This publication was made possible by funding from grant T32ES019851 (to C. Fritsch), R01AG054641, and an AFAR young investigator grant (to M. Vermulst).

<ol>
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K., Smith, Z., Okamoto, K., Lynch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+detection+of+in+vivo+transcription+errors.">Large-scale detection of in vivo transcription errors.</a> Proceedings of the National Academy of Science of the United States of America. 110, 18584-18589 (2013).</li><li>Viswanathan, A., You, H. J., Doetsch, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+change+caused+by+transcriptional+bypass+of+uracil+in+nondividing+cells.">Phenotypic change caused by transcriptional bypass of uracil in nondividing cells.</a> Science. 284, 159-162 (1999).</li><li>Bregeon, D., Doddridge, Z. A., You, H. J., Weiss, B., Doetsch, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+mutagenesis+induced+by+uracil+and+8-oxoguanine+in+Escherichia+coli.">Transcriptional mutagenesis induced by uracil and 8-oxoguanine in Escherichia coli.</a> Molecular Cell. 12, 959-970 (2003).</li><li>Saxowsky, T. T., Doetsch, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+polymerase+encounters+with+DNA+damage:+transcription-coupled+repair+or+transcriptional+mutagenesis.">RNA polymerase encounters with DNA damage: transcription-coupled repair or transcriptional mutagenesis.</a> Chemical Reviews. 106, 474-488 (2006).</li><li>Saxowsky, T. T., Meadows, K. L., Klungland, A., Doetsch, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=8-Oxoguanine-mediated+transcriptional+mutagenesis+causes+Ras+activation+in+mammalian+cells.">8-Oxoguanine-mediated transcriptional mutagenesis causes Ras activation in mammalian cells.</a> Proceedings of the National Academy of Science of the United States of America. 105, 18877-18882 (2008).</li><li>Vermulst, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+errors+induce+proteotoxic+stress+and+shorten+cellular+lifespan.">Transcription errors induce proteotoxic stress and shorten cellular lifespan.</a> Nature Communications. 6, 8065 (2015).</li><li>van Leeuwen, F. W., Burbach, J. P., Hol, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+RNA:+a+first+example+of+molecular+misreading+in+Alzheimer's+disease.">Mutations in RNA: a first example of molecular misreading in Alzheimer's disease.</a> Trends in Neurosciences. 21, 331-335 (1998).</li><li>van Leeuwen, F. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frameshift+mutants+of+beta+amyloid+precursor+protein+and+ubiquitin-B+in+Alzheimer's+and+Down+patients.">Frameshift mutants of beta amyloid precursor protein and ubiquitin-B in Alzheimer's and Down patients.</a> Science. 279, 242-247 (1998).</li><li>Morreall, J. F., Petrova, L., Doetsch, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+mutagenesis+and+its+potential+roles+in+the+etiology+of+cancer+and+bacterial+antibiotic+resistance.">Transcriptional mutagenesis and its potential roles in the etiology of cancer and bacterial antibiotic resistance.</a> Journal of Cellular Physiology. 228, 2257-2261 (2013).</li><li>Strathern, J. N., Jin, D. J., Court, D. L., Kashlev, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+transcription+fidelity+mutants.">Isolation and characterization of transcription fidelity mutants.</a> Biochimica et Biophysica Acta. 1819, 694-699 (2012).</li><li>Acevedo, A., Andino, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Library+preparation+for+highly+accurate+population+sequencing+of+RNA+viruses.">Library preparation for highly accurate population sequencing of RNA viruses.</a> Nature Protocols. 9, 1760-1769 (2014).</li><li>Traverse, C. C., Ochman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-Wide+Spectra+of+Transcription+Insertions+and+Deletions+Reveal+That+Slippage+Depends+on+RNA:DNA+Hybrid+Complementarity.">Genome-Wide Spectra of Transcription Insertions and Deletions Reveal That Slippage Depends on RNA:DNA Hybrid Complementarity.</a> mBio. 8, (2017).</li><li>Martin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutadapt+Removes+Adapter+Sequences+From+High-Throughput+Sequencing+Reads.">Cutadapt Removes Adapter Sequences From High-Throughput Sequencing Reads.</a> EMBnet.journal. 17 (1), 10-12 (2011).</li><li>Kent, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLAT--the+BLAST-like+alignment+tool.">BLAT--the BLAST-like alignment tool.</a> Genome Research. 12, 656-664 (2002).</li><li>Kim, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopHat2:+accurate+alignment+of+transcriptomes+in+the+presence+of+insertions,+deletions+and+gene+fusions.">TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions.</a> Genome Biology. 14, R36 (2013).</li><li>Zhou, Y. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+RNA+polymerase+rpoB+mutations+that+alter+transcription+slippage+during+elongation+in+Escherichia+coli.">Isolation and characterization of RNA polymerase rpoB mutations that alter transcription slippage during elongation in Escherichia coli.</a> The Journal of Biological Chemistry. 288, 2700-2710 (2013).</li><li>Reid-Bayliss, K. S., Loeb, L. 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The authors have nothing to disclose.

