CRISPRi enables studies of enterococcal biofilm initiation, maturation and maintenance.

Among the Enterococci, Enterococcus faecalis is most frequently associated with human infections ranging from the urinary tract and wound infection to endocarditis and bacteraemia. These infections are often multidrug-resistant and, hence, life-threatening. Moreover, E. faecalis are often co-isolated with other pathogenic bacteria from polymicrobial biofilm-associated infections contributing to disease progression and poorer patient outcomes. Genetic tools to dissect complex interactions in biofilms and mixed microbial communities are largely limited to transposon mutagenesis and traditional allelic exchange methods requiring time- and labour-intensive two-step integration and excision screening that can take a week or more to make a single mutant. We built upon the well-characterized CRISPR interference system using streptococcal dCas9 to develop an easily-modifiable, inducible system for E. faecalis that can efficiently silence single and multiple genes in a matter of hours. We show that this system can silence genes involved in biofilm formation, antibiotic resistance, and can be used to interrogate gene essentiality. Uniquely, this tool is optimized to study genes important for biofilm initiation, maturation, and maintenance, and can be used to perturb pre-formed biofilms. This inducible CRISPRi system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal regulation networks within the biofilms, including polymicrobial biofilms.