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Barriers to genome editing with CRISPR in bacteria

  • Justin M. Vento
  • Nathan Crook
  • Chase L. BeiselEmail author
Metabolic Engineering and Synthetic Biology - Review

Abstract

Genome editing is essential for probing genotype–phenotype relationships and for enhancing chemical production and phenotypic robustness in industrial bacteria. Currently, the most popular tools for genome editing couple recombineering with DNA cleavage by the CRISPR nuclease Cas9 from Streptococcus pyogenes. Although successful in some model strains, CRISPR-based genome editing has been slow to extend to the multitude of industrially relevant bacteria. In this review, we analyze existing barriers to implementing CRISPR-based editing across diverse bacterial species. We first compare the efficacy of current CRISPR-based editing strategies. Next, we discuss alternatives when the S. pyogenes Cas9 does not yield colonies. Finally, we describe different ways bacteria can evade editing and how elucidating these failure modes can improve CRISPR-based genome editing across strains. Together, this review highlights existing obstacles to CRISPR-based editing in bacteria and offers guidelines to help achieve and enhance editing in a wider range of bacterial species, including non-model strains.

Keywords

Bacteria Nuclease Genome editing CRISPR Recombineering 

Notes

Acknowledgements

This work was supported by the National Science Foundation (MCB-1452902 to CLB), the National Institutes of Health (5T32GM008776 to JMV) and start-up funds from NCSU (to NCC). We also gratefully acknowledge Jie Sun for assistance with figure preparation.

