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Berlin Brandenburg

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  • 1
    Language: English
    In: Science (New York, N.Y.), 28 November 2014, Vol.346(6213), pp.1258096
    Description: The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics.
    Keywords: Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Engineering -- Methods ; Genome, Human -- Genetics
    ISSN: 00368075
    E-ISSN: 1095-9203
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  • 2
    In: Nature, 2013, Vol.495(7439), p.50
    Keywords: Animals–Enzymology ; Bacteria–Methods ; Biotechnology–Genetics ; DNA–Metabolism ; DNA Cleavage–Metabolism ; Endonucleases–Methods ; Genetic Engineering–Genetics ; Humans–Genetics ; Inverted Repeat Sequences–Genetics ; RNA, Bacterial–Genetics ; RNA, Guide–Genetics ; RNA, Bacterial ; RNA, Guide ; DNA ; Endonucleases;
    ISSN: 0028-0836
    E-ISSN: 14764687
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  • 3
    Language: English
    In: Science (New York, N.Y.), 17 August 2012, Vol.337(6096), pp.816-21
    Description: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
    Keywords: DNA Breaks, Double-Stranded ; DNA Cleavage ; Inverted Repeat Sequences ; Bacteriophages -- Immunology ; Deoxyribonucleases, Type II Site-Specific -- Metabolism ; RNA -- Metabolism ; Streptococcus Pyogenes -- Enzymology
    ISSN: 00368075
    E-ISSN: 1095-9203
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  • 4
    Language: English
    In: Nature, March 7, 2013, Vol.495(7439), p.50(2)
    Keywords: DNA Sequencing – Observations ; RNA – Properties ; Genetic Engineering – Research
    ISSN: 0028-0836
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  • 5
    Language: English
    In: Nature, 3/2013, Vol.495(7439), pp.50-51
    Description: When the double-stranded breaks are repaired by standard cellular repair mechanisms, either by homologous recombination (the exchange of genetic information between DNA molecules with similar sequences) or non-homologous end joining (NHEJ; the introduction of insertions or deletions into the sequence), the sequence at the repair site can be modified or new genetic information inserted. When the researchers expressed the 'humanized' Cas9 together with these guide RNAs in various human cell lines, including induced pluripotent stem cells, they observed the expected alterations to the target DNA - achieved through the introduction of double-stranded breaks followed by repair.
    Keywords: Deoxyribonucleic Acid–DNA ; Genes ; Proteins ; Studies ; Bacteria ; Enzymes ; Genetic Engineering ; Editing ; Genomes;
    ISSN: 0028-0836
    E-ISSN: 1476-4687
    Source: Nature Publishing Group (via CrossRef)
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  • 6
    Language: English
    In: Nature, 28 April 2016, Vol.532(7600), pp.517-21
    Description: CRISPR-Cas systems that provide defence against mobile genetic elements in bacteria and archaea have evolved a variety of mechanisms to target and cleave RNA or DNA. The well-studied types I, II and III utilize a set of distinct CRISPR-associated (Cas) proteins for production of mature CRISPR RNAs (crRNAs) and interference with invading nucleic acids. In types I and III, Cas6 or Cas5d cleaves precursor crRNA (pre-crRNA) and the mature crRNAs then guide a complex of Cas proteins (Cascade-Cas3, type I; Csm or Cmr, type III) to target and cleave invading DNA or RNA. In type II systems, RNase III cleaves pre-crRNA base-paired with trans-activating crRNA (tracrRNA) in the presence of Cas9 (refs 13, 14). The mature tracrRNA-crRNA duplex then guides Cas9 to cleave target DNA. Here, we demonstrate a novel mechanism in CRISPR-Cas immunity. We show that type V-A Cpf1 from Francisella novicida is a dual-nuclease that is specific to crRNA biogenesis and target DNA interference. Cpf1 cleaves pre-crRNA upstream of a hairpin structure formed within the CRISPR repeats and thereby generates intermediate crRNAs that are processed further, leading to mature crRNAs. After recognition of a 5'-YTN-3' protospacer adjacent motif on the non-target DNA strand and subsequent probing for an eight-nucleotide seed sequence, Cpf1, guided by the single mature repeat-spacer crRNA, introduces double-stranded breaks in the target DNA to generate a 5' overhang. The RNase and DNase activities of Cpf1 require sequence- and structure-specific binding to the hairpin of crRNA repeats. Cpf1 uses distinct active domains for both nuclease reactions and cleaves nucleic acids in the presence of magnesium or calcium. This study uncovers a new family of enzymes with specific dual endoribonuclease and endonuclease activities, and demonstrates that type V-A constitutes the most minimalistic of the CRISPR-Cas systems so far described.
