Introduction of a CRISPR–nCas9 Gene-Targeting Plasmid intoStaphylococcus aureusfor Gene Disruption

Abstract

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 second part. Specifically, we describe (1) how to introduce the gene-targeting pnCasSA-BEC plasmid intoStaphylococcus aureus, (2) how the gene inactivation inS. aureuscan be confirmed by PCR and sequencing, and (3) how, following successful gene inactivation, the strain can be cured of the pnCasSA-BEC plasmid. To better illustrate the method, and as specific example, two differentgehgene-inactivation mutations are generated here inS. aureusRN4220. The protocol, however, can easily be adapted to generate other gene-inactivating mutations.