LAUSR

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.