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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 118,445 papers from 510,944 authors.

Most downloaded biology preprints, all time

in category molecular biology

3,576 results found. For more information, click each entry to expand.

41: Evolutionary persistence of DNA methylation for millions of years after ancient loss of a de novo methyltransferase
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Posted 13 Jun 2017

Evolutionary persistence of DNA methylation for millions of years after ancient loss of a de novo methyltransferase
4,176 downloads bioRxiv molecular biology

Sandra Catania, Phillip A. Dumesic, Harold Pimentel, Ammar Nasif, Caitlin I Stoddard, Jordan E Burke, Jolene K. Diedrich, Sophie Cook, Terrance Shea, Elizabeth Geinger, Robert Lintner, John R Yates, Petra Hajkova, Geeta J. Narlikar, Christina Cuomo, Jonathan K. Pritchard, Hiten D. Madhani

Cytosine methylation of DNA is a widespread modification of DNA that plays numerous critical roles, yet has been lost many times in diverse eukaryotic lineages. In the yeast Cryptococcus neoformans , CG methylation occurs in transposon-rich repeats and requires the DNA methyltransferase, Dnmt5. We show that Dnmt5 displays exquisite maintenance-type specificity in vitro and in vivo and utilizes similar in vivo cofactors as the metazoan maintenance methylase Dnmt1. Remarkably, phylogenetic and functional analysis revealed that the ancestral species lost the gene for a de novo methylase, DnmtX, between 50-150 MYA. We examined how methylation has persisted since the ancient loss of DnmtX. Experimental and comparative studies reveal efficient replication of methylation patterns in C. neoformans , rare stochastic methylation loss and gain events, and the action of natural selection. We propose that an epigenome has been propagated for >50 MY through a process analogous to Darwinian evolution of the genome.

42: Rapid, field-deployable nucleobase detection and identification using FnCas9
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Posted 09 Apr 2020

Rapid, field-deployable nucleobase detection and identification using FnCas9
4,159 downloads bioRxiv molecular biology

Mohd. Azhar, Rhythm Phutela, Asgar Hussain Ansari, Dipanjali Sinha, Namrata Sharma, Manoj Kumar, Meghali Aich, Saumya Sharma, Riya Rauthan, Khushboo Singhal, Harsha Lad, Pradeep Kumar Patra, Govind Makharia, Giriraj Ratan Chandak, Debojyoti Chakraborty, Souvik Maiti

Detection of pathogenic sequences or variants in DNA and RNA through a point-of-care diagnostic approach is valuable for rapid clinical prognosis. In recent times, CRISPR based detection of nucleic acids have provided an economical and quicker alternative to sequencing-based platforms which are often difficult to implement in the field. Here, we present FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that employs a highly accurate enzymatic readout for detecting nucleotide sequences, identifying nucleobase identity and inferring zygosity with precision. We demonstrate that FELUDA output can be adapted to multiple signal detection platforms and can be quickly designed and deployed for versatile applications including rapid diagnosis during infectious disease outbreaks like COVID-19. ### Competing Interest Statement D.C., S.M., M.A., R.P., A.S.A, D.S., N.S. and S.S. are authors in provisional patent applications that have been filed in relation to this work.

43: “Unexpected mutations after CRISPR-Cas9 editing in vivo” are most likely pre-existing sequence variants and not nuclease-induced mutations
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Posted 05 Jul 2017

“Unexpected mutations after CRISPR-Cas9 editing in vivo” are most likely pre-existing sequence variants and not nuclease-induced mutations
4,118 downloads bioRxiv molecular biology

Caleb A. Lareau, Kendell Clement, Jonathan Y Hsu, Vikram Pattanayak, J. Keith Joung, Martin J Aryee, Luca Pinello

