Team:Freiburg/Project/crrna

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</p>
</p>
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<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/1"> Overview </a></p>
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<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/1"> Abstract & Intro </a></p>
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<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/effector"> Effector </a></p>
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<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/induction"> Induction </a> </p>
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<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/crrna" class="active"> Targeting </a></p>
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/crrna" class="active"> Targeting </a></p>
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<p class="second_order"> <a href="#design_tool"> crRNA design tool </a> </p>
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<p class="second_order"> <a href="#rnaimer"> RNAimer </a> </p>
<p class="second_order"> <a href="#multiple_targeting"> Multiple targeting </a> </p>
<p class="second_order"> <a href="#multiple_targeting"> Multiple targeting </a> </p>
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<p class="second_order"> <a href="#design_tool"> crRNA design tool </a> </p>
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<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/effector"> Effectors </a></p>
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<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/induction"> Effector Control </a> </p>
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<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/modeling"> Modeling </a></p>
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<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/truncation"> Truncation </a></p>
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/method"> uniBAss </a></p>
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/method"> uniBAss </a></p>
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<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/toolkit"> Toolkit </a></p>
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<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/unibox"> uniBOX </a></p>
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<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/modeling"> Modeling </a></p>
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<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/toolkit"> Manual </a></p>
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<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Project/application" > Application </a></p>
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</div>
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</p>
</p>
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<div id="design_tool">
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<p>
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<p id="h2">
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<p> In our uniCAS Toolkit we engineered the <b><a id="link" href="https://2013.igem.org/Team:Freiburg/Project/1#background">CRISPR/Cas9</a></b> system for future applications to regulate gene expression. The key components for the binding of the catalytically inactive Cas9 (dCas9) to its targets are two small, non-coding RNAs: the CRISPR-RNA (crRNA) and the tracrRNA. These RNAs that guide our dCas9 to specific DNA sequences have to be cotransfected with the dCas9-plasmids. Therefore we designed an RNA plasmid, termed <b><a id="link" href="https://2013.igem.org/Team:Freiburg/Project/crrna#rnaimer">RNAimer</a></b>, which contains all required RNAs for efficiently guiding the dCas9 protein to the required DNA - even if multiple DNA sites should be targeted.
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crRNA design tool
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</p>
</p>
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<p>
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As an essential part of our toolkit is the binding of our protein to DNA, we evaluated this in detail: Various DNA target sequences were compared to evaluate the best guiding crRNAs. Evaluated parameters were varying loci and GC-content. Based on these results, we tested if dCas9 is able to bind to <b><a id="link" href="https://2013.igem.org/Team:Freiburg/Project/crrna#multiple_targeting">multiple targets</a></b>.
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</p>
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<form id="form1" action="" method="get">
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<p>
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<legend id="h3">Enter Target Sequence</legend>
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In order to simplify the search for potential target sequences, we programmed the <b><a id="link" href="https://2013.igem.org/Team:Freiburg/Project/crrna#design_tool">crRNA design tool</a></b> and provide it to the iGEM community. As the binding of the RNA-guided Cas9 requires a protospacer adjacent motif (PAM), the tool determines all possible regions of the desired DNA sequence that is pasted into the tool. Eventually, you obtain the position of your target sites and the sequences of the oligos that have to be inserted into the RNAimer - ready to order!
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<textarea name="Sequenz" type="text" rows="8" cols="40">
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</p>
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</textarea>
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<fieldset>
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<p id="h4">Choose Program</p>
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<p>
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Which strand should be targeted?<br>
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<p>
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<input type="radio" name="rad" value="1"> non coding strand
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<input type="radio" name="rad" value="2"> coding strand
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</p>
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<p>
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<button id="button1" type="button"> Submit </button>
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<input type="reset" value="reset">  
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</p>
 
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</fieldset>
 
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<div id="rnaimer"></div>
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<div id="explanation">
 
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<p>This tool helps you to design a crRNA-insert for dCAS RNA plasmid: "uniCAS RNaimer" (<a style="color:white" href="https://2013.igem.org/Team:Freiburg/parts/BBa_K1150034 "> BBa_K1150034 </a>). <br>
 
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Using this tool you do not have to do this on your own. Just insert the desired target sequence and you get all different oligo possibilities and their positions. The oligos contain overhangs which fit to this plasmid's BbsI-overhangs and are ready to use. <br>
 
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The two different target possibilities are the coding and non-coding strand, depending on the desired target sequence. </p>
 
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<p> a) For repression of gene transcription by targeting the coding sequence it´s crucial to target the non template (= coding) DNA strand. <br>  
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<table id="headline_pic">
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Therefore the oligos must be designed as follows: </p>
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<tr>
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<td style="width:1000px;">
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<ol>
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<p id="h2">
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    <li>Search at your desired target sequence for a CCN (reverse complement of the PAM sequence) at the coding strand.</li>
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RNAimer - targeting dCas9 to its destination
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  <li> Extract the following (3') 30 nucleotides.</li>
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  <li> Extract the reverse complement.</li>
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  <li>Add AAAC at the 5' end and GT at the 3' end. This will be your fist oligo.</li>
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    <li>Take the sequence from step 2 and add TAAAAC at the 5' end. This will be your second oligo.</li>
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    </ol>
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<p style="padding-top:10px"> b) For repression of gene transcription by targeting the non-coding sequence it´s crucial to target the template DNA strand. <br>  
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Therefore the oligos must be designed as follows: </p>
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<ol>
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-
    <li>Search at your desired target sequence for a NGG (the PAM sequence) at the non-coding strand.</li>
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  <li> Extract the 30 nucleotides before (5') the PAM sequence.</li>
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<li> Extract the reverse complement.</li>
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-
  <li> Add TAAAAC at the 5' end. This will be your second oligo. </li>
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    <li>Take the sequence from step 2 and add AAAC at the 5' end and GT at the 3' end. This will be your first oligo.</li>
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    </ol>
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<div><img src="https://static.igem.org/mediawiki/2013/3/3a/Freiburg2013_CrRNA_Design.png" ></div>
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<p style="padding-top:10px"> <i>(the oligos are designed analog to: Cong L, Ran FA, Cox D, Lin S, Barretto R,Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex (2013 Jan 3). Genome Engineering using CRISPR/Cas Systems. Science. DOI: 10.1126/science.1231143 )</i><br>
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</p>
</p>
 +
</td>
 +
<td>
 +
<img src="https://static.igem.org/mediawiki/2013/b/bb/Freiburg_2013_main2_Multiple_Targeting.png" style="width:100px;">
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</td>
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</tr>
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</table>
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<p id = "h3"> technical information</p>
 
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<a style="color:white" href="" >view source code</a>
 
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</div>
 
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<div id="multiple_targeting">
 
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<p id="h2">
 
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Multiple targeting
 
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</p>
 
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</div>
 
<p id="h3">
<p id="h3">
Introduction
Introduction
</p>
</p>
 +
<p>
<p>
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One of the biggest advantages of the CRISPR-Cas system compared to other transcription activators (e.g. Zn fingers, TALEs) is that only one protein is required for targeting several DNA sites: For a new target there has to be just another crRNA. We designed an RNA plasmid containing the tracrRNA, where the crRNA can be introduced easily by digesting with BbsI and inserting two previous annealed oligos. Two of these RNA plasmids (with different crRNAs) can be fused using the iGEM biobrick system. This way it is possible to get two or more crRNAs on one plasmid.
+
As dCas9 requires two small, non-coding RNAs to mediate binding to the DNA, we designed an RNA plasmid containing the tracrRNA and a site where the crRNA can be introduced. This is achieved by digestion using BbsI and annealed oligos corresponding to respective crRNA sequence. Two (or more) of these RNA plasmids (with different crRNAs) can be fused using the iGEM BioBrick cloning strategy thereby allowing for a RNAimer plasmid carrying multiple crRNAs.<br>
</p>
</p>
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<div>
<div>
<table class="imgtxt" width="860px">
<table class="imgtxt" width="860px">
<tr>  
<tr>  
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<td style="padding-top:10px; padding-bottom:10px; padding-left:5px; padding-right:5px;"> <img class="imgtxt" width="860px" src="https://static.igem.org/mediawiki/2013/6/6a/Multiple_targeting_Freiburg_2013_%281%29.png"> </td>
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<td style="padding-top:10px; padding-bottom:10px; padding-left:5px; padding-right:5px;"> <img class="imgtxt" width="860px"  
 +
 
