We thank H. Chang, T. Oro, S. Tavazoie, R. Flynn, P. Batista, E. Calo, and the entire Wang laboratory for technical support and critical reading of the manuscript. S.L.M. has been supported in this work through the NSFGRF (DGE-114747), NDSEGF (FA9550-11-C-0028), and the National Cancer Institute (1F99CA222541-01). K.C.W. is supported by a Career Award for Medical Scientists from the Burroughs Wellcome Fund, and is a Donald E. and Delia B. Baxter Foundation Faculty Scholar.

1. gRNA Design
<ol>
	<li>Select a standard gRNA design tool to design the guide RNAs10. Use complementary pairs of the previously developed CLOuD9 constructs9 (i.e., at least 1 S. aureus (CSA) and 1 S. pyogenes (CSP) construct) for each experiment.
	<ol>
		<li>Within the selected online design tool, use the Protospacer Adjacent Motif (PAM) sequence &#34;NGG&#34; with a guide length of 20 bp for CSP and the PAM sequence &#34;NNGRRT&#34; with a guide length of 21 for CSA.</li>
		<li>Choose guides based on specificity score and design guides on both strands to maximize chances of obtaining a working guide.</li>
		<li>Order 2 oligonucleotides per guide from an oligo manufacturer: for the forward oligo, add &#34;caccg&#34; to the 5&#39; end of the guide sequence, and for the reverse oligo, add &#34;aaac&#34; to the 5&#39; end and &#34;c&#34; to the 3&#39; end before ordering.</li>
	</ol>
	</li>
	<li>Initially, design 3-5 gRNAs for each region of interest, spread over a 250-1000 bp region. Assess the gRNA targeting efficiency as follows:
	<ol>
		<li>Clone identified gRNAs into an active Cas9 plasmid (see step 3) and transiently transfect into 293T cells (see step 4)11.</li>
		<li>Grow cells for 2-3 d in Dulbecco&#39;s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (pen strep), then harvest and extract genomic DNA12.</li>
		<li>Amplify targeted regions by polymerase chain reaction (PCR). Run products on a 1.5% agarose gel and purify appropriate bands.</li>
		<li>Perform a T7 endonuclease assay as described in the Guschin, et al. protocol13,14. 
		NOTE: Efficient guides are those determined to cut DNA at the target site.</li>
	</ol>
	</li>
</ol>
2. Cell Culture
<ol>
	<li>Maintain cells in a healthy and actively dividing state.
	<ol>
		<li>For K562s, culture in Roswell Park Memorial Institute (RPMI) 1640 media with 10% FBS and 1% pen strep.
		<ol>
			<li>Grow K562s in a 25 cm2 canted neck flask for&#160;maintenance and adjust the cell density to 400,000 cells per mL each day.</li>
			<li>After K562s have been transduced with CLOuD9 constructs, add 2 &#181;g/mL puromycin and 100 &#181;g/mL hygromycin to maintenance media.</li>
		</ol>
		</li>
		<li>For 293Ts, culture in Dulbecco&#39;s Modified Eagle Medium (DMEM) with 10% FBS and 1% pen strep.
		<ol>
			<li>Grow 293Ts in 10 cm2&#160;plates and split when confluent.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Plasmid Preparation and gRNA Insertion15
Note: Plasmid maps are available in the appendix and primers utilized in the example experiments are available in Supplementary Table 1.
<ol>
	<li>Mix 5 &#181;g of the lentiviral CRISPR plasmid with 3 &#181;L of BsmBI, 3 &#181;L of Alkaline Phosphatase, 6 &#181;L of 10x Buffer, and 0.6 &#181;L of freshly prepared 100 mM DTT. Bring the total volume to 60 &#181;L with ddH2O and incubate at 37 &#176;C for 30 min to digest and dephosphorylate the plasmid.</li>
	<li>Gel purify the digested plasmid and elute in ddH2O.</li>
	<li>Mix 1 &#181;L of each of the paired guides at 100 &#181;M with 1 &#181;L of 10x T4 Ligation Buffer, 6.5 &#181;L of ddH2O, and 0.5 &#181;L T4 PNK, to reach 10 &#181;L total volume. Incubate at 37 &#176;C for 30 min, then 95 &#176;C for 5 min, then ramp down to 25 &#176;C at 5 &#176;C/min. 
	Note: This will anneal the pair of guides.</li>
	<li>Dilute the annealed guides (from step 3.3) 1:200 in ddH2O.</li>
	<li>Mix 1 &#181;L of the digested plasmid from step 3.2 with 0.5 &#181;L of the diluted ligated guides from step 3.4, 2.5 &#181;L of 2x Buffer, 1 &#181;l of ddH2O, and 0.5 &#181;L of Ligase, to make a 5.5 &#181;L total reaction. Incubate this at room temperature for 10 min to ligate the guides to the BsmBI digested plasmid.</li>
	<li>Transform the newly ligated plasmid from step 3.5 into Stbl3 bacteria and amplify using any plasmid preparation method.</li>
</ol>
4. Lentivirus Production
<ol>
	<li>Count 750,000 293T cells per well for a six-well plate using a hemocytometer and seed them in DMEM with 10% FBS and 1% pen strep. Use one well for each construct.</li>
	<li>24 h after seeding, change media to fresh antibiotic-free DMEM with 10% FBS.</li>
	<li>In one tube, dilute 11 &#181;L of lipid-based transfection reagent with 150 &#181;L of the OptiMEM medium, per reaction11.</li>
	<li>In a separate tube, dilute 2 &#181;g of the CLOuD9 lentiviral vector plasmid from step 3.6, 0.35 &#181;g of pMDLg/pRRE, 1 &#181;g of pRSV-Rev, and 0.65 &#181;g of pMD2.G with 150 &#181;L of the OptiMEM medium, per reaction11.</li>
	<li>For each reaction, add the diluted lentiviral plasmid mixtures to the diluted lipid-based transfection reagent at a 1:1 ratio, and incubate for 5 min11.</li>
	<li>Add the entire volume of each mixed complex from step 4.5 into the respective wells of the antibiotic-free 293T cells from step 4.2.</li>