Schaefer et al. recently advanced the provocative conclusion that CRISPR-Cas9 nuclease can induce off-target alterations at genomic loci that do not resemble the intended on-target site. Using high-coverage whole genome sequencing (WGS), these authors reported finding SNPs and indels in two CRISPR-Cas9-treated mice that were not present in a single untreated control mouse. On the basis of this association, Schaefer et al. concluded that these sequence variants were caused by CRISPR-Cas9. This new proposed CRISPR-Cas9 off-target activity runs contrary to previously published work and, if the authors are correct, could have profound implications for research and therapeutic applications. Here we demonstrate that the simplest interpretation of Schaefer et al.'s data is that the two CRISPR-Cas9-treated mice are actually more closely related genetically to each other than to the control mouse. This strongly suggests that the so-called “unexpected mutations” simply represent SNPs and indels shared in common by these mice prior to nuclease treatment. In addition, given the genomic and sequence distribution profiles of these variants, we show that it is challenging to explain how CRISPR-Cas9 might be expected to induce such changes. Finally, we argue that the lack of appropriate controls in Schaefer et al.'s experimental design precludes assignment of causality to CRISPR-Cas9. Given these substantial issues, we urge Schaefer et al. to revise or re-state the original conclusions of their published work so as to avoid leaving misleading and unsupported statements to persist in the literature.

44: An improved auxin-inducible degron system preserves native protein levels and enables rapid and specific protein depletion
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Posted 22 Mar 2019

An improved auxin-inducible degron system preserves native protein levels and enables rapid and specific protein depletion
4,083 downloads bioRxiv molecular biology

Kizhakke Mattada Sathyan, Brian D McKenna, Warren D. Anderson, Fabiana M. Duarte, Leighton Core, Michael J. Guertin

Rapid perturbation of protein function permits the ability to define primary molecular responses while avoiding down-stream cumulative effects of protein dysregulation. The auxin-inducible degron (AID) system was developed as a tool to achieve rapid and inducible protein degradation in non-plant systems. However, tagging proteins at their endogenous loci results in chronic, auxin-independent degradation by the proteasome. To correct this deficiency, we expressed the Auxin Response Transcription Factor (ARF) in an improved inducible degron system. ARF is absent from previously engineered AID systems, but ARF is a critical component of native auxin signaling. In plants, ARF directly interacts with AID in the absence of auxin and we found that expression of the ARF Phox and Bem1 (PB1) domain suppresses constitutive degradation of AID-tagged proteins. Moreover, the rate of auxin-induced AID degradation is substantially faster in the ARF-AID system. To test the ARF-AID system in a quantitative and sensitive manner, we measured genome-wide changes in nascent transcription after rapidly depleting the ZNF143 transcription factor. Transciptional profiling indicates that ZNF143 activates transcription in cis and ZNF143 regulates promoter-proximal paused RNA Polymerase density. Rapidly inducible degradation systems that preserve the target protein’s native expression levels and patterns will revolutionize the study of biological systems by enabling specific and temporally defined protein dysregulation.

45: Validation of a Lysis Buffer Containing 4 M Guanidinium Thiocyanate (GITC)/ Triton X-100 for Extraction of SARS-CoV-2 RNA for COVID-19 Testing: Comparison of Formulated Lysis Buffers Containing 4 to 6 M GITC, Roche External Lysis Buffer and Qiagen RTL Lysis Buffer
more details view paper

Posted 08 Apr 2020

Validation of a Lysis Buffer Containing 4 M Guanidinium Thiocyanate (GITC)/ Triton X-100 for Extraction of SARS-CoV-2 RNA for COVID-19 Testing: Comparison of Formulated Lysis Buffers Containing 4 to 6 M GITC, Roche External Lysis Buffer and Qiagen RTL Lysis Buffer
4,022 downloads bioRxiv molecular biology

Martina F Scallan, Catherine Dempsey, John MacSharry, Isabelle O’Callaghan, Paula M. O’Connor, Conor P. Horgan, Edel Durack, Paul D Cotter, Sarah Hudson, Humphrey A. Moynihan, Brigid Lucey

The COVID-19 pandemic has resulted in increased need for diagnostic testing using reverse transcriptase real-time PCR (RT-PCR). An exponential increase in demand has resulted in a shortage of numerous reagents in particular those associated with the lysis buffer required to extract the viral RNA. Herein, we describe a rapid collective effort by hospital laboratory scientists, academic researchers and the biopharma industry to generate a validated lysis buffer. We have formulated a 4M Guanidinium thiocyanate (GITC)/ Triton X-100 Lysis buffer which provides comparable results with the recommended reagents. This buffer will ease the burden on hospital labs in their heroic efforts to diagnose a large population of patients. ### Competing Interest Statement The authors have declared no competing interest.