 +
src="https://static.igem.org/mediawiki/2013/0/0f/Multiple_targeting_Freiburg_2013_28129.png"> </td>
</tr>
</tr>
<tr>
<tr>
-
<td> <p> <b>Fig. 1: RNA plasmid (BBa_K1150034)</b><br>
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<td> <p> <b>Figure 1: RNAimer (BBa_K1150034)</b><br>
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Our RNA plasmid contains the tracrRNA and a site where the desired crRNA can be inserted. Both RNAs are driven by different human RNA polymerase III promoters. Assembly of multiple crRNAs can be easily done by digestion with enzymes of prefix and suffix and combining these parts according to iGEM standard assembly. </p>
+
Our RNA plasmid, termed the RNAimer, contains the tracrRNA and a site where the desired crRNA can be inserted. Transcription of both RNAs are driven by the H1 and the U6 promoter RNA polymerase III promoters, respectively. Assembly of multiple crRNAs can be easily done according to iGEM standard assembly.</p>
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</div>
</div>
<p>
<p>
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As it is important that the RNAs are not being marked for protein expression the RNA polymerase III is required for transcription. RNA polymerase III mainly synthesize small non-coding RNAs (e.g. tRNAs or rRNAs) whereas the commonly used polymerase II is responsible for transcription of mainly mRNAs <span id="refer"> <a href="#(1)"> [1,2] </a></span>. We chose the human U6- and H1-promoter to drive the RNAs as they are exclusively recognized by polymerase III <span id="refer"> <a href="#(2)"> [2] </a></span>.
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As it is important that the RNAs are not being marked for protein expression and nuclear export, the RNA polymerase III is required for transcription. RNA polymerase III mainly synthesizes small non-coding RNAs (e.g. tRNAs or rRNAs) whereas the commonly used polymerase II is responsible for transcription of mainly mRNAs.  
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<br>
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With this RNA plasmid and another plasmid containing the Cas9-effector fusion protein it is possible to target several DNA sites at once by transfecting only two plasmids. This could mean the simultaneous regulation of different genes or a stricter controlling of one gen by bringing more effector domains to this gene.
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</p>
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<p id="h3">
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-
Results
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</p>
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<p id="h4">Activation of different genes at once</p>
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<span id="refer"> <a href="#(1)"> [1, 2]</a></span>. We chose the human U6- and H1-promoter to drive transcription of the RNAs as they are exclusively recognized by
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In order to test the simultaneously activation of several genes we assembled 3 plasmids containing different fluorescent proteins. Every protein is fused to a different signal for intracellular localization. Thus, we were able to distinguish better between the different fluorescent proteins, because there will be no interference of the emitted light.
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<div>
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polymerase III <span id="refer"> <a href="#(2)"> [2]</a></span>.
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<table class="imgtxt" width="860px">
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 +
In order to reduce the amount of plasmids for transfection when intending to target several genes or target sites at once, we wanted to join the required
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 +
crRNAs on one RNA plasmid. This was easily manageable by using the iGEM standard assembly: Oligos are inserted separately into the RNAimer plasmid (BBa_K1150034) by
 +
 
 +
digesting using BbsI and ligation. Afterwards the inserts located within the respective RNAimer plasmids can be combined by using the restriction enzymes of the prefix and suffix of the iGEM standard (Figure 6).
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 +
<div id="RNAimer-principle"></div>
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<center><div>
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<table class="imgtxt" width="500px">
<tr>  
<tr>  
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<td> <img class="imgtxt" width="860px" src="https://static.igem.org/mediawiki/2013/b/bc/Multiple_targeting_Freiburg_2013_%282%29.png"> </td>
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<td> <EMBED  HEIGHT=604 WIDTH=550 TYPE="video/quicktime"
 +
PLUGINSPAGE="http://www.apple.com/quicktime/download/"  
 +
SRC="https://static.igem.org/mediawiki/2013/b/b9/RNAimer.mov"
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AUTOPLAY="false"/></OBJECT> </td>
</tr>
</tr>
<tr>
<tr>
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<td> <b>Fig. 2: Plasmids encoding the fluorescent proteins</b><br>
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<td> <b>Figure 6: Assembly and function of the RNAimer </b><br>
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Each fluorescent protein is driven by a CMV minimal promoter, that can be switched on by binding of TetR-VP16 to the TetO sequence. Between TetO and CMV<sub>min</sub> there is a target site for Cas9 binding, a different on each plasmid. The fluorescent were fused to signal sequences for subcellular localization, so mCherry will be in the nucleus, GFP in the Golgi apparatus and BFP at the membrane. T: terminator.
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For each desired crRNA, one RNAimer plasmid (containing the crRNA after ligation) has to be digested with BbsI. Afterwards the annealed crRNA oligos will be ligated. The different RNA inserts can be assembled using the idempotent iGEM cloning strategy. Three different crRNAs (red, yellow, blue) will be transcribed.
</td>
</td>
</tr>
</tr>
</table>
</table>
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</div>
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</div></center>
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<div>
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<p><br></p>
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<table class="imgtxt" width="860px">
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<p id="h3">
 +
Results
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</p>
 +
 
 +
 
 +
 
 +
To test this strategy functionally we performed SEAP (Secreted Embryonic Alkaline Phosphatase) assays with <b><a id="link" href="https://2013.igem.org/Team:Freiburg/Project/effector#activation">dCas9-VP16</a></b>. HEK-293T cells were transfected with dCas9-VP16 and different RNAimer plasmids containing different crRNAs. We either co-transfected two crRNAs or a single plasmid carrying both crRNAs. This resulted in the same activation of SEAP expression (Figure 7).
 +
 
