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<p><small><strong>Figure 1. Western blot analysis using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />Western blot was performed on protein extracts from HEK293T cells transfected with Cas9 (lane 1) or from untransfected cells (lane 2) using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15200216), diluted 1:5,000 in PBS-T containing 0.5% NFDM. The marker is shown on the left, position of the Cas9 protein is indicated on the right.</small></p>
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<p><small><strong>Figure 2. IP using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />IP was performed on whole cell extracts from HEK293T cells transfected with a Cas9 expression vector (lane 1, 3 and 5), or untransfected cells (lane 2 and 4) using 5 µg of the Diagenode antibody against CRISPR/Cas9 (cat. No. C15200216, lane 3 and 4) or with an equal amount of IgG, used as a negative control (lane 5). The immunoprecipitated proteins were subsequently analysed by Western blot with the polyclonal Cas9 antibody (Cat. No. C15310258, diluted 1:5,000). Lane 1 and 2 show the result of the input.</small></p>
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<p><small><strong>Figure 1. Western blot analysis using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />Western blot was performed on protein extracts from HEK293T cells transfected with Cas9 (lane 1) or from untransfected cells (lane 2) using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15200216), diluted 1:5,000 in PBS-T containing 0.5% NFDM. The marker is shown on the left, position of the Cas9 protein is indicated on the right.</small></p>
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<p><small><strong>Figure 2. IP using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />IP was performed on whole cell extracts from HEK293T cells transfected with a Cas9 expression vector (lane 1, 3 and 5), or untransfected cells (lane 2 and 4) using 5 µg of the Diagenode antibody against CRISPR/Cas9 (cat. No. C15200216, lane 3 and 4) or with an equal amount of IgG, used as a negative control (lane 5). The immunoprecipitated proteins were subsequently analysed by Western blot with the polyclonal Cas9 antibody (Cat. No. C15310258, diluted 1:5,000). Lane 1 and 2 show the result of the input.</small></p>
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<p><small><strong>Figure 3. Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />HEK293T cells were transiently transfected with a Cas9 expression vector. The cells were fixed with 4% formaldehyde, permeabilized in 0,1% Triton X-100 and blocked in PBS containing 5% BSA. The cells were stained with the Cas9 antibody diluted 1;400 at 4°C o/n, followed by incubation with an anti mouse secondary antibody coupled to AF596 for 1 h at RT (left). Nuclei were counter-stained with DAPI (middle). A merge of the two stainings is shown on the right.</small></p>
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<p><small><strong>Figure 1. Western blot analysis using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />Western blot was performed on protein extracts from HEK293T cells transfected with Cas9 (lane 1) or from untransfected cells (lane 2) using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15200216), diluted 1:5,000 in PBS-T containing 0.5% NFDM. The marker is shown on the left, position of the Cas9 protein is indicated on the right.</small></p>
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<p><small><strong>Figure 2. IP using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />IP was performed on whole cell extracts from HEK293T cells transfected with a Cas9 expression vector (lane 1, 3 and 5), or untransfected cells (lane 2 and 4) using 5 µg of the Diagenode antibody against CRISPR/Cas9 (cat. No. C15200216, lane 3 and 4) or with an equal amount of IgG, used as a negative control (lane 5). The immunoprecipitated proteins were subsequently analysed by Western blot with the polyclonal Cas9 antibody (Cat. No. C15310258, diluted 1:5,000). Lane 1 and 2 show the result of the input.</small></p>
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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15200216-IF.png" alt="CRISPR/Cas9 Antibody for IF" caption="false" width="284" height="93" /></p>
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<p><small><strong>Figure 3. Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />HEK293T cells were transiently transfected with a Cas9 expression vector. The cells were fixed with 4% formaldehyde, permeabilized in 0,1% Triton X-100 and blocked in PBS containing 5% BSA. The cells were stained with the Cas9 antibody diluted 1;400 at 4°C o/n, followed by incubation with an anti mouse secondary antibody coupled to AF596 for 1 h at RT (left). Nuclei were counter-stained with DAPI (middle). A merge of the two stainings is shown on the right.</small></p>
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<p><span><strong>Specifically optimized for ChIP-seq</strong></span><br /><br /><span>The MicroPlex Library Preparation™ kit is the only kit on the market which is validated for ChIP-seq and which allows the preparation of indexed libraries from just picogram inputs. In combination with the </span><a href="./true-microchip-kit-x16-16-rxns">True MicroChIP kit</a><span>, it allows for performing ChIP-seq on as few as 10,000 cells. Less input, fewer steps, fewer supplies, faster time to results! </span></p>
<p>The MicroPlex v2 kit (Cat. No. C05010012) contains all necessary reagents including single indexes for multiplexing up to 12 samples using single barcoding. For higher multiplexing (using dual indexes) check <a href="https://www.diagenode.com/en/p/microplex-lib-prep-kit-v3-48-rxns">MicroPlex Library Preparation Kits v3</a>.</p>',
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<li><strong>1 tube, 2 hours, 3 steps</strong> protocol</li>
<li><strong>Input: </strong>50 pg – 50 ng</li>
<li><strong>Reduce potential bias</strong> - few PCR amplification cycles needed</li>
<li><strong>High sensitivity ChIP-seq</strong> - low PCR duplication rate</li>
<li><strong>Great multiplexing flexibility</strong> with 12 barcodes (8 nt) included</li>
<li><strong>Validated with the <a href="https://www.diagenode.com/p/sx-8g-ip-star-compact-automated-system-1-unit" title="IP-Star Automated System">IP-Star<sup>®</sup> Automated Platform</a></strong></li>
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<h3>How it works</h3>
<center><img src="https://www.diagenode.com/img/product/kits/microplex-method-overview-v2.png" /></center>
<p style="margin-bottom: 0;"><small><strong>Microplex workflow - protocol with single indexes</strong><br />An input of 50 pg to 50 ng of fragmented dsDNA is converted into sequencing-ready libraries for Illumina® NGS platforms using a fast and simple 3-step protocol</small></p>
<ul class="accordion" data-accordion="" id="readmore" style="margin-left: 0;">
<li class="accordion-navigation"><a href="#first" style="background: #ffffff; padding: 0rem; margin: 0rem; color: #13b2a2;"><small>Read more about MicroPlex workflow</small></a>
<div id="first" class="content">
<p><small><strong>Step 1. Template Preparation</strong> provides efficient repair of the fragmented double-stranded DNA input.</small></p>
<p><small>In this step, the DNA is repaired and yields molecules with blunt ends.</small></p>
<p><small><strong>Step 2. Library Synthesis.</strong> enables ligation of MicroPlex patented stem- loop adapters.</small></p>
<p><small>In the next step, stem-loop adaptors with blocked 5’ ends are ligated with high efficiency to the 5’ end of the genomic DNA, leaving a nick at the 3’ end. The adaptors cannot ligate to each other and do not have single- strand tails, both of which contribute to non-specific background found with many other NGS preparations.</small></p>
<p><small><strong>Step 3. Library Amplification</strong> enables extension of the template, cleavage of the stem-loop adaptors, and amplification of the library. Illumina- compatible indexes are also introduced using a high-fidelity, highly- processive, low-bias DNA polymerase.</small></p>
<p><small>In the final step, the 3’ ends of the genomic DNA are extended to complete library synthesis and Illumina-compatible indexes are added through a high-fidelity amplification. Any remaining free adaptors are destroyed. Hands-on time and the risk of contamination are minimized by using a single tube and eliminating intermediate purifications.</small></p>
<p><small>Obtained libraries are purified, quantified and sized. The libraries pooling can be performed as well before sequencing.</small></p>
</div>
</li>
</ul>
<p></p>
<h3>Reliable detection of enrichments in ChIP-seq</h3>
<p><img src="https://www.diagenode.com/img/product/kits/microplex-library-prep-kit-figure-a.png" alt="Reliable detection of enrichments in ChIP-seq figure 1" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure A.</strong> ChIP has been peformed with H3K4me3 antibody, amplification of 17 pg of DNA ChIP'd from 10.000 cells and amplification of 35 pg of DNA ChIP'd from 100.000 cells (control experiment). The IP'd DNA was amplified and transformed into a sequencing-ready preparation for the Illumina plateform with the MicroPlex Library Preparation kit. The library was then analysed on an Illumina<sup>®</sup> Genome Analyzer. Cluster generation and sequencing were performed according to the manufacturer's instructions.</p>
<p><img src="https://www.diagenode.com/img/product/kits/microplex-library-prep-kit-figure-b.png" alt="Reliable detection of enrichments in ChIP-seq figure 2" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure B.</strong> We observed a perfect match between the top 40% of True MicroChIP peaks and the reference dataset. Based on the NIH Encode project criterion, ChIP-seq results are considered reproducible between an original and reproduced dataset if the top 40% of peaks have at least an 80% overlap ratio with the compared dataset.</p>',
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<p>Learn more about: <a href="https://www.diagenode.com/applications/western-blot">Loading control, MW marker visualization</a><em>. <br /></em></p>
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<p>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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<p><span style="font-weight: 400;">Diagenode’s highly validated antibodies:</span></p>
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<li>Highly sensitive and specific</li>
<li>Cost-effective (requires less antibody per reaction)</li>
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'description' => '<p>Diagenode offers the broad range of antibodies raised against the N- or C-terminus of the Cas9 nuclease from <em>Streptococcus <g class="gr_ gr_5 gr-alert gr_spell gr_disable_anim_appear ContextualSpelling ins-del multiReplace" id="5" data-gr-id="5">pyogenes</g></em>. These highly specific polyclonal and monoclonal antibodies are validated in Western blot, immunoprecipitation, immunofluorescence and in chromatin immunoprecipitation.</p>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h2><em><a name="pyogenes"></a>S. pyogenes</em> CRISPR/Cas9 antibodies<a></a></h2>
<div class="panel">
<h2>Discover our first monoclonal CRISPR/Cas9 antibody validated in ChIP<br /><br /></h2>
<div class="row">
<div class="small-5 medium-5 large-5 columns"><img src="/img/landing-pages/crispr-cas9-chip-on-hih3t3.jpg" alt="" /></div>
<div class="small-7 medium-7 large-7 columns">
<ul>
<li>Validated in chromatin immunoprecipitation</li>
<li>Performs better than FLAG antibody</li>
<li>Excellent for WB, IF and IP</li>
</ul>
<p><small><strong>ChIP</strong> was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50 µg chromatin was incubated overnight at 4°C with 5 or 10 µg of either an anti-FLAG antibody or the Diagenode antibody against Cas9 (Cat. No. C15200229). Mouse IgG was used as a negative IP control. qPCR was performed with primers specific for the GFP gene, and for a non-targeted region (protein kinase C delta, Prkcd), used as negative control. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).</small></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns text-right"><a href="/p/crispr-cas9-monoclonal-antibody-50-ug-25-μl" class="tiny details button radius">Learn more</a></div>
</div>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>First ChIP-grade CRISPR/Cas9 polyclonal antibody</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/landing-pages/c_a_s9-chip-grade-antibody.png" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Excellent polyclonal antibody for chromatin immunoprecipitation</li>
<li>Optimized for highest ChIP specificity and yields</li>
<li>Validated for all applications including immunoblotting, immunofluorescence and western blot</li>
</ul>
<p><small><strong>ChIP</strong> was performed on NIH3T3 cells stably expressing GFP- H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50 μg chromatin was incubated with either 5 μg of an anti-FLAG antibody or 2 μl of the Diagenode antibody against Cas9. The pre-immune serum (PPI) was used as negative IP control. Then qPCR was performed with primers specific for the GFP gene, and for two non-targeted regions: Ppap2c and Prkcd, used as negative controls. This figure shows the recovery, expressed as a % of input.</small></p>
<p class="text-right"><a href="../p/crispr-cas9-polyclonal-antibody" class="details tiny button">Learn more</a></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>CRISPR/Cas9 monoclonal antibody 4G10</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/landing-pages/cas9_4g10_fig1.png" width="170" height="302" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Antibody raised against N-terminus of Cas9 nuclease</li>
<li>Validated for western blot, IP and immunofluorescence</li>
</ul>
<p><small><strong>Immunofluorescence</strong>: Hela cells were transiently transfected with a Cas9 expression vector. The cells were fixed in 3.7% formaldehyde, permeabilized in 0.5% Triton-X-100 and blocked in PBS containing 2% BSA. The cells were stained with the Cas9 antibody at 4°C o/n, followed by incubation with an anti mouse secondary antibody coupled to AF488 for 1 h at RT. Nuclei were counter-stained with Hoechst 33342 (right).</small></p>
<p class="text-right"><a href="../p/crispr-cas9-monoclonal-antibody-4g10-50-ug" class="details tiny button">Learn more</a></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>CRISPR/Cas9 C-terminal monoclonal antibody</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/product/antibodies/C15200223-IP.png" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Antibody raised against C-terminus of Cas9 nuclease</li>
<li>Validated for western blot, IP and immunofluorescence</li>
</ul>
<p><small><strong> Western blot</strong> was performed on 20 μg protein extracts from Cas9 expressing HeLa cells (lane 1) and on negative control HeLa cells (lane 2) with the Diagenode antibody against Cas9. The antibody was diluted 1:4,000. The marker is shown on the left, position of the Cas9 protein is indicated on the right. </small></p>
<p class="text-right"><a href="../p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug" class="details tiny button">Learn more</a></p>
</div>
<div class="small-12 medium-12 large-12 columns">
<h3>Which CRISPR/Cas9 antibody is the best for your application?</h3>
<a name="table"></a>
<table>
<thead>
<tr>
<th>Antibody</th>
<th>WB</th>
<th>IF</th>
<th>IP</th>
<th>ChIP</th>
<th>Antibody raised against</th>
</tr>
</thead>
<tbody>
<tr>
<td><a href="../p/crispr-cas9-monoclonal-antibody-50-ug-25-μl"><strong class="diacol">CRISPR/Cas9 monoclonal antibody</strong></a></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><span class="diacol">N-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-polyclonal-antibody">CRISPR/Cas9 polyclonal antibody</a></td>
<td>++</td>
<td>++</td>
<td>++</td>
<td><strong class="diacol">+++</strong></td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-monoclonal-antibody-4g10-50-ug">CRISPR/Cas9 monoclonal antibody 4G10</a></td>
<td>+++</td>
<td>+++</td>
<td>++</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="../p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug-23-ul"><strong class="diacol">CRISPR/Cas9 C-terminal monoclonal antibody</strong></a> <span class="label alert" style="font-size: 0.9rem;">NEW!</span></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">no</strong></td>
<td><strong class="diacol">+</strong></td>
