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| − | <!-- Part summary -->
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| − | <section id="1">
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| − | <h1>Staple subunit: dMbCas12a-Nucleoplasmin NLS</h1>
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| − | <p>dMbCas12a is one crucial part of our PICasSO tool box, being able to function as a Cas staple with the fgRNA
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| − | (BBa_K5237000) and the dSpCas9 (BBa_K5237002). Transactivation has been shown using this part proving the proper
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| − | formation of our Cas staple. Easy employment through swift reprogramming of the fgRNA.</p>
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| − | <p> </p>
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| − | </section>
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| − | <div id="toc" class="toc" style="width:30%;">
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| − | <div id="toctitle">
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| − | <h1>Contents</h1>
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| − | </div>
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| − | <ul>
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| − | <li class="toclevel-1 tocsection-1"><a href="#1"><span class="tocnumber">1</span> <span class="toctext">Sequence
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| − | overview</span></a>
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| − | </li>
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| − | <li class="toclevel-1 tocsection-2"><a href="#2"><span class="tocnumber">2</span> <span class="toctext">Usage and
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| − | Biology</span></a>
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| − | </li>
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| − | <li class="toclevel-1 tocsetction-3"><a href="#3"><span class="tocnumber">3</span> <span class="toctext">Assembly
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| − | and part evolution</span></a>
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| − | <ul>
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| − | <li class="toclevel-2 tocsection-3.1"><a href="#3.1"><span class="tocnumber">3.1</span> <span
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| − | class="toctext">Qualtitative assesment of Cas12a orthologs</span></a>
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| − | </li>
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| − | <li class="toclevel-2 tocsection-3.2"><a href="#3.2"><span class="tocnumber">3.2</span> <span
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| − | class="toctext">Quantitative comparison between AsCas12a and MbCas12a</span></a>
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| − | </li>
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| − | <li class="toclevel-2 tocsection-3.3"><a href="#3.3"><span class="tocnumber">3.3</span> <span
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| − | class="toctext">MbCas12a tolerates co-transfection and
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| − | simultaneous editing of different Cas proteins</span></a>
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| − | </li>
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| − | <li class="toclevel-2 tocsection-3.4"><a href="#3.4"><span class="tocnumber">3.4</span> <span
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| − | class="toctext"></span>MbCas12a shows editing with fgRNA</a>
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| − | </li>
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| − | <li class="toclevel-2 tocsection-3.5"><a href="#3.5"><span class="tocnumber">3.5</span> <span
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| − | class="toctext">MbCas12a withstanding fusion to SpCas9 while staying functional</span></a>
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| − | </li>
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| − | <li class="toclevel-2 tocsection-3.6"><a href="#3.6"><span class="tocnumber">3.6</span> <span
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| − | class="toctext">MbCas12a fused to SpCas9 editing utilizing a fgRNA</span></a>
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| − | </ul>
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| − | </li>
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| − | <li class="toclevel-1 tocsection-5"><a href="#4"><span class="tocnumber">4</span> <span
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| − | class="toctext">Results</span></a>
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| − | </li>
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| − | <li class="toclevel-1 tocsection-8"><a href="#5"><span class="tocnumber">5</span> <span
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| − | class="toctext">References</span></a>
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| − | </li>
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| − | </ul>
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| − | </div>
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| − | <section>
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| − | <font size="5"><b>The PICasSO Toolbox </b> </font>
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| − | <p><br></p>
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| − | <div class="thumb"></div>
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| − | <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">
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| − | <div class="thumbcaption">
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| − | <i><b>Figure 1: Example how the part collection can be used to engineer new staples</b></i>
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| − | </div>
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| − | </div>
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| − | </div>
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| − |
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| − |
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| − | <p>
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| − | <br>
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| − | The 3D organization of the genome plays a crucial role in regulating gene expression in eukaryotic cells,
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| − | impacting cellular behavior, evolution, and disease. Beyond the linear DNA sequence, the spatial arrangement of
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| − | chromatin, influenced by DNA-DNA interactions, shapes pathways of gene regulation. However, the tools to precisely
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| − | manipulate this genomic architecture remain limited, rendering it challenging to explore the full potential of the
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| − | 3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular
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| − | toolbox based on various DNA-binding proteins to address this issue.
