Development of a CRISPR/Cas9 System for Methylococcus capsulatus In Vivo Gene Editing

Tapscott, Timothy; Guarnieri, Michael T.; Henard, Calvin A.; Kelly, Robert M.

doi:10.1128/AEM.00340-19

Title: Development of a CRISPR/Cas9 System for Methylococcus capsulatus In Vivo Gene Editing

Journal Article · Fri Mar 29 00:00:00 EDT 2019 · Applied and Environmental Microbiology

DOI:https://doi.org/10.1128/AEM.00340-19· OSTI ID:1505930

Tapscott, Timothy ^[1]; Guarnieri, Michael T. ^[1];

^[1]; Kelly, Robert M. ^[2]

National Renewable Energy Lab. (NREL), Golden, CO (United States)
North Carolina State Univ., Raleigh, NC (United States)

Methanotrophic bacteria play a crucial role in the Earth’s biogeochemical cycle and have the potential to be employed in industrial biomanufacturing processes due to their capacity to use natural gas- and biogas-derived methane as a sole carbon and energy source. Advanced gene-editing systems have the potential to enable rapid, high-throughput methanotrophic genetics and biocatalyst development. To this end, we employed a series of broad-host-range expression plasmids to construct a conjugatable clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene-editing system in Methylococcus capsulatus (Bath). Heterologous coexpression of the Streptococcus pyogenes Cas9 endonuclease and a synthetic single guide RNA (gRNA) showed efficient Cas9 DNA targeting and double-stranded DNA (dsDNA) cleavage that resulted in cell death. We demonstrated effective in vivo editing of plasmid DNA using both Cas9 and Cas9^D10A nickase to convert green fluorescent protein (GFP)- to blue fluorescent protein (BFP)-expressing cells with 71% efficiency. Further, we successfully introduced a premature stop codon into the soluble methane monooxygenase (sMMO) hydroxylase component-encoding mmoX gene with the Cas9^D10A nickase, disrupting sMMO function. These data provide proof of concept for CRISPR/Cas9-mediated gene editing in M. capsulatus. Given the broad-host-range replicons and conjugation capability of these CRISPR/Cas9 tools, they have potential utility in other methanotrophs and a wide array of Gram-negative microorganisms.

View Accepted Manuscript (DOE)

Cite

Export

Save

Research Organization:: National Renewable Energy Lab. (NREL), Golden, CO (United States)

Sponsoring Organization:: USDOE National Renewable Energy Laboratory (NREL), Laboratory Directed Research and Development (LDRD) Program

Grant/Contract Number:: AC36-08GO28308

OSTI ID:: 1505930

Report Number(s):: NREL/JA-5100-73621

Journal Information:: Applied and Environmental Microbiology, Vol. 85, Issue 11; ISSN 0099-2240

Publisher:: American Society for MicrobiologyCopyright Statement

Country of Publication:: United States

Language:: English

Citation Metrics:

Cited by: 27 works

Citation information provided by
Web of Science

References (45)

