Difference between revisions of "Team:BGU Israel/test/Description"

Line 129: Line 129:
 
<ul>
 
<ul>
  
−
<h2> Detailed design </h2><br />
+
<h2> Overview </h2><br />
−
<h3><i>Design 1: Cancer-specific CRISPR/Cas9-mediated gene knock-out </i></h3><br />
+
<h3><i>Motivation</i></h3><br />
−
This design utilizes “classical” CRISPR-Cas9 system for knock-out of a cancer-essential gene.
+
−
 
+
−
 
+
 
<p>
 
<p>
−
<i>Illustration similar to 2nd design </i><br/><br/>
+
Although it is one of the most researched and funded fields in medicine, cancer is still a major cause of morbidity and mortality worldwide, with 14 million new cases and over 8 million deaths per year.
−
The system includes two parts: one AAV expression cassette with the Cas9 gene, and the other with a gRNA designed to target 3 sequence repeats in the second exon of Ubb.<br/><br/>
+
It is the second cause of death worldwide, and it’s responsible for quarter of the death cases among developed countries. If current trends continue, cancer will soon surpass heart disease as the leading cause of death in the U.S
−
<b>Cas9</b> – the Cas9 endonuclease was assembled into an expression vector under hTERT promoter. Therefore Cas9 should be expressed predominantly in cells in which the promoter is highly active, namely – cancer cells (1). We utilized Staphylococcus aureus Cas9 version (SaCas9) (4). <br/><br/>
+
<br />
−
<b>gRNA</b> – the guide RNA is a hundred base-long molecule with a unique two dimensional structure which binds Cas9 and guides it to a dsDNA sequence complementary to 21-22 base pairs on the 5' end of the molecule. The gRNA was also assembled into a AAV vector, under the control of human survivin promoter. gRNA scaffold sequence for SaCas9 was used (5). In order to utilize the cancer specific promoter hyperactivation we used an RGR (Ribozyme gRNA Ribozyme). This design allows for gRNAs to be transcribed and processed using RNA polymerase II promoters, since these are the main promoters controlling gene activation (6). <br/><br/>
+
The failure of current therapies to cure cancer is due to a few reasons:<br />
−
 
+
1. Most treatments cannot distinguish precisely enough between cancer and healthy cells. Low specificity means higher toxicity and high rate of adverse effects.<br /><br />
−
When both conditions are met, meaning the system is in a cancer cell in which both promoters are highly active, the SaCas9 is guided by the gRNA to the target DNA, and introduces double strand breaks (DSB) at the target site. This then leads to activation of intrinsic DNA damage repair mechanism - predominantly error-prone non-homologous end joining (NHEJ), which introduces insertion/deletion mutations. This, in turn, can significantly disrupt a coding sequence, eliminating partially or completely a target protein function.
+
2. Cancer cells have an extremely complex pathophysiology with multiple biological pathways allowing their infinite growth and resistance to treatment. Thus, intervening with only one of this pathways, as most current therapies do, is doomed to fail.<br /><br />
−
<br/><br/>
+
3. Cancer is not a single disease, but a collection of diseases arising from different genetic mutations, involving abnormal cell growth.
−
For a proof-of-concept of the knock-out system, we chose Ubiquitin B (Ubb) gene which encodes for poly-Ubiquitin, as a target for gRNA-guided SaCas9. Ubiquitin levels are elevated in most, if not all human cancer cells, it is essential to the growth of cancer cells, and the protein product of the gene is thought to help cancer cells adapt to increased stress (7). Ubb emerges as one of the promising targets for cancer therapy. For example, Ubb downregulation by siRNA has shown a high decrease in tumor proliferation and increased apoptosis, both in vitro and in vivo (7).
+
−
</p>
+
 
<br /><br />
 
<br /><br />
  
 +
<b> Our aim </b>, therefore, is to develop the ideal cancer therapy that is both highly specific for cancer cells, efficient, and personalized for each tumor and patient genetics. <br />
 +
</ p>
  
 +
<h3><i>Boomerang</i></h3><br />
  
−
<h3><i>Design 1: Cancer-specific CRISPR/Cas9-mediated gene knock-out </i></h3><br />
 
