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Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 65,752 bioRxiv papers from 291,212 authors.

Most downloaded bioRxiv papers, all time

in category molecular biology

2,046 results found. For more information, click each entry to expand.

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

Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq
3,273 downloads 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.

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

Resolving microbial microdiversity with high accuracy full length 16S rRNA Illumina sequencing
3,240 downloads 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.

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

An improved auxin-inducible degron system preserves native protein levels and enables rapid and specific protein depletion
3,229 downloads 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 downstream 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.

24: RNA polymerase II clustering through CTD phase separation
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Posted to bioRxiv 07 May 2018

RNA polymerase II clustering through CTD phase separation
3,176 downloads 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.

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

A comparison of DNA stains and staining methods for Agarose Gel Electrophoresis
3,090 downloads 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%.

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

Evolutionary persistence of DNA methylation for millions of years after ancient loss of a de novo methyltransferase
3,023 downloads 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 A 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.

27: 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 to bioRxiv 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
2,947 downloads 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.

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

P53 toxicity is a hurdle to CRISPR/CAS9 screening and engineering in human pluripotent stem cells
2,942 downloads 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.

29: Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers and Nanopore sequencing
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Posted to bioRxiv 24 May 2019

Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers and Nanopore sequencing
2,932 downloads molecular biology

Soeren Michael Karst, Ryan Ziels, Rasmus Hansen Kirkegaard, Mads Albertsen

High-throughput amplicon sequencing of large genomic regions represents a challenge for existing short-read technologies. Long-read technologies can in theory sequence large genomic regions, but they currently suffer from high error rates. Here, we report a high-throughput amplicon sequencing approach that combines unique molecular identifiers (UMIs) with Oxford Nanopore sequencing to generate single-molecule consensus sequences of large genomic regions. We demonstrate the approach by generating nearly 10,000 full-length ribosomal RNA (rRNA) operons of roughly 4,400 bp in length from a mock microbial community consisting of eight bacterial species using a single Oxford Nanopore MinION flowcell. The mean error rate of the consensus sequences was 0.03%, with no detectable chimeras due to a rigorous UMI-barcode filtering strategy. The simplicity and accessibility of this method paves way for widespread use of high-accuracy amplicon sequencing in a variety of genomic applications.

30: 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 to bioRxiv 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).
2,882 downloads 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.

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

High-precision CRISPR-Cas9 base editors with minimized bystander and off-target mutations
2,847 downloads 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.

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

Human co-transcriptional splicing kinetics and coordination revealed by direct nascent RNA sequencing
2,784 downloads 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.

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

Sampling the conformational space of the catalytic subunit of human γ-secretase
2,757 downloads 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.

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

Characterization and Validation of a Novel Group of Type V, Class 2 Nucleases for in vivo Genome Editing
2,729 downloads 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.

35: Neighborhood regulation by lncRNA promoters, transcription, and splicing
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Posted to bioRxiv 29 Apr 2016

Neighborhood regulation by lncRNA promoters, transcription, and splicing
2,675 downloads molecular biology

Jesse M Engreitz, Jenna E. Haines, Glen Munson, Jenny Chen, Elizabeth M. Perez, Michael Kane, Patrick E McDonel, Mitchell Guttman, Eric S Lander

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.

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

Large-scale, quantitative protein assays on a high-throughput DNA sequencing chip
2,641 downloads 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.

37: A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture
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Posted to bioRxiv 22 May 2014

A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture
2,639 downloads molecular biology

Henriette O’Geen, Isabelle M. Henry, Mital S. Bhakta, Joshua F. Meckler, David J. Segal

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.

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

Tagmentation-Based Mapping (TagMap) of Mobile DNA Genomic Insertion Sites
2,633 downloads 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.

39: Sub-tomogram averaging in RELION
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Posted to bioRxiv 04 Nov 2015

Sub-tomogram averaging in RELION
2,600 downloads molecular biology

Tanmay A.M. Bharat, Sjors H.W. 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.

40: Inducible, tunable and multiplex human gene regulation using CRISPR-Cpf1-based transcription factors
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Posted to bioRxiv 15 Jun 2017

Inducible, tunable and multiplex human gene regulation using CRISPR-Cpf1-based transcription factors
2,588 downloads molecular biology

Yu Gyoung Tak, Benjamin P. Kleinstiver, James K. Nuñez, Jonathan Y Hsu, Jingyi Gong, Jonathan S Weissman, J. Keith Joung

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.

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