Engineering and characterization of a superfolder green fluorescent protein Pédelacq, Jean-Denis; Cabantous, Stéphanie; Tran, Timothy Nature Biotechnology, Vol. 24, Issue 1, p. 79-88 https://doi.org/10.1038/nbt1172	journal	December 2005
GFP to BFP Conversion: A Versatile Assay for the Quantification of CRISPR/Cas9-mediated Genome Editing Glaser, Astrid; McColl, Bradley; Vadolas, Jim Molecular Therapy - Nucleic Acids, Vol. 5 https://doi.org/10.1038/mtna.2016.48	journal	January 2016
Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression Qi, Lei S.; Larson, Matthew H.; Gilbert, Luke A. Cell, Vol. 152, Issue 5, p. 1173-1183 https://doi.org/10.1016/j.cell.2013.02.022	journal	February 2013
Discovery, taxonomic distribution, and phenotypic characterization of a gene required for 3-methylhopanoid production Welander, P. V.; Summons, R. E. Proceedings of the National Academy of Sciences, Vol. 109, Issue 32 https://doi.org/10.1073/pnas.1208255109	journal	July 2012
Efficient Counterselection for Methylococcus capsulatus (Bath) by Using a Mutated pheS Gene Ishikawa, Masahito; Yokoe, Sho; Kato, Souichiro Applied and Environmental Microbiology, Vol. 84, Issue 23 https://doi.org/10.1128/AEM.01875-18	journal	September 2018
Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria Gasiunas, G.; Barrangou, R.; Horvath, P. Proceedings of the National Academy of Sciences, Vol. 109, Issue 39, p. E2579-E2586 https://doi.org/10.1073/pnas.1208507109	journal	September 2012
Copper-dependent reciprocal transcriptional regulation of methane monooxygenase genes in Methylococcus capsulatus and Methylosinus trichosporium Nielsen, Allan K.; Gerdes, Kenn; Murrell, J. Colin Molecular Microbiology, Vol. 25, Issue 02 https://doi.org/10.1046/j.1365-2958.1997.4801846.x	journal	July 1997
Single-strand nicks induce homologous recombination with less toxicity than double-strand breaks using an AAV vector template Metzger, Michael J.; McConnell-Smith, Audrey; Stoddard, Barry L. Nucleic Acids Research, Vol. 39, Issue 3 https://doi.org/10.1093/nar/gkq826	journal	September 2010
Genes involved in the copper-dependent regulation of soluble methane monooxygenase of Methylococcus capsulatus (Bath): cloning, sequencing and mutational analysis Csaki, R. Microbiology, Vol. 149, Issue 7 https://doi.org/10.1099/mic.0.26061-0	journal	July 2003
Expression of Individual Copies of Methylococcus capsulatus Bath Particulate Methane Monooxygenase Genes Stolyar, S.; Franke, M.; Lidstrom, M. E. Journal of Bacteriology, Vol. 183, Issue 5 https://doi.org/10.1128/JB.183.5.1810-1812.2001	journal	March 2001
A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity Jinek, M.; Chylinski, K.; Fonfara, I. Science, Vol. 337, Issue 6096, p. 816-821 https://doi.org/10.1126/science.1225829	journal	June 2012
Enrichment, Isolation and Some Properties of Methane-utilizing Bacteria Whittenbury, R.; Phillips, K. C.; Wilkinson, J. F. Journal of General Microbiology, Vol. 61, Issue 2 https://doi.org/10.1099/00221287-61-2-205	journal	May 1970
Development and validation of promoter-probe vectors for the study of methane monooxygenase gene expression in Methylococcus capsulatus Bath Ali, H.; Murrell, J. C. Microbiology, Vol. 155, Issue 3 https://doi.org/10.1099/mic.0.021816-0	journal	March 2009
Metabolic engineering of Escherichia coli using CRISPR–Cas9 meditated genome editing Li, Yifan; Lin, Zhenquan; Huang, Can Metabolic Engineering, Vol. 31, p. 13-21 https://doi.org/10.1016/j.ymben.2015.06.006	journal	September 2015
CRISPR-Cas9 ^D10A Nickase-Assisted Genome Editing in Lactobacillus casei Song, Xin; Huang, He; Xiong, Zhiqiang Applied and Environmental Microbiology, Vol. 83, Issue 22 https://doi.org/10.1128/AEM.01259-17	journal	September 2017
Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes Kovach, Michael E.; Elzer, Philip H.; Steven Hill, D. Gene, Vol. 166, Issue 1 https://doi.org/10.1016/0378-1119(95)00584-1	journal	December 1995
Bioconversion of methane to C-4 carboxylic acids using carbon flux through acetyl-CoA in engineered Methylomicrobium buryatense 5GB1C Garg, Shivani; Wu, Hao; Clomburg, James M. Metabolic Engineering, Vol. 48 https://doi.org/10.1016/j.ymben.2018.06.001	journal	July 2018
Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage Komor, Alexis C.; Kim, Yongjoo B.; Packer, Michael S. Nature, Vol. 533, Issue 7603 https://doi.org/10.1038/nature17946	journal	April 2016
A modular approach for high-flux lactic acid production from methane in an industrial medium using engineered Methylomicrobium buryatense 5GB1 Garg, Shivani; Clomburg, James M.; Gonzalez, Ramon Journal of Industrial Microbiology & Biotechnology, Vol. 45, Issue 6 https://doi.org/10.1007/s10295-018-2035-3	journal	April 2018
Mycobacteria exploit three genetically distinct DNA double-strand break repair pathways: DSB repair in mycobacterium Gupta, Richa; Barkan, Daniel; Redelman-Sidi, Gil Molecular Microbiology, Vol. 79, Issue 2 https://doi.org/10.1111/j.1365-2958.2010.07463.x	journal	November 2010
A new recombineering system for precise genome-editing in Shewanella oneidensis strain MR-1 using single-stranded oligonucleotides Corts, Anna D.; Thomason, Lynn C.; Gill, Ryan T. Scientific Reports, Vol. 9, Issue 1 https://doi.org/10.1038/s41598-018-37025-4	journal	January 2019
