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<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-fig1.png" alt="CRISPR/Cas9 Antibody ChIP Grade" caption="false" width="241" height="286" /></p>
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<p><small><strong> Figure 1. ChIP using the Diagenode antibody directed against Cas9 </strong><br />ChIP was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50μg chromatin was incubated overnight at 4°C with either 5 μg of an anti-FLAG antibody or 2 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The pre-immune serum (Cas9, PPI) was used as negative IP control. qPCR was performed with primers specific for the GFP gene, and for two non-targeted regions phosphatidic acid phosphatase type 2C (Ppap2c) and protein kinase C delta (Prkcd), used as negative controls. 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 antibody directed against Cas9 </strong><br />Western blot was performed on protein extracts from HeLa cells transfected with Cas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:1,000 (lane 2) or 1:5,000 (lane 3). Lane 1 shows the result with the pre-immune serum. The marker is shown on the left, the 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 (500 μg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 μg) is shown in lane 1 and 2. </small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" alt="CRISPR/Cas9 Antibody Validation in IF" caption="false" width="278" height="123" /></p>
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<p><small><strong> Figure 4. Immunofluorescence using the Diagenode antibody directed against Cas9 </strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counterstained with Hoechst 33342. Figure 4 shows the result in the presence (left) or absence (right) of doxycycline. </small></p>
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<td>ChIP/ChIP-seq <sup>*</sup></td>
<td>2-5 µl/ChIP</td>
<td>Fig 1, 2</td>
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<td>Western Blotting</td>
<td>1:5,000</td>
<td>Fig 3</td>
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<td>1 µl/IP</td>
<td>Fig 4</td>
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<td>1:1,000</td>
<td>Fig 5</td>
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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 µl per IP.</small></p>',
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'description' => '<p>Polyclonal antibody raised in rabbit against the <strong>Cas9</strong> nuclease (<strong>CRISPR</strong>-associated protein 9) using a recombinant protein. </p>',
'label1' => 'Validation data',
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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-fig1.png" alt="CRISPR/Cas9 Antibody ChIP Grade" caption="false" width="241" height="286" /></p>
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<p><small><strong> Figure 1. ChIP using the Diagenode antibody directed against Cas9 </strong><br />ChIP was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50μg chromatin was incubated overnight at 4°C with either 5 μg of an anti-FLAG antibody or 2 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The pre-immune serum (Cas9, PPI) was used as negative IP control. qPCR was performed with primers specific for the GFP gene, and for two non-targeted regions phosphatidic acid phosphatase type 2C (Ppap2c) and protein kinase C delta (Prkcd), used as negative controls. 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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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-WB.png" alt="CRISPR/Cas9 Antibody Validation in WB " caption="false" width="171" height="192" /></p>
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<p><small><strong> Figure 2. Western blot analysis using the Diagenode antibody directed against Cas9 </strong><br />Western blot was performed on protein extracts from HeLa cells transfected with Cas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:1,000 (lane 2) or 1:5,000 (lane 3). Lane 1 shows the result with the pre-immune serum. The marker is shown on the left, the position of the Cas9 protein is indicated on the right. </small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IP.png" alt="CRISPR/Cas9 Antibody Validation in IP" caption="false" width="252" height="286" /></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 (500 μg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 μg) is shown in lane 1 and 2. </small></p>
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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" alt="CRISPR/Cas9 Antibody Validation in IF" caption="false" width="278" height="123" /></p>
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<div class="small-8 columns">
<p><small><strong> Figure 4. Immunofluorescence using the Diagenode antibody directed against Cas9 </strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counterstained with Hoechst 33342. Figure 4 shows the result in the presence (left) or absence (right) of doxycycline. </small></p>
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'clonality' => '',
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'lot' => 'A2508-004',
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'type' => 'Polyclonal, <strong>ChIP grade, ChIP-seq grade</strong>',
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<thead>
<tr>
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<th>Suggested dilution</th>
<th>References</th>
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<tbody>
<tr>
<td>ChIP/ChIP-seq <sup>*</sup></td>
<td>2-5 µl/ChIP</td>
<td>Fig 1, 2</td>
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<tr>
<td>Western Blotting</td>
<td>1:5,000</td>
<td>Fig 3</td>
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<tr>
<td>Immunoprecipitation</td>
<td>1 µl/IP</td>
<td>Fig 4</td>
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<tr>
<td>Immunofluorescence</td>
<td>1:1,000</td>
<td>Fig 5</td>
</tr>
</tbody>
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<p></p>
<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 µl per IP.</small></p>',
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'name' => 'CRISPR/Cas9 Antibody',
'description' => '<p>Polyclonal antibody raised in rabbit against the <strong>Cas9</strong> nuclease (<strong>CRISPR</strong>-associated protein 9) using a recombinant protein. </p>',
'label1' => 'Validation data',
'info1' => '<div class="row">
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-fig1.png" /></center></div>
<div class="small-6 columns">
<p><strong>Figure 1. ChIP using the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing nuclease dead Cas9 and sgRNA targeting a sequence in intron 8 of the GAPDH gene, using the iDeal ChIP-seq kit for transcription factors. A titration consisting of 1, 2, 5 and 10 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) was tested. IgG (2 µg/IP) was used as negative IP control. qPCR was performed with primers specific for the targeted sequence in the GAPDH gene, and for the MYOD1 gene, 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).</p>
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<div class="row">
<div class="small-12 columns"><center>A.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-a.jpg" width="700" /></center><center>B.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-b.jpg" width="700" /></center></div>
</div>
<div class="row">
<div class="small-12 columns">
<p><strong>Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing dCas9 and a GAPDH sgRNA cells using 2 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) as described above. The IP'd DNA was subsequently analysed on an Illumina NovaSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the ChIP-seq profile in a region of chromosome 12 surrounding the GAPDH gene (fig 2B) and in a region of chromosome 2 surrounding an off-target peak in the YIPF4 gene.</p>
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</div>
<div class="row">
<div class="small-4 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-WB.png" /></center></div>
<div class="small-8 columns">
<p><strong>Figure 3. Western blot analysis using the Diagenode antibody directed against Cas9</strong><br />Western blot was performed on protein extracts from HEK293 cells transfected with dCas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:5,000. The marker is shown on the left, the position of the Cas9 protein is indicated on the right.</p>
</div>
</div>
<div class="row">
<div class="small-4 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IP.png" /></center></div>
<div class="small-8 columns">
<p><strong>Figure 4. IP using the Diagenode antibody directed against Cas9</strong><br />IP was performed on whole cell extracts (500 µg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 µl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 µg) is shown in lane 1 and 2.</p>
</div>
</div>
<div class="row">
<div class="small-12 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" width="500" /></center></div>
</div>
<div class="row">
<div class="small-12 columns">
<p><strong>Figure 5. Immunofluorescence using the Diagenode antibody directed against Cas9</strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counter-stained with Hoechst 33342. Figure 5 shows the result in the presence (left) or absence (right) of doxycycline.</p>
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'info2' => '<p style="text-align: justify;">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 <strong>CRISPR/Cas9</strong> (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 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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'meta_description' => 'S. pyogenes CRISPR/Cas9 Polyclonal Antibody validated in ChIP-seq, ChIP-qPCR, IP, IF and WB. Batch-specific data available on the website. Sample size available.',
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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' => '<h1><strong>Validated epigenetics antibodies</strong> – care for a sample?<br /> </h1>
<p>Diagenode has partnered with leading epigenetics experts and numerous epigenetics consortiums to bring to you a validated and comprehensive collection of epigenetic antibodies. As an expert in epigenetics, we are committed to offering highly-specific antibodies validated for ChIP/ChIP-seq and many other applications. All batch-specific validation data is available on our website.<br /><a href="../categories/antibodies">Read about our expertise in antibody production</a>.</p>
<ul>
<li><strong>Focused</strong> - Diagenode's selection of antibodies is exclusively dedicated for epigenetic research. <a title="See the full collection." href="../categories/all-antibodies">See the full collection.</a></li>
<li><strong>Strict quality standards</strong> with rigorous QC and validation</li>
<li><strong>Classified</strong> based on level of validation for flexibility of application</li>
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<p>Existing sample sizes are listed below. We will soon expand our collection. Are you looking for a sample size of another antibody? Just <a href="mailto:agnieszka.zelisko@diagenode.com?Subject=Sample%20Size%20Request" target="_top">Contact us</a>.</p>',
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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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'meta_title' => 'Diagenode's selection of Antibodies is exclusively dedicated for Epigenetic Research | Diagenode',
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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>
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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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'description' => '<p><span>The fluorescence ubiquitination cell cycle inhibitor (FUCCI) has been introduced to monitor cell cycle activity in living cells, including human induced pluripotent stem cells (hiPSC) and derived cell types. We have recently developed hiPSC with stable expression of dCas9VPR for endogenous gene activation and a Citrine-tagged ACTN2 cell line to monitor sarcomere development and function in muscle cells. Here, we present dual and triple transgenic hiPSC lines developed by genomic integration of FUCCI with and without dCas9VPR into the ROSA26 and AAVS1 loci, respectively, in the previously introduced ACTN2-Citrine line. Functionality of the transgenes was demonstrated in the novel hiPSC line, which we introduce as Myo-CCER and CraCCER.</span></p>',
'date' => '2024-08-05',
'pmid' => 'https://www.sciencedirect.com/science/article/pii/S1873506124002290',
'doi' => 'https://doi.org/10.1016/j.scr.2024.103531',
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'name' => 'Methyltransferase Inhibition Enables Tgf Driven Induction of and in Cancer Cells.',
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'description' => '<p>deletion or silencing is common across human cancer, reinforcing the general importance of bypassing its tumor suppression in cancer formation or progression. In rhabdomyosarcoma (RMS) and neuroblastoma, two common childhood cancers, the three transcripts are independently expressed to varying degrees, but one, is uniformly silenced. Although TGFβ induces certain transcripts in HeLa cells, it was unable to do so in five tested RMS lines unless the cells were pretreated with a broadly acting methyltransferase inhibitor, DZNep, or one targeting EZH2. induction by TGFβ correlated with de novo appearance of three H3K27Ac peaks within a 20 kb element ∼150 kb proximal to . Deleting that segment prevented their induction by TGFβ but not a basal increase driven by methyltransferase inhibition alone. Expression of two transcripts was enhanced by dCas9/CRISPR activation targeting either the relevant promoter or the 20 kb elements, and this "precise" manipulation diminished RMS cell propagation in vitro. Our findings show crosstalk between methyltransferase inhibition and TGFβ-dependent activation of a remote enhancer to reverse silencing. Though focused on here, such crosstalk may apply to other TGFβ-responsive genes and perhaps govern this signaling protein's complex effects promoting or blocking cancer.</p>',
'date' => '2023-01-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/36941772',
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'name' => 'Massively parallel multi-target CRISPR system interrogates Cas9-basedtarget recognition, DNA cleavage, and DNA repair',
'authors' => 'Zou Roger S. et al.',
'description' => '<p>CRISPR-Cas9 nucleases, and particularly Streptococcus pyogenes Cas9, are widespread tools for genome editing. However, many aspects of intracellular Cas9 activity and the ensuing DNA damage response remain incompletely characterized. In order to address these issues, we developed a multiplexed CRISPR approach, where a single, degenerate multi-target gRNA (mgRNA) directs the Cas9 enzyme to target hundred endogenous sites at once. When combined with next-generation sequencing readouts, this system enables interrogation of Cas9 activity and DNA double-strand break (DSB) repair response in high-throughput. Here, we present a step-by-step protocol to deliver a Cas9:mgRNA ribonucleoprotein complex into cultured cells and measure key processes related to Cas9 activity and DSB repair.</p>',
'date' => '2022-09-01',
'pmid' => 'https://europepmc.org/article/ppr/ppr540155',
'doi' => '10.21203/rs.3.pex-1938/v1',
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(int) 3 => array(
'id' => '4437',
'name' => 'Analysis of estrogen-regulated enhancer RNAs identifies a functionalmotif required for enhancer assembly and gene expression.',
'authors' => 'Hou Tim Y and Kraus W Lee',
'description' => '<p>To better understand the functions of non-coding enhancer RNAs (eRNAs), we annotated the estrogen-regulated eRNA transcriptome in estrogen receptor α (ERα)-positive breast cancer cells using PRO-cap and RNA sequencing. We then cloned a subset of the eRNAs identified, fused them to single guide RNAs, and targeted them to their ERα enhancers of origin using CRISPR/dCas9. Some of the eRNAs tested modulated the expression of cognate, but not heterologous, target genes after estrogen treatment by increasing ERα recruitment and stimulating p300-catalyzed H3K27 acetylation at the enhancer. We identified a ∼40 nucleotide functional eRNA regulatory motif (FERM) present in many eRNAs that was necessary and sufficient to modulate gene expression, but not the specificity of activation, after estrogen treatment. The FERM interacted with BCAS2, an RNA-binding protein amplified in breast cancers. The ectopic expression of a targeted eRNA controlling the expression of an oncogene resulted in increased cell proliferation, demonstrating the regulatory potential of eRNAs in breast cancer.</p>',
'date' => '2022-06-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/35705040',
'doi' => '10.1016/j.celrep.2022.110944',
'modified' => '2022-09-28 09:20:34',
'created' => '2022-09-08 16:32:20',
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(int) 4 => array(
'id' => '4560',
'name' => 'Avian influenza viruses suppress innate immunity by inducingtrans-transcriptional readthrough via SSU72.',
'authors' => 'Zhao Y. et al.',
'description' => '<p>Innate immunity plays critical antiviral roles. The highly virulent avian influenza viruses (AIVs) H5N1, H7N9, and H5N6 can better escape host innate immune responses than the less virulent seasonal H1N1 virus. Here, we report a mechanism by which transcriptional readthrough (TRT)-mediated suppression of innate immunity occurs post AIV infection. By using cell lines, mouse lungs, and patient PBMCs, we showed that genes on the complementary strand ("trans" genes) influenced by TRT were involved in the disruption of host antiviral responses during AIV infection. The trans-TRT enhanced viral lethality, and TRT abolishment increased cell viability and STAT1/2 expression. The viral NS1 protein directly bound to SSU72, and degradation of SSU72 induced TRT. SSU72 overexpression reduced TRT and alleviated mouse lung injury. Our results suggest that AIVs infection induce TRT by reducing SSU72 expression, thereby impairing host immune responses, a molecular mechanism acting through the NS1-SSU72-trans-TRT-STAT1/2 axis. Thus, restoration of SSU72 expression might be a potential strategy for preventing AIV pandemics.</p>',
'date' => '2022-03-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/35332300',
'doi' => '10.1038/s41423-022-00843-8',
'modified' => '2022-11-24 10:02:04',
'created' => '2022-11-24 08:49:52',
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'id' => '4365',
'name' => 'Antisense non-coding transcription represses the <i>PHO5</i> model gene<i>via</i> remodelling of promoter chromatin structure',
'authors' => 'Novačić A. et al. ',
'description' => '<p>Pervasive transcription of eukaryotic genomes generates non-coding transcripts with regulatory potential. We examined the effects of non-coding antisense transcription on the regulation of expression of the yeast PHO5 gene, a paradigmatic case for gene regulation through promoter chromatin remodeling. By enhancing or impairing the level of overlapping antisense transcription through specific mutant backgrounds and the use of the CRISPRi system, we demonstrated a negative role for antisense transcription at the PHO5 gene locus. Furthermore, enhanced elongation of PHO5 antisense leads to a more repressive chromatin configuration at the PHO5 gene promoter, which is remodeled more slowly upon gene induction. The negative effect of antisense transcription on PHO5 gene transcription is mitigated by inactivation of the histone deacetylase Rpd3, showing that PHO5 antisense RNA acts via histone deacetylation. This regulatory pathway leads to Rpd3-dependent decreased recruitment of the RSC chromatin remodeling complex to the PHO5 gene promoter upon induction of antisense transcription. Overall, we extend the model of PHO5 gene regulation by demonstrating a gene silencing function of antisense transcription through a chromatin-based mechanism.</p>',
'date' => '2022-02-01',
'pmid' => 'https://doi.org/10.1101%2F2022.02.21.481265',
'doi' => '10.1101/2022.02.21.481265',
'modified' => '2022-08-04 15:52:12',
'created' => '2022-08-04 14:55:36',
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(int) 6 => array(
'id' => '4237',
'name' => 'A predominant enhancer co-amplified with the oncogene is necessary andsufficient for its expression in squamous cancer',
'authors' => 'Liu Y. et al.',
'description' => '<p>Amplification and overexpression of the SOX2 oncogene represent a hallmark of squamous cancers originating from diverse tissue types. Here, we find that squamous cancers selectively amplify a 3’ noncoding region together with SOX2, which harbors squamous cancer-specific chromatin accessible regions. We identify a single enhancer e1 that predominantly drives SOX2 expression. Repression of e1 in SOX2-high cells causes collapse of the surrounding enhancers, remarkable reduction in SOX2 expression, and a global transcriptional change reminiscent of SOX2 knockout. The e1 enhancer is driven by a combination of transcription factors including SOX2 itself and the AP-1 complex, which facilitates recruitment of the co-activator BRD4. CRISPR-mediated activation of e1 in SOX2-low cells is sufficient to rebuild the e1-SOX2 loop and activate SOX2 expression. Our study shows that squamous cancers selectively amplify a predominant enhancer to drive SOX2 overexpression, uncovering functional links among enhancer activation, chromatin looping, and lineage-specific copy number amplifications of oncogenes.</p>',
'date' => '2021-12-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/34880227',
'doi' => '10.1038/s41467-021-27055-4',
'modified' => '2022-05-19 17:05:00',
'created' => '2022-05-19 10:41:50',
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(int) 7 => array(
'id' => '4301',
'name' => 'Establishment of a second generation homozygous CRISPRa human inducedpluripotent stem cell (hiPSC) line for enhanced levels of endogenous geneactivation.',
'authors' => 'Schoger Eric et al.',
'description' => '<p>CRISPR/Cas9 technology based on nuclease inactive dCas9 and fused to the heterotrimeric VPR transcriptional activator is a powerful tool to enhance endogenous transcription by targeting defined genomic loci. We generated homozygous human induced pluripotent stem cell (hiPSC) lines carrying dCas9 fused to VPR along with a WPRE element at the AAVS1 locus (CRISPRa2). We demonstrated pluripotency, genomic integrity and differentiation potential into all three germ layers. CRISPRa2 cells showed increased transgene expression and higher transcriptional induction in hiPSC-derived cardiomyocytes compared to a previously described CRISPRa line. Both lines allow studying endogenous transcriptional modulation with lower and higher transcript abundance.</p>',
'date' => '2021-10-01',
'pmid' => 'https://doi.org/10.1016%2Fj.scr.2021.102518',
'doi' => '10.1016/j.scr.2021.102518',
'modified' => '2022-05-30 09:56:01',
'created' => '2022-05-19 10:41:50',
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(int) 8 => array(
'id' => '4328',
'name' => 'Establishment of two homozygous CRISPR interference (CRISPRi)knock-in human induced pluripotent stem cell (hiPSC) lines for titratableendogenous gene repression.',
'authors' => 'Schoger Eric et al.',
'description' => '<p>Using nuclease-deficient dead (d)Cas9 without enzymatic activity fused to transcriptional inhibitors (CRISPRi) allows for transcriptional interference and results in a powerful tool for the elucidation of developmental, homeostatic and disease mechanisms. We inserted dCas9KRAB (CRISPRi) cassette into the AAVS1 locus of hiPSC lines, which resulted in homozygous knock-in with an otherwise unaltered genome. Expression of dCas9KRAB protein, pluripotency and the ability to differentiate into all three embryonic germ layers were validated. Furthermore, functional cardiomyocyte generation was tested. The hiPSC-CRISPRi cell lines offer a valuable tool for studying endogenous transcriptional repression with single and multiplexed possibilities in all human cell types.</p>',
'date' => '2021-08-01',
'pmid' => 'https://doi.org/10.1016%2Fj.scr.2021.102473',
'doi' => '10.1016/j.scr.2021.102473',
'modified' => '2022-06-22 09:28:49',
'created' => '2022-05-19 10:41:50',
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[maximum depth reached]
)
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(int) 9 => array(
'id' => '4209',
'name' => 'TGFβ promotes widespread enhancer chromatin opening and operates ongenomic regulatory domains.',
'authors' => 'Guerrero-Martínez J. et al. ',
