Difference between revisions of "Part:BBa K5237021"

 
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===Usage and Biology===
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  <!-- Part summary -->
 +
  <section id="1">
 +
    <h1>NLS-Gal4-VP64</h1>
 +
    <p>
 +
      This part of our simulated enhancer hijacking assay system binds to the upstream activation sites (UAS) next to
 +
      the
 +
      Oct1 sites (<a href="https://parts.igem.org/Part:BBa_K5237023">BBa_K5237023</a>), resulting in transactivation of
 +
      a gene on another plasmid, e.g. a firefly luciferase gene
 +
      (<a href="https://parts.igem.org/Part:BBa_K5237024">BBa_K5237024</a>). Furthermore allowing for swift testing of DNA
 +
      brought into proximity, which can be adapted by
 +
      other iGEMers for other assay systems.
 +
    </p>
 +
    <p>&nbsp;</p>
 +
  </section>
 +
  <div id="toc" class="toc">
 +
    <div id="toctitle">
 +
      <h1>Contents</h1>
 +
    </div>
 +
    <ul>
 +
      <li class="toclevel-1 tocsection-1"><a href="#1"><span class="tocnumber">1</span> <span class="toctext">Sequence
 +
            overview</span></a>
 +
      </li>
 +
      <li class="toclevel-1 tocsection-2"><a href="#2"><span class="tocnumber">2</span> <span class="toctext">Usage and
 +
            Biology</span></a>
 +
      </li>
 +
      <li class="toclevel-1 tocsetction-3"><a href="#3"><span class="tocnumber">3</span> <span class="toctext">Assembly
 +
            and part evolution</span></a>
 +
      </li>
 +
      <li class="toclevel-1 tocsection-5"><a href="#4"><span class="tocnumber">4</span> <span
 +
            class="toctext">Results</span></a>
 +
      </li>
 +
      <li class="toclevel-1 tocsection-8"><a href="#5"><span class="tocnumber">5</span> <span
 +
            class="toctext">References</span></a>
 +
      </li>
 +
    </ul>
 +
  </div>
 +
  <section>
 +
    <p><br><br></p>
 +
    <font size="5"><b>The PICasSO Toolbox </b> </font>
 +
 
 +
    <div class="thumb" style="margin-top:10px;"></div>
 +
    <div class="thumbinner" style="width:550px"><img alt=""
 +
        src="https://static.igem.wiki/teams/5237/wetlab-results/registry-part-collection-engineering-cycle-example-overview.svg"
 +
        style="width:99%;" class="thumbimage">
 +
      <div class="thumbcaption">
 +
        <i><b>Figure 1: How our part collection can be used to engineer new staples</b></i>
 +
      </div>
 +
    </div>
 +
    </div>
 +
 
 +
 
 +
    <p>
 +
      <br>
 +
      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.
 +
    </p>
 +
    <p>
 +
      The <b>PICasSO</b> 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.
 +
      Beyond its versatility, PICasSO includes robust assay 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
 +
      parts crucial for testing every step of the cycle (design, build, test, learn) when engineering new parts.
 +
    </p>
 +
 
 +
    <p>At its heart, the PICasSO part collection consists of three categories. <br><b>(i)</b> Our <b>DNA-binding
 +
        proteins</b>
 +
      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. <br>
 +
      <b>(ii)</b> As <b>functional elements</b>, 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 <i>in vivo</i>.
 +
      Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs
 +
      with our
 +
      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
 +
        readout
 +
        systems</b>. 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.
 +
    </p>
 +
    <p>
 +
      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
 +
        exceptional performance as described on our iGEM wiki and can serve as a reference.</mark> 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.<br>
 +
    </p>
 +
    <p>
 +
      <font size="4"><b>Our part collection includes:</b></font><br>
 +
    </p>
 +
 
