Difference between revisions of "Part:BBa K5237000"

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<body>
 
<body>
  <!-- Part summary -->
+
<!-- Part summary -->
  <section id="1">
+
<section id="1">
    <h1>fgRNA Entryvector MbCas12a-SpCas9</h1>
+
<h1>fgRNA Entry Vector MbCas12a-SpCas9</h1>
    <p>
+
<p>
 
       This part integrates the crRNA of MbCas12a (<a href="https://parts.igem.org/Part:BBa_K5237206">BBa_K5237206</a>) and the sgRNA of SpCas9 (<a href="https://parts.igem.org/Part:BBa_K5237209">BBa_K5237209</a>) into a single
 
       This part integrates the crRNA of MbCas12a (<a href="https://parts.igem.org/Part:BBa_K5237206">BBa_K5237206</a>) and the sgRNA of SpCas9 (<a href="https://parts.igem.org/Part:BBa_K5237209">BBa_K5237209</a>) into a single
 
       fusion
 
       fusion
       guide RNA (fgRNA). The fgRNA is functional meaning that the MbCas12a (<a
+
       guide RNA (fgRNA). The fgRNA is functional, meaning that the MbCas12a (<a href="https://parts.igem.org/Part:BBa_K5237001">BBa_K5237001</a>),
        href="https://parts.igem.org/Part:BBa_K5237001">BBa_K5237000</a>),
+
       SpCas9 (<a href="https://parts.igem.org/Part:BBa_K5237002">BBa_K5237002</a>) and the fusion dCas (<a href="https://parts.igem.org/Part:BBa_K5237003">BBa_K5237003</a>)
       SpCas9 (<a href="https://parts.igem.org/Part:BBa_K5237002">BBa_K5237002</a>) and the Cas-Staple (<a
+
       can utilize the fgRNA to target two loci simultaneously. The fgRNA also works in combination with the catalyitcally inactive Cas
        href="https://parts.igem.org/Part:BBa_K5237003">BBa_K5237003</a>)
+
       can utilize the fgRNA to target two loci simultaneously. The fgRNA also works with the catalyitcally inactive
+
 
       versions.
 
       versions.
       We could show editing with the active version and induced proximity of to loci with the inactive version.
+
       We successfully showed genome editing using active SpCas9 and Cas12a and induced proximity of two loci with the inactive dSpCas9 and dMbCas12a.<br/>
 +
      For our part collection, the PICasSO toolbox, this part has a crucial role in formation of our CRISPR/Cas staples.
 
     </p>
 
     </p>
    <p> </p>
+
<p> </p>
  </section>
+
</section>
  <div class="toc" id="toc">
+
<div class="toc" id="toc">
    <div id="toctitle">
+
<div id="toctitle">
      <h1>Contents</h1>
+
<h1>Contents</h1>
    </div>
+
</div>
    <ul>
+
<ul>
      <li class="toclevel-1 tocsection-1"><a href="#1"><span class="tocnumber">1</span> <span class="toctext">Sequence
+
<li class="toclevel-1 tocsection-1"><a href="#1"><span class="tocnumber">1</span> <span class="toctext">Sequence
 
             overview</span></a>
 
             overview</span></a>
      </li>
+
</li>
      <li class="toclevel-1 tocsection-2"><a href="#2"><span class="tocnumber">2</span> <span class="toctext">Usage and
+
<li class="toclevel-1 tocsection-2"><a href="#2"><span class="tocnumber">2</span> <span class="toctext">Usage and
 
             Biology</span></a>
 
             Biology</span></a>
        <ul>
+
<ul>
          <li class="toclevel-2 tocsection-2.1">
+
<li class="toclevel-2 tocsection-2.1">
            <a href="#2.1"><span class="tocnumber">2.1</span> <span class="toctext">Discovery and Mechanism of
+
<a href="#2.1"><span class="tocnumber">2.1</span> <span class="toctext">Discovery and Mechanism of
 
                 CRISPR/Cas9</span></a>
 
                 CRISPR/Cas9</span></a>
          </li>
+
</li>
          <li class="toclevel-2 tocsection-2.2">
+
<li class="toclevel-2 tocsection-2.2">
            <a href="#2.2"><span class="tocnumber">2.2</span> <span class="toctext">Differences between Cas9 and
+
<a href="#2.2"><span class="tocnumber">2.2</span> <span class="toctext">Differences between Cas9 and
 
                 Cas12a</span></a>
 
                 Cas12a</span></a>
          </li>
+
</li>
          <li class="toclevel-2 tocsection-2.3">
+
<li class="toclevel-2 tocsection-2.3">
            <a href="#2.3"><span class="tocnumber">2.3</span> <span class="toctext">Dead Cas Proteins and their
+
<a href="#2.3"><span class="tocnumber">2.3</span> <span class="toctext">Dead Cas Proteins and their
 
                 Application</span></a>
 
                 Application</span></a>
          </li>
+
</li>
          <li class="toclevel-2 tocsection-2.4">
+
<li class="toclevel-2 tocsection-2.4">
            <a href="#2.4"><span class="tocnumber">2.4</span> <span class="toctext">fgRNA and CHyMErA System</span></a>
+
<a href="#2.4"><span class="tocnumber">2.4</span> <span class="toctext">fgRNA and CHyMErA System</span></a>
          </li>
+
</li>
        </ul>
+
</ul>
      </li>
+
</li>
      <li class="toclevel-1 tocsetction-3"><a href="#3"><span class="tocnumber">3</span> <span class="toctext">Assembly
+
<li class="toclevel-1 tocsetction-3"><a href="#3"><span class="tocnumber">3</span> <span class="toctext">Assembly
 
             and part evolution</span></a>
 
             and part evolution</span></a>
      </li>
+
</li>
      <li class="toclevel-1 tocsection-4"><a href="#4"><span class="tocnumber">4</span> <span
+
<li class="toclevel-1 tocsection-4"><a href="#4"><span class="tocnumber">4</span> <span class="toctext">Results</span></a>
            class="toctext">Results</span></a>
+
<ul>
        <ul>
+
<li class="toclevel-2 tocsection-4.1">
          <li class="toclevel-2 tocsection-4.1">
+
<a href="#4.1"><span class="tocnumber">4.1</span> <span class="toctext">Editing endogenous loci with
            <a href="#4.1"><span class="tocnumber">4.1</span> <span class="toctext">Editing endogenous loci with
+
 
                 fgRNAs</span></a>
 
                 fgRNAs</span></a>
          </li>
+
</li>
          <li class="toclevel-2 tocsection-4.2">
+
<li class="toclevel-2 tocsection-4.2">
            <a href="#4.2"><span class="tocnumber">4.2</span> <span class="toctext">Proximity assay with inactive Cas
+
<a href="#4.2"><span class="tocnumber">4.2</span> <span class="toctext">Proximity assay with inactive Cas
 
                 proteins</span></a>
 
                 proteins</span></a>
          </li>
+
</li>
          <li class="toclevel-2 tocsection-4.3">
+
<li class="toclevel-2 tocsection-4.3">
            <a href="#4.3"><span class="tocnumber">4.3</span> <span class="toctext">The Inclusion of a Linker Does Not
+
<a href="#4.3"><span class="tocnumber">4.3</span> <span class="toctext">The Inclusion of a Linker Does Not
 
                 Lower Editing Rates</span></a>
 
                 Lower Editing Rates</span></a>
          </li>
+
</li>
          <li class="toclevel-2 tocsection-4.4">
+
<li class="toclevel-2 tocsection-4.4">
            <a href="#4.4"><span class="tocnumber">4.4</span> <span class="toctext">fgRNAs can be Used for
+
<a href="#4.4"><span class="tocnumber">4.4</span> <span class="toctext">fgRNAs can be Used for
 
                 CRISPRa</span></a>
 
                 CRISPRa</span></a>
          </li>
+
</li>
          <li class="toclevel-2 tocsection-4.5">
+
<li class="toclevel-2 tocsection-4.5">
            <a href="#4.5"><span class="tocnumber">4.5</span> <span class="toctext">Stapling Two DNA Strands Together
+
<a href="#4.5"><span class="tocnumber">4.5</span> <span class="toctext">Stapling Two DNA Strands Together
 
                 Using fgRNAs</span></a>
 
                 Using fgRNAs</span></a>
          </li>
+
</li>
        </ul>
+
</ul>
      </li>
+
</li>
      <li class="toclevel-1 tocsection-8"><a href="#5"><span class="tocnumber">5</span> <span
+
<li class="toclevel-1 tocsection-8"><a href="#5"><span class="tocnumber">5</span> <span class="toctext">References</span></a>
            class="toctext">References</span></a>
+
</li>
      </li>
+
</ul>
    </ul>
+
</div>
  </div>
+
<section><p><br/><br/></p>
  <section>
+
<font size="5"><b>The PICasSO Toolbox </b> </font>
    <p><br /><br /></p>
+
<div class="thumb" style="margin-top:10px;"></div>
    <font size="5"><b>The PICasSO Toolbox </b> </font>
+
<div class="thumbinner" style="width:550px"><img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/registry-part-collection-engineering-cycle-example-overview.svg" style="width:99%;"/>
    <div class="thumb" style="margin-top:10px;"></div>
+
<div class="thumbcaption">
    <div class="thumbinner" style="width:550px"><img alt="" class="thumbimage"
+
<i><b>Figure 1: How our part collection can be used to engineer new staples</b></i>
        src="https://static.igem.wiki/teams/5237/wetlab-results/registry-part-collection-engineering-cycle-example-overview.svg"
+
</div>
        style="width:99%;" />
+
</div>
      <div class="thumbcaption">
+
        <i><b>Figure 1: How our part collection can be used to engineer new staples</b></i>
+
      </div>
+
    </div>
+
  
    <p>
+
<p>
      <br />
+
<br/>
       Next to the well-studied linear DNA sequence, the 3D spatial organization of DNA plays a crucial role in gene
+
       Next to the well-studied linear DNA sequence, the <b>3D spatial organization</b> of DNA plays a crucial role in
       regulation,
+
       gene regulation,
       cell fate, disease development and more. However, the tools to precisely manipulate this genomic architecture
+
       cell fate, disease development and more. However, the tools to precisely manipulate this genomic
       remain limited, rendering it challenging to explore the full potential of the
+
       architecture remain limited, rendering it challenging to explore the full potential of the
       3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular
+
       3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a <b>powerful
       toolbox based on various DNA-binding proteins to address this issue.
+
       molecular toolbox</b> based on various DNA-binding proteins to address this issue.
 
     </p>
 
     </p>
    <p>
+
<p>
 
       The <b>PICasSO</b> part collection offers a comprehensive, modular platform for precise manipulation and
 
       The <b>PICasSO</b> part collection offers a comprehensive, modular platform for precise manipulation and
       re-programming
+
       <b>re-programming
       of DNA-DNA interactions using protein staples in living cells, enabling researchers to recreate natural 3D genomic
+
       of DNA-DNA interactions</b> using protein staples in living cells, enabling researchers to recreate natural 3D genomic
 
       interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation.
 
       interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation.
       Beyond its versatility, PICasSO includes robust assay systems to support the engineering, optimization, and
+
       Specifically, the fusion of two DNA binding proteins enables to artifically bring distant genomic loci into
 +
      proximty.
 +
      To unlock the system's full potential, we introduce versatile <b>chimeric CRISPR/Cas complexes</b>, connected either on
 +
      the protein or the guide RNA level. These1 complexes are reffered to as protein- or Cas staples. Beyond its
 +
      versatility, PICasSO includes <b>robust assay</b> systems to support the engineering, optimization, and
 
       testing of new staples, ensuring functionality <i>in vitro</i> and <i>in vivo</i>. We took special care to include
 
       testing of new staples, ensuring functionality <i>in vitro</i> and <i>in vivo</i>. We took special care to include
       parts crucial for testing every step of the cycle (design, build, test, learn) when engineering new parts.
+
       parts crucial for testing every step of the cycle (design, build, test, learn) when <b>engineering new parts</b>.
 