 
<p>
 
<p>
−
<i>Take updated figure </i><br/><br/>
+
This summer we have set our goal to design and test a synthetic machine which could distinguish individual cancer cells from healthy tissue. Our design makes sure that the function of our machine will be limited exclusively to cancer cells. Our machine does so by being operated by 2 separate cancer-specific promoters, which are highly and predominantly activated in cancer cells (1)+ <a href="">(link to Results figure of TERT and survivin)</a>.
−
This design utilizes modified CRISPR-Cas9 system for transcriptional activation of any gene of interest.<br/>
+
By using two separate promoters we ensure that our system will be exclusively activated only in cancer cells, with minimal, if any, expression in healthy cells. Simply by changing the promoters that control the system parts, our modular system makes it easy to design the system to fit the genetic profile of each individual malignancy. <br />
−
The system includes 3 parts: one AAV expression cassette with the activator Cas9 (dCas9-VP64), the second with a gRNA designed to guide activator Cas9 to synthetic promoter, and the third with the gene of interest (modelled by GFP) under the control of synthetic activation promoter. <br/>
+
<br />
  
−
<b>dCas9-VP64</b> – dCas9-VP64 was engineered so that it lacks endonuclease activity (“dead” Cas9) and has 4 VP16 activation domains fused to the protein. When guided to a specific promoter, dCas9-VP64 activates transcription of genes downstream of its binding site (8). dCas9-VP64 was assembled into an expression vector under hTERT promoter.
+
There were several ways in which we can deliver our system in the body, and we chose AAV (Adeno Associated Virus) because of its many advantages, including low pathogenicity and mild immune response. AAV is used today in advanced clinical trials for gene therapy. The efficacy of our system will be dependent on the development of effective delivery approaches. (3).
−
<br/><br/>
+
<br /><br />
−
<b>gRNA</b>- The gRNA (using the scaffold for Staphylococcus pyogenes Cas9) was also assembled into a AAV vector, under the control of human survivin promoter, using previously described ribozyme design. The gRNA sequence is used to guide dCas9-V64 to a specific synthetic promoter.
+
−
<br/><br/>
+
−
<b>Synthetic activation promoter</b>- The third part  of the system is an expression AAV cassette with GFP under the control of synthetic promoter (9). The synthetic promoter has 3 complementary sites for the gRNA, which, upon binding of dCas9-VP64 in a tandem, should promote transcription of a downstream gene.  
+
−
<br/><br/>
+
  
−
For a proof-of-concept, we utilized GFP as our target gene. The design allows for an expression of any desired protein: 1) to induce cancer cell apoptosis or cell death by using reversed caspase-3, which can lead to apoptosis when expressed in cells, or diphteria toxin A, which can kill a cell (10,11), 2) label the tumor for complete surgical removal by using chromoproteins; and 3) produce a biomarker detectable in the blood or urine, for cancer diagnosis, for example, by using SEAP (Secreted embryonic alkaline phosphatase), which can be excreted out of the cells and its levels  monitored easily (12) (figure 2).
+
In our specific design for the prototype/proof-of-concept studies we use promoters which are linked to tumor proliferation (human telomerase-reverse transcriptase (hTERT) promoter) and enhanced survival (human survivin promoter), both known to be highly active in multiple cancer cell types.  
−
<br /><br />
+
−
<table>
+
−
  <tr>
+
−
    <th> <b>Figure 2 - </b></th>
+
−
  </tr>
+
−
  <tr>
+
−
    <th> <img src="https://static.igem.org/mediawiki/2015/7/72/BGUigem_project_design1.png" alt="Smiley face" height="310" width="473"></th>
+
−
</tr>
+
−
</table>
+
 
</p>
 
</p>
−
<br /><br />
 
−
 
−
 
−
 
−
 
−
 
−
 
−
 
−
<b> <h2> Detailed design and cloning program</h2></b><br />
 
−
1)
 
−
 
−
2) Design of Boomerang components
 
−
    Our basic components include prompters, Cas9 proteins and guideRNAs. Most have been synthesized by IDT and Synthezza Bioscience and some were ordered from addgene.
 