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| − |
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| − | </p>
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| − | <p>
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| − | The <b>PICasSO</b> part collection offers a comprehensive, modular platform for precise manipulation and
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| − | re-programming
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| − | of DNA-DNA interactions using protein staples in living cells, enabling researchers to recreate natural 3D genomic
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| − | interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation.
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| − | Beyond its versatility, PICasSO includes robust assay systems to support the engineering, optimization, and
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| − | testing of new staples, ensuring functionality <i>in vitro</i> and <i>in vivo</i>. We took special care to include
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| − | parts crucial for testing every step of the cycle (design, build, test, learn) when engineering new parts
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| − | </p>
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| − |
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| − | <p>At its heart, the PICasSO part collection consists of three categories. <br><b>(i)</b> Our <b>DNA-binding proteins</b>
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| − | include our
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| − | finalized enhancer hijacking Cas staple as well as half staples that can be used by scientists to compose entirely
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| − | new Cas staples in the future. We also include our simple staples that serve as controls for successful stapling
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| − | and can be further engineered to create alternative, simpler and more compact staples. <br>
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| − | <b>(ii)</b> As <b>functional elements</b>, we list additional parts that enhance the functionality of our Cas and Basic staples. These
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| − | consist of
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| − | protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling <i>in vivo</i>.
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| − | Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs with our
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| − | interkingdom conjugation system. <br>
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| − | <b>(iii)</b> As the final component of our collection, we provide parts that support the use of our <b>custom readout
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| − | systems</b>. These include components of our established FRET-based proximity assay system, enabling users to
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| − | confirm
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| − | accurate stapling. Additionally, we offer a complementary, application-oriented testing system for functional
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| − | readout via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking.
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| − | </p>
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| − | <p>
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| − | The following table gives a complete overview of all parts in our PICasSO toolbox. The highlighted parts showed
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| − | exceptional performance as described on our iGEM wiki and can serve as a reference. The other parts in the
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| − | collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their
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| − | own custom Cas staples, enabling further optimization and innovation.<br>
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| − | </p>
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| − | <p>
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| − | <font size="4"><b>Our part collection includes:</b></font><br>
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| − | </p>
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| − |
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| − | <table style="width: 90%;">
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| − | <td colspan="3" align="left"><b>DNA-binding proteins: </b>
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| − | The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring
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| − | easy assembly.</td>
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| − | <tbody>
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| − | <tr bgcolor="#FFD700">
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a></td>
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| − | <td>fgRNA Entryvector MbCas12a-SpCas9</td>
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| − | <td>Entryvector for simple fgRNA cloning via SapI</td>
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| − | </tr>
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| − | <tr>
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237001" target="_blank">BBa_K5237001</a></td>
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| − | <td>Staple subunit: dMbCas12a-Nucleoplasmin NLS</td>
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| − | <td>Staple subunit that can be combined to form a functional staple, for example with fgRNA and dCas9 </td>
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| − | </tr>
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| − | <tr>
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237002" target="_blank">BBa_K5237002</a></td>
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| − | <td>Staple subunit: SV40 NLS-dSpCas9-SV40 NLS</td>
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| − | <td>Staple subunit that can be combined to form a functional staple, for example with our fgRNA or dCas12a
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| − | </td>
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| − | </tr>
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| − | <tr>
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a></td>
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| − | <td>Cas-Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS</td>
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| − | <td>Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands in close proximity
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| − | </td>
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| − | </tr>
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| − | <tr>
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237004" target="_blank">BBa_K5237004</a></td>
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| − | <td>Staple subunit: Oct1-DBD</td>
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| − | <td>Staple subunit that can be combined to form a functional staple, for example with TetR.<br>
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| − | Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
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| − | </tr>
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| − | <tr>
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237005" target="_blank">BBa_K5237005</a></td>
