Systematic investigation of CRISPR–Cas9 configurations for flexible and efficient genome editing in Corynebacterium glutamicum NRRL-B11474

  • R. Cameron Coates
  • Stephen Blaskowski
  • Shawn Szyjka
  • Harmen M. van Rossum
  • Jim Vallandingham
  • Kedar Patel
  • Zach Serber
  • Jed DeanEmail author
Biotechnology Methods - Original Paper


This study details a reliable and efficient method for CRISPR–Cas9 genome engineering in the high amino acid-producing strain of Corynebacterium glutamicum, NRRL-B11474. Our investigation demonstrates that a plasmid-encoded single-guide RNA paired with different edit-encoding fragments is sufficient to generate edits without the addition of an exogenous recombinase. This approach leverages a genome-integrated copy of the cas9 gene for reduced toxicity, in combination with a single plasmid carrying the targeting guide RNA and matching edit fragment. Our study systematically investigated the impact of homology arm length on editing efficiency and demonstrates genome editing with homology arm lengths as small as 25 bp for single-nucleotide polymorphisms and 75 bp for 100 bp sequence swaps. These homology arm lengths are smaller than previously reported for other strains of C. glutamicum. Our study finds that C. glutamicum NRRL-B11474 is not amenable to efficient transformation with plasmids containing the BL1, NG2, or CC1 origins of replication. This finding differs from all previously reported approaches to plasmid-based CRISPR–Cas9 or Cpf1 editing in other strains of C. glutamicum. Two alternative origins of replication (CG1 and CASE1) can be used to successfully introduce genome edits; furthermore, our data demonstrate improved editing efficiency when guide RNAs and edit fragments are encoded on plasmids carrying the CASE1 origin of replication (compared to plasmids carrying CG1). In addition, this study demonstrates that efficient editing can be done using an integrated Cas9 without the need for a recombinase. We demonstrate that the specifics of CRISPR–Cas9 editing configurations may need to be tailored to enable different edit types in a particular strain background. Refining configuration parameters such as edit type, homology arm length, and plasmid origin of replication enables robust, flexible, and efficient CRISPR–Cas9 editing in differing genetic strain contexts.


CRISPR–Cas9 Corynebacterium glutamicum Genetic engineering Genome editing 



This study was partly supported by the DARPA Living Foundries initiative. The authors would like to thank Dr. Brian Chaikind, Dr. Carrie Cizauskas, Michael Flashman, Dr. Mike Hamady, Dr. Sharon Hoover, Dr. Stefan de Kok, Michael Martyn III, Dr. Aaron Miller, and Dr. Solomon Stonebloom for their assistance with the project.

