Abstract
CRISPR-Cas9 has been explored as a transformative genome engineering tool for many eukaryotic organisms. However, its utilization in bacteria remains limited and ineffective. This chapter, taking Clostridium beijerinckii as an example, describes the use of Streptococcus pyogenes CRISPR-Cas9 system guided by the single chimeric guide RNA (gRNA) for diverse genome-editing purposes, including chromosomal gene deletion, integration, single nucleotide modification, as well as “clean” mutant selection. The general principle is to use CRISPR-Cas9 as an efficient selection tool for the edited mutant (whose CRISPR-Cas9 target site has been disrupted through a homologous recombination event and thus can survive selection) against? the wild type background cells. This protocol is broadly applicable to other microorganisms for genome-editing purposes.
Keywords
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Chen Y, Indurthi DC, Jones SW, Papoutsakis ET (2011) Small RNAs in the genus Clostridium. MBio 2:e00340–e00310. https://doi.org/10.1128/mBio.00340-10
Mermelstein LD, Papoutsakis ET (1993) In vivo methylation in Escherichia coli by the Bacillus subtilis phage phi 3T I methyltransferase to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol 59:1077–1081
Green EM, Boynton ZL, Harris LM et al (1996) Genetic manipulation of acid formation pathways by gene inactivation in Clostridium acetobutylicum ATCC 824. Microbiology 142:2079–2086. https://doi.org/10.1099/13500872-142-8-2079
Harris LM, Welker NE, Papoutsakis ET (2002) Northern, morphological, and fermentation analysis of spo0A inactivation and overexpression in Clostridium acetobutylicum ATCC 824. J Bacteriol 184:3586–3597. https://doi.org/10.1128/JB.184.13.3586-3597.2002
Al-Hinai MA, Fast AG, Papoutsakis ET (2012) Novel system for efficient isolation of Clostridium double-crossover allelic exchange mutants enabling markerless chromosomal gene geletions and DNA integration. Appl Environ Microbiol 78:8112–8121. https://doi.org/10.1128/AEM.02214-12
Heap JT, Pennington OJ, Cartman ST et al (2007) The clostron: a universal gene knock-out system for the genus Clostridium. J Microbiol Methods 70:452–464. https://doi.org/10.1016/j.mimet.2007.05.021
Wang Y, Li X, Milne CB et al (2013) Development of a gene knockout system using mobile group II introns (targetron) and genetic disruption of acid production pathways in Clostridium beijerinckii. Appl Environ Microbiol 79:5853–5863. https://doi.org/10.1128/AEM.00971-13
Marraffini LA, Sontheimer EJ (2010) CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat Rev Genet 11:181–190. https://doi.org/10.1038/nrg2749
Cong L, Ran FA, Cox D et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823. https://doi.org/10.1126/science.1231143
Jiang W, Bikard D, Cox D et al (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31:233–239. https://doi.org/10.1038/nbt.2508
Oh J-H, van Pijkeren J-P (2014) CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri. Nucleic Acids Res 42:e131. https://doi.org/10.1093/nar/gku623
Cobb RE, Wang Y, Zhao H (2015) High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system. ACS Synth Biol 4:723–728. https://doi.org/10.1021/sb500351f
Huang H, Zheng G, Jiang W et al (2015) One-step high-efficiency CRISPR/Cas9-mediated genome editing in Streptomyces. Acta Biochim Biophys Sin Shanghai 47:231–243. https://doi.org/10.1093/abbs/gmv007
Tong Y, Charusanti P, Zhang L et al (2015) CRISPR-Cas9 based engineering of actinomycetal genomes. ACS Synth Biol 4:1020–1029. https://doi.org/10.1021/acssynbio.5b00038
Jiang Y, Chen B, Duan C et al (2015) Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 81:2506–2514. https://doi.org/10.1128/AEM.04023-14
