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Efficient programmable gene silencing by Cascade
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
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.
Visa övriga samt affilieringar
2015 (Engelska)Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, nr 1, s. 237-246Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Methods that permit controlled changes in the expression of genes are important tools for biological and medical research, and for biotechnological applications. Conventional methods are directed at individually changing each gene, its regulatory elements or its mRNA's translation rate. We demonstrate that the CRISPR-associated DNA-binding Cascade complex can be used for efficient, long-lasting and programmable gene silencing. When Cascade is targeted to a promoter sequence the transcription of the downstream gene is inhibited, resulting in dramatically reduced expression. The specificity of Cascade binding is provided by the integral crRNA component, which is easily designed to target virtually any stretch of DNA. Cascade targeted to the ORF sequence of the gene can also silence expression, albeit at lower efficiency. The system can be used to silence plasmid and chromosome targets, simultaneously target several genes and is active in different bacterial species and strains. The findings described here are an addition to the expanding range of CRISPR-based technologies and may be adapted to additional organisms and cell systems.

Ort, förlag, år, upplaga, sidor
2015. Vol. 43, nr 1, s. 237-246
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:uu:diva-249042DOI: 10.1093/nar/gku1257ISI: 000350207100026PubMedID: 25435544OAI: oai:DiVA.org:uu-249042DiVA, id: diva2:807442
Tillgänglig från: 2015-04-23 Skapad: 2015-04-10 Senast uppdaterad: 2018-02-28Bibliografiskt granskad
Ingår i avhandling
1. The type I-E CRISPR-Cas system: Biology and applications of an adaptive immune system in bacteria
Öppna denna publikation i ny flik eller fönster >>The type I-E CRISPR-Cas system: Biology and applications of an adaptive immune system in bacteria
2017 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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. 

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2017. s. 61
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1466
Nyckelord
CRISPR, CRISPR-Cas, virus defense, bacteria, bacteriophage, adaptation, spacer integration, interference, gene silencing, fluorescent reporter, Escherichia coli
Nationell ämneskategori
Mikrobiologi
Forskningsämne
Mikrobiologi
Identifikatorer
urn:nbn:se:uu:diva-312234 (URN)978-91-554-9787-3 (ISBN)
Disputation
2017-02-24, A1:111a, BMC, Husargatan 3, Uppsala, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2017-02-02 Skapad: 2017-01-08 Senast uppdaterad: 2017-02-07
2. 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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