     </p>
 
     </p>
    <p>At its heart, the PICasSO part collection consists of three categories. <br /><b>(i)</b> Our <b>DNA-binding
+
<p>At its heart, the PICasSO part collection consists of three categories. <br/><b>(i)</b> Our <b>DNA-binding
 
         proteins</b>
 
         proteins</b>
 
       include our
 
       include our
 
       finalized enhancer hijacking Cas staple as well as half staples that can be used by scientists to compose entirely
 
       finalized enhancer hijacking Cas staple as well as half staples that can be used by scientists to compose entirely
 
       new Cas staples in the future. We also include our Simple staples that serve as controls for successful stapling
 
       new Cas staples in the future. We also include our Simple staples that serve as controls for successful stapling
       and can be further engineered to create alternative, simpler and more compact staples. <br />
+
       and can be further engineered to create alternative, simpler and more compact staples. <br/>
      <b>(ii)</b> As <b>functional elements</b>, we list additional parts that enhance the functionality of our Cas and
+
<b>(ii)</b> As <b>functional elements</b>, we list additional parts that enhance the functionality of our Cas and
 
       Basic staples. These
 
       Basic staples. These
 
       consist of
 
       consist of
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       Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs
 
       Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs
 
       with our
 
       with our
       interkingdom conjugation system. <br />
+
       interkingdom conjugation system. <br/>
      <b>(iii)</b> As the final category of our collection, we provide parts that support the use of our <b>custom
+
<b>(iii)</b> As the final category of our collection, we provide parts that support the use of our <b>custom
 
         readout
 
         readout
 
         systems</b>. These include components of our established FRET-based proximity assay system, enabling users to
 
         systems</b>. These include components of our established FRET-based proximity assay system, enabling users to
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       in mammalian cells.
 
       in mammalian cells.
 
     </p>
 
     </p>
    <p>
+
<p>
       The following table gives a comprehensive overview of all parts in our PICasSO toolbox. <mark
+
       The following table gives a comprehensive overview of all parts in our PICasSO toolbox. <mark style="background-color: #FFD700; color: black;">The highlighted parts showed
        style="background-color: #FFD700; color: black;">The highlighted parts showed
+
 
         exceptional performance as described on our iGEM wiki and can serve as a reference.</mark> The other parts in
 
         exceptional performance as described on our iGEM wiki and can serve as a reference.</mark> The other parts in
 
       the
 
       the
 
       collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their
 
       collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their
       own custom Cas staples, enabling further optimization and innovation.<br />
+
       own custom Cas staples, enabling further optimization and innovation.<br/>
    </p>
+
</p>
    <p>
+
<p>
      <font size="4"><b>Our part collection includes:</b></font><br />
+
<font size="4"><b>Our part collection includes:</b></font><br/>
    </p>
+
</p>
    <table style="width: 90%; padding-right:10px;">
+
<table style="width: 90%; padding-right:10px;">
      <td align="left" colspan="3"><b>DNA-binding proteins: </b>
+
<td align="left" colspan="3"><b>DNA-binding proteins: </b>
 
         The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring
 
         The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring
 
         easy assembly.</td>
 
         easy assembly.</td>
      <tbody>
+
<tbody>
        <tr bgcolor="#FFD700">
+
<tr bgcolor="#FFD700">
          <td><a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a></td>
          <td>fgRNA Entry vector MbCas12a-SpCas9</td>
+
<td>fgRNA Entry vector MbCas12a-SpCas9</td>
          <td>Entryvector for simple fgRNA cloning via SapI</td>
+
<td>Entryvector for simple fgRNA cloning via SapI</td>
        </tr>
+
</tr>
        <tr bgcolor="#FFD700">
+
<tr bgcolor="#FFD700">
          <td><a href="https://parts.igem.org/Part:BBa_K5237001" target="_blank">BBa_K5237001</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237001" target="_blank">BBa_K5237001</a></td>
          <td>Staple subunit: dMbCas12a-Nucleoplasmin NLS</td>
+
<td>Staple subunit: dMbCas12a-Nucleoplasmin NLS</td>
          <td>Staple subunit that can be combined with sgRNA or fgRNA and dCas9 to form a functional staple</td>
+
<td>Staple subunit that can be combined with sgRNA or fgRNA and dCas9 to form a functional staple</td>
        </tr>
+
</tr>
        <tr bgcolor="#FFD700">
+
<tr bgcolor="#FFD700">
          <td><a href="https://parts.igem.org/Part:BBa_K5237002" target="_blank">BBa_K5237002</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237002" target="_blank">BBa_K5237002</a></td>
          <td>Staple subunit: SV40 NLS-dSpCas9-SV40 NLS</td>
+
<td>Staple subunit: SV40 NLS-dSpCas9-SV40 NLS</td>
          <td>Staple subunit that can be combined witha sgRNA or fgRNA and dCas12avto form a functional staple
+
<td>Staple subunit that can be combined witha sgRNA or fgRNA and dCas12avto form a functional staple
 
           </td>
 
           </td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a></td>
          <td>Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS</td>
+
<td>Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS</td>
          <td>Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands into close
+
<td>Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands into close
 
             proximity
 
             proximity
 
           </td>
 
           </td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237004" target="_blank">BBa_K5237004</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237004" target="_blank">BBa_K5237004</a></td>
          <td>Staple subunit: Oct1-DBD</td>
+
<td>Staple subunit: Oct1-DBD</td>
          <td>Staple subunit that can be combined to form a functional staple, for example with TetR.<br />
+
<td>Staple subunit that can be combined to form a functional staple, for example with TetR.<br/>
 
             Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
 
             Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237005" target="_blank">BBa_K5237005</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237005" target="_blank">BBa_K5237005</a></td>
          <td>Staple subunit: TetR</td>
+
<td>Staple subunit: TetR</td>
          <td>Staple subunit that can be combined to form a functional staple, for example with Oct1.<br />
+
<td>Staple subunit that can be combined to form a functional staple, for example with Oct1.<br/>
 
             Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
 
             Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237006" target="_blank">BBa_K5237006</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237006" target="_blank">BBa_K5237006</a></td>
          <td>Simple staple: TetR-Oct1</td>
+
<td>Simple staple: TetR-Oct1</td>
          <td>Functional staple that can be used to bring two DNA strands in close proximity</td>
+
<td>Functional staple that can be used to bring two DNA strands in close proximity</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237007" target="_blank">BBa_K5237007</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237007" target="_blank">BBa_K5237007</a></td>
          <td>Staple subunit: GCN4</td>
+
<td>Staple subunit: GCN4</td>
          <td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td>
+
<td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237008" target="_blank">BBa_K5237008</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237008" target="_blank">BBa_K5237008</a></td>
          <td>Staple subunit: rGCN4</td>
+
<td>Staple subunit: rGCN4</td>
          <td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td>
+
<td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237009" target="_blank">BBa_K5237009</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237009" target="_blank">BBa_K5237009</a></td>
          <td>Mini staple: bGCN4</td>
+
<td>Mini staple: bGCN4</td>
          <td>
+
<td>
 
             Assembled staple with minimal size that can be further engineered</td>
 
             Assembled staple with minimal size that can be further engineered</td>
        </tr>
+
</tr>
      </tbody>
+
</tbody>
      <td align="left" colspan="3"><b>Functional elements: </b>
+
<td align="left" colspan="3"><b>Functional elements: </b>
 
         Protease-cleavable peptide linkers and inteins are used to control and modify staples for further optimization
 
         Protease-cleavable peptide linkers and inteins are used to control and modify staples for further optimization
 
         for custom applications</td>
 
         for custom applications</td>
      <tbody>
+
<tbody>
        <tr bgcolor="#FFD700">
+
<tr bgcolor="#FFD700">
          <td><a href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a></td>
          <td>Cathepsin B-cleavable Linker: GFLG</td>
+
<td>Cathepsin B-cleavable Linker: GFLG</td>
          <td>Cathepsin B-cleavable peptide linker that can be used to combine two staple subunits to make responsive
+
<td>Cathepsin B-cleavable peptide linker that can be used to combine two staple subunits to make responsive
 
             staples</td>
 
             staples</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237011" target="_blank">BBa_K5237011</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237011" target="_blank">BBa_K5237011</a></td>
          <td>Cathepsin B Expression Cassette</td>
+
<td>Cathepsin B Expression Cassette</td>
          <td>Expression Cassette for the overexpression of cathepsin B</td>
+
<td>Expression Cassette for the overexpression of cathepsin B</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237012" target="_blank">BBa_K5237012</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237012" target="_blank">BBa_K5237012</a></td>
          <td>Caged NpuN Intein</td>
+
<td>Caged NpuN Intein</td>
          <td>A caged NpuN split intein fragment that undergoes protein <i>trans</i>-splicing after protease activation.
+
<td>A caged NpuN split intein fragment that undergoes protein <i>trans</i>-splicing after protease activation.
 
             Can be used to create functionalized staples
 
             Can be used to create functionalized staples
 
             units</td>
 
             units</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237013" target="_blank">BBa_K5237013</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237013" target="_blank">BBa_K5237013</a></td>
          <td>Caged NpuC Intein</td>
+
<td>Caged NpuC Intein</td>
          <td>A caged NpuC split intein fragment that undergoes protein <i>trans</i>-splicing after protease activation.
+
<td>A caged NpuC split intein fragment that undergoes protein <i>trans</i>-splicing after protease activation.
 
