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  • 1
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    Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR proteins (2015)
    Nature Publishing Group (NPG)
    Details
    Publication Date: 2015-09-30
    Description: The battle for survival between bacteria and the viruses that infect them (phages) has led to the evolution of many bacterial defence systems and phage-encoded antagonists of these systems. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated (cas) genes comprise an adaptive immune system that is one of the most widespread means by which bacteria defend themselves against phages. We identified the first examples of proteins produced by phages that inhibit a CRISPR-Cas system. Here we performed biochemical and in vivo investigations of three of these anti-CRISPR proteins, and show that each inhibits CRISPR-Cas activity through a distinct mechanism. Two block the DNA-binding activity of the CRISPR-Cas complex, yet do this by interacting with different protein subunits, and using steric or non-steric modes of inhibition. The third anti-CRISPR protein operates by binding to the Cas3 helicase-nuclease and preventing its recruitment to the DNA-bound CRISPR-Cas complex. In vivo, this anti-CRISPR can convert the CRISPR-Cas system into a transcriptional repressor, providing the first example-to our knowledge-of modulation of CRISPR-Cas activity by a protein interactor. The diverse sequences and mechanisms of action of these anti-CRISPR proteins imply an independent evolution, and foreshadow the existence of other means by which proteins may alter CRISPR-Cas function.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Bondy-Denomy, Joseph -- Garcia, Bianca -- Strum, Scott -- Du, Mingjian -- Rollins, MaryClare F -- Hidalgo-Reyes, Yurima -- Wiedenheft, Blake -- Maxwell, Karen L -- Davidson, Alan R -- MOP-130482/Canadian Institutes of Health Research/Canada -- MOP-136845/Canadian Institutes of Health Research/Canada -- P20GM103500/GM/NIGMS NIH HHS/ -- R01GM108888/GM/NIGMS NIH HHS/ -- England -- Nature. 2015 Oct 1;526(7571):136-9. doi: 10.1038/nature15254. Epub 2015 Sep 23.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada. ; Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada. ; Department of Microbiology and Immunology, Montana State University, Bozeman, Montana 59717, USA. ; Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/26416740" target="_blank"〉PubMed〈/a〉
    Keywords: Bacteria/*metabolism/*virology ; Bacteriophages/*metabolism ; CRISPR-Associated Proteins/*antagonists & inhibitors/metabolism ; CRISPR-Cas Systems/genetics/*physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Helicases/antagonists & inhibitors/metabolism ; DNA, Viral/metabolism ; DNA-Binding Proteins/antagonists & inhibitors/metabolism ; Endonucleases/antagonists & inhibitors/metabolism ; *Evolution, Molecular ; Protein Binding ; Protein Subunits/antagonists & inhibitors/metabolism ; Repressor Proteins/genetics/metabolism ; Substrate Specificity ; Viral Proteins/*metabolism
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Published by Nature Publishing Group (NPG)
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  • 2
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    Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system (2012)
    Nature Publishing Group (NPG)
    Details
    Publication Date: 2012-12-18
    Description: A widespread system used by bacteria for protection against potentially dangerous foreign DNA molecules consists of the clustered regularly interspaced short palindromic repeats (CRISPR) coupled with cas (CRISPR-associated) genes. Similar to RNA interference in eukaryotes, these CRISPR/Cas systems use small RNAs for sequence-specific detection and neutralization of invading genomes. Here we describe the first examples of genes that mediate the inhibition of a CRISPR/Cas system. Five distinct 'anti-CRISPR' genes were found in the genomes of bacteriophages infecting Pseudomonas aeruginosa. Mutation of the anti-CRISPR gene of a phage rendered it unable to infect bacteria with a functional CRISPR/Cas system, and the addition of the same gene to the genome of a CRISPR/Cas-targeted phage allowed it to evade the CRISPR/Cas system. Phage-encoded anti-CRISPR genes may represent a widespread mechanism for phages to overcome the highly prevalent CRISPR/Cas systems. The existence of anti-CRISPR genes presents new avenues for the elucidation of CRISPR/Cas functional mechanisms and provides new insight into the co-evolution of phages and bacteria.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Bondy-Denomy, Joe -- Pawluk, April -- Maxwell, Karen L -- Davidson, Alan R -- MOP-6279/Canadian Institutes of Health Research/Canada -- England -- Nature. 2013 Jan 17;493(7432):429-32. doi: 10.1038/nature11723. Epub 2012 Dec 16.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/23242138" target="_blank"〉PubMed〈/a〉
    Keywords: Bacteriophages/*genetics ; Biological Evolution ; Gene Expression Regulation, Viral ; Genes, Bacterial/*genetics ; Genes, Viral/*genetics ; Genome, Viral/genetics ; Inverted Repeat Sequences/*genetics ; Molecular Sequence Data ; Pseudomonas aeruginosa/genetics/*immunology/*virology
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Published by Nature Publishing Group (NPG)
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  • 3
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    The diversity-generating benefits of a prokaryotic adaptive immune system (2016)
    Nature Publishing Group (NPG)
    Details
    Publication Date: 2016-04-14
