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<p><small><strong>Figure 1. Western blot analysis using the Diagenode monoclonal antibody directed against Cas9<br /></strong>Western blot was performed on protein extracts from HeLa cells transfected with a flag-tagged Cas9 using the Diagenode antibody against Cas9 (cat. No. C15200203). The antibody was used at different dilutions. The marker is shown on the left, position of the flag-tagged Cas9 protein is indicated on the right.</small></p>
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<p><small><strong>Figure 2. Western blot analysis using the Diagenode monoclonal antibody directed against Cas9<br /></strong>Western blot was performed on protein extracts from HeLa cells (lane 1) and on HeLa cells spiked with 1 ng of recombinant Cas9 protein (lane 2) using the Diagenode antibody against Cas9 (cat. No. C15200203). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left.</small></p>
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<p><small><strong>Figure 4. Immunofluorescence using the Diagenode monoclonal antibody directed against Cas9<br /></strong>Hela cells were transiently transfected with a Flag-tagged Cas9 expression vector. 48 hours post transfection the cells were fixed in 3.7% formaldehyde, permeabilized in 0.5% Triton-X-100 and blocked in PBS containing 2% BSA for 2 hours at RT. 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 (left). Nuclei were counter-stained with DAPI (right).</small></p>
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<p><small><strong>Figure 2. Western blot analysis using the Diagenode monoclonal antibody directed against Cas9<br /></strong>Western blot was performed on protein extracts from HeLa cells (lane 1) and on HeLa cells spiked with 1 ng of recombinant Cas9 protein (lane 2) using the Diagenode antibody against Cas9 (cat. No. C15200203). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left.</small></p>
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<p><small><strong>Figure 1. Western blot analysis using the Diagenode monoclonal antibody directed against Cas9<br /></strong>Western blot was performed on protein extracts from HeLa cells transfected with a flag-tagged Cas9 using the Diagenode antibody against Cas9 (cat. No. C15200203). The antibody was used at different dilutions. The marker is shown on the left, position of the flag-tagged Cas9 protein is indicated on the right.</small></p>
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<p><small><strong>Figure 2. Western blot analysis using the Diagenode monoclonal antibody directed against Cas9<br /></strong>Western blot was performed on protein extracts from HeLa cells (lane 1) and on HeLa cells spiked with 1 ng of recombinant Cas9 protein (lane 2) using the Diagenode antibody against Cas9 (cat. No. C15200203). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left.</small></p>
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<p><small><strong>Figure 3. IP using the Diagenode monoclonal antibody directed against Cas9<br /></strong>IP was performed on whole cell extracts (100 µg) from HEK293 cells transfected with a Flag-tagged Cas9 using the Diagenode antibody against Cas9 (Cat. No. C15200203). The immunoprecipitated proteins were subsequently analysed by Western blot with the antibody. Lane 3 and 4 show the result of the IP; a negative IP control (IP on untransfected cells) and the input (15 µg) are shown in lane 2 and 1, respectively.</small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15200203_IF.png" alt="CRISPR/Cas9 Antibody validated in IF" caption="false" width="201" height="99" /></p>
</div>
<div class="small-9 columns">
<p><small><strong>Figure 4. Immunofluorescence using the Diagenode monoclonal antibody directed against Cas9<br /></strong>Hela cells were transiently transfected with a Flag-tagged Cas9 expression vector. 48 hours post transfection the cells were fixed in 3.7% formaldehyde, permeabilized in 0.5% Triton-X-100 and blocked in PBS containing 2% BSA for 2 hours at RT. 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 (left). Nuclei were counter-stained with DAPI (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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<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>
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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>
<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>
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<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>
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<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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'type' => 'Brochure',
'url' => 'files/brochures/Epigenetic_Antibodies_Brochure.pdf',
'slug' => 'epigenetic-antibodies-brochure',
'meta_keywords' => '',
'meta_description' => '',
'modified' => '2016-06-15 11:24:06',
'created' => '2015-07-03 16:05:27',
'ProductsDocument' => array(
[maximum depth reached]
)
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(int) 2 => array(
'id' => '384',
'name' => 'Datasheet CRISPR/Cas9 monoclonal antibody',
'description' => '<p>This antibody has been raised against the N-terminal region of the CRISP/Cas9 protein.</p>',
'image_id' => null,
'type' => 'Datasheet',
'url' => 'files/products/antibodies/Datasheet_CRISPR_Cas9_monoclonal_antibody.pdf',
'slug' => 'datasheet-crispr-cas9-monoclonal-antibody',
'meta_keywords' => '',
'meta_description' => '',
'modified' => '2019-05-15 11:20:01',
'created' => '2015-07-07 11:47:44',
'ProductsDocument' => array(
[maximum depth reached]
)
),
(int) 3 => array(
'id' => '991',
'name' => ' Accurate QC to optimize CRISPR/Cas9 genome editing specificity',
'description' => '<p>The CRISPR/Cas9 technology is delivering superior genetic models for fundamental disease research, drug screening, therapy development, rapid diagnostics, and transcriptional modulation. Although CRISPR/Cas9 enables rapid genome editing, several aspects affect its efficiency and specificity including guide RNA design, delivery methods, and off-targets effects. Diagenode has developed strategies to overcome these common pitfalls and has optimized CRISPR/Cas9 genome editing specificity</p>',
'image_id' => null,
'type' => 'Poster',
'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(
[maximum depth reached]
)
)
),
'Feature' => array(),
'Image' => array(
(int) 0 => array(
'id' => '1782',
'name' => 'product/antibodies/cas9-icon.png',
'alt' => 'CRISPR/Cas9 Antibody ',
'modified' => '2020-11-27 07:01:20',
'created' => '2018-03-15 15:53:46',
'ProductsImage' => array(
[maximum depth reached]
)
)
),
'Promotion' => array(),
'Protocol' => array(),
'Publication' => array(
(int) 0 => array(
'id' => '4953',
'name' => 'Loss of tumor suppressors promotes inflammatory tumor microenvironment and enhances LAG3+T cell mediated immune suppression',
'authors' => 'Zahraeifard, S. et al.',
'description' => '<p><span>Low response rate, treatment relapse, and resistance remain key challenges for cancer treatment with immune checkpoint blockade (ICB). Here we report that loss of specific tumor suppressors (TS) induces an inflammatory response and promotes an immune suppressive tumor microenvironment. Importantly, low expression of these TSs is associated with a higher expression of immune checkpoint inhibitory mediators. Here we identify, by using in vivo CRISPR/Cas9 based loss-of-function screening, that NF1, TSC1, and TGF-β RII as TSs regulating immune composition. Loss of each of these three TSs leads to alterations in chromatin accessibility and enhances IL6-JAK3-STAT3/6 inflammatory pathways. This results in an immune suppressive landscape, characterized by increased numbers of LAG3+ CD8 and CD4 T cells. ICB targeting LAG3 and PD-L1 simultaneously inhibits metastatic progression in preclinical triple negative breast cancer (TNBC) mouse models of NF1-, TSC1- or TGF-β RII- deficient tumors. Our study thus reveals a role of TSs in regulating metastasis via non-cell-autonomous modulation of the immune compartment and provides proof-of-principle for ICB targeting LAG3 for patients with NF1-, TSC1- or TGF-β RII-inactivated cancers.</span></p>',
'date' => '2024-07-12',
'pmid' => 'https://www.nature.com/articles/s41467-024-50262-8',
'doi' => 'https://doi.org/10.1038/s41467-024-50262-8',
'modified' => '2024-07-29 10:47:10',
'created' => '2024-07-29 10:47:10',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 1 => array(
'id' => '4784',
'name' => 'A kinesin-based approach for inducing chromosome-specific mis-segregationin human cells.',
'authors' => 'Truong M.A. et al.',
'description' => '<p>Various cancer types exhibit characteristic and recurrent aneuploidy patterns. The origins of these cancer type-specific karyotypes are still unknown, partly because introducing or eliminating specific chromosomes in human cells still poses a challenge. Here, we describe a novel strategy to induce mis-segregation of specific chromosomes in different human cell types. We employed Tet repressor or nuclease-dead Cas9 to link a microtubule minus-end-directed kinesin (Kinesin14VIb) from Physcomitrella patens to integrated Tet operon repeats and chromosome-specific endogenous repeats, respectively. By live- and fixed-cell imaging, we observed poleward movement of the targeted loci during (pro)metaphase. Kinesin14VIb-mediated pulling forces on the targeted chromosome were counteracted by forces from kinetochore-attached microtubules. This tug-of-war resulted in chromosome-specific segregation errors during anaphase and revealed that spindle forces can heavily stretch chromosomal arms. By single-cell whole-genome sequencing, we established that kinesin-induced targeted mis-segregations predominantly result in chromosomal arm aneuploidies after a single cell division. Our kinesin-based strategy opens the possibility to investigate the immediate cellular responses to specific aneuploidies in different cell types; an important step toward understanding how tissue-specific aneuploidy patterns evolve.</p>',
'date' => '2023-04-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/37038978',
'doi' => '10.15252/embj.2022111559',
'modified' => '2023-06-13 09:21:25',
'created' => '2023-05-05 12:34:24',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 2 => array(
'id' => '4344',
'name' => 'MICAL1 regulates actin cytoskeleton organization, directional cellmigration and the growth of human breast cancer cells as orthotopicxenograft tumours.',
'authors' => 'McGarry David J et al.',
'description' => '<p>The Molecule Interacting with CasL 1 (MICAL1) monooxygenase has emerged as an important regulator of cytoskeleton organization via actin oxidation. Although filamentous actin (F-actin) increases MICAL1 monooxygenase activity, hydrogen peroxide (HO) is also generated in the absence of F-actin, suggesting that diffusible HO might have additional functions. MICAL1 gene disruption by CRISPR/Cas9 in MDA MB 231 human breast cancer cells knocked out (KO) protein expression, which affected F-actin organization, cell size and motility. Transcriptomic profiling revealed that MICAL1 deletion significantly affected the expression of over 700 genes, with the majority being reduced in their expression levels. In addition, the absolute magnitudes of reduced gene expression were significantly greater than the magnitudes of increased gene expression. Gene set enrichment analysis (GSEA) identified receptor regulator activity as the most significant negatively enriched molecular function gene set. The prominent influence exerted by MICAL1 on F-actin structures was also associated with changes in the expression of several serum-response factor (SRF) regulated genes in KO cells. Moreover, MICAL1 disruption attenuated breast cancer tumour growth in vivo. Elevated MICAL1 gene expression was observed in invasive breast cancer samples from human patients relative to normal tissue, while MICAL1 amplification or point mutations were associated with reduced progression free survival. Collectively, these results demonstrate that MICAL1 gene disruption altered cytoskeleton organization, cell morphology and migration, gene expression, and impaired tumour growth in an orthotopic in vivo breast cancer model, suggesting that pharmacological MICAL1 inhibition could have therapeutic benefits for cancer patients.</p>',
'date' => '2021-10-01',
'pmid' => 'https://doi.org/10.1016%2Fj.canlet.2021.07.039',
'doi' => '10.1016/j.canlet.2021.07.039',
'modified' => '2022-06-21 16:56:09',
'created' => '2022-05-19 10:41:50',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 3 => array(
'id' => '3925',
'name' => 'CRISPR-based gene knockout screens reveal deubiquitinases involved in HIV-1 latency in two Jurkat cell models.',
'authors' => 'Rathore A, Iketani S, Wang P, Jia M, Sahi V, Ho DD',
'description' => '<p>The major barrier to a HIV-1 cure is the persistence of latent genomes despite treatment with antiretrovirals. To investigate host factors which promote HIV-1 latency, we conducted a genome-wide functional knockout screen using CRISPR-Cas9 in a HIV-1 latency cell line model. This screen identified IWS1, POLE3, POLR1B, PSMD1, and TGM2 as potential regulators of HIV-1 latency, of which PSMD1 and TMG2 could be confirmed pharmacologically. Further investigation of PSMD1 revealed that an interacting enzyme, the deubiquitinase UCH37, was also involved in HIV-1 latency. We therefore conducted a comprehensive evaluation of the deubiquitinase family by gene knockout, identifying several deubiquitinases, UCH37, USP14, OTULIN, and USP5 as possible HIV-1 latency regulators. A specific inhibitor of USP14, IU1, reversed HIV-1 latency and displayed synergistic effects with other latency reversal agents. IU1 caused degradation of TDP-43, a negative regulator of HIV-1 transcription. Collectively, this study is the first comprehensive evaluation of deubiquitinases in HIV-1 latency and establishes that they may hold a critical role.</p>',
'date' => '2020-03-24',
'pmid' => 'http://www.pubmed.gov/32210344',
'doi' => '10.1038/s41598-020-62375-3',
'modified' => '2020-08-17 10:51:24',
'created' => '2020-08-10 12:12:25',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 4 => array(
'id' => '3821',
'name' => 'DAPK1 loss triggers tumor invasion in colorectal tumor cells.',
'authors' => 'Steinmann S, Kunze P, Hampel C, Eckstein M, Bertram Bramsen J, Muenzner JK, Carlé B, Ndreshkjana B, Kemenes S, Gasparini P, Friedrich O, Andersen C, Geppert C, Wang S, Eyupoglu I, Bäuerle T, Hartmann A, Schneider-Stock R',
'description' => '<p>Colorectal cancer (CRC) is one of the leading cancer-related causes of death worldwide. Despite the improvement of surgical and chemotherapeutic treatments, as of yet, the disease has not been overcome due to metastasis to distant organs. Hence, it is of great relevance to understand the mechanisms responsible for metastasis initiation and progression and to identify novel metastatic markers for a higher chance of preventing the metastatic disease. The Death-associated protein kinase 1 (DAPK1), recently, has been shown to be a potential candidate for regulating metastasis in CRC. Hence, the aim of the study was to investigate the impact of DAPK1 protein on CRC aggressiveness. Using CRISPR/Cas9 technology, we generated DAPK1-deficient HCT116 monoclonal cell lines and characterized their knockout phenotype in vitro and in vivo. We show that loss of DAPK1 implemented changes in growth pattern and enhanced tumor budding in vivo in the chorioallantoic membrane (CAM) model. Further, we observed more tumor cell dissemination into chicken embryo organs and increased invasion capacity using rat brain 3D in vitro model. The novel identified DAPK1-loss gene expression signature showed a stroma typical pattern and was associated with a gained ability for remodeling the extracellular matrix. Finally, we suggest the DAPK1-ERK1 signaling axis being involved in metastatic progression of CRC. Our results highlight DAPK1 as an anti-metastatic player in CRC and suggest DAPK1 as a potential predictive biomarker for this cancer type.</p>',
'date' => '2019-11-26',
'pmid' => 'http://www.pubmed.gov/31772156',
'doi' => '10.1038/s41419-019-2122-z',
'modified' => '2020-02-25 13:45:15',
'created' => '2020-02-13 10:02:44',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(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',
'created' => '2019-06-06 12:11:18',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 6 => array(
'id' => '3565',
'name' => 'Can Mitochondrial DNA be CRISPRized: Pro and Contra.',
'authors' => 'Loutre R, Heckel AM, Smirnova A, Entelis N, Tarassov I',
'description' => '<p>Mitochondria represent a chimera of macromolecules encoded either in the organellar genome, mtDNA, or in the nuclear one. If the pathway of protein targeting to different sub-compartments of mitochondria was relatively well studied, import of small noncoding RNAs into mammalian mitochondria still awaits mechanistic explanations and its functional issues are often not understood thus raising polemics. At the same time, RNA mitochondrial import pathway has an obvious attractiveness as it appears as a unique natural mechanism permitting to address nucleic acids into the organelles. Deciphering the function(s) of imported RNAs inside the mitochondria is extremely complicated due to their relatively low abundance, which suggests their regulatory role. We previously demonstrated that mitochondrial targeting of small noncoding RNAs able to specifically anneal with the mutant mitochondrial DNA led to a decrease of the mtDNA heteroplasmy level by inhibiting mutant mtDNA replication. We then demonstrated that increasing level of expression of such antireplicative recombinant RNAs increases significantly the antireplicative effect. In this report, we present a new data investigating the possibility to establish a CRISPR-Cas9 system targeting mtDNA exploiting of the pathway of RNA import into mitochondria. Mitochondrially addressed Cas9 versions and a set of mitochondrially targeted guide RNAs were tested in vitro and in vivo and their effect on mtDNA copy number was demonstrated. So far, the system appeared as more complicated for use than previously found for nuclear DNA, because only application of a pair of guide RNAs produced the effect of mtDNA depletion. We discuss, in a critical way, these results and put them in a broader context of polemics concerning the possibilities of manipulation of mtDNA in mammalians. The findings described here prove the potential of the RNA import pathway as a tool for studying mtDNA and for future therapy of mitochondrial disorders. © The Authors. IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 70(12):1233-1239, 2018.</p>',
'date' => '2018-12-01',
'pmid' => 'http://www.pubmed.gov/30184317',
'doi' => '10.1002/iub.1919',