<td><span class="diacol">C-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug">CRISPR/Cas9 C-terminal monoclonal antibody</a></td>
<td>++</td>
<td>++</td>
<td>+</td>
<td>no</td>
<td><span class="diacol">C-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-monoclonal-antibody-7A9-50-mg">CRISPR/Cas9 monoclonal antibody 7A9</a></td>
<td>++</td>
<td>++</td>
<td>++</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-hrp-monoclonal-antibody-50-ul">CRISPR/Cas9 - HRP monoclonal antibody 7A9</a></td>
<td>+++</td>
<td>no</td>
<td>no</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
</tbody>
</table>
</div>
</div>
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'description' => '<p style="text-align: justify;"><span>Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of large sets of genome-wide sequencing data. ChIP sequencing (ChIP-seq) has now become the gold standard method for chromatin studies, given the accuracy and coverage scale of the approach over other methods. Successful ChIP-seq, however, requires a higher level of experimental accuracy and consistency in all steps of ChIP than ever before. Particularly crucial is the quality of ChIP antibodies. </span></p>',
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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>Tissue-specific gene expression is fundamental in development and evolution, and is mediated by transcription factors and by the </span><i>cis</i><span>-regulatory regions (enhancers) that they control. Transcription factors and their respective tissue-specific enhancers are essential components of gene regulatory networks responsible for the development of tissues and organs. Although numerous transcription factors have been characterized from different organisms, the knowledge of the enhancers responsible for their tissue-specific expression remains fragmentary. Here we use<span> </span></span><i>Ciona</i><span><span> </span>to study the enhancers associated with ten transcription factors expressed in the notochord, an evolutionary hallmark of the chordate phylum. Our results illustrate how two evolutionarily conserved transcription factors, Brachyury and Foxa2, coordinate the deployment of other notochord transcription factors. The results of these detailed<span> </span></span><i>cis</i><span>-regulatory analyses delineate a high-resolution view of the essential notochord gene regulatory network of<span> </span></span><i>Ciona</i><span>, and provide a reference for studies of transcription factors, enhancers, and their roles in development, disease, and evolution.</span></p>',
'date' => '2024-04-08',
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'name' => 'Monitoring autochthonous lung tumors induced by somatic CRISPR geneediting in mice using a secreted luciferase.',
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'description' => '<p>BACKGROUND: In vivo gene editing of somatic cells with CRISPR nucleases has facilitated the generation of autochthonous mouse tumors, which are initiated by genetic alterations relevant to the human disease and progress along a natural timeline as in patients. However, the long and variable, orthotopic tumor growth in inner organs requires sophisticated, time-consuming and resource-intensive imaging for longitudinal disease monitoring and impedes the use of autochthonous tumor models for preclinical studies. METHODS: To facilitate a more widespread use, we have generated a reporter mouse that expresses a Cre-inducible luciferase from Gaussia princeps (GLuc), which is secreted by cells in an energy-consuming process and can be measured quantitatively in the blood as a marker for the viable tumor load. In addition, we have developed a flexible, complementary toolkit to rapidly assemble recombinant adenoviruses (AVs) for delivering Cre recombinase together with CRISPR nucleases targeting cancer driver genes. RESULTS: We demonstrate that intratracheal infection of GLuc reporter mice with CRISPR-AVs efficiently induces lung tumors driven by mutations in the targeted cancer genes and simultaneously activates the GLuc transgene, resulting in GLuc secretion into the blood by the growing tumor. GLuc blood levels are easily and robustly quantified in small-volume blood samples with inexpensive equipment, enable tumor detection already several months before the humane study endpoint and precisely mirror the kinetics of tumor development specified by the inducing gene combination. CONCLUSIONS: Our study establishes blood-based GLuc monitoring as an inexpensive, rapid, high-throughput and animal-friendly method to longitudinally monitor autochthonous tumor growth in preclinical studies.</p>',
'date' => '2022-10-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/36192757',
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'name' => 'Systematic comparison of CRISPR-based transcriptional activatorsuncovers gene-regulatory features of enhancer-promoter interactions.',
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'description' => '<p>Nuclease-inactivated CRISPR/Cas-based (dCas-based) systems have emerged as powerful technologies to synthetically reshape the human epigenome and gene expression. Despite the increasing adoption of these platforms, their relative potencies and mechanistic differences are incompletely characterized, particularly at human enhancer-promoter pairs. Here, we systematically compared the most widely adopted dCas9-based transcriptional activators, as well as an activator consisting of dCas9 fused to the catalytic core of the human CBP protein, at human enhancer-promoter pairs. We find that these platforms display variable relative expression levels in different human cell types and that their transactivation efficacies vary based upon the effector domain, effector recruitment architecture, targeted locus and cell type. We also show that each dCas9-based activator can induce the production of enhancer RNAs (eRNAs) and that this eRNA induction is positively correlated with downstream mRNA expression from a cognate promoter. Additionally, we use dCas9-based activators to demonstrate that an intrinsic transcriptional and epigenetic reciprocity can exist between human enhancers and promoters and that enhancer-mediated tracking and engagement of a downstream promoter can be synthetically driven by targeting dCas9-based transcriptional activators to an enhancer. Collectively, our study provides new insights into the enhancer-mediated control of human gene expression and the use of dCas9-based activators.</p>',
'date' => '2022-07-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/35849129',
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'description' => '<p>Tools for actively targeted DNA demethylation are required to increase our knowledge about regulation and specific functions of this important epigenetic modification. DNA demethylation in mammals involves TET-mediated oxidation of 5-methylcytosine (5-meC), which may promote its replication-dependent dilution and/or active removal through base excision repair (BER). However, it is still unclear whether oxidized derivatives of 5-meC are simply DNA demethylation intermediates or rather epigenetic marks on their own. Unlike animals, plants have evolved enzymes that directly excise 5-meC without previous modification. In this work, we have fused the catalytic domain of Arabidopsis ROS1 5-meC DNA glycosylase to a CRISPR-associated null-nuclease (dCas9) and analyzed its capacity for targeted reactivation of methylation-silenced genes, in comparison to other dCas9-effectors. We found that dCas9-ROS1, but not dCas9-TET1, is able to reactivate methylation-silenced genes and induce partial demethylation in a replication-independent manner. We also found that reactivation induced by dCas9-ROS1, as well as that achieved by two different CRISPR-based chromatin effectors (dCas9-VP160 and dCas9-p300), generally decreases with methylation density. Our results suggest that plant 5-meC DNA glycosylases are a valuable addition to the CRISPR-based toolbox for epigenetic editing.</p>',
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'description' => '<p>Editing the β-globin locus in hematopoietic stem cells is an alternative therapeutic approach for gene therapy of β-thalassemia and sickle cell disease. Using the CRISPR/Cas9 system, we genetically modified human hematopoietic stem and progenitor cells (HSPCs) to mimic the large rearrangements in the β-globin locus associated with hereditary persistence of fetal hemoglobin (HPFH), a condition that mitigates the clinical phenotype of patients with β-hemoglobinopathies. We optimized and compared the efficiency of plasmid-, lentiviral vector (LV)-, RNA-, and ribonucleoprotein complex (RNP)-based methods to deliver the CRISPR/Cas9 system into HSPCs. Plasmid delivery of Cas9 and gRNA pairs targeting two HPFH-like regions led to high frequency of genomic rearrangements and HbF reactivation in erythroblasts derived from sorted, Cas9 HSPCs but was associated with significant cell toxicity. RNA-mediated delivery of CRISPR/Cas9 was similarly toxic but much less efficient in editing the β-globin locus. Transduction of HSPCs by LVs expressing Cas9 and gRNA pairs was robust and minimally toxic but resulted in poor genome-editing efficiency. Ribonucleoprotein (RNP)-based delivery of CRISPR/Cas9 exhibited a good balance between cytotoxicity and efficiency of genomic rearrangements as compared to the other delivery systems and resulted in HbF upregulation in erythroblasts derived from unselected edited HSPCs.</p>',
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<p><span><strong>Specifically optimized for ChIP-seq</strong></span><br /><br /><span>The MicroPlex Library Preparation™ kit is the only kit on the market which is validated for ChIP-seq and which allows the preparation of indexed libraries from just picogram inputs. In combination with the </span><a href="./true-microchip-kit-x16-16-rxns">True MicroChIP kit</a><span>, it allows for performing ChIP-seq on as few as 10,000 cells. Less input, fewer steps, fewer supplies, faster time to results! </span></p>
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<li><strong>1 tube, 2 hours, 3 steps</strong> protocol</li>
<li><strong>Input: </strong>50 pg – 50 ng</li>
<li><strong>Reduce potential bias</strong> - few PCR amplification cycles needed</li>
<li><strong>High sensitivity ChIP-seq</strong> - low PCR duplication rate</li>
<li><strong>Great multiplexing flexibility</strong> with 12 barcodes (8 nt) included</li>
<li><strong>Validated with the <a href="https://www.diagenode.com/p/sx-8g-ip-star-compact-automated-system-1-unit" title="IP-Star Automated System">IP-Star<sup>®</sup> Automated Platform</a></strong></li>
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<h3>How it works</h3>
<center><img src="https://www.diagenode.com/img/product/kits/microplex-method-overview-v2.png" /></center>
<p style="margin-bottom: 0;"><small><strong>Microplex workflow - protocol with single indexes</strong><br />An input of 50 pg to 50 ng of fragmented dsDNA is converted into sequencing-ready libraries for Illumina® NGS platforms using a fast and simple 3-step protocol</small></p>
<ul class="accordion" data-accordion="" id="readmore" style="margin-left: 0;">
<li class="accordion-navigation"><a href="#first" style="background: #ffffff; padding: 0rem; margin: 0rem; color: #13b2a2;"><small>Read more about MicroPlex workflow</small></a>
<div id="first" class="content">
<p><small><strong>Step 1. Template Preparation</strong> provides efficient repair of the fragmented double-stranded DNA input.</small></p>
<p><small>In this step, the DNA is repaired and yields molecules with blunt ends.</small></p>
<p><small><strong>Step 2. Library Synthesis.</strong> enables ligation of MicroPlex patented stem- loop adapters.</small></p>
<p><small>In the next step, stem-loop adaptors with blocked 5’ ends are ligated with high efficiency to the 5’ end of the genomic DNA, leaving a nick at the 3’ end. The adaptors cannot ligate to each other and do not have single- strand tails, both of which contribute to non-specific background found with many other NGS preparations.</small></p>
<p><small><strong>Step 3. Library Amplification</strong> enables extension of the template, cleavage of the stem-loop adaptors, and amplification of the library. Illumina- compatible indexes are also introduced using a high-fidelity, highly- processive, low-bias DNA polymerase.</small></p>
<p><small>In the final step, the 3’ ends of the genomic DNA are extended to complete library synthesis and Illumina-compatible indexes are added through a high-fidelity amplification. Any remaining free adaptors are destroyed. Hands-on time and the risk of contamination are minimized by using a single tube and eliminating intermediate purifications.</small></p>
<p><small>Obtained libraries are purified, quantified and sized. The libraries pooling can be performed as well before sequencing.</small></p>
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<p></p>
<h3>Reliable detection of enrichments in ChIP-seq</h3>
<p><img src="https://www.diagenode.com/img/product/kits/microplex-library-prep-kit-figure-a.png" alt="Reliable detection of enrichments in ChIP-seq figure 1" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure A.</strong> ChIP has been peformed with H3K4me3 antibody, amplification of 17 pg of DNA ChIP'd from 10.000 cells and amplification of 35 pg of DNA ChIP'd from 100.000 cells (control experiment). The IP'd DNA was amplified and transformed into a sequencing-ready preparation for the Illumina plateform with the MicroPlex Library Preparation kit. The library was then analysed on an Illumina<sup>®</sup> Genome Analyzer. Cluster generation and sequencing were performed according to the manufacturer's instructions.</p>
<p><img src="https://www.diagenode.com/img/product/kits/microplex-library-prep-kit-figure-b.png" alt="Reliable detection of enrichments in ChIP-seq figure 2" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure B.</strong> We observed a perfect match between the top 40% of True MicroChIP peaks and the reference dataset. Based on the NIH Encode project criterion, ChIP-seq results are considered reproducible between an original and reproduced dataset if the top 40% of peaks have at least an 80% overlap ratio with the compared dataset.</p>',
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<p><small><strong>Figure 2. IP using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />IP was performed on whole cell extracts from HEK293T cells transfected with a Cas9 expression vector (lane 1, 3 and 5), or untransfected cells (lane 2 and 4) using 5 µg of the Diagenode antibody against CRISPR/Cas9 (cat. No. C15200216, lane 3 and 4) or with an equal amount of IgG, used as a negative control (lane 5). The immunoprecipitated proteins were subsequently analysed by Western blot with the polyclonal Cas9 antibody (Cat. No. C15310258, diluted 1:5,000). Lane 1 and 2 show the result of the input.</small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15200216-IF.png" alt="CRISPR/Cas9 Antibody for IF" caption="false" width="284" height="93" /></p>
</div>
<div class="small-8 columns">
<p><small><strong>Figure 3. Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />HEK293T cells were transiently transfected with a Cas9 expression vector. The cells were fixed with 4% formaldehyde, permeabilized in 0,1% Triton X-100 and blocked in PBS containing 5% BSA. The cells were stained with the Cas9 antibody diluted 1;400 at 4°C o/n, followed by incubation with an anti mouse secondary antibody coupled to AF596 for 1 h at RT (left). Nuclei were counter-stained with DAPI (middle). A merge of the two stainings is shown on the right.</small></p>
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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 to induce indel mutations, specific sequence replacements or insertions and large deletions or genomic rearrangements at any desired location in the genome. In addition, Cas9 can also be used to mediate upregulation of specific endogenous genes or to alter histone modifications or DNA methylation.',
'clonality' => '4G10',
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'lot' => '003',
'concentration' => '2 μg/μl ',
'reactivity' => 'Streptococcus pyogenes',
'type' => 'Monoclonal',
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'application_table' => '<table>
<thead>
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<th>Suggested dilution <sup>*</sup></th>
<th>References</th>
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<tr>
<td>Western Blotting</td>
<td>1:5,000</td>
<td>Fig 1</td>