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| − | <td>Staple subunit: TetR</td>
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| − | <td>Staple subunit that can be combined to form a functional staple, for example with Oct1.<br>
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| − | Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
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| − | </tr>
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| − | <tr>
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237006" target="_blank">BBa_K5237006</a></td>
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| − | <td>Simple taple: TetR-Oct1</td>
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| − | <td>Functional staple that can be used to bring two DNA strands in close proximity</td>
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| − | </tr>
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| − | <tr>
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237007" target="_blank">BBa_K5237007</a></td>
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| − | <td>Staple subunit: GCN4</td>
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| − | <td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td>
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| − | </tr>
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| − | <tr>
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237008" target="_blank">BBa_K5237008</a></td>
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| − | <td>Staple subunit: rGCN4</td>
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| − | <td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td>
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| − | </tr>
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| − | <tr>
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237009" target="_blank">BBa_K5237009</a></td>
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| − | <td>Mini staple: bGCN4</td>
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| − | <td>
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| − | Assembled staple with minimal size that can be further engineered</td>
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| − | </tr>
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| − | </tbody>
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| − | <td colspan="3" align="left"><b>Functional elements: </b>
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| − | Protease cleavable peptide linkers and inteins are used to control and modify staples for further optimization
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| − | for custom applications.</td>
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| − | <tbody>
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| − | <tr bgcolor="#FFD700">
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a></td>
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| − | <td>Cathepsin B-Cleavable Linker (GFLG)</td>
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| − | <td>Cathepsin B cleavable peptide linker, that can be used to combine two staple subunits ,to make responsive
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| − | staples</td>
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| − | </tr>
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| − | <tr>
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237011" target="_blank">BBa_K5237011</a></td>
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| − | <td>Cathepsin B Expression Cassette</td>
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| − | <td>Cathepsin B which can be selectively express to cut the cleavable linker</td>
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| − | </tr>
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| − | <tr>
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237012" target="_blank">BBa_K5237012</a></td>
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| − | <td>Caged NpuN Intein</td>
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| − | <td>Undergoes protein transsplicing after protease activation, can be used to create functionalized staple
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| − | units</td>
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| − | </tr>
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| − | <tr>
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237013" target="_blank">BBa_K5237013</a></td>
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| − | <td>Caged NpuC Intein</td>
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| − | <td>Undergoes protein transsplicing after protease activation, can be used to create functionalized staple
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| − | units</td>
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| − | </tr>
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| − | <tr>
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237014" target="_blank">BBa_K5237014</a></td>
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| − | <td>fgRNA processing casette</td>
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| − | <td>Processing casette to produce multiple fgRNAs from one transcript, can be used for multiplexing</td>
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| − | </tr>
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| − | <tr>
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237015" target="_blank">BBa_K5237015</a></td>
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| − | <td>Intimin anti-EGFR Nanobody</td>
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| − | <td>Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large
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| − | constructs</td>
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| − | </tr>
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| − | </tbody>
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| − | <td colspan="3" align="left"><b>Readout Systems: </b>
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| − | FRET and enhancer recruitment to measure proximity of stapled DNA in bacterial and mammalian living cells
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| − | enabling swift testing and easy development for new systems.</td>
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| − | <tbody>
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| − | <tr bgcolor="#FFD700">
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237016" target="_blank">BBa_K5237016</a></td>
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| − | <td>FRET-Donor: mNeonGreen-Oct1</td>
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| − | <td>Donor part for the FRET assay binding the Oct1 binding cassette. Can be used to visualize DNA-DNA
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| − | proximity</td>
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| − | </tr>
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| − | <tr bgcolor="#FFD700">
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237017" target="_blank">BBa_K5237017</a></td>
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| − | <td>FRET-Acceptor: TetR-mScarlet-I</td>
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| − | <td>Acceptor part for the FRET assay binding the TetR binding cassette. Can be used to visualize DNA-DNA
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| − | proximity</td>
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| − | </tr>
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| − | <tr>
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237018" target="_blank">BBa_K5237018</a></td>
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| − | <td>Oct1 Binding Casette</td>
