Difference between revisions of "Team:BGU Israel/Design"
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| − | < | + | <ul> |
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| + | <h2> Detailed design </h2><br /> | ||
| + | <br /> | ||
| + | <h3><i>Design 1: Cancer-specific CRISPR/Cas9-mediated activation of the gene of interest </i></h3><br /><br /> | ||
| + | <p>This design utilizes modified CRISPR-Cas9 system for transcriptional activation of any gene of interest.</p> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/e/e4/BGU_Activ2%28full%29.jpg" height="500px" width="800px" /> | ||
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| − | + | 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/> | |
| − | + | <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. We designed dCas9-VP64 to be expressed under the control of human TERT promoter. Therefore dCas9-VP64 should be expressed predominantly in cells in which the promoter is highly active, namely – cancer cells (1). When guided to a specific promoter, dCas9-VP64 activates transcription of genes downstream of its binding site (2). dCas9-VP64 was assembled into an expression vector under hTERT promoter. | |
| − | + | <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 (using the scaffold sequence for Staphylococcus pyogenes Cas9) was assembled into a AAV vector, under the control of human survivin promoter (3). In order to utilize the cancer specific promoter hyperactivation we used an RGR (Ribozyme gRNA Ribozyme) design. This design allows for gRNAs to be transcribed and processed using RNA polymerase II promoters, since these are the main promoters controlling gene activation (4). The gRNA sequence is used to guide dCas9-VP64 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 (5). 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. | |
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| − | + | 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 (6), or diphteria toxin A, which can kill a cell (7), 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 (8) (Figure 1). | |
| − | + | <br /><br /> | |
| − | + | <table style="margin:0 !important;"> | |
| − | + | <tr> | |
| − | + | <td> <b>Figure 1. Possible applications of cancer-specific CRISPR-mediated gene activation </b></td> | |
| − | + | </tr> | |
| − | + | <tr> | |
| − | + | <td> <img src="https://static.igem.org/mediawiki/2015/3/31/BGUigem_design_design2.png" alt="Smiley face" height="500" width="800"></td> | |
| − | + | </tr> | |
| − | + | </table> | |
| − | + | </p> | |
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| − | + | <b>A functional prototype of this design working in human cancer cells is shown <a href="https://2015.igem.org/Team:BGU_Israel/Results#prototype">here.</a></b> | |
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| − | + | <h3><i>Design 2: Cancer-specific CRISPR/Cas9-mediated gene knock-out </i></h3><br /><br /> | |
| − | + | <p>This design utilizes “classical” CRISPR-Cas9 system for knock-out of a cancer-essential gene.</p> | |
| − | + | <br /> | |
| − | + | <img src="https://static.igem.org/mediawiki/2015/3/3f/Kill2%28full%29.jpg" height="500px" width="800px" /> | |
| − | + | <!--img src="https://static.igem.org/mediawiki/2015/1/18/BGU_Kill2.jpg" height="500px" width="800px" /--> | |
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| − | < | + | <p> |
| + | 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/> | ||
| + | <b>Cas9</b> – the Cas9 endonuclease was assembled into an expression vector under hTERT promoter. We utilized Staphylococcus aureus Cas9 version (SaCas9) (9). <br/><br/> | ||
| + | <b>gRNA</b> – The gRNA (using the scaffold sequence for Staphylococcus aureus Cas9) was also assembled into a AAV vector, under the control of human survivin promoter, using previously described ribozyme design. <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. | ||
| + | <br/><br/> | ||
| + | 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 (10). 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 (10). | ||
| + | </p> | ||
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| + | <b> <h2> Detailed design and cloning program</h2></b><br /> | ||
| + | <p><b>1. Design of “master” template with specific restriction sites for subcloning</b> | ||
| + | <br /> | ||
</p> | </p> | ||
