Difference between revisions of "Part:BBa K5237001"

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<!-- Part summary -->
 
<!-- Part summary -->
 
<section id="1">
 
<section id="1">
<h1>Staple subunit: dMbCas12a-Nucleoplasmin NLS</h1>
+
<h1>Staple Subunit: dMbCas12a-Nucleoplasmin NLS</h1>
 
<p>dMbCas12a is one crucial part of our PICasSO tool box, being able to function as a Cas staple with the fgRNA
 
<p>dMbCas12a is one crucial part of our PICasSO tool box, being able to function as a Cas staple with the fgRNA
       (BBa_K5237000) and the dSpCas9 (BBa_K5237002). Transactivation has been shown using this part proving the proper
+
       (<a href="https://parts.igem.org/Part:BBa_K5237000">BBa_K5237000</a>) and the dSpCas9 (<a href="https://parts.igem.org/Part:BBa_K5237002">BBa_K5237002</a>). Transactivation has been shown using this part proving the proper
 
       formation of our Cas staple. Easy employment through swift reprogramming of the fgRNA.</p>
 
       formation of our Cas staple. Easy employment through swift reprogramming of the fgRNA.</p>
 
<p> </p>
 
<p> </p>
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</li>
 
</li>
 
<li class="toclevel-2 tocsection-3.6"><a href="#3.6"><span class="tocnumber">3.6</span> <span class="toctext">MbCas12a fused to SpCas9 editing utilizing a fgRNA</span></a>
 
<li class="toclevel-2 tocsection-3.6"><a href="#3.6"><span class="tocnumber">3.6</span> <span class="toctext">MbCas12a fused to SpCas9 editing utilizing a fgRNA</span></a>
</li></ul>
+
</li>
 +
</ul>
 
</li>
 
</li>
 
<li class="toclevel-1 tocsection-5"><a href="#4"><span class="tocnumber">4</span> <span class="toctext">Results</span></a>
 
<li class="toclevel-1 tocsection-5"><a href="#4"><span class="tocnumber">4</span> <span class="toctext">Results</span></a>
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</div>
 
</div>
 
</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 regulation,
+
       While synthetic biology has in the past focused on engineering the genomic sequence of organisms, the <b>3D
       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
+
        spatial organization</b> of DNA is well-known to be an important layer of information encoding in
       3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular
+
      particular in eukaryotes, playing a crucial role in
       toolbox based on various DNA-binding proteins to address this issue.
+
      gene regulation and hence
 +
       cell fate, disease development, evolution, and more. However, tools to precisely manipulate and control the
 +
      genomic spatial
 +
      architecture are limited, hampering the exploration of
 +
       3D genome engineering in synthetic biology. We - the iGEM Team Heidelberg 2024 - have developed PICasSO, a
 +
       <b>powerful
 +
        molecular toolbox for rationally engineering genome 3D architectures</b> in living cells, based on
 +
      various DNA-binding proteins.
 
     </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 engineered "protein staples" in living cells. This enables
       interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation.
+
      researchers to recreate naturally occurring alterations of 3D genomic
       Beyond its versatility, PICasSO includes robust assay systems to support the engineering, optimization, and
+
       interactions, such as enhancer hijacking in cancer, or to design entirely new spatial architectures for
       testing of new staples, ensuring functionality <i>in vitro</i> and <i>in vivo</i>. We took special care to include
+
      artificial gene regulation and cell function control.
       parts crucial for testing every step of the cycle (design, build, test, learn) when engineering new parts.
+
       Specifically, the fusion of two DNA binding proteins enables to artificially bring otherwise distant genomic
 +
      loci into
 +
      spatial proximity.
 +
      To unlock the system's full potential, we introduce versatile <b>chimeric CRISPR/Cas complexes</b>,
 +
      connected either at
 +
      the protein or - in the case of CRISPR/Cas-based DNA binding moieties - the guide RNA level. These complexes are
 +
      referred to as protein- or Cas staples, respectively. Beyond its
 +
      versatility with regard to the staple constructs themselves, PICasSO includes <b>robust assay</b> systems to
 +
      support the engineering, optimization, and
 +
       testing of new staples <i>in vitro</i> and <i>in vivo</i>. Notably, the PICasSO toolbox was developed in a
 +
       design-build-test-learn <b>engineering cycle closely intertwining wet lab experiments and computational
 +
        modeling</b> and iterated several times, yielding a collection of well-functioning and -characterized
 +
      parts.
 
     </p>
 
     </p>
<p>At its heart, the PICasSO part collection consists of three categories. <br/><b>(i)</b> Our <b>DNA-binding proteins</b>
+
<p>At its heart, the PICasSO part collection consists of three categories. <br/><b>(i)</b> Our <b>DNA-binding
 +
        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 Cas staple experimentally validated using an artificial "enhancer hijacking" system as well as
       new Cas staples in the future. We also include our Simple staples that serve as controls for successful stapling
+
      "half staples" that can be combined by scientists to compose entirely
       and can be further engineered to create alternative, simpler and more compact staples. <br/>
+
       new Cas staples in the future. We also include our Simple staples comprised of particularly small, simple
<b>(ii)</b> As <b>functional elements</b>, we list additional parts that enhance the functionality of our Cas and Basic staples. These
+
      and robust DNA binding domains well-known to the synthetic biology community, which serve as controls for
       consist of
+
      successful stapling
       protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling <i>in vivo</i>.
+
       and can be further engineered to create alternative, simpler, and more compact staples. <br/>
       Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs with our
+
<b>(ii)</b> As <b>functional elements</b>, we list additional parts that enhance and expand the
 +
      functionality of our Cas and
 +
      Basic staples. These
 +
       consist of staples dependent on
 +
       cleavable peptide linkers targeted by cancer-specific proteases or inteins that allow condition-specific,
 +
      dynamic stapling <i>in vivo</i>.
 +
       We also include several engineered parts that enable the efficient delivery of PICasSO's constructs into
 +
      target cells, including mammalian cells,
 +
      with our new
 
