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Synthetic Metabolic Pathways pp 163–184Cite as

CRISPR-Cas9 Toolkit for Actinomycete Genome Editing

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1671))

Abstract

Bacteria of the order Actinomycetales are one of the most important sources of bioactive natural products, which are the source of many drugs. However, many of them still lack efficient genome editing methods, some strains even cannot be manipulated at all. This restricts systematic metabolic engineering approaches for boosting known and discovering novel natural products. In order to facilitate the genome editing for actinomycetes, we developed a CRISPR-Cas9 toolkit with high efficiency for actinomyces genome editing. This basic toolkit includes a software for spacer (sgRNA) identification, a system for in-frame gene/gene cluster knockout, a system for gene loss-of-function study, a system for generating a random size deletion library, and a system for gene knockdown. For the latter, a uracil-specific excision reagent (USER) cloning technology was adapted to simplify the CRISPR vector construction process. The application of this toolkit was successfully demonstrated by perturbation of genomes of Streptomyces coelicolor A3(2) and Streptomyces collinus Tü 365. The CRISPR-Cas9 toolkit and related protocol described here can be widely used for metabolic engineering of actinomycetes.

The authors have filed a patent (EP15160126.7) on the actinomycete CRISPR toolkit.

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References

  1. Berdy J (2005) Bioactive microbial metabolites – a personal view. J Antibiot 58(1):1–26

    Article  CAS  PubMed  Google Scholar 

  2. Berdy J (2012) Thoughts and facts about antibiotics: where we are now and where we are heading. J Antibiot 65(8):385–395. doi:10.1038/Ja.2012.27

    Article  CAS  PubMed  Google Scholar 

  3. Hwang KS, Kim HU, Charusanti P et al (2014) Systems biology and biotechnology of Streptomyces species for the production of secondary metabolites. Biotechnol Adv 32(2):255–268. doi:10.1016/J.Biotechadv.2013.10.008

    Article  CAS  PubMed  Google Scholar 

  4. Blin K, Medema MH, Kazempour D et al (2013) antiSMASH 2.0-a versatile platform for genome mining of secondary metabolite producers. Nucleic Acids Res 41(W1):W204–W212. doi:10.1093/nar/gkt449

    Article  PubMed  PubMed Central  Google Scholar 

  5. Weber T, Blin K, Duddela S et al (2015) antiSMASH 3.0-a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res 43(W1):W237–W243. doi:10.1093/nar/gkv437

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Weber T, Kim HU (2016) The secondary metabolite bioinformatics portal: computational tools to facilitate synthetic biology of secondary metabolite production. Synthetic Syst Biotechnol 1(2):69–79

    Article  Google Scholar 

  7. Ziemert N, Alanjary M, Weber T (2016) The evolution of genome mining in microbes – a review. Nat Prod Rep 33(8):988–1005. doi:10.1039/c6np00025h

    Article  CAS  PubMed  Google Scholar 

  8. Weber T, Charusanti P, Musiol-Kroll EM et al (2015) Metabolic engineering of antibiotic factories: new tools for antibiotic production in actinomycetes. Trends Biotechnol 33(1):15–26. doi:10.1016/j.tibtech.2014.10.009

    Article  CAS  PubMed  Google Scholar 

  9. Deveau H, Garneau JE, Moineau S (2010) CRISPR/Cas system and its role in phage-bacteria interactions. Annu Rev Microbiol 64:475–493. doi:10.1146/annurev.micro.112408.134123

    Article  CAS  PubMed  Google Scholar 

  10. Koonin EV, Makarova KS (2013) CRISPR-Cas: evolution of an RNA-based adaptive immunity system in prokaryotes. RNA Biol 10(5):679–686. doi:10.4161/rna.24022

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Barrangou R, Marraffini LA (2014) CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity. Mol Cell 54(2):234–244. doi:10.1016/j.molcel.2014.03.011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Abudayyeh OO, Gootenberg JS, Konermann S et al (2016) C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353(6299):aaf5573. doi:10.1126/science.aaf5573

    Article  PubMed  PubMed Central  Google Scholar 

  13. Makarova KS, Wolf YI, Alkhnbashi OS et al (2015) An updated evolutionary classification of CRISPR-Cas systems. Nat Rev Microbiol 13(11):722–736. doi:10.1038/nrmicro3569

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Barrangou R, Fremaux C, Deveau H et al (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315(5819):1709–1712. doi:10.1126/science.1138140

    Article  CAS  PubMed  Google Scholar 

  15. Bhaya D, Davison M, Barrangou R (2011) CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu Rev Genet 45:273–297. doi:10.1146/annurev-genet-110410-132430

    Article  CAS  PubMed  Google Scholar 

  16. Horvath P, Barrangou R (2010) CRISPR/Cas, the immune system of bacteria and archaea. Science 327(5962):167–170. doi:10.1126/science.1179555