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| − | <td>DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET
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| − | proximity assay</td>
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| − | </tr>
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| − | <tr>
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a></td>
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| − | <td>TetR Binding Cassette</td>
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| − | <td>DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET
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| − | proximity assay</td>
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| − | </tr>
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237020" target="_blank">BBa_K5237020</a></td>
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| − | <td>Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64</td>
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| − | <td>Readout system that responds to protease activity. It was used to test Cathepsin-B cleavable linker.</td>
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| − | </tr>
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| − | <tr>
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237021" target="_blank">BBa_K5237021</a></td>
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| − | <td>NLS-Gal4-VP64</td>
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| − | <td>Trans-activating enhancer, that can be used to simulate enhancer hijacking. </td>
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| − | </tr>
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237022" target="_blank">BBa_K5237022</a></td>
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| − | <td>mCherry Expression Cassette: UAS, minimal Promotor, mCherry</td>
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| − | <td>Readout system for enhancer binding. It was used to test Cathepsin-B cleavable linker.</td>
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| − | </tr>
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| − | <tr>
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237023" target="_blank">BBa_K5237023</a></td>
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| − | <td>Oct1 - 5x UAS binding casette</td>
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| − | <td>Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay.</td>
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| − | </tr>
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| − | <tr>
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| − | <td><a href="https://parts.igem.org/Part:BBa_K5237024" target="_blank">BBa_K5237024</a></td>
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| − | <td>TRE-minimal promoter- firefly luciferase</td>
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| − | <td>Contains Firefly luciferase controlled by a minimal promoter. It was used as a luminescence readout for
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| − | simulated enhancer hijacking.</td>
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| − | </tr>
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| − | </tbody>
| |
| − | </table>
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| − | </p>
| |
| − | </section>
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| − | <section id="1">
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| − | <h1>1. Sequence overview</h1>
| |
| − | </section>
| |
| − | </body>
| |
| − |
| |
| − | </html>
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| − |
| |
| − | <!--################################-->
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| − | <span class='h3bb'>Sequence and Features</span>
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| − | <partinfo>BBa_K5237001 SequenceAndFeatures</partinfo>
| |
| − | <!--################################-->
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| − |
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| − | <html>
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| − |
| |
| − |
| |
| − | <section id="2">
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| − | <h1>2. Usage and Biology</h1>
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| − | <p>
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| − | In 2012, Jinek <i>et al.</i> discovered the use of the Clustered Regularly Interspaced Short Palindromic Repeats
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| − | (CRISPR)/Cas system to induce double-strand breaks in DNA. Since then, the system has been well established as a
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| − | tool for genome editing. The CRISPR/Cas system, which originates from the bacterial immune system, is constituted
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| − | by a ribonucleoprotein complex. For class 1 CRISPR systems, the RNA is complexed by multiple Cas proteins, whereas
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| − | class 2 systems consist of a singular protein and RNA. The class 2 type II system describes all ribonucleoprotein
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| − | complexes with Cas9 (Pacesa <i>et al.</i>, 2024). They include a CRISPR RNA (crRNA), which specifies the target with a 20
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| − | nucleotide (nt) spacer sequence, and a transactivating CRISPR RNA (tracrRNA), which induces the processing by the
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| − | Cas protein (Jinek <i>et al.</i>, 2012) (Figure 2 A). Furthermore, a specific three
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| − | nucleotide sequence (NGG) on the 3' end in the targeted DNA is needed for binding and cleavage. This is referred
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| − | to as the protospacer adjacent motif (PAM) (Sternberg <i>et al.</i>, 2014). The most commonly used Cas9 protein is SpCas9
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| − | or SpyCas9, which originates from Streptococcus pyogenes (Pacesa <i>et al.</i>, 2024).
| |
| − | </p>
| |
| − | <div class="thumb">
| |
| − | <div class="thumbinner" style="width:60%;">
| |
| − | <img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/cas-staple-svg/background-cas9-cas12a-principle.svg"
| |
| − | style="width:99%;" class="thumbimage">
| |
| − | <div class="thumbcaption">
| |
| − | <i>
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| − | <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 PAM.
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| − | 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 bind to the Cas protein. The cut sites by the cleaving domains, RuvC and HNH, are
| |
| − | symbolized by the scissors
| |
| − | </i>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | <p>
| |
| − | A significant enhancement of this system was the introduction of single guide RNA (sgRNA)s, which combine the
| |
| − | 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 sequence accordingly.
| |
| − | </p>
| |
| − | </section>
| |
| − |
| |
| − | <section id="3">
| |
| − | <h1>3. Assembly and part evolution</h1>
| |
| − | <section id="3.1">
| |
| − | <h2>3.1 Qualtitative assesment of Cas12a orthologs</h2>
| |
| − | <p>
| |
| − | To select a suitable Cas12a ortholog for cronstructing the Cas sstaple, three different orhtologs were ordered from Addgene:
| |
| − | AsCas12a (<a href="https://www.addgene.org/69982/" target="_blank">#69982</a>), LbCas12a (<a href="https://www.addgene.org/69988/" target="_blank">#69988</a>),
| |
| − | and MbCas12a (<a href="https://www.addgene.org/115142/" target="_blank">#115142</a>).
