Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

Multiplex CRISPRi-Cas9 silencing of planktonic and stage-specific biofilm genes in Enterococcus faecalis

Irina Afonina, June Ong, Jerome Chua, Timothy Lu, View ORCID ProfileKimberly A. Kline
doi: https://doi.org/10.1101/2020.04.30.071571
Irina Afonina
aSingapore–MIT Alliance for Research and Technology, Antimicrobial Drug Resistance Interdisciplinary Research Group, Singapore 138602
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
June Ong
bSchool of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jerome Chua
bSchool of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Timothy Lu
aSingapore–MIT Alliance for Research and Technology, Antimicrobial Drug Resistance Interdisciplinary Research Group, Singapore 138602
cElectrical Engineering and Computer Science, MIT, Cambridge, MA 02139, USA
dDepartment of Biological Engineering, MIT, Cambridge, MA 02139, USA.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Kimberly A. Kline
aSingapore–MIT Alliance for Research and Technology, Antimicrobial Drug Resistance Interdisciplinary Research Group, Singapore 138602
bSchool of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551
dDepartment of Biological Engineering, MIT, Cambridge, MA 02139, USA.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Kimberly A. Kline
  • For correspondence: kkline@ntu.edu.sg
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Preview PDF
Loading

ABSTRACT

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 multi-level regulation. Compared to established knockout methodologies, CRISPRi approaches enable 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 non-template and template strand targeting. Since 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-bacteria interactions, and inter-species communication.

IMPORTANCE Enterococcus faecalis causes multidrug resistant life-threatening infections, and is often co-isolated 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 labour-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, 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 system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal biology.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
Back to top
PreviousNext
Posted May 01, 2020.
Download PDF
Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
Multiplex CRISPRi-Cas9 silencing of planktonic and stage-specific biofilm genes in Enterococcus faecalis
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Multiplex CRISPRi-Cas9 silencing of planktonic and stage-specific biofilm genes in Enterococcus faecalis
Irina Afonina, June Ong, Jerome Chua, Timothy Lu, Kimberly A. Kline
bioRxiv 2020.04.30.071571; doi: https://doi.org/10.1101/2020.04.30.071571
Digg logo Reddit logo Twitter logo Facebook logo Google logo LinkedIn logo Mendeley logo
Citation Tools
Multiplex CRISPRi-Cas9 silencing of planktonic and stage-specific biofilm genes in Enterococcus faecalis
Irina Afonina, June Ong, Jerome Chua, Timothy Lu, Kimberly A. Kline
bioRxiv 2020.04.30.071571; doi: https://doi.org/10.1101/2020.04.30.071571

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Microbiology
Subject Areas
All Articles
  • Animal Behavior and Cognition (3686)
  • Biochemistry (7774)
  • Bioengineering (5668)
  • Bioinformatics (21245)
  • Biophysics (10563)
  • Cancer Biology (8162)
  • Cell Biology (11915)
  • Clinical Trials (138)
  • Developmental Biology (6738)
  • Ecology (10388)
  • Epidemiology (2065)
  • Evolutionary Biology (13843)
  • Genetics (9694)
  • Genomics (13056)
  • Immunology (8123)
  • Microbiology (19956)
  • Molecular Biology (7833)
  • Neuroscience (42973)
  • Paleontology (318)
  • Pathology (1276)
  • Pharmacology and Toxicology (2256)
  • Physiology (3350)
  • Plant Biology (7208)
  • Scientific Communication and Education (1309)
  • Synthetic Biology (1999)
  • Systems Biology (5528)
  • Zoology (1126)