Abstract
Clostridium difficile is often the primary cause of nosocomial diarrhea, leading to thousands of deaths annually worldwide. The availability of an efficient genome editing tool for C. difficile is essential to understanding its pathogenic mechanism and physiological behavior. Here, we describe a streamlined CRISPR-Cpf1-based protocol to achieve precise genome editing in C. difficile with high efficiencies. Our work highlighted the first application of CRISPR-Cpf1 for genome editing in C. difficile, which are both crucial for understanding pathogenic mechanism of C. difficile and developing strategies to fight against C. difficile infection (CDI). In addition, for the DNA cloning, we developed a one-step-assembly protocol along with a Python-based algorithm for automatic primer design, shortening the time for plasmid construction to half that of conventional procedures. Approaches we developed herein are easily and broadly applicable to other microorganisms. Our results provide valuable guidance for establishing CRISPR-Cpf1 as a versatile genome engineering tool in prokaryotic cells.
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Acknowledgments
This work was supported by Guizhou Provincial Natural Science Foundation (Grant No. [2020]1Z067), the United States Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) Hatch project (ALA014-1017025), and the Alabama Agricultural Experiment Station. Dr. Wei Hong is a recipient of scholarship for visiting scholars offered by the China Scholarship Council (CSC). We thank Dr. Shonna McBride (Emory University) for providing C. difficile 630 strain, Dr. Hans Blaschek (University of Illinois at Urbana−Champaign) for providing pYW51, Dr. Mike Young (Aberystwyth University, UK) for providing E. coli CA434, and Dr. Nigel Minton (University of Nottingham, UK) for providing pMTL82151.
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Hong, W., Zhang, J., Cui, G., Zhou, Q., Wang, P., Wang, Y. (2022). Highly Efficient Genome Editing in Clostridium difficile Using the CRISPR-Cpf1 System. In: Reisch, C.R. (eds) Recombineering. Methods in Molecular Biology, vol 2479. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2233-9_12
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DOI: https://doi.org/10.1007/978-1-0716-2233-9_12
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