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<p><small> <strong>Figure 1. ChIP using the Diagenode monoclonal antibody directed against Cas9</strong><br />ChIP was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50 μg chromatin was incubated overnight at 4°C with 5 or 10 μg of either an anti-FLAG antibody or the Diagenode antibody against Cas9 (Cat. No. C15200229). Mouse IgG was used as a negative IP control. qPCR was performed with primers specific for the GFP gene, and for a non-targeted region (protein kinase C delta, Prkcd), used as negative control. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).</small></p>
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<p><small><strong>Figure 2. Western blot analysis using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />Figure 1A: Western blot was performed on protein extracts from HeLa cells transfected with Cas9 using the Diagenode antibody against CRISPR/Cas9 (Cat. No. C15200229). The antibody was used at different dilutions. The marker is shown on the left, position of the Cas9 protein is indicated on the right. Figure 1B: Western blot was performed on protein extracts from HeLa cells transfected with Cas9 (lane 1) or from untransfected cells (lane 2) using the Diagenode antibody against CRISPR/Cas9 (Cat. No. C15200229), diluted 1:4,000 in PBS-T containing 3% NFDM. The marker is shown on the left, position of the Cas9 protein is indicated on the right.</small></p>
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<p><small> <strong>Figure 3. IP using the Diagenode monoclonal antibody directed against Cas9</strong> <br /> IP was performed on whole cell extracts (300 μg) from HEK293 cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 6 μg of the Diagenode antibody against Cas9 (Cat. No. C15200229). The immunoprecipitated proteins were subsequently analysed by Western blot with the polyclonal Cas9 antibody (Cat. No. C15310258, diluted 1:8,000). Lane 3 and 4 show the result of the IP, the input (15 μg) is shown in lane 1 and 2.</small></p>
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<p><small> <strong>Figure 4. Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong> <br /> HeLa cells transfected with a Cas9 expression vector (left) or untransfected cells (right) were fixed in methanol at -20°C, permeabilized with acetone at -20°C and blocked with PBS containing 2% BSA. The cells were stained with the Cas9 N-terminal antibody (Cat. No. C15200229) diluted 1:400, followed by incubation with an anti-mouse secondary antibody coupled to AF488. The bottom images show counter-staining of the nuclei with Hoechst 33342.</small></p>
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<p><small><strong>Figure 2. Western blot analysis using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />Figure 1A: Western blot was performed on protein extracts from HeLa cells transfected with Cas9 using the Diagenode antibody against CRISPR/Cas9 (Cat. No. C15200229). The antibody was used at different dilutions. The marker is shown on the left, position of the Cas9 protein is indicated on the right. Figure 1B: Western blot was performed on protein extracts from HeLa cells transfected with Cas9 (lane 1) or from untransfected cells (lane 2) using the Diagenode antibody against CRISPR/Cas9 (Cat. No. C15200229), diluted 1:4,000 in PBS-T containing 3% NFDM. The marker is shown on the left, position of the Cas9 protein is indicated on the right.</small></p>
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<p><small> <strong>Figure 3. IP using the Diagenode monoclonal antibody directed against Cas9</strong> <br /> IP was performed on whole cell extracts (300 μg) from HEK293 cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 6 μg of the Diagenode antibody against Cas9 (Cat. No. C15200229). The immunoprecipitated proteins were subsequently analysed by Western blot with the polyclonal Cas9 antibody (Cat. No. C15310258, diluted 1:8,000). Lane 3 and 4 show the result of the IP, the input (15 μg) is shown in lane 1 and 2.</small></p>
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<p><small><strong>Figure 2. Western blot analysis using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />Figure 1A: Western blot was performed on protein extracts from HeLa cells transfected with Cas9 using the Diagenode antibody against CRISPR/Cas9 (Cat. No. C15200229). The antibody was used at different dilutions. The marker is shown on the left, position of the Cas9 protein is indicated on the right. Figure 1B: Western blot was performed on protein extracts from HeLa cells transfected with Cas9 (lane 1) or from untransfected cells (lane 2) using the Diagenode antibody against CRISPR/Cas9 (Cat. No. C15200229), diluted 1:4,000 in PBS-T containing 3% NFDM. The marker is shown on the left, position of the Cas9 protein is indicated on the right.</small></p>
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<p><small> <strong>Figure 3. IP using the Diagenode monoclonal antibody directed against Cas9</strong> <br /> IP was performed on whole cell extracts (300 μg) from HEK293 cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 6 μg of the Diagenode antibody against Cas9 (Cat. No. C15200229). The immunoprecipitated proteins were subsequently analysed by Western blot with the polyclonal Cas9 antibody (Cat. No. C15310258, diluted 1:8,000). Lane 3 and 4 show the result of the IP, the input (15 μg) is shown in lane 1 and 2.</small></p>
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<p><small> <strong>Figure 4. Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong> <br /> HeLa cells transfected with a Cas9 expression vector (left) or untransfected cells (right) were fixed in methanol at -20°C, permeabilized with acetone at -20°C and blocked with PBS containing 2% BSA. The cells were stained with the Cas9 N-terminal antibody (Cat. No. C15200229) diluted 1:400, followed by incubation with an anti-mouse secondary antibody coupled to AF488. The bottom images show counter-staining of the nuclei with Hoechst 33342.</small></p>
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'description' => '<p><strong>Western blot</strong> : The quality of antibodies used in this technique is crucial for correct and specific protein identification. Diagenode offers huge selection of highly sensitive and specific western blot-validated antibodies.</p>
<p>Learn more about: <a href="https://www.diagenode.com/applications/western-blot">Loading control, MW marker visualization</a><em>. <br /></em></p>
<p><em></em>Check our selection of antibodies validated in Western blot.</p>',
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<p>Diagenode offers huge selection of highly sensitive antibodies validated in IF.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15200229-IF.jpg" alt="" height="245" width="256" /></p>
<p><sup><strong>Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong></sup></p>
<p><sup>HeLa cells transfected with a Cas9 expression vector (left) or untransfected cells (right) were fixed in methanol at -20°C, permeabilized with acetone at -20°C and blocked with PBS containing 2% BSA. The cells were stained with the Cas9 C-terminal antibody (Cat. No. C15200229) diluted 1:400, followed by incubation with an anti-mouse secondary antibody coupled to AF488. The bottom images show counter-staining of the nuclei with Hoechst 33342.</sup></p>
<h5><sup>Check our selection of antibodies validated in IF.</sup></h5>',
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'description' => '<p><span style="font-weight: 400;">All Diagenode’s antibodies are listed below. Please, use our Quick search field to find the antibody of interest by target name, application, purity.</span></p>
<p><span style="font-weight: 400;">Diagenode’s highly validated antibodies:</span></p>
<ul>
<li>Highly sensitive and specific</li>
<li>Cost-effective (requires less antibody per reaction)</li>
<li>Batch-specific data is available on the website</li>
<li>Expert technical support</li>
<li>Sample sizes available</li>
<li>100% satisfaction guarantee</li>
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'name' => '<em>S. pyogenes</EM> CRISPR/Cas9 antibodies',
'description' => '<p>Diagenode offers the broad range of antibodies raised against the N- or C-terminus of the Cas9 nuclease from <em>Streptococcus <g class="gr_ gr_5 gr-alert gr_spell gr_disable_anim_appear ContextualSpelling ins-del multiReplace" id="5" data-gr-id="5">pyogenes</g></em>. These highly specific polyclonal and monoclonal antibodies are validated in Western blot, immunoprecipitation, immunofluorescence and in chromatin immunoprecipitation.</p>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h2><em><a name="pyogenes"></a>S. pyogenes</em> CRISPR/Cas9 antibodies<a></a></h2>
<div class="panel">
<h2>Discover our first monoclonal CRISPR/Cas9 antibody validated in ChIP<br /><br /></h2>
<div class="row">
<div class="small-5 medium-5 large-5 columns"><img src="/img/landing-pages/crispr-cas9-chip-on-hih3t3.jpg" alt="" /></div>
<div class="small-7 medium-7 large-7 columns">
<ul>
<li>Validated in chromatin immunoprecipitation</li>
<li>Performs better than FLAG antibody</li>
<li>Excellent for WB, IF and IP</li>
</ul>
<p><small><strong>ChIP</strong> was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50 µg chromatin was incubated overnight at 4°C with 5 or 10 µg of either an anti-FLAG antibody or the Diagenode antibody against Cas9 (Cat. No. C15200229). Mouse IgG was used as a negative IP control. qPCR was performed with primers specific for the GFP gene, and for a non-targeted region (protein kinase C delta, Prkcd), used as negative control. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).</small></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns text-right"><a href="/p/crispr-cas9-monoclonal-antibody-50-ug-25-μl" class="tiny details button radius">Learn more</a></div>
</div>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>First ChIP-grade CRISPR/Cas9 polyclonal antibody</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/landing-pages/c_a_s9-chip-grade-antibody.png" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Excellent polyclonal antibody for chromatin immunoprecipitation</li>
<li>Optimized for highest ChIP specificity and yields</li>
<li>Validated for all applications including immunoblotting, immunofluorescence and western blot</li>
</ul>
<p><small><strong>ChIP</strong> was performed on NIH3T3 cells stably expressing GFP- H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50 μg chromatin was incubated with either 5 μg of an anti-FLAG antibody or 2 μl of the Diagenode antibody against Cas9. The pre-immune serum (PPI) was used as negative IP control. Then qPCR was performed with primers specific for the GFP gene, and for two non-targeted regions: Ppap2c and Prkcd, used as negative controls. This figure shows the recovery, expressed as a % of input.</small></p>
<p class="text-right"><a href="../p/crispr-cas9-polyclonal-antibody" class="details tiny button">Learn more</a></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>CRISPR/Cas9 monoclonal antibody 4G10</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/landing-pages/cas9_4g10_fig1.png" width="170" height="302" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Antibody raised against N-terminus of Cas9 nuclease</li>
<li>Validated for western blot, IP and immunofluorescence</li>
</ul>
<p><small><strong>Immunofluorescence</strong>: Hela cells were transiently transfected with a Cas9 expression vector. The cells were fixed in 3.7% formaldehyde, permeabilized in 0.5% Triton-X-100 and blocked in PBS containing 2% BSA. The cells were stained with the Cas9 antibody at 4°C o/n, followed by incubation with an anti mouse secondary antibody coupled to AF488 for 1 h at RT. Nuclei were counter-stained with Hoechst 33342 (right).</small></p>
<p class="text-right"><a href="../p/crispr-cas9-monoclonal-antibody-4g10-50-ug" class="details tiny button">Learn more</a></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>CRISPR/Cas9 C-terminal monoclonal antibody</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/product/antibodies/C15200223-IP.png" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Antibody raised against C-terminus of Cas9 nuclease</li>
<li>Validated for western blot, IP and immunofluorescence</li>
</ul>
<p><small><strong> Western blot</strong> was performed on 20 μg protein extracts from Cas9 expressing HeLa cells (lane 1) and on negative control HeLa cells (lane 2) with the Diagenode antibody against Cas9. The antibody was diluted 1:4,000. The marker is shown on the left, position of the Cas9 protein is indicated on the right. </small></p>
<p class="text-right"><a href="../p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug" class="details tiny button">Learn more</a></p>
</div>
<div class="small-12 medium-12 large-12 columns">
<h3>Which CRISPR/Cas9 antibody is the best for your application?</h3>
<a name="table"></a>
<table>
<thead>
<tr>
<th>Antibody</th>
<th>WB</th>
<th>IF</th>
<th>IP</th>
<th>ChIP</th>
<th>Antibody raised against</th>
</tr>
</thead>
<tbody>
<tr>
<td><a href="../p/crispr-cas9-monoclonal-antibody-50-ug-25-μl"><strong class="diacol">CRISPR/Cas9 monoclonal antibody</strong></a></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><span class="diacol">N-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-polyclonal-antibody">CRISPR/Cas9 polyclonal antibody</a></td>
<td>++</td>
<td>++</td>
<td>++</td>
<td><strong class="diacol">+++</strong></td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-monoclonal-antibody-4g10-50-ug">CRISPR/Cas9 monoclonal antibody 4G10</a></td>
<td>+++</td>
<td>+++</td>
<td>++</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="../p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug-23-ul"><strong class="diacol">CRISPR/Cas9 C-terminal monoclonal antibody</strong></a> <span class="label alert" style="font-size: 0.9rem;">NEW!</span></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">no</strong></td>
<td><strong class="diacol">+</strong></td>
<td><span class="diacol">C-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug">CRISPR/Cas9 C-terminal monoclonal antibody</a></td>