| − | < | + | <img src="https://static.igem.org/mediawiki/2015/7/72/BGUigem_project_design1.png" ><br /><br /><br /> |
| − | < | + | <p><b>2. Design of Boomerang components</b>. |
| − | + | <br /> | |
| + | Our basic components include promoters, Cas9 proteins and guide RNAs. | ||
| + | |||
| + | </p> | ||
| + | <br /><br /> | ||
| + | |||
| + | <table width="800" style="margin:0 !important; text-align:left !important;"> | ||
| + | <tbody> | ||
| + | <tr> | ||
| + | <th colspan="3"> | ||
| + | <p dir="LTR"> | ||
| + | <b>Synthesized Components</b> | ||
| + | </p> | ||
| + | </th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td width="130"> | ||
| + | <b>Name</b> | ||
| + | </td> | ||
| + | <td width="370"> | ||
| + | <b>Description</b> | ||
| + | </td> | ||
| + | <td width="300"> | ||
| + | <b>Source</b> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> | ||
| + | dCas9-VP64 | ||
| + | </td> | ||
| + | <td> | ||
| + | Activator Cas9 for transcriptional activation of genes | ||
| + | </td> | ||
| + | <td> | ||
| + | Sequence from (2). Ordered from Addgene. | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> | ||
| + | SaCas9 | ||
| + | </td> | ||
| + | <td> | ||
| + | "Classical" Cas9 endonuclease for knock-out of target genes | ||
| + | </td> | ||
| + | <td> | ||
| + | Sequence from (9) Ordered from Addgene. | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> | ||
| + | gMLP | ||
| + | </td> | ||
| + | <td> | ||
| + | Ribozyme-flanked guide RNA leading dCas9-VP64 to the synthetic promoter | ||
| + | </td> | ||
| + | <td> | ||
| + | Sequence from (4). Synthesized by IDT. | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> | ||
| + | gUBB | ||
| + | </td> | ||
| + | <td> | ||
| + | Ribozyme-flanked guide RNA leading SaCas9 to UBB gene (exon 2) | ||
| + | </td> | ||
| + | <td> | ||
| + | Designed in Benchling. Synthesized by IDT. | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> | ||
| + | U6 promoter | ||
| + | </td> | ||
| + | <td> | ||
| + | RNA polymerase III promoter, positive control for RGR design | ||
| + | </td> | ||
| + | <td> | ||
| + | Sequence from (9). Synthesized by IDT. | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> | ||
| + | pSurvivin | ||
| + | </td> | ||
| + | <td> | ||
| + | Promoter for human survivin gene | ||
| + | </td> | ||
| + | <td> | ||
| + | Synthesized by Syntezza Bioscience | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> | ||
| + | phTERT | ||
| + | </td> | ||
| + | <td> | ||
| + | Promoter for human TERT gene | ||
| + | </td> | ||
| + | <td> | ||
| + | Synthesized by Syntezza Bioscience | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> | ||
| + | pMLPm | ||
| + | </td> | ||
| + | <td> | ||
| + | Synthetic activation promoter | ||
| + | </td> | ||
| + | <td> | ||
| + | Sequence from (5). Synthesized by Syntezza Bioscience | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> | ||
| + | hTERT eGFP polyA - MASTER | ||
| + | </td> | ||
| + | <td> | ||
| + | Master template. Cloned into delivery vector as a cloning template for all other inserts. | ||
| + | </td> | ||
| + | <td> | ||
| + | Synthesized by Syntezza | ||
| + | Bioscience | ||
| + | </td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table> | ||
| + | |||
| + | <!-- Map & Sequences | ||
| + | <table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 width=602 | ||
| + | style='width:451.45pt;margin-left:5.0pt;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; | ||
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| + | <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; | ||
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| + | <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; | ||
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| + | <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> | ||
| + | |||
| + | </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: | ||
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| + | <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 transcriptional activation of genes </span><o:p></o:p></p> | ||
| + | </td> | ||
| + | <td width=97 valign=top style='width:72.75pt;border-top:none;border-left: | ||
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| + | <p> Sequence from <a href="10.1038/nmeth.2600">(8)</a>. Ordered from Addgene.</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; | ||