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<tr>
<td>Immunoprecipitation</td>
<td>5 μg/IP</td>
<td>Fig 2</td>
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<td>Immunofluorescence</td>
<td>1:400</td>
<td>Fig 3</td>
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'description' => '<p><span>Alternative name: <strong>Csn1</strong></span></p>
<p><span>Monoclonal </span>antibody raised in mouse against the N-terminus of the Cas9 nuclease (<strong>CRISPR-associated protein 9</strong>) using a recombinant protein.</p>',
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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15200216-WB.png" alt="CRISPR/Cas9 Antibody for Western blot" caption="false" width="187" height="152" /></p>
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<div class="small-8 columns">
<p><small><strong>Figure 1. Western blot analysis using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />Western blot was performed on protein extracts from HEK293T cells transfected with Cas9 (lane 1) or from untransfected cells (lane 2) using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15200216), diluted 1:5,000 in PBS-T containing 0.5% NFDM. The marker is shown on the left, position of the Cas9 protein is indicated on the right.</small></p>
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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15200216-IP.png" alt="CRISPR/Cas9 Antibody validated in IP" caption="false" width="284" height="99" /></p>
</div>
<div class="small-8 columns">
<p><small><strong>Figure 2. IP using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />IP was performed on whole cell extracts from HEK293T cells transfected with a Cas9 expression vector (lane 1, 3 and 5), or untransfected cells (lane 2 and 4) using 5 µg of the Diagenode antibody against CRISPR/Cas9 (cat. No. C15200216, lane 3 and 4) or with an equal amount of IgG, used as a negative control (lane 5). The immunoprecipitated proteins were subsequently analysed by Western blot with the polyclonal Cas9 antibody (Cat. No. C15310258, diluted 1:5,000). Lane 1 and 2 show the result of the input.</small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15200216-IF.png" alt="CRISPR/Cas9 Antibody for IF" caption="false" width="284" height="93" /></p>
</div>
<div class="small-8 columns">
<p><small><strong>Figure 3. Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />HEK293T cells were transiently transfected with a Cas9 expression vector. The cells were fixed with 4% formaldehyde, permeabilized in 0,1% Triton X-100 and blocked in PBS containing 5% BSA. The cells were stained with the Cas9 antibody diluted 1;400 at 4°C o/n, followed by incubation with an anti mouse secondary antibody coupled to AF596 for 1 h at RT (left). Nuclei were counter-stained with DAPI (middle). A merge of the two stainings is shown on the right.</small></p>
</div>
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'description' => '<p><a href="https://www.diagenode.com/files/products/kits/MicroPlex-Libary-Prep-Kit-v2-manual.pdf"><img src="https://www.diagenode.com/img/buttons/bt-manual.png" /></a></p>
<p><span><strong>Specifically optimized for ChIP-seq</strong></span><br /><br /><span>The MicroPlex Library Preparation™ kit is the only kit on the market which is validated for ChIP-seq and which allows the preparation of indexed libraries from just picogram inputs. In combination with the </span><a href="./true-microchip-kit-x16-16-rxns">True MicroChIP kit</a><span>, it allows for performing ChIP-seq on as few as 10,000 cells. Less input, fewer steps, fewer supplies, faster time to results! </span></p>
<p>The MicroPlex v2 kit (Cat. No. C05010012) contains all necessary reagents including single indexes for multiplexing up to 12 samples using single barcoding. For higher multiplexing (using dual indexes) check <a href="https://www.diagenode.com/en/p/microplex-lib-prep-kit-v3-48-rxns">MicroPlex Library Preparation Kits v3</a>.</p>',
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<li><strong>High sensitivity ChIP-seq</strong> - low PCR duplication rate</li>
<li><strong>Great multiplexing flexibility</strong> with 12 barcodes (8 nt) included</li>
<li><strong>Validated with the <a href="https://www.diagenode.com/p/sx-8g-ip-star-compact-automated-system-1-unit" title="IP-Star Automated System">IP-Star<sup>®</sup> Automated Platform</a></strong></li>
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<h3>How it works</h3>
<center><img src="https://www.diagenode.com/img/product/kits/microplex-method-overview-v2.png" /></center>
<p style="margin-bottom: 0;"><small><strong>Microplex workflow - protocol with single indexes</strong><br />An input of 50 pg to 50 ng of fragmented dsDNA is converted into sequencing-ready libraries for Illumina® NGS platforms using a fast and simple 3-step protocol</small></p>
<ul class="accordion" data-accordion="" id="readmore" style="margin-left: 0;">
<li class="accordion-navigation"><a href="#first" style="background: #ffffff; padding: 0rem; margin: 0rem; color: #13b2a2;"><small>Read more about MicroPlex workflow</small></a>
<div id="first" class="content">
<p><small><strong>Step 1. Template Preparation</strong> provides efficient repair of the fragmented double-stranded DNA input.</small></p>
<p><small>In this step, the DNA is repaired and yields molecules with blunt ends.</small></p>
<p><small><strong>Step 2. Library Synthesis.</strong> enables ligation of MicroPlex patented stem- loop adapters.</small></p>
<p><small>In the next step, stem-loop adaptors with blocked 5’ ends are ligated with high efficiency to the 5’ end of the genomic DNA, leaving a nick at the 3’ end. The adaptors cannot ligate to each other and do not have single- strand tails, both of which contribute to non-specific background found with many other NGS preparations.</small></p>
<p><small><strong>Step 3. Library Amplification</strong> enables extension of the template, cleavage of the stem-loop adaptors, and amplification of the library. Illumina- compatible indexes are also introduced using a high-fidelity, highly- processive, low-bias DNA polymerase.</small></p>
<p><small>In the final step, the 3’ ends of the genomic DNA are extended to complete library synthesis and Illumina-compatible indexes are added through a high-fidelity amplification. Any remaining free adaptors are destroyed. Hands-on time and the risk of contamination are minimized by using a single tube and eliminating intermediate purifications.</small></p>
<p><small>Obtained libraries are purified, quantified and sized. The libraries pooling can be performed as well before sequencing.</small></p>
</div>
</li>
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<p></p>
<h3>Reliable detection of enrichments in ChIP-seq</h3>
<p><img src="https://www.diagenode.com/img/product/kits/microplex-library-prep-kit-figure-a.png" alt="Reliable detection of enrichments in ChIP-seq figure 1" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure A.</strong> ChIP has been peformed with H3K4me3 antibody, amplification of 17 pg of DNA ChIP'd from 10.000 cells and amplification of 35 pg of DNA ChIP'd from 100.000 cells (control experiment). The IP'd DNA was amplified and transformed into a sequencing-ready preparation for the Illumina plateform with the MicroPlex Library Preparation kit. The library was then analysed on an Illumina<sup>®</sup> Genome Analyzer. Cluster generation and sequencing were performed according to the manufacturer's instructions.</p>
<p><img src="https://www.diagenode.com/img/product/kits/microplex-library-prep-kit-figure-b.png" alt="Reliable detection of enrichments in ChIP-seq figure 2" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure B.</strong> We observed a perfect match between the top 40% of True MicroChIP peaks and the reference dataset. Based on the NIH Encode project criterion, ChIP-seq results are considered reproducible between an original and reproduced dataset if the top 40% of peaks have at least an 80% overlap ratio with the compared dataset.</p>',
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'description' => '<p><strong>Western blot</strong> : The quality of antibodies used in this technique is crucial for correct and specific protein identification. Diagenode offers huge selection of highly sensitive and specific western blot-validated antibodies.</p>
<p>Learn more about: <a href="https://www.diagenode.com/applications/western-blot">Loading control, MW marker visualization</a><em>. <br /></em></p>
<p><em></em>Check our selection of antibodies validated in Western blot.</p>',
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<p>Diagenode offers huge selection of highly sensitive antibodies validated in IF.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15200229-IF.jpg" alt="" height="245" width="256" /></p>
<p><sup><strong>Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong></sup></p>
<p><sup>HeLa cells transfected with a Cas9 expression vector (left) or untransfected cells (right) were fixed in methanol at -20°C, permeabilized with acetone at -20°C and blocked with PBS containing 2% BSA. The cells were stained with the Cas9 C-terminal antibody (Cat. No. C15200229) diluted 1:400, followed by incubation with an anti-mouse secondary antibody coupled to AF488. The bottom images show counter-staining of the nuclei with Hoechst 33342.</sup></p>
<h5><sup>Check our selection of antibodies validated in IF.</sup></h5>',
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<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>
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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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'description' => '<p style="text-align: justify;"><span>Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of large sets of genome-wide sequencing data. ChIP sequencing (ChIP-seq) has now become the gold standard method for chromatin studies, given the accuracy and coverage scale of the approach over other methods. Successful ChIP-seq, however, requires a higher level of experimental accuracy and consistency in all steps of ChIP than ever before. Particularly crucial is the quality of ChIP antibodies. </span></p>',
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'name' => ' Accurate QC to optimize CRISPR/Cas9 genome editing specificity',
'description' => '<p>The CRISPR/Cas9 technology is delivering superior genetic models for fundamental disease research, drug screening, therapy development, rapid diagnostics, and transcriptional modulation. Although CRISPR/Cas9 enables rapid genome editing, several aspects affect its efficiency and specificity including guide RNA design, delivery methods, and off-targets effects. Diagenode has developed strategies to overcome these common pitfalls and has optimized CRISPR/Cas9 genome editing specificity</p>',
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'name' => 'Cis-regulatory interfaces reveal the molecular mechanisms underlying the notochord gene regulatory network of Ciona',
'authors' => 'Negrón-Piñeiro L. J. et al.',
'description' => '<p><span>Tissue-specific gene expression is fundamental in development and evolution, and is mediated by transcription factors and by the </span><i>cis</i><span>-regulatory regions (enhancers) that they control. Transcription factors and their respective tissue-specific enhancers are essential components of gene regulatory networks responsible for the development of tissues and organs. Although numerous transcription factors have been characterized from different organisms, the knowledge of the enhancers responsible for their tissue-specific expression remains fragmentary. Here we use<span> </span></span><i>Ciona</i><span><span> </span>to study the enhancers associated with ten transcription factors expressed in the notochord, an evolutionary hallmark of the chordate phylum. Our results illustrate how two evolutionarily conserved transcription factors, Brachyury and Foxa2, coordinate the deployment of other notochord transcription factors. The results of these detailed<span> </span></span><i>cis</i><span>-regulatory analyses delineate a high-resolution view of the essential notochord gene regulatory network of<span> </span></span><i>Ciona</i><span>, and provide a reference for studies of transcription factors, enhancers, and their roles in development, disease, and evolution.</span></p>',
'date' => '2024-04-08',
'pmid' => 'https://www.nature.com/articles/s41467-024-46850-3',
'doi' => 'https://doi.org/10.1038/s41467-024-46850-3',
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'name' => 'Monitoring autochthonous lung tumors induced by somatic CRISPR geneediting in mice using a secreted luciferase.',
'authors' => 'Merle N. et al.',
'description' => '<p>BACKGROUND: In vivo gene editing of somatic cells with CRISPR nucleases has facilitated the generation of autochthonous mouse tumors, which are initiated by genetic alterations relevant to the human disease and progress along a natural timeline as in patients. However, the long and variable, orthotopic tumor growth in inner organs requires sophisticated, time-consuming and resource-intensive imaging for longitudinal disease monitoring and impedes the use of autochthonous tumor models for preclinical studies. METHODS: To facilitate a more widespread use, we have generated a reporter mouse that expresses a Cre-inducible luciferase from Gaussia princeps (GLuc), which is secreted by cells in an energy-consuming process and can be measured quantitatively in the blood as a marker for the viable tumor load. In addition, we have developed a flexible, complementary toolkit to rapidly assemble recombinant adenoviruses (AVs) for delivering Cre recombinase together with CRISPR nucleases targeting cancer driver genes. RESULTS: We demonstrate that intratracheal infection of GLuc reporter mice with CRISPR-AVs efficiently induces lung tumors driven by mutations in the targeted cancer genes and simultaneously activates the GLuc transgene, resulting in GLuc secretion into the blood by the growing tumor. GLuc blood levels are easily and robustly quantified in small-volume blood samples with inexpensive equipment, enable tumor detection already several months before the humane study endpoint and precisely mirror the kinetics of tumor development specified by the inducing gene combination. CONCLUSIONS: Our study establishes blood-based GLuc monitoring as an inexpensive, rapid, high-throughput and animal-friendly method to longitudinally monitor autochthonous tumor growth in preclinical studies.</p>',
'date' => '2022-10-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/36192757',
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'name' => 'Systematic comparison of CRISPR-based transcriptional activatorsuncovers gene-regulatory features of enhancer-promoter interactions.',
'authors' => 'Wang K. et al.',
'description' => '<p>Nuclease-inactivated CRISPR/Cas-based (dCas-based) systems have emerged as powerful technologies to synthetically reshape the human epigenome and gene expression. Despite the increasing adoption of these platforms, their relative potencies and mechanistic differences are incompletely characterized, particularly at human enhancer-promoter pairs. Here, we systematically compared the most widely adopted dCas9-based transcriptional activators, as well as an activator consisting of dCas9 fused to the catalytic core of the human CBP protein, at human enhancer-promoter pairs. We find that these platforms display variable relative expression levels in different human cell types and that their transactivation efficacies vary based upon the effector domain, effector recruitment architecture, targeted locus and cell type. We also show that each dCas9-based activator can induce the production of enhancer RNAs (eRNAs) and that this eRNA induction is positively correlated with downstream mRNA expression from a cognate promoter. Additionally, we use dCas9-based activators to demonstrate that an intrinsic transcriptional and epigenetic reciprocity can exist between human enhancers and promoters and that enhancer-mediated tracking and engagement of a downstream promoter can be synthetically driven by targeting dCas9-based transcriptional activators to an enhancer. Collectively, our study provides new insights into the enhancer-mediated control of human gene expression and the use of dCas9-based activators.</p>',
