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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: 2013-05-31
    Description: Female mosquitoes of some species are generalists and will blood-feed on a variety of vertebrate hosts, whereas others display marked host preference. Anopheles gambiae and Aedes aegypti have evolved a strong preference for humans, making them dangerously efficient vectors of malaria and Dengue haemorrhagic fever. Specific host odours probably drive this strong preference because other attractive cues, including body heat and exhaled carbon dioxide (CO2), are common to all warm-blooded hosts. Insects sense odours via several chemosensory receptor families, including the odorant receptors (ORs), membrane proteins that form heteromeric odour-gated ion channels comprising a variable ligand-selective subunit and an obligate co-receptor called Orco (ref. 6). Here we use zinc-finger nucleases to generate targeted mutations in the orco gene of A. aegypti to examine the contribution of Orco and the odorant receptor pathway to mosquito host selection and sensitivity to the insect repellent DEET (N,N-diethyl-meta-toluamide). orco mutant olfactory sensory neurons have greatly reduced spontaneous activity and lack odour-evoked responses. Behaviourally, orco mutant mosquitoes have severely reduced attraction to honey, an odour cue related to floral nectar, and do not respond to human scent in the absence of CO2. However, in the presence of CO2, female orco mutant mosquitoes retain strong attraction to both human and animal hosts, but no longer strongly prefer humans. orco mutant females are attracted to human hosts even in the presence of DEET, but are repelled upon contact, indicating that olfactory- and contact-mediated effects of DEET are mechanistically distinct. We conclude that the odorant receptor pathway is crucial for an anthropophilic vector mosquito to discriminate human from non-human hosts and to be effectively repelled by volatile DEET.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3696029/" 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/PMC3696029/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉DeGennaro, Matthew -- McBride, Carolyn S -- Seeholzer, Laura -- Nakagawa, Takao -- Dennis, Emily J -- Goldman, Chloe -- Jasinskiene, Nijole -- James, Anthony A -- Vosshall, Leslie B -- AI29746/AI/NIAID NIH HHS/ -- DC012069/DC/NIDCD NIH HHS/ -- R01 AI029746/AI/NIAID NIH HHS/ -- R37 AI029746/AI/NIAID NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2013 Jun 27;498(7455):487-91. doi: 10.1038/nature12206. Epub 2013 May 29.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Laboratory of Neurogenetics and Behavior, and Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10065, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/23719379" target="_blank"〉PubMed〈/a〉
    Keywords: Aedes/drug effects/*genetics/*physiology ; Amino Acid Sequence ; Animals ; Base Sequence ; DEET/administration & dosage/*pharmacology ; Drug Resistance/drug effects ; Female ; Genes, Insect/*genetics ; Honey ; Host Specificity/drug effects/*genetics ; Humans ; Insect Repellents/administration & dosage/*pharmacology ; Male ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Mutation/*genetics ; Neurons/cytology/drug effects ; Odors/analysis ; Olfactory Pathways/cytology/drug effects ; Volatilization
    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: 2015-10-28
    Description: Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30-40-base-pair lengths into clustered regularly interspaced short palindromic repeat (CRISPR) loci as spacer segments. The universally conserved Cas1-Cas2 integrase complex catalyses spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases. How the Cas1-Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1-Cas2 complex bound to cognate 33-nucleotide protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3'-OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo. Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1-Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4662619/" 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/PMC4662619/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Nunez, James K -- Harrington, Lucas B -- Kranzusch, Philip J -- Engelman, Alan N -- Doudna, Jennifer A -- AI070042/AI/NIAID NIH HHS/ -- R01 AI070042/AI/NIAID NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2015 Nov 26;527(7579):535-8. doi: 10.1038/nature15760. Epub 2015 Oct 21.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA. ; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California 94720, USA. ; Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA. ; Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA. ; Department of Chemistry, University of California, Berkeley, Berkeley, California 94720, USA. ; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA. ; Innovative Genomics Initiative, University of California, Berkeley, Berkeley, California 94720, USA. ; Center for RNA Systems Biology, University of California, Berkeley, Berkeley, California 94720, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/26503043" target="_blank"〉PubMed〈/a〉
    Keywords: *Adaptive Immunity ; Bacteriophage M13/genetics/immunology ; Base Sequence ; CRISPR-Associated Proteins/chemistry/*metabolism ; Catalytic Domain ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Crystallography, X-Ray ; DNA, Viral/chemistry/*genetics/*immunology/metabolism ; Escherichia coli/enzymology/genetics/immunology/virology ; Integrases/chemistry/metabolism ; Models, Molecular ; *Virus Integration/genetics/immunology