  
 +
<h4> The Design </h4>
 +
<p>
 
We have constructed two separate designs, both utilizing different versions of CRISPR/Cas9 system: <br />
 
We have constructed two separate designs, both utilizing different versions of CRISPR/Cas9 system: <br />
 
<ul>
 
<ul>
−
   <li> Design of “master” template with specific restriction sites for subcloning
+
   <li> Knock-out of genes essential for cancer cell survival (e.g., to inhibit tumor proliferation and induce apoptosis) </li>
−
    <img src="https://static.igem.org/mediawiki/2015/7/72/BGUigem_project_design1.png" >
+
   <li>Expression of exogenous proteins which could: 1) label the tumor in a way which would enable surgeons to identify its edges for its complete removal (e.g., a chromophore), 2) lead to cancer cell death (e.g., by expression of an apoptotic protein); and 3) to produce a biomarker detectable in blood and/or urine for cancer diagnosis</li>
−
</li>
+
−
   <li> Design of Boomerang components<br />
+
−
      Our basic components include prompters, Cas9 proteins and guideRNAs.  
+
−
      Most have been synthesized by IDT and Synthezza Bioscience and some were ordered from addgene.
+
−
  </li>
+
 
</ul>
 
</ul>
 
</p>
 
</p>
−
<br /><br /><br />
 
  
−
<!-- Map & Sequences -->
+
<table>
−
<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 width=602
+
   <tr>
−
style='width:451.45pt;margin-left:5.0pt;border-collapse:collapse;border:none;
+
    <th> <img src="https://static.igem.org/mediawiki/2015/7/72/BGUigem_project_design1.png" alt="Smiley face" height="310" width="473"></th>
−
mso-border-alt:solid black 1.0pt;mso-yfti-tbllook:1536;mso-padding-alt:0cm 5.4pt 0cm 5.4pt;
+
    <th>TBD</th>
−
mso-border-insideh:1.0pt solid black;mso-border-insidev:1.0pt solid black'>
+
   </tr>
−
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes;height:21.0pt'>
+
−
   <td width=602 colspan=4 valign=top style='width:451.45pt;border:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt;height:21.0pt'>
+
−
  <p class=MsoNormal align=center dir=LTR style='text-align:center;line-height:
+
−
  normal;mso-pagination:none'><b style='mso-bidi-font-weight:normal'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>Synthesized Components</span></b><o:p></o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:1'>
+
−
  <td width=98 valign=top style='width:73.5pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><b
+
−
  style='mso-bidi-font-weight:normal'><span style='font-family:"Calibri","sans-serif";
+
−
  mso-fareast-font-family:Calibri;mso-bidi-font-family:Calibri'>Name</span></b><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=329 valign=top style='width:246.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><b
+
−
  style='mso-bidi-font-weight:normal'><span style='font-family:"Calibri","sans-serif";
+
−
  mso-fareast-font-family:Calibri;mso-bidi-font-family:Calibri'>Description</span></b><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=97 valign=top style='width:72.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><b
+
−
  style='mso-bidi-font-weight:normal'><span style='font-family:"Calibri","sans-serif";
+
−
  mso-fareast-font-family:Calibri;mso-bidi-font-family:Calibri'>Source</span></b><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=78 valign=top style='width:58.45pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><b
+
−
  style='mso-bidi-font-weight:normal'><span style='font-family:"Calibri","sans-serif";
+
−
  mso-fareast-font-family:Calibri;mso-bidi-font-family:Calibri'>Map, Sequence</span></b><o:p></o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:2'>
+
−
  <td width=98 valign=top style='width:73.5pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>dCas9-VP64</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=329 valign=top style='width:246.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>Activator Cas9 for promotion of exogenous genes
+
−
  expression</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=97 valign=top style='width:72.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><a
+
−
  href="https://www.addgene.org/47107/"><span style='font-size:9.5pt;
+
−
  color:#1155CC;background:white;mso-highlight:white'>https://www.addgene.org/47107/</span></a><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=78 valign=top style='width:58.45pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:3'>
+
−
  <td width=98 valign=top style='width:73.5pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>SaCas9</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=329 valign=top style='width:246.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>“Classical” Cas9 endonuclease for knock-down of
