Most downloaded biology preprints, all time
in category molecular biology
3,874 results found. For more information, click each entry to expand.
3,156 downloads bioRxiv molecular biology
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.
3,146 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.
3,133 downloads bioRxiv molecular biology
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 the subject of 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 domains participate in DNA cleavage activities of NgAgo. We showed that NgAgo can be programmed with guides to cleave specific DNA in vitro and in E.coli. We also found that these endonuclease activities are essential for enhanced NgAgo-guided homologous recombination, or gene-editing, in E. coli. Collectively, our results demonstrate the potential of NgAgo for gene-editing and reconciles seemingly contradictory reports.
3,067 downloads bioRxiv molecular biology
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.
3,042 downloads bioRxiv molecular biology
Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady-state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq), an orthogonal chemistry- based epitranscriptomics-sequencing technology that uncovers 4-thiouridine (s4U)- incorporation in RNA species at single-nucleotide resolution. In combination with well- established metabolic RNA labeling protocols and coupled to standard, low-input, high- throughput RNA sequencing methods, SLAM-seq enables rapid access to RNA polymerase II-dependent gene expression dynamics in the context of total RNA. When applied to mouse embryonic stem cells, SLAM-seq provides global and transcript- specific insights into pluripotency-associated gene expression. We validated the method by showing that the RNA-polymerase II-dependent transcriptional output scales with Oct4/Sox2/Nanog-defined enhancer activity; and we provide quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post- transcriptional gene regulatory pathways initiated by microRNAs and N6-methyladenosine. SLAM-seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective, and scalable manner.
3,034 downloads bioRxiv molecular biology
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.
3,028 downloads bioRxiv molecular biology
Benjamin Ng, Jinrui Dong, Sivakumar Viswanathan, Giuseppe D’Agostino, Anissa Anindya Widjaja, Wei-Wen Lim, Nicole SJ. Ko, Jessie Tan, Sonia P. Chothani, Benjamin Huang, Chen Xie, Ann-Marie Chacko, Nuno Guimarães-Camboa, Sylvia M Evans, Adam J. Byrne, Toby M Maher, Jiurong Liang, Paul W. Noble, Sebastian Schafer, Stuart A Cook
Idiopathic pulmonary fibrosis (IPF) remains a progressive disease despite best medical management. We previously identified IL-11 as a critical factor for cardiovascular fibrosis and examine here its role in pulmonary fibrosis. IL-11 is consistently upregulated in IPF genomic datasets, which we confirmed by histology. Pulmonary fibroblasts stimulated with IL-11 transform into invasive myofibroblasts whereas fibroblasts from Il11ra deleted mice did not respond to pro-fibrotic stimuli. In the mouse, injection of recombinant Il-11 or fibroblast-specific expression of Il-11 caused pulmonary fibrosis. We then generated a neutralising IL-11 binding antibody that blocks lung fibroblast activation across species. In a mouse model of IPF, anti-IL-11 therapy attenuated lung fibrosis and specifically blocked ERK activation. These data prioritise IL-11 as an accessible drug target in IPF.
2,998 downloads bioRxiv molecular biology
Cannabis sativa is listed as a Schedule I substance by the United States Drug Enforcement Agency and has been federally illegal in the United States since 1937. However, the majority of states in the United States, as well as several countries, now have various levels of legal Cannabis. Products are labeled with identifying strain names but there is no official mechanism to register Cannabis strains, therefore the potential exists for incorrect identification or labeling. This study uses genetic analyses to investigate strain reliability from the consumer point of view. Ten microsatellite regions were used to examine samples from strains obtained from dispensaries in three states. Samples were examined for genetic similarity within strains, and also a possible genetic distinction between Sativa, Indica, or Hybrid types. The analyses revealed genetic inconsistencies within strains. Additionally, although there was strong statistical support dividing the samples into two genetic groups, the groups did not correspond to commonly reported Sativa/Hybrid/Indica types. Genetic differences have the potential to lead to phenotypic differences and unexpected effects, which could be surprising for the recreational user, but have more serious implications for patients relying on strains that alleviate specific medical symptoms.
