step high-efficiency CRISPR/Cas9-mediated genome editing in Streptomyces. Acta Biochim. Biophys. Sin. 47, 231–243 (2015).

    CAS  PubMed  Google Scholar 

  12. 12.

    Zeng, H. et al. Highly efficient editing of the actinorhodin polyketide chain length factor gene in Streptomyces coelicolor M145 using CRISPR/Cas9-CodA(sm) combined system. Appl. Microbiol. Biotechnol. 99, 10575–10585 (2015).

    CAS  PubMed  Google Scholar 

  13. 13.

    Tong, Y., Weber, T. & Lee, S. Y. CRISPR/Cas-based genome engineering in natural product discovery. Nat. Prod. Rep. 36, 1262–1280 (2018).

    Google Scholar 

  14. 14.

    Alberti, F. & Corre, C. Editing streptomycete genomes in the CRISPR/Cas9 age. Nat. Prod. Rep. 36, 1237–1248 (2019).

    CAS  PubMed  Google Scholar 

  15. 15.

    Tong, Y. et al. Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST. Proc. Natl Acad. Sci. USA 116, 20366–20375 (2019).

    CAS  PubMed  Google Scholar 

  16. 16.

    Tong, Y., Robertsen, H. L., Blin, K., Weber, T. & Lee, S. Y. CRISPR-Cas9 toolkit for actinomycete genome editing. Methods Mol. Biol. 1671, 163–184 (2018).

    CAS  PubMed  Google Scholar 

  17. 17.

    Haurwitz, R. E., Jinek, M., Wiedenheft, B., Zhou, K. & Doudna, J. A. Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 329, 1355–1358 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Blin, K., Pedersen, L. E., Weber, T. & Lee, S. Y. CRISPy-web: an online resource to design sgRNAs for CRISPR applications. Synth. Syst. Biotechnol. 1, 118–121 (2016).

    PubMed  PubMed Central  Google Scholar 

  19. 19.

    Blin, K., Shaw, S., Tong, Y. & Weber, T. Designing sgRNAs for CRISPR-BEST base editing applications with CRISPy-web 2.0. Synth. Syst. Biotechnol. 5, 99–102 (2020).

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    Bibb, M. J., Ward, J. M. & Hopwood, D. A. Transformation of plasmid DNA into Streptomyces at high frequency. Nature 274, 398–400 (1978).

    CAS  PubMed  Google Scholar 

  21. 21.

    Kieser, T., Bibb, M., Buttner, M., Chater, K. & Hopwood, D. Practical Streptomyces Genetics (John Innes Foundation, 2000).

  22. 22.

    Gust, B., Challis, G. L., Fowler, K., Kieser, T. & Chater, K. F. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc. Natl Acad. Sci. USA 100, 1541–1546 (2003).

    CAS  PubMed  Google Scholar 

  23. 23.

    Fernandez-Martinez, L. T. & Bibb, M. J. Use of the meganuclease I-SceI of Saccharomyces cerevisiae to select for gene deletions in actinomycetes. Sci. Rep. 4, 7100 (2014).

    PubMed  PubMed Central  Google Scholar 

  24. 24.

    Volff, J. N. & Altenbuchner, J. Genetic instability of the Streptomyces chromosome. Mol. Microbiol. 27, 239–246 (1998).

    CAS  PubMed  Google Scholar 

  25. 25.

    Hoff, G., Bertrand, C., Piotrowski, E., Thibessard, A. & Leblond, P. Genome plasticity is governed by double strand break DNA repair in Streptomyces. Sci. Rep. 8, 5272 (2018).

    PubMed  PubMed Central  Google Scholar 

  26. 26.

    Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, aaf8729 (2016).

    PubMed  Google Scholar 

  27. 27.

    Gaudelli, N. M. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Lovett, S. T. Encoded errors: mutations and rearrangements mediated by misalignment at repetitive DNA sequences. Mol. Microbiol. 52, 1243–1253 (2004).

    CAS  PubMed  Google Scholar 

  30. 30.