46: High-resolution interrogation of functional elements in the noncoding genome
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Posted 18 Apr 2016

High-resolution interrogation of functional elements in the noncoding genome
3,971 downloads bioRxiv molecular biology

Neville E Sanjana, Jason Wright, Kaijie Zheng, Ophir Shalem, Pierre Fontanillas, Julia Joung, Christine Cheng, Aviv Regev, Feng Zhang

The noncoding genome plays a major role in gene regulation and disease yet we lack tools for rapid identification and manipulation of noncoding elements. Here, we develop a large-scale CRISPR screen employing ~18,000 sgRNAs targeting >700 kb of noncoding sequence in an unbiased manner surrounding three genes (NF1, NF2, and CUL3) involved in resistance to the BRAF inhibitor vemurafenib in the BRAF-mutant melanoma cell line A375. We identify specific noncoding locations near genes that modulate drug resistance when mutated. These sites have predictive hallmarks of noncoding function, such as physical interaction with gene promoters, evolutionary conservation and tissue-specific chromatin accessibility. At a subset of identified elements at the CUL3 locus, we show that engineered mutations lead to a loss of gene expression associated with changes in transcription factor occupancy and in long-range and local epigenetic environments, implicating these sites in gene regulation and chemotherapeutic resistance. This demonstration of an unbiased mutagenesis screen across large noncoding regions expands the potential of pooled CRISPR screens for fundamental genomic discovery and for elucidating biologically relevant mechanisms of gene regulation.

47: DNA Break-Induced Epigenetic Drift as a Cause of Mammalian Aging
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Posted 21 Oct 2019

DNA Break-Induced Epigenetic Drift as a Cause of Mammalian Aging
3,903 downloads bioRxiv molecular biology

Motoshi Hayano, Jae-Hyun Yang, Michael S. Bonkowski, Joao A. Amorim, Jaime M. Ross, Giuseppe Coppotelli, Patrick T. Griffin, Yap Ching Chew, Wei Guo, Xiaojing Yang, Daniel L. Vera, Elias L. Salfati, Abhirup Das, Sachin Thakur, Alice E Kane, Sarah J. Mitchell, Yasuaki Mohri, Emi K. Nishimura, Laura Schaevitz, Neha Garg, Ana-Maria Balta, Meghan A Rego, Meredith Gregory-Ksander, Tatjana C. Jakobs, Lei Zhong, Hiroko Wakimoto, Raul Mostoslavsky, Amy J Wagers, Kazuo Tsubota, Stephen J Bonasera, Carlos M. Palmeira, Jonathan G Seidman, Christine E. Seidman, Norman S. Wolf, Jill A. Kreiling, John M Sedivy, George F. Murphy, Philipp Oberdoerffer, Bruce R. Ksander, Luis A. Rajman, David A. Sinclair

There are numerous hallmarks of aging in mammals, but no unifying cause has been identified. In budding yeast, aging is associated with a loss of epigenetic information that occurs in response to genome instability, particularly DNA double-strand breaks (DSBs). Mammals also undergo predictable epigenetic changes with age, including alterations to DNA methylation patterns that serve as epigenetic "age" clocks, but what drives these changes is not known. Using a transgenic mouse system called "ICE" (for inducible changes to the epigenome), we show that a tissue's response to non-mutagenic DSBs reorganizes the epigenome and accelerates physiological, cognitive, and molecular changes normally seen in older mice, including advancement of the epigenetic clock. These findings implicate DSB-induced epigenetic drift as a conserved cause of aging from yeast to mammals.

