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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 118,853 papers from 512,610 authors.

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in category molecular biology

3,582 results found. For more information, click each entry to expand.

61: Resolving microbial microdiversity with high accuracy full length 16S rRNA Illumina sequencing
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Posted 06 Nov 2014

Resolving microbial microdiversity with high accuracy full length 16S rRNA Illumina sequencing
3,372 downloads bioRxiv molecular biology

Catherine M Burke, Aaron E. Darling

We describe a method for sequencing full-length 16S rRNA gene amplicons using the high throughput Illumina MiSeq platform. The resulting sequences have about 100-fold higher accuracy than standard Illumina reads and are chimera filtered using information from a single molecule dual tagging scheme that boosts the signal available for chimera detection. We demonstrate that the data provides fine scale phylogenetic resolution not available from Illumina amplicon methods targeting smaller variable regions of the 16S rRNA gene.

62: Sub-tomogram averaging in RELION
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Posted 04 Nov 2015

Sub-tomogram averaging in RELION
3,370 downloads bioRxiv molecular biology

Tanmay A.M. Bharat, Sjors Scheres

Electron cryo-tomography (cryo-ET) and sub-tomogram averaging allow structure determination of macromolecules in situ, and are gaining in popularity for initial model generation for single- particle analysis. We describe herein, a protocol for sub-tomogram averaging from cryo-ET data using the RELION software. We describe how to calculate newly developed three-dimensional models for the contrast transfer function and the missing wedge of each sub-tomogram, and how to use these models for regularized-likelihood refinement. This approach has been implemented in the existing workflow for single-particle analysis, so that users may conveniently tap into existing capabilities of the RELION software. As example applications, we present analyses of purified hepatitis B capsid particles and S. cerevisiae 80S ribosomes. In both cases, we show that following initial classification, sub-tomogram averaging in RELION allows de novo generation of initial models, and provides high-resolution maps where secondary structure elements are resolved.

63: Super-resolution Imaging Reveals 3D Structure and Organizing Mechanism of Accessible Chromatin
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Posted 21 Jun 2019

Super-resolution Imaging Reveals 3D Structure and Organizing Mechanism of Accessible Chromatin
3,333 downloads bioRxiv molecular biology

Liangqi Xie, Peng Dong, Yifeng Qi, Margherita De Marzio, Xingqi Chen, Sambashiva Banala, Wesley R Legant, Brian P. English, Anders S. Hansen, Anton Schulmann, Luke D. Lavis, Eric Betzig, Rafael Casellas, Howard Y. Chang, Bin Zhang, Robert Tjian, Zhe Liu

Access to cis-regulatory elements packaged in chromatin is essential for directing gene expression and cell viability. Here, we report a super-resolution imaging strategy, 3D ATAC-PALM, that enables direct visualization of the entire accessible genome. We found that active chromosomal segments are organized into spatially-segregated accessible chromatin domains (ACDs). Rapid depletion of CTCF or Cohesin (RAD21 subunit) induced enhanced ACD clustering, reduced physical separation between intrachromosomal ACDs, and differentially regulated ACD compaction. Experimental perturbations and polymer modeling suggest that dynamic protein-protein and protein-DNA interactions within ACDs couple with loop extrusion to organize ACD topology. Dysorganization of ACDs upon CTCF or Cohesin loss alters transcription factor binding and target search dynamics in living cells. These results uncover fundamental mechanisms underpinning the formation of 3D genome architecture and its pivotal function in transcriptional regulation.

64: Discovery of baicalin and baicalein as novel, natural product inhibitors of SARS-CoV-2 3CL protease in vitro
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Posted 14 Apr 2020

Discovery of baicalin and baicalein as novel, natural product inhibitors of SARS-CoV-2 3CL protease in vitro
3,301 downloads bioRxiv molecular biology

Haixia Su, Sheng Yao, Wenfeng Zhao, Minjun Li, Jia Liu, WeiJuan Shang, Hang Xie, Changqiang Ke, Meina Gao, Kunqian Yu, Hong Liu, Jingshan Shen, Wei Tang, Leike Zhang, Jianping Zuo, Ruotian Jiang, Fang Bai, Yan Wu, Yang Ye, Yechun Xu

