Difference between revisions of "Part:BBa K5237000"

 
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<body>
 
<!-- Part summary -->
 
<!-- Part summary -->
<section id="1">
+
<section>
 
<h1>fgRNA Entry Vector MbCas12a-SpCas9</h1>
 
<h1>fgRNA Entry Vector MbCas12a-SpCas9</h1>
 
<p>
 
<p>
      This part integrates the crRNA of MbCas12a (<a href="https://parts.igem.org/Part:BBa_K5237206">BBa_K5237206</a>) and the sgRNA of SpCas9 (<a href="https://parts.igem.org/Part:BBa_K5237209">BBa_K5237209</a>) into a single
+
            This part integrates the crRNA of MbCas12a (<a href="https://parts.igem.org/Part:BBa_K5237206">BBa_K5237206</a>) and the sgRNA of SpCas9 (<a href="https://parts.igem.org/Part:BBa_K5237209">BBa_K5237209</a>) into a single
      fusion
+
            fusion
      guide RNA (fgRNA). The fgRNA is functional, meaning that the MbCas12a (<a href="https://parts.igem.org/Part:BBa_K5237001">BBa_K5237001</a>),
+
            guide RNA (fgRNA). This fgRNA was functionally validated (see detailed characterization data below). MbCas12a (<a href="https://parts.igem.org/Part:BBa_K5237001">BBa_K5237001</a>),
      SpCas9 (<a href="https://parts.igem.org/Part:BBa_K5237002">BBa_K5237002</a>) and the fusion dCas (<a href="https://parts.igem.org/Part:BBa_K5237003">BBa_K5237003</a>)
+
            SpCas9 (<a href="https://parts.igem.org/Part:BBa_K5237002">BBa_K5237002</a>) and the novel fusion MbdCas12a-SpdCas9 (<a href="https://parts.igem.org/Part:BBa_K5237003">BBa_K5237003</a>)
      can utilize the fgRNA to target two loci simultaneously. The fgRNA also works in combination with the catalyitcally inactive Cas
+
            can all utilize the fgRNA to target/bind two different genomic loci simultaneously. The fgRNA works in combination
      versions.
+
            with both, the catalytically active as well as inactive Cas9 and Cas12a
      We successfully showed genome editing using active SpCas9 and Cas12a and induced proximity of two loci with the inactive dSpCas9 and dMbCas12a.<br/>
+
            versions, facilitating multiplexed genome editing (with catalytically active Cas) as well as DNA-DNA stapling and hence 3D genome engineering in eukaryotes (with catalytically inactive Cas).
      For our part collection, the PICasSO toolbox, this part has a crucial role in formation of our CRISPR/Cas staples.
+
            Employing the fgRNA design described here, we successfully showed simultaneous genome editing at two different loci in human cells. Furthermore, the fgRNA enabled us to induce spatial proximity of otherwise separate gene regulatory elements (enhancer and promoter) with the catalytically inactive dSpCas9 and dMbCas12a.<br/>
    </p>
+
            In context of our part collection, the PICasSO toolbox, part BBa_K5237000 is the core component, since it enables the
 +
            creation and programming of our so-called CRISPR/Cas staples: An innovative, trimeric complex comprised of a fgRNA, dCas9 and dCas12a employed
 +
            for <b> tethering two distinct genomic loci (see section 4.5 below)</b>, hence enabling rational engineering of the 3D genome conformation in living cells.
 +
        </p>
 
<p> </p>
 
<p> </p>
 
</section>
 
</section>
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<ul>
 
<ul>
 
<li class="toclevel-1 tocsection-1"><a href="#1"><span class="tocnumber">1</span> <span class="toctext">Sequence
 
<li class="toclevel-1 tocsection-1"><a href="#1"><span class="tocnumber">1</span> <span class="toctext">Sequence
            overview</span></a>
+
                        Overview</span></a>
 
</li>
 
</li>
 
<li class="toclevel-1 tocsection-2"><a href="#2"><span class="tocnumber">2</span> <span class="toctext">Usage and
 