    Article  CAS  PubMed  Google Scholar 

  17. Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821. doi:10.1126/science.1225829

    Article  CAS  PubMed  Google Scholar 

  18. Deltcheva E, Chylinski K, Sharma CM et al (2011) CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471(7340):602–607. doi:10.1038/Nature09886

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Nishimasu H, Ran FA, Hsu PD et al (2014) Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156(5):935–949. doi:10.1016/j.cell.2014.02.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Sternberg SH, Redding S, Jinek M et al (2014) DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507(7490):62–67. doi:10.1038/Nature13011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Iliakis G, Wang H, Perrault AR et al (2004) Mechanisms of DNA double strand break repair and chromosome aberration formation. Cytogenet Genome Res 104(1–4):14–20. doi:10.1159/000077461

    Article  CAS  PubMed  Google Scholar 

  22. Kanaar R, Hoeijmakers JH, van Gent DC (1998) Molecular mechanisms of DNA double strand break repair. Trends Cell Biol 8(12):483–489

    Article  CAS  PubMed  Google Scholar 

  23. Bassett AR, Tibbit C, Ponting CP et al (2013) Highly efficient targeted mutagenesis of drosophila with the CRISPR/Cas9 system. Cell Rep 4(1):220–228. doi:10.1016/J.Celrep.2013.06.020

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. DiCarlo JE, Norville JE, Mali P et al (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res 41(7):4336–4343

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Friedland AE, Tzur YB, Esvelt KM et al (2013) Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat Methods 10(8):741–743. doi:10.1038/Nmeth.2532

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Li DL, Qiu ZW, Shao YJ et al (2013) Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat Biotechnol 31(8):681–683. doi:10.1038/Nbt.2661

    Article  CAS  PubMed  Google Scholar 

  27. Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339(6121):823–826. doi:10.1126/science.1232033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Ronda C, Pedersen LE, Hansen HG et al (2014) Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy, a web-based target finding tool. Biotechnol Bioeng 111(8):1604–1616. doi:10.1002/Bit.25233

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Wang HY, Yang H, Shivalila CS et al (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153(4):910–918. doi:10.1016/J.Cell.2013.04.025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Xie KB, Yang YN (2013) RNA-guided genome editing in plants using a CRISPRCas system. Mol Plant 6(6):1975–1983. doi:10.1093/Mp/Sst119

    Article  CAS  PubMed  Google Scholar 

  31. Yang DS, Xu J, Zhu TQ et al (2014) Effective gene targeting in rabbits using RNA-guided Cas9 nucleases. J Mol Cell Biol 6(1):97–99. doi:10.1093/Jmcb/Mjt047

    Article  PubMed  PubMed Central  Google Scholar 

  32. Lieber MR (1999) The biochemistry and biological significance of nonhomologous DNA end joining: an essential repair process in multicellular eukaryotes. Genes Cells 4(2):77–85

    Article  CAS  PubMed  Google Scholar 

  33. Deriano L, Roth DB (2013) Modernizing the nonhomologous end-joining repertoire: alternative and classical NHEJ share the stage. Annu Rev Genet 47:433–455. doi:10.1146/annurev-genet-110711-155540

    Article  CAS  PubMed  Google Scholar 

  34. Aravind L, Koonin EV (2001) Prokaryotic homologs of the eukaryotic DNA-end-binding protein Ku, novel domains in the Ku protein and prediction of a prokaryotic double-strand break repair system. Genome Res 11(8):1365–1374. doi:10.1101/gr.181001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Bowater R, Doherty AJ (2006) Making ends meet: repairing breaks in bacterial DNA by non-homologous end-joining. PLoS Genet 2(2):e8. doi:10.1371/journal.pgen.0020008

    Article  PubMed  PubMed Central  Google Scholar 

  36. Tong Y, Charusanti P, Zhang L et al (2015) CRISPR-Cas9 based engineering of actinomycetal genomes. ACS Synth Biol 4(9):1020–1029. doi:10.1021/acssynbio.5b00038

    Article  CAS  PubMed  Google Scholar 

  37. Tong Y, Weber, T, Lee, SY (2015) CRISPR/Cas9-based system. EU Patent EP15160126.7, 25 Mar 2015

    Google Scholar 

  38. Qi LS, Larson MH, Gilbert LA et al (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152(5):1173–1183. doi:10.1016/J.Cell.2013.02.022

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Czar MJ, Anderson JC, Bader JS et al (2009) Gene synthesis demystified. Trends Biotechnol 27(2):63–72. doi:10.1016/j.tibtech.2008.10.007