+
−
  target genes</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=97 valign=top style='width:72.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><a
+
−
  href="https://www.addgene.org/61591/"><span style='font-size:9.5pt;
+
−
  color:#1155CC;background:white;mso-highlight:white'>https://www.addgene.org/61591/</span></a><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=78 valign=top style='width:58.45pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:4'>
+
−
  <td width=98 valign=top style='width:73.5pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>gMLP</span></span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=329 valign=top style='width:246.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>Ribozyme flanked guide RNA leading dCas9-VP64
+
−
  to the synthetic promoter</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=97 valign=top style='width:72.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>IDT</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=78 valign=top style='width:58.45pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:5'>
+
−
  <td width=98 valign=top style='width:73.5pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>gUBB</span></span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=329 valign=top style='width:246.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>Ribozyme flaked guide RNA leading Cas9 to cut
+
−
  UBB gene</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=97 valign=top style='width:72.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>IDT</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=78 valign=top style='width:58.45pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:6'>
+
−
  <td width=98 valign=top style='width:73.5pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>U6</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=329 valign=top style='width:246.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>As positive control for RGR design</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=97 valign=top style='width:72.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
  </td>
+
−
  <td width=78 valign=top style='width:58.45pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:7'>
+
−
  <td width=98 valign=top style='width:73.5pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>pSurvivin</span></span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=329 valign=top style='width:246.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>Promoter for <span class=SpellE>survivin</span>
+
−
  gene</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=97 valign=top style='width:72.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>Synthezza</span></span><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'> Bioscience</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=78 valign=top style='width:58.45pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:8'>
+
−
  <td width=98 valign=top style='width:73.5pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>phTERT</span></span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=329 valign=top style='width:246.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>Promoter for <span class=SpellE>hTERT</span>
+
−
  gene</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=97 valign=top style='width:72.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>Synthezza</span></span><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'> Bioscience</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=78 valign=top style='width:58.45pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:9;mso-yfti-lastrow:yes'>
+
−
  <td width=98 valign=top style='width:73.5pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>hTERT</span></span><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'> <span class=SpellE>eGFP</span> poly A - MASTER</span><o:p></o:p></p>
+
−
   </td>
+
−
  <td width=329 valign=top style='width:246.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>Master. Cloned into delivery vector as a
+
−
  cloning template for all other inserts.</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=97 valign=top style='width:72.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>Synthezza</span></span><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'> Bioscience</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=78 valign=top style='width:58.45pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
  </td>
+
−
</tr>
+
 