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| + | 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-out 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; | ||
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| + | padding:5.0pt 5.0pt 5.0pt 5.0pt'> | ||
| + | <p class=MsoNormal dir=LTR style='line-height:normal;mso-pagination:none'><span style='font-size:9.5pt; | ||
| + | color:#1155CC;background:white;mso-highlight:white'></span><o:p>Sequence from <a href="10.1038/nature14299">(5)</a> Ordered from Addgene.</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'>Sequence from <a href="http://pubs.acs.org/doi/full/10.1021/sb400081r">(9)</a>. Synthesized by IDT.</span><o:p></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-flanked guide RNA leading SaCas9 to UBB gene (exon 2)</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'>Designed in Benchling. Synthesized by IDT.</span><o:p></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 promoter</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'>RNA polymerase III promoter, 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>Sequence from <a href="10.1038/nature14299">(5). </a>Synthesized by IDT. </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>human 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'>Synthesized by Syntezza </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> | ||
| + | |||
| + | </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> human TERT</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'>Synthesized by Syntezza</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> | ||
| + | |||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:9'> | ||
| + | <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'>pMLPm</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'>Synthetic <span class=SpellE> activation</span> | ||
| + | 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 | ||
| + | class=SpellE><span style='font-family:"Calibri","sans-serif";mso-fareast-font-family: | ||
| + | Calibri;mso-bidi-font-family:Calibri'> Sequence from <a href="http://pubs.acs.org/doi/full/10.1021/sb400081r">(9)</a>. Synthesized by Syntezza</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> | ||
| + | |||
| + | </tr> | ||
| + | <tr style='mso-yfti-irow:10;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> polyA - 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 template. 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'>Synthesized by Syntezza</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> | ||
| + | |||
| + | </tr> | ||
| + | </table> | ||
| + | --> | ||
| + | |||
| + | |||
| + | |||
| + | <br /> | ||
| + | |||
| + | <B><p>3. Cloning of “master” into AAV vector</p></B> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/8/82/MainPlasmid.png" height="725" width="800" > | ||
<br /><br /> | <br /><br /> | ||
| + | <b><p>4. Cloning of various Boomerang components into “MASTER-AAV”</p></b> | ||
| − | + | <table width="800" style="margin:0 !important; text-align:left !important;"> | |
| + | <tbody> | ||
| + | <tr> | ||
| + | <th colspan="3"> | ||
| + | <b>Activation System</b> | ||
| + | </th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td width="230"> | ||
| + | <b>Name</b> | ||
| + | </td> | ||
| + | <td width="500"> | ||
| + | <b>Description</b> | ||
| + | </td> | ||
| + | <td width="70"> | ||
| + | <b>Map</b> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> | ||
| + | phTERT-dCas9-VP64-polyA-pAAV | ||
| + | </td> | ||
| + | <td> | ||
| + | dCas9-VP64 under hTERT promoter. System part. | ||
| + | </td> | ||
| + | <td> | ||
| + | <a href="https://static.igem.org/mediawiki/2015/f/f9/PhTERT-dCas9-VP64-polyA-pAAV-sequence.pdf" target="_blank">Map</a> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> | ||
| + | CMV-dCas9-VP64-polyA-pAAV | ||
| + | </td> | ||
| + | <td> | ||
| + | dCas9 under CMV promoter. Positive control for phTERT-dCas9-VP64 construct. | ||
| + | </td> | ||
| + | <td> | ||
| + | <a href="https://static.igem.org/mediawiki/2015/4/43/CMV-dCas9-VP64-polyA-pAAV-sequence.pdf" target="_blank">Map</a> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> | ||
| + | pSurvivin-gMLP-polyA-pAAV | ||
| + | </td> | ||
| + | <td> | ||