 +
    <table style="width: 90%; padding-right:10px;">
 +
      <td colspan="3" align="left"><b>DNA-binding proteins: </b>
 +
        The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring
 +
        easy assembly.</td>
 +
      <tbody>
 +
        <tr bgcolor="#FFD700">
 +
          <td><a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a></td>
 +
          <td>fgRNA Entry vector MbCas12a-SpCas9</td>
 +
          <td>Entryvector for simple fgRNA cloning via SapI</td>
 +
        </tr>
 +
        <tr bgcolor="#FFD700">
 +
          <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 that can be combined with sgRNA or fgRNA and dCas9 to form a functional staple</td>
 +
        </tr>
 +
        <tr bgcolor="#FFD700">
 +
          <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 that can be combined witha sgRNA or fgRNA and dCas12avto form a functional staple
 +
          </td>
 +
        </tr>
 +
        <tr>
 +
          <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>Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands into close
 +
            proximity
 +
          </td>
 +
        </tr>
 +
        <tr>
 +
          <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 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>
 +
        </tr>
 +
        <tr>
 +
          <td><a href="https://parts.igem.org/Part:BBa_K5237005" target="_blank">BBa_K5237005</a></td>
 +
          <td>Staple subunit: TetR</td>
 +
          <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>
 +
        </tr>
 +
        <tr>
 +
          <td><a href="https://parts.igem.org/Part:BBa_K5237006" target="_blank">BBa_K5237006</a></td>
 +
          <td>Simple staple: TetR-Oct1</td>
 +
          <td>Functional staple that can be used to bring two DNA strands in close proximity</td>
 +
        </tr>
 +
        <tr>
 +
          <td><a href="https://parts.igem.org/Part:BBa_K5237007" target="_blank">BBa_K5237007</a></td>
 +
          <td>Staple subunit: GCN4</td>
 +
          <td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td>
 +
        </tr>
 +
        <tr>
 +
          <td><a href="https://parts.igem.org/Part:BBa_K5237008" target="_blank">BBa_K5237008</a></td>
 +
          <td>Staple subunit: rGCN4</td>
 +
          <td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td>
 +
        </tr>
 +
        <tr>
 +
          <td><a href="https://parts.igem.org/Part:BBa_K5237009" target="_blank">BBa_K5237009</a></td>
 +
          <td>Mini staple: bGCN4</td>
 +
          <td>
 +
            Assembled staple with minimal size that can be further engineered</td>
 +
        </tr>
 +
      </tbody>
 +
      <td colspan="3" align="left"><b>Functional elements: </b>
 +
        Protease-cleavable peptide linkers and inteins are used to control and modify staples for further optimization
 +
        for custom applications</td>
 +
      <tbody>
 +
        <tr bgcolor="#FFD700">
 +
          <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 peptide linker that can be used to combine two staple subunits to make responsive
 +
            staples</td>
 +
        </tr>
 +
        <tr>
 +
          <td><a href="https://parts.igem.org/Part:BBa_K5237011" target="_blank">BBa_K5237011</a></td>
 +
          <td>Cathepsin B Expression Cassette</td>
 +
          <td>Expression Cassette for the overexpression of cathepsin B</td>
 +
        </tr>
 +
        <tr>
 +
          <td><a href="https://parts.igem.org/Part:BBa_K5237012" target="_blank">BBa_K5237012</a></td>
 +
          <td>Caged NpuN Intein</td>
 +