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<center><div>
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<table class="imgtxt" width="400px">
<tr>  
<tr>  
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<td> <img class="imgtxt" width="860px" src="https://static.igem.org/mediawiki/2013/e/e9/Multiple_targeting_Freiburg_2013_%283%29.png"> </td>
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<td> <img class="imgtxt" width="400px" src="https://static.igem.org/mediawiki/2013/4/49/Thomastargeting_freiburg_13.png"> </td>
</tr>
</tr>
<tr>
<tr>
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<td> <b>Fig. 3: Microscopy pictures of fluorescent proteins expressed in HeLa cells</b><br>
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<td> <b>Figure 7: SEAP activation with dCas9-VP16 and RNAimer.</b><br>
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Fluorescence pictures were taken of fixed HeLa cells transfected with Golgi-GFP and mCherry-NLS. Channels of GFP and mCherry were merged. All pictures have the same scale.  
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HEK-293T cells were seeded at 65,000 cells in a 24-well format. After 24 hours cells were transfected following the standard procedure. 48 h post-transfection, supernatant was harvested and SEAP activity was evaluated following the standard protocol. Activation by dCas9-VP16 was performed using two targets at once. In the case of T3+4 the loci were on the same plasmid, whereas in the case of T3&T4 two separate plasmids were co-transfected. Both combinations display a strong activation of the CMV<sub>min</sub> promoter, driving the SEAP reporter expression. The activation of the two conditions is comparable. This is a hint, that there is no decrease in efficiency when combining several loci on one plasmid.
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</td>
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</tr>
</tr>
</table>
</table>
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</div></center>
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 +
<p><br></p>
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<p id="h3">
 +
Discussion
 +
</p>
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 +
<p>We have shown that our RNA plasmid "RNAimer" works as expected. The combination of two targets does not seem to decrease the efficiency of
 +
 +
targeting as the activation is comparable when transfecting two separate plasmids or combining both targets in one plasmid. With our toolkit it is possible to
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 +
induce gene expression by co-transfecting two plasmids (plasmid coding for the dCas9-effector fusion protein and the RNAimer plasmid carrying the crRNA and tracrRNA).<br>
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For future application researchers will be able to alter gene expression by transfecting only the plasmid containing the crRNA. This could be possible when the <i>dCas9</i> will be
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 +
stably integrated into the genome of cells or model organisms, as recently done by Gilbert et al. <span id="refer"> <a href="#(4)"> [4]</a></span>.
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</p>
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<p><br></p>
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<p id="h4"> References </p>
 +
<small>
 +
<div id="(1)">(1) Dieci G.,<i>et al.</i> (2007). The expanding RNA polymerase III transcriptome. Trends Genet. 23, <i>614-622</i>. <br></div>
 +
<div id="(2)">(2) Myslinski E., <i>et al.</i> (2001). An unusually compact external promoter for RNA polymerase III transcription of the human H1RNA gene. Nucleic Acids Res. 29, <i>2502-2509</i>. <br></div>
 +
<div id="(3)">(3) Cheng AW., <i>et al.</i> (2013). Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. <br></div>
 +
<div id="(4)">(4) Gilbert LA.,<i>et al.</i> (2013). CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes. Cell 154, <i>442-451</i>. <br></div>
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</small>
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<div id="multiple_targeting">
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<p id="h2">
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Multiple Targeting
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</p>
</div>
</div>
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<p id="h3">
 +
Introduction
 +
</p>
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HEK cells were transfected with different combinations of these plasmids and Cas9-VP16 or TetR-VP16 (4 fold amount of effector DNA)  After 2 days of expression the cells were analysed by flow cytometry. This way the fluorescence intensity of every cell could be determined (Fig. 4).
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<p>
 +
One of the greatest advantages of the CRISPR/Cas9 system compared to other transcription activators (e.g. Zinc fingers (ZFNs) or transcription-activator like effectors (TALEs)) is that only one protein is
 +
 
 +
required for targeting several DNA loci: For a new target just another crRNA has to be expressed. So a <a id="link" href="#rnaimer">RNA plasmid</a> was
 +
 
 +
designed containing the tracrRNA, where the crRNA can be easily introduced.<br>
 +
With this RNA plasmid and another plasmid containing the dCas9-effector fusion gene it is possible to target several DNA sites at once by co-transfecting two plasmids. This allows the simultaneous regulation of different genes or a stricter control of one gene by targeting several loci of this gene the same time.
 +
</p>
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<p>
 +
First of all, we had to evaluate different DNA sequences for targeting, since the results of our first experiments (see labjournal for details) showed that various crRNAs resulted in different SEAP levels. Thus, we cloned five different target sites 26 bp upstream of the CMV<sub>min</sub> promoter of a SEAP reporter plasmid.
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Additionally we designed crRNAs in various distances upstream of this promoter (Tab. 1). These target sites were evaluated by activating SEAP expression
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with dCas9-VP16.
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</p>
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<p><br></p>
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<div>
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<table>
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<tr>
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<td colspan="4">
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<b><div id="tabelle1">Table 1: Different target sites</div></b><br>
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The VEGF and EMX1 target sites were cloned into the SEAP reporter plasmid. T2-T4 are original sequences found on this plasmid. All target loci are upstream of
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the CMV<sub>min</sub> promoter. VEGF and EMX1 are parts of the sequence upstream of the human VEGF or EMX1 gene, respectively.
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</td>
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</tr>
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<tr> <th> Name </th> <th> Distance to promoter </th> <th> Sequence </th> <th>
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 +
GC content [%] </th> </tr>
 +
<tr> <td> VEGF VZ-573 </td> <td> <center> -26 bp </center> </td> <td> GTGTGCAGACGGCAGTCACTAGGGGGCGCT
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</td> <td> <center> 70 </center> </td> </tr>
 +
<tr> <td> VEGF VZ-475 </td> <td> <center> -26 bp </center> </td> <td> GTGAGTGTGTGCGTGTGGGGTTGAGGGCGT
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 +
</td> <td> <center> 63 </center> </td> </tr>
 +
<tr> <td> VEGF VZ-8 </td> <td> <center> -26 bp </center> </td> <td> TTAAAAGTCGGCTGGTAGCGGGGAGGATCG
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 +
</td> <td> <center> 57 </center> </td> </tr>
 +
<tr> <td> VEGF VZ+434 </td> <td> <center> -26 bp </center> </td> <td> GACCTGCTTTTGGGGGTGACCGCCGGAGCG
 +
 
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</td> <td> <center> 70 </center> </td> </tr>
 +
<tr> <td> EMX1 </td> <td> <center> -26 bp </center> </td> <td> GGAAGGGCCTGAGTCCGAGCAGAAGAAGAA
 +
 
 +
</td> <td> <center> 57 </center> </td> </tr>
 +
<tr> <td> T2 </td> <td> <center> -148 bp </center> </td> <td> AAGCATTTATCAGGGTTATTGTCTCATGAG </td>
 +
 
 +
<td> <center> 37 </center> </td> </tr>
 +
<tr> <td> T3 </td> <td> <center> -222 bp </center> </td> <td> AATGCCGCAAAAAAGGGAATAAGGGCGACA
 +
 
 +
</td> <td> <center> 47 </center> </td> </tr>
 +
<tr> <td> T4 </td> <td> <center> -443 bp </center> </td> <td> GACCGAGTTGCTCTTGCCCGGCGTCAATAC
 +
 
 +
</td> <td> <center> 60 </center> </td> </tr>
 +
</table>
 +
</div>
 +
 
 +
<p><br></p>
 +
 
 +
<p id="h3">
 +
Results
 +
</p>
 +
 
 +
<p id="h4">Evaluation of different target sequences</p>
 +
 
 +
At first we tested different target sequences at the same distance from the promoter. To do so we had to insert the target sequences into the SEAP reporter plasmid in front of the CMV<sub>min</sub>.<br>
 +
HEK-293T cells were transfected with one of these SEAP plasmids and a plasmid containing dCas9-VP16, the tracrRNA and the appropriate crRNA. The
 +
 
 +
results show different activation efficiencies (Figure 8) in which no activation (VEGF VZ -573 and +434) and up to a 5 fold increase of SEAP activation could be observed. With the
 +
 
 +
tested target sequences that have a GC content of 70 % no activation was observed whereas induction of SEAP expression with the target
 +
 
 +
sequences with a GC content of about 60 % (compare Tab. 1) could be shown.
<center><div>
<center><div>
-
<table class="imgtxt" width="600px">
+
<table class="imgtxt" width="700px">
<tr>  
<tr>  
-
<td> <img class="imgtxt" width="600px" src="https://static.igem.org/mediawiki/2013/a/a2/Multiple_targeting_Freiburg_2013_%284%29.png"> </td>
+
<td> <img class="imgtxt" width="700px" src="https://static.igem.org/mediawiki/2013/f/f6/Targets_Freiburg_2013_%281%29.png">  
 +
 
