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Results 1 through 20 out of 3582

in category molecular biology


1: The novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells

Markus Hoffmann, Hannah Kleine-Weber et al.

39,313 downloads (posted 31 Jan 2020)

The emergence of a novel, highly pathogenic coronavirus, 2019-nCoV, in China, and its rapid national and international spread pose a global health emergency. Coronaviruses use their spike proteins to select and enter target cells and insights into nCoV-2019 spike (S)-driven entry might facilitate assessment of pandemic potential and reveal therapeutic targets. Here, we demonstrate that 2019-nCoV-S uses the SARS-coronavirus receptor, ACE2, for entry and the cellular protease TMPRSS2 for 2019-nCoV-S priming. A TMPRSS2 inhibitor blocked entry and might constitute a treatment option. Finally, we show that the serum from a convalescent SARS patient neutralized 2019-nCoV-S-driven entry. Our results reveal important commonalities between 2019-nCoV and SARS-coronavirus infection, which might translate into similar transmissibility and disease pathogenesis. Moreover, they identify a target for antiviral intervention.


2: Protocol: Genome-scale CRISPR-Cas9 Knockout and Transcriptional Activation Screening

Julia Joung, Silvana Konermann et al.

26,252 downloads (posted 18 Jun 2016)

Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial immune system CRISPR (clustered regularly interspaced short palindromic repeats) has been adapted for genome-scale screening by combining Cas9 with guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 syste...


3: Index Switching Causes “Spreading-Of-Signal” Among Multiplexed Samples In Illumina HiSeq 4000 DNA Sequencing

Rahul Sinha, Geoff Stanley et al.

23,453 downloads (posted 09 Apr 2017)

Illumina-based next generation sequencing (NGS) has accelerated biomedical discovery through its ability to generate thousands of gigabases of sequencing output per run at a fraction of the time and cost of conventional technologies. The process typically involves four basic steps: library preparation, cluster generation, sequencing, and data analysis. In 2015, a new chemistry of cluster generation was introduced in the newer Illumina machines (HiSeq 3000/4000/X Ten) called exclusion amplification (ExAmp), which was a f...


4: Reversal of ageing- and injury-induced vision loss by Tet-dependent epigenetic reprogramming

Yuancheng Lu, Anitha Krishnan et al.

16,075 downloads (posted 31 Jul 2019)

Ageing is a degenerative process leading to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise, which disrupts youthful gene expression patterns that are required for cells to function optimally and recover from damage. Changes to DNA methylation patterns over time form the basis of an 'ageing clock', but whether old individuals retain information to reset the clock and, if so, whether this would improve tissue function is not known. Of all the tissues in the body, the centr...


5: Shotgun Transcriptome and Isothermal Profiling of SARS-CoV-2 Infection Reveals Unique Host Responses, Viral Diversification, and Drug Interactions

Daniel J Butler, Christopher Mozsary et al.

15,869 downloads (posted 20 Apr 2020)

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused thousands of deaths worldwide, including >18,000 in New York City (NYC) alone. The sudden emergence of this pandemic has highlighted a pressing clinical need for rapid, scalable diagnostics that can detect infection, interrogate strain evolution, and identify novel patient biomarkers. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs, plus a large-scale sho...


6: LAMP-Seq: Population-Scale COVID-19 Diagnostics Using Combinatorial Barcoding

Jonathan L. Schmid-Burgk, Ricarda Maria Schmithausen et al.

15,622 downloads (posted 08 Apr 2020)

The ongoing SARS-CoV-2 pandemic has already caused devastating losses. Exponential spread can be slowed by social distancing and population-wide isolation measures, but those place a tremendous burden on society, and, once lifted, exponential spread can re-emerge. Regular population-scale testing, combined with contact tracing and case isolation, should help break the cycle of transmission, but current detection strategies are not capable of such large-scale processing. Here we present a protocol for LAMP-Seq, a barcode...


7: Mammalian Y RNAs are modified at discrete guanosine residues with N-glycans

Ryan A. Flynn, Benjamin A. H. Smith et al.