	<li>48 h later, collect viral production media by pipetting into a fresh tube and spin down at 300 x g for 5 min. After centrifugation, move the supernatant to a fresh tube to remove residual cell debris.</li>
	<li>Use viral production media immediately for the transduction of target cells or freeze at -80 &#176;C for future use.</li>
</ol>
5. Lentivirus Transduction of Cells
<ol>
	<li>Add 250 &#181;L of each viral construct of interest (comprised of complementary CLOuD9 plasmids) to a 15 mL conical tube containing 80,000 cells for the CLOuD9 application.</li>
	<li>Bring the total volume of media in each conical to 1 mL with antibiotic-free media, and add polybrene to a final concentration of between 1-8 &#181;g/mL (the maximum concentration of polybrene tolerated by the cell type utilized will need to be determined experimentally).</li>
	<li>Spin cells at 800 x g for 30 min at room temperature, then resuspend by pipetting without removing the viral supernatant. Move the entire cell suspension to a cell culture plate.</li>
	<li>24 h post-transduction, spin cells down at 300 x g for 5 min at room temperature.</li>
	<li>Aspirate viral supernatant and resuspend cells in regular cell culture media.</li>
	<li>On the next day, add puromycin (1 &#181;g/mL for 293T cells) and hygromycin (25 &#181;g/mL for 293T cells) to the cell culture medium to select for doubly transduced cells. Experimentally determine the appropriate concentrations of puromycin and hygromycin for a given cell type prior to beginning the experiments.</li>
	<li>Keep cells in selection media for at least 3 d before any downstream experiments and maintain in selection media for the duration of all experiments.</li>
</ol>
6. Cell Dimerization and Wash Out
<ol>
	<li>Add 1 mM Abscisic Acid (ABA) or an equivalent volume of dimethyl sulfoxide (DMSO) for controls to cell culture to dimerize the CLOuD9 selected and transduced cells. Use ABA within 6 months of date of receipt, keep cold, and protect it from light throughout use. 2 mM ABA can be utilized if needed.
	<ol>
		<li>Change media daily to provide fresh ABA (or DMSO for controls) for the duration of the experiments. 
		NOTE: No limit on the length of dimerization has yet been observed.</li>
	</ol>
	</li>
	<li>Reverse dimerization through the removal of media containing ABA and washing cells with enough phosphate buffered saline (PBS) to cover the surface of the plate, twice. Thereafter, culture cells in ABA-free media to maintain an un-dimerized state.</li>
</ol>
7. Immunoprecipitation and Co-immunoprecipitations
NOTE: Make all buffers fresh and immediately prior to use.
<ol>
	<li>Following completion of dimerization experiments, collect and spin down cells, then aspirate supernatant.</li>
	<li>Crosslink and freeze down cells
	<ol>
		<li>Make a fresh stock of 1% formaldehyde in PBS at room temperature using fresh materials. For every 2 million cells, gently resuspend with 1 mL of 1% formaldehyde at room temperature for exactly 10 min, while rotating. 
		NOTE: Use a minimum volume of 10 mL for crosslinking less than 10 million cells.</li>
		<li>Quench crosslinking with the addition of glycine to a final concentration of 0.125 M, followed by incubation for 5 min at room temperature, while rotating.</li>
		<li>Spin down cells and wash 1-2x with ice-cold PBS. Cell pellets can then be snap frozen in liquid nitrogen and stored at -80 &#176;C or used immediately. 
		NOTE: Heat will reverse formaldehyde crosslinks. If rushed, cells can be stored directly in -80 &#176;C without the snap freeze.</li>
	</ol>
	</li>
	<li>Prepare antibody/bead conjugate 
	Note: Optimizing conditions for chosen antibody (i.e, testing different lysis buffers for cell lysis and performing IP) may be worthwhile. Two common buffers, Farnham lysis buffer and Modified RIPA buffer, can have a significant effect on the IP efficiency and sonication efficiency. All buffer compositions can be found in the Table of Materials. Additionally, keep in mind that chromatin sonication may take several hours to complete.
	<ol>
		<li>Resuspend beads by vortexing briefly as appropriate for the chosen antibody.</li>
		<li>Transfer an appropriate amount of beads for the experiment to a 1.5 mL tube. As a starting point, use 20 &#181;L of beads for every 1 &#181;g of antibody. Do not add antibody yet. 
		NOTE: Use 10 &#181;g antibody with 200 &#181;L of beads for 1 mg of total lysate as a starting point, unless it is known that the antibody is more or less efficient than this would imply. For low abundance proteins, this ratio may need to be adjusted.</li>
		<li>If using Farnham lysis buffer, wash 3x in IP dilution buffer. If using RIPA lysis buffer, wash 3x in RIPA lysis buffer.</li>
		<li>Resuspend beads in a final volume of the same buffer from step 7.3.3 (either IP dilution or RIPA buffer) that is &#62;1x and &#60;5x the initial volume. i.e. For 100 &#181;L initial bead volume, use between 100 and 500 &#181;L final resuspension volume.</li>
		<li>Add antibody to washed beads now at an appropriate concentration. For a good HA or Flag antibody to IP CLOuD9 constructs, use antibodies at 1:50 (&#181;g protein: &#181;g protein lysate) and incubate between 8+ h and overnight at 4 &#176;C, while rotating. Alternatively, incubate for 2-4 h at room temperature.</li>
		<li>Remove the supernatant from beads and discard.</li>
		<li>Wash beads 3x with the Wash buffer.</li>