             Can be used to create functionalized staples
 
             Can be used to create functionalized staples
 
             units</td>
 
             units</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237014" target="_blank">BBa_K5237014</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237014" target="_blank">BBa_K5237014</a></td>
          <td>fgRNA processing casette</td>
+
<td>fgRNA processing casette</td>
          <td>Processing casette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D
+
<td>Processing casette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D
 
             genome reprograming</td>
 
             genome reprograming</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237015" target="_blank">BBa_K5237015</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237015" target="_blank">BBa_K5237015</a></td>
          <td>Intimin anti-EGFR Nanobody</td>
+
<td>Intimin anti-EGFR Nanobody</td>
          <td>Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large
+
<td>Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large
 
             constructs</td>
 
             constructs</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K4643003" target="_blank">BBa_K4643003</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K4643003" target="_blank">BBa_K4643003</a></td>
          <td>incP origin of transfer</td>
+
<td>incP origin of transfer</td>
          <td>Origin of transfer that can be cloned into the plasmid vector and used for conjugation as a means of
+
<td>Origin of transfer that can be cloned into the plasmid vector and used for conjugation as a means of
 
             delivery</td>
 
             delivery</td>
        </tr>
+
</tr>
      </tbody>
+
</tbody>
      <td align="left" colspan="3"><b>Readout Systems: </b>
+
<td align="left" colspan="3"><b>Readout Systems: </b>
 
         FRET and enhancer recruitment to measure proximity of stapled DNA in bacterial and mammalian living cells
 
         FRET and enhancer recruitment to measure proximity of stapled DNA in bacterial and mammalian living cells
 
         enabling swift testing and easy development for new systems</td>
 
         enabling swift testing and easy development for new systems</td>
      <tbody>
+
<tbody>
        <tr bgcolor="#FFD700">
+
<tr bgcolor="#FFD700">
          <td><a href="https://parts.igem.org/Part:BBa_K5237016" target="_blank">BBa_K5237016</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237016" target="_blank">BBa_K5237016</a></td>
          <td>FRET-Donor: mNeonGreen-Oct1</td>
+
<td>FRET-Donor: mNeonGreen-Oct1</td>
          <td>FRET Donor-Fluorpohore fused to Oct1-DBD that binds to the Oct1 binding cassette. Can be used to visualize
+
<td>FRET Donor-Fluorpohore fused to Oct1-DBD that binds to the Oct1 binding cassette. Can be used to visualize
 
             DNA-DNA
 
             DNA-DNA
 
             proximity</td>
 
             proximity</td>
        </tr>
+
</tr>
        <tr bgcolor="#FFD700">
+
<tr bgcolor="#FFD700">
          <td><a href="https://parts.igem.org/Part:BBa_K5237017" target="_blank">BBa_K5237017</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237017" target="_blank">BBa_K5237017</a></td>
          <td>FRET-Acceptor: TetR-mScarlet-I</td>
+
<td>FRET-Acceptor: TetR-mScarlet-I</td>
          <td>Acceptor part for the FRET assay binding the TetR binding cassette. Can be used to visualize DNA-DNA
+
<td>Acceptor part for the FRET assay binding the TetR binding cassette. Can be used to visualize DNA-DNA
 
             proximity</td>
 
             proximity</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237018" target="_blank">BBa_K5237018</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237018" target="_blank">BBa_K5237018</a></td>
          <td>Oct1 Binding Casette</td>
+
<td>Oct1 Binding Casette</td>
          <td>DNA sequence containing 12 Oct1 binding motifs, compatible with various assays such as the FRET
+
<td>DNA sequence containing 12 Oct1 binding motifs, compatible with various assays such as the FRET
 
             proximity assay</td>
 
             proximity assay</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a></td>
          <td>TetR Binding Cassette</td>
+
<td>TetR Binding Cassette</td>
          <td>DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET
+
<td>DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET
 
             proximity assay</td>
 
             proximity assay</td>
        </tr>
+
</tr>
        <td><a href="https://parts.igem.org/Part:BBa_K5237020" target="_blank">BBa_K5237020</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237020" target="_blank">BBa_K5237020</a></td>
        <td>Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64</td>
+
<td>Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64</td>
        <td>Readout system that responds to protease activity. It was used to test cathepsin B-cleavable linker</td>
+
<td>Readout system that responds to protease activity. It was used to test cathepsin B-cleavable linker</td>
  
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237021" target="_blank">BBa_K5237021</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237021" target="_blank">BBa_K5237021</a></td>
          <td>NLS-Gal4-VP64</td>
+
<td>NLS-Gal4-VP64</td>
          <td>Trans-activating enhancer, that can be used to simulate enhancer hijacking</td>
+
<td>Trans-activating enhancer, that can be used to simulate enhancer hijacking</td>
        </tr>
+
</tr>
        <td><a href="https://parts.igem.org/Part:BBa_K5237022" target="_blank">BBa_K5237022</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237022" target="_blank">BBa_K5237022</a></td>
        <td>mCherry Expression Cassette: UAS, minimal Promotor, mCherry</td>
+
<td>mCherry Expression Cassette: UAS, minimal Promotor, mCherry</td>
        <td>Readout system for enhancer binding. It was used to test cathepsin B-cleavable linker</td>
+
<td>Readout system for enhancer binding. It was used to test cathepsin B-cleavable linker</td>
  
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237023" target="_blank">BBa_K5237023</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237023" target="_blank">BBa_K5237023</a></td>
          <td>Oct1 - 5x UAS binding casette</td>
+
<td>Oct1 - 5x UAS binding casette</td>
          <td>Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay</td>
+
<td>Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237024" target="_blank">BBa_K5237024</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237024" target="_blank">BBa_K5237024</a></td>
          <td>TRE-minimal promoter- firefly luciferase</td>
+
<td>TRE-minimal promoter- firefly luciferase</td>
          <td>Contains Firefly luciferase controlled by a minimal promoter. It was used as a luminescence readout for
+
<td>Contains Firefly luciferase controlled by a minimal promoter. It was used as a luminescence readout for
 
             simulated enhancer hijacking</td>
 
             simulated enhancer hijacking</td>
        </tr>
+
</tr>
      </tbody>
+
</tbody>
    </table>
+
</table>
  </section>
+
</section>
  <section id="1">
+
<section id="1">
    <h1>1. Sequence overview</h1>
+
<h1>1. Sequence overview</h1>
  </section>
+
</section>
 
</body>
 
</body>
 
 
</html>
 
</html>
 
<span class="h3bb">Sequence and Features</span>
 
<span class="h3bb">Sequence and Features</span>
 
<partinfo>BBa_K5237000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5237000 SequenceAndFeatures</partinfo>
 
<html>
 
<html>
 
 
<body>
 
<body>
  <section id="2">
+
<section id="2">
    <h1>2. Usage and Biology</h1>
+
<h1>2. Usage and Biology</h1>
    <section id="2.1">
+
<section id="2.1">
      <h2>2.1 Discovery and Mechanism of CRISPR/Cas9</h2>
+
<h2>2.1 Discovery and Mechanism of CRISPR/Cas9</h2>
 
+
<div class="thumb tright" style="margin:0;">
      <div class="thumb tright" style="margin:0;">
+
<div class="thumbinner" style="width:450px;">
        <div class="thumbinner" style="width:450px;">
+
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/cas-staple-svg/background-cas9-cas12a-principle.svg" style="width:99%;"/>
          <img alt="" class="thumbimage"
+
<div class="thumbcaption">
            src="https://static.igem.wiki/teams/5237/wetlab-results/cas-staple-svg/background-cas9-cas12a-principle.svg"
+
<i>
            style="width:99%;" />
+
<b>Figure 2: The CRISPR/Cas system </b>
          <div class="thumbcaption">
+
            <i>
+
              <b>Figure 2: The CRISPR/Cas system (adapted from Pacesa <i>et al.</i> (2024))</b>
+
 
               A and B, schematic structure of Cas9 and Cas12a with their sgRNA/crRNA, sitting on a DNA strand with the
 
               A and B, schematic structure of Cas9 and Cas12a with their sgRNA/crRNA, sitting on a DNA strand with the
 
               PAM.
 
               PAM.
Line 374: Line 364:
 
               spacer is located at the 5' prime end, for Cas12a at the 3' end of the gRNA. The scaffold of the gRNA
 
               spacer is located at the 5' prime end, for Cas12a at the 3' end of the gRNA. The scaffold of the gRNA
 
               forms a specific
 
               forms a specific
               secondary structure enabling it to bind to the Cas protein. The cut sites by the cleaving domains, RuvC
+
               secondary structure enabling it to be bound by the Cas protein. DNA cleavage sites are indicated by the scissors.
              and HNH, are
+
              symbolized by the scissors
+
 
             </i>
 
             </i>
          </div>
+
</div>
        </div>
+
</div>
      </div>
+
</div>
      <p>
+
<p>
 
         In 2012, Jinek <i>et al.</i> discovered the use of the Clustered Regularly Interspaced Short Palindromic Repeats
 
         In 2012, Jinek <i>et al.</i> discovered the use of the Clustered Regularly Interspaced Short Palindromic Repeats
 
         (CRISPR)/Cas system to induce double-strand breaks in DNA. Since then, the system has been well established as a
 
         (CRISPR)/Cas system to induce double-strand breaks in DNA. Since then, the system has been well established as a
Line 395: Line 383:
 
         the
 
         the
 
         Cas protein (Jinek <i>et al.</i>, 2012) (Fig. 2 A). Furthermore, a specific three
 
         Cas protein (Jinek <i>et al.</i>, 2012) (Fig. 2 A). Furthermore, a specific three
         nucleotide sequence (NGG) on the 3' end in the targeted DNA is needed for binding and cleavage. This is referred
+
         nucleotide sequence (NGG) at the 3' end in the targeted DNA is needed for binding and cleavage. This is referred
 
         to as the protospacer adjacent motif (PAM) (Sternberg <i>et al.</i>, 2014). The most commonly used Cas9 protein
 
         to as the protospacer adjacent motif (PAM) (Sternberg <i>et al.</i>, 2014). The most commonly used Cas9 protein
 
         is SpCas9
 
         is SpCas9
 
         or SpyCas9, which originates from Streptococcus pyogenes (Pacesa <i>et al.</i>, 2024).
 
         or SpyCas9, which originates from Streptococcus pyogenes (Pacesa <i>et al.</i>, 2024).
 
       </p>
 
       </p>
      <p>
+
<p>
         A significant enhancement of this system was the introduction of single guide RNA (sgRNA)s, which combine the
+
         A significant enhancement of this system was the introduction of single guide RNAs (sgRNA[s]), which combine the
 
         functions of a tracrRNA and crRNA (Mali <i>et al.</i>, 2013).
 
         functions of a tracrRNA and crRNA (Mali <i>et al.</i>, 2013).
 
         Moreover, Cong (2013) established precise targeting of human endogenous loci by designing the 20 nt spacer
 
         Moreover, Cong (2013) established precise targeting of human endogenous loci by designing the 20 nt spacer
 
         sequence accordingly.
 
         sequence accordingly.
 
       </p>
 
       </p>
    </section>
+
</section>
    <section id="2.2">
+
<section id="2.2">
      <h2>2.2 Differences between Cas9 and Cas12a</h2>
+
<h2>2.2 Differences between Cas9 and Cas12a</h2>
      <p>
+
<p>
 
         Over the following years, further CRISPR/Cas systems have been discovered, including the Cpf1 system, which has
 
         Over the following years, further CRISPR/Cas systems have been discovered, including the Cpf1 system, which has
 
         been
 
         been
Line 427: Line 415:
 
         2020).
 
         2020).
 
       </p>
 
       </p>
    </section>
+
</section>
    <section id="2.3">
+
<section id="2.3">
      <h2>2.3 Dead Cas Proteins and their Application</h2>
+
<h2>2.3 Dead Cas Proteins and their Application</h2>
      <p>
+
<p>
 
         Specific mutations of these domains result in catalytic inactivity and therefore allow for the creation of
 
         Specific mutations of these domains result in catalytic inactivity and therefore allow for the creation of
 
         nickases that only cut one of the DNA strands, or completely inactive Cas proteins (Koonin <i>et al.</i>, 2023)
 
         nickases that only cut one of the DNA strands, or completely inactive Cas proteins (Koonin <i>et al.</i>, 2023)
Line 440: Line 428:
 
         involves fusing Cas9 with the transcriptional activator VP64 (Kampmann, 2017).
 
         involves fusing Cas9 with the transcriptional activator VP64 (Kampmann, 2017).
 