2,986 downloads bioRxiv molecular biology
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.
2,905 downloads bioRxiv molecular biology
CRISPR genome editing experiments offer enormous potential for the evaluation of genomic loci using arrayed single guide RNAs (sgRNAs) or pooled sgRNA libraries. Numerous computational tools are available to help design sgRNAs with optimal on-target efficiency and minimal off-target potential. In addition, computational tools have been developed to analyze deep sequencing data resulting from genome editing experiments. However, these tools are typically developed in isolation and oftentimes not readily translatable into laboratory-based experiments. Here we present a protocol that describes in detail both the computational and benchtop implementation of an arrayed and/or pooled CRISPR genome editing experiment. This protocol provides instructions for sgRNA design with CRISPOR, experimental implementation, and analysis of the resulting high-throughput sequencing data with CRISPResso. This protocol allows for design and execution of arrayed and pooled CRISPR experiments in 4-5 weeks by non-experts as well as computational data analysis in 1-2 days that can be performed by both computational and non-computational biologists alike.
2,851 downloads bioRxiv molecular biology
Mathias Uhlén, Hanna Tegel, Åsa Sivertsson, Chih-Chung Kuo, Jahir M. Gutierrez, Nathan E Lewis, Björn Forsström, Melanie Dannemeyer, Linn Fagerberg, Magdalena Malm, Helian Vunk, Fredrik Edfors, Andreas Hober, Evelina Sjöstedt, David Kotol, Jan Mulder, Adil Mardinoglu, Jochen Schwenk, Peter Nilsson, Martin Zwahlen, Jenny Ottosson Takanen, Kalle von Feilitzen, Charlotte Stadler, Cecilia Lindskog, Fredrik Ponten, Jens Nielsen, Bernhard O Palsson, Anna-Luisa Volk, Magnus Lundqvist, Anna Berling, Anne-Sophie Svensson, Sara Kanje, Henric Enstedt, Delaram Afshari, Siri Ekblad, Julia Scheffel, Borbala Katona, Jimmy Vuu, Emil Lindström, LanLan Xu, Roxana Mihai, Lucas Bremer, Malin Westin, Muna Muse, Lorenz M Mayr, Sinead Knight, Sven Göpel, Rick Davies, Paul Varley, Diane Hatton, Ray Fields, Bjørn G Voldborg, Johan Rockberg, Lovisa Holmberg Schiavone, Sophia Hober
The proteins secreted by human tissues (the secretome) are important for the basic understanding of human biology, but also for identification of potential targets for future diagnosis and therapy. Here, we present an annotated list of all predicted secreted proteins (n=2,623) with information about cellular origin and spatial distribution in the human body. A high-throughput mammalian cell factory was established to create a resource of recombinant full-length proteins. This resource was used for phenotypic assays involving β-cell dedifferentiation and for development of targeted proteomics assays. A comparison between host cells, including omics analysis, shows that many of the proteins that failed to be generated in CHO cells could be rescued in human HEK 293 cells. In conclusion, the human secretome has been mapped and characterized and a resource has been generated to facilitate further exploration of the human secretome.
2,844 downloads bioRxiv molecular biology
Coronavirus disease 2019 (COVID-19) can be screened and diagnosed through the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by real-time reverse transcription polymerase chain reaction. SARS-CoV-2 nucleic acid amplification tests (NAATs) have been rapidly developed and quickly applied to clinical testing during the pandemic. However, studies evaluating the performance of these NAAT assays are limited. We evaluated the performance of four NAATs, which were marked by the Conformité Européenne and widely used in China during the pandemic. Results showed that the analytical sensitivity of the four assays was significantly lower than that claimed by the NAAT manufacturers. The limit of detection (LOD) of Daan, Sansure, and Hybribio NAATs was 3000 copies/mL, whereas the LOD of Bioperfectus NAATs was 4000 copies/mL. The results of the consistency test using 46 samples showed that Daan, Sansure, and Hybribio NAATs could detect the samples with a specificity of 100% (30/30) and a sensitivity of 100% (16 /16), whereas Bioperfectus NAAT detected the samples with a specificity of 100% (30/30) and a sensitivity 81.25% (13/16). The sensitivity of Bioperfectus NAAT was lower than that of the three other NAATs; this finding was consistent with the result that Bioperfectus NAAT had a higher LOD than the three other kinds of NAATs. The four above mentioned reagents presented high specificity; however, for the detection of the samples with low virus concentration, Bioperfectus reagent had the risk of missing detection. Therefore, the LOD should be considered in the selection of SARS-CoV-2 NAATs.