		<li>Resuspend in a minimal volume of the buffer utilized for overnight incubation with the antibody (IP dilution or RIPA buffer). Keep cold until combined with protein lysate.</li>
	</ol>
	</li>
	<li>Sonicate Chromatin
	<ol>
		<li>First lyse cells with the addition of Swelling buffer to cell pellets on ice for 10 min, flicking cells every few min to prevent settling. 
		NOTE: Generally, 500 &#181;L of Swelling buffer is added to 25 million cells and scaled from there, but do not use less than 500 &#181;L buffer for &#60;25 million cells.</li>
		<li>Dounce manually both clockwise and counterclockwise, 13x each.</li>
		<li>Then spin nuclei down at 4 &#176;C for 5 min at 1,500 x g. Aspirate the supernatant completely and repeat to remove remaining supernatant, then weigh the cell pellet in mg.</li>
		<li>Add protease inhibitor to nuclei lysis buffer (Farnham buffer or RIPA buffer, above, choose one).</li>
		<li>Add 10x amount of nuclei lysis buffer to pelleted nuclei to resuspend (for example, add 1 mL of nuclei lysis buffer to a 0.100 g pellet). Briefly vortex and lyse for 10 min on ice. 
		NOTE: Keep in mind the size of the sonication tube. Ideal volume is often &#60;1 mL, so samples may need to be divided if pellets are very large.
		<ol>
			<li>For Co-IP, sonicate nuclei briefly to solubilize material and quench SDS to a level that permits immunoprecipitation with 2-3 volumes of dilution buffer, then proceed to step 7.4.6. For very sensitive antibodies, add more dilution buffer to further reduce the concentration of SDS. 
			NOTE: Stop sonication when lysate clears; it will change from an opaque/translucent white to completely transparent. Keep samples as cold as possible and do not over sonicate.</li>
			<li>For ChIP, thoroughly sonicate DNA to obtain 150-1000 bp DNA fragments, then proceed to step 7.4.6.</li>
		</ol>
		</li>
		<li>Spin out insoluble material at maximum speed for 10 min at 4 &#176;C.</li>
		<li>Transfer soluble material to a fresh tube.</li>
		<li>For Co-IP, measure the concentration of protein and proceed to the pulldown in step 7.5.</li>
		<li>For ChIP, remove a 10 &#181;L aliquot of cleared lysate and add 40 &#181;L IP elution buffer and 6 &#181;L 5 M NaCl to that for a total of 56ul. Boil for 15 min at 95 &#176;C, add 5-10 &#181;L of 3 M NaOAc pH 5, and clean up on a PCR purification column. Measure the concentration of DNA by measuring the absorbance at 260 nm and standardize across samples. Then proceed to pulldown in step 7.5. 
		NOTE: Extra lysate can be snap frozen at -80 &#176;C and used at a later time, but best results are obtained from the fresh lysate. If freezing, do not freeze/thaw lysate more than once.</li>
	</ol>
	</li>
	<li>Pulldown
	<ol>
		<li>Transfer an appropriate amount of protein lysate to a fresh tube. If using Farnham lysis buffer, dilute in 2.1 volumes of IP dilution buffer (above).</li>
		<li>Set aside 100 &#181;L of supernatant for input control, and store at 4 &#176;C until the next day.</li>
		<li>Add bead/antibody conjugate from step 7.3.8 to samples now.</li>
		<li>Rotate samples at 4 &#176;C overnight and ensure there is enough volume for liquid movement in 1.5 mL tubes.</li>
	</ol>
	</li>
	<li>Wash and Elute
	<ol>
		<li>After overnight rotation, save the supernatant as the nonbinding fraction. Then wash beads 3-5x in IP dilution buffer.</li>
		<li>Prepare IP elution buffer at room temperature, without inhibitors.</li>
		<li>Elute complexes with 50 &#181;L elution buffer shaken on a vortex at 67 &#176;C for 15 min. Transfer elution to a new tube and repeat with another 50 &#181;L. Combine them both for a total 100 &#181;L elution. 
		NOTE: If there is too much background from this elution procedure, heat can be reduced or eliminated during shaking/incubation. This generally reduces the yield of target protein but does significantly decrease the background.</li>
	</ol>
	</li>
	<li>Reverse crosslinks by heating at 67 &#176;C for &#62;4 h. 
	NOTE: This is not strictly necessary for Co-IPs but can be helpful.
	<ol>
		<li>For Co-IP, to ensure full dimerization between the targets of interest, run eluates on an SDS-page gel and probe with antibodies against the HA tag or Flag tag, as indicated, all at 1: 1,000. Then continue to step 8.</li>
		<li>For ChIP-qPCR, to ensure correct localization and targeting of each CRISPR-dCas9 component, clean elution product on a column and elute in 10 &#181;L. Perform real-time quantitative polymerase chain reaction (qPCR) of purified DNA. Primers utilized in the presented data are available in Supplementary Table 1. Then continue to step 8.</li>
	</ol>
	</li>
</ol>
8. RNA Extraction and Quantitative PCR
<ol>
	<li>To investigate the CLOuD9-induced changes in gene expression, first, isolate and purify total RNA from both control cell pellets and dimerized cell pellets.</li>
	<li>Make complementary DNA (cDNA) from the purified RNA by reverse transcription17 and perform qPCR analyses. Primers are available in Supplementary Table 1.</li>
</ol>
9. Chromosome Conformation Capture Assay
<ol>
	<li>To observe changes in the frequency of genomic loci contacts induced by CLOuD9, perform chromosome conformation capture (3C) assays8.</li>
	<li>Quantify 3C ligation products in two sets of duplicates for each of three biological replicates by quantitative real-time PCR. Normalize samples to the 3C signals from the tubulin locus. Primers are available in Supplementary Table 1.</li>
</ol>