'description' => '<p>The Transforming Growth Factor-β (TGFβ) signaling pathway controls transcription by regulating enhancer activity. How TGFβ-regulated enhancers are selected and what chromatin changes are associated with TGFβ-dependent enhancers regulation are still unclear. Here we report that TGFβ treatment triggers fast and widespread increase in chromatin accessibility in about 80\% of the enhancers of normal mouse mammary epithelial-gland cells, irrespective of whether they are activated, repressed or not regulated by TGFβ. This enhancer opening depends on both the canonical and non-canonical TGFβ pathways. Most TGFβ-regulated genes are located around enhancers regulated in the same way, often creating domains of several co-regulated genes that we term TGFβ regulatory domains (TRD). CRISPR-mediated inactivation of enhancers within TRDs impairs TGFβ-dependent regulation of all co-regulated genes, demonstrating that enhancer targeting is more promiscuous than previously anticipated. The area of TRD influence is restricted by topologically associating domains (TADs) borders, causing a bias towards co-regulation within TADs.</p>',
'date' => '2020-12-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/33273453',
'doi' => '10.1038/s41467-020-19877-5',
'modified' => '2022-01-13 14:59:41',
'created' => '2021-12-06 15:53:19',
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'id' => '4013',
'name' => 'A gene therapy for inherited blindness using dCas9-VPR–mediatedtranscriptional activation',
'authors' => 'Böhm, Sybille and Splith, Victoria and Riedmayr, Lisa Maria and Rötzer,René Dominik and Gasparoni, Gilles and Nordström, Karl J. V. and Wagner,Johanna Elisabeth and Hinrichsmeyer, Klara Sonnie and Walter, Jörn andWahl-Schott, Christian and Fenske, Stef',
'description' => '<p>Catalytically inactive dCas9 fused to transcriptional activators (dCas9-VPR) enables activation of silent genes. Many disease genes have counterparts, which serve similar functions but are expressed in distinct cell types. One attractive option to compensate for the missing function of a defective gene could be to transcriptionally activate its functionally equivalent counterpart via dCas9-VPR. Key challenges of this approach include the delivery of dCas9-VPR, activation efficiency, long-term expression of the target gene, and adverse effects in vivo. Using dual adeno-associated viral vectors expressing split dCas9-VPR, we show efficient transcriptional activation and long-term expression of cone photoreceptor-specific M-opsin (Opn1mw) in a rhodopsin-deficient mouse model for retinitis pigmentosa. One year after treatment, this approach yields improved retinal function and attenuated retinal degeneration with no apparent adverse effects. Our study demonstrates that dCas9-VPR–mediated transcriptional activation of functionally equivalent genes has great potential for the treatment of genetic disorders.</p>',
'date' => '2020-08-19',
'pmid' => 'https://advances.sciencemag.org/content/6/34/eaba5614',
'doi' => '10.1126/sciadv.aba5614',
'modified' => '2020-12-16 17:32:46',
'created' => '2020-10-12 14:54:59',
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[maximum depth reached]
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(int) 11 => array(
'id' => '3631',
'name' => 'Guidelines for optimized gene knockout using CRISPR/Cas9',
'authors' => 'Campenhout CV et al.',
'description' => '<p>CRISPR/Cas9 technology has evolved as the most powerful approach to generate genetic models both for fundamental and preclinical research. Despite its apparent simplicity, the outcome of a genome-editing experiment can be substantially impacted by technical parameters and biological considerations. Here, we present guidelines and tools to optimize CRISPR/Cas9 genome-targeting efficiency and specificity. The nature of the target locus, the design of the single guide RNA and the choice of the delivery method should all be carefully considered prior to a genome-editing experiment. Different methods can also be used to detect off-target cleavages and decrease the risk of unwanted mutations. Together, these optimized tools and proper controls are essential to the assessment of CRISPR/Cas9 genome-editing experiments.</p>',
'date' => '2019-05-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/31039627',
'doi' => '10.2144/btn-2018-0187',
'modified' => '2019-05-09 15:37:50',
'created' => '2019-05-09 15:37:50',
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(int) 12 => array(
'id' => '3653',
'name' => 'CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency.',
'authors' => 'Matharu N, Rattanasopha S, Tamura S, Maliskova L, Wang Y, Bernard A, Hardin A, Eckalbar WL, Vaisse C, Ahituv N',
'description' => '<p>A wide range of human diseases result from haploinsufficiency, where the function of one of the two gene copies is lost. Here, we targeted the remaining functional copy of a haploinsufficient gene using CRISPR-mediated activation (CRISPRa) in and heterozygous mouse models to rescue their obesity phenotype. Transgenic-based CRISPRa targeting of the promoter or its distant hypothalamic enhancer up-regulated its expression from the endogenous functional allele in a tissue-specific manner, rescuing the obesity phenotype in heterozygous mice. To evaluate the therapeutic potential of CRISPRa, we injected CRISPRa-recombinant adeno-associated virus into the hypothalamus, which led to reversal of the obesity phenotype in and haploinsufficient mice. Our results suggest that endogenous gene up-regulation could be a potential strategy to treat altered gene dosage diseases.</p>',
'date' => '2019-01-18',
'pmid' => 'http://www.pubmed.gov/30545847',
'doi' => '10.1126/science.aau0629',
'modified' => '2019-06-07 09:05:53',
'created' => '2019-06-06 12:11:18',
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[maximum depth reached]
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(int) 13 => array(
'id' => '3327',
'name' => '(Po)STAC (Polycistronic SunTAg modified CRISPR) enables live-cell and fixed-cell super-resolution imaging of multiple genes',
'authors' => 'Neguembor M.V. et al.',
'description' => '<p>CRISPR/dCas9-based labeling has allowed direct visualization of genomic regions in living cells. However, poor labeling efficiency and signal-to-background ratio have limited its application to visualize genome organization using super-resolution microscopy. We developed (Po)STAC (Polycistronic SunTAg modified CRISPR) by combining CRISPR/dCas9 with SunTag labeling and polycistronic vectors. (Po)STAC enhances both labeling efficiency and fluorescence signal detected from labeled loci enabling live cell imaging as well as super-resolution fixed-cell imaging of multiple genes with high spatiotemporal resolution.</p>',
'date' => '2017-12-23',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/29294098',
'doi' => '',
'modified' => '2018-02-07 10:03:26',
'created' => '2018-02-07 10:03:26',
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(int) 14 => array(
'id' => '4399',
'name' => 'A self-inactivating system for AAV-mediated in vivo base editing',
'authors' => 'Zuo Y. et al.',
'description' => '<p>DNA base editors have been harnessed as an exciting therapeutic platform for human diseases and are rapidly progressing into human clinical trials. However, persistent expression of base editors delivered via adeno-associated virus (AAV) poses concerns with specificity and immunogenicity. Here we develop selfinactivating base editor (siBE) systems with a negative feedback loop where one guide RNA (gRNA) targets the gene of interest and the other targets the deaminase domain itself. We demonstrate that siBE confers efficient on-target editing with time-dependent self-inactivation and increased editing specificity. For the in vivo utilization, we further employ the intein split approach to package siBE targeting mouse Angptl3 into AAV9. Systemic delivery of AAV9-siBE confer efficient editing of Angptl3 in liver, resulting in reduced serum levels of ANGPTL3, triglyceride and total cholesterol, with the active base editor undetectable at 8 weeks after administration. These self-inactivating base editing systems are highly promising for future therapeutic applications.</p>',
'date' => '0000-00-00',
'pmid' => 'https://doi.org/10.21203%2Frs.3.rs-1663604%2Fv1',
'doi' => '10.21203/rs.3.rs-1663604/v1',
'modified' => '2022-08-11 15:24:23',
'created' => '2022-08-11 12:14:50',
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'name' => 'Researcher from the University of Manchester',
'description' => '<p>I used ChIP-qPCR with the Diagenode CRISPR/Cas9 polyclonal antibody to successfully show that Cas9 binds to the target region of my sgRNA, validating my CRISPR experiment. The antibody produced minimal background signal at non-specific genomic regions. I am now using the antibody to validate further sgRNA in different CRISPR cell lines.</p>
<center><img src="../../img/categories/antibodies/cas9-jurkat-vp64-result.png" /></center>
<p><small>ChIP was performed on Jurkat cells expressing dCas9-VP64-mCherry and a sgRNA targeting the IL1RN promoter. Each IP was performed using 4 million cells and 2 µL CRISPR/Cas9 polyclonal antibody (Diagenode C15310258) or 1 µg rabbit IgG control antibody (Diagenode C15410206). qPCR was carried out on undiluted ChIP DNA using SYBR green and PCR primers directed against the sgRNA binding site at IL1RN, as well as two non-target regions at the SLC4A1 and TP53 promoters. ChIP enrichment was measured using the percent input method.</small></p>',
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'description' => '<p><strong>Diagenode’s CRISPR/Cas9 polyclonal antibody</strong> shows superior signal than the original <strong>clone 7A9</strong>: a researcher from EPFL in Lausanne, Switzerland has compared these two antibodies in Western blot.</p>
<center><img src="../../emailing/images/cas9-fig.png" /></center>
<p><small>Western blot was performed using HCT116 DKO cells transduced with Krab-dCas9 (2) or non-transduced (1) cells. Then, 100,000 cells were lysed in sample buffer 2x and boiled 5 min at 95°C before loading in a 15% acrylamide gel. The same sample was loaded 3x in the same gel. The membrane was cut in 3 parts for each antibody. Membrane was blocked 1h with 3% BSA at RT. Antibodies were diluted 1:1,000 in 3% BSA and incubated overnight at 4°C. Secondary incubation was done for 1h at RT (1:10,000 dilution). Anti-hnRNPA1 was used as a loading control.</small></p>',
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'name' => 'CRISPR/Cas9 Antibody',
'description' => '<p>Polyclonal antibody raised in rabbit against the <strong>Cas9</strong> nuclease (<strong>CRISPR</strong>-associated protein 9) using a recombinant protein. </p>',
'label1' => 'Validation data',
'info1' => '<div class="row">
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-fig1.png" /></center></div>
<div class="small-6 columns">
<p><strong>Figure 1. ChIP using the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing nuclease dead Cas9 and sgRNA targeting a sequence in intron 8 of the GAPDH gene, using the iDeal ChIP-seq kit for transcription factors. A titration consisting of 1, 2, 5 and 10 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) was tested. IgG (2 µg/IP) was used as negative IP control. qPCR was performed with primers specific for the targeted sequence in the GAPDH gene, and for the MYOD1 gene, 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).</p>
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<div class="row">
<div class="small-12 columns"><center>A.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-a.jpg" width="700" /></center><center>B.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-b.jpg" width="700" /></center></div>
</div>
<div class="row">
<div class="small-12 columns">
<p><strong>Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing dCas9 and a GAPDH sgRNA cells using 2 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) as described above. The IP'd DNA was subsequently analysed on an Illumina NovaSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the ChIP-seq profile in a region of chromosome 12 surrounding the GAPDH gene (fig 2B) and in a region of chromosome 2 surrounding an off-target peak in the YIPF4 gene.</p>
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<div class="small-4 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-WB.png" /></center></div>
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<p><strong>Figure 3. Western blot analysis using the Diagenode antibody directed against Cas9</strong><br />Western blot was performed on protein extracts from HEK293 cells transfected with dCas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:5,000. The marker is shown on the left, the position of the Cas9 protein is indicated on the right.</p>
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<p><strong>Figure 4. IP using the Diagenode antibody directed against Cas9</strong><br />IP was performed on whole cell extracts (500 µg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 µl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 µg) is shown in lane 1 and 2.</p>
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<div class="small-12 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" width="500" /></center></div>
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<p><strong>Figure 5. Immunofluorescence using the Diagenode antibody directed against Cas9</strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counter-stained with Hoechst 33342. Figure 5 shows the result in the presence (left) or absence (right) of doxycycline.</p>
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<p><small><strong> Figure 1. ChIP using the Diagenode antibody directed against Cas9 </strong><br />ChIP was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50μg chromatin was incubated overnight at 4°C with either 5 μg of an anti-FLAG antibody or 2 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The pre-immune serum (Cas9, PPI) was used as negative IP control. qPCR was performed with primers specific for the GFP gene, and for two non-targeted regions phosphatidic acid phosphatase type 2C (Ppap2c) and protein kinase C delta (Prkcd), used as negative controls. 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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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-WB.png" alt="CRISPR/Cas9 Antibody Validation in WB " caption="false" width="171" height="192" /></p>
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<p><small><strong> Figure 2. Western blot analysis using the Diagenode antibody directed against Cas9 </strong><br />Western blot was performed on protein extracts from HeLa cells transfected with Cas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:1,000 (lane 2) or 1:5,000 (lane 3). Lane 1 shows the result with the pre-immune serum. The marker is shown on the left, the position of the Cas9 protein is indicated on the right. </small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IP.png" alt="CRISPR/Cas9 Antibody Validation in IP" caption="false" width="252" height="286" /></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 (500 μg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 μg) is shown in lane 1 and 2. </small></p>
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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" alt="CRISPR/Cas9 Antibody Validation in IF" caption="false" width="278" height="123" /></p>
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<p><small><strong> Figure 4. Immunofluorescence using the Diagenode antibody directed against Cas9 </strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counterstained with Hoechst 33342. Figure 4 shows the result in the presence (left) or absence (right) of doxycycline. </small></p>
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$testimonials = '<blockquote><p><strong>Diagenode’s CRISPR/Cas9 polyclonal antibody</strong> shows superior signal than the original <strong>clone 7A9</strong>: a researcher from EPFL in Lausanne, Switzerland has compared these two antibodies in Western blot.</p>
<center><img src="../../emailing/images/cas9-fig.png" /></center>
<p><small>Western blot was performed using HCT116 DKO cells transduced with Krab-dCas9 (2) or non-transduced (1) cells. Then, 100,000 cells were lysed in sample buffer 2x and boiled 5 min at 95°C before loading in a 15% acrylamide gel. The same sample was loaded 3x in the same gel. The membrane was cut in 3 parts for each antibody. Membrane was blocked 1h with 3% BSA at RT. Antibodies were diluted 1:1,000 in 3% BSA and incubated overnight at 4°C. Secondary incubation was done for 1h at RT (1:10,000 dilution). Anti-hnRNPA1 was used as a loading control.</small></p><cite>EPFL in Lausanne</cite></blockquote>
'
$featured_testimonials = '<blockquote><span class="label-green" style="margin-bottom:16px;margin-left:-22px">TESTIMONIAL</span><p>I used ChIP-qPCR with the Diagenode CRISPR/Cas9 polyclonal antibody to successfully show that Cas9 binds to the target region of my sgRNA, validating my CRISPR experiment. The antibody produced minimal background signal at non-specific genomic regions. I am now using the antibody to validate further sgRNA in different CRISPR cell lines.</p>
<center><img src="../../img/categories/antibodies/cas9-jurkat-vp64-result.png" /></center>
<p><small>ChIP was performed on Jurkat cells expressing dCas9-VP64-mCherry and a sgRNA targeting the IL1RN promoter. Each IP was performed using 4 million cells and 2 µL CRISPR/Cas9 polyclonal antibody (Diagenode C15310258) or 1 µg rabbit IgG control antibody (Diagenode C15410206). qPCR was carried out on undiluted ChIP DNA using SYBR green and PCR primers directed against the sgRNA binding site at IL1RN, as well as two non-target regions at the SLC4A1 and TP53 promoters. ChIP enrichment was measured using the percent input method.</small></p><cite>Researcher from the University of Manchester</cite></blockquote>
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'description' => '<p><strong>Diagenode’s CRISPR/Cas9 polyclonal antibody</strong> shows superior signal than the original <strong>clone 7A9</strong>: a researcher from EPFL in Lausanne, Switzerland has compared these two antibodies in Western blot.</p>
<center><img src="../../emailing/images/cas9-fig.png" /></center>
<p><small>Western blot was performed using HCT116 DKO cells transduced with Krab-dCas9 (2) or non-transduced (1) cells. Then, 100,000 cells were lysed in sample buffer 2x and boiled 5 min at 95°C before loading in a 15% acrylamide gel. The same sample was loaded 3x in the same gel. The membrane was cut in 3 parts for each antibody. Membrane was blocked 1h with 3% BSA at RT. Antibodies were diluted 1:1,000 in 3% BSA and incubated overnight at 4°C. Secondary incubation was done for 1h at RT (1:10,000 dilution). Anti-hnRNPA1 was used as a loading control.</small></p>',
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'description' => '<p>Polyclonal antibody raised in rabbit against the <strong>Cas9</strong> nuclease (<strong>CRISPR</strong>-associated protein 9) using a recombinant protein. </p>',
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<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-fig1.png" /></center></div>
<div class="small-6 columns">
<p><strong>Figure 1. ChIP using the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing nuclease dead Cas9 and sgRNA targeting a sequence in intron 8 of the GAPDH gene, using the iDeal ChIP-seq kit for transcription factors. A titration consisting of 1, 2, 5 and 10 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) was tested. IgG (2 µg/IP) was used as negative IP control. qPCR was performed with primers specific for the targeted sequence in the GAPDH gene, and for the MYOD1 gene, 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).</p>
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<div class="small-12 columns"><center>A.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-a.jpg" width="700" /></center><center>B.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-b.jpg" width="700" /></center></div>
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<p><strong>Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing dCas9 and a GAPDH sgRNA cells using 2 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) as described above. The IP'd DNA was subsequently analysed on an Illumina NovaSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the ChIP-seq profile in a region of chromosome 12 surrounding the GAPDH gene (fig 2B) and in a region of chromosome 2 surrounding an off-target peak in the YIPF4 gene.</p>
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<div class="small-4 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-WB.png" /></center></div>
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<p><strong>Figure 3. Western blot analysis using the Diagenode antibody directed against Cas9</strong><br />Western blot was performed on protein extracts from HEK293 cells transfected with dCas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:5,000. The marker is shown on the left, the position of the Cas9 protein is indicated on the right.</p>
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<div class="small-4 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IP.png" /></center></div>
<div class="small-8 columns">
<p><strong>Figure 4. IP using the Diagenode antibody directed against Cas9</strong><br />IP was performed on whole cell extracts (500 µg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 µl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 µg) is shown in lane 1 and 2.</p>
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<div class="small-12 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" width="500" /></center></div>
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<p><strong>Figure 5. Immunofluorescence using the Diagenode antibody directed against Cas9</strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counter-stained with Hoechst 33342. Figure 5 shows the result in the presence (left) or absence (right) of doxycycline.</p>
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'name' => 'Optimize the selection of guide RNA by ChIP to keep CRISPR on-target',
'description' => '<p>The mechanisms of target recognition and target specificity of the Cas9 protein is still not completely understood. A major hurdle of this technology is the introduction of double-strand breaks (DSBs) at sites other than the intended on-target site (off-target effects). All CRISPR/Cas9 applications require the verification of the specific binding of the sgRNA at the locus of interest. Chromatin immunoprecipitation followed by real-time PCR (ChIP-qPCR) is a technique of choice for studying protein-DNA interactions. In this study, we show a successful ChIP-qPCR method to verify the binding efficiency of the dCas9/sgRNA complex in the targeted region; and ChIP-seq – to monitor off-target bindings of the dCas9/sgRNA complex in the genome.</p>',
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'name' => 'A self-inactivating system for AAV-mediated in vivo base editing',
'authors' => 'Zuo Y. et al.',
'description' => '<p>DNA base editors have been harnessed as an exciting therapeutic platform for human diseases and are rapidly progressing into human clinical trials. However, persistent expression of base editors delivered via adeno-associated virus (AAV) poses concerns with specificity and immunogenicity. Here we develop selfinactivating base editor (siBE) systems with a negative feedback loop where one guide RNA (gRNA) targets the gene of interest and the other targets the deaminase domain itself. We demonstrate that siBE confers efficient on-target editing with time-dependent self-inactivation and increased editing specificity. For the in vivo utilization, we further employ the intein split approach to package siBE targeting mouse Angptl3 into AAV9. Systemic delivery of AAV9-siBE confer efficient editing of Angptl3 in liver, resulting in reduced serum levels of ANGPTL3, triglyceride and total cholesterol, with the active base editor undetectable at 8 weeks after administration. These self-inactivating base editing systems are highly promising for future therapeutic applications.</p>',
'date' => '0000-00-00',
'pmid' => 'https://doi.org/10.21203%2Frs.3.rs-1663604%2Fv1',
'doi' => '10.21203/rs.3.rs-1663604/v1',
'modified' => '2022-08-11 15:24:23',
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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
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Dispatcher::dispatch() - CORE/Cake/Routing/Dispatcher.php, line 167
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-fig1.png" alt="CRISPR/Cas9 Antibody ChIP Grade" caption="false" width="241" height="286" /></p>
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<p><small><strong> Figure 1. ChIP using the Diagenode antibody directed against Cas9 </strong><br />ChIP was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50μg chromatin was incubated overnight at 4°C with either 5 μg of an anti-FLAG antibody or 2 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The pre-immune serum (Cas9, PPI) was used as negative IP control. qPCR was performed with primers specific for the GFP gene, and for two non-targeted regions phosphatidic acid phosphatase type 2C (Ppap2c) and protein kinase C delta (Prkcd), used as negative controls. 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/C15310258-CRISPR-WB.png" alt="CRISPR/Cas9 Antibody Validation in WB " caption="false" width="171" height="192" /></p>
