Skip to main content
SpringerLink
Log in

Genome Editing of Corynebacterium glutamicum Using CRISPR-Cpf1 System

  • Protocol
  • First Online:
Recombineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2479))

Abstract

Corynebacterium glutamicum, as an important microbial chassis, has great potential in industrial application. However, complicated genetic modification is severely slowed by lack of efficient genome editing tools. The Streptococcus pyogenes (Sp) CRISPR-Cas9 system has been verified as a very powerful tool for mediating genome alteration in many microorganisms but cannot work well in C. glutamicum. We recently developed two Francisella novicida (Fn) CRISPR-Cpf1 assisted systems for genome editing via homologous recombination in C. glutamicum. Here, we describe the protocols and demonstrated that N iterative rounds of genome editing can be achieved in 3 N + 4 or 3 N + 2 days, respectively.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Lee J-Y, Na Y-A, Kim E, Lee H-S, Kim P (2016) The Actinobacterium Corynebacterium glutamicum, an industrial workhorse. J Microbiol Biotechnol 26(5):807–822. https://doi.org/10.4014/jmb.1601.01053

    Article  CAS  PubMed  Google Scholar 

  2. Wendisch VF, Jorge JMP, Perez-Garcia F, Sgobba E (2016) Updates on industrial production of amino acids using Corynebacterium glutamicum. World J Microbiol Biotechnol 32(6):105. https://doi.org/10.1007/s11274-016-2060-1

    Article  CAS  PubMed  Google Scholar 

  3. Heider SAE, Wendisch VF (2015) Engineering microbial cell factories: metabolic engineering of Corynebacterium glutamicum with a focus on non-natural products. Biotechnol J 10(8):1170–1184. https://doi.org/10.1002/biot.201400590

    Article  CAS  PubMed  Google Scholar 

  4. Eggeling L, Bott M (2015) A giant market and a powerful metabolism: L-lysine provided by Corynebacterium glutamicum. Appl Microbiol Biotechnol 99(8):3387–3394. https://doi.org/10.1007/s00253-015-6508-2

    Article  CAS  PubMed  Google Scholar 

  5. Tan Y, Xu D, Li Y, Wang X (2012) Construction of a novel sacB-based system for marker-free gene deletion in Corynebacterium glutamicum. Plasmid 67(1):44–52. https://doi.org/10.1016/j.plasmid.2011.11.001

    Article  CAS  PubMed  Google Scholar 

  6. Schwarzer A, Puhler A (1991) Manipulation of Corynebacterium glutamicum by gene disruption and replacement. Bio/Technology 9(1):84–87. https://doi.org/10.1038/nbt0191-84

    Article  CAS  Google Scholar 

  7. Schafer A, Tauch A, Jager W, Kalinowski J, Thierbach G, Puhler A (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145(1):69–73. https://doi.org/10.1016/0378-1119(94)90324-7

    Article  CAS  PubMed  Google Scholar 

  8. Okibe N, Suzuki N, Inui M, Yukawa H (2011) Efficient markerless gene replacement in Corynebacterium glutamicum using a new temperature-sensitive plasmid. J Microbiol Methods 85(2):155–163. https://doi.org/10.1016/j.mimet.2011.02.012

    Article  CAS  PubMed  Google Scholar 

  9. Nesvera J, Patek M (2011) Tools for genetic manipulations in Corynebacterium glutamicum and their applications. Appl Microbiol Biotechnol 90(5):1641–1654. https://doi.org/10.1007/s00253-011-3272-9

    Article  CAS  PubMed  Google Scholar 

  10. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152(5):1173–1183. https://doi.org/10.1016/j.cell.2013.02.022

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339(6121):823–826. https://doi.org/10.1126/science.1232033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31(3):233–239. https://doi.org/10.1038/nbt.2508

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Doudna JA, Charpentier E (2014) The new frontier of genome engineering with CRISPR-Cas9. Science 346(6213):1077. https://doi.org/10.1126/science.1258096

    Article  CAS  Google Scholar 

  14. Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA (2013) Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res 41(15):7429–7437. https://doi.org/10.1093/nar/gkt520

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Jiang Y, Qian F, Yang J, Liu Y, Dong F, Xu C, Sun B, Chen B, Xu X, Li Y, Wang R, Yang S (2017) CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum. Nat Commun 8:15179. https://doi.org/10.1038/ncomms15179

    Article  PubMed  PubMed Central  Google Scholar 

  16. Cleto S, Jensen JVK, Wendisch VF, Lu TK (2016) Corynebacterium glutamicum metabolic engineering with CRISPR Interference (CRISPRi). ACS Synth Biol 5(5):375–385. https://doi.org/10.1021/acssynbio.5b00216

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F (2015) Cpf1 is a single RNA-guided endonuclease of a Class 2 CRISPR-Cas system. Cell 163(3):759–771. https://doi.org/10.1016/j.cell.2015.09.038

