Introduction of a Recombineering Oligonucleotide and a CRISPR–Cas9 Gene-Targeting Plasmid into Staphylococcus aureus for Generating a Gene-Deletion Strain
- 1Section of Molecular Microbiology and Medical Research Council Center for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom
- 2Department of Laboratory Medicine and Pathology, School of Medicine, University of Washington, Seattle, Washington 98195, USA
- ↵3Correspondence: a.grundling{at}imperial.ac.uk; stevesal{at}uw.edu
Abstract
Gene deletions can be generated in Staphylococcus aureus using recombineering in combination with a CRISPR–Cas9 counterselection approach. The method involves first designing the recombineering oligonucleotides and generating the relevant plasmids, and then introducing these elements into S. aureus to generate the desired gene deletion. Here, we describe the second part of this workflow; the introduction of the gene-targeting plasmid and the recombineering oligonucleotide(s) into S. aureus to generate the gene-deletion strain. Specifically, we outline the steps to (1) generate the S. aureus recipient strain for the recombineering CRISPR–Cas9 counterselection method by introducing plasmid pCN-EF2132tet, (2) introduce the recombineering oligonucleotide(s) and gene-targeting plasmid into the pCN-EF2132tet plasmid-containing S. aureus strain, (3) confirm the gene deletion in S. aureus by colony PCR and sequencing, and (4) curate the plasmids following successful gene deletion. To illustrate the method, we give a specific example of how to generate a 55-bp deletion in the geh gene of S. aureus strain RN4220. The protocol, however, can be easily adapted to other strain backgrounds and to generate deletions in other genes.
Footnotes
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From the Experiments in Bacterial Genetics collection, edited by Lionello Bossi, Andrew Camilli, and Angelika Gründling.