       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 readout
+
<b>(iii)</b> As the final category of our collection, we provide parts that underlie our <b>custom
         systems</b>. These include components of our established FRET-based proximity assay system, enabling users to
+
        readout
 +
         systems</b>. These include components of our established FRET-based proximity assay system, enabling
 +
      users to
 
       confirm
 
       confirm
       accurate stapling. Additionally, we offer a complementary, application-oriented testing system for functional
+
       accurate stapling. Additionally, we offer a complementary, application-oriented testing system based on a
       readouts via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking in mammalian cells.
+
       luciferase reporter, which allows for straightforward experimental assessment of functional enhancer
 +
      hijacking events
 +
      in mammalian cells.
 
     </p>
 
     </p>
 
<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
 
       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
+
        exceptional performance as described on our iGEM wiki and can serve as a reference.</mark> The other
       collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their
+
      parts in
       own custom Cas staples, enabling further optimization and innovation.<br/>
+
      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 in the new field of 3D genome
 +
      engineering.<br/>
 
</p>
 
</p>
 
<p>
 
<p>
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</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
+
         Modular building blocks for engineering of custom staples to mediate defined DNA-DNA interactions <i>in vivo</i></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>Fusion Guide RNA Entry Vector MbCas12a-SpCas9</td>
<td>Entryvector for simple fgRNA cloning via SapI</td>
+
<td>Entry vector 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 crRNA or fgRNA and dSpCas9 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 with a sgRNA or fgRNA and dMbCas12a to form a functional staple
 
           </td>
 
           </td>
 
</tr>
 
</tr>
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<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 proximity
+
<td>Functional Cas staple that can be combined with sgRNA and crRNA or fgRNA to bring two DNA strands into
 +
            close
 +
            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>
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<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>
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<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>
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<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>
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<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. Can be used to create functionalized staples
+
<td>A caged NpuN split intein fragment that undergoes protein <i>trans</i>-splicing after protease
             units</td>
+
            activation, which can be used to create functionalized staple
 +
             subunits</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. Can be used to create functionalized staples
+
<td>A caged NpuC split intein fragment that undergoes protein <i>trans</i>-splicing after protease
             units</td>
+
            activation, which can be used to create functionalized staple
 +
             subunits</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>Fusion Guide RNA Processing Casette</td>
<td>Processing casette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D genome reprograming</td>
+
<td>Processing cassette to produce multiple fgRNAs from one transcript, that can be used for
 +
            multiplexed 3D
 +
            genome reprogramming</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>Interkingdom conjugation between bacteria and mammalian cells, as an 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 delivery</td>
+
<td>Origin of transfer that can be cloned into the plasmid vector and used for conjugation as a
 +
            means of
 +
            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 readout systems to rapidly assess successful DNA stapling in bacterial and
        enabling swift testing and easy development for new systems</td>
+
        mammalian cells
 +
      </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 DNA-DNA
+
<td>FRET donor-fluorophore fused to Oct1-DBD that binds to the Oct1 binding cassette, which can be used to
 +
            visualize
 +
            DNA-DNA
 
             proximity</td>
 
             proximity</td>
 
</tr>
 
</tr>
Line 249: Line 300:
 
<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, which can be used to visualize
 +
            DNA-DNA
 
             proximity</td>
 
             proximity</td>
 
</tr>
 
</tr>
Line 261: Line 313:
 
<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, which 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>
Line 274: Line 327:
 
</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 Promoter, mCherry</td>
<td>Readout system for enhancer binding. It was used to test cathepsin B-cleavable linker</td>
+
<td>Readout system for enhancer binding, which 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, which 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>
Line 304: Line 356:
 
<h1>2. Usage and Biology</h1>
 
<h1>2. Usage and Biology</h1>
 
<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
      tool for genome editing. The CRISPR/Cas system, which originates from the bacterial immune system, is constituted
+
    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
+
    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
+
    class 2 systems consist of a singular protein and RNA. The class 2 type II system describes all ribonucleoprotein
      complexes with Cas9 (Pacesa <i>et al.</i>, 2024). They include a CRISPR RNA (crRNA), which specifies the target with a 20
+
    complexes with Cas9 (Pacesa <i>et al.</i>, 2024). They include a CRISPR RNA (crRNA), which specifies the target with
      nucleotide (nt) spacer sequence, and a transactivating CRISPR RNA (tracrRNA), which induces the processing by the
+
    a 20
      Cas protein (Jinek <i>et al.</i>, 2012) (Figure 2 A). Furthermore, a specific three
+
    nucleotide (nt) spacer sequence, and a transactivating CRISPR RNA (tracrRNA), which induces the processing by the
      nucleotide sequence (NGG) on the 3' end in the targeted DNA is needed for binding and cleavage. This is referred
+
    Cas protein (Jinek <i>et al.</i>, 2012) (Figure 2 A). Furthermore, a specific three
      to as the protospacer adjacent motif (PAM) (Sternberg <i>et al.</i>, 2014). The most commonly used Cas9 protein is SpCas9
+
    nucleotide sequence (NGG) on the 3' end in the targeted DNA is needed for binding and cleavage. This is referred
      or SpyCas9, which originates from Streptococcus pyogenes (Pacesa <i>et al.</i>, 2024).
+
    to as the protospacer adjacent motif (PAM) (Sternberg <i>et al.</i>, 2014). The most commonly used Cas9 protein is
    </p>
+
    SpCas9
 +
    or SpyCas9, which originates from Streptococcus pyogenes (Pacesa <i>et al.</i>, 2024).
 +
  </p>
 