    Article  CAS  PubMed  Google Scholar 

  40. Nour-Eldin HH, Hansen BG, Norholm MHH et al (2006) Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments. Nucleic Acids Res 34(18):e122. doi:10.1093/nar/gkl635

    Article  PubMed  PubMed Central  Google Scholar 

  41. Bitinaite J, Rubino M, Varma KH et al (2007) USER (TM) friendly DNA engineering and cloning method by uracil excision. Nucleic Acids Res 35(6):1992–2002. doi:10.1093/nar/gkm041

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Genee HJ, Bonde MT, Bagger FO et al (2015) Software-supported USER cloning strategies for site-directed mutagenesis and DNA assembly. ACS Synth Biol 4(3):342–349. doi:10.1021/sb500194z

    Article  CAS  PubMed  Google Scholar 

  43. Norholm MHH (2010) A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnol 10:21. doi:10.1186/1472-6750-10-21

    Article  PubMed  PubMed Central  Google Scholar 

  44. Salomonsen BMUH, Halkier BA (2014) USER-derived cloning methods and their primer design. In: SLR V (ed) DNA cloning and assembly methods. Methods in molecular biology, vol 1116. Springer, New York

    Google Scholar 

  45. Cavaleiro AM, Kim SH, Seppala S et al (2015) Accurate DNA assembly and genome engineering with optimized uracil excision cloning. ACS Synth Biol 4(9):1042–1046. doi:10.1021/acssynbio.5b00113

    Article  CAS  PubMed  Google Scholar 

  46. Cobb RE, Wang Y, Zhao H (2014) High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system. ACS Synth Biol. doi:10.1021/sb500351f

  47. Huang H, Zheng GS, Jiang WH et al (2015) One-step high-efficiency CRISPR/Cas9-mediated genome editing in Streptomyces. Acta Biochim Biophys Sin (Shanghai) 47(4). doi:10.1093/abbs/gmv007

  48. Zeng H, Wen SS, Xu W et al (2015) 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(24):10575–10585. doi:10.1007/s00253-015-6931-4

    Article  CAS  PubMed  Google Scholar 

  49. Kieser T, Bibb M, Buttner M et al (2000) Practical Streptomyces genetics. Norwich, UK, The John Innes Foundation

    Google Scholar 

  50. Muth G, Nussbaumer B, Wohlleben W et al (1989) A vector system with temperature-sensitive replication for gene disruption and mutational cloning in streptomycetes. Mol Gen Genet 219(3):341–348. doi:10.1007/Bf00259605

    Article  CAS  Google Scholar 

  51. Xie SS, Shen B, Zhang CB et al (2014) sgRNAcas9: a software package for designing CRISPR sgRNA and evaluating potential off-target cleavage sites. PLoS One 9(6). doi:10.1371/journal.pone.0100448

  52. Blin K, Pedersen LE, Weber T et al (2016) CRISPy-web: an online resource to design sgRNAs for CRISPR applications. Synthetic Syst Biotechnol 1(2):4

    Google Scholar 

  53. Smithies O (2001) Forty years with homologous recombination. Nat Med 7(10):1083–1086. doi:10.1038/nm1001-1083

    Article  CAS  PubMed  Google Scholar 

  54. Nour-Eldin HH, Geu-Flores F, Halkier BA (2010) USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories. In: AG F-N (ed) Plant secondary metabolism engineering. Methods in molecular biology, vol 643. Springer, New York

    Google Scholar 

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Acknowledgments

This work was funded by grants from the Novo Nordisk Foundation to S.Y.L. and T.W. (NNF15OC0016226). The authors thank Günther Muth from the University of Tübingen for providing the pGM1190 plasmid.

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Authors and Affiliations

  1. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs. Lyngby, 2800, Denmark

    Yaojun Tong, Helene Lunde Robertsen, Kai Blin, 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, BioInformatics Research Center, and BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 305-701, Republic of Korea

    Sang Yup Lee

Authors
  1. Yaojun Tong
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  2. Helene Lunde Robertsen
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  3. Kai Blin
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  4. Tilmann Weber
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  5. Sang Yup Lee
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Corresponding authors

Correspondence to Tilmann Weber or Sang Yup Lee .

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Editors and Affiliations

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

    Michael Krogh Jensen

  2. Joint BioEnergy Institute, Emeryville, California, USA

    Jay D. Keasling

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Tong, Y., Robertsen, H.L., Blin, K., Weber, T., Lee, S.Y. (2018). CRISPR-Cas9 Toolkit for Actinomycete Genome Editing. In: Jensen, M.K., Keasling, J.D. (eds) Synthetic Metabolic Pathways. Methods in Molecular Biology, vol 1671. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7295-1_11

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  • DOI: https://doi.org/10.1007/978-1-4939-7295-1_11

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7294-4

  • Online ISBN: 978-1-4939-7295-1

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