       </p>
 
       </p>
    </section>
+
</section>
    <section id="2.4">
+
</section>
      <h2>2.4 fgRNA and CHyMErA System</h2>
+
<section id="3" style="clear:both;">
      <div class="thumb tright" style="margin:0;">
+
<h1>3. Assembly and part evolution</h1>
        <div class="thumbinner" style="width:400px;">
+
<p>
          <img alt="" class="thumbimage"
+
            src="https://static.igem.wiki/teams/5237/wetlab-results/cas-staple-svg/background-crispr-cas-system-fgrna-past.svg"
+
            style="width:99%;" />
+
          <div class="thumbcaption">
+
            <i>
+
              <b>Figure 3: Applications of the Fusion Guide RNA (adapted from Kweon <i>et al.</i> (2017)).</b>
+
              Fusion Guide RNAs can be used for multiplex genome editing by guidingactive Cas12a and Cas9 to two
+
              distinct loci. Similarly, fgRNAs allow for CRISPRa, by guiding the Cas9-VP64 transcriptional activator
+
              towards a
+
              target locus.
+
            </i>
+
          </div>
+
        </div>
+
      </div>
+
   
+
      <p>
+
        Kweon <i>et al.</i> (2017) further expanded the ways in which the CRISPR/Cas system could be used by introducing
+
        the concept of fusion guide RNA
+
        (fgRNA)s. By fusing the 3' end of a Cas12a gRNA to the 5' end of a Cas9 gRNA, the newly created fgRNA could be
+
        used by both proteins independently for either multiplex genome editing or transcriptional regulation and genome
+
        editing in parallel (Fig. 3). Similarly, this is also
+
        possible using the Cas Hybrid for Multiplexed Editing and screening Applications (CHyMErA) system
+
        (Gonatopoulos-Pournatzis <i>et al.</i>, 2020).
+
        In this instance, the gRNAs of Cas12a and Cas9 are connected in the
+
        opposite direction (3' Cas9 gRNA to 5' Cas12a gRNA), allowing for Cas12a to process the RNA into individual
+
        units
+
        (Fig. 3). Amongst other things, this allows for the analysis
+
        of the interaction between different genes by targeting them simultaneously with the two distinct spacers
+
        (Aregger
+
        <i>et al.</i>, 2021) (Fig. 3).
+
      </p>
+
 
+
    </section>
+
  </section >
+
  <section style="clear:both;" id="3">
+
    <h1>3. Assembly and part evolution</h1>
+
    <p>
+
 
       Building on insights of our fusion Cas engineering cycle and findings from Kweon (2017), fgRNAs were designed by
 
       Building on insights of our fusion Cas engineering cycle and findings from Kweon (2017), fgRNAs were designed by
       combining the sgRNA from SpCas9 with the crRNA from MbCas12a, specifically the 3'-end of the MbCas12a gRNA was
+
       combining the sgRNA from SpCas9 with the crRNA from MbCas12a. Specifically the 3'-end of the MbCas12a gRNA was
       fused to the 5'-end of the SpCas9 gRNA. Via this approach the two spacer sequences are fused directly, ensuring a
+
       linked to the 5'-end of the SpCas9 gRNA. Via this approach, the two spacer sequences are fused directly, ensuring a
 
       minimal distance between the two DNA strands.This also facilitates efficient cloning of different spacer
 
       minimal distance between the two DNA strands.This also facilitates efficient cloning of different spacer
       sequences. Linking the crRNA and sgRNA comes with the advantage of easily multiplexing the system, while still
+
       sequences, as both spacers can be exchangeed as one consecutive sequence. Linking the crRNA and sgRNA further enables
       guaranteeing that specific pairs of genomic loci are connected. The entry vector includes a U6 promoter, the
+
       multiplexing as Cas12a can inherently process gRNA repeats that are expressed from one single transcript enabling multiplexing. The entry vector includes a U6 promoter, the
 
       MbCas12a scaffold, a bacterial promoter driving <b>ccdB</b> expression, and the SpCas9 scaffold. Successful spacer
 
       MbCas12a scaffold, a bacterial promoter driving <b>ccdB</b> expression, and the SpCas9 scaffold. Successful spacer
 
       integration leads to the removal of the <b>ccdB</b> gene, allowing bacterial growth to be used as an indicator for
 
       integration leads to the removal of the <b>ccdB</b> gene, allowing bacterial growth to be used as an indicator for
       cloning success.<br />
+
       cloning success.<br/>
       An existing vector containing the U6 promoter and the MbCas12a scaffold was selected as the basis
+
       A conventional gRNA expression vector containing an MbCas12a crRNA scoffold under the control of an U6 promoter was selected as the basis
 
       for entry vector cloning. The vector and a ccdB-SpCas9 scaffold construct were PCR amplified and fitting overhangs
 
       for entry vector cloning. The vector and a ccdB-SpCas9 scaffold construct were PCR amplified and fitting overhangs
       for SapI were introduced. Golden Gate assembly (GGA) with Esp3I was used to create the final plasmid. The
+
       for SapI were introduced (Fig. 3). Golden Gate assembly (GGA) with Esp3I was used to create the final plasmid. The
       transformation was carried out in the ccdB-resistant XL1 Blue<i>E. Coli </i> strain.
+
       transformation was carried out in the ccdB-resistant XL1 Blue <i>E. Coli</i> strain.
 
     </p>
 
     </p>
    <div class="thumb">
+
<div class="thumb">
      <div class="thumbinner" style="width:80%;">
+
<div class="thumbinner" style="width:80%;">
        <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/entry-vector.svg"
+
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/entry-vector.svg" style="width:99%;"/>
          style="width:99%;" />
+
<div class="thumbcaption">
        <div class="thumbcaption">
+
<i>
          <i>
+
<b>Figure 3: Construction process of fgRNAs using the entry vector.</b> The ccdB gene excised using
            <b>Figure 4: Construction process of fgRNAs using the entry vector.</b> The ccdB gene can be cut out using
+
             SapI in a Golden Gate
             SapI in the Golden Gate
+
 
             assembly. By inserting oligonucleotides with the desired spacer sequences and matching overhangs, the
 
             assembly. By inserting oligonucleotides with the desired spacer sequences and matching overhangs, the
 
             complete fgRNA
 
             complete fgRNA
             can be expressed. Due to the cytotoxic nature of ccdB, only cells with the oligonucleotides as inserts
+
             can be assembled into the entry vector. Due to the cytotoxic nature of ccdB, only cells with the oligonucleotides as inserts
 
             survive.
 
             survive.
 
           </i>
 
           </i>
        </div>
+
</div>
      </div>
+
</div>
      <p>
+
<p>
 
         The first goal after assembly was to prove the editing activity of both proteins using fgRNA. The genes
 
         The first goal after assembly was to prove the editing activity of both proteins using fgRNA. The genes
 
         VEGFA and FANCF were selected as targets for Cas12a and Cas9, each target was tested with each Cas protein.
 
         VEGFA and FANCF were selected as targets for Cas12a and Cas9, each target was tested with each Cas protein.
         Editing efficiency will be analyzed with the T7 Endonuclease I (T7EI) assay. Controls will include crRNAs and
+
         Editing efficiency was analyzed with the T7 Endonuclease I (T7EI) assay. Controls included crRNAs and
         sgRNAs as positive controls, and non-targeting guides as negative controls. Desired spacer sequences can be
+
         sgRNAs as positive controls, and non-targeting guides as negative controls. Desired spacer sequences were
 
         ordered as oligos, annealed, and cloned in via GGA utilizing SapI.
 
         ordered as oligos, annealed, and cloned in via GGA utilizing SapI.
 
       </p>
 
       </p>
      <table style="width:40%; margin-top:20px; margin-bottom:20px;">
+
<div class="thumb tright" style="margin:0;"></div>
        <thead>
+
<div class="thumbinner" style="width:400px;">
          <td align="left" colspan="2">
+
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/cas-staple-svg/background-crispr-cas-system-fgrna-past.svg" style="width:99%;"/>
            <b>Table 1:</b> A list of all the different spacers we cloned and tested within the fgRNA
+
<div class="thumbcaption">
 +
<i>
 +
<b>Figure 4: Applications of the Fusion Guide RNA</b>
 +
              Fusion Guide RNAs can be used for multiplex genome editing by guidingactive Cas12a and Cas9 to two
 +
              distinct loci. Similarly, fgRNAs allow for CRISPRa, by guiding the Cas9-VP64 transcriptional activator
 +
              towards a
 +
              target locus.
 +
            </i>
 +
</div>
 +
</div>
 +
</div>
 +
<table style="width:40%; margin-top:20px; margin-bottom:20px;">
 +
<thead>
 +
<td align="left" colspan="2">
 +
<b>Table 1:</b> A list of all the different spacers we cloned and tested within the fgRNA
 
           </td>
 
           </td>
        </thead>
+
</thead>
        <tbody>
+
<tbody>
          <tr>
+
<tr>
            <td>VEGFA</td>
+
<td>VEGFA</td>
            <td>ctaggaatattgaagggggc</td>
+
<td>ctaggaatattgaagggggc</td>
          </tr>
+
</tr>
          <tr>
+
<tr>
            <td>FANCF</td>
+
<td>FANCF</td>
            <td>ggcggggtccagttccggga</td>
+
<td>ggcggggtccagttccggga</td>
          </tr>
+
</tr>
          <tr>
+
<tr>
            <td>CCR5</td>
+
<td>CCR5</td>
            <td>tgacatcaattattatacat</td>
+
<td>tgacatcaattattatacat</td>
          </tr>
+
</tr>
          <tr>
+
<tr>
            <td>TetO (<a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a>)</td>
+
<td>TetO (<a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a>)</td>
            <td>tctctatcactgatagggag</td>
+
<td>tctctatcactgatagggag</td>
          </tr>
+
</tr>
          <tr>
+
<tr>
            <td>Oct1-B (<a href="https://parts.igem.org/Part:BBa_K5237018" target="_blank">BBa_K5237018</a>)</td>
+
<td>Oct1-B (<a href="https://parts.igem.org/Part:BBa_K5237018" target="_blank">BBa_K5237018</a>)</td>
            <td>atgcaaatactgcactagtg</td>
+
<td>atgcaaatactgcactagtg</td>
          </tr>
+
</tr>
        </tbody>
+
</tbody>
      </table>
+
</table>
      <p>
+
<p>
 
         We constructed a second entry vector incorporating an AsCas12a scaffold (5' taatttctactcttgtagat 3') instead of
 
         We constructed a second entry vector incorporating an AsCas12a scaffold (5' taatttctactcttgtagat 3') instead of
 
         MbCas12a.
 
         MbCas12a.
         The sequence of the AsCas12a scaffold, was the only modification in the composite part. This vector was tested
+
         The sequence of the AsCas12a scaffold was the only modification in the composite part. This vector was tested
 
         on the
 
         on the
 
         loci VEGFA and FANCF to assess its functionality.
 
         loci VEGFA and FANCF to assess its functionality.
 
