Harnessing CRISPR interference for controlled gene expression in Group A Streptococcus
Essential genes in the important human pathogen Streptococcus pyogenes (Group A Strep or GAS) are very poorly characterised, partly due to the limited molecular toolbox available to control GAS gene expression combined with its low genetic transformability. CRISPR interference (CRISPRi) employing catalytically inactive S. pyogenes Cas9 (dCas9) has proven a powerful approach to specifically repress gene expression in both bacteria and eukaryotes. Here, we created a highly transformable, fully virulent serotype M1T1 GAS strain and introduced a doxycycline-inducible CRISPRi system to efficiently repress gene expression. The tools and approaches described here will facilitate fundamental research in GAS, vaccine development and antibiotic target discovery.