| + | Ribozyme-flanked gRNA for the synthetic promoter under Survivin promoter. System part. | ||
| + | </td> | ||
| + | <td> | ||
| + | <a href="https://static.igem.org/mediawiki/2015/a/aa/PSurvivin-gMLP-polyA-pAAV-sequence.pdf" target="_blank">Map</a> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> | ||
| + | U6-gMLP-pAAV | ||
| + | </td> | ||
| + | <td> | ||
| + | gRNA for the synthetic promoter under human U6 promoter. Positive control for RGR design of gMLP. | ||
| + | </td> | ||
| + | <td> | ||
| + | <a href="https://static.igem.org/mediawiki/2015/4/4d/U6-gMLP-pAAV-sequence.pdf" target="_blank">Map</a> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> | ||
| + | pMLPm-eGFP-polyA-pAAV | ||
| + | </td> | ||
| + | <td> | ||
| + | GFP under the synthetic activation promoter. System part. | ||
| + | </td> | ||
| + | <td> | ||
| + | <a href="https://static.igem.org/mediawiki/2015/e/e6/PMLPm-eGFP-polyA-pAAV-sequence.pdf" target="_blank">Map</a> | ||
| + | </td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table> | ||
| − | + | <!-- | |
| − | + | <table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 width=659 | |
| − | + | style='width:494.25pt;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=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</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> | ||
| + | <a href="https://static.igem.org/mediawiki/2015/f/f9/PhTERT-dCas9-VP64-polyA-pAAV-sequence.pdf" target="_blank">Map</a> | ||
| + | </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 | ||
| + | phTERT-dCas9-VP64 construct.</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> | ||
| + | |||
| + | <a href="https://static.igem.org/mediawiki/2015/4/43/CMV-dCas9-VP64-polyA-pAAV-sequence.pdf" target="_blank">Map</a> | ||
| + | </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> | ||
| + | <a href="https://static.igem.org/mediawiki/2015/a/aa/PSurvivin-gMLP-polyA-pAAV-sequence.pdf" target="_blank">Map</a> | ||
| − | </ | + | </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 human U6 promoter. | ||
| + | Positive control for <span class=SpellE>RGR design of 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> | ||
| + | <a href="https://static.igem.org/mediawiki/2015/4/4d/U6-gMLP-pAAV-sequence.pdf" target="_blank">Map</a> | ||
| + | </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 activation 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> | ||
| + | <a href="https://static.igem.org/mediawiki/2015/e/e6/PMLPm-eGFP-polyA-pAAV-sequence.pdf" target="_blank">Map</a> | ||
| + | </o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | --> | ||
| + | <br /><br /> | ||
| + | <table width="800" style="margin:0 !important; text-align:left !important;"> | ||
| + | <tbody> | ||
| + | <tr> | ||
| + | <th colspan="3"> | ||
| + | <b>Knock-out System</b> | ||
| + | </th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td width="230"> | ||
| + | <b>Name</b> | ||
| + | </td> | ||
| + | <td width="500"> | ||
| + | <b>Description</b> | ||
| + | </td> | ||
| + | <td width="70"> | ||
| + | <b>Map</b> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> | ||
| + | phTERT-SaCas9-polyA-pAAV | ||
| + | </td> | ||
| + | <td> | ||
| + | SaCas9 under hTERT promoter. System part. | ||
| + | </td> | ||
| + | <td> | ||
| + | <a href="https://static.igem.org/mediawiki/2015/b/b0/PhTERT-SaCas9-polyA-pAAV-sequence_%281%29.pdf" target="_blank">Map</a> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> | ||
| + | CMV-SaCas9-polyA-pAAV | ||
| + | </td> | ||
| + | <td> | ||
| + | SaCas9 under CMV. Positive control for phTERT-SaCas9 construct. | ||
| + | </td> | ||
| + | <td> | ||
| + | <a href="https://static.igem.org/mediawiki/2015/3/35/CMV-SaCas9-polyA-pAAV-sequence.pdf" target="_blank">Map</a> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> | ||
| + | pSurvivin-gUBB-polyA-pAAV | ||
| + | </td> | ||
| + | <td> | ||
| + | Ribozyme-flanked gRNA for UBB gene under human Survivin promoter. System part. | ||
| + | </td> | ||
| + | <td> | ||
| + | <a href="https://static.igem.org/mediawiki/2015/0/06/PSurvivin-gUBB-polyA-pAAV-sequence.pdf" target="_blank">Map</a> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> | ||
| + | U6-gUBB-pAAV | ||
| + | </td> | ||
| + | <td> | ||
| + | gRNA for UBB gene under human U6 promoter. Positive control for RGR design of gUBB. | ||