</table>
 
</table>
  
Line 504: Line 177:
  
  
−
<br /> <!-- missing data & photos -->
+
<br />
  
−
<div align=left dir=ltr>
+
<h4>Why Boomerang?</h4>
  
−
<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 width=659
+
<p>
−
style='width:494.25pt;margin-left:-15.0pt;border-collapse:collapse;border:
+
<table>
−
none;mso-border-alt:solid black 1.0pt;mso-yfti-tbllook:1536;mso-padding-alt:
+
   <tr>
−
0cm 5.4pt 0cm 5.4pt;mso-border-insideh:1.0pt solid black;mso-border-insidev:
+
    <th> <img src="https://static.igem.org/mediawiki/2015/8/83/Igem_bgu_logo.png" height="60px" width="245px"></th>
−
1.0pt solid black'>
+
    <th>Like a boomerang (boomerang logo) thrown by a person which flies back instantly, our synthetic machine uses cancer cells' own genetic alterations against them.</th>
−
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes;height:21.0pt'>
+
   </tr>
−
  <td width=659 colspan=4 valign=top style='width:494.25pt;border:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt;height:21.0pt'>
+
−
  <p class=MsoNormal align=center dir=LTR style='text-align:center;line-height:
+
−
  normal;mso-pagination:none'><b style='mso-bidi-font-weight:normal'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>Activation System</span></b><o:p></o:p></p>
+
−
   </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:1'>
+
−
  <td width=133 valign=top style='width:99.75pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><b
+
−
  style='mso-bidi-font-weight:normal'><span style='font-family:"Calibri","sans-serif";
+
−
  mso-fareast-font-family:Calibri;mso-bidi-font-family:Calibri'>Name</span></b><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=303 valign=top style='width:227.25pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><b
+
−
  style='mso-bidi-font-weight:normal'><span style='font-family:"Calibri","sans-serif";
+
−
  mso-fareast-font-family:Calibri;mso-bidi-font-family:Calibri'>Description</span></b><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=145 valign=top style='width:108.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><b
+
−
  style='mso-bidi-font-weight:normal'><span style='font-family:"Calibri","sans-serif";
+
−
  mso-fareast-font-family:Calibri;mso-bidi-font-family:Calibri'>Source</span></b><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=78 valign=top style='width:58.5pt;border-top:none;border-left:none;
+
−
  border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+
−
  solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><b
+
−
  style='mso-bidi-font-weight:normal'><span style='font-family:"Calibri","sans-serif";
+
−
  mso-fareast-font-family:Calibri;mso-bidi-font-family:Calibri'>Map, Sequence</span></b><o:p></o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:2'>
+
−
  <td width=133 valign=top style='width:99.75pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>phTERT-dCas9-VP64-polyA-pAAV</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=303 valign=top style='width:227.25pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=GramE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>dCas9-VP64</span></span><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'> under <span class=SpellE>hTERT</span>
+
−
  promoter. System part.</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=145 valign=top style='width:108.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>Synthezza</span></span><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'> Bioscience</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=78 valign=top style='width:58.5pt;border-top:none;border-left:none;
+
−
  border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+
−
  solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:3'>
+
−
  <td width=133 valign=top style='width:99.75pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>CMV-dCas9-VP64-polyA-pAAV</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=303 valign=top style='width:227.25pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=GramE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>dCas9</span></span><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'> under CMV promoter. Positive control for
+
−
  dCas9.</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=145 valign=top style='width:108.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>Self cloning</span></span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=78 valign=top style='width:58.5pt;border-top:none;border-left:none;
+
−
  border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+
−
  solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:4'>
+
−
  <td width=133 valign=top style='width:99.75pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>CMV-<span class=SpellE>gMLP</span>-<span
+
−
  class=SpellE>polyA-pAAV</span></span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=303 valign=top style='width:227.25pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>Ribozyme flanked <span class=SpellE>gRNA</span>
+
−
  for the synthetic promoter under CMV. Positive control for RGR design and
+
−
  system activation.</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=145 valign=top style='width:108.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
   </td>
+
−
  <td width=78 valign=top style='width:58.5pt;border-top:none;border-left:none;
+
−
  border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+
−
  solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:5'>
+
−
  <td width=133 valign=top style='width:99.75pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>pSurvivin-gMLP-polyA-pAAV</span></span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=303 valign=top style='width:227.25pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>Ribozyme flanked <span class=SpellE>gRNA</span>
+
−
  for the synthetic promoter under <span class=SpellE>Survivin</span> promoter.
+
−
  System part.</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=145 valign=top style='width:108.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>Synthezza</span></span><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'> Bioscience</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=78 valign=top style='width:58.5pt;border-top:none;border-left:none;
+
−
  border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+
−
  solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:6'>
+
−
  <td width=133 valign=top style='width:99.75pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>U6-gMLP-pAAV</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=303 valign=top style='width:227.25pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span class=GramE><span style='font-family:"Calibri","sans-serif";
+
−
  mso-fareast-font-family:Calibri;mso-bidi-font-family:Calibri'>gRNA</span></span></span><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'> for the synthetic promoter under U6 promoter.
+
−
  Positive control for <span class=SpellE>gMLP</span>.</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=145 valign=top style='width:108.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>Self cloning</span></span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=78 valign=top style='width:58.5pt;border-top:none;border-left:none;
+
−
  border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+
−
  solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:7;mso-yfti-lastrow:yes'>
+
−
  <td width=133 valign=top style='width:99.75pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>pMLPm-eGFP-polyA-pAAV</span></span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=303 valign=top style='width:227.25pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>GFP under the synthetic promoter. System part.</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=145 valign=top style='width:108.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>Synthezza</span></span><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'> Bioscience</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=78 valign=top style='width:58.5pt;border-top:none;border-left:none;
+
−
  border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+
−
  solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
  </td>
+
−
</tr>
+
 
</table>
 
</table>
−
 
+
</p>
−
</div>
+
−
 
+
−
<p class=MsoNormal dir=LTR style='line-height:107%'><o:p>&nbsp;</o:p></p>
+
−
 
+
−
<p class=MsoNormal dir=LTR style='line-height:107%'><o:p>&nbsp;</o:p></p>
+
−
 
+
−
<p class=MsoNormal dir=LTR style='line-height:107%'><o:p>&nbsp;</o:p></p>
+
−
 
+
−
<p class=MsoNormal dir=LTR style='line-height:107%'><o:p>&nbsp;</o:p></p>
+
−
 