12,562 downloads (posted 30 Sep 2019)

Glycans modify lipids and proteins to mediate inter- and intramolecular interactions across all domains of life. RNA, another multifaceted biopolymer, is not thought to be a major target of glycosylation. Here, we challenge this view with evidence that mammalian cells use RNA as a third scaffold for glycosylation in the secretory pathway. Using a battery of chemical and biochemical approaches, we find that a select group of small noncoding RNAs including Y RNAs are modified with complex, sialylated N-glycans (glycoRNAs)...


8: Design and specificity of long ssDNA donors for CRISPR-based knock-in

Han Li, Kyle A. Beckman et al.

11,666 downloads (posted 21 Aug 2017)

CRISPR/Cas technologies have transformed our ability to manipulate genomes for research and gene-based therapy. In particular, homology-directed repair after genomic cleavage allows for precise modification of genes using exogenous donor sequences as templates. While both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) forms of donors have been used as repair templates, a systematic comparison of the performance and specificity of repair using ssDNA versus dsDNA donors is still lacking. Here, we describe an ...


9: Improved CUT&RUN chromatin profiling and analysis tools

Michael P. Meers, Terri Bryson et al.

11,505 downloads (posted 06 Mar 2019)

We previously described a novel alternative to Chromatin Immunoprecipitation, Cleavage Under Targets & Release Using Nuclease (CUT&RUN), in which unfixed permeabilized cells are incubated with antibody, followed by binding of a Protein A-Micrococcal Nuclease (pA/MNase) fusion protein ([1][1]). Upon activation of tethered MNase, the bound complex is excised and released into the supernatant for DNA extraction and sequencing. Here we introduce four enhancements to CUT&RUN: 1) a hybrid Protein A-Protein G-MNase construct t...


10: Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing

Søren M Karst, Ryan M. Ziels et al.

10,963 downloads (posted 24 May 2019)

High-throughput amplicon sequencing of large genomic regions remains challenging for short-read technologies. Here, we report a high-throughput amplicon sequencing approach combining unique molecular identifiers (UMIs) with Oxford Nanopore Technologies or Pacific Biosciences CCS sequencing, yielding high accuracy single-molecule consensus sequences of large genomic regions. Our approach generates amplicon and genomic sequences of >10,000 bp in length with a mean error-rate of 0.0049-0.0006% and chimera rate <0.022%.


11: A comparison of DNA stains and staining methods for Agarose Gel Electrophoresis

Andie C. Hall

10,350 downloads (posted 06 Mar 2019)

Nucleic acid stains are necessary for Agarose Gel Electrophoresis (AGE). The commonly used but mutagenic Ethidium Bromide is being usurped by a range of safer but more expensive alternatives. These safe stains vary in cost, sensitivity and the impedance of DNA as it migrates through the gel. Modified protocols developed to reduce cost increase this variability. In this study, five Gel stains (GelRed™, GelGreen™, SYBR™ safe, SafeView and EZ-Vision®In-Gel Solution) two premixed loading dyes (SafeWhite, EZ-Vision®One) and ...


12: An ultrasensitive, rapid, and portable coronavirus SARS-CoV-2 sequence detection method based on CRISPR-Cas12

Curti Lucia, Pereyra-Bonnet Federico et al.

10,134 downloads (posted 02 Mar 2020)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has received global attention due to the recent outbreak in China. In this work, we report a CRISPR-Cas12 based diagnostic tool to detect synthetic SARS-CoV-2 RNA sequences in a proof-of-principle evaluation. The test proved to be sensitive, rapid, and potentially portable. These key traits of the CRISPR method are critical for virus detection in regions that lack resources to use the currently available methods.


13: A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing

Max J. Kellner, James J Ross et al.

8,799 downloads (posted 23 Jun 2020)

Global efforts to combat the Covid-19 pandemic caused by the beta coronavirus SARS-CoV-2 are currently based on RT-qPCR-based diagnostic tests. However, their high cost, moderate throughput and reliance on sophisticated equipment limit widespread implementation. Loop-mediated isothermal amplification after reverse transcription (RT-LAMP) is an alternative detection method that has the potential to overcome these limitations. Here we present a rapid, robust, highly sensitive and versatile RT-LAMP based SARS-CoV-2 detecti...


14: Background free tracking of single RNA in living cells using catalytically inactive CasE

Feng Gao, Yue Sun et al.