'modified' => '2019-03-21 17:20:49',
'created' => '2019-03-21 14:12:08',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 7 => array(
'id' => '3268',
'name' => 'In trans paired nicking triggers seamless genome editing without double-stranded DNA cutting',
'authors' => 'Chen X. et al.',
'description' => '<p>Precise genome editing involves homologous recombination between donor DNA and chromosomal sequences subjected to double-stranded DNA breaks made by programmable nucleases. Ideally, genome editing should be efficient, specific, and accurate. However, besides constituting potential translocation-initiating lesions, double-stranded DNA breaks (targeted or otherwise) are mostly repaired through unpredictable and mutagenic non-homologous recombination processes. Here, we report that the coordinated formation of paired single-stranded DNA breaks, or nicks, at donor plasmids and chromosomal target sites by RNA-guided nucleases based on CRISPR-Cas9 components, triggers seamless homology-directed gene targeting of large genetic payloads in human cells, including pluripotent stem cells. Importantly, in addition to significantly reducing the mutagenicity of the genome modification procedure, this in trans paired nicking strategy achieves multiplexed, single-step, gene targeting, and yields higher frequencies of accurately edited cells when compared to the standard double-stranded DNA break-dependent approach.</p>',
'date' => '2017-09-22',
'pmid' => 'https://www.nature.com/articles/s41467-017-00687-1',
'doi' => '',
'modified' => '2017-10-09 16:27:58',
'created' => '2017-10-09 16:27:58',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 8 => array(
'id' => '3244',
'name' => 'Highly efficient gene inactivation by adenoviral CRISPR/Cas9 in human primary cells',
'authors' => 'Voets O. et al.',
'description' => '<p>Phenotypic assays using human primary cells are highly valuable tools for target discovery and validation in drug discovery. Expression knockdown (KD) of such targets in these assays allows the investigation of their role in models of disease processes. Therefore, efficient and fast modes of protein KD in phenotypic assays are required. The CRISPR/Cas9 system has been shown to be a versatile and efficient means of gene inactivation in immortalized cell lines. Here we describe the use of adenoviral (AdV) CRISPR/Cas9 vectors for efficient gene inactivation in two human primary cell types, normal human lung fibroblasts and human bronchial epithelial cells. The effects of gene inactivation were studied in the TGF-β-induced fibroblast to myofibroblast transition assay (FMT) and the epithelial to mesenchymal transition assay (EMT), which are SMAD3 dependent and reflect pathogenic mechanisms observed in fibrosis. Co-transduction (co-TD) of AdV Cas9 with SMAD3-targeting guide RNAs (gRNAs) resulted in fast and efficient genome editing judged by insertion/deletion (indel) formation, as well as significant reduction of SMAD3 protein expression and nuclear translocation. This led to phenotypic changes downstream of SMAD3 inhibition, including substantially decreased alpha smooth muscle actin and fibronectin 1 expression, which are markers for FMT and EMT, respectively. A direct comparison between co-TD of separate Cas9 and gRNA AdV, versus TD with a single "all-in-one" Cas9/gRNA AdV, revealed that both methods achieve similar levels of indel formation. These data demonstrate that AdV CRISPR/Cas9 is a useful and efficient tool for protein KD in human primary cell phenotypic assays. The use of AdV CRISPR/Cas9 may offer significant advantages over the current existing tools and should enhance target discovery and validation opportunities.</p>',
'date' => '2017-08-11',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/28800587',
'doi' => '',
'modified' => '2017-09-19 17:26:46',
'created' => '2017-09-19 17:26:46',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 9 => array(
'id' => '3040',
'name' => 'Noncoding somatic and inherited single-nucleotide variants converge to promote ESR1 expression in breast cancer',
'authors' => 'Bailey SD et al.',
'description' => '<p>Sustained expression of the estrogen receptor-<span class="mb">α</span> (ESR1) drives two-thirds of breast cancer and defines the ESR1-positive subtype. ESR1 engages enhancers upon estrogen stimulation to establish an oncogenic expression program<sup><a href="http://www.nature.com/ng/journal/v48/n10/full/ng.3650.html#ref1" title="Green, K.A. & Carroll, J.S. Oestrogen-receptor-mediated transcription and the influence of co-factors and chromatin state. Nat. Rev. Cancer 7, 713-722 (2007)." id="ref-link-5">1</a></sup>. Somatic copy number alterations involving the <i>ESR1</i> gene occur in approximately 1% of ESR1-positive breast cancers<sup><a href="http://www.nature.com/ng/journal/v48/n10/full/ng.3650.html#ref2" title="Vincent-Salomon, A., Raynal, V., Lucchesi, C., Gruel, N. & Delattre, O. ESR1 gene amplification in breast cancer: a common phenomenon? Nat. Genet. 40, 809, author reply 810-812 (2008)." id="ref-link-6">2</a>, <a href="http://www.nature.com/ng/journal/v48/n10/full/ng.3650.html#ref3" title="Brown, L.A. et al. ESR1 gene amplification in breast cancer: a common phenomenon? Nat. Genet. 40, 806-807, author reply 810-812 (2008)." id="ref-link-7">3</a>, <a href="http://www.nature.com/ng/journal/v48/n10/full/ng.3650.html#ref4" title="Horlings, H.M. et al. ESR1 gene amplification in breast cancer: a common phenomenon? Nat. Genet. 40, 807-808, author reply 810-812 (2008)." id="ref-link-8">4</a>, <a href="http://www.nature.com/ng/journal/v48/n10/full/ng.3650.html#ref5" title="Reis-Filho, J.S. et al. ESR1 gene amplification in breast cancer: a common phenomenon? Nat. Genet. 40, 809-810, author reply 810-812 (2008)." id="ref-link-9">5</a></sup>, suggesting that other mechanisms underlie the persistent expression of <i>ESR1</i>. We report significant enrichment of somatic mutations within the set of regulatory elements (SRE) regulating <i>ESR1</i> in 7% of ESR1-positive breast cancers. These mutations regulate <i>ESR1</i> expression by modulating transcription factor binding to the DNA. The SRE includes a recurrently mutated enhancer whose activity is also affected by rs9383590, a functional inherited single-nucleotide variant (SNV) that accounts for several breast cancer risk–associated loci. Our work highlights the importance of considering the combinatorial activity of regulatory elements as a single unit to delineate the impact of noncoding genetic alterations on single genes in cancer.</p>',
'date' => '2016-08-29',
'pmid' => 'http://www.nature.com/ng/journal/v48/n10/full/ng.3650.html',
'doi' => '',
'modified' => '2016-11-03 11:59:03',
'created' => '2016-10-07 11:08:31',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 10 => array(
'id' => '3038',
'name' => 'The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing.',
'authors' => 'de Solis CA et al.',
'description' => '<p>The RNA-guided Cas9 nuclease, from the type II prokaryotic Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) adaptive immune system, has been adapted and utilized by scientists to edit the genomes of eukaryotic cells. Here, we report the development of a viral mediated CRISPR/Cas9 system that can be rendered inducible utilizing doxycycline (Dox) and can be delivered to cells in vitro and in vivo utilizing adeno-associated virus (AAV). Specifically, we developed an inducible gRNA (gRNAi) AAV vector that is designed to express the gRNA from a H1/TO promoter. This AAV vector is also designed to express the Tet repressor (TetR) to regulate the expression of the gRNAi in a Dox dependent manner. We show that H1/TO promoters of varying length and a U6/TO promoter can edit DNA with similar efficiency in vitro, in a Dox dependent manner. We also demonstrate that our inducible gRNAi vector can be used to edit the genomes of neurons in vivo within the mouse brain in a Dox dependent manner. Genome editing can be induced in vivo with this system by supplying animals Dox containing food for as little as 1 day. This system might be cross compatible with many existing S. pyogenes Cas9 systems (i.e., Cas9 mouse, CRISPRi, etc.), and therefore it likely can be used to render these systems inducible as well.</p>',
'date' => '2016-08-18',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/27587996',
'doi' => '',
'modified' => '2016-10-05 15:48:14',
'created' => '2016-10-05 15:48:14',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 11 => 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(
[maximum depth reached]
)
),
(int) 12 => array(
'id' => '2823',
'name' => 'A geminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing',
'authors' => 'Yin K, Han T, Liu G, Chen T, Wang Y, Yu AY, Liu Y',
'description' => '<p>CRISPR/Cas has emerged as potent genome editing technology and has successfully been applied in many organisms, including several plant species. However, delivery of genome editing reagents remains a challenge in plants. Here, we report a <u>vi</u>rus-based guide RNA (gRNA) delivery system for CRISPR/Cas9 mediated plant <u>g</u>enome <u>e</u>diting (VIGE) that can be used to precisely target genome locations and cause mutations. VIGE is performed by using a modified Cabbage Leaf Curl virus (CaLCuV) vector to express gRNAs in stable transgenic plants expressing Cas9. DNA sequencing confirmed VIGE of endogenous <i>NbPDS3</i> and <i>NbIspH</i> genes in non-inoculated leaves because CaLCuV can infect plants systemically. Moreover, VIGE of <i>NbPDS3</i> and <i>NbIspH</i> in newly developed leaves caused photo-bleached phenotype. These results demonstrate that geminivirus-based VIGE could be a powerful tool in plant genome editing.</p>',
'date' => '2015-10-09',
'pmid' => 'http://www.ncbi.nlm.nih.gov/pubmed/26450012',
'doi' => '10.1038/srep14926',
'modified' => '2016-02-16 13:55:32',
'created' => '2016-02-16 13:55:32',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 13 => array(
'id' => '2765',
'name' => 'Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection.',
'authors' => 'Liang X, Potter J, Kumar S, Zou Y, Quintanilla R, Sridharan M, Carte J, Chen W, Roark N, Ranganathan S, Ravinder N, Chesnut JD',
'description' => '<p>CRISPR-Cas9 systems provide a platform for high efficiency genome editing that are enabling innovative applications of mammalian cell engineering. However, the delivery of Cas9 and synthesis of guide RNA (gRNA) remain as steps that can limit overall efficiency and ease of use. Here we describe methods for rapid synthesis of gRNA and for delivery of Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs) into a variety of mammalian cells through liposome-mediated transfection or electroporation. Using these methods, we report nuclease-mediated indel rates of up to 94% in Jurkat T cells and 87% in induced pluripotent stem cells (iPSC) for a single target. When we used this approach for multigene targeting in Jurkat cells we found that two-locus and three-locus indels were achieved in approximately 93% and 65% of the resulting isolated cell lines, respectively. Further, we found that the off-target cleavage rate is reduced using Cas9 protein when compared to plasmid DNA transfection. Taken together, we present a streamlined cell engineering workflow that enables gRNA design to analysis of edited cells in as little as four days and results in highly efficient genome modulation in hard-to-transfect cells. The reagent preparation and delivery to cells is amenable to high throughput, multiplexed genome-wide cell engineering.</p>',
'date' => '2015-08-20',
'pmid' => 'http://www.ncbi.nlm.nih.gov/pubmed/26003884',
'doi' => '10.1016/j.jbiotec.2015.04.024',
'modified' => '2016-09-21 16:28:13',
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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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<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>
<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>Diagenode, a dedicated supplier of high quality Cas9 antibodies, was<strong> the first company</strong> that offered<strong> the antibody Cas9 (clone 7A9)</strong>. This CRISPR/Cas antibody has been validated in a number of different applications including WB, IF, and IP. Our long history of expertise with CRISPR/Cas9 will guarantee your experimental success.</p>
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'description' => '<p>Diagenode, a dedicated supplier of high quality Cas9 antibodies, was<strong> the first company</strong> that offered<strong> the antibody Cas9 (clone 7A9)</strong>. This CRISPR/Cas antibody has been validated in a number of different applications including WB, IF, and IP. Our long history of expertise with <strong>CRISPR/Cas9</strong> will guarantee your experimental success.</p>
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<p><small><strong>Figure 2. Western blot analysis using the Diagenode monoclonal antibody directed against Cas9<br /></strong>Western blot was performed on protein extracts from HeLa cells (lane 1) and on HeLa cells spiked with 1 ng of recombinant Cas9 protein (lane 2) using the Diagenode antibody against Cas9 (cat. No. C15200203). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left.</small></p>
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<p><small><strong>Figure 3. IP using the Diagenode monoclonal antibody directed against Cas9<br /></strong>IP was performed on whole cell extracts (100 µg) from HEK293 cells transfected with a Flag-tagged Cas9 using the Diagenode antibody against Cas9 (Cat. No. C15200203). The immunoprecipitated proteins were subsequently analysed by Western blot with the antibody. Lane 3 and 4 show the result of the IP; a negative IP control (IP on untransfected cells) and the input (15 µg) are shown in lane 2 and 1, respectively.</small></p>
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<p><small><strong>Figure 4. Immunofluorescence using the Diagenode monoclonal antibody directed against Cas9<br /></strong>Hela cells were transiently transfected with a Flag-tagged Cas9 expression vector. 48 hours post transfection the cells were fixed in 3.7% formaldehyde, permeabilized in 0.5% Triton-X-100 and blocked in PBS containing 2% BSA for 2 hours at RT. 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 (left). Nuclei were counter-stained with DAPI (right).</small></p>
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<p><small><strong>Figure 2. Western blot analysis using the Diagenode monoclonal antibody directed against Cas9<br /></strong>Western blot was performed on protein extracts from HeLa cells (lane 1) and on HeLa cells spiked with 1 ng of recombinant Cas9 protein (lane 2) using the Diagenode antibody against Cas9 (cat. No. C15200203). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left.</small></p>
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<p><small><strong>Figure 4. Immunofluorescence using the Diagenode monoclonal antibody directed against Cas9<br /></strong>Hela cells were transiently transfected with a Flag-tagged Cas9 expression vector. 48 hours post transfection the cells were fixed in 3.7% formaldehyde, permeabilized in 0.5% Triton-X-100 and blocked in PBS containing 2% BSA for 2 hours at RT. 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 (left). Nuclei were counter-stained with DAPI (right).</small></p>
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<p><small><strong>Figure 3. IP using the Diagenode monoclonal antibody directed against Cas9<br /></strong>IP was performed on whole cell extracts (100 µg) from HEK293 cells transfected with a Flag-tagged Cas9 using the Diagenode antibody against Cas9 (Cat. No. C15200203). The immunoprecipitated proteins were subsequently analysed by Western blot with the antibody. Lane 3 and 4 show the result of the IP; a negative IP control (IP on untransfected cells) and the input (15 µg) are shown in lane 2 and 1, respectively.</small></p>
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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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'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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'description' => '<p><strong>Immunofluorescence</strong>:</p>
<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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'id' => '30',
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'meta_description' => 'Diagenode offers a wide range of antibodies and technical support for Immunoprecipitation applications',
'meta_title' => 'Immunoprecipitation - Monoclonal antibody - Polyclonal antibody | Diagenode',
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'id' => '103',
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'name' => 'All antibodies',
'description' => '<p><span style="font-weight: 400;">All Diagenode’s antibodies are listed below. Please, use our Quick search field to find the antibody of interest by target name, application, purity.</span></p>
<p><span style="font-weight: 400;">Diagenode’s highly validated antibodies:</span></p>
<ul>
<li>Highly sensitive and specific</li>
<li>Cost-effective (requires less antibody per reaction)</li>
<li>Batch-specific data is available on the website</li>
<li>Expert technical support</li>
<li>Sample sizes available</li>
<li>100% satisfaction guarantee</li>
</ul>',
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'meta_description' => 'Diagenode Offers Strict quality standards with Rigorous QC and validated Antibodies. Classified based on level of validation for flexibility of Application. Comprehensive selection of histone and non-histone Antibodies',
'meta_title' => 'Diagenode's selection of Antibodies is exclusively dedicated for Epigenetic Research | Diagenode',
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(int) 1 => array(
'id' => '122',
'position' => '1',
'parent_id' => '100',
'name' => '<em>S. pyogenes</EM> CRISPR/Cas9 antibodies',
'description' => '<p>Diagenode offers the broad range of antibodies raised against the N- or C-terminus of the Cas9 nuclease from <em>Streptococcus <g class="gr_ gr_5 gr-alert gr_spell gr_disable_anim_appear ContextualSpelling ins-del multiReplace" id="5" data-gr-id="5">pyogenes</g></em>. These highly specific polyclonal and monoclonal antibodies are validated in Western blot, immunoprecipitation, immunofluorescence and in chromatin immunoprecipitation.</p>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h2><em><a name="pyogenes"></a>S. pyogenes</em> CRISPR/Cas9 antibodies<a></a></h2>
<div class="panel">
<h2>Discover our first monoclonal CRISPR/Cas9 antibody validated in ChIP<br /><br /></h2>
<div class="row">
<div class="small-5 medium-5 large-5 columns"><img src="/img/landing-pages/crispr-cas9-chip-on-hih3t3.jpg" alt="" /></div>
<div class="small-7 medium-7 large-7 columns">
<ul>