 +
</td>
</tr>
</tr>
<tr>
<tr>
-
<td> <b>Fig. 4: Activation of expression of different fluorescent proteins</b><br>
+
<td> <b>Figure 8: Targets with different sequences</b><br>
-
The fluorescence intensity of each cell was analysed by flow cytometry. The mean fluorescence intensity was calculated with the intensities of the cells which were brighter than untransfected cells. The bars represent the mean with standard deviation of these mean fluorescences of three different cell populations.<br>
+
The grey bars represent the SEAP activity of HEK-293T cells transfected with dCas9-VP16 but no crRNA (normalized to value 1). The blue bars show fold induction of SEAP activity by tansfecting dCas9-VP16 with crRNAs in comparison to the appropriate control without crRNA. An activation is visible for nearly all targets, but the fold-induction is differing strongly. All values are means of three biological replicates. Error bars represent standard deviation.
-
blue: only the plasmid containing the fluorescent proteins with minimal promoter were transfected; green: the minimal promoter driven fluorescent proteins were cotransfected with TetR-VP16; yellow: the minimal promoter driven fluorescent proteins were cotransfected with Cas9-VP16.
+
For the experiment HEK-293T cells were seeded at 65,000 cells in a 24-well format. After 24 h cells were transfected following standard procedure. After 48 h of expression supernatant was harvested and SEAP activity was evaluated following the standard protocol.
</td>
</td>
</tr>
</tr>
Line 465: Line 579:
</div></center>
</div></center>
-
Unfortunately Cas9-VP16 was not able to increase the intensity of any fluorescent protein at all, even when transfected exclucively, whereas TetR-VP16 strongly activates the expression of fluorescent proteins. This may be due to the higher number of binding sites for TetR (16 in comparison with 1 for Cas9).
+
Next, we evaluated different distances upstream of the promoter. Thus, we targeted four different loci on the reporter plasmid (EMX1, T2, T3 & T4; for sequence see Tab. 1).<br>
 +
HEK-293T cells were co-transfected with a CMV<sub>min</sub>:SEAP plasmid that contains EMX1, CMV:dCas9-VP16 and the RNA plasmid, respectively. The results show an significant increase in  
 +
SEAP expression when EMX1, which is the closest target site to the promoter, is targeted (Figure 2). Activation decreases, the larger the distance
-
<p id="h4">Stricter gen regulation by targeting different loci simultaneously</p>
+
between promoter and target sites becomes (compare Tab. 1).  
-
 
+
-
As we wanted to improve the efficiency of our gene regulation tool kit, we tried to target several loci upstream of the reporter gene's promoter at once. Thus, we ordered crRNAs that are complementary to sequences on the SEAP reporter plasmid with different distances to the promoter.
+
-
 
+
-
<br><br>TABELLE: Targets on SEAP reporter plasmid<br><br>
+
-
 
+
-
HEK cells were transfected with the SEAP reporter plasmid, Cas9-VP16 (iGEM standard), one or two RNA plasmids and a plasmid coding for Renilla luciferase for a internal standard (to eliminate variabilities of different cell numbers or expression levels). The total amount of RNA plasmids was always the same, so it can be exclude that an increase of SEAP expression is due to more available crRNA.
+
-
When combining the Targets EMX1 and T2 there could be observed a higher SEAP activation than the sum of the single targets (Fig. 5).
+
<center><div>
<center><div>
-
<table class="imgtxt" width="600px">
+
<table class="imgtxt" width="550px">
<tr>  
<tr>  
-
<td> <img class="imgtxt" width="600px" src="https://static.igem.org/mediawiki/2013/5/5d/Multiple_Targeting_Freiburg_2013_%285%29.png"> </td>
+
<td> <img class="imgtxt" width="550px" src="https://static.igem.org/mediawiki/2013/2/29/Targets_Freiburg_2013_%282%29.png">  
 +
 
 +
</td>
</tr>
</tr>
<tr>
<tr>
-
<td> <b>Fig. 5: Effects of different target numbers</b><br>
+
<td> <b>Figure 9: Targets with different distances to the promoter</b><br>
-
SEAP activity was divided through luminescence intensity of Renilla luciferase. The bars represent the mean of biological triplicates with standard deviation. All samples were transfected with CMV::Cas9-VP16 in pSB1C3 and with no (left), one (middle) or two RNA plasmids (right).
+
Activation of CMV<sub>min</sub>:SEAP via dCas9-VP16 with different distances to the promoter in HEK-293T cells. The grey bar represents the SEAP level without crRNA, the blue bars represent the activation using different crRNAs in increasing distance to the promoter (EMX1 the closest, T4 with the greatest distance). A clear decrease in activation is observed with increasing distance from promoter. All epxeriments were performed in biological triplicates. Error bars represent standard deviation. HEK-293T cells were seeded at 65,000 cells in 24-well format and 24 hours later transfected following the standard procedure. After 48 h of expression supernatant was harvested and SEAP activity was evaluated following the standard protocol.
</td>
</td>
</tr>
</tr>
Line 490: Line 601:
</div></center>
</div></center>
 +
<p><br></p>
-
<p id="h4">RNAimer - the multiple RNA plasmid</p>
 
-
In order to reduce the amount of plasmids for transfection when intending to target several genes or target sites at once, we wanted to join the required crRNAs on one RNA plasmid. This was easily manageable by using the iGEM standard assembly: We inserted the oligos seperately into BBa_K1150034 by digesting with BbsI and ligation. Afterwards the whole inserts can be combined by using the restriction enzymes of the prefix and suffix.
 
-
<br><br>FIGURE: RNAimer (mit Verweis!)<br><br>
+
<p id="h4">Stricter gene regulation by targeting different loci simultaneously</p>
-
When compared to two RNA plasmids containing the same crRNAs the RNAimer causes the same SEAP activation in HEK cells transfected with this plasmid(s), CMV::Cas9-VP16, Renilla luciferase and the SEAP reporter plasmid (Fig. 7).
+
<p>
 +
In order to improve the efficiency of our gene regulation toolkit, we tried to target several loci upstream of the promoter of the reporter gene at once.
 +
 
 +
Thus, we designed crRNAs that are complementary to sequences on the SEAP reporter plasmid with different distances to the promoter (<a id="link"
 +
 
 +
href="#tabelle1">Tab. 1</a>).
 +
</p>
 +
<p>
 +
HEK-293T cells were transfected with the CMV<sub>min</sub>:SEAP reporter plasmid, dCas9-VP16 (iGEM standard), one or two RNA plasmids and a plasmid coding for Renilla  
 +
 