2,841 downloads bioRxiv molecular biology
Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided nucleases have gathered considerable excitement as a tool for genome engineering. However, questions remain about the specificity of their target site recognition. Most previous studies have examined predicted off-target binding sites that differ from the perfect target site by one to four mismatches, which represent only a subset of genomic regions. Here, we use ChIP-seq to examine genome-wide CRISPR binding specificity at gRNA-specific and gRNA-independent sites. For two guide RNAs targeting the murine Snurf gene promoter, we observed very high binding specificity at the intended target site while off-target binding was observed at 2- to 6-fold lower intensities. We also identified significant gRNA-independent off-target binding. Interestingly, we found that these regions are highly enriched in the PAM site, a sequence required for target site recognition by CRISPR. To determine the relationship between Cas9 binding and endonuclease activity, we used targeted sequence capture as a high-throughput approach to survey a large number of the potential off-target sites identified by ChIP-seq or computational prediction. A high frequency of indels was observed at both target sites and one off-target site, while no cleavage activity could be detected at other ChIP-bound regions. Our data is consistent with recent finding that most interactions between the CRISPR nuclease complex and genomic PAM sites are transient and do not lead to DNA cleavage. The interactions are stabilized by gRNAs with good matches to the target sequence adjacent to the PAM site, resulting in target cleavage activity.
2,820 downloads bioRxiv molecular biology
Mammalian genomes are pervasively transcribed to produce thousands of spliced long noncoding RNAs (lncRNAs), whose functions remain poorly understood. Because recent evidence has implicated several specific lncRNA loci in the local regulation of gene expression, we sought to determine whether such local regulation is a property of many lncRNA loci. We used genetic manipulations to dissect 12 genomic loci that produce lncRNAs and found that 5 of these loci influence the expression of a neighboring gene in cis. Surprisingly, however, none of these effects required the specific lncRNA transcripts themselves and instead involved general processes associated with their production, including enhancer-like activity of gene promoters, the process of transcription, and the splicing of the transcript. Interestingly, such effects are not limited to lncRNA loci: we found similar effects on local gene expression at 4 of 6 protein-coding loci. These results demonstrate that 'crosstalk' among neighboring genes is a prevalent phenomenon that can involve multiple mechanisms and cis regulatory signals, including a novel role for RNA splicing. These mechanisms may explain the function and evolution of some genomic loci that produce lncRNAs.
2,792 downloads bioRxiv molecular biology
Phil Oberacker, Peter Stepper, Donna M Bond, Sven Höhn, Jule Focken, Vivien Meyer, Luca Schelle, Victoria J Sugrue, Gert-Jan Jeunen, Tim Moser, Timothy Hore, Ferdinand von Meyenn, Katharina Hipp, Timothy A Hore, Tomasz Piotr Jurkowski
Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge- and column-based protocols require specialised equipment, often use toxic reagents and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA and total RNA from different sources, as well as environmental TNA and PCR amplicons. We also provide a bead-based protocol for bisulfite conversion, and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility and extreme cost-effectiveness of using magnetic beads. These open source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement.