<li>Validated in chromatin immunoprecipitation</li>
<li>Performs better than FLAG antibody</li>
<li>Excellent for WB, IF and IP</li>
</ul>
<p><small><strong>ChIP</strong> was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50 µg chromatin was incubated overnight at 4°C with 5 or 10 µg of either an anti-FLAG antibody or the Diagenode antibody against Cas9 (Cat. No. C15200229). Mouse IgG was used as a negative IP control. qPCR was performed with primers specific for the GFP gene, and for a non-targeted region (protein kinase C delta, Prkcd), used as negative control. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).</small></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns text-right"><a href="/p/crispr-cas9-monoclonal-antibody-50-ug-25-μl" class="tiny details button radius">Learn more</a></div>
</div>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>First ChIP-grade CRISPR/Cas9 polyclonal antibody</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/landing-pages/c_a_s9-chip-grade-antibody.png" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Excellent polyclonal antibody for chromatin immunoprecipitation</li>
<li>Optimized for highest ChIP specificity and yields</li>
<li>Validated for all applications including immunoblotting, immunofluorescence and western blot</li>
</ul>
<p><small><strong>ChIP</strong> was performed on NIH3T3 cells stably expressing GFP- H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50 μg chromatin was incubated with either 5 μg of an anti-FLAG antibody or 2 μl of the Diagenode antibody against Cas9. The pre-immune serum (PPI) was used as negative IP control. Then qPCR was performed with primers specific for the GFP gene, and for two non-targeted regions: Ppap2c and Prkcd, used as negative controls. This figure shows the recovery, expressed as a % of input.</small></p>
<p class="text-right"><a href="../p/crispr-cas9-polyclonal-antibody" class="details tiny button">Learn more</a></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>CRISPR/Cas9 monoclonal antibody 4G10</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/landing-pages/cas9_4g10_fig1.png" width="170" height="302" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Antibody raised against N-terminus of Cas9 nuclease</li>
<li>Validated for western blot, IP and immunofluorescence</li>
</ul>
<p><small><strong>Immunofluorescence</strong>: Hela cells were transiently transfected with a Cas9 expression vector. The cells were fixed in 3.7% formaldehyde, permeabilized in 0.5% Triton-X-100 and blocked in PBS containing 2% BSA. The cells were stained with the Cas9 antibody at 4°C o/n, followed by incubation with an anti mouse secondary antibody coupled to AF488 for 1 h at RT. Nuclei were counter-stained with Hoechst 33342 (right).</small></p>
<p class="text-right"><a href="../p/crispr-cas9-monoclonal-antibody-4g10-50-ug" class="details tiny button">Learn more</a></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>CRISPR/Cas9 C-terminal monoclonal antibody</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/product/antibodies/C15200223-IP.png" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Antibody raised against C-terminus of Cas9 nuclease</li>
<li>Validated for western blot, IP and immunofluorescence</li>
</ul>
<p><small><strong> Western blot</strong> was performed on 20 μg protein extracts from Cas9 expressing HeLa cells (lane 1) and on negative control HeLa cells (lane 2) with the Diagenode antibody against Cas9. The antibody was diluted 1:4,000. The marker is shown on the left, position of the Cas9 protein is indicated on the right. </small></p>
<p class="text-right"><a href="../p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug" class="details tiny button">Learn more</a></p>
</div>
<div class="small-12 medium-12 large-12 columns">
<h3>Which CRISPR/Cas9 antibody is the best for your application?</h3>
<a name="table"></a>
<table>
<thead>
<tr>
<th>Antibody</th>
<th>WB</th>
<th>IF</th>
<th>IP</th>
<th>ChIP</th>
<th>Antibody raised against</th>
</tr>
</thead>
<tbody>
<tr>
<td><a href="../p/crispr-cas9-monoclonal-antibody-50-ug-25-μl"><strong class="diacol">CRISPR/Cas9 monoclonal antibody</strong></a></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><span class="diacol">N-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-polyclonal-antibody">CRISPR/Cas9 polyclonal antibody</a></td>
<td>++</td>
<td>++</td>
<td>++</td>
<td><strong class="diacol">+++</strong></td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-monoclonal-antibody-4g10-50-ug">CRISPR/Cas9 monoclonal antibody 4G10</a></td>
<td>+++</td>
<td>+++</td>
<td>++</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="../p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug-23-ul"><strong class="diacol">CRISPR/Cas9 C-terminal monoclonal antibody</strong></a> <span class="label alert" style="font-size: 0.9rem;">NEW!</span></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">no</strong></td>
<td><strong class="diacol">+</strong></td>
<td><span class="diacol">C-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug">CRISPR/Cas9 C-terminal monoclonal antibody</a></td>
<td>++</td>
<td>++</td>
<td>+</td>
<td>no</td>
<td><span class="diacol">C-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-monoclonal-antibody-7A9-50-mg">CRISPR/Cas9 monoclonal antibody 7A9</a></td>
<td>++</td>
<td>++</td>
<td>++</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-hrp-monoclonal-antibody-50-ul">CRISPR/Cas9 - HRP monoclonal antibody 7A9</a></td>
<td>+++</td>
<td>no</td>
<td>no</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
</tbody>
</table>
</div>
</div>
</div>',
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'slug' => 's-pyogenes-crispr-cas9-antibodies',
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'meta_description' => 'S.pyogenes CRISPR/Cas9 antibodies',
'meta_title' => 'S.pyogenes CRISPR/Cas9 antibodies',
'modified' => '2018-03-29 11:58:22',
'created' => '2016-09-09 10:39:20',
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(int) 0 => array(
'id' => '11',
'name' => 'Antibodies you can trust',
'description' => '<p style="text-align: justify;"><span>Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of large sets of genome-wide sequencing data. ChIP sequencing (ChIP-seq) has now become the gold standard method for chromatin studies, given the accuracy and coverage scale of the approach over other methods. Successful ChIP-seq, however, requires a higher level of experimental accuracy and consistency in all steps of ChIP than ever before. Particularly crucial is the quality of ChIP antibodies. </span></p>',
'image_id' => null,
'type' => 'Poster',
'url' => 'files/posters/Antibodies_you_can_trust_Poster.pdf',
'slug' => 'antibodies-you-can-trust-poster',
'meta_keywords' => '',
'meta_description' => '',
'modified' => '2015-10-01 20:18:31',
'created' => '2015-07-03 16:05:15',
'ProductsDocument' => array(
[maximum depth reached]
)
),
(int) 1 => array(
'id' => '38',
'name' => 'Epigenetic Antibodies Brochure',
'description' => '<p>More than in any other immuoprecipitation assays, quality antibodies are critical tools in many epigenetics experiments. Since 10 years, Diagenode has developed the most stringent quality production available on the market for antibodies exclusively focused on epigenetic uses. All our antibodies have been qualified to work in epigenetic applications.</p>',
'image_id' => null,
'type' => 'Brochure',
'url' => 'files/brochures/Epigenetic_Antibodies_Brochure.pdf',
'slug' => 'epigenetic-antibodies-brochure',
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'meta_description' => '',
'modified' => '2016-06-15 11:24:06',
'created' => '2015-07-03 16:05:27',
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[maximum depth reached]
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(int) 2 => array(
'id' => '384',
'name' => 'Datasheet CRISPR/Cas9 monoclonal antibody',
'description' => '<p>This antibody has been raised against the N-terminal region of the CRISP/Cas9 protein.</p>',
'image_id' => null,
'type' => 'Datasheet',
'url' => 'files/products/antibodies/Datasheet_CRISPR_Cas9_monoclonal_antibody.pdf',
'slug' => 'datasheet-crispr-cas9-monoclonal-antibody',
'meta_keywords' => '',
'meta_description' => '',
'modified' => '2019-05-15 11:20:01',
'created' => '2015-07-07 11:47:44',
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[maximum depth reached]
)
),
(int) 3 => array(
'id' => '991',
'name' => ' Accurate QC to optimize CRISPR/Cas9 genome editing specificity',
'description' => '<p>The CRISPR/Cas9 technology is delivering superior genetic models for fundamental disease research, drug screening, therapy development, rapid diagnostics, and transcriptional modulation. Although CRISPR/Cas9 enables rapid genome editing, several aspects affect its efficiency and specificity including guide RNA design, delivery methods, and off-targets effects. Diagenode has developed strategies to overcome these common pitfalls and has optimized CRISPR/Cas9 genome editing specificity</p>',
'image_id' => null,
'type' => 'Poster',
'url' => 'files/posters/CRISPR-Cas9-Poster-Accurate_QC.pdf',
'slug' => 'crispr-cas9-accurate-qc',
'meta_keywords' => '',
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'modified' => '2018-02-12 15:36:31',
'created' => '2018-02-12 13:15:37',
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'Feature' => array(),
'Image' => array(
(int) 0 => array(
'id' => '1782',
'name' => 'product/antibodies/cas9-icon.png',
'alt' => 'CRISPR/Cas9 Antibody ',
'modified' => '2020-11-27 07:01:20',
'created' => '2018-03-15 15:53:46',
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'Publication' => array(
(int) 0 => array(
'id' => '4953',
'name' => 'Loss of tumor suppressors promotes inflammatory tumor microenvironment and enhances LAG3+T cell mediated immune suppression',
'authors' => 'Zahraeifard, S. et al.',
'description' => '<p><span>Low response rate, treatment relapse, and resistance remain key challenges for cancer treatment with immune checkpoint blockade (ICB). Here we report that loss of specific tumor suppressors (TS) induces an inflammatory response and promotes an immune suppressive tumor microenvironment. Importantly, low expression of these TSs is associated with a higher expression of immune checkpoint inhibitory mediators. Here we identify, by using in vivo CRISPR/Cas9 based loss-of-function screening, that NF1, TSC1, and TGF-β RII as TSs regulating immune composition. Loss of each of these three TSs leads to alterations in chromatin accessibility and enhances IL6-JAK3-STAT3/6 inflammatory pathways. This results in an immune suppressive landscape, characterized by increased numbers of LAG3+ CD8 and CD4 T cells. ICB targeting LAG3 and PD-L1 simultaneously inhibits metastatic progression in preclinical triple negative breast cancer (TNBC) mouse models of NF1-, TSC1- or TGF-β RII- deficient tumors. Our study thus reveals a role of TSs in regulating metastasis via non-cell-autonomous modulation of the immune compartment and provides proof-of-principle for ICB targeting LAG3 for patients with NF1-, TSC1- or TGF-β RII-inactivated cancers.</span></p>',
'date' => '2024-07-12',
'pmid' => 'https://www.nature.com/articles/s41467-024-50262-8',
'doi' => 'https://doi.org/10.1038/s41467-024-50262-8',
'modified' => '2024-07-29 10:47:10',
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'id' => '4784',
'name' => 'A kinesin-based approach for inducing chromosome-specific mis-segregationin human cells.',
'authors' => 'Truong M.A. et al.',
'description' => '<p>Various cancer types exhibit characteristic and recurrent aneuploidy patterns. The origins of these cancer type-specific karyotypes are still unknown, partly because introducing or eliminating specific chromosomes in human cells still poses a challenge. Here, we describe a novel strategy to induce mis-segregation of specific chromosomes in different human cell types. We employed Tet repressor or nuclease-dead Cas9 to link a microtubule minus-end-directed kinesin (Kinesin14VIb) from Physcomitrella patens to integrated Tet operon repeats and chromosome-specific endogenous repeats, respectively. By live- and fixed-cell imaging, we observed poleward movement of the targeted loci during (pro)metaphase. Kinesin14VIb-mediated pulling forces on the targeted chromosome were counteracted by forces from kinetochore-attached microtubules. This tug-of-war resulted in chromosome-specific segregation errors during anaphase and revealed that spindle forces can heavily stretch chromosomal arms. By single-cell whole-genome sequencing, we established that kinesin-induced targeted mis-segregations predominantly result in chromosomal arm aneuploidies after a single cell division. Our kinesin-based strategy opens the possibility to investigate the immediate cellular responses to specific aneuploidies in different cell types; an important step toward understanding how tissue-specific aneuploidy patterns evolve.</p>',
'date' => '2023-04-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/37038978',
'doi' => '10.15252/embj.2022111559',
'modified' => '2023-06-13 09:21:25',
'created' => '2023-05-05 12:34:24',
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(int) 2 => array(
'id' => '4344',
'name' => 'MICAL1 regulates actin cytoskeleton organization, directional cellmigration and the growth of human breast cancer cells as orthotopicxenograft tumours.',
'authors' => 'McGarry David J et al.',
'description' => '<p>The Molecule Interacting with CasL 1 (MICAL1) monooxygenase has emerged as an important regulator of cytoskeleton organization via actin oxidation. Although filamentous actin (F-actin) increases MICAL1 monooxygenase activity, hydrogen peroxide (HO) is also generated in the absence of F-actin, suggesting that diffusible HO might have additional functions. MICAL1 gene disruption by CRISPR/Cas9 in MDA MB 231 human breast cancer cells knocked out (KO) protein expression, which affected F-actin organization, cell size and motility. Transcriptomic profiling revealed that MICAL1 deletion significantly affected the expression of over 700 genes, with the majority being reduced in their expression levels. In addition, the absolute magnitudes of reduced gene expression were significantly greater than the magnitudes of increased gene expression. Gene set enrichment analysis (GSEA) identified receptor regulator activity as the most significant negatively enriched molecular function gene set. The prominent influence exerted by MICAL1 on F-actin structures was also associated with changes in the expression of several serum-response factor (SRF) regulated genes in KO cells. Moreover, MICAL1 disruption attenuated breast cancer tumour growth in vivo. Elevated MICAL1 gene expression was observed in invasive breast cancer samples from human patients relative to normal tissue, while MICAL1 amplification or point mutations were associated with reduced progression free survival. Collectively, these results demonstrate that MICAL1 gene disruption altered cytoskeleton organization, cell morphology and migration, gene expression, and impaired tumour growth in an orthotopic in vivo breast cancer model, suggesting that pharmacological MICAL1 inhibition could have therapeutic benefits for cancer patients.</p>',
'date' => '2021-10-01',
'pmid' => 'https://doi.org/10.1016%2Fj.canlet.2021.07.039',
'doi' => '10.1016/j.canlet.2021.07.039',
'modified' => '2022-06-21 16:56:09',
'created' => '2022-05-19 10:41:50',
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(int) 3 => array(
'id' => '3925',
'name' => 'CRISPR-based gene knockout screens reveal deubiquitinases involved in HIV-1 latency in two Jurkat cell models.',
'authors' => 'Rathore A, Iketani S, Wang P, Jia M, Sahi V, Ho DD',
'description' => '<p>The major barrier to a HIV-1 cure is the persistence of latent genomes despite treatment with antiretrovirals. To investigate host factors which promote HIV-1 latency, we conducted a genome-wide functional knockout screen using CRISPR-Cas9 in a HIV-1 latency cell line model. This screen identified IWS1, POLE3, POLR1B, PSMD1, and TGM2 as potential regulators of HIV-1 latency, of which PSMD1 and TMG2 could be confirmed pharmacologically. Further investigation of PSMD1 revealed that an interacting enzyme, the deubiquitinase UCH37, was also involved in HIV-1 latency. We therefore conducted a comprehensive evaluation of the deubiquitinase family by gene knockout, identifying several deubiquitinases, UCH37, USP14, OTULIN, and USP5 as possible HIV-1 latency regulators. A specific inhibitor of USP14, IU1, reversed HIV-1 latency and displayed synergistic effects with other latency reversal agents. IU1 caused degradation of TDP-43, a negative regulator of HIV-1 transcription. Collectively, this study is the first comprehensive evaluation of deubiquitinases in HIV-1 latency and establishes that they may hold a critical role.</p>',
'date' => '2020-03-24',
'pmid' => 'http://www.pubmed.gov/32210344',
'doi' => '10.1038/s41598-020-62375-3',
'modified' => '2020-08-17 10:51:24',
'created' => '2020-08-10 12:12:25',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 4 => array(
'id' => '3821',
'name' => 'DAPK1 loss triggers tumor invasion in colorectal tumor cells.',
'authors' => 'Steinmann S, Kunze P, Hampel C, Eckstein M, Bertram Bramsen J, Muenzner JK, Carlé B, Ndreshkjana B, Kemenes S, Gasparini P, Friedrich O, Andersen C, Geppert C, Wang S, Eyupoglu I, Bäuerle T, Hartmann A, Schneider-Stock R',
'description' => '<p>Colorectal cancer (CRC) is one of the leading cancer-related causes of death worldwide. Despite the improvement of surgical and chemotherapeutic treatments, as of yet, the disease has not been overcome due to metastasis to distant organs. Hence, it is of great relevance to understand the mechanisms responsible for metastasis initiation and progression and to identify novel metastatic markers for a higher chance of preventing the metastatic disease. The Death-associated protein kinase 1 (DAPK1), recently, has been shown to be a potential candidate for regulating metastasis in CRC. Hence, the aim of the study was to investigate the impact of DAPK1 protein on CRC aggressiveness. Using CRISPR/Cas9 technology, we generated DAPK1-deficient HCT116 monoclonal cell lines and characterized their knockout phenotype in vitro and in vivo. We show that loss of DAPK1 implemented changes in growth pattern and enhanced tumor budding in vivo in the chorioallantoic membrane (CAM) model. Further, we observed more tumor cell dissemination into chicken embryo organs and increased invasion capacity using rat brain 3D in vitro model. The novel identified DAPK1-loss gene expression signature showed a stroma typical pattern and was associated with a gained ability for remodeling the extracellular matrix. Finally, we suggest the DAPK1-ERK1 signaling axis being involved in metastatic progression of CRC. Our results highlight DAPK1 as an anti-metastatic player in CRC and suggest DAPK1 as a potential predictive biomarker for this cancer type.</p>',