Supplementary material

10295_2018_2112_MOESM1_ESM.eps (36 kb)
Supplementary material 1 Transformation efficiency for 100 bp sequence swaps (S1A) and SNPs (S1B) is higher for control plasmids and editing constructs transformed into the cas9-integrated strain of NRRL-B11474 C. glutamicum (blue points) as compared to the wild-type reference strain (black points). Data points are jittered. Transformation efficiency is broken down by target locus, plasmid origin of replication, and edit fragment homology arm length. The gRNA control plasmids contain a gRNA targeted to the respective test locus, but no edit fragment. Each point represents data from a unique transformation event (EPS 35 kb)
10295_2018_2112_MOESM2_ESM.eps (32 kb)
Supplementary material 2 (EPS 32 kb)
10295_2018_2112_MOESM3_ESM.eps (12 kb)
Supplementary material 3 Differential editing efficiency with CASE1 and CG1 plasmid origins of replication at various edit fragment homology arm lengths. We analyzed combined editing efficiency data to examine the effect of plasmid origin of replication on percent colonies edited. Data is broken out by 100 bp sequence swaps (top panel) and SNPs (bottom panel), by edit fragment homology arm length, and by plasmid origin of replication. Error bars represent 95% confidence intervals around the mean. At every homology arm length except 2000 bp for sequence swaps, the mean editing efficiency resulting from plasmids containing the CASE1 origin of replication was greater than or equal to the mean editing efficiency of CG1-containing plasmids. A two-tailed t test revealed significant differences in editing efficiency for sequence swaps at 500 bp homology arm lengths (CASE1: M = 91.8%, SD= 10.1%; CG1: M = 46.2%, SD= 18.8%; t(7.68) = − 5.24, p = 0.0004), and at 125 bp homology arm lengths (CASE1: M = 27.6%, SD= 22.6%; CG1: M = 0.0%, SD= 0.0%; t(4) = − 2.74, p = 0.0261) (EPS 11 kb)
10295_2018_2112_MOESM4_ESM.eps (63 kb)
Supplementary material 4 Coverage plots showing representative analysis of NGS-sequencing data for a successful 100 bp sequence swap edit at the cg3031 test locus (S3A), an unsuccessful SNP edit at the cg0167 test locus (S3B), and a partial SNP edit at the cg3404 test locus (S3C). In every case, the same library of sequencing reads is aligned to a WT reference sequence (bottom alignments) and an edited reference sequence (top alignments). The coverage plots displayed are truncated segments of the full alignments, zoomed-in on the region of interest. Figure S3A displays a successful edit in which the edit alignment shows no mismatches or gaps in coverage depth, while the WT alignment shows a gap in coverage, where the sample’s parental WT sequence has been replaced by the 100 bp edit sequence. S3B displays an unsuccessfully edited SNP, where the WT alignment shows no mismatches or gaps in coverage depth, and the edit alignment shows two mismatches, where the intended SNPs failed to be incorporated and WT sequence remains. Figure S3C displays a partial edit, where the edit alignment is correct at the site, where the first SNP was incorporated, but there is a mismatch at the site, where the second SNP failed to be incorporated, and the wild-type alignment inversely shows a mismatch at the site of the first successful SNP and a correct alignment at the site of the second unsuccessful SNP (EPS 62 kb)
10295_2018_2112_MOESM5_ESM.eps (53 kb)
Supplementary material 5 (EPS 53 kb)
10295_2018_2112_MOESM6_ESM.eps (53 kb)
Supplementary material 6 (EPS 53 kb)
10295_2018_2112_MOESM7_ESM.xlsx (13 kb)
Supplementary material 7 (XLSX 12 kb)
10295_2018_2112_MOESM8_ESM.xlsx (12 kb)
Supplementary material 8 (XLSX 12 kb)


