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https://www.readbyqxmd.com/read/27783955/a-cas9-ribonucleoprotein-platform-for-functional-genetic-studies-of-hiv-host-interactions-in-primary-human-t-cells
#1
Judd F Hultquist, Kathrin Schumann, Jonathan M Woo, Lara Manganaro, Michael J McGregor, Jennifer Doudna, Viviana Simon, Nevan J Krogan, Alexander Marson
New genetic tools are needed to understand the functional interactions between HIV and human host factors in primary cells. We recently developed a method to edit the genome of primary CD4(+) T cells by electroporation of CRISPR/Cas9 ribonucleoproteins (RNPs). Here, we adapted this methodology to a high-throughput platform for the efficient, arrayed editing of candidate host factors. CXCR4 or CCR5 knockout cells generated with this method are resistant to HIV infection in a tropism-dependent manner, whereas knockout of LEDGF or TNPO3 results in a tropism-independent reduction in infection...
October 25, 2016: Cell Reports
https://www.readbyqxmd.com/read/27749837/atac-see-reveals-the-accessible-genome-by-transposase-mediated-imaging-and-sequencing
#2
Xingqi Chen, Ying Shen, Will Draper, Jason D Buenrostro, Ulrike Litzenburger, Seung Woo Cho, Ansuman T Satpathy, Ava C Carter, Rajarshi P Ghosh, Alexandra East-Seletsky, Jennifer A Doudna, William J Greenleaf, Jan T Liphardt, Howard Y Chang
Spatial organization of the genome plays a central role in gene expression, DNA replication, and repair. But current epigenomic approaches largely map DNA regulatory elements outside of the native context of the nucleus. Here we report assay of transposase-accessible chromatin with visualization (ATAC-see), a transposase-mediated imaging technology that employs direct imaging of the accessible genome in situ, cell sorting, and deep sequencing to reveal the identity of the imaged elements. ATAC-see revealed the cell-type-specific spatial organization of the accessible genome and the coordinated process of neutrophil chromatin extrusion, termed NETosis...
October 17, 2016: Nature Methods
https://www.readbyqxmd.com/read/27731797/insights-into-hiv-1-proviral-transcription-from-integrative-structure-and-dynamics-of-the-tat-aff4-p-tefb-tar-complex
#3
Ursula Schulze-Gahmen, Ignacia Echeverria, Goran Stjepanovic, Yun Bai, Huasong Lu, Dina Schneidman-Duhovny, Jennifer A Doudna, Qiang Zhou, Andrej Sali, James H Hurley
HIV-1 Tat hijacks the human superelongation complex (SEC) to promote proviral transcription. Here we report the 5.9 Å structure of HIV-1 TAR in complex with HIV-1 Tat and human AFF4, CDK9, and CycT1. The TAR central loop contacts the CycT1 Tat-TAR recognition motif (TRM) and the second Tat Zn(2+)-binding loop. Hydrogen-deuterium exchange (HDX) shows that AFF4 helix 2 is stabilized in the TAR complex despite not touching the RNA, explaining how it enhances TAR binding to the SEC 50-fold. RNA SHAPE and SAXS data were used to help model the extended (Tat Arginine-Rich Motif) ARM, which enters the TAR major groove between the bulge and the central loop...
October 12, 2016: ELife
https://www.readbyqxmd.com/read/27669025/two-distinct-rnase-activities-of-crispr-c2c2-enable-guide-rna-processing-and-rna-detection
#4
Alexandra East-Seletsky, Mitchell R O'Connell, Spencer C Knight, David Burstein, Jamie H D Cate, Robert Tjian, Jennifer A Doudna
Bacterial adaptive immune systems use CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage. Although most prokaryotic adaptive immune systems generally target DNA substrates, type III and VI CRISPR systems direct interference complexes against single-stranded RNA substrates. In type VI systems, the single-subunit C2c2 protein functions as an RNA-guided RNA endonuclease (RNase). How this enzyme acquires mature CRISPR RNAs (crRNAs) that are essential for immune surveillance and how it carries out crRNA-mediated RNA cleavage remain unclear...