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<p><small><strong> Figure 2. Western blot analysis using the Diagenode antibody directed against Cas9 </strong><br />Western blot was performed on protein extracts from HeLa cells transfected with Cas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:1,000 (lane 2) or 1:5,000 (lane 3). Lane 1 shows the result with the pre-immune serum. The marker is shown on the left, the position of the Cas9 protein is indicated on the right. </small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IP.png" alt="CRISPR/Cas9 Antibody Validation in IP" caption="false" width="252" height="286" /></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 (500 μg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 μg) is shown in lane 1 and 2. </small></p>
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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" alt="CRISPR/Cas9 Antibody Validation in IF" caption="false" width="278" height="123" /></p>
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<p><small><strong> Figure 4. Immunofluorescence using the Diagenode antibody directed against Cas9 </strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counterstained with Hoechst 33342. Figure 4 shows the result in the presence (left) or absence (right) of doxycycline. </small></p>
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'info2' => '<p style="text-align: justify;">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 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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<td>ChIP/ChIP-seq <sup>*</sup></td>
<td>2-5 µl/ChIP</td>
<td>Fig 1, 2</td>
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<tr>
<td>Western Blotting</td>
<td>1:5,000</td>
<td>Fig 3</td>
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<tr>
<td>Immunoprecipitation</td>
<td>1 µl/IP</td>
<td>Fig 4</td>
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<td>Immunofluorescence</td>
<td>1:1,000</td>
<td>Fig 5</td>
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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 µl per IP.</small></p>',
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'description' => '<p>Polyclonal antibody raised in rabbit against the <strong>Cas9</strong> nuclease (<strong>CRISPR</strong>-associated protein 9) using a recombinant protein. </p>',
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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-fig1.png" alt="CRISPR/Cas9 Antibody ChIP Grade" caption="false" width="241" height="286" /></p>
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<p><small><strong> Figure 1. ChIP using the Diagenode antibody directed against Cas9 </strong><br />ChIP was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50μg chromatin was incubated overnight at 4°C with either 5 μg of an anti-FLAG antibody or 2 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The pre-immune serum (Cas9, PPI) was used as negative IP control. qPCR was performed with primers specific for the GFP gene, and for two non-targeted regions phosphatidic acid phosphatase type 2C (Ppap2c) and protein kinase C delta (Prkcd), used as negative controls. 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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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-WB.png" alt="CRISPR/Cas9 Antibody Validation in WB " caption="false" width="171" height="192" /></p>
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<p><small><strong> Figure 2. Western blot analysis using the Diagenode antibody directed against Cas9 </strong><br />Western blot was performed on protein extracts from HeLa cells transfected with Cas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:1,000 (lane 2) or 1:5,000 (lane 3). Lane 1 shows the result with the pre-immune serum. The marker is shown on the left, the position of the Cas9 protein is indicated on the right. </small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IP.png" alt="CRISPR/Cas9 Antibody Validation in IP" caption="false" width="252" height="286" /></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 (500 μg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 μg) is shown in lane 1 and 2. </small></p>
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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" alt="CRISPR/Cas9 Antibody Validation in IF" caption="false" width="278" height="123" /></p>
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<div class="small-8 columns">
<p><small><strong> Figure 4. Immunofluorescence using the Diagenode antibody directed against Cas9 </strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counterstained with Hoechst 33342. Figure 4 shows the result in the presence (left) or absence (right) of doxycycline. </small></p>
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'label2' => 'Background',
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'meta_title' => 'CRISPR/Cas9 polyclonal antibody (sample size)',
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'description' => '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 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.',
'clonality' => '',
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'lot' => 'A2508-004',
'concentration' => 'Not determined',
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'type' => 'Polyclonal, <strong>ChIP grade, ChIP-seq grade</strong>',
'purity' => 'Whole antiserum from rabbit containing 0.05% azide.',
'classification' => '',
'application_table' => '<table>
<thead>
<tr>
<th>Applications</th>
<th>Suggested dilution</th>
<th>References</th>
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<tbody>
<tr>
<td>ChIP/ChIP-seq <sup>*</sup></td>
<td>2-5 µl/ChIP</td>
<td>Fig 1, 2</td>
</tr>
<tr>
<td>Western Blotting</td>
<td>1:5,000</td>
<td>Fig 3</td>
</tr>
<tr>
<td>Immunoprecipitation</td>
<td>1 µl/IP</td>
<td>Fig 4</td>
</tr>
<tr>
<td>Immunofluorescence</td>
<td>1:1,000</td>
<td>Fig 5</td>
</tr>
</tbody>
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<p></p>
<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 µl per IP.</small></p>',
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'name' => 'CRISPR/Cas9 Antibody',
'description' => '<p>Polyclonal antibody raised in rabbit against the <strong>Cas9</strong> nuclease (<strong>CRISPR</strong>-associated protein 9) using a recombinant protein. </p>',
'label1' => 'Validation data',
'info1' => '<div class="row">
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-fig1.png" /></center></div>
<div class="small-6 columns">
<p><strong>Figure 1. ChIP using the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing nuclease dead Cas9 and sgRNA targeting a sequence in intron 8 of the GAPDH gene, using the iDeal ChIP-seq kit for transcription factors. A titration consisting of 1, 2, 5 and 10 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) was tested. IgG (2 µg/IP) was used as negative IP control. qPCR was performed with primers specific for the targeted sequence in the GAPDH gene, and for the MYOD1 gene, 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).</p>
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<div class="row">
<div class="small-12 columns"><center>A.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-a.jpg" width="700" /></center><center>B.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-b.jpg" width="700" /></center></div>
</div>
<div class="row">
<div class="small-12 columns">
<p><strong>Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing dCas9 and a GAPDH sgRNA cells using 2 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) as described above. The IP'd DNA was subsequently analysed on an Illumina NovaSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the ChIP-seq profile in a region of chromosome 12 surrounding the GAPDH gene (fig 2B) and in a region of chromosome 2 surrounding an off-target peak in the YIPF4 gene.</p>
</div>
</div>
<div class="row">
<div class="small-4 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-WB.png" /></center></div>
<div class="small-8 columns">
<p><strong>Figure 3. Western blot analysis using the Diagenode antibody directed against Cas9</strong><br />Western blot was performed on protein extracts from HEK293 cells transfected with dCas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:5,000. The marker is shown on the left, the position of the Cas9 protein is indicated on the right.</p>
</div>
</div>
<div class="row">
<div class="small-4 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IP.png" /></center></div>
<div class="small-8 columns">
<p><strong>Figure 4. IP using the Diagenode antibody directed against Cas9</strong><br />IP was performed on whole cell extracts (500 µg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 µl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 µg) is shown in lane 1 and 2.</p>
</div>
</div>
<div class="row">
<div class="small-12 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" width="500" /></center></div>
</div>
<div class="row">
<div class="small-12 columns">
<p><strong>Figure 5. Immunofluorescence using the Diagenode antibody directed against Cas9</strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counter-stained with Hoechst 33342. Figure 5 shows the result in the presence (left) or absence (right) of doxycycline.</p>
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'info2' => '<p style="text-align: justify;">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 <strong>CRISPR/Cas9</strong> (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 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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'meta_description' => 'S. pyogenes CRISPR/Cas9 Polyclonal Antibody validated in ChIP-seq, ChIP-qPCR, IP, IF and WB. Batch-specific data available on the website. Sample size available.',
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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' => '<h1><strong>Validated epigenetics antibodies</strong> – care for a sample?<br /> </h1>
<p>Diagenode has partnered with leading epigenetics experts and numerous epigenetics consortiums to bring to you a validated and comprehensive collection of epigenetic antibodies. As an expert in epigenetics, we are committed to offering highly-specific antibodies validated for ChIP/ChIP-seq and many other applications. All batch-specific validation data is available on our website.<br /><a href="../categories/antibodies">Read about our expertise in antibody production</a>.</p>
<ul>
<li><strong>Focused</strong> - Diagenode's selection of antibodies is exclusively dedicated for epigenetic research. <a title="See the full collection." href="../categories/all-antibodies">See the full collection.</a></li>
<li><strong>Strict quality standards</strong> with rigorous QC and validation</li>
<li><strong>Classified</strong> based on level of validation for flexibility of application</li>
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<p>Existing sample sizes are listed below. We will soon expand our collection. Are you looking for a sample size of another antibody? Just <a href="mailto:agnieszka.zelisko@diagenode.com?Subject=Sample%20Size%20Request" target="_top">Contact us</a>.</p>',
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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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'meta_title' => 'Diagenode's selection of Antibodies is exclusively dedicated for Epigenetic Research | Diagenode',
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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>
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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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'description' => '<p><span>The fluorescence ubiquitination cell cycle inhibitor (FUCCI) has been introduced to monitor cell cycle activity in living cells, including human induced pluripotent stem cells (hiPSC) and derived cell types. We have recently developed hiPSC with stable expression of dCas9VPR for endogenous gene activation and a Citrine-tagged ACTN2 cell line to monitor sarcomere development and function in muscle cells. Here, we present dual and triple transgenic hiPSC lines developed by genomic integration of FUCCI with and without dCas9VPR into the ROSA26 and AAVS1 loci, respectively, in the previously introduced ACTN2-Citrine line. Functionality of the transgenes was demonstrated in the novel hiPSC line, which we introduce as Myo-CCER and CraCCER.</span></p>',
'date' => '2024-08-05',
'pmid' => 'https://www.sciencedirect.com/science/article/pii/S1873506124002290',
'doi' => 'https://doi.org/10.1016/j.scr.2024.103531',
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'name' => 'Methyltransferase Inhibition Enables Tgf Driven Induction of and in Cancer Cells.',
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'description' => '<p>deletion or silencing is common across human cancer, reinforcing the general importance of bypassing its tumor suppression in cancer formation or progression. In rhabdomyosarcoma (RMS) and neuroblastoma, two common childhood cancers, the three transcripts are independently expressed to varying degrees, but one, is uniformly silenced. Although TGFβ induces certain transcripts in HeLa cells, it was unable to do so in five tested RMS lines unless the cells were pretreated with a broadly acting methyltransferase inhibitor, DZNep, or one targeting EZH2. induction by TGFβ correlated with de novo appearance of three H3K27Ac peaks within a 20 kb element ∼150 kb proximal to . Deleting that segment prevented their induction by TGFβ but not a basal increase driven by methyltransferase inhibition alone. Expression of two transcripts was enhanced by dCas9/CRISPR activation targeting either the relevant promoter or the 20 kb elements, and this "precise" manipulation diminished RMS cell propagation in vitro. Our findings show crosstalk between methyltransferase inhibition and TGFβ-dependent activation of a remote enhancer to reverse silencing. Though focused on here, such crosstalk may apply to other TGFβ-responsive genes and perhaps govern this signaling protein's complex effects promoting or blocking cancer.</p>',
'date' => '2023-01-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/36941772',
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'name' => 'Massively parallel multi-target CRISPR system interrogates Cas9-basedtarget recognition, DNA cleavage, and DNA repair',
'authors' => 'Zou Roger S. et al.',
'description' => '<p>CRISPR-Cas9 nucleases, and particularly Streptococcus pyogenes Cas9, are widespread tools for genome editing. However, many aspects of intracellular Cas9 activity and the ensuing DNA damage response remain incompletely characterized. In order to address these issues, we developed a multiplexed CRISPR approach, where a single, degenerate multi-target gRNA (mgRNA) directs the Cas9 enzyme to target hundred endogenous sites at once. When combined with next-generation sequencing readouts, this system enables interrogation of Cas9 activity and DNA double-strand break (DSB) repair response in high-throughput. Here, we present a step-by-step protocol to deliver a Cas9:mgRNA ribonucleoprotein complex into cultured cells and measure key processes related to Cas9 activity and DSB repair.</p>',
'date' => '2022-09-01',
'pmid' => 'https://europepmc.org/article/ppr/ppr540155',
'doi' => '10.21203/rs.3.pex-1938/v1',
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(int) 3 => array(
'id' => '4437',
'name' => 'Analysis of estrogen-regulated enhancer RNAs identifies a functionalmotif required for enhancer assembly and gene expression.',
'authors' => 'Hou Tim Y and Kraus W Lee',
'description' => '<p>To better understand the functions of non-coding enhancer RNAs (eRNAs), we annotated the estrogen-regulated eRNA transcriptome in estrogen receptor α (ERα)-positive breast cancer cells using PRO-cap and RNA sequencing. We then cloned a subset of the eRNAs identified, fused them to single guide RNAs, and targeted them to their ERα enhancers of origin using CRISPR/dCas9. Some of the eRNAs tested modulated the expression of cognate, but not heterologous, target genes after estrogen treatment by increasing ERα recruitment and stimulating p300-catalyzed H3K27 acetylation at the enhancer. We identified a ∼40 nucleotide functional eRNA regulatory motif (FERM) present in many eRNAs that was necessary and sufficient to modulate gene expression, but not the specificity of activation, after estrogen treatment. The FERM interacted with BCAS2, an RNA-binding protein amplified in breast cancers. The ectopic expression of a targeted eRNA controlling the expression of an oncogene resulted in increased cell proliferation, demonstrating the regulatory potential of eRNAs in breast cancer.</p>',
'date' => '2022-06-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/35705040',
'doi' => '10.1016/j.celrep.2022.110944',
'modified' => '2022-09-28 09:20:34',
'created' => '2022-09-08 16:32:20',
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(int) 4 => array(
'id' => '4560',
'name' => 'Avian influenza viruses suppress innate immunity by inducingtrans-transcriptional readthrough via SSU72.',
'authors' => 'Zhao Y. et al.',
'description' => '<p>Innate immunity plays critical antiviral roles. The highly virulent avian influenza viruses (AIVs) H5N1, H7N9, and H5N6 can better escape host innate immune responses than the less virulent seasonal H1N1 virus. Here, we report a mechanism by which transcriptional readthrough (TRT)-mediated suppression of innate immunity occurs post AIV infection. By using cell lines, mouse lungs, and patient PBMCs, we showed that genes on the complementary strand ("trans" genes) influenced by TRT were involved in the disruption of host antiviral responses during AIV infection. The trans-TRT enhanced viral lethality, and TRT abolishment increased cell viability and STAT1/2 expression. The viral NS1 protein directly bound to SSU72, and degradation of SSU72 induced TRT. SSU72 overexpression reduced TRT and alleviated mouse lung injury. Our results suggest that AIVs infection induce TRT by reducing SSU72 expression, thereby impairing host immune responses, a molecular mechanism acting through the NS1-SSU72-trans-TRT-STAT1/2 axis. Thus, restoration of SSU72 expression might be a potential strategy for preventing AIV pandemics.</p>',
'date' => '2022-03-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/35332300',
'doi' => '10.1038/s41423-022-00843-8',
'modified' => '2022-11-24 10:02:04',
'created' => '2022-11-24 08:49:52',
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'id' => '4365',
'name' => 'Antisense non-coding transcription represses the <i>PHO5</i> model gene<i>via</i> remodelling of promoter chromatin structure',
'authors' => 'Novačić A. et al. ',
'description' => '<p>Pervasive transcription of eukaryotic genomes generates non-coding transcripts with regulatory potential. We examined the effects of non-coding antisense transcription on the regulation of expression of the yeast PHO5 gene, a paradigmatic case for gene regulation through promoter chromatin remodeling. By enhancing or impairing the level of overlapping antisense transcription through specific mutant backgrounds and the use of the CRISPRi system, we demonstrated a negative role for antisense transcription at the PHO5 gene locus. Furthermore, enhanced elongation of PHO5 antisense leads to a more repressive chromatin configuration at the PHO5 gene promoter, which is remodeled more slowly upon gene induction. The negative effect of antisense transcription on PHO5 gene transcription is mitigated by inactivation of the histone deacetylase Rpd3, showing that PHO5 antisense RNA acts via histone deacetylation. This regulatory pathway leads to Rpd3-dependent decreased recruitment of the RSC chromatin remodeling complex to the PHO5 gene promoter upon induction of antisense transcription. Overall, we extend the model of PHO5 gene regulation by demonstrating a gene silencing function of antisense transcription through a chromatin-based mechanism.</p>',
'date' => '2022-02-01',
'pmid' => 'https://doi.org/10.1101%2F2022.02.21.481265',
'doi' => '10.1101/2022.02.21.481265',
'modified' => '2022-08-04 15:52:12',
'created' => '2022-08-04 14:55:36',
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(int) 6 => array(
'id' => '4237',
'name' => 'A predominant enhancer co-amplified with the oncogene is necessary andsufficient for its expression in squamous cancer',
'authors' => 'Liu Y. et al.',
'description' => '<p>Amplification and overexpression of the SOX2 oncogene represent a hallmark of squamous cancers originating from diverse tissue types. Here, we find that squamous cancers selectively amplify a 3’ noncoding region together with SOX2, which harbors squamous cancer-specific chromatin accessible regions. We identify a single enhancer e1 that predominantly drives SOX2 expression. Repression of e1 in SOX2-high cells causes collapse of the surrounding enhancers, remarkable reduction in SOX2 expression, and a global transcriptional change reminiscent of SOX2 knockout. The e1 enhancer is driven by a combination of transcription factors including SOX2 itself and the AP-1 complex, which facilitates recruitment of the co-activator BRD4. CRISPR-mediated activation of e1 in SOX2-low cells is sufficient to rebuild the e1-SOX2 loop and activate SOX2 expression. Our study shows that squamous cancers selectively amplify a predominant enhancer to drive SOX2 overexpression, uncovering functional links among enhancer activation, chromatin looping, and lineage-specific copy number amplifications of oncogenes.</p>',
'date' => '2021-12-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/34880227',
'doi' => '10.1038/s41467-021-27055-4',
'modified' => '2022-05-19 17:05:00',
'created' => '2022-05-19 10:41:50',
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(int) 7 => array(
'id' => '4301',
'name' => 'Establishment of a second generation homozygous CRISPRa human inducedpluripotent stem cell (hiPSC) line for enhanced levels of endogenous geneactivation.',
'authors' => 'Schoger Eric et al.',
'description' => '<p>CRISPR/Cas9 technology based on nuclease inactive dCas9 and fused to the heterotrimeric VPR transcriptional activator is a powerful tool to enhance endogenous transcription by targeting defined genomic loci. We generated homozygous human induced pluripotent stem cell (hiPSC) lines carrying dCas9 fused to VPR along with a WPRE element at the AAVS1 locus (CRISPRa2). We demonstrated pluripotency, genomic integrity and differentiation potential into all three germ layers. CRISPRa2 cells showed increased transgene expression and higher transcriptional induction in hiPSC-derived cardiomyocytes compared to a previously described CRISPRa line. Both lines allow studying endogenous transcriptional modulation with lower and higher transcript abundance.</p>',
'date' => '2021-10-01',
'pmid' => 'https://doi.org/10.1016%2Fj.scr.2021.102518',
'doi' => '10.1016/j.scr.2021.102518',
'modified' => '2022-05-30 09:56:01',
'created' => '2022-05-19 10:41:50',
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(int) 8 => array(
'id' => '4328',
'name' => 'Establishment of two homozygous CRISPR interference (CRISPRi)knock-in human induced pluripotent stem cell (hiPSC) lines for titratableendogenous gene repression.',
'authors' => 'Schoger Eric et al.',
'description' => '<p>Using nuclease-deficient dead (d)Cas9 without enzymatic activity fused to transcriptional inhibitors (CRISPRi) allows for transcriptional interference and results in a powerful tool for the elucidation of developmental, homeostatic and disease mechanisms. We inserted dCas9KRAB (CRISPRi) cassette into the AAVS1 locus of hiPSC lines, which resulted in homozygous knock-in with an otherwise unaltered genome. Expression of dCas9KRAB protein, pluripotency and the ability to differentiate into all three embryonic germ layers were validated. Furthermore, functional cardiomyocyte generation was tested. The hiPSC-CRISPRi cell lines offer a valuable tool for studying endogenous transcriptional repression with single and multiplexed possibilities in all human cell types.</p>',
'date' => '2021-08-01',
'pmid' => 'https://doi.org/10.1016%2Fj.scr.2021.102473',
'doi' => '10.1016/j.scr.2021.102473',
'modified' => '2022-06-22 09:28:49',
'created' => '2022-05-19 10:41:50',
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[maximum depth reached]
)
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(int) 9 => array(
'id' => '4209',
'name' => 'TGFβ promotes widespread enhancer chromatin opening and operates ongenomic regulatory domains.',
'authors' => 'Guerrero-Martínez J. et al. ',