Here, we describe an optimized protocol for the preparation of C-seq libraries for the detection of transcription errors in Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol has numerous advantages over existing protocols, as well as alternative techniques.
Over the past 15 years, numerous reporter systems have been developed that rely on luciferase7,8 or Cre-Lox rec...

The genome provides a precise biological blueprint of life. To implement this blueprint correctly, it is important for the genome to be transcribed with great precision. However, transcription is unlikely to be error free. For example, RNA polymerases have long been known to be error-prone in vitro1,2, and recently it was shown that they commit errors in vivo as well3,5,6, particularly when confronted with DNA damage7,8,9,10. Taken together, these observations indicate that transcription errors occur continuously in all living cells, suggesting that they could be a potent source of mutated proteins.
This process, termed transcriptional mutagenesis, differs from classical mutagenesis in two ways. First, in contrast to genetic mutations, transcription errors affect both mitotic and post-mitotic cells, as they do not depend on DNA replication. Studying the mechanisms that impact the fidelity of transcription will, therefore, provide valuable insight into the mutation load of both mitotic and post-mitotic cells. Interestingly, transcription errors have recently been implicated in the promotion of protein aggregation11,12,13 and have been hypothesized to contribute to both carcinogenesis10 and the development of antibiotic resistance in bacteria14.
Second, in contrast to genetic mutations, transcription errors are transient in nature. Their temporary existence is particularly challenging because it makes transcription errors exceedingly difficult to detect. For example, while several labs have devised valuable reporter assays for the study of transcriptional mutagenesis, these assays are only able to measure transcription errors in a limited number of contexts and model organisms4,15. To overcome these limitations, many researchers have turned to RNA sequencing technology (RNA-seq), which theoretically allows transcription errors to be recorded throughout the transcriptome of any species. However, these studies are easily confounded by library construction artifacts, such as reverse transcription errors, PCR amplification errors, and the error-prone nature of sequencing itself. For example, reverse transcriptases commit approximately one error every ~20,000 bases, while RNA polymerases (RNAPs) are expected to make only one error every 300,000 bases5,6. Because the error rate of reverse transcription alone dwarfs the error rate of RNA polymerases inside cells, it is virtually impossible to distinguish true transcription errors from artifacts caused by the library preparation in traditional RNA-Seq data (Figure 1a).
To solve this problem, we developed an optimized version of the Circle-Sequencing (Cirseq, or C-seq henceforth) assay5,16. This assay allows the user to detect transcription errors and other rare variants in RNA throughout the transcriptome5. The circular-sequencing assay carries this name because a key step in this assay revolves around RNA circularization. Once the RNA targets are circularized, they are reverse transcribed in a rolling circle fashion, to produce linear cDNA molecules that contain numerous copies of the same RNA template. If an error was present in one of these templates, this error would also be present in every single repeat contained within the cDNA molecule. In contrast, errors introduced by reverse transcription, PCR amplification, or sequencing tend to arise randomly, and will thus be present in only one or two repeats. Thus, by generating a consensus sequence for each cDNA molecule, and distinguishing random errors from errors that occur in all repeats, library construction artifacts can effectively be separated from true transcription errors (Figure 1b).
If used properly, the C-seq assay can be used to accurately detect the rate of base substitutions, insertions, and deletions in RNA throughout the transcriptome of any species (for example, see Traverse and Ochman17). For example, we have used the C-seq assay to provide genome-wide measurements of the error rate of transcription in Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans with a single base resolution5 (unpublished observations). Originally used to accurately sequence RNA virus populations, this optimized version of the C-seq assay has been streamlined to minimize harsh conditions during the library preparation that contribute to library construction artifacts. In addition, by using a number of commercially available kits, the throughput of the assay is greatly improved, as well as its user-friendliness. If used properly, this assay can accurately detect thousands of transcription errors per replicate, thereby greatly improving on previous studies6. Overall, this method provides a powerful tool to study transcriptional mutagenesis and will allow the user to gain novel insights into the mechanisms that control the fidelity of transcription in a wide range of organisms.