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The authors have nothing to disclose.

The most critical steps in CLOuD9 chromatin looping are: 1) Designing or using the correct gRNAs, 2) changing media daily on CLOuD9-transduced cells, including ABA or DMSO, 3) maintaining freshness of ABA, and 4) performing accurate and careful assessments of chromatin conformation.
The limits of CLOuD9 primarily reside in the ability to design guides to the target region of choice. Guide RNAs perform the important task of localizing the dCas9 components to target DNA regions to be dimerized and the efficacy of the guides are based on their specific target site. Without the proper gRNA components, the CLOuD9 system will not be able to form reversibly induced loops. Thus, by designing multiple guides for each region of interest and spreading the guides over a region of 250-1000 bp, at least one successful guide will be ensured. Guide location is also integral to accurate results. It is important to avoid guides located in transcription factor binding sites or other critical regions to prevent background effects such as up or down regulation of transcription. Additionally, the precise location of the CLOuD9 construct can slightly impact transcription of the target gene. This emphasizes the importance of testing multiple pairs of guides for each target region, to identify the most robust pair for experimental purposes. Further, in each pair of target regions, the CSA construct should be targeted with gRNAs for S. aureus, and the CSP construct should be targeted with gRNAs for S. pyogenes for targeting specificity.
To ensure accurate results and correct dimerization, it is also important to maintain the freshness of the cellular environments following the transduction of the CLOuD9 constructs. Daily media change and the addition of fresh dimerizer (or control) ensures that the complementary constructs will remain in proximity and preserve the altered chromatin conformation. Furthermore, guaranteeing the ABA is fresh and has been stored appropriately according to the manufacturer&#39;s protocol (opened within 6 months, kept cold, protected from light) is essential to obtaining authentic results.
Notably, the ABA dimerizer for CLOuD9 was used with the ABI and PYL dimerization proteins, rather than the more commonly utilized FRB and FKBP system. The necessity of a rapalog for the FRB/FKBP system would have limited the applicability of CLOuD9, due to the toxicity to cancer cells. The alternative ABI/PYL system circumvented this limitation, effectively enabling CLOuD9 to be more broadly utilizable.
Collectively, we have developed CLOuD9, a unique and robust technology that can forcibly but reversibly create contacts between long-range target genomic loci. Through inducing chromatin loops, we also demonstrate that CLOuD9 can be utilized to modify gene expression in the appropriate cellular context. The adaptability of the technology allows for the unrestricted study of the interactions between any two genomic loci, without requiring prior knowledge of the looping regions or looping mechanisms. In addition, CLOuD9&#39;s unique demonstrated reversibility enables further examination of the looping mechanisms in disease and development. While the on-target effects of chromatin looping have been demonstrated clearly, there is yet to be data offering insight into the effects of off-target looping and the subsequent impact on the on-target loops.
Our data illustrates only a few applications of this tool but implies the major underlying idea that chromatin arrangement is indicative of gene expression. Our technology can be used to study and reveal the nuances of chromatin structure in gene regulation, thereby improving the overall comprehension of the role of chromatin folding in transcription of genes. A better understanding of the subtleties of transcriptional dynamics can lead the way in research and treatment of cancer, hereditary diseases, and congenital disorders, in which distinct chromatin assembly undoubtedly alters gene expression20,21,22,23. Subsequent work utilizing the CLOuD9 technology will illuminate further details about the arrangement and dynamics of chromatin domains and how they drive folding to sustain stable gene expression in both development and disease.