'description' => '<p>The Transforming Growth Factor-β (TGFβ) signaling pathway controls transcription by regulating enhancer activity. How TGFβ-regulated enhancers are selected and what chromatin changes are associated with TGFβ-dependent enhancers regulation are still unclear. Here we report that TGFβ treatment triggers fast and widespread increase in chromatin accessibility in about 80\% of the enhancers of normal mouse mammary epithelial-gland cells, irrespective of whether they are activated, repressed or not regulated by TGFβ. This enhancer opening depends on both the canonical and non-canonical TGFβ pathways. Most TGFβ-regulated genes are located around enhancers regulated in the same way, often creating domains of several co-regulated genes that we term TGFβ regulatory domains (TRD). CRISPR-mediated inactivation of enhancers within TRDs impairs TGFβ-dependent regulation of all co-regulated genes, demonstrating that enhancer targeting is more promiscuous than previously anticipated. The area of TRD influence is restricted by topologically associating domains (TADs) borders, causing a bias towards co-regulation within TADs.</p>',
'date' => '2020-12-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/33273453',
'doi' => '10.1038/s41467-020-19877-5',
'modified' => '2022-01-13 14:59:41',
'created' => '2021-12-06 15:53:19',
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'id' => '4013',
'name' => 'A gene therapy for inherited blindness using dCas9-VPR–mediatedtranscriptional activation',
'authors' => 'Böhm, Sybille and Splith, Victoria and Riedmayr, Lisa Maria and Rötzer,René Dominik and Gasparoni, Gilles and Nordström, Karl J. V. and Wagner,Johanna Elisabeth and Hinrichsmeyer, Klara Sonnie and Walter, Jörn andWahl-Schott, Christian and Fenske, Stef',
'description' => '<p>Catalytically inactive dCas9 fused to transcriptional activators (dCas9-VPR) enables activation of silent genes. Many disease genes have counterparts, which serve similar functions but are expressed in distinct cell types. One attractive option to compensate for the missing function of a defective gene could be to transcriptionally activate its functionally equivalent counterpart via dCas9-VPR. Key challenges of this approach include the delivery of dCas9-VPR, activation efficiency, long-term expression of the target gene, and adverse effects in vivo. Using dual adeno-associated viral vectors expressing split dCas9-VPR, we show efficient transcriptional activation and long-term expression of cone photoreceptor-specific M-opsin (Opn1mw) in a rhodopsin-deficient mouse model for retinitis pigmentosa. One year after treatment, this approach yields improved retinal function and attenuated retinal degeneration with no apparent adverse effects. Our study demonstrates that dCas9-VPR–mediated transcriptional activation of functionally equivalent genes has great potential for the treatment of genetic disorders.</p>',
'date' => '2020-08-19',
'pmid' => 'https://advances.sciencemag.org/content/6/34/eaba5614',
'doi' => '10.1126/sciadv.aba5614',
'modified' => '2020-12-16 17:32:46',
'created' => '2020-10-12 14:54:59',
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[maximum depth reached]
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(int) 11 => array(
'id' => '3631',
'name' => 'Guidelines for optimized gene knockout using CRISPR/Cas9',
'authors' => 'Campenhout CV et al.',
'description' => '<p>CRISPR/Cas9 technology has evolved as the most powerful approach to generate genetic models both for fundamental and preclinical research. Despite its apparent simplicity, the outcome of a genome-editing experiment can be substantially impacted by technical parameters and biological considerations. Here, we present guidelines and tools to optimize CRISPR/Cas9 genome-targeting efficiency and specificity. The nature of the target locus, the design of the single guide RNA and the choice of the delivery method should all be carefully considered prior to a genome-editing experiment. Different methods can also be used to detect off-target cleavages and decrease the risk of unwanted mutations. Together, these optimized tools and proper controls are essential to the assessment of CRISPR/Cas9 genome-editing experiments.</p>',
'date' => '2019-05-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/31039627',
'doi' => '10.2144/btn-2018-0187',
'modified' => '2019-05-09 15:37:50',
'created' => '2019-05-09 15:37:50',
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(int) 12 => array(
'id' => '3653',
'name' => 'CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency.',
'authors' => 'Matharu N, Rattanasopha S, Tamura S, Maliskova L, Wang Y, Bernard A, Hardin A, Eckalbar WL, Vaisse C, Ahituv N',
'description' => '<p>A wide range of human diseases result from haploinsufficiency, where the function of one of the two gene copies is lost. Here, we targeted the remaining functional copy of a haploinsufficient gene using CRISPR-mediated activation (CRISPRa) in and heterozygous mouse models to rescue their obesity phenotype. Transgenic-based CRISPRa targeting of the promoter or its distant hypothalamic enhancer up-regulated its expression from the endogenous functional allele in a tissue-specific manner, rescuing the obesity phenotype in heterozygous mice. To evaluate the therapeutic potential of CRISPRa, we injected CRISPRa-recombinant adeno-associated virus into the hypothalamus, which led to reversal of the obesity phenotype in and haploinsufficient mice. Our results suggest that endogenous gene up-regulation could be a potential strategy to treat altered gene dosage diseases.</p>',
'date' => '2019-01-18',
'pmid' => 'http://www.pubmed.gov/30545847',
'doi' => '10.1126/science.aau0629',
'modified' => '2019-06-07 09:05:53',
'created' => '2019-06-06 12:11:18',
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[maximum depth reached]
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(int) 13 => array(
'id' => '3327',
'name' => '(Po)STAC (Polycistronic SunTAg modified CRISPR) enables live-cell and fixed-cell super-resolution imaging of multiple genes',
'authors' => 'Neguembor M.V. et al.',
'description' => '<p>CRISPR/dCas9-based labeling has allowed direct visualization of genomic regions in living cells. However, poor labeling efficiency and signal-to-background ratio have limited its application to visualize genome organization using super-resolution microscopy. We developed (Po)STAC (Polycistronic SunTAg modified CRISPR) by combining CRISPR/dCas9 with SunTag labeling and polycistronic vectors. (Po)STAC enhances both labeling efficiency and fluorescence signal detected from labeled loci enabling live cell imaging as well as super-resolution fixed-cell imaging of multiple genes with high spatiotemporal resolution.</p>',
'date' => '2017-12-23',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/29294098',
'doi' => '',
'modified' => '2018-02-07 10:03:26',
'created' => '2018-02-07 10:03:26',
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(int) 14 => array(
'id' => '4399',
'name' => 'A self-inactivating system for AAV-mediated in vivo base editing',
'authors' => 'Zuo Y. et al.',
'description' => '<p>DNA base editors have been harnessed as an exciting therapeutic platform for human diseases and are rapidly progressing into human clinical trials. However, persistent expression of base editors delivered via adeno-associated virus (AAV) poses concerns with specificity and immunogenicity. Here we develop selfinactivating base editor (siBE) systems with a negative feedback loop where one guide RNA (gRNA) targets the gene of interest and the other targets the deaminase domain itself. We demonstrate that siBE confers efficient on-target editing with time-dependent self-inactivation and increased editing specificity. For the in vivo utilization, we further employ the intein split approach to package siBE targeting mouse Angptl3 into AAV9. Systemic delivery of AAV9-siBE confer efficient editing of Angptl3 in liver, resulting in reduced serum levels of ANGPTL3, triglyceride and total cholesterol, with the active base editor undetectable at 8 weeks after administration. These self-inactivating base editing systems are highly promising for future therapeutic applications.</p>',
'date' => '0000-00-00',
'pmid' => 'https://doi.org/10.21203%2Frs.3.rs-1663604%2Fv1',
'doi' => '10.21203/rs.3.rs-1663604/v1',
'modified' => '2022-08-11 15:24:23',
'created' => '2022-08-11 12:14:50',
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'name' => 'Researcher from the University of Manchester',
'description' => '<p>I used ChIP-qPCR with the Diagenode CRISPR/Cas9 polyclonal antibody to successfully show that Cas9 binds to the target region of my sgRNA, validating my CRISPR experiment. The antibody produced minimal background signal at non-specific genomic regions. I am now using the antibody to validate further sgRNA in different CRISPR cell lines.</p>
<center><img src="../../img/categories/antibodies/cas9-jurkat-vp64-result.png" /></center>
<p><small>ChIP was performed on Jurkat cells expressing dCas9-VP64-mCherry and a sgRNA targeting the IL1RN promoter. Each IP was performed using 4 million cells and 2 µL CRISPR/Cas9 polyclonal antibody (Diagenode C15310258) or 1 µg rabbit IgG control antibody (Diagenode C15410206). qPCR was carried out on undiluted ChIP DNA using SYBR green and PCR primers directed against the sgRNA binding site at IL1RN, as well as two non-target regions at the SLC4A1 and TP53 promoters. ChIP enrichment was measured using the percent input method.</small></p>',
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'description' => '<p><strong>Diagenode’s CRISPR/Cas9 polyclonal antibody</strong> shows superior signal than the original <strong>clone 7A9</strong>: a researcher from EPFL in Lausanne, Switzerland has compared these two antibodies in Western blot.</p>
<center><img src="../../emailing/images/cas9-fig.png" /></center>
<p><small>Western blot was performed using HCT116 DKO cells transduced with Krab-dCas9 (2) or non-transduced (1) cells. Then, 100,000 cells were lysed in sample buffer 2x and boiled 5 min at 95°C before loading in a 15% acrylamide gel. The same sample was loaded 3x in the same gel. The membrane was cut in 3 parts for each antibody. Membrane was blocked 1h with 3% BSA at RT. Antibodies were diluted 1:1,000 in 3% BSA and incubated overnight at 4°C. Secondary incubation was done for 1h at RT (1:10,000 dilution). Anti-hnRNPA1 was used as a loading control.</small></p>',
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'name' => 'CRISPR/Cas9 Antibody',
'description' => '<p>Polyclonal antibody raised in rabbit against the <strong>Cas9</strong> nuclease (<strong>CRISPR</strong>-associated protein 9) using a recombinant protein. </p>',
'label1' => 'Validation data',
'info1' => '<div class="row">
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-fig1.png" /></center></div>
<div class="small-6 columns">
<p><strong>Figure 1. ChIP using the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing nuclease dead Cas9 and sgRNA targeting a sequence in intron 8 of the GAPDH gene, using the iDeal ChIP-seq kit for transcription factors. A titration consisting of 1, 2, 5 and 10 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) was tested. IgG (2 µg/IP) was used as negative IP control. qPCR was performed with primers specific for the targeted sequence in the GAPDH gene, and for the MYOD1 gene, 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).</p>
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<div class="row">
<div class="small-12 columns"><center>A.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-a.jpg" width="700" /></center><center>B.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-b.jpg" width="700" /></center></div>
</div>
<div class="row">
<div class="small-12 columns">
<p><strong>Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing dCas9 and a GAPDH sgRNA cells using 2 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) as described above. The IP'd DNA was subsequently analysed on an Illumina NovaSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the ChIP-seq profile in a region of chromosome 12 surrounding the GAPDH gene (fig 2B) and in a region of chromosome 2 surrounding an off-target peak in the YIPF4 gene.</p>
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<div class="small-4 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-WB.png" /></center></div>
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<p><strong>Figure 3. Western blot analysis using the Diagenode antibody directed against Cas9</strong><br />Western blot was performed on protein extracts from HEK293 cells transfected with dCas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:5,000. The marker is shown on the left, the position of the Cas9 protein is indicated on the right.</p>
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<p><strong>Figure 4. IP using the Diagenode antibody directed against Cas9</strong><br />IP was performed on whole cell extracts (500 µg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 µl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 µg) is shown in lane 1 and 2.</p>
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<div class="small-12 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" width="500" /></center></div>
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<p><strong>Figure 5. Immunofluorescence using the Diagenode antibody directed against Cas9</strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counter-stained with Hoechst 33342. Figure 5 shows the result in the presence (left) or absence (right) of doxycycline.</p>
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<p><small><strong> Figure 1. ChIP using the Diagenode antibody directed against Cas9 </strong><br />ChIP was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50μg chromatin was incubated overnight at 4°C with either 5 μg of an anti-FLAG antibody or 2 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The pre-immune serum (Cas9, PPI) was used as negative IP control. qPCR was performed with primers specific for the GFP gene, and for two non-targeted regions phosphatidic acid phosphatase type 2C (Ppap2c) and protein kinase C delta (Prkcd), used as negative controls. 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/C15310258-CRISPR-WB.png" alt="CRISPR/Cas9 Antibody Validation in WB " caption="false" width="171" height="192" /></p>
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<p><small><strong> Figure 2. Western blot analysis using the Diagenode antibody directed against Cas9 </strong><br />Western blot was performed on protein extracts from HeLa cells transfected with Cas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:1,000 (lane 2) or 1:5,000 (lane 3). Lane 1 shows the result with the pre-immune serum. The marker is shown on the left, the position of the Cas9 protein is indicated on the right. </small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IP.png" alt="CRISPR/Cas9 Antibody Validation in IP" caption="false" width="252" height="286" /></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 (500 μg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 μg) is shown in lane 1 and 2. </small></p>
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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" alt="CRISPR/Cas9 Antibody Validation in IF" caption="false" width="278" height="123" /></p>
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<p><small><strong> Figure 4. Immunofluorescence using the Diagenode antibody directed against Cas9 </strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counterstained with Hoechst 33342. Figure 4 shows the result in the presence (left) or absence (right) of doxycycline. </small></p>
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$testimonials = '<blockquote><p><strong>Diagenode’s CRISPR/Cas9 polyclonal antibody</strong> shows superior signal than the original <strong>clone 7A9</strong>: a researcher from EPFL in Lausanne, Switzerland has compared these two antibodies in Western blot.</p>
<center><img src="../../emailing/images/cas9-fig.png" /></center>
<p><small>Western blot was performed using HCT116 DKO cells transduced with Krab-dCas9 (2) or non-transduced (1) cells. Then, 100,000 cells were lysed in sample buffer 2x and boiled 5 min at 95°C before loading in a 15% acrylamide gel. The same sample was loaded 3x in the same gel. The membrane was cut in 3 parts for each antibody. Membrane was blocked 1h with 3% BSA at RT. Antibodies were diluted 1:1,000 in 3% BSA and incubated overnight at 4°C. Secondary incubation was done for 1h at RT (1:10,000 dilution). Anti-hnRNPA1 was used as a loading control.</small></p><cite>EPFL in Lausanne</cite></blockquote>
'
$featured_testimonials = '<blockquote><span class="label-green" style="margin-bottom:16px;margin-left:-22px">TESTIMONIAL</span><p>I used ChIP-qPCR with the Diagenode CRISPR/Cas9 polyclonal antibody to successfully show that Cas9 binds to the target region of my sgRNA, validating my CRISPR experiment. The antibody produced minimal background signal at non-specific genomic regions. I am now using the antibody to validate further sgRNA in different CRISPR cell lines.</p>
<center><img src="../../img/categories/antibodies/cas9-jurkat-vp64-result.png" /></center>
<p><small>ChIP was performed on Jurkat cells expressing dCas9-VP64-mCherry and a sgRNA targeting the IL1RN promoter. Each IP was performed using 4 million cells and 2 µL CRISPR/Cas9 polyclonal antibody (Diagenode C15310258) or 1 µg rabbit IgG control antibody (Diagenode C15410206). qPCR was carried out on undiluted ChIP DNA using SYBR green and PCR primers directed against the sgRNA binding site at IL1RN, as well as two non-target regions at the SLC4A1 and TP53 promoters. ChIP enrichment was measured using the percent input method.</small></p><cite>Researcher from the University of Manchester</cite></blockquote>
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'description' => '<p><strong>Diagenode’s CRISPR/Cas9 polyclonal antibody</strong> shows superior signal than the original <strong>clone 7A9</strong>: a researcher from EPFL in Lausanne, Switzerland has compared these two antibodies in Western blot.</p>
<center><img src="../../emailing/images/cas9-fig.png" /></center>
<p><small>Western blot was performed using HCT116 DKO cells transduced with Krab-dCas9 (2) or non-transduced (1) cells. Then, 100,000 cells were lysed in sample buffer 2x and boiled 5 min at 95°C before loading in a 15% acrylamide gel. The same sample was loaded 3x in the same gel. The membrane was cut in 3 parts for each antibody. Membrane was blocked 1h with 3% BSA at RT. Antibodies were diluted 1:1,000 in 3% BSA and incubated overnight at 4°C. Secondary incubation was done for 1h at RT (1:10,000 dilution). Anti-hnRNPA1 was used as a loading control.</small></p>',
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'description' => '<p>Polyclonal antibody raised in rabbit against the <strong>Cas9</strong> nuclease (<strong>CRISPR</strong>-associated protein 9) using a recombinant protein. </p>',
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<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-fig1.png" /></center></div>
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<p><strong>Figure 1. ChIP using the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing nuclease dead Cas9 and sgRNA targeting a sequence in intron 8 of the GAPDH gene, using the iDeal ChIP-seq kit for transcription factors. A titration consisting of 1, 2, 5 and 10 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) was tested. IgG (2 µg/IP) was used as negative IP control. qPCR was performed with primers specific for the targeted sequence in the GAPDH gene, and for the MYOD1 gene, 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).</p>
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<div class="small-12 columns"><center>A.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-a.jpg" width="700" /></center><center>B.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-b.jpg" width="700" /></center></div>
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<p><strong>Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing dCas9 and a GAPDH sgRNA cells using 2 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) as described above. The IP'd DNA was subsequently analysed on an Illumina NovaSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the ChIP-seq profile in a region of chromosome 12 surrounding the GAPDH gene (fig 2B) and in a region of chromosome 2 surrounding an off-target peak in the YIPF4 gene.</p>
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<div class="small-4 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-WB.png" /></center></div>
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<p><strong>Figure 3. Western blot analysis using the Diagenode antibody directed against Cas9</strong><br />Western blot was performed on protein extracts from HEK293 cells transfected with dCas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:5,000. The marker is shown on the left, the position of the Cas9 protein is indicated on the right.</p>
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<div class="small-4 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IP.png" /></center></div>
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<p><strong>Figure 4. IP using the Diagenode antibody directed against Cas9</strong><br />IP was performed on whole cell extracts (500 µg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 µl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 µg) is shown in lane 1 and 2.</p>
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<div class="small-12 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" width="500" /></center></div>
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<p><strong>Figure 5. Immunofluorescence using the Diagenode antibody directed against Cas9</strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counter-stained with Hoechst 33342. Figure 5 shows the result in the presence (left) or absence (right) of doxycycline.</p>
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'name' => 'Optimize the selection of guide RNA by ChIP to keep CRISPR on-target',
'description' => '<p>The mechanisms of target recognition and target specificity of the Cas9 protein is still not completely understood. A major hurdle of this technology is the introduction of double-strand breaks (DSBs) at sites other than the intended on-target site (off-target effects). All CRISPR/Cas9 applications require the verification of the specific binding of the sgRNA at the locus of interest. Chromatin immunoprecipitation followed by real-time PCR (ChIP-qPCR) is a technique of choice for studying protein-DNA interactions. In this study, we show a successful ChIP-qPCR method to verify the binding efficiency of the dCas9/sgRNA complex in the targeted region; and ChIP-seq – to monitor off-target bindings of the dCas9/sgRNA complex in the genome.</p>',
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'name' => 'A self-inactivating system for AAV-mediated in vivo base editing',
'authors' => 'Zuo Y. et al.',
'description' => '<p>DNA base editors have been harnessed as an exciting therapeutic platform for human diseases and are rapidly progressing into human clinical trials. However, persistent expression of base editors delivered via adeno-associated virus (AAV) poses concerns with specificity and immunogenicity. Here we develop selfinactivating base editor (siBE) systems with a negative feedback loop where one guide RNA (gRNA) targets the gene of interest and the other targets the deaminase domain itself. We demonstrate that siBE confers efficient on-target editing with time-dependent self-inactivation and increased editing specificity. For the in vivo utilization, we further employ the intein split approach to package siBE targeting mouse Angptl3 into AAV9. Systemic delivery of AAV9-siBE confer efficient editing of Angptl3 in liver, resulting in reduced serum levels of ANGPTL3, triglyceride and total cholesterol, with the active base editor undetectable at 8 weeks after administration. These self-inactivating base editing systems are highly promising for future therapeutic applications.</p>',
'date' => '0000-00-00',
'pmid' => 'https://doi.org/10.21203%2Frs.3.rs-1663604%2Fv1',
'doi' => '10.21203/rs.3.rs-1663604/v1',
'modified' => '2022-08-11 15:24:23',
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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
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Dispatcher::dispatch() - CORE/Cake/Routing/Dispatcher.php, line 167
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-fig1.png" alt="CRISPR/Cas9 Antibody ChIP Grade" caption="false" width="241" height="286" /></p>
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<p><small><strong> Figure 1. ChIP using the Diagenode antibody directed against Cas9 </strong><br />ChIP was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50μg chromatin was incubated overnight at 4°C with either 5 μg of an anti-FLAG antibody or 2 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The pre-immune serum (Cas9, PPI) was used as negative IP control. qPCR was performed with primers specific for the GFP gene, and for two non-targeted regions phosphatidic acid phosphatase type 2C (Ppap2c) and protein kinase C delta (Prkcd), used as negative controls. 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/C15310258-CRISPR-WB.png" alt="CRISPR/Cas9 Antibody Validation in WB " caption="false" width="171" height="192" /></p>
