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J Biotechnol. 2015 Apr 20;200:1-5. doi: 10.1016/j.jbiotec.2015.02.005. Epub 2015 Feb 11.

Markerless chromosomal gene deletion in Clostridium beijerinckii using CRISPR/Cas9 system.

Author information

1
Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
2
Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
3
Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
4
Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
5
Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; The Integrated Bioprocessing Research Laboratory (IBRL), University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. Electronic address: blaschek@illinois.edu.

Abstract

The anaerobic spore-forming, gram-positive, solventogenic clostridia are notorious for being difficult to genetically engineer. Based on CRISPR/Cas9 assisted homologous recombination, we demonstrated that clean markerless gene deletion from the chromosome can be easily achieved with a high efficiency through a single-step transformation in Clostridium beijerinckii NCIMB 8052, one of the most prominent strains for acetone, butanol and ethanol (ABE) production. This highly efficient genome engineering system can be further explored for multiplex genome engineering purposes. The protocols and principles developed in this study provided valuable references for genome engineering in other microorganisms lacking developed genetic engineering tools.

KEYWORDS:

Butanol; CRISPR/Cas9; Clostridium; Genome engineering; Homologous recombination

PMID:
25680931
DOI:
10.1016/j.jbiotec.2015.02.005
[Indexed for MEDLINE]

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