The relationship between chromatin folding in the nucleus and the specific organization of the genome has garnered significant interest in recent years, as it has been shown to be closely associated with gene expression1,2. While the precise relationship between gene activity and modulation of chromatin structure remains unclear, it has been hypothesized that the interactions between chromosomal contacts as a result of dynamic three-dimensional chromatin organization serve a gene regulatory function3. Indeed, such an effect has been well demonstrated at the human globin gene locus, where the locus control region (LCR) regulates the activity of the globin genes in a developmentally specific manner by creating a chromatin loop between the two regions4. However, in both this and other regions, it is unclear whether chromatin looping is a cause or consequence of alterations in gene expression.
Until now, the challenges in studying this phenomenon remained unresolved. For example, other attempts at inducing chromatin loops involved modifying the linear DNA sequence or complicated procedures requiring an abundance of background knowledge on specific elements that facilitate looping5,6,7,8. Additionally, while previous work has suggested that chromatin loops drive gene expression in a specific and restricted context7,8, the level at which chromatin looping affects transcription globally is uncertain. Though interest in the impact of long-range looping on gene expression has grown continually in recent years, unanswered questions about establishing and retaining chromatin contacts to change gene activity persist.
The technology that we have engineered employs the nuclease deficient clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (dCas9), to allow for the broadly applicable targeting of any genomic loci9. This technology eliminates the complex issues related to modifications of the linear DNA sequence and is accessible without significant prior knowledge of particular looping components. Most notably, the tool is universal and broadly applicable to chromatin loops recognized in development as well as in a variety of diseases, such as cancer. The power of CLOuD9 is demonstrated by reversibly altering the structure of loops to effectively modulate gene expression.