+
−
<div align=left dir=ltr>
+
−
 
+
−
<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 width=657
+
−
style='width:492.75pt;margin-left:-15.0pt;border-collapse:collapse;border:
+
−
none;mso-border-alt:solid black 1.0pt;mso-yfti-tbllook:1536;mso-padding-alt:
+
−
0cm 5.4pt 0cm 5.4pt;mso-border-insideh:1.0pt solid black;mso-border-insidev:
+
−
1.0pt solid black'>
+
−
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes;height:21.0pt'>
+
−
  <td width=657 colspan=4 valign=top style='width:492.75pt;border:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt;height:21.0pt'>
+
−
  <p class=MsoNormal align=center dir=LTR style='text-align:center;line-height:
+
−
  normal;mso-pagination:none'><b style='mso-bidi-font-weight:normal'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>Knock-out System</span></b><o:p></o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:1'>
+
−
  <td width=128 valign=top style='width:96.0pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><b
+
−
  style='mso-bidi-font-weight:normal'><span style='font-family:"Calibri","sans-serif";
+
−
  mso-fareast-font-family:Calibri;mso-bidi-font-family:Calibri'>Name</span></b><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=306 valign=top style='width:229.5pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><b
+
−
  style='mso-bidi-font-weight:normal'><span style='font-family:"Calibri","sans-serif";
+
−
  mso-fareast-font-family:Calibri;mso-bidi-font-family:Calibri'>Description</span></b><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=145 valign=top style='width:108.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><b
+
−
  style='mso-bidi-font-weight:normal'><span style='font-family:"Calibri","sans-serif";
+
−
  mso-fareast-font-family:Calibri;mso-bidi-font-family:Calibri'>Source</span></b><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=78 valign=top style='width:58.5pt;border-top:none;border-left:none;
+
−
  border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+
−
  solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><b
+
−
  style='mso-bidi-font-weight:normal'><span style='font-family:"Calibri","sans-serif";
+
−
  mso-fareast-font-family:Calibri;mso-bidi-font-family:Calibri'>Map, Sequence</span></b><o:p></o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:2'>
+
−
  <td width=128 valign=top style='width:96.0pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>phTERT-SaCas9-polyA-pAAV</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=306 valign=top style='width:229.5pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>SaCas9 under <span class=SpellE>hTERT</span>
+
−
  promoter.</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=145 valign=top style='width:108.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>Synthezza</span></span><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'> Bioscience</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=78 valign=top style='width:58.5pt;border-top:none;border-left:none;
+
−
  border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+
−
  solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:3'>
+
−
  <td width=128 valign=top style='width:96.0pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>CMV-SaCas9-polyA-pAAV</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=306 valign=top style='width:229.5pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>SaCas9 under CMV. Positive control for SaCas9.</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=145 valign=top style='width:108.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>Self cloning</span></span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=78 valign=top style='width:58.5pt;border-top:none;border-left:none;
+
−
  border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+
−
  solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:4'>
+
−
  <td width=128 valign=top style='width:96.0pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>CMV-<span class=SpellE>gUBB</span>-<span
+
−
  class=SpellE>polyA-pAAV</span></span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=306 valign=top style='width:229.5pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>Ribozyme flanked <span class=SpellE>gRNA</span>
+
−
  for UBB gene. Positive control for RGR design and system activation.</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=145 valign=top style='width:108.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
  </td>
+
−
  <td width=78 valign=top style='width:58.5pt;border-top:none;border-left:none;
+
−
  border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+
−
  solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:5'>
+
−
  <td width=128 valign=top style='width:96.0pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>pSurvivin-gUBB-polyA-pAAV</span></span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=306 valign=top style='width:229.5pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span class=GramE><span style='font-family:"Calibri","sans-serif";
+
−
  mso-fareast-font-family:Calibri;mso-bidi-font-family:Calibri'>gRNA</span></span></span><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'> for UBB gene under <span class=SpellE>Survivin</span>.
+
−
  System part.</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=145 valign=top style='width:108.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>Synthezza</span></span><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'> Bioscience</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=78 valign=top style='width:58.5pt;border-top:none;border-left:none;
+
−
  border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+
−
  solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:6;mso-yfti-lastrow:yes'>
+
−
  <td width=128 valign=top style='width:96.0pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>U6-gUBB-pAAV</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=306 valign=top style='width:229.5pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span class=GramE><span style='font-family:"Calibri","sans-serif";
+
−
  mso-fareast-font-family:Calibri;mso-bidi-font-family:Calibri'>gRNA</span></span></span><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'> for UBB gene under U6 promoter. Control for
+
−
  RGR design and <span class=SpellE>gUBB</span>.</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=145 valign=top style='width:108.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>Self cloning</span></span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=78 valign=top style='width:58.5pt;border-top:none;border-left:none;
+
−
  border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+
−
  solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
  </td>
+
−
</tr>
+
−
</table>
+
−
 