48: A Scalable, Easy-to-Deploy, Protocol for Cas13-Based Detection of SARS-CoV-2 Genetic Material
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Posted 21 Apr 2020

A Scalable, Easy-to-Deploy, Protocol for Cas13-Based Detection of SARS-CoV-2 Genetic Material
3,869 downloads bioRxiv molecular biology

Jennifer N. Rauch, Eric Valois, Sabrina C Solley, Friederike Braig, Ryan S Lach, Morgane Audouard, Jose Carlos Ponce-Rojas, Michael S. Costello, Naomi J Baxter, Kenneth S. Kosik, Carolina Arias, Diego Acosta-Alvear, Maxwell Z. Wilson

The COVID-19 pandemic has created massive demand for widespread, distributed tools for detecting SARS-CoV-2 genetic material. The hurdles to scalable testing include reagent and instrument accessibility, availability of highly-trained personnel, and large upfront investment. Here we showcase an orthogonal pipeline we call CREST (Cas13-based, Rugged, Equitable, Scalable Testing) that addresses some of these hurdles. Specifically, CREST pairs commonplace and reliable biochemical methods (PCR) with low-cost instrumentation, without sacrificing detection sensitivity. By taking advantage of simple fluorescence visualizers, CREST allows for a binary interpretation of results. CREST may provide a point- of-care solution to increase the distribution of COVID-19 surveillance. ### Competing Interest Statement The authors have declared no competing interest.

49: A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for reagent production
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Posted 31 Mar 2020

A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for reagent production
3,822 downloads bioRxiv molecular biology

Sanchita Bhadra, Andre C Maranhao, Andrew D. Ellington

Given the scale of the ongoing COVID-19 pandemic, the need for reliable, scalable testing, and the likelihood of reagent shortages, especially in resource-poor settings, we have developed a RT-qPCR assay that relies on an alternative to conventional viral reverse transcriptases, a thermostable reverse transcriptase / DNA polymerase (RTX). Here we show that RTX performs comparably to the other assays sanctioned by the CDC and validated in kit format. We demonstrate two modes of RTX use - (i) dye-based RT-qPCR assays that require only RTX polymerase, and (ii) TaqMan RT-qPCR assays that use a combination of RTX and Taq DNA polymerases (as the RTX exonuclease does not degrade a TaqMan probe). We also provide straightforward recipes for the purification of this alternative reagent. We anticipate that in low resource or point-of-need settings researchers could obtain the available constructs from Addgene or our lab and begin to develop their own assays, within whatever regulatory framework exists for them. ### Competing Interest Statement The authors have declared no competing interest.

50: Single-cell isoform RNA sequencing (ScISOr-Seq) across thousands of cells reveals isoforms of cerebellar cell types.
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Posted 08 Jul 2018

Single-cell isoform RNA sequencing (ScISOr-Seq) across thousands of cells reveals isoforms of cerebellar cell types.
3,814 downloads bioRxiv molecular biology

Ishaan Gupta, Paul G Collier, Bettina Haase, Ahmed Mahfouz, Anoushka Joglekar, Taylor Floyd, Frank Koopmans, Ben Barres, August B Smit, Steven Sloan, Wenjie Luo, Olivier Fedrigo, M Elizabeth Ross, Hagen U Tilgner

Full-length isoform sequencing has advanced our knowledge of isoform biology. However, apart from applying full-length isoform sequencing to very few single cells, isoform sequencing has been limited to bulk tissue, cell lines, or sorted cells. Single splicing events have been described for <=200 single cells with great statistical success, but these methods do not describe full-length mRNAs. Single cell short-read 3' sequencing has allowed identification of many cell sub-types, but full-length isoforms for these cell types have not been profiled. Using our new method of single-cell-isoform-RNA-sequencing (ScISOr-Seq) we determine isoform-expression in thousands of individual cells from a heterogeneous bulk tissue (cerebellum), without specific antibody-fluorescence activated cell sorting. We elucidate isoform usage in high-level cell types such as neurons, astrocytes and microglia and finer sub-types, such as Purkinje cells and Granule cells, including the combination patterns of distant splice sites, which for individual molecules requires long reads. We produce an enhanced genome annotation revealing cell-type specific expression of known and 16,872 novel (with respect to mouse Gencode version 10) isoforms (see isoformatlas.com).