<table><tbody><tr><td>RPMI 1640&amp;nbsp; media</td><td>Life Technologies</td><td>11875-119</td><td>For K562 cell culture</td></tr><tr><td>DMEM media</td><td>Life Technologies</td><td>11995-065</td><td>1X, for 293T cell culture</td></tr><tr><td>lentiCRISPR v2</td><td>Addgene plasmid</td><td>#52961</td><td>For CLOuD9 plasmid development</td></tr><tr><td>pRSV-Rev</td><td>Addgene plasmid</td><td>#12253</td><td>For lentivirus production</td></tr><tr><td>pMD2.G</td><td>Addgene plasmid</td><td>#12259</td><td>For lentivirus production</td></tr><tr><td>pMDLg/pRRE</td><td>Addgene plasmid</td><td>#12251</td><td>For lentivirus production</td></tr><tr><td>Lipofectamine 2000</td><td>Thermo Fisher Scientific</td><td>11668-019</td><td>For lentivirus production</td></tr><tr><td>anti-HA antibody</td><td>Cell Signaling</td><td>3724</td><td>For immunoprecipitation</td></tr><tr><td>anti-Flag antibody</td><td>Sigma</td><td>F1804</td><td>For immunoprecipitation</td></tr><tr><td>DNeasy Blood and Tissue Kit</td><td>Qiagen</td><td>69504</td><td>For DNA extraction</td></tr><tr><td>TRIzol</td><td>Life Technologies</td><td>15596-018</td><td>For RNA extraction</td></tr><tr><td>RNeasy Kit</td><td>Qiagen</td><td>74106</td><td>For RNA extraction</td></tr><tr><td>Superscript VILO</td><td>Life Technologies</td><td>11754-050</td><td>For cDNA&amp;nbsp;</td></tr><tr><td>SYBR Green I MasterMix</td><td>Roche</td><td>4707516001</td><td>For qPCR analysis</td></tr><tr><td>Light Cycler 480II</td><td>Roche</td><td></td><td>For qPCR analysis</td></tr><tr><td>anti-H3K4me3 antibody</td><td>AbCam</td><td>ab8580</td><td>For ChIP-qPCR</td></tr><tr><td>anti-RNA Pol-II antibody</td><td>Active Motif</td><td>61083</td><td>For ChIP-qPCR</td></tr><tr><td>EDTA free protease inhibitor</td><td>Roche</td><td>11873580001</td><td>For protein extraction</td></tr><tr><td>4-12% Tris Glycine gel</td><td>Biorad</td><td></td><td>Any size, For western blot</td></tr><tr><td>anti-Rabbit HRP antibody</td><td>Santa Cruz</td><td>sc-2030</td><td>For western blot</td></tr><tr><td>anti-mouse HRP antibody</td><td>Cell Signaling</td><td>7076S</td><td>For western blot</td></tr><tr><td>K562 and H3K293 ChIP-Seq data</td><td>Encode</td><td>ENCSR000AKU</td><td>For ChIP-seq analysis</td></tr><tr><td>K562 and H3K293 ChIP-Seq data</td><td>Encode</td><td>ENCSR000APE</td><td>For ChIP-seq analysis</td></tr><tr><td>K562 and H3K293 ChIP-Seq data</td><td>Encode</td><td>ENCSR000FCJ</td><td>For ChIP-seq analysis</td></tr><tr><td>K562 and H3K293 ChIP-Seq data</td><td>GEO</td><td>GSM1479215</td><td>For ChIP-seq analysis</td></tr><tr><td>Dynabeads Protein A for Immunoprecipitation</td><td>Thermo Fisher Scientific</td><td>10001D</td><td>For immunoprecipitation</td></tr><tr><td>Dynabeads Protein G for Immunoprecipitation</td><td>Thermo Fisher Scientific</td><td>10004D</td><td>For immunoprecipitation</td></tr><tr><td>RNA Clean &amp;amp; Concentrator-5</td><td>Zymo Research</td><td>R1015</td><td>For RNA purification</td></tr><tr><td>Pierce 16% Formaldehyde Methanol-free</td><td>Thermo Fisher Scientific</td><td>28908</td><td>For crosslinking</td></tr><tr><td>PX458 Plasmid</td><td>Addgene</td><td>48138</td><td>Suggested active Cas9 plasmid for gRNA cloning, but any active Cas9 plasmid will do</td></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td>For PCR purification</td></tr><tr><td>FastDigest BsmBI</td><td>Thermo Fisher Scientific</td><td>FD0454</td><td>For cloning guide RNAs</td></tr><tr><td>FastAP</td><td>Thermo Fisher Scientific</td><td>EF0651</td><td>For cloning guide RNAs</td></tr><tr><td>10X FastDigest Buffer</td><td>Thermo Fisher Scientific</td><td>B64</td><td>For cloning guide RNAs</td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td>For cloning guide RNAs</td></tr><tr><td>10X T4 Ligation Buffer</td><td>NEB</td><td>B0202S</td><td>For cloning guide RNAs</td></tr><tr><td>T4 PNK</td><td>NEB</td><td>M0201S</td><td>For cloning guide RNAs</td></tr><tr><td>2X Quick Ligase Buffer</td><td>NEB</td><td>B2200S</td><td>For cloning guide RNAs</td></tr><tr><td>Quick Ligase</td><td>NEB</td><td>M2200S</td><td>For cloning guide RNAs</td></tr><tr><td>&lt;strong&gt;Buffers&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Farnham lysis buffer</td><td></td><td></td><td>1% Tris-Cl pH 8.0, 1% SDS, 1% protease inhibitor water solution (non-EDTA), and 1 mM EDTA in water</td></tr><tr><td>Modified RIPA buffer</td><td></td><td></td><td>1% NP40/Igepal, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, and 1% protease inhibitor water solution (non-EDTA) in PBS pH 7.8 or 7.4</td></tr><tr><td>IP dilution buffer</td><td></td><td></td><td>0.01% SDS, 1.1% Triton-X 100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.0, 167 mM NaCl, 0.1x protease inhibitor</td></tr><tr><td>Wash buffer</td><td></td><td></td><td>100 mM Tris pH 9, 100 mM LiCl, 1% NP-40, and 1% sodium deoxycholate</td></tr><tr><td>Swelling buffer</td><td></td><td></td><td>0.1 M Tris pH 7.5, 10 mM potassium acetate, 15 mM magnesium acetate, 1% NP-40</td></tr><tr><td>Dilution buffer</td><td></td><td></td><td>0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris pH 8 and 167 mM NaCl</td></tr><tr><td>IP elution buffer</td><td></td><td></td><td>1% SDS, 10% NaHCO&lt;sub&gt;3 &lt;/sub&gt;&amp;nbsp;</td></tr></tbody></table>

crispr-mediated reorganization of chromatin loop structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Watch this Scientific Journal Video about CRISPR-Mediated Reorganization of Chromatin Loop Structure at JoVE.com

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

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12.5K Views.  Stanford University School of Medicine. Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

Video: CRISPR-Mediated Reorganization of Chromatin Loop Structure

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

A Method to Study de novo Formation of Chromatin Domains

The organization and structure of chromatin domains are unique to individual cell lineages. Their misregulation might lead to a loss in cellular identity and/or disease. Despite tremendous efforts, our understanding of the formation and propagation of chromatin domains is still limited. Chromatin domains have been studied under steady-state conditions, which are not conducive to following the initial events during their establishment. Here, we present a method to inducibly reconstruct chromatin domains and follow their re-formation as a function of time. Although, first applied to the case of PRC2-mediated repressive chromatin domain formation, it could be easily adapted to other chromatin domains. The modification of and/or the combination of this method with genomics and imaging technologies will provide invaluable tools to study the establishment of chromatin domains in great detail. We believe that this method will revolutionize our understanding of how chromatin domains form and interact with each other.