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<p><small><strong> Figure 2. Western blot analysis using the Diagenode antibody directed against Cas9 </strong><br />Western blot was performed on protein extracts from HeLa cells transfected with Cas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:1,000 (lane 2) or 1:5,000 (lane 3). Lane 1 shows the result with the pre-immune serum. The marker is shown on the left, the position of the Cas9 protein is indicated on the right. </small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IP.png" alt="CRISPR/Cas9 Antibody Validation in IP" caption="false" width="252" height="286" /></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 (500 μg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 μg) is shown in lane 1 and 2. </small></p>
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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" alt="CRISPR/Cas9 Antibody Validation in IF" caption="false" width="278" height="123" /></p>
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<p><small><strong> Figure 4. Immunofluorescence using the Diagenode antibody directed against Cas9 </strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counterstained with Hoechst 33342. Figure 4 shows the result in the presence (left) or absence (right) of doxycycline. </small></p>
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'info2' => '<p style="text-align: justify;">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 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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<td>ChIP/ChIP-seq <sup>*</sup></td>
<td>2-5 µl/ChIP</td>
<td>Fig 1, 2</td>
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<tr>
<td>Western Blotting</td>
<td>1:5,000</td>
<td>Fig 3</td>
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<tr>
<td>Immunoprecipitation</td>
<td>1 µl/IP</td>
<td>Fig 4</td>
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<td>Immunofluorescence</td>
<td>1:1,000</td>
<td>Fig 5</td>
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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 µl per IP.</small></p>',
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'description' => '<p>Polyclonal antibody raised in rabbit against the <strong>Cas9</strong> nuclease (<strong>CRISPR</strong>-associated protein 9) using a recombinant protein. </p>',
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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-fig1.png" alt="CRISPR/Cas9 Antibody ChIP Grade" caption="false" width="241" height="286" /></p>
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<p><small><strong> Figure 1. ChIP using the Diagenode antibody directed against Cas9 </strong><br />ChIP was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50μg chromatin was incubated overnight at 4°C with either 5 μg of an anti-FLAG antibody or 2 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The pre-immune serum (Cas9, PPI) was used as negative IP control. qPCR was performed with primers specific for the GFP gene, and for two non-targeted regions phosphatidic acid phosphatase type 2C (Ppap2c) and protein kinase C delta (Prkcd), used as negative controls. 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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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-WB.png" alt="CRISPR/Cas9 Antibody Validation in WB " caption="false" width="171" height="192" /></p>
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<p><small><strong> Figure 2. Western blot analysis using the Diagenode antibody directed against Cas9 </strong><br />Western blot was performed on protein extracts from HeLa cells transfected with Cas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:1,000 (lane 2) or 1:5,000 (lane 3). Lane 1 shows the result with the pre-immune serum. The marker is shown on the left, the position of the Cas9 protein is indicated on the right. </small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IP.png" alt="CRISPR/Cas9 Antibody Validation in IP" caption="false" width="252" height="286" /></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 (500 μg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 μg) is shown in lane 1 and 2. </small></p>
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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" alt="CRISPR/Cas9 Antibody Validation in IF" caption="false" width="278" height="123" /></p>
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<div class="small-8 columns">
<p><small><strong> Figure 4. Immunofluorescence using the Diagenode antibody directed against Cas9 </strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counterstained with Hoechst 33342. Figure 4 shows the result in the presence (left) or absence (right) of doxycycline. </small></p>
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'label2' => 'Background',
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'meta_title' => 'CRISPR/Cas9 polyclonal antibody (sample size)',
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'description' => '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 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.',
'clonality' => '',
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'lot' => 'A2508-004',
'concentration' => 'Not determined',
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'type' => 'Polyclonal, <strong>ChIP grade, ChIP-seq grade</strong>',
'purity' => 'Whole antiserum from rabbit containing 0.05% azide.',
'classification' => '',
'application_table' => '<table>
<thead>
<tr>
<th>Applications</th>
<th>Suggested dilution</th>
<th>References</th>
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<tbody>
<tr>
<td>ChIP/ChIP-seq <sup>*</sup></td>
<td>2-5 µl/ChIP</td>
<td>Fig 1, 2</td>
</tr>
<tr>
<td>Western Blotting</td>
<td>1:5,000</td>
<td>Fig 3</td>
</tr>
<tr>
<td>Immunoprecipitation</td>
<td>1 µl/IP</td>
<td>Fig 4</td>
</tr>
<tr>
<td>Immunofluorescence</td>
<td>1:1,000</td>
<td>Fig 5</td>
</tr>
</tbody>
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<p></p>
<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 µl per IP.</small></p>',
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'name' => 'CRISPR/Cas9 Antibody',
'description' => '<p>Polyclonal antibody raised in rabbit against the <strong>Cas9</strong> nuclease (<strong>CRISPR</strong>-associated protein 9) using a recombinant protein. </p>',
'label1' => 'Validation data',
'info1' => '<div class="row">
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-fig1.png" /></center></div>
<div class="small-6 columns">
<p><strong>Figure 1. ChIP using the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing nuclease dead Cas9 and sgRNA targeting a sequence in intron 8 of the GAPDH gene, using the iDeal ChIP-seq kit for transcription factors. A titration consisting of 1, 2, 5 and 10 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) was tested. IgG (2 µg/IP) was used as negative IP control. qPCR was performed with primers specific for the targeted sequence in the GAPDH gene, and for the MYOD1 gene, 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).</p>
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<div class="row">
<div class="small-12 columns"><center>A.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-a.jpg" width="700" /></center><center>B.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-b.jpg" width="700" /></center></div>
</div>
<div class="row">
<div class="small-12 columns">
<p><strong>Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing dCas9 and a GAPDH sgRNA cells using 2 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) as described above. The IP'd DNA was subsequently analysed on an Illumina NovaSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the ChIP-seq profile in a region of chromosome 12 surrounding the GAPDH gene (fig 2B) and in a region of chromosome 2 surrounding an off-target peak in the YIPF4 gene.</p>
</div>
</div>
<div class="row">
<div class="small-4 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-WB.png" /></center></div>
<div class="small-8 columns">
<p><strong>Figure 3. Western blot analysis using the Diagenode antibody directed against Cas9</strong><br />Western blot was performed on protein extracts from HEK293 cells transfected with dCas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:5,000. The marker is shown on the left, the position of the Cas9 protein is indicated on the right.</p>
</div>
</div>
<div class="row">
<div class="small-4 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IP.png" /></center></div>
<div class="small-8 columns">
<p><strong>Figure 4. IP using the Diagenode antibody directed against Cas9</strong><br />IP was performed on whole cell extracts (500 µg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 µl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 µg) is shown in lane 1 and 2.</p>
</div>
</div>
<div class="row">
<div class="small-12 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" width="500" /></center></div>
</div>
<div class="row">
<div class="small-12 columns">
<p><strong>Figure 5. Immunofluorescence using the Diagenode antibody directed against Cas9</strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counter-stained with Hoechst 33342. Figure 5 shows the result in the presence (left) or absence (right) of doxycycline.</p>
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'info2' => '<p style="text-align: justify;">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 <strong>CRISPR/Cas9</strong> (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 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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'meta_description' => 'S. pyogenes CRISPR/Cas9 Polyclonal Antibody validated in ChIP-seq, ChIP-qPCR, IP, IF and WB. Batch-specific data available on the website. Sample size available.',
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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' => '<h1><strong>Validated epigenetics antibodies</strong> – care for a sample?<br /> </h1>
<p>Diagenode has partnered with leading epigenetics experts and numerous epigenetics consortiums to bring to you a validated and comprehensive collection of epigenetic antibodies. As an expert in epigenetics, we are committed to offering highly-specific antibodies validated for ChIP/ChIP-seq and many other applications. All batch-specific validation data is available on our website.<br /><a href="../categories/antibodies">Read about our expertise in antibody production</a>.</p>
<ul>
<li><strong>Focused</strong> - Diagenode's selection of antibodies is exclusively dedicated for epigenetic research. <a title="See the full collection." href="../categories/all-antibodies">See the full collection.</a></li>
<li><strong>Strict quality standards</strong> with rigorous QC and validation</li>
<li><strong>Classified</strong> based on level of validation for flexibility of application</li>
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<p>Existing sample sizes are listed below. We will soon expand our collection. Are you looking for a sample size of another antibody? Just <a href="mailto:agnieszka.zelisko@diagenode.com?Subject=Sample%20Size%20Request" target="_top">Contact us</a>.</p>',
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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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'meta_title' => 'Diagenode's selection of Antibodies is exclusively dedicated for Epigenetic Research | Diagenode',
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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>
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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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'description' => '<p><span>The fluorescence ubiquitination cell cycle inhibitor (FUCCI) has been introduced to monitor cell cycle activity in living cells, including human induced pluripotent stem cells (hiPSC) and derived cell types. We have recently developed hiPSC with stable expression of dCas9VPR for endogenous gene activation and a Citrine-tagged ACTN2 cell line to monitor sarcomere development and function in muscle cells. Here, we present dual and triple transgenic hiPSC lines developed by genomic integration of FUCCI with and without dCas9VPR into the ROSA26 and AAVS1 loci, respectively, in the previously introduced ACTN2-Citrine line. Functionality of the transgenes was demonstrated in the novel hiPSC line, which we introduce as Myo-CCER and CraCCER.</span></p>',
'date' => '2024-08-05',
'pmid' => 'https://www.sciencedirect.com/science/article/pii/S1873506124002290',
'doi' => 'https://doi.org/10.1016/j.scr.2024.103531',
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'name' => 'Methyltransferase Inhibition Enables Tgf Driven Induction of and in Cancer Cells.',
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'description' => '<p>deletion or silencing is common across human cancer, reinforcing the general importance of bypassing its tumor suppression in cancer formation or progression. In rhabdomyosarcoma (RMS) and neuroblastoma, two common childhood cancers, the three transcripts are independently expressed to varying degrees, but one, is uniformly silenced. Although TGFβ induces certain transcripts in HeLa cells, it was unable to do so in five tested RMS lines unless the cells were pretreated with a broadly acting methyltransferase inhibitor, DZNep, or one targeting EZH2. induction by TGFβ correlated with de novo appearance of three H3K27Ac peaks within a 20 kb element ∼150 kb proximal to . Deleting that segment prevented their induction by TGFβ but not a basal increase driven by methyltransferase inhibition alone. Expression of two transcripts was enhanced by dCas9/CRISPR activation targeting either the relevant promoter or the 20 kb elements, and this "precise" manipulation diminished RMS cell propagation in vitro. Our findings show crosstalk between methyltransferase inhibition and TGFβ-dependent activation of a remote enhancer to reverse silencing. Though focused on here, such crosstalk may apply to other TGFβ-responsive genes and perhaps govern this signaling protein's complex effects promoting or blocking cancer.</p>',
'date' => '2023-01-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/36941772',
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'name' => 'Massively parallel multi-target CRISPR system interrogates Cas9-basedtarget recognition, DNA cleavage, and DNA repair',
'authors' => 'Zou Roger S. et al.',
'description' => '<p>CRISPR-Cas9 nucleases, and particularly Streptococcus pyogenes Cas9, are widespread tools for genome editing. However, many aspects of intracellular Cas9 activity and the ensuing DNA damage response remain incompletely characterized. In order to address these issues, we developed a multiplexed CRISPR approach, where a single, degenerate multi-target gRNA (mgRNA) directs the Cas9 enzyme to target hundred endogenous sites at once. When combined with next-generation sequencing readouts, this system enables interrogation of Cas9 activity and DNA double-strand break (DSB) repair response in high-throughput. Here, we present a step-by-step protocol to deliver a Cas9:mgRNA ribonucleoprotein complex into cultured cells and measure key processes related to Cas9 activity and DSB repair.</p>',
'date' => '2022-09-01',
'pmid' => 'https://europepmc.org/article/ppr/ppr540155',
'doi' => '10.21203/rs.3.pex-1938/v1',
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(int) 3 => array(
'id' => '4437',
'name' => 'Analysis of estrogen-regulated enhancer RNAs identifies a functionalmotif required for enhancer assembly and gene expression.',
'authors' => 'Hou Tim Y and Kraus W Lee',
'description' => '<p>To better understand the functions of non-coding enhancer RNAs (eRNAs), we annotated the estrogen-regulated eRNA transcriptome in estrogen receptor α (ERα)-positive breast cancer cells using PRO-cap and RNA sequencing. We then cloned a subset of the eRNAs identified, fused them to single guide RNAs, and targeted them to their ERα enhancers of origin using CRISPR/dCas9. Some of the eRNAs tested modulated the expression of cognate, but not heterologous, target genes after estrogen treatment by increasing ERα recruitment and stimulating p300-catalyzed H3K27 acetylation at the enhancer. We identified a ∼40 nucleotide functional eRNA regulatory motif (FERM) present in many eRNAs that was necessary and sufficient to modulate gene expression, but not the specificity of activation, after estrogen treatment. The FERM interacted with BCAS2, an RNA-binding protein amplified in breast cancers. The ectopic expression of a targeted eRNA controlling the expression of an oncogene resulted in increased cell proliferation, demonstrating the regulatory potential of eRNAs in breast cancer.</p>',
'date' => '2022-06-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/35705040',
'doi' => '10.1016/j.celrep.2022.110944',
'modified' => '2022-09-28 09:20:34',
'created' => '2022-09-08 16:32:20',
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(int) 4 => array(
'id' => '4560',
'name' => 'Avian influenza viruses suppress innate immunity by inducingtrans-transcriptional readthrough via SSU72.',
'authors' => 'Zhao Y. et al.',
'description' => '<p>Innate immunity plays critical antiviral roles. The highly virulent avian influenza viruses (AIVs) H5N1, H7N9, and H5N6 can better escape host innate immune responses than the less virulent seasonal H1N1 virus. Here, we report a mechanism by which transcriptional readthrough (TRT)-mediated suppression of innate immunity occurs post AIV infection. By using cell lines, mouse lungs, and patient PBMCs, we showed that genes on the complementary strand ("trans" genes) influenced by TRT were involved in the disruption of host antiviral responses during AIV infection. The trans-TRT enhanced viral lethality, and TRT abolishment increased cell viability and STAT1/2 expression. The viral NS1 protein directly bound to SSU72, and degradation of SSU72 induced TRT. SSU72 overexpression reduced TRT and alleviated mouse lung injury. Our results suggest that AIVs infection induce TRT by reducing SSU72 expression, thereby impairing host immune responses, a molecular mechanism acting through the NS1-SSU72-trans-TRT-STAT1/2 axis. Thus, restoration of SSU72 expression might be a potential strategy for preventing AIV pandemics.</p>',
'date' => '2022-03-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/35332300',
'doi' => '10.1038/s41423-022-00843-8',
'modified' => '2022-11-24 10:02:04',
'created' => '2022-11-24 08:49:52',
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'id' => '4365',
'name' => 'Antisense non-coding transcription represses the <i>PHO5</i> model gene<i>via</i> remodelling of promoter chromatin structure',
'authors' => 'Novačić A. et al. ',
'description' => '<p>Pervasive transcription of eukaryotic genomes generates non-coding transcripts with regulatory potential. We examined the effects of non-coding antisense transcription on the regulation of expression of the yeast PHO5 gene, a paradigmatic case for gene regulation through promoter chromatin remodeling. By enhancing or impairing the level of overlapping antisense transcription through specific mutant backgrounds and the use of the CRISPRi system, we demonstrated a negative role for antisense transcription at the PHO5 gene locus. Furthermore, enhanced elongation of PHO5 antisense leads to a more repressive chromatin configuration at the PHO5 gene promoter, which is remodeled more slowly upon gene induction. The negative effect of antisense transcription on PHO5 gene transcription is mitigated by inactivation of the histone deacetylase Rpd3, showing that PHO5 antisense RNA acts via histone deacetylation. This regulatory pathway leads to Rpd3-dependent decreased recruitment of the RSC chromatin remodeling complex to the PHO5 gene promoter upon induction of antisense transcription. Overall, we extend the model of PHO5 gene regulation by demonstrating a gene silencing function of antisense transcription through a chromatin-based mechanism.</p>',
'date' => '2022-02-01',
'pmid' => 'https://doi.org/10.1101%2F2022.02.21.481265',
'doi' => '10.1101/2022.02.21.481265',
'modified' => '2022-08-04 15:52:12',
'created' => '2022-08-04 14:55:36',
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(int) 6 => array(
'id' => '4237',
'name' => 'A predominant enhancer co-amplified with the oncogene is necessary andsufficient for its expression in squamous cancer',
'authors' => 'Liu Y. et al.',
'description' => '<p>Amplification and overexpression of the SOX2 oncogene represent a hallmark of squamous cancers originating from diverse tissue types. Here, we find that squamous cancers selectively amplify a 3’ noncoding region together with SOX2, which harbors squamous cancer-specific chromatin accessible regions. We identify a single enhancer e1 that predominantly drives SOX2 expression. Repression of e1 in SOX2-high cells causes collapse of the surrounding enhancers, remarkable reduction in SOX2 expression, and a global transcriptional change reminiscent of SOX2 knockout. The e1 enhancer is driven by a combination of transcription factors including SOX2 itself and the AP-1 complex, which facilitates recruitment of the co-activator BRD4. CRISPR-mediated activation of e1 in SOX2-low cells is sufficient to rebuild the e1-SOX2 loop and activate SOX2 expression. Our study shows that squamous cancers selectively amplify a predominant enhancer to drive SOX2 overexpression, uncovering functional links among enhancer activation, chromatin looping, and lineage-specific copy number amplifications of oncogenes.</p>',
'date' => '2021-12-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/34880227',
'doi' => '10.1038/s41467-021-27055-4',
'modified' => '2022-05-19 17:05:00',
'created' => '2022-05-19 10:41:50',
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(int) 7 => array(
'id' => '4301',
'name' => 'Establishment of a second generation homozygous CRISPRa human inducedpluripotent stem cell (hiPSC) line for enhanced levels of endogenous geneactivation.',
'authors' => 'Schoger Eric et al.',
'description' => '<p>CRISPR/Cas9 technology based on nuclease inactive dCas9 and fused to the heterotrimeric VPR transcriptional activator is a powerful tool to enhance endogenous transcription by targeting defined genomic loci. We generated homozygous human induced pluripotent stem cell (hiPSC) lines carrying dCas9 fused to VPR along with a WPRE element at the AAVS1 locus (CRISPRa2). We demonstrated pluripotency, genomic integrity and differentiation potential into all three germ layers. CRISPRa2 cells showed increased transgene expression and higher transcriptional induction in hiPSC-derived cardiomyocytes compared to a previously described CRISPRa line. Both lines allow studying endogenous transcriptional modulation with lower and higher transcript abundance.</p>',
'date' => '2021-10-01',
'pmid' => 'https://doi.org/10.1016%2Fj.scr.2021.102518',
'doi' => '10.1016/j.scr.2021.102518',
'modified' => '2022-05-30 09:56:01',
'created' => '2022-05-19 10:41:50',
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(int) 8 => array(
'id' => '4328',
'name' => 'Establishment of two homozygous CRISPR interference (CRISPRi)knock-in human induced pluripotent stem cell (hiPSC) lines for titratableendogenous gene repression.',
'authors' => 'Schoger Eric et al.',
'description' => '<p>Using nuclease-deficient dead (d)Cas9 without enzymatic activity fused to transcriptional inhibitors (CRISPRi) allows for transcriptional interference and results in a powerful tool for the elucidation of developmental, homeostatic and disease mechanisms. We inserted dCas9KRAB (CRISPRi) cassette into the AAVS1 locus of hiPSC lines, which resulted in homozygous knock-in with an otherwise unaltered genome. Expression of dCas9KRAB protein, pluripotency and the ability to differentiate into all three embryonic germ layers were validated. Furthermore, functional cardiomyocyte generation was tested. The hiPSC-CRISPRi cell lines offer a valuable tool for studying endogenous transcriptional repression with single and multiplexed possibilities in all human cell types.</p>',
'date' => '2021-08-01',
'pmid' => 'https://doi.org/10.1016%2Fj.scr.2021.102473',
'doi' => '10.1016/j.scr.2021.102473',
'modified' => '2022-06-22 09:28:49',
'created' => '2022-05-19 10:41:50',
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[maximum depth reached]
)
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(int) 9 => array(
'id' => '4209',
'name' => 'TGFβ promotes widespread enhancer chromatin opening and operates ongenomic regulatory domains.',
'authors' => 'Guerrero-Martínez J. et al. ',