    Keywords: DNA Cleavage ; RNA Processing, Post-Transcriptional ; Bacterial Proteins -- Metabolism ; Crispr-Associated Proteins -- Metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats -- Genetics ; RNA Precursors -- Genetics
    ISSN: 00280836
    E-ISSN: 1476-4687
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  • 7
    In: Nature, 2011, Vol.471(7340), p.602
    Description: CRISPR/Cas systems constitute a widespread class of immunity systems that protect bacteria and archaea against phages and plasmids, and commonly use repeat/spacer-derived short crRNAs to silence foreign nucleic acids in a sequence-specific manner. Although the maturation of crRNAs represents a key event in CRISPR activation, the responsible endoribonucleases (CasE, Cas6, Csy4) are missing in many CRISPR/Cas subtypes. Here, differential RNA sequencing of the human pathogen Streptococcus pyogenes uncovered tracrRNA, a trans-encoded small RNA with 24-nucleotide complementarity to the repeat regions of crRNA precursor transcripts. We show that tracrRNA directs the maturation of crRNAs by the activities of the widely conserved endogenous RNase III and the CRISPR-associated Csn1 protein; all these components are essential to protect S. pyogenes against prophage-derived DNA. Our study reveals a novel pathway of small guide RNA maturation and the first example of a host factor (RNase III) required for bacterial RNA-mediated immunity against invaders. [PUBLICATION ]
    Keywords: Bacterial Proteins–Chemistry ; Bacterial Proteins–Genetics ; Bacterial Proteins–Immunology ; Bacterial Proteins–Metabolism ; Conserved Sequence–Genetics ; DNA, Viral–Metabolism ; DNA, Viral–Genetics ; Escherichia Coli–Genetics ; Models, Biological–Metabolism ; Prophages–Biosynthesis ; RNA Precursors–Genetics ; RNA Precursors–Immunology ; RNA Processing, Post-Transcriptional–Metabolism ; RNA, Bacterial–Genetics ; RNA, Bacterial–Metabolism ; RNA, Bacterial–Genetics ; RNA, Bacterial–Immunology ; RNA, Guide–Metabolism ; Ribonuclease III–Virology ; Streptococcus Pyogenes–Virology ; Streptococcus Pyogenes–Virology ; Streptococcus Pyogenes–Virology ; Streptococcus Pyogenes–Virology ; E Coli ; Bacteria ; Bacteriology ; Plasmids ; Proteins ; Bacterial Proteins ; DNA, Viral ; RNA Precursors ; RNA, Bacterial ; RNA, Guide ; Ribonuclease III;
    ISSN: 0028-0836
    E-ISSN: 14764687
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  • 8
    Language: English
    In: EMBO Molecular Medicine, 2015, Vol. 7(4), pp. 363-365
    Description: To purchase or authenticate to the full-text of this article, please visit this link: http://onlinelibrary.wiley.com/doi/10.15252/emmm.201504847/abstract Byline: Emmanuelle Charpentier ***** No abstract is available for this article. *****
    Keywords: Engineering And Technology ; Industrial Biotechnology ; Medical Biotechnology ; Teknik Och Teknologier ; Industriell Bioteknik ; Medicinsk Bioteknik
    ISSN: 1757-4676
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  • 9
    Language: English
    In: Science, 17 August 2012, Vol.337(6096), pp.816-821
    Description: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
    Keywords: Physical sciences -- Chemistry -- Chemical compounds ; Biological sciences -- Biology -- Genetics ; Physical sciences -- Chemistry -- Chemical compounds ; Biological sciences -- Biology -- Genetics ; Biological sciences -- Biochemistry -- Biomolecules ; Physical sciences -- Chemistry -- Chemical compounds ; Physical sciences -- Chemistry -- Chemical compounds ; Biological sciences -- Biology -- Genetics ; Physical sciences -- Physics -- Microphysics ; Applied sciences -- Computer science -- Computer programming
    ISSN: 00368075
    E-ISSN: 10959203
    Source: Archival Journals (JSTOR)
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  • 10
    Language: English
    In: Cellular and Molecular Life Sciences (CMLS), Jan, 2010, Vol.67(1), p.217(21)
    Description: Byline: Pascale Romby (1), Emmanuelle Charpentier (2,3) Keywords: Regulatory RNAs; Small RNAs; Noncoding RNAs; Riboswitches; CRISPR; Gene expression regulation; Virulence; Gram-positive bacteria Abstract: During the last decade, RNA molecules with regulatory functions on gene expression have benefited from a renewed interest. In bacteria, recent high throughput computational and experimental approaches have led to the discovery that 10--20% of all genes code for RNAs with critical regulatory roles in metabolic, physiological and pathogenic processes. The trans-acting RNAs comprise the noncoding RNAs, RNAs with a short open reading frame and antisense RNAs. Many of these RNAs act through binding to their target mRNAs while others modulate protein activity or target DNA. The cis-acting RNAs include regulatory regions of mRNAs that can respond to various signals. These RNAs often provide the missing link between sensing changing conditions in the environment and fine-tuning the subsequent biological responses. Information on their various functions and modes of action has been well documented for gram-negative bacteria. Here, we summarize the current knowledge of regulatory RNAs in gram-positive bacteria. Author Affiliation: (1) Architecture et Reactivite de l'ARN, Universite de Strasbourg, CNRS, IBMC, 15 rue Rene Descartes, 67084, Strasbourg, France (2) Max F. Perutz Laboratories, University of Vienna, Dr. Bohrgasse 9, 1030, Vienna, Austria (3) The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umea University, 90187, Umea, Sweden Article History: Registration Date: 23/09/2009 Received Date: 29/06/2009 Accepted Date: 23/09/2009 Online Date: 27/10/2009
    Keywords: Messenger Rna ; Gene Expression ; Anopheles ; Dna
    ISSN: 1420-682X
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