Using CRISPR–Cas9-Based Methods for Genome Editing inStaphylococcus aureus

Chromosomal mutations and targeted gene deletions and inactivations inStaphylococcus aureusare typically generated using the allelic exchange method. In recent years, however, more rapid methods have been developed, often using CRISPR–Cas9-based systems. Here, we describe recently developed CRISPR–Cas9-based plasmid systems for use inS. aureus,and discuss their use for targeted gene mutation and inactivation. First, we describe how a CRISPR–Cas9 counterselection strategy can be combined with a recombineering strategy to generate gene deletions inS. aureus. We then introduce dead Cas9 (dCas9) and Cas9 nickase (nCas9) enzymes, and discuss how the nCas9 enzyme fused to different nucleoside deaminases can be used to introduce specific base changes in target genes. We then discuss how the nCas9-deaminase fusion enzymes can be used for targeted gene inactivation via the introduction of premature stop codons or by mutating the start codon. Together, these tools highlight the power and potential of CRISPR–Cas9-based methods for genome editing inS. aureus.