'description' => '<p>The Transforming Growth Factor-β (TGFβ) signaling pathway controls transcription by regulating enhancer activity. How TGFβ-regulated enhancers are selected and what chromatin changes are associated with TGFβ-dependent enhancers regulation are still unclear. Here we report that TGFβ treatment triggers fast and widespread increase in chromatin accessibility in about 80\% of the enhancers of normal mouse mammary epithelial-gland cells, irrespective of whether they are activated, repressed or not regulated by TGFβ. This enhancer opening depends on both the canonical and non-canonical TGFβ pathways. Most TGFβ-regulated genes are located around enhancers regulated in the same way, often creating domains of several co-regulated genes that we term TGFβ regulatory domains (TRD). CRISPR-mediated inactivation of enhancers within TRDs impairs TGFβ-dependent regulation of all co-regulated genes, demonstrating that enhancer targeting is more promiscuous than previously anticipated. The area of TRD influence is restricted by topologically associating domains (TADs) borders, causing a bias towards co-regulation within TADs.</p>',
'date' => '2020-12-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/33273453',
'doi' => '10.1038/s41467-020-19877-5',
'modified' => '2022-01-13 14:59:41',
'created' => '2021-12-06 15:53:19',
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'id' => '4013',
'name' => 'A gene therapy for inherited blindness using dCas9-VPR–mediatedtranscriptional activation',
'authors' => 'Böhm, Sybille and Splith, Victoria and Riedmayr, Lisa Maria and Rötzer,René Dominik and Gasparoni, Gilles and Nordström, Karl J. V. and Wagner,Johanna Elisabeth and Hinrichsmeyer, Klara Sonnie and Walter, Jörn andWahl-Schott, Christian and Fenske, Stef',
'description' => '<p>Catalytically inactive dCas9 fused to transcriptional activators (dCas9-VPR) enables activation of silent genes. Many disease genes have counterparts, which serve similar functions but are expressed in distinct cell types. One attractive option to compensate for the missing function of a defective gene could be to transcriptionally activate its functionally equivalent counterpart via dCas9-VPR. Key challenges of this approach include the delivery of dCas9-VPR, activation efficiency, long-term expression of the target gene, and adverse effects in vivo. Using dual adeno-associated viral vectors expressing split dCas9-VPR, we show efficient transcriptional activation and long-term expression of cone photoreceptor-specific M-opsin (Opn1mw) in a rhodopsin-deficient mouse model for retinitis pigmentosa. One year after treatment, this approach yields improved retinal function and attenuated retinal degeneration with no apparent adverse effects. Our study demonstrates that dCas9-VPR–mediated transcriptional activation of functionally equivalent genes has great potential for the treatment of genetic disorders.</p>',
'date' => '2020-08-19',
'pmid' => 'https://advances.sciencemag.org/content/6/34/eaba5614',
'doi' => '10.1126/sciadv.aba5614',
'modified' => '2020-12-16 17:32:46',
'created' => '2020-10-12 14:54:59',
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[maximum depth reached]
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(int) 11 => array(
'id' => '3631',
'name' => 'Guidelines for optimized gene knockout using CRISPR/Cas9',
'authors' => 'Campenhout CV et al.',
'description' => '<p>CRISPR/Cas9 technology has evolved as the most powerful approach to generate genetic models both for fundamental and preclinical research. Despite its apparent simplicity, the outcome of a genome-editing experiment can be substantially impacted by technical parameters and biological considerations. Here, we present guidelines and tools to optimize CRISPR/Cas9 genome-targeting efficiency and specificity. The nature of the target locus, the design of the single guide RNA and the choice of the delivery method should all be carefully considered prior to a genome-editing experiment. Different methods can also be used to detect off-target cleavages and decrease the risk of unwanted mutations. Together, these optimized tools and proper controls are essential to the assessment of CRISPR/Cas9 genome-editing experiments.</p>',
'date' => '2019-05-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/31039627',
'doi' => '10.2144/btn-2018-0187',
'modified' => '2019-05-09 15:37:50',
'created' => '2019-05-09 15:37:50',
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(int) 12 => array(
'id' => '3653',
'name' => 'CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency.',
'authors' => 'Matharu N, Rattanasopha S, Tamura S, Maliskova L, Wang Y, Bernard A, Hardin A, Eckalbar WL, Vaisse C, Ahituv N',
'description' => '<p>A wide range of human diseases result from haploinsufficiency, where the function of one of the two gene copies is lost. Here, we targeted the remaining functional copy of a haploinsufficient gene using CRISPR-mediated activation (CRISPRa) in and heterozygous mouse models to rescue their obesity phenotype. Transgenic-based CRISPRa targeting of the promoter or its distant hypothalamic enhancer up-regulated its expression from the endogenous functional allele in a tissue-specific manner, rescuing the obesity phenotype in heterozygous mice. To evaluate the therapeutic potential of CRISPRa, we injected CRISPRa-recombinant adeno-associated virus into the hypothalamus, which led to reversal of the obesity phenotype in and haploinsufficient mice. Our results suggest that endogenous gene up-regulation could be a potential strategy to treat altered gene dosage diseases.</p>',
'date' => '2019-01-18',
'pmid' => 'http://www.pubmed.gov/30545847',
'doi' => '10.1126/science.aau0629',
'modified' => '2019-06-07 09:05:53',
'created' => '2019-06-06 12:11:18',
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[maximum depth reached]
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(int) 13 => array(
'id' => '3327',
'name' => '(Po)STAC (Polycistronic SunTAg modified CRISPR) enables live-cell and fixed-cell super-resolution imaging of multiple genes',
'authors' => 'Neguembor M.V. et al.',
'description' => '<p>CRISPR/dCas9-based labeling has allowed direct visualization of genomic regions in living cells. However, poor labeling efficiency and signal-to-background ratio have limited its application to visualize genome organization using super-resolution microscopy. We developed (Po)STAC (Polycistronic SunTAg modified CRISPR) by combining CRISPR/dCas9 with SunTag labeling and polycistronic vectors. (Po)STAC enhances both labeling efficiency and fluorescence signal detected from labeled loci enabling live cell imaging as well as super-resolution fixed-cell imaging of multiple genes with high spatiotemporal resolution.</p>',
'date' => '2017-12-23',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/29294098',
'doi' => '',
'modified' => '2018-02-07 10:03:26',
'created' => '2018-02-07 10:03:26',
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(int) 14 => array(
'id' => '4399',
'name' => 'A self-inactivating system for AAV-mediated in vivo base editing',
'authors' => 'Zuo Y. et al.',
'description' => '<p>DNA base editors have been harnessed as an exciting therapeutic platform for human diseases and are rapidly progressing into human clinical trials. However, persistent expression of base editors delivered via adeno-associated virus (AAV) poses concerns with specificity and immunogenicity. Here we develop selfinactivating base editor (siBE) systems with a negative feedback loop where one guide RNA (gRNA) targets the gene of interest and the other targets the deaminase domain itself. We demonstrate that siBE confers efficient on-target editing with time-dependent self-inactivation and increased editing specificity. For the in vivo utilization, we further employ the intein split approach to package siBE targeting mouse Angptl3 into AAV9. Systemic delivery of AAV9-siBE confer efficient editing of Angptl3 in liver, resulting in reduced serum levels of ANGPTL3, triglyceride and total cholesterol, with the active base editor undetectable at 8 weeks after administration. These self-inactivating base editing systems are highly promising for future therapeutic applications.</p>',
'date' => '0000-00-00',
'pmid' => 'https://doi.org/10.21203%2Frs.3.rs-1663604%2Fv1',
'doi' => '10.21203/rs.3.rs-1663604/v1',
'modified' => '2022-08-11 15:24:23',
'created' => '2022-08-11 12:14:50',
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'name' => 'Researcher from the University of Manchester',
'description' => '<p>I used ChIP-qPCR with the Diagenode CRISPR/Cas9 polyclonal antibody to successfully show that Cas9 binds to the target region of my sgRNA, validating my CRISPR experiment. The antibody produced minimal background signal at non-specific genomic regions. I am now using the antibody to validate further sgRNA in different CRISPR cell lines.</p>
<center><img src="../../img/categories/antibodies/cas9-jurkat-vp64-result.png" /></center>
<p><small>ChIP was performed on Jurkat cells expressing dCas9-VP64-mCherry and a sgRNA targeting the IL1RN promoter. Each IP was performed using 4 million cells and 2 µL CRISPR/Cas9 polyclonal antibody (Diagenode C15310258) or 1 µg rabbit IgG control antibody (Diagenode C15410206). qPCR was carried out on undiluted ChIP DNA using SYBR green and PCR primers directed against the sgRNA binding site at IL1RN, as well as two non-target regions at the SLC4A1 and TP53 promoters. ChIP enrichment was measured using the percent input method.</small></p>',
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'description' => '<p><strong>Diagenode’s CRISPR/Cas9 polyclonal antibody</strong> shows superior signal than the original <strong>clone 7A9</strong>: a researcher from EPFL in Lausanne, Switzerland has compared these two antibodies in Western blot.</p>
<center><img src="../../emailing/images/cas9-fig.png" /></center>
<p><small>Western blot was performed using HCT116 DKO cells transduced with Krab-dCas9 (2) or non-transduced (1) cells. Then, 100,000 cells were lysed in sample buffer 2x and boiled 5 min at 95°C before loading in a 15% acrylamide gel. The same sample was loaded 3x in the same gel. The membrane was cut in 3 parts for each antibody. Membrane was blocked 1h with 3% BSA at RT. Antibodies were diluted 1:1,000 in 3% BSA and incubated overnight at 4°C. Secondary incubation was done for 1h at RT (1:10,000 dilution). Anti-hnRNPA1 was used as a loading control.</small></p>',
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'name' => 'CRISPR/Cas9 Antibody',
'description' => '<p>Polyclonal antibody raised in rabbit against the <strong>Cas9</strong> nuclease (<strong>CRISPR</strong>-associated protein 9) using a recombinant protein. </p>',
'label1' => 'Validation data',
'info1' => '<div class="row">
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-fig1.png" /></center></div>
<div class="small-6 columns">
<p><strong>Figure 1. ChIP using the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing nuclease dead Cas9 and sgRNA targeting a sequence in intron 8 of the GAPDH gene, using the iDeal ChIP-seq kit for transcription factors. A titration consisting of 1, 2, 5 and 10 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) was tested. IgG (2 µg/IP) was used as negative IP control. qPCR was performed with primers specific for the targeted sequence in the GAPDH gene, and for the MYOD1 gene, 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).</p>
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<div class="row">
<div class="small-12 columns"><center>A.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-a.jpg" width="700" /></center><center>B.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-b.jpg" width="700" /></center></div>
</div>
<div class="row">
<div class="small-12 columns">
<p><strong>Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing dCas9 and a GAPDH sgRNA cells using 2 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) as described above. The IP'd DNA was subsequently analysed on an Illumina NovaSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the ChIP-seq profile in a region of chromosome 12 surrounding the GAPDH gene (fig 2B) and in a region of chromosome 2 surrounding an off-target peak in the YIPF4 gene.</p>
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<div class="small-4 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-WB.png" /></center></div>
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<p><strong>Figure 3. Western blot analysis using the Diagenode antibody directed against Cas9</strong><br />Western blot was performed on protein extracts from HEK293 cells transfected with dCas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:5,000. The marker is shown on the left, the position of the Cas9 protein is indicated on the right.</p>
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<p><strong>Figure 4. IP using the Diagenode antibody directed against Cas9</strong><br />IP was performed on whole cell extracts (500 µg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 µl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 µg) is shown in lane 1 and 2.</p>
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<div class="small-12 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" width="500" /></center></div>
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<p><strong>Figure 5. Immunofluorescence using the Diagenode antibody directed against Cas9</strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counter-stained with Hoechst 33342. Figure 5 shows the result in the presence (left) or absence (right) of doxycycline.</p>
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<p><small><strong> Figure 1. ChIP using the Diagenode antibody directed against Cas9 </strong><br />ChIP was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50μg chromatin was incubated overnight at 4°C with either 5 μg of an anti-FLAG antibody or 2 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The pre-immune serum (Cas9, PPI) was used as negative IP control. qPCR was performed with primers specific for the GFP gene, and for two non-targeted regions phosphatidic acid phosphatase type 2C (Ppap2c) and protein kinase C delta (Prkcd), used as negative controls. 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/C15310258-CRISPR-WB.png" alt="CRISPR/Cas9 Antibody Validation in WB " caption="false" width="171" height="192" /></p>
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<p><small><strong> Figure 2. Western blot analysis using the Diagenode antibody directed against Cas9 </strong><br />Western blot was performed on protein extracts from HeLa cells transfected with Cas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:1,000 (lane 2) or 1:5,000 (lane 3). Lane 1 shows the result with the pre-immune serum. The marker is shown on the left, the position of the Cas9 protein is indicated on the right. </small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IP.png" alt="CRISPR/Cas9 Antibody Validation in IP" caption="false" width="252" height="286" /></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 (500 μg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 μg) is shown in lane 1 and 2. </small></p>
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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" alt="CRISPR/Cas9 Antibody Validation in IF" caption="false" width="278" height="123" /></p>
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<p><small><strong> Figure 4. Immunofluorescence using the Diagenode antibody directed against Cas9 </strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counterstained with Hoechst 33342. Figure 4 shows the result in the presence (left) or absence (right) of doxycycline. </small></p>
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$testimonials = '<blockquote><p><strong>Diagenode’s CRISPR/Cas9 polyclonal antibody</strong> shows superior signal than the original <strong>clone 7A9</strong>: a researcher from EPFL in Lausanne, Switzerland has compared these two antibodies in Western blot.</p>
<center><img src="../../emailing/images/cas9-fig.png" /></center>
<p><small>Western blot was performed using HCT116 DKO cells transduced with Krab-dCas9 (2) or non-transduced (1) cells. Then, 100,000 cells were lysed in sample buffer 2x and boiled 5 min at 95°C before loading in a 15% acrylamide gel. The same sample was loaded 3x in the same gel. The membrane was cut in 3 parts for each antibody. Membrane was blocked 1h with 3% BSA at RT. Antibodies were diluted 1:1,000 in 3% BSA and incubated overnight at 4°C. Secondary incubation was done for 1h at RT (1:10,000 dilution). Anti-hnRNPA1 was used as a loading control.</small></p><cite>EPFL in Lausanne</cite></blockquote>
'
$featured_testimonials = '<blockquote><span class="label-green" style="margin-bottom:16px;margin-left:-22px">TESTIMONIAL</span><p>I used ChIP-qPCR with the Diagenode CRISPR/Cas9 polyclonal antibody to successfully show that Cas9 binds to the target region of my sgRNA, validating my CRISPR experiment. The antibody produced minimal background signal at non-specific genomic regions. I am now using the antibody to validate further sgRNA in different CRISPR cell lines.</p>
<center><img src="../../img/categories/antibodies/cas9-jurkat-vp64-result.png" /></center>
<p><small>ChIP was performed on Jurkat cells expressing dCas9-VP64-mCherry and a sgRNA targeting the IL1RN promoter. Each IP was performed using 4 million cells and 2 µL CRISPR/Cas9 polyclonal antibody (Diagenode C15310258) or 1 µg rabbit IgG control antibody (Diagenode C15410206). qPCR was carried out on undiluted ChIP DNA using SYBR green and PCR primers directed against the sgRNA binding site at IL1RN, as well as two non-target regions at the SLC4A1 and TP53 promoters. ChIP enrichment was measured using the percent input method.</small></p><cite>Researcher from the University of Manchester</cite></blockquote>
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'description' => '<p><strong>Diagenode’s CRISPR/Cas9 polyclonal antibody</strong> shows superior signal than the original <strong>clone 7A9</strong>: a researcher from EPFL in Lausanne, Switzerland has compared these two antibodies in Western blot.</p>
<center><img src="../../emailing/images/cas9-fig.png" /></center>
<p><small>Western blot was performed using HCT116 DKO cells transduced with Krab-dCas9 (2) or non-transduced (1) cells. Then, 100,000 cells were lysed in sample buffer 2x and boiled 5 min at 95°C before loading in a 15% acrylamide gel. The same sample was loaded 3x in the same gel. The membrane was cut in 3 parts for each antibody. Membrane was blocked 1h with 3% BSA at RT. Antibodies were diluted 1:1,000 in 3% BSA and incubated overnight at 4°C. Secondary incubation was done for 1h at RT (1:10,000 dilution). Anti-hnRNPA1 was used as a loading control.</small></p>',
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'description' => '<p>Polyclonal antibody raised in rabbit against the <strong>Cas9</strong> nuclease (<strong>CRISPR</strong>-associated protein 9) using a recombinant protein. </p>',
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<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-fig1.png" /></center></div>
<div class="small-6 columns">
<p><strong>Figure 1. ChIP using the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing nuclease dead Cas9 and sgRNA targeting a sequence in intron 8 of the GAPDH gene, using the iDeal ChIP-seq kit for transcription factors. A titration consisting of 1, 2, 5 and 10 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) was tested. IgG (2 µg/IP) was used as negative IP control. qPCR was performed with primers specific for the targeted sequence in the GAPDH gene, and for the MYOD1 gene, 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).</p>
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<div class="small-12 columns"><center>A.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-a.jpg" width="700" /></center><center>B.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-b.jpg" width="700" /></center></div>
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<p><strong>Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing dCas9 and a GAPDH sgRNA cells using 2 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) as described above. The IP'd DNA was subsequently analysed on an Illumina NovaSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the ChIP-seq profile in a region of chromosome 12 surrounding the GAPDH gene (fig 2B) and in a region of chromosome 2 surrounding an off-target peak in the YIPF4 gene.</p>
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<div class="small-4 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-WB.png" /></center></div>
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<p><strong>Figure 3. Western blot analysis using the Diagenode antibody directed against Cas9</strong><br />Western blot was performed on protein extracts from HEK293 cells transfected with dCas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:5,000. The marker is shown on the left, the position of the Cas9 protein is indicated on the right.</p>
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<div class="small-4 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IP.png" /></center></div>
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<p><strong>Figure 4. IP using the Diagenode antibody directed against Cas9</strong><br />IP was performed on whole cell extracts (500 µg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 µl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 µg) is shown in lane 1 and 2.</p>
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<div class="small-12 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" width="500" /></center></div>
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<p><strong>Figure 5. Immunofluorescence using the Diagenode antibody directed against Cas9</strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counter-stained with Hoechst 33342. Figure 5 shows the result in the presence (left) or absence (right) of doxycycline.</p>
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'name' => 'Optimize the selection of guide RNA by ChIP to keep CRISPR on-target',
'description' => '<p>The mechanisms of target recognition and target specificity of the Cas9 protein is still not completely understood. A major hurdle of this technology is the introduction of double-strand breaks (DSBs) at sites other than the intended on-target site (off-target effects). All CRISPR/Cas9 applications require the verification of the specific binding of the sgRNA at the locus of interest. Chromatin immunoprecipitation followed by real-time PCR (ChIP-qPCR) is a technique of choice for studying protein-DNA interactions. In this study, we show a successful ChIP-qPCR method to verify the binding efficiency of the dCas9/sgRNA complex in the targeted region; and ChIP-seq – to monitor off-target bindings of the dCas9/sgRNA complex in the genome.</p>',
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'name' => 'A self-inactivating system for AAV-mediated in vivo base editing',
'authors' => 'Zuo Y. et al.',
'description' => '<p>DNA base editors have been harnessed as an exciting therapeutic platform for human diseases and are rapidly progressing into human clinical trials. However, persistent expression of base editors delivered via adeno-associated virus (AAV) poses concerns with specificity and immunogenicity. Here we develop selfinactivating base editor (siBE) systems with a negative feedback loop where one guide RNA (gRNA) targets the gene of interest and the other targets the deaminase domain itself. We demonstrate that siBE confers efficient on-target editing with time-dependent self-inactivation and increased editing specificity. For the in vivo utilization, we further employ the intein split approach to package siBE targeting mouse Angptl3 into AAV9. Systemic delivery of AAV9-siBE confer efficient editing of Angptl3 in liver, resulting in reduced serum levels of ANGPTL3, triglyceride and total cholesterol, with the active base editor undetectable at 8 weeks after administration. These self-inactivating base editing systems are highly promising for future therapeutic applications.</p>',
'date' => '0000-00-00',
'pmid' => 'https://doi.org/10.21203%2Frs.3.rs-1663604%2Fv1',
'doi' => '10.21203/rs.3.rs-1663604/v1',
'modified' => '2022-08-11 15:24:23',
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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
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Dispatcher::dispatch() - CORE/Cake/Routing/Dispatcher.php, line 167
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-fig1.png" alt="CRISPR/Cas9 Antibody ChIP Grade" caption="false" width="241" height="286" /></p>
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<p><small><strong> Figure 1. ChIP using the Diagenode antibody directed against Cas9 </strong><br />ChIP was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50μg chromatin was incubated overnight at 4°C with either 5 μg of an anti-FLAG antibody or 2 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The pre-immune serum (Cas9, PPI) was used as negative IP control. qPCR was performed with primers specific for the GFP gene, and for two non-targeted regions phosphatidic acid phosphatase type 2C (Ppap2c) and protein kinase C delta (Prkcd), used as negative controls. 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/C15310258-CRISPR-WB.png" alt="CRISPR/Cas9 Antibody Validation in WB " caption="false" width="171" height="192" /></p>