| |
| − | <br><br>
| |
| − | We assessed their performance using a T7 Endonuclease I (T7EI) assay on three genomic loci: RUNX1, DNMT1, and FANCF. For
| |
| − | comparison, we also included SpCas9 editing the CCR5 locus (Fig. 3). Editing was only observed at the
| |
| − | RUNX1 locus, where we used the qualitative assay to compare the editing efficiencies of the Cas12a orthologs.
| |
| − | </p>
| |
| − | <div class="thumb">
| |
| − | <div class="thumbinner" style="width:60%;">
| |
| − | <img alt="" src="https://static.igem.wiki/teams/5237/engineering/fuscas-engi-fig1-1.svg"
| |
| − | style="width:99%; border:none;" class="thumbimage">
| |
| − | <img alt="" src="https://static.igem.wiki/teams/5237/engineering/fuscas-engi-fig1-2.svg"
| |
| − | style="width:99%; border:none;" class="thumbimage">
| |
| − | <div class="thumbcaption">
| |
| − | <i>
| |
| − | <b>Figure 3: Preliminary T7 Endonuclease I testing of Cas12a orthologs.</b>
| |
| − | T7EI samples were run on a 2% agarose gel in 1x TBE buffer dyed with gel red. SpCas9 targeting CCR5 functions as a
| |
| − | benchmark for proper editing. For the Cas12a orthologs, LbCas12a, AsCas12a and MbCas12a, the three loci RUNX1, DNMT1 and
| |
| − | FANCF were targeted. Editing is indicated by an extra band compared to the negative control.
| |
| − | </i>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </section>
| |
| − | <section id="3.2"><h2>3.2 Quantitative comparison between AsCas12a and MbCas12a</h2>
| |
| − | <p>
| |
| − | Since the T7EI assay can only be used to qualitatively compare editing efficiencies, it was hard to distinguish
| |
| − | between the better editing ortholog.<br>
| |
| − | To accurately quantify the editing efficiency, we concted a dual luciferase assay. This assay measures the luminescence
| |
| − | of Firefly luciferase, which decreases proportionally to the editing efficiency at the target site.
| |
| − | To account for variations in cell count and transfection efficiency, the luminescence is normalized
| |
| − | to Renilla luciferase, which acts as an internal control (Fig. 4).
| |
| − | The results revealed a clear difference, with MbCas12a demonstrating significantly higher editing efficiency compared to
| |
| − | AsCas12a (p=0.005). Based on these findings, we decided to proceed with MbCas12a.
| |
| − | </p>
| |
| − | <div class="thumb">
| |
| − | <div class="thumbinner" style="width:60%;">
| |
| − | <img alt="" src="https://static.igem.wiki/teams/5237/engineering/cas12-decision.svg"
| |
| − | style="width:99%;" class="thumbimage">
| |
| − | <div class="thumbcaption">
| |
| − | <i>
| |
| − | <b>Figure 4: Comparison of AsCas12a and MbCas12a with a dual luciferae assay.</b>
| |
| − | 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
| |
| − | </i>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </section>
| |
| − |
| |
| − | <section id="3.3"><h2>3.3 MbCas12a tolerates co-transfection and simultaneous editing of different Cas proteins</h2>
| |
| − | <p>
| |
| − | Wanting to employ the MbCas12a as part of a Cas staple, our goal was to find out how well MbCas12a can stay active while
| |
| − | being transfected together with SpCas9. Therefore we engineered the dual luciferase assay to allow us for testing two
| |
| − | catalytically active Cas proteins at once. Including a well established protospacer, CCR5, into the renilla luciferase
| |
| − | gene enables us to test the editing rates of two Cas proteins simultaneously (Fig.5 ).
| |
| − | </p>
| |
| − | <div class="thumb">
| |
| − | <div class="thumbinner" style="width:60%">
| |
| − | <img alt="" src="https://static.igem.wiki/teams/5237/engineering/co-transf-registry.svg"
| |
| − | style="width:99%;" class="thumbimage">
| |
| − | <div class="thumbcaption">
| |
| − | <i>
| |
| − | <b>Figure 5: Testing for Simultaneous Editing with Double Cut Luciferae Assay</b>
| |
| − | Firefly and renilla luminescence intensity is plotted, both measured 48 h after transfection. Co-transformed contains
| |
| − | MbCas12a and SpCas9. Cas12a are the Firefly relative luminescence units (RLUs), while Cas9 are the Renilla RLUs.
| |
| − | Data is depicted as the mean +/- SD (n=3).