Human infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause coronavirus disease 19 (COVID-19) and there is currently no cure. The 3C-like protease (3CLpro), a highly conserved protease indispensable for replication of coronaviruses, is a promising target for development of broad-spectrum antiviral drugs. To advance the speed of drug discovery and development, we investigated the inhibition of SARS-CoV-2 3CLpro by natural products derived from Chinese traditional medicines. Baicalin and baicalein were identified as the first non-covalent, non-peptidomimetic inhibitors of SARS-CoV-2 3CLpro and exhibited potent antiviral activities in a cell-based system. Remarkably, the binding mode of baicalein with SARS-CoV-2 3CLpro determined by X-ray protein crystallography is distinctly different from those of known inhibitors. Baicalein is perfectly ensconced in the core of the substrate-binding pocket by interacting with two catalytic residues, the crucial S1/S2 subsites and the oxyanion loop, acting as a 'shield' in front of the catalytic dyad to prevent the peptide substrate approaching the active site. The simple chemical structure, unique mode of action, and potent antiviral activities in vitro, coupled with the favorable safety data from clinical trials, emphasize that baicalein provides a great opportunity for the development of critically needed anti-coronaviral drugs. ### Competing Interest Statement The authors have declared no competing interest.

65: Erosion of the Epigenetic Landscape and Loss of Cellular Identity as a Cause of Aging in Mammals
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Posted 19 Oct 2019

Erosion of the Epigenetic Landscape and Loss of Cellular Identity as a Cause of Aging in Mammals
3,260 downloads bioRxiv molecular biology

Jae-Hyun Yang, Patrick T. Griffin, Daniel L. Vera, John K. Apostolides, Motoshi Hayano, Margarita V. Meer, Elias L. Salfati, Qiao Su, Elizabeth M Munding, Marco Blanchette, Mital Bhakta, Zhixun Dou, Caiyue Xu, Jeffrey W. Pippin, Michael L. Creswell, Brendan L. O’Connell, Richard E. Green, Benjamin A Garcia, Shelley L Berger, Philipp Oberdoerffer, Stuart J. Shankland, Vadim N Gladyshev, Luis A. Rajman, Andreas R Pfenning, David A. Sinclair

All living things experience entropy, manifested as a loss of inherited genetic and epigenetic information over time. As budding yeast cells age, epigenetic changes result in a loss of cell identity and sterility, both hallmarks of yeast aging. In mammals, epigenetic information is also lost over time, but what causes it to be lost and whether it is a cause or a consequence of aging is not known. Here we show that the transient induction of genomic instability, in the form of a low number of non-mutagenic DNA breaks, accelerates many of the chromatin and tissue changes seen during aging, including the erosion of the epigenetic landscape, a loss of cellular identity, advancement of the DNA methylation clock and cellular senescence. These data support a model in which a loss of epigenetic information is a cause of aging in mammals.

66: High-precision CRISPR-Cas9 base editors with minimized bystander and off-target mutations
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Posted 01 Mar 2018

High-precision CRISPR-Cas9 base editors with minimized bystander and off-target mutations
3,228 downloads bioRxiv molecular biology

Jason M. Gehrke, Oliver Cervantes, M. Kendell Clement, Luca Pinello, J. Keith Joung

Recently described base editor (BE) technology, which uses CRISPR-Cas9 to direct cytidine deaminase enzymatic activity to specific genomic loci, enables the highly efficient introduction of precise cytidine-to-thymidine (C to T) DNA alterations in many different cell types and organisms. In contrast to genome-editing nucleases, BEs avoid the need to introduce double-strand breaks or exogenous donor DNA templates and induce lower levels of unwanted variable-length insertion/deletion mutations (indels). However, existing BEs can also efficiently create unwanted C to T alterations when more than one C is present within the five base pair "editing window" of these proteins, a lack of precision that can cause potentially deleterious bystander mutations. Mutations in the cytidine deaminase enzyme can shorten the length of the editing window and thereby partially address this limitation but these BE variants still do not discriminate among multiple cytidines within the narrowed window and also possess a more limited targeting range. Here, we describe an alternative strategy for reducing bystander mutations using a novel BE architecture that harbors an engineered human APOBEC3A (eA3A) domain, which preferentially deaminates cytidines according to a TCR>TCY>VCN (V = G, A, C, Y = C, T) hierarchy. In direct comparisons with the widely used BE3 fusion in human cells, our eA3A-BE3 fusion exhibits comparable activities on cytidines in TC motifs but greatly reduced or no significant editing on cytidines in other sequence contexts. Importantly, we show that eA3A-BE3 can correct a human beta-thalassemia promoter mutation with much higher (>40-fold) precision than BE3, substantially minimizing the creation of an undesirable bystander mutation. Surprisingly, we also found that eA3A-BE3 shows reduced mutation frequencies on known off-target sites of BE3, even when targeting promiscuous homopolymeric sites. Our results validate a general strategy to improve the precision of base editors by engineering their cytidine deaminases to possess greater sequence specificity, an important proof-of-principle that should motivate the development of a larger suite of new base editors with such properties.