<td>++</td>
<td>++</td>
<td>+</td>
<td>no</td>
<td><span class="diacol">C-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-monoclonal-antibody-7A9-50-mg">CRISPR/Cas9 monoclonal antibody 7A9</a></td>
<td>++</td>
<td>++</td>
<td>++</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-hrp-monoclonal-antibody-50-ul">CRISPR/Cas9 - HRP monoclonal antibody 7A9</a></td>
<td>+++</td>
<td>no</td>
<td>no</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
</tbody>
</table>
</div>
</div>
</div>',
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'name' => 'ChIP-grade antibodies',
'description' => '<div class="row">
<div class="small-12 columns"><center></center>
<p><br />Chromatin immunoprecipitation (<b>ChIP</b>) is a technique to study the associations of proteins with the specific genomic regions in intact cells. One of the most important steps of this protocol is the immunoprecipitation of targeted protein using the antibody specifically recognizing it. The quality of antibodies used in ChIP is essential for the success of the experiment. Diagenode offers extensively validated ChIP-grade antibodies, confirmed for their specificity, and high level of performance in ChIP. Each batch is validated, and batch-specific data are available on the website.</p>
<p></p>
</div>
</div>
<p><strong>ChIP results</strong> obtained with the antibody directed against H3K4me3 (Cat. No. <a href="../p/h3k4me3-polyclonal-antibody-premium-50-ug-50-ul">C15410003</a>). </p>
<div class="row">
<div class="small-12 medium-6 large-6 columns"><img src="https://www.diagenode.com/img/product/antibodies/C15410003-fig1-ChIP.jpg" alt="" width="400" height="315" /> </div>
<div class="small-12 medium-6 large-6 columns">
<p></p>
<p></p>
<p></p>
</div>
</div>
<p></p>
<p>Our aim at Diagenode is to offer the largest collection of highly specific <strong>ChIP-grade antibodies</strong>. We add new antibodies monthly. Find your ChIP-grade antibody in the list below and check more information about tested applications, extensive validation data, and product information.</p>',
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'meta_description' => 'Diagenode Offers Extensively Validated ChIP-Grade Antibodies, Confirmed for their Specificity, and high level of Performance in Chromatin Immunoprecipitation ChIP',
'meta_title' => 'Chromatin immunoprecipitation ChIP-grade antibodies | Diagenode',
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'description' => '<p style="text-align: justify;"><span>Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of large sets of genome-wide sequencing data. ChIP sequencing (ChIP-seq) has now become the gold standard method for chromatin studies, given the accuracy and coverage scale of the approach over other methods. Successful ChIP-seq, however, requires a higher level of experimental accuracy and consistency in all steps of ChIP than ever before. Particularly crucial is the quality of ChIP antibodies. </span></p>',
'image_id' => null,
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(int) 1 => array(
'id' => '38',
'name' => 'Epigenetic Antibodies Brochure',
'description' => '<p>More than in any other immuoprecipitation assays, quality antibodies are critical tools in many epigenetics experiments. Since 10 years, Diagenode has developed the most stringent quality production available on the market for antibodies exclusively focused on epigenetic uses. All our antibodies have been qualified to work in epigenetic applications.</p>',
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'id' => '864',
'name' => 'CRISPR/Cas9 monoclonal antibody C15200229',
'description' => '<p>CRISPR/Cas9 monoclonal antibody datasheet</p>',
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'type' => 'Datasheet',
'url' => 'files/products/antibodies/C15200229-CRISPR-Cas9-4G10.pdf',
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(int) 3 => array(
'id' => '991',
'name' => ' Accurate QC to optimize CRISPR/Cas9 genome editing specificity',
'description' => '<p>The CRISPR/Cas9 technology is delivering superior genetic models for fundamental disease research, drug screening, therapy development, rapid diagnostics, and transcriptional modulation. Although CRISPR/Cas9 enables rapid genome editing, several aspects affect its efficiency and specificity including guide RNA design, delivery methods, and off-targets effects. Diagenode has developed strategies to overcome these common pitfalls and has optimized CRISPR/Cas9 genome editing specificity</p>',
'image_id' => null,
'type' => 'Poster',
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(int) 0 => array(
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'name' => 'product/antibodies/cas9-icon.png',
'alt' => 'CRISPR/Cas9 Antibody ',
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'name' => 'Genome-wide CRISPR screen identifies protein pathways modulating tauprotein levels in neurons',
'authors' => 'Sanchez C. G. et al. ',
'description' => '<p>Aggregates of hyperphosphorylated tau protein are a pathological hallmark of more than 20 distinct neurodegenerative diseases, including Alzheimer’s disease, progressive supranuclear palsy, and frontotemporal dementia. While the exact mechanism of tau aggregation is unknown, the accumulation of aggregates correlates with disease progression. Here we report a genome-wide CRISPR screen to identify modulators of endogenous tau protein for the first time. Primary screens performed in SH-SY5Y cells, identified positive and negative regulators of tau protein levels. Hit validation of the top 43 candidate genes was performed using Ngn2-induced human cortical excitatory neurons. Using this approach, genes and pathways involved in modulation of endogenous tau levels were identified, including chromatin modifying enzymes, neddylation and ubiquitin pathway members, and components of the mTOR pathway. TSC1, a critical component of the mTOR pathway, was further validated in vivo, demonstrating the relevance of this screening strategy. These findings may have implications for treating neurodegenerative diseases in the future.</p>',
'date' => '2021-06-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/34127790',
'doi' => '10.1038/s42003-021-02272-1',
'modified' => '2022-08-02 17:02:10',
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(int) 1 => array(
'id' => '4141',
'name' => 'Transgenic mice for in vivo epigenome editing with CRISPR-based systems',
'authors' => 'Gemberling, M. et al.',
'description' => '<p>The discovery, characterization, and adaptation of the RNA-guided clustered regularly interspersed short palindromic repeat (CRISPR)-Cas9 system has greatly increased the ease with which genome and epigenome editing can be performed. Fusion of chromatin-modifying domains to the nuclease-deactivated form of Cas9 (dCas9) has enabled targeted gene activation or repression in both cultured cells and in vivo in animal models. However, delivery of the large dCas9 fusion proteins to target cell types and tissues is an obstacle to widespread adoption of these tools for in vivo studies. Here we describe the generation and validation of two conditional transgenic mouse lines for targeted gene regulation, Rosa26:LSL-dCas9-p300 for gene activation and Rosa26:LSL-dCas9-KRAB for gene repression. Using the dCas9p300 and dCas9KRAB transgenic mice we demonstrate activation or repression of genes in both the brain and liver in vivo, and T cells and fibroblasts ex vivo. We show gene regulation and targeted epigenetic modification with gRNAs targeting either transcriptional start sites (TSS) or distal enhancer elements, as well as corresponding changes to downstream phenotypes. These mouse lines are convenient and valuable tools for facile, temporally controlled, and tissue-restricted epigenome editing and manipulation of gene expression in vivo.</p>',
'date' => '2021-03-01',
'pmid' => 'https://doi.org/10.1101%2F2021.03.08.434430',
'doi' => '10.1101/2021.03.08.434430',
'modified' => '2021-12-13 09:23:10',
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(int) 2 => array(
'id' => '4047',
'name' => 'Circuit-specific hippocampal ΔFosB underlies resilience to
stress-induced social avoidance.',
'authors' => 'Eagle, Andrew L and Manning, Claire E and Williams, Elizabeth S and Bastle,
Ryan M and Gajewski, Paula A and Garrison, Amber and Wirtz, Alexis J and
Akguen, Seda and Brandel-Ankrapp, Katie and Endege, Wilson and Boyce,
Frederick M and Ohnishi, Yoshinori N',
'description' => 'Chronic stress is a key risk factor for mood disorders like depression, but
the stress-induced changes in brain circuit function and gene expression
underlying depression symptoms are not completely understood, hindering
development of novel treatments. Because of its projections to brain
regions regulating reward and anxiety, the ventral hippocampus is uniquely
poised to translate the experience of stress into altered brain function
and pathological mood, though the cellular and molecular mechanisms of this
process are not fully understood. Here, we use a novel method of
circuit-specific gene editing to show that the transcription factor
ΔFosB drives projection-specific activity of ventral hippocampus
glutamatergic neurons causing behaviorally diverse responses to stress. We
establish molecular, cellular, and circuit-level mechanisms for depression-
and anxiety-like behavior in response to stress and use circuit-specific
gene expression profiling to uncover novel downstream targets as potential
sites of therapeutic intervention in depression.',
'date' => '2020-09-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/32901027',
'doi' => '10.1038/s41467-020-17825-x',
'modified' => '2021-02-18 10:21:53',
'created' => '2021-02-18 10:21:53',
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'id' => '3930',
'name' => 'Endogenous retroviruses are a source of enhancers with oncogenic potential in acute myeloid leukaemia',
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'description' => '<p>Acute myeloid leukemia (AML) is a highly aggressive hematopoietic malignancy, defined by a series of genetic and epigenetic alterations, which result in deregulation of transcriptional networks. One understudied but important source of transcriptional regulators are transposable elements (TEs), which are widespread throughout the human genome. Aberrant usage of these sequences could therefore contribute to oncogenic transcriptional circuits. However, the regulatory influence of TEs and their links to disease pathogenesis remain unexplored in AML. Using epigenomic data from AML primary samples and leukemia cell lines, we identified six endogenous retrovirus (ERV) families with AML-associated enhancer chromatin signatures that are enriched in binding of key regulators of hematopoiesis and AML pathogenesis. Using both CRISPR-mediated locus-specific genetic editing and simultaneous epigenetic silencing of multiple ERVs, we demonstrate that ERV deregulation directly alters the expression of adjacent genes in AML. Strikingly, deletion or epigenetic silencing of an ERV-derived enhancer suppressed cell growth by inducing apoptosis in leukemia cell lines. Our work reveals that ERVs are a previously unappreciated source of AML enhancers that have the potential to play key roles in leukemogenesis. We suggest that ERV activation provides an additional layer of gene regulation in AML that may be exploited by cancer cells to help drive tumour heterogeneity and evolution.</p>',
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'description' => '<p>Current gene editing approaches for treatment of recessive dystrophic epidermolysis bullosa (RDEB), an inherited, severe form of blistering skin disease, suffer from low efficiencies and safety concerns that complicate implementation in clinical settings. We present a strategy for efficient and precise repair of RDEB-associated mutations in the COL7A1 gene. We compared the efficacy of double-strand breaks (induced by CRISPR/Cas9), single-nicks, or double-nicks (induced by Cas9n) in mediating repair of a COL7A1 splice-site mutation in exon 3 by homologous recombination (HR). We accomplished remarkably high HR frequencies of 89% with double-nicking while at the same time kept unwanted repair outcomes, such as non-homologous end joining (NHEJ), at a minimum (11%). We also investigated the effects of subtle differences in repair template design on HR-rates and found that strategic template nicking can enhance COL7A1 editing efficiency. In RDEB patient keratinocytes, application of double-nicking led to restoration and subsequent secretion of type VII collagen at high efficiency. Comprehensive analysis of 25 putative off-target sites revealed no off-target activity for double-nicking, while usage of Cas9 resulted in 54% modified alleles at one site. Taken together, our work provides a framework for efficient, precise, and safe repair of COL7A1, which lies at the heart of a future curative therapy of RDEB.</p>',
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'description' => '<p>Current gene-editing approaches for treatment of recessive dystrophic epidermolysis bullosa (RDEB), an inherited, severe form of blistering skin disease, suffer from low efficiencies and safety concerns that complicate implementation in clinical settings. We present a strategy for efficient and precise repair of RDEB-associated mutations in the COL7A1 gene. We compared the efficacy of double-strand breaks (induced by CRISPR/Cas9), single nicks, or double nicks (induced by Cas9n) in mediating repair of a COL7A1 splice-site mutation in exon 3 by homologous recombination (HR). We accomplished remarkably high HR frequencies of 89% with double nicking while at the same time keeping unwanted repair outcomes, such as non-homologous end joining (NHEJ), at a minimum (11%). We also investigated the effects of subtle differences in repair template design on HR rates and found that strategic template-nicking can enhance COL7A1-editing efficiency. In RDEB patient keratinocytes, application of double-nicking led to restoration and subsequent secretion of type VII collagen at high efficiency. Comprehensive analysis of 25 putative off-target sites revealed no off-target activity for double-nicking, while usage of Cas9 resulted in 54% modified alleles at one site. Taken together, our work provides a framework for efficient, precise, and safe repair of COL7A1, which lies at the heart of a future curative therapy of RDEB.</p>',
