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https://www.readbyqxmd.com/read/29668265/receptor-mediated-delivery-of-crispr-cas9-endonuclease-for-cell-type-specific-gene-editing
#1
Romain Rouet, Benjamin A Thuma, Marc D Roy, Nathanael G Lintner, David M Rubitski, James E Finley, Hanna M Wisniewska, Rima Mendonsa, Ariana Hirsh, Lorena de Oñate, Joan Compte Barrón, Thomas J McLellan, Justin Bellenger, Xidong Feng, Alison Varghese, Boris A Chrunyk, Kris Borzilleri, Kevin D Hesp, Kaihong Zhou, Nannan Ma, Meihua Tu, Robert Dullea, Kim F McClure, Ross C Wilson, Spiros Liras, Vincent Mascitti, Jennifer A Doudna
CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface...
May 30, 2018: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/29664898/correction-selective-stalling-of-human-translation-through-small-molecule-engagement-of-the-ribosome-nascent-chain
#2
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
[This corrects the article DOI: 10.1371/journal.pbio.2001882.].
April 2018: PLoS Biology
https://www.readbyqxmd.com/read/29531059/programmable-rna-recognition-using-a-crispr-associated-argonaute
#3
Audrone Lapinaite, Jennifer A Doudna, Jamie H D Cate
Argonaute proteins (Agos) are present in all domains of life. Although the physiological function of eukaryotic Agos in regulating gene expression is well documented, the biological roles of many of their prokaryotic counterparts remain enigmatic. In some bacteria, Agos are associated with CRISPR (clustered regularly interspaced short palindromic repeats) loci and use noncanonical 5'-hydroxylated guide RNAs (gRNAs) for nucleic acid targeting. Here we show that using 5-bromo-2'-deoxyuridine (BrdU) as the 5' nucleotide of gRNAs stabilizes in vitro reconstituted CRISPR-associated Marinitoga piezophila Argonaute-gRNA complexes (MpAgo RNPs) and significantly improves their specificity and affinity for RNA targets...
March 27, 2018: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29449511/crispr-cas12a-target-binding-unleashes-indiscriminate-single-stranded-dnase-activity
#4
Janice S Chen, Enbo Ma, Lucas B Harrington, Maria Da Costa, Xinran Tian, Joel M Palefsky, Jennifer A Doudna
CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a has been harnessed for genome editing on the basis of its ability to generate targeted, double-stranded DNA breaks. Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules. We find that target-activated, nonspecific single-stranded deoxyribonuclease (ssDNase) cleavage is also a property of other type V CRISPR-Cas12 enzymes...
April 27, 2018: Science
https://www.readbyqxmd.com/read/29448761/spotlight-a-conversation-with-laura-kiessling-and-jennifer-doudna
#5
Laura L Kiessling, Jennifer A Doudna
ACS Chemical Biology recorded a special podcast, in which Editor-in-Chief Laura Kiessling (Massachusetts Institute of Technology) interviews CRISPR investigator and former Associate Editor Jennifer Doudna (University of California, Berkeley). Listen to the podcast here . A transcript of the interview, which has been lightly edited, is published here as part of our Special Issue on the Chemical Biology of CRISPR.
February 16, 2018: ACS Chemical Biology
https://www.readbyqxmd.com/read/29303478/rna-dependent-rna-targeting-by-crispr-cas9
#6
Steven C Strutt, Rachel M Torrez, Emine Kaya, Oscar A Negrete, Jennifer A Doudna
Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo...
January 5, 2018: ELife
https://www.readbyqxmd.com/read/29222508/crispr-cpf1-mediates-efficient-homology-directed-repair-and-temperature-controlled-genome-editing
#7
Miguel A Moreno-Mateos, Juan P Fernandez, Romain Rouet, Charles E Vejnar, Maura A Lane, Emily Mis, Mustafa K Khokha, Jennifer A Doudna, Antonio J Giraldez
Cpf1 is a novel class of CRISPR-Cas DNA endonucleases, with a wide range of activity across different eukaryotic systems. Yet, the underlying determinants of this variability are poorly understood. Here, we demonstrate that LbCpf1, but not AsCpf1, ribonucleoprotein complexes allow efficient mutagenesis in zebrafish and Xenopus. We show that temperature modulates Cpf1 activity by controlling its ability to access genomic DNA. This effect is stronger on AsCpf1, explaining its lower efficiency in ectothermic organisms...
