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Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
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2017 (Engelska)Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, artikel-id 10392Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
NATURE PUBLISHING GROUP , 2017. Vol. 7, artikel-id 10392
Nationell ämneskategori
Cell- och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:uu:diva-335401DOI: 10.1038/s41598-017-10876-zISI: 000408997700091PubMedID: 28871175OAI: oai:DiVA.org:uu-335401DiVA, id: diva2:1162842
Forskningsfinansiär
VetenskapsrådetWenner-Gren StiftelsernaKungliga VetenskapsakademienScience for Life Laboratory - a national resource center for high-throughput molecular bioscienceTillgänglig från: 2017-12-05 Skapad: 2017-12-05 Senast uppdaterad: 2020-02-04Bibliografiskt granskad
Ingår i avhandling
1. Small RNAs, Big Consequences: Post-transcriptional Regulation and Adaptive Immunity in Bacteria
Öppna denna publikation i ny flik eller fönster >>Small RNAs, Big Consequences: Post-transcriptional Regulation and Adaptive Immunity in Bacteria
2018 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2018. s. 83
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1637
Nyckelord
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
Nationell ämneskategori
Mikrobiologi
Forskningsämne
Biologi med inriktning mot mikrobiologi
Identifikatorer
urn:nbn:se:uu:diva-343526 (URN)978-91-513-0252-2 (ISBN)
Disputation
2018-04-20, A1:111a, BMC, Husargatan 3, Uppsala, 09:30 (Engelska)
Opponent
Handledare
Tillgänglig från: 2018-03-26 Skapad: 2018-02-28 Senast uppdaterad: 2018-04-24

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