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'date' => '2015-11-04',
'pmid' => 'http://onlinelibrary.wiley.com/doi/10.1002/9780470151808.sc05a08s35/abstract',
'doi' => '10.1002/9780470151808.sc05a08s35',
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<p><small><strong>Figure 2. Western blot analysis using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />Figure 1A: Western blot was performed on protein extracts from HeLa cells transfected with Cas9 using the Diagenode antibody against CRISPR/Cas9 (Cat. No. C15200229). The antibody was used at different dilutions. The marker is shown on the left, position of the Cas9 protein is indicated on the right. Figure 1B: Western blot was performed on protein extracts from HeLa cells transfected with Cas9 (lane 1) or from untransfected cells (lane 2) using the Diagenode antibody against CRISPR/Cas9 (Cat. No. C15200229), diluted 1:4,000 in PBS-T containing 3% NFDM. The marker is shown on the left, position of the Cas9 protein is indicated on the right.</small></p>
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'info2' => '<p>CRISPR systems are adaptable immune mechanisms which are present in many bacteria to protect themselves from foreign nucleic acids, such as viruses, transposable elements or plasmids. Recently, the CRISPR/Cas9 (CRISPR-associated protein 9 nuclease, UniProtKB/Swiss-Prot entry Q99ZW2) system from S. pyogenes has been adapted for inducing sequence-specific double stranded breaks and targeted genome editing. This system is unique and flexible due to its dependence on RNA as the moiety that targets the nuclease to a desired DNA sequence and can be used to induce indel mutations, specific sequence replacements or insertions and large deletions or genomic rearrangements at any desired location in the genome. In addition, Cas9 can also be used to mediate upregulation of specific endogenous genes or to alter histone modifications or DNA methylation.</p>',
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'description' => '<p><strong>Western blot</strong> : The quality of antibodies used in this technique is crucial for correct and specific protein identification. Diagenode offers huge selection of highly sensitive and specific western blot-validated antibodies.</p>
<p>Learn more about: <a href="https://www.diagenode.com/applications/western-blot">Loading control, MW marker visualization</a><em>. <br /></em></p>
<p><em></em>Check our selection of antibodies validated in Western blot.</p>',
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<p>Diagenode offers huge selection of highly sensitive antibodies validated in IF.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15200229-IF.jpg" alt="" height="245" width="256" /></p>
<p><sup><strong>Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong></sup></p>
<p><sup>HeLa cells transfected with a Cas9 expression vector (left) or untransfected cells (right) were fixed in methanol at -20°C, permeabilized with acetone at -20°C and blocked with PBS containing 2% BSA. The cells were stained with the Cas9 C-terminal antibody (Cat. No. C15200229) diluted 1:400, followed by incubation with an anti-mouse secondary antibody coupled to AF488. The bottom images show counter-staining of the nuclei with Hoechst 33342.</sup></p>
<h5><sup>Check our selection of antibodies validated in IF.</sup></h5>',
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'description' => '<p><span style="font-weight: 400;">All Diagenode’s antibodies are listed below. Please, use our Quick search field to find the antibody of interest by target name, application, purity.</span></p>
<p><span style="font-weight: 400;">Diagenode’s highly validated antibodies:</span></p>
<ul>
<li>Highly sensitive and specific</li>
<li>Cost-effective (requires less antibody per reaction)</li>
<li>Batch-specific data is available on the website</li>
<li>Expert technical support</li>
<li>Sample sizes available</li>
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'description' => '<p>Diagenode offers the broad range of antibodies raised against the N- or C-terminus of the Cas9 nuclease from <em>Streptococcus <g class="gr_ gr_5 gr-alert gr_spell gr_disable_anim_appear ContextualSpelling ins-del multiReplace" id="5" data-gr-id="5">pyogenes</g></em>. These highly specific polyclonal and monoclonal antibodies are validated in Western blot, immunoprecipitation, immunofluorescence and in chromatin immunoprecipitation.</p>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h2><em><a name="pyogenes"></a>S. pyogenes</em> CRISPR/Cas9 antibodies<a></a></h2>
<div class="panel">
<h2>Discover our first monoclonal CRISPR/Cas9 antibody validated in ChIP<br /><br /></h2>
<div class="row">
<div class="small-5 medium-5 large-5 columns"><img src="/img/landing-pages/crispr-cas9-chip-on-hih3t3.jpg" alt="" /></div>
<div class="small-7 medium-7 large-7 columns">
<ul>
<li>Validated in chromatin immunoprecipitation</li>
<li>Performs better than FLAG antibody</li>
<li>Excellent for WB, IF and IP</li>
</ul>
<p><small><strong>ChIP</strong> was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50 µg chromatin was incubated overnight at 4°C with 5 or 10 µg of either an anti-FLAG antibody or the Diagenode antibody against Cas9 (Cat. No. C15200229). Mouse IgG was used as a negative IP control. qPCR was performed with primers specific for the GFP gene, and for a non-targeted region (protein kinase C delta, Prkcd), used as negative control. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).</small></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns text-right"><a href="/p/crispr-cas9-monoclonal-antibody-50-ug-25-μl" class="tiny details button radius">Learn more</a></div>
</div>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>First ChIP-grade CRISPR/Cas9 polyclonal antibody</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/landing-pages/c_a_s9-chip-grade-antibody.png" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Excellent polyclonal antibody for chromatin immunoprecipitation</li>
<li>Optimized for highest ChIP specificity and yields</li>
<li>Validated for all applications including immunoblotting, immunofluorescence and western blot</li>
</ul>
<p><small><strong>ChIP</strong> was performed on NIH3T3 cells stably expressing GFP- H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50 μg chromatin was incubated with either 5 μg of an anti-FLAG antibody or 2 μl of the Diagenode antibody against Cas9. The pre-immune serum (PPI) was used as negative IP control. Then qPCR was performed with primers specific for the GFP gene, and for two non-targeted regions: Ppap2c and Prkcd, used as negative controls. This figure shows the recovery, expressed as a % of input.</small></p>
<p class="text-right"><a href="../p/crispr-cas9-polyclonal-antibody" class="details tiny button">Learn more</a></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>CRISPR/Cas9 monoclonal antibody 4G10</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/landing-pages/cas9_4g10_fig1.png" width="170" height="302" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Antibody raised against N-terminus of Cas9 nuclease</li>
<li>Validated for western blot, IP and immunofluorescence</li>
</ul>
<p><small><strong>Immunofluorescence</strong>: Hela cells were transiently transfected with a Cas9 expression vector. The cells were fixed in 3.7% formaldehyde, permeabilized in 0.5% Triton-X-100 and blocked in PBS containing 2% BSA. The cells were stained with the Cas9 antibody at 4°C o/n, followed by incubation with an anti mouse secondary antibody coupled to AF488 for 1 h at RT. Nuclei were counter-stained with Hoechst 33342 (right).</small></p>
<p class="text-right"><a href="../p/crispr-cas9-monoclonal-antibody-4g10-50-ug" class="details tiny button">Learn more</a></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>CRISPR/Cas9 C-terminal monoclonal antibody</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/product/antibodies/C15200223-IP.png" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Antibody raised against C-terminus of Cas9 nuclease</li>
<li>Validated for western blot, IP and immunofluorescence</li>
</ul>
<p><small><strong> Western blot</strong> was performed on 20 μg protein extracts from Cas9 expressing HeLa cells (lane 1) and on negative control HeLa cells (lane 2) with the Diagenode antibody against Cas9. The antibody was diluted 1:4,000. The marker is shown on the left, position of the Cas9 protein is indicated on the right. </small></p>
<p class="text-right"><a href="../p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug" class="details tiny button">Learn more</a></p>
</div>
<div class="small-12 medium-12 large-12 columns">
<h3>Which CRISPR/Cas9 antibody is the best for your application?</h3>
<a name="table"></a>
<table>
<thead>
<tr>
<th>Antibody</th>
<th>WB</th>
<th>IF</th>
<th>IP</th>
<th>ChIP</th>
<th>Antibody raised against</th>
</tr>
</thead>
<tbody>
<tr>
<td><a href="../p/crispr-cas9-monoclonal-antibody-50-ug-25-μl"><strong class="diacol">CRISPR/Cas9 monoclonal antibody</strong></a></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><span class="diacol">N-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-polyclonal-antibody">CRISPR/Cas9 polyclonal antibody</a></td>
<td>++</td>
<td>++</td>
<td>++</td>
<td><strong class="diacol">+++</strong></td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-monoclonal-antibody-4g10-50-ug">CRISPR/Cas9 monoclonal antibody 4G10</a></td>
<td>+++</td>
<td>+++</td>
<td>++</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="../p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug-23-ul"><strong class="diacol">CRISPR/Cas9 C-terminal monoclonal antibody</strong></a> <span class="label alert" style="font-size: 0.9rem;">NEW!</span></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">no</strong></td>
<td><strong class="diacol">+</strong></td>
<td><span class="diacol">C-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug">CRISPR/Cas9 C-terminal monoclonal antibody</a></td>
<td>++</td>
<td>++</td>
<td>+</td>
<td>no</td>
<td><span class="diacol">C-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-monoclonal-antibody-7A9-50-mg">CRISPR/Cas9 monoclonal antibody 7A9</a></td>
<td>++</td>
<td>++</td>
<td>++</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-hrp-monoclonal-antibody-50-ul">CRISPR/Cas9 - HRP monoclonal antibody 7A9</a></td>
<td>+++</td>
<td>no</td>
<td>no</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
</tbody>
</table>
</div>
</div>
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'description' => '<div class="row">
<div class="small-12 columns"><center></center>
<p><br />Chromatin immunoprecipitation (<b>ChIP</b>) is a technique to study the associations of proteins with the specific genomic regions in intact cells. One of the most important steps of this protocol is the immunoprecipitation of targeted protein using the antibody specifically recognizing it. The quality of antibodies used in ChIP is essential for the success of the experiment. Diagenode offers extensively validated ChIP-grade antibodies, confirmed for their specificity, and high level of performance in ChIP. Each batch is validated, and batch-specific data are available on the website.</p>
<p></p>
</div>
</div>
<p><strong>ChIP results</strong> obtained with the antibody directed against H3K4me3 (Cat. No. <a href="../p/h3k4me3-polyclonal-antibody-premium-50-ug-50-ul">C15410003</a>). </p>
<div class="row">
<div class="small-12 medium-6 large-6 columns"><img src="https://www.diagenode.com/img/product/antibodies/C15410003-fig1-ChIP.jpg" alt="" width="400" height="315" /> </div>
<div class="small-12 medium-6 large-6 columns">
<p></p>
<p></p>
<p></p>
</div>
</div>
<p></p>
<p>Our aim at Diagenode is to offer the largest collection of highly specific <strong>ChIP-grade antibodies</strong>. We add new antibodies monthly. Find your ChIP-grade antibody in the list below and check more information about tested applications, extensive validation data, and product information.</p>',
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'name' => 'CRISPR/Cas9 monoclonal antibody C15200229',
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'name' => ' Accurate QC to optimize CRISPR/Cas9 genome editing specificity',
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'description' => '<p>Aggregates of hyperphosphorylated tau protein are a pathological hallmark of more than 20 distinct neurodegenerative diseases, including Alzheimer’s disease, progressive supranuclear palsy, and frontotemporal dementia. While the exact mechanism of tau aggregation is unknown, the accumulation of aggregates correlates with disease progression. Here we report a genome-wide CRISPR screen to identify modulators of endogenous tau protein for the first time. Primary screens performed in SH-SY5Y cells, identified positive and negative regulators of tau protein levels. Hit validation of the top 43 candidate genes was performed using Ngn2-induced human cortical excitatory neurons. Using this approach, genes and pathways involved in modulation of endogenous tau levels were identified, including chromatin modifying enzymes, neddylation and ubiquitin pathway members, and components of the mTOR pathway. TSC1, a critical component of the mTOR pathway, was further validated in vivo, demonstrating the relevance of this screening strategy. These findings may have implications for treating neurodegenerative diseases in the future.</p>',
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'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/34127790',
'doi' => '10.1038/s42003-021-02272-1',
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'name' => 'Transgenic mice for in vivo epigenome editing with CRISPR-based systems',
'authors' => 'Gemberling, M. et al.',
