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  • 1
    Language: English
    In: Nature, 01 October 2015, Vol.526(7571), pp.55-61
    Description: Prokaryotic organisms are threatened by a large array of viruses and have developed numerous defence strategies. Among these, only clustered, regularly interspaced short palindromic repeat (CRISPR)-Cas systems provide adaptive immunity against foreign elements. Upon viral injection, a small sequence of the viral genome, known as a spacer, is integrated into the CRISPR locus to immunize the host cell. Spacers are transcribed into small RNA guides that direct the cleavage of the viral DNA by Cas nucleases. Immunization through spacer acquisition enables a unique form of evolution whereby a population not only rapidly acquires resistance to its predators but also passes this resistance mechanism vertically to its progeny.
    Keywords: Crispr-Cas Systems -- Immunology ; Prokaryotic Cells -- Immunology
    ISSN: 00280836
    E-ISSN: 1476-4687
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  • 2
    Article
    Article
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 03 January 2017, Vol.114(1), pp.15-16
    Description: Author contributions: L.A.M. wrote the paper.
    Keywords: Sciences (General);
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 3
    In: Nature, 2010, Vol.463(7280), p.568
    Description: All immune systems must distinguish self from non-self to repel invaders without inducing autoimmunity. C lustered, r egularly i nterspaced, s hort p alindromic r epeat (CRISPR) loci protect bacteria and archaea from invasion by phage and plasmid DNA through a genetic interference pathway 1 – 9 . CRISPR loci are present in ~ 40% and ~90% of sequenced bacterial and archaeal genomes respectively 10 and evolve rapidly, acquiring new spacer sequences to adapt to highly dynamic viral populations 1 , 11 – 13 . Immunity requires a sequence match between the invasive DNA and the spacers that lie between CRISPR repeats 1 – 9 . Each cluster is genetically linked to a subset of the cas (CRISPR-associated) genes 14 – 16 that collectively encode 〉40 families of proteins involved in adaptation and interference. CRISPR loci encode small CRISPR RNAs (crRNAs) that contain a full spacer flanked by partial repeat sequences 2 , 17 – 19 . CrRNA spacers are thought to identify targets by direct Watson-Crick pairing with invasive “protospacer” DNA 2 , 3 , but how they avoid targeting the spacer DNA within the encoding CRISPR locus itself is unknown. Here we have defined the mechanism of CRISPR self/non-self discrimination. In Staphylococcus epidermidis , target/crRNA mismatches at specific positions outside of the spacer sequence license foreign DNA for interference, whereas extended pairing between crRNA and CRISPR DNA repeats prevents autoimmunity. Hence, this CRISPR system uses the base-pairing potential of crRNAs not only to specify a target but also to spare the bacterial chromosome from interference. Differential complementarity outside of the spacer sequence is a built-in feature of all CRISPR systems, suggesting that this mechanism is a broadly applicable solution to the self/non-self dilemma that confronts all immune pathways.
    Keywords: Article;
    ISSN: 0028-0836
    E-ISSN: 14764687
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  • 4
    Language: English
    In: Nature, Nov 4, 2010, Vol.468(7320), p.45(2)
    Keywords: Bacterial Genetics -- Research ; Rna Interference -- Research
    ISSN: 0028-0836
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  • 5
    In: Nature, 2017
    Description: Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems provide protection against viral and plasmid infection by capturing short DNA sequences from these invaders and integrating them into the CRISPR locus of the prokaryotic host. These sequences, known as spacers, are transcribed into short CRISPR RNA guides that specify the cleavage site of Cas nucleases in the genome of the invader. It is not known when spacer sequences are acquired during viral infection. Here, to investigate this, we tracked spacer acquisition in Staphylococcus aureus cells harbouring a type II CRISPR-Cas9 system after infection with the staphylococcal bacteriophage ϕ12. We found that new spacers were acquired immediately after infection preferentially from the cos site, the viral free DNA end that is first injected into the cell. Analysis of spacer acquisition after infection with mutant phages demonstrated that most spacers are acquired during DNA injection, but not during other stages of the viral cycle that produce free DNA ends, such as DNA replication or packaging. Finally, we showed that spacers acquired from early-injected genomic regions, which direct Cas9 cleavage of the viral DNA immediately after infection, provide better immunity than spacers acquired from late-injected regions. Our results reveal that CRISPR-Cas systems exploit the phage life cycle to generate a pattern of spacer acquisition that ensures a successful CRISPR immune response.