'date' => '2019-11-26',
'pmid' => 'http://www.pubmed.gov/31772156',
'doi' => '10.1038/s41419-019-2122-z',
'modified' => '2020-02-25 13:45:15',
'created' => '2020-02-13 10:02:44',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(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',
'created' => '2019-06-06 12:11:18',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 6 => array(
'id' => '3565',
'name' => 'Can Mitochondrial DNA be CRISPRized: Pro and Contra.',
'authors' => 'Loutre R, Heckel AM, Smirnova A, Entelis N, Tarassov I',
'description' => '<p>Mitochondria represent a chimera of macromolecules encoded either in the organellar genome, mtDNA, or in the nuclear one. If the pathway of protein targeting to different sub-compartments of mitochondria was relatively well studied, import of small noncoding RNAs into mammalian mitochondria still awaits mechanistic explanations and its functional issues are often not understood thus raising polemics. At the same time, RNA mitochondrial import pathway has an obvious attractiveness as it appears as a unique natural mechanism permitting to address nucleic acids into the organelles. Deciphering the function(s) of imported RNAs inside the mitochondria is extremely complicated due to their relatively low abundance, which suggests their regulatory role. We previously demonstrated that mitochondrial targeting of small noncoding RNAs able to specifically anneal with the mutant mitochondrial DNA led to a decrease of the mtDNA heteroplasmy level by inhibiting mutant mtDNA replication. We then demonstrated that increasing level of expression of such antireplicative recombinant RNAs increases significantly the antireplicative effect. In this report, we present a new data investigating the possibility to establish a CRISPR-Cas9 system targeting mtDNA exploiting of the pathway of RNA import into mitochondria. Mitochondrially addressed Cas9 versions and a set of mitochondrially targeted guide RNAs were tested in vitro and in vivo and their effect on mtDNA copy number was demonstrated. So far, the system appeared as more complicated for use than previously found for nuclear DNA, because only application of a pair of guide RNAs produced the effect of mtDNA depletion. We discuss, in a critical way, these results and put them in a broader context of polemics concerning the possibilities of manipulation of mtDNA in mammalians. The findings described here prove the potential of the RNA import pathway as a tool for studying mtDNA and for future therapy of mitochondrial disorders. © The Authors. IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 70(12):1233-1239, 2018.</p>',
'date' => '2018-12-01',
'pmid' => 'http://www.pubmed.gov/30184317',
'doi' => '10.1002/iub.1919',
'modified' => '2019-03-21 17:20:49',
'created' => '2019-03-21 14:12:08',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 7 => array(
'id' => '3268',
'name' => 'In trans paired nicking triggers seamless genome editing without double-stranded DNA cutting',
'authors' => 'Chen X. et al.',
'description' => '<p>Precise genome editing involves homologous recombination between donor DNA and chromosomal sequences subjected to double-stranded DNA breaks made by programmable nucleases. Ideally, genome editing should be efficient, specific, and accurate. However, besides constituting potential translocation-initiating lesions, double-stranded DNA breaks (targeted or otherwise) are mostly repaired through unpredictable and mutagenic non-homologous recombination processes. Here, we report that the coordinated formation of paired single-stranded DNA breaks, or nicks, at donor plasmids and chromosomal target sites by RNA-guided nucleases based on CRISPR-Cas9 components, triggers seamless homology-directed gene targeting of large genetic payloads in human cells, including pluripotent stem cells. Importantly, in addition to significantly reducing the mutagenicity of the genome modification procedure, this in trans paired nicking strategy achieves multiplexed, single-step, gene targeting, and yields higher frequencies of accurately edited cells when compared to the standard double-stranded DNA break-dependent approach.</p>',
'date' => '2017-09-22',
'pmid' => 'https://www.nature.com/articles/s41467-017-00687-1',
'doi' => '',
'modified' => '2017-10-09 16:27:58',
'created' => '2017-10-09 16:27:58',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 8 => array(
'id' => '3244',
'name' => 'Highly efficient gene inactivation by adenoviral CRISPR/Cas9 in human primary cells',
'authors' => 'Voets O. et al.',
'description' => '<p>Phenotypic assays using human primary cells are highly valuable tools for target discovery and validation in drug discovery. Expression knockdown (KD) of such targets in these assays allows the investigation of their role in models of disease processes. Therefore, efficient and fast modes of protein KD in phenotypic assays are required. The CRISPR/Cas9 system has been shown to be a versatile and efficient means of gene inactivation in immortalized cell lines. Here we describe the use of adenoviral (AdV) CRISPR/Cas9 vectors for efficient gene inactivation in two human primary cell types, normal human lung fibroblasts and human bronchial epithelial cells. The effects of gene inactivation were studied in the TGF-β-induced fibroblast to myofibroblast transition assay (FMT) and the epithelial to mesenchymal transition assay (EMT), which are SMAD3 dependent and reflect pathogenic mechanisms observed in fibrosis. Co-transduction (co-TD) of AdV Cas9 with SMAD3-targeting guide RNAs (gRNAs) resulted in fast and efficient genome editing judged by insertion/deletion (indel) formation, as well as significant reduction of SMAD3 protein expression and nuclear translocation. This led to phenotypic changes downstream of SMAD3 inhibition, including substantially decreased alpha smooth muscle actin and fibronectin 1 expression, which are markers for FMT and EMT, respectively. A direct comparison between co-TD of separate Cas9 and gRNA AdV, versus TD with a single "all-in-one" Cas9/gRNA AdV, revealed that both methods achieve similar levels of indel formation. These data demonstrate that AdV CRISPR/Cas9 is a useful and efficient tool for protein KD in human primary cell phenotypic assays. The use of AdV CRISPR/Cas9 may offer significant advantages over the current existing tools and should enhance target discovery and validation opportunities.</p>',
'date' => '2017-08-11',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/28800587',
'doi' => '',
'modified' => '2017-09-19 17:26:46',
'created' => '2017-09-19 17:26:46',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 9 => array(
'id' => '3040',
'name' => 'Noncoding somatic and inherited single-nucleotide variants converge to promote ESR1 expression in breast cancer',
'authors' => 'Bailey SD et al.',
'description' => '<p>Sustained expression of the estrogen receptor-<span class="mb">α</span> (ESR1) drives two-thirds of breast cancer and defines the ESR1-positive subtype. ESR1 engages enhancers upon estrogen stimulation to establish an oncogenic expression program<sup><a href="http://www.nature.com/ng/journal/v48/n10/full/ng.3650.html#ref1" title="Green, K.A. & Carroll, J.S. Oestrogen-receptor-mediated transcription and the influence of co-factors and chromatin state. Nat. Rev. Cancer 7, 713-722 (2007)." id="ref-link-5">1</a></sup>. Somatic copy number alterations involving the <i>ESR1</i> gene occur in approximately 1% of ESR1-positive breast cancers<sup><a href="http://www.nature.com/ng/journal/v48/n10/full/ng.3650.html#ref2" title="Vincent-Salomon, A., Raynal, V., Lucchesi, C., Gruel, N. & Delattre, O. ESR1 gene amplification in breast cancer: a common phenomenon? Nat. Genet. 40, 809, author reply 810-812 (2008)." id="ref-link-6">2</a>, <a href="http://www.nature.com/ng/journal/v48/n10/full/ng.3650.html#ref3" title="Brown, L.A. et al. ESR1 gene amplification in breast cancer: a common phenomenon? Nat. Genet. 40, 806-807, author reply 810-812 (2008)." id="ref-link-7">3</a>, <a href="http://www.nature.com/ng/journal/v48/n10/full/ng.3650.html#ref4" title="Horlings, H.M. et al. ESR1 gene amplification in breast cancer: a common phenomenon? Nat. Genet. 40, 807-808, author reply 810-812 (2008)." id="ref-link-8">4</a>, <a href="http://www.nature.com/ng/journal/v48/n10/full/ng.3650.html#ref5" title="Reis-Filho, J.S. et al. ESR1 gene amplification in breast cancer: a common phenomenon? Nat. Genet. 40, 809-810, author reply 810-812 (2008)." id="ref-link-9">5</a></sup>, suggesting that other mechanisms underlie the persistent expression of <i>ESR1</i>. We report significant enrichment of somatic mutations within the set of regulatory elements (SRE) regulating <i>ESR1</i> in 7% of ESR1-positive breast cancers. These mutations regulate <i>ESR1</i> expression by modulating transcription factor binding to the DNA. The SRE includes a recurrently mutated enhancer whose activity is also affected by rs9383590, a functional inherited single-nucleotide variant (SNV) that accounts for several breast cancer risk–associated loci. Our work highlights the importance of considering the combinatorial activity of regulatory elements as a single unit to delineate the impact of noncoding genetic alterations on single genes in cancer.</p>',
'date' => '2016-08-29',
'pmid' => 'http://www.nature.com/ng/journal/v48/n10/full/ng.3650.html',
'doi' => '',
'modified' => '2016-11-03 11:59:03',
'created' => '2016-10-07 11:08:31',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 10 => array(
'id' => '3038',
'name' => 'The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing.',
'authors' => 'de Solis CA et al.',
'description' => '<p>The RNA-guided Cas9 nuclease, from the type II prokaryotic Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) adaptive immune system, has been adapted and utilized by scientists to edit the genomes of eukaryotic cells. Here, we report the development of a viral mediated CRISPR/Cas9 system that can be rendered inducible utilizing doxycycline (Dox) and can be delivered to cells in vitro and in vivo utilizing adeno-associated virus (AAV). Specifically, we developed an inducible gRNA (gRNAi) AAV vector that is designed to express the gRNA from a H1/TO promoter. This AAV vector is also designed to express the Tet repressor (TetR) to regulate the expression of the gRNAi in a Dox dependent manner. We show that H1/TO promoters of varying length and a U6/TO promoter can edit DNA with similar efficiency in vitro, in a Dox dependent manner. We also demonstrate that our inducible gRNAi vector can be used to edit the genomes of neurons in vivo within the mouse brain in a Dox dependent manner. Genome editing can be induced in vivo with this system by supplying animals Dox containing food for as little as 1 day. This system might be cross compatible with many existing S. pyogenes Cas9 systems (i.e., Cas9 mouse, CRISPRi, etc.), and therefore it likely can be used to render these systems inducible as well.</p>',
'date' => '2016-08-18',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/27587996',
'doi' => '',
'modified' => '2016-10-05 15:48:14',
'created' => '2016-10-05 15:48:14',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 11 => 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(
[maximum depth reached]
)
),
(int) 12 => array(
'id' => '2823',
'name' => 'A geminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing',
'authors' => 'Yin K, Han T, Liu G, Chen T, Wang Y, Yu AY, Liu Y',
'description' => '<p>CRISPR/Cas has emerged as potent genome editing technology and has successfully been applied in many organisms, including several plant species. However, delivery of genome editing reagents remains a challenge in plants. Here, we report a <u>vi</u>rus-based guide RNA (gRNA) delivery system for CRISPR/Cas9 mediated plant <u>g</u>enome <u>e</u>diting (VIGE) that can be used to precisely target genome locations and cause mutations. VIGE is performed by using a modified Cabbage Leaf Curl virus (CaLCuV) vector to express gRNAs in stable transgenic plants expressing Cas9. DNA sequencing confirmed VIGE of endogenous <i>NbPDS3</i> and <i>NbIspH</i> genes in non-inoculated leaves because CaLCuV can infect plants systemically. Moreover, VIGE of <i>NbPDS3</i> and <i>NbIspH</i> in newly developed leaves caused photo-bleached phenotype. These results demonstrate that geminivirus-based VIGE could be a powerful tool in plant genome editing.</p>',
'date' => '2015-10-09',
'pmid' => 'http://www.ncbi.nlm.nih.gov/pubmed/26450012',
'doi' => '10.1038/srep14926',
'modified' => '2016-02-16 13:55:32',
'created' => '2016-02-16 13:55:32',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 13 => array(
'id' => '2765',
'name' => 'Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection.',
'authors' => 'Liang X, Potter J, Kumar S, Zou Y, Quintanilla R, Sridharan M, Carte J, Chen W, Roark N, Ranganathan S, Ravinder N, Chesnut JD',
'description' => '<p>CRISPR-Cas9 systems provide a platform for high efficiency genome editing that are enabling innovative applications of mammalian cell engineering. However, the delivery of Cas9 and synthesis of guide RNA (gRNA) remain as steps that can limit overall efficiency and ease of use. Here we describe methods for rapid synthesis of gRNA and for delivery of Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs) into a variety of mammalian cells through liposome-mediated transfection or electroporation. Using these methods, we report nuclease-mediated indel rates of up to 94% in Jurkat T cells and 87% in induced pluripotent stem cells (iPSC) for a single target. When we used this approach for multigene targeting in Jurkat cells we found that two-locus and three-locus indels were achieved in approximately 93% and 65% of the resulting isolated cell lines, respectively. Further, we found that the off-target cleavage rate is reduced using Cas9 protein when compared to plasmid DNA transfection. Taken together, we present a streamlined cell engineering workflow that enables gRNA design to analysis of edited cells in as little as four days and results in highly efficient genome modulation in hard-to-transfect cells. The reagent preparation and delivery to cells is amenable to high throughput, multiplexed genome-wide cell engineering.</p>',
'date' => '2015-08-20',
'pmid' => 'http://www.ncbi.nlm.nih.gov/pubmed/26003884',
'doi' => '10.1016/j.jbiotec.2015.04.024',
'modified' => '2016-09-21 16:28:13',
'created' => '2015-07-24 15:39:05',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 14 => array(
'id' => '2792',
'name' => 'A localized nucleolar DNA damage response facilitates recruitment of the homology-directed repair machinery independent of cell cycle stage.',
'authors' => 'van Sluis M, McStay B',
'description' => 'DNA double-strand breaks (DSBs) are repaired by two main pathways: nonhomologous end-joining and homologous recombination (HR). Repair pathway choice is thought to be determined by cell cycle timing and chromatin context. Nucleoli, prominent nuclear subdomains and sites of ribosome biogenesis, form around nucleolar organizer regions (NORs) that contain rDNA arrays located on human acrocentric chromosome p-arms. Actively transcribed rDNA repeats are positioned within the interior of the nucleolus, whereas sequences proximal and distal to NORs are packaged as heterochromatin located at the nucleolar periphery. NORs provide an opportunity to investigate the DSB response at highly transcribed, repetitive, and essential loci. Targeted introduction of DSBs into rDNA, but not abutting sequences, results in ATM-dependent inhibition of their transcription by RNA polymerase I. This is coupled with movement of rDNA from the nucleolar interior to anchoring points at the periphery. Reorganization renders rDNA accessible to repair factors normally excluded from nucleoli. Importantly, DSBs within rDNA recruit the HR machinery throughout the cell cycle. Additionally, unscheduled DNA synthesis, consistent with HR at damaged NORs, can be observed in G1 cells. These results suggest that HR can be templated in cis and suggest a role for chromosomal context in the maintenance of NOR genomic stability.',
'date' => '2015-06-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/26019174',
'doi' => '',
'modified' => '2015-07-24 15:39:05',
'created' => '2015-07-24 15:39:05',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 15 => array(
'id' => '2645',
'name' => 'Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis.',
'authors' => 'Chen S, Sanjana NE, Zheng K, Shalem O, Lee K, Shi X, Scott DA, Song J, Pan JQ, Weissleder R, Lee H, Zhang F, Sharp PA',
'description' => 'Genetic screens are powerful tools for identifying genes responsible for diverse phenotypes. Here we describe a genome-wide CRISPR/Cas9-mediated loss-of-function screen in tumor growth and metastasis. We mutagenized a non-metastatic mouse cancer cell line using a genome-scale library with 67,405 single-guide RNAs (sgRNAs). The mutant cell pool rapidly generates metastases when transplanted into immunocompromised mice. Enriched sgRNAs in lung metastases and late-stage primary tumors were found to target a small set of genes, suggesting that specific loss-of-function mutations drive tumor growth and metastasis. Individual sgRNAs and a small pool of 624 sgRNAs targeting the top-scoring genes from the primary screen dramatically accelerate metastasis. In all of these experiments, the effect of mutations on primary tumor growth positively correlates with the development of metastases. Our study demonstrates Cas9-based screening as a robust method to systematically assay gene phenotypes in cancer evolution in vivo.',
'date' => '2015-03-12',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/25748654',
'doi' => '',
'modified' => '2015-07-24 15:39:05',
'created' => '2015-07-24 15:39:05',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 16 => array(
'id' => '2679',
'name' => 'Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells.',
'authors' => 'Liao HK, Gu Y, Diaz A, Marlett J, Takahashi Y, Li M, Suzuki K, Xu R, Hishida T, Chang CJ, Esteban CR, Young J, Izpisua Belmonte JC',
'description' => '<p>To combat hostile viruses, bacteria and archaea have evolved a unique antiviral defense system composed of clustered regularly interspaced short palindromic repeats (CRISPRs), together with CRISPR-associated genes (Cas). The CRISPR/Cas9 system develops an adaptive immune resistance to foreign plasmids and viruses by creating site-specific DNA double-stranded breaks (DSBs). Here we adapt the CRISPR/Cas9 system to human cells for intracellular defense against foreign DNA and viruses. Using HIV-1 infection as a model, our results demonstrate that the CRISPR/Cas9 system disrupts latently integrated viral genome and provides long-term adaptive defense against new viral infection, expression and replication in human cells. We show that engineered human-induced pluripotent stem cells stably expressing HIV-targeted CRISPR/Cas9 can be efficiently differentiated into HIV reservoir cell types and maintain their resistance to HIV-1 challenge. These results unveil the potential of the CRISPR/Cas9 system as a new therapeutic strategy against viral infections.</p>',