<div class="thumb">
 
<div class="thumb">
 
<div class="thumbinner" style="width:60%;">
 
<div class="thumbinner" style="width:60%;">
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<div class="thumbcaption">
 
<div class="thumbcaption">
 
<i>
 
<i>
<b>Figure 2: The CRISPR/Cas system (adapted from Pacesa <i>et al.</i> (2024))</b>  
+
<b>Figure 2: The CRISPR/Cas System.</b>
            A and B, schematic structure of Cas9 and Cas12a with their sgRNA/crRNA, sitting on a DNA strand with the PAM.  
+
          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
+
          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
+
          spacer is located at the 5' prime end, for Cas12a at the 3' end of the gRNA. The scaffold of the gRNA forms a
            secondary structure enabling it to bind to the Cas protein. The cut sites by the cleaving domains, RuvC and HNH, are
+
          specific
            symbolized by the scissors
+
          secondary structure enabling it to bind to the Cas protein. The cut sites by the cleaving domains, RuvC and
          </i>
+
          HNH, are
 +
          symbolized by the scissors
 +
        </i>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
<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 RNA (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 sequence accordingly.  
+
    Moreover, Cong (2013) established precise targeting of human endogenous loci by designing the 20 nt spacer sequence
    </p>
+
    accordingly.
 +
  </p>
 
</section>
 
</section>
 
<section id="3">
 
<section id="3">
<h1>3. Assembly and part evolution</h1>
+
<h1>3. Assembly and Part Evolution</h1>
 
<section id="3.1">
 
<section id="3.1">
<h2>3.1 Qualtitative assesment of Cas12a orthologs</h2>
+
<h2>3.1 Qualitatively Assessing Gene Editing of Cas12a Orthologs</h2>
 
<p>
 
<p>
        To select a suitable Cas12a ortholog for cronstructing the Cas sstaple, three different orhtologs were ordered from Addgene:  
+
      To select a suitable Cas12a ortholog for constructing the Cas staples, three different orthologs were ordered
        AsCas12a (<a href="https://www.addgene.org/69982/" target="_blank">#69982</a>), LbCas12a (<a href="https://www.addgene.org/69988/" target="_blank">#69988</a>),  
+
      from Addgene:
        and MbCas12a (<a href="https://www.addgene.org/115142/" target="_blank">#115142</a>).
+
      AsCas12a (<a href="https://www.addgene.org/69982/" target="_blank">#69982</a>), LbCas12a (<a href="https://www.addgene.org/69988/" target="_blank">#69988</a>),
        <br/><br/>
+
      and MbCas12a (<a href="https://www.addgene.org/115142/" target="_blank">#115142</a>).
        We assessed their performance using a T7 Endonuclease I (T7EI) assay on three genomic loci: RUNX1, DNMT1, and FANCF. For
+
      <br/><br/>
        comparison, we also included SpCas9 editing the CCR5 locus (Fig. 3). Editing was only observed at the
+
      We assessed their performance using a T7 Endonuclease I (T7EI) assay on three genomic loci: RUNX1, DNMT1, and
        RUNX1 locus, where we used the qualitative assay to compare the editing efficiencies of the Cas12a orthologs.
+
      FANCF. For
      </p>
+
      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="thumb">
 
<div class="thumbinner" style="width:60%;">
 
<div class="thumbinner" style="width:60%;">
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<div class="thumbcaption">
 
<div class="thumbcaption">
 
<i>
 
<i>
<b>Figure 3: Preliminary T7 Endonuclease I testing of Cas12a orthologs.</b>
+
<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
+
            T7EI samples were run on a 2% agarose gel in 1x TBE buffer dyed with gel red. SpCas9 targeting CCR5
              benchmark for proper editing. For the Cas12a orthologs, LbCas12a, AsCas12a and MbCas12a, the three loci RUNX1, DNMT1 and
+
            functions as a
              FANCF were targeted. Editing is indicated by an extra band compared to the negative control.
+
            benchmark for proper editing. For the Cas12a orthologs, LbCas12a, AsCas12a and MbCas12a, the three loci
            </i>
+
            RUNX1, DNMT1 and
 +
            FANCF were targeted. Editing is indicated by an extra band compared to the negative control.
 +
          </i>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
</section>
 
</section>
<section id="3.2"><h2>3.2 Quantitative comparison between AsCas12a and MbCas12a</h2>
+
<section id="3.2">
 +
<h2>3.2 Quantitative Comparison Between AsCas12a and MbCas12a</h2>
 
<p>
 
<p>
        Since the T7EI assay can only be used to qualitatively compare editing efficiencies, it was hard to distinguish
+
      Since the T7EI assay can only be used to qualitatively compare editing efficiencies, it was hard to distinguish
        between the better editing ortholog.<br/>
+
      between the better editing ortholog.<br/>
        To accurately quantify the editing efficiency, we concted a dual luciferase assay. This assay measures the luminescence
+
      To accurately quantify the editing efficiency, we conducted a dual luciferase assay. This assay measures the
        of Firefly luciferase, which decreases proportionally to the editing efficiency at the target site.  
+
      luminescence
        To account for variations in cell count and transfection efficiency, the luminescence is normalized
+
      of firefly luciferase, which decreases proportionally to the editing efficiency at the target site.
        to Renilla luciferase, which acts as an internal control (Fig. 4).
+
      To account for variations in cell count and transfection efficiency, the luminescence is normalized
        The results revealed a clear difference, with MbCas12a demonstrating significantly higher editing efficiency compared to
+
      to Renilla luciferase, which acts as an internal control (Fig. 4).
        AsCas12a (p=0.005). Based on these findings, we decided to proceed with MbCas12a.
+
      The results revealed a clear difference, with MbCas12a demonstrating significantly higher editing efficiency
      </p>
+
      compared to
 +
      AsCas12a (p=0.005). Based on these findings, we decided to proceed with MbCas12a.
 +
    </p>
 