| |
| − | Statistical analysis was performed using 2way ANOVA with Tukey's multiple comparisons test. For better clarity, only
| |
| − | significant differences within a group are shown.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
| |
| − | </i>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | <p>
| |
| − | For the MbCas12a we have a highly significant editing rate in the single cut, meaning both Cas proteins are transfected,
| |
| − | but only MbCas12a has a targeting gRNA. When introducing a targeting gRNA for SpCas9 we see no reduction in the highly
| |
| − | significant editing of MbCas12a, strongly suggesting both Cas proteins to be able to edit simultaneously.
| |
| − | </p>
| |
| − | </section>
| |
| − | <section id="3.4"><h2>3.4 MbCas12a shows editing with fgRNA</h2>
| |
| − | <p>
| |
| − | To further confirm if MbCas12a is compatible with our Cas staples, editing rates were tested using a fusion guide RNA
| |
| − | (fgRNA, <a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a>)
| |
| − | targeting two different loci: <i>FANCF</i> and <i>VEGFA</i>. 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.<br>
| |
| − | Having the sgRNA with single Cas proteins in the same sample resulted in no clear
| |
| − | difference in the editing rates (Fig. 6). 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. Nonetheless we were able to show MbCas12a editing utilizing a fgRNA.
| |
| − | </p>
| |
| − | <div class="thumb">
| |
| − | <div class="thumbinner" style="width:60%;">
| |
| − | <img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/results-mbcas-2.svg"
| |
| − | style="width:99%;" class="thumbimage">
| |
| − | <div class="thumbcaption">
| |
| − | <i>
| |
| − | <b>Figure 6: 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 assay. Editing % was
| |
| − | determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band))<sup>1/2</sup>. 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. <i class="italic">A</i> and <i class="italic">B</i> display both
| |
| − | orientations of the two spacers for VEGFA and FANCF.
| |
| − | </i>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | <p>
| |
| − | 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. 7).
| |
| − | </p>
| |
| − | <div class="thumb">
| |
| − | <div class="thumbinner" style="width:60%;">
| |
| − | <img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/results-ccr5-2.svg"
| |
| − | style="width:99%;" class="thumbimage">
| |
| − | <div class="thumbcaption">
| |
| − | <i>
| |
| − | <b>Figure 7: 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 measuring band
| |
| − | intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band)) <sup>1/2</sup>. 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.
| |
| − | </i>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </section>
| |
| − | <section id="3.5"><h2>3.5 MbCas12a tolerates fusion to SpCas9</h2>
| |
| − | <p>
| |
| − | Testing showed simultaneous editing of MbCas12a and SpCas9. Therefore we wanted to test protein-protein fusion,
| |
| − | potentially giving us another way of Cas stapling. Our goal was to find out how well MbCas12a can stay active while
| |
| − | being fused to SpCas9. Therefore we employed the previously engineered dual luciferase assay to allow us for testing two
| |
| − | catalytically active Cas proteins at once, this time being fused to each other (Fig. 8).
| |
| − | </p>
| |
| − | <div class="thumb">
| |
| − | <div class="thumbinner" style="width:60%;">
| |
| − | <img alt="" src="https://static.igem.wiki/teams/5237/engineering/fusioncas-registry.svg"
| |
| − | style="width:99%;" class="thumbimage">
| |
| − | <div class="thumbcaption">
| |
| − | <i>
| |
| − | <b>Figure 8: Double cut dual luciferase assay testing Fusion Cas simultaneous editing.</b>
| |
| − | Firefly and renilla luminescence intensity is plotted, both measured 48 h after transfection. On the x-axis the negative
| |
| − | control, single edit and simultaneous edit, are grouped by the two Cas proteins in the fusion Cas. The fusion Cas
| |
| − | contains MbCas12a and SpCas9. MbCas12a are the Firefly relative luminescence units (RLUs), while Cas9 are the Renilla
| |
| − | RLUs. Data is depicted as the mean +/- SD (n=3). Statistical analysis was performed using 2way ANOVA with Tukey's multiple
| |
| − | comparisons test. For better clarity, only significant differences within a group are shown.*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001
| |
| − | </i>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | <p>
| |
| − | For the MbCas12a we have a highly significant editing rate in the single cut, meaning only MbCas12a has a targeting
| |
| − | gRNA. When introducing a targeting gRNA for SpCas9 we see no reduction in the highly significant editing of MbCas12a,
| |
| − | strongly suggesting both Cas proteins in the fusion Cas to be able to edit simultaneously.
