Abstract
CRISPR-Cas9 has been explored as a transformative genome engineering tool for many eukaryotic organisms. However, its utilization in bacteria remains limited and ineffective. This chapter, taking Clostridium beijerinckii as an example, describes the use of Streptococcus pyogenes CRISPR-Cas9 system guided by the single chimeric guide RNA (gRNA) for diverse genome-editing purposes, including chromosomal gene deletion, integration, single nucleotide modification, as well as “clean” mutant selection. The general principle is to use CRISPR-Cas9 as an efficient selection tool for the edited mutant (whose CRISPR-Cas9 target site has been disrupted through a homologous recombination event and thus can survive selection) against? the wild type background cells. This protocol is broadly applicable to other microorganisms for genome-editing purposes.
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Acknowledgments
This work was supported by Department of Energy (DOE) grant #2011-01219 to HPB. We thank Dr. Terry Papoutsakis for providing the pKO_mazF plasmid. We also thank Dr. Wenyan Jiang (from Dr. Luciano A. Marraffini’s group at The Rockefeller University), Dr. Esteban Toro (from Dr. Adam P. Arkin’s group at UC-Berkeley), Dr. Jason Peters (from Dr. Carol Gross’ group at UC-San Francisco), and Dr. Martin Jinek (from Dr. Jennifer Doudna’s group at UC-Berkeley) for their helpful discussions. We acknowledge www.somersault1824.com for allowing us to use their library to generate Figs. 1 and 4.
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Zhang, ZT. et al. (2018). Bacterial Genome Editing with CRISPR-Cas9: Taking Clostridium beijerinckii as an Example. In: Braman, J. (eds) Synthetic Biology. Methods in Molecular Biology, vol 1772. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7795-6_17
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DOI: https://doi.org/10.1007/978-1-4939-7795-6_17
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