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https://www.readbyqxmd.com/read/28384323/correction-rna-and-dna-targeting-by-a-reconstituted-thermus-thermophilus-type-iii-a-crispr-cas-system
#1
Tina Y Liu, Anthony T Iavarone, Jennifer A Doudna
[This corrects the article DOI: 10.1371/journal.pone.0170552.].
2017: PloS One
https://www.readbyqxmd.com/read/28375731/crispr-cas9-structures-and-mechanisms
#2
Fuguo Jiang, Jennifer A Doudna
Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes...
March 30, 2017: Annual Review of Biophysics
https://www.readbyqxmd.com/read/28334779/targeted-gene-knock-in-by-homology-directed-genome-editing-using-cas9-ribonucleoprotein-and-aav-donor-delivery
#3
Thomas Gaj, Brett T Staahl, Gonçalo M C Rodrigues, Prajit Limsirichai, Freja K Ekman, Jennifer A Doudna, David V Schaffer
Realizing the full potential of genome editing requires the development of efficient and broadly applicable methods for delivering programmable nucleases and donor templates for homology-directed repair (HDR). The RNA-guided Cas9 endonuclease can be introduced into cells as a purified protein in complex with a single guide RNA (sgRNA). Such ribonucleoproteins (RNPs) can facilitate the high-fidelity introduction of single-base substitutions via HDR following co-delivery with a single-stranded DNA oligonucleotide...
March 2, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28323820/selective-stalling-of-human-translation-through-small-molecule-engagement-of-the-ribosome-nascent-chain
#4
Nathanael G Lintner, Kim F McClure, Donna Petersen, Allyn T Londregan, David W Piotrowski, Liuqing Wei, Jun Xiao, Michael Bolt, Paula M Loria, Bruce Maguire, Kieran F Geoghegan, Austin Huang, Tim Rolph, Spiros Liras, Jennifer A Doudna, Robert G Dullea, Jamie H D Cate
Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating the levels of plasma low-density lipoprotein cholesterol (LDL-C). Here, we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling, we demonstrate that PF-06446846 is highly selective for the inhibition of PCSK9 translation...
March 2017: PLoS Biology
https://www.readbyqxmd.com/read/28196981/rna-based-recognition-and-targeting-sowing-the-seeds-of-specificity
#5
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
#6
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
#7
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
#8
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
#9
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
#10
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
#11
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...
January 11, 2017: 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
#12
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
#13
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...
December 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
#14
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
#15
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
#16
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
#17
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
#18
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
#19
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
#20
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
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