'date' => '2015-03-10',
'pmid' => 'http://www.ncbi.nlm.nih.gov/pubmed/25752527',
'doi' => '',
'modified' => '2016-09-21 16:29:13',
'created' => '2015-07-24 15:39:05',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 17 => array(
'id' => '2637',
'name' => 'An inducible lentiviral guide RNA platform enables the identification of tumor-essential genes and tumor-promoting mutations in vivo.',
'authors' => 'Aubrey BJ, Kelly GL, Kueh AJ, Brennan MS, O'Connor L, Milla L, Wilcox S, Tai L, Strasser A, Herold MJ',
'description' => '<p>The CRISPR/Cas9 technology enables the introduction of genomic alterations into almost any organism; however, systems for efficient and inducible gene modification have been lacking, especially for deletion of essential genes. Here, we describe a drug-inducible small guide RNA (sgRNA) vector system allowing for ubiquitous and efficient gene deletion in murine and human cells. This system mediates the efficient, temporally controlled deletion of MCL-1, both in vitro and in vivo, in human Burkitt lymphoma cell lines that require this anti-apoptotic BCL-2 protein for sustained survival and growth. Unexpectedly, repeated induction of the same sgRNA generated similar inactivating mutations in the human Mcl-1 gene due to low mutation variability exerted by the accompanying non-homologous end-joining (NHEJ) process. Finally, we were able to generate hematopoietic cell compartment-restricted Trp53-knockout mice, leading to the identification of cancer-promoting mutants of this critical tumor suppressor.</p>',
'date' => '2015-03-03',
'pmid' => 'http://www.ncbi.nlm.nih.gov/pubmed/25732831',
'doi' => '',
'modified' => '2016-09-21 16:26:37',
'created' => '2015-07-24 15:39:05',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 18 => array(
'id' => '2961',
'name' => 'An inducible lentiviral guide RNA platform enables the identification of tumor-essential genes and tumor-promoting mutations in vivo',
'authors' => 'Aubrey BJ et al.',
'description' => '<p>The CRISPR/Cas9 technology enables the introduction of genomic alterations into almost any organism; however, systems for efficient and inducible gene modification have been lacking, especially for deletion of essential genes. Here, we describe a drug-inducible small guide RNA (sgRNA) vector system allowing for ubiquitous and efficient gene deletion in murine and human cells. This system mediates the efficient, temporally controlled deletion of MCL-1, both in vitro and in vivo, in human Burkitt lymphoma cell lines that require this anti-apoptotic BCL-2 protein for sustained survival and growth. Unexpectedly, repeated induction of the same sgRNA generated similar inactivating mutations in the human Mcl-1 gene due to low mutation variability exerted by the accompanying non-homologous end-joining (NHEJ) process. Finally, we were able to generate hematopoietic cell compartment-restricted Trp53-knockout mice, leading to the identification of cancer-promoting mutants of this critical tumor suppressor.</p>',
'date' => '2015-03-03',
'pmid' => 'http://www.ncbi.nlm.nih.gov/pubmed/25732831',
'doi' => '10.1016/j.celrep.2015.02.002',
'modified' => '2016-06-23 11:21:30',
'created' => '2016-06-23 11:21:30',
'ProductsPublication' => array(
[maximum depth reached]
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'id' => '4',
'name' => 'Testimonial Vel',
'description' => '<p>The antibody worked very well, even during our first attempt. We did a 1 hour incubation on a shaker at 4 degrees instead of overnight. We wanted to verify expression of Cas9 in various cell lines I had made (immunofluorescence). We have used the antibody a number of times and it works every time.</p>',
'author' => 'Researcher at Harvard University',
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'modified' => '2015-07-29 10:48:39',
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'name' => 'CRISPR/Cas9 antibody 7A9 SDS US en',
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'url' => 'files/SDS/CRISPR-Cas9/SDS-C15200203-CRISPR_Cas9_Antibody_7A9-US-en-GHS_2_0.pdf',
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'modified' => '2020-06-09 12:52:19',
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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>
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<li><strong>1 tube, 2 hours, 3 steps</strong> protocol</li>
<li><strong>Input: </strong>50 pg – 50 ng</li>
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<li><strong>High sensitivity ChIP-seq</strong> - low PCR duplication rate</li>
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<h3>How it works</h3>
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<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>
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<p><small><strong>Step 1. Template Preparation</strong> provides efficient repair of the fragmented double-stranded DNA input.</small></p>
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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>
<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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'name' => 'CRISPR/Cas9 Antibody 7A9',
'description' => '<p>Diagenode, a dedicated supplier of high quality Cas9 antibodies, was<strong> the first company</strong> that offered<strong> the antibody Cas9 (clone 7A9)</strong>. This CRISPR/Cas antibody has been validated in a number of different applications including WB, IF, and IP. Our long history of expertise with <strong>CRISPR/Cas9</strong> will guarantee your experimental success.</p>
<p><strong>Other name:</strong> Csn1</p>
<p>Monoclonal antibody raised in mouse against the N-terminus of the Cas9 nuclease (CRISPR-associated protein 9).</p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15200203_WB_fig1.png" alt="CRISPR/Cas9 Antibody validated in WB " caption="false" width="145" height="188" /></p>
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<p><small><strong>Figure 1. Western blot analysis using the Diagenode monoclonal antibody directed against Cas9<br /></strong>Western blot was performed on protein extracts from HeLa cells transfected with a flag-tagged Cas9 using the Diagenode antibody against Cas9 (cat. No. C15200203). The antibody was used at different dilutions. The marker is shown on the left, position of the flag-tagged Cas9 protein is indicated on the right.</small></p>
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<p><small><strong>Figure 2. Western blot analysis using the Diagenode monoclonal antibody directed against Cas9<br /></strong>Western blot was performed on protein extracts from HeLa cells (lane 1) and on HeLa cells spiked with 1 ng of recombinant Cas9 protein (lane 2) using the Diagenode antibody against Cas9 (cat. No. C15200203). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left.</small></p>
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<p><small><strong>Figure 3. IP using the Diagenode monoclonal antibody directed against Cas9<br /></strong>IP was performed on whole cell extracts (100 µg) from HEK293 cells transfected with a Flag-tagged Cas9 using the Diagenode antibody against Cas9 (Cat. No. C15200203). The immunoprecipitated proteins were subsequently analysed by Western blot with the antibody. Lane 3 and 4 show the result of the IP; a negative IP control (IP on untransfected cells) and the input (15 µg) are shown in lane 2 and 1, respectively.</small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15200203_IF.png" alt="CRISPR/Cas9 Antibody validated in IF" caption="false" width="201" height="99" /></p>
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<p><small><strong>Figure 4. Immunofluorescence using the Diagenode monoclonal antibody directed against Cas9<br /></strong>Hela cells were transiently transfected with a Flag-tagged Cas9 expression vector. 48 hours post transfection the cells were fixed in 3.7% formaldehyde, permeabilized in 0.5% Triton-X-100 and blocked in PBS containing 2% BSA for 2 hours at RT. 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 (left). Nuclei were counter-stained with DAPI (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>Great multiplexing flexibility</strong> with 12 barcodes (8 nt) included</li>
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<center><img src="https://www.diagenode.com/img/product/kits/microplex-method-overview-v2.png" /></center>
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<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>
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<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>Learn more about: <a href="https://www.diagenode.com/applications/western-blot">Loading control, MW marker visualization</a><em>. <br /></em></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15200229-IF.jpg" alt="" height="245" width="256" /></p>
<p><sup><strong>Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong></sup></p>
<p><sup>HeLa cells transfected with a Cas9 expression vector (left) or untransfected cells (right) were fixed in methanol at -20°C, permeabilized with acetone at -20°C and blocked with PBS containing 2% BSA. The cells were stained with the Cas9 C-terminal antibody (Cat. No. C15200229) diluted 1:400, followed by incubation with an anti-mouse secondary antibody coupled to AF488. The bottom images show counter-staining of the nuclei with Hoechst 33342.</sup></p>
<h5><sup>Check our selection of antibodies validated in IF.</sup></h5>',
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<p><span style="font-weight: 400;">Diagenode’s highly validated antibodies:</span></p>
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'name' => '<em>S. pyogenes</EM> CRISPR/Cas9 antibodies',
'description' => '<p>Diagenode offers the broad range of antibodies raised against the N- or C-terminus of the Cas9 nuclease from <em>Streptococcus <g class="gr_ gr_5 gr-alert gr_spell gr_disable_anim_appear ContextualSpelling ins-del multiReplace" id="5" data-gr-id="5">pyogenes</g></em>. These highly specific polyclonal and monoclonal antibodies are validated in Western blot, immunoprecipitation, immunofluorescence and in chromatin immunoprecipitation.</p>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h2><em><a name="pyogenes"></a>S. pyogenes</em> CRISPR/Cas9 antibodies<a></a></h2>
<div class="panel">
<h2>Discover our first monoclonal CRISPR/Cas9 antibody validated in ChIP<br /><br /></h2>
<div class="row">
<div class="small-5 medium-5 large-5 columns"><img src="/img/landing-pages/crispr-cas9-chip-on-hih3t3.jpg" alt="" /></div>
<div class="small-7 medium-7 large-7 columns">
<ul>
<li>Validated in chromatin immunoprecipitation</li>
<li>Performs better than FLAG antibody</li>
<li>Excellent for WB, IF and IP</li>
</ul>
<p><small><strong>ChIP</strong> was performed on NIH3T3 cells stably expressing GFP-H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50 µg chromatin was incubated overnight at 4°C with 5 or 10 µg of either an anti-FLAG antibody or the Diagenode antibody against Cas9 (Cat. No. C15200229). Mouse IgG was used as a negative IP control. qPCR was performed with primers specific for the GFP gene, and for a non-targeted region (protein kinase C delta, Prkcd), used as negative control. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).</small></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns text-right"><a href="/p/crispr-cas9-monoclonal-antibody-50-ug-25-μl" class="tiny details button radius">Learn more</a></div>
</div>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>First ChIP-grade CRISPR/Cas9 polyclonal antibody</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/landing-pages/c_a_s9-chip-grade-antibody.png" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Excellent polyclonal antibody for chromatin immunoprecipitation</li>
<li>Optimized for highest ChIP specificity and yields</li>
<li>Validated for all applications including immunoblotting, immunofluorescence and western blot</li>
</ul>
<p><small><strong>ChIP</strong> was performed on NIH3T3 cells stably expressing GFP- H2B, nuclease dead Cas9, and a GFP-targeting gRNA. 50 μg chromatin was incubated with either 5 μg of an anti-FLAG antibody or 2 μl of the Diagenode antibody against Cas9. The pre-immune serum (PPI) was used as negative IP control. Then qPCR was performed with primers specific for the GFP gene, and for two non-targeted regions: Ppap2c and Prkcd, used as negative controls. This figure shows the recovery, expressed as a % of input.</small></p>
<p class="text-right"><a href="../p/crispr-cas9-polyclonal-antibody" class="details tiny button">Learn more</a></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>CRISPR/Cas9 monoclonal antibody 4G10</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/landing-pages/cas9_4g10_fig1.png" width="170" height="302" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Antibody raised against N-terminus of Cas9 nuclease</li>
<li>Validated for western blot, IP and immunofluorescence</li>
</ul>
<p><small><strong>Immunofluorescence</strong>: Hela cells were transiently transfected with a Cas9 expression vector. The cells were fixed in 3.7% formaldehyde, permeabilized in 0.5% Triton-X-100 and blocked in PBS containing 2% BSA. The cells were stained with the Cas9 antibody at 4°C o/n, followed by incubation with an anti mouse secondary antibody coupled to AF488 for 1 h at RT. Nuclei were counter-stained with Hoechst 33342 (right).</small></p>
<p class="text-right"><a href="../p/crispr-cas9-monoclonal-antibody-4g10-50-ug" class="details tiny button">Learn more</a></p>
</div>
</div>
<div class="row">
<div class="small-12 medium-12 large-12 columns">
<h3>CRISPR/Cas9 C-terminal monoclonal antibody</h3>
</div>
<div class="small-4 medium-4 large-4 columns"><img src="https://www.diagenode.com/img/product/antibodies/C15200223-IP.png" /></div>
<div class="small-8 medium-8 large-8 columns">
<ul>
<li>Antibody raised against C-terminus of Cas9 nuclease</li>
<li>Validated for western blot, IP and immunofluorescence</li>
</ul>
<p><small><strong> Western blot</strong> was performed on 20 μg protein extracts from Cas9 expressing HeLa cells (lane 1) and on negative control HeLa cells (lane 2) with the Diagenode antibody against Cas9. The antibody was diluted 1:4,000. The marker is shown on the left, position of the Cas9 protein is indicated on the right. </small></p>
<p class="text-right"><a href="../p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug" class="details tiny button">Learn more</a></p>
</div>
<div class="small-12 medium-12 large-12 columns">
<h3>Which CRISPR/Cas9 antibody is the best for your application?</h3>
<a name="table"></a>
<table>
<thead>
<tr>
<th>Antibody</th>
<th>WB</th>
<th>IF</th>
<th>IP</th>
<th>ChIP</th>
<th>Antibody raised against</th>
</tr>
</thead>
<tbody>
<tr>
<td><a href="../p/crispr-cas9-monoclonal-antibody-50-ug-25-μl"><strong class="diacol">CRISPR/Cas9 monoclonal antibody</strong></a></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><span class="diacol">N-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-polyclonal-antibody">CRISPR/Cas9 polyclonal antibody</a></td>
<td>++</td>
<td>++</td>
<td>++</td>
<td><strong class="diacol">+++</strong></td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-monoclonal-antibody-4g10-50-ug">CRISPR/Cas9 monoclonal antibody 4G10</a></td>
<td>+++</td>
<td>+++</td>
<td>++</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="../p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug-23-ul"><strong class="diacol">CRISPR/Cas9 C-terminal monoclonal antibody</strong></a> <span class="label alert" style="font-size: 0.9rem;">NEW!</span></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">+++</strong></td>
<td><strong class="diacol">no</strong></td>
<td><strong class="diacol">+</strong></td>
<td><span class="diacol">C-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-c-terminal-monoclonal-antibody-50-ug">CRISPR/Cas9 C-terminal monoclonal antibody</a></td>
<td>++</td>
<td>++</td>
<td>+</td>
<td>no</td>
<td><span class="diacol">C-terminus of Cas9 nuclease</span></td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-monoclonal-antibody-7A9-50-mg">CRISPR/Cas9 monoclonal antibody 7A9</a></td>
<td>++</td>
<td>++</td>
<td>++</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
<tr>
<td><a href="/p/crispr-cas9-hrp-monoclonal-antibody-50-ul">CRISPR/Cas9 - HRP monoclonal antibody 7A9</a></td>
<td>+++</td>
<td>no</td>
<td>no</td>
<td>no</td>
<td>N-terminus of Cas9 nuclease</td>
</tr>
</tbody>
</table>
</div>
</div>
</div>',
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'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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'image_id' => null,
'type' => 'Datasheet',
'url' => 'files/products/antibodies/Datasheet_CRISPR_Cas9_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,
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'id' => '4953',
'name' => 'Loss of tumor suppressors promotes inflammatory tumor microenvironment and enhances LAG3+T cell mediated immune suppression',
'authors' => 'Zahraeifard, S. et al.',
'description' => '<p><span>Low response rate, treatment relapse, and resistance remain key challenges for cancer treatment with immune checkpoint blockade (ICB). Here we report that loss of specific tumor suppressors (TS) induces an inflammatory response and promotes an immune suppressive tumor microenvironment. Importantly, low expression of these TSs is associated with a higher expression of immune checkpoint inhibitory mediators. Here we identify, by using in vivo CRISPR/Cas9 based loss-of-function screening, that NF1, TSC1, and TGF-β RII as TSs regulating immune composition. Loss of each of these three TSs leads to alterations in chromatin accessibility and enhances IL6-JAK3-STAT3/6 inflammatory pathways. This results in an immune suppressive landscape, characterized by increased numbers of LAG3+ CD8 and CD4 T cells. ICB targeting LAG3 and PD-L1 simultaneously inhibits metastatic progression in preclinical triple negative breast cancer (TNBC) mouse models of NF1-, TSC1- or TGF-β RII- deficient tumors. Our study thus reveals a role of TSs in regulating metastasis via non-cell-autonomous modulation of the immune compartment and provides proof-of-principle for ICB targeting LAG3 for patients with NF1-, TSC1- or TGF-β RII-inactivated cancers.</span></p>',
'date' => '2024-07-12',
'pmid' => 'https://www.nature.com/articles/s41467-024-50262-8',