| |
| − | </p>
| |
| − | </section>
| |
| − | <section id="3.6"><h2>3.6 MbCas12a fused to SpCas9 editing utilizing a fgRNA</h2>
| |
| − | <p>
| |
| − | The capability of MbCas12 in the fusion Cas was tested by assessing the editing rates via a T7EI assay. For this, the
| |
| − | same target sequences as before were used, namely FANCF and VEGFA in both configurations. We included biological
| |
| − | duplicates in this assay.<br>
| |
| − | MbCas12a editing rates were significantly lower, dropping to about 1%. At the same time, when targeting VEGFA they
| |
| − | resulted in a higher editing efficiency than FANCF.
| |
| − | </p>
| |
| − | <div class="thumb">
| |
| − | <div class="thumbinner" style="width:60%;">
| |
| − | <img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/results-fcas-2.svg"
| |
| − | style="width:99%;" class="thumbimage">
| |
| − | <div>
| |
| − | <i>
| |
| − | <b>Figure 9: Editing rates for fusion guide RNAs with fusion Cas proteins.</b>
| |
| − | 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 band/uncleaved band))<sup>1/2</sup>. 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. Cas proteins linked by a dash ("-") were fused to each other.
| |
| − | Biological replicates are marked as individual dots.
| |
| − | </i>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </section>
| |
| − | </section>
| |
| − | <section id="4">
| |
| − | <h1>4. Results</h1>
| |
| − | <p>
| |
| − | We extensively characterized the catalytically active MbCas12a in several assays determining the best ortholog out of
| |
| − | three for our Cas staples, assert functional editing with a fusion guide RNA (BBa_K5237000), showing high activity
| |
| − | while being co-transfected with SpCas9, our second staple protein's active version, and lastly a functioning fusion to
| |
| − | SpCas9.<br>
| |
| − | After all these successful test we were confident to test the Cas staples in action.
| |
| − | </p>
| |
| − | <section><h2 id="4.1">4.1 dMbCas12a Transactivation as Part of Cas Staple</h2>
| |
| − | <p>
| |
| − | The next step was to use the MbCas12a as part of a Cas staple 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 A).
| |
| − | Different linker lengths were tested. Cells were again normalized against ubiquitous renilla expression. Further
| |
| − | information on our learnings from this assay can be found in the Cas staple engineering cycle iteration 2.
| |
| − | Using no linker between the two spacers showed similar relative luciferase activity to the baseline control (Fig. 10 B).
| |
| − | An extension of the linker from 20 nt up to 40 nt resulted in an increasingly higher expression of the reporter gene.
| |
| − | </p>
| |
| − | <div class="thumb">
| |
| − | <div class="thumbinner" style="width:60%;">
| |
| − | <img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/results-eh-2.svg"
| |
| − | style="width:99%;" class="thumbimage">
| |
| − | <div class="thumbcaption">
| |
| − | <i>
| |
| − | <b>Figure 10: 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 <
| |
| − | 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.
| |
| − | </i>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </section>
| |
| − | </section>
| |
| − | <section id="5">
| |
| − | <h1>5. References</h1>
| |
| − | <p>Kampmann, M. (2017). CRISPRi and CRISPRa Screens in Mammalian Cells for Precision Biology and Medicine. <i>ACS Chemical Biology, 13</i>, 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., and Joung, J. K. (2019). Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. <i>Nature Biotechnology, 37</i>, 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>
| |
| − |
| |
| − | <p>Koonin, E. V., Gootenberg, J. S., and Abudayyeh, O. O. (2023). Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox. <i>Biochemistry, 62</i>, 3465–3487. <a href="https://doi.org/10.1021/acs.biochem.3c00159" target="_blank">https://doi.org/10.1021/acs.biochem.3c00159</a>.</p>
| |
| − |
| |
| − | <p>Pacesa, M., Pelea, O., and Jinek, M. (2024). Past, present, and future of CRISPR genome editing technologies. <i>Cell, 187</i>, 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>
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| − |
| |
| − | <p>Paul, B., and Montoya, G. (2020). CRISPR-Cas12a: Functional overview and applications. <i>Biomedical Journal, 43</i>, 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>
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| − |
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| − | <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., and Zhang, F. (2015). Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. <i>Cell, 163</i>, 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>.</p>
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| − |
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| − | </section>
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| − | </body>
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| − |
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| − | </html>
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