References

  1. 1.
    Altenbuchner J (2016) Editing of the Bacillus subtilis genome by the CRISPR-Cas9 System. Appl Environ Microbiol 82:5421–5427Google Scholar
  2. 2.
    Aparicio T, de Lorenzo V, Martínez-García E (2018) CRISPR/Cas9-based counterselection boosts recombineering efficiency in Pseudomonas putida. Biotechnol J 13:e1700161Google Scholar
  3. 3.
    Banno S, Nishida K, Arazoe T et al (2018) Deaminase-mediated multiplex genome editing in Escherichia coli. Nat Microbiol 3:423–429Google Scholar
  4. 4.
    Bassalo MC, Garst AD, Halweg-Edwards AL et al (2016) Rapid and efficient one-step metabolic pathway integration in E. coli. ACS Synth Biol 5:561–568Google Scholar
  5. 5.
    Börner RA, Kandasamy V, Axelsen AM et al (2019) Genome editing of lactic acid bacteria: opportunities for food, feed, pharma and biotech. FEMS Microbiol Lett 366:291.  https://doi.org/10.1093/femsle/fny291 Google Scholar
  6. 6.
    Chen W, Zhang Y, Yeo W-S et al (2017) Rapid and efficient genome editing in Staphylococcus aureus by using an engineered CRISPR/Cas9 system. J Am Chem Soc 139:3790–3795Google Scholar
  7. 7.
    Chen W, Zhang Y, Zhang Y et al (2018) CRISPR/Cas9-based genome editing in Pseudomonas aeruginosa and cytidine deaminase-mediated base editing in Pseudomonas species. IScience 6:222–231Google Scholar
  8. 8.
    Cho S, Choe D, Lee E et al (2018) High-level dCas9 expression induces abnormal cell morphology in Escherichia coli. ACS Synth Biol 7:1085–1094Google Scholar
  9. 9.
    Cobb RE, Wang Y, Zhao H (2015) High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system. ACS Synth Biol 4:723–728Google Scholar
  10. 10.
    Codner GF, Mianné J, Caulder A et al (2018) Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants. BMC Biol 16:70Google Scholar
  11. 11.
    Court DL, Sawitzke JA, Thomason LC (2002) Genetic engineering using homologous recombination. Annu Rev Genet 36:361–388Google Scholar
  12. 12.
    Cui L, Bikard D (2016) Consequences of Cas9 cleavage in the chromosome of Escherichia coli. Nucleic Acids Res 44:4243–4251Google Scholar
  13. 13.
    Cui L, Vigouroux A, Rousset F et al (2018) A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9. Nature Commun 9:1912Google Scholar
  14. 14.
    de Lorenzo V, Herrero M, Jakubzik U, Timmis KN (1990) Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol 172:6568–6572Google Scholar
  15. 15.
    Deltcheva E, Chylinski K, Sharma CM et al (2011) CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471:602–607Google Scholar
  16. 16.
    DiCarlo JE, Norville JE, Mali P et al (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res 41:4336–4343Google Scholar
  17. 17.
    Donohoue PD, Barrangou R, May AP (2018) Advances in industrial biotechnology using CRISPR-Cas systems. Trends Biotechnol 36:134–146Google Scholar
  18. 18.
    Fischer S, Maier L-K, Stoll B et al (2012) An archaeal immune system can detect multiple protospacer adjacent motifs (PAMs) to target invader DNA. J Biol Chem 287:33351–33363Google Scholar
  19. 19.
    Gasiunas G, Barrangou R, Horvath P, Siksnys V (2012) Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci USA 109:E2579–E2586Google Scholar
  20. 20.
    Gomaa AA, Klumpe HE, Luo ML et al (2014) Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems. MBio 5:e00928-13Google Scholar
  21. 21.
    Grissa I, Vergnaud G, Pourcel C (2007) The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics 8:172Google Scholar
  22. 22.
    Guo T, Xin Y, Zhang Y et al (2019) A rapid and versatile tool for genomic engineering in Lactococcus lactis. Microb Cell Fact 18:22Google Scholar
  23. 23.
    Hidalgo-Cantabrana C, Goh YJ, Barrangou R (2019) Characterization and repurposing of type I and type II CRISPR-Cas systems in bacteria. J Mol Biol 431:21–33Google Scholar
  24. 24.
    Hong W, Zhang J, Cui G et al (2018) Multiplexed CRISPR-Cpf1-mediated genome editing in Clostridium difficile toward the understanding of pathogenesis of C. difficile infection. ACS Synth Biol 7:1588–1600Google Scholar
  25. 25.
    Hu JH, Miller SM, Geurts MH et al (2018) Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556:57–63Google Scholar
  26. 26.
    Huang H, Zheng G, Jiang W et al (2015) One-step high-efficiency CRISPR/Cas9-mediated genome editing in Streptomyces. Acta Biochim Biophys Sin 47:231–243Google Scholar
  27. 27.
    Huang H, Chai C, Li N et al (2016) CRISPR/Cas9-based efficient genome editing in Clostridium ljungdahlii, an autotrophic gas-fermenting bacterium. ACS Synth Biol 5:1355–1361Google Scholar
  28. 28.