67: P53 toxicity is a hurdle to CRISPR/CAS9 screening and engineering in human pluripotent stem cells
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Posted 26 Jul 2017

P53 toxicity is a hurdle to CRISPR/CAS9 screening and engineering in human pluripotent stem cells
3,218 downloads bioRxiv molecular biology

Robert J Ihry, Kathleen A Worringer, Max R Salick, Elizabeth Frias, Daniel Ho, Kraig Theriault, Sravya Kommineni, Julie Chen, Marie Sondey, Chaoyang Ye, Ranjit Randhawa, Tripti Kulkarni, Zinger Yang, Gregory McAllister, Carsten Russ, John Reece-Hoyes, William Forrester, Gregory R Hoffman, Ricardo Dolmetsch, Ajamete Kaykas

CRISPR/Cas9 has revolutionized our ability to engineer genomes and to conduct genome-wide screens in human cells. While some cell types are easily modified with Cas9, human pluripotent stem cells (hPSCs) poorly tolerate Cas9 and are difficult to engineer. Using a stable Cas9 cell line or transient delivery of ribonucleoproteins (RNPs) we achieved an average insertion or deletion efficiency greater than 80%. This high efficiency made it apparent that double strand breaks (DSBs) induced by Cas9 are toxic and kill most treated hPSCs. Cas9 toxicity creates an obstacle to the high-throughput use CRISPR/Cas9 for genome-engineering and screening in hPSCs. We demonstrated the toxic response is tp53-dependent and the toxic effect of tp53 severely reduces the efficiency of precise genome-engineering in hPSCs. Our results highlight that CRISPR-based therapies derived from hPSCs should proceed with caution. Following engineering, it is critical to monitor for tp53 function, especially in hPSCs which spontaneously acquire tp53 mutations.

68: Characterization and Validation of a Novel Group of Type V, Class 2 Nucleases for in vivo Genome Editing
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Posted 23 Sep 2017

Characterization and Validation of a Novel Group of Type V, Class 2 Nucleases for in vivo Genome Editing
3,217 downloads bioRxiv molecular biology

Matthew B Begemann, Benjamin N Gray, Emma January, Anna Singer, Dylan C Kesler, Yonghua He, Haijun Liu, Hongjie Guo, Alex Jordan, Thomas P. Brutnell, Todd C. Mockler, Mohammed Oufattole

CRISPR-based genome editing is an enabling technology with potential to dramatically transform multiple industries. Identification of additional editing tools will be imperative for broad adoption and application of this technology. A novel Type V, Class 2 CRISPR nuclease system was identified from Microgenomates and Smithella bacterial species (CRISPR from Microgenomates and Smithella, Cms1). This system was shown to efficiently generate indel mutations in the major crop plant rice (Oryza sativa). Cms1 are distinct from other Type V nucleases, are smaller than most other CRISPR nucleases, do not require a tracrRNA, and have an AT-rich protospacer-adjacent motif site requirement. A total of four novel Cms1 nucleases across multiple bacterial species were shown to be functional in a eukaryotic system. This is a major expansion of the Type V CRISPR effector protein toolbox and increases the diversity of options available to researchers.