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Wang H, La Russa M, Qi LS (2016) CRISPR/Cas9 in genome editing and beyond. Annu Rev Biochem 85:227–264. https://doi.org/10.1146/annurev-biochem-060815-014607

    Article  CAS  PubMed  Google Scholar 

  19. Zetsche B, Heidenreich M, Mohanraju P, Fedorova I, Kneppers J, DeGennaro EM, Winblad N, Choudhury SR, Abudayyeh OO, Gootenberg JS, Wu WY, Scott DA, Severinov K, van der Oost J, Zhang F (2017) Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. Nat Biotechnol 35(1):31–34. https://doi.org/10.1038/nbt.3737

    Article  CAS  PubMed  Google Scholar 

  20. Wang B, Hu Q, Zhang Y, Shi R, Chai X, Liu Z, Shang X, Zhang Y, Wen T (2018) A RecET-assisted CRISPR-Cas9 genome editing in Corynebacterium glutamicum. Microb Cell Factories 17(1):63. https://doi.org/10.1186/s12934-018-0910-2

    Article  CAS  Google Scholar 

  21. Niu T-C, Lin G-M, Xie L-R, Wang Z-Q, Xing W-Y, Zhang J-Y, Zhang C-C (2019) Expanding the potential of CRISPR-Cpf1-based genome editing technology in the Cyanobacterium Anabaena PCC 7120. ACS Synth Biol 8(1):170–180. https://doi.org/10.1021/acssynbio.8b00437

    Article  CAS  PubMed  Google Scholar 

  22. Zhang J, Yang F, Yang Y, Jiang Y, Huo Y-X (2019) Optimizing a CRISPR-Cpf1-based genome engineering system for Corynebacterium glutamicum. Microb Cell Factories 18(1):60. https://doi.org/10.1186/s12934-019-1109-x

    Article  Google Scholar 

  23. Becker J, Zelder O, Haefner S, Schroeder H, Wittmann C (2011) From zero to hero-design-based systems metabolic engineering of Corynebacterium glutamicum for L-lysine production. Metab Eng 13(2):159–168. https://doi.org/10.1016/j.ymben.2011.01.003

    Article  CAS  PubMed  Google Scholar 

  24. Gao L, Cox DBT, Yan WX, Manteiga JC, Schneider MW, Yamano T, Nishimasu H, Nureki O, Crosetto N, Zhang F (2017) Engineered Cpf1 variants with altered PAM specificities. Nat Biotechnol 35(8):789–792. https://doi.org/10.1038/nbt.3900

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Li Q, Chen J, Minton NP, Zhang Y, Wen Z, Liu J, Yang H, Zeng Z, Ren X, Yang J, Gu Y, Jiang W, Jiang Y, Yang S (2016) CRISPR-based genome editing and expression control systems in Clostridium acetobutylicum and Clostridium beijerinckii. Biotechnol J 11(7):961–972. https://doi.org/10.1002/biot.201600053

    Article  CAS  PubMed  Google Scholar 

  26. Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA III, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5):343–U341. https://doi.org/10.1038/nmeth.1318

    Article  CAS  PubMed  Google Scholar 

  27. Nakamura J, Kanno S, Kimura E, Matsui K, Nakamatsu T, Wachi M (2006) Temperature-sensitive cloning vector for Corynebacterium glutamicum. Plasmid 56(3):179–186. https://doi.org/10.1016/j.plasmid.2006.05.003

    Article  CAS  PubMed  Google Scholar 

  28. Chen T, Zhu N, Xia H (2014) Aerobic production of succinate from arabinose by metabolically engineered Corynebacterium glutamicum. Bioresour Technol 151:411–414. https://doi.org/10.1016/j.biortech.2013.10.017

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work is supported by grants from the National Natural Science Foundation of China (21825804, 31670094, 31971343, and 21706133).

Author information

Authors and Affiliations

  1. School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing, China

    Zhiqiang Wen

  2. Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, China

    Fenghui Qian

  3. Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China

    Jiao Zhang & Sheng Yang

  4. College of Life Science, University of Chinese Academy of Sciences, Beijing, China

    Jiao Zhang

  5. Shanghai Taoyusheng Biotechnology Co., Ltd., Shanghai, China

    Yu Jiang

  6. Huzhou Center of Industrial Biotechnology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Zhejiang, China

    Sheng Yang

Authors
  1. Zhiqiang Wen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fenghui Qian
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jiao Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yu Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sheng Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sheng Yang .

Editor information

Editors and Affiliations

  1. Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, USA

    Christopher R. Reisch

Rights and permissions

Reprints and permissions

Copyright information

© 2022 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Wen, Z., Qian, F., Zhang, J., Jiang, Y., Yang, S. (2022). Genome Editing of Corynebacterium glutamicum Using CRISPR-Cpf1 System. In: Reisch, C.R. (eds) Recombineering. Methods in Molecular Biology, vol 2479. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2233-9_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2233-9_13

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2232-2

  • Online ISBN: 978-1-0716-2233-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation