Abstract
The clustered regularly interspersed short palindromic repeat (CRISPR)/Cas9 system has emerged as an efficient genome engineering method attributed to its high efficiency and versatility. By generating a lethal double-strand DNA break in the target genome, the CRISPR/Cas9 system is capable of selecting the separated crossover events occurring in the traditional genetic manipulation methods in one step, therefore enabling rapid and efficient genome editing in Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA). By engineering the fusion of a cytidine deaminase APOBEC1 and a Cas9 nickase, a base editor was further developed as a highly efficient gene inactivation and point mutation tool in S. aureus. Here we describe a detailed protocol for CRISPR/Cas9-based genome editing in S. aureus, including genome modification and base editing. This protocol outlines in detail the design of primers, the construction and transformation of editing plasmids, as well as the verification of sequence-specific CRISPR/Cas9-mediated mutagenesis in S. aureus.
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Acknowledgment
This work was financially supported by the National Natural Science Foundation of China (91753127 and 31700123) to Q.J. and the Shanghai Sailing Program (18YF1416500) to W.C.
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Chen, W., Ji, Q. (2020). Genetic Manipulation of MRSA Using CRISPR/Cas9 Technology. In: Ji, Y. (eds) Methicillin-Resistant Staphylococcus Aureus (MRSA) Protocols. Methods in Molecular Biology, vol 2069. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9849-4_9
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DOI: https://doi.org/10.1007/978-1-4939-9849-4_9
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Publisher Name: Humana, New York, NY
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