       </p>
 
       </p>
    </div>
+
 
  </section>
+
</section>
  <section id="4">
+
<section id="4">
    <h1>4. Results</h1>
+
<h1>4. Results</h1>
    <section id="4.1">
+
<section id="4.1">
      <h2>4.1 Editing endogenous loci with fgRNAs</h2>
+
<h2>4.1 Editing endogenous loci with fgRNAs</h2>
      <p>
+
<p>
 
         To prove that our fusion gRNAs still result in active ribonucleoproteins, a series of different fgRNAs were
 
         To prove that our fusion gRNAs still result in active ribonucleoproteins, a series of different fgRNAs were
 
         created, each carrying spacers specific to the VEGFA and FANCF genes.HEK293-T cells were transfected with the
 
         created, each carrying spacers specific to the VEGFA and FANCF genes.HEK293-T cells were transfected with the
 
         Cas
 
         Cas
 
         protein and gRNA constructs. The editing rate was tested 72h after transfection via a T7 endonuclease I
 
         protein and gRNA constructs. The editing rate was tested 72h after transfection via a T7 endonuclease I
         assay.<br />
+
         assay.<br/>
  
 
         AsCas12a and SpCas9 were used. The AsCas12a spacer targets VEGFA, while the SpCas9 spacer targets FANCF. The
 
         AsCas12a and SpCas9 were used. The AsCas12a spacer targets VEGFA, while the SpCas9 spacer targets FANCF. The
Line 582: Line 546:
 
         fgRNAs.
 
         fgRNAs.
 
       </p>
 
       </p>
      <div class="thumb">
+
<div class="thumb">
        <div class="thumbinner" style="width:60%;">
+
<div class="thumbinner" style="width:60%;">
          <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/results-ascas-2.svg"
+
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/results-ascas-2.svg" style="width:99%;"/>
            style="width:99%;" />
+
<div class="thumbcaption">
          <div class="thumbcaption">
+
<i>
            <i>
+
<b>Figure 5: fgRNAs Enable Efficient Editing of Endogenous Loci.</b>
              <b>Figure 5: fgRNAs Enable Efficient Editing of Endogenous Loci.</b>
+
 
               The editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by
 
               The editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by
 
               measuring band
 
               measuring band
Line 597: Line 560:
 
               each sample.
 
               each sample.
 
             </i>
 
             </i>
          </div>
+
</div>
        </div>
+
</div>
      </div>
+
</div>
    </section>
+
</section>
    <section id="4.2">
+
<section id="4.2">
      <h2>4.2 Fusion Guide RNAs Allow for Editing Rates With Variant Cas Orthologs</h2>
+
<h2>4.2 Efficient Fusion Guide RNA-Mediated Editing With Various Cas Orthologs</h2>
      <p>
+
<p>
         To further evaluate the capabilities of the fgRNAs, we tested them in combination
+
         After showing efficient editing, the next step was to evaluate the capabilities of the fgRNAs, we tested them in combination
 
         with different Cas12a orthologs. After some initial testing, we decided on using MbCas12a together with SpCas9,
 
         with different Cas12a orthologs. After some initial testing, we decided on using MbCas12a together with SpCas9,
 
         because we found AsCas12a to be less active in a dual luciferase assay when co-transfected with SpCas9 compared
 
         because we found AsCas12a to be less active in a dual luciferase assay when co-transfected with SpCas9 compared
Line 611: Line 574:
 
         SpCas9 editing has not been significantly different.
 
         SpCas9 editing has not been significantly different.
 
       </p>
 
       </p>
      <div class="thumb">
+
<div class="thumb">
        <div class="thumbinner" style="width:60%;">
+
<div class="thumbinner" style="width:60%;">
          <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/engineering/cas12-decision.svg"
+
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/engineering/cas12-decision.svg" style="width:99%;"/>
            style="width:99%;" />
+
<div class="thumbcaption">
          <div class="thumbcaption">
+
<i>
            <i>
+
<b>Figure 6: Comparison of AsCas12a and MbCas12a with a dual luciferase assay.</b>
              <b>Figure 6: Comparison of AsCas12a and MbCas12a with a dual luciferae assay.</b>
+
 
               Firefly luminescence intensity measured 48 h after transfection. Normalized against renilla luminescence.
 
               Firefly luminescence intensity measured 48 h after transfection. Normalized against renilla luminescence.
 
               On the x-axis
 
               On the x-axis
Line 628: Line 590:
 
               ****p&lt;0.0001
 
               ****p&lt;0.0001
 
             </i>
 
             </i>
          </div>
+
</div>
        </div>
+
</div>
      </div>
+
</div>
      <p>
+
<p>
 
         Additionally, to test if the differences in editing rates from the preliminary assay resulted from the targeted
 
         Additionally, to test if the differences in editing rates from the preliminary assay resulted from the targeted
 
         loci or the different Cas orthologs, the spacers were tested in both arrangements. Once with Cas12a targeting
 
         loci or the different Cas orthologs, the spacers were tested in both arrangements. Once with Cas12a targeting
 
         FANCF and SpCas9 targeting VEGFA and once vice versa. To better assess the impact that the utilization of a
 
         FANCF and SpCas9 targeting VEGFA and once vice versa. To better assess the impact that the utilization of a
 
         fgRNA
 
         fgRNA
         has on the editing rates, the sgRNAs were tested separately and in one sample.<br />
+
         has on the editing rates, the sgRNAs were tested separately and in one sample.<br/>
 
         Having the sgRNA with single Cas
 
         Having the sgRNA with single Cas
 
         proteins in the same sample resulted in no clear difference in the editing rates (Fig. 7). The fusion of the
 
         proteins in the same sample resulted in no clear difference in the editing rates (Fig. 7). The fusion of the
Line 644: Line 606:
 
         the same conditions, the editing rates for MbCas12a were overall lower than the ones from SpCas9.
 
         the same conditions, the editing rates for MbCas12a were overall lower than the ones from SpCas9.
 
       </p>
 
       </p>
      <div class="thumb">
+
<div class="thumb">
        <div class="thumbinner" style="width:90%;">
+
<div class="thumbinner" style="width:90%;">
          <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/results-mbcas-2.svg"
+
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/results-mbcas-2.svg" style="width:99%;"/>
            style="width:99%;" />
+
<div class="thumbcaption">
          <div class="thumbcaption">
+
<i>
            <i>
+
<b>Figure 7: Fusion gRNA Editing Rates In Combination with MbCas12a.</b>
              <b>Figure 7: Fusion gRNA Editing Rates In Combination with MbCas12a.</b>
+
 
               In <b>A</b> and <b>B</b> the editing rates were determined 72h after transfection via T7EI
 
               In <b>A</b> and <b>B</b> the editing rates were determined 72h after transfection via T7EI
 
               assay. Editing % was determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved
 
               assay. Editing % was determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved
Line 659: Line 620:
 
               display both orientations of the two spacers for VEGFA and FANCF.
 
               display both orientations of the two spacers for VEGFA and FANCF.
 
             </i>
 
             </i>
          </div>
+
</div>
        </div>
+
</div>
      </div>
+
</div>
    </section>
+
</section>
    <section id="4.3">
+
<section id="4.3">
      <h2>4.3 The Inclusion of a Linker Does Not Lower Editing Rates</h2>
+
<h2>4.3 The Inclusion of a Linker Does Not Lower Editing Rates</h2>
      <p>
+
<p>
 
         To further assess the effect of the genomic locus on the editing rate, we included CCR5 as an additional
 
         To further assess the effect of the genomic locus on the editing rate, we included CCR5 as an additional
 
         gene target. For this assay, a fgRNA with a 20 nt long linker was included between the two spacers. The editing
 
         gene target. For this assay, a fgRNA with a 20 nt long linker was included between the two spacers. The editing
Line 673: Line 634:
 
         addition of the 20 nt linker had no effect on the editing rates compared to no linker.
 
         addition of the 20 nt linker had no effect on the editing rates compared to no linker.
 
       </p>
 
       </p>
      <div class="thumb">
+
<div class="thumb">
        <div class="thumbinner" style="width:60%;">
+
<div class="thumbinner" style="width:60%;">
          <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/results-ccr5-2.svg"
+
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/results-ccr5-2.svg" style="width:99%;"/>
            style="width:99%;" />
+
<div class="thumbcaption">
          <div class="thumbcaption">
+
<i>
            <i>
+
<b>Figure 8: Fusion gRNA Editing Rates for Multiplexing CCR5 and VEGFA</b>
              <b>Figure 8: Fusion gRNA Editing Rates for Multiplexing CCR5 and VEGFA</b>
+
 
               The editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by
 
               The editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by
 
               measuring band
 
               measuring band
Line 688: Line 648:
 
               each sample. Cas12a targets VEGFA and Cas9 targets CCR5.
 
               each sample. Cas12a targets VEGFA and Cas9 targets CCR5.
 
             </i>
 
             </i>
          </div>
+
</div>
        </div>
+
</div>
      </div>
+
</div>
    </section>
+
</section>
    <section id="4.4">
+
<section id="4.4">
      <h2>4.4 fgRNAs can be used for CRISPRa</h2>
+
<h2>4.4 fgRNAs can be used for CRISPRa</h2>
      <p>
+
<p>
 
         To establish the foundation for their use as protein scaffolds, we identified the next step as demonstrating the
 
         To establish the foundation for their use as protein scaffolds, we identified the next step as demonstrating the
 
         use
 
         use
Line 706: Line 666:
 
         to a sgRNA (Fig. 9).
 
         to a sgRNA (Fig. 9).
 
       </p>
 
       </p>
      <div class="thumb">
+
<div class="thumb">
        <div class="thumbinner" style="width:40%;">
+
<div class="thumbinner" style="width:40%;">
          <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/results-crispra-2.svg"
+
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/results-crispra-2.svg" style="width:99%;"/>
            style="width:99%;" />
+
<div class="thumbcaption">
          <div class="thumbcaption">
+
<i>
            <i>
+
<b>Figure 9: CRISPRa Induced Luciferase Expression for sgRNAs and fgRNAs.</b>
              <b>Figure 9: CRISPRa Induced Luciferase Expression for sgRNAs and fgRNAs.</b>
+
 
               Firefly luciferase activity was measured 48h after transfection. Normalized against ubiquitously expressed
 
               Firefly luciferase activity was measured 48h after transfection. Normalized against ubiquitously expressed
 
               Renilla
 
               Renilla
Line 721: Line 680:
 
               spacer is the targeted gene. The symbols below indicate which parts are included in each sample.
 
               spacer is the targeted gene. The symbols below indicate which parts are included in each sample.
 
             </i>
 
             </i>
          </div>
+
</div>
        </div>
+
</div>
      </div>
+
</div>
    </section>
+
</section>
    <section id="4.5">
+
<section id="4.5">
      <h2>4.5 Stapling Two DNA Strands Together Using fgRNAs</h2>
+
<h2>4.5 Stapling Two DNA Strands Together Using fgRNAs</h2>
      <p>
+
<p>
 
         After showing the general capability of the fgRNA
 
         After showing the general capability of the fgRNA
 
         to work for editing and for CRISPR activation, the next step was to use it to staple two DNA loci together, and
 
         to work for editing and for CRISPR activation, the next step was to use it to staple two DNA loci together, and
Line 736: Line 695:
 
         introducing
 
         introducing
 
         a fgRNA staple and a Gal4-VP64, expression of the luciferase is induced (Fig. 10, Panel A).
 
         a fgRNA staple and a Gal4-VP64, expression of the luciferase is induced (Fig. 10, Panel A).
         Different linker lengths were tested. Cells were again normalized against ubiquitous renilla expression.<br />
+
         Different linker lengths were tested. Cells were again normalized against ubiquitous renilla expression.<br/>
 
         Using no linker between the two spacers showed similar relative luciferase activity to the baseline control
 
         Using no linker between the two spacers showed similar relative luciferase activity to the baseline control
 
         (Fig. 10, Panel B). An extension of the linker from 20 nt up to 40 nt resulted in an increasingly higher
 
         (Fig. 10, Panel B). An extension of the linker from 20 nt up to 40 nt resulted in an increasingly higher
Line 743: Line 702:
 
         hijacking an enhancer/activator.
 
         hijacking an enhancer/activator.
 