<table><tbody><tr><td>RiboPure RNA purification kit</td><td>ThermoFisher</td><td>AM1926</td><td>Total RNA purification</td></tr><tr><td>Genelute mRNA purification kit</td><td>Sigma-Aldrich</td><td>MRN70-1KT</td><td>mRNA purification</td></tr><tr><td>Nuclease-free Water</td><td>Ambion</td><td>AM9937</td><td>Elution and dilution</td></tr><tr><td>Ambion RNase III</td><td>ThermoFisher</td><td>AM2290</td><td>RNA fragmentation</td></tr><tr><td>T4 RNA Ligase 1 (ssRNA Ligase)</td><td>New England Biolabs</td><td>M0204S</td><td>RNA circularization</td></tr><tr><td>Ribolock</td><td>ThermoFisher</td><td>EO0381</td><td>RNase inhibitor</td></tr><tr><td>SuperScript III Reverse Transcriptase</td><td>ThermoFisher</td><td>18080044</td><td>Rolling circle reverse transcription</td></tr><tr><td>10 mM dNTP mix</td><td>ThermoFisher</td><td>18427013</td><td>Rolling circle reverse transcription</td></tr><tr><td>Random hexamers (50 ng/&amp;micro;L)</td><td>ThermoFisher</td><td>N8080127</td><td>Rolling circle reverse transcription</td></tr><tr><td>NEB Second Strand Synthesis Module</td><td>New England Biolabs</td><td>E6111S</td><td>Second Strand Synthesis</td></tr><tr><td>NEBNext Ultra DNA Library Prep Kit for Illumina</td><td>New England Biolabs</td><td>E7370S</td><td>cDNA library preparation</td></tr><tr><td>NEB Next index primers</td><td>NEB</td><td>E7335S</td><td>Multiplex PCR primers</td></tr><tr><td>Oligo Clean &amp;amp; Concentrator</td><td>Zymo Research</td><td>D4061</td><td>Clean up of RNA and DNA samples</td></tr><tr><td>DynaMag-2 Magnet</td><td>ThermoFisher</td><td>12321D</td><td>Magnetic bead purification</td></tr><tr><td>AMPure XP beads</td><td>Beckman Coulter</td><td>A63881</td><td>Magnetic bead purification</td></tr><tr><td>Eppendorf 5424 Microcentrifuge</td><td>FisherScientific</td><td>05-403-93</td><td>centrifugation</td></tr><tr><td>INCU-Shaker 10 L</td><td>Benchmark Scientific</td><td>H1010</td><td>Cell culture</td></tr><tr><td>T100 Thermal Cycler</td><td>BIO RAD</td><td>1861096</td><td>Medium to High temperature cycling conditions</td></tr><tr><td>PTC-200 Thermal Cycler</td><td>GMI</td><td>8252-30-0001&amp;nbsp;</td><td>Low temperature cycling conditions</td></tr><tr><td>RNase Away</td><td>Molecular Bioproducts</td><td>700S-11</td><td>Sterilization</td></tr><tr><td>50 mL Centrifuge Tube</td><td>Corning</td><td>430290</td><td>Nuclease-free</td></tr><tr><td>15 mL Centrifuge Tube</td><td>Corning</td><td>430052</td><td>Nuclease-free</td></tr><tr><td>Eppendorf tubes</td><td>USA Scientific</td><td>1615-5500</td><td>Nuclease-free</td></tr><tr><td>4200 Tapestation System</td><td>Agilent</td><td>G2991AA</td><td>Nucleotide analysis instrument for quality control of RNA and single stranded DNA samples</td></tr><tr><td>High Sensitivity RNA Screen Tape</td><td>Agilent</td><td>5067-5579</td><td>Quality control of RNA and single stranded DNA samples</td></tr><tr><td>RNA ScreenTape Sample Buffer</td><td>Agilent</td><td>5067-5577</td><td>Quality control of RNA and single stranded DNA samples</td></tr><tr><td>RNA ScreenTape Ladder</td><td>Agilent</td><td>5067-5578</td><td>Quality control of RNA and single stranded DNA samples</td></tr><tr><td>2100 Bioanalyzer Instrument</td><td>Agilent</td><td>G2939BA</td><td>Double stranded DNA quality control</td></tr><tr><td>High Sensitivity DNA Kit</td><td>Agilent</td><td>5067-4626</td><td>Quality control for double stranded cDNA samples</td></tr><tr><td>Water Bath</td><td>VWR</td><td>462-0244</td><td>Incubation</td></tr><tr><td>NanoDrop 2000/2000C Spectrophotometer</td><td>ThermoFisher</td><td>ND-2000C</td><td>Determination of RNA concentration</td></tr></tbody></table>