Development of an Efficient Genome Editing Tool in Bacillus licheniformis Using CRISPR-Cas9 Nickase Li, Kaifeng; Cai, Dongbo; Wang, Zhangqian Applied and Environmental Microbiology, Vol. 84, Issue 6 https://doi.org/10.1128/AEM.02608-17	journal	January 2018
Bioconversion of methane to lactate by an obligate methanotrophic bacterium Henard, Calvin A.; Smith, Holly; Dowe, Nancy Scientific Reports, Vol. 6, Issue 1 https://doi.org/10.1038/srep21585	journal	February 2016
Evolved Cas9 variants with broad PAM compatibility and high DNA specificity Hu, Johnny H.; Miller, Shannon M.; Geurts, Maarten H. Nature, Vol. 556, Issue 7699 https://doi.org/10.1038/nature26155	journal	February 2018
Systematic metabolic engineering of Methylomicrobium alcaliphilum 20Z for 2,3-butanediol production from methane Nguyen, Anh Duc; Hwang, In Yeub; Lee, Ok Kyung Metabolic Engineering, Vol. 47 https://doi.org/10.1016/j.ymben.2018.04.010	journal	May 2018
Consequences of Cas9 cleavage in the chromosome of Escherichia coli Cui, Lun; Bikard, David Nucleic Acids Research, Vol. 44, Issue 9 https://doi.org/10.1093/nar/gkw223	journal	April 2016
CRISPR EnAbled Trackable genome Engineering for isopropanol production in Escherichia coli Liang, Liya; Liu, Rongming; Garst, Andrew D. Metabolic Engineering, Vol. 41 https://doi.org/10.1016/j.ymben.2017.02.009	journal	May 2017
Role of multiple gene copies in particulate methane monooxygenase activity in the methane-oxidizing bacterium Methylococcus capsulatus Bath Stolyar, S.; Costello, A. M.; Peeples, T. L. Microbiology, Vol. 145, Issue 5 https://doi.org/10.1099/13500872-145-5-1235	journal	May 1999
High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects Kleinstiver, Benjamin P.; Pattanayak, Vikram; Prew, Michelle S. Nature, Vol. 529, Issue 7587 https://doi.org/10.1038/nature16526	journal	January 2016
Rapid and Efficient One-Step Metabolic Pathway Integration in E. coli Bassalo, Marcelo C.; Garst, Andrew D.; Halweg-Edwards, Andrea L. ACS Synthetic Biology, Vol. 5, Issue 7 https://doi.org/10.1021/acssynbio.5b00187	journal	April 2016
Transcriptomic profiling of Methylococcus capsulatus (Bath) during growth with two different methane monooxygenases Larsen, Øivind; Karlsen, Odd A. MicrobiologyOpen, Vol. 5, Issue 2 https://doi.org/10.1002/mbo3.324	journal	December 2015
Rationally engineered Cas9 nucleases with improved specificity Slaymaker, I. M.; Gao, L.; Zetsche, B. Science, Vol. 351, Issue 6268 https://doi.org/10.1126/science.aad5227	journal	December 2015
Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering Garst, Andrew D.; Bassalo, Marcelo C.; Pines, Gur Nature Biotechnology, Vol. 35, Issue 1, p. 48-55 https://doi.org/10.1038/nbt.3718	journal	December 2016
Genetic Tools for the Industrially Promising Methanotroph Methylomicrobium buryatense Puri, Aaron W.; Owen, Sarah; Chu, Frances Applied and Environmental Microbiology, Vol. 81, Issue 5 https://doi.org/10.1128/AEM.03795-14	journal	December 2014
RNA-guided editing of bacterial genomes using CRISPR-Cas systems Jiang, Wenyan; Bikard, David; Cox, David Nature Biotechnology, Vol. 31, Issue 3, p. 233-239 https://doi.org/10.1038/nbt.2508	journal	January 2013
Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage Gaudelli, Nicole M.; Komor, Alexis C.; Rees, Holly A. Nature, Vol. 551, Issue 7681 https://doi.org/10.1038/nature24644	journal	October 2017
Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification Guilinger, John P.; Thompson, David B.; Liu, David R. Nature Biotechnology, Vol. 32, Issue 6 https://doi.org/10.1038/nbt.2909	journal	April 2014
The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA Garneau, Josiane E.; Dupuis, Marie-Ève; Villion, Manuela Nature, Vol. 468, Issue 7320 https://doi.org/10.1038/nature09523	journal	November 2010
Efficient Genome Editing in Clostridium cellulolyticum via CRISPR-Cas9 Nickase Xu, Tao; Li, Yongchao; Shi, Zhou Applied and Environmental Microbiology, Vol. 81, Issue 13 https://doi.org/10.1128/AEM.00873-15	journal	April 2015
Enhanced proofreading governs CRISPR–Cas9 targeting accuracy Chen, Janice S.; Dagdas, Yavuz S.; Kleinstiver, Benjamin P. Nature, Vol. 550, Issue 7676 https://doi.org/10.1038/nature24268	journal	September 2017
Molecular Nature of Two Nonconjugative Plasmids Carrying Drug Resistance Genes Guerry, Patricia; Van Embden, Jan; Falkow, Stanley Journal of Bacteriology, Vol. 117, Issue 2 https://doi.org/10.1128/jb.117.2.619-630.1974	journal	February 1974
Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene dose-sensitive AD suppressor in human brain Alić, Ivan; Goh, Pollyanna A.; Murray, Aoife Molecular Psychiatry https://doi.org/10.1038/s41380-020-0806-5	journal	July 2020
Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses Levy, Jonathan M.; Yeh, Wei-Hsi; Pendse, Nachiket Nature Biomedical Engineering, Vol. 4, Issue 1 https://doi.org/10.1038/s41551-019-0501-5	journal	January 2020
The Ethanologenic Bacterium Zymomonas mobilis Divides Asymmetrically and Exhibits Heterogeneity in DNA Content Fuchino, Katsuya; Chan, Helena; Hwang, Ling Chin Applied and Environmental Microbiology, Vol. 87, Issue 6 https://doi.org/10.1128/aem.02441-20	journal	February 2021
Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression Qi, Lei S.; Larson, Matthew H.; Gilbert, Luke A. Cell, Vol. 184, Issue 3 https://doi.org/10.1016/j.cell.2021.01.019	journal	February 2021