 +
luciferase for an internal standard (to eliminate variabilities of different cell numbers or expression levels). The total amount of RNA plasmids is kept constant, so an increase of SEAP expression due to more available crRNA can be excluded.
 +
By combining the targets EMX1 and T2 a higher SEAP activity than the sum of the single targets can be observed (Figure 13).
 +
</p>
<center><div>
<center><div>
-
<table class="imgtxt" width="400px">
+
<table class="imgtxt" width="550px">
<tr>  
<tr>  
-
<td> <img class="imgtxt" width="400px" src="https://static.igem.org/mediawiki/2013/1/15/Multiple_Targeting_Freiburg_2013_%287%29.png"> </td>
+
<td> <img class="imgtxt" width="550px" src="https://static.igem.org/mediawiki/2013/2/24/Thomastargeting2_freiburg_13.png"> </td>
</tr>
</tr>
<tr>
<tr>
-
<td> <b>Figure 7: RNAimer in comparison to two RNA plasmids</b><br>
+
<td> <b>Figure 13: Effects of different target numbers</b><br>
-
SEAP activity was divided through luminescence intensity of Renilla luciferase. The bars represent the mean of biological triplicates with standard deviation. All samples were transfected with CMV::Cas9-VP16 in pSB1C3 and with two (left) or one RNA plasmid (right), each time coding for the same crRNAs.
+
Activation of CMV<sub>min</sub>:SEAP was evaluated by the usage of single targets and combined targets. Every target itself displays an activation of the minimal promoter. By combining two targets (EMX1 & T2), an even stronger activation is visible, that exceeds the additive effect of single plasmids. <br> HEK-293T cells were seeded at 65,000 cells in 24-well format. After 24 hours cells were transfected following standard procedure. After 48 h of expression supernatant was harvested and SEAP activity was evaluated following the standard protocol.
 +
</td>
</tr>
</tr>
</table>
</table>
Line 511: Line 634:
-
<p id="h3">
+
<p><br></p>
-
Summary
+
 
 +
<p id="h3">Discussion</p>
 +
 
 +
<p>
 +
From the few target sites we evaluated we could draw the conclusion that a target should be chosen like this:
 +
<ul>
 +
<li>The distance to the promoter should be kept at minimum.</li>
 +
<li>Target sites with a GC content around 60 % should be preferred.</li>
 +
</ul>
 +
As we tested our target sequences with dCas9-VP16, stricly speaking, we can only make a statement about the best target sites for gene activation.<br>
 +
Moreover there may be other parameters that influence the effects of dCas9-VP16 on SEAP (e.g. the secondary structure of the crRNA). But for the
 +
 
 +
distance to the promoter Mali et al. <span id="refer"> <a href="#(1.1)"> [1]</a></span> came to the same conclusion: Only the target site that had the shortest distance to the
 +
 
 +
promoter showed a high increase of activation.
</p>
</p>
 +
<p>
 +
By targeting different loci upstream of a promoter simultaneously the efficiency of transcription activation can be enhanced. While Cheng et al. <span
-
<p>We have shown, that the efficiency of transcription activation can be enhanced by targeting different loci upstream of a promoter simultaneously. While Cheng et al. <span id="refer"> <a href="#(3)"> [3] </a></span> yielded an up to 8 fold activation by using at least three different targets in comparison to the highest activation of a single target, we were able to achieve a three fold increase by using only two different targets. <br>
+
id="refer"> <a href="#(1.2)"> [2]</a></span> yielded an activation up to 8-fold by using at least three different targets in comparison to the highest  
-
With our toolkit it is possible to induce a gene regulation by transfecting only two plasmids (Cas9 with effector and RNAimer) that can be combinied with no effort in accordance to the experimental setup. <br>
+
-
For future application researchers may be able to render gene expression by transfecting even only plasmid containing the crRNA: when Cas9 will be stable integrated into the genome of cells or model organisms.</p>
+
-
<p>About the ability of activating multiple genes we could not make a statement as the activation of our reporter plasmids could not be detected at all with Cas9-VP16. This may be due to the high background expression of CMV<sub>min</sub>.
+
activation of a single target, we were able to achieve a three fold increase by using only two different targets. <br>  
-
<p id="h3"> References </p>
 
-
<div id="(1)">[1] Dieci G,<i>et al.</i> (2007). The expanding RNA polymerase III transcriptome. <i>Trends Genet. 2007 Dec;23(12):614-22</i>. <br></div>  
+
<p><br></p>
-
<div id="(2)">[2] Myslinski E, <i>et al.</i> (2001). An unusually compact external promoter for RNA polymerase III transcription of the human H1RNA gene. <i>Nucleic Acids Res. 2001 Jun 15;29(12):2502-9</i>. <br></div>  
+
<p id="h4"> References </p>
 +
<small>
-
<div id="(3)">[3] Cheng AW, <i>et al.</i> (2013). Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. <i>Cell Res. 2013 Aug 27. doi: 10.1038/cr.2013.122</i>. <br></div>  
+
<div id="(1.1)">(1) Mali P.,<i>et al.</i> (2013). Cas9 transcriptional activators for target specificity screening and paired nickases for cooperative genome  engineering. Nat Biotechnol. <br></div>
 +
<div id="(1.2)">(2) Cheng AW., <i>et al.</i> (2013). Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator  
 +
system. Cell Res. <br></div>  
 +
</small>
 +
<div id="design_tool">
 +
<p id="h2">
 +
crRNA design tool
 +
</p>
 +
 +
 +
<form id="form1" action="" method="get">
 +
<legend id="h3">Enter Target Sequence</legend>
 +
<textarea name="Sequenz" type="text" rows="8" cols="40">
 +
</textarea>
 +
 +
 +
<fieldset>
 +
<p id="h4">Choose Program</p>
 +
<p>
 +
Which strand should be targeted?<br>
 +
<p>
 +
<input type="radio" name="rad" value="1"> non coding strand
 +
<input type="radio" name="rad" value="2"> coding strand
 +
</p>
 +
 +
<p>
 +
<button id="button1" type="button"> Submit </button>
 +
<input type="reset" value="reset">
 +
 +
</p>
 +
 +
</fieldset>
 +
 +
</form>
 +
 +
 +
 +
 +
<div id="explanation">
 +
<p>This tool helps you to design a crRNA-insert for dCas9 RNA plasmid: "uniCAS RNAimer" (<a id="link" href="http://parts.igem.org/Part:BBa_K1150034">BBa_K1150034</a>). <br>
 +
Using this tool you do not have to do this on your own. Just insert the desired target sequence and you get all different oligo possibilities and their positions. The oligos contain overhangs which fit to this plasmid's BbsI-overhangs and are ready to use. <br>
 +
The two different target possibilities are the coding and non-coding strand, depending on the desired target sequence. </p>
 +
 +
 +
 +
<p> a) For repression of gene transcription by targeting the coding strand the oligos must be designed as follows: </p>
 +
 +
<ol>
 +
    <li>Search at your desired target sequence for a CCN (reverse complement of the PAM sequence) at the coding strand.</li>
 +
  <li> Extract the following (3') 30 nucleotides.</li>
 +
  <li> Extract the reverse complement.</li>
 +
  <li>Add AAAC at the 5' end and GT at the 3' end. This will be your fist oligo.</li>
 +
    <li>Take the sequence from step 2 and add TAAAAC at the 5' end. This will be your second oligo.</li>
 +
    </ol>
 +
 +
<div id="coding"><img src="https://static.igem.org/mediawiki/2013/3/3a/Freiburg2013_CrRNA_Design.png" ></div>
 +
 +
<p style="padding-top:10px"> b) For repression of gene transcription by targeting the non-coding strand the oligos must be designed as follows: </p>
 +
 +
<ol>
 +
    <li>Search at your desired target sequence for a NGG (the PAM sequence) at the coding strand.</li>
 +
  <li> Extract the 30 nucleotides before (5') the PAM sequence.</li>
 +
<li> Extract the reverse complement.</li>
 +
  <li> Add TAAAAC at the 5' end. This will be your second oligo. </li>
 +
    <li>Take the sequence from step 2 and add AAAC at the 5' end and GT at the 3' end. This will be your first oligo.</li>
 +
    <li><b>Advice: To prevent BioBrick cloning from failing, avoid illegal iGEM restriction sites within the crRNAs. </b></li>
 +
</ol>
 +
 +
 +
<div id="coding"><img src="https://static.igem.org/mediawiki/2013/5/5d/Non_coding.png" ></div>
 +
 +
 +
 +
<p style="padding-top:10px"> <small>(the oligos are designed analog to: Cong, L., <i>et al.</i> (2013). Multiplex Genome Engineering using CRISPR/Cas Systems. Science.)</small><br>
 +
</p>
 +
<p><br></p>
 +
<p><br></p>
 +
</div>
 +
 +
 +
<p id = "h3"> Technical details about the crRNA design tool</p>
 +
<a style="color:white" href="javascript://" onclick="anz('news1');return false;">Source Code</a>
 +
 +
<div id="news1" style="display: none;">
 +
 +
<code>
 +
<pre>
 +
jQuery(document).ready( function() {
 +
document.getElementById('button1').onclick = function() {
 +
 +
var TestVar = document.getElementById('form1').Sequenz.value;
 +
var length = TestVar.length;
 +
  TestVar = TestVar.trim();
 +
  TestVar = TestVar.toUpperCase();
 +
if(length & lt ; 32){alert("too short");return;}
 +
var nonNuc = TestVar.search(/[^T|^A|^G|^C]/);
 +
if (nonNuc != -1){alert("There are non nucleotide signs in your sequence.
 +
                Please check your sequence!");return;
 +
                }
 +
 +
for (Count = 0; Count & lt ; 2; Count++) {
 +
    if (document.getElementById('form1').rad[Count].checked){ break; }
 +
  }
 +
 +
if (Count==0) {
 +
  var pattern = /.GG/g;
 +
  var m;
 +
  var oligo4 = [];
 +
  var index1 = [];
 +
  var k = 0;
 +
 +
while (m = pattern.exec(TestVar)){
 +
  oligo4.push(m);
 +
  index1.push(m.index);
 +
  }
 +
 
