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https://www.readbyqxmd.com/read/29222508/crispr-cpf1-mediates-efficient-homology-directed-repair-and-temperature-controlled-genome-editing
#1
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
#2
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
#3
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
#4
Spencer Knight, Robert Tjian, Jennifer 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 genomic 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 new mechanistic questions about chromosome dynamics and three-dimensional genome organization in vivo...
October 27, 2017: Angewandte Chemie
https://www.readbyqxmd.com/read/28931002/enhanced-proofreading-governs-crispr-cas9-targeting-accuracy
#5
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(1-4). 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(5-9). Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state(10) when bound to mismatched targets...
September 20, 2017: Nature
https://www.readbyqxmd.com/read/28892041/guide-bound-structures-of-an-rna-targeting-a-cleaving-crispr-cas13a-enzyme
#6
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
#7
https://www.readbyqxmd.com/read/28844692/a-broad-spectrum-inhibitor-of-crispr-cas9
#8
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
#9
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
#10
(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
#11
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
#12
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
#13
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
#14
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
https://www.readbyqxmd.com/read/28495970/high-throughput-biochemical-profiling-reveals-sequence-determinants-of-dcas9-off-target-binding-and-unbinding
#15
Evan A Boyle, Johan O L Andreasson, Lauren M Chircus, Samuel H Sternberg, Michelle J Wu, Chantal K Guegler, Jennifer A Doudna, William J Greenleaf
The bacterial adaptive immune system CRISPR-Cas9 has been appropriated as a versatile tool for editing genomes, controlling gene expression, and visualizing genetic loci. To analyze Cas9's ability to bind DNA rapidly and specifically, we generated multiple libraries of potential binding partners for measuring the kinetics of nuclease-dead Cas9 (dCas9) interactions. Using a massively parallel method to quantify protein-DNA interactions on a high-throughput sequencing flow cell, we comprehensively assess the effects of combinatorial mismatches between guide RNA (gRNA) and target nucleotides, both in the seed and in more distal nucleotides, plus disruption of the protospacer adjacent motif (PAM)...
May 23, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28475872/rna-targeting-by-functionally-orthogonal-type-vi-a-crispr-cas-enzymes
#16
Alexandra East-Seletsky, Mitchell R O'Connell, David Burstein, Gavin J Knott, Jennifer A Doudna
CRISPR adaptive immunity pathways protect prokaryotic cells against foreign nucleic acids using CRISPR RNA (crRNA)-guided nucleases. In type VI-A CRISPR-Cas systems, the signature protein Cas13a (formerly C2c2) contains two separate ribonuclease activities that catalyze crRNA maturation and ssRNA degradation. The Cas13a protein family occurs across different bacterial phyla and varies widely in both protein sequence and corresponding crRNA sequence conservation. Although grouped phylogenetically together, we show that the Cas13a enzyme family comprises two distinct functional groups that recognize orthogonal sets of crRNAs and possess different ssRNA cleavage specificities...
May 4, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28384323/correction-rna-and-dna-targeting-by-a-reconstituted-thermus-thermophilus-type-iii-a-crispr-cas-system
#17
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
#18
REVIEW
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...
May 22, 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
#19
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...
June 20, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28323820/selective-stalling-of-human-translation-through-small-molecule-engagement-of-the-ribosome-nascent-chain
#20
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
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