  1. 1.
    Archer JA, Sinskey AJ (1993) The DNA sequence and minimal replicon of the Corynebacterium glutamicum plasmid pSR1: evidence of a common ancestry with plasmids from C. diphtheriae. J Gen Microbiol 139(8):1753–1759. CrossRefGoogle Scholar
  2. 2.
    Becker J, Rohles CM, Wittmann C (2018) Metabolically engineered Corynebacterium glutamicum for bio-based production of chemicals, fuels, materials, and healthcare products. Metab Eng. (In-Press) Google Scholar
  3. 3.
    Binder S, Siedler S, Marienhagen J, Bott M, Eggeling L (2013) Recombineering in Corynebacterium glutamicum combined with optical nanosensors: a general strategy for fast producer strain generation. Nucleic Acids Res 41(12):6360–6369. CrossRefGoogle Scholar
  4. 4.
    Chen W, Zhang Y, Zhang Y, Pi Y, Gu T, Song L, Wang Y, Ji Q (2018) CRISPR/Cas9-based genome editing in Pseudomonas aeruginosa and cytidine deaminase-mediated base editing in pseudomonas species. IScience 6:222–231. CrossRefGoogle Scholar
  5. 5.
    Cho JS, Choi KR, Prabowo CPS, Shin JH, Yang D, Jang J, Lee SY (2017) CRISPR/Cas9-coupled recombineering for metabolic engineering of Corynebacterium glutamicum. Metab Eng 42:157–167. CrossRefGoogle Scholar
  6. 6.
    Cobb RE, Wang Y, Zhao H (2015) High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system. ACS Synth Biol 4(6):723–728. CrossRefGoogle Scholar
  7. 7.
    Cui L, Vigouroux A, Rousset F, Varet H, Khanna V, Bikard D (2018) A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9. Nat Commun 9(1):1912. CrossRefGoogle Scholar
  8. 8.
    Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97(12):6640–6645. CrossRefGoogle Scholar
  9. 9.
    Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I, Sullender M, Ebert BL, Xavier RJ, Root DE (2014) Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation. Nat Biotechnol 32(12):1262–1267. CrossRefGoogle Scholar
  10. 10.
    Entian K-D, Kötter P (2007) 25 yeast genetic strain and plasmid collections. Methods Microbiol 36:629–666. CrossRefGoogle Scholar
  11. 11.
    Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5):343–345. CrossRefGoogle Scholar
  12. 12.
    Griffith KL, Grossman AD (2008) Inducible protein degradation in Bacillus subtilis using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP. Mol Microbiol 70(4):1012–1025. Google Scholar
  13. 13.
    Hockemeyer D, Wang H, Kiani S, Lai CS, Gao Q, Cassady JP, Cost GJ, Zhang L, Santiago Y, Miller JC, Zeitler B, Cherone JM, Meng X (2011) Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol 29(8):731–734. CrossRefGoogle Scholar
  14. 14.
    Jia H, Zhang L, Wang T, Han J, Tang H, Zhang L (2017) Development of a CRISPR/Cas9-mediated gene-editing tool in Streptomyces rimosus. Microbiology 163(8):1148–1155CrossRefGoogle Scholar
  15. 15.
    Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S (2015) Multigene editing in the <span class = "named-content genus-species" id = "named-content-1">Escherichia coli</span> genome via the CRISPR-Cas9 system. Appl Environ Microbiol 81(7):2506–2514CrossRefGoogle Scholar
  16. 16.
    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(May):1–11. Google Scholar
  17. 17.
    Karberg M, Guo H, Zhong J, Coon R, Perutka J, Lambowitz AM (2001) Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria. Nat Biotechnol 19(12):1162–1167. CrossRefGoogle Scholar
  18. 18.
    Kilby NJ, Snaith MR, Murray JA (1993) Site-specific recombinases: tools for genome engineering. Trends Genet 9(12):413–421. CrossRefGoogle Scholar
  19. 19.
    Kuhlman TE, Cox EC (2010) Site-specific chromosomal integration of large synthetic constructs. Nucleic Acids Res 38(6):e92–e92. CrossRefGoogle Scholar
  20. 20.
    Leenay RT, Vento JM, Shah M, Martino ME, Leulier F, Beisel CL (2018) Genome editing with CRISPR-Cas9 in Lactobacillus plantarum revealed that editing outcomes can vary across strains and between methods. Biotechnol J. Google Scholar
  21. 21.
    Li H, Durbin R (2009) Fast and accurate short read alignment with burrows-wheeler transform. Bioinformatics 25(14):1754–1760CrossRefGoogle Scholar
  22. 22.
    Li K, Cai D, Wang Z, He Z, Chen S (2018) Development of an efficient genome editing tool in Bacillus licheniformis using CRISPR-Cas9 nickase. Appl Environ Microbiol 84(6):e02608–17-e02608-17. CrossRefGoogle Scholar
  23. 23.
    Li M, Niu T, Zhang J, Su Z, Kong D (2010) High electroporation efficiency of Corynebacterium glutamicum with xenogeneic plasmid DNA. 2010 4th International Conference on Bioinformatics and Biomedical Engineering, pp. 1–4Google Scholar
  24. 24.
    Li T, Huang S, Zhao X, Wright DA, Carpenter S, Spalding MH, Weeks DP, Yang B (2011) Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Nucleic Acids Res 39(14):6315–6325. CrossRefGoogle Scholar
  25. 25.