| + | </td> | ||
| + | <td> | ||
| + | <a href="https://static.igem.org/mediawiki/2015/5/5d/U6-gUBB-pAAV-sequence.pdf" target="_blank">Map</a> | ||
| + | </td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table> | ||
| − | < | + | <br /><br /> |
| − | + | ||
| + | <table width="800" style="margin:0 !important; text-align:left !important;"> | ||
| + | <tbody> | ||
| + | <tr> | ||
| + | <th colspan="3"> | ||
| + | <b>General Controls</b> | ||
| + | </th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td width="230"> | ||
| + | <b>Name</b> | ||
| + | </td> | ||
| + | <td width="500"> | ||
| + | <b>Description</b> | ||
| + | </td> | ||
| + | <td width="70"> | ||
| + | <b>Map</b> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> | ||
| + | phTERT-eGFP-polyA master-pAAV | ||
| + | </td> | ||
| + | <td> | ||
| + | GFP under hTERT promoter. Validation control for hTERT promoter. | ||
| + | </td> | ||
| + | <td> | ||
| + | <a href="https://static.igem.org/mediawiki/2015/9/9e/PhTERT-eGFP-polyA-master-pAAV-sequence.pdf" target="_blank">Map</a> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> | ||
| + | pSurvivin-mCherry-polyA-pAAV | ||
| + | </td> | ||
| + | <td> | ||
| + | mCherry under human Survivin promoter. Validation control for Survivin promoter. | ||
| + | </td> | ||
| + | <td> | ||
| + | <a href="https://static.igem.org/mediawiki/2015/a/a2/PSurvivin-mCherry-polyA-pAAV-sequence.pdf" target="_blank">Map</a> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> | ||
| + | eGFP-AAV | ||
| + | </td> | ||
| + | <td> | ||
| + | eGFP under the control of constitutive CMV promoter. Transfection/transduction control. | ||
| + | </td> | ||
| + | <td> | ||
| + | <a href="https://static.igem.org/mediawiki/2015/3/30/PAAV-GFP-sequence.pdf" target="_blank">Map</a> | ||
| + | </td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table> | ||
| + | <!-- | ||
| + | <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</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. 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> | ||
| − | + | <a href="https://static.igem.org/mediawiki/2015/b/b0/PhTERT-SaCas9-polyA-pAAV-sequence_%281%29.pdf" target="_blank">Map</a> | |
| − | + | ||
| − | + | ||
| − | <a | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| + | </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 phTERT-SaCas9 construct.</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> | ||
| + | <a href="https://static.igem.org/mediawiki/2015/3/35/CMV-SaCas9-polyA-pAAV-sequence.pdf" target="_blank">Map</a> | ||
| + | </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'>Ribozyme-flanked 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 human <span class=SpellE>Survivin promoter</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> | ||
| + | <a href="https://static.igem.org/mediawiki/2015/0/06/PSurvivin-gUBB-polyA-pAAV-sequence.pdf" target="_blank">Map</a> | ||
| − | < | + | </o:p></p> |
| − | < | + | </td> |
| − | <p> < | + | </tr> |
| − | <p> < | + | <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 human U6 promoter. Positive control for | ||
| + | RGR design of <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> | ||
| − | </ | + | <a href="https://static.igem.org/mediawiki/2015/5/5d/U6-gUBB-pAAV-sequence.pdf" target="_blank">Map</a> |
| + | </o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | </table> | ||
</div> | </div> | ||
| + | <p class=MsoNormal dir=LTR style='line-height:107%'><o:p> </o:p></p> | ||
| − | </p> | + | <p class=MsoNormal dir=LTR style='margin-bottom:8.0pt;line-height:107%'><o:p> </o:p></p> |
| + | <p class=MsoNormal dir=LTR style='line-height:107%'><o:p> </o:p></p> | ||
| − | + | <div align=left dir=ltr> | |
| − | <div | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | <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</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. Validation control for <span class=SpellE>hTERT promoter</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> | ||
| − | + | <a href="https://static.igem.org/mediawiki/2015/9/9e/PhTERT-eGFP-polyA-master-pAAV-sequence.pdf" target="_blank">Map</a> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| + | </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>human Survivin</span> | ||