       </p>
 
       </p>
      <div class="thumb">
+
<div class="thumb">
        <div class="thumbinner" style="width:60%;">
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<div class="thumbinner" style="width:60%;">
          <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/results-eh-2.svg"
+
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/results-eh-2.svg" style="width:99%;"/>
            style="width:99%;" />
+
<div class="thumbcaption">
          <div class="thumbcaption">
+
<i>
            <i>
+
<b>Figure 10: Applying Fusion Guide RNAs for Cas staples.</b> <b>A</b>, schematic overview of the assay.
              <b>Figure 10: Applying Fusion Guide RNAs for Cas staples.</b> <b>A</b>, schematic overview of the assay.
+
 
               An enhancer
 
               An enhancer
 
               plasmid and a reporter plasmid are brought into proximity by a fgRNA Cas staple complex binding both
 
               plasmid and a reporter plasmid are brought into proximity by a fgRNA Cas staple complex binding both
Line 765: Line 723:
 
               to 40 nt.
 
               to 40 nt.
 
             </i>
 
             </i>
          </div>
+
</div>
        </div>
+
</div>
      </div>
+
</div>
    </section>
+
</section>
  </section>
+
</section>
  <section id="5">
+
<section id="5">
    <h1>5. References</h1>
+
<h1>5. References</h1>
    <p>Aregger, M., Xing, K., &amp; Gonatopoulos-Pournatzis, T. (2021). Application of CHyMErA Cas9-Cas12a combinatorial
+
<p>Aregger, M., Xing, K., &amp; Gonatopoulos-Pournatzis, T. (2021). Application of CHyMErA Cas9-Cas12a combinatorial
 
       genome-editing platform for genetic interaction mapping and gene fragment deletion screening. <i>Nature
 
       genome-editing platform for genetic interaction mapping and gene fragment deletion screening. <i>Nature
         Protocols</i>, 16, 4722-4765. <a href="https://doi.org/10.1038/s41596-021-00595-1"
+
         Protocols</i>, 16, 4722-4765. <a href="https://doi.org/10.1038/s41596-021-00595-1" target="_blank">https://doi.org/10.1038/s41596-021-00595-1</a></p>
        target="_blank">https://doi.org/10.1038/s41596-021-00595-1</a></p>
+
<p>Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L.
    <p>Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L.
+
 
       A., &amp; Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. <i>Science</i>, 339, 819-823.
 
       A., &amp; Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. <i>Science</i>, 339, 819-823.
 
       <a href="https://doi.org/10.1126/science.1231143" target="_blank">https://doi.org/10.1126/science.1231143</a></p>
 
       <a href="https://doi.org/10.1126/science.1231143" target="_blank">https://doi.org/10.1126/science.1231143</a></p>
    <p>Gonatopoulos-Pournatzis, T., Aregger, M., Brown, K. R., Farhangmehr, S., Braunschweig, U., Ward, H. N., Ha, K. C.
+
<p>Gonatopoulos-Pournatzis, T., Aregger, M., Brown, K. R., Farhangmehr, S., Braunschweig, U., Ward, H. N., Ha, K. C.
 
       H., Weiss, A., Billmann, M., Durbic, T., Myers, C. L., Blencowe, B. J., &amp; Moffat, J. (2020). Genetic
 
       H., Weiss, A., Billmann, M., Durbic, T., Myers, C. L., Blencowe, B. J., &amp; Moffat, J. (2020). Genetic
 
       interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform. <i>Nature
 
       interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform. <i>Nature
         Biotechnology</i>, 38, 638-648. <a href="https://doi.org/10.1038/s41587-020-0437-z"
+
         Biotechnology</i>, 38, 638-648. <a href="https://doi.org/10.1038/s41587-020-0437-z" target="_blank">https://doi.org/10.1038/s41587-020-0437-z</a></p>
        target="_blank">https://doi.org/10.1038/s41587-020-0437-z</a></p>
+
<p>Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., &amp; Charpentier, E. (2012). A programmable
    <p>Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., &amp; Charpentier, E. (2012). A programmable
+
       dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. <i>Science</i>, 337, 816-821. <a href="https://doi.org/10.1126/science.1225829" target="_blank">https://doi.org/10.1126/science.1225829</a></p>
       dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. <i>Science</i>, 337, 816-821. <a
+
<p>Kampmann, M. (2017). CRISPRi and CRISPRa screens in mammalian cells for precision biology and medicine. <i>ACS
        href="https://doi.org/10.1126/science.1225829" target="_blank">https://doi.org/10.1126/science.1225829</a></p>
+
         Chemical Biology</i>, 13, 406-416. <a href="https://doi.org/10.1021/acschembio.7b00657" target="_blank">https://doi.org/10.1021/acschembio.7b00657</a></p>
    <p>Kampmann, M. (2017). CRISPRi and CRISPRa screens in mammalian cells for precision biology and medicine. <i>ACS
+
<p>Kleinstiver, B. P., Sousa, A. A., Walton, R. T., Tak, Y. E., Hsu, J. Y., Clement, K., Welch, M. M., Horng, J. E.,
         Chemical Biology</i>, 13, 406-416. <a href="https://doi.org/10.1021/acschembio.7b00657"
+
        target="_blank">https://doi.org/10.1021/acschembio.7b00657</a></p>
+
    <p>Kleinstiver, B. P., Sousa, A. A., Walton, R. T., Tak, Y. E., Hsu, J. Y., Clement, K., Welch, M. M., Horng, J. E.,
+
 
       Malagon-Lopez, J., Scarfò, I., Maus, M. V., Pinello, L., Aryee, M. J., &amp; Joung, J. K. (2019). Engineered
 
       Malagon-Lopez, J., Scarfò, I., Maus, M. V., Pinello, L., Aryee, M. J., &amp; Joung, J. K. (2019). Engineered
 
       CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base
 
       CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base
       editing. <i>Nature Biotechnology</i>, 37, 276-282. <a href="https://doi.org/10.1038/s41587-018-0011-0"
+
       editing. <i>Nature Biotechnology</i>, 37, 276-282. <a href="https://doi.org/10.1038/s41587-018-0011-0" target="_blank">https://doi.org/10.1038/s41587-018-0011-0</a></p>
        target="_blank">https://doi.org/10.1038/s41587-018-0011-0</a></p>
+
<p>Koonin, E. V., Gootenberg, J. S., &amp; Abudayyeh, O. O. (2023). Discovery of diverse CRISPR-Cas systems and
    <p>Koonin, E. V., Gootenberg, J. S., &amp; Abudayyeh, O. O. (2023). Discovery of diverse CRISPR-Cas systems and
+
       expansion of the genome engineering toolbox. <i>Biochemistry</i>, 62, 3465-3487. <a href="https://doi.org/10.1021/acs.biochem.3c00159" target="_blank">https://doi.org/10.1021/acs.biochem.3c00159</a></p>
       expansion of the genome engineering toolbox. <i>Biochemistry</i>, 62, 3465-3487. <a
+
<p>Kweon, J., Jang, A.-H., Kim, D.-e., Yang, J. W., Yoon, M., Rim Shin, H., Kim, J.-S., &amp; Kim, Y. (2017). Fusion
        href="https://doi.org/10.1021/acs.biochem.3c00159"
+
       guide RNAs for orthogonal gene manipulation with Cas9 and Cpf1. <i>Nature Communications</i>, 8. <a href="https://doi.org/10.1038/s41467-017-01650-w" target="_blank">https://doi.org/10.1038/s41467-017-01650-w</a>
        target="_blank">https://doi.org/10.1021/acs.biochem.3c00159</a></p>
+
</p>
    <p>Kweon, J., Jang, A.-H., Kim, D.-e., Yang, J. W., Yoon, M., Rim Shin, H., Kim, J.-S., &amp; Kim, Y. (2017). Fusion
+
<p>Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E., &amp; Church, G. M.
       guide RNAs for orthogonal gene manipulation with Cas9 and Cpf1. <i>Nature Communications</i>, 8. <a
+
       (2013). RNA-guided human genome engineering via Cas9. <i>Science</i>, 339, 823-826. <a href="https://doi.org/10.1126/science.1232033" target="_blank">https://doi.org/10.1126/science.1232033</a></p>
        href="https://doi.org/10.1038/s41467-017-01650-w" target="_blank">https://doi.org/10.1038/s41467-017-01650-w</a>
+
<p>Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I., Dohmae, N., Ishitani, R., Zhang, F., &amp;
    </p>
+
    <p>Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E., &amp; Church, G. M.
+
       (2013). RNA-guided human genome engineering via Cas9. <i>Science</i>, 339, 823-826. <a
+
        href="https://doi.org/10.1126/science.1232033" target="_blank">https://doi.org/10.1126/science.1232033</a></p>
+
    <p>Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I., Dohmae, N., Ishitani, R., Zhang, F., &amp;
+
 
       Nureki, O. (2014). Crystal structure of Cas9 in complex with guide RNA and target DNA. <i>Cell</i>, 156, 935-949.
 
