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Rxivist combines biology preprints from bioRxiv and medRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 136,194 papers from 580,789 authors.

Most downloaded biology preprints, all time

in category molecular biology

4,046 results found. For more information, click each entry to expand.

1: The novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells
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Posted 31 Jan 2020

The novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells
40,924 downloads bioRxiv molecular biology

Markus Hoffmann, Hannah Kleine-Weber, Nadine Krüger, Marcel Müller, Christian Drosten, Stefan Poehlmann

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: Neutralization of UK-variant VUI-202012/01 with COVAXIN vaccinated human serum
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Posted 26 Jan 2021

Neutralization of UK-variant VUI-202012/01 with COVAXIN vaccinated human serum
29,757 downloads bioRxiv molecular biology

Gajanan N Sapkal, Pragya Yadav, Raches Ella, Gururaj Deshpande, Rima Sahay, Nivedita Gupta, V Krishna Mohan, Priya Abraham, Samiran Panda, Balram Bhargava

We performed the plaque reduction neutralization test (PRNT50) using sera collected from the 26 recipients of BBV152/COVAXINTM against hCoV-19/India/20203522 (UK-variant) and hCoV27 19/India/2020Q111 (heterologous strain). A comparable neutralization activity of sera of the vaccinated individuals showed against UK-variant and the heterologous strain with similar efficiency, dispel the uncertainty of possible neutralization escape.

3: Protocol: Genome-scale CRISPR-Cas9 Knockout and Transcriptional Activation Screening
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Posted 18 Jun 2016

Protocol: Genome-scale CRISPR-Cas9 Knockout and Transcriptional Activation Screening
27,202 downloads bioRxiv molecular biology

Julia Joung, Silvana Konermann, Jonathan S Gootenberg, Omar O Abudayyeh, Randall J Platt, Mark D Brigham, Neville E Sanjana, Feng Zhang

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 system. Custom- or ready-made guide RNA libraries are constructed and packaged into lentivirus for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified from the screen, we further describe strategies for confirming the screening phenotype as well as genetic perturbation through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 6-10 weeks followed by 3-4 weeks of validation.

4: Index Switching Causes “Spreading-Of-Signal” Among Multiplexed Samples In Illumina HiSeq 4000 DNA Sequencing
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Posted 09 Apr 2017

Index Switching Causes “Spreading-Of-Signal” Among Multiplexed Samples In Illumina HiSeq 4000 DNA Sequencing
23,901 downloads bioRxiv molecular biology

Rahul Sinha, Geoff Stanley, Gunsagar S. Gulati, Camille Ezran, Kyle J. Travaglini, Eric Wei, Charles K.F. Chan, Ahmad N. Nabhan, Tianying Su, Rachel M. Morganti, Stephanie D Conley, Hassan Chaib, Kristy Red-Horse, Michael T. Longaker, Michael P. Snyder, Mark A Krasnow, Irving L. Weissman

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 fundamental shift from the earlier method of random cluster generation by bridge amplification on a non-patterned flow cell. The ExAmp chemistry, in conjunction with patterned flow cells containing nanowells at fixed locations, increases cluster density on the flow cell, thereby reducing the cost per run. It also increases sequence read quality, especially for longer read lengths (up to 150 base pairs). This advance has been widely adopted for genome sequencing because greater sequencing depth can be achieved for lower cost without compromising the quality of longer reads. We show that this promising chemistry is problematic, however, when multiplexing samples. We discovered that up to 5-10% of sequencing reads (or signals) are incorrectly assigned from a given sample to other samples in a multiplexed pool. We provide evidence that this “spreading-of-signals” arises from low levels of free index primers present in the pool. These index primers can prime pooled library fragments at random via complementary 3′ ends, and get extended by DNA polymerase, creating a new library molecule with a new index before binding to the patterned flow cell to generate a cluster for sequencing. This causes the resulting read from that cluster to be assigned to a different sample, causing the spread of signals within multiplexed samples. We show that low levels of free index primers persist after the most common library purification procedure recommended by Illumina, and that the amount of signal spreading among samples is proportional to the level of free index primer present in the library pool. This artifact causes homogenization and misclassification of cells in single cell RNA-seq experiments. Therefore, all data generated in this way must now be carefully re-examined to ensure that “spreading-of-signals” has not compromised data analysis and conclusions. Re-sequencing samples using an older technology that uses conventional bridge amplification for cluster generation, or improved library cleanup strategies to remove free index primers, can minimize or eliminate this signal spreading artifact.