'doi' => 'https://doi.org/10.1038/s41467-024-50262-8',
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'name' => 'A kinesin-based approach for inducing chromosome-specific mis-segregationin human cells.',
'authors' => 'Truong M.A. et al.',
'description' => '<p>Various cancer types exhibit characteristic and recurrent aneuploidy patterns. The origins of these cancer type-specific karyotypes are still unknown, partly because introducing or eliminating specific chromosomes in human cells still poses a challenge. Here, we describe a novel strategy to induce mis-segregation of specific chromosomes in different human cell types. We employed Tet repressor or nuclease-dead Cas9 to link a microtubule minus-end-directed kinesin (Kinesin14VIb) from Physcomitrella patens to integrated Tet operon repeats and chromosome-specific endogenous repeats, respectively. By live- and fixed-cell imaging, we observed poleward movement of the targeted loci during (pro)metaphase. Kinesin14VIb-mediated pulling forces on the targeted chromosome were counteracted by forces from kinetochore-attached microtubules. This tug-of-war resulted in chromosome-specific segregation errors during anaphase and revealed that spindle forces can heavily stretch chromosomal arms. By single-cell whole-genome sequencing, we established that kinesin-induced targeted mis-segregations predominantly result in chromosomal arm aneuploidies after a single cell division. Our kinesin-based strategy opens the possibility to investigate the immediate cellular responses to specific aneuploidies in different cell types; an important step toward understanding how tissue-specific aneuploidy patterns evolve.</p>',
'date' => '2023-04-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/37038978',
'doi' => '10.15252/embj.2022111559',
'modified' => '2023-06-13 09:21:25',
'created' => '2023-05-05 12:34:24',
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(int) 2 => array(
'id' => '4344',
'name' => 'MICAL1 regulates actin cytoskeleton organization, directional cellmigration and the growth of human breast cancer cells as orthotopicxenograft tumours.',
'authors' => 'McGarry David J et al.',
'description' => '<p>The Molecule Interacting with CasL 1 (MICAL1) monooxygenase has emerged as an important regulator of cytoskeleton organization via actin oxidation. Although filamentous actin (F-actin) increases MICAL1 monooxygenase activity, hydrogen peroxide (HO) is also generated in the absence of F-actin, suggesting that diffusible HO might have additional functions. MICAL1 gene disruption by CRISPR/Cas9 in MDA MB 231 human breast cancer cells knocked out (KO) protein expression, which affected F-actin organization, cell size and motility. Transcriptomic profiling revealed that MICAL1 deletion significantly affected the expression of over 700 genes, with the majority being reduced in their expression levels. In addition, the absolute magnitudes of reduced gene expression were significantly greater than the magnitudes of increased gene expression. Gene set enrichment analysis (GSEA) identified receptor regulator activity as the most significant negatively enriched molecular function gene set. The prominent influence exerted by MICAL1 on F-actin structures was also associated with changes in the expression of several serum-response factor (SRF) regulated genes in KO cells. Moreover, MICAL1 disruption attenuated breast cancer tumour growth in vivo. Elevated MICAL1 gene expression was observed in invasive breast cancer samples from human patients relative to normal tissue, while MICAL1 amplification or point mutations were associated with reduced progression free survival. Collectively, these results demonstrate that MICAL1 gene disruption altered cytoskeleton organization, cell morphology and migration, gene expression, and impaired tumour growth in an orthotopic in vivo breast cancer model, suggesting that pharmacological MICAL1 inhibition could have therapeutic benefits for cancer patients.</p>',
'date' => '2021-10-01',
'pmid' => 'https://doi.org/10.1016%2Fj.canlet.2021.07.039',
'doi' => '10.1016/j.canlet.2021.07.039',
'modified' => '2022-06-21 16:56:09',
'created' => '2022-05-19 10:41:50',
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(int) 3 => array(
'id' => '3925',
'name' => 'CRISPR-based gene knockout screens reveal deubiquitinases involved in HIV-1 latency in two Jurkat cell models.',
'authors' => 'Rathore A, Iketani S, Wang P, Jia M, Sahi V, Ho DD',
'description' => '<p>The major barrier to a HIV-1 cure is the persistence of latent genomes despite treatment with antiretrovirals. To investigate host factors which promote HIV-1 latency, we conducted a genome-wide functional knockout screen using CRISPR-Cas9 in a HIV-1 latency cell line model. This screen identified IWS1, POLE3, POLR1B, PSMD1, and TGM2 as potential regulators of HIV-1 latency, of which PSMD1 and TMG2 could be confirmed pharmacologically. Further investigation of PSMD1 revealed that an interacting enzyme, the deubiquitinase UCH37, was also involved in HIV-1 latency. We therefore conducted a comprehensive evaluation of the deubiquitinase family by gene knockout, identifying several deubiquitinases, UCH37, USP14, OTULIN, and USP5 as possible HIV-1 latency regulators. A specific inhibitor of USP14, IU1, reversed HIV-1 latency and displayed synergistic effects with other latency reversal agents. IU1 caused degradation of TDP-43, a negative regulator of HIV-1 transcription. Collectively, this study is the first comprehensive evaluation of deubiquitinases in HIV-1 latency and establishes that they may hold a critical role.</p>',
'date' => '2020-03-24',
'pmid' => 'http://www.pubmed.gov/32210344',
'doi' => '10.1038/s41598-020-62375-3',
'modified' => '2020-08-17 10:51:24',
'created' => '2020-08-10 12:12:25',
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),
(int) 4 => array(
'id' => '3821',
'name' => 'DAPK1 loss triggers tumor invasion in colorectal tumor cells.',
'authors' => 'Steinmann S, Kunze P, Hampel C, Eckstein M, Bertram Bramsen J, Muenzner JK, Carlé B, Ndreshkjana B, Kemenes S, Gasparini P, Friedrich O, Andersen C, Geppert C, Wang S, Eyupoglu I, Bäuerle T, Hartmann A, Schneider-Stock R',
'description' => '<p>Colorectal cancer (CRC) is one of the leading cancer-related causes of death worldwide. Despite the improvement of surgical and chemotherapeutic treatments, as of yet, the disease has not been overcome due to metastasis to distant organs. Hence, it is of great relevance to understand the mechanisms responsible for metastasis initiation and progression and to identify novel metastatic markers for a higher chance of preventing the metastatic disease. The Death-associated protein kinase 1 (DAPK1), recently, has been shown to be a potential candidate for regulating metastasis in CRC. Hence, the aim of the study was to investigate the impact of DAPK1 protein on CRC aggressiveness. Using CRISPR/Cas9 technology, we generated DAPK1-deficient HCT116 monoclonal cell lines and characterized their knockout phenotype in vitro and in vivo. We show that loss of DAPK1 implemented changes in growth pattern and enhanced tumor budding in vivo in the chorioallantoic membrane (CAM) model. Further, we observed more tumor cell dissemination into chicken embryo organs and increased invasion capacity using rat brain 3D in vitro model. The novel identified DAPK1-loss gene expression signature showed a stroma typical pattern and was associated with a gained ability for remodeling the extracellular matrix. Finally, we suggest the DAPK1-ERK1 signaling axis being involved in metastatic progression of CRC. Our results highlight DAPK1 as an anti-metastatic player in CRC and suggest DAPK1 as a potential predictive biomarker for this cancer type.</p>',
'date' => '2019-11-26',
'pmid' => 'http://www.pubmed.gov/31772156',
'doi' => '10.1038/s41419-019-2122-z',
'modified' => '2020-02-25 13:45:15',
'created' => '2020-02-13 10:02:44',
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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',
'created' => '2019-06-06 12:11:18',
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(int) 6 => array(
'id' => '3565',
'name' => 'Can Mitochondrial DNA be CRISPRized: Pro and Contra.',
'authors' => 'Loutre R, Heckel AM, Smirnova A, Entelis N, Tarassov I',
'description' => '<p>Mitochondria represent a chimera of macromolecules encoded either in the organellar genome, mtDNA, or in the nuclear one. If the pathway of protein targeting to different sub-compartments of mitochondria was relatively well studied, import of small noncoding RNAs into mammalian mitochondria still awaits mechanistic explanations and its functional issues are often not understood thus raising polemics. At the same time, RNA mitochondrial import pathway has an obvious attractiveness as it appears as a unique natural mechanism permitting to address nucleic acids into the organelles. Deciphering the function(s) of imported RNAs inside the mitochondria is extremely complicated due to their relatively low abundance, which suggests their regulatory role. We previously demonstrated that mitochondrial targeting of small noncoding RNAs able to specifically anneal with the mutant mitochondrial DNA led to a decrease of the mtDNA heteroplasmy level by inhibiting mutant mtDNA replication. We then demonstrated that increasing level of expression of such antireplicative recombinant RNAs increases significantly the antireplicative effect. In this report, we present a new data investigating the possibility to establish a CRISPR-Cas9 system targeting mtDNA exploiting of the pathway of RNA import into mitochondria. Mitochondrially addressed Cas9 versions and a set of mitochondrially targeted guide RNAs were tested in vitro and in vivo and their effect on mtDNA copy number was demonstrated. So far, the system appeared as more complicated for use than previously found for nuclear DNA, because only application of a pair of guide RNAs produced the effect of mtDNA depletion. We discuss, in a critical way, these results and put them in a broader context of polemics concerning the possibilities of manipulation of mtDNA in mammalians. The findings described here prove the potential of the RNA import pathway as a tool for studying mtDNA and for future therapy of mitochondrial disorders. © The Authors. IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 70(12):1233-1239, 2018.</p>',
'date' => '2018-12-01',
'pmid' => 'http://www.pubmed.gov/30184317',
'doi' => '10.1002/iub.1919',
'modified' => '2019-03-21 17:20:49',
'created' => '2019-03-21 14:12:08',
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[maximum depth reached]
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),
(int) 7 => array(
'id' => '3268',
'name' => 'In trans paired nicking triggers seamless genome editing without double-stranded DNA cutting',
'authors' => 'Chen X. et al.',
'description' => '<p>Precise genome editing involves homologous recombination between donor DNA and chromosomal sequences subjected to double-stranded DNA breaks made by programmable nucleases. Ideally, genome editing should be efficient, specific, and accurate. However, besides constituting potential translocation-initiating lesions, double-stranded DNA breaks (targeted or otherwise) are mostly repaired through unpredictable and mutagenic non-homologous recombination processes. Here, we report that the coordinated formation of paired single-stranded DNA breaks, or nicks, at donor plasmids and chromosomal target sites by RNA-guided nucleases based on CRISPR-Cas9 components, triggers seamless homology-directed gene targeting of large genetic payloads in human cells, including pluripotent stem cells. Importantly, in addition to significantly reducing the mutagenicity of the genome modification procedure, this in trans paired nicking strategy achieves multiplexed, single-step, gene targeting, and yields higher frequencies of accurately edited cells when compared to the standard double-stranded DNA break-dependent approach.</p>',
'date' => '2017-09-22',
'pmid' => 'https://www.nature.com/articles/s41467-017-00687-1',
'doi' => '',
'modified' => '2017-10-09 16:27:58',
'created' => '2017-10-09 16:27:58',
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(int) 8 => array(
'id' => '3244',
'name' => 'Highly efficient gene inactivation by adenoviral CRISPR/Cas9 in human primary cells',
'authors' => 'Voets O. et al.',
'description' => '<p>Phenotypic assays using human primary cells are highly valuable tools for target discovery and validation in drug discovery. Expression knockdown (KD) of such targets in these assays allows the investigation of their role in models of disease processes. Therefore, efficient and fast modes of protein KD in phenotypic assays are required. The CRISPR/Cas9 system has been shown to be a versatile and efficient means of gene inactivation in immortalized cell lines. Here we describe the use of adenoviral (AdV) CRISPR/Cas9 vectors for efficient gene inactivation in two human primary cell types, normal human lung fibroblasts and human bronchial epithelial cells. The effects of gene inactivation were studied in the TGF-β-induced fibroblast to myofibroblast transition assay (FMT) and the epithelial to mesenchymal transition assay (EMT), which are SMAD3 dependent and reflect pathogenic mechanisms observed in fibrosis. Co-transduction (co-TD) of AdV Cas9 with SMAD3-targeting guide RNAs (gRNAs) resulted in fast and efficient genome editing judged by insertion/deletion (indel) formation, as well as significant reduction of SMAD3 protein expression and nuclear translocation. This led to phenotypic changes downstream of SMAD3 inhibition, including substantially decreased alpha smooth muscle actin and fibronectin 1 expression, which are markers for FMT and EMT, respectively. A direct comparison between co-TD of separate Cas9 and gRNA AdV, versus TD with a single "all-in-one" Cas9/gRNA AdV, revealed that both methods achieve similar levels of indel formation. These data demonstrate that AdV CRISPR/Cas9 is a useful and efficient tool for protein KD in human primary cell phenotypic assays. The use of AdV CRISPR/Cas9 may offer significant advantages over the current existing tools and should enhance target discovery and validation opportunities.</p>',
'date' => '2017-08-11',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/28800587',
'doi' => '',
'modified' => '2017-09-19 17:26:46',
'created' => '2017-09-19 17:26:46',
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[maximum depth reached]
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(int) 9 => array(
'id' => '3040',
'name' => 'Noncoding somatic and inherited single-nucleotide variants converge to promote ESR1 expression in breast cancer',
'authors' => 'Bailey SD et al.',
'description' => '<p>Sustained expression of the estrogen receptor-<span class="mb">α</span> (ESR1) drives two-thirds of breast cancer and defines the ESR1-positive subtype. ESR1 engages enhancers upon estrogen stimulation to establish an oncogenic expression program<sup><a href="http://www.nature.com/ng/journal/v48/n10/full/ng.3650.html#ref1" title="Green, K.A. & Carroll, J.S. Oestrogen-receptor-mediated transcription and the influence of co-factors and chromatin state. Nat. Rev. Cancer 7, 713-722 (2007)." id="ref-link-5">1</a></sup>. Somatic copy number alterations involving the <i>ESR1</i> gene occur in approximately 1% of ESR1-positive breast cancers<sup><a href="http://www.nature.com/ng/journal/v48/n10/full/ng.3650.html#ref2" title="Vincent-Salomon, A., Raynal, V., Lucchesi, C., Gruel, N. & Delattre, O. ESR1 gene amplification in breast cancer: a common phenomenon? Nat. Genet. 40, 809, author reply 810-812 (2008)." id="ref-link-6">2</a>, <a href="http://www.nature.com/ng/journal/v48/n10/full/ng.3650.html#ref3" title="Brown, L.A. et al. ESR1 gene amplification in breast cancer: a common phenomenon? Nat. Genet. 40, 806-807, author reply 810-812 (2008)." id="ref-link-7">3</a>, <a href="http://www.nature.com/ng/journal/v48/n10/full/ng.3650.html#ref4" title="Horlings, H.M. et al. ESR1 gene amplification in breast cancer: a common phenomenon? Nat. Genet. 40, 807-808, author reply 810-812 (2008)." id="ref-link-8">4</a>, <a href="http://www.nature.com/ng/journal/v48/n10/full/ng.3650.html#ref5" title="Reis-Filho, J.S. et al. ESR1 gene amplification in breast cancer: a common phenomenon? Nat. Genet. 40, 809-810, author reply 810-812 (2008)." id="ref-link-9">5</a></sup>, suggesting that other mechanisms underlie the persistent expression of <i>ESR1</i>. We report significant enrichment of somatic mutations within the set of regulatory elements (SRE) regulating <i>ESR1</i> in 7% of ESR1-positive breast cancers. These mutations regulate <i>ESR1</i> expression by modulating transcription factor binding to the DNA. The SRE includes a recurrently mutated enhancer whose activity is also affected by rs9383590, a functional inherited single-nucleotide variant (SNV) that accounts for several breast cancer risk–associated loci. Our work highlights the importance of considering the combinatorial activity of regulatory elements as a single unit to delineate the impact of noncoding genetic alterations on single genes in cancer.</p>',
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'name' => 'The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing.',
'authors' => 'de Solis CA et al.',
'description' => '<p>The RNA-guided Cas9 nuclease, from the type II prokaryotic Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) adaptive immune system, has been adapted and utilized by scientists to edit the genomes of eukaryotic cells. Here, we report the development of a viral mediated CRISPR/Cas9 system that can be rendered inducible utilizing doxycycline (Dox) and can be delivered to cells in vitro and in vivo utilizing adeno-associated virus (AAV). Specifically, we developed an inducible gRNA (gRNAi) AAV vector that is designed to express the gRNA from a H1/TO promoter. This AAV vector is also designed to express the Tet repressor (TetR) to regulate the expression of the gRNAi in a Dox dependent manner. We show that H1/TO promoters of varying length and a U6/TO promoter can edit DNA with similar efficiency in vitro, in a Dox dependent manner. We also demonstrate that our inducible gRNAi vector can be used to edit the genomes of neurons in vivo within the mouse brain in a Dox dependent manner. Genome editing can be induced in vivo with this system by supplying animals Dox containing food for as little as 1 day. This system might be cross compatible with many existing S. pyogenes Cas9 systems (i.e., Cas9 mouse, CRISPRi, etc.), and therefore it likely can be used to render these systems inducible as well.</p>',
'date' => '2016-08-18',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/27587996',
