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.
Bibliographical noteFunding Information:
This work was supported by U.S. Department of Energy (DOE) grant no. 2011-01219 to HPB. We thank Dr. Roderick I. Mackie for sharing his lab facilities. We also thank Mr. Wenyan Jiang (Dr. Luciano A. Marraffini group at The Rockefeller University), Dr. Esteban Toro (Dr. Adam P. Arkin group at UC-Berkeley), Dr. Jason Peters (Dr. Carol Gross group at UC-San Francisco), and Dr. Martin Jinek (Dr. Jennifer Doudna group at UC-Berkeley) for their helpful discussions.
- Genome engineering
- Homologous recombination