    Keywords: Sciences (General) ; Physics;
    ISSN: 0028-0836
    E-ISSN: 1476-4687
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  • 6
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 27 December 2011, Vol.108(52), pp.21218-22
    Description: Precise RNA processing is fundamental to all small RNA-mediated interference pathways. In prokaryotes, clustered, regularly interspaced, short palindromic repeats (CRISPR) loci encode small CRISPR RNAs (crRNAs) that protect against invasive genetic elements by antisense targeting. CRISPR loci are transcribed as a long precursor that is cleaved within repeat sequences by CRISPR-associated (Cas) proteins. In many organisms, this primary processing generates crRNA intermediates that are subject to additional nucleolytic trimming to render mature crRNAs of specific lengths. The molecular mechanisms underlying this maturation event remain poorly understood. Here, we defined the genetic requirements for crRNA primary processing and maturation in Staphylococcus epidermidis. We show that changes in the position of the primary processing site result in extended or diminished maturation to generate mature crRNAs of constant length. These results indicate that crRNA maturation occurs by a ruler mechanism anchored at the primary processing site. We also show that maturation is mediated by specific cas genes distinct from those genes involved in primary processing, showing that this event is directed by CRISPR/Cas loci.
    Keywords: Models, Genetic ; Inverted Repeat Sequences -- Genetics ; RNA Processing, Post-Transcriptional -- Physiology ; RNA, Bacterial -- Genetics ; Staphylococcus Epidermidis -- Physiology
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 7
    Language: English
    In: Nature, 11/2010, Vol.468(7320), pp.45-46
    Description: Intriguingly, proteins encoded by 'RAMP module' genes, which accompany core CRISPR/Cas loci in a subset of mostly archaeal species, have been shown to catalyse crRNA-guided RNA cleavage in vitro7. [...] as with RNAi, multiple branches of the CRISPR interference pathway probably coexist. [...] Garneau and colleagues' demonstration6 of crRNAdirected DNA cleavage could have practical as well as biological import.
    Keywords: Deoxyribonucleic Acid–DNA ; Bacteriology ; Plasmids;
    ISSN: 0028-0836
    E-ISSN: 1476-4687
    Source: Nature Publishing Group (via CrossRef)
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  • 8
    In: Nature, 2014
    Description: A fundamental feature of immune systems is the ability to distinguish pathogenic from self and commensal elements, and to attack the former but tolerate the latter. Prokaryotic CRISPR-Cas immune systems defend against phage infection by using Cas nucleases and small RNA guides that specify one or more target sites for cleavage of the viral genome. Temperate phages include viruses that can integrate into the bacterial chromosome, and they can carry genes that provide a fitness advantage to the lysogenic host. However, CRISPR-Cas targeting that relies strictly on DNA sequence recognition provides indiscriminate immunity both to lytic and lysogenic infection by temperate phages-compromising the genetic stability of these potentially beneficial elements altogether. Here we show that the Staphylococcus epidermidis CRISPR-Cas system can prevent lytic infection but tolerate lysogenization by temperate phages. Conditional tolerance is achieved through transcription-dependent DNA targeting, and ensures that targeting is resumed upon induction of the prophage lytic cycle. Our results provide evidence for the functional divergence of CRISPR-Cas systems and highlight the importance of targeting mechanism diversity. In addition, they extend the concept of 'tolerance to non-self' to the prokaryotic branch of adaptive immunity.
    Keywords: Bacteriology ; Staphylococcus Infections ; Deoxyribonucleic Acid–DNA ; Genomes ; Efficiency;
    ISSN: 0028-0836
    E-ISSN: 14764687
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  • 9
    In: Nature Reviews Genetics, 2010, Vol.11(3), p.181
    Description: Sequence-directed genetic interference pathways control gene expression and preserve genome integrity in all kingdoms of life. The importance of such pathways is highlighted by the extensive study of RNA interference (RNAi) and related processes in eukaryotes. In many bacteria and most archaea, clustered, regularly interspaced short palindromic repeats (CRISPRs) are involved in a more recently discovered interference pathway that protects cells from bacteriophages and conjugative plasmids. CRISPR sequences provide an adaptive, heritable record of past infections and express CRISPR RNAs - small RNAs that target invasive nucleic acids. Here, we review the mechanisms of CRISPR interference and its roles in microbial physiology and evolution. We also discuss potential applications of this novel interference pathway.
    Keywords: Archaea -- Genetics ; Bacteria -- Genetics;
    ISSN: 1471-0056
    E-ISSN: 14710064
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  • 10
    Language: English
    In: Science (New York, N.Y.), 15 February 2013, Vol.339(6121), pp.819-23
    Description: Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
    Keywords: Crispr-Cas Systems ; DNA Cleavage ; Genetic Engineering -- Methods ; Genome -- Genetics ; Inverted Repeat Sequences -- Genetics ; Microarray Analysis -- Methods
    ISSN: 00368075
    E-ISSN: 1095-9203
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