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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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'name' => 'A geminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing',
'authors' => 'Yin K, Han T, Liu G, Chen T, Wang Y, Yu AY, Liu Y',
'description' => '<p>CRISPR/Cas has emerged as potent genome editing technology and has successfully been applied in many organisms, including several plant species. However, delivery of genome editing reagents remains a challenge in plants. Here, we report a <u>vi</u>rus-based guide RNA (gRNA) delivery system for CRISPR/Cas9 mediated plant <u>g</u>enome <u>e</u>diting (VIGE) that can be used to precisely target genome locations and cause mutations. VIGE is performed by using a modified Cabbage Leaf Curl virus (CaLCuV) vector to express gRNAs in stable transgenic plants expressing Cas9. DNA sequencing confirmed VIGE of endogenous <i>NbPDS3</i> and <i>NbIspH</i> genes in non-inoculated leaves because CaLCuV can infect plants systemically. Moreover, VIGE of <i>NbPDS3</i> and <i>NbIspH</i> in newly developed leaves caused photo-bleached phenotype. These results demonstrate that geminivirus-based VIGE could be a powerful tool in plant genome editing.</p>',
'date' => '2015-10-09',
'pmid' => 'http://www.ncbi.nlm.nih.gov/pubmed/26450012',
'doi' => '10.1038/srep14926',
'modified' => '2016-02-16 13:55:32',
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'name' => 'Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection.',
'authors' => 'Liang X, Potter J, Kumar S, Zou Y, Quintanilla R, Sridharan M, Carte J, Chen W, Roark N, Ranganathan S, Ravinder N, Chesnut JD',
'description' => '<p>CRISPR-Cas9 systems provide a platform for high efficiency genome editing that are enabling innovative applications of mammalian cell engineering. However, the delivery of Cas9 and synthesis of guide RNA (gRNA) remain as steps that can limit overall efficiency and ease of use. Here we describe methods for rapid synthesis of gRNA and for delivery of Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs) into a variety of mammalian cells through liposome-mediated transfection or electroporation. Using these methods, we report nuclease-mediated indel rates of up to 94% in Jurkat T cells and 87% in induced pluripotent stem cells (iPSC) for a single target. When we used this approach for multigene targeting in Jurkat cells we found that two-locus and three-locus indels were achieved in approximately 93% and 65% of the resulting isolated cell lines, respectively. Further, we found that the off-target cleavage rate is reduced using Cas9 protein when compared to plasmid DNA transfection. Taken together, we present a streamlined cell engineering workflow that enables gRNA design to analysis of edited cells in as little as four days and results in highly efficient genome modulation in hard-to-transfect cells. The reagent preparation and delivery to cells is amenable to high throughput, multiplexed genome-wide cell engineering.</p>',
'date' => '2015-08-20',
'pmid' => 'http://www.ncbi.nlm.nih.gov/pubmed/26003884',
'doi' => '10.1016/j.jbiotec.2015.04.024',
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'description' => 'DNA double-strand breaks (DSBs) are repaired by two main pathways: nonhomologous end-joining and homologous recombination (HR). Repair pathway choice is thought to be determined by cell cycle timing and chromatin context. Nucleoli, prominent nuclear subdomains and sites of ribosome biogenesis, form around nucleolar organizer regions (NORs) that contain rDNA arrays located on human acrocentric chromosome p-arms. Actively transcribed rDNA repeats are positioned within the interior of the nucleolus, whereas sequences proximal and distal to NORs are packaged as heterochromatin located at the nucleolar periphery. NORs provide an opportunity to investigate the DSB response at highly transcribed, repetitive, and essential loci. Targeted introduction of DSBs into rDNA, but not abutting sequences, results in ATM-dependent inhibition of their transcription by RNA polymerase I. This is coupled with movement of rDNA from the nucleolar interior to anchoring points at the periphery. Reorganization renders rDNA accessible to repair factors normally excluded from nucleoli. Importantly, DSBs within rDNA recruit the HR machinery throughout the cell cycle. Additionally, unscheduled DNA synthesis, consistent with HR at damaged NORs, can be observed in G1 cells. These results suggest that HR can be templated in cis and suggest a role for chromosomal context in the maintenance of NOR genomic stability.',
'date' => '2015-06-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/26019174',
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'name' => 'Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis.',
'authors' => 'Chen S, Sanjana NE, Zheng K, Shalem O, Lee K, Shi X, Scott DA, Song J, Pan JQ, Weissleder R, Lee H, Zhang F, Sharp PA',
'description' => 'Genetic screens are powerful tools for identifying genes responsible for diverse phenotypes. Here we describe a genome-wide CRISPR/Cas9-mediated loss-of-function screen in tumor growth and metastasis. We mutagenized a non-metastatic mouse cancer cell line using a genome-scale library with 67,405 single-guide RNAs (sgRNAs). The mutant cell pool rapidly generates metastases when transplanted into immunocompromised mice. Enriched sgRNAs in lung metastases and late-stage primary tumors were found to target a small set of genes, suggesting that specific loss-of-function mutations drive tumor growth and metastasis. Individual sgRNAs and a small pool of 624 sgRNAs targeting the top-scoring genes from the primary screen dramatically accelerate metastasis. In all of these experiments, the effect of mutations on primary tumor growth positively correlates with the development of metastases. Our study demonstrates Cas9-based screening as a robust method to systematically assay gene phenotypes in cancer evolution in vivo.',
'date' => '2015-03-12',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/25748654',
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'description' => '<p>To combat hostile viruses, bacteria and archaea have evolved a unique antiviral defense system composed of clustered regularly interspaced short palindromic repeats (CRISPRs), together with CRISPR-associated genes (Cas). The CRISPR/Cas9 system develops an adaptive immune resistance to foreign plasmids and viruses by creating site-specific DNA double-stranded breaks (DSBs). Here we adapt the CRISPR/Cas9 system to human cells for intracellular defense against foreign DNA and viruses. Using HIV-1 infection as a model, our results demonstrate that the CRISPR/Cas9 system disrupts latently integrated viral genome and provides long-term adaptive defense against new viral infection, expression and replication in human cells. We show that engineered human-induced pluripotent stem cells stably expressing HIV-targeted CRISPR/Cas9 can be efficiently differentiated into HIV reservoir cell types and maintain their resistance to HIV-1 challenge. These results unveil the potential of the CRISPR/Cas9 system as a new therapeutic strategy against viral infections.</p>',
'date' => '2015-03-10',
'pmid' => 'http://www.ncbi.nlm.nih.gov/pubmed/25752527',
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'description' => '<p>The CRISPR/Cas9 technology enables the introduction of genomic alterations into almost any organism; however, systems for efficient and inducible gene modification have been lacking, especially for deletion of essential genes. Here, we describe a drug-inducible small guide RNA (sgRNA) vector system allowing for ubiquitous and efficient gene deletion in murine and human cells. This system mediates the efficient, temporally controlled deletion of MCL-1, both in vitro and in vivo, in human Burkitt lymphoma cell lines that require this anti-apoptotic BCL-2 protein for sustained survival and growth. Unexpectedly, repeated induction of the same sgRNA generated similar inactivating mutations in the human Mcl-1 gene due to low mutation variability exerted by the accompanying non-homologous end-joining (NHEJ) process. Finally, we were able to generate hematopoietic cell compartment-restricted Trp53-knockout mice, leading to the identification of cancer-promoting mutants of this critical tumor suppressor.</p>',
'date' => '2015-03-03',
'pmid' => 'http://www.ncbi.nlm.nih.gov/pubmed/25732831',
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'description' => '<p>The CRISPR/Cas9 technology enables the introduction of genomic alterations into almost any organism; however, systems for efficient and inducible gene modification have been lacking, especially for deletion of essential genes. Here, we describe a drug-inducible small guide RNA (sgRNA) vector system allowing for ubiquitous and efficient gene deletion in murine and human cells. This system mediates the efficient, temporally controlled deletion of MCL-1, both in vitro and in vivo, in human Burkitt lymphoma cell lines that require this anti-apoptotic BCL-2 protein for sustained survival and growth. Unexpectedly, repeated induction of the same sgRNA generated similar inactivating mutations in the human Mcl-1 gene due to low mutation variability exerted by the accompanying non-homologous end-joining (NHEJ) process. Finally, we were able to generate hematopoietic cell compartment-restricted Trp53-knockout mice, leading to the identification of cancer-promoting mutants of this critical tumor suppressor.</p>',
'date' => '2015-03-03',
'pmid' => 'http://www.ncbi.nlm.nih.gov/pubmed/25732831',
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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>
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<li><strong>High sensitivity ChIP-seq</strong> - low PCR duplication rate</li>
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<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>
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<p><small><strong>Step 1. Template Preparation</strong> provides efficient repair of the fragmented double-stranded DNA input.</small></p>
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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><small><strong>Figure 1. Western blot analysis using the Diagenode monoclonal antibody directed against Cas9<br /></strong>Western blot was performed on protein extracts from HeLa cells transfected with a flag-tagged Cas9 using the Diagenode antibody against Cas9 (cat. No. C15200203). The antibody was used at different dilutions. The marker is shown on the left, position of the flag-tagged Cas9 protein is indicated on the right.</small></p>
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<p><small><strong>Figure 2. Western blot analysis using the Diagenode monoclonal antibody directed against Cas9<br /></strong>Western blot was performed on protein extracts from HeLa cells (lane 1) and on HeLa cells spiked with 1 ng of recombinant Cas9 protein (lane 2) using the Diagenode antibody against Cas9 (cat. No. C15200203). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left.</small></p>
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<p><small><strong>Figure 3. IP using the Diagenode monoclonal antibody directed against Cas9<br /></strong>IP was performed on whole cell extracts (100 µg) from HEK293 cells transfected with a Flag-tagged Cas9 using the Diagenode antibody against Cas9 (Cat. No. C15200203). The immunoprecipitated proteins were subsequently analysed by Western blot with the antibody. Lane 3 and 4 show the result of the IP; a negative IP control (IP on untransfected cells) and the input (15 µg) are shown in lane 2 and 1, respectively.</small></p>
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'meta_title' => 'CRISPR/Cas9 Antibody – clone 7A9 (C15200203) | Diagenode',
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'meta_description' => 'The first on the market S. pyogenes CRISPR/Cas9 Monoclonal Antibody, clone 7A9. Validated in IP, IF and WB. Batch-specific data available on the website. Other name: Csn1',
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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>
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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>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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<p><span style="font-weight: 400;">Diagenode’s highly validated antibodies:</span></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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'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(
[maximum depth reached]
)
)
),
'Feature' => array(),
'Image' => array(
(int) 0 => array(
'id' => '1782',
'name' => 'product/antibodies/cas9-icon.png',
'alt' => 'CRISPR/Cas9 Antibody ',
'modified' => '2020-11-27 07:01:20',
'created' => '2018-03-15 15:53:46',
'ProductsImage' => array(
[maximum depth reached]
)
)
),
'Promotion' => array(),
'Protocol' => array(),
'Publication' => array(
(int) 0 => array(
'id' => '4953',
'name' => 'Loss of tumor suppressors promotes inflammatory tumor microenvironment and enhances LAG3+T cell mediated immune suppression',
'authors' => 'Zahraeifard, S. et al.',
'description' => '<p><span>Low response rate, treatment relapse, and resistance remain key challenges for cancer treatment with immune checkpoint blockade (ICB). Here we report that loss of specific tumor suppressors (TS) induces an inflammatory response and promotes an immune suppressive tumor microenvironment. Importantly, low expression of these TSs is associated with a higher expression of immune checkpoint inhibitory mediators. Here we identify, by using in vivo CRISPR/Cas9 based loss-of-function screening, that NF1, TSC1, and TGF-β RII as TSs regulating immune composition. Loss of each of these three TSs leads to alterations in chromatin accessibility and enhances IL6-JAK3-STAT3/6 inflammatory pathways. This results in an immune suppressive landscape, characterized by increased numbers of LAG3+ CD8 and CD4 T cells. ICB targeting LAG3 and PD-L1 simultaneously inhibits metastatic progression in preclinical triple negative breast cancer (TNBC) mouse models of NF1-, TSC1- or TGF-β RII- deficient tumors. Our study thus reveals a role of TSs in regulating metastasis via non-cell-autonomous modulation of the immune compartment and provides proof-of-principle for ICB targeting LAG3 for patients with NF1-, TSC1- or TGF-β RII-inactivated cancers.</span></p>',
'date' => '2024-07-12',
'pmid' => 'https://www.nature.com/articles/s41467-024-50262-8',
'doi' => 'https://doi.org/10.1038/s41467-024-50262-8',
'modified' => '2024-07-29 10:47:10',
'created' => '2024-07-29 10:47:10',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 1 => array(
'id' => '4784',
'name' => 'A kinesin-based approach for inducing chromosome-specific mis-segregationin human cells.',
'authors' => 'Truong M.A. et al.',
'description' => '<p>Various cancer types exhibit characteristic and recurrent aneuploidy patterns. The origins of these cancer type-specific karyotypes are still unknown, partly because introducing or eliminating specific chromosomes in human cells still poses a challenge. Here, we describe a novel strategy to induce mis-segregation of specific chromosomes in different human cell types. We employed Tet repressor or nuclease-dead Cas9 to link a microtubule minus-end-directed kinesin (Kinesin14VIb) from Physcomitrella patens to integrated Tet operon repeats and chromosome-specific endogenous repeats, respectively. By live- and fixed-cell imaging, we observed poleward movement of the targeted loci during (pro)metaphase. Kinesin14VIb-mediated pulling forces on the targeted chromosome were counteracted by forces from kinetochore-attached microtubules. This tug-of-war resulted in chromosome-specific segregation errors during anaphase and revealed that spindle forces can heavily stretch chromosomal arms. By single-cell whole-genome sequencing, we established that kinesin-induced targeted mis-segregations predominantly result in chromosomal arm aneuploidies after a single cell division. Our kinesin-based strategy opens the possibility to investigate the immediate cellular responses to specific aneuploidies in different cell types; an important step toward understanding how tissue-specific aneuploidy patterns evolve.</p>',
'date' => '2023-04-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/37038978',
'doi' => '10.15252/embj.2022111559',
'modified' => '2023-06-13 09:21:25',
'created' => '2023-05-05 12:34:24',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 2 => array(
'id' => '4344',
'name' => 'MICAL1 regulates actin cytoskeleton organization, directional cellmigration and the growth of human breast cancer cells as orthotopicxenograft tumours.',
'authors' => 'McGarry David J et al.',
'description' => '<p>The Molecule Interacting with CasL 1 (MICAL1) monooxygenase has emerged as an important regulator of cytoskeleton organization via actin oxidation. Although filamentous actin (F-actin) increases MICAL1 monooxygenase activity, hydrogen peroxide (HO) is also generated in the absence of F-actin, suggesting that diffusible HO might have additional functions. MICAL1 gene disruption by CRISPR/Cas9 in MDA MB 231 human breast cancer cells knocked out (KO) protein expression, which affected F-actin organization, cell size and motility. Transcriptomic profiling revealed that MICAL1 deletion significantly affected the expression of over 700 genes, with the majority being reduced in their expression levels. In addition, the absolute magnitudes of reduced gene expression were significantly greater than the magnitudes of increased gene expression. Gene set enrichment analysis (GSEA) identified receptor regulator activity as the most significant negatively enriched molecular function gene set. The prominent influence exerted by MICAL1 on F-actin structures was also associated with changes in the expression of several serum-response factor (SRF) regulated genes in KO cells. Moreover, MICAL1 disruption attenuated breast cancer tumour growth in vivo. Elevated MICAL1 gene expression was observed in invasive breast cancer samples from human patients relative to normal tissue, while MICAL1 amplification or point mutations were associated with reduced progression free survival. Collectively, these results demonstrate that MICAL1 gene disruption altered cytoskeleton organization, cell morphology and migration, gene expression, and impaired tumour growth in an orthotopic in vivo breast cancer model, suggesting that pharmacological MICAL1 inhibition could have therapeutic benefits for cancer patients.</p>',
'date' => '2021-10-01',
'pmid' => 'https://doi.org/10.1016%2Fj.canlet.2021.07.039',
'doi' => '10.1016/j.canlet.2021.07.039',
'modified' => '2022-06-21 16:56:09',
'created' => '2022-05-19 10:41:50',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 3 => array(