    Huang H, Song X, Yang S (2019) Development of a RecE/T-assisted CRISPR-Cas9 toolbox for Lactobacillus. Biotechnol J 2019:e1800690Google Scholar
  29. 29.
    Jiang F, Doudna JA (2017) CRISPR-Cas9 structures and mechanisms. Annu Rev Biophys 46:505–529Google Scholar
  30. 30.
    Jiang W, Bikard D, Cox D et al (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31:233–239Google Scholar
  31. 31.
    Jiang W, Zhou H, Bi H et al (2013) Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res 41:e188Google Scholar
  32. 32.
    Jiang Y, Chen B, Duan C et al (2015) Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 81:2506–2514Google Scholar
  33. 33.
    Jiang Y, Qian F, Yang J et al (2017) CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum. Nat Commun 8:15179Google Scholar
  34. 34.
    Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821Google Scholar
  35. 35.
    Kaboli S, Babazada H (2018) CRISPR mediated genome engineering and its application in industry. Curr Issues Mol Biol 26:81–92Google Scholar
  36. 36.
    Kleinstiver BP, Pattanayak V, Prew MS et al (2016) High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529:490–495Google Scholar
  37. 37.
    Komor AC, Kim YB, Packer MS et al (2016) Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533:420–424Google Scholar
  38. 38.
    Komor AC, Badran AH, Liu DR (2017) Editing the genome without double-stranded DNA breaks. ACS Chem Biol 13:383–388Google Scholar
  39. 39.
    Koonin EV, Makarova KS, Zhang F (2017) Diversity, classification and evolution of CRISPR-Cas systems. Curr Opin Microbiol 37:67–78Google Scholar
  40. 40.
    Kurtz CB, Millet YA, Puurunen MK et al (2019) An engineered E. coli Nissle improves hyperammonemia and survival in mice and shows dose-dependent exposure in healthy humans. Sci Transl Med 11:eaau7975Google Scholar
  41. 41.
    Leenay RT, Beisel CL (2017) Deciphering, communicating, and engineering the CRISPR PAM. J Mol Biol 429:177–191Google Scholar
  42. 42.
    Leenay RT, Vento JM, Shah M et al (2019) Genome editing with CRISPR-Cas9 in Lactobacillus plantarum revealed that editing outcomes can vary across strains and between methods. Biotechnol J 14:e1700583Google Scholar
  43. 43.
    Li H, Shen CR, Huang C-H et al (2016) CRISPR-Cas9 for the genome engineering of cyanobacteria and succinate production. Metab Eng 38:293–302Google Scholar
  44. 44.
    Li K, Cai D, Wang Z et al (2018) Development of an efficient genome editing tool in Bacillus licheniformis using CRISPR-Cas9 nickase. Appl Environ Microbiol 84:e02608–e02617Google Scholar
  45. 45.
    Li L, Wei K, Zheng G et al (2018) CRISPR-Cpf1-assisted multiplex genome editing and transcriptional repression in Streptomyces. Appl Environ Microbiol 84:e00827-18Google Scholar
  46. 46.
    Li Q, Chen J, Minton NP et al (2016) CRISPR-based genome editing and expression control systems in Clostridium acetobutylicum and Clostridium beijerinckii. Biotechnol J 11:961–972Google Scholar
  47. 47.
    Li Q, Seys FM, Minton NP et al (2019) CRISPR-Cas9D10A nickase-assisted base editing in solvent producer Clostridium beijerinckii. Biotechnol Bioeng 116:1475–1483Google Scholar
  48. 48.
    Li X-T, Thomason LC, Sawitzke JA et al (2013) Positive and negative selection using the tetA-sacB cassette: recombineering and P1 transduction in Escherichia coli. Nucleic Acids Res 41:e204Google Scholar
  49. 49.
    Li Y, Lin Z, Huang C et al (2015) Metabolic engineering of Escherichia coli using CRISPR-Cas9 mediated genome editing. Metab Eng 31:13–21Google Scholar
  50. 50.
    Liang L, Liu R, Garst AD et al (2017) CRISPR EnAbled trackable genome engineering for isopropanol production in Escherichia coli. Metab Eng 41:1–10Google Scholar
  51. 51.
    Lin J-L, Wagner JM, Alper HS (2017) Enabling tools for high-throughput detection of metabolites: metabolic engineering and directed evolution applications. Biotechnol Adv 35:950–970Google Scholar
  52. 52.
    Liu P, Jenkins NA, Copeland NG (2003) A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res 13:476–484Google Scholar
  53. 53.
    Luo ML, Mullis AS, Leenay RT, Beisel CL (2015) Repurposing endogenous Type I CRISPR-Cas systems for programmable gene repression. Nucleic Acids Res 43:674–681Google Scholar
  54. 54.
    Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826Google Scholar
  55. 55.
    Malzahn AA, Tang X, Lee K et al (2019) Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis. BMC Biol 17:9Google Scholar
  56. 56.