          <td>A caged NpuN split intein fragment that undergoes protein <i>trans</i>-splicing after protease activation.
 +
            Can be used to create functionalized staples
 +
            units</td>
 +
        </tr>
 +
        <tr>
 +
          <td><a href="https://parts.igem.org/Part:BBa_K5237013" target="_blank">BBa_K5237013</a></td>
 +
          <td>Caged NpuC Intein</td>
 +
          <td>A caged NpuC split intein fragment that undergoes protein <i>trans</i>-splicing after protease activation.
 +
            Can be used to create functionalized staples
 +
            units</td>
 +
        </tr>
 +
        <tr>
 +
          <td><a href="https://parts.igem.org/Part:BBa_K5237014" target="_blank">BBa_K5237014</a></td>
 +
          <td>fgRNA processing casette</td>
 +
          <td>Processing casette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D
 +
            genome reprograming</td>
 +
        </tr>
 +
        <tr>
 +
          <td><a href="https://parts.igem.org/Part:BBa_K5237015" target="_blank">BBa_K5237015</a></td>
 +
          <td>Intimin anti-EGFR Nanobody</td>
 +
          <td>Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large
 +
            constructs</td>
 +
        </tr>
 +
        <tr>
 +
          <td><a href="https://parts.igem.org/Part:BBa_K4643003" target="_blank">BBa_K4643003</a></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
 +
            delivery</td>
 +
        </tr>
 +
      </tbody>
 +
      <td colspan="3" align="left"><b>Readout Systems: </b>
 +
        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>
 +
      <tbody>
 +
        <tr bgcolor="#FFD700">
 +
          <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-Fluorpohore fused to Oct1-DBD that binds to the Oct1 binding cassette. Can be used to visualize
 +
            DNA-DNA
 +
            proximity</td>
 +
        </tr>
 +
        <tr bgcolor="#FFD700">
 +
          <td><a href="https://parts.igem.org/Part:BBa_K5237017" target="_blank">BBa_K5237017</a></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
 +
            proximity</td>
 +
        </tr>
 +
        <tr>
 +
          <td><a href="https://parts.igem.org/Part:BBa_K5237018" target="_blank">BBa_K5237018</a></td>
 +
          <td>Oct1 Binding Casette</td>
 +
          <td>DNA sequence containing 12 Oct1 binding motifs, compatible with various assays such as the FRET
 +
            proximity assay</td>
 +
        </tr>
 +
        <tr>
 +
          <td><a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a></td>
 +
          <td>TetR Binding Cassette</td>
 +
          <td>DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET
 +
            proximity assay</td>
 +
        </tr>
 +
        <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>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>NLS-Gal4-VP64</td>
 +
          <td>Trans-activating enhancer, that can be used to simulate enhancer hijacking</td>
 +
        </tr>
 +
        <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>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>Oct1 - 5x UAS binding casette</td>
 +
          <td>Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay</td>
 +
        </tr>
 +
        <tr>
 +
          <td><a href="https://parts.igem.org/Part:BBa_K5237024" target="_blank">BBa_K5237024</a></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
 +
            simulated enhancer hijacking</td>
 +
        </tr>
 +
      </tbody>
 +
    </table>
 +
    </p>
 +
  </section>
 +
  <section id="1">
 +
    <h1>1. Sequence overview</h1>
 +
  </section>
 +
</body>
 +
 