September 26, 2016: Nature
https://www.readbyqxmd.com/read/27624851/real-time-observation-of-dna-recognition-and-rejection-by-the-rna-guided-endonuclease-cas9
#5
Digvijay Singh, Samuel H Sternberg, Jingyi Fei, Jennifer A Doudna, Taekjip Ha
Binding specificity of Cas9-guide RNA complexes to DNA is important for genome-engineering applications; however, how mismatches influence target recognition/rejection kinetics is not well understood. Here we used single-molecule FRET to probe real-time interactions between Cas9-RNA and DNA targets. The bimolecular association rate is only weakly dependent on sequence; however, the dissociation rate greatly increases from <0.006 s(-1) to >2 s(-1) upon introduction of mismatches proximal to protospacer-adjacent motif (PAM), demonstrating that mismatches encountered early during heteroduplex formation induce rapid rejection of off-target DNA...
September 14, 2016: Nature Communications
https://www.readbyqxmd.com/read/27606440/applications-of-crispr-technologies-in-research-and-beyond
#6
Rodolphe Barrangou, Jennifer A Doudna
Programmable DNA cleavage using CRISPR-Cas9 enables efficient, site-specific genome engineering in single cells and whole organisms. In the research arena, versatile CRISPR-enabled genome editing has been used in various ways, such as controlling transcription, modifying epigenomes, conducting genome-wide screens and imaging chromosomes. CRISPR systems are already being used to alleviate genetic disorders in animals and are likely to be employed soon in the clinic to treat human diseases of the eye and blood...
September 8, 2016: Nature Biotechnology
https://www.readbyqxmd.com/read/27595346/protecting-genome-integrity-during-crispr-immune-adaptation
#7
Addison V Wright, Jennifer A Doudna
Bacterial CRISPR-Cas systems include genomic arrays of short repeats flanking foreign DNA sequences and provide adaptive immunity against viruses. Integration of foreign DNA must occur specifically to avoid damaging the genome or the CRISPR array, but surprisingly promiscuous activity occurs in vitro. Here we reconstituted full-site DNA integration and show that the Streptococcus pyogenes type II-A Cas1-Cas2 integrase maintains specificity in part through limitations on the second integration step. At non-CRISPR sites, integration stalls at the half-site intermediate, thereby enabling reaction reversal...
October 2016: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/27588603/dna-targeting-by-a-minimal-crispr-rna-guided-cascade
#8
Megan L Hochstrasser, David W Taylor, Jack E Kornfeld, Eva Nogales, Jennifer A Doudna
Bacteria employ surveillance complexes guided by CRISPR (clustered, regularly interspaced, short palindromic repeats) RNAs (crRNAs) to target foreign nucleic acids for destruction. Although most type I and type III CRISPR systems require four or more distinct proteins to form multi-subunit surveillance complexes, the type I-C systems use just three proteins to achieve crRNA maturation and double-stranded DNA target recognition. We show that each protein plays multiple functional and structural roles: Cas5c cleaves pre-crRNAs and recruits Cas7 to position the RNA guide for DNA binding and unwinding by Cas8c...
September 1, 2016: Molecular Cell
https://www.readbyqxmd.com/read/27462815/eif3d-is-an-mrna-cap-binding-protein-that-is-required-for-specialized-translation-initiation
#9
Amy S Lee, Philip J Kranzusch, Jennifer A Doudna, Jamie H D Cate
Eukaryotic mRNAs contain a 5′ cap structure that is crucial for recruitment of the translation machinery and initiation of protein synthesis. mRNA recognition is thought to require direct interactions between eukaryotic initiation factor 4E (eIF4E) and the mRNA cap. However, translation of numerous capped mRNAs remains robust during cellular stress, early development, and cell cycle progression despite inactivation of eIF4E. Here we describe a cap-dependent pathway of translation initiation in human cells that relies on a previously unknown cap-binding activity of eIF3d, a subunit of the 800-kilodalton eIF3 complex...