    Marshall R, Maxwell CS, Collins SP et al (2018) Rapid and scalable characterization of CRISPR technologies using an E. coli cell-free transcription-translation system. Mol Cell 69:146–157Google Scholar
  57. 57.
    McAllister KN, Bouillaut L, Kahn JN et al (2017) Using CRISPR-Cas9-mediated genome editing to generate C. difficile mutants defective in selenoproteins synthesis. Sci Rep 7:14672Google Scholar
  58. 58.
    McGovern S, Baconnais S, Roblin P et al (2016) C-terminal region of bacterial Ku controls DNA bridging, DNA threading and recruitment of DNA ligase D for double strand breaks repair. Nucleic Acids Res 44:4785–4806Google Scholar
  59. 59.
    Moreb EA, Hoover B, Yaseen A et al (2017) Managing the SOS response for enhanced CRISPR-Cas-based recombineering in E. coli through transient inhibition of host RecA activity. ACS Synth Biol 6:2209–2218Google Scholar
  60. 60.
    Mougiakos I, Bosma EF, Weenink K et al (2017) Efficient genome editing of a facultative thermophile using mesophilic spCas9. ACS Synth Biol 6:849–861Google Scholar
  61. 61.
    Murphy KC (1998) Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. J Bacteriol 180:2063–2071Google Scholar
  62. 62.
    Nihongaki Y, Otabe T, Sato M (2018) Emerging approaches for spatiotemporal control of targeted genome with inducible CRISPR-Cas9. Anal Chem 90:429–439Google Scholar
  63. 63.
    Oh J-H, van Pijkeren J-P (2014) CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri. Nucleic Acids Res 42:e131Google Scholar
  64. 64.
    Penewit K, Holmes EA, McLean K et al (2018) Efficient and scalable precision genome editing in Staphylococcus aureus through conditional recombineering and CRISPR/Cas9-mediated counterselection. MBio 9:e00067-18Google Scholar
  65. 65.
    Polstein LR, Gersbach CA (2015) A light-inducible CRISPR-Cas9 system for control of endogenous gene activation. Nat Chem Biol 11:198–200Google Scholar
  66. 66.
    Pyne ME, Bruder MR, Moo-Young M et al (2016) Harnessing heterologous and endogenous CRISPR-Cas machineries for efficient markerless genome editing in Clostridium. Sci Rep 6:25666Google Scholar
  67. 67.
    Reisch CR, Prather KLJ (2015) The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli. Sci Rep 5:15096Google Scholar
  68. 68.
    Rock JM, Hopkins FF, Chavez A et al (2017) Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform. Nat Microbiol 2:16274Google Scholar
  69. 69.
    Ronda C, Pedersen LE, Sommer MOA, Nielsen AT (2016) CRMAGE: cRISPR optimized MAGE recombineering. Sci Rep 6:19452Google Scholar
  70. 70.
    Selle K, Barrangou R (2015) Harnessing CRISPR–Cas systems for bacterial genome editing. Trends Microbiol 23:225–232Google Scholar
  71. 71.
    Selle K, Klaenhammer TR, Barrangou R (2015) CRISPR-based screening of genomic island excision events in bacteria. Proc Natl Acad Sci USA 112:8076–8081Google Scholar
  72. 72.
    Shuman S, Glickman MS (2007) Bacterial DNA repair by non-homologous end joining. Nat Rev Microbiol 5:852–861Google Scholar
  73. 73.
    Smith BT, Walker GC (1998) Mutagenesis and more: umuDC and the Escherichia coli SOS response. Genetics 148:1599–1610Google Scholar
  74. 74.
    So Y, Park S-Y, Park E-H et al (2017) A highly efficient CRISPR-Cas9-mediated large genomic deletion in Bacillus subtilis. Front Microbiol 8:1167.  https://doi.org/10.3389/fmicb.2017.01167 Google Scholar
  75. 75.
    Song X, Huang H, Xiong Z et al (2017) CRISPR-Cas9 nickase-assisted genome editing in Lactobacillus casei. Appl Environ Microbiol 83:e01259-17Google Scholar
  76. 76.
    Stachler A-E, Marchfelder A (2016) Gene repression in Haloarchaea using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas I-B system. J Biol Chem 291:15226–15242Google Scholar
  77. 77.
    Stachler A-E, Turgeman-Grott I, Shtifman-Segal E et al (2017) High tolerance to self-targeting of the genome by the endogenous CRISPR-Cas system in an archaeon. Nucleic Acids Res 45:5208–5216Google Scholar
  78. 78.
    Standage-Beier K, Zhang Q, Wang X (2015) Targeted large-scale deletion of bacterial genomes using CRISPR-nickases. ACS Synth Biol 4:1217–1225Google Scholar
  79. 79.
    Sun B, Yang J, Yang S et al (2018) A CRISPR-Cpf1-assisted non-homologous end joining genome editing system of Mycobacterium smegmatis. Biotechnol J 13:e1700588Google Scholar
  80. 80.
    Sun J, Wang Q, Jiang Y et al (2018) Genome editing and transcriptional repression in Pseudomonas putida KT2440 via the type II CRISPR system. Microb Cell Fact 17:41Google Scholar
  81. 81.
    Tapscott T, Guarnieri MT, Henard CA (2019) Development of a CRISPR/Cas9 system for Methylococcus capsulatus in vivo gene editing. Appl Environ Microbiol 85:e00340-19Google Scholar