+
−
</div>
+
−
 
+
−
<p class=MsoNormal dir=LTR style='line-height:107%'><o:p>&nbsp;</o:p></p>
+
−
 
+
−
<p class=MsoNormal dir=LTR style='margin-bottom:8.0pt;line-height:107%'><o:p>&nbsp;</o:p></p>
+
−
 
+
−
<p class=MsoNormal dir=LTR style='line-height:107%'><o:p>&nbsp;</o:p></p>
+
−
 
+
−
<div align=left dir=ltr>
+
−
 
+
−
<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 width=653
+
−
style='width:489.75pt;margin-left:-15.0pt;border-collapse:collapse;border:
+
−
none;mso-border-alt:solid black 1.0pt;mso-yfti-tbllook:1536;mso-padding-alt:
+
−
0cm 5.4pt 0cm 5.4pt;mso-border-insideh:1.0pt solid black;mso-border-insidev:
+
−
1.0pt solid black'>
+
−
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes;height:21.0pt'>
+
−
  <td width=653 colspan=4 valign=top style='width:489.75pt;border:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt;height:21.0pt'>
+
−
  <p class=MsoNormal align=center dir=LTR style='text-align:center;line-height:
+
−
  normal;mso-pagination:none'><b style='mso-bidi-font-weight:normal'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>General Controls</span></b><o:p></o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:1'>
+
−
  <td width=128 valign=top style='width:96.0pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><b
+
−
  style='mso-bidi-font-weight:normal'><span style='font-family:"Calibri","sans-serif";
+
−
  mso-fareast-font-family:Calibri;mso-bidi-font-family:Calibri'>Name</span></b><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=304 valign=top style='width:228.0pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><b
+
−
  style='mso-bidi-font-weight:normal'><span style='font-family:"Calibri","sans-serif";
+
−
  mso-fareast-font-family:Calibri;mso-bidi-font-family:Calibri'>Description</span></b><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=145 valign=top style='width:108.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><b
+
−
  style='mso-bidi-font-weight:normal'><span style='font-family:"Calibri","sans-serif";
+
−
  mso-fareast-font-family:Calibri;mso-bidi-font-family:Calibri'>Source</span></b><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=76 valign=top style='width:57.0pt;border-top:none;border-left:none;
+
−
  border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+
−
  solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><b
+
−
  style='mso-bidi-font-weight:normal'><span style='font-family:"Calibri","sans-serif";
+
−
  mso-fareast-font-family:Calibri;mso-bidi-font-family:Calibri'>Map, Sequence</span></b><o:p></o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:2'>
+
−
  <td width=128 valign=top style='width:96.0pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>phTERT-eGFP-polyA</span></span><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'> master-<span class=SpellE>pAAV</span></span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=304 valign=top style='width:228.0pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'>GFP under <span class=SpellE>hTERT</span>
+
−
  promoter. Positive control for <span class=SpellE>hTERT</span>.</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=145 valign=top style='width:108.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>Synthezza</span></span><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'> Bioscience</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=76 valign=top style='width:57.0pt;border-top:none;border-left:none;
+
−
  border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+
−
  solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
  </td>
+
−
</tr>
+
−
<tr style='mso-yfti-irow:3;mso-yfti-lastrow:yes'>
+
−
  <td width=128 valign=top style='width:96.0pt;border:solid black 1.0pt;
+
−
  border-top:none;mso-border-top-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>pSurvivin-mCherry-polyA-pAAV</span></span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=304 valign=top style='width:228.0pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span class=GramE><span style='font-family:"Calibri","sans-serif";
+
−
  mso-fareast-font-family:Calibri;mso-bidi-font-family:Calibri'>mCherry</span></span></span><span
+
−
  style='font-family:"Calibri","sans-serif";mso-fareast-font-family:Calibri;
+
−
  mso-bidi-font-family:Calibri'> under <span class=SpellE>Survivin</span>
+
−
  promoter. Positive control for <span class=SpellE>Survivin</span>.</span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=145 valign=top style='width:108.75pt;border-top:none;border-left:
+
−
  none;border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;
+
−
  mso-border-top-alt:solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;
+
−
  padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span
+
−
  class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family:
+
−
  Calibri;mso-bidi-font-family:Calibri'>Self cloning</span></span><o:p></o:p></p>
+
−
  </td>
+
−
  <td width=76 valign=top style='width:57.0pt;border-top:none;border-left:none;
+
−
  border-bottom:solid black 1.0pt;border-right:solid black 1.0pt;mso-border-top-alt:
+
−
  solid black 1.0pt;mso-border-left-alt:solid black 1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt'>
+
−
  <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><o:p>&nbsp;</o:p></p>
+
−
  </td>
+
−
</tr>
+
−
</table>
+
−
 