'id' => '3925',
'name' => 'CRISPR-based gene knockout screens reveal deubiquitinases involved in HIV-1 latency in two Jurkat cell models.',
'authors' => 'Rathore A, Iketani S, Wang P, Jia M, Sahi V, Ho DD',
'description' => '<p>The major barrier to a HIV-1 cure is the persistence of latent genomes despite treatment with antiretrovirals. To investigate host factors which promote HIV-1 latency, we conducted a genome-wide functional knockout screen using CRISPR-Cas9 in a HIV-1 latency cell line model. This screen identified IWS1, POLE3, POLR1B, PSMD1, and TGM2 as potential regulators of HIV-1 latency, of which PSMD1 and TMG2 could be confirmed pharmacologically. Further investigation of PSMD1 revealed that an interacting enzyme, the deubiquitinase UCH37, was also involved in HIV-1 latency. We therefore conducted a comprehensive evaluation of the deubiquitinase family by gene knockout, identifying several deubiquitinases, UCH37, USP14, OTULIN, and USP5 as possible HIV-1 latency regulators. A specific inhibitor of USP14, IU1, reversed HIV-1 latency and displayed synergistic effects with other latency reversal agents. IU1 caused degradation of TDP-43, a negative regulator of HIV-1 transcription. Collectively, this study is the first comprehensive evaluation of deubiquitinases in HIV-1 latency and establishes that they may hold a critical role.</p>',
'date' => '2020-03-24',
'pmid' => 'http://www.pubmed.gov/32210344',
'doi' => '10.1038/s41598-020-62375-3',
'modified' => '2020-08-17 10:51:24',
'created' => '2020-08-10 12:12:25',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 4 => array(
'id' => '3821',
'name' => 'DAPK1 loss triggers tumor invasion in colorectal tumor cells.',
'authors' => 'Steinmann S, Kunze P, Hampel C, Eckstein M, Bertram Bramsen J, Muenzner JK, Carlé B, Ndreshkjana B, Kemenes S, Gasparini P, Friedrich O, Andersen C, Geppert C, Wang S, Eyupoglu I, Bäuerle T, Hartmann A, Schneider-Stock R',
'description' => '<p>Colorectal cancer (CRC) is one of the leading cancer-related causes of death worldwide. Despite the improvement of surgical and chemotherapeutic treatments, as of yet, the disease has not been overcome due to metastasis to distant organs. Hence, it is of great relevance to understand the mechanisms responsible for metastasis initiation and progression and to identify novel metastatic markers for a higher chance of preventing the metastatic disease. The Death-associated protein kinase 1 (DAPK1), recently, has been shown to be a potential candidate for regulating metastasis in CRC. Hence, the aim of the study was to investigate the impact of DAPK1 protein on CRC aggressiveness. Using CRISPR/Cas9 technology, we generated DAPK1-deficient HCT116 monoclonal cell lines and characterized their knockout phenotype in vitro and in vivo. We show that loss of DAPK1 implemented changes in growth pattern and enhanced tumor budding in vivo in the chorioallantoic membrane (CAM) model. Further, we observed more tumor cell dissemination into chicken embryo organs and increased invasion capacity using rat brain 3D in vitro model. The novel identified DAPK1-loss gene expression signature showed a stroma typical pattern and was associated with a gained ability for remodeling the extracellular matrix. Finally, we suggest the DAPK1-ERK1 signaling axis being involved in metastatic progression of CRC. Our results highlight DAPK1 as an anti-metastatic player in CRC and suggest DAPK1 as a potential predictive biomarker for this cancer type.</p>',
'date' => '2019-11-26',
'pmid' => 'http://www.pubmed.gov/31772156',
'doi' => '10.1038/s41419-019-2122-z',
'modified' => '2020-02-25 13:45:15',
'created' => '2020-02-13 10:02:44',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(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',
'created' => '2019-06-06 12:11:18',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 6 => array(
'id' => '3565',
'name' => 'Can Mitochondrial DNA be CRISPRized: Pro and Contra.',
'authors' => 'Loutre R, Heckel AM, Smirnova A, Entelis N, Tarassov I',
'description' => '<p>Mitochondria represent a chimera of macromolecules encoded either in the organellar genome, mtDNA, or in the nuclear one. If the pathway of protein targeting to different sub-compartments of mitochondria was relatively well studied, import of small noncoding RNAs into mammalian mitochondria still awaits mechanistic explanations and its functional issues are often not understood thus raising polemics. At the same time, RNA mitochondrial import pathway has an obvious attractiveness as it appears as a unique natural mechanism permitting to address nucleic acids into the organelles. Deciphering the function(s) of imported RNAs inside the mitochondria is extremely complicated due to their relatively low abundance, which suggests their regulatory role. We previously demonstrated that mitochondrial targeting of small noncoding RNAs able to specifically anneal with the mutant mitochondrial DNA led to a decrease of the mtDNA heteroplasmy level by inhibiting mutant mtDNA replication. We then demonstrated that increasing level of expression of such antireplicative recombinant RNAs increases significantly the antireplicative effect. In this report, we present a new data investigating the possibility to establish a CRISPR-Cas9 system targeting mtDNA exploiting of the pathway of RNA import into mitochondria. Mitochondrially addressed Cas9 versions and a set of mitochondrially targeted guide RNAs were tested in vitro and in vivo and their effect on mtDNA copy number was demonstrated. So far, the system appeared as more complicated for use than previously found for nuclear DNA, because only application of a pair of guide RNAs produced the effect of mtDNA depletion. We discuss, in a critical way, these results and put them in a broader context of polemics concerning the possibilities of manipulation of mtDNA in mammalians. The findings described here prove the potential of the RNA import pathway as a tool for studying mtDNA and for future therapy of mitochondrial disorders. © The Authors. IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 70(12):1233-1239, 2018.</p>',
'date' => '2018-12-01',
'pmid' => 'http://www.pubmed.gov/30184317',
'doi' => '10.1002/iub.1919',
'modified' => '2019-03-21 17:20:49',
'created' => '2019-03-21 14:12:08',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 7 => array(
'id' => '3268',
'name' => 'In trans paired nicking triggers seamless genome editing without double-stranded DNA cutting',
'authors' => 'Chen X. et al.',
'description' => '<p>Precise genome editing involves homologous recombination between donor DNA and chromosomal sequences subjected to double-stranded DNA breaks made by programmable nucleases. Ideally, genome editing should be efficient, specific, and accurate. However, besides constituting potential translocation-initiating lesions, double-stranded DNA breaks (targeted or otherwise) are mostly repaired through unpredictable and mutagenic non-homologous recombination processes. Here, we report that the coordinated formation of paired single-stranded DNA breaks, or nicks, at donor plasmids and chromosomal target sites by RNA-guided nucleases based on CRISPR-Cas9 components, triggers seamless homology-directed gene targeting of large genetic payloads in human cells, including pluripotent stem cells. Importantly, in addition to significantly reducing the mutagenicity of the genome modification procedure, this in trans paired nicking strategy achieves multiplexed, single-step, gene targeting, and yields higher frequencies of accurately edited cells when compared to the standard double-stranded DNA break-dependent approach.</p>',
'date' => '2017-09-22',
'pmid' => 'https://www.nature.com/articles/s41467-017-00687-1',
'doi' => '',
'modified' => '2017-10-09 16:27:58',
'created' => '2017-10-09 16:27:58',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 8 => array(
'id' => '3244',
'name' => 'Highly efficient gene inactivation by adenoviral CRISPR/Cas9 in human primary cells',
'authors' => 'Voets O. et al.',
'description' => '<p>Phenotypic assays using human primary cells are highly valuable tools for target discovery and validation in drug discovery. Expression knockdown (KD) of such targets in these assays allows the investigation of their role in models of disease processes. Therefore, efficient and fast modes of protein KD in phenotypic assays are required. The CRISPR/Cas9 system has been shown to be a versatile and efficient means of gene inactivation in immortalized cell lines. Here we describe the use of adenoviral (AdV) CRISPR/Cas9 vectors for efficient gene inactivation in two human primary cell types, normal human lung fibroblasts and human bronchial epithelial cells. The effects of gene inactivation were studied in the TGF-β-induced fibroblast to myofibroblast transition assay (FMT) and the epithelial to mesenchymal transition assay (EMT), which are SMAD3 dependent and reflect pathogenic mechanisms observed in fibrosis. Co-transduction (co-TD) of AdV Cas9 with SMAD3-targeting guide RNAs (gRNAs) resulted in fast and efficient genome editing judged by insertion/deletion (indel) formation, as well as significant reduction of SMAD3 protein expression and nuclear translocation. This led to phenotypic changes downstream of SMAD3 inhibition, including substantially decreased alpha smooth muscle actin and fibronectin 1 expression, which are markers for FMT and EMT, respectively. A direct comparison between co-TD of separate Cas9 and gRNA AdV, versus TD with a single "all-in-one" Cas9/gRNA AdV, revealed that both methods achieve similar levels of indel formation. These data demonstrate that AdV CRISPR/Cas9 is a useful and efficient tool for protein KD in human primary cell phenotypic assays. The use of AdV CRISPR/Cas9 may offer significant advantages over the current existing tools and should enhance target discovery and validation opportunities.</p>',
'date' => '2017-08-11',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/28800587',
'doi' => '',
'modified' => '2017-09-19 17:26:46',
'created' => '2017-09-19 17:26:46',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 9 => array(
'id' => '3040',
'name' => 'Noncoding somatic and inherited single-nucleotide variants converge to promote ESR1 expression in breast cancer',
'authors' => 'Bailey SD et al.',
'description' => '<p>Sustained expression of the estrogen receptor-<span class="mb">α</span> (ESR1) drives two-thirds of breast cancer and defines the ESR1-positive subtype. ESR1 engages enhancers upon estrogen stimulation to establish an oncogenic expression program<sup><a href="http://www.nature.com/ng/journal/v48/n10/full/ng.3650.html#ref1" title="Green, K.A. & Carroll, J.S. Oestrogen-receptor-mediated transcription and the influence of co-factors and chromatin state. Nat. Rev. Cancer 7, 713-722 (2007)." id="ref-link-5">1</a></sup>. Somatic copy number alterations involving the <i>ESR1</i> gene occur in approximately 1% of ESR1-positive breast cancers<sup><a href="http://www.nature.com/ng/journal/v48/n10/full/ng.3650.html#ref2" title="Vincent-Salomon, A., Raynal, V., Lucchesi, C., Gruel, N. & Delattre, O. ESR1 gene amplification in breast cancer: a common phenomenon? Nat. Genet. 40, 809, author reply 810-812 (2008)." id="ref-link-6">2</a>, <a href="http://www.nature.com/ng/journal/v48/n10/full/ng.3650.html#ref3" title="Brown, L.A. et al. ESR1 gene amplification in breast cancer: a common phenomenon? Nat. Genet. 40, 806-807, author reply 810-812 (2008)." id="ref-link-7">3</a>, <a href="http://www.nature.com/ng/journal/v48/n10/full/ng.3650.html#ref4" title="Horlings, H.M. et al. ESR1 gene amplification in breast cancer: a common phenomenon? Nat. Genet. 40, 807-808, author reply 810-812 (2008)." id="ref-link-8">4</a>, <a href="http://www.nature.com/ng/journal/v48/n10/full/ng.3650.html#ref5" title="Reis-Filho, J.S. et al. ESR1 gene amplification in breast cancer: a common phenomenon? Nat. Genet. 40, 809-810, author reply 810-812 (2008)." id="ref-link-9">5</a></sup>, suggesting that other mechanisms underlie the persistent expression of <i>ESR1</i>. We report significant enrichment of somatic mutations within the set of regulatory elements (SRE) regulating <i>ESR1</i> in 7% of ESR1-positive breast cancers. These mutations regulate <i>ESR1</i> expression by modulating transcription factor binding to the DNA. The SRE includes a recurrently mutated enhancer whose activity is also affected by rs9383590, a functional inherited single-nucleotide variant (SNV) that accounts for several breast cancer risk–associated loci. Our work highlights the importance of considering the combinatorial activity of regulatory elements as a single unit to delineate the impact of noncoding genetic alterations on single genes in cancer.</p>',
'date' => '2016-08-29',
'pmid' => 'http://www.nature.com/ng/journal/v48/n10/full/ng.3650.html',
'doi' => '',
'modified' => '2016-11-03 11:59:03',
'created' => '2016-10-07 11:08:31',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 10 => array(
'id' => '3038',
'name' => 'The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing.',
'authors' => 'de Solis CA et al.',
'description' => '<p>The RNA-guided Cas9 nuclease, from the type II prokaryotic Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) adaptive immune system, has been adapted and utilized by scientists to edit the genomes of eukaryotic cells. Here, we report the development of a viral mediated CRISPR/Cas9 system that can be rendered inducible utilizing doxycycline (Dox) and can be delivered to cells in vitro and in vivo utilizing adeno-associated virus (AAV). Specifically, we developed an inducible gRNA (gRNAi) AAV vector that is designed to express the gRNA from a H1/TO promoter. This AAV vector is also designed to express the Tet repressor (TetR) to regulate the expression of the gRNAi in a Dox dependent manner. We show that H1/TO promoters of varying length and a U6/TO promoter can edit DNA with similar efficiency in vitro, in a Dox dependent manner. We also demonstrate that our inducible gRNAi vector can be used to edit the genomes of neurons in vivo within the mouse brain in a Dox dependent manner. Genome editing can be induced in vivo with this system by supplying animals Dox containing food for as little as 1 day. This system might be cross compatible with many existing S. pyogenes Cas9 systems (i.e., Cas9 mouse, CRISPRi, etc.), and therefore it likely can be used to render these systems inducible as well.</p>',
'date' => '2016-08-18',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/27587996',
'doi' => '',
'modified' => '2016-10-05 15:48:14',
'created' => '2016-10-05 15:48:14',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 11 => 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(
[maximum depth reached]
)
),
(int) 12 => array(
'id' => '2823',
'name' => 'A geminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing',
'authors' => 'Yin K, Han T, Liu G, Chen T, Wang Y, Yu AY, Liu Y',
'description' => '<p>CRISPR/Cas has emerged as potent genome editing technology and has successfully been applied in many organisms, including several plant species. However, delivery of genome editing reagents remains a challenge in plants. Here, we report a <u>vi</u>rus-based guide RNA (gRNA) delivery system for CRISPR/Cas9 mediated plant <u>g</u>enome <u>e</u>diting (VIGE) that can be used to precisely target genome locations and cause mutations. VIGE is performed by using a modified Cabbage Leaf Curl virus (CaLCuV) vector to express gRNAs in stable transgenic plants expressing Cas9. DNA sequencing confirmed VIGE of endogenous <i>NbPDS3</i> and <i>NbIspH</i> genes in non-inoculated leaves because CaLCuV can infect plants systemically. Moreover, VIGE of <i>NbPDS3</i> and <i>NbIspH</i> in newly developed leaves caused photo-bleached phenotype. These results demonstrate that geminivirus-based VIGE could be a powerful tool in plant genome editing.</p>',
'date' => '2015-10-09',
'pmid' => 'http://www.ncbi.nlm.nih.gov/pubmed/26450012',
'doi' => '10.1038/srep14926',
'modified' => '2016-02-16 13:55:32',
'created' => '2016-02-16 13:55:32',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 13 => array(
'id' => '2765',
'name' => 'Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection.',
'authors' => 'Liang X, Potter J, Kumar S, Zou Y, Quintanilla R, Sridharan M, Carte J, Chen W, Roark N, Ranganathan S, Ravinder N, Chesnut JD',
'description' => '<p>CRISPR-Cas9 systems provide a platform for high efficiency genome editing that are enabling innovative applications of mammalian cell engineering. However, the delivery of Cas9 and synthesis of guide RNA (gRNA) remain as steps that can limit overall efficiency and ease of use. Here we describe methods for rapid synthesis of gRNA and for delivery of Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs) into a variety of mammalian cells through liposome-mediated transfection or electroporation. Using these methods, we report nuclease-mediated indel rates of up to 94% in Jurkat T cells and 87% in induced pluripotent stem cells (iPSC) for a single target. When we used this approach for multigene targeting in Jurkat cells we found that two-locus and three-locus indels were achieved in approximately 93% and 65% of the resulting isolated cell lines, respectively. Further, we found that the off-target cleavage rate is reduced using Cas9 protein when compared to plasmid DNA transfection. Taken together, we present a streamlined cell engineering workflow that enables gRNA design to analysis of edited cells in as little as four days and results in highly efficient genome modulation in hard-to-transfect cells. The reagent preparation and delivery to cells is amenable to high throughput, multiplexed genome-wide cell engineering.</p>',
'date' => '2015-08-20',
'pmid' => 'http://www.ncbi.nlm.nih.gov/pubmed/26003884',
'doi' => '10.1016/j.jbiotec.2015.04.024',
'modified' => '2016-09-21 16:28:13',
'created' => '2015-07-24 15:39:05',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 14 => array(
'id' => '2792',
'name' => 'A localized nucleolar DNA damage response facilitates recruitment of the homology-directed repair machinery independent of cell cycle stage.',
'authors' => 'van Sluis M, McStay B',
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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>
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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>Diagenode, a dedicated supplier of high quality Cas9 antibodies, was<strong> the first company</strong> that offered<strong> the antibody Cas9 (clone 7A9)</strong>. This CRISPR/Cas antibody has been validated in a number of different applications including WB, IF, and IP. Our long history of expertise with CRISPR/Cas9 will guarantee your experimental success.</p>
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