Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
Show others and affiliations
2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 10392Article in journal (Refereed) Published
Abstract [en]

CRISPR-Cas systems are adaptive prokaryotic immune systems protecting against horizontally transferred DNA or RNA such as viruses and other mobile genetic elements. Memory of past invaders is stored as spacers in CRISPR loci in a process called adaptation. Here we developed a novel assay where spacer integration results in fluorescence, enabling detection of memory formation in single cells and quantification of as few as 0.05% cells with expanded CRISPR arrays in a bacterial population. Using this fluorescent CRISPR Adaptation Reporter (f-CAR), we quantified adaptation of the two CRISPR arrays of the type I-E CRISPR-Cas system in Escherichia coli, and confirmed that more integration events are targeted to CRISPR-II than to CRISPR-I. The f-CAR conveniently analyzes and compares many samples, allowing new insights into adaptation. For instance, we show that in an E. coli culture the majority of acquisition events occur in late exponential phase.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP , 2017. Vol. 7, article id 10392
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-335401DOI: 10.1038/s41598-017-10876-zISI: 000408997700091PubMedID: 28871175OAI: oai:DiVA.org:uu-335401DiVA, id: diva2:1162842
Funder
Swedish Research CouncilWenner-Gren FoundationsThe Royal Swedish Academy of SciencesScience for Life Laboratory - a national resource center for high-throughput molecular bioscienceAvailable from: 2017-12-05 Created: 2017-12-05 Last updated: 2018-02-28Bibliographically approved
In thesis
1. Small RNAs, Big Consequences: Post-transcriptional Regulation and Adaptive Immunity in Bacteria
Open this publication in new window or tab >>Small RNAs, Big Consequences: Post-transcriptional Regulation and Adaptive Immunity in Bacteria
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

It is nowadays widely accepted that non-coding RNAs play important roles in post-transcriptional regulation of genes in all kingdoms of life. In bacteria, the largest group of RNA regulators are the small RNAs (sRNAs). Almost all sRNAs act through anti-sense base-pairing with target mRNAs, and by doing so regulate their translation and/or stability. As important modulators of gene expression, sRNAs are involved in all aspects of bacterial physiology. My studies aimed to deepen our understanding of the mechanisms behind sRNA-mediated gene regulation. We have shown that translation of the di-guanylate-cyclase YdaM, a major player in the biofilm regulatory cascade, is repressed by the sRNAs OmrA and OmrB. OmrAB require the RNA chaperone protein Hfq for efficient regulation. Interestingly, our results suggest a non-canonical mechanism for Hfq-mediated ydaM-OmrA/B base-pairing. Instead of serving as RNA interaction platform, Hfq restructures the ydaM mRNA to enable sRNA binding. We also addressed the question of how bacteria utilize regulatory RNAs to create phenotypic heterogeneity by studying the role of the tisB/istR-1 type 1 toxin-antitoxin system in SOS-induced persister cell formation in E. coli.

In addition, I have investigated the prokaryotic CRISPR-Cas immune system, which has led to the development of two molecular tools. The CRISPR-Cas adaptive immune system consists of a CRISPR array, where palindromic repeats are interspaced by unique spacer sequences derived from foreign genetic elements, and the CRISPR-associated (Cas) proteins. In the adaptation stage, memory is created by insertion of spacer sequences into the CRISPR array. We developed a fluorescent reporter that accurately and sensitively detects spacer integration events (denoted: “acquisition”) in single cells and in real-time. In the effector stage of immunity, crRNAs, consisting of one spacer-repeat unit, associate with the Cas proteins to form a ribonucleoprotein complex that surveys the cell for invader DNA. Target identification occurs by base-pairing between the crRNA and the complementary sequence in the target nucleic acid, which triggers degradation. We repurposed the E. coli type I-E CRISPR-Cas effector complex Cascade for specific reprogrammable transcriptional gene silencing.

The studies presented herein thus contributes to our understanding of RNA-based target identification for gene regulation and adaptive immunity.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 83
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1637
Keywords
small RNA, sRNA, non-coding RNA, Hfq, gene regulation, post-transcriptional regulation, biofilm, OmrA, OmrB, toxin-antitoxin, TisB, IstR-1, Persisters, CRISPR-Cas, CRISPR-Cas adaptation reporter, Escherichia coli
National Category
Microbiology
Research subject
Biology with specialization in Microbiology
Identifiers
urn:nbn:se:uu:diva-343526 (URN)978-91-513-0252-2 (ISBN)
Public defence
2018-04-20, A1:111a, BMC, Husargatan 3, Uppsala, 09:30 (English)
Opponent
Supervisors
Available from: 2018-03-26 Created: 2018-02-28 Last updated: 2018-04-24

Open Access in DiVA

fulltext(3335 kB)9 downloads
File information
File name FULLTEXT01.pdfFile size 3335 kBChecksum SHA-512
52ba9bbc59bd8310d1cf721364c7b92b0b086070ae1cf3ab11f354df9f17945033fdd890badc5f6c618ac8fd6976340bbc2ce9ef13bf621b3958cc3e53130909
Type fulltextMimetype application/pdf

Other links

Publisher's full textPubMed

Search in DiVA

By author/editor
Amlinger, LinaHoekzema, MirtheWagner, Gerhart E. H.Koskiniemi, SannaLundgren, Magnus
By organisation
Microbiology
In the same journal
Scientific Reports
Biological Sciences

Search outside of DiVA

GoogleGoogle Scholar
Total: 9 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 91 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf