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  • 1
    Publication Date: 2014-10-03
    Description: The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4268322/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4268322/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉O'Connell, Mitchell R -- Oakes, Benjamin L -- Sternberg, Samuel H -- East-Seletsky, Alexandra -- Kaplan, Matias -- Doudna, Jennifer A -- P50 GM102706/GM/NIGMS NIH HHS/ -- P50GM102706-03/GM/NIGMS NIH HHS/ -- T32 GM007232/GM/NIGMS NIH HHS/ -- T32 GM066698/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2014 Dec 11;516(7530):263-6. doi: 10.1038/nature13769. Epub 2014 Sep 28.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA. ; Department of Chemistry, University of California, Berkeley, California 94720, USA. ; 1] Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA [2] Department of Agricultural and Biological Engineering, University of Florida, Gainesville, Florida 32611, USA. ; 1] Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA [2] Department of Chemistry, University of California, Berkeley, California 94720, USA [3] Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA [4] Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/25274302" target="_blank"〉PubMed〈/a〉
    Keywords: Base Sequence ; CRISPR-Associated Proteins/*metabolism ; CRISPR-Cas Systems/*physiology ; Cell Extracts ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/chemistry/genetics/metabolism ; Genetic Engineering/*methods ; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/genetics ; HeLa Cells ; Humans ; Nucleotide Motifs ; Oligonucleotides/chemistry/genetics/metabolism ; RNA/chemistry/genetics/*metabolism ; RNA, Guide/chemistry/genetics/metabolism ; RNA, Messenger/genetics/isolation & purification/metabolism ; Substrate Specificity
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 2
    Publication Date: 2014-01-31
    Description: The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106473/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106473/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Sternberg, Samuel H -- Redding, Sy -- Jinek, Martin -- Greene, Eric C -- Doudna, Jennifer A -- GM074739/GM/NIGMS NIH HHS/ -- R01 GM073794/GM/NIGMS NIH HHS/ -- R01 GM074739/GM/NIGMS NIH HHS/ -- T32 GM066698/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2014 Mar 6;507(7490):62-7. doi: 10.1038/nature13011. Epub 2014 Jan 29.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉1] Department of Chemistry, University of California, Berkeley, California 94720, USA [2]. ; 1] Department of Chemistry, Columbia University, New York, New York 10032, USA [2]. ; 1] Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA [2] Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland. ; Department of Biochemistry and Molecular Biophysics and Howard Hughes Medical Institute, Columbia University, New York, New York 10032, USA. ; 1] Department of Chemistry, University of California, Berkeley, California 94720, USA [2] Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA [3] Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA [4] Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/24476820" target="_blank"〉PubMed〈/a〉
    Keywords: Apoenzymes/metabolism ; *Base Pairing ; Base Sequence ; Biocatalysis ; CRISPR-Associated Proteins/*metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA/chemistry/genetics/metabolism ; *DNA Cleavage ; Diffusion ; Endonucleases/*metabolism ; Enzyme Activation ; Genetic Engineering/methods ; Genome/genetics ; Nucleic Acid Denaturation ; Nucleic Acid Heteroduplexes/chemistry/genetics/metabolism ; Nucleotide Motifs ; RNA/chemistry/*genetics/metabolism ; Streptococcus pyogenes/enzymology/immunology ; Substrate Specificity ; Thermodynamics
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 3
    Publication Date: 2012-02-18
    Description: Clustered regularly interspaced short palindromic repeat (CRISPR) are essential components of nucleic-acid-based adaptive immune systems that are widespread in bacteria and archaea. Similar to RNA interference (RNAi) pathways in eukaryotes, CRISPR-mediated immune systems rely on small RNAs for sequence-specific detection and silencing of foreign nucleic acids, including viruses and plasmids. However, the mechanism of RNA-based bacterial immunity is distinct from RNAi. Understanding how small RNAs are used to find and destroy foreign nucleic acids will provide new insights into the diverse mechanisms of RNA-controlled genetic silencing systems.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Wiedenheft, Blake -- Sternberg, Samuel H -- Doudna, Jennifer A -- Howard Hughes Medical Institute/ -- England -- Nature. 2012 Feb 15;482(7385):331-8. doi: 10.1038/nature10886.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, Maryland 20815-6789, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/22337052" target="_blank"〉PubMed〈/a〉
    Keywords: Archaea/*genetics ; Bacteria/*genetics ; *Gene Expression Regulation, Bacterial ; *Gene Silencing ; Models, Molecular ; RNA Interference ; RNA, Archaeal/biosynthesis/chemistry/genetics/*metabolism ; RNA, Bacterial/biosynthesis/chemistry/genetics/*metabolism
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 4
    Publication Date: 2015-11-03
    Description: Cas9 is an RNA-guided DNA endonuclease that targets foreign DNA for destruction as part of a bacterial adaptive immune system mediated by clustered regularly interspaced short palindromic repeats (CRISPR). Together with single-guide RNAs, Cas9 also functions as a powerful genome engineering tool in plants and animals, and efforts are underway to increase the efficiency and specificity of DNA targeting for potential therapeutic applications. Studies of off-target effects have shown that DNA binding is far more promiscuous than DNA cleavage, yet the molecular cues that govern strand scission have not been elucidated. Here we show that the conformational state of the HNH nuclease domain directly controls DNA cleavage activity. Using intramolecular Forster resonance energy transfer experiments to detect relative orientations of the Cas9 catalytic domains when associated with on- and off-target DNA, we find that DNA cleavage efficiencies scale with the extent to which the HNH domain samples an activated conformation. We furthermore uncover a surprising mode of allosteric communication that ensures concerted firing of both Cas9 nuclease domains. Our results highlight a proofreading mechanism beyond initial protospacer adjacent motif (PAM) recognition and RNA-DNA base-pairing that serves as a final specificity checkpoint before DNA double-strand break formation.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Sternberg, Samuel H -- LaFrance, Benjamin -- Kaplan, Matias -- Doudna, Jennifer A -- T32GM007232/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2015 Nov 5;527(7576):110-3. doi: 10.1038/nature15544. Epub 2015 Oct 28.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Chemistry, University of California, Berkeley, California 94720, USA. ; Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA. ; Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA. ; Innovative Genomics Initiative, University of California, Berkeley, California 94720, USA. ; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/26524520" target="_blank"〉PubMed〈/a〉
    Keywords: Allosteric Regulation ; Bacterial Proteins/chemistry/metabolism ; Base Pairing ; Binding Sites ; CRISPR-Associated Proteins/*chemistry/*metabolism ; *CRISPR-Cas Systems ; Catalytic Domain ; DNA/chemistry/*metabolism ; DNA Breaks, Double-Stranded ; *DNA Cleavage ; Endonucleases/chemistry/*metabolism ; Fluorescence Resonance Energy Transfer ; *Genetic Engineering ; Models, Molecular ; RNA, Guide/chemistry/metabolism ; Streptococcus pyogenes
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 5
    Publication Date: 2015-03-21