| + | promoter. Validation control for <span class=SpellE>Survivin promoter</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> | ||
| + | <a href="https://static.igem.org/mediawiki/2015/a/a2/PSurvivin-mCherry-polyA-pAAV-sequence.pdf" target="_blank">Map</a> | ||
| + | </o:p></p> | ||
| + | </td> | ||
| + | </tr> | ||
| + | </table> | ||
| − | </ | + | </div>--> |
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| + | <br /> | ||
| + | <h4>References</h4> | ||
| + | |||
| + | <br />(1) The telomerase reverse transcriptase promoter drives efficacious tumor suicide gene therapy while preventing hepatotoxicity encountered with constitutive promoters. Majumdar AS, Hughes DE, Lichtsteiner SP, Wang Z, Lebkowski JS, Vasserot AP. Gene Ther. 2001 Apr;8(7):568-78. | ||
| + | <br /><a href="http://www.ncbi.nlm.nih.gov/pubmed/11319624">http://www.ncbi.nlm.nih.gov/pubmed/11319624</a> | ||
| + | |||
| + | <br /><br />(2) RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM, Polstein LR, Thakore PI, Glass KA, Ousterout DG, Leong KW, Guilak F, Crawford GE, Reddy TE, Gersbach CA. Nat Methods. 2013 Oct;10(10):973-6. doi: 10.1038/nmeth.2600. Epub 2013 Jul 25. | ||
| + | <br /><a href="http://www.ncbi.nlm.nih.gov/pubmed/23892895">http://www.ncbi.nlm.nih.gov/pubmed/23892895</a> | ||
| + | |||
| + | <br /><br />(3) Targeting of tumor radioiodine therapy by expression of the sodium iodide symporter under control of the survivin promoter. Huang R, Zhao Z, Ma X, Li S, Gong R, Kuang A. Cancer Gene Ther. 2011 Feb;18(2):144-52. doi: 10.1038/cgt.2010.66. Epub 2010 Oct 29. | ||
| + | <br /><a href="http://www.ncbi.nlm.nih.gov/pubmed/21037556">http://www.ncbi.nlm.nih.gov/pubmed/21037556</a> | ||
| + | |||
| + | <br /><br />(4) Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. Gao Y, Zhao Y. J Integr Plant Biol. 2014 Apr;56(4):343-9. doi: 10.1111/jipb.12152. Epub 2014 Mar 6. | ||
| + | <br /><a href="http://www.ncbi.nlm.nih.gov/pubmed/24373158">http://www.ncbi.nlm.nih.gov/pubmed/24373158</a> | ||
| + | |||
| + | <br /><br />(5) Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas. Farzadfard F, Perli SD, Lu TK. ACS Synth Biol. 2013 Oct 18;2(10):604-13. doi: 10.1021/sb400081r. Epub 2013 Sep 11. | ||
| + | <br /><a href="http://www.ncbi.nlm.nih.gov/pubmed/23977949">http://www.ncbi.nlm.nih.gov/pubmed/23977949</a> | ||
| + | |||
| + | <br /><br />(6) Generation of constitutively active recombinant caspases-3 and -6 by rearrangement of their subunits. Srinivasula SM, Ahmad M, MacFarlane M, Luo Z, Huang Z, Fernandes-Alnemri T, Alnemri ES. J Biol Chem. 1998 Apr 24;273(17):10107-11. | ||
| + | <br /><a href="http://www.ncbi.nlm.nih.gov/pubmed/9553057">http://www.ncbi.nlm.nih.gov/pubmed/9553057</a> | ||
| + | |||
| + | <br /><br />(7) One molecule of diphtheria toxin fragment A introduced into a cell can kill the cell. Yamaizumi M, Mekada E, Uchida T, Okada Y. Cell. 1978 Sep;15(1):245-50. | ||
| + | <br /><a href="http://www.ncbi.nlm.nih.gov/pubmed/699044">http://www.ncbi.nlm.nih.gov/pubmed/699044</a> | ||
| + | |||
| + | <br /><br />(8) SEAP expression in transiently transfected mammalian cells grown in serum-free suspension culture. Schlaeger EJ, Kitas EA, Dorn A. Cytotechnology. 2003 May;42(1):47-55. doi: 10.1023/A:1026125016602. | ||
| + | <br /><a href="http://www.ncbi.nlm.nih.gov/pubmed/19002927">http://www.ncbi.nlm.nih.gov/pubmed/19002927</a> | ||
| + | |||
| + | <br /><br />(9) In vivo genome editing using Staphylococcus aureus Cas9. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F. Nature. 2015 Apr 9;520(7546):186-91. doi: 10.1038/nature14299. Epub 2015 Apr 1. | ||
| + | <br /><a href="http://www.ncbi.nlm.nih.gov/pubmed/25830891">http://www.ncbi.nlm.nih.gov/pubmed/25830891</a> | ||
| + | |||
| + | <br /><br />(10) Downregulation of ubiquitin level via knockdown of polyubiquitin gene Ubb as potential cancer therapeutic intervention. Oh C, Park S, Lee EK, Yoo YJ. Sci Rep. 2013;3:2623. doi: 10.1038/srep02623. | ||
| + | <br /><a href="http://www.ncbi.nlm.nih.gov/pubmed/24022007">http://www.ncbi.nlm.nih.gov/pubmed/24022007</a> | ||
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Latest revision as of 15:54, 18 September 2015
|
Detailed design
Design 1: Cancer-specific CRISPR/Cas9-mediated activation of the gene of interest
This design utilizes modified CRISPR-Cas9 system for transcriptional activation of any gene of interest.