Cited By (4)

Muconic acid production from methane using rationally-engineered methanotrophic biocatalysts Henard, Calvin A.; Akberdin, Ilya R.; Kalyuzhnaya, Marina G. Green Chemistry, Vol. 21, Issue 24 https://doi.org/10.1039/c9gc03722e	journal	January 2019
Barriers to genome editing with CRISPR in bacteria Vento, Justin M.; Crook, Nathan; Beisel, Chase L. Journal of Industrial Microbiology & Biotechnology, Vol. 46, Issue 9-10 https://doi.org/10.1007/s10295-019-02195-1	journal	June 2019
Strategies for Developing CRISPR‐Based Gene Editing Methods in Bacteria Wu, Zhaowei; Wang, Yujue; Zhang, Yifei Small Methods, Vol. 4, Issue 2 https://doi.org/10.1002/smtd.201900560	journal	November 2019
Bioproduction of Isoprenoids and Other Secondary Metabolites Using Methanotrophic Bacteria as an Alternative Microbial Cell Factory Option: Current Stage and Future Aspects Jeon, Young Chan; Nguyen, Anh Duc; Lee, Eun Yeol Catalysts, Vol. 9, Issue 11 https://doi.org/10.3390/catal9110883	journal	October 2019

Similar Records

Identification of putative methanol dehydrogenase (moxF) structural genes in methylotrophs and cloning of moxF genes from methylococcus capsulatus bath and Methylomonas albus BG8

Journal Article · Sun May 01 00:00:00 EDT 1988 · J. Bacteriol.; (United States) · OSTI ID:1505930

Stephens, R L; Haygood, M G; Lidstrom, M E

Genomic Insights into Methanotrophy: The Complete Genome Sequence of Methylococcus capsulatus (Bath)

Journal Article · Tue Sep 21 00:00:00 EDT 2004 · PLoS Biology (Online) · OSTI ID:1505930

Ward, Naomi; Larsen, Øivind; Sakwa, James; +35 more

Transformation and gene editing in the bioenergy grass Miscanthus

Journal Article · Wed Dec 28 00:00:00 EST 2022 · Biotechnology for Biofuels and Bioproducts · OSTI ID:1505930

Trieu, Anthony; Belaffif, Mohammad B.; Hirannaiah, Pradeepa; +10 more

Related Subjects

09 BIOMASS FUELS
59 BASIC BIOLOGICAL SCIENCES
CRISPR/Cas9
methanotroph
methane monooxygenase
gene editing
Methylococcus capsulatus
methane biocatalyst

Title: Development of a CRISPR/Cas9 System for Methylococcus capsulatus In Vivo Gene Editing

Citation Formats

References (45)

Cited By (4)

Similar Records

Related Subjects