    Description: Prokaryotic CRISPR-Cas adaptive immune systems insert spacers derived from viruses and other parasitic DNA elements into CRISPR loci to provide sequence-specific immunity. This frequently results in high within-population spacer diversity, but it is unclear if and why this is important. Here we show that, as a result of this spacer diversity, viruses can no longer evolve to overcome CRISPR-Cas by point mutation, which results in rapid virus extinction. This effect arises from synergy between spacer diversity and the high specificity of infection, which greatly increases overall population resistance. We propose that the resulting short-lived nature of CRISPR-dependent bacteria-virus coevolution has provided strong selection for the evolution of sophisticated virus-encoded anti-CRISPR mechanisms.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉van Houte, Stineke -- Ekroth, Alice K E -- Broniewski, Jenny M -- Chabas, Helene -- Ashby, Ben -- Bondy-Denomy, Joseph -- Gandon, Sylvain -- Boots, Mike -- Paterson, Steve -- Buckling, Angus -- Westra, Edze R -- DP5-OD021344/OD/NIH HHS/ -- Biotechnology and Biological Sciences Research Council/United Kingdom -- Wellcome Trust/United Kingdom -- England -- Nature. 2016 Apr 21;532(7599):385-8. doi: 10.1038/nature17436. Epub 2016 Apr 13.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉ESI and CEC, Biosciences, University of Exeter, Cornwall Campus, Penryn TR10 9EZ, UK. ; CEFE UMR 5175, CNRS-Universite de Montpellier, Universite Paul-Valery Montpellier, EPHE, 1919, route de Mende 34293, Montpellier Cedex 5, France. ; Department of Integrative Biology, University of California, Berkeley, California 94720, USA. ; CEC, Biosciences, University of Exeter, Cornwall Campus, Penryn TR10 9EZ, UK. ; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California 94158, USA. ; Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, UK.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/27074511" target="_blank"〉PubMed〈/a〉
    Keywords: Bacteriophages/genetics/immunology/physiology ; *Biological Evolution ; CRISPR-Cas Systems/*genetics/*immunology ; Extinction, Biological ; Genetic Fitness/genetics/physiology ; Point Mutation/genetics ; Pseudomonas aeruginosa/*genetics/*immunology/virology
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Published by Nature Publishing Group (NPG)
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  • 4
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    Foreign DNA acquisition by the I-F CRISPR-Cas system requires all components of the interference machinery (2015)
    Oxford University Press
    Details
    Publication Date: 2015-12-16
    Description: CRISPR immunity depends on acquisition of fragments of foreign DNA into CRISPR arrays. For type I-E CRISPR–Cas systems two modes of spacer acquisition, naïve and primed adaptation, were described. Naïve adaptation requires just two most conserved Cas1 and Cas2 proteins; it leads to spacer acquisition from both foreign and bacterial DNA and results in multiple spacers incapable of immune response. Primed adaptation requires all Cas proteins and a CRISPR RNA recognizing a partially matching target. It leads to selective acquisition of spacers from DNA molecules recognized by priming CRISPR RNA, with most spacers capable of protecting the host. Here, we studied spacer acquisition by a type I-F CRISPR–Cas system. We observe both naïve and primed adaptation. Both processes require not just Cas1 and Cas2, but also intact Csy complex and CRISPR RNA. Primed adaptation shows a gradient of acquisition efficiency as a function of distance from the priming site and a strand bias that is consistent with existence of single-stranded adaption intermediates. The results provide new insights into the mechanism of spacer acquisition and illustrate surprising mechanistic diversity of related CRISPR–Cas systems.
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 5
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    Discovery of widespread type I and type V CRISPR-Cas inhibitors (2018)
    American Association for the Advancement of Science (AAAS)
    In: Science
    Details
    Publication Date: 2018-10-12
    Description: Bacterial CRISPR-Cas systems protect their host from bacteriophages and other mobile genetic elements. Mobile elements, in turn, encode various anti-CRISPR (Acr) proteins to inhibit the immune function of CRISPR-Cas. To date, Acr proteins have been discovered for type I (subtypes I-D, I-E, and I-F) and type II (II-A and II-C) but not other CRISPR systems. Here, we report the discovery of 12 acr genes, including inhibitors of type V-A and I-C CRISPR systems. AcrVA1 inhibits a broad spectrum of Cas12a (Cpf1) orthologs—including MbCas12a, Mb3Cas12a, AsCas12a, and LbCas12a—when assayed in human cells. The acr genes reported here provide useful biotechnological tools and mark the discovery of acr loci in many bacteria and phages.
    Keywords: Microbiology, Molecular Biology
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Geosciences , Computer Science , Medicine , Natural Sciences in General , Physics
    Published by American Association for the Advancement of Science (AAAS)
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  • 6
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    Disabling Cas9 by an anti-CRISPR DNA mimic (2017)
    American Association for the Advancement of Science (AAAS)
    Details
    Publication Date: 2017-07-13
    Description: CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known, and the potential applications for Cas9 inhibitor proteins in mammalian cells have not been fully established. We show that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9–single-guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo–electron microscopy structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with a 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif. Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on preformed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.
    Electronic ISSN: 2375-2548
    Topics: Natural Sciences in General
    Published by American Association for the Advancement of Science (AAAS)
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