 +
  var oligo1 = [];
 +
  var index = [];
 +
 
 +
  var i = 0;
 +
  for (var k = 0; k& lt ;index1.length; k++){
 +
  if (index1[k]  & gt;  31){
 +
  oligo1[i] = TestVar.substring(index1[k]-30 ,index1[k]);
 +
  index[i] = index1[k]-30;
 +
  i++;
 +
  }
 +
  }
 +
 
 +
if (oligo1 == null ) {
 +
  alert("There are no matches found!");return;
 +
  }
 +
 
 +
  for ( var k = 0; k& lt ;oligo1.length; k++){
 +
  oligo1[k] = oligo1[k].substring(0,30);
 +
  }
 +
 
 +
  var oligo2 = oligo1.slice();
 +
  var index = oligo1.slice();
 +
   
 +
  for( var k=0; k& lt ;oligo1.length; k++ ) {
 +
  index[k] = TestVar.indexOf(oligo1[k]);
 +
  }
 +
 
 +
  for( var k=0; k& lt ;oligo2.length; k++ ) {
 +
  oligo2[k] = oligo2[k].split("").reverse().join("");
 +
  oligo2[k]=oligo2[k].replace(/A/gi,"X");
 +
  oligo2[k]=oligo2[k].replace(/T/gi,"A");
 +
  oligo2[k]=oligo2[k].replace(/X/gi,"T");
 +
  oligo2[k]=oligo2[k].replace(/G/gi,"Y");
 +
  oligo2[k]=oligo2[k].replace(/C/gi,"G");
 +
  oligo2[k]=oligo2[k].replace(/Y/gi,"C");
 +
  oligo2[k]="TAAAAC" + oligo2[k] ;
 +
  }
 +
 
 +
  for( var k=0; k& lt ;oligo1.length; k++ ) {
 +
  oligo1[k]="AAAC"+ oligo1[k] +"GT";
 +
  }
 +
 
 +
var j = 0;
 +
  for( var k=0; k& lt ;oligo2.length; k++ ) {
 +
  TestVar = TestVar.substring(0,index[k]+30+j) +
 +
                                TestVar.substring(index[k]+30+j,index[k]+33+j).fontcolor("red") +
 +
                                TestVar.substring(index[k]+33+j,TestVar.length);
 +
  j = j+25;
 +
  }
 +
 +
} else {
 +
 
 +
var pattern = /CC./g;
 +
  var m;
 +
  var oligo4 = [];
 +
  var index1 = [];
 +
  var k = 0;
 +
 
 +
  while (m = pattern.exec(TestVar)){
 +
  oligo4.push(m);
 +
  index1.push(m.index);
 +
  }
 +
 +
var oligo1 = [];
 +
  var index = [];
 +
 
 +
  var i = 0;
 +
  for (var k = 0; k& lt ;index1.length; k++){
 +
  if (index1[k] & lt ; TestVar.length-33){
 +
  oligo1[i] = TestVar.substring(index1[k]+3 ,index1[k]+33);
 +
  index[i] = index1[k];
 +
  i++;
 +
  }
 +
  }
 +
 +
  var oligo2 = oligo1.slice();
 +
 
 +
  for( var k=0; k& lt ;oligo2.length; k++ ) {
 +
  oligo2[k]="TAAAAC" + oligo2[k] ;
 +
  }
 +
 
 +
  for( var k=0; k& lt ;oligo1.length; k++ ) {
 +
  oligo1[k] = oligo1[k].split("").reverse().join("");
 +
  oligo1[k]=oligo1[k].replace(/A/gi,"X");
 +
  oligo1[k]=oligo1[k].replace(/T/gi,"A");
 +
  oligo1[k]=oligo1[k].replace(/X/gi,"T");
 +
  oligo1[k]=oligo1[k].replace(/G/gi,"Y");
 +
  oligo1[k]=oligo1[k].replace(/C/gi,"G");
 +
  oligo1[k]=oligo1[k].replace(/Y/gi,"C");
 +
  oligo1[k]="AAAC"+ oligo1[k] +"GT";
 +
  }
 +
 
 +
  var j = 0;
 +
  for( var k=0; k& lt ;oligo2.length; k++ ) {
 +
  TestVar = TestVar.substring(0,index[k]+j) + 
 +
                                TestVar.substring(index[k]+j,index[k]+3+j).
 +
                                fontcolor("red") + TestVar.substring
 +
                              (index[k]+3+j,TestVar.length);
 +
  j = j+25;
 +
  }
 +
  }
 +
 +
var oligo2rev = oligo1.slice();
 +
for( var k=0; k& lt ;oligo1.length; k++ ) {
 +
  oligo2rev[k] = oligo2[k].split("").reverse().join("");
 +
}
 +
 +
TestVar = TestVar.replace(/& lt ;/g,"\n & lt ;"); 
 +
  var w = window.open("blank.html", "blank", "toolbar=yes, location=yes,
 +
                        directories=no, status=no, menubar=yes, scrollbars=yes, resizable=no,
 +
                        copyhistory=yes, width=1000, height=700");
 +
w.document.writeln("& lt;h2 & gt; The inserted sequence is: & lt ;/h2 & gt; ");
 +
w.document.writeln(TestVar);
 +
w.document.writeln("& lt ;h1 & gt; possible crRNAs are:
 +
                                  & lt ;/h1 & gt; & lt ;div & gt; ");
 +
w.document.writeln("& lt ;table & gt; ");
 +
w.document.writeln("& lt ;tr & gt; & lt ;td & gt; ");
 +
 +
for (i=0; i& lt ;oligo2.length; i++ ) {   
 +
w.document.writeln("& lt ;h3 & gt;  Oligo number: " +
 +
                        (parseInt(i, 10) + parseInt(1, 10)) + " was found at position " +
 +
                        (parseInt(index[i], 10) + parseInt(1, 10))  + "& lt ;/h3 & gt; ");
 +
    w.document.writeln("& lt ;p & gt; Oligo1: " + oligo1[i] +
 +
                        "& lt ;/p & gt; & lt ;p & gt; Oligo2: " + oligo2[i] + "& lt;
 +
                        /p & gt; ");
 +
    w.document.writeln("& lt ;pre & gt; Oligo1: " + oligo1[i] +
 +
                        "& lt ;br & gt;           
 +
                        ||||||||||||||||||||||||||||||||& lt ;br & gt; Oligo2:    " +
 +
                        oligo2rev[i] + "& lt ;/pre & gt; ");
 +
    }
 +
 +
w.document.writeln("& lt ;/td & gt; ");
 +
  w.document.writeln("& lt ;td & gt; ");
 +
  w.document.writeln("& lt ;/td & gt; & lt ;/tr & gt; & lt ;/table & gt; ");
 +
  w.document.writeln("& lt ;/div & gt; ");
 +
  w.document.close(); 
 +
}
 +
});
 +
</pre>
 +
</code>
 +
 +
</div>