       Nureki, O. (2014). Crystal structure of Cas9 in complex with guide RNA and target DNA. <i>Cell</i>, 156, 935-949.
       <a href="https://doi.org/10.1016/j.cell.2014.02.001"
+
       <a href="https://doi.org/10.1016/j.cell.2014.02.001" target="_blank">https://doi.org/10.1016/j.cell.2014.02.001</a></p>
        target="_blank">https://doi.org/10.1016/j.cell.2014.02.001</a></p>
+
<p>Pacesa, M., Pelea, O., &amp; Jinek, M. (2024). Past, present, and future of CRISPR genome editing technologies.
    <p>Pacesa, M., Pelea, O., &amp; Jinek, M. (2024). Past, present, and future of CRISPR genome editing technologies.
+
       <i>Cell</i>, 187, 1076-1100. <a href="https://doi.org/10.1016/j.cell.2024.01.042" target="_blank">https://doi.org/10.1016/j.cell.2024.01.042</a></p>
       <i>Cell</i>, 187, 1076-1100. <a href="https://doi.org/10.1016/j.cell.2024.01.042"
+
<p>Paul, B., &amp; Montoya, G. (2020). CRISPR-Cas12a: Functional overview and applications. <i>Biomedical
        target="_blank">https://doi.org/10.1016/j.cell.2024.01.042</a></p>
+
         Journal</i>, 43, 8-17. <a href="https://doi.org/10.1016/j.bj.2019.10.005" target="_blank">https://doi.org/10.1016/j.bj.2019.10.005</a></p>
    <p>Paul, B., &amp; Montoya, G. (2020). CRISPR-Cas12a: Functional overview and applications. <i>Biomedical
+
<p>Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C., &amp; Doudna, J. A. (2014). DNA interrogation by the
         Journal</i>, 43, 8-17. <a href="https://doi.org/10.1016/j.bj.2019.10.005"
+
       CRISPR RNA-guided endonuclease Cas9. <i>Nature</i>, 507, 62-67. <a href="https://doi.org/10.1038/nature13011" target="_blank">https://doi.org/10.1038/nature13011</a></p>
        target="_blank">https://doi.org/10.1016/j.bj.2019.10.005</a></p>
+
<p>Zetsche, B., Gootenberg, J. S., Abudayyeh, O. O., Slaymaker, I. M., Makarova, K. S., Essletzbichler, P., Volz, S.
    <p>Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C., &amp; Doudna, J. A. (2014). DNA interrogation by the
+
       CRISPR RNA-guided endonuclease Cas9. <i>Nature</i>, 507, 62-67. <a href="https://doi.org/10.1038/nature13011"
+
        target="_blank">https://doi.org/10.1038/nature13011</a></p>
+
    <p>Zetsche, B., Gootenberg, J. S., Abudayyeh, O. O., Slaymaker, I. M., Makarova, K. S., Essletzbichler, P., Volz, S.
+
 
       E., Joung, J., van der Oost, J., Regev, A., Koonin, E. V., &amp; Zhang, F. (2015). Cpf1 is a single RNA-guided
 
       E., Joung, J., van der Oost, J., Regev, A., Koonin, E. V., &amp; Zhang, F. (2015). Cpf1 is a single RNA-guided
       endonuclease of a class 2 CRISPR-Cas system. <i>Cell</i>, 163, 759-771. <a
+
       endonuclease of a class 2 CRISPR-Cas system. <i>Cell</i>, 163, 759-771. <a href="https://doi.org/10.1016/j.cell.2015.09.038" target="_blank">https://doi.org/10.1016/j.cell.2015.09.038</a>
        href="https://doi.org/10.1016/j.cell.2015.09.038" target="_blank">https://doi.org/10.1016/j.cell.2015.09.038</a>
+
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Revision as of 21:42, 1 October 2024

BBa_K5237000

fgRNA Entry Vector MbCas12a-SpCas9

This part integrates the crRNA of MbCas12a (BBa_K5237206) and the sgRNA of SpCas9 (BBa_K5237209) into a single fusion guide RNA (fgRNA). The fgRNA is functional, meaning that the MbCas12a (BBa_K5237001), SpCas9 (BBa_K5237002) and the fusion dCas (BBa_K5237003) can utilize the fgRNA to target two loci simultaneously. The fgRNA also works in combination with the catalyitcally inactive Cas versions. We successfully showed genome editing using active SpCas9 and Cas12a and induced proximity of two loci with the inactive dSpCas9 and dMbCas12a.
For our part collection, the PICasSO toolbox, this part has a crucial role in formation of our CRISPR/Cas staples.

 



The PICasSO Toolbox
Figure 1: How our part collection can be used to engineer new staples


Next to the well-studied linear DNA sequence, the 3D spatial organization of DNA plays a crucial role in gene regulation, cell fate, disease development and more. However, the tools to precisely manipulate this genomic architecture remain limited, rendering it challenging to explore the full potential of the 3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular toolbox based on various DNA-binding proteins to address this issue.

The PICasSO part collection offers a comprehensive, modular platform for precise manipulation and re-programming of DNA-DNA interactions using protein staples in living cells, enabling researchers to recreate natural 3D genomic interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation. Specifically, the fusion of two DNA binding proteins enables to artifically bring distant genomic loci into proximty. To unlock the system's full potential, we introduce versatile chimeric CRISPR/Cas complexes, connected either on the protein or the guide RNA level. These1 complexes are reffered to as protein- or Cas staples. Beyond its versatility, PICasSO includes robust assay systems to support the engineering, optimization, and testing of new staples, ensuring functionality in vitro and in vivo. We took special care to include parts crucial for testing every step of the cycle (design, build, test, learn) when engineering new parts.

At its heart, the PICasSO part collection consists of three categories.
(i) Our DNA-binding proteins include our finalized enhancer hijacking Cas staple as well as half staples that can be used by scientists to compose entirely new Cas staples in the future. We also include our Simple staples that serve as controls for successful stapling and can be further engineered to create alternative, simpler and more compact staples.
(ii) As functional elements, we list additional parts that enhance the functionality of our Cas and Basic staples. These consist of protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling in vivo. Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs with our interkingdom conjugation system.
(iii) As the final category of our collection, we provide parts that support the use of our custom readout systems. These include components of our established FRET-based proximity assay system, enabling users to confirm accurate stapling. Additionally, we offer a complementary, application-oriented testing system for functional readouts via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking in mammalian cells.

The following table gives a comprehensive overview of all parts in our PICasSO toolbox. The highlighted parts showed exceptional performance as described on our iGEM wiki and can serve as a reference. The other parts in the collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their own custom Cas staples, enabling further optimization and innovation.

Our part collection includes:

DNA-binding proteins: The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring easy assembly.
BBa_K5237000 fgRNA Entry vector MbCas12a-SpCas9 Entryvector for simple fgRNA cloning via SapI
BBa_K5237001 Staple subunit: dMbCas12a-Nucleoplasmin NLS Staple subunit that can be combined with sgRNA or fgRNA and dCas9 to form a functional staple
BBa_K5237002 Staple subunit: SV40 NLS-dSpCas9-SV40 NLS Staple subunit that can be combined witha sgRNA or fgRNA and dCas12avto form a functional staple
BBa_K5237003 Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands into close proximity
BBa_K5237004 Staple subunit: Oct1-DBD Staple subunit that can be combined to form a functional staple, for example with TetR.
Can also be combined with a fluorescent protein as part of the FRET proximity assay
BBa_K5237005 Staple subunit: TetR Staple subunit that can be combined to form a functional staple, for example with Oct1.
Can also be combined with a fluorescent protein as part of the FRET proximity assay
BBa_K5237006 Simple staple: TetR-Oct1 Functional staple that can be used to bring two DNA strands in close proximity
BBa_K5237007 Staple subunit: GCN4 Staple subunit that can be combined to form a functional staple, for example with rGCN4
BBa_K5237008 Staple subunit: rGCN4 Staple subunit that can be combined to form a functional staple, for example with rGCN4
BBa_K5237009 Mini staple: bGCN4 Assembled staple with minimal size that can be further engineered
Functional elements: Protease-cleavable peptide linkers and inteins are used to control and modify staples for further optimization for custom applications
BBa_K5237010 Cathepsin B-cleavable Linker: GFLG Cathepsin B-cleavable peptide linker that can be used to combine two staple subunits to make responsive staples
BBa_K5237011 Cathepsin B Expression Cassette Expression Cassette for the overexpression of cathepsin B
BBa_K5237012 Caged NpuN Intein A caged NpuN split intein fragment that undergoes protein trans-splicing after protease activation. Can be used to create functionalized staples units
BBa_K5237013 Caged NpuC Intein A caged NpuC split intein fragment that undergoes protein trans-splicing after protease activation. Can be used to create functionalized staples units
BBa_K5237014 fgRNA processing casette Processing casette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D genome reprograming
BBa_K5237015 Intimin anti-EGFR Nanobody Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large constructs
BBa_K4643003 incP origin of transfer Origin of transfer that can be cloned into the plasmid vector and used for conjugation as a means of delivery
Readout Systems: FRET and enhancer recruitment to measure proximity of stapled DNA in bacterial and mammalian living cells enabling swift testing and easy development for new systems
BBa_K5237016 FRET-Donor: mNeonGreen-Oct1 FRET Donor-Fluorpohore fused to Oct1-DBD that binds to the Oct1 binding cassette. Can be used to visualize DNA-DNA proximity
BBa_K5237017 FRET-Acceptor: TetR-mScarlet-I Acceptor part for the FRET assay binding the TetR binding cassette. Can be used to visualize DNA-DNA proximity
BBa_K5237018 Oct1 Binding Casette DNA sequence containing 12 Oct1 binding motifs, compatible with various assays such as the FRET proximity assay
BBa_K5237019 TetR Binding Cassette DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET proximity assay
BBa_K5237020 Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64 Readout system that responds to protease activity. It was used to test cathepsin B-cleavable linker
BBa_K5237021 NLS-Gal4-VP64 Trans-activating enhancer, that can be used to simulate enhancer hijacking
BBa_K5237022 mCherry Expression Cassette: UAS, minimal Promotor, mCherry Readout system for enhancer binding. It was used to test cathepsin B-cleavable linker
BBa_K5237023 Oct1 - 5x UAS binding casette Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay
BBa_K5237024 TRE-minimal promoter- firefly luciferase Contains Firefly luciferase controlled by a minimal promoter. It was used as a luminescence readout for simulated enhancer hijacking

1. Sequence overview

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 339
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 571
    Illegal SapI site found at 662
    Illegal SapI.rc site found at 280

2. Usage and Biology

2.1 Discovery and Mechanism of CRISPR/Cas9

Figure 2: The CRISPR/Cas system A and B, schematic structure of Cas9 and Cas12a with their sgRNA/crRNA, sitting on a DNA strand with the PAM. The spacer sequence forms base pairings with the dsDNA. In case of Cas9 the spacer is located at the 5' prime end, for Cas12a at the 3' end of the gRNA. The scaffold of the gRNA forms a specific secondary structure enabling it to be bound by the Cas protein. DNA cleavage sites are indicated by the scissors.

In 2012, Jinek et al. discovered the use of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system to induce double-strand breaks in DNA. Since then, the system has been well established as a tool for genome editing. The CRISPR/Cas system, which originates from the bacterial immune system, is constituted by a ribonucleoprotein complex. For class 1 CRISPR systems, the RNA is complexed by multiple Cas proteins, whereas class 2 systems consist of a singular protein and RNA. The class 2 type II system describes all ribonucleoprotein complexes with Cas9 (Pacesa et al., 2024). They include a CRISPR RNA (crRNA), which specifies the target with a 20 nucleotide (nt) spacer sequence, and a transactivating CRISPR RNA (tracrRNA), which induces the processing by the Cas protein (Jinek et al., 2012) (Fig. 2 A). Furthermore, a specific three nucleotide sequence (NGG) at the 3' end in the targeted DNA is needed for binding and cleavage. This is referred to as the protospacer adjacent motif (PAM) (Sternberg et al., 2014). The most commonly used Cas9 protein is SpCas9 or SpyCas9, which originates from Streptococcus pyogenes (Pacesa et al., 2024).

A significant enhancement of this system was the introduction of single guide RNAs (sgRNA[s]), which combine the functions of a tracrRNA and crRNA (Mali et al., 2013). Moreover, Cong (2013) established precise targeting of human endogenous loci by designing the 20 nt spacer sequence accordingly.