'description' => '<p>The discovery, characterization, and adaptation of the RNA-guided clustered regularly interspersed short palindromic repeat (CRISPR)-Cas9 system has greatly increased the ease with which genome and epigenome editing can be performed. Fusion of chromatin-modifying domains to the nuclease-deactivated form of Cas9 (dCas9) has enabled targeted gene activation or repression in both cultured cells and in vivo in animal models. However, delivery of the large dCas9 fusion proteins to target cell types and tissues is an obstacle to widespread adoption of these tools for in vivo studies. Here we describe the generation and validation of two conditional transgenic mouse lines for targeted gene regulation, Rosa26:LSL-dCas9-p300 for gene activation and Rosa26:LSL-dCas9-KRAB for gene repression. Using the dCas9p300 and dCas9KRAB transgenic mice we demonstrate activation or repression of genes in both the brain and liver in vivo, and T cells and fibroblasts ex vivo. We show gene regulation and targeted epigenetic modification with gRNAs targeting either transcriptional start sites (TSS) or distal enhancer elements, as well as corresponding changes to downstream phenotypes. These mouse lines are convenient and valuable tools for facile, temporally controlled, and tissue-restricted epigenome editing and manipulation of gene expression in vivo.</p>',
'date' => '2021-03-01',
'pmid' => 'https://doi.org/10.1101%2F2021.03.08.434430',
'doi' => '10.1101/2021.03.08.434430',
'modified' => '2021-12-13 09:23:10',
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'id' => '4047',
'name' => 'Circuit-specific hippocampal ΔFosB underlies resilience to
stress-induced social avoidance.',
'authors' => 'Eagle, Andrew L and Manning, Claire E and Williams, Elizabeth S and Bastle,
Ryan M and Gajewski, Paula A and Garrison, Amber and Wirtz, Alexis J and
Akguen, Seda and Brandel-Ankrapp, Katie and Endege, Wilson and Boyce,
Frederick M and Ohnishi, Yoshinori N',
'description' => 'Chronic stress is a key risk factor for mood disorders like depression, but
the stress-induced changes in brain circuit function and gene expression
underlying depression symptoms are not completely understood, hindering
development of novel treatments. Because of its projections to brain
regions regulating reward and anxiety, the ventral hippocampus is uniquely
poised to translate the experience of stress into altered brain function
and pathological mood, though the cellular and molecular mechanisms of this
process are not fully understood. Here, we use a novel method of
circuit-specific gene editing to show that the transcription factor
ΔFosB drives projection-specific activity of ventral hippocampus
glutamatergic neurons causing behaviorally diverse responses to stress. We
establish molecular, cellular, and circuit-level mechanisms for depression-
and anxiety-like behavior in response to stress and use circuit-specific
gene expression profiling to uncover novel downstream targets as potential
sites of therapeutic intervention in depression.',
'date' => '2020-09-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/32901027',
'doi' => '10.1038/s41467-020-17825-x',
'modified' => '2021-02-18 10:21:53',
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'id' => '3930',
'name' => 'Endogenous retroviruses are a source of enhancers with oncogenic potential in acute myeloid leukaemia',
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'description' => '<p>Acute myeloid leukemia (AML) is a highly aggressive hematopoietic malignancy, defined by a series of genetic and epigenetic alterations, which result in deregulation of transcriptional networks. One understudied but important source of transcriptional regulators are transposable elements (TEs), which are widespread throughout the human genome. Aberrant usage of these sequences could therefore contribute to oncogenic transcriptional circuits. However, the regulatory influence of TEs and their links to disease pathogenesis remain unexplored in AML. Using epigenomic data from AML primary samples and leukemia cell lines, we identified six endogenous retrovirus (ERV) families with AML-associated enhancer chromatin signatures that are enriched in binding of key regulators of hematopoiesis and AML pathogenesis. Using both CRISPR-mediated locus-specific genetic editing and simultaneous epigenetic silencing of multiple ERVs, we demonstrate that ERV deregulation directly alters the expression of adjacent genes in AML. Strikingly, deletion or epigenetic silencing of an ERV-derived enhancer suppressed cell growth by inducing apoptosis in leukemia cell lines. Our work reveals that ERVs are a previously unappreciated source of AML enhancers that have the potential to play key roles in leukemogenesis. We suggest that ERV activation provides an additional layer of gene regulation in AML that may be exploited by cancer cells to help drive tumour heterogeneity and evolution.</p>',
'date' => '2020-04-04',
'pmid' => 'https://www.biorxiv.org/content/10.1101/772954v2',
'doi' => 'https://doi.org/10.1101/772954',
'modified' => '2020-08-17 10:43:25',
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'name' => 'Improved double-nicking strategies for COL7A1 editing by homologous recombination',
'authors' => 'Kocher Thomas, Wagner Roland N., Klausegger Alfred, Guttmann-Gruber Christina, Hainzl Stefan, Bauer Johann W., Reichelt Julia, Koller Ulrich',
'description' => '<p>Current gene editing approaches for treatment of recessive dystrophic epidermolysis bullosa (RDEB), an inherited, severe form of blistering skin disease, suffer from low efficiencies and safety concerns that complicate implementation in clinical settings. We present a strategy for efficient and precise repair of RDEB-associated mutations in the COL7A1 gene. We compared the efficacy of double-strand breaks (induced by CRISPR/Cas9), single-nicks, or double-nicks (induced by Cas9n) in mediating repair of a COL7A1 splice-site mutation in exon 3 by homologous recombination (HR). We accomplished remarkably high HR frequencies of 89% with double-nicking while at the same time kept unwanted repair outcomes, such as non-homologous end joining (NHEJ), at a minimum (11%). We also investigated the effects of subtle differences in repair template design on HR-rates and found that strategic template nicking can enhance COL7A1 editing efficiency. In RDEB patient keratinocytes, application of double-nicking led to restoration and subsequent secretion of type VII collagen at high efficiency. Comprehensive analysis of 25 putative off-target sites revealed no off-target activity for double-nicking, while usage of Cas9 resulted in 54% modified alleles at one site. Taken together, our work provides a framework for efficient, precise, and safe repair of COL7A1, which lies at the heart of a future curative therapy of RDEB.</p>',
'date' => '2019-09-12',
'pmid' => 'https://www.cell.com/molecular-therapy-family/nucleic-acids/fulltext/S2162-2531(19)30258-6',
'doi' => '10.1016/j.omtn.2019.09.011',
'modified' => '2022-05-18 19:30:10',
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'name' => 'Improved Double-Nicking Strategies for COL7A1-Editing by Homologous Recombination.',
'authors' => 'Kocher T, Wagner RN, Klausegger A, Guttmann-Gruber C, Hainzl S, Bauer JW, Reichelt J, Koller U',
'description' => '<p>Current gene-editing approaches for treatment of recessive dystrophic epidermolysis bullosa (RDEB), an inherited, severe form of blistering skin disease, suffer from low efficiencies and safety concerns that complicate implementation in clinical settings. We present a strategy for efficient and precise repair of RDEB-associated mutations in the COL7A1 gene. We compared the efficacy of double-strand breaks (induced by CRISPR/Cas9), single nicks, or double nicks (induced by Cas9n) in mediating repair of a COL7A1 splice-site mutation in exon 3 by homologous recombination (HR). We accomplished remarkably high HR frequencies of 89% with double nicking while at the same time keeping unwanted repair outcomes, such as non-homologous end joining (NHEJ), at a minimum (11%). We also investigated the effects of subtle differences in repair template design on HR rates and found that strategic template-nicking can enhance COL7A1-editing efficiency. In RDEB patient keratinocytes, application of double-nicking led to restoration and subsequent secretion of type VII collagen at high efficiency. Comprehensive analysis of 25 putative off-target sites revealed no off-target activity for double-nicking, while usage of Cas9 resulted in 54% modified alleles at one site. Taken together, our work provides a framework for efficient, precise, and safe repair of COL7A1, which lies at the heart of a future curative therapy of RDEB.</p>',
'date' => '2019-09-12',
'pmid' => 'http://www.pubmed.gov/31670199',
'doi' => '10.1016/j.omtn.2019.09.011.',
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'name' => 'Essential Gene Profiles for Human Pluripotent Stem Cells Identify Uncharacterized Genes and Substrate Dependencies.',
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'description' => '<p>Human pluripotent stem cells (hPSCs) provide an invaluable tool for modeling diseases and hold promise for regenerative medicine. For understanding pluripotency and lineage differentiation mechanisms, a critical first step involves systematically cataloging essential genes (EGs) that are indispensable for hPSC fitness, defined as cell reproduction in this study. To map essential genetic determinants of hPSC fitness, we performed genome-scale loss-of-function screens in an inducible Cas9 H1 hPSC line cultured on feeder cells and laminin to identify EGs. Among these, we found FOXH1 and VENTX, genes that encode transcription factors previously implicated in stem cell biology, as well as an uncharacterized gene, C22orf43/DRICH1. hPSC EGs are substantially different from other human model cell lines, and EGs in hPSCs are highly context dependent with respect to different growth substrates. Our CRISPR screens establish parameters for genome-wide screens in hPSCs, which will facilitate the characterization of unappreciated genetic regulators of hPSC biology.</p>',
'date' => '2019-04-09',
'pmid' => 'http://www.pubmed.gov/30970261',
'doi' => '10.1016/j.celrep.2019.02.041',
'modified' => '2019-08-07 09:17:29',
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'name' => 'RNA-Based dCas9–VP64 System Improves the Viability of Cryopreserved Mammalian Cells',
'authors' => 'Hu Yong, Li Lei, Yu Yin, Huang Haishui, Uygun Basak E., Yarmush Martin L.',
'description' => '<p>Regenerative therapies require availability of an abundant healthy cell source which can be achieved by e±cient cryopreservation techniques. Here, we established a novel approach for improved cell cryopreservation using an mRNA-based dCas9-VP64 gene activation system for transient, yet highly e±cient expression of epigenetic related genes in mammalian cells for repression of metabolic activity. Before freezing, mammalian cells were treated by dCas9-VP64- modi¯ed mRNA and guide RNAs for upregulation of histone deacetylase (HDAC), DNA methyltransferase (DNMT) and transcriptional co-repressor Sin3A genes. Cell viability, karyotype, pluripotency, and other cell speci¯c functions were analyzed during post-thaw culture. Using conventional cryopreservation protocols, we found improvement of viability in dCas9- VP64 pretreated cells (P < 0:05) compared to untreated cells. Combined with dCas9-VP64 system, a reduced amount of cryoprotectant (5% DMSO) did not negatively a®ect the post-thaw viability. Co-delivering chemically modi¯ed dCas9-VP64 mRNA with gRNAs is an e±cient gene delivery method compared to DNA-based strategies, without the associated safety concerns. This approach is a simple, yet e®ective way to accelerate a wide array of cellular research and translational medical applications.</p>',
'date' => '2018-01-01',
'pmid' => 'https://www.worldscientific.com/doi/10.1142/S1793984418500046',
'doi' => '10.1142/S1793984418500046',
'modified' => '2019-02-18 11:55:37',
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'name' => 'CRISPR-Mediated Gene Targeting of Human Induced Pluripotent Stem Cells',
'authors' => 'Susan M. Byrne, George M. Church',
'description' => '<p><span>CRISPR/Cas9 nuclease systems can create double-stranded DNA breaks at specific sequences to efficiently and precisely disrupt, excise, mutate, insert, or replace genes. However, human embryonic stem cells and induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, an optimized protocol is described for genome engineering of human iPSCs using simple transient transfection of plasmids and/or single-stranded oligonucleotides without any further selection or enrichment steps. This protocol achieves transfection efficiencies >60%, with gene disruption efficiencies of 1-25% and gene insertion/replacement efficiencies of 0.5-10%. Details are also provided for designing optimal sgRNA target sites and donor targeting vectors, cloning individual iPSCs by single-cell FACS sorting, and genotyping successfully edited cells.</span></p>',
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<p><small> <strong>Figure 1. ChIP using the Diagenode monoclonal antibody directed against Cas9</strong><br />ChIP was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50 μg chromatin was incubated overnight at 4°C with 5 or 10 μg of either an anti-FLAG antibody or the Diagenode antibody against Cas9 (Cat. No. C15200229). Mouse IgG was used as a negative IP control. qPCR was performed with primers specific for the GFP gene, and for a non-targeted region (protein kinase C delta, Prkcd), used as negative control. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).</small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15200229-WB-A.jpg" alt="CRISPR/Cas9 Antibody for Western Blot " /> <img src="https://www.diagenode.com/img/product/antibodies/C15200229-WB-B.jpg" alt="CRISPR/Cas9 Antibody for Western Blot " /></p>
</div>
<div class="small-8 columns">