<div class="thumb">
 
<div class="thumb">
 
<div class="thumbinner" style="width:60%;">
 
<div class="thumbinner" style="width:60%;">
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<div class="thumbcaption">
 
<div class="thumbcaption">
 
<i>
 
<i>
<b>Figure 4: Comparison of AsCas12a and MbCas12a with a dual luciferae assay.</b>
+
<b>Figure 4: Comparison of AsCas12a and MbCas12a with a Dual Luciferase Assay.</b>
              Firefly luminescence intensity measured 48 h after transfection. Normalized against renilla luminescence. On the x-axis
+
            Firefly luminescence intensity measured 48 h after transfection. Normalized against renilla luminescence. On
              the samples Cas9 + AsCas12a , Cas9 + MbCas12a, AsCas12a and MbCas12a are depicted.
+
            the x-axis
              Data is depicted as the mean +/- SD (n=3).
+
            the samples Cas9 + AsCas12a , Cas9 + MbCas12a, AsCas12a and MbCas12a are depicted.
              Statistical analysis was performed using 1way ANOVA with Tukey's multiple comparisons test. For better clarity, only
+
            Data is depicted as the mean +/- SD (n=3).
              significant differences within a group between the same Cas proteins are shown.*p&lt;0.05, **p&lt;0.01, ***p&lt;0.001,
+
            Statistical analysis was performed using 1way ANOVA with Tukey's multiple comparisons test. For better
                ****p&lt;0.0001
+
            clarity, only
            </i>
+
            significant differences within a group between the same Cas proteins are shown.*p&lt;0.05, **p&lt;0.01,
 +
            ***p&lt;0.001,
 +
            ****p&lt;0.0001
 +
          </i>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
</section>
 
</section>
<section id="3.3"><h2>3.3 MbCas12a tolerates co-transfection and simultaneous editing of different Cas proteins</h2>
+
<section id="3.3">
 +
<h2>3.3 Multiplex Gene Editing Using MbCas12a and SpCas9</h2>
 
<p>
 
<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
+
      To employ the MbCas12a as part of a Cas staple, we sought to determine MbCas12a's activity while
        being transfected together with SpCas9. Therefore we engineered the dual luciferase assay to allow us for testing two
+
      being transfected together with SpCas9. Therefore we engineered the dual luciferase assay to allow us for testing
        catalytically active Cas proteins at once. Including a well established protospacer, CCR5, into the renilla luciferase
+
      two
        gene enables us to test the editing rates of two Cas proteins simultaneously (Fig.5 ).
+
      catalytically active Cas proteins at once. Including a well established protospacer, CCR5, into the renilla
      </p>
+
      luciferase
 +
      gene enables us to test the editing rates of two Cas proteins simultaneously (Fig. 5).
 +
    </p>
 
<div class="thumb">
 
<div class="thumb">
 
<div class="thumbinner" style="width:60%">
 
<div class="thumbinner" style="width:60%">
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<div class="thumbcaption">
 
<div class="thumbcaption">
 
<i>
 
<i>
<b>Figure 5: Testing for Simultaneous Editing with Double Cut Luciferae Assay</b>
+
<b>Figure 5: Testing for Simultaneous Editing with Double Cut Luciferase Assay.</b>
                Firefly and renilla luminescence intensity is plotted, both measured 48 h after transfection. Co-transformed contains
+
            Firefly and renilla luminescence intensity is plotted, both measured 48 h after transfection. Co-transformed
                MbCas12a and SpCas9. Cas12a are the Firefly relative luminescence units (RLUs), while Cas9 are the Renilla RLUs.
+
            contains
                Data is depicted as the mean +/- SD (n=3).
+
            MbCas12a and SpCas9. Cas12a are the firefly relative luminescence units (RLUs), while Cas9 are the Renilla
                Statistical analysis was performed using 2way ANOVA with Tukey's multiple comparisons test. For better clarity, only
+
            RLUs.
                significant differences within a group are shown.*p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001
+
            Data is depicted as the mean +/- SD (n=3).
              </i>
+
            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 &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt;
 +
            0.0001
 +
          </i>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
<p>
 
<p>
        For the MbCas12a we have a highly significant editing rate in the single cut, meaning both Cas proteins are transfected,
+
      For the MbCas12a we have a highly significant editing rate in the single cut, meaning both Cas proteins are
        but only MbCas12a has a targeting gRNA. When introducing a targeting gRNA for SpCas9 we see no reduction in the highly
+
      transfected,
        significant editing of MbCas12a, strongly suggesting both Cas proteins to be able to edit simultaneously.
+
      but only MbCas12a has a targeting gRNA. When introducing a targeting gRNA for SpCas9 we did not observe reduction in the
      </p>
+
      highly
 +
      significant editing of MbCas12a, strongly suggesting both Cas proteins to be able to edit simultaneously.
 +
    </p>
 
</section>
 
</section>
<section id="3.4"><h2>3.4 MbCas12a shows editing with fgRNA</h2>
+
<section id="3.4">
 +
<h2>3.4 Fusion Guide RNA Enabled Editing with MbCas12a</h2>
 