2,758 downloads bioRxiv molecular biology
Many of the proteins produced by SARS-CoV-2 have related counterparts across the Severe Acute Respiratory Syndrome (SARS-CoV) family. One such protein is non-structural protein 9 (Nsp9), which is thought to mediate both viral replication and virulence. Current understanding suggests that Nsp9 is involved in viral genomic RNA reproduction. Nsp9 is thought to bind RNA via a fold that is unique to this class of betacoronoaviruses although the molecular basis for this remains ill-defined. We sought to better characterise the SARS-CoV-2 Nsp9 protein and subsequently solved its X-ray crystal structure, in an apo-form and, unexpectedly, in a peptide-bound form with a sequence originating from a rhinoviral 3C protease sequence (LEVL). The structure of the SARS-CoV-2 Nsp9 revealed the high level of structural conservation within the Nsp9 family. The exogenous peptide binding site is close to the dimer interface and impacted on the relative juxtaposition of the monomers within the homodimer. Together we have established a protocol for the production of SARS-CoV-2 Nsp9, determined its structure and identified a peptide-binding site that may warrant further study from the perspective of understanding Nsp9 function.
2,755 downloads bioRxiv molecular biology
Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach that directly addresses the underlying genetic basis of disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit the number of donor molecules available to the HDR machinery, thus limiting HDR efficacy. Here, we explore modifications to both double-stranded and single-stranded repair template DNAs and describe simple 5′ end modifications that consistently and dramatically increase donor potency and HDR efficacy across cell types and species.
2,739 downloads bioRxiv molecular biology
Recent advances in genome editing have greatly improved knock-in (KI) efficiency. Searching for factors to further improve KI efficiency for therapeutic use and generation of non-human primate (NHP) models, we found that the strand exchange protein RAD51 can significantly increase homozygous KI using CRISPR/Cas9 in mouse embryos through an interhomolog repair (IHR) mechanism. IHR is well-described in the context of meiosis, but only occurs at low frequencies in mitotic cells and its existence in zygotes is controversial. Using a variety of approaches, we provide evidence for an endogenous IHR mechanism in zygotes that can be enhanced by RAD51. We show that this process can be harnessed for generating homozygous KI animals from wildtype zygotes based on exogenous donors and for converting heterozygous alleles into homozygous alleles without exogenous templates. Furthermore, we elucidate additional factors that contribute to zygotic IHR and identify a RAD51 mutant capable of insertion-deletion (indel)-free stimulation of IHR. Thus, our study provides conclusive evidence for the existence of zygotic IHR and demonstrates methods to enhance IHR for potential use in gene drives, gene therapy, and biotechnology.
2,738 downloads bioRxiv molecular biology
Targeted and inducible regulation of mammalian gene expression is a broadly important research capability that may also enable development of novel therapeutics for treating human diseases. Here we demonstrate that a catalytically inactive RNA-guided CRISPR-Cpf1 nuclease fused to transcriptional activation domains can up-regulate endogenous human gene expression. We engineered drug-inducible Cpf1-based activators and show how this system can be used to tune the regulation of endogenous gene transcription in human cells. Leveraging the simpler multiplex capability of the Cpf1 platform, we show that we can induce both synergistic and combinatorial gene expression in human cells. Our work should enable the creation of other Cpf1-based gene regulatory fusion proteins and the development of multiplex gene perturbation library screens for understanding complex cellular phenotypes.
2,734 downloads bioRxiv molecular biology
Ralph Stadhouders, Enrique Vidal, Francois Serra, Bruno Di Stefano, François le Dily, Javier Quilez, Antonio Gomez, Samuel Collombet, Clara Berenguer, Yasmina Cuartero, Jochen Hecht, Guillaume Filion, Miguel Beato, Marc A. Marti-Renom, Thomas Graf
Chromosomal architecture is known to influence gene expression, yet its role in controlling cell fate remains poorly understood. Reprogramming of somatic cells into pluripotent stem cells by the transcription factors (TFs) Oct4, Sox2, Klf4 and Myc offers an opportunity to address this question but is severely limited by the low proportion of responding cells. We recently developed a highly efficient reprogramming protocol that synchronously converts somatic into pluripotent stem cells. Here, we employ this system to integrate time-resolved changes in genome topology with gene expression, TF binding and chromatin state dynamics. This revealed that TFs drive topological genome reorganization at multiple architectural levels, which often precedes changes in gene expression. Removal of locus-specific topological barriers can explain why pluripotency genes are activated sequentially, instead of simultaneously, during reprogramming. Taken together, our study implicates genome topology as an instructive force for implementing transcriptional programs and cell fate in mammals.
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