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'description' => '<p>Tools for actively targeted DNA demethylation are required to increase our knowledge about regulation and specific functions of this important epigenetic modification. DNA demethylation in mammals involves TET-mediated oxidation of 5-methylcytosine (5-meC), which may promote its replication-dependent dilution and/or active removal through base excision repair (BER). However, it is still unclear whether oxidized derivatives of 5-meC are simply DNA demethylation intermediates or rather epigenetic marks on their own. Unlike animals, plants have evolved enzymes that directly excise 5-meC without previous modification. In this work, we have fused the catalytic domain of Arabidopsis ROS1 5-meC DNA glycosylase to a CRISPR-associated null-nuclease (dCas9) and analyzed its capacity for targeted reactivation of methylation-silenced genes, in comparison to other dCas9-effectors. We found that dCas9-ROS1, but not dCas9-TET1, is able to reactivate methylation-silenced genes and induce partial demethylation in a replication-independent manner. We also found that reactivation induced by dCas9-ROS1, as well as that achieved by two different CRISPR-based chromatin effectors (dCas9-VP160 and dCas9-p300), generally decreases with methylation density. Our results suggest that plant 5-meC DNA glycosylases are a valuable addition to the CRISPR-based toolbox for epigenetic editing.</p>',
'date' => '2020-02-19',
'pmid' => 'http://www.pubmed.gov/32087201',
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'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',
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'name' => 'Optimization of CRISPR/Cas9 Delivery to Human Hematopoietic Stem and Progenitor Cells for Therapeutic Genomic Rearrangements.',
'authors' => 'Lattanzi A, Meneghini V, Pavani G, Amor F, Ramadier S, Felix T, Antoniani C, Masson C, Alibeu O, Lee C, Porteus MH, Bao G, Amendola M, Mavilio F, Miccio A',
'description' => '<p>Editing the β-globin locus in hematopoietic stem cells is an alternative therapeutic approach for gene therapy of β-thalassemia and sickle cell disease. Using the CRISPR/Cas9 system, we genetically modified human hematopoietic stem and progenitor cells (HSPCs) to mimic the large rearrangements in the β-globin locus associated with hereditary persistence of fetal hemoglobin (HPFH), a condition that mitigates the clinical phenotype of patients with β-hemoglobinopathies. We optimized and compared the efficiency of plasmid-, lentiviral vector (LV)-, RNA-, and ribonucleoprotein complex (RNP)-based methods to deliver the CRISPR/Cas9 system into HSPCs. Plasmid delivery of Cas9 and gRNA pairs targeting two HPFH-like regions led to high frequency of genomic rearrangements and HbF reactivation in erythroblasts derived from sorted, Cas9 HSPCs but was associated with significant cell toxicity. RNA-mediated delivery of CRISPR/Cas9 was similarly toxic but much less efficient in editing the β-globin locus. Transduction of HSPCs by LVs expressing Cas9 and gRNA pairs was robust and minimally toxic but resulted in poor genome-editing efficiency. Ribonucleoprotein (RNP)-based delivery of CRISPR/Cas9 exhibited a good balance between cytotoxicity and efficiency of genomic rearrangements as compared to the other delivery systems and resulted in HbF upregulation in erythroblasts derived from unselected edited HSPCs.</p>',
'date' => '2019-01-02',
'pmid' => 'http://www.pubmed.gov/30424953',
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'name' => 'Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo',
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'description' => '<p>The advent of CRISPR/Cas9 has made genome editing possible in virtually any organism, including those not previously amenable to genetic manipulations. Here, we present an optimization of CRISPR/Cas9 for application to early avian embryos with improved efficiency via a three-fold strategy. First, we employed Cas9 protein flanked with two nuclear localization signal sequences for improved nuclear localization. Second, we used a modified guide RNA (gRNA) scaffold that obviates premature termination of transcription and unstable Cas9-gRNA interactions. Third, we used a chick-specific U6 promoter that yields 4-fold higher gRNA expression than the previously utilized human U6. For rapid screening of gRNAs for in vivo applications, we also generated a chicken fibroblast cell line that constitutively expresses Cas9. As proof of principle, we performed electroporation-based loss-of-function studies in the early chick embryo to knock out Pax7 and Sox10, key transcription factors with known functions in neural crest development. The results show that CRISPR/Cas9-mediated deletion causes loss of their respective proteins and transcripts, as well as predicted downstream targets. Taken together, the results reveal the utility of this optimized CRISPR/Cas9 method for targeted gene knockout in chicken embryos in a manner that is reproducible, robust and specific.</p>',
'date' => '2017-12-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/29150011',
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'name' => 'Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA',
'authors' => 'Liang W. et al.',
'description' => '<p>While CRISPR-based gene knock out in mammalian cells has proven to be very efficient, precise insertion of genetic elements via the cellular homology directed repair (HDR) pathway remains a rate-limiting step to seamless genome editing. Under the conditions described here, we achieved up to 56% targeted integration efficiency with up to a six-nucleotide insertion in HEK293 cells. In induced pluripotent stem cells (iPSCs), we achieved precise genome editing rates of up to 45% by co-delivering the Cas9 RNP and donor DNA. In addition, the use of a short double stranded DNA oligonucleotide with 3' overhangs allowed integration of a longer FLAG epitope tag along with a restriction site at rates of up to 50%. We propose a model that favors the design of donor DNAs with the change as close to the cleavage site as possible. For small changes such as SNPs or short insertions, asymmetric single stranded donor molecules with 30 base homology arms 3' to the insertion/repair cassette and greater than 40 bases of homology on the 5' end seems to be favored. For larger insertions such as an epitope tag, a dsDNA donor with protruding 3' homology arms of 30 bases is favored. In both cases, protecting the ends of the donor DNA with phosphorothioate modifications improves the editing efficiency.</p>',
'date' => '2016-11-11',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/27845164',
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'description' => '<p><span>CRISPR/Cas9 nuclease systems can create double-stranded DNA breaks at specific sequences to efficiently and precisely disrupt, excise, mutate, insert, or replace genes. However, human embryonic stem cells and induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, an optimized protocol is described for genome engineering of human iPSCs using simple transient transfection of plasmids and/or single-stranded oligonucleotides without any further selection or enrichment steps. This protocol achieves transfection efficiencies >60%, with gene disruption efficiencies of 1-25% and gene insertion/replacement efficiencies of 0.5-10%. Details are also provided for designing optimal sgRNA target sites and donor targeting vectors, cloning individual iPSCs by single-cell FACS sorting, and genotyping successfully edited cells.</span></p>',
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'description' => '<p>When doing CRISPR experiments, it is essential to verify the expression levels of the Cas9 to ensure high-quality results. Diagenode's new CRISPR/Cas9 4G10 antibody is <strong>extremely specific</strong>, able to <strong>recognize different Cas9 fusion proteins</strong> (even those used for CRISPRi experiments) and compatible with many biochemical assays, such as <strong>immunofluorescence, western blot</strong> and <strong>immunoprecipitation</strong>. Among the different antibodies in the market, Diagenode's 4G10 CRISPR/Cas9 is our antibody of choice.</p>',
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<span class="success label" style="">C05010012</span>
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<h6 style="height:60px">MicroPlex Library Preparation Kit v2 (12 indexes)</h6>
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<p><span><strong>Specifically optimized for ChIP-seq</strong></span><br /><br /><span>The MicroPlex Library Preparation™ kit is the only kit on the market which is validated for ChIP-seq and which allows the preparation of indexed libraries from just picogram inputs. In combination with the </span><a href="./true-microchip-kit-x16-16-rxns">True MicroChIP kit</a><span>, it allows for performing ChIP-seq on as few as 10,000 cells. Less input, fewer steps, fewer supplies, faster time to results! </span></p>
<p>The MicroPlex v2 kit (Cat. No. C05010012) contains all necessary reagents including single indexes for multiplexing up to 12 samples using single barcoding. For higher multiplexing (using dual indexes) check <a href="https://www.diagenode.com/en/p/microplex-lib-prep-kit-v3-48-rxns">MicroPlex Library Preparation Kits v3</a>.</p>',
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<li><strong>Input: </strong>50 pg – 50 ng</li>
<li><strong>Reduce potential bias</strong> - few PCR amplification cycles needed</li>
<li><strong>High sensitivity ChIP-seq</strong> - low PCR duplication rate</li>
<li><strong>Great multiplexing flexibility</strong> with 12 barcodes (8 nt) included</li>
<li><strong>Validated with the <a href="https://www.diagenode.com/p/sx-8g-ip-star-compact-automated-system-1-unit" title="IP-Star Automated System">IP-Star<sup>®</sup> Automated Platform</a></strong></li>
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<h3>How it works</h3>
<center><img src="https://www.diagenode.com/img/product/kits/microplex-method-overview-v2.png" /></center>
<p style="margin-bottom: 0;"><small><strong>Microplex workflow - protocol with single indexes</strong><br />An input of 50 pg to 50 ng of fragmented dsDNA is converted into sequencing-ready libraries for Illumina® NGS platforms using a fast and simple 3-step protocol</small></p>
<ul class="accordion" data-accordion="" id="readmore" style="margin-left: 0;">
<li class="accordion-navigation"><a href="#first" style="background: #ffffff; padding: 0rem; margin: 0rem; color: #13b2a2;"><small>Read more about MicroPlex workflow</small></a>
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<p><small><strong>Step 1. Template Preparation</strong> provides efficient repair of the fragmented double-stranded DNA input.</small></p>
<p><small>In this step, the DNA is repaired and yields molecules with blunt ends.</small></p>
<p><small><strong>Step 2. Library Synthesis.</strong> enables ligation of MicroPlex patented stem- loop adapters.</small></p>
<p><small>In the next step, stem-loop adaptors with blocked 5’ ends are ligated with high efficiency to the 5’ end of the genomic DNA, leaving a nick at the 3’ end. The adaptors cannot ligate to each other and do not have single- strand tails, both of which contribute to non-specific background found with many other NGS preparations.</small></p>
<p><small><strong>Step 3. Library Amplification</strong> enables extension of the template, cleavage of the stem-loop adaptors, and amplification of the library. Illumina- compatible indexes are also introduced using a high-fidelity, highly- processive, low-bias DNA polymerase.</small></p>
<p><small>In the final step, the 3’ ends of the genomic DNA are extended to complete library synthesis and Illumina-compatible indexes are added through a high-fidelity amplification. Any remaining free adaptors are destroyed. Hands-on time and the risk of contamination are minimized by using a single tube and eliminating intermediate purifications.</small></p>
<p><small>Obtained libraries are purified, quantified and sized. The libraries pooling can be performed as well before sequencing.</small></p>
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<h3>Reliable detection of enrichments in ChIP-seq</h3>
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<p><strong>Figure A.</strong> ChIP has been peformed with H3K4me3 antibody, amplification of 17 pg of DNA ChIP'd from 10.000 cells and amplification of 35 pg of DNA ChIP'd from 100.000 cells (control experiment). The IP'd DNA was amplified and transformed into a sequencing-ready preparation for the Illumina plateform with the MicroPlex Library Preparation kit. The library was then analysed on an Illumina<sup>®</sup> Genome Analyzer. Cluster generation and sequencing were performed according to the manufacturer's instructions.</p>
<p><img src="https://www.diagenode.com/img/product/kits/microplex-library-prep-kit-figure-b.png" alt="Reliable detection of enrichments in ChIP-seq figure 2" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure B.</strong> We observed a perfect match between the top 40% of True MicroChIP peaks and the reference dataset. Based on the NIH Encode project criterion, ChIP-seq results are considered reproducible between an original and reproduced dataset if the top 40% of peaks have at least an 80% overlap ratio with the compared dataset.</p>',
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'description' => '<p><span>CRISPR/Cas9 nuclease systems can create double-stranded DNA breaks at specific sequences to efficiently and precisely disrupt, excise, mutate, insert, or replace genes. However, human embryonic stem cells and induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, an optimized protocol is described for genome engineering of human iPSCs using simple transient transfection of plasmids and/or single-stranded oligonucleotides without any further selection or enrichment steps. This protocol achieves transfection efficiencies >60%, with gene disruption efficiencies of 1-25% and gene insertion/replacement efficiencies of 0.5-10%. Details are also provided for designing optimal sgRNA target sites and donor targeting vectors, cloning individual iPSCs by single-cell FACS sorting, and genotyping successfully edited cells.</span></p>',
'date' => '2015-11-04',
'pmid' => 'http://onlinelibrary.wiley.com/doi/10.1002/9780470151808.sc05a08s35/abstract',
'doi' => '10.1002/9780470151808.sc05a08s35',
'modified' => '2016-02-10 16:29:28',
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<p><span><strong>Specifically optimized for ChIP-seq</strong></span><br /><br /><span>The MicroPlex Library Preparation™ kit is the only kit on the market which is validated for ChIP-seq and which allows the preparation of indexed libraries from just picogram inputs. In combination with the </span><a href="./true-microchip-kit-x16-16-rxns">True MicroChIP kit</a><span>, it allows for performing ChIP-seq on as few as 10,000 cells. Less input, fewer steps, fewer supplies, faster time to results! </span></p>
<p>The MicroPlex v2 kit (Cat. No. C05010012) contains all necessary reagents including single indexes for multiplexing up to 12 samples using single barcoding. For higher multiplexing (using dual indexes) check <a href="https://www.diagenode.com/en/p/microplex-lib-prep-kit-v3-48-rxns">MicroPlex Library Preparation Kits v3</a>.</p>',
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<li><strong>High sensitivity ChIP-seq</strong> - low PCR duplication rate</li>
<li><strong>Great multiplexing flexibility</strong> with 12 barcodes (8 nt) included</li>
<li><strong>Validated with the <a href="https://www.diagenode.com/p/sx-8g-ip-star-compact-automated-system-1-unit" title="IP-Star Automated System">IP-Star<sup>®</sup> Automated Platform</a></strong></li>
</ul>
<h3>How it works</h3>
<center><img src="https://www.diagenode.com/img/product/kits/microplex-method-overview-v2.png" /></center>
<p style="margin-bottom: 0;"><small><strong>Microplex workflow - protocol with single indexes</strong><br />An input of 50 pg to 50 ng of fragmented dsDNA is converted into sequencing-ready libraries for Illumina® NGS platforms using a fast and simple 3-step protocol</small></p>
<ul class="accordion" data-accordion="" id="readmore" style="margin-left: 0;">
<li class="accordion-navigation"><a href="#first" style="background: #ffffff; padding: 0rem; margin: 0rem; color: #13b2a2;"><small>Read more about MicroPlex workflow</small></a>
<div id="first" class="content">
<p><small><strong>Step 1. Template Preparation</strong> provides efficient repair of the fragmented double-stranded DNA input.</small></p>
<p><small>In this step, the DNA is repaired and yields molecules with blunt ends.</small></p>
<p><small><strong>Step 2. Library Synthesis.</strong> enables ligation of MicroPlex patented stem- loop adapters.</small></p>
