Oligonucleotide Design and Construction of a Gene-Targeting CRISPR–Cas9 Plasmid inEscherichia colifor Generating a Gene-Deletion Strain inStaphylococcus aureus

Abstract

Gene deletions can be constructed 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 first part of this workflow, oligonucleotide design and plasmid generation. To better illustrate the method and oligonucleotide design, the construction of a 55-bp out-of-frame deletion in theS. aureus gehgene will be presented as a specific example. To this end, we describe the use ofgehgene–specific recombineering oligonucleotides and the construction of agehgene–targeting CRISPR–Cas9 plasmid. The protocol is divided into three parts: (1) design of the gene-specific targeting spacer oligonucleotides for introduction into the CRISPR–Cas9 plasmid pCas9-counter, (2) design of 90-mer recombineering oligonucleotides to generate a 55-bp out-of-frame gene deletion, and (3) construction of the gene-targeting CRISPR–Cas9 plasmid pCas9-geh, plasmid recovery inEscherichia coli, and confirmation by colony PCR and sequencing. The method can easily be adapted to design deletions for otherS. aureusgenes.