CRISPR-Cas Genome Editing in the Cellulolytic Bacterium Clostridium thermocellum (C. thermocellum)

Stettner, Sean; Eckert, Carrie

doi:10.1007/978-1-0716-1657-4_22

CRISPR-Cas Genome Editing in the Cellulolytic Bacterium Clostridium thermocellum (C. thermocellum)

Book · Sat Jul 31 04:00:00 EDT 2021

DOI:https://doi.org/10.1007/978-1-0716-1657-4_22· OSTI ID:1986067

Stettner, Sean;

Clostridium thermocellum is an anaerobic thermophile that can efficiently degrade lignocellulosic biomass and directly convert it into value-added products such as ethanol. The cellulolytic and ethanologenic capabilities of C. thermocellum make it an excellent candidate for consolidated bioprocessing (CBP) for industrial production, where biomass degradation and fermentation occur simultaneously. In this organism, strain development for effective CBP has traditionally been hindered by the lack of genetic tools. Here, we detail our efficient two-step CRISPR-Cas genome editing protocol for C. thermocellum, using both the native Type I-B CRISPR-Cas system and an exogenous Type-II CRISPR system from Geobacillus stearothermophilus. As recombination is limiting in C. thermocellum genome engineering, we highlight effective thermophilic recombinases necessary to improve genome editing in these systems. We additionally provide design rules for the repair template and synthetic guide RNA (gRNA) for each system. Using these newly developed CRISPR and recombineering tools, targeted C. thermocellum engineering will substantiate efforts toward CBP strain development in industrial applications.

🛈

OSTI does not have a digital full text copy available. For more information, please see document availability, search WorldCat, or search Google Scholar.

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

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

DOE Contract Number:: AC36-08GO28308

OSTI ID:: 1986067

Report Number(s):: NREL/CH-2700-78415; MainId:32332; UUID:f3e674f1-13d3-4a45-b65f-28996ad2e90a; MainAdminID:18858

Country of Publication:: United States

Language:: English

References (26)

