Construction And Application Of A DCas9-based Gene Knockdown System In Staphylococcus Aureus

Staphylococcus aureus is an important opportunistic pathogen of both human and animal. It is frequently found in the environment and on the skin and mucous membrane of humans, causing a wide spectrum of nosocomial and community-associated infections with high morbidity and mortality. Genetic studies of S. aureus has provided an avenue for the molecular understanding of its virulence, pathogenesis and drug resistance, contributing to the discovery of new drug targets and new therapies to treat staphylococcal infections.However, methodologies developed for genetic manipulation of S. aureus usually involve either low efficiency or laborious procedures. Moreover, there has been constant absence of a both easy and efficient gene knockdown method in S. aureus.In this study, we constructed an efficient and simple gene knockdown system called CRISPRi (CRISPR inteference) in S. aureus based on a RNA-guided DNA binding protein, dCas9. Specific gene knockdown can be achieved with the co-expression of dCas9 and a small guide RNA (sgRNA) complementary to the target gene. An anhydrotetracycline (ATc) inducible promoter is used for dcas9 expression to make the gene knockdown inducible and a special restriction enzyme Sapl has been chosen to profoundly simplify the cloning of knockdown plasmid.With CRISPRi system, we have successfully knocked down diverse sets of genes varying in size and expression level in different S. aureus strains. We also show that this system can be adapted for functional studies of essential genes and operon genes by selectively repressing an entire operon or part of it. Besides, by expressing multiple sgRNAs together, this system can knock down multiple genes simultaneously or achieve combinatorial repression of a single gene. We have also explored the possibility of using Cas9, a RNA-guided nuclease for genome editing in S. aureus, but this method seems not applicable since the low efficiency of the DNA repair system in S.aureus.CRISPRi system can be used for study of both essential and nonessential genes in S. aureus with incomparable advantage over other methods in both simplicity and efficiency. We believe that this simple, rapid and affordable selective gene knockdown system will serve as a promising tool for functional gene study in S. aureus, thus facilitating the understanding of this pathogen and fighting against its infections.