Latest revision as of 03:59, 29 October 2013


CrispR

Targeting

In our uniCAS Toolkit we engineered the CRISPR/Cas9 system for future applications to regulate gene expression. The key components for the binding of the catalytically inactive Cas9 (dCas9) to its targets are two small, non-coding RNAs: the CRISPR-RNA (crRNA) and the tracrRNA. These RNAs that guide our dCas9 to specific DNA sequences have to be cotransfected with the dCas9-plasmids. Therefore we designed an RNA plasmid, termed RNAimer, which contains all required RNAs for efficiently guiding the dCas9 protein to the required DNA - even if multiple DNA sites should be targeted.

As an essential part of our toolkit is the binding of our protein to DNA, we evaluated this in detail: Various DNA target sequences were compared to evaluate the best guiding crRNAs. Evaluated parameters were varying loci and GC-content. Based on these results, we tested if dCas9 is able to bind to multiple targets.

In order to simplify the search for potential target sequences, we programmed the crRNA design tool and provide it to the iGEM community. As the binding of the RNA-guided Cas9 requires a protospacer adjacent motif (PAM), the tool determines all possible regions of the desired DNA sequence that is pasted into the tool. Eventually, you obtain the position of your target sites and the sequences of the oligos that have to be inserted into the RNAimer - ready to order!

RNAimer - targeting dCas9 to its destination

Introduction

As dCas9 requires two small, non-coding RNAs to mediate binding to the DNA, we designed an RNA plasmid containing the tracrRNA and a site where the crRNA can be introduced. This is achieved by digestion using BbsI and annealed oligos corresponding to respective crRNA sequence. Two (or more) of these RNA plasmids (with different crRNAs) can be fused using the iGEM BioBrick cloning strategy thereby allowing for a RNAimer plasmid carrying multiple crRNAs.

Figure 1: RNAimer (BBa_K1150034)
Our RNA plasmid, termed the RNAimer, contains the tracrRNA and a site where the desired crRNA can be inserted. Transcription of both RNAs are driven by the H1 and the U6 promoter RNA polymerase III promoters, respectively. Assembly of multiple crRNAs can be easily done according to iGEM standard assembly.

As it is important that the RNAs are not being marked for protein expression and nuclear export, the RNA polymerase III is required for transcription. RNA polymerase III mainly synthesizes small non-coding RNAs (e.g. tRNAs or rRNAs) whereas the commonly used polymerase II is responsible for transcription of mainly mRNAs. [1, 2]. We chose the human U6- and H1-promoter to drive transcription of the RNAs as they are exclusively recognized by polymerase III [2]. In order to reduce the amount of plasmids for transfection when intending to target several genes or target sites at once, we wanted to join the required crRNAs on one RNA plasmid. This was easily manageable by using the iGEM standard assembly: Oligos are inserted separately into the RNAimer plasmid (BBa_K1150034) by digesting using BbsI and ligation. Afterwards the inserts located within the respective RNAimer plasmids can be combined by using the restriction enzymes of the prefix and suffix of the iGEM standard (Figure 6).

Figure 6: Assembly and function of the RNAimer
For each desired crRNA, one RNAimer plasmid (containing the crRNA after ligation) has to be digested with BbsI. Afterwards the annealed crRNA oligos will be ligated. The different RNA inserts can be assembled using the idempotent iGEM cloning strategy. Three different crRNAs (red, yellow, blue) will be transcribed.


Results

To test this strategy functionally we performed SEAP (Secreted Embryonic Alkaline Phosphatase) assays with dCas9-VP16. HEK-293T cells were transfected with dCas9-VP16 and different RNAimer plasmids containing different crRNAs. We either co-transfected two crRNAs or a single plasmid carrying both crRNAs. This resulted in the same activation of SEAP expression (Figure 7).
Figure 7: SEAP activation with dCas9-VP16 and RNAimer.
HEK-293T cells were seeded at 65,000 cells in a 24-well format. After 24 hours cells were transfected following the standard procedure. 48 h post-transfection, supernatant was harvested and SEAP activity was evaluated following the standard protocol. Activation by dCas9-VP16 was performed using two targets at once. In the case of T3+4 the loci were on the same plasmid, whereas in the case of T3&T4 two separate plasmids were co-transfected. Both combinations display a strong activation of the CMVmin promoter, driving the SEAP reporter expression. The activation of the two conditions is comparable. This is a hint, that there is no decrease in efficiency when combining several loci on one plasmid.


Discussion

We have shown that our RNA plasmid "RNAimer" works as expected. The combination of two targets does not seem to decrease the efficiency of targeting as the activation is comparable when transfecting two separate plasmids or combining both targets in one plasmid. With our toolkit it is possible to induce gene expression by co-transfecting two plasmids (plasmid coding for the dCas9-effector fusion protein and the RNAimer plasmid carrying the crRNA and tracrRNA).
For future application researchers will be able to alter gene expression by transfecting only the plasmid containing the crRNA. This could be possible when the dCas9 will be stably integrated into the genome of cells or model organisms, as recently done by Gilbert et al. [4].


References

(1) Dieci G.,et al. (2007). The expanding RNA polymerase III transcriptome. Trends Genet. 23, 614-622.
(2) Myslinski E., et al. (2001). An unusually compact external promoter for RNA polymerase III transcription of the human H1RNA gene. Nucleic Acids Res. 29, 2502-2509.
(3) Cheng AW., et al. (2013). Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res.
(4) Gilbert LA.,et al. (2013). CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes. Cell 154, 442-451.

Multiple Targeting

Introduction

One of the greatest advantages of the CRISPR/Cas9 system compared to other transcription activators (e.g. Zinc fingers (ZFNs) or transcription-activator like effectors (TALEs)) is that only one protein is required for targeting several DNA loci: For a new target just another crRNA has to be expressed. So a RNA plasmid was designed containing the tracrRNA, where the crRNA can be easily introduced.
With this RNA plasmid and another plasmid containing the dCas9-effector fusion gene it is possible to target several DNA sites at once by co-transfecting two plasmids. This allows the simultaneous regulation of different genes or a stricter control of one gene by targeting several loci of this gene the same time.

First of all, we had to evaluate different DNA sequences for targeting, since the results of our first experiments (see labjournal for details) showed that various crRNAs resulted in different SEAP levels. Thus, we cloned five different target sites 26 bp upstream of the CMVmin promoter of a SEAP reporter plasmid. Additionally we designed crRNAs in various distances upstream of this promoter (Tab. 1). These target sites were evaluated by activating SEAP expression with dCas9-VP16.


