CRISPRDetect: A flexible algorithm to define CRISPR arrays

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Abstract

Background

CRISPR (clustered regularly interspaced short palindromic repeats) RNAs provide the specificity for noncoding RNA-guided adaptive immune defence systems in prokaryotes. CRISPR arrays consist of repeat sequences separated by specific spacer sequences. CRISPR arrays have previously been identified in a large proportion of prokaryotic genomes. However, currently available detection algorithms do not utilise recently discovered features regarding CRISPR loci.

Results

We have developed a new approach to automatically detect, predict and interactively refine CRISPR arrays. It is available as a web program and command line from bioanalysis.otago.ac.nz/CRISPRDetect. CRISPRDetect discovers putative arrays, extends the array by detecting additional variant repeats, corrects the direction of arrays, refines the repeat/spacer boundaries, and annotates different types of sequence variations (e.g. insertion/deletion) in near identical repeats. Due to these features, CRISPRDetect has significant advantages when compared to existing identification tools. As well as further support for small medium and large repeats, CRISPRDetect identified a class of arrays with ‘extra-large’ repeats in bacteria (repeats 44–50 nt). The CRISPRDetect output is integrated with other analysis tools. Notably, the predicted spacers can be directly utilised by CRISPRTarget to predict targets.

Conclusion

CRISPRDetect enables more accurate detection of arrays and spacers and its gff output is suitable for inclusion in genome annotation pipelines and visualisation. It has been used to analyse all complete bacterial and archaeal reference genomes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-016-2627-0) contains supplementary material, which is available to authorized users.

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Most cited references 41

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The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats

Ibtissem Grissa, Gilles Vergnaud, Christine Pourcel (2007)

Background In Archeae and Bacteria, the repeated elements called CRISPRs for "clustered regularly interspaced short palindromic repeats" are believed to participate in the defence against viruses. Short sequences called spacers are stored in-between repeated elements. In the current model, motifs comprising spacers and repeats may target an invading DNA and lead to its degradation through a proposed mechanism similar to RNA interference. Analysis of intra-species polymorphism shows that new motifs (one spacer and one repeated element) are added in a polarised fashion. Although their principal characteristics have been described, a lot remains to be discovered on the way CRISPRs are created and evolve. As new genome sequences become available it appears necessary to develop automated scanning tools to make available CRISPRs related information and to facilitate additional investigations. Description We have produced a program, CRISPRFinder, which identifies CRISPRs and extracts the repeated and unique sequences. Using this software, a database is constructed which is automatically updated monthly from newly released genome sequences. Additional tools were created to allow the alignment of flanking sequences in search for similarities between different loci and to build dictionaries of unique sequences. To date, almost six hundred CRISPRs have been identified in 475 published genomes. Two Archeae out of thirty-seven and about half of Bacteria do not possess a CRISPR. Fine analysis of repeated sequences strongly supports the current view that new motifs are added at one end of the CRISPR adjacent to the putative promoter. Conclusion It is hoped that availability of a public database, regularly updated and which can be queried on the web will help in further dissecting and understanding CRISPR structure and flanking sequences evolution. Subsequent analyses of the intra-species CRISPR polymorphism will be facilitated by CRISPRFinder and the dictionary creator. CRISPRdb is accessible at

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Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence.

Ekaterina Semenova, Matthijs Jore, Kirill A. Datsenko … (2011)

Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)/Cas (CRISPR-associated sequences) systems provide adaptive immunity against viruses when a spacer sequence of small CRISPR RNA (crRNA) matches a protospacer sequence in the viral genome. Viruses that escape CRISPR/Cas resistance carry point mutations in protospacers, though not all protospacer mutations lead to escape. Here, we show that in the case of Escherichia coli subtype CRISPR/Cas system, the requirements for crRNA matching are strict only for a seven-nucleotide seed region of a protospacer immediately following the essential protospacer-adjacent motif. Mutations in the seed region abolish CRISPR/Cas mediated immunity by reducing the binding affinity of the crRNA-guided Cascade complex to protospacer DNA. We propose that the crRNA seed sequence plays a role in the initial scanning of invader DNA for a match, before base pairing of the full-length spacer occurs, which may enhance the protospacer locating efficiency of the E. coli Cascade complex. In agreement with this proposal, single or multiple mutations within the protospacer but outside the seed region do not lead to escape. The relaxed specificity of the CRISPR/Cas system limits escape possibilities and allows a single crRNA to effectively target numerous related viruses.

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Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli

Ido Yosef, Moran Goren, Udi Qimron (2012)

The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR/Cas) constitute a recently identified prokaryotic defense mechanism against invading nucleic acids. Activity of the CRISPR/Cas system comprises of three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called ‘adaptation’, (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the alien nucleic acid. Here we describe a robust assay in Escherichia coli to explore the hitherto least-studied process, adaptation. We identify essential genes and DNA elements in the leader sequence and in the array which are essential for the adaptation step. We also provide mechanistic insights on the insertion of the repeat-spacer unit by showing that the first repeat serves as the template for the newly inserted repeat. Taken together, our results elucidate fundamental steps in the adaptation process of the CRISPR/Cas system.

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Author and article information

Contributors

Chris M. Brown: +64 3 479 5201 , +64 3 479 7866 , chris.brown@otago.ac.nz

Journal

Journal ID (nlm-ta): BMC Genomics

Journal ID (iso-abbrev): BMC Genomics

Title: BMC Genomics

Publisher: BioMed Central (London )

ISSN (Electronic): 1471-2164

Publication date (Electronic): 17 May 2016

Publication date PMC-release: 17 May 2016

Publication date Collection: 2016

Volume: 17

Electronic Location Identifier: 356

Affiliations

[ ]Department of Biochemistry, University of Otago, PO Box 56, Dunedin, 9054 New Zealand

[ ]Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin, 9054 New Zealand

[ ]Genetics Otago, University of Otago, PO Box 56, Dunedin, 9054 New Zealand

Article

Publisher ID: 2627

DOI: 10.1186/s12864-016-2627-0

PMC ID: 4869251

PubMed ID: 27184979

SO-VID: 497b6a4c-be6f-4418-9973-4564b63a5247

License:

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

History

Date received : 18 November 2015

Date accepted : 16 April 2016

Funding

Funded by: Marsden fund

Funded by: FundRef http://dx.doi.org/10.13039/100008247, University of Otago;

Funded by: Human Frontier Science

Custom metadata

ScienceOpen disciplines: Genetics

Keywords: phage resistance,plasmids,horizontal gene transfer,cas,crispr,small rna targets,crrna,bioinformatics,repeat elements

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ScienceOpen disciplines: Genetics

Keywords: phage resistance, plasmids, horizontal gene transfer, cas, crispr, small rna targets, crrna, bioinformatics, repeat elements

CRISPRDetect: A flexible algorithm to define CRISPR arrays

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Abstract

Background

Results

Conclusion

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Genome Engineering using CRISPR

Most cited references 41

The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats

Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence.

Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli

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