+
−
</div>
+
−
 
+
−
 
+
−
 
+
−
 
+
−
 
+
  
  

Revision as of 08:56, 3 September 2015


<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">

Team:BGU Israel





    Overview


    Motivation


    Although it is one of the most researched and funded fields in medicine, cancer is still a major cause of morbidity and mortality worldwide, with 14 million new cases and over 8 million deaths per year. It is the second cause of death worldwide, and it’s responsible for quarter of the death cases among developed countries. If current trends continue, cancer will soon surpass heart disease as the leading cause of death in the U.S
    The failure of current therapies to cure cancer is due to a few reasons:
    1. Most treatments cannot distinguish precisely enough between cancer and healthy cells. Low specificity means higher toxicity and high rate of adverse effects.

    2. Cancer cells have an extremely complex pathophysiology with multiple biological pathways allowing their infinite growth and resistance to treatment. Thus, intervening with only one of this pathways, as most current therapies do, is doomed to fail.

    3. Cancer is not a single disease, but a collection of diseases arising from different genetic mutations, involving abnormal cell growth.

    Our aim , therefore, is to develop the ideal cancer therapy that is both highly specific for cancer cells, efficient, and personalized for each tumor and patient genetics.

    Boomerang


    This summer we have set our goal to design and test a synthetic machine which could distinguish individual cancer cells from healthy tissue. Our design makes sure that the function of our machine will be limited exclusively to cancer cells. Our machine does so by being operated by 2 separate cancer-specific promoters, which are highly and predominantly activated in cancer cells (1)+ (link to Results figure of TERT and survivin). By using two separate promoters we ensure that our system will be exclusively activated only in cancer cells, with minimal, if any, expression in healthy cells. Simply by changing the promoters that control the system parts, our modular system makes it easy to design the system to fit the genetic profile of each individual malignancy.

    There were several ways in which we can deliver our system in the body, and we chose AAV (Adeno Associated Virus) because of its many advantages, including low pathogenicity and mild immune response. AAV is used today in advanced clinical trials for gene therapy. The efficacy of our system will be dependent on the development of effective delivery approaches. (3).

    In our specific design for the prototype/proof-of-concept studies we use promoters which are linked to tumor proliferation (human telomerase-reverse transcriptase (hTERT) promoter) and enhanced survival (human survivin promoter), both known to be highly active in multiple cancer cell types.

    The Design

    We have constructed two separate designs, both utilizing different versions of CRISPR/Cas9 system:

    • Knock-out of genes essential for cancer cell survival (e.g., to inhibit tumor proliferation and induce apoptosis)
    • Expression of exogenous proteins which could: 1) label the tumor in a way which would enable surgeons to identify its edges for its complete removal (e.g., a chromophore), 2) lead to cancer cell death (e.g., by expression of an apoptotic protein); and 3) to produce a biomarker detectable in blood and/or urine for cancer diagnosis

    Smiley face TBD

Why Boomerang?

Like a boomerang (boomerang logo) thrown by a person which flies back instantly, our synthetic machine uses cancer cells' own genetic alterations against them.


References

(1) The telomerase reverse transcriptase promoter drives efficacious tumor suicide gene therapy while preventing hepatotoxicity encountered with constitutive promoters

(2) Applications of the CRISPR–Cas9 system in cancer biology

(3) Oncolytic viruses: a new class of immunotherapy drugs

(4) Targeting of tumor radioiodine therapy by expression of the sodium iodide symporter under control of the survivin promoter

(5) In vivo genome editing using Staphylococcus aureus Cas9.

(6) Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing

(7) DDownregulation of ubiquitin level via knockdown of polyubiquitin gene Ubb as potential cancer therapeutic intervention

(8) RNA-guided gene activation by CRISPR-Cas9-based transcription factors.

(9) Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas

(10) Generation of Constitutively Active Recombinant Caspases-3 and -6 by Rearrangement of Their Subunits

(11) One molecule of diphtheria toxin fragment a introduced into a cell can kill the cell

(12) SEAP expression in transiently transfected mammalian cells grown in serum-free suspension culture







Retrieved from "https://2015.igem.org/Team:BGU_Israel/Team"