5: Reversal of ageing- and injury-induced vision loss by Tet-dependent epigenetic reprogramming
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Posted 31 Jul 2019

Reversal of ageing- and injury-induced vision loss by Tet-dependent epigenetic reprogramming
17,989 downloads bioRxiv molecular biology

Yuancheng Lu, Anitha Krishnan, Benedikt Brommer, Xiao Tian, Margarita Meer, Daniel L. Vera, Chen Wang, Qiurui Zeng, Doudou Yu, Michael S. Bonkowski, Jae-Hyun Yang, Emma M. Hoffmann, Songlin Zhou, Ekaterina Korobkina, Noah Davidsohn, Michael B. Schultz, Karolina Chwalek, Luis A. Rajman, George M. Church, Konrad Hochedlinger, Vadim N Gladyshev, Steve Horvath, Meredith S. Gregory-Ksander, Bruce R. Ksander, Zhigang He, David A. Sinclair

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 central nervous system (CNS) is one of the first to lose regenerative capacity. Using the eye as a model tissue, we show that expression of Oct4, Sox2, and Klf4 genes (OSK) in mice resets youthful gene expression patterns and the DNA methylation age of retinal ganglion cells, promotes axon regeneration after optic nerve crush injury, and restores vision in a mouse model of glaucoma and in normal old mice. This process, which we call recovery of information via epigenetic reprogramming or REVIVER, requires the DNA demethylases Tet1 and Tet2, indicating that DNA methylation patterns don't just indicate age, they participate in ageing. Thus, old tissues retain a faithful record of youthful epigenetic information that can be accessed for functional age reversal.

6: LAMP-Seq: Population-Scale COVID-19 Diagnostics Using Combinatorial Barcoding
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Posted 08 Apr 2020

LAMP-Seq: Population-Scale COVID-19 Diagnostics Using Combinatorial Barcoding
17,170 downloads bioRxiv molecular biology

Jonathan L. Schmid-Burgk, Ricarda Maria Schmithausen, David Li, Ronja Hollstein, Amir Ben-Shmuel, Ofir Israeli, Shay Weiss, Nir Paran, Gero Wilbring, Jana Liebing, David Feldman, Mikołaj Słabicki, Bärbel Lippke, Esther Sib, Jacob Borrajo, Jonathan Strecker, Julia Reinhardt, Per Hoffmann, Brian Cleary, Michael Hölzel, Markus M. Nöthen, Martin Exner, Kerstin U Ludwig, Aviv Regev, Feng Zhang

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 barcoded Reverse-Transcription Loop-mediated Isothermal Amplification (RT-LAMP) method that is highly scalable. Individual samples are stabilized, inactivated, and amplified in three isothermal heat steps, generating barcoded amplicons that can be pooled and analyzed en masse by sequencing. Using unique barcode combinations per sample from a compressed barcode space enables extensive pooling, potentially further reducing cost and simplifying logistics. We validated LAMP-Seq on 28 clinical samples, empirically optimized the protocol and barcode design, and performed initial safety evaluation. Relying on world-wide infrastructure for next-generation sequencing, and in the context of population-wide sample collection, LAMP-Seq could be scaled to analyze millions of samples per day. ### Competing Interest Statement J.S.-B., D.L., and F.Z. are inventors on a patent application filed by the Broad Institute related to this work with the specific aim of ensuring this technology can be made freely, widely, and rapidly available for research and deployment. F.Z. is a co-founder of Editas Medicine, Beam Therapeutics, Pairwise Plants, Arbor Biotechnologies, and Sherlock Biosciences. A.R. is a founder of Celsius Therapeutics, equity holder in Immunitas, and an SAB member for ThermoFisher Scientific, Syros Pharmaceuticals, Asimov, and Neogene Therapeutics. P.H. and M.M.N. are SAB members of HMG Systems Bioengineering GmbH. M.M.N. served on SABs for Lundbeck Foundation and Robert-Bosch-Stiftung, was reimbursed travel expenses by Shire GmbH, receives salary from and holds shares in Life & Brain GmbH.

