Construction of a Gene Knockdown System Based on Catalytically Inactive (“Dead”) Cas9 (dCas9) in Staphylococcus aureus

Abstract

ABSTRACT There has been an absence of an efficient method of gene knockdown in the important human pathogen Staphylococcus aureus like RNA interference in eukaryotes. The previously developed antisense RNA technology is mainly applied for forward genetic screening but is rather limited in specific gene knockdown because of the lack of rational antisense RNA design strategies. Here we report an efficient and specific system for gene knockdown in S. aureus based on the type II clustered regularly interspaced short palindromic repeat (CRISPR) system from Streptococcus pyogenes . We can achieve gene silencing with the coexpression of dCas9, an RNA-guided DNA binding protein, and a small guide RNA complementary to the target gene. With this system, we have successfully silenced diverse sets of genes varying in size and expression level in different S. aureus strains. This system exhibited high-efficiency knockdown of both essential and nonessential genes, and its effect is inducible and reversible. In addition, the system can repress the expression of multiple genes simultaneously and silence an entire operon or part of it. This RNA-guided DNA targeting system thus provides a simple, rapid, and affordable method for selective gene knockdown in S. aureus . IMPORTANCE Staphylococcus aureus is an important human and animal pathogen that can cause a diversity of infectious diseases. Molecular genetic study of S. aureus has provided an avenue for the understanding of its virulence, pathogenesis, and drug resistance, leading to the discovery of new therapies for the treatment of staphylococcal infections. However, methodologies developed for genetic manipulation of S. aureus usually involve either low efficiency or laborious procedures. Here we report an RNA-guided system for gene knockdown in S. aureus and show its high efficiency and simplicity for selective gene silencing in different strains of S. aureus . This simple, rapid, and affordable system may serve as a promising tool for functional gene study in S. aureus , especially for the study of essential genes, thus facilitating the understanding of this pathogen and its interaction with its hosts.