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      A programmable dual RNA-guided DNA endonuclease in adaptive bacterial immunity

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          Abstract

          CRISPR/Cas systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using crRNAs to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA base-paired to trans-activating 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 while the Cas9 RuvC-like domain cleaves the non-complementary 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.

          One-Sentence Summary:

          A two-RNA structure directs an endonuclease to cleave target DNA.

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          Author and article information

          Journal
          0404511
          7473
          Science
          Science
          Science (New York, N.Y.)
          0036-8075
          1095-9203
          26 November 2018
          28 June 2012
          17 August 2012
          07 December 2018
          : 337
          : 6096
          : 816-821
          Affiliations
          [1 ]Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA.
          [2 ]Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
          [3 ]Max F. Perutz Laboratories, University of Vienna, A-1030 Vienna, Austria.
          [4 ]The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå Centre for Microbial Research (UCMR), Department of Molecular Biology, Umeå University, S-90187 Umeå, Sweden.
          [5 ]Present address: Friedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland.
          [6 ]Department of Chemistry, University of California, Berkeley, California 94720, USA.
          [7 ]Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
          Author notes
          [* ]Correspondence should be addressed to J.A.D. ( doudna@ 123456berkeley.edu ) and E.C. ( emmanuelle.charpentier@ 123456mims.umu.se
          Article
          PMC6286148 PMC6286148 6286148 hhmipa995853
          10.1126/science.1225829
          6286148
          22745249
          ff2d0fa4-1c69-49fd-8e22-a19dfa4262d7
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