December 8, 2017: Nature Communications
https://www.readbyqxmd.com/read/29141229/widespread-translational-remodeling-during-human-neuronal-differentiation
#8
John D Blair, Dirk Hockemeyer, Jennifer A Doudna, Helen S Bateup, Stephen N Floor
Faithful cellular differentiation requires temporally precise activation of gene expression programs, which are coordinated at the transcriptional and translational levels. Neurons express the most complex set of mRNAs of any human tissue, but translational changes during neuronal differentiation remain incompletely understood. Here, we induced forebrain neuronal differentiation of human embryonic stem cells (hESCs) and measured genome-wide RNA and translation levels with transcript-isoform resolution. We found that thousands of genes change translation status during differentiation without a corresponding change in RNA level...
November 14, 2017: Cell Reports
https://www.readbyqxmd.com/read/29127284/a-thermostable-cas9-with-increased-lifetime-in-human-plasma
#9
Lucas B Harrington, David Paez-Espino, Brett T Staahl, Janice S Chen, Enbo Ma, Nikos C Kyrpides, Jennifer A Doudna
CRISPR-Cas9 is a powerful technology that has enabled genome editing in a wide range of species. However, the currently developed Cas9 homologs all originate from mesophilic bacteria, making them susceptible to degradation and unsuitable for applications requiring cleavage at elevated temperatures. Here, we show that the Cas9 protein from the thermophilic bacterium Geobacillus stearothermophilus (GeoCas9) catalyzes RNA-guided DNA cleavage at elevated temperatures. GeoCas9 is active at temperatures up to 70 °C, compared to 45 °C for Streptococcus pyogenes Cas9 (SpyCas9), which expands the temperature range for CRISPR-Cas9 applications...
November 10, 2017: Nature Communications
https://www.readbyqxmd.com/read/29080263/genomes-in-focus-development-and-applications-of-crispr-cas9-imaging-technologies
#10
REVIEW
Spencer C Knight, Robert Tjian, Jennifer A Doudna
The discovery of the CRISPR-Cas9 endonuclease has enabled facile genome editing in living cells and organisms. Catalytically inactive Cas9 (dCas9) retains the ability to bind DNA in an RNA-guided fashion, and has additionally been explored as a tool for transcriptional modulation, epigenetic editing, and genome imaging. This Review highlights recent progress and challenges in the development of dCas9 for imaging genomic loci. The emergence and maturation of this technology offers the potential to answer mechanistic questions about chromosome dynamics and three-dimensional genome organization in vivo...
April 9, 2018: Angewandte Chemie
https://www.readbyqxmd.com/read/28931002/enhanced-proofreading-governs-crispr-cas9-targeting-accuracy
#11
Janice S Chen, Yavuz S Dagdas, Benjamin P Kleinstiver, Moira M Welch, Alexander A Sousa, Lucas B Harrington, Samuel H Sternberg, J Keith Joung, Ahmet Yildiz, Jennifer A Doudna
The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity are unknown. Here, using single-molecule Förster resonance energy transfer experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state when bound to mismatched targets...
October 19, 2017: Nature
https://www.readbyqxmd.com/read/28892041/guide-bound-structures-of-an-rna-targeting-a-cleaving-crispr-cas13a-enzyme
#12
Gavin J Knott, Alexandra East-Seletsky, Joshua C Cofsky, James M Holton, Emeric Charles, Mitchell R O'Connell, Jennifer A Doudna
CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR-Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation...
