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Quantification of CRISPR-Cas spacer integration using a fluorescent reporter
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.
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(English)Manuscript (preprint) (Other academic)
National Category
Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-312231OAI: oai:DiVA.org:uu-312231DiVA: diva2:1062723
Available from: 2017-01-08 Created: 2017-01-08 Last updated: 2017-01-09
In thesis
1. The type I-E CRISPR-Cas system: Biology and applications of an adaptive immune system in bacteria
Open this publication in new window or tab >>The type I-E CRISPR-Cas system: Biology and applications of an adaptive immune system in bacteria
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

CRISPR-Cas systems are adaptive immune systems in bacteria and archaea, consisting of a clustered regularly interspaced short palindromic repeats (CRISPR) array and CRISPR associated (Cas) proteins. In this work, the type I-E CRISPR-Cas system of Escherichia coli was studied.

CRISPR-Cas immunity is divided into three stages. In the first stage, adaptation, Cas1 and Cas2 store memory of invaders in the CRISPR array as short intervening sequences, called spacers. During the expression stage, the array is transcribed, and subsequently processed into small CRISPR RNAs (crRNA), each consisting of one spacer and one repeat. The crRNAs are bound by the Cascade multi-protein complex. During the interference step, Cascade searches for DNA molecules complementary to the crRNA spacer. When a match is found, the target DNA is degraded by the recruited Cas3 nuclease.

Host factors required for integration of new spacers into the CRISPR array were first investigated. Deleting recD, involved in DNA repair, abolished memory formation by reducing the concentration of the Cas1-Cas2 expression plasmid, leading to decreased amounts of Cas1 to levels likely insufficient for spacer integration. Deletion of RecD has an indirect effect on adaptation. To facilitate detection of adaptation, a sensitive fluorescent reporter was developed where an out-of-frame yfp reporter gene is moved into frame when a new spacer is integrated, enabling fluorescent detection of adaptation. Integration can be detected in single cells by a variety of fluorescence-based methods. A second aspect of this thesis aimed at investigating spacer elements affecting target interference. Spacers with predicted secondary structures in the crRNA impaired the ability of the CRISPR-Cas system to prevent transformation of targeted plasmids. Lastly, in absence of Cas3, Cascade was successfully used to inhibit transcription of specific genes by preventing RNA polymerase access to the promoter.

The CRISPR-Cas field has seen rapid development since the first demonstration of immunity almost ten years ago. However, much research remains to fully understand these interesting adaptive immune systems and the research presented here increases our understanding of the type I-E CRISPR-Cas system. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. 61 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1466
Keyword
CRISPR, CRISPR-Cas, virus defense, bacteria, bacteriophage, adaptation, spacer integration, interference, gene silencing, fluorescent reporter, Escherichia coli
National Category
Microbiology
Research subject
Microbiology
Identifiers
urn:nbn:se:uu:diva-312234 (URN)978-91-554-9787-3 (ISBN)
Public defence
2017-02-24, A1:111a, BMC, Husargatan 3, Uppsala, 09:00 (English)
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Supervisors
Available from: 2017-02-02 Created: 2017-01-08 Last updated: 2017-02-07

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