Multiplex CRISPRi System Enables the Study of Stage-Specific Biofilm Genetic Requirements in Enterococcus faecalis
| العنوان: | Multiplex CRISPRi System Enables the Study of Stage-Specific Biofilm Genetic Requirements in |
|---|---|
| المؤلفون: | June Ong, Irina Afonina, Kimberly A. Kline, Jerome Chua, Timothy K. Lu |
| المساهمون: | School of Biological Sciences, Singapore Centre for Environmental Life Sciences and Engineering (SCELSE) |
| المصدر: | mBio, Vol 11, Iss 5 (2020) mBio |
| بيانات النشر: | American Society for Microbiology, 2020. |
| سنة النشر: | 2020 |
| مصطلحات موضوعية: | Molecular Biology and Physiology, Computational biology, Enterococcus Faecalis, medicine.disease_cause, Microbiology, Enterococcus faecalis, Antibiotic resistance, Bacterial Proteins, Virology, medicine, Gene silencing, Gene, CRISPR interference, Genes, Essential, biology, Biofilm, Biological sciences [Science], food and beverages, Pathogenic bacteria, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, QR1-502, CRISPR Interference, gene essentiality, Ebp pili, bacteria, Transposon mutagenesis, CRISPR-Cas Systems, biofilms, Research Article |
| الوصف: | Enterococcus faecalis causes multidrug-resistant life-threatening infections and is often coisolated with other pathogenic bacteria from polymicrobial biofilm-associated infections. Genetic tools to dissect complex interactions in mixed microbial communities are largely limited to transposon mutagenesis and traditional time- and labor-intensive allelic-exchange methods. Built upon streptococcal dCas9, we developed an easily modifiable, inducible CRISPRi system for E. faecalis that can efficiently silence single and multiple genes. This system can silence genes involved in biofilm formation and 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 preformed biofilms. This system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal biology. Enterococcus faecalis is an opportunistic pathogen, which can cause multidrug-resistant life-threatening infections. Gaining a complete understanding of enterococcal pathogenesis is a crucial step in identifying a strategy to effectively treat enterococcal infections. However, bacterial pathogenesis is a complex process often involving a combination of genes and multilevel regulation. Compared to established knockout methodologies, CRISPR interference (CRISPRi) approaches enable the rapid and efficient silencing of genes to interrogate gene products and pathways involved in pathogenesis. As opposed to traditional gene inactivation approaches, CRISPRi can also be quickly repurposed for multiplexing or used to study essential genes. Here, we have developed a novel dual-vector nisin-inducible CRISPRi system in E. faecalis that can efficiently silence via both nontemplate and template strand targeting. Since the nisin-controlled gene expression system is functional in various Gram-positive bacteria, the developed CRISPRi tool can be extended to other genera. This system can be applied to study essential genes, genes involved in antimicrobial resistance, and genes involved in biofilm formation and persistence. The system is robust and can be scaled up for high-throughput screens or combinatorial targeting. This tool substantially enhances our ability to study enterococcal biology and pathogenesis, host-bacterium interactions, and interspecies communication. |
| وصف الملف: | application/pdf |
| اللغة: | English |
| تدمد: | 2150-7511 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::5db2e336f148716f53777485da7e0ae5 https://journals.asm.org/doi/10.1128/mBio.01101-20 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....5db2e336f148716f53777485da7e0ae5 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 21507511 |
|---|