<p>
 
<p>
        To further confirm if MbCas12a is compatible with our Cas staples, editing rates were tested using a fusion guide RNA
+
      To further confirm that MbCas12a can be combined with fgRNAs in a functional Cas staple, editing rates were tested
        (fgRNA, <a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a>)
+
      using a fusion guide RNA
        targeting two different loci: <i>FANCF</i> and <i>VEGFA</i>. To better assess the impact that the utilization of a
+
      (fgRNA, <a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a>)
        fgRNA has on the editing rates, the sgRNAs were tested separately and in one sample.<br/>
+
      targeting two different loci: <i>FANCF</i> and <i>VEGFA</i>. To better assess the impact that the utilization of a
        Having the sgRNA with single Cas proteins in the same sample resulted in no clear
+
      fgRNA has on the editing rates, the sgRNAs were tested separately and in one sample.<br/>
        difference in the editing rates (Fig. 6). The fusion of the gRNAs resulted in a lower editing rate
+
      Having the sgRNA with single Cas proteins in the same sample resulted in no clear
        overall. While the editing for VEGFA stayed at about 20% in all
+
      difference in the editing rates (Fig. 6). The fusion of the gRNAs resulted in a lower editing rate
        cases, the editing for FANCF dropped significantly. Nonetheless we were able to show MbCas12a editing utilizing a fgRNA.
+
      overall. While the editing for VEGFA stayed at about 20% in all
      </p>
+
      cases, the editing for FANCF dropped significantly. Nonetheless we were able to show MbCas12a editing utilizing a
 +
      fgRNA.
 +
    </p>
 
<div class="thumb">
 
<div class="thumb">
 
<div class="thumbinner" style="width:95%;">
 
<div class="thumbinner" style="width:95%;">
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<div class="thumbcaption">
 
<div class="thumbcaption">
 
<i>
 
<i>
<b>Figure 6: Fusion gRNA Editing Rates In Combination with MbCas12a.</b>  
+
<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
+
            In <b>A</b> and <b>B</b> the editing rates were determined 72h after transfection via T7EI assay. Editing %
              determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band))<sup>1/2</sup>. The
+
            was
              schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below
+
            determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved
              indicate which parts are included in each sample. <i class="italic">A</i> and <i class="italic">B</i> display both
+
            band))<sup>1/2</sup>. The
              orientations of the two spacers for VEGFA and FANCF.
+
            schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols
            </i>
+
            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>
 
</div>
 
</div>
 
</div>
 
<p>
 
<p>
        To further assess the effect of the genomic locus on the editing rate, we included CCR5 as an additional gene target.
+
      To further assess the effect of the genomic locus on the editing rate, we included CCR5 as an additional gene
        For this assay, a fgRNA with a 20 nt long linker was included between the two spacers.
+
      target.
        The editing rate for VEGFA was again relatively consistent throughout the samples (Fig. 7).
+
      For this assay, a fgRNA with a 20 nt long linker was included between the two spacers.
      </p>
+
      The editing rate for VEGFA was again relatively consistent throughout the samples (Fig. 7).
 +
    </p>
 
<div class="thumb">
 
<div class="thumb">
 
<div class="thumbinner" style="width:60%;">
 
<div class="thumbinner" style="width:60%;">
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<div class="thumbcaption">
 
<div class="thumbcaption">
 
<i>
 
<i>
<b>Figure 7: Fusion gRNA Editing Rates for Multiplexing CCR5 and VEGFA</b>  
+
<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
+
            The editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by
              intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band)) <sup>1/2</sup>. The schematic at the top shows the
+
            measuring band
              composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts are included in
+
            intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band)) <sup>1/2</sup>. The schematic at the
              each sample. Cas12a targets VEGFA and Cas9 targets CCR5.
+
            top shows the
            </i>
+
            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>
 
</div>
 
</div>
 
</div>
 
</section>
 
</section>
<section id="3.5"><h2>3.5 MbCas12a tolerates fusion to SpCas9</h2>
+
<section id="3.5">
 +
<h2>3.5 Establishing Functional Fusion Cas Proteins with MbCas12a and SpCas9</h2>
 
<p>
 
<p>
        Testing showed simultaneous editing of MbCas12a and SpCas9. Therefore we wanted to test protein-protein fusion,
+
      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
+
      potentially giving us another way of Cas stapling. Our goal was to find out how well MbCas12a can stay active
        being fused to SpCas9. Therefore we employed the previously engineered dual luciferase assay to allow us for testing two
+
      while
        catalytically active Cas proteins at once, this time being fused to each other (Fig. 8).
+
      being fused to SpCas9. Therefore we employed the previously engineered dual luciferase assay to allow us for
      </p>
+
      testing two
 +
      catalytically active Cas proteins at once, this time being fused to each other (Fig. 8).
 +
    </p>
 
<div class="thumb">
 
<div class="thumb">
 
<div class="thumbinner" style="width:60%;">
 
<div class="thumbinner" style="width:60%;">
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<div class="thumbcaption">
 
<div class="thumbcaption">
 
<i>
 
<i>
<b>Figure 8: Double cut dual luciferase assay testing Fusion Cas simultaneous editing.</b>
+
<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
+
            Firefly and renilla luminescence intensity is plotted, both measured 48 h after transfection. On the x-axis
              control, single edit and simultaneous edit, are grouped by the two Cas proteins in the fusion Cas. The fusion Cas
+
            the negative
              contains MbCas12a and SpCas9. MbCas12a are the Firefly relative luminescence units (RLUs), while Cas9 are the Renilla
+
            control, single edit and simultaneous edit, are grouped by the two Cas proteins in the fusion Cas. The
              RLUs. Data is depicted as the mean +/- SD (n=3). Statistical analysis was performed using 2way ANOVA with Tukey's multiple  
+
            fusion Cas
              comparisons test. For better clarity, only significant differences within a group are shown.*p&lt;0.05, **p&lt;0.01, ***p&lt;0.001, ****p&lt;0.0001
+
            contains MbCas12a and SpCas9. MbCas12a are the firefly relative luminescence units (RLUs), while Cas9 are
            </i>
+
            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&lt;0.05,
 +
            **p&lt;0.01, ***p&lt;0.001, ****p&lt;0.0001
 +
          </i>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
<p>
 