7: Shotgun Transcriptome and Isothermal Profiling of SARS-CoV-2 Infection Reveals Unique Host Responses, Viral Diversification, and Drug Interactions
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Posted 20 Apr 2020

Shotgun Transcriptome and Isothermal Profiling of SARS-CoV-2 Infection Reveals Unique Host Responses, Viral Diversification, and Drug Interactions
17,155 downloads bioRxiv molecular biology

Daniel J Butler, Christopher Mozsary, Cem Meydan, David C Danko, Jonathan Foox, Joel Rosiene, Alon Shaiber, Ebrahim Afshinnekoo, Matthew MacKay, Fritz J Sedlazeck, Nikolay A Ivanov, Maria Sierra, Diana Pohle, Michael Zietz, Undina Gisladottir, Vijendra Ramlall, Craig D Westover, Krista Ryon, Benjamin Young, Chandrima Bhattacharya, Phyllis Ruggiero, Bradley W Langhorst, Nathan Tanner, Justyna Gawrys, Dmitry Meleshko, Dong Xu, Peter A D Steel, Amos J Shemesh, Jenny Xiang, Jean Thierry-Mieg, Danielle Thierry-Mieg, Robert E. Schwartz, Angelika Iftner, Daniela Bezdan, John Sipley, Lin Cong, Arryn Craney, Priya Velu, Ari M. Melnick, Iman Hajirasouliha, Stacy M. Horner, Thomas Iftner, Mirella Salvatore, Massimo Loda, Lars F Westblade, Melissa Cushing, Shawn E. Levy, Shixiu Wu, Nicholas P Tatonetti, Marcin Imielinski, Hanna Rennert, Christopher E. Mason

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 shotgun metatranscriptomics platform (total-RNA-seq) for host, bacterial, and viral profiling. We applied both technologies across 857 SARS-CoV-2 clinical specimens and 86 NYC subway samples, providing a broad molecular portrait of the COVID-19 NYC outbreak. Our results define new features of SARS-CoV-2 evolution, nominate a novel, NYC-enriched viral subclade, reveal specific host responses in ACE, interferon, hematological, and olfaction pathways, and examine risks associated with use of ACE inhibitors and angiotensin receptor blockers. Together, these findings have immediate applications to SARS-CoV-2 diagnostics, public health monitoring, and new therapeutic targets. ### Competing Interest Statement N.T. and B.L. are employees at New England Biolabs.

8: A comparison of DNA stains and staining methods for Agarose Gel Electrophoresis
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Posted 06 Mar 2019

A comparison of DNA stains and staining methods for Agarose Gel Electrophoresis
14,044 downloads bioRxiv molecular biology

Andie C. Hall

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 four methods (pre-loading at 100x, pre-loading at 10x, precasting and post-staining) are evaluated for sensitivity and effect on DNA migration. GelRed™ was found to be the most sensitive while the EZ-Vision® dyes and SafeWhite had no discernible effect on DNA migration. Homemade loading dyes were as effective as readymade ones at less than 4% of the price. This method used less than 1% of the dye needed for the manufacturer recommended protocols. Thus, with careful consideration of stain and method, Gel stain expenditure can be reduced by over 99%.

9: Mammalian Y RNAs are modified at discrete guanosine residues with N-glycans
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Posted 30 Sep 2019

Mammalian Y RNAs are modified at discrete guanosine residues with N-glycans
13,298 downloads bioRxiv molecular biology

Ryan E Flynn, Benjamin A. H. Smith, Alex G. Johnson, Kayvon Pedram, Benson M. George, Stacy A Malaker, Karim Majzoub, Jan E. Carette, Carolyn R Bertozzi

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). These glycoRNA are present in multiple cell types and mammalian species, both in cultured cells and in vivo. Finally, we find that RNA glycosylation depends on the canonical N-glycan biosynthetic machinery within the ER/Golgi luminal spaces. Collectively, these findings suggest the existence of a ubiquitous interface of RNA biology and glycobiology suggesting an expanded role for glycosylation beyond canonical lipid and protein scaffolds.