<p><small><strong>Figure 2. Western blot analysis using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />Figure 1A: Western blot was performed on protein extracts from HeLa cells transfected with Cas9 using the Diagenode antibody against CRISPR/Cas9 (Cat. No. C15200229). The antibody was used at different dilutions. The marker is shown on the left, position of the Cas9 protein is indicated on the right. Figure 1B: Western blot was performed on protein extracts from HeLa cells transfected with Cas9 (lane 1) or from untransfected cells (lane 2) using the Diagenode antibody against CRISPR/Cas9 (Cat. No. C15200229), diluted 1:4,000 in PBS-T containing 3% NFDM. The marker is shown on the left, position of the Cas9 protein is indicated on the right.</small></p>
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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15200229-IP.jpg" alt="CRISPR/Cas9 Antibody for Immunoprecipitation" /></p>
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<div class="small-8 columns">
<p><small> <strong>Figure 3. IP using the Diagenode monoclonal antibody directed against Cas9</strong> <br /> IP was performed on whole cell extracts (300 μg) from HEK293 cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 6 μg of the Diagenode antibody against Cas9 (Cat. No. C15200229). The immunoprecipitated proteins were subsequently analysed by Western blot with the polyclonal Cas9 antibody (Cat. No. C15310258, diluted 1:8,000). Lane 3 and 4 show the result of the IP, the input (15 μg) is shown in lane 1 and 2.</small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15200229-IF.jpg" alt="CRISPR/Cas9 Antibody for Immunofluorescence" /></p>
</div>
<div class="small-8 columns">
<p><small> <strong>Figure 4. Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong> <br /> HeLa cells transfected with a Cas9 expression vector (left) or untransfected cells (right) were fixed in methanol at -20°C, permeabilized with acetone at -20°C and blocked with PBS containing 2% BSA. The cells were stained with the Cas9 N-terminal antibody (Cat. No. C15200229) diluted 1:400, followed by incubation with an anti-mouse secondary antibody coupled to AF488. The bottom images show counter-staining of the nuclei with Hoechst 33342.</small></p>
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<p>Learn more about: <a href="https://www.diagenode.com/applications/western-blot">Loading control, MW marker visualization</a><em>. <br /></em></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15200229-IF.jpg" alt="" height="245" width="256" /></p>
<p><sup><strong>Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong></sup></p>
<p><sup>HeLa cells transfected with a Cas9 expression vector (left) or untransfected cells (right) were fixed in methanol at -20°C, permeabilized with acetone at -20°C and blocked with PBS containing 2% BSA. The cells were stained with the Cas9 C-terminal antibody (Cat. No. C15200229) diluted 1:400, followed by incubation with an anti-mouse secondary antibody coupled to AF488. The bottom images show counter-staining of the nuclei with Hoechst 33342.</sup></p>
<h5><sup>Check our selection of antibodies validated in IF.</sup></h5>',
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<p><span style="font-weight: 400;">Diagenode’s highly validated antibodies:</span></p>
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<li>Highly sensitive and specific</li>
<li>Cost-effective (requires less antibody per reaction)</li>
<li>Batch-specific data is available on the website</li>
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'description' => '<p>Diagenode offers the broad range of antibodies raised against the N- or C-terminus of the Cas9 nuclease from <em>Streptococcus <g class="gr_ gr_5 gr-alert gr_spell gr_disable_anim_appear ContextualSpelling ins-del multiReplace" id="5" data-gr-id="5">pyogenes</g></em>. These highly specific polyclonal and monoclonal antibodies are validated in Western blot, immunoprecipitation, immunofluorescence and in chromatin immunoprecipitation.</p>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h2><em><a name="pyogenes"></a>S. pyogenes</em> CRISPR/Cas9 antibodies<a></a></h2>
<div class="panel">
<h2>Discover our first monoclonal CRISPR/Cas9 antibody validated in ChIP<br /><br /></h2>
<div class="row">
<div class="small-5 medium-5 large-5 columns"><img src="/img/landing-pages/crispr-cas9-chip-on-hih3t3.jpg" alt="" /></div>
<div class="small-7 medium-7 large-7 columns">
<ul>
<li>Validated in chromatin immunoprecipitation</li>
<li>Performs better than FLAG antibody</li>
<li>Excellent for WB, IF and IP</li>
</ul>
<p><small><strong>ChIP</strong> was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50 µg chromatin was incubated overnight at 4°C with 5 or 10 µg of either an anti-FLAG antibody or the Diagenode antibody against Cas9 (Cat. No. C15200229). Mouse IgG was used as a negative IP control. qPCR was performed with primers specific for the GFP gene, and for a non-targeted region (protein kinase C delta, Prkcd), used as negative control. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).</small></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns text-right"><a href="/p/crispr-cas9-monoclonal-antibody-50-ug-25-μl" class="tiny details button radius">Learn more</a></div>
</div>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>First ChIP-grade CRISPR/Cas9 polyclonal antibody</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/landing-pages/c_a_s9-chip-grade-antibody.png" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Excellent polyclonal antibody for chromatin immunoprecipitation</li>
<li>Optimized for highest ChIP specificity and yields</li>
<li>Validated for all applications including immunoblotting, immunofluorescence and western blot</li>
</ul>
<p><small><strong>ChIP</strong> was performed on NIH3T3 cells stably expressing GFP- H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50 μg chromatin was incubated with either 5 μg of an anti-FLAG antibody or 2 μl of the Diagenode antibody against Cas9. The pre-immune serum (PPI) was used as negative IP control. Then qPCR was performed with primers specific for the GFP gene, and for two non-targeted regions: Ppap2c and Prkcd, used as negative controls. This figure shows the recovery, expressed as a % of input.</small></p>
<p class="text-right"><a href="../p/crispr-cas9-polyclonal-antibody" class="details tiny button">Learn more</a></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>CRISPR/Cas9 monoclonal antibody 4G10</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/landing-pages/cas9_4g10_fig1.png" width="170" height="302" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Antibody raised against N-terminus of Cas9 nuclease</li>
<li>Validated for western blot, IP and immunofluorescence</li>
</ul>
<p><small><strong>Immunofluorescence</strong>: Hela cells were transiently transfected with a Cas9 expression vector. The cells were fixed in 3.7% formaldehyde, permeabilized in 0.5% Triton-X-100 and blocked in PBS containing 2% BSA. The cells were stained with the Cas9 antibody at 4°C o/n, followed by incubation with an anti mouse secondary antibody coupled to AF488 for 1 h at RT. Nuclei were counter-stained with Hoechst 33342 (right).</small></p>
<p class="text-right"><a href="../p/crispr-cas9-monoclonal-antibody-4g10-50-ug" class="details tiny button">Learn more</a></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>CRISPR/Cas9 C-terminal monoclonal antibody</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/product/antibodies/C15200223-IP.png" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Antibody raised against C-terminus of Cas9 nuclease</li>
<li>Validated for western blot, IP and immunofluorescence</li>
</ul>
<p><small><strong> Western blot</strong> was performed on 20 μg protein extracts from Cas9 expressing HeLa cells (lane 1) and on negative control HeLa cells (lane 2) with the Diagenode antibody against Cas9. The antibody was diluted 1:4,000. The marker is shown on the left, position of the Cas9 protein is indicated on the right. </small></p>
<p class="text-right"><a href="../p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug" class="details tiny button">Learn more</a></p>
</div>
<div class="small-12 medium-12 large-12 columns">
<h3>Which CRISPR/Cas9 antibody is the best for your application?</h3>
<a name="table"></a>
<table>
<thead>
<tr>
<th>Antibody</th>
<th>WB</th>
<th>IF</th>
<th>IP</th>
<th>ChIP</th>
<th>Antibody raised against</th>
</tr>
</thead>
<tbody>
<tr>
<td><a href="../p/crispr-cas9-monoclonal-antibody-50-ug-25-μl"><strong class="diacol">CRISPR/Cas9 monoclonal antibody</strong></a></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><span class="diacol">N-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-polyclonal-antibody">CRISPR/Cas9 polyclonal antibody</a></td>
<td>++</td>
<td>++</td>
<td>++</td>
<td><strong class="diacol">+++</strong></td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-monoclonal-antibody-4g10-50-ug">CRISPR/Cas9 monoclonal antibody 4G10</a></td>
<td>+++</td>
<td>+++</td>
<td>++</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="../p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug-23-ul"><strong class="diacol">CRISPR/Cas9 C-terminal monoclonal antibody</strong></a> <span class="label alert" style="font-size: 0.9rem;">NEW!</span></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">no</strong></td>
<td><strong class="diacol">+</strong></td>
<td><span class="diacol">C-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug">CRISPR/Cas9 C-terminal monoclonal antibody</a></td>
<td>++</td>
<td>++</td>
<td>+</td>
<td>no</td>
<td><span class="diacol">C-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-monoclonal-antibody-7A9-50-mg">CRISPR/Cas9 monoclonal antibody 7A9</a></td>
<td>++</td>
<td>++</td>
<td>++</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-hrp-monoclonal-antibody-50-ul">CRISPR/Cas9 - HRP monoclonal antibody 7A9</a></td>
<td>+++</td>
<td>no</td>
<td>no</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
</tbody>
</table>
</div>
</div>
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<div class="small-12 columns"><center></center>
<p><br />Chromatin immunoprecipitation (<b>ChIP</b>) is a technique to study the associations of proteins with the specific genomic regions in intact cells. One of the most important steps of this protocol is the immunoprecipitation of targeted protein using the antibody specifically recognizing it. The quality of antibodies used in ChIP is essential for the success of the experiment. Diagenode offers extensively validated ChIP-grade antibodies, confirmed for their specificity, and high level of performance in ChIP. Each batch is validated, and batch-specific data are available on the website.</p>
<p></p>
</div>
</div>
<p><strong>ChIP results</strong> obtained with the antibody directed against H3K4me3 (Cat. No. <a href="../p/h3k4me3-polyclonal-antibody-premium-50-ug-50-ul">C15410003</a>). </p>
<div class="row">
<div class="small-12 medium-6 large-6 columns"><img src="https://www.diagenode.com/img/product/antibodies/C15410003-fig1-ChIP.jpg" alt="" width="400" height="315" /> </div>
<div class="small-12 medium-6 large-6 columns">
<p></p>
<p></p>
<p></p>
</div>
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<p></p>
<p>Our aim at Diagenode is to offer the largest collection of highly specific <strong>ChIP-grade antibodies</strong>. We add new antibodies monthly. Find your ChIP-grade antibody in the list below and check more information about tested applications, extensive validation data, and product information.</p>',
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'name' => ' Accurate QC to optimize CRISPR/Cas9 genome editing specificity',
'description' => '<p>The CRISPR/Cas9 technology is delivering superior genetic models for fundamental disease research, drug screening, therapy development, rapid diagnostics, and transcriptional modulation. Although CRISPR/Cas9 enables rapid genome editing, several aspects affect its efficiency and specificity including guide RNA design, delivery methods, and off-targets effects. Diagenode has developed strategies to overcome these common pitfalls and has optimized CRISPR/Cas9 genome editing specificity</p>',
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'name' => 'Genome-wide CRISPR screen identifies protein pathways modulating tauprotein levels in neurons',
'authors' => 'Sanchez C. G. et al. ',
'description' => '<p>Aggregates of hyperphosphorylated tau protein are a pathological hallmark of more than 20 distinct neurodegenerative diseases, including Alzheimer’s disease, progressive supranuclear palsy, and frontotemporal dementia. While the exact mechanism of tau aggregation is unknown, the accumulation of aggregates correlates with disease progression. Here we report a genome-wide CRISPR screen to identify modulators of endogenous tau protein for the first time. Primary screens performed in SH-SY5Y cells, identified positive and negative regulators of tau protein levels. Hit validation of the top 43 candidate genes was performed using Ngn2-induced human cortical excitatory neurons. Using this approach, genes and pathways involved in modulation of endogenous tau levels were identified, including chromatin modifying enzymes, neddylation and ubiquitin pathway members, and components of the mTOR pathway. TSC1, a critical component of the mTOR pathway, was further validated in vivo, demonstrating the relevance of this screening strategy. These findings may have implications for treating neurodegenerative diseases in the future.</p>',
'date' => '2021-06-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/34127790',
'doi' => '10.1038/s42003-021-02272-1',
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'name' => 'Transgenic mice for in vivo epigenome editing with CRISPR-based systems',
'authors' => 'Gemberling, M. et al.',