 +
</html>
 +
 
 +
<!--################################-->
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
−
<partinfo>BBa_K5237021 SequenceAndFeatures</partinfo>
+
<partinfo>BBa_K5237023 SequenceAndFeatures</partinfo>
 +
<!--################################-->
 +
 
 +
<html>
 +
 
 +
 
 +
<section id="2">
 +
  <h1>2. Usage and Biology</h1>
 +
  <p>
 +
    Gal4 is a well-known transcription factor from Saccharomyces cerevisiae that binds specifically to UAS regions on DNA,
 +
    activating transcription of downstream genes. Its DNA-binding domain has been widely utilized in synthetic biology and
 +
    gene regulation studies due to its specificity and ability to recruit transcriptional machinery (Kakidani and Ptashne
 +
    (1988)).<br>
 +
    VP64 is a synthetic transcriptional activator composed of four tandem repeats of the Herpes Simplex Virus VP16
 +
    transcriptional activation domain. VP64 is commonly used in CRISPR-based gene activation strategies, where it recruits
 +
    transcriptional machinery to target genes, enhancing transcription (Wang <i> et al.<i> 2016).<br>
 +
      Fusions of Gal4 and VP64 create a potent transactivation system. When Gal4 is fused with VP64, the chimeric protein
 +
      retains Gal4's DNA-binding specificity and gains the strong transactivation capability of VP64, enabling robust gene
 +
      expression (Lowder <i> et al.<i> 2017).
 +
  </p>
 +
</section>
 +
<section id="3">
 +
  <h1>3. Assembly and part evolution</h1>
 +
  <p>
 +
    The construct was provided by our PI, used for the assembly of our Cathepsin B-Cleavable
 +
    trans-Activator (<a href="https://parts.igem.org/Part:BBa_K5237020">BBa_K5237020</a>)
 +
    and mainly used in the enhancer hijacking assay for the Cas staples (Fig. 2)
 +
  </p>
 +
</section>
 +
<section id="4">
 +
  <h1>4. Results</h1>
 +
  <p>
 +
    To show that the Cas staple can staple two DNA loci togther, and thereby induce proximity between two separate
 +
    functional elements, we employed the NLS-Gal4-VP64 fusion as the transactivator.<br>
 +
    For this, an enhancer plasmid (containing <a href="https://parts.igem.org/Part:BBa_K5237023">BBa_K5237023</a>) and a
 +
    reporter plasmid (containing <a href="https://parts.igem.org/Part:BBa_K5237024">BBa_K5237024</a>) 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 the NLS-Gal4-VP64 fusion,
 +
    expression of the luciferase is induced (Fig. 2 A).
 +
  </p>
 +
  <div class="thumb">
 +
    <div class="thumbinner" style="width:60%;">
 +
      <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/results-eh-2.svg"
 +
        style="width:99%;" />
 +
      <div class="thumbcaption">
 +
        <i>
 +
          <b>Figure 2: Applying Fusion Guide RNAs for Cas staples.</b> <b>A</b>, 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>B</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 &lt;
 +
          0.05; **p &lt; 0.01; ***p &lt; 0.001; mean +/- SD). The assay included sgRNAs and fgRNAs with linker
 +
          lengths from 0 nt
 +
          to 40 nt.
 +
        </i>
 +
      </div>
 +
    </div>
 +
  </div>
 +
  <p>
 +
    This transactivation has also been shown using our fusion dCas protein (<a href="https://parts.igem.org/BBa_K5237003">BBa_K5237003</a>
 +
    in a Cas staple with fgRNAs of different linker lengths (Fig. 3)
 +
  </p>
 +
  <div class="thumb">
 +
    <div class="thumbinner" style="width:60%;">
 +
      <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/results-fcas-eh-2.svg"
 +
        style="width:99%;" />
 +
      <div class="thumbcaption">
 +
        <i>
 +
          <b>Figure 3: : Results of Implementing Fusion Cas Proteins in Trans Activation</b> <b>A</b>, 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>B</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 &lt;
 +
          0.05; **p &lt; 0.01; ***p &lt; 0.001; mean +/- SD). The assay included sgRNAs and fgRNAs with linker
 +
          lengths from 0 nt
 +
          to 40 nt.
 +
        </i>
 +
      </div>
 +
    </div>
 +
  </div>
 +
  </section>
 +
<section id="5">
 +
  <h1>5. References</h1>
 +
  <p>Kakidani, H., & Ptashne, M. (1988). GAL4 activates gene expression in mammalian cells. <i>Cell</i>, <b>52</b>, 161-167. <a href="https://doi.org/10.1016/0092-8674(88)90504-1" target="_blank">https://doi.org/10.1016/0092-8674(88)90504-1</a>.</p>
  
 +
  <p>Lowder, L., Zhou, J., Zhang, Y., Malzahn, A., Zhong, Z., Hsieh, T., Voytas, D., Zhang, Y., & Qi, Y. (2017). Robust Transcriptional Activation in Plants Using Multiplexed CRISPR-Act2.0 and mTALE-Act Systems. <i>Molecular Plant</i>, <b>11</b>(2), 245-256. <a href="https://doi.org/10.1016/j.molp.2017.11.010" target="_blank">https://doi.org/10.1016/j.molp.2017.11.010</a>.</p>
 +
 
 +
  <p>Wang, J., Wu, F., Zhu, S., Xu, Y., Cheng, Z., Wang, J., Li, C., Sheng, P., Zhang, H., Cai, M., Guo, X., Zhang, X., Wang, C., & Wan, J. (2016). Overexpression of OsMYB1R1–VP64 fusion protein increases grain yield in rice by delaying flowering time. <i>FEBS Letters</i>, <b>590</b>. <a href="https://doi.org/10.1002/1873-3468.12374" target="_blank">https://doi.org/10.1002/1873-3468.12374</a>.</p>
 +
 
