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Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems
Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig, Germany.
Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig, Germany.
Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Deptartment of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, Austria.
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2014 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 4, 2577-2590 p.Article in journal (Refereed) Published
Abstract [en]

The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA: crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA: Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool.

Place, publisher, year, edition, pages
2014. Vol. 42, no 4, 2577-2590 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-87653DOI: 10.1093/nar/gkt1074ISI: 000332381000048OAI: oai:DiVA.org:umu-87653DiVA: diva2:710912
Available from: 2014-04-08 Created: 2014-04-07 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Multifaceted RNA-mediated regulatory mechanisms in Streptococcus pyogenes
Open this publication in new window or tab >>Multifaceted RNA-mediated regulatory mechanisms in Streptococcus pyogenes
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Bacterial pathogens rely on precise regulation of gene expression to coordinate host infection processes and resist invasion by mobile genetic elements. An interconnected network of protein and RNA regulators dynamically controls the expression of virulence factors using a variety of mechanisms. In this thesis, the role of selected regulators, belonging to the class of small RNAs (sRNAs), is investigated.

Streptococcus pyogenes is a pathogen responsible for a wide range of human diseases. Genome-wide screenings have indicated that S. pyogenes encodes numerous sRNAs, yet only a limited number have been characterized. A major goal of this study was to identify and characterize novel sRNAs and antisense RNAs (asRNAs) using RNA sequencing analysis. We validated 30 novel sRNAs and asRNAs, and identified 9 sRNAs directly cleaved by the ribonucleases RNase III and/or RNase Y.

Previous work from the laboratory has highlighted the role of sRNAs from the type II Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins (CRISPR-Cas) systems in S. pyogenes. CRISPR-Cas systems provide adaptive immunity to prokaryotes against infection by mobile genetic elements. Two sRNAs, forming a complementary duplex (dual-RNA), are effectors of this system: the mature CRISPR RNAs (crRNAs) and the trans-activating crRNA (tracrRNA). The dual-RNA guides the Cas9 endonuclease to cleave both strands of the invading DNA in a sequence-specific manner. This RNA-programmable CRISPR-Cas9 system is now utilized for genome editing and engineering in a wide range of cells and organisms. To expand the potentialities of this tool, we both, searched for Cas9 orthologs and predicted numerous tracrRNA orthologs. We defined tracrRNA as a new family of sRNAs sharing the ability to base-pair to cognate crRNAs, without conservation of structure, sequence or location. We show that Cas9 and the dual tracrRNA:crRNAs are only interchangeable between closely related type II CRISPR-Cas systems.

In summary, this thesis presents new insights into RNA-mediated regulatory mechanisms in S. pyogenes. We identified and described the expression of novel sRNAs, highlighting potential antisense RNAs. Focusing on the dual-RNA programmable type II CRISPR-Cas system, we provided evidence for co-evolution of the Cas9 enzyme with tracrRNA:crRNA, a basis for Cas9 multiplexing in genome editing.

Place, publisher, year, edition, pages
Umeå: Umeå university, 2015. 75 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1732
Keyword
Streptococcus pyogenes, small RNAs, CRISPR, Cas9, tracrRNA, RNases, gene expression, RNA sequencing
National Category
Microbiology Biochemistry and Molecular Biology
Research subject
Infectious Diseases; Microbiology
Identifiers
urn:nbn:se:umu:diva-111090 (URN)978-91-7601-304-5 (ISBN)
Public defence
2015-12-14, Unod R1, Hörsal E04, Byggnad 6E, NUS - Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2015-11-10 Created: 2015-11-04 Last updated: 2015-11-09Bibliographically approved

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Fonfara, InesLe Rhun, AnaïsChylinski, KrzysztofLécrivain, Anne-LaureBzdrenga, JanekCharpentier, Emmanuelle
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