A Method to Study de novo Formation of Chromatin Domains

This method is designed to follow formation of PRC2-mediated chromatin domains in cell lines, and the method can be adapted to many other systems.

The organization and structure of chromatin domains are unique to individual cell lineages. Their misregulation might lead to a loss in cellular identity ...

R-Loop Analysis by Dot-Blot

The three-stranded nucleic acid structure, R-loop, is increasingly recognized for its role in gene regulation. Initially, R-loops were thought to be the by-products of transcription; but recent findings of fewer R-loops in diseased cells made it clear that R-loops have functional roles in a variety of human cells. Next, it is critical to understand the roles of R-loops and how cells balance their abundance. A challenge in the field is the quantitation of R-loops since much of the work relies on the S9.6 monoclonal antibody whose specificity for RNA-DNA hybrids has been questioned. Here, we use dot-blots with the S9.6 antibody to quantify R-loops and show the sensitivity and specificity of this assay with RNase H, RNase T1, and RNase III that cleave RNA-DNA hybrids, single-stranded RNA, and double-stranded RNA, respectively. This method is highly reproducible, uses general laboratory equipment and reagents, and provides results within two days. This assay can be used in research and clinical settings to quantify R-loops and assess the effect of mutations in genes such as senataxin on R-loop abundance.

This protocol details a simple method that quantifies R-loop, a three-stranded nucleic acid structure that comprises of an RNA-DNA hybrid and a displaced DNA strand.

The three-stranded nucleic acid structure, R-loop, is increasingly recognized for its role in gene regulation. Initially, R-loops were thought to be the ...

In-Nucleus Hi-C in Drosophila Cells

The genome is organized into topologically associating domains (TADs) delimited by boundaries that isolate interactions between domains. In Drosophila, the mechanisms underlying TAD formation and boundaries are still under investigation. The application of the in-nucleus Hi-C method described here helped to dissect the function of architectural protein (AP)-binding sites at TAD boundaries isolating the Notch gene. Genetic modification of domain boundaries that cause loss of APs results in TAD fusion, transcriptional defects, and long-range topological alterations. These results provided evidence demonstrating the contribution of genetic elements to domain boundary formation and gene expression control in Drosophila. Here, the in-nucleus Hi-C method has been described in detail, which provides important checkpoints to assess the quality of the experiment along with the protocol. Also shown are the required numbers of sequencing reads and valid Hi-C pairs to analyze genomic interactions at different genomic scales. CRISPR/Cas9-mediated genetic editing of regulatory elements and high-resolution profiling of genomic interactions using this in-nucleus Hi-C protocol could be a powerful combination for the investigation of the structural function of genetic elements.

The genome is organized in the nuclear space into different structures that can be revealed through chromosome conformation capture technologies. The in-nucleus Hi-C method provides a genome-wide collection of chromatin interactions in Drosophila cell lines, which generates contact maps that can be explored at megabase resolution at restriction fragment level.

The genome is organized into topologically associating domains (TADs) delimited by boundaries that isolate interactions between domains. In Drosophila, ...

Mapping R-Loops and RNA:DNA Hybrids with S9.6-Based Immunoprecipitation Methods

R-loops constitute a prevalent class of transcription-driven non-B DNA structures that occur in all genomes depending on both DNA sequence and topological favorability. In recent years, R-loops have been implicated in a variety of adaptive and maladaptive roles and have been linked to genomic instability in the context of human disorders. As a consequence, the accurate mapping of these structures in genomes is of high interest to many investigators. DRIP-seq (DNA:RNA Immunoprecipitation followed by high throughput sequencing) is described here. It is a robust and reproducible technique that permits accurate and semi-quantitative mapping of R-loops. A recent iteration of the method is also described in which fragmentation is accomplished using sonication (sDRIP-seq), which allows strand-specific and high-resolution mapping of R-loops. sDRIP-seq thus addresses some of the common limitations of the DRIP-seq method in terms of resolution and strandedness, making it a method of choice for R-loop mapping.

R-loops constitute a prevalent class of transcription-driven non-B DNA structures that occur in all genomes depending on both DNA sequence and topological ...