'description' => '<p>The discovery, characterization, and adaptation of the RNA-guided clustered regularly interspersed short palindromic repeat (CRISPR)-Cas9 system has greatly increased the ease with which genome and epigenome editing can be performed. Fusion of chromatin-modifying domains to the nuclease-deactivated form of Cas9 (dCas9) has enabled targeted gene activation or repression in both cultured cells and in vivo in animal models. However, delivery of the large dCas9 fusion proteins to target cell types and tissues is an obstacle to widespread adoption of these tools for in vivo studies. Here we describe the generation and validation of two conditional transgenic mouse lines for targeted gene regulation, Rosa26:LSL-dCas9-p300 for gene activation and Rosa26:LSL-dCas9-KRAB for gene repression. Using the dCas9p300 and dCas9KRAB transgenic mice we demonstrate activation or repression of genes in both the brain and liver in vivo, and T cells and fibroblasts ex vivo. We show gene regulation and targeted epigenetic modification with gRNAs targeting either transcriptional start sites (TSS) or distal enhancer elements, as well as corresponding changes to downstream phenotypes. These mouse lines are convenient and valuable tools for facile, temporally controlled, and tissue-restricted epigenome editing and manipulation of gene expression in vivo.</p>',
'date' => '2021-03-01',
'pmid' => 'https://doi.org/10.1101%2F2021.03.08.434430',
'doi' => '10.1101/2021.03.08.434430',
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'name' => 'Circuit-specific hippocampal ΔFosB underlies resilience to
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'authors' => 'Eagle, Andrew L and Manning, Claire E and Williams, Elizabeth S and Bastle,
Ryan M and Gajewski, Paula A and Garrison, Amber and Wirtz, Alexis J and
Akguen, Seda and Brandel-Ankrapp, Katie and Endege, Wilson and Boyce,
Frederick M and Ohnishi, Yoshinori N',
'description' => 'Chronic stress is a key risk factor for mood disorders like depression, but
the stress-induced changes in brain circuit function and gene expression
underlying depression symptoms are not completely understood, hindering
development of novel treatments. Because of its projections to brain
regions regulating reward and anxiety, the ventral hippocampus is uniquely
poised to translate the experience of stress into altered brain function
and pathological mood, though the cellular and molecular mechanisms of this
process are not fully understood. Here, we use a novel method of
circuit-specific gene editing to show that the transcription factor
ΔFosB drives projection-specific activity of ventral hippocampus
glutamatergic neurons causing behaviorally diverse responses to stress. We
establish molecular, cellular, and circuit-level mechanisms for depression-
and anxiety-like behavior in response to stress and use circuit-specific
gene expression profiling to uncover novel downstream targets as potential
sites of therapeutic intervention in depression.',
'date' => '2020-09-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/32901027',
'doi' => '10.1038/s41467-020-17825-x',
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'name' => 'Endogenous retroviruses are a source of enhancers with oncogenic potential in acute myeloid leukaemia',
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'description' => '<p>Acute myeloid leukemia (AML) is a highly aggressive hematopoietic malignancy, defined by a series of genetic and epigenetic alterations, which result in deregulation of transcriptional networks. One understudied but important source of transcriptional regulators are transposable elements (TEs), which are widespread throughout the human genome. Aberrant usage of these sequences could therefore contribute to oncogenic transcriptional circuits. However, the regulatory influence of TEs and their links to disease pathogenesis remain unexplored in AML. Using epigenomic data from AML primary samples and leukemia cell lines, we identified six endogenous retrovirus (ERV) families with AML-associated enhancer chromatin signatures that are enriched in binding of key regulators of hematopoiesis and AML pathogenesis. Using both CRISPR-mediated locus-specific genetic editing and simultaneous epigenetic silencing of multiple ERVs, we demonstrate that ERV deregulation directly alters the expression of adjacent genes in AML. Strikingly, deletion or epigenetic silencing of an ERV-derived enhancer suppressed cell growth by inducing apoptosis in leukemia cell lines. Our work reveals that ERVs are a previously unappreciated source of AML enhancers that have the potential to play key roles in leukemogenesis. We suggest that ERV activation provides an additional layer of gene regulation in AML that may be exploited by cancer cells to help drive tumour heterogeneity and evolution.</p>',
'date' => '2020-04-04',
'pmid' => 'https://www.biorxiv.org/content/10.1101/772954v2',
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'name' => 'Improved double-nicking strategies for COL7A1 editing by homologous recombination',
'authors' => 'Kocher Thomas, Wagner Roland N., Klausegger Alfred, Guttmann-Gruber Christina, Hainzl Stefan, Bauer Johann W., Reichelt Julia, Koller Ulrich',
'description' => '<p>Current gene editing approaches for treatment of recessive dystrophic epidermolysis bullosa (RDEB), an inherited, severe form of blistering skin disease, suffer from low efficiencies and safety concerns that complicate implementation in clinical settings. We present a strategy for efficient and precise repair of RDEB-associated mutations in the COL7A1 gene. We compared the efficacy of double-strand breaks (induced by CRISPR/Cas9), single-nicks, or double-nicks (induced by Cas9n) in mediating repair of a COL7A1 splice-site mutation in exon 3 by homologous recombination (HR). We accomplished remarkably high HR frequencies of 89% with double-nicking while at the same time kept unwanted repair outcomes, such as non-homologous end joining (NHEJ), at a minimum (11%). We also investigated the effects of subtle differences in repair template design on HR-rates and found that strategic template nicking can enhance COL7A1 editing efficiency. In RDEB patient keratinocytes, application of double-nicking led to restoration and subsequent secretion of type VII collagen at high efficiency. Comprehensive analysis of 25 putative off-target sites revealed no off-target activity for double-nicking, while usage of Cas9 resulted in 54% modified alleles at one site. Taken together, our work provides a framework for efficient, precise, and safe repair of COL7A1, which lies at the heart of a future curative therapy of RDEB.</p>',
'date' => '2019-09-12',
'pmid' => 'https://www.cell.com/molecular-therapy-family/nucleic-acids/fulltext/S2162-2531(19)30258-6',
'doi' => '10.1016/j.omtn.2019.09.011',
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'name' => 'Improved Double-Nicking Strategies for COL7A1-Editing by Homologous Recombination.',
'authors' => 'Kocher T, Wagner RN, Klausegger A, Guttmann-Gruber C, Hainzl S, Bauer JW, Reichelt J, Koller U',
'description' => '<p>Current gene-editing approaches for treatment of recessive dystrophic epidermolysis bullosa (RDEB), an inherited, severe form of blistering skin disease, suffer from low efficiencies and safety concerns that complicate implementation in clinical settings. We present a strategy for efficient and precise repair of RDEB-associated mutations in the COL7A1 gene. We compared the efficacy of double-strand breaks (induced by CRISPR/Cas9), single nicks, or double nicks (induced by Cas9n) in mediating repair of a COL7A1 splice-site mutation in exon 3 by homologous recombination (HR). We accomplished remarkably high HR frequencies of 89% with double nicking while at the same time keeping unwanted repair outcomes, such as non-homologous end joining (NHEJ), at a minimum (11%). We also investigated the effects of subtle differences in repair template design on HR rates and found that strategic template-nicking can enhance COL7A1-editing efficiency. In RDEB patient keratinocytes, application of double-nicking led to restoration and subsequent secretion of type VII collagen at high efficiency. Comprehensive analysis of 25 putative off-target sites revealed no off-target activity for double-nicking, while usage of Cas9 resulted in 54% modified alleles at one site. Taken together, our work provides a framework for efficient, precise, and safe repair of COL7A1, which lies at the heart of a future curative therapy of RDEB.</p>',
'date' => '2019-09-12',
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'name' => 'Essential Gene Profiles for Human Pluripotent Stem Cells Identify Uncharacterized Genes and Substrate Dependencies.',
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'description' => '<p>Human pluripotent stem cells (hPSCs) provide an invaluable tool for modeling diseases and hold promise for regenerative medicine. For understanding pluripotency and lineage differentiation mechanisms, a critical first step involves systematically cataloging essential genes (EGs) that are indispensable for hPSC fitness, defined as cell reproduction in this study. To map essential genetic determinants of hPSC fitness, we performed genome-scale loss-of-function screens in an inducible Cas9 H1 hPSC line cultured on feeder cells and laminin to identify EGs. Among these, we found FOXH1 and VENTX, genes that encode transcription factors previously implicated in stem cell biology, as well as an uncharacterized gene, C22orf43/DRICH1. hPSC EGs are substantially different from other human model cell lines, and EGs in hPSCs are highly context dependent with respect to different growth substrates. Our CRISPR screens establish parameters for genome-wide screens in hPSCs, which will facilitate the characterization of unappreciated genetic regulators of hPSC biology.</p>',
'date' => '2019-04-09',
'pmid' => 'http://www.pubmed.gov/30970261',
'doi' => '10.1016/j.celrep.2019.02.041',
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'authors' => 'Hu Yong, Li Lei, Yu Yin, Huang Haishui, Uygun Basak E., Yarmush Martin L.',
'description' => '<p>Regenerative therapies require availability of an abundant healthy cell source which can be achieved by e±cient cryopreservation techniques. Here, we established a novel approach for improved cell cryopreservation using an mRNA-based dCas9-VP64 gene activation system for transient, yet highly e±cient expression of epigenetic related genes in mammalian cells for repression of metabolic activity. Before freezing, mammalian cells were treated by dCas9-VP64- modi¯ed mRNA and guide RNAs for upregulation of histone deacetylase (HDAC), DNA methyltransferase (DNMT) and transcriptional co-repressor Sin3A genes. Cell viability, karyotype, pluripotency, and other cell speci¯c functions were analyzed during post-thaw culture. Using conventional cryopreservation protocols, we found improvement of viability in dCas9- VP64 pretreated cells (P < 0:05) compared to untreated cells. Combined with dCas9-VP64 system, a reduced amount of cryoprotectant (5% DMSO) did not negatively a®ect the post-thaw viability. Co-delivering chemically modi¯ed dCas9-VP64 mRNA with gRNAs is an e±cient gene delivery method compared to DNA-based strategies, without the associated safety concerns. This approach is a simple, yet e®ective way to accelerate a wide array of cellular research and translational medical applications.</p>',
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'pmid' => 'https://www.worldscientific.com/doi/10.1142/S1793984418500046',
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<p><small><sup>*</sup> Please note that the optimal antibody amount per IP should be determined by the end-user. We recommend testing 1-10 μg per IP.</small></p>',
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'description' => '<p>Monoclonal antibody raised in mouse against the <strong>N-terminus of the</strong> <strong>Cas9</strong> <strong>nuclease</strong> (<strong>CRISPR</strong>-<strong>associated protein 9</strong>) using a recombinant protein. </p>',
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<div class="small-8 columns">
<p><small> <strong>Figure 1. ChIP using the Diagenode monoclonal antibody directed against Cas9</strong><br />ChIP was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50 μg chromatin was incubated overnight at 4°C with 5 or 10 μg of either an anti-FLAG antibody or the Diagenode antibody against Cas9 (Cat. No. C15200229). Mouse IgG was used as a negative IP control. qPCR was performed with primers specific for the GFP gene, and for a non-targeted region (protein kinase C delta, Prkcd), used as negative control. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).</small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15200229-WB-A.jpg" alt="CRISPR/Cas9 Antibody for Western Blot " /> <img src="https://www.diagenode.com/img/product/antibodies/C15200229-WB-B.jpg" alt="CRISPR/Cas9 Antibody for Western Blot " /></p>
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<div class="small-8 columns">
<p><small><strong>Figure 2. Western blot analysis using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />Figure 1A: Western blot was performed on protein extracts from HeLa cells transfected with Cas9 using the Diagenode antibody against CRISPR/Cas9 (Cat. No. C15200229). The antibody was used at different dilutions. The marker is shown on the left, position of the Cas9 protein is indicated on the right. Figure 1B: Western blot was performed on protein extracts from HeLa cells transfected with Cas9 (lane 1) or from untransfected cells (lane 2) using the Diagenode antibody against CRISPR/Cas9 (Cat. No. C15200229), diluted 1:4,000 in PBS-T containing 3% NFDM. The marker is shown on the left, position of the Cas9 protein is indicated on the right.</small></p>
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<div class="small-8 columns">
<p><small> <strong>Figure 3. IP using the Diagenode monoclonal antibody directed against Cas9</strong> <br /> IP was performed on whole cell extracts (300 μg) from HEK293 cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 6 μg of the Diagenode antibody against Cas9 (Cat. No. C15200229). The immunoprecipitated proteins were subsequently analysed by Western blot with the polyclonal Cas9 antibody (Cat. No. C15310258, diluted 1:8,000). Lane 3 and 4 show the result of the IP, the input (15 μg) is shown in lane 1 and 2.</small></p>