genome-wide surveillance of transcription errors in eukaryotic organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

Like all massively parallel sequencing approaches, each C-seq experiment produces an unwieldy, large dataset. For first-time users, it can be difficult to handle these datasets; thus, it is recommended that all users contact an experienced bio-informatician prior to the experimentation. On average, the expectation is that users will generate approximately 55&#8211;70 Giga bases (Gbases) per run on most massively parallel sequencing platforms. For this protocol, typically, 12&#8211;30 samp...

Watch this Scientific Journal Video about Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms at JoVE.com

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

genome-wide-surveillance-transcription-errors-eukaryotic

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

9.4K Views.  Children's Hospital of Philadelphia. This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

Video: Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach

This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here is a powerful experimental approach for analyzing the transcription termination defect under physiological conditions. Like the traditional TRO assay, it relies on the presence of a transcriptionally active polymerase beyond the 3&#8242; end of the gene as an indicator of a transcription termination defect1. It overcomes two major problems encountered with the traditional TRO assay. First, it can detect if the polymerase reading through the termination signal is the one that initiated transcription from the promoter-proximal region, or if it is simply representing a pervasively transcribing polymerase that initiated non-specifically from somewhere in the body or the 3&#8242; end of the gene. Secondly, it can distinguish if the transcriptionally active polymerase signal beyond the terminator region is truly the readthrough sense mRNA transcribing polymerase or a terminator-initiated non-coding anti-sense RNA signal. Briefly, the protocol involves permeabilizing the exponentially growing yeast cells, allowing the transcripts that initiated in vivo to elongate in the presence of the BrUTP nucleotide, purifying BrUTP-labelled RNA by the affinity approach, reverse transcribing the purified nascent RNA and amplifying the cDNA using strand-specific primers flanking the promoter and the terminator regions of the gene2.

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on TRO Approach

We describe a basic experimental approach for analysis of termination of transcription by RNA polymerase II in vivo using BrUTP by the strand-specific transcription run-on (TRO) approach in budding yeast. This protocol can be extended to study transcription termination by other RNA polymerases both in yeast and higher eukaryotes.

This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here ...

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using &#967;CRAC

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be bound by many different RBPs that will regulate its production, stability, activity, and degradation. As such, much has been done to understand the dynamics that exist between these two types of molecules. A particularly important breakthrough came with the emergence of &#8216;cross-linking and immunoprecipitation&#8217; (CLIP). This technique allowed stringent investigation into which RNAs are bound by a particular RBP. In short, the protein of interest is UV cross-linked to its RNA substrates in vivo, purified under highly stringent conditions, and then the RNAs covalently cross-linked to the protein are converted into cDNA libraries and sequenced. Since its conception, many derivative techniques have been developed in order to make CLIP amenable to particular fields of study. However, cross-linking using ultraviolet light is notoriously inefficient. This results in extended exposure times that make the temporal study of RBP-RNA interactions impossible. To overcome this issue, we recently designed and built much-improved UV irradiation and cell harvesting devices. Using these new tools, we developed a protocol for time-resolved analyses of RBP-RNA interactions in living cells at high temporal resolution: Kinetic CRoss-linking and Analysis of cDNAs (&#967;CRAC). We recently used this technique to study the role of yeast RBPs in nutrient stress adaptation. This manuscript provides a detailed overview of the &#967;CRAC method and presents recent results obtained with the Nrd1 RBP.