8,194 downloads (posted 13 May 2019)

RNAs have important and diverse functions. Visualizing an isolated RNA in living cells provide us essential information of its roles. By now, there are two kinds of live RNA imaging systems invented, one is the MS2 system and the other is the Cas13a system. In this study, we show that when fused with split-Fp, CasE can be engineered into a live RNA tracking tool.


15: A proximity biotinylation map of a human cell

Christopher D Go, James D.R. Knight et al.

7,900 downloads (posted 07 Oct 2019)

Compartmentalization is an essential characteristic of eukaryotic cells, ensuring that cellular processes are partitioned to defined subcellular locations. High throughput microscopy and biochemical fractionation coupled with mass spectrometry have helped to define the proteomes of multiple organelles and macromolecular structures. However, many compartments have remained refractory to such methods, partly due to lysis and purification artefacts and poor subcompartment resolution. Recently developed proximity-dependent ...


16: Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites

Sisi Kang, Mei Yang et al.

7,484 downloads (posted 07 Mar 2020)

The outbreak of coronavirus disease (COVID-19) in China caused by SARS-CoV-2 virus continually lead to worldwide human infections and deaths. It is currently no specific viral protein targeted therapeutics yet. Viral nucleocapsid protein is a potential antiviral drug target, serving multiple critical functions during the viral life cycle. However, the structural information of SARS-CoV-2 nucleocapsid protein is yet to be clear. Herein, we have determined the 2.7 Å crystal structure of the N-terminal RNA binding domain o...


17: Structural Basis for the Inhibition of the RNA-Dependent RNA Polymerase from SARS-CoV-2 by Remdesivir

Wanchao Yin, Chunyou Mao et al.

7,105 downloads (posted 09 Apr 2020)

The pandemic of Corona Virus Disease 2019 (COVID-19) caused by SARS-CoV-2 has become a global crisis. The replication of SARS-CoV-2 requires the viral RNA-dependent RNA polymerase (RdRp), a direct target of the antiviral drug, Remdesivir. Here we report the structure of the SARS-CoV-2 RdRp either in the apo form or in complex with a 50-base template-primer RNA and Remdesivir at a resolution range of 2.5-2.8 Å. The complex structure reveals that the partial double-stranded RNA template is inserted into the central channe...


18: The SARS-CoV-2 exerts a distinctive strategy for interacting with the ACE2 human receptor

Esther S Brielle, Dina Schneidman-Duhovny et al.

6,231 downloads (posted 12 Mar 2020)

The COVID-19 disease has plagued over 110 countries and has resulted in over 4,000 deaths within 10 weeks. We compare the interaction between the human ACE2 receptor and the SARS-CoV-2 spike protein with that of other pathogenic coronaviruses using molecular dynamics simulations. SARS-CoV, SARS-CoV-2, and HCoV-NL63 recognize ACE2 as the natural receptor but present a distinct binding interface to ACE2 and a different network of residue-residue contacts. SARS-CoV and SARS-CoV-2 have comparable binding affinities achieved...


19: Structural and Evolutionary Analysis Indicate that the SARS-CoV-2 Mpro is an Inconvenient Target for Small-Molecule Inhibitors Design

Maria Bzówka, Karolina Mitusińska et al.

6,227 downloads (posted 02 Mar 2020)

The novel coronavirus whose outbreak took place in December 2019 continues to spread at a rapid rate worldwide. In the absence of an effective vaccine, inhibitor repurposing or de novo drug design may offer a longer-term strategy to combat this and future infections due to similar viruses. Here, we report on detailed classical and mix-solvent molecular dynamics simulations of the main protease (Mpro) enriched by evolutionary and stability analysis of the protein. The results were compared with those for a highly similar...


20: Crystal structure of Nsp15 endoribonuclease NendoU from SARS-CoV-2

Youngchang Kim, Robert Jedrzejczak et al.

6,171 downloads (posted 03 Mar 2020)

Severe Acute Respiratory Syndrome Coronavirus 2 is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS- and MERS-CoVs the detailed information about SARS-CoV-2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies and antivirals. We applied high-throughput protein production and structure determination pipeline at the Center for Structura...