</div>
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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15200229-IF.jpg" alt="CRISPR/Cas9 Antibody for Immunofluorescence" /></p>
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<div class="small-8 columns">
<p><small> <strong>Figure 4. Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong> <br /> HeLa cells transfected with a Cas9 expression vector (left) or untransfected cells (right) were fixed in methanol at -20°C, permeabilized with acetone at -20°C and blocked with PBS containing 2% BSA. The cells were stained with the Cas9 N-terminal antibody (Cat. No. C15200229) diluted 1:400, followed by incubation with an anti-mouse secondary antibody coupled to AF488. The bottom images show counter-staining of the nuclei with Hoechst 33342.</small></p>
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<p>Learn more about: <a href="https://www.diagenode.com/applications/western-blot">Loading control, MW marker visualization</a><em>. <br /></em></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15200229-IF.jpg" alt="" height="245" width="256" /></p>
<p><sup><strong>Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong></sup></p>
<p><sup>HeLa cells transfected with a Cas9 expression vector (left) or untransfected cells (right) were fixed in methanol at -20°C, permeabilized with acetone at -20°C and blocked with PBS containing 2% BSA. The cells were stained with the Cas9 C-terminal antibody (Cat. No. C15200229) diluted 1:400, followed by incubation with an anti-mouse secondary antibody coupled to AF488. The bottom images show counter-staining of the nuclei with Hoechst 33342.</sup></p>
<h5><sup>Check our selection of antibodies validated in IF.</sup></h5>',
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<p><span style="font-weight: 400;">Diagenode’s highly validated antibodies:</span></p>
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<li>Highly sensitive and specific</li>
<li>Cost-effective (requires less antibody per reaction)</li>
<li>Batch-specific data is available on the website</li>
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'description' => '<p>Diagenode offers the broad range of antibodies raised against the N- or C-terminus of the Cas9 nuclease from <em>Streptococcus <g class="gr_ gr_5 gr-alert gr_spell gr_disable_anim_appear ContextualSpelling ins-del multiReplace" id="5" data-gr-id="5">pyogenes</g></em>. These highly specific polyclonal and monoclonal antibodies are validated in Western blot, immunoprecipitation, immunofluorescence and in chromatin immunoprecipitation.</p>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h2><em><a name="pyogenes"></a>S. pyogenes</em> CRISPR/Cas9 antibodies<a></a></h2>
<div class="panel">
<h2>Discover our first monoclonal CRISPR/Cas9 antibody validated in ChIP<br /><br /></h2>
<div class="row">
<div class="small-5 medium-5 large-5 columns"><img src="/img/landing-pages/crispr-cas9-chip-on-hih3t3.jpg" alt="" /></div>
<div class="small-7 medium-7 large-7 columns">
<ul>
<li>Validated in chromatin immunoprecipitation</li>
<li>Performs better than FLAG antibody</li>
<li>Excellent for WB, IF and IP</li>
</ul>
<p><small><strong>ChIP</strong> was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50 µg chromatin was incubated overnight at 4°C with 5 or 10 µg of either an anti-FLAG antibody or the Diagenode antibody against Cas9 (Cat. No. C15200229). Mouse IgG was used as a negative IP control. qPCR was performed with primers specific for the GFP gene, and for a non-targeted region (protein kinase C delta, Prkcd), used as negative control. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).</small></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns text-right"><a href="/p/crispr-cas9-monoclonal-antibody-50-ug-25-μl" class="tiny details button radius">Learn more</a></div>
</div>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>First ChIP-grade CRISPR/Cas9 polyclonal antibody</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/landing-pages/c_a_s9-chip-grade-antibody.png" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Excellent polyclonal antibody for chromatin immunoprecipitation</li>
<li>Optimized for highest ChIP specificity and yields</li>
<li>Validated for all applications including immunoblotting, immunofluorescence and western blot</li>
</ul>
<p><small><strong>ChIP</strong> was performed on NIH3T3 cells stably expressing GFP- H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50 μg chromatin was incubated with either 5 μg of an anti-FLAG antibody or 2 μl of the Diagenode antibody against Cas9. The pre-immune serum (PPI) was used as negative IP control. Then qPCR was performed with primers specific for the GFP gene, and for two non-targeted regions: Ppap2c and Prkcd, used as negative controls. This figure shows the recovery, expressed as a % of input.</small></p>
<p class="text-right"><a href="../p/crispr-cas9-polyclonal-antibody" class="details tiny button">Learn more</a></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>CRISPR/Cas9 monoclonal antibody 4G10</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/landing-pages/cas9_4g10_fig1.png" width="170" height="302" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Antibody raised against N-terminus of Cas9 nuclease</li>
<li>Validated for western blot, IP and immunofluorescence</li>
</ul>
<p><small><strong>Immunofluorescence</strong>: Hela cells were transiently transfected with a Cas9 expression vector. The cells were fixed in 3.7% formaldehyde, permeabilized in 0.5% Triton-X-100 and blocked in PBS containing 2% BSA. The cells were stained with the Cas9 antibody at 4°C o/n, followed by incubation with an anti mouse secondary antibody coupled to AF488 for 1 h at RT. Nuclei were counter-stained with Hoechst 33342 (right).</small></p>
<p class="text-right"><a href="../p/crispr-cas9-monoclonal-antibody-4g10-50-ug" class="details tiny button">Learn more</a></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>CRISPR/Cas9 C-terminal monoclonal antibody</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/product/antibodies/C15200223-IP.png" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Antibody raised against C-terminus of Cas9 nuclease</li>
<li>Validated for western blot, IP and immunofluorescence</li>
</ul>
<p><small><strong> Western blot</strong> was performed on 20 μg protein extracts from Cas9 expressing HeLa cells (lane 1) and on negative control HeLa cells (lane 2) with the Diagenode antibody against Cas9. The antibody was diluted 1:4,000. The marker is shown on the left, position of the Cas9 protein is indicated on the right. </small></p>
<p class="text-right"><a href="../p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug" class="details tiny button">Learn more</a></p>
</div>
<div class="small-12 medium-12 large-12 columns">
<h3>Which CRISPR/Cas9 antibody is the best for your application?</h3>
<a name="table"></a>
<table>
<thead>
<tr>
<th>Antibody</th>
<th>WB</th>
<th>IF</th>
<th>IP</th>
<th>ChIP</th>
<th>Antibody raised against</th>
</tr>
</thead>
<tbody>
<tr>
<td><a href="../p/crispr-cas9-monoclonal-antibody-50-ug-25-μl"><strong class="diacol">CRISPR/Cas9 monoclonal antibody</strong></a></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><span class="diacol">N-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-polyclonal-antibody">CRISPR/Cas9 polyclonal antibody</a></td>
<td>++</td>
<td>++</td>
<td>++</td>
<td><strong class="diacol">+++</strong></td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-monoclonal-antibody-4g10-50-ug">CRISPR/Cas9 monoclonal antibody 4G10</a></td>
<td>+++</td>
<td>+++</td>
<td>++</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="../p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug-23-ul"><strong class="diacol">CRISPR/Cas9 C-terminal monoclonal antibody</strong></a> <span class="label alert" style="font-size: 0.9rem;">NEW!</span></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">no</strong></td>
<td><strong class="diacol">+</strong></td>
<td><span class="diacol">C-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug">CRISPR/Cas9 C-terminal monoclonal antibody</a></td>
<td>++</td>
<td>++</td>
<td>+</td>
<td>no</td>
<td><span class="diacol">C-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-monoclonal-antibody-7A9-50-mg">CRISPR/Cas9 monoclonal antibody 7A9</a></td>
<td>++</td>
<td>++</td>
<td>++</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-hrp-monoclonal-antibody-50-ul">CRISPR/Cas9 - HRP monoclonal antibody 7A9</a></td>
<td>+++</td>
<td>no</td>
<td>no</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
</tbody>
</table>
</div>
</div>
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'description' => '<div class="row">
<div class="small-12 columns"><center></center>
<p><br />Chromatin immunoprecipitation (<b>ChIP</b>) is a technique to study the associations of proteins with the specific genomic regions in intact cells. One of the most important steps of this protocol is the immunoprecipitation of targeted protein using the antibody specifically recognizing it. The quality of antibodies used in ChIP is essential for the success of the experiment. Diagenode offers extensively validated ChIP-grade antibodies, confirmed for their specificity, and high level of performance in ChIP. Each batch is validated, and batch-specific data are available on the website.</p>
<p></p>
</div>
</div>
<p><strong>ChIP results</strong> obtained with the antibody directed against H3K4me3 (Cat. No. <a href="../p/h3k4me3-polyclonal-antibody-premium-50-ug-50-ul">C15410003</a>). </p>
<div class="row">
<div class="small-12 medium-6 large-6 columns"><img src="https://www.diagenode.com/img/product/antibodies/C15410003-fig1-ChIP.jpg" alt="" width="400" height="315" /> </div>
<div class="small-12 medium-6 large-6 columns">
<p></p>
<p></p>
<p></p>
</div>
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<p></p>
<p>Our aim at Diagenode is to offer the largest collection of highly specific <strong>ChIP-grade antibodies</strong>. We add new antibodies monthly. Find your ChIP-grade antibody in the list below and check more information about tested applications, extensive validation data, and product information.</p>',
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'name' => ' Accurate QC to optimize CRISPR/Cas9 genome editing specificity',
'description' => '<p>The CRISPR/Cas9 technology is delivering superior genetic models for fundamental disease research, drug screening, therapy development, rapid diagnostics, and transcriptional modulation. Although CRISPR/Cas9 enables rapid genome editing, several aspects affect its efficiency and specificity including guide RNA design, delivery methods, and off-targets effects. Diagenode has developed strategies to overcome these common pitfalls and has optimized CRISPR/Cas9 genome editing specificity</p>',
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'name' => 'Genome-wide CRISPR screen identifies protein pathways modulating tauprotein levels in neurons',
'authors' => 'Sanchez C. G. et al. ',
'description' => '<p>Aggregates of hyperphosphorylated tau protein are a pathological hallmark of more than 20 distinct neurodegenerative diseases, including Alzheimer’s disease, progressive supranuclear palsy, and frontotemporal dementia. While the exact mechanism of tau aggregation is unknown, the accumulation of aggregates correlates with disease progression. Here we report a genome-wide CRISPR screen to identify modulators of endogenous tau protein for the first time. Primary screens performed in SH-SY5Y cells, identified positive and negative regulators of tau protein levels. Hit validation of the top 43 candidate genes was performed using Ngn2-induced human cortical excitatory neurons. Using this approach, genes and pathways involved in modulation of endogenous tau levels were identified, including chromatin modifying enzymes, neddylation and ubiquitin pathway members, and components of the mTOR pathway. TSC1, a critical component of the mTOR pathway, was further validated in vivo, demonstrating the relevance of this screening strategy. These findings may have implications for treating neurodegenerative diseases in the future.</p>',
'date' => '2021-06-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/34127790',
'doi' => '10.1038/s42003-021-02272-1',
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'name' => 'Transgenic mice for in vivo epigenome editing with CRISPR-based systems',
'authors' => 'Gemberling, M. et al.',
'description' => '<p>The discovery, characterization, and adaptation of the RNA-guided clustered regularly interspersed short palindromic repeat (CRISPR)-Cas9 system has greatly increased the ease with which genome and epigenome editing can be performed. Fusion of chromatin-modifying domains to the nuclease-deactivated form of Cas9 (dCas9) has enabled targeted gene activation or repression in both cultured cells and in vivo in animal models. However, delivery of the large dCas9 fusion proteins to target cell types and tissues is an obstacle to widespread adoption of these tools for in vivo studies. Here we describe the generation and validation of two conditional transgenic mouse lines for targeted gene regulation, Rosa26:LSL-dCas9-p300 for gene activation and Rosa26:LSL-dCas9-KRAB for gene repression. Using the dCas9p300 and dCas9KRAB transgenic mice we demonstrate activation or repression of genes in both the brain and liver in vivo, and T cells and fibroblasts ex vivo. We show gene regulation and targeted epigenetic modification with gRNAs targeting either transcriptional start sites (TSS) or distal enhancer elements, as well as corresponding changes to downstream phenotypes. These mouse lines are convenient and valuable tools for facile, temporally controlled, and tissue-restricted epigenome editing and manipulation of gene expression in vivo.</p>',
'date' => '2021-03-01',
'pmid' => 'https://doi.org/10.1101%2F2021.03.08.434430',
'doi' => '10.1101/2021.03.08.434430',
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'id' => '4047',
'name' => 'Circuit-specific hippocampal ΔFosB underlies resilience to