<p>
        For the MbCas12a we have a highly significant editing rate in the single cut, meaning only MbCas12a has a targeting
+
      For the MbCas12a we have a highly significant editing rate in the single cut, meaning only MbCas12a has a
        gRNA. When introducing a targeting gRNA for SpCas9 we see no reduction in the highly significant editing of MbCas12a,
+
      targeting
        strongly suggesting both Cas proteins in the fusion Cas to be able to edit simultaneously.
+
      gRNA. When introducing a targeting gRNA for SpCas9 we see no reduction in the highly significant editing of
      </p>
+
      MbCas12a,
 +
      strongly suggesting both Cas proteins in the fusion Cas to be able to edit simultaneously.
 +
    </p>
 
</section>
 
</section>
<section id="3.6"><h2>3.6 MbCas12a fused to SpCas9 editing utilizing a fgRNA</h2>
+
<section id="3.6">
 +
<h2>3.6 The Combination of Fusion Guide RNAs and Fusion Cas Proteins</h2>
 
<p>
 
<p>
        The capability of MbCas12 in the fusion Cas was tested by assessing the editing rates via a T7EI assay. For this, the
+
      The gene editing efficiency of MbCas12a in the fusion Cas was tested by assessing the editing rates via a T7EI
        same target sequences as before were used, namely FANCF and VEGFA in both configurations. We included biological
+
      assay. For this, the
        duplicates in this assay.<br/>
+
      same target sequences as before were used, namely FANCF and VEGFA in both configurations. We included biological
        MbCas12a editing rates were significantly lower, dropping to about 1%. At the same time, when targeting VEGFA they
+
      duplicates in this assay.<br/>
        resulted in a higher editing efficiency than FANCF.
+
      MbCas12a editing rates were significantly lower, dropping to about 1%. At the same time, when targeting VEGFA they
      </p>
+
      resulted in a higher editing efficiency than FANCF.
 +
    </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-fcas-2.svg" style="width:99%;"/>
 
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/results-fcas-2.svg" style="width:99%;"/>
<div>
+
<div class="thumbcaption">
 
<i>
 
<i>
<b>Figure 9: Editing rates for fusion guide RNAs with fusion Cas proteins.</b>
+
<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
+
            In <b>A</b> and <b>B</b> the editing rates were determined 72h after transfection via T7EI assay. Editing %
                determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band))<sup>1/2</sup>. The
+
            was
                schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below
+
            determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved
                indicate which parts are included in each sample. Cas proteins linked by a dash ("-") were fused to each other.
+
            band))<sup>1/2</sup>. The
                Biological replicates are marked as individual dots.
+
            schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols
              </i>
+
            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>
 
</div>
Line 523: Line 626:
 
<h1>4. Results</h1>
 
<h1>4. Results</h1>
 
<p>
 
<p>
      We extensively characterized the catalytically active MbCas12a in several assays determining the best ortholog out of
+
    We extensively characterized the catalytically active MbCas12a in several assays determining the best ortholog out
      three for our Cas staples, assert functional editing with a fusion guide RNA (BBa_K5237000), showing high activity
+
    of
      while being co-transfected with SpCas9, our second staple protein's active version, and lastly a functioning fusion to
+
    three for our Cas staples, assert functional editing with a fusion guide RNA (BBa_K5237000), showing high activity
      SpCas9.<br/>
+
    while being co-transfected with SpCas9, our second staple protein's active version, and lastly a functioning fusion
      After all these successful test we were confident to test the Cas staples in action.
+
    to
    </p>
+
    SpCas9.<br/>
<section><h2 id="4.1">4.1 dMbCas12a Transactivation as Part of Cas Staple</h2>
+
    After all these successful test we were confident to test the Cas staples in action.
 +
  </p>
 +
<section>
 +
<h2 id="4.1">4.1 dMbCas12a as Part of a Cas Staple Establishes DNA-DNA Proximity</h2>
 
<p>
 
<p>
        The next step was to use the MbCas12a as part of a Cas staple to staple two DNA loci together, and thereby induce
+
      The next step was to use the MbCas12a as part of a Cas staple to bring 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
+
      proximity between two separate functional elements. Mimicking the phenomenon of enhancer hijacking, in which an enhancer
        reporter plasmid has firefly luciferase behind several repeats of a Cas9 targeted sequence. The enhancer plasmid has a
+
      activates an otherwise distant promoter, we constructed artificial enhancer and reporter plasmids. The
        Gal4 binding site behind several repeats of a Cas12a targeted sequence. By introducing a fgRNA staple and a Gal4-VP64,
+
      reporter plasmid encodes a firefly luciferase downstream of several repeats of a Cas9 targeted sequence. The enhancer plasmid
        expression of the luciferase is induced (Fig. 10 A).
+
      has a
        Different linker lengths were tested. Cells were again normalized against ubiquitous renilla expression. Further
+
      Gal4 binding site behind several repeats of a Cas12a targeted sequence. By introducing a fgRNA staple and a
        information on our learnings from this assay can be found in the Cas staple engineering cycle iteration 2.
+
      Gal4-VP64,
        Using no linker between the two spacers showed similar relative luciferase activity to the baseline control (Fig. 10 B).  
+
      expression of the luciferase is induced (Fig. 10A).
        An extension of the linker from 20 nt up to 40 nt resulted in an increasingly higher expression of the reporter gene.
+
      Different linker lengths were tested. Cells were again normalized against ubiquitous renilla expression. Further
      </p>
+
      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.
 +
      10B).
 +
      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="thumb">
 