 +
</section>
 +
</body>
  
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===Functional Parameters===
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Revision as of 04:34, 1 October 2024

BBa_K5237021

NLS-Gal4-VP64

This part of our simulated enhancer hijacking assay system binds to the upstream activation sites (UAS) next to the Oct1 sites (BBa_K5237023), resulting in transactivation of a gene on another plasmid, e.g. a firefly luciferase gene (BBa_K5237024). Furthermore allowing for swift testing of DNA brought into proximity, which can be adapted by other iGEMers for other assay systems.

 



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. 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
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 103
    Illegal SpeI site found at 144
    Illegal SpeI site found at 175
    Illegal SpeI site found at 206
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 144
    Illegal SpeI site found at 175
    Illegal SpeI site found at 206
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 215
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 103
    Illegal SpeI site found at 144
    Illegal SpeI site found at 175
    Illegal SpeI site found at 206
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 103
    Illegal SpeI site found at 144
    Illegal SpeI site found at 175
    Illegal SpeI site found at 206
  • 1000
    COMPATIBLE WITH RFC[1000]

2. Usage and Biology

Gal4 is a well-known transcription factor from Saccharomyces cerevisiae that binds specifically to UAS regions on DNA, activating transcription of downstream genes. Its DNA-binding domain has been widely utilized in synthetic biology and gene regulation studies due to its specificity and ability to recruit transcriptional machinery (Kakidani and Ptashne (1988)).
VP64 is a synthetic transcriptional activator composed of four tandem repeats of the Herpes Simplex Virus VP16 transcriptional activation domain. VP64 is commonly used in CRISPR-based gene activation strategies, where it recruits transcriptional machinery to target genes, enhancing transcription (Wang et al. 2016).
Fusions of Gal4 and VP64 create a potent transactivation system. When Gal4 is fused with VP64, the chimeric protein retains Gal4's DNA-binding specificity and gains the strong transactivation capability of VP64, enabling robust gene expression (Lowder et al. 2017).

3. Assembly and part evolution

The construct was provided by our PI, used for the assembly of our Cathepsin B-Cleavable trans-Activator (BBa_K5237020) and mainly used in the enhancer hijacking assay for the Cas staples (Fig. 2)

4. Results

To show that the Cas staple can staple two DNA loci togther, and thereby induce proximity between two separate functional elements, we employed the NLS-Gal4-VP64 fusion as the transactivator.
For this, an enhancer plasmid (containing BBa_K5237023) and a reporter plasmid (containing BBa_K5237024) 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 the NLS-Gal4-VP64 fusion, expression of the luciferase is induced (Fig. 2 A).

Figure 2: 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.

This transactivation has also been shown using our fusion dCas protein (BBa_K5237003 in a Cas staple with fgRNAs of different linker lengths (Fig. 3)

Figure 3: : Results of Implementing Fusion Cas Proteins in Trans Activation 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.

5. References

Kakidani, H., & Ptashne, M. (1988). GAL4 activates gene expression in mammalian cells. Cell, 52, 161-167. https://doi.org/10.1016/0092-8674(88)90504-1.

Lowder, L., Zhou, J., Zhang, Y., Malzahn, A., Zhong, Z., Hsieh, T., Voytas, D., Zhang, Y., & Qi, Y. (2017). Robust Transcriptional Activation in Plants Using Multiplexed CRISPR-Act2.0 and mTALE-Act Systems. Molecular Plant, 11(2), 245-256. https://doi.org/10.1016/j.molp.2017.11.010.

Wang, J., Wu, F., Zhu, S., Xu, Y., Cheng, Z., Wang, J., Li, C., Sheng, P., Zhang, H., Cai, M., Guo, X., Zhang, X., Wang, C., & Wan, J. (2016). Overexpression of OsMYB1R1–VP64 fusion protein increases grain yield in rice by delaying flowering time. FEBS Letters, 590. https://doi.org/10.1002/1873-3468.12374.