    Jack, B. R. et al. Predicting the genetic stability of engineered DNA sequences with the EFM calculator. ACS Synth. Biol. 4, 939–943 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Brophy, J. A. & Voigt, C. A. Principles of genetic circuit design. Nat. Methods 11, 508–520 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Alberti, F. et al. Triggering the expression of a silent gene cluster from genetically intractable bacteria results in scleric acid discovery. Chem. Sci. 10, 453–463 (2019).

    CAS  PubMed  Google Scholar 

  33. 33.

    Low, Z. J. et al. Identification of a biosynthetic gene cluster for the polyene macrolactam sceliphrolactam in a Streptomyces strain isolated from mangrove sediment. Sci. Rep. 8, 1594 (2018).

    PubMed  PubMed Central  Google Scholar 

  34. 34.

    Culp, E. J. et al. Hidden antibiotics in actinomycetes can be identified by inactivation of gene clusters for common antibiotics. Nat. Biotechnol. 37, 1149–1154 (2019).

    CAS  PubMed  Google Scholar 

  35. 35.

    Cohen, D. R. & Townsend, C. A. A dual role for a polyketide synthase in dynemicin enediyne and anthraquinone biosynthesis. Nat. Chem. 10, 231–236 (2018).

    CAS  PubMed  Google Scholar 

  36. 36.

    Cho, J. S. et al. CRISPR/Cas9-coupled recombineering for metabolic engineering of Corynebacterium glutamicum. Metab. Eng. 42, 157–167 (2017).

    CAS  PubMed  Google Scholar 

  37. 37.

    Mo, J. et al. Efficient editing DNA regions with high sequence identity in actinomycetal genomes by a CRISPR-Cas9 system. Synth. Syst. Biotechnol. 4, 86–91 (2019).

    PubMed  PubMed Central  Google Scholar 

  38. 38.

    Wang, Q. et al. Dual-function chromogenic screening-based CRISPR/Cas9 genome editing system for actinomycetes. Appl. Microbiol. Biotechnol. 104, 225–239 (2020).

    CAS  PubMed  Google Scholar 

  39. 39.

    Zhang, M. M. et al. CRISPR-Cas9 strategy for activation of silent Streptomyces biosynthetic gene clusters. Nat. Chem. Biol. 13, 607–609 (2017).

    CAS  Google Scholar 

  40. 40.

    Dow, L. E. et al. Inducible in vivo genome editing with CRISPR-Cas9. Nat. Biotechnol. 33, 390–394 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Wu, X. et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat. Biotechnol. 32, 670–676 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Muth, G. The pSG5-based thermosensitive vector family for genome editing and gene expression in actinomycetes. Appl. Microbiol. Biotechnol. 102, 9067–9080 (2018).

    CAS  PubMed  Google Scholar 

  44. 44.

    MacNeil, D. J. et al. Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene 111, 61–68 (1992).

    CAS  PubMed  Google Scholar 

  45. 45.

    Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Bentley, S. D. et al. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417, 141–147 (2002).

    PubMed  Google Scholar 

  47. 47.

    Schubert, M., Lindgreen, S. & Orlando, L. AdapterRemoval v2: rapid adapter trimming, identification, and read merging. BMC Res. Notes 9, 88 (2016).

    PubMed  PubMed Central  Google Scholar 

  48. 48.

    Deatherage, D. E. & Barrick, J. E. Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq. Methods Mol. Biol. 1151, 165–188 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Blin, K. et al. antiSMASH 5.0: updates to the secondary metabolite genome mining pipeline. Nucleic Acids Res. 47, W81–W87 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Walker, B. J. et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One 9, e112963 (2014).

    PubMed  PubMed Central  Google Scholar 

  51. 51.

    Bai, C. et al. Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces. Proc. Natl Acad. Sci. USA 112, 12181–12186 (2015).

    CAS  PubMed  Google Scholar 

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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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Affiliations

Authors

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.

Ethics declarations

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.

Related links

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. coliStreptomyces 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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