<p><small>In the next step, stem-loop adaptors with blocked 5’ ends are ligated with high efficiency to the 5’ end of the genomic DNA, leaving a nick at the 3’ end. The adaptors cannot ligate to each other and do not have single- strand tails, both of which contribute to non-specific background found with many other NGS preparations.</small></p>
<p><small><strong>Step 3. Library Amplification</strong> enables extension of the template, cleavage of the stem-loop adaptors, and amplification of the library. Illumina- compatible indexes are also introduced using a high-fidelity, highly- processive, low-bias DNA polymerase.</small></p>
<p><small>In the final step, the 3’ ends of the genomic DNA are extended to complete library synthesis and Illumina-compatible indexes are added through a high-fidelity amplification. Any remaining free adaptors are destroyed. Hands-on time and the risk of contamination are minimized by using a single tube and eliminating intermediate purifications.</small></p>
<p><small>Obtained libraries are purified, quantified and sized. The libraries pooling can be performed as well before sequencing.</small></p>
</div>
</li>
</ul>
<p></p>
<h3>Reliable detection of enrichments in ChIP-seq</h3>
<p><img src="https://www.diagenode.com/img/product/kits/microplex-library-prep-kit-figure-a.png" alt="Reliable detection of enrichments in ChIP-seq figure 1" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure A.</strong> ChIP has been peformed with H3K4me3 antibody, amplification of 17 pg of DNA ChIP'd from 10.000 cells and amplification of 35 pg of DNA ChIP'd from 100.000 cells (control experiment). The IP'd DNA was amplified and transformed into a sequencing-ready preparation for the Illumina plateform with the MicroPlex Library Preparation kit. The library was then analysed on an Illumina<sup>®</sup> Genome Analyzer. Cluster generation and sequencing were performed according to the manufacturer's instructions.</p>
<p><img src="https://www.diagenode.com/img/product/kits/microplex-library-prep-kit-figure-b.png" alt="Reliable detection of enrichments in ChIP-seq figure 2" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure B.</strong> We observed a perfect match between the top 40% of True MicroChIP peaks and the reference dataset. Based on the NIH Encode project criterion, ChIP-seq results are considered reproducible between an original and reproduced dataset if the top 40% of peaks have at least an 80% overlap ratio with the compared dataset.</p>',
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<p>Learn more about: <a href="https://www.diagenode.com/applications/western-blot">Loading control, MW marker visualization</a><em>. <br /></em></p>
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<p><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>
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'description' => '<p>Diagenode offers the broad range of antibodies raised against the N- or C-terminus of the Cas9 nuclease from <em>Streptococcus <g class="gr_ gr_5 gr-alert gr_spell gr_disable_anim_appear ContextualSpelling ins-del multiReplace" id="5" data-gr-id="5">pyogenes</g></em>. These highly specific polyclonal and monoclonal antibodies are validated in Western blot, immunoprecipitation, immunofluorescence and in chromatin immunoprecipitation.</p>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h2><em><a name="pyogenes"></a>S. pyogenes</em> CRISPR/Cas9 antibodies<a></a></h2>
<div class="panel">
<h2>Discover our first monoclonal CRISPR/Cas9 antibody validated in ChIP<br /><br /></h2>
<div class="row">
<div class="small-5 medium-5 large-5 columns"><img src="/img/landing-pages/crispr-cas9-chip-on-hih3t3.jpg" alt="" /></div>
<div class="small-7 medium-7 large-7 columns">
<ul>
<li>Validated in chromatin immunoprecipitation</li>
<li>Performs better than FLAG antibody</li>
<li>Excellent for WB, IF and IP</li>
</ul>
<p><small><strong>ChIP</strong> was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50 µg chromatin was incubated overnight at 4°C with 5 or 10 µg of either an anti-FLAG antibody or the Diagenode antibody against Cas9 (Cat. No. C15200229). Mouse IgG was used as a negative IP control. qPCR was performed with primers specific for the GFP gene, and for a non-targeted region (protein kinase C delta, Prkcd), used as negative control. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).</small></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns text-right"><a href="/p/crispr-cas9-monoclonal-antibody-50-ug-25-μl" class="tiny details button radius">Learn more</a></div>
</div>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>First ChIP-grade CRISPR/Cas9 polyclonal antibody</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/landing-pages/c_a_s9-chip-grade-antibody.png" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Excellent polyclonal antibody for chromatin immunoprecipitation</li>
<li>Optimized for highest ChIP specificity and yields</li>
<li>Validated for all applications including immunoblotting, immunofluorescence and western blot</li>
</ul>
<p><small><strong>ChIP</strong> was performed on NIH3T3 cells stably expressing GFP- H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50 μg chromatin was incubated with either 5 μg of an anti-FLAG antibody or 2 μl of the Diagenode antibody against Cas9. The pre-immune serum (PPI) was used as negative IP control. Then qPCR was performed with primers specific for the GFP gene, and for two non-targeted regions: Ppap2c and Prkcd, used as negative controls. This figure shows the recovery, expressed as a % of input.</small></p>
<p class="text-right"><a href="../p/crispr-cas9-polyclonal-antibody" class="details tiny button">Learn more</a></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>CRISPR/Cas9 monoclonal antibody 4G10</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/landing-pages/cas9_4g10_fig1.png" width="170" height="302" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Antibody raised against N-terminus of Cas9 nuclease</li>
<li>Validated for western blot, IP and immunofluorescence</li>
</ul>
<p><small><strong>Immunofluorescence</strong>: Hela cells were transiently transfected with a Cas9 expression vector. The cells were fixed in 3.7% formaldehyde, permeabilized in 0.5% Triton-X-100 and blocked in PBS containing 2% BSA. The cells were stained with the Cas9 antibody at 4°C o/n, followed by incubation with an anti mouse secondary antibody coupled to AF488 for 1 h at RT. Nuclei were counter-stained with Hoechst 33342 (right).</small></p>
<p class="text-right"><a href="../p/crispr-cas9-monoclonal-antibody-4g10-50-ug" class="details tiny button">Learn more</a></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>CRISPR/Cas9 C-terminal monoclonal antibody</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/product/antibodies/C15200223-IP.png" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Antibody raised against C-terminus of Cas9 nuclease</li>
<li>Validated for western blot, IP and immunofluorescence</li>
</ul>
<p><small><strong> Western blot</strong> was performed on 20 μg protein extracts from Cas9 expressing HeLa cells (lane 1) and on negative control HeLa cells (lane 2) with the Diagenode antibody against Cas9. The antibody was diluted 1:4,000. The marker is shown on the left, position of the Cas9 protein is indicated on the right. </small></p>
<p class="text-right"><a href="../p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug" class="details tiny button">Learn more</a></p>
</div>
<div class="small-12 medium-12 large-12 columns">
<h3>Which CRISPR/Cas9 antibody is the best for your application?</h3>
<a name="table"></a>
<table>
<thead>
<tr>
<th>Antibody</th>
<th>WB</th>
<th>IF</th>
<th>IP</th>
<th>ChIP</th>
<th>Antibody raised against</th>
</tr>
</thead>
<tbody>
<tr>
<td><a href="../p/crispr-cas9-monoclonal-antibody-50-ug-25-μl"><strong class="diacol">CRISPR/Cas9 monoclonal antibody</strong></a></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><span class="diacol">N-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-polyclonal-antibody">CRISPR/Cas9 polyclonal antibody</a></td>
<td>++</td>
<td>++</td>
<td>++</td>
<td><strong class="diacol">+++</strong></td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-monoclonal-antibody-4g10-50-ug">CRISPR/Cas9 monoclonal antibody 4G10</a></td>
<td>+++</td>
<td>+++</td>
<td>++</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="../p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug-23-ul"><strong class="diacol">CRISPR/Cas9 C-terminal monoclonal antibody</strong></a> <span class="label alert" style="font-size: 0.9rem;">NEW!</span></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">no</strong></td>
<td><strong class="diacol">+</strong></td>
<td><span class="diacol">C-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug">CRISPR/Cas9 C-terminal monoclonal antibody</a></td>
<td>++</td>
<td>++</td>
<td>+</td>
<td>no</td>
<td><span class="diacol">C-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-monoclonal-antibody-7A9-50-mg">CRISPR/Cas9 monoclonal antibody 7A9</a></td>
<td>++</td>
<td>++</td>
<td>++</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-hrp-monoclonal-antibody-50-ul">CRISPR/Cas9 - HRP monoclonal antibody 7A9</a></td>
<td>+++</td>
<td>no</td>
<td>no</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
</tbody>
</table>
</div>
</div>
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'name' => 'Monitoring autochthonous lung tumors induced by somatic CRISPR geneediting in mice using a secreted luciferase.',
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'description' => '<p>BACKGROUND: In vivo gene editing of somatic cells with CRISPR nucleases has facilitated the generation of autochthonous mouse tumors, which are initiated by genetic alterations relevant to the human disease and progress along a natural timeline as in patients. However, the long and variable, orthotopic tumor growth in inner organs requires sophisticated, time-consuming and resource-intensive imaging for longitudinal disease monitoring and impedes the use of autochthonous tumor models for preclinical studies. METHODS: To facilitate a more widespread use, we have generated a reporter mouse that expresses a Cre-inducible luciferase from Gaussia princeps (GLuc), which is secreted by cells in an energy-consuming process and can be measured quantitatively in the blood as a marker for the viable tumor load. In addition, we have developed a flexible, complementary toolkit to rapidly assemble recombinant adenoviruses (AVs) for delivering Cre recombinase together with CRISPR nucleases targeting cancer driver genes. RESULTS: We demonstrate that intratracheal infection of GLuc reporter mice with CRISPR-AVs efficiently induces lung tumors driven by mutations in the targeted cancer genes and simultaneously activates the GLuc transgene, resulting in GLuc secretion into the blood by the growing tumor. GLuc blood levels are easily and robustly quantified in small-volume blood samples with inexpensive equipment, enable tumor detection already several months before the humane study endpoint and precisely mirror the kinetics of tumor development specified by the inducing gene combination. CONCLUSIONS: Our study establishes blood-based GLuc monitoring as an inexpensive, rapid, high-throughput and animal-friendly method to longitudinally monitor autochthonous tumor growth in preclinical studies.</p>',
'date' => '2022-10-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/36192757',
'doi' => '10.1186/s12943-022-01661-2',
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'name' => 'Systematic comparison of CRISPR-based transcriptional activatorsuncovers gene-regulatory features of enhancer-promoter interactions.',
'authors' => 'Wang K. et al.',
'description' => '<p>Nuclease-inactivated CRISPR/Cas-based (dCas-based) systems have emerged as powerful technologies to synthetically reshape the human epigenome and gene expression. Despite the increasing adoption of these platforms, their relative potencies and mechanistic differences are incompletely characterized, particularly at human enhancer-promoter pairs. Here, we systematically compared the most widely adopted dCas9-based transcriptional activators, as well as an activator consisting of dCas9 fused to the catalytic core of the human CBP protein, at human enhancer-promoter pairs. We find that these platforms display variable relative expression levels in different human cell types and that their transactivation efficacies vary based upon the effector domain, effector recruitment architecture, targeted locus and cell type. We also show that each dCas9-based activator can induce the production of enhancer RNAs (eRNAs) and that this eRNA induction is positively correlated with downstream mRNA expression from a cognate promoter. Additionally, we use dCas9-based activators to demonstrate that an intrinsic transcriptional and epigenetic reciprocity can exist between human enhancers and promoters and that enhancer-mediated tracking and engagement of a downstream promoter can be synthetically driven by targeting dCas9-based transcriptional activators to an enhancer. Collectively, our study provides new insights into the enhancer-mediated control of human gene expression and the use of dCas9-based activators.</p>',
'date' => '2022-07-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/35849129',
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'name' => 'DNA Methylation Editing by CRISPR-guided Excision of 5-Methylcytosine.',
'authors' => 'Devesa-Guerra I, Morales-Ruiz T, Pérez-Roldán J, Parrilla-Doblas JT, Dorado-León M, García-Ortiz MV, Ariza RR, Roldán-Arjona T',
'description' => '<p>Tools for actively targeted DNA demethylation are required to increase our knowledge about regulation and specific functions of this important epigenetic modification. DNA demethylation in mammals involves TET-mediated oxidation of 5-methylcytosine (5-meC), which may promote its replication-dependent dilution and/or active removal through base excision repair (BER). However, it is still unclear whether oxidized derivatives of 5-meC are simply DNA demethylation intermediates or rather epigenetic marks on their own. Unlike animals, plants have evolved enzymes that directly excise 5-meC without previous modification. In this work, we have fused the catalytic domain of Arabidopsis ROS1 5-meC DNA glycosylase to a CRISPR-associated null-nuclease (dCas9) and analyzed its capacity for targeted reactivation of methylation-silenced genes, in comparison to other dCas9-effectors. We found that dCas9-ROS1, but not dCas9-TET1, is able to reactivate methylation-silenced genes and induce partial demethylation in a replication-independent manner. We also found that reactivation induced by dCas9-ROS1, as well as that achieved by two different CRISPR-based chromatin effectors (dCas9-VP160 and dCas9-p300), generally decreases with methylation density. Our results suggest that plant 5-meC DNA glycosylases are a valuable addition to the CRISPR-based toolbox for epigenetic editing.</p>',
'date' => '2020-02-19',
'pmid' => 'http://www.pubmed.gov/32087201',
'doi' => '10.1016/j.jmb.2020.02.007',
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'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',
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'name' => 'Optimization of CRISPR/Cas9 Delivery to Human Hematopoietic Stem and Progenitor Cells for Therapeutic Genomic Rearrangements.',
'authors' => 'Lattanzi A, Meneghini V, Pavani G, Amor F, Ramadier S, Felix T, Antoniani C, Masson C, Alibeu O, Lee C, Porteus MH, Bao G, Amendola M, Mavilio F, Miccio A',
'description' => '<p>Editing the β-globin locus in hematopoietic stem cells is an alternative therapeutic approach for gene therapy of β-thalassemia and sickle cell disease. Using the CRISPR/Cas9 system, we genetically modified human hematopoietic stem and progenitor cells (HSPCs) to mimic the large rearrangements in the β-globin locus associated with hereditary persistence of fetal hemoglobin (HPFH), a condition that mitigates the clinical phenotype of patients with β-hemoglobinopathies. We optimized and compared the efficiency of plasmid-, lentiviral vector (LV)-, RNA-, and ribonucleoprotein complex (RNP)-based methods to deliver the CRISPR/Cas9 system into HSPCs. Plasmid delivery of Cas9 and gRNA pairs targeting two HPFH-like regions led to high frequency of genomic rearrangements and HbF reactivation in erythroblasts derived from sorted, Cas9 HSPCs but was associated with significant cell toxicity. RNA-mediated delivery of CRISPR/Cas9 was similarly toxic but much less efficient in editing the β-globin locus. Transduction of HSPCs by LVs expressing Cas9 and gRNA pairs was robust and minimally toxic but resulted in poor genome-editing efficiency. Ribonucleoprotein (RNP)-based delivery of CRISPR/Cas9 exhibited a good balance between cytotoxicity and efficiency of genomic rearrangements as compared to the other delivery systems and resulted in HbF upregulation in erythroblasts derived from unselected edited HSPCs.</p>',