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.
dCas9-VP64 – dCas9-VP64 was engineered so that it lacks endonuclease activity (“dead” Cas9) and has 4 VP16 activation domains fused to the protein. We designed dCas9-VP64 to be expressed under the control of human TERT promoter. Therefore dCas9-VP64 should be expressed predominantly in cells in which the promoter is highly active, namely – cancer cells (1). When guided to a specific promoter, dCas9-VP64 activates transcription of genes downstream of its binding site (2). dCas9-VP64 was assembled into an expression vector under hTERT promoter.
gRNA- 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 (using the scaffold sequence for Staphylococcus pyogenes Cas9) was assembled into a AAV vector, under the control of human survivin promoter (3). In order to utilize the cancer specific promoter hyperactivation we used an RGR (Ribozyme gRNA Ribozyme) design. This design allows for gRNAs to be transcribed and processed using RNA polymerase II promoters, since these are the main promoters controlling gene activation (4). The gRNA sequence is used to guide dCas9-VP64 to a specific synthetic promoter
Synthetic activation promoter- The third part of the system is an expression AAV cassette with GFP under the control of synthetic promoter (5). 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.
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 (6), or diphteria toxin A, which can kill a cell (7), 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 (8) (Figure 1).
| Figure 1. Possible applications of cancer-specific CRISPR-mediated gene activation |
 |
A functional prototype of this design working in human cancer cells is shown here.
Design 2: Cancer-specific CRISPR/Cas9-mediated gene knock-out
This design utilizes “classical” CRISPR-Cas9 system for knock-out of a cancer-essential gene.
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.
Cas9 – the Cas9 endonuclease was assembled into an expression vector under hTERT promoter. We utilized Staphylococcus aureus Cas9 version (SaCas9) (9).
gRNA – The gRNA (using the scaffold sequence for Staphylococcus aureus Cas9) was also assembled into a AAV vector, under the control of human survivin promoter, using previously described ribozyme design.
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.
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 (10). 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 (10).
Detailed design and cloning program
1. Design of “master” template with specific restriction sites for subcloning
2. Design of Boomerang components.
Our basic components include promoters, Cas9 proteins and guide RNAs.
|
Synthesized Components |
||
|---|---|---|
| Name | Description | Source |
| dCas9-VP64 | Activator Cas9 for transcriptional activation of genes | Sequence from (2). Ordered from Addgene. |
| SaCas9 | "Classical" Cas9 endonuclease for knock-out of target genes | Sequence from (9) Ordered from Addgene. |
| gMLP | Ribozyme-flanked guide RNA leading dCas9-VP64 to the synthetic promoter | Sequence from (4). Synthesized by IDT. |
| gUBB | Ribozyme-flanked guide RNA leading SaCas9 to UBB gene (exon 2) | Designed in Benchling. Synthesized by IDT. |
| U6 promoter | RNA polymerase III promoter, positive control for RGR design | Sequence from (9). Synthesized by IDT. |
| pSurvivin | Promoter for human survivin gene | Synthesized by Syntezza Bioscience |
| phTERT | Promoter for human TERT gene | Synthesized by Syntezza Bioscience |
| pMLPm | Synthetic activation promoter | Sequence from (5). Synthesized by Syntezza Bioscience |
| hTERT eGFP polyA - MASTER | Master template. Cloned into delivery vector as a cloning template for all other inserts. | Synthesized by Syntezza Bioscience |
3. Cloning of “master” into AAV vector
4. Cloning of various Boomerang components into “MASTER-AAV”
| Activation System | ||
|---|---|---|
| Name | Description | Map |
| phTERT-dCas9-VP64-polyA-pAAV | dCas9-VP64 under hTERT promoter. System part. | Map |