August 4, 2016: Nature
https://www.readbyqxmd.com/read/27343348/reconstitution-of-selective-hiv-1-rna-packaging-in-vitro-by-membrane-bound-gag-assemblies
#10
Lars-Anders Carlson, Yun Bai, Sarah C Keane, Jennifer A Doudna, James H Hurley
HIV-1 Gag selects and packages a dimeric, unspliced viral RNA in the context of a large excess of cytosolic human RNAs. As Gag assembles on the plasma membrane, the HIV-1 genome is enriched relative to cellular RNAs by an unknown mechanism. We used a minimal system consisting of purified RNAs, recombinant HIV-1 Gag and giant unilamellar vesicles to recapitulate the selective packaging of the 5' untranslated region of the HIV-1 genome in the presence of excess competitor RNA. Mutations in the CA-CTD domain of Gag which subtly affect the self-assembly of Gag abrogated RNA selectivity...
2016: ELife
https://www.readbyqxmd.com/read/27211867/crispr-immunological-memory-requires-a-host-factor-for-specificity
#11
James K Nuñez, Lawrence Bai, Lucas B Harrington, Tracey L Hinder, Jennifer A Doudna
Bacteria and archaea employ adaptive immunity against foreign genetic elements using CRISPR-Cas systems. To generate immunological memory, the Cas1-Cas2 protein complex captures 30-40 base pair segments of foreign DNA and catalyzes their integration into the host genome as unique spacer sequences. Although spacers are inserted strictly at the A-T-rich leader end of CRISPR loci in vivo, the molecular mechanism of leader-specific spacer integration remains poorly understood. Here we show that the E. coli integration host factor (IHF) protein is required for spacer acquisition in vivo and for integration into linear DNA in vitro...
June 16, 2016: Molecular Cell
https://www.readbyqxmd.com/read/27136077/profiling-of-engineering-hotspots-identifies-an-allosteric-crispr-cas9-switch
#12
Benjamin L Oakes, Dana C Nadler, Avi Flamholz, Christof Fellmann, Brett T Staahl, Jennifer A Doudna, David F Savage
The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease with widespread utility for genome modification. However, the structural constraints limiting the engineering of Cas9 have not been determined. Here we experimentally profile Cas9 using randomized insertional mutagenesis and delineate hotspots in the structure capable of tolerating insertions of a PDZ domain without disruption of the enzyme's binding and cleavage functions...
June 2016: Nature Biotechnology
https://www.readbyqxmd.com/read/27130520/nucleosome-breathing-and-remodeling-constrain-crispr-cas9-function
#13
R Stefan Isaac, Fuguo Jiang, Jennifer A Doudna, Wendell A Lim, Geeta J Narlikar, Ricardo Almeida
The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad...
2016: ELife
https://www.readbyqxmd.com/read/27092008/qnas-with-jennifer-doudna
#14
COMMENT
Prashant Nair
No abstract text is available yet for this article.
May 3, 2016: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/27058758/medulloblastoma-associated-ddx3-variant-selectively-alters-the-translational-response-to-stress
#15
Sekyung Oh, Ryan A Flynn, Stephen N Floor, James Purzner, Lance Martin, Brian T Do, Simone Schubert, Dedeepya Vaka, Sorana Morrissy, Yisu Li, Marcel Kool, Volker Hovestadt, David T W Jones, Paul A Northcott, Thomas Risch, Hans-Jörg Warnatz, Marie-Laure Yaspo, Christopher M Adams, Ryan D Leib, Marcus Breese, Marco A Marra, David Malkin, Peter Lichter, Jennifer A Doudna, Stefan M Pfister, Michael D Taylor, Howard Y Chang, Yoon-Jae Cho
DDX3X encodes a DEAD-box family RNA helicase (DDX3) commonly mutated in medulloblastoma, a highly aggressive cerebellar tumor affecting both children and adults. Despite being implicated in several facets of RNA metabolism, the nature and scope of DDX3's interactions with RNA remain unclear. Here, we show DDX3 collaborates extensively with the translation initiation machinery through direct binding to 5'UTRs of nearly all coding RNAs, specific sites on the 18S rRNA, and multiple components of the translation initiation complex...
May 10, 2016: Oncotarget
https://www.readbyqxmd.com/read/27035975/a-bacterial-argonaute-with-noncanonical-guide-rna-specificity
#16
Emine Kaya, Kevin W Doxzen, Kilian R Knoll, Ross C Wilson, Steven C Strutt, Philip J Kranzusch, Jennifer A Doudna
Eukaryotic Argonaute proteins induce gene silencing by small RNA-guided recognition and cleavage of mRNA targets. Although structural similarities between human and prokaryotic Argonautes are consistent with shared mechanistic properties, sequence and structure-based alignments suggested that Argonautes encoded within CRISPR-cas [clustered regularly interspaced short palindromic repeats (CRISPR)-associated] bacterial immunity operons have divergent activities. We show here that the CRISPR-associated Marinitoga piezophila Argonaute (MpAgo) protein cleaves single-stranded target sequences using 5'-hydroxylated guide RNAs rather than the 5'-phosphorylated guides used by all known Argonautes...