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in response to environmental stimuli. These changes are essential for genomic integrity, epigenetic regulation, and DNA metabolic reactions such as replication, transcription, and repair. Chromatin assembly is crucial for chromatin dynamics and is catalyzed by histone chaperones. Despite extensive studies, the mechanisms by which histone chaperones enable chromatin assembly remains elusive. Moreover, the global features of nucleosomes organized by histone chaperones are poorly understood. To address these problems, this work describes a unique single-molecule imaging technique named DNA curtain, which facilitates the investigation of the molecular details of nucleosome assembly by histone chaperones. DNA curtain is a hybrid technique that combines lipid fluidity, microfluidics, and total internal reflection fluorescence microscopy (TIRFM) to provide a universal platform for real-time imaging of diverse protein-DNA interactions.Using DNA curtain, the histone chaperone function of Abo1, the Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase, is investigated, and the molecular mechanism underlying histone assembly of Abo1 is revealed. DNA curtain provides a unique approach for studying chromatin dynamics.

DNA curtain, a high-throughput single-molecule imaging technique, provides a platform for real-time visualization of diverse protein-DNA interactions. The present protocol utilizes the DNA curtain technique to investigate the biological role and molecular mechanism of Abo1, a Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase.

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in ...

Biochemistry

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Bioengineering

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

Biology

Chromatin Extraction from Frozen Chimeric Liver Tissue for Chromatin Immunoprecipitation Analysis

Crosslinking Chromatin Immunoprecipitation (X-ChIP) is a widely used technique to assess levels of histone marks and occupancy of transcription factors on host and/or pathogen chromatin. Chromatin preparation from tissues creates additional challenges that need to be overcome to obtain reproducible and reliable protocols comparable to those used for cell culture. Tissue disruption and fixation are critical steps to achieve efficient shearing of chromatin. Coexistence of different cell types and clusters may also require different shearing times to reach optimal fragment size and hinders shearing reproducibility. The purpose of this method is to achieve reliable and reproducible host chromatin preparations from frozen tissue (liver) suitable for both ChIP-qPCR and next generation sequencing (NGS) applications. We observed that the combination of liquid nitrogen tissue pulverization followed by homogenization leads to increased reproducibility compared to homogenization only, since it provides a suspension consisting mostly of dissociated single cells that can be efficiently sheared. Moreover, the fixation step should be performed under mild rotation to provide homogeneous crosslinking. The fixed material is then suitable for buffer-based nuclei isolation, to reduce contamination of cytoplasmic protein and pathogen DNAs and RNAs (when applicable), avoiding time-consuming centrifugation gradients. Subsequent sonication will complete nuclear lysis and shear the chromatin, producing a specific size range according to the chosen shearing conditions. The size range should fall between 100 and 300 nt for NGS applications, while it could be higher (300-700 nt) for ChIP-qPCR analysis. Such protocol adaptations can greatly improve chromatin analyses from frozen tissue specimens.

This protocol focuses on chromatin preparation from snap frozen tissues and it is suitable for Crosslinking Chromatin Immunoprecipitation (X-ChIP) followed by either quantitative PCR analysis (X-ChIP-qPCR) or next generation sequencing approaches (X-ChIP-seq).

Crosslinking Chromatin Immunoprecipitation (X-ChIP) is a widely used technique to assess levels of histone marks and occupancy of transcription factors on ...

Duplication of Chromatin Structure

The process of chromosome duplication during cell division requires genome-wide disruption and re-assembly of chromatin. The chromatin structure must be accurately inherited, reassembled, and maintained in the daughter cells to ensure lineage propagation.
The basic unit of the chromatin is the nucleosome, consisting of DNA wrapped around octameric histone proteins and short stretches of linker DNA separating individual nucleosomes. The histone proteins within the nucleosome have their N-terminal ends protruding out of the core, providing sites for various covalent modifications that regulate chromatin structure and function.
During replication, as the DNA unwinds, parental nucleosomes are disrupted, and histone proteins are released. As replication progresses and daughter strands form, the parental histones and the additional histone proteins synthesized during S-phase are assembled, allowing the formation of nucleosomes.
The post translational modifications of the histones and other epigenetic domains in the DNA are also faithfully reproduced in the daughter genome.
<h4>Chromatin structure influences gene expression</h4>
Within a cell, the major portion of the genome remains inaccessible to transcription factors, as the regulatory and coding DNA sequences exist mostly concealed within the nucleosomes. For a gene to express, it is necessary to create accessible sites for transcription factors to bind and also to modify the histones to reorganize the chromatin structure and create an environment permissive for transcription.
Specific regulatory factor complexes are involved in opening up localized regions of the chromatin by displacement or disruption of nucleosomes. Post-translational modifications of the histone tail serve to maintain either an active or inactive transcriptional state. Specific modifications at the histone tail can ease the level of DNA compaction, facilitating the destabilization and displacement of nucleosomes, providing access for transcription machinery to influence gene expression.

The process of chromosome duplication during cell division requires genome-wide disruption and re-assembly of chromatin. The chromatin structure must be ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Summary

Explore More Videos

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

A Method to Study de novo Formation of Chromatin Domains

R-Loop Analysis by Dot-Blot

In-Nucleus Hi-C in Drosophila Cells

Mapping R-Loops and RNA:DNA Hybrids with S9.6-Based Immunoprecipitation Methods

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Extraction from Frozen Chimeric Liver Tissue for Chromatin Immunoprecipitation Analysis

Duplication of Chromatin Structure