Kinetic cross-linking and analysis of cDNA is a method that allows investigation of the dynamics of protein-RNA interactions in living cells at high temporal resolution. Here the protocol is described in detail, including the growth of yeast cells, UV cross-linking, harvesting, protein purification, and next generation sequencing library preparation steps.

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be ...

Biochemistry

Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. Conflicts between the replication and transcription machinery, termed transcription-replication conflicts (TRCs), pose a considerable risk to genome stability, a critical factor in cancer development. While several factors regulating these collisions have been identified, pinpointing primary causes remains difficult due to limited tools for direct visualization and clear interpretation. In this study, we directly visualize TRCs using a proximity ligation assay (PLA), leveraging antibodies specific to PCNA and phosphorylated CTD of RNA polymerase II. This approach allows precise measurement of TRCs between replication and transcription processes mediated by RNA polymerase II. The method is further enhanced through DNA primers conjugated covalently to these antibodies, coupled with PCR amplification using fluorescent probes, providing a highly sensitive and specific means of detecting endogenous TRCs. Fluorescence microscopy enables the visualization of these conflicts, offering a powerful tool to study genome instability mechanisms associated with cancer. This technique addresses the gap in direct TRC visualization, allowing for a more comprehensive analysis and understanding of the underlying processes driving genome instability in cells.

Here, we present a sensitive and specific method to detect oncogene-induced transcription-replication conflicts (TRCs) using the proximity ligation assay (PLA). This approach leverages specific antibodies targeting PCNA and phospho-CTD RNAPII, enabling the assessment of TRC prevalence between RNA polymerase II transcription and DNA replication machinery.

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. ...

Cancer Research

Capturing Common Fragile Site Breaks by Native &#947;H2A.X ChIP

Replication stress induced by exposure to extrinsic agents can lead to DNA breaks at common fragile sites, which are regions in the genome known to be prone to structural instability. The &#947;H2A.X chromatin immunoprecipitation (ChIP) assay serves as a powerful tool in genotoxicity studies, as &#947;H2A.X phosphorylation is a well-established marker for DNA double-strand breaks. Traditional &#947;H2A.X ChIP assays, however, are often labor-intensive and involve multiple, time-consuming steps. In this study, we present a simplified yet effective method that combines subcellular fractionation with native ChIP to isolate &#947;H2A.X-associated complexes. This approach is particularly suitable for analyzing &#947;H2A.X-chromatin interactions with enhanced specificity and efficiency. Using subcellular fractionation, chromatin-unbound materials are effectively removed, resulting in a purified chromatin fraction. Subsequent micrococcal nuclease (MNase) digestion under mild conditions allows chromatin fragmentation while preserving physiological interactions between &#947;H2A.X and its associated protein complexes. This preservation is essential for studying native interaction partners involved in DNA damage response pathways. This optimized native ChIP protocol substantially reduces the time and labor associated with conventional &#947;H2A.X ChIP assays. The streamlined procedure not only simplifies the workflow but also yields highly reproducible results, making it particularly advantageous in settings where high-throughput processing of multiple samples is required. This method has broad applicability in studies focused on genome stability, DNA repair, and chromatin biology, where accurate and efficient detection of DNA damage sites is critical. By employing optimized protocols and streamlined steps, this method enables the detection of DNA damage at fragile sites with improved sensitivity and minimal sample handling, making it a valuable tool for studies on genome stability and DNA damage response.

We present a rapid and efficient method to detect common fragile site breaks through native &#947;H2A.X chromatin immunoprecipitation (ChIP). This approach significantly reduces both the time and labor associated with traditional &#947;H2A.X ChIP assays while maintaining high reproducibility and reliability of results.

Replication stress induced by exposure to extrinsic agents can lead to DNA breaks at common fragile sites, which are regions in the genome known to be ...

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e. total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.
We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.

Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene ...

Biology

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Summary

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Chapters in this video

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using χCRAC

Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts

Capturing Common Fragile Site Breaks by Native γH2A.X ChIP

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)