Consolidated bioprocessing of cellulosic biomass: an update Lynd, Lee R.; van Zyl, Willem H.; McBride, John E. Current Opinion in Biotechnology, Vol. 16, Issue 5, p. 577-583 https://doi.org/10.1016/j.copbio.2005.08.009	journal	October 2005
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
Generation of a fully erythromycin-sensitive strain of Clostridioides difficile using a novel CRISPR-Cas9 genome editing system Ingle, Patrick; Groothuis, Daphne; Rowe, Peter Scientific Reports, Vol. 9, Issue 1 https://doi.org/10.1038/s41598-019-44458-y	journal	May 2019
The CRISPR tool kit for genome editing and beyond Adli, Mazhar Nature Communications, Vol. 9, Issue 1 https://doi.org/10.1038/s41467-018-04252-2	journal	May 2018
Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System Jiang, Yu; Chen, Biao; Duan, Chunlan Applied and Environmental Microbiology, Vol. 81, Issue 7, p. 2506-2514 https://doi.org/10.1128/AEM.04023-14	journal	January 2015
Harnessing heterologous and endogenous CRISPR-Cas machineries for efficient markerless genome editing in Clostridium Pyne, Michael E.; Bruder, Mark R.; Moo-Young, Murray Scientific Reports, Vol. 6, Issue 1 https://doi.org/10.1038/srep25666	journal	May 2016
Recent progress in consolidated bioprocessing Olson, Daniel G.; McBride, John E.; Joe Shaw, A. Current Opinion in Biotechnology, Vol. 23, Issue 3, p. 396-405 https://doi.org/10.1016/j.copbio.2011.11.026	journal	June 2012
Applications of CRISPR Technologies Across the Food Supply Chain Brandt, Katelyn; Barrangou, Rodolphe Annual Review of Food Science and Technology, Vol. 10, Issue 1 https://doi.org/10.1146/annurev-food-032818-121204	journal	March 2019
CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes Barrangou, R.; Fremaux, C.; Deveau, H. Science, Vol. 315, Issue 5819 https://doi.org/10.1126/science.1138140	journal	March 2007
Synthetic biology approaches for chromosomal integration of genes and pathways in industrial microbial systems Li, Lei; Liu, Xiaocao; Wei, Keke Biotechnology Advances, Vol. 37, Issue 5 https://doi.org/10.1016/j.biotechadv.2019.04.002	journal	September 2019
RNA-Guided Human Genome Engineering via Cas9 Mali, P.; Yang, L.; Esvelt, K. M. Science, Vol. 339, Issue 6121, p. 823-826 https://doi.org/10.1126/science.1232033	journal	January 2013
Development of both type I–B and type II CRISPR/Cas genome editing systems in the cellulolytic bacterium Clostridium thermocellum Walker, Julie E.; Lanahan, Anthony A.; Zheng, Tianyong Metabolic Engineering Communications, Vol. 10 https://doi.org/10.1016/j.mec.2019.e00116	journal	June 2020
Multiplex Genome Engineering Using CRISPR/Cas Systems Cong, L.; Ran, F. A.; Cox, D. Science, Vol. 339, Issue 6121, p. 819-823 https://doi.org/10.1126/science.1231143	journal	January 2013
Developing Riboswitch-Mediated Gene Regulatory Controls in Thermophilic Bacteria Marcano-Velazquez, Joan G.; Lo, Jonathan; Nag, Ambarish ACS Synthetic Biology, Vol. 8, Issue 4 https://doi.org/10.1021/acssynbio.8b00487	journal	April 2019
Small CRISPR RNAs Guide Antiviral Defense in Prokaryotes Brouns, S. J. J.; Jore, M. M.; Lundgren, M. Science, Vol. 321, Issue 5891, p. 960-964 https://doi.org/10.1126/science.1159689	journal	August 2008
Advances in Industrial Biotechnology Using CRISPR-Cas Systems Donohoue, Paul D.; Barrangou, Rodolphe; May, Andrew P. Trends in Biotechnology, Vol. 36, Issue 2 https://doi.org/10.1016/j.tibtech.2017.07.007	journal	February 2018
Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellum Guss, Adam M.; Olson, Daniel G.; Caiazza, Nicky C. Biotechnology for Biofuels, Vol. 5, Issue 1 https://doi.org/10.1186/1754-6834-5-30	journal	January 2012
Low-mutation-rate, reduced-genome Escherichia coli: An improved host for faithful maintenance of engineered genetic constructs Csorgo, Balint; Feher, Tamas; Timar, Edit Microbial Cell Factories, Vol. 11, Issue 1 https://doi.org/10.1186/1475-2859-11-11	journal	January 2012
Simultaneous achievement of high ethanol yield and titer in Clostridium thermocellum Tian, Liang; Papanek, Beth; Olson, Daniel G. Biotechnology for Biofuels, Vol. 9, Issue 1 https://doi.org/10.1186/s13068-016-0528-8	journal	June 2016
Directed Evolution of Clostridium thermocellum β-Glucosidase A Towards Enhanced Thermostability Yoav, Shahar; Stern, Johanna; Salama-Alber, Orly International Journal of Molecular Sciences, Vol. 20, Issue 19 https://doi.org/10.3390/ijms20194701	journal	September 2019
Transcriptomic and proteomic changes from medium supplementation and strain evolution in high-yielding Clostridium thermocellum strains Papanek, Beth; O’Dell, Kaela B.; Manga, Punita Journal of Industrial Microbiology and Biotechnology, Vol. 45, Issue 11 https://doi.org/10.1007/s10295-018-2073-x	journal	November 2018
Transformation of Clostridium Thermocellum by Electroporation Olson, Daniel G.; Lynd, Lee R. Cellulases https://doi.org/10.1016/B978-0-12-415931-0.00017-3	book	January 2012
Implementation of the CRISPR-Cas9 system in fission yeast Jacobs, Jake Z.; Ciccaglione, Keith M.; Tournier, Vincent Nature Communications, Vol. 5, Issue 1 https://doi.org/10.1038/ncomms6344	journal	October 2014
The emergence of Clostridium thermocellum as a high utility candidate for consolidated bioprocessing applications Akinosho, Hannah; Yee, Kelsey; Close, Dan Frontiers in Chemistry, Vol. 2 https://doi.org/10.3389/fchem.2014.00066	journal	August 2014
Exploiting endogenous CRISPR-Cas system for multiplex genome editing in Clostridium tyrobutyricum and engineer the strain for high-level butanol production Zhang, Jie; Zong, Wenming; Hong, Wei Metabolic Engineering, Vol. 47 https://doi.org/10.1016/j.ymben.2018.03.007	journal	May 2018
Using an Endogenous CRISPR-Cas System for Genome Editing in the Human Pathogen Clostridium difficile Maikova, Anna; Kreis, Victor; Boutserin, Anaïs Applied and Environmental Microbiology, Vol. 85, Issue 20 https://doi.org/10.1128/AEM.01416-19	journal	August 2019

Similar Records

Recombineering machinery to increase homology directed genome editing in thermophilic microbes

Patent · Mon Jan 22 23:00:00 EST 2024 · OSTI ID:2541706

Engineering the Cellulolytic Bacterium, Clostridium Thermocellum, to Co-Utilize Hemicellulose

Journal Article · Mon Apr 15 00:00:00 EDT 2024 · Metabolic Engineering · OSTI ID:2349291

Related Subjects

BIOMASS FUELS
CRISPR-Cas
Clostridium thermocellum
anaerobe
cellulose
consolidated bioprocessing
recombinase
thermophile

CRISPR-Cas Genome Editing in the Cellulolytic Bacterium Clostridium thermocellum (C. thermocellum)

Citation Formats

References (26)

Similar Records

Related Subjects