'date' => '2019-01-02',
'pmid' => 'http://www.pubmed.gov/30424953',
'doi' => '10.1016/j.ymthe.2018.10.008',
'modified' => '2019-06-07 10:25:31',
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'name' => 'Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo',
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'description' => '<p>The advent of CRISPR/Cas9 has made genome editing possible in virtually any organism, including those not previously amenable to genetic manipulations. Here, we present an optimization of CRISPR/Cas9 for application to early avian embryos with improved efficiency via a three-fold strategy. First, we employed Cas9 protein flanked with two nuclear localization signal sequences for improved nuclear localization. Second, we used a modified guide RNA (gRNA) scaffold that obviates premature termination of transcription and unstable Cas9-gRNA interactions. Third, we used a chick-specific U6 promoter that yields 4-fold higher gRNA expression than the previously utilized human U6. For rapid screening of gRNAs for in vivo applications, we also generated a chicken fibroblast cell line that constitutively expresses Cas9. As proof of principle, we performed electroporation-based loss-of-function studies in the early chick embryo to knock out Pax7 and Sox10, key transcription factors with known functions in neural crest development. The results show that CRISPR/Cas9-mediated deletion causes loss of their respective proteins and transcripts, as well as predicted downstream targets. Taken together, the results reveal the utility of this optimized CRISPR/Cas9 method for targeted gene knockout in chicken embryos in a manner that is reproducible, robust and specific.</p>',
'date' => '2017-12-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/29150011',
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'name' => 'Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA',
'authors' => 'Liang W. et al.',
'description' => '<p>While CRISPR-based gene knock out in mammalian cells has proven to be very efficient, precise insertion of genetic elements via the cellular homology directed repair (HDR) pathway remains a rate-limiting step to seamless genome editing. Under the conditions described here, we achieved up to 56% targeted integration efficiency with up to a six-nucleotide insertion in HEK293 cells. In induced pluripotent stem cells (iPSCs), we achieved precise genome editing rates of up to 45% by co-delivering the Cas9 RNP and donor DNA. In addition, the use of a short double stranded DNA oligonucleotide with 3' overhangs allowed integration of a longer FLAG epitope tag along with a restriction site at rates of up to 50%. We propose a model that favors the design of donor DNAs with the change as close to the cleavage site as possible. For small changes such as SNPs or short insertions, asymmetric single stranded donor molecules with 30 base homology arms 3' to the insertion/repair cassette and greater than 40 bases of homology on the 5' end seems to be favored. For larger insertions such as an epitope tag, a dsDNA donor with protruding 3' homology arms of 30 bases is favored. In both cases, protecting the ends of the donor DNA with phosphorothioate modifications improves the editing efficiency.</p>',
'date' => '2016-11-11',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/27845164',
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'name' => 'CRISPR-Mediated Gene Targeting of Human Induced Pluripotent Stem Cells',
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'description' => '<p><span>CRISPR/Cas9 nuclease systems can create double-stranded DNA breaks at specific sequences to efficiently and precisely disrupt, excise, mutate, insert, or replace genes. However, human embryonic stem cells and induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, an optimized protocol is described for genome engineering of human iPSCs using simple transient transfection of plasmids and/or single-stranded oligonucleotides without any further selection or enrichment steps. This protocol achieves transfection efficiencies >60%, with gene disruption efficiencies of 1-25% and gene insertion/replacement efficiencies of 0.5-10%. Details are also provided for designing optimal sgRNA target sites and donor targeting vectors, cloning individual iPSCs by single-cell FACS sorting, and genotyping successfully edited cells.</span></p>',
'date' => '2015-11-04',
'pmid' => 'http://onlinelibrary.wiley.com/doi/10.1002/9780470151808.sc05a08s35/abstract',
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<h6 style="height:60px">MicroPlex Library Preparation Kit v2 (12 indexes)</h6>
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<p><span><strong>Specifically optimized for ChIP-seq</strong></span><br /><br /><span>The MicroPlex Library Preparation™ kit is the only kit on the market which is validated for ChIP-seq and which allows the preparation of indexed libraries from just picogram inputs. In combination with the </span><a href="./true-microchip-kit-x16-16-rxns">True MicroChIP kit</a><span>, it allows for performing ChIP-seq on as few as 10,000 cells. Less input, fewer steps, fewer supplies, faster time to results! </span></p>
<p>The MicroPlex v2 kit (Cat. No. C05010012) contains all necessary reagents including single indexes for multiplexing up to 12 samples using single barcoding. For higher multiplexing (using dual indexes) check <a href="https://www.diagenode.com/en/p/microplex-lib-prep-kit-v3-48-rxns">MicroPlex Library Preparation Kits v3</a>.</p>',
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<li><strong>Great multiplexing flexibility</strong> with 12 barcodes (8 nt) included</li>
<li><strong>Validated with the <a href="https://www.diagenode.com/p/sx-8g-ip-star-compact-automated-system-1-unit" title="IP-Star Automated System">IP-Star<sup>®</sup> Automated Platform</a></strong></li>
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<h3>How it works</h3>
<center><img src="https://www.diagenode.com/img/product/kits/microplex-method-overview-v2.png" /></center>
<p style="margin-bottom: 0;"><small><strong>Microplex workflow - protocol with single indexes</strong><br />An input of 50 pg to 50 ng of fragmented dsDNA is converted into sequencing-ready libraries for Illumina® NGS platforms using a fast and simple 3-step protocol</small></p>
<ul class="accordion" data-accordion="" id="readmore" style="margin-left: 0;">
<li class="accordion-navigation"><a href="#first" style="background: #ffffff; padding: 0rem; margin: 0rem; color: #13b2a2;"><small>Read more about MicroPlex workflow</small></a>
<div id="first" class="content">
<p><small><strong>Step 1. Template Preparation</strong> provides efficient repair of the fragmented double-stranded DNA input.</small></p>
<p><small>In this step, the DNA is repaired and yields molecules with blunt ends.</small></p>
<p><small><strong>Step 2. Library Synthesis.</strong> enables ligation of MicroPlex patented stem- loop adapters.</small></p>
<p><small>In the next step, stem-loop adaptors with blocked 5’ ends are ligated with high efficiency to the 5’ end of the genomic DNA, leaving a nick at the 3’ end. The adaptors cannot ligate to each other and do not have single- strand tails, both of which contribute to non-specific background found with many other NGS preparations.</small></p>
<p><small><strong>Step 3. Library Amplification</strong> enables extension of the template, cleavage of the stem-loop adaptors, and amplification of the library. Illumina- compatible indexes are also introduced using a high-fidelity, highly- processive, low-bias DNA polymerase.</small></p>
<p><small>In the final step, the 3’ ends of the genomic DNA are extended to complete library synthesis and Illumina-compatible indexes are added through a high-fidelity amplification. Any remaining free adaptors are destroyed. Hands-on time and the risk of contamination are minimized by using a single tube and eliminating intermediate purifications.</small></p>
<p><small>Obtained libraries are purified, quantified and sized. The libraries pooling can be performed as well before sequencing.</small></p>
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<h3>Reliable detection of enrichments in ChIP-seq</h3>
<p><img src="https://www.diagenode.com/img/product/kits/microplex-library-prep-kit-figure-a.png" alt="Reliable detection of enrichments in ChIP-seq figure 1" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure A.</strong> ChIP has been peformed with H3K4me3 antibody, amplification of 17 pg of DNA ChIP'd from 10.000 cells and amplification of 35 pg of DNA ChIP'd from 100.000 cells (control experiment). The IP'd DNA was amplified and transformed into a sequencing-ready preparation for the Illumina plateform with the MicroPlex Library Preparation kit. The library was then analysed on an Illumina<sup>®</sup> Genome Analyzer. Cluster generation and sequencing were performed according to the manufacturer's instructions.</p>
<p><img src="https://www.diagenode.com/img/product/kits/microplex-library-prep-kit-figure-b.png" alt="Reliable detection of enrichments in ChIP-seq figure 2" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure B.</strong> We observed a perfect match between the top 40% of True MicroChIP peaks and the reference dataset. Based on the NIH Encode project criterion, ChIP-seq results are considered reproducible between an original and reproduced dataset if the top 40% of peaks have at least an 80% overlap ratio with the compared dataset.</p>',
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<p><small><strong>Figure 1. Western blot analysis using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />Western blot was performed on protein extracts from HEK293T cells transfected with Cas9 (lane 1) or from untransfected cells (lane 2) using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15200216), diluted 1:5,000 in PBS-T containing 0.5% NFDM. The marker is shown on the left, position of the Cas9 protein is indicated on the right.</small></p>
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<p><small><strong>Figure 2. IP using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />IP was performed on whole cell extracts from HEK293T cells transfected with a Cas9 expression vector (lane 1, 3 and 5), or untransfected cells (lane 2 and 4) using 5 µg of the Diagenode antibody against CRISPR/Cas9 (cat. No. C15200216, lane 3 and 4) or with an equal amount of IgG, used as a negative control (lane 5). The immunoprecipitated proteins were subsequently analysed by Western blot with the polyclonal Cas9 antibody (Cat. No. C15310258, diluted 1:5,000). Lane 1 and 2 show the result of the input.</small></p>
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<p><small><strong>Figure 3. Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />HEK293T cells were transiently transfected with a Cas9 expression vector. The cells were fixed with 4% formaldehyde, permeabilized in 0,1% Triton X-100 and blocked in PBS containing 5% BSA. The cells were stained with the Cas9 antibody diluted 1;400 at 4°C o/n, followed by incubation with an anti mouse secondary antibody coupled to AF596 for 1 h at RT (left). Nuclei were counter-stained with DAPI (middle). A merge of the two stainings is shown on the right.</small></p>
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<p><small><strong>Figure 1. Western blot analysis using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />Western blot was performed on protein extracts from HEK293T cells transfected with Cas9 (lane 1) or from untransfected cells (lane 2) using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15200216), diluted 1:5,000 in PBS-T containing 0.5% NFDM. The marker is shown on the left, position of the Cas9 protein is indicated on the right.</small></p>
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<p><small><strong>Figure 2. IP using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />IP was performed on whole cell extracts from HEK293T cells transfected with a Cas9 expression vector (lane 1, 3 and 5), or untransfected cells (lane 2 and 4) using 5 µg of the Diagenode antibody against CRISPR/Cas9 (cat. No. C15200216, lane 3 and 4) or with an equal amount of IgG, used as a negative control (lane 5). The immunoprecipitated proteins were subsequently analysed by Western blot with the polyclonal Cas9 antibody (Cat. No. C15310258, diluted 1:5,000). Lane 1 and 2 show the result of the input.</small></p>
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<p><small><strong>Figure 3. Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />HEK293T cells were transiently transfected with a Cas9 expression vector. The cells were fixed with 4% formaldehyde, permeabilized in 0,1% Triton X-100 and blocked in PBS containing 5% BSA. The cells were stained with the Cas9 antibody diluted 1;400 at 4°C o/n, followed by incubation with an anti mouse secondary antibody coupled to AF596 for 1 h at RT (left). Nuclei were counter-stained with DAPI (middle). A merge of the two stainings is shown on the right.</small></p>
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<p><small><strong>Figure 1. Western blot analysis using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />Western blot was performed on protein extracts from HEK293T cells transfected with Cas9 (lane 1) or from untransfected cells (lane 2) using the Diagenode antibody against CRISPR/Cas9 (cat. No. C15200216), diluted 1:5,000 in PBS-T containing 0.5% NFDM. The marker is shown on the left, position of the Cas9 protein is indicated on the right.</small></p>
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<p><small><strong>Figure 2. IP using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />IP was performed on whole cell extracts from HEK293T cells transfected with a Cas9 expression vector (lane 1, 3 and 5), or untransfected cells (lane 2 and 4) using 5 µg of the Diagenode antibody against CRISPR/Cas9 (cat. No. C15200216, lane 3 and 4) or with an equal amount of IgG, used as a negative control (lane 5). The immunoprecipitated proteins were subsequently analysed by Western blot with the polyclonal Cas9 antibody (Cat. No. C15310258, diluted 1:5,000). Lane 1 and 2 show the result of the input.</small></p>
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<p><small><strong>Figure 3. Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong><br />HEK293T cells were transiently transfected with a Cas9 expression vector. The cells were fixed with 4% formaldehyde, permeabilized in 0,1% Triton X-100 and blocked in PBS containing 5% BSA. The cells were stained with the Cas9 antibody diluted 1;400 at 4°C o/n, followed by incubation with an anti mouse secondary antibody coupled to AF596 for 1 h at RT (left). Nuclei were counter-stained with DAPI (middle). A merge of the two stainings is shown on the right.</small></p>
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<p><span><strong>Specifically optimized for ChIP-seq</strong></span><br /><br /><span>The MicroPlex Library Preparation™ kit is the only kit on the market which is validated for ChIP-seq and which allows the preparation of indexed libraries from just picogram inputs. In combination with the </span><a href="./true-microchip-kit-x16-16-rxns">True MicroChIP kit</a><span>, it allows for performing ChIP-seq on as few as 10,000 cells. Less input, fewer steps, fewer supplies, faster time to results! </span></p>
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<div id="first" class="content">
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<p><small><strong>Step 3. Library Amplification</strong> enables extension of the template, cleavage of the stem-loop adaptors, and amplification of the library. Illumina- compatible indexes are also introduced using a high-fidelity, highly- processive, low-bias DNA polymerase.</small></p>
<p><small>In the final step, the 3’ ends of the genomic DNA are extended to complete library synthesis and Illumina-compatible indexes are added through a high-fidelity amplification. Any remaining free adaptors are destroyed. Hands-on time and the risk of contamination are minimized by using a single tube and eliminating intermediate purifications.</small></p>
