Oligonucleotide Design and Construction of a Gene-Targeting CRISPR-Cas9 Plasmid in Escherichia coli for Generating a Gene-Deletion Strain in Staphylococcus aureus .

Gene deletions can be constructed in 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 to 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 the gene will be presented as a specific example. To this end, we describe the use of gene-specific recombineering oligonucleotides and the construction of a gene-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-, plasmid recovery in , and confirmation by colony PCR and sequencing. The method can easily be adapted to design deletions for other genes.