    Li Y, Lin Z, Huang C, Zhang Y, Wang Z, Tang Y, Chen T, Zhao X (2015) Metabolic engineering of Escherichia coli using CRISPR–Cas9 meditated genome editing. Metab Eng 31:13–21. CrossRefGoogle Scholar
  26. 26.
    Liu J, Wang Y, Lu Y, Zheng P, Sun J, Ma Y (2017) Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum. Microb Cell Fact 16(1):205. CrossRefGoogle Scholar
  27. 27.
    Ma H, Kunes S, Schatz PJ, Botstein D (1987) Plasmid construction by homologous recombination in yeast. Gene 58(2–3):201–216. CrossRefGoogle Scholar
  28. 28.
    Mans R, van Rossum HM, Wijsman M, Backx A, Kuijpers NGA, van den Broek M, Daran-Lapujade P, Pronk JT, van Maris AJA, Daran JMG (2015) CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae. FEMS Yeast Res. Google Scholar
  29. 29.
    Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ, Dulay GP, Hua KL, Ankoudinova I, Cost GJ, Urnov FD, Zhang HS, Holmes MC, Zhang L, Gregory PD, Rebar EJ (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29(2):143–148. CrossRefGoogle Scholar
  30. 30.
    Mosberg JA, Gregg CJ, Lajoie MJ, Wang HH, Church GM (2012) Improving lambda red genome engineering in Escherichia coli via rational removal of endogenous nucleases. PLoS One 7(9):e44638–e44638. CrossRefGoogle Scholar
  31. 31.
    Nakamura Y, Nishio Y, Ikeo K, Gojobori T (2003) The genome stability in Corynebacterium species due to lack of the recombinational repair system. Gene 317(1–2):149–155. CrossRefGoogle Scholar
  32. 32.
    Oh J-H, van Pijkeren J-P (2014) CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri. Nucleic Acids Res 42(17):e131. CrossRefGoogle Scholar
  33. 33.
    Patek M, Nesvera J (2013) Promoters and plasmid vectors of Corynebacterium glutamicum. In: Yukawa H, Inui M (eds) Microbiology Monographs, vol 23. Springer, BerlinGoogle Scholar
  34. 34.
    Peng F, Wang X, Sun Y, Dong G, Yang Y, Liu X, Bai Z (2017) Efficient gene editing in Corynebacterium glutamicum using the CRISPR/Cas9 system. Microb Cell Fact 16(1):1–13. CrossRefGoogle Scholar
  35. 35.
    Rayapati PJ, Crafton CM (1999) Metabolic engineering of amino acid production, 1999Google Scholar
  36. 36.
    Reisch CR, Prather KLJ (2015) The no-SCAR (scarless Cas9 assisted recombineering) system for genome editing in Escherichia coli. Sci Rep 5:15096CrossRefGoogle Scholar
  37. 37.
    Resende BC, Rebelato AB, D’Afonseca V, Santos AR, Stutzman T, Azevedo VA, Santos LL, Miyoshi A, Lopes DO (2011) DNA repair in Corynebacterium model. Gene 482(1–2):1–7. CrossRefGoogle Scholar
  38. 38.
    Sternberg N, Hamilton D, Austin S, Yarmolinsky M, Hoess R (1981) Site-specific recombination and its role in the life cycle of bacteriophage P1. Cold Spring Harb Symp Quant Biol 45:297–309. CrossRefGoogle Scholar
  39. 39.
    Tao W, Yang A, Deng Z, Sun Y (2018) CRISPR/Cas9-based editing of Streptomyces for discovery, characterization, and production of natural products. Front Microbiol 9:1660. CrossRefGoogle Scholar
  40. 40.
    Tong Y, Charusanti P, Zhang L, Weber T, Lee SY (2015) CRISPR-Cas9 based engineering of actinomycetal genomes. ACS Synth Biol 4(9):1020–1029. CrossRefGoogle Scholar
  41. 41.
    Tsuchida Y, Kimura S, Suzuki N, Inui M, Yukawa H (2010) Characterization of a 24-kb plasmid pCGR2 newly isolated from Corynebacterium glutamicum. Appl Microbiol Biotechnol 87(5):1855–1866. CrossRefGoogle Scholar
  42. 42.
    Urnov FD, Miller JC, Lee Y-L, Beausejour CM, Rock JM, Augustus S, Jamieson AC, Porteus MH, Gregory PD, Holmes MC (2005) Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435(7042):646–651. CrossRefGoogle Scholar
  43. 43.
    Venkova-Canova T, Patek M, Nesvera J (2004) Characterization of the cryptic plasmid pCC1 from Corynebacterium callunae and its use for vector construction. Plasmid 51(1):54–60. CrossRefGoogle Scholar
  44. 44.
    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 Fact 17(1):63. CrossRefGoogle Scholar
  45. 45.
    Wang HH, Isaacs FJ, Carr PA, Sun ZZ, Xu G, Forest CR, Church GM (2009) Programming cells by multiplex genome engineering and accelerated evolution. Nature 460:894CrossRefGoogle Scholar
  46. 46.
    Wu X, Kriz A, Sharp P (2014) Target specificity of the CRISPR-Cas9 system. Quant Biol 2(2):59–70. CrossRefGoogle Scholar
  47. 47.
    Yao J, Lambowitz AM (2007) Gene targeting in gram-negative bacteria by use of a mobile group II Intron (“Targetron”) expressed from a broad-host-range vector. Appl Environ Microbiol 73(8):2735–2743. CrossRefGoogle Scholar
  48. 48.
    Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci USA 97(11):5978–5983. CrossRefGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2018

Authors and Affiliations

  • R. Cameron Coates
    • 1
  • Stephen Blaskowski
    • 1
  • Shawn Szyjka
    • 1
  • Harmen M. van Rossum
    • 1
  • Jim Vallandingham
    • 1
  • Kedar Patel
    • 1
  • Zach Serber
    • 1
  • Jed Dean
    • 1
    Email author
  1. 1.Zymergen Inc.EmeryvilleUSA

Personalised recommendations