| CMV-dCas9-VP64-polyA-pAAV | dCas9 under CMV promoter. Positive control for phTERT-dCas9-VP64 construct. | Map |
| pSurvivin-gMLP-polyA-pAAV | Ribozyme-flanked gRNA for the synthetic promoter under Survivin promoter. System part. | Map |
| U6-gMLP-pAAV | gRNA for the synthetic promoter under human U6 promoter. Positive control for RGR design of gMLP. | Map |
| pMLPm-eGFP-polyA-pAAV | GFP under the synthetic activation promoter. System part. | Map |
| Knock-out System | ||
|---|---|---|
| Name | Description | Map |
| phTERT-SaCas9-polyA-pAAV | SaCas9 under hTERT promoter. System part. | Map |
| CMV-SaCas9-polyA-pAAV | SaCas9 under CMV. Positive control for phTERT-SaCas9 construct. | Map |
| pSurvivin-gUBB-polyA-pAAV | Ribozyme-flanked gRNA for UBB gene under human Survivin promoter. System part. | Map |
| U6-gUBB-pAAV | gRNA for UBB gene under human U6 promoter. Positive control for RGR design of gUBB. | Map |
| General Controls | ||
|---|---|---|
| Name | Description | Map |
| phTERT-eGFP-polyA master-pAAV | GFP under hTERT promoter. Validation control for hTERT promoter. | Map |
| pSurvivin-mCherry-polyA-pAAV | mCherry under human Survivin promoter. Validation control for Survivin promoter. | Map |
| eGFP-AAV | eGFP under the control of constitutive CMV promoter. Transfection/transduction control. | Map |
References
(1) The telomerase reverse transcriptase promoter drives efficacious tumor suicide gene therapy while preventing hepatotoxicity encountered with constitutive promoters. Majumdar AS, Hughes DE, Lichtsteiner SP, Wang Z, Lebkowski JS, Vasserot AP. Gene Ther. 2001 Apr;8(7):568-78.
http://www.ncbi.nlm.nih.gov/pubmed/11319624
(2) RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM, Polstein LR, Thakore PI, Glass KA, Ousterout DG, Leong KW, Guilak F, Crawford GE, Reddy TE, Gersbach CA. Nat Methods. 2013 Oct;10(10):973-6. doi: 10.1038/nmeth.2600. Epub 2013 Jul 25.
http://www.ncbi.nlm.nih.gov/pubmed/23892895
(3) Targeting of tumor radioiodine therapy by expression of the sodium iodide symporter under control of the survivin promoter. Huang R, Zhao Z, Ma X, Li S, Gong R, Kuang A. Cancer Gene Ther. 2011 Feb;18(2):144-52. doi: 10.1038/cgt.2010.66. Epub 2010 Oct 29.
http://www.ncbi.nlm.nih.gov/pubmed/21037556
(4) Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. Gao Y, Zhao Y. J Integr Plant Biol. 2014 Apr;56(4):343-9. doi: 10.1111/jipb.12152. Epub 2014 Mar 6.
http://www.ncbi.nlm.nih.gov/pubmed/24373158
(5) Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas. Farzadfard F, Perli SD, Lu TK. ACS Synth Biol. 2013 Oct 18;2(10):604-13. doi: 10.1021/sb400081r. Epub 2013 Sep 11.
http://www.ncbi.nlm.nih.gov/pubmed/23977949
(6) Generation of constitutively active recombinant caspases-3 and -6 by rearrangement of their subunits. Srinivasula SM, Ahmad M, MacFarlane M, Luo Z, Huang Z, Fernandes-Alnemri T, Alnemri ES. J Biol Chem. 1998 Apr 24;273(17):10107-11.
http://www.ncbi.nlm.nih.gov/pubmed/9553057
(7) One molecule of diphtheria toxin fragment A introduced into a cell can kill the cell. Yamaizumi M, Mekada E, Uchida T, Okada Y. Cell. 1978 Sep;15(1):245-50.
http://www.ncbi.nlm.nih.gov/pubmed/699044
(8) SEAP expression in transiently transfected mammalian cells grown in serum-free suspension culture. Schlaeger EJ, Kitas EA, Dorn A. Cytotechnology. 2003 May;42(1):47-55. doi: 10.1023/A:1026125016602.
http://www.ncbi.nlm.nih.gov/pubmed/19002927
(9) In vivo genome editing using Staphylococcus aureus Cas9. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F. Nature. 2015 Apr 9;520(7546):186-91. doi: 10.1038/nature14299. Epub 2015 Apr 1.
http://www.ncbi.nlm.nih.gov/pubmed/25830891
(10) Downregulation of ubiquitin level via knockdown of polyubiquitin gene Ubb as potential cancer therapeutic intervention. Oh C, Park S, Lee EK, Yoo YJ. Sci Rep. 2013;3:2623. doi: 10.1038/srep02623.
http://www.ncbi.nlm.nih.gov/pubmed/24022007
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