Introduction of a Recombineering Oligonucleotide and a CRISPR–Cas9 Gene-Targeting Plasmid intoStaphylococcus aureusfor Generating a Gene-Deletion Strain

Abstract

Gene deletions can be generated inStaphylococcus aureususing 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 intoS. aureusto 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) intoS. aureusto generate the gene-deletion strain. Specifically, we outline the steps to (1) generate theS. aureusrecipient 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-containingS. aureusstrain, (3) confirm the gene deletion inS. aureusby 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 thegehgene ofS. aureusstrain RN4220. The protocol, however, can be easily adapted to other strain backgrounds and to generate deletions in other genes.