2.2 Differences between Cas9 and Cas12a

Over the following years, further CRISPR/Cas systems have been discovered, including the Cpf1 system, which has been classified as Cas12a since then (Zetsche et al., 2015). Cas12a forms a class 2 type V system with its RNA, that in comparison to the type II systems, only requires a crRNA for targeting and activation. Cas12a is capable of processing the precursor crRNA into crRNA independently, whereas Cas9 requires the RNase III enzyme and tracrRNA for this process (Paul and Montoya, 2020). This crRNA is often also referred to as a guide RNA (gRNA). However, the stem loop that is formed when binding the Cas protein is structurally distinct to the Cas9 gRNA and positioned on the 5' side of the crRNA (Fig. 2 B). Similarly, the PAM (TTTV) is also on the 5' side (Pacesa et al., 2024). Cas9 possesses RuvC and HNH domains that are catalytically active, each of which cleaves one of the DNA strands at the same site, resulting in the formation of blunt end cuts (Nishimasu et al., 2014). Cas12a possesses one RuvC-like domain that creates staggered cuts with overhangs that are about 5nt long (Paul and Montoya, 2020).

2.3 Dead Cas Proteins and their Application

Specific mutations of these domains result in catalytic inactivity and therefore allow for the creation of nickases that only cut one of the DNA strands, or completely inactive Cas proteins (Koonin et al., 2023) (Kleinstiver et al., 2019). These are referred to as dead Cas proteins or dCas9 and dCas12a. These Cas proteins can be used to activate (CRISPRa) or inhibit (CRISPRi) the expression of genes by fusing them to effector domains and targeting the respective gene with the spacer sequence (Kampmann, 2017). A common approach for CRISPRa involves fusing Cas9 with the transcriptional activator VP64 (Kampmann, 2017).

3. Assembly and part evolution

Building on insights of our fusion Cas engineering cycle and findings from Kweon (2017), fgRNAs were designed by combining the sgRNA from SpCas9 with the crRNA from MbCas12a. Specifically the 3'-end of the MbCas12a gRNA was linked to the 5'-end of the SpCas9 gRNA. Via this approach, the two spacer sequences are fused directly, ensuring a minimal distance between the two DNA strands.This also facilitates efficient cloning of different spacer sequences, as both spacers can be exchangeed as one consecutive sequence. Linking the crRNA and sgRNA further enables multiplexing as Cas12a can inherently process gRNA repeats that are expressed from one single transcript enabling multiplexing. The entry vector includes a U6 promoter, the MbCas12a scaffold, a bacterial promoter driving ccdB expression, and the SpCas9 scaffold. Successful spacer integration leads to the removal of the ccdB gene, allowing bacterial growth to be used as an indicator for cloning success.
A conventional gRNA expression vector containing an MbCas12a crRNA scoffold under the control of an U6 promoter was selected as the basis for entry vector cloning. The vector and a ccdB-SpCas9 scaffold construct were PCR amplified and fitting overhangs for SapI were introduced (Fig. 3). Golden Gate assembly (GGA) with Esp3I was used to create the final plasmid. The transformation was carried out in the ccdB-resistant XL1 Blue E. Coli strain.

Figure 3: Construction process of fgRNAs using the entry vector. The ccdB gene excised using SapI in a Golden Gate assembly. By inserting oligonucleotides with the desired spacer sequences and matching overhangs, the complete fgRNA can be assembled into the entry vector. Due to the cytotoxic nature of ccdB, only cells with the oligonucleotides as inserts survive.

The first goal after assembly was to prove the editing activity of both proteins using fgRNA. The genes VEGFA and FANCF were selected as targets for Cas12a and Cas9, each target was tested with each Cas protein. Editing efficiency was analyzed with the T7 Endonuclease I (T7EI) assay. Controls included crRNAs and sgRNAs as positive controls, and non-targeting guides as negative controls. Desired spacer sequences were ordered as oligos, annealed, and cloned in via GGA utilizing SapI.

Figure 4: Applications of the Fusion Guide RNA Fusion Guide RNAs can be used for multiplex genome editing by guidingactive Cas12a and Cas9 to two distinct loci. Similarly, fgRNAs allow for CRISPRa, by guiding the Cas9-VP64 transcriptional activator towards a target locus.
Table 1: A list of all the different spacers we cloned and tested within the fgRNA
VEGFA ctaggaatattgaagggggc
FANCF ggcggggtccagttccggga
CCR5 tgacatcaattattatacat
TetO (BBa_K5237019) tctctatcactgatagggag
Oct1-B (BBa_K5237018) atgcaaatactgcactagtg

We constructed a second entry vector incorporating an AsCas12a scaffold (5' taatttctactcttgtagat 3') instead of MbCas12a. The sequence of the AsCas12a scaffold was the only modification in the composite part. This vector was tested on the loci VEGFA and FANCF to assess its functionality.

4. Results

4.1 Editing endogenous loci with fgRNAs

To prove that our fusion gRNAs still result in active ribonucleoproteins, a series of different fgRNAs were created, each carrying spacers specific to the VEGFA and FANCF genes.HEK293-T cells were transfected with the Cas protein and gRNA constructs. The editing rate was tested 72h after transfection via a T7 endonuclease I assay.
AsCas12a and SpCas9 were used. The AsCas12a spacer targets VEGFA, while the SpCas9 spacer targets FANCF. The samples included standard single gRNAs with the corresponding Cas protein, the fgRNA with only one of the two Cas proteins and the fgRNA with both Cas proteins simultaneously (Fig. 5). The sgRNAs allowed for the highest editing rates for both genes (45% for VEGFA and 15% for FANCF), while the editing rates for FANCF were consistently lower in all experiments. Importantly, targeting FANCF with fgRNAs resulted in noticeable editing of about 10%, with just the SpCas9 and both Cas proteins in the sample. For VEGFA, the AsCas12a only sample resulted in approximately 20% editing rate in combination with the fgRNA, while adding both Cas proteins led to approximately 40%. These initial results confirmed our engineering approach proving efficient genome editing with fgRNAs.

Figure 5: fgRNAs Enable Efficient Editing of Endogenous Loci. The editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band))1/2. The schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts are included in each sample.

4.2 Efficient Fusion Guide RNA-Mediated Editing With Various Cas Orthologs

After showing efficient editing, the next step was to evaluate the capabilities of the fgRNAs, we tested them in combination with different Cas12a orthologs. After some initial testing, we decided on using MbCas12a together with SpCas9, because we found AsCas12a to be less active in a dual luciferase assay when co-transfected with SpCas9 compared to MbCas12a (Fig. 6). Between these two co-transfections the SpCas9 editing has not been significantly different.

Figure 6: Comparison of AsCas12a and MbCas12a with a dual luciferase assay. Firefly luminescence intensity measured 48 h after transfection. Normalized against renilla luminescence. On the x-axis the samples Cas9 + AsCas12a , Cas9 + MbCas12a, AsCas12a and MbCas12a are depicted. Data is depicted as the mean +/- SD (n=3). Statistical analysis was performed using 1way ANOVA with Tukey's multiple comparisons test. For better clarity, only significant differences within a group between the same Cas proteins are shown.*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Additionally, to test if the differences in editing rates from the preliminary assay resulted from the targeted loci or the different Cas orthologs, the spacers were tested in both arrangements. Once with Cas12a targeting FANCF and SpCas9 targeting VEGFA and once vice versa. To better assess the impact that the utilization of a fgRNA has on the editing rates, the sgRNAs were tested separately and in one sample.
Having the sgRNA with single Cas proteins in the same sample resulted in no clear difference in the editing rates (Fig. 7). The fusion of the gRNAs resulted in a lower editing rate overall. While the editing for VEGFA stayed at about 20% in all cases, the editing for FANCF dropped significantly. When targeting the same gene under the same conditions, the editing rates for MbCas12a were overall lower than the ones from SpCas9.

Figure 7: Fusion gRNA Editing Rates In Combination with MbCas12a. In A and B the editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band) 1/2). The schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts are included in each sample. A and B display both orientations of the two spacers for VEGFA and FANCF.

4.3 The Inclusion of a Linker Does Not Lower Editing Rates

To further assess the effect of the genomic locus on the editing rate, we included CCR5 as an additional gene target. For this assay, a fgRNA with a 20 nt long linker was included between the two spacers. The editing rate for VEGFA was again relatively consistent throughout the samples (Fig. 8). For CCR5, the editing rate with sgRNAs was approximately the same at about 30%. However, it dropped below 10% for the fgRNA. The addition of the 20 nt linker had no effect on the editing rates compared to no linker.

Figure 8: Fusion gRNA Editing Rates for Multiplexing CCR5 and VEGFA The editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band))1/2. The schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts are included in each sample. Cas12a targets VEGFA and Cas9 targets CCR5.

4.4 fgRNAs can be used for CRISPRa

To establish the foundation for their use as protein scaffolds, we identified the next step as demonstrating the use of fgRNAs for CRISPR activation. For this, we intend to recruit the transcriptional activator VP64 to a firefly luciferase gene to induce expression. The VP64 protein is attached to the catalytically inactive Cas9 protein, which is then guided by gRNAs to the luciferase gene. The gRNAs target a TetO sequence, which is positioned in front of the luciferase gene in multiple repeats. The firefly luciferase activity was then quantified as photon counts and normalized against Renilla luciferase, which is expressed on a separate plasmid under an ubiquitous promoter. In two biological replicates we saw similar Relative luciferase activity with fgRNA as a guide compared to a sgRNA (Fig. 9).

Figure 9: CRISPRa Induced Luciferase Expression for sgRNAs and fgRNAs. Firefly luciferase activity was measured 48h after transfection. Normalized against ubiquitously expressed Renilla luciferase. The tetO repeats were targeted by Cas9-VP64, once with a sgRNA and once with a fgRNA that had a non-targeting sequence for the Cas12a spacer. The schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts are included in each sample.

4.5 Stapling Two DNA Strands Together Using fgRNAs

After showing the general capability of the fgRNA to work for editing and for CRISPR activation, the next step was to use it to staple two DNA loci together, and thereby induce proximity between two separate functional elements. For this, an enhancer plasmid and a reporter plasmid was used. The reporter plasmid has firefly luciferase behind several repeats of a Cas9 targeted sequence. The enhancer plasmid has a Gal4 binding site behind several repeats of a Cas12a targeted sequence. By introducing a fgRNA staple and a Gal4-VP64, expression of the luciferase is induced (Fig. 10, Panel A). Different linker lengths were tested. Cells were again normalized against ubiquitous renilla expression.
Using no linker between the two spacers showed similar relative luciferase activity to the baseline control (Fig. 10, Panel B). An extension of the linker from 20 nt up to 40 nt resulted in an increasingly higher expression of the reporter gene. These results suggest an extension of the linker might lead to better transactivation when hijacking an enhancer/activator.

Figure 10: Applying Fusion Guide RNAs for Cas staples. A, schematic overview of the assay. An enhancer plasmid and a reporter plasmid are brought into proximity by a fgRNA Cas staple complex binding both plasmids. Target sequences were included in multiple repeats prior to the functional elements. Firefly luciferase serves as the reporter gene, the enhancer is constituted by multiple Gal4 repeats that are bound by a Gal4-VP64 fusion. B, results of using a fgRNA Cas staple for trans activation of firefly luciferase. Firefly luciferase activity was measured 48h after transfection. Normalized against ubiquitously expressed Renilla luciferase. Statistical significance was calculated with ordinary One-way ANOVA with Dunn's method for multiple comparisons (*p < 0.05; **p < 0.01; ***p < 0.001; mean +/- SD). The assay included sgRNAs and fgRNAs with linker lengths from 0 nt to 40 nt.

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