Table 1: Different target sites

The VEGF and EMX1 target sites were cloned into the SEAP reporter plasmid. T2-T4 are original sequences found on this plasmid. All target loci are upstream of the CMVmin promoter. VEGF and EMX1 are parts of the sequence upstream of the human VEGF or EMX1 gene, respectively.
Name Distance to promoter Sequence GC content [%]
VEGF VZ-573
-26 bp
GTGTGCAGACGGCAGTCACTAGGGGGCGCT
70
VEGF VZ-475
-26 bp
GTGAGTGTGTGCGTGTGGGGTTGAGGGCGT
63
VEGF VZ-8
-26 bp
TTAAAAGTCGGCTGGTAGCGGGGAGGATCG
57
VEGF VZ+434
-26 bp
GACCTGCTTTTGGGGGTGACCGCCGGAGCG
70
EMX1
-26 bp
GGAAGGGCCTGAGTCCGAGCAGAAGAAGAA
57
T2
-148 bp
AAGCATTTATCAGGGTTATTGTCTCATGAG
37
T3
-222 bp
AATGCCGCAAAAAAGGGAATAAGGGCGACA
47
T4
-443 bp
GACCGAGTTGCTCTTGCCCGGCGTCAATAC
60


Results

Evaluation of different target sequences

At first we tested different target sequences at the same distance from the promoter. To do so we had to insert the target sequences into the SEAP reporter plasmid in front of the CMVmin.
HEK-293T cells were transfected with one of these SEAP plasmids and a plasmid containing dCas9-VP16, the tracrRNA and the appropriate crRNA. The results show different activation efficiencies (Figure 8) in which no activation (VEGF VZ -573 and +434) and up to a 5 fold increase of SEAP activation could be observed. With the tested target sequences that have a GC content of 70 % no activation was observed whereas induction of SEAP expression with the target sequences with a GC content of about 60 % (compare Tab. 1) could be shown.
Figure 8: Targets with different sequences
The grey bars represent the SEAP activity of HEK-293T cells transfected with dCas9-VP16 but no crRNA (normalized to value 1). The blue bars show fold induction of SEAP activity by tansfecting dCas9-VP16 with crRNAs in comparison to the appropriate control without crRNA. An activation is visible for nearly all targets, but the fold-induction is differing strongly. All values are means of three biological replicates. Error bars represent standard deviation. For the experiment HEK-293T cells were seeded at 65,000 cells in a 24-well format. After 24 h cells were transfected following standard procedure. After 48 h of expression supernatant was harvested and SEAP activity was evaluated following the standard protocol.
Next, we evaluated different distances upstream of the promoter. Thus, we targeted four different loci on the reporter plasmid (EMX1, T2, T3 & T4; for sequence see Tab. 1).
HEK-293T cells were co-transfected with a CMVmin:SEAP plasmid that contains EMX1, CMV:dCas9-VP16 and the RNA plasmid, respectively. The results show an significant increase in SEAP expression when EMX1, which is the closest target site to the promoter, is targeted (Figure 2). Activation decreases, the larger the distance between promoter and target sites becomes (compare Tab. 1).
Figure 9: Targets with different distances to the promoter
Activation of CMVmin:SEAP via dCas9-VP16 with different distances to the promoter in HEK-293T cells. The grey bar represents the SEAP level without crRNA, the blue bars represent the activation using different crRNAs in increasing distance to the promoter (EMX1 the closest, T4 with the greatest distance). A clear decrease in activation is observed with increasing distance from promoter. All epxeriments were performed in biological triplicates. Error bars represent standard deviation. HEK-293T cells were seeded at 65,000 cells in 24-well format and 24 hours later transfected following the standard procedure. After 48 h of expression supernatant was harvested and SEAP activity was evaluated following the standard protocol.


Stricter gene regulation by targeting different loci simultaneously

In order to improve the efficiency of our gene regulation toolkit, we tried to target several loci upstream of the promoter of the reporter gene at once. Thus, we designed crRNAs that are complementary to sequences on the SEAP reporter plasmid with different distances to the promoter (Tab. 1).

HEK-293T cells were transfected with the CMVmin:SEAP reporter plasmid, dCas9-VP16 (iGEM standard), one or two RNA plasmids and a plasmid coding for Renilla luciferase for an internal standard (to eliminate variabilities of different cell numbers or expression levels). The total amount of RNA plasmids is kept constant, so an increase of SEAP expression due to more available crRNA can be excluded. By combining the targets EMX1 and T2 a higher SEAP activity than the sum of the single targets can be observed (Figure 13).

Figure 13: Effects of different target numbers
Activation of CMVmin:SEAP was evaluated by the usage of single targets and combined targets. Every target itself displays an activation of the minimal promoter. By combining two targets (EMX1 & T2), an even stronger activation is visible, that exceeds the additive effect of single plasmids.
HEK-293T cells were seeded at 65,000 cells in 24-well format. After 24 hours cells were transfected following standard procedure. After 48 h of expression supernatant was harvested and SEAP activity was evaluated following the standard protocol.


Discussion

From the few target sites we evaluated we could draw the conclusion that a target should be chosen like this:

  • The distance to the promoter should be kept at minimum.
  • Target sites with a GC content around 60 % should be preferred.
As we tested our target sequences with dCas9-VP16, stricly speaking, we can only make a statement about the best target sites for gene activation.
Moreover there may be other parameters that influence the effects of dCas9-VP16 on SEAP (e.g. the secondary structure of the crRNA). But for the distance to the promoter Mali et al. [1] came to the same conclusion: Only the target site that had the shortest distance to the promoter showed a high increase of activation.

By targeting different loci upstream of a promoter simultaneously the efficiency of transcription activation can be enhanced. While Cheng et al. [2] yielded an activation up to 8-fold by using at least three different targets in comparison to the highest activation of a single target, we were able to achieve a three fold increase by using only two different targets.


References

(1) Mali P.,et al. (2013). Cas9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol.
(2) Cheng AW., et al. (2013). Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res.

crRNA design tool

Enter Target Sequence

Choose Program

Which strand should be targeted?

non coding strand coding strand

This tool helps you to design a crRNA-insert for dCas9 RNA plasmid: "uniCAS RNAimer" (BBa_K1150034).
Using this tool you do not have to do this on your own. Just insert the desired target sequence and you get all different oligo possibilities and their positions. The oligos contain overhangs which fit to this plasmid's BbsI-overhangs and are ready to use.
The two different target possibilities are the coding and non-coding strand, depending on the desired target sequence.

a) For repression of gene transcription by targeting the coding strand the oligos must be designed as follows:

  1. Search at your desired target sequence for a CCN (reverse complement of the PAM sequence) at the coding strand.
  2. Extract the following (3') 30 nucleotides.
  3. Extract the reverse complement.
  4. Add AAAC at the 5' end and GT at the 3' end. This will be your fist oligo.
  5. Take the sequence from step 2 and add TAAAAC at the 5' end. This will be your second oligo.

b) For repression of gene transcription by targeting the non-coding strand the oligos must be designed as follows:

  1. Search at your desired target sequence for a NGG (the PAM sequence) at the coding strand.
  2. Extract the 30 nucleotides before (5') the PAM sequence.
  3. Extract the reverse complement.
  4. Add TAAAAC at the 5' end. This will be your second oligo.
  5. Take the sequence from step 2 and add AAAC at the 5' end and GT at the 3' end. This will be your first oligo.
  6. Advice: To prevent BioBrick cloning from failing, avoid illegal iGEM restriction sites within the crRNAs.

(the oligos are designed analog to: Cong, L., et al. (2013). Multiplex Genome Engineering using CRISPR/Cas Systems. Science.)



Technical details about the crRNA design tool

Source Code