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https://www.readbyqxmd.com/read/28196981/rna-based-recognition-and-targeting-sowing-the-seeds-of-specificity
#1
REVIEW
Stanislaw A Gorski, Jörg Vogel, Jennifer A Doudna
RNA is involved in the regulation of multiple cellular processes, often by forming sequence-specific base pairs with cellular RNA or DNA targets that must be identified among the large number of nucleic acids in a cell. Several RNA-based regulatory systems in eukaryotes, bacteria and archaea, including microRNAs (miRNAs), small interfering RNAs (siRNAs), CRISPR RNAs (crRNAs) and small RNAs (sRNAs) that are dependent on the RNA chaperone protein Hfq, achieve specificity using similar strategies. Central to their function is the presentation of short 'seed sequences' within a ribonucleoprotein complex to facilitate the search for and recognition of targets...
February 15, 2017: Nature Reviews. Molecular Cell Biology
https://www.readbyqxmd.com/read/28191903/efficient-genome-editing-in-the-mouse-brain-by-local-delivery-of-engineered-cas9-ribonucleoprotein-complexes
#2
Brett T Staahl, Madhurima Benekareddy, Claire Coulon-Bainier, Ashwin A Banfal, Stephen N Floor, Jennifer K Sabo, Cole Urnes, Gabriela Acevedo Munares, Anirvan Ghosh, Jennifer A Doudna
We demonstrate editing of post-mitotic neurons in the adult mouse brain following injection of Cas9 ribonucleoprotein (RNP) complexes in the hippocampus, striatum and cortex. Engineered variants of Cas9 with multiple SV40 nuclear localization sequences enabled a tenfold increase in the efficiency of neuronal editing in vivo. These advances indicate the potential of genome editing in the brain to correct or inactivate the underlying genetic causes of neurological diseases.
February 13, 2017: Nature Biotechnology
https://www.readbyqxmd.com/read/28114398/rna-and-dna-targeting-by-a-reconstituted-thermus-thermophilus-type-iii-a-crispr-cas-system
#3
Tina Y Liu, Anthony T Iavarone, Jennifer A Doudna
CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are RNA-guided adaptive immunity pathways used by bacteria and archaea to defend against phages and plasmids. Type III-A systems use a multisubunit interference complex called Csm, containing Cas proteins and a CRISPR RNA (crRNA) to target cognate nucleic acids. The Csm complex is intriguing in that it mediates RNA-guided targeting of both RNA and transcriptionally active DNA, but the mechanism is not well understood...
2017: PloS One
https://www.readbyqxmd.com/read/28017588/mutations-in-cas9-enhance-the-rate-of-acquisition-of-viral-spacer-sequences-during-the-crispr-cas-immune-response
#4
Robert Heler, Addison V Wright, Marija Vucelja, David Bikard, Jennifer A Doudna, Luciano A Marraffini
CRISPR loci and their associated (Cas) proteins encode a prokaryotic immune system that protects against viruses and plasmids. Upon infection, a low fraction of cells acquire short DNA sequences from the invader. These sequences (spacers) are integrated in between the repeats of the CRISPR locus and immunize the host against the matching invader. Spacers specify the targets of the CRISPR immune response through transcription into short RNA guides that direct Cas nucleases to the invading DNA molecules. Here we performed random mutagenesis of the RNA-guided Cas9 nuclease to look for variants that provide enhanced immunity against viral infection...
January 5, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28008168/cornerstones-of-crispr-cas-in-drug-discovery-and-therapy
#5
REVIEW
Christof Fellmann, Benjamin G Gowen, Pei-Chun Lin, Jennifer A Doudna, Jacob E Corn
The recent development of CRISPR-Cas systems as easily accessible and programmable tools for genome editing and regulation is spurring a revolution in biology. Paired with the rapid expansion of reference and personalized genomic sequence information, technologies based on CRISPR-Cas are enabling nearly unlimited genetic manipulation, even in previously difficult contexts, including human cells. Although much attention has focused on the potential of CRISPR-Cas to cure Mendelian diseases, the technology also holds promise to transform the development of therapies to treat complex heritable and somatic disorders...
2017: Nature Reviews. Drug Discovery
https://www.readbyqxmd.com/read/28005056/new-crispr-cas-systems-from-uncultivated-microbes
#6
David Burstein, Lucas B Harrington, Steven C Strutt, Alexander J Probst, Karthik Anantharaman, Brian C Thomas, Jennifer A Doudna, Jillian F Banfield
CRISPR-Cas systems provide microbes with adaptive immunity by employing short DNA sequences, termed spacers, that guide Cas proteins to cleave foreign DNA. Class 2 CRISPR-Cas systems are streamlined versions, in which a single RNA-bound Cas protein recognizes and cleaves target sequences. The programmable nature of these minimal systems has enabled researchers to repurpose them into a versatile technology that is broadly revolutionizing biological and clinical research. However, current CRISPR-Cas technologies are based solely on systems from isolated bacteria, leaving the vast majority of enzymes from organisms that have not been cultured untapped...
February 9, 2017: Nature
https://www.readbyqxmd.com/read/27958730/rna-scanning-of-a-molecular-machine-with-a-built-in-ruler
#7
Hye Ran Koh, Mary Anne Kidwell, Jennifer Doudna, Sua Myong
Advanced single-molecule techniques have enabled tracking of nanometer-scale movements of DNA and RNA motor proteins in real time. Previously, we reported an ATP-independent diffusion of transactivation response RNA binding protein (TRBP) on dsRNA, yet the mechanistic details remain elusive. Using single-molecule fluorescence assays, we demonstrate that the diffusion activity of TRBP is coordinated by an independent movement of two subdomains, dsRBD1 and dsRBD2, in which the diffusion distance is determined by the length of a flexible linker domain that connects the two dsRBDs...
December 29, 2016: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/27783955/a-cas9-ribonucleoprotein-platform-for-functional-genetic-studies-of-hiv-host-interactions-in-primary-human-t-cells
#8
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
#9
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
#10
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
#11
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...
October 13, 2016: Nature
https://www.readbyqxmd.com/read/27624851/real-time-observation-of-dna-recognition-and-rejection-by-the-rna-guided-endonuclease-cas9
#12
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
#13
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
#14
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
#15
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
#16
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
#17
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
#18
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
#19
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
#20
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
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