10: Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing
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Posted 24 May 2019

Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing
13,113 downloads bioRxiv molecular biology

Søren M Karst, Ryan M. Ziels, Rasmus H Kirkegaard, Emil A. Sørensen, Daniel McDonald, Qiyun Zhu, Rob Knight, Mads Albertsen

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: Improved CUT&RUN chromatin profiling and analysis tools
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Posted 06 Mar 2019

Improved CUT&RUN chromatin profiling and analysis tools
12,556 downloads bioRxiv molecular biology

Michael P. Meers, Terri Bryson, Steven Henikoff

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 that expands antibody compatibility and simplifies purification; 2) a modified digestion protocol that inhibits premature release of the nuclease-bound complex; 3) a calibration strategy based on carry-over of E. coli DNA introduced with the fusion protein; and 4) a novel peak-calling strategy customized for the low-background profiles obtained using CUT&RUN. These new features, coupled with the previously described low-cost, high efficiency, high reproducibility and high-throughput capability of CUT&RUN make it the method of choice for routine epigenomic profiling. [1]: #ref-1

12: Design and specificity of long ssDNA donors for CRISPR-based knock-in
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Posted 21 Aug 2017

Design and specificity of long ssDNA donors for CRISPR-based knock-in
12,524 downloads bioRxiv molecular biology

Han Li, Kyle A. Beckman, Veronica Pessino, Bo Huang, Jonathan S. Weissman, Manuel D. Leonetti

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 optimized method for the synthesis of long ssDNA templates and demonstrate that ssDNA donors can drive efficient integration of gene-sized reporters in human cell lines. We next define a set of rules to maximize the efficiency of ssDNA-mediated knock-in by optimizing donor design. Finally, by comparing ssDNA donors with equivalent dsDNA sequences (PCR products or plasmids), we demonstrate that ssDNA templates have a unique advantage in terms of repair specificity while dsDNA donors can lead to a high rate of off-target integration. Our results provide a framework for designing high-fidelity CRISPR-based knock-in experiments, in both research and therapeutic settings. Update: November 12th, 2019 Dear bioRxiv community, The conclusions of this pre-print (originally posted in August 2017) are outdated. While the experiments we present here are accurate, a recent and more systematic analysis revealed that the integration outcomes driven by different forms of HDR donors are more complex than our methods could originally identify. We initially analyzed donor integration only in FACS-selected cells, which under-estimates alleles where the mis-integration of payload leads to non-functional selection markers, and we quantified integration by ddPCR, which is an indirect read-out of sequence properties. These approaches could not capture the full details of donor integration events in our experiments. To address this, we have now developed a new framework based on long-read amplicon sequencing and an integrated computational pipeline to precisely analyze knock-in repair outcomes across a wide range of experimental parameters. Our new data uncover a complex repair landscape in which both single-stranded and double-stranded donors can lead to high rates of imprecise integration in some cell types. Please read our new bioRxiv pre-print entitled “Deep profiling reveals substantial heterogeneity of integration outcomes in CRISPR knock-in experiments” for further information. I hope that this example highlights one of the powers of pre-prints: the ability to update scientific discussions (and set records straight) as new results are obtained, often fueled by the availability of new technologies. Please do not hesitate to contact me directly for any questions or comments.

13: A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing
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Posted 23 Jun 2020

A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing
11,219 downloads bioRxiv molecular biology

Max J. Kellner, James J Ross, Jakob Schnabl, Marcus P.S. Dekens, Robert Heinen, Irina Grishkovskaya, Benedikt Bauer, Johannes Stadlmann, Luis Menéndez-Arias, Robert Fritsche-Polanz, Marianna Traugott, Tamara Seitz, Alexander Zoufaly, Manuela Födinger, Christoph Wenisch, Johannes Zuber, Vienna Covid-19 Diagnostics Initiative (VCDI), Andrea Pauli, Julius Brennecke

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 detection assay. Our forty-minute procedure bypasses a dedicated RNA isolation step, is insensitive to carry-over contamination, and uses a hydroxynaphthol blue (HNB)-based colorimetric readout, which allows robust SARS-CoV-2 detection from various sample types. Based on this assay we have substantially increased sensitivity and scalability by a simple nucleic acid enrichment step (bead-LAMP), established a pipette-free version for home testing (HomeDip-LAMP), and developed a version with open source enzymes that could be produced in any molecular biology setting. Our advanced, universally applicable RT-LAMP assay is a major step towards population-scale SARS-CoV-2 testing. ### Competing Interest Statement The authors have declared no competing interest.