51: Ferrets not infected by SARS-CoV-2 in a high-exposure domestic setting
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Posted 22 Aug 2020

Ferrets not infected by SARS-CoV-2 in a high-exposure domestic setting
3,768 downloads bioRxiv molecular biology

Kaitlin Sawatzki, Nichola Hill, Wendy Puryear, Alexa Foss, Jonathon Stone, Jonathan Runstadler

Ferrets ( Mustela putorius furo ) are mustelids of special relevance to laboratory studies of respiratory viruses and have been shown to be susceptible to SARS-CoV-2 infection and onward transmission. Here, we report the results of a natural experiment where 29 ferrets in one home had prolonged, direct contact and constant environmental exposure to two humans with symptomatic COVID-19. We observed no evidence of SARS-CoV-2 transmission from humans to ferrets based on RT-PCR and ELISA. To better understand this discrepancy in experimental and natural infection in ferrets, we compared SARS-CoV-2 sequences from natural and experimental mustelid infections and identified two surface glycoprotein (Spike) mutations associated with mustelids. While we found evidence that ACE2 provides a weak host barrier, one mutation only seen in ferrets is located in the novel S1/S2 cleavage site and is computationally predicted to decrease furin activity. These data support that host factors interacting with the novel S1/S2 cleavage site may be a barrier in ferret SARS-CoV-2 susceptibility and that domestic ferrets are at low risk of natural infection from currently circulating SARS-CoV-2. This may be overcome in laboratory settings using concentrated viral inoculum, but the effects of ferret host-adaptations require additional investigation. ### Competing Interest Statement The authors have declared no competing interest.

52: RNA-protein interaction mapping via MS2 or Cas13-based APEX targeting
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Posted 28 Feb 2020

RNA-protein interaction mapping via MS2 or Cas13-based APEX targeting
3,765 downloads bioRxiv molecular biology

Shuo Han, Boxuan Simen Zhao, Samuel A. Myers, Steven A Carr, Chuan He, Alice Y Ting

RNA-protein interactions underlie a wide range of cellular processes. Improved methods are needed to systematically map RNA-protein interactions in living cells in an unbiased manner. Capitalizing on the ability of the engineered peroxidase APEX2 to identify protein interaction partners via proximity-dependent biotinylation, we used two approaches to target APEX2 to specific cellular RNAs. Both an MS2-MCP system and an engineered CRISPR-Cas13 system were able to deliver APEX2 to the human telomerase RNA hTR with high specificity. One-minute proximity biotinylation captured endogenous protein interaction partners of hTR, including more than a dozen proteins not previously linked to hTR. We validated the unexpected interaction between hTR and the N6-methyladenosine (m6A) demethylase ALKBH5. Further investigation showed that endogenous hTR is modified by m6A, which can be erased by ALKBH5, and that ALKBH5 influences both telomerase complex assembly and activity. These results highlight the ability of MS2- and Cas13-targeted APEX2 to identify novel RNA-protein interactions in living cells.

53: SARS-CoV-2 detection from nasopharyngeal swab samples without RNA extraction
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Posted 30 Mar 2020

SARS-CoV-2 detection from nasopharyngeal swab samples without RNA extraction
3,763 downloads bioRxiv molecular biology

Carolina Beltrán-Pavez, Chantal L. Márquez, Gabriela Muñoz, Fernando Valiente-Echeverría, Aldo Gaggero, Ricardo Soto-Rifo, Gonzalo P. Barriga

The ongoing COVID-19 pandemic has reached more than 200 countries and territories worldwide. Given the large requirement of SARS-CoV-2 diagnosis and considering that RNA extraction kits are in short supply, we investigated whether two commercial RT-qPCR kits were compatible with direct SARS-CoV-2 detection from nasopharyngeal swab samples. We show that one of the tested kits is fully compatible with direct SARS-CoV-2 detection suggesting that omission of RNA extraction step should be considered in SARS-CoV-2 diagnosis.