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<h3>Reliable detection of enrichments in ChIP-seq</h3>
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<p><strong>Figure A.</strong> ChIP has been peformed with H3K4me3 antibody, amplification of 17 pg of DNA ChIP'd from 10.000 cells and amplification of 35 pg of DNA ChIP'd from 100.000 cells (control experiment). The IP'd DNA was amplified and transformed into a sequencing-ready preparation for the Illumina plateform with the MicroPlex Library Preparation kit. The library was then analysed on an Illumina<sup>®</sup> Genome Analyzer. Cluster generation and sequencing were performed according to the manufacturer's instructions.</p>
<p><img src="https://www.diagenode.com/img/product/kits/microplex-library-prep-kit-figure-b.png" alt="Reliable detection of enrichments in ChIP-seq figure 2" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure B.</strong> We observed a perfect match between the top 40% of True MicroChIP peaks and the reference dataset. Based on the NIH Encode project criterion, ChIP-seq results are considered reproducible between an original and reproduced dataset if the top 40% of peaks have at least an 80% overlap ratio with the compared dataset.</p>',
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<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>
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<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>
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<ul>
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<li>Performs better than FLAG antibody</li>
<li>Excellent for WB, IF and IP</li>
</ul>
<p><small><strong>ChIP</strong> was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50 µg chromatin was incubated overnight at 4°C with 5 or 10 µg of either an anti-FLAG antibody or the Diagenode antibody against Cas9 (Cat. No. C15200229). Mouse IgG was used as a negative IP control. qPCR was performed with primers specific for the GFP gene, and for a non-targeted region (protein kinase C delta, Prkcd), used as negative control. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).</small></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns text-right"><a href="/p/crispr-cas9-monoclonal-antibody-50-ug-25-μl" class="tiny details button radius">Learn more</a></div>
</div>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>First ChIP-grade CRISPR/Cas9 polyclonal antibody</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/landing-pages/c_a_s9-chip-grade-antibody.png" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Excellent polyclonal antibody for chromatin immunoprecipitation</li>
<li>Optimized for highest ChIP specificity and yields</li>
<li>Validated for all applications including immunoblotting, immunofluorescence and western blot</li>
</ul>
<p><small><strong>ChIP</strong> was performed on NIH3T3 cells stably expressing GFP- H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50 μg chromatin was incubated with either 5 μg of an anti-FLAG antibody or 2 μl of the Diagenode antibody against Cas9. The pre-immune serum (PPI) was used as negative IP control. Then qPCR was performed with primers specific for the GFP gene, and for two non-targeted regions: Ppap2c and Prkcd, used as negative controls. This figure shows the recovery, expressed as a % of input.</small></p>
<p class="text-right"><a href="../p/crispr-cas9-polyclonal-antibody" class="details tiny button">Learn more</a></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>CRISPR/Cas9 monoclonal antibody 4G10</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/landing-pages/cas9_4g10_fig1.png" width="170" height="302" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Antibody raised against N-terminus of Cas9 nuclease</li>
<li>Validated for western blot, IP and immunofluorescence</li>
</ul>
<p><small><strong>Immunofluorescence</strong>: Hela cells were transiently transfected with a Cas9 expression vector. The cells were fixed in 3.7% formaldehyde, permeabilized in 0.5% Triton-X-100 and blocked in PBS containing 2% BSA. The cells were stained with the Cas9 antibody at 4°C o/n, followed by incubation with an anti mouse secondary antibody coupled to AF488 for 1 h at RT. Nuclei were counter-stained with Hoechst 33342 (right).</small></p>
<p class="text-right"><a href="../p/crispr-cas9-monoclonal-antibody-4g10-50-ug" class="details tiny button">Learn more</a></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>CRISPR/Cas9 C-terminal monoclonal antibody</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/product/antibodies/C15200223-IP.png" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Antibody raised against C-terminus of Cas9 nuclease</li>
<li>Validated for western blot, IP and immunofluorescence</li>
</ul>
<p><small><strong> Western blot</strong> was performed on 20 μg protein extracts from Cas9 expressing HeLa cells (lane 1) and on negative control HeLa cells (lane 2) with the Diagenode antibody against Cas9. The antibody was diluted 1:4,000. The marker is shown on the left, position of the Cas9 protein is indicated on the right. </small></p>
<p class="text-right"><a href="../p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug" class="details tiny button">Learn more</a></p>
</div>
<div class="small-12 medium-12 large-12 columns">
<h3>Which CRISPR/Cas9 antibody is the best for your application?</h3>
<a name="table"></a>
<table>
<thead>
<tr>
<th>Antibody</th>
<th>WB</th>
<th>IF</th>
<th>IP</th>
<th>ChIP</th>
<th>Antibody raised against</th>
</tr>
</thead>
<tbody>
<tr>
<td><a href="../p/crispr-cas9-monoclonal-antibody-50-ug-25-μl"><strong class="diacol">CRISPR/Cas9 monoclonal antibody</strong></a></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><span class="diacol">N-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-polyclonal-antibody">CRISPR/Cas9 polyclonal antibody</a></td>
<td>++</td>
<td>++</td>
<td>++</td>
<td><strong class="diacol">+++</strong></td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-monoclonal-antibody-4g10-50-ug">CRISPR/Cas9 monoclonal antibody 4G10</a></td>
<td>+++</td>
<td>+++</td>
<td>++</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="../p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug-23-ul"><strong class="diacol">CRISPR/Cas9 C-terminal monoclonal antibody</strong></a> <span class="label alert" style="font-size: 0.9rem;">NEW!</span></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">no</strong></td>
<td><strong class="diacol">+</strong></td>
<td><span class="diacol">C-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug">CRISPR/Cas9 C-terminal monoclonal antibody</a></td>
<td>++</td>
<td>++</td>
<td>+</td>
<td>no</td>
<td><span class="diacol">C-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-monoclonal-antibody-7A9-50-mg">CRISPR/Cas9 monoclonal antibody 7A9</a></td>
<td>++</td>
<td>++</td>
<td>++</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-hrp-monoclonal-antibody-50-ul">CRISPR/Cas9 - HRP monoclonal antibody 7A9</a></td>
<td>+++</td>
<td>no</td>
<td>no</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
</tbody>
</table>
</div>
</div>
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'description' => '<p style="text-align: justify;"><span>Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of large sets of genome-wide sequencing data. ChIP sequencing (ChIP-seq) has now become the gold standard method for chromatin studies, given the accuracy and coverage scale of the approach over other methods. Successful ChIP-seq, however, requires a higher level of experimental accuracy and consistency in all steps of ChIP than ever before. Particularly crucial is the quality of ChIP antibodies. </span></p>',
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'description' => '<p>More than in any other immuoprecipitation assays, quality antibodies are critical tools in many epigenetics experiments. Since 10 years, Diagenode has developed the most stringent quality production available on the market for antibodies exclusively focused on epigenetic uses. All our antibodies have been qualified to work in epigenetic applications.</p>',
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'name' => 'Datasheet CRISPR Cas9 4G10 monoclonal antibody',
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'image_id' => null,
'type' => 'Datasheet',
'url' => 'files/products/antibodies/Datasheet_CRISPR_Cas9_4G10_monoclonal_antibody.pdf',
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'id' => '991',
'name' => ' Accurate QC to optimize CRISPR/Cas9 genome editing specificity',
'description' => '<p>The CRISPR/Cas9 technology is delivering superior genetic models for fundamental disease research, drug screening, therapy development, rapid diagnostics, and transcriptional modulation. Although CRISPR/Cas9 enables rapid genome editing, several aspects affect its efficiency and specificity including guide RNA design, delivery methods, and off-targets effects. Diagenode has developed strategies to overcome these common pitfalls and has optimized CRISPR/Cas9 genome editing specificity</p>',
'image_id' => null,
'type' => 'Poster',
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'id' => '4937',
'name' => 'Cis-regulatory interfaces reveal the molecular mechanisms underlying the notochord gene regulatory network of Ciona',
'authors' => 'Negrón-Piñeiro L. J. et al.',
'description' => '<p><span>Tissue-specific gene expression is fundamental in development and evolution, and is mediated by transcription factors and by the </span><i>cis</i><span>-regulatory regions (enhancers) that they control. Transcription factors and their respective tissue-specific enhancers are essential components of gene regulatory networks responsible for the development of tissues and organs. Although numerous transcription factors have been characterized from different organisms, the knowledge of the enhancers responsible for their tissue-specific expression remains fragmentary. Here we use<span> </span></span><i>Ciona</i><span><span> </span>to study the enhancers associated with ten transcription factors expressed in the notochord, an evolutionary hallmark of the chordate phylum. Our results illustrate how two evolutionarily conserved transcription factors, Brachyury and Foxa2, coordinate the deployment of other notochord transcription factors. The results of these detailed<span> </span></span><i>cis</i><span>-regulatory analyses delineate a high-resolution view of the essential notochord gene regulatory network of<span> </span></span><i>Ciona</i><span>, and provide a reference for studies of transcription factors, enhancers, and their roles in development, disease, and evolution.</span></p>',
'date' => '2024-04-08',
'pmid' => 'https://www.nature.com/articles/s41467-024-46850-3',
'doi' => 'https://doi.org/10.1038/s41467-024-46850-3',
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'id' => '4497',
'name' => 'Monitoring autochthonous lung tumors induced by somatic CRISPR geneediting in mice using a secreted luciferase.',
'authors' => 'Merle N. et al.',
'description' => '<p>BACKGROUND: In vivo gene editing of somatic cells with CRISPR nucleases has facilitated the generation of autochthonous mouse tumors, which are initiated by genetic alterations relevant to the human disease and progress along a natural timeline as in patients. However, the long and variable, orthotopic tumor growth in inner organs requires sophisticated, time-consuming and resource-intensive imaging for longitudinal disease monitoring and impedes the use of autochthonous tumor models for preclinical studies. METHODS: To facilitate a more widespread use, we have generated a reporter mouse that expresses a Cre-inducible luciferase from Gaussia princeps (GLuc), which is secreted by cells in an energy-consuming process and can be measured quantitatively in the blood as a marker for the viable tumor load. In addition, we have developed a flexible, complementary toolkit to rapidly assemble recombinant adenoviruses (AVs) for delivering Cre recombinase together with CRISPR nucleases targeting cancer driver genes. RESULTS: We demonstrate that intratracheal infection of GLuc reporter mice with CRISPR-AVs efficiently induces lung tumors driven by mutations in the targeted cancer genes and simultaneously activates the GLuc transgene, resulting in GLuc secretion into the blood by the growing tumor. GLuc blood levels are easily and robustly quantified in small-volume blood samples with inexpensive equipment, enable tumor detection already several months before the humane study endpoint and precisely mirror the kinetics of tumor development specified by the inducing gene combination. CONCLUSIONS: Our study establishes blood-based GLuc monitoring as an inexpensive, rapid, high-throughput and animal-friendly method to longitudinally monitor autochthonous tumor growth in preclinical studies.</p>',
'date' => '2022-10-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/36192757',
'doi' => '10.1186/s12943-022-01661-2',
'modified' => '2022-11-21 10:27:46',
'created' => '2022-11-15 09:26:20',
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(int) 2 => array(
'id' => '4517',
'name' => 'Systematic comparison of CRISPR-based transcriptional activatorsuncovers gene-regulatory features of enhancer-promoter interactions.',
'authors' => 'Wang K. et al.',
'description' => '<p>Nuclease-inactivated CRISPR/Cas-based (dCas-based) systems have emerged as powerful technologies to synthetically reshape the human epigenome and gene expression. Despite the increasing adoption of these platforms, their relative potencies and mechanistic differences are incompletely characterized, particularly at human enhancer-promoter pairs. Here, we systematically compared the most widely adopted dCas9-based transcriptional activators, as well as an activator consisting of dCas9 fused to the catalytic core of the human CBP protein, at human enhancer-promoter pairs. We find that these platforms display variable relative expression levels in different human cell types and that their transactivation efficacies vary based upon the effector domain, effector recruitment architecture, targeted locus and cell type. We also show that each dCas9-based activator can induce the production of enhancer RNAs (eRNAs) and that this eRNA induction is positively correlated with downstream mRNA expression from a cognate promoter. Additionally, we use dCas9-based activators to demonstrate that an intrinsic transcriptional and epigenetic reciprocity can exist between human enhancers and promoters and that enhancer-mediated tracking and engagement of a downstream promoter can be synthetically driven by targeting dCas9-based transcriptional activators to an enhancer. Collectively, our study provides new insights into the enhancer-mediated control of human gene expression and the use of dCas9-based activators.</p>',
'date' => '2022-07-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/35849129',
'doi' => '10.1093/nar/gkac582',
'modified' => '2022-11-24 08:53:06',
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'id' => '3886',
'name' => 'DNA Methylation Editing by CRISPR-guided Excision of 5-Methylcytosine.',
'authors' => 'Devesa-Guerra I, Morales-Ruiz T, Pérez-Roldán J, Parrilla-Doblas JT, Dorado-León M, García-Ortiz MV, Ariza RR, Roldán-Arjona T',
'description' => '<p>Tools for actively targeted DNA demethylation are required to increase our knowledge about regulation and specific functions of this important epigenetic modification. DNA demethylation in mammals involves TET-mediated oxidation of 5-methylcytosine (5-meC), which may promote its replication-dependent dilution and/or active removal through base excision repair (BER). However, it is still unclear whether oxidized derivatives of 5-meC are simply DNA demethylation intermediates or rather epigenetic marks on their own. Unlike animals, plants have evolved enzymes that directly excise 5-meC without previous modification. In this work, we have fused the catalytic domain of Arabidopsis ROS1 5-meC DNA glycosylase to a CRISPR-associated null-nuclease (dCas9) and analyzed its capacity for targeted reactivation of methylation-silenced genes, in comparison to other dCas9-effectors. We found that dCas9-ROS1, but not dCas9-TET1, is able to reactivate methylation-silenced genes and induce partial demethylation in a replication-independent manner. We also found that reactivation induced by dCas9-ROS1, as well as that achieved by two different CRISPR-based chromatin effectors (dCas9-VP160 and dCas9-p300), generally decreases with methylation density. Our results suggest that plant 5-meC DNA glycosylases are a valuable addition to the CRISPR-based toolbox for epigenetic editing.</p>',