69: Potent neutralization of clinical isolates of SARS-CoV-2 D614 and G614 variants by a monomeric, sub-nanomolar affinity Nanobody
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Posted 10 Jun 2020

Potent neutralization of clinical isolates of SARS-CoV-2 D614 and G614 variants by a monomeric, sub-nanomolar affinity Nanobody
3,192 downloads bioRxiv molecular biology

Guillermo Valenzuela Nieto, Ronald Jara, Daniel Watterson, Naphak Modhiran, Alberto A Amarilla, Johanna Himelreichs, Alexander A Khromykh, Constanza Salinas, Teresa Pinto, Yorka Cheuquemilla, Yago Margolles, Natalia López González del Rey, Zaray Miranda-Chacon, Alexei Cuevas, Anne Berking, Camila Deride, Sebastián González-Moraga, Héctor Mancilla, Daniel Maturana, Andreas Langer, Juan Pablo Toledo, Ananda Müller, Benjamín Uberti, Paola Krall, Pamela Ehrenfeld, Javier Blesa, Pedro Chana-Cuevas, German Rehren, David Schwefel, Luis Ángel Fernandez, Alejandro Rojas-Fernandez

Despite unprecedented global efforts to rapidly develop SARS-CoV-2 treatments, in order to reduce the burden placed on health systems, the situation remains critical. Effective diagnosis, treatment, and prophylactic measures are urgently required to meet global demand: recombinant antibodies fulfill these requirements and have marked clinical potential. Here, we describe the fast-tracked development of an alpaca Nanobody specific for the receptor-binding-domain (RBD) of the SARS-CoV-2 Spike protein with therapeutic potential applicability. We present a rapid method for nanobody isolation that includes an optimized immunization regimen coupled with VHH library E. coli surface display, which allows single-step selection of high-affinity nanobodies using a simple density gradient centrifugation of the bacterial library. The selected single and monomeric Nanobody, W25, binds to the SARS-CoV-2 S RBD with sub-nanomolar affinity and efficiently competes with ACE-2 receptor binding. Furthermore, W25 potently neutralizes SARS-CoV-2 wild type and the D614G variant with IC50 values in the nanomolar range, demonstrating its potential as antiviral agent. ### Competing Interest Statement Conflict of interest statement The Austral University of Chile claiming priority to U.S. Provisional Patent Application No. US Serial No. 63/025534, filed MAY-2020.

70: Tagmentation-Based Mapping (TagMap) of Mobile DNA Genomic Insertion Sites
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Posted 22 Jan 2016

Tagmentation-Based Mapping (TagMap) of Mobile DNA Genomic Insertion Sites
3,121 downloads bioRxiv molecular biology

David L. Stern

Multiple methods have been introduced over the past 30 years to identify the genomic insertion sites of transposable elements and other DNA elements that integrate into genomes. However, each of these methods suffer from limitations that can frustrate attempts to map multiple insertions in a single genome and to map insertions in genomes of high complexity that contain extensive repetitive DNA. I introduce a new method for transposon mapping that is simple to perform, can accurately map multiple insertions per genome, and generates long sequence reads that facilitate mapping to complex genomes. The method, called TagMap, for Tagmentation-based Mapping, relies on a modified Tn5 tagmentation protocol with a single tagmentation adaptor followed by PCR using primers specific to the tranposable element and the adaptor sequence. Several minor modifications to normal tagmentation reagents and protocols allow easy and rapid preparation of TagMap libraries. Short read sequencing starting from the adaptor sequence generates oriented reads that flank and are oriented toward the transposable element insertion site. The convergent orientation of adjacent reads at the insertion site allows straightforward prediction of the precise insertion site(s). A Linux shell script is provided to identify insertion sites from fastq files.

71: Structural and Functional Characterization of G Protein-Coupled Receptors with Deep Mutational Scanning
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Posted 30 Apr 2019

Structural and Functional Characterization of G Protein-Coupled Receptors with Deep Mutational Scanning
3,114 downloads bioRxiv molecular biology

Eric M Jones, Nathan B. Lubock, AJ Venkatakrishnan, Jeffrey Wang, Alex M Tseng, Joseph Paggi, Naomi R. Latorraca, Daniel Cancilla, Megan Satyadi, Jessica E Davis, M. Madan Babu, Ron O. Dror, Sriram Kosuri