54: Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq
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Posted 14 Nov 2018

Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq
3,629 downloads bioRxiv molecular biology

Beeke Wienert, Stacia K. Wyman, Christopher D Richardson, Charles D Yeh, Pinar Akcakaya, Michelle J. Porritt, Michaela Morlock, Jonathan T. Vu, Katelynn R. Kazane, Hannah L Watry, Luke M Judge, Bruce R. Conklin, Marcello Maresca, Jacob E. Corn

Genome editing using nucleases such as CRISPR-Cas induces programmable DNA damage at a target genomic site but can also affect off-target sites. Here, we develop a powerful, sensitive assay for the unbiased identification of off-target sites that we term DISCOVER-Seq. This approach takes advantage of the recruitment of endogenous DNA repair factors for genome-wide identification of Cas-induced double-strand breaks. One such factor, MRE11, is recruited precisely to double-strand breaks, enabling molecular characterization of nuclease cut sites with single-base resolution. DISCOVER-Seq detects off-targets in cellular models and in vivo upon adenoviral gene editing of mouse livers, paving the way for real-time off-target discovery during therapeutic gene editing. DISCOVER-Seq is furthermore applicable to multiple types of Cas nucleases and provides an unprecedented view of events that precede repair of the affected sites.

55: Spatial single-cell profiling of intracellular metabolomes in situ
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Posted 02 Jan 2019

Spatial single-cell profiling of intracellular metabolomes in situ
3,613 downloads bioRxiv molecular biology

Luca Rappez, Mira Stadler, Sergio Triana, Prasad Phapale, Mathias Heikenwalder, Theodore Alexandrov

The recently unveiled extent of cellular heterogeneity demands for single-cell investigations of intracellular metabolomes to reveal their roles in intracellular processes, molecular microenvironment and cell-cell interactions. To address this, we developed SpaceM, a method for in situ spatial single-cell metabolomics of cell monolayers which detects >100 metabolites in >10000 individual cells together with fluorescence and morpho-spatial cellular features. We discovered that the intracellular metabolomes of co-cultured human HeLa cells and mouse NIH3T3 fibroblasts predict the cell type with 90.4% accuracy and revealed a short-distance metabolic intermixing between HeLa and NIH3T3. We characterized lipid classes composing lipid droplets in steatotic differentiated human hepatocytes, and discovered a preferential accumulation of long-chain phospholipids, a co-regulation of oleic and linoleic acids, and an association of phosphatidylinositol monophosphate with high cell-cell contact. SpaceM provides single-cell metabolic, phenotypic, and spatial information and enables spatio-molecular investigations of intracellular metabolomes in a variety of cellular models.

56: RNA polymerase II clustering through CTD phase separation
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Posted 07 May 2018

RNA polymerase II clustering through CTD phase separation
3,599 downloads bioRxiv molecular biology

M. Boehning, C. Dugast-Darzacq, M. Rankovic, A. S. Hansen, T. Yu, H. Marie-Nelly, D.T. McSwiggen, G. Kokic, G. M. Dailey, P. Cramer, X. Darzacq, M. Zweckstetter

The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is an intrinsically disordered low-complexity region that is critical for pre-mRNA transcription and processing. The CTD consists of hepta-amino acid repeats varying in number from 52 in humans to 26 in yeast. Here we report that human and yeast CTDs undergo cooperative liquid phase separation at increasing protein concentration, with the shorter yeast CTD forming less stable droplets. In human cells, truncation of the CTD to the length of the yeast CTD decreases Pol II clustering and chromatin association whereas CTD extension has the opposite effect. CTD droplets can incorporate intact Pol II and are dissolved by CTD phosphorylation with the transcription initiation factor IIH kinase CDK7. Together with published data, our results suggest that Pol II forms clusters/hubs at active genes through interactions between CTDs and with activators, and that CTD phosphorylation liberates Pol II enzymes from hubs for promoter escape and transcription elongation.

57: Simplifying the combined use of CRISPR-Cas9 and Cre-loxP technologies for the efficient generation of targeted conditional gene knockouts in mammalian cells
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Posted 19 Dec 2017

Simplifying the combined use of CRISPR-Cas9 and Cre-loxP technologies for the efficient generation of targeted conditional gene knockouts in mammalian cells
3,577 downloads bioRxiv molecular biology

Tzahi Noiman, Chaim Kahana

Gene knockout technologies have contributed fundamentally to our understanding of the cellular functions of various genes. Two prevalent systems used for the efficient elimination of the expression of specific genes are the Cre-LoxP system and the CRISPR-Cas9 system. Here we present a simple method that combines the use of CRISPR-Cas9 and Cre-loxP for the conditional deletion of essential genes in mammalian cells. First, an inducible Cre recombinase is stably expressed in the cells. Next CRISPR-Cas9 is used to knockout an essential gene, whose function is complemented by stable expression of a FLAG-tagged version of the same protein encoded from a floxed transcription unit containing silent mutations, making it refractory to the CRISPR-Cas9 guide. This FLAG-tagged protein can be deleted by activating the expressed Cre protein enabling evaluation of the cellular consequences of its deletion. We have further used this system to evaluate potential mutants of the tested gene.