14: An ultrasensitive, rapid, and portable coronavirus SARS-CoV-2 sequence detection method based on CRISPR-Cas12
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Posted 02 Mar 2020

An ultrasensitive, rapid, and portable coronavirus SARS-CoV-2 sequence detection method based on CRISPR-Cas12
11,207 downloads bioRxiv molecular biology

Curti Lucia, Pereyra-Bonnet Federico, Gimenez Carla Alejandra

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.

15: A proximity-dependent biotinylation map of a human cell: an interactive web resource
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Posted 07 Oct 2019

A proximity-dependent biotinylation map of a human cell: an interactive web resource
9,516 downloads bioRxiv molecular biology

Christopher D Go, James D R Knight, Archita Rajasekharan, Bhavisha Rathod, Geoffrey G Hesketh, Kento T Abe, Ji-Young Youn, Payman Samavarchi-Tehrani, Hui Zhang, Lucie Y Zhu, Evelyn Popiel, Jean-Philippe Lambert, Étienne Coyaud, Sally W T Cheung, Dushyandi Rajendran, Cassandra J Wong, Hana Antonicka, Laurence Pelletier, Alexander F Palazzo, Eric A Shoubridge, Brian Raught, Anne-Claude Gingras

Compartmentalization is a defining characteristic of eukaryotic cells, partitioning cellular processes into discrete subcellular locations. High throughput microscopy and biochemical fractionation coupled with mass spectrometry have helped to define the proteomes of a variety of organelles and macromolecular structures. However, many other intracellular compartments have remained refractory to such approaches, due for example to difficulty in purifying non-membrane bound structures. Proximity-dependent biotinylation techniques such as BioID provide an alternative approach for defining the composition of cellular compartments in living cells. Here we present a BioID-based map of a human cell based on 192 markers from 32 different subcellular compartments, comprising 35,902 high confidence proximity interactions, and defining the intracellular locations of 4,145 unique proteins in HEK 293 cells. Our localization predictions meet or exceed previous approaches, with higher specificity, and enabled the discovery of proteins at the mitochondrial outer membrane-endoplasmic reticulum (ER) interface that are critical for mitochondrial homeostasis. Based on this dataset, we have established humancellmap.org as a community resource that provides online tools for localization analysis of user BioID data, and demonstrate how this resource can be used to better understand BioID datasets.

16: Background free tracking of single RNA in living cells using catalytically inactive CasE
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Posted 13 May 2019

Background free tracking of single RNA in living cells using catalytically inactive CasE
8,391 downloads bioRxiv molecular biology

Feng Gao, Yue Sun, Feng Jiang, Xiaoyue Bai, Chunyu Han

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.

17: Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites
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Posted 07 Mar 2020

Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites
8,148 downloads bioRxiv molecular biology

Sisi Kang, Mei Yang, Zhongsi Hong, Liping Zhang, Zhaoxia Huang, Xiaoxue Chen, Suhua He, Ziliang Zhou, Zhechong Zhou, Qiuyue Chen, Yan Yan, Changsheng Zhang, Hong Shan, Shoudeng Chen

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 of SARS-CoV-2 nucleocapsid protein. Although overall structure is similar with other reported coronavirus nucleocapsid protein N-terminal domain, the surface electrostatic potential characteristics between them are distinct. Further comparison with mild virus type HCoV-OC43 equivalent domain demonstrates a unique potential RNA binding pocket alongside the β-sheet core. Complemented by in vitro binding studies, our data provide several atomic resolution features of SARS-CoV-2 nucleocapsid protein N-terminal domain, guiding the design of novel antiviral agents specific targeting to SARS-CoV-2.