April 12, 2016: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/26997482/programmable-rna-tracking-in-live-cells-with-crispr-cas9
#17
David A Nelles, Mark Y Fang, Mitchell R O'Connell, Jia L Xu, Sebastian J Markmiller, Jennifer A Doudna, Gene W Yeo
RNA-programmed genome editing using CRISPR/Cas9 from Streptococcus pyogenes has enabled rapid and accessible alteration of specific genomic loci in many organisms. A flexible means to target RNA would allow alteration and imaging of endogenous RNA transcripts analogous to CRISPR/Cas-based genomic tools, but most RNA targeting methods rely on incorporation of exogenous tags. Here, we demonstrate that nuclease-inactive S. pyogenes CRISPR/Cas9 can bind RNA in a nucleic-acid-programmed manner and allow endogenous RNA tracking in living cells...
April 7, 2016: Cell
https://www.readbyqxmd.com/read/26963549/voices-of-biotech
#18
Ido Amit, David Baker, Roger Barker, Bonnie Berger, Carolyn Bertozzi, Sangeeta Bhatia, Alessandra Biffi, Francesca Demichelis, Jennifer Doudna, Steven F Dowdy, Drew Endy, Moritz Helmstaedter, Howard Junca, Carl June, Sasha Kamb, Anastasia Khvorova, Dae-Hyeong Kim, Jin-Soo Kim, Yamuna Krishnan, Melike Lakadamyali, Tuuli Lappalainen, Sharon Lewin, James Liao, Nick Loman, Emma Lundberg, Lee Lynd, Cathie Martin, Ira Mellman, Atsushi Miyawaki, Christine Mummery, Karen Nelson, Jeanne Paz, Pamela Peralta-Yahya, Paola Picotti, Kornelia Polyak, Kristala Prather, Jun Qin, Stephen Quake, Aviv Regev, John A Rogers, Reshma Shetty, Morten Sommer, Molly Stevens, Gustavo Stolovitzky, Masayo Takahashi, Fuchou Tang, Sarah Teichmann, Maria-Elena Torres-Padilla, Leena Tripathi, Praveen Vemula, Greg Verdine, Frank Vollmer, Jun Wang, Jackie Y Ying, Feng Zhang, Tian Zhang
No abstract text is available yet for this article.
March 2016: Nature Biotechnology
https://www.readbyqxmd.com/read/26874259/energy-biotechnology-in-the-crispr-cas9-era
#19
REVIEW
Raissa Estrela, Jamie Harrison Doudna Cate
The production of bioenergy from plant biomass previously relied on using microorganisms that rapidly and efficiently convert simple sugars into fuels and chemicals. However, to exploit the far more abundant carbon fixed in plant cell walls, future industrial production hosts will need to be engineered to leverage the most efficient biochemical pathways and most robust traits that can be found in nature. The CRISPR-Cas9 genome editing technology now enables writing the genome at will, which will allow biotechnology to become an 'information science...
April 2016: Current Opinion in Biotechnology
https://www.readbyqxmd.com/read/26857072/chemical-and-biophysical-modulation-of-cas9-for-tunable-genome-engineering
#20
James K Nuñez, Lucas B Harrington, Jennifer A Doudna
The application of the CRISPR-Cas9 system for genome engineering has revolutionized the ability to interrogate genomes of mammalian cells. Programming the Cas9 endonuclease to induce DNA breaks at specified sites is achieved by simply modifying the sequence of its cognate guide RNA. Although Cas9-mediated genome editing has been shown to be highly specific, cleavage events at off-target sites have also been reported. Minimizing, and eventually abolishing, unwanted off-target cleavage remains a major goal of the CRISPR-Cas9 technology before its implementation for therapeutic use...
March 18, 2016: ACS Chemical Biology
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