Wang Y, Zhang Z-T, Seo S-O et al (2015) Markerless chromosomal gene deletion in Clostridium beijerinckii using CRISPR/Cas9 system. J Biotechnol 200:1–5. https://doi.org/10.1016/j.jbiotec.2015.02.005
Wang Y, Zhang Z-T, Seo S-O et al (2016) Bacterial genome editing with CRISPR-Cas9: deletion, integration, single nucleotide modification, and desirable “clean mutant” selection in Clostridium beijerinckii as an example. ACS Synth Biol 5:721–732. https://doi.org/10.1021/acssynbio.6b00060
Xu T, Li Y, Shi Z et al (2015) Efficient genome editing in Clostridium cellulolyticum via CRISPR-Cas9 nickase. Appl Environ Microbiol 81:4423–4431. https://doi.org/10.1128/AEM.00873-15
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:e00233–17. https://doi.org/10.1128/AEM.00233-17
Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826. https://doi.org/10.1126/science.1232033
Qi L, Larson M, Gilbert L et al (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152:1173–1183. https://doi.org/10.1016/j.cell.2013.02.022
Qiagen (2008) .QIAquick® spin handbook. http://sevierlab.vet.cornell.edu/resources/EN-QIAquick-Spin-Handbook.pdf. Accessed 15 Jun 2017
NEB Protocol: Optimizing restriction endonuclease reactions. https://www.neb.com/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions. Accessed 15 Jun 2017
NEB Gibson assembly® master mix instructions manual. https://www.neb.com/~/media/Catalog/All-Products/0AA961B294E444AFBEDD5C4A904C76E6/Datacards or Manuals/ManualE2611.pdf. Accessed 15 Jun 2017
Gibson DG, Young L, Chuang R-Y et al (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345. https://doi.org/10.1038/nmeth.1318
Zymo Research Corp. Instruction manual quick-RNA™ miniprep. https://www.zymoresearch.com/downloads/dl/file/id/152/r1054i.pdf. Accessed 15 Jun 2017
NEB ProtoScript® first strand cDNA synthesis kit instruction manual. https://www.neb.com/~/media/Catalog/All-Products/8DC78EF3331D476F9C263B572910651B/Datacards or Manuals/manualE6300.pdf. Accessed 15 Jun 2017
Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821. https://doi.org/10.1126/science.1225829
NEB HiScribe™ T7 quick high yield RNA synthesis kit instructions manuals. https://www.neb.com/~/media/Catalog/All-Products/9D7209047C8E4B34ABE47B635EB600E2/Datacards or Manuals/manualE2050.pdf. Accessed 15 Jun 2017
NEB Protocol: In vitro digestion of DNA with Cas9 nuclease, S. pyogenes. (M0386). https://www.neb.com/protocols/2014/05/01/in-vitro-digestion-of-dna-with-cas9-nuclease-s-pyogenes-m0386. Accessed 15 Jun 2017
Heap JT, Kuehne SA, Ehsaan M et al (2010) The ClosTron: Mutagenesis in Clostridium refined and streamlined. J Microbiol Methods 80:49–55. https://doi.org/10.1016/j.mimet.2009.10.018
Jesse TW (2003) Genetic characterization and manipulation of solvent-producing Clostridia. University of Illinois at Urbana-Champaign, Dissertation
Acknowledgments
This work was supported by Department of Energy (DOE) grant #2011-01219 to HPB. We thank Dr. Terry Papoutsakis for providing the pKO_mazF plasmid. We also thank Dr. Wenyan Jiang (from Dr. Luciano A. Marraffini’s group at The Rockefeller University), Dr. Esteban Toro (from Dr. Adam P. Arkin’s group at UC-Berkeley), Dr. Jason Peters (from Dr. Carol Gross’ group at UC-San Francisco), and Dr. Martin Jinek (from Dr. Jennifer Doudna’s group at UC-Berkeley) for their helpful discussions. We acknowledge www.somersault1824.com for allowing us to use their library to generate Figs. 1 and 4.
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Zhang, ZT. et al. (2018). Bacterial Genome Editing with CRISPR-Cas9: Taking Clostridium beijerinckii as an Example. In: Braman, J. (eds) Synthetic Biology. Methods in Molecular Biology, vol 1772. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7795-6_17
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DOI: https://doi.org/10.1007/978-1-4939-7795-6_17
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