In humans, the 813 G protein-coupled receptors (GPCRs) are responsible for transducing diverse chemical stimuli to alter cell state, and are the largest class of drug targets. Their myriad structural conformations and various modes of signaling make it challenging to understand their structure and function. Here we developed a platform to characterize large libraries of GPCR variants in human cell lines with a barcoded transcriptional reporter of G-protein signal transduction. We tested 7,800 of 7,828 possible single amino acid substitutions to the beta-2 adrenergic receptor (β2AR) at four concentrations of the agonist isoproterenol. We identified residues specifically important for β2AR signaling, mutations in the human population that are potentially loss of function, and residues that modulate basal activity. Using unsupervised learning, we resolve residues critical for signaling, including all major structural motifs and molecular interfaces. We also find a previously uncharacterized structural latch spanning the first two extracellular loops that is highly conserved across Class A GPCRs and is conformationally rigid in both the inactive and active states of the receptor. More broadly, by linking deep mutational scanning with engineered transcriptional reporters, we establish a generalizable method for exploring pharmacogenomics, structure and function across broad classes of drug receptors.

72: The Histone H3-H4 Tetramer is a Copper Reductase Enzyme
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Posted 19 Jun 2018

The Histone H3-H4 Tetramer is a Copper Reductase Enzyme
3,107 downloads bioRxiv molecular biology

Narsis Attar, Oscar A Campos, Maria Vogelauer, Chen Cheng, Yong Xue, Stefan Schmollinger, Nathan V Mallipeddi, Brandon A Boone, Linda Yen, Sichen Yang, Shannon Zikovich, Jade Dardine, Michael F Carey, Sabeeha S Merchant, Siavash K. Kurdistani

Ancestral histones were present in organisms with small genomes, no nucleus, and little evidence for epigenetic regulation, suggesting histones may have additional older functions. We report that the histone H3-H4 tetramer is an enzyme that catalyzes the reduction of Cu2+ to Cu1+ when assembled in vitro from recombinant histones. Mutations of residues in the putative active site at the interface of the apposing H3 proteins alter the enzymatic activity and cellular processes such as Sod1 function or mitochondrial respiration that depend on availability of reduced copper. These effects are not due to altered gene expression or copper abundance but are consistent with decreased levels of cuprous ions. We propose that the H3-H4 tetramer is an oxidoreductase that provides biousable copper for cellular and mitochondrial chemistry. As the emergence of eukaryotes coincided with the Great Oxidation Event and decreased biousability of metals, the histone enzymatic function may have facilitated eukaryogenesis.

73: Large-scale, quantitative protein assays on a high-throughput DNA sequencing chip
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Posted 08 Jun 2018

Large-scale, quantitative protein assays on a high-throughput DNA sequencing chip
3,101 downloads bioRxiv molecular biology

Curtis J Layton, Peter L McMahon, William J Greenleaf

High-throughput DNA sequencing techniques have enabled diverse approaches for linking DNA sequence to biochemical function. In contrast, assays of protein function have substantial limitations in terms of throughput, automation, and widespread availability. We have adapted a widely-used high-throughput sequencing chip to display an immense diversity of ribosomally-translated proteins and peptides, then carry out fluorescence-based functional assays directly on this flow cell. We quantified the binding of the M2 anti-FLAG antibody to a library of 1.3x10^4 variant FLAG peptides, discovering non-additive effects of combinations of mutations, as well as a "superFLAG" epitope variant. We also measured the enzymatic activity of 1.56x10^5 molecular variants of full-length of human O6-alkylguanine-DNA alkyltransferase (SNAP-tag). This comprehensive corpus of catalytic rates linked to amino acid sequence perturbations revealed amino acid interaction networks and cooperativity, linked positive cooperativity to structural proximity, and revealed ubiquitous positively cooperative interactions with histidine residues.

74: Localization microscopy at doubled precision with patterned illumination
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Posted 20 Feb 2019

Localization microscopy at doubled precision with patterned illumination
3,071 downloads bioRxiv molecular biology

Jelmer Cnossen, Taylor Hinsdale, Rasmus Ø. Thorsen, Florian Schueder, Ralf Jungmann, Carlas S. Smith, Bernd Rieger, Sjoerd Stallinga

MINFLUX offers a breakthrough in single molecule localization precision, but suffers from a tiny field of-view and a lack of practical parallelism. Here, we combine centroid estimation and illumination pattern induced photon count variations in a conventional widefield imaging setup to extract position information over a typical micron sized field-of-view. We show a near twofold improvement in precision over standard localization with the same photon count on DNA-origami nano-structures.

