Identification of Editable Sites, Spacer Oligonucleotide Design, and Generation of the Gene-Targeting CRISPR–nCas9 Plasmid for Gene Disruption inStaphylococcus aureusUsing the CRISPR–nCas9 and Cytidine Deaminase System

Methods for gene disruption are essential for functional genomics, and there are multiple approaches for altering gene function in bacteria. One of these methods involves introducing a premature stop codon in a gene of interest, which can be achieved by using the CRISPR–nCas9-cytidine deaminase system. The approach involves the mutation of editable cytidines to thymidines, with the goal of generating a novel stop codon that ultimately results in a nonfunctional gene product. The workflow involves two major sections, one for the identification of editable cytidines, the design of the targeting spacer oligonucleotides for introduction into the CRISPR–nCas9 cytidine deaminase plasmid, and the construction of the gene-targeting CRISPR–nCas9 cytosine deaminase plasmids, and one for the actual introduction of the mutation in the species of interest. Here, we describe the steps for the first part. To better illustrate the method and oligonucleotide design, we describe the construction of Staphylococcus aureus RN4220 geh mutants with C to T base changes at two different positions, leading to the construction of strains RN4220- geh (160stop) and RN4220 -geh (712stop). We outline the steps for (1) the identification of editable cytidines within genes using the CRISPR-CBEI toolkit website, and (2) the design of the targeting spacer oligonucleotides for introduction into the CRISPR–nCas9 cytidine deaminase plasmid pnCasSA-BEC, followed by (3) the construction of the gene-targeting (in this example, geh gene-targeting) CRISPR–nCas9 cytosine deaminase plasmids pnCasSA-BEC- gehC160T and pnCasSA-BEC- gehC712T using the Golden Gate assembly method, plasmid recovery in Escherichia coli , and confirmation by colony PCR and sequencing. The method can be easily adapted to construct gene-inactivation mutants in other S. aureus genes.