58: In-cell architecture of an actively transcribing-translating expressome
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Posted 28 Feb 2020

In-cell architecture of an actively transcribing-translating expressome
3,562 downloads bioRxiv molecular biology

Francis J. O’Reilly, Liang Xue, Andrea Graziadei, Ludwig Sinn, Swantje Lenz, Dimitry Tegunov, Cedric Blötz, Wim J. H. Hagen, Patrick Cramer, Jörg Stülke, Julia Mahamid, Juri Rappsilber

Structural biology performed inside cells can capture molecular machines in action within their native context. Here we develop an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae. We combine whole-cell crosslinking mass spectrometry, cellular cryo-electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at sub-nanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpoint NusA at the interface between a NusG-bound elongating RNAP and the ribosome, and propose it could mediate transcription-translation coupling. Translation inhibition dissociates the expressome, whereas transcription inhibition stalls and rearranges it, demonstrating that the active expressome architecture requires both translation and transcription elongation within the cell.

59: High-surety isothermal amplification and detection of SARS-CoV-2, including with crude enzymes
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Posted 14 Apr 2020

High-surety isothermal amplification and detection of SARS-CoV-2, including with crude enzymes
3,522 downloads bioRxiv molecular biology

Sanchita Bhadra, Timothy E Riedel, Simren Lakhotia, Nicholas D Tran, Andrew D. Ellington

Isothermal nucleic acid amplification tests (iNAT), such as loop-mediated isothermal amplification (LAMP), are good alternatives to polymerase chain reaction (PCR)-based amplification assays, especially for point-of-care and low resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can generate spurious amplicons, especially in the absence of target sequences, resulting in false positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets, and can accumulate mutations that will lead to inefficient primer binding and thus false negative results. Internally redundant assays targeting different regions of the target sequence can help to reduce such false negatives. Here we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on non-sequence-specific readout into assays that can be visually read using sequence-specific fluorogenic oligonucleotide strand exchange (OSD) probes. We evaluate one-pot operation of both individual and multiplex LAMP-OSD assays and demonstrate detection of SARS-CoV-2 virions in crude human saliva. ### Competing Interest Statement The authors have declared no competing interest.

60: Human co-transcriptional splicing kinetics and coordination revealed by direct nascent RNA sequencing
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Posted 16 Apr 2019

Human co-transcriptional splicing kinetics and coordination revealed by direct nascent RNA sequencing
3,518 downloads bioRxiv molecular biology

Heather L Drexler, Karine Choquet, L. Stirling Churchman

Human genes have numerous exons that are differentially spliced within pre-mRNA. Understanding how multiple splicing events are coordinated across nascent transcripts requires quantitative analyses of transient RNA processing events in living cells. We developed nanopore analysis of CO-transcriptional Processing (nano-COP), in which nascent RNAs are directly sequenced through nanopores, exposing the dynamics and patterns of RNA splicing without biases introduced by amplification. nano-COP showed that in both human and Drosophila cells, co-transcriptional splicing occurs after RNA polymerase II transcribes several kilobases of pre-mRNA, suggesting that metazoan splicing transpires distally from the transcription machinery. Inhibition of the branch-site recognition complex SF3B globally abolished co-transcriptional splicing in both species. Our findings revealed that splicing order does not strictly follow the order of transcription and is influenced by cis-regulatory elements. In human cells, introns with delayed splicing frequently neighbor alternative exons and are associated with RNA-binding factors. Moreover, neighboring introns in human cells tend to be spliced concurrently, implying that splicing occurs cooperatively. Thus, nano-COP unveils the organizational complexity of metazoan RNA processing.

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