    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: 2011-07-22
    Description: Recombination, together with mutation, gives rise to genetic variation in populations. Here we leverage the recent mixture of people of African and European ancestry in the Americas to build a genetic map measuring the probability of crossing over at each position in the genome, based on about 2.1 million crossovers in 30,000 unrelated African Americans. At intervals of more than three megabases it is nearly identical to a map built in Europeans. At finer scales it differs significantly, and we identify about 2,500 recombination hotspots that are active in people of West African ancestry but nearly inactive in Europeans. The probability of a crossover at these hotspots is almost fully controlled by the alleles an individual carries at PRDM9 (P value 〈 10(-245)). We identify a 17-base-pair DNA sequence motif that is enriched in these hotspots, and is an excellent match to the predicted binding target of PRDM9 alleles common in West Africans and rare in Europeans. Sites of this motif are predicted to be risk loci for disease-causing genomic rearrangements in individuals carrying these alleles. More generally, this map provides a resource for research in human genetic variation and evolution.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3154982/" 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/PMC3154982/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Hinch, Anjali G -- Tandon, Arti -- Patterson, Nick -- Song, Yunli -- Rohland, Nadin -- Palmer, Cameron D -- Chen, Gary K -- Wang, Kai -- Buxbaum, Sarah G -- Akylbekova, Ermeg L -- Aldrich, Melinda C -- Ambrosone, Christine B -- Amos, Christopher -- Bandera, Elisa V -- Berndt, Sonja I -- Bernstein, Leslie -- Blot, William J -- Bock, Cathryn H -- Boerwinkle, Eric -- Cai, Qiuyin -- Caporaso, Neil -- Casey, Graham -- Cupples, L Adrienne -- Deming, Sandra L -- Diver, W Ryan -- Divers, Jasmin -- Fornage, Myriam -- Gillanders, Elizabeth M -- Glessner, Joseph -- Harris, Curtis C -- Hu, Jennifer J -- Ingles, Sue A -- Isaacs, William -- John, Esther M -- Kao, W H Linda -- Keating, Brendan -- Kittles, Rick A -- Kolonel, Laurence N -- Larkin, Emma -- Le Marchand, Loic -- McNeill, Lorna H -- Millikan, Robert C -- Murphy, Adam -- Musani, Solomon -- Neslund-Dudas, Christine -- Nyante, Sarah -- Papanicolaou, George J -- Press, Michael F -- Psaty, Bruce M -- Reiner, Alex P -- Rich, Stephen S -- Rodriguez-Gil, Jorge L -- Rotter, Jerome I -- Rybicki, Benjamin A -- Schwartz, Ann G -- Signorello, Lisa B -- Spitz, Margaret -- Strom, Sara S -- Thun, Michael J -- Tucker, Margaret A -- Wang, Zhaoming -- Wiencke, John K -- Witte, John S -- Wrensch, Margaret -- Wu, Xifeng -- Yamamura, Yuko -- Zanetti, Krista A -- Zheng, Wei -- Ziegler, Regina G -- Zhu, Xiaofeng -- Redline, Susan -- Hirschhorn, Joel N -- Henderson, Brian E -- Taylor, Herman A Jr -- Price, Alkes L -- Hakonarson, Hakon -- Chanock, Stephen J -- Haiman, Christopher A -- Wilson, James G -- Reich, David -- Myers, Simon R -- 090532/Wellcome Trust/United Kingdom -- CA060691/CA/NCI NIH HHS/ -- CA092447/CA/NCI NIH HHS/ -- CA100374/CA/NCI NIH HHS/ -- CA100598/CA/NCI NIH HHS/ -- CA1116460/CA/NCI NIH HHS/ -- CA1116460S1/CA/NCI NIH HHS/ -- CA121197/CA/NCI NIH HHS/ -- CA121197S2/CA/NCI NIH HHS/ -- CA127219/CA/NCI NIH HHS/ -- CA1326792/CA/NCI NIH HHS/ -- CA140388/CA/NCI NIH HHS/ -- CA141716/CA/NCI NIH HHS/ -- CA148085/CA/NCI NIH HHS/ -- CA148127/CA/NCI NIH HHS/ -- CA22453/CA/NCI NIH HHS/ -- CA54281/CA/NCI NIH HHS/ -- CA55769/CA/NCI NIH HHS/ -- CA58223/CA/NCI NIH HHS/ -- CA63464/CA/NCI NIH HHS/ -- CA68485/CA/NCI NIH HHS/ -- CA68578/CA/NCI NIH HHS/ -- CA77305/CA/NCI NIH HHS/ -- CA87895/CA/NCI NIH HHS/ -- CA88164/CA/NCI NIH HHS/ -- ES007784/ES/NIEHS NIH HHS/ -- ES011126/ES/NIEHS NIH HHS/ -- ES06717/ES/NIEHS NIH HHS/ -- ES10126/ES/NIEHS NIH HHS/ -- GM08016/GM/NIGMS NIH HHS/ -- GM091332/GM/NIGMS NIH HHS/ -- HD33175/HD/NICHD NIH HHS/ -- HG004726/HG/NHGRI NIH HHS/ -- HHSN268200960009C/PHS HHS/ -- HL084107/HL/NHLBI NIH HHS/ -- N01-HC-65226/HC/NHLBI NIH HHS/ -- P30 ES010126/ES/NIEHS NIH HHS/ -- R01 CA052689/CA/NCI NIH HHS/ -- R01 CA092447/CA/NCI NIH HHS/ -- R01 HG006399/HG/NHGRI NIH HHS/ -- R01 HL084107-04/HL/NHLBI NIH HHS/ -- R01-CA73629/CA/NCI NIH HHS/ -- U01 HG004168/HG/NHGRI NIH HHS/ -- U01 HG004168-03/HG/NHGRI NIH HHS/ -- Intramural NIH HHS/ -- Wellcome Trust/United Kingdom -- England -- Nature. 2011 Jul 20;476(7359):170-5. doi: 10.1038/nature10336.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Wellcome Trust Centre for Human Genetics, Oxford University, Roosevelt Drive, Oxford OX3 7BN, UK.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/21775986" target="_blank"〉PubMed〈/a〉
    Keywords: Africa, Western/ethnology ; African Americans/*genetics ; Alleles ; Amino Acid Motifs ; Base Sequence ; Chromosome Mapping ; Crossing Over, Genetic/*genetics ; Europe/ethnology ; European Continental Ancestry Group/genetics ; Evolution, Molecular ; Female ; Gene Frequency ; Genetics, Population ; Genome, Human/*genetics ; Genomics ; Haplotypes/genetics ; Histone-Lysine N-Methyltransferase/chemistry/genetics/metabolism ; Humans ; Male ; Molecular Sequence Data ; Pedigree ; Polymorphism, Single Nucleotide/genetics ; Probability
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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