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<p><small><strong> Figure 2. Western blot analysis using the Diagenode antibody directed against Cas9 </strong><br />Western blot was performed on protein extracts from HeLa cells transfected with Cas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:1,000 (lane 2) or 1:5,000 (lane 3). Lane 1 shows the result with the pre-immune serum. The marker is shown on the left, the position of the Cas9 protein is indicated on the right. </small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IP.png" alt="CRISPR/Cas9 Antibody Validation in IP" caption="false" width="252" height="286" /></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 (500 μg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 μg) is shown in lane 1 and 2. </small></p>
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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" alt="CRISPR/Cas9 Antibody Validation in IF" caption="false" width="278" height="123" /></p>
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<p><small><strong> Figure 4. Immunofluorescence using the Diagenode antibody directed against Cas9 </strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counterstained with Hoechst 33342. Figure 4 shows the result in the presence (left) or absence (right) of doxycycline. </small></p>
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'info2' => '<p style="text-align: justify;">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 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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<td>ChIP/ChIP-seq <sup>*</sup></td>
<td>2-5 µl/ChIP</td>
<td>Fig 1, 2</td>
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<tr>
<td>Western Blotting</td>
<td>1:5,000</td>
<td>Fig 3</td>
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<tr>
<td>Immunoprecipitation</td>
<td>1 µl/IP</td>
<td>Fig 4</td>
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<td>Immunofluorescence</td>
<td>1:1,000</td>
<td>Fig 5</td>
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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 µl per IP.</small></p>',
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'description' => '<p>Polyclonal antibody raised in rabbit against the <strong>Cas9</strong> nuclease (<strong>CRISPR</strong>-associated protein 9) using a recombinant protein. </p>',
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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-fig1.png" alt="CRISPR/Cas9 Antibody ChIP Grade" caption="false" width="241" height="286" /></p>
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<p><small><strong> Figure 1. ChIP using the Diagenode antibody directed against Cas9 </strong><br />ChIP was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50μg chromatin was incubated overnight at 4°C with either 5 μg of an anti-FLAG antibody or 2 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The pre-immune serum (Cas9, PPI) was used as negative IP control. qPCR was performed with primers specific for the GFP gene, and for two non-targeted regions phosphatidic acid phosphatase type 2C (Ppap2c) and protein kinase C delta (Prkcd), used as negative controls. 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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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-WB.png" alt="CRISPR/Cas9 Antibody Validation in WB " caption="false" width="171" height="192" /></p>
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<p><small><strong> Figure 2. Western blot analysis using the Diagenode antibody directed against Cas9 </strong><br />Western blot was performed on protein extracts from HeLa cells transfected with Cas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:1,000 (lane 2) or 1:5,000 (lane 3). Lane 1 shows the result with the pre-immune serum. The marker is shown on the left, the position of the Cas9 protein is indicated on the right. </small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IP.png" alt="CRISPR/Cas9 Antibody Validation in IP" caption="false" width="252" height="286" /></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 (500 μg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 μg) is shown in lane 1 and 2. </small></p>
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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" alt="CRISPR/Cas9 Antibody Validation in IF" caption="false" width="278" height="123" /></p>
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<div class="small-8 columns">
<p><small><strong> Figure 4. Immunofluorescence using the Diagenode antibody directed against Cas9 </strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counterstained with Hoechst 33342. Figure 4 shows the result in the presence (left) or absence (right) of doxycycline. </small></p>
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'label2' => 'Background',
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'meta_title' => 'CRISPR/Cas9 polyclonal antibody (sample size)',
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'description' => '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 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.',
'clonality' => '',
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'lot' => 'A2508-004',
'concentration' => 'Not determined',
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'type' => 'Polyclonal, <strong>ChIP grade, ChIP-seq grade</strong>',
'purity' => 'Whole antiserum from rabbit containing 0.05% azide.',
'classification' => '',
'application_table' => '<table>
<thead>
<tr>
<th>Applications</th>
<th>Suggested dilution</th>
<th>References</th>
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<tbody>
<tr>
<td>ChIP/ChIP-seq <sup>*</sup></td>
<td>2-5 µl/ChIP</td>
<td>Fig 1, 2</td>
</tr>
<tr>
<td>Western Blotting</td>
<td>1:5,000</td>
<td>Fig 3</td>
</tr>
<tr>
<td>Immunoprecipitation</td>
<td>1 µl/IP</td>
<td>Fig 4</td>
</tr>
<tr>
<td>Immunofluorescence</td>
<td>1:1,000</td>
<td>Fig 5</td>
</tr>
</tbody>
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<p></p>
<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 µl per IP.</small></p>',
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'name' => 'CRISPR/Cas9 Antibody',
'description' => '<p>Polyclonal antibody raised in rabbit against the <strong>Cas9</strong> nuclease (<strong>CRISPR</strong>-associated protein 9) using a recombinant protein. </p>',
'label1' => 'Validation data',
'info1' => '<div class="row">
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-fig1.png" /></center></div>
<div class="small-6 columns">
<p><strong>Figure 1. ChIP using the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing nuclease dead Cas9 and sgRNA targeting a sequence in intron 8 of the GAPDH gene, using the iDeal ChIP-seq kit for transcription factors. A titration consisting of 1, 2, 5 and 10 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) was tested. IgG (2 µg/IP) was used as negative IP control. qPCR was performed with primers specific for the targeted sequence in the GAPDH gene, and for the MYOD1 gene, 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).</p>
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<div class="row">
<div class="small-12 columns"><center>A.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-a.jpg" width="700" /></center><center>B.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-b.jpg" width="700" /></center></div>
</div>
<div class="row">
<div class="small-12 columns">
<p><strong>Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing dCas9 and a GAPDH sgRNA cells using 2 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) as described above. The IP'd DNA was subsequently analysed on an Illumina NovaSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the ChIP-seq profile in a region of chromosome 12 surrounding the GAPDH gene (fig 2B) and in a region of chromosome 2 surrounding an off-target peak in the YIPF4 gene.</p>
</div>
</div>
<div class="row">
<div class="small-4 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-WB.png" /></center></div>
<div class="small-8 columns">
<p><strong>Figure 3. Western blot analysis using the Diagenode antibody directed against Cas9</strong><br />Western blot was performed on protein extracts from HEK293 cells transfected with dCas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:5,000. The marker is shown on the left, the position of the Cas9 protein is indicated on the right.</p>
</div>
</div>
<div class="row">
<div class="small-4 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IP.png" /></center></div>
<div class="small-8 columns">
<p><strong>Figure 4. IP using the Diagenode antibody directed against Cas9</strong><br />IP was performed on whole cell extracts (500 µg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 µl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 µg) is shown in lane 1 and 2.</p>
</div>
</div>
<div class="row">
<div class="small-12 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" width="500" /></center></div>
</div>
<div class="row">
<div class="small-12 columns">
<p><strong>Figure 5. Immunofluorescence using the Diagenode antibody directed against Cas9</strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counter-stained with Hoechst 33342. Figure 5 shows the result in the presence (left) or absence (right) of doxycycline.</p>
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'info2' => '<p style="text-align: justify;">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 <strong>CRISPR/Cas9</strong> (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 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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'meta_description' => 'S. pyogenes CRISPR/Cas9 Polyclonal Antibody validated in ChIP-seq, ChIP-qPCR, IP, IF and WB. Batch-specific data available on the website. Sample size available.',
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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' => '<h1><strong>Validated epigenetics antibodies</strong> – care for a sample?<br /> </h1>
<p>Diagenode has partnered with leading epigenetics experts and numerous epigenetics consortiums to bring to you a validated and comprehensive collection of epigenetic antibodies. As an expert in epigenetics, we are committed to offering highly-specific antibodies validated for ChIP/ChIP-seq and many other applications. All batch-specific validation data is available on our website.<br /><a href="../categories/antibodies">Read about our expertise in antibody production</a>.</p>
<ul>
<li><strong>Focused</strong> - Diagenode's selection of antibodies is exclusively dedicated for epigenetic research. <a title="See the full collection." href="../categories/all-antibodies">See the full collection.</a></li>
<li><strong>Strict quality standards</strong> with rigorous QC and validation</li>
<li><strong>Classified</strong> based on level of validation for flexibility of application</li>
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<p>Existing sample sizes are listed below. We will soon expand our collection. Are you looking for a sample size of another antibody? Just <a href="mailto:agnieszka.zelisko@diagenode.com?Subject=Sample%20Size%20Request" target="_top">Contact us</a>.</p>',
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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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'meta_title' => 'Diagenode's selection of Antibodies is exclusively dedicated for Epigenetic Research | Diagenode',
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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>
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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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'description' => '<p><span>The fluorescence ubiquitination cell cycle inhibitor (FUCCI) has been introduced to monitor cell cycle activity in living cells, including human induced pluripotent stem cells (hiPSC) and derived cell types. We have recently developed hiPSC with stable expression of dCas9VPR for endogenous gene activation and a Citrine-tagged ACTN2 cell line to monitor sarcomere development and function in muscle cells. Here, we present dual and triple transgenic hiPSC lines developed by genomic integration of FUCCI with and without dCas9VPR into the ROSA26 and AAVS1 loci, respectively, in the previously introduced ACTN2-Citrine line. Functionality of the transgenes was demonstrated in the novel hiPSC line, which we introduce as Myo-CCER and CraCCER.</span></p>',
'date' => '2024-08-05',
'pmid' => 'https://www.sciencedirect.com/science/article/pii/S1873506124002290',
'doi' => 'https://doi.org/10.1016/j.scr.2024.103531',
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'name' => 'Methyltransferase Inhibition Enables Tgf Driven Induction of and in Cancer Cells.',
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'description' => '<p>deletion or silencing is common across human cancer, reinforcing the general importance of bypassing its tumor suppression in cancer formation or progression. In rhabdomyosarcoma (RMS) and neuroblastoma, two common childhood cancers, the three transcripts are independently expressed to varying degrees, but one, is uniformly silenced. Although TGFβ induces certain transcripts in HeLa cells, it was unable to do so in five tested RMS lines unless the cells were pretreated with a broadly acting methyltransferase inhibitor, DZNep, or one targeting EZH2. induction by TGFβ correlated with de novo appearance of three H3K27Ac peaks within a 20 kb element ∼150 kb proximal to . Deleting that segment prevented their induction by TGFβ but not a basal increase driven by methyltransferase inhibition alone. Expression of two transcripts was enhanced by dCas9/CRISPR activation targeting either the relevant promoter or the 20 kb elements, and this "precise" manipulation diminished RMS cell propagation in vitro. Our findings show crosstalk between methyltransferase inhibition and TGFβ-dependent activation of a remote enhancer to reverse silencing. Though focused on here, such crosstalk may apply to other TGFβ-responsive genes and perhaps govern this signaling protein's complex effects promoting or blocking cancer.</p>',
'date' => '2023-01-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/36941772',
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'name' => 'Massively parallel multi-target CRISPR system interrogates Cas9-basedtarget recognition, DNA cleavage, and DNA repair',
'authors' => 'Zou Roger S. et al.',
'description' => '<p>CRISPR-Cas9 nucleases, and particularly Streptococcus pyogenes Cas9, are widespread tools for genome editing. However, many aspects of intracellular Cas9 activity and the ensuing DNA damage response remain incompletely characterized. In order to address these issues, we developed a multiplexed CRISPR approach, where a single, degenerate multi-target gRNA (mgRNA) directs the Cas9 enzyme to target hundred endogenous sites at once. When combined with next-generation sequencing readouts, this system enables interrogation of Cas9 activity and DNA double-strand break (DSB) repair response in high-throughput. Here, we present a step-by-step protocol to deliver a Cas9:mgRNA ribonucleoprotein complex into cultured cells and measure key processes related to Cas9 activity and DSB repair.</p>',
'date' => '2022-09-01',
'pmid' => 'https://europepmc.org/article/ppr/ppr540155',
'doi' => '10.21203/rs.3.pex-1938/v1',
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(int) 3 => array(
'id' => '4437',
'name' => 'Analysis of estrogen-regulated enhancer RNAs identifies a functionalmotif required for enhancer assembly and gene expression.',
'authors' => 'Hou Tim Y and Kraus W Lee',
'description' => '<p>To better understand the functions of non-coding enhancer RNAs (eRNAs), we annotated the estrogen-regulated eRNA transcriptome in estrogen receptor α (ERα)-positive breast cancer cells using PRO-cap and RNA sequencing. We then cloned a subset of the eRNAs identified, fused them to single guide RNAs, and targeted them to their ERα enhancers of origin using CRISPR/dCas9. Some of the eRNAs tested modulated the expression of cognate, but not heterologous, target genes after estrogen treatment by increasing ERα recruitment and stimulating p300-catalyzed H3K27 acetylation at the enhancer. We identified a ∼40 nucleotide functional eRNA regulatory motif (FERM) present in many eRNAs that was necessary and sufficient to modulate gene expression, but not the specificity of activation, after estrogen treatment. The FERM interacted with BCAS2, an RNA-binding protein amplified in breast cancers. The ectopic expression of a targeted eRNA controlling the expression of an oncogene resulted in increased cell proliferation, demonstrating the regulatory potential of eRNAs in breast cancer.</p>',
'date' => '2022-06-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/35705040',
'doi' => '10.1016/j.celrep.2022.110944',
'modified' => '2022-09-28 09:20:34',
'created' => '2022-09-08 16:32:20',
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(int) 4 => array(
'id' => '4560',
'name' => 'Avian influenza viruses suppress innate immunity by inducingtrans-transcriptional readthrough via SSU72.',
'authors' => 'Zhao Y. et al.',
'description' => '<p>Innate immunity plays critical antiviral roles. The highly virulent avian influenza viruses (AIVs) H5N1, H7N9, and H5N6 can better escape host innate immune responses than the less virulent seasonal H1N1 virus. Here, we report a mechanism by which transcriptional readthrough (TRT)-mediated suppression of innate immunity occurs post AIV infection. By using cell lines, mouse lungs, and patient PBMCs, we showed that genes on the complementary strand ("trans" genes) influenced by TRT were involved in the disruption of host antiviral responses during AIV infection. The trans-TRT enhanced viral lethality, and TRT abolishment increased cell viability and STAT1/2 expression. The viral NS1 protein directly bound to SSU72, and degradation of SSU72 induced TRT. SSU72 overexpression reduced TRT and alleviated mouse lung injury. Our results suggest that AIVs infection induce TRT by reducing SSU72 expression, thereby impairing host immune responses, a molecular mechanism acting through the NS1-SSU72-trans-TRT-STAT1/2 axis. Thus, restoration of SSU72 expression might be a potential strategy for preventing AIV pandemics.</p>',
'date' => '2022-03-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/35332300',
'doi' => '10.1038/s41423-022-00843-8',
'modified' => '2022-11-24 10:02:04',
'created' => '2022-11-24 08:49:52',
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'id' => '4365',
'name' => 'Antisense non-coding transcription represses the <i>PHO5</i> model gene<i>via</i> remodelling of promoter chromatin structure',
'authors' => 'Novačić A. et al. ',
'description' => '<p>Pervasive transcription of eukaryotic genomes generates non-coding transcripts with regulatory potential. We examined the effects of non-coding antisense transcription on the regulation of expression of the yeast PHO5 gene, a paradigmatic case for gene regulation through promoter chromatin remodeling. By enhancing or impairing the level of overlapping antisense transcription through specific mutant backgrounds and the use of the CRISPRi system, we demonstrated a negative role for antisense transcription at the PHO5 gene locus. Furthermore, enhanced elongation of PHO5 antisense leads to a more repressive chromatin configuration at the PHO5 gene promoter, which is remodeled more slowly upon gene induction. The negative effect of antisense transcription on PHO5 gene transcription is mitigated by inactivation of the histone deacetylase Rpd3, showing that PHO5 antisense RNA acts via histone deacetylation. This regulatory pathway leads to Rpd3-dependent decreased recruitment of the RSC chromatin remodeling complex to the PHO5 gene promoter upon induction of antisense transcription. Overall, we extend the model of PHO5 gene regulation by demonstrating a gene silencing function of antisense transcription through a chromatin-based mechanism.</p>',
'date' => '2022-02-01',
'pmid' => 'https://doi.org/10.1101%2F2022.02.21.481265',
'doi' => '10.1101/2022.02.21.481265',
'modified' => '2022-08-04 15:52:12',
'created' => '2022-08-04 14:55:36',
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(int) 6 => array(
'id' => '4237',
'name' => 'A predominant enhancer co-amplified with the oncogene is necessary andsufficient for its expression in squamous cancer',
'authors' => 'Liu Y. et al.',
'description' => '<p>Amplification and overexpression of the SOX2 oncogene represent a hallmark of squamous cancers originating from diverse tissue types. Here, we find that squamous cancers selectively amplify a 3’ noncoding region together with SOX2, which harbors squamous cancer-specific chromatin accessible regions. We identify a single enhancer e1 that predominantly drives SOX2 expression. Repression of e1 in SOX2-high cells causes collapse of the surrounding enhancers, remarkable reduction in SOX2 expression, and a global transcriptional change reminiscent of SOX2 knockout. The e1 enhancer is driven by a combination of transcription factors including SOX2 itself and the AP-1 complex, which facilitates recruitment of the co-activator BRD4. CRISPR-mediated activation of e1 in SOX2-low cells is sufficient to rebuild the e1-SOX2 loop and activate SOX2 expression. Our study shows that squamous cancers selectively amplify a predominant enhancer to drive SOX2 overexpression, uncovering functional links among enhancer activation, chromatin looping, and lineage-specific copy number amplifications of oncogenes.</p>',
'date' => '2021-12-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/34880227',
'doi' => '10.1038/s41467-021-27055-4',
'modified' => '2022-05-19 17:05:00',
'created' => '2022-05-19 10:41:50',
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(int) 7 => array(
'id' => '4301',
'name' => 'Establishment of a second generation homozygous CRISPRa human inducedpluripotent stem cell (hiPSC) line for enhanced levels of endogenous geneactivation.',
'authors' => 'Schoger Eric et al.',
'description' => '<p>CRISPR/Cas9 technology based on nuclease inactive dCas9 and fused to the heterotrimeric VPR transcriptional activator is a powerful tool to enhance endogenous transcription by targeting defined genomic loci. We generated homozygous human induced pluripotent stem cell (hiPSC) lines carrying dCas9 fused to VPR along with a WPRE element at the AAVS1 locus (CRISPRa2). We demonstrated pluripotency, genomic integrity and differentiation potential into all three germ layers. CRISPRa2 cells showed increased transgene expression and higher transcriptional induction in hiPSC-derived cardiomyocytes compared to a previously described CRISPRa line. Both lines allow studying endogenous transcriptional modulation with lower and higher transcript abundance.</p>',
'date' => '2021-10-01',
'pmid' => 'https://doi.org/10.1016%2Fj.scr.2021.102518',
'doi' => '10.1016/j.scr.2021.102518',
'modified' => '2022-05-30 09:56:01',
'created' => '2022-05-19 10:41:50',
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(int) 8 => array(
'id' => '4328',
'name' => 'Establishment of two homozygous CRISPR interference (CRISPRi)knock-in human induced pluripotent stem cell (hiPSC) lines for titratableendogenous gene repression.',
'authors' => 'Schoger Eric et al.',
'description' => '<p>Using nuclease-deficient dead (d)Cas9 without enzymatic activity fused to transcriptional inhibitors (CRISPRi) allows for transcriptional interference and results in a powerful tool for the elucidation of developmental, homeostatic and disease mechanisms. We inserted dCas9KRAB (CRISPRi) cassette into the AAVS1 locus of hiPSC lines, which resulted in homozygous knock-in with an otherwise unaltered genome. Expression of dCas9KRAB protein, pluripotency and the ability to differentiate into all three embryonic germ layers were validated. Furthermore, functional cardiomyocyte generation was tested. The hiPSC-CRISPRi cell lines offer a valuable tool for studying endogenous transcriptional repression with single and multiplexed possibilities in all human cell types.</p>',
'date' => '2021-08-01',
'pmid' => 'https://doi.org/10.1016%2Fj.scr.2021.102473',
'doi' => '10.1016/j.scr.2021.102473',
'modified' => '2022-06-22 09:28:49',
'created' => '2022-05-19 10:41:50',
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[maximum depth reached]
)
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(int) 9 => array(
'id' => '4209',
'name' => 'TGFβ promotes widespread enhancer chromatin opening and operates ongenomic regulatory domains.',
'authors' => 'Guerrero-Martínez J. et al. ',