75: No evidence for genome editing of the endogenous DNA in mouse zygotes and HEK293T human cell line using the DNA-guided Natronobacterium gregoryi Argonaute (NgAgo).
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Posted 06 Dec 2016

No evidence for genome editing of the endogenous DNA in mouse zygotes and HEK293T human cell line using the DNA-guided Natronobacterium gregoryi Argonaute (NgAgo).
3,048 downloads bioRxiv molecular biology

Nay Chi Khin, Jenna L. Lowe, Lora M. Jensen, Gaetan Burgio

A recently published research article reported that the extreme halophile archaebacterium Natronobacterium gregoryi Argonaute enzyme (NgAgo) could cleave the cellular DNA under physiological temperature conditions in cell line and be implemented as an alternative to CRISPR/Cas9 genome editing technology. We assessed this claim in mouse zygotes for four loci (Sptb, Tet-1, Tet-2 and Tet-3) and in the human HEK293T cell line for the EMX1 locus. Over 100 zygotes were microinjected with nls-NgAgo-GK plasmid provided from Addgene and various concentrations of 5-phosphorylated guide DNA (gDNA) from 2.5 ng/microl to 50 ng/microl and cultured to blastocyst stage of development. The presence of indels was verified using T7 endonuclease 1 assay (T7E1) and Sanger sequencing. We reported no evidence of successful editing of the mouse genome. We then assessed the lack of editing efficiency in HEK293T cell line for the EMX1 endogenous locus by monitoring the NgAgo protein expression level and the editing efficiency by T7E1 assay and Sanger sequencing. We reported that the NgAgo protein was expressed from 8 hours to a maximum expression at 48 hours post-transfection, confirming the efficient delivery of the plasmid and the gDNA but no evidence of successful editing of EMX1 target in all transfected samples. Together our findings indicate that we failed to edit using NgAgo.

76: In situ structural analysis of SARS-CoV-2 spike reveals flexibility mediated by three hinges
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Posted 26 Jun 2020

In situ structural analysis of SARS-CoV-2 spike reveals flexibility mediated by three hinges
3,025 downloads bioRxiv molecular biology

Beata Turoňová, Mateusz Sikora, Christoph Schürmann, Wim J. H. Hagen, Sonja Welsch, Florian E. C. Blanc, Sören von Bülow, Michael Gecht, Katrin Bagola, Cindy Hörner, Ger van Zandbergen, Shyamal Mosalaganti, Andre Schwarz, Roberto Covino, Michael D. Mühlebach, Gerhard Hummer, Jacomine Krijnse Locker, Martin Beck

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the major focus for vaccine development. We combine cryo electron tomography, subtomogram averaging and molecular dynamics simulations to structurally analyze S in situ . Compared to recombinant S, the viral S is more heavily glycosylated and occurs predominantly in a closed pre-fusion conformation. We show that the stalk domain of S contains three hinges that give the globular domain unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and the development of safe vaccines. The large scale tomography data set of SARS-CoV-2 used for this study is therefore sufficient to resolve structural features to below 5 Ångstrom, and is publicly available at EMPIAR-10453. ### Competing Interest Statement The authors have declared no competing interest.

77: CRISPR-Cas12b-assisted nucleic acid detection platform
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Posted 06 Jul 2018

CRISPR-Cas12b-assisted nucleic acid detection platform
2,991 downloads bioRxiv molecular biology

Linxian Li, Shiyuan Li, Jin Wang

Rapid molecular diagnostic technology is very useful in many areas, including public health, environmental testing and criminal investigation. We recently showed that Cas12a had trans-cleavage activity upon collateral single-stranded DNA (ssDNA), with which the HOLMES platform (one-HOur Low-cost Multipurpose highly Efficient System) was developed. Here, we combine the thermophilic Cas12b, which also has the ssDNA trans-cleavage activity, with Loop-Mediated Isothermal Amplification (LAMP), and create HOLMESv2. In HOLMESv2, LAMP amplification and Cas12b trans-cleavage can be integrated into a one-step system with a constant temperature, which therefore brings much convenience in nucleic acid detection. Moreover, we also simplify the RNA detection procedures in HOLMESv2, using an RNA-dependent DNA polymerase for amplification and therefore omitting an extra reverse transcription step.

