Abstract
In recent years, the clustered regularly interspaced palindromic repeats-Cas (CRISPR-Cas) technology has become the method of choice for precision genome editing in many organisms due to its simplicity and efficacy. Multiplex genome editing, point mutations, and large genomic modifications are attractive features of the CRISPR-Cas9 system. These applications facilitate both the ease and velocity of genetic manipulations and the discovery of novel functions. In this protocol chapter, we describe the use of a CRISPR-Cas9 system for multiplex integration and deletion modifications, and deletions of large genomic regions by the use of a single guide RNA (sgRNA), and, finally, targeted point mutation modifications in Paenibacillus polymyxa.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Jeong H, Choi SK, Ryu CM et al (2019) Chronicle of a soil bacterium: Paenibacillus polymyxa E681 as a Tiny Guardian of plant and human health. Front Microbiol 10:467
Rütering M, Schmid J, Rühmann B et al (2016) Controlled production of polysaccharides-exploiting nutrient supply for levan and heteropolysaccharide formation in Paenibacillus sp. Carbohydr Polym 148:326–334
Schilling C, Ciccone R, Sieber V et al (2020) Engineering of the 2,3-butanediol pathway of Paenibacillus polymyxa DSM 365. Metab Eng 61:381–388
Zarschler K, Janesch B, Zayni S et al (2009) Construction of a gene knockout system for application in Paenibacillus alvei CCM 2051T, exemplified by the S-layer glycan biosynthesis initiation enzyme WsfP. Appl Environ Microbiol 75:3077–3085
Bin KS, Timmusk S (2013) A simplified method for gene knockout and direct screening of recombinant clones for application in Paenibacillus polymyxa. PLoS One 8:e68092
Choi SK, Park SY, Kim R et al (2008) Identification and functional analysis of the fusaricidin biosynthetic gene of Paenibacillus polymyxa E681. Biochem Biophys Res Commun 365:89–95
Rütering M, Cress BF, Schilling M et al (2017) Tailor-made exopolysaccharides-CRISPR-Cas9 mediated genome editing in Paenibacillus polymyxa. Synth Biol 2:ysx007
**ek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science (1979) 337:816–821
Garneau JE, Dupuis MÈ, Villion M et al (2010) The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468:67–71
Gasiunas G, Barrangou R, Horvath P et al (2012) Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A 109:E2579–E2586
Szostak JW, Orr-Weaver TL, Rothstein RJ et al (1983) The Double-Strand-Break repair model for recombination. Cell 33:25–35
Chayot R, Montagne B, Mazel D et al (2010) An end-joining repair mechanism in Escherichia coli. Proc Natl Acad Sci U S A 107:2141–2146
Lieber MR (2010) The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu Rev Biochem 79:181–211
Bikard D, Jiang W, Samai P et al (2013) Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res 41:7429–7437
Qi LS, Larson MH, Gilbert LA et al (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152:1173–1183
Komor AC, Kim YB, Packer MS et al (2016) Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533:420–424
Adiego-Pérez B, Randazzo P, Daran JM et al (2019) Multiplex genome editing of microorganisms using CRISPR-Cas. FEMS Microbiol Lett 366:fnz086
Tian J, **ng B, Li M et al (2022) Efficient large-scale and scarless genome engineering enables the construction and screening of Bacillus subtilis biofuel overproducers. Int J Mol Sci 23:4853
Baumgart M, Unthan S, Kloß R et al (2018) Corynebacterium glutamicum Chassis C1∗: building and testing a novel platform host for synthetic biology and industrial biotechnology. ACS Synth Biol 7:132–144
Fan X, Zhang Y, Zhao F et al (2020) Genome reduction enhances production of polyhydroxyalkanoate and alginate oligosaccharide in Pseudomonas mendocina. Int J Biol Macromol 163:2023–2031
Zhang F, Huo K, Song X et al (2020) Engineering of a genome-reduced strain Bacillus amyloliquefaciens for enhancing surfactin production. Microb Cell Factories 19:223
Milunovic B, diCenzo GC, Morton RA et al (2014) Cell growth inhibition upon deletion of four toxin-antitoxin loci from the megaplasmids of Sinorhizobium meliloti. J Bacteriol 196:811–824
diCenzo GC, Zamani M, Milunovic B et al (2016) Genomic resources for identification of the minimal N2-fixing symbiotic genome. Environ Microbiol 18:2534–2547
Schilling C, Koffas MAG, Sieber V et al (2021) Novel prokaryotic CRISPR-Cas12a-based tool for programmable transcriptional activation and repression. ACS Synth Biol 9:3353–3363
Kim MS, Kim HR, Jeong DE et al (2021) Cytosine base editor-mediated multiplex genome editing to accelerate discovery of novel antibiotics in Bacillus subtilis and Paenibacillus polymyxa. Front Microbiol 12:691839
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
Meliawati M, Teckentrup C, Schmid J (2022) CRISPR-Cas9-mediated large cluster deletion and multiplex genome editing in Paenibacillus polymyxa. ACS Synth Biol 11:77–84
Meliawati M, May T, Eckerlin J et al (2022) Insights in the complex DegU, DegS, and Spo0A regulation system of Paenibacillus polymyxa by CRISPR-Cas9-based targeted point mutations. Appl Environ Microbiol 88:e0016422
NEB protocol: PCR Using Q5® High-Fidelity DNA Polymerase (M0491). https://international.neb.com/protocols/2013/12/13/pcr-using-q5-high-fidelity-dna-polymerase-m0491. Accessed March 2023
Bioline protocol: Accuzyme DNA polymerase. https://www.bioline.com/mwdownloads/download/link/id/2703/accuzyme_dna_polymerase_product_manual.pdf. Accessed March 2023
Thermo Fischer Scientific protocol: GeneJET PCR purification kit. https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2FMAN0012662_GeneJET_PCR_Purification_UG.pdf. Accessed March 2023
NEB protocol: Monarch DNA Gel extraction kit protocol card https://international.neb.com/protocols/2015/11/23/monarch-dna-gel-extraction-kit-protocol-t1020. Accessed March 2023
Microsynth protocol: Sanger sequencing. https://www.microsynth.com/files/Inhalte/PDFs/Sanger/UserGuide_EconomyRun.pdf. Accessed March 2023
Promega protocol: GoTaq G2 green master mix. https://www.promega.de/-/media/files/resources/protocols/product-information-sheets/g/gotaq-g2-green-master-mix-protocol.pdf?rev=ba2e5156136b43068889f3c33c344240&la=en. Accessed March 2023
Qiagen protocol: DNeasy Blood & Tissue Handbook. https://www.qiagen.com/us/resources/resourcedetail?id=68f29296-5a9f-40fa-8b3d-1c148d0b3030&lang=en. Accessed March 2023
Acknowledgments
This study is part of the German Federal Ministry of Education and Research (BMBF) funded project Polymore with the no. 031B0855A and by BASF SE, Germany.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Ravagnan, G., Meliawati, M., Schmid, J. (2024). CRISPR-Cas9-Mediated Genome Editing in Paenibacillus polymyxa. In: Braman, J.C. (eds) Synthetic Biology. Methods in Molecular Biology, vol 2760. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3658-9_16
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3658-9_16
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3657-2
Online ISBN: 978-1-0716-3658-9
eBook Packages: Springer Protocols