Genome Editing in Staphylococcus aureus by Conditional Recombineering and CRISPR/Cas9-Mediated Counterselection

Methods for the genetic manipulation of S. aureus have historically proven challenging, which has hindered experimental studies of this organism. We recently developed a system for recombineering and CRISPR/Cas9-mediated counterselection in S. aureus which utilizes commercially synthesized synthetic DNA oligonucleotides as substrates for introducing precise genomic modifications into the organism and for performing lethal counterselection of unedited cells. These techniques make it possible to scalably and inexpensively engineer desired genomic changes into laboratory or clinical S. aureus strains, using electroporation to introduce the effector plasmid vectors and oligonucleotides. Here we describe detailed protocols for performing genome editing of S. aureus in order to produce isogenic strains using this system and detail general principles which are broadly applicable across a range of organisms for which equivalent systems have been established.