stress-induced social avoidance.',
'authors' => 'Eagle, Andrew L and Manning, Claire E and Williams, Elizabeth S and Bastle,
Ryan M and Gajewski, Paula A and Garrison, Amber and Wirtz, Alexis J and
Akguen, Seda and Brandel-Ankrapp, Katie and Endege, Wilson and Boyce,
Frederick M and Ohnishi, Yoshinori N',
'description' => 'Chronic stress is a key risk factor for mood disorders like depression, but
the stress-induced changes in brain circuit function and gene expression
underlying depression symptoms are not completely understood, hindering
development of novel treatments. Because of its projections to brain
regions regulating reward and anxiety, the ventral hippocampus is uniquely
poised to translate the experience of stress into altered brain function
and pathological mood, though the cellular and molecular mechanisms of this
process are not fully understood. Here, we use a novel method of
circuit-specific gene editing to show that the transcription factor
ΔFosB drives projection-specific activity of ventral hippocampus
glutamatergic neurons causing behaviorally diverse responses to stress. We
establish molecular, cellular, and circuit-level mechanisms for depression-
and anxiety-like behavior in response to stress and use circuit-specific
gene expression profiling to uncover novel downstream targets as potential
sites of therapeutic intervention in depression.',
'date' => '2020-09-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/32901027',
'doi' => '10.1038/s41467-020-17825-x',
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'name' => 'Endogenous retroviruses are a source of enhancers with oncogenic potential in acute myeloid leukaemia',
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'description' => '<p>Acute myeloid leukemia (AML) is a highly aggressive hematopoietic malignancy, defined by a series of genetic and epigenetic alterations, which result in deregulation of transcriptional networks. One understudied but important source of transcriptional regulators are transposable elements (TEs), which are widespread throughout the human genome. Aberrant usage of these sequences could therefore contribute to oncogenic transcriptional circuits. However, the regulatory influence of TEs and their links to disease pathogenesis remain unexplored in AML. Using epigenomic data from AML primary samples and leukemia cell lines, we identified six endogenous retrovirus (ERV) families with AML-associated enhancer chromatin signatures that are enriched in binding of key regulators of hematopoiesis and AML pathogenesis. Using both CRISPR-mediated locus-specific genetic editing and simultaneous epigenetic silencing of multiple ERVs, we demonstrate that ERV deregulation directly alters the expression of adjacent genes in AML. Strikingly, deletion or epigenetic silencing of an ERV-derived enhancer suppressed cell growth by inducing apoptosis in leukemia cell lines. Our work reveals that ERVs are a previously unappreciated source of AML enhancers that have the potential to play key roles in leukemogenesis. We suggest that ERV activation provides an additional layer of gene regulation in AML that may be exploited by cancer cells to help drive tumour heterogeneity and evolution.</p>',
'date' => '2020-04-04',
'pmid' => 'https://www.biorxiv.org/content/10.1101/772954v2',
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'name' => 'Improved double-nicking strategies for COL7A1 editing by homologous recombination',
'authors' => 'Kocher Thomas, Wagner Roland N., Klausegger Alfred, Guttmann-Gruber Christina, Hainzl Stefan, Bauer Johann W., Reichelt Julia, Koller Ulrich',
'description' => '<p>Current gene editing approaches for treatment of recessive dystrophic epidermolysis bullosa (RDEB), an inherited, severe form of blistering skin disease, suffer from low efficiencies and safety concerns that complicate implementation in clinical settings. We present a strategy for efficient and precise repair of RDEB-associated mutations in the COL7A1 gene. We compared the efficacy of double-strand breaks (induced by CRISPR/Cas9), single-nicks, or double-nicks (induced by Cas9n) in mediating repair of a COL7A1 splice-site mutation in exon 3 by homologous recombination (HR). We accomplished remarkably high HR frequencies of 89% with double-nicking while at the same time kept unwanted repair outcomes, such as non-homologous end joining (NHEJ), at a minimum (11%). We also investigated the effects of subtle differences in repair template design on HR-rates and found that strategic template nicking can enhance COL7A1 editing efficiency. In RDEB patient keratinocytes, application of double-nicking led to restoration and subsequent secretion of type VII collagen at high efficiency. Comprehensive analysis of 25 putative off-target sites revealed no off-target activity for double-nicking, while usage of Cas9 resulted in 54% modified alleles at one site. Taken together, our work provides a framework for efficient, precise, and safe repair of COL7A1, which lies at the heart of a future curative therapy of RDEB.</p>',
'date' => '2019-09-12',
'pmid' => 'https://www.cell.com/molecular-therapy-family/nucleic-acids/fulltext/S2162-2531(19)30258-6',
'doi' => '10.1016/j.omtn.2019.09.011',
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'name' => 'Improved Double-Nicking Strategies for COL7A1-Editing by Homologous Recombination.',
'authors' => 'Kocher T, Wagner RN, Klausegger A, Guttmann-Gruber C, Hainzl S, Bauer JW, Reichelt J, Koller U',
'description' => '<p>Current gene-editing approaches for treatment of recessive dystrophic epidermolysis bullosa (RDEB), an inherited, severe form of blistering skin disease, suffer from low efficiencies and safety concerns that complicate implementation in clinical settings. We present a strategy for efficient and precise repair of RDEB-associated mutations in the COL7A1 gene. We compared the efficacy of double-strand breaks (induced by CRISPR/Cas9), single nicks, or double nicks (induced by Cas9n) in mediating repair of a COL7A1 splice-site mutation in exon 3 by homologous recombination (HR). We accomplished remarkably high HR frequencies of 89% with double nicking while at the same time keeping unwanted repair outcomes, such as non-homologous end joining (NHEJ), at a minimum (11%). We also investigated the effects of subtle differences in repair template design on HR rates and found that strategic template-nicking can enhance COL7A1-editing efficiency. In RDEB patient keratinocytes, application of double-nicking led to restoration and subsequent secretion of type VII collagen at high efficiency. Comprehensive analysis of 25 putative off-target sites revealed no off-target activity for double-nicking, while usage of Cas9 resulted in 54% modified alleles at one site. Taken together, our work provides a framework for efficient, precise, and safe repair of COL7A1, which lies at the heart of a future curative therapy of RDEB.</p>',
'date' => '2019-09-12',
'pmid' => 'http://www.pubmed.gov/31670199',
'doi' => '10.1016/j.omtn.2019.09.011.',
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'name' => 'Essential Gene Profiles for Human Pluripotent Stem Cells Identify Uncharacterized Genes and Substrate Dependencies.',
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'description' => '<p>Human pluripotent stem cells (hPSCs) provide an invaluable tool for modeling diseases and hold promise for regenerative medicine. For understanding pluripotency and lineage differentiation mechanisms, a critical first step involves systematically cataloging essential genes (EGs) that are indispensable for hPSC fitness, defined as cell reproduction in this study. To map essential genetic determinants of hPSC fitness, we performed genome-scale loss-of-function screens in an inducible Cas9 H1 hPSC line cultured on feeder cells and laminin to identify EGs. Among these, we found FOXH1 and VENTX, genes that encode transcription factors previously implicated in stem cell biology, as well as an uncharacterized gene, C22orf43/DRICH1. hPSC EGs are substantially different from other human model cell lines, and EGs in hPSCs are highly context dependent with respect to different growth substrates. Our CRISPR screens establish parameters for genome-wide screens in hPSCs, which will facilitate the characterization of unappreciated genetic regulators of hPSC biology.</p>',
'date' => '2019-04-09',
'pmid' => 'http://www.pubmed.gov/30970261',
'doi' => '10.1016/j.celrep.2019.02.041',
'modified' => '2019-08-07 09:17:29',
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'name' => 'RNA-Based dCas9–VP64 System Improves the Viability of Cryopreserved Mammalian Cells',
'authors' => 'Hu Yong, Li Lei, Yu Yin, Huang Haishui, Uygun Basak E., Yarmush Martin L.',
'description' => '<p>Regenerative therapies require availability of an abundant healthy cell source which can be achieved by e±cient cryopreservation techniques. Here, we established a novel approach for improved cell cryopreservation using an mRNA-based dCas9-VP64 gene activation system for transient, yet highly e±cient expression of epigenetic related genes in mammalian cells for repression of metabolic activity. Before freezing, mammalian cells were treated by dCas9-VP64- modi¯ed mRNA and guide RNAs for upregulation of histone deacetylase (HDAC), DNA methyltransferase (DNMT) and transcriptional co-repressor Sin3A genes. Cell viability, karyotype, pluripotency, and other cell speci¯c functions were analyzed during post-thaw culture. Using conventional cryopreservation protocols, we found improvement of viability in dCas9- VP64 pretreated cells (P < 0:05) compared to untreated cells. Combined with dCas9-VP64 system, a reduced amount of cryoprotectant (5% DMSO) did not negatively a®ect the post-thaw viability. Co-delivering chemically modi¯ed dCas9-VP64 mRNA with gRNAs is an e±cient gene delivery method compared to DNA-based strategies, without the associated safety concerns. This approach is a simple, yet e®ective way to accelerate a wide array of cellular research and translational medical applications.</p>',
'date' => '2018-01-01',
'pmid' => 'https://www.worldscientific.com/doi/10.1142/S1793984418500046',
'doi' => '10.1142/S1793984418500046',
'modified' => '2019-02-18 11:55:37',
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'name' => 'CRISPR-Mediated Gene Targeting of Human Induced Pluripotent Stem Cells',
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'description' => '<p><span>CRISPR/Cas9 nuclease systems can create double-stranded DNA breaks at specific sequences to efficiently and precisely disrupt, excise, mutate, insert, or replace genes. However, human embryonic stem cells and induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, an optimized protocol is described for genome engineering of human iPSCs using simple transient transfection of plasmids and/or single-stranded oligonucleotides without any further selection or enrichment steps. This protocol achieves transfection efficiencies >60%, with gene disruption efficiencies of 1-25% and gene insertion/replacement efficiencies of 0.5-10%. Details are also provided for designing optimal sgRNA target sites and donor targeting vectors, cloning individual iPSCs by single-cell FACS sorting, and genotyping successfully edited cells.</span></p>',
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'name' => ' Accurate QC to optimize CRISPR/Cas9 genome editing specificity',
'description' => '<p>The CRISPR/Cas9 technology is delivering superior genetic models for fundamental disease research, drug screening, therapy development, rapid diagnostics, and transcriptional modulation. Although CRISPR/Cas9 enables rapid genome editing, several aspects affect its efficiency and specificity including guide RNA design, delivery methods, and off-targets effects. Diagenode has developed strategies to overcome these common pitfalls and has optimized CRISPR/Cas9 genome editing specificity</p>',
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'name' => 'CRISPR-Mediated Gene Targeting of Human Induced Pluripotent Stem Cells',
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'description' => '<p><span>CRISPR/Cas9 nuclease systems can create double-stranded DNA breaks at specific sequences to efficiently and precisely disrupt, excise, mutate, insert, or replace genes. However, human embryonic stem cells and induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, an optimized protocol is described for genome engineering of human iPSCs using simple transient transfection of plasmids and/or single-stranded oligonucleotides without any further selection or enrichment steps. This protocol achieves transfection efficiencies >60%, with gene disruption efficiencies of 1-25% and gene insertion/replacement efficiencies of 0.5-10%. Details are also provided for designing optimal sgRNA target sites and donor targeting vectors, cloning individual iPSCs by single-cell FACS sorting, and genotyping successfully edited cells.</span></p>',
'date' => '2015-11-04',
'pmid' => 'http://onlinelibrary.wiley.com/doi/10.1002/9780470151808.sc05a08s35/abstract',
'doi' => '10.1002/9780470151808.sc05a08s35',
'modified' => '2016-02-10 16:29:28',
'created' => '2016-02-10 16:29:28',
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$externalLink = ' <a href="http://onlinelibrary.wiley.com/doi/10.1002/9780470151808.sc05a08s35/abstract" target="_blank"><i class="fa fa-external-link"></i></a>'include - APP/View/Products/view.ctp, line 755
View::_evaluate() - CORE/Cake/View/View.php, line 971
View::_render() - CORE/Cake/View/View.php, line 933
View::render() - CORE/Cake/View/View.php, line 473
Controller::render() - CORE/Cake/Controller/Controller.php, line 963
ProductsController::slug() - APP/Controller/ProductsController.php, line 1052
ReflectionMethod::invokeArgs() - [internal], line ??
Controller::invokeAction() - CORE/Cake/Controller/Controller.php, line 491
Dispatcher::_invoke() - CORE/Cake/Routing/Dispatcher.php, line 193
Dispatcher::dispatch() - CORE/Cake/Routing/Dispatcher.php, line 167
[main] - APP/webroot/index.php, line 118
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