<div class="thumbinner" style="width:60%;">
 
<div class="thumbinner" style="width:60%;">
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<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. An enhancer
+
<b>Figure 10: Applying Fusion Guide RNAs for Cas staples.</b> <b>A</b>, schematic overview of the assay. An
              plasmid and a reporter plasmid are brought into proximity by a fgRNA Cas staple complex binding both plasmids. Target
+
            enhancer
              sequences were included in multiple repeats prior to the functional elements. Firefly luciferase serves as the reporter
+
            plasmid and a reporter plasmid are brought into proximity by a fgRNA Cas staple complex binding both
              gene, the enhancer is constituted by multiple Gal4 repeats that are bound by a Gal4-VP64 fusion.  
+
            plasmids. Target
              <b>B</b>, results of using a fgRNA Cas staple for trans activation of firefly luciferase. Firefly
+
            sequences were included in multiple repeats prior to the functional elements. Firefly luciferase serves as
              luciferase activity was measured 48h after transfection. Normalized against ubiquitously expressed Renilla luciferase.
+
            the reporter
              Statistical significance was calculated with ordinary One-way ANOVA with Dunn's method for multiple comparisons (*p &lt;
+
            gene, the enhancer is constituted by multiple Gal4 repeats that are bound by a Gal4-VP64 fusion.
              0.05; **p &lt; 0.01; ***p &lt; 0.001; mean +/- SD). The assay included sgRNAs and fgRNAs with linker lengths from 0 nt
+
            <b>B</b>, results of using a fgRNA Cas staple for trans activation of firefly luciferase. Firefly
              to 40 nt.
+
            luciferase activity was measured 48h after transfection. Normalized against ubiquitously expressed Renilla
            </i>
+
            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>
 
</div>
Line 563: Line 680:
 
<section id="5">
 
<section id="5">
 
<h1>5. References</h1>
 
<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>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., 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>
+
      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>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>Kleinstiver, B. P., Sousa, A. A., Walton, R. T., Tak, Y. E., Hsu, J. Y., Clement, K., Welch, M. M., Horng, J. E.,
<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>
+
    Malagon-Lopez, J., Scarfò, I., Maus, M. V., Pinello, L., Aryee, M. J., and Joung, J. K. (2019). Engineered
<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>
+
    CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base
<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>
+
    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>
 +
<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>
 +
<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>
 
</section>
 
</section>
 
</html>
 
</html>

Revision as of 06:51, 2 October 2024

BBa_K5237001

Staple Subunit: dMbCas12a-Nucleoplasmin NLS

dMbCas12a is one crucial part of our PICasSO tool box, being able to function as a Cas staple with the fgRNA (BBa_K5237000) and the dSpCas9 (BBa_K5237002). Transactivation has been shown using this part proving the proper formation of our Cas staple. Easy employment through swift reprogramming of the fgRNA.

 



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


While synthetic biology has in the past focused on engineering the genomic sequence of organisms, the 3D spatial organization of DNA is well-known to be an important layer of information encoding in particular in eukaryotes, playing a crucial role in gene regulation and hence cell fate, disease development, evolution, and more. However, tools to precisely manipulate and control the genomic spatial architecture are limited, hampering the exploration of 3D genome engineering in synthetic biology. We - the iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular toolbox for rationally engineering genome 3D architectures in living cells, based on various DNA-binding proteins.

The PICasSO part collection offers a comprehensive, modular platform for precise manipulation and re-programming of DNA-DNA interactions using engineered "protein staples" in living cells. This enables researchers to recreate naturally occurring alterations of 3D genomic interactions, such as enhancer hijacking in cancer, or to design entirely new spatial architectures for artificial gene regulation and cell function control. Specifically, the fusion of two DNA binding proteins enables to artificially bring otherwise distant genomic loci into spatial proximity. To unlock the system's full potential, we introduce versatile chimeric CRISPR/Cas complexes, connected either at the protein or - in the case of CRISPR/Cas-based DNA binding moieties - the guide RNA level. These complexes are referred to as protein- or Cas staples, respectively. Beyond its versatility with regard to the staple constructs themselves, PICasSO includes robust assay systems to support the engineering, optimization, and testing of new staples in vitro and in vivo. Notably, the PICasSO toolbox was developed in a design-build-test-learn engineering cycle closely intertwining wet lab experiments and computational modeling and iterated several times, yielding a collection of well-functioning and -characterized parts.

At its heart, the PICasSO part collection consists of three categories.
(i) Our DNA-binding proteins include our finalized Cas staple experimentally validated using an artificial "enhancer hijacking" system as well as "half staples" that can be combined by scientists to compose entirely new Cas staples in the future. We also include our Simple staples comprised of particularly small, simple and robust DNA binding domains well-known to the synthetic biology community, which 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 and expand the functionality of our Cas and Basic staples. These consist of staples dependent on cleavable peptide linkers targeted by cancer-specific proteases or inteins that allow condition-specific, dynamic stapling in vivo. We also include several engineered parts that enable the efficient delivery of PICasSO's constructs into target cells, including mammalian cells, with our new interkingdom conjugation system.
(iii) As the final category of our collection, we provide parts that underlie 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 based on a luciferase reporter, which allows for straightforward experimental assessment of functional enhancer hijacking events 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 in the new field of 3D genome engineering.

