| 1. |
- Makarova, Kira S, et al.
(författare)
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An updated evolutionary classification of CRISPR-Cas systems
- 2015
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Ingår i: Nature Reviews Microbiology. - : Springer Science and Business Media LLC. - 1740-1526 .- 1740-1534. ; 13:11, s. 722-736
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Tidskriftsartikel (refereegranskat)abstract
- The evolution of CRISPR-cas loci, which encode adaptive immune systems in archaea and bacteria, involves rapid changes, in particular numerous rearrangements of the locus architecture and horizontal transfer of complete loci or individual modules. These dynamics complicate straightforward phylogenetic classification, but here we present an approach combining the analysis of signature protein families and features of the architecture of cas loci that unambiguously partitions most CRISPR-cas loci into distinct classes, types and subtypes. The new classification retains the overall structure of the previous version but is expanded to now encompass two classes, five types and 16 subtypes. The relative stability of the classification suggests that the most prevalent variants of CRISPR-Cas systems are already known. However, the existence of rare, currently unclassifiable variants implies that additional types and subtypes remain to be characterized.
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| 2. |
- Brouns, Stan J, et al.
(författare)
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Small CRISPR RNAs guide antiviral defense in prokaryotes
- 2008
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 321:5891, s. 960-964
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Tidskriftsartikel (refereegranskat)abstract
- Prokaryotes acquire virus resistance by integrating short fragments of viral nucleic acid into clusters of regularly interspaced short palindromic repeats (CRISPRs). Here we show how virus-derived sequences contained in CRISPRs are used by CRISPR-associated (Cas) proteins from the host to mediate an antiviral response that counteracts infection. After transcription of the CRISPR, a complex of Cas proteins termed Cascade cleaves a CRISPR RNA precursor in each repeat and retains the cleavage products containing the virus-derived sequence. Assisted by the helicase Cas3, these mature CRISPR RNAs then serve as small guide RNAs that enable Cascade to interfere with virus proliferation. Our results demonstrate that the formation of mature guide RNAs by the CRISPR RNA endonuclease subunit of Cascade is a mechanistic requirement for antiviral defense.
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| 3. |
- Chylinski, Krzysztof, et al.
(författare)
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Classification and evolution of type II CRISPR-Cas systems
- 2014
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 42:10, s. 6091-6105
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Tidskriftsartikel (refereegranskat)abstract
- The CRISPR-Cas systems of archaeal and bacterial adaptive immunity are classified into three types that differ by the repertoires of CRISPR-associated (cas) genes, the organization of cas operons and the structure of repeats in the CRISPR arrays. The simplest among the CRISPR-Cas systems is type II in which the endonuclease activities required for the interference with foreign deoxyribonucleic acid (DNA) are concentrated in a single multidomain protein, Cas9, and are guided by a co-processed dual-tracrRNA: crRNA molecule. This compact enzymatic machinery and readily programmable site-specific DNA targeting make type II systems top candidates for a new generation of powerful tools for genomic engineering. Here we report an updated census of CRISPR-Cas systems in bacterial and archaeal genomes. Type II systems are the rarest, missing in archaea, and represented in similar to 5% of bacterial genomes, with an over-representation among pathogens and commensals. Phylogenomic analysis suggests that at least three cas genes, cas1, cas2 and cas4, and the CRISPR repeats of the type II-B system were acquired via recombination with a type I CRISPR-Cas locus. Distant homologs of Cas9 were identified among proteins encoded by diverse transposons, suggesting that type II CRISPR-Cas evolved via recombination of mobile nuclease genes with type I loci.
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| 4. |
- Drobysheva, Arina V., et al.
(författare)
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Structure and function of virion RNA polymerase of a crAss-like phage
- 2021
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Ingår i: Nature. - : Nature Publishing Group. - 0028-0836 .- 1476-4687. ; 589:7841, s. 306-309
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Tidskriftsartikel (refereegranskat)abstract
- The RNA polymerase from the crAss-like bacteriophage phi14:2, which is translocated into the host cell with phage DNA and transcribes early phage genes, is structurally most similar to eukaryotic RNA interference polymerases, suggesting that the latter have a phage origin. CrAss-like phages are a recently described expansive group of viruses that includes the most abundant virus in the human gut(1-3). The genomes of all crAss-like phages encode a large virion-packaged protein(2,4) that contains a DFDxD sequence motif, which forms the catalytic site in cellular multisubunit RNA polymerases (RNAPs)(5). Here, using Cellulophaga baltica crAss-like phage phi14:2 as a model system, we show that this protein is a DNA-dependent RNAP that is translocated into the host cell along with the phage DNA and transcribes early phage genes. We determined the crystal structure of this 2,180-residue enzyme in a self-inhibited state, which probably occurs before virion packaging. This conformation is attained with the help of a cleft-blocking domain that interacts with the active site and occupies the cavity in which the RNA-DNA hybrid binds. Structurally, phi14:2 RNAP is most similar to eukaryotic RNAPs that are involved in RNA interference(6,7), although most of the phi14:2 RNAP structure (nearly 1,600 residues) maps to a new region of the protein fold space. Considering this structural similarity, we propose that eukaryal RNA interference polymerases have their origins in phage, which parallels the emergence of the mitochondrial transcription apparatus(8).
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| 5. |
- Fonfara, Ines, et al.
(författare)
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Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems
- 2014
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 42:4, s. 2577-2590
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Makarova, Kira S, et al.
(författare)
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Evolution and classification of the CRISPR-Cas systems
- 2011
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Ingår i: Nature Reviews Microbiology. - : Nature Publishing Group. - 1740-1526 .- 1740-1534. ; 9:6, s. 467-477
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Tidskriftsartikel (refereegranskat)abstract
- The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) modules are adaptive immunity systems that are present in many archaea and bacteria. These defence systems are encoded by operons that have an extraordinarily diverse architecture and a high rate of evolution for both the cas genes and the unique spacer content. Here, we provide an updated analysis of the evolutionary relationships between CRISPR-Cas systems and Cas proteins. Three major types of CRISPR-Cas system are delineated, with a further division into several subtypes and a few chimeric variants. Given the complexity of the genomic architectures and the extremely dynamic evolution of the CRISPR-Cas systems, a unified classification of these systems should be based on multiple criteria. Accordingly, we propose a 'polythetic' classification that integrates the phylogenies of the most common cas genes, the sequence and organization of the CRISPR repeats and the architecture of the CRISPR-cas loci.
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| 7. |
- Quax, Tessa E. F., et al.
(författare)
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Differential Translation Tunes Uneven Production of Operon-Encoded Proteins
- 2013
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 4:5, s. 938-944
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Tidskriftsartikel (refereegranskat)abstract
- Clustering of functionally related genes in operons allows for coregulated gene expression in prokaryotes. This is advantageous when equal amounts of gene products are required. Production of protein complexes with an uneven stoichiometry, however, requires tuning mechanisms to generate subunits in appropriate relative quantities. Using comparative genomic analysis, we show that differential translation is a key determinant of modulated expression of genes clustered in operons and that codon bias generally is the best in silico indicator of unequal protein production. Variable ribosome density profiles of polycistronic transcripts correlate strongly with differential translation patterns. In addition, we provide experimental evidence that de novo initiation of translation can occur at intercistronic sites, allowing for differential translation of any gene irrespective of its position on a polycistronic messenger. Thus, modulation of translation efficiency appears to be a universal mode of control in bacteria and archaea that allows for differential production of operon-encoded proteins.
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