18: Performance comparison of reverse transcriptases for single-cell studies
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Posted 07 May 2019

Performance comparison of reverse transcriptases for single-cell studies
7,496 downloads bioRxiv molecular biology

Zucha Daniel, Androvic Peter, Kubista Mikael, Valihrach Lukas

Background: Recent technical advances allowing quantification of RNA from single cells are revolutionizing biology and medicine. Currently, almost all single-cell transcriptomic protocols rely on conversion of RNA to cDNA by reverse transcription (RT). However, RT is recognized as highly limiting step due to its inherent variability and suboptimal sensitivity, especially at minute amounts of RNA. Primary factor influencing RT outcome is reverse transcriptase (RTase). Recently, several new RTases with potential to decrease the loss of information during RT have been developed, but the thorough assessment of their performance is missing. Methods: We have compared the performance of 11 RTases in RT-qPCR on single-cell and 100-cell bulk templates using two priming strategies: conventional mixture of random hexamers with oligo(dT)s and reduced concentration of oligo(dT)s mimicking common single-cell RNA-Seq library preparation protocols. Based on the performance, two RTases were further tested in high-throughput single-cell experiment. Results: All RTases tested reverse transcribed low-concentration templates with high accuracy (R2 > 0.9445) but variable reproducibility (median CVRT = 40.1 %). The most pronounced differences were found in the ability to capture rare transcripts (0 - 90% reaction positivity rate) as well as in the rate of RNA conversion to cDNA (7.3 - 124.5 % absolute yield). Finally, RTase performance and reproducibility across all tested parameters were compared using Z-scores and validity of obtained results was confirmed in a single-cell model experiment. The better performing RTase provided higher positive reaction rate and expression levels and improved resolution in clustering analysis. Conclusions: We performed a comprehensive comparison of 11 RTases in low RNA concentration range and identified two best-performing enzymes (Maxima H-; SuperScript IV). We found that using better-performing enzyme (Maxima H-) over commonly-used below-average performer (SuperScript II) increases the sensitivity of single-cell experiment. Our results provide a reference for the improvement of current single-cell quantification protocols.

19: Structural Basis for the Inhibition of the RNA-Dependent RNA Polymerase from SARS-CoV-2 by Remdesivir
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Posted 09 Apr 2020

Structural Basis for the Inhibition of the RNA-Dependent RNA Polymerase from SARS-CoV-2 by Remdesivir
7,347 downloads bioRxiv molecular biology

Wanchao Yin, Chunyou Mao, Xiaodong Luan, Dan-Dan Shen, Qingya Shen, Haixia Su, Xiaoxi Wang, Fulai Zhou, Wenfeng Zhao, Minqi Gao, Shenghai Chang, Yuan-Chao Xie, Guanghui Tian, He-Wei Jiang, Sheng-Ce Tao, Jingshan Shen, Yi Jiang, Ruotian Jiang, H. Eric Xu, Shuyang Zhang, Yan Zhang, H. Eric Xu

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 channel of the RdRp where Remdesivir is incorporated into the first replicated base pair and terminates the chain elongation. Our structures provide critical insights into the working mechanism of viral RNA replication and a rational template for drug design to combat the viral infection. ### Competing Interest Statement The authors have declared no competing interest.

20: Characterization of the SARS-CoV-2 Spike in an Early Prefusion Conformation
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Posted 17 Mar 2020

Characterization of the SARS-CoV-2 Spike in an Early Prefusion Conformation
6,971 downloads bioRxiv molecular biology

Tingting Li, Qingbing Zheng, Hai Yu, Dinghui Wu, Wenhui Xue, Yuyun Zhang, Xiaofen Huang, Lizhi Zhou, Zhigang Zhang, Zhenghui Zha, Tingting Chen, Zhiping Wang, Jie Chen, Hui Sun, Tingting Deng, Yingbin Wang, Yixin Chen, Qinjian Zhao, Jun Zhang, Ying Gu, Shaowei Li, Ningshao Xia

Pandemic coronavirus disease 2019 (COVID-19) is caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for which there are no efficacious vaccines or therapeutics that are urgently needed. We expressed three versions of spike (S) proteins—receptor binding domain (RBD), S1 subunit and S ectodomain—in insect cells. RBD appears monomer in solutions, whereas S1 and S associate into homotrimer with substantial glycosylation. The three proteins confer excellent antigenicity with six convalescent COVID-19 patient sera. Cryo-electron microscopy (cryo-EM) analyses indicate that the SARS-CoV-2 S trimer dominate in a unique conformation distinguished from the classic prefusion conformation of coronaviruses by the upper S1 region at lower position ~15 Å proximal to viral membrane. Such conformation is proposed as an early prefusion state for the SARS-CoV-2 spike that may broaden the knowledge of coronavirus and facilitate vaccine development.

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