78: A Survey of Genome Editing Activity for 16 Cpf1 orthologs
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Posted 04 May 2017

A Survey of Genome Editing Activity for 16 Cpf1 orthologs
2,986 downloads bioRxiv molecular biology

Bernd Zetsche, Jonathan Strecker, Omar O Abudayyeh, Jonathan S Gootenberg, David A Scott, Feng Zhang

The recently discovered class 2 CRISPR-Cas endonuclease Cpf1 offers several advantages over Cas9, including the ability to process its own array and requirement for just a single RNA guide. These attributes make Cpf1 promising for many genome engineering applications. To further expand the suite of Cpf1 tools available, we tested 16 Cpf1 orthologs for activity in eukaryotic cells. Four of these new enzymes demonstrated targeted activity, one of which, from Moraxella bovoculi AAX11_00205 (Mb3Cpf1), exhibited robust indel formation. We also show that Mb3Cpf1 displays some tolerance for a shortened PAM (TTN versus the canonical Cpf1 PAM TTTV). The addition of these enzymes to the genome editing toolbox will further expand the utility of this powerful technology.

79: Sampling the conformational space of the catalytic subunit of human γ-secretase
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Posted 01 Sep 2015

Sampling the conformational space of the catalytic subunit of human γ-secretase
2,967 downloads bioRxiv molecular biology

Xiao-chen Bai, Eeson Rajendra, Guanghui Yang, Yigong Shi, Sjors H. W Scheres

Human γ-secretase is an intra-membrane protease that cleaves many different substrates. Aberrant cleavage of Notch is implicated in cancer, while abnormalities in cutting amyloid precursor protein lead to Alzheimer's disease. Our previous cryo-EM structure of γ-secretase revealed considerable disorder in its catalytic subunit presenilin. Here, we introduce an image classification procedure that characterizes molecular plasticity at the secondary structure level, and apply this method to identify three distinct conformations in our previous sample. In one of these conformations, an additional transmembrane helix is visible that cannot be attributed to the known components of γ-secretase. In addition, we present a γ-secretase structure in complex with the dipeptidic inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). Our results reveal how conformational mobility in the second and sixth transmembrane helices of presenilin is greatly reduced upon binding of DAPT or the additional helix, and form the basis for a new model of how substrate enters the transmembrane domain.

80: NgAgo-enhanced homologous recombination in E. coli is mediated by DNA endonuclease activity
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Posted 04 Apr 2019

NgAgo-enhanced homologous recombination in E. coli is mediated by DNA endonuclease activity
2,959 downloads bioRxiv molecular biology

Kok Zhi Lee, Michael A Mechikoff, Archana Kikla, Arren Liu, Paula Pandolfi, Frederick S Gimble, Kevin V. Solomon

Prokaryotic Argonautes (pAgos) have been proposed as more flexible tools for gene-editing as they do not require sequence motifs adjacent to their targets for function, unlike popular CRISPR/Cas systems. One promising pAgo candidate, from the halophilic archaeon Natronobacterium gregoryi (NgAgo), however, has been subject to intense debate regarding its potential in eukaryotic systems. Here, we revisit this enzyme and characterize its function in prokaryotes. NgAgo expresses poorly in non-halophilic hosts with the majority of protein being insoluble and inactive even after refolding. However, we report that the soluble fraction does indeed act as a DNA endonuclease. Structural homology modelling revealed that NgAgo shares canonical domains with other catalytically active pAgos but also contains a previously unrecognized single stranded DNA binding domain (repA). Both repA and the canonical PIWI domain participate in DNA cleavage activities. We also found that these endonuclease activities are essential for enhanced NgAgo-guided homologous recombination, or gene-editing, in E. coli. Collectively, our results provide insight into the poorly characterized NgAgo for subsequent gene-editing tool development and sheds new light on seemingly contradictory reports.

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