October 2017: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/28859601/book-review-a-crack-in-creation-gene-editing-and-the-unthinkable-power-to-control-evolution-a-crack-in-creation-gene-editing-and-the-unthinkable-power-to-control-evolution-doudna-jennifer-a-sternberg-samuel-h-isbn-13-978-0544716940-2017-houghton-mifflin-harcourt
#13
https://www.readbyqxmd.com/read/28844692/a-broad-spectrum-inhibitor-of-crispr-cas9
#14
Lucas B Harrington, Kevin W Doxzen, Enbo Ma, Jun-Jie Liu, Gavin J Knott, Alireza Edraki, Bianca Garcia, Nadia Amrani, Janice S Chen, Joshua C Cofsky, Philip J Kranzusch, Erik J Sontheimer, Alan R Davidson, Karen L Maxwell, Jennifer A Doudna
CRISPR-Cas9 proteins function within bacterial immune systems to target and destroy invasive DNA and have been harnessed as a robust technology for genome editing. Small bacteriophage-encoded anti-CRISPR proteins (Acrs) can inactivate Cas9, providing an efficient off switch for Cas9-based applications. Here, we show that two Acrs, AcrIIC1 and AcrIIC3, inhibit Cas9 by distinct strategies. AcrIIC1 is a broad-spectrum Cas9 inhibitor that prevents DNA cutting by multiple divergent Cas9 orthologs through direct binding to the conserved HNH catalytic domain of Cas9...
September 7, 2017: Cell
https://www.readbyqxmd.com/read/28808686/a-conformational-checkpoint-between-dna-binding-and-cleavage-by-crispr-cas9
#15
Yavuz S Dagdas, Janice S Chen, Samuel H Sternberg, Jennifer A Doudna, Ahmet Yildiz
The Cas9 endonuclease is widely used for genome engineering applications by programming its single-guide RNA, and ongoing work is aimed at improving the accuracy and efficiency of DNA targeting. DNA cleavage of Cas9 is controlled by the conformational state of the HNH nuclease domain, but the mechanism that governs HNH activation at on-target DNA while reducing cleavage activity at off-target sites remains poorly understood. Using single-molecule Förster resonance energy transfer, we identified an intermediate state of Streptococcus pyogenes Cas9, representing a conformational checkpoint between DNA binding and cleavage...
August 2017: Science Advances
https://www.readbyqxmd.com/read/28741483/jennifer-doudna-tailoring-the-genome
#16
(no author information available yet)
No abstract text is available yet for this article.
October 2016: Trends in Cancer
https://www.readbyqxmd.com/read/28729350/structures-of-the-crispr-genome-integration-complex
#17
Addison V Wright, Jun-Jie Liu, Gavin J Knott, Kevin W Doxzen, Eva Nogales, Jennifer A Doudna
CRISPR-Cas systems depend on the Cas1-Cas2 integrase to capture and integrate short foreign DNA fragments into the CRISPR locus, enabling adaptation to new viruses. We present crystal structures of Cas1-Cas2 bound to both donor and target DNA in intermediate and product integration complexes, as well as a cryo-electron microscopy structure of the full CRISPR locus integration complex, including the accessory protein IHF (integration host factor). The structures show unexpectedly that indirect sequence recognition dictates integration site selection by favoring deformation of the repeat and the flanking sequences...
September 15, 2017: Science
https://www.readbyqxmd.com/read/28706995/disabling-cas9-by-an-anti-crispr-dna-mimic
#18
Jiyung Shin, Fuguo Jiang, Jun-Jie Liu, Nicolas L Bray, Benjamin J Rauch, Seung Hyun Baik, Eva Nogales, Joseph Bondy-Denomy, Jacob E Corn, Jennifer A Doudna
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known, and the potential applications for Cas9 inhibitor proteins in mammalian cells have not been fully established...
July 2017: Science Advances
https://www.readbyqxmd.com/read/28703186/gene-editing-crispr-book-review-doudna-responds
#19
Jennifer Doudna
No abstract text is available yet for this article.
July 12, 2017: Nature
https://www.readbyqxmd.com/read/28520746/dna-recognition-by-an-rna-guided-bacterial-argonaute
#20
Kevin W Doxzen, Jennifer A Doudna
Argonaute (Ago) proteins are widespread in prokaryotes and eukaryotes and share a four-domain architecture capable of RNA- or DNA-guided nucleic acid recognition. Previous studies identified a prokaryotic Argonaute protein from the eubacterium Marinitoga piezophila (MpAgo), which binds preferentially to 5'-hydroxylated guide RNAs and cleaves single-stranded RNA (ssRNA) and DNA (ssDNA) targets. Here we present a 3.2 Å resolution crystal structure of MpAgo bound to a 21-nucleotide RNA guide and a complementary 21-nucleotide ssDNA substrate...
2017: PloS One
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