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The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems
Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
2013 (English)In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 10, no 5, 726-737 p.Article in journal (Refereed) Published
Abstract [en]

CRISPR-Cas is a rapidly evolving RNA-mediated adaptive immune system that protects bacteria and archaea against mobile genetic elements. The system relies on the activity of short mature CRISPR RNAs (crRNAs) that guide Cas protein(s) to silence invading nucleic acids. A set of CRISPR-Cas, type II, requires a trans-activating small RNA, tracrRNA, for maturation of precursor crRNA (pre-crRNA) and interference with invading sequences. Following co-processing of tracrRNA and pre-crRNA by RNase III, dual-tracrRNA:crRNA guides the CRISPR-associated endonuclease Cas9 (Csn1) to cleave site-specifically cognate target DNA. Here, we screened available genomes for type II CRISPR-Cas loci by searching for Cas9 orthologs. We analyzed 75 representative loci, and for 56 of them we predicted novel tracrRNA orthologs. Our analysis demonstrates a high diversity in cas operon architecture and position of the tracrRNA gene within CRISPR-Cas loci. We observed a correlation between locus heterogeneity and Cas9 sequence diversity, resulting in the identification of various type II CRISPR-Cas subgroups. We validated the expression and co-processing of predicted tracrRNAs and pre-crRNAs by RNA sequencing in five bacterial species. This study reveals tracrRNA family as an atypical, small RNA family with no obvious conservation of structure, sequence or localization within type II CRISPR-Cas loci. The tracrRNA family is however characterized by the conserved feature to base-pair to cognate pre-crRNA repeats, an essential function for crRNA maturation and DNA silencing by dual-RNA:Cas9. The large panel of tracrRNA and Cas9 ortholog sequences should constitute a useful database to improve the design of RNA-programmable Cas9 as genome editing tool.

Place, publisher, year, edition, pages
Landes Bioscience , 2013. Vol. 10, no 5, 726-737 p.
Keyword [en]
tracrRNA, CRISPR-Cas, type II system, Cas9 (Csn1), RNA processing, RNA maturation, small non-coding RNA, bacteria, adaptive immunity, mobile genetic elements
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-80075DOI: 10.4161/rna.24321ISI: 000323176900011OAI: oai:DiVA.org:umu-80075DiVA: diva2:646637
Available from: 2013-09-09 Created: 2013-09-09 Last updated: 2017-12-06Bibliographically 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)
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Available from: 2015-11-10 Created: 2015-11-04 Last updated: 2015-11-09Bibliographically approved

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Chylinski, KrzysztofLe Rhun, AnaïsCharpentier, Emmanuelle
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Molecular Infection Medicine Sweden (MIMS)Umeå Centre for Microbial Research (UCMR)Department of Molecular Biology (Faculty of Medicine)
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RNA Biology
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