'description' => '<p>The Transforming Growth Factor-β (TGFβ) signaling pathway controls transcription by regulating enhancer activity. How TGFβ-regulated enhancers are selected and what chromatin changes are associated with TGFβ-dependent enhancers regulation are still unclear. Here we report that TGFβ treatment triggers fast and widespread increase in chromatin accessibility in about 80\% of the enhancers of normal mouse mammary epithelial-gland cells, irrespective of whether they are activated, repressed or not regulated by TGFβ. This enhancer opening depends on both the canonical and non-canonical TGFβ pathways. Most TGFβ-regulated genes are located around enhancers regulated in the same way, often creating domains of several co-regulated genes that we term TGFβ regulatory domains (TRD). CRISPR-mediated inactivation of enhancers within TRDs impairs TGFβ-dependent regulation of all co-regulated genes, demonstrating that enhancer targeting is more promiscuous than previously anticipated. The area of TRD influence is restricted by topologically associating domains (TADs) borders, causing a bias towards co-regulation within TADs.</p>',
'date' => '2020-12-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/33273453',
'doi' => '10.1038/s41467-020-19877-5',
'modified' => '2022-01-13 14:59:41',
'created' => '2021-12-06 15:53:19',
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'id' => '4013',
'name' => 'A gene therapy for inherited blindness using dCas9-VPR–mediatedtranscriptional activation',
'authors' => 'Böhm, Sybille and Splith, Victoria and Riedmayr, Lisa Maria and Rötzer,René Dominik and Gasparoni, Gilles and Nordström, Karl J. V. and Wagner,Johanna Elisabeth and Hinrichsmeyer, Klara Sonnie and Walter, Jörn andWahl-Schott, Christian and Fenske, Stef',
'description' => '<p>Catalytically inactive dCas9 fused to transcriptional activators (dCas9-VPR) enables activation of silent genes. Many disease genes have counterparts, which serve similar functions but are expressed in distinct cell types. One attractive option to compensate for the missing function of a defective gene could be to transcriptionally activate its functionally equivalent counterpart via dCas9-VPR. Key challenges of this approach include the delivery of dCas9-VPR, activation efficiency, long-term expression of the target gene, and adverse effects in vivo. Using dual adeno-associated viral vectors expressing split dCas9-VPR, we show efficient transcriptional activation and long-term expression of cone photoreceptor-specific M-opsin (Opn1mw) in a rhodopsin-deficient mouse model for retinitis pigmentosa. One year after treatment, this approach yields improved retinal function and attenuated retinal degeneration with no apparent adverse effects. Our study demonstrates that dCas9-VPR–mediated transcriptional activation of functionally equivalent genes has great potential for the treatment of genetic disorders.</p>',
'date' => '2020-08-19',
'pmid' => 'https://advances.sciencemag.org/content/6/34/eaba5614',
'doi' => '10.1126/sciadv.aba5614',
'modified' => '2020-12-16 17:32:46',
'created' => '2020-10-12 14:54:59',
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[maximum depth reached]
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(int) 11 => array(
'id' => '3631',
'name' => 'Guidelines for optimized gene knockout using CRISPR/Cas9',
'authors' => 'Campenhout CV et al.',
'description' => '<p>CRISPR/Cas9 technology has evolved as the most powerful approach to generate genetic models both for fundamental and preclinical research. Despite its apparent simplicity, the outcome of a genome-editing experiment can be substantially impacted by technical parameters and biological considerations. Here, we present guidelines and tools to optimize CRISPR/Cas9 genome-targeting efficiency and specificity. The nature of the target locus, the design of the single guide RNA and the choice of the delivery method should all be carefully considered prior to a genome-editing experiment. Different methods can also be used to detect off-target cleavages and decrease the risk of unwanted mutations. Together, these optimized tools and proper controls are essential to the assessment of CRISPR/Cas9 genome-editing experiments.</p>',
'date' => '2019-05-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/31039627',
'doi' => '10.2144/btn-2018-0187',
'modified' => '2019-05-09 15:37:50',
'created' => '2019-05-09 15:37:50',
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(int) 12 => array(
'id' => '3653',
'name' => 'CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency.',
'authors' => 'Matharu N, Rattanasopha S, Tamura S, Maliskova L, Wang Y, Bernard A, Hardin A, Eckalbar WL, Vaisse C, Ahituv N',
'description' => '<p>A wide range of human diseases result from haploinsufficiency, where the function of one of the two gene copies is lost. Here, we targeted the remaining functional copy of a haploinsufficient gene using CRISPR-mediated activation (CRISPRa) in and heterozygous mouse models to rescue their obesity phenotype. Transgenic-based CRISPRa targeting of the promoter or its distant hypothalamic enhancer up-regulated its expression from the endogenous functional allele in a tissue-specific manner, rescuing the obesity phenotype in heterozygous mice. To evaluate the therapeutic potential of CRISPRa, we injected CRISPRa-recombinant adeno-associated virus into the hypothalamus, which led to reversal of the obesity phenotype in and haploinsufficient mice. Our results suggest that endogenous gene up-regulation could be a potential strategy to treat altered gene dosage diseases.</p>',
'date' => '2019-01-18',
'pmid' => 'http://www.pubmed.gov/30545847',
'doi' => '10.1126/science.aau0629',
'modified' => '2019-06-07 09:05:53',
'created' => '2019-06-06 12:11:18',
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[maximum depth reached]
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(int) 13 => array(
'id' => '3327',
'name' => '(Po)STAC (Polycistronic SunTAg modified CRISPR) enables live-cell and fixed-cell super-resolution imaging of multiple genes',
'authors' => 'Neguembor M.V. et al.',
'description' => '<p>CRISPR/dCas9-based labeling has allowed direct visualization of genomic regions in living cells. However, poor labeling efficiency and signal-to-background ratio have limited its application to visualize genome organization using super-resolution microscopy. We developed (Po)STAC (Polycistronic SunTAg modified CRISPR) by combining CRISPR/dCas9 with SunTag labeling and polycistronic vectors. (Po)STAC enhances both labeling efficiency and fluorescence signal detected from labeled loci enabling live cell imaging as well as super-resolution fixed-cell imaging of multiple genes with high spatiotemporal resolution.</p>',
'date' => '2017-12-23',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/29294098',
'doi' => '',
'modified' => '2018-02-07 10:03:26',
'created' => '2018-02-07 10:03:26',
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(int) 14 => array(
'id' => '4399',
'name' => 'A self-inactivating system for AAV-mediated in vivo base editing',
'authors' => 'Zuo Y. et al.',
'description' => '<p>DNA base editors have been harnessed as an exciting therapeutic platform for human diseases and are rapidly progressing into human clinical trials. However, persistent expression of base editors delivered via adeno-associated virus (AAV) poses concerns with specificity and immunogenicity. Here we develop selfinactivating base editor (siBE) systems with a negative feedback loop where one guide RNA (gRNA) targets the gene of interest and the other targets the deaminase domain itself. We demonstrate that siBE confers efficient on-target editing with time-dependent self-inactivation and increased editing specificity. For the in vivo utilization, we further employ the intein split approach to package siBE targeting mouse Angptl3 into AAV9. Systemic delivery of AAV9-siBE confer efficient editing of Angptl3 in liver, resulting in reduced serum levels of ANGPTL3, triglyceride and total cholesterol, with the active base editor undetectable at 8 weeks after administration. These self-inactivating base editing systems are highly promising for future therapeutic applications.</p>',
'date' => '0000-00-00',
'pmid' => 'https://doi.org/10.21203%2Frs.3.rs-1663604%2Fv1',
'doi' => '10.21203/rs.3.rs-1663604/v1',
'modified' => '2022-08-11 15:24:23',
'created' => '2022-08-11 12:14:50',
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'name' => 'Researcher from the University of Manchester',
'description' => '<p>I used ChIP-qPCR with the Diagenode CRISPR/Cas9 polyclonal antibody to successfully show that Cas9 binds to the target region of my sgRNA, validating my CRISPR experiment. The antibody produced minimal background signal at non-specific genomic regions. I am now using the antibody to validate further sgRNA in different CRISPR cell lines.</p>
<center><img src="../../img/categories/antibodies/cas9-jurkat-vp64-result.png" /></center>
<p><small>ChIP was performed on Jurkat cells expressing dCas9-VP64-mCherry and a sgRNA targeting the IL1RN promoter. Each IP was performed using 4 million cells and 2 µL CRISPR/Cas9 polyclonal antibody (Diagenode C15310258) or 1 µg rabbit IgG control antibody (Diagenode C15410206). qPCR was carried out on undiluted ChIP DNA using SYBR green and PCR primers directed against the sgRNA binding site at IL1RN, as well as two non-target regions at the SLC4A1 and TP53 promoters. ChIP enrichment was measured using the percent input method.</small></p>',
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'description' => '<p><strong>Diagenode’s CRISPR/Cas9 polyclonal antibody</strong> shows superior signal than the original <strong>clone 7A9</strong>: a researcher from EPFL in Lausanne, Switzerland has compared these two antibodies in Western blot.</p>
<center><img src="../../emailing/images/cas9-fig.png" /></center>
<p><small>Western blot was performed using HCT116 DKO cells transduced with Krab-dCas9 (2) or non-transduced (1) cells. Then, 100,000 cells were lysed in sample buffer 2x and boiled 5 min at 95°C before loading in a 15% acrylamide gel. The same sample was loaded 3x in the same gel. The membrane was cut in 3 parts for each antibody. Membrane was blocked 1h with 3% BSA at RT. Antibodies were diluted 1:1,000 in 3% BSA and incubated overnight at 4°C. Secondary incubation was done for 1h at RT (1:10,000 dilution). Anti-hnRNPA1 was used as a loading control.</small></p>',
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'name' => 'CRISPR/Cas9 Antibody',
'description' => '<p>Polyclonal antibody raised in rabbit against the <strong>Cas9</strong> nuclease (<strong>CRISPR</strong>-associated protein 9) using a recombinant protein. </p>',
'label1' => 'Validation data',
'info1' => '<div class="row">
<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-fig1.png" /></center></div>
<div class="small-6 columns">
<p><strong>Figure 1. ChIP using the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing nuclease dead Cas9 and sgRNA targeting a sequence in intron 8 of the GAPDH gene, using the iDeal ChIP-seq kit for transcription factors. A titration consisting of 1, 2, 5 and 10 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) was tested. IgG (2 µg/IP) was used as negative IP control. qPCR was performed with primers specific for the targeted sequence in the GAPDH gene, and for the MYOD1 gene, 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).</p>
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<div class="row">
<div class="small-12 columns"><center>A.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-a.jpg" width="700" /></center><center>B.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-b.jpg" width="700" /></center></div>
</div>
<div class="row">
<div class="small-12 columns">
<p><strong>Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing dCas9 and a GAPDH sgRNA cells using 2 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) as described above. The IP'd DNA was subsequently analysed on an Illumina NovaSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the ChIP-seq profile in a region of chromosome 12 surrounding the GAPDH gene (fig 2B) and in a region of chromosome 2 surrounding an off-target peak in the YIPF4 gene.</p>
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<div class="small-4 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-WB.png" /></center></div>
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<p><strong>Figure 3. Western blot analysis using the Diagenode antibody directed against Cas9</strong><br />Western blot was performed on protein extracts from HEK293 cells transfected with dCas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:5,000. The marker is shown on the left, the position of the Cas9 protein is indicated on the right.</p>
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<p><strong>Figure 4. IP using the Diagenode antibody directed against Cas9</strong><br />IP was performed on whole cell extracts (500 µg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 µl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 µg) is shown in lane 1 and 2.</p>
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<div class="small-12 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" width="500" /></center></div>
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<p><strong>Figure 5. Immunofluorescence using the Diagenode antibody directed against Cas9</strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counter-stained with Hoechst 33342. Figure 5 shows the result in the presence (left) or absence (right) of doxycycline.</p>
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<p><small><strong> Figure 1. ChIP using the Diagenode antibody directed against Cas9 </strong><br />ChIP was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50μg chromatin was incubated overnight at 4°C with either 5 μg of an anti-FLAG antibody or 2 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The pre-immune serum (Cas9, PPI) was used as negative IP control. qPCR was performed with primers specific for the GFP gene, and for two non-targeted regions phosphatidic acid phosphatase type 2C (Ppap2c) and protein kinase C delta (Prkcd), used as negative controls. 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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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-WB.png" alt="CRISPR/Cas9 Antibody Validation in WB " caption="false" width="171" height="192" /></p>
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<p><small><strong> Figure 2. Western blot analysis using the Diagenode antibody directed against Cas9 </strong><br />Western blot was performed on protein extracts from HeLa cells transfected with Cas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:1,000 (lane 2) or 1:5,000 (lane 3). Lane 1 shows the result with the pre-immune serum. The marker is shown on the left, the position of the Cas9 protein is indicated on the right. </small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IP.png" alt="CRISPR/Cas9 Antibody Validation in IP" caption="false" width="252" height="286" /></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 (500 μg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 μl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 μg) is shown in lane 1 and 2. </small></p>
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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" alt="CRISPR/Cas9 Antibody Validation in IF" caption="false" width="278" height="123" /></p>
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<p><small><strong> Figure 4. Immunofluorescence using the Diagenode antibody directed against Cas9 </strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counterstained with Hoechst 33342. Figure 4 shows the result in the presence (left) or absence (right) of doxycycline. </small></p>
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$testimonials = '<blockquote><p><strong>Diagenode’s CRISPR/Cas9 polyclonal antibody</strong> shows superior signal than the original <strong>clone 7A9</strong>: a researcher from EPFL in Lausanne, Switzerland has compared these two antibodies in Western blot.</p>
<center><img src="../../emailing/images/cas9-fig.png" /></center>
<p><small>Western blot was performed using HCT116 DKO cells transduced with Krab-dCas9 (2) or non-transduced (1) cells. Then, 100,000 cells were lysed in sample buffer 2x and boiled 5 min at 95°C before loading in a 15% acrylamide gel. The same sample was loaded 3x in the same gel. The membrane was cut in 3 parts for each antibody. Membrane was blocked 1h with 3% BSA at RT. Antibodies were diluted 1:1,000 in 3% BSA and incubated overnight at 4°C. Secondary incubation was done for 1h at RT (1:10,000 dilution). Anti-hnRNPA1 was used as a loading control.</small></p><cite>EPFL in Lausanne</cite></blockquote>
'
$featured_testimonials = '<blockquote><span class="label-green" style="margin-bottom:16px;margin-left:-22px">TESTIMONIAL</span><p>I used ChIP-qPCR with the Diagenode CRISPR/Cas9 polyclonal antibody to successfully show that Cas9 binds to the target region of my sgRNA, validating my CRISPR experiment. The antibody produced minimal background signal at non-specific genomic regions. I am now using the antibody to validate further sgRNA in different CRISPR cell lines.</p>
<center><img src="../../img/categories/antibodies/cas9-jurkat-vp64-result.png" /></center>
<p><small>ChIP was performed on Jurkat cells expressing dCas9-VP64-mCherry and a sgRNA targeting the IL1RN promoter. Each IP was performed using 4 million cells and 2 µL CRISPR/Cas9 polyclonal antibody (Diagenode C15310258) or 1 µg rabbit IgG control antibody (Diagenode C15410206). qPCR was carried out on undiluted ChIP DNA using SYBR green and PCR primers directed against the sgRNA binding site at IL1RN, as well as two non-target regions at the SLC4A1 and TP53 promoters. ChIP enrichment was measured using the percent input method.</small></p><cite>Researcher from the University of Manchester</cite></blockquote>
'
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'description' => '<p><strong>Diagenode’s CRISPR/Cas9 polyclonal antibody</strong> shows superior signal than the original <strong>clone 7A9</strong>: a researcher from EPFL in Lausanne, Switzerland has compared these two antibodies in Western blot.</p>
<center><img src="../../emailing/images/cas9-fig.png" /></center>
<p><small>Western blot was performed using HCT116 DKO cells transduced with Krab-dCas9 (2) or non-transduced (1) cells. Then, 100,000 cells were lysed in sample buffer 2x and boiled 5 min at 95°C before loading in a 15% acrylamide gel. The same sample was loaded 3x in the same gel. The membrane was cut in 3 parts for each antibody. Membrane was blocked 1h with 3% BSA at RT. Antibodies were diluted 1:1,000 in 3% BSA and incubated overnight at 4°C. Secondary incubation was done for 1h at RT (1:10,000 dilution). Anti-hnRNPA1 was used as a loading control.</small></p>',
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'description' => '<p>Polyclonal antibody raised in rabbit against the <strong>Cas9</strong> nuclease (<strong>CRISPR</strong>-associated protein 9) using a recombinant protein. </p>',
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<div class="small-6 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-fig1.png" /></center></div>
<div class="small-6 columns">
<p><strong>Figure 1. ChIP using the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing nuclease dead Cas9 and sgRNA targeting a sequence in intron 8 of the GAPDH gene, using the iDeal ChIP-seq kit for transcription factors. A titration consisting of 1, 2, 5 and 10 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) was tested. IgG (2 µg/IP) was used as negative IP control. qPCR was performed with primers specific for the targeted sequence in the GAPDH gene, and for the MYOD1 gene, 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).</p>
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<div class="small-12 columns"><center>A.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-a.jpg" width="700" /></center><center>B.<img src="https://www.diagenode.com/img/product/antibodies/C15310258-chipseq-b.jpg" width="700" /></center></div>
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<div class="small-12 columns">
<p><strong>Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against Cas9</strong><br />ChIP was performed on sheared chromatin from 4 million HEK293T cells stably expressing dCas9 and a GAPDH sgRNA cells using 2 µl of the Diagenode antibody against Cas9 (cat. No. C15310258) as described above. The IP'd DNA was subsequently analysed on an Illumina NovaSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the ChIP-seq profile in a region of chromosome 12 surrounding the GAPDH gene (fig 2B) and in a region of chromosome 2 surrounding an off-target peak in the YIPF4 gene.</p>
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<div class="small-4 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-WB.png" /></center></div>
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<p><strong>Figure 3. Western blot analysis using the Diagenode antibody directed against Cas9</strong><br />Western blot was performed on protein extracts from HEK293 cells transfected with dCas9 using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15310258). The antibody was diluted 1:5,000. The marker is shown on the left, the position of the Cas9 protein is indicated on the right.</p>
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<div class="small-4 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IP.png" /></center></div>
<div class="small-8 columns">
<p><strong>Figure 4. IP using the Diagenode antibody directed against Cas9</strong><br />IP was performed on whole cell extracts (500 µg) from HeLa cells transfected with a Cas9 expression vector (lane 1 and 3), or untransfected cells (lane 2 and 4) using 1 µl of the Diagenode antibody against Cas9 (cat. No. C15310258). The immunoprecipitated proteins were subsequently analysed by Western blot. Lane 3 and 4 show the result of the IP, the input (25 µg) is shown in lane 1 and 2.</p>
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<div class="small-12 columns"><center><img src="https://www.diagenode.com/img/product/antibodies/C15310258-CRISPR-IF.png" width="500" /></center></div>
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<p><strong>Figure 5. Immunofluorescence using the Diagenode antibody directed against Cas9</strong><br />HeLa cells expressing Cas9 under the control of the tight TRE promoter 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 antibody (cat. No. C15310258) diluted 1:1000, followed by incubation with a goat anti-rabbit secondary antibody coupled to AF594. Nuclei were counter-stained with Hoechst 33342. Figure 5 shows the result in the presence (left) or absence (right) of doxycycline.</p>
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'name' => 'Optimize the selection of guide RNA by ChIP to keep CRISPR on-target',
'description' => '<p>The mechanisms of target recognition and target specificity of the Cas9 protein is still not completely understood. A major hurdle of this technology is the introduction of double-strand breaks (DSBs) at sites other than the intended on-target site (off-target effects). All CRISPR/Cas9 applications require the verification of the specific binding of the sgRNA at the locus of interest. Chromatin immunoprecipitation followed by real-time PCR (ChIP-qPCR) is a technique of choice for studying protein-DNA interactions. In this study, we show a successful ChIP-qPCR method to verify the binding efficiency of the dCas9/sgRNA complex in the targeted region; and ChIP-seq – to monitor off-target bindings of the dCas9/sgRNA complex in the genome.</p>',
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'name' => 'A self-inactivating system for AAV-mediated in vivo base editing',
'authors' => 'Zuo Y. et al.',
'description' => '<p>DNA base editors have been harnessed as an exciting therapeutic platform for human diseases and are rapidly progressing into human clinical trials. However, persistent expression of base editors delivered via adeno-associated virus (AAV) poses concerns with specificity and immunogenicity. Here we develop selfinactivating base editor (siBE) systems with a negative feedback loop where one guide RNA (gRNA) targets the gene of interest and the other targets the deaminase domain itself. We demonstrate that siBE confers efficient on-target editing with time-dependent self-inactivation and increased editing specificity. For the in vivo utilization, we further employ the intein split approach to package siBE targeting mouse Angptl3 into AAV9. Systemic delivery of AAV9-siBE confer efficient editing of Angptl3 in liver, resulting in reduced serum levels of ANGPTL3, triglyceride and total cholesterol, with the active base editor undetectable at 8 weeks after administration. These self-inactivating base editing systems are highly promising for future therapeutic applications.</p>',
'date' => '0000-00-00',
'pmid' => 'https://doi.org/10.21203%2Frs.3.rs-1663604%2Fv1',
'doi' => '10.21203/rs.3.rs-1663604/v1',
'modified' => '2022-08-11 15:24:23',
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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
×