Our part collection includes:

DNA-Binding Proteins: Modular building blocks for engineering of custom staples to mediate defined DNA-DNA interactions in vivo
BBa_K5237000 Fusion Guide RNA Entry Vector MbCas12a-SpCas9 Entry vector for simple fgRNA cloning via SapI
BBa_K5237001 Staple Subunit: dMbCas12a-Nucleoplasmin NLS Staple subunit that can be combined with crRNA or fgRNA and dSpCas9 to form a functional staple
BBa_K5237002 Staple Subunit: SV40 NLS-dSpCas9-SV40 NLS Staple subunit that can be combined with a sgRNA or fgRNA and dMbCas12a to form a functional staple
BBa_K5237003 Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS Functional Cas staple that can be combined with sgRNA and crRNA 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, which can be used to create functionalized staple subunits
BBa_K5237013 Caged NpuC Intein A caged NpuC split intein fragment that undergoes protein trans-splicing after protease activation, which can be used to create functionalized staple subunits
BBa_K5237014 Fusion Guide RNA Processing Casette Processing cassette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D genome reprogramming
BBa_K5237015 Intimin anti-EGFR Nanobody Interkingdom conjugation between bacteria and mammalian cells, as an 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 readout systems to rapidly assess successful DNA stapling in bacterial and mammalian cells
BBa_K5237016 FRET-Donor: mNeonGreen-Oct1 FRET donor-fluorophore fused to Oct1-DBD that binds to the Oct1 binding cassette, which 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, which 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, which 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 Promoter, mCherry Readout system for enhancer binding, which 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, which was used as a luminescence readout for simulated enhancer hijacking

1. Sequence overview

Sequence and Features


Assembly Compatibility:
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    Illegal PstI site found at 274
    Illegal PstI site found at 295
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    Illegal PstI site found at 3049
    Illegal PstI site found at 3324
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    Illegal PstI site found at 274
    Illegal PstI site found at 295
    Illegal PstI site found at 607
    Illegal PstI site found at 1762
    Illegal PstI site found at 3049
    Illegal PstI site found at 3324
    Illegal NgoMIV site found at 355
    Illegal NgoMIV site found at 787
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2. Usage and Biology

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) (Figure 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 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).

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 bind to the Cas protein. The cut sites by the cleaving domains, RuvC and HNH, are symbolized by the scissors

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 et al., 2013). Moreover, Cong (2013) established precise targeting of human endogenous loci by designing the 20 nt spacer sequence accordingly.

3. Assembly and Part Evolution

3.1 Qualitatively Assessing Gene Editing of Cas12a Orthologs

To select a suitable Cas12a ortholog for constructing the Cas staples, three different orthologs were ordered from Addgene: AsCas12a (#69982), LbCas12a (#69988), and MbCas12a (#115142).

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.

Figure 3: Preliminary T7 Endonuclease I Testing of Cas12a Orthologs. 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.

3.2 Quantitative Comparison Between AsCas12a and MbCas12a

Since the T7EI assay can only be used to qualitatively compare editing efficiencies, it was hard to distinguish between the better editing ortholog.
To accurately quantify the editing efficiency, we conducted 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.

Figure 4: 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

3.3 Multiplex Gene Editing Using MbCas12a and SpCas9

To employ the MbCas12a as part of a Cas staple, we sought to determine MbCas12a's activity 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).

Figure 5: Testing for Simultaneous Editing with Double Cut Luciferase Assay. 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

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 did not observe reduction in the highly significant editing of MbCas12a, strongly suggesting both Cas proteins to be able to edit simultaneously.

3.4 Fusion Guide RNA Enabled Editing with MbCas12a

To further confirm that MbCas12a can be combined with fgRNAs in a functional Cas staple, editing rates were tested using a fusion guide RNA (fgRNA, BBa_K5237000) targeting two different loci: FANCF and VEGFA. 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. 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.

Figure 6: 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.

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).

Figure 7: 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.

3.5 Establishing Functional Fusion Cas Proteins with MbCas12a and SpCas9

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).

Figure 8: Double Cut Dual Luciferase Assay Testing Fusion Cas Simultaneous Editing. 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

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.

3.6 The Combination of Fusion Guide RNAs and Fusion Cas Proteins

The gene editing efficiency of MbCas12a 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.
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.

Figure 9: Editing Rates for Fusion Guide RNAs with Fusion Cas Proteins. 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. Cas proteins linked by a dash ("-") were fused to each other. Biological replicates are marked as individual dots.

4. Results

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.
After all these successful test we were confident to test the Cas staples in action.

4.1 dMbCas12a as Part of a Cas Staple Establishes DNA-DNA Proximity

The next step was to use the MbCas12a as part of a Cas staple to bring two DNA loci together, and thereby induce proximity between two separate functional elements. Mimicking the phenomenon of enhancer hijacking, in which an enhancer activates an otherwise distant promoter, we constructed artificial enhancer and reporter plasmids. The reporter plasmid encodes a firefly luciferase downstream of 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. 10A). 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. 10B). An extension of the linker from 20 nt up to 40 nt resulted in an increasingly higher expression of the reporter gene.

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.

5. References

Kampmann, M. (2017). CRISPRi and CRISPRa Screens in Mammalian Cells for Precision Biology and Medicine. ACS Chemical Biology, 13, 406–416. https://doi.org/10.1021/acschembio.7b00657.

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. Nature Biotechnology, 37, 276–282. https://doi.org/10.1038/s41587-018-0011-0.

Koonin, E. V., Gootenberg, J. S., and Abudayyeh, O. O. (2023). Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox. Biochemistry, 62, 3465–3487. https://doi.org/10.1021/acs.biochem.3c00159.

Pacesa, M., Pelea, O., and Jinek, M. (2024). Past, present, and future of CRISPR genome editing technologies. Cell, 187, 1076–1100. https://doi.org/10.1016/j.cell.2024.01.042.

Paul, B., and Montoya, G. (2020). CRISPR-Cas12a: Functional overview and applications. Biomedical Journal, 43, 8–17. https://doi.org/10.1016/j.bj.2019.10.005.

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. Cell, 163, 759–771. https://doi.org/10.1016/j.cell.2015.09.038.