  82. 82.
    Teng F, Cui T, Feng G et al (2018) Repurposing CRISPR-Cas12b for mammalian genome engineering. Cell Discov 4:63Google Scholar
  83. 83.
    Tong Y, Charusanti P, Zhang L et al (2015) CRISPR-Cas9 based engineering of Actinomycetal genomes. ACS Synth Biol 4:1020–1029Google Scholar
  84. 84.
    van der Els S, James JK, Kleerebezem M, Bron PA (2018) Versatile Cas9-driven subpopulation selection toolbox for Lactococcus lactis. Appl Environ Microbiol 84:e02752-17Google Scholar
  85. 85.
    Vercoe RB, Chang JT, Dy RL et al (2013) Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands. PLoS Genet 9:e1003454Google Scholar
  86. 86.
    Waller MC, Bober JR, Nair NU, Beisel CL (2017) Toward a genetic tool development pipeline for host-associated bacteria. Curr Opin Microbiol 38:156–164Google Scholar
  87. 87.
    Wang S, Dong S, Wang P et al (2017) Genome editing in Clostridium saccharoperbutylacetonicum N1-4 with the CRISPR-Cas9 system. Appl Environ Microbiol 83:e00233-17Google Scholar
  88. 88.
    Wang S, Hong W, Dong S et al (2018) Genome engineering of Clostridium difficile using the CRISPR-Cas9 system. Clin Microbiol Infect 24:1095–1099Google Scholar
  89. 89.
    Wang Y, Wang S, Chen W et al (2018) CRISPR-Cas9 and CRISPR-assisted cytidine deaminase enable precise and efficient genome editing in Klebsiella pneumoniae. Appl Environ Microbiol 84:e01834-18Google Scholar
  90. 90.
    Warming S, Costantino N, Court DL et al (2005) Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res 33:e36Google Scholar
  91. 91.
    Wasels F, Jean-Marie J, Collas F et al (2017) A two-plasmid inducible CRISPR/Cas9 genome editing tool for Clostridium acetobutylicum. J Microbiol Methods 140:5–11Google Scholar
  92. 92.
    Wirth NT, Kozaeva E, Nikel PI (2019) Accelerated genome engineering of Pseudomonas putida by I-SceI-mediated recombination and CRISPR-Cas9 counterselection. Microb Biotechnol.  https://doi.org/10.1111/1751-7915.13396 Google Scholar
  93. 93.
    Wu Y, Hao Y, Wei X et al (2017) Impairment of NADH dehydrogenase and regulation of anaerobic metabolism by the small RNA RyhB and NadE for improved biohydrogen production in Enterobacter aerogenes. Biotechnol Biofuels 10:248Google Scholar
  94. 94.
    Wu Z, Chen Z, Gao X et al (2019) Combination of ssDNA recombineering and CRISPR-Cas9 for Pseudomonas putida KT2440 genome editing. Appl Microbiol Biotechnol.  https://doi.org/10.1007/s00253-019-09654-w Google Scholar
  95. 95.
    Xin Y, Guo T, Mu Y, Kong J (2017) Identification and functional analysis of potential prophage-derived recombinases for genome editing in Lactobacillus casei. FEMS Microbiol Lett 364:fnx243Google Scholar
  96. 96.
    Xu T, Li Y, Shi Z et al (2015) Efficient genome editing in Clostridium cellulolyticum via CRISPR-Cas9 nickase. Appl Environ Microbiol 81:4423–4431Google Scholar
  97. 97.
    Yan M-Y, Yan H-Q, Ren G-X et al (2017) CRISPR-Cas12a-assisted recombineering in bacteria. Appl Environ Microbiol 83:e00947-17Google Scholar
  98. 98.
    Yan WX, Hunnewell P, Alfonse LE et al (2019) Functionally diverse type V CRISPR-Cas systems. Science 363:88–91Google Scholar
  99. 99.
    Zerbini F, Zanella I, Fraccascia D et al (2017) Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli. Microb Cell Fact 16:68Google Scholar
  100. 100.
    Zetsche B, Gootenberg JS, Abudayyeh OO et al (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163:759–771Google Scholar
  101. 101.
    Zhang J, Zong W, Hong W et al (2018) Exploiting endogenous CRISPR-Cas system for multiplex genome editing in Clostridium tyrobutyricum and engineer the strain for high-level butanol production. Metab Eng 47:49–59Google Scholar
  102. 102.
    Zhang J, Yang F, Yang Y et al (2019) Optimizing a CRISPR-Cpf1-based genome engineering system for Corynebacterium glutamicum. Microb Cell Fact 18:60Google Scholar
  103. 103.
    Zhou XX, Zou X, Chung HK et al (2018) A single-chain photoswitchable CRISPR-Cas9 architecture for light-inducible gene editing and transcription. ACS Chem Biol 13:443–448Google Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2019

Authors and Affiliations

  1. 1.Department of Chemical and Biomolecular EngineeringNorth Carolina State UniversityRaleighUSA
  2. 2.Helmholtz Institute for RNA-based Infection Research (HIRI)Helmholtz-Centre for Infection Research (HZI)WürzburgGermany
  3. 3.Medical FacultyUniversity of WürzburgWürzburgGermany

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