'date' => '2020-02-19',
'pmid' => 'http://www.pubmed.gov/32087201',
'doi' => '10.1016/j.jmb.2020.02.007',
'modified' => '2020-03-20 17:23:27',
'created' => '2020-03-13 13:45:54',
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(int) 4 => 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',
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(int) 5 => array(
'id' => '3637',
'name' => 'Optimization of CRISPR/Cas9 Delivery to Human Hematopoietic Stem and Progenitor Cells for Therapeutic Genomic Rearrangements.',
'authors' => 'Lattanzi A, Meneghini V, Pavani G, Amor F, Ramadier S, Felix T, Antoniani C, Masson C, Alibeu O, Lee C, Porteus MH, Bao G, Amendola M, Mavilio F, Miccio A',
'description' => '<p>Editing the β-globin locus in hematopoietic stem cells is an alternative therapeutic approach for gene therapy of β-thalassemia and sickle cell disease. Using the CRISPR/Cas9 system, we genetically modified human hematopoietic stem and progenitor cells (HSPCs) to mimic the large rearrangements in the β-globin locus associated with hereditary persistence of fetal hemoglobin (HPFH), a condition that mitigates the clinical phenotype of patients with β-hemoglobinopathies. We optimized and compared the efficiency of plasmid-, lentiviral vector (LV)-, RNA-, and ribonucleoprotein complex (RNP)-based methods to deliver the CRISPR/Cas9 system into HSPCs. Plasmid delivery of Cas9 and gRNA pairs targeting two HPFH-like regions led to high frequency of genomic rearrangements and HbF reactivation in erythroblasts derived from sorted, Cas9 HSPCs but was associated with significant cell toxicity. RNA-mediated delivery of CRISPR/Cas9 was similarly toxic but much less efficient in editing the β-globin locus. Transduction of HSPCs by LVs expressing Cas9 and gRNA pairs was robust and minimally toxic but resulted in poor genome-editing efficiency. Ribonucleoprotein (RNP)-based delivery of CRISPR/Cas9 exhibited a good balance between cytotoxicity and efficiency of genomic rearrangements as compared to the other delivery systems and resulted in HbF upregulation in erythroblasts derived from unselected edited HSPCs.</p>',
'date' => '2019-01-02',
'pmid' => 'http://www.pubmed.gov/30424953',
'doi' => '10.1016/j.ymthe.2018.10.008',
'modified' => '2019-06-07 10:25:31',
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'id' => '3323',
'name' => 'Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo',
'authors' => 'Gandhi S. et al.',
'description' => '<p>The advent of CRISPR/Cas9 has made genome editing possible in virtually any organism, including those not previously amenable to genetic manipulations. Here, we present an optimization of CRISPR/Cas9 for application to early avian embryos with improved efficiency via a three-fold strategy. First, we employed Cas9 protein flanked with two nuclear localization signal sequences for improved nuclear localization. Second, we used a modified guide RNA (gRNA) scaffold that obviates premature termination of transcription and unstable Cas9-gRNA interactions. Third, we used a chick-specific U6 promoter that yields 4-fold higher gRNA expression than the previously utilized human U6. For rapid screening of gRNAs for in vivo applications, we also generated a chicken fibroblast cell line that constitutively expresses Cas9. As proof of principle, we performed electroporation-based loss-of-function studies in the early chick embryo to knock out Pax7 and Sox10, key transcription factors with known functions in neural crest development. The results show that CRISPR/Cas9-mediated deletion causes loss of their respective proteins and transcripts, as well as predicted downstream targets. Taken together, the results reveal the utility of this optimized CRISPR/Cas9 method for targeted gene knockout in chicken embryos in a manner that is reproducible, robust and specific.</p>',
'date' => '2017-12-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/29150011',
'doi' => '',
'modified' => '2018-02-06 09:21:38',
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(int) 7 => array(
'id' => '3074',
'name' => 'Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA',
'authors' => 'Liang W. et al.',
'description' => '<p>While CRISPR-based gene knock out in mammalian cells has proven to be very efficient, precise insertion of genetic elements via the cellular homology directed repair (HDR) pathway remains a rate-limiting step to seamless genome editing. Under the conditions described here, we achieved up to 56% targeted integration efficiency with up to a six-nucleotide insertion in HEK293 cells. In induced pluripotent stem cells (iPSCs), we achieved precise genome editing rates of up to 45% by co-delivering the Cas9 RNP and donor DNA. In addition, the use of a short double stranded DNA oligonucleotide with 3' overhangs allowed integration of a longer FLAG epitope tag along with a restriction site at rates of up to 50%. We propose a model that favors the design of donor DNAs with the change as close to the cleavage site as possible. For small changes such as SNPs or short insertions, asymmetric single stranded donor molecules with 30 base homology arms 3' to the insertion/repair cassette and greater than 40 bases of homology on the 5' end seems to be favored. For larger insertions such as an epitope tag, a dsDNA donor with protruding 3' homology arms of 30 bases is favored. In both cases, protecting the ends of the donor DNA with phosphorothioate modifications improves the editing efficiency.</p>',
'date' => '2016-11-11',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/27845164',
'doi' => '',
'modified' => '2016-11-25 09:30:26',
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'id' => '2818',
'name' => 'CRISPR-Mediated Gene Targeting of Human Induced Pluripotent Stem Cells',
'authors' => 'Susan M. Byrne, George M. Church',
'description' => '<p><span>CRISPR/Cas9 nuclease systems can create double-stranded DNA breaks at specific sequences to efficiently and precisely disrupt, excise, mutate, insert, or replace genes. However, human embryonic stem cells and induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, an optimized protocol is described for genome engineering of human iPSCs using simple transient transfection of plasmids and/or single-stranded oligonucleotides without any further selection or enrichment steps. This protocol achieves transfection efficiencies >60%, with gene disruption efficiencies of 1-25% and gene insertion/replacement efficiencies of 0.5-10%. Details are also provided for designing optimal sgRNA target sites and donor targeting vectors, cloning individual iPSCs by single-cell FACS sorting, and genotyping successfully edited cells.</span></p>',
'date' => '2015-11-04',
'pmid' => 'http://onlinelibrary.wiley.com/doi/10.1002/9780470151808.sc05a08s35/abstract',
'doi' => '10.1002/9780470151808.sc05a08s35',
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<span class="success label" style="">C05010012</span>
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<h6 style="height:60px">MicroPlex Library Preparation Kit v2 (12 indexes)</h6>
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<p><span><strong>Specifically optimized for ChIP-seq</strong></span><br /><br /><span>The MicroPlex Library Preparation™ kit is the only kit on the market which is validated for ChIP-seq and which allows the preparation of indexed libraries from just picogram inputs. In combination with the </span><a href="./true-microchip-kit-x16-16-rxns">True MicroChIP kit</a><span>, it allows for performing ChIP-seq on as few as 10,000 cells. Less input, fewer steps, fewer supplies, faster time to results! </span></p>
<p>The MicroPlex v2 kit (Cat. No. C05010012) contains all necessary reagents including single indexes for multiplexing up to 12 samples using single barcoding. For higher multiplexing (using dual indexes) check <a href="https://www.diagenode.com/en/p/microplex-lib-prep-kit-v3-48-rxns">MicroPlex Library Preparation Kits v3</a>.</p>',
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<li><strong>1 tube, 2 hours, 3 steps</strong> protocol</li>
<li><strong>Input: </strong>50 pg – 50 ng</li>
<li><strong>Reduce potential bias</strong> - few PCR amplification cycles needed</li>
<li><strong>High sensitivity ChIP-seq</strong> - low PCR duplication rate</li>
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<li><strong>Validated with the <a href="https://www.diagenode.com/p/sx-8g-ip-star-compact-automated-system-1-unit" title="IP-Star Automated System">IP-Star<sup>®</sup> Automated Platform</a></strong></li>
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<h3>How it works</h3>
<center><img src="https://www.diagenode.com/img/product/kits/microplex-method-overview-v2.png" /></center>
<p style="margin-bottom: 0;"><small><strong>Microplex workflow - protocol with single indexes</strong><br />An input of 50 pg to 50 ng of fragmented dsDNA is converted into sequencing-ready libraries for Illumina® NGS platforms using a fast and simple 3-step protocol</small></p>
<ul class="accordion" data-accordion="" id="readmore" style="margin-left: 0;">
<li class="accordion-navigation"><a href="#first" style="background: #ffffff; padding: 0rem; margin: 0rem; color: #13b2a2;"><small>Read more about MicroPlex workflow</small></a>
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<p><small><strong>Step 1. Template Preparation</strong> provides efficient repair of the fragmented double-stranded DNA input.</small></p>
<p><small>In this step, the DNA is repaired and yields molecules with blunt ends.</small></p>
<p><small><strong>Step 2. Library Synthesis.</strong> enables ligation of MicroPlex patented stem- loop adapters.</small></p>
<p><small>In the next step, stem-loop adaptors with blocked 5’ ends are ligated with high efficiency to the 5’ end of the genomic DNA, leaving a nick at the 3’ end. The adaptors cannot ligate to each other and do not have single- strand tails, both of which contribute to non-specific background found with many other NGS preparations.</small></p>
<p><small><strong>Step 3. Library Amplification</strong> enables extension of the template, cleavage of the stem-loop adaptors, and amplification of the library. Illumina- compatible indexes are also introduced using a high-fidelity, highly- processive, low-bias DNA polymerase.</small></p>
<p><small>In the final step, the 3’ ends of the genomic DNA are extended to complete library synthesis and Illumina-compatible indexes are added through a high-fidelity amplification. Any remaining free adaptors are destroyed. Hands-on time and the risk of contamination are minimized by using a single tube and eliminating intermediate purifications.</small></p>
<p><small>Obtained libraries are purified, quantified and sized. The libraries pooling can be performed as well before sequencing.</small></p>
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<h3>Reliable detection of enrichments in ChIP-seq</h3>
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<p><strong>Figure A.</strong> ChIP has been peformed with H3K4me3 antibody, amplification of 17 pg of DNA ChIP'd from 10.000 cells and amplification of 35 pg of DNA ChIP'd from 100.000 cells (control experiment). The IP'd DNA was amplified and transformed into a sequencing-ready preparation for the Illumina plateform with the MicroPlex Library Preparation kit. The library was then analysed on an Illumina<sup>®</sup> Genome Analyzer. Cluster generation and sequencing were performed according to the manufacturer's instructions.</p>
<p><img src="https://www.diagenode.com/img/product/kits/microplex-library-prep-kit-figure-b.png" alt="Reliable detection of enrichments in ChIP-seq figure 2" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure B.</strong> We observed a perfect match between the top 40% of True MicroChIP peaks and the reference dataset. Based on the NIH Encode project criterion, ChIP-seq results are considered reproducible between an original and reproduced dataset if the top 40% of peaks have at least an 80% overlap ratio with the compared dataset.</p>',
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'id' => '4858',
'product_id' => '2958',
'application_id' => '30'
)
)
$slugs = array(
(int) 0 => 'immunoprecipitation'
)
$applications = array(
'id' => '30',
'position' => '10',
'parent_id' => '40',
'name' => 'IP',
'description' => '<p>Immunoprecipitation</p>',
'in_footer' => false,
'in_menu' => false,
'online' => true,
'tabular' => true,
'slug' => 'immunoprecipitation',
'meta_keywords' => 'Immunoprecipitation,Monoclonal antibody,Polyclonal antibody',
'meta_description' => 'Diagenode offers a wide range of antibodies and technical support for Immunoprecipitation applications',
'meta_title' => 'Immunoprecipitation - Monoclonal antibody - Polyclonal antibody | Diagenode',
'modified' => '2016-01-13 12:23:07',
'created' => '2015-07-08 13:46:50',
'locale' => 'eng'
)
$description = '<p>Immunoprecipitation</p>'
$name = 'IP'
$document = array(
'id' => '991',
'name' => ' Accurate QC to optimize CRISPR/Cas9 genome editing specificity',
'description' => '<p>The CRISPR/Cas9 technology is delivering superior genetic models for fundamental disease research, drug screening, therapy development, rapid diagnostics, and transcriptional modulation. Although CRISPR/Cas9 enables rapid genome editing, several aspects affect its efficiency and specificity including guide RNA design, delivery methods, and off-targets effects. Diagenode has developed strategies to overcome these common pitfalls and has optimized CRISPR/Cas9 genome editing specificity</p>',
'image_id' => null,
'type' => 'Poster',
'url' => 'files/posters/CRISPR-Cas9-Poster-Accurate_QC.pdf',
'slug' => 'crispr-cas9-accurate-qc',
'meta_keywords' => '',
'meta_description' => '',
'modified' => '2018-02-12 15:36:31',
'created' => '2018-02-12 13:15:37',
'ProductsDocument' => array(
'id' => '2563',
'product_id' => '2958',
'document_id' => '991'
)
)
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'id' => '498',
'name' => 'CRISPR/Cas9 Antibody 4G10 SDS BE nl',
'language' => 'nl',
'url' => 'files/SDS/CRISPR-Cas9/SDS-C15200216-CRISPR_Cas9_Antibody_4G10-BE-nl-GHS_1_0.pdf',
'countries' => 'BE',
'modified' => '2020-07-01 12:14:00',
'created' => '2020-07-01 12:14:00',
'ProductsSafetySheet' => array(
'id' => '947',
'product_id' => '2958',
'safety_sheet_id' => '498'
)
)
$publication = array(
'id' => '2818',
'name' => 'CRISPR-Mediated Gene Targeting of Human Induced Pluripotent Stem Cells',
'authors' => 'Susan M. Byrne, George M. Church',
'description' => '<p><span>CRISPR/Cas9 nuclease systems can create double-stranded DNA breaks at specific sequences to efficiently and precisely disrupt, excise, mutate, insert, or replace genes. However, human embryonic stem cells and induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, an optimized protocol is described for genome engineering of human iPSCs using simple transient transfection of plasmids and/or single-stranded oligonucleotides without any further selection or enrichment steps. This protocol achieves transfection efficiencies >60%, with gene disruption efficiencies of 1-25% and gene insertion/replacement efficiencies of 0.5-10%. Details are also provided for designing optimal sgRNA target sites and donor targeting vectors, cloning individual iPSCs by single-cell FACS sorting, and genotyping successfully edited cells.</span></p>',
'date' => '2015-11-04',
'pmid' => 'http://onlinelibrary.wiley.com/doi/10.1002/9780470151808.sc05a08s35/abstract',
'doi' => '10.1002/9780470151808.sc05a08s35',
'modified' => '2016-02-10 16:29:28',
'created' => '2016-02-10 16:29:28',
'ProductsPublication' => array(
'id' => '2686',
'product_id' => '2958',
'publication_id' => '2818'
)
)
$externalLink = ' <a href="http://onlinelibrary.wiley.com/doi/10.1002/9780470151808.sc05a08s35/abstract" target="_blank"><i class="fa fa-external-link"></i></a>'include - APP/View/Products/view.ctp, line 755
View::_evaluate() - CORE/Cake/View/View.php, line 971
View::_render() - CORE/Cake/View/View.php, line 933
View::render() - CORE/Cake/View/View.php, line 473
Controller::render() - CORE/Cake/Controller/Controller.php, line 963
ProductsController::slug() - APP/Controller/ProductsController.php, line 1052
ReflectionMethod::invokeArgs() - [internal], line ??
Controller::invokeAction() - CORE/Cake/Controller/Controller.php, line 491
Dispatcher::_invoke() - CORE/Cake/Routing/Dispatcher.php, line 193
Dispatcher::dispatch() - CORE/Cake/Routing/Dispatcher.php, line 167
[main] - APP/webroot/index.php, line 118
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