    Description: 〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4394183/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4394183/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Baltimore, David -- Berg, Paul -- Botchan, Michael -- Carroll, Dana -- Charo, R Alta -- Church, George -- Corn, Jacob E -- Daley, George Q -- Doudna, Jennifer A -- Fenner, Marsha -- Greely, Henry T -- Jinek, Martin -- Martin, G Steven -- Penhoet, Edward -- Puck, Jennifer -- Sternberg, Samuel H -- Weissman, Jonathan S -- Yamamoto, Keith R -- P50 HG005550/HG/NHGRI NIH HHS/ -- T32 GM066698/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- New York, N.Y. -- Science. 2015 Apr 3;348(6230):36-8. doi: 10.1126/science.aab1028. Epub 2015 Mar 19.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉California Institute of Technology, Mail Code 147-75, Pasadena, CA 91125, USA. ; Stanford University School of Medicine, 291 Campus Drive, Stanford, CA 94305, USA. ; University of California, Berkeley, 450 Li Ka Shing no. 3370, Berkeley, CA 94720-3370, USA. Innovative Genomics Initiative, University of California, Berkeley, 188 Li Ka Shing Center, Berkeley, CA 94720-3370, USA. ; Department of Biochemistry, University of Utah School of Medicine, 15 North Medical Drive East, Room 4100, Salt Lake City, UT 84112-5650, USA. ; Department of Medical History and Bioethics, School of Medicine and Public Health, University of Wisconsin Law School, 975 Bascom Mall, Madison, WI 53706, USA. ; Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. ; Innovative Genomics Initiative, University of California, Berkeley, 188 Li Ka Shing Center, Berkeley, CA 94720-3370, USA. ; Boston Children's Hospital, 300 Longwood Avenue, Karp Family Building, 7th Floor, Boston, MA 02115, USA. Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, MD 20815, USA. ; Innovative Genomics Initiative, University of California, Berkeley, 188 Li Ka Shing Center, Berkeley, CA 94720-3370, USA. Departments of Molecular and Cell Biology and Chemistry, Howard Hughes Medical Institute, 731 Stanley Hall, MS 3220, University of California, Berkeley, Berkeley, CA 94720-3220, USA. doudna@berkeley.edu. ; Center for Law and the Biosciences, Crown Quadrangle 559 Nathan Abbott Way Stanford, CA 94305-8610, USA. ; Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. ; Department of Molecular and Cell Biology, College of Letters and Science, University of California, Berkeley, 210K Durant Hall, Berkeley, CA 94720-2920, USA. ; Alta Partners, One Embarcadero Center, 37th Floor, San Francisco, CA 94111, USA. ; Department of Pediatrics UCSF School of Medicine, 513 Parnassus Avenue, San Francisco, CA 94143, USA. ; Department of Chemistry, 731 Stanley Hall, MS 3220, University of California, Berkeley, CA 94720-3220, USA. ; Innovative Genomics Initiative, University of California, Berkeley, 188 Li Ka Shing Center, Berkeley, CA 94720-3370, USA. Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, Byers Hall, 1700 4th Street, San Francisco, CA 94158-2330, USA. ; Innovative Genomics Initiative, University of California, Berkeley, 188 Li Ka Shing Center, Berkeley, CA 94720-3370, USA. UCSF School of Medicine, 600 16th Street, San Francisco, CA 94158, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/25791083" target="_blank"〉PubMed〈/a〉
    Keywords: Biotechnology/ethics ; *Caspase 9 ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Transfer, Horizontal ; Genetic Engineering/*ethics ; Genetic Predisposition to Disease/*prevention & control ; Genome, Human/genetics ; Genomics ; *Germ Cells ; Humans ; Risk Management ; Targeted Gene Repair/*ethics
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
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  • 6
    Publication Date: 2014-02-08
    Description: Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4184034/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4184034/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Jinek, Martin -- Jiang, Fuguo -- Taylor, David W -- Sternberg, Samuel H -- Kaya, Emine -- Ma, Enbo -- Anders, Carolin -- Hauer, Michael -- Zhou, Kaihong -- Lin, Steven -- Kaplan, Matias -- Iavarone, Anthony T -- Charpentier, Emmanuelle -- Nogales, Eva -- Doudna, Jennifer A -- T32 GM066698/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- New York, N.Y. -- Science. 2014 Mar 14;343(6176):1247997. doi: 10.1126/science.1247997. Epub 2014 Feb 6.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/24505130" target="_blank"〉PubMed〈/a〉
    Keywords: Actinomyces/*enzymology ; Amino Acid Sequence ; Bacterial Proteins/*chemistry ; Caspase 9/*chemistry ; Crystallography, X-Ray ; DNA Cleavage ; Molecular Sequence Data ; Nucleic Acid Conformation ; Protein Structure, Secondary ; Protein Structure, Tertiary ; RNA/*chemistry ; Streptococcus pyogenes/*enzymology
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
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