<li class="toclevel-1 tocsection-2"><a href="#2"><span class="tocnumber">2</span> <span class="toctext">Usage and
            Biology</span></a>
+
                        Biology</span></a>
 
<ul>
 
<ul>
 
<li class="toclevel-2 tocsection-2.1">
 
<li class="toclevel-2 tocsection-2.1">
<a href="#2.1"><span class="tocnumber">2.1</span> <span class="toctext">Discovery and Mechanism of
+
<a href="#2.1"><span class="tocnumber">2.1</span> <span class="toctext">Discovery and Mechanism
                CRISPR/Cas9</span></a>
+
                                of
 +
                                CRISPR/Cas9</span></a>
 
</li>
 
</li>
 
<li class="toclevel-2 tocsection-2.2">
 
<li class="toclevel-2 tocsection-2.2">
<a href="#2.2"><span class="tocnumber">2.2</span> <span class="toctext">Differences between Cas9 and
+
<a href="#2.2"><span class="tocnumber">2.2</span> <span class="toctext">Differences between Cas9
                Cas12a</span></a>
+
                                and
 +
                                Cas12a</span></a>
 
</li>
 
</li>
 
<li class="toclevel-2 tocsection-2.3">
 
<li class="toclevel-2 tocsection-2.3">
<a href="#2.3"><span class="tocnumber">2.3</span> <span class="toctext">Dead Cas Proteins and their
+
<a href="#2.3"><span class="tocnumber">2.3</span> <span class="toctext">Dead Cas Proteins and
                Application</span></a>
+
                                their
</li>
+
                                Application</span></a>
<li class="toclevel-2 tocsection-2.4">
+
<a href="#2.4"><span class="tocnumber">2.4</span> <span class="toctext">fgRNA and CHyMErA System</span></a>
+
 
</li>
 
</li>
 
</ul>
 
</ul>
 
</li>
 
</li>
 
<li class="toclevel-1 tocsetction-3"><a href="#3"><span class="tocnumber">3</span> <span class="toctext">Assembly
 
<li class="toclevel-1 tocsetction-3"><a href="#3"><span class="tocnumber">3</span> <span class="toctext">Assembly
            and part evolution</span></a>
+
                        and part evolution</span></a>
 
</li>
 
</li>
 
<li class="toclevel-1 tocsection-4"><a href="#4"><span class="tocnumber">4</span> <span class="toctext">Results</span></a>
 
<li class="toclevel-1 tocsection-4"><a href="#4"><span class="tocnumber">4</span> <span class="toctext">Results</span></a>
 
<ul>
 
<ul>
 
<li class="toclevel-2 tocsection-4.1">
 
<li class="toclevel-2 tocsection-4.1">
<a href="#4.1"><span class="tocnumber">4.1</span> <span class="toctext">Editing endogenous loci with
+
<a href="#4.1"><span class="tocnumber">4.1</span> <span class="toctext">Editing Endogenous Loci
                fgRNAs</span></a>
+
                                With
 +
                                fgRNAs</span></a>
 
</li>
 
</li>
 
<li class="toclevel-2 tocsection-4.2">
 
<li class="toclevel-2 tocsection-4.2">
<a href="#4.2"><span class="tocnumber">4.2</span> <span class="toctext">Proximity assay with inactive Cas
+
<a href="#4.2"><span class="tocnumber">4.2</span> <span class="toctext">Efficient Fusion Guide RNA-Mediated Editing With Various Cas Orthologs</span></a>
                proteins</span></a>
+
 
</li>
 
</li>
 
<li class="toclevel-2 tocsection-4.3">
 
<li class="toclevel-2 tocsection-4.3">
<a href="#4.3"><span class="tocnumber">4.3</span> <span class="toctext">The Inclusion of a Linker Does Not
+
<a href="#4.3"><span class="tocnumber">4.3</span> <span class="toctext">Fusion Guide RNAs are Compatible with Linkers of Various lengths</span></a>
                Lower Editing Rates</span></a>
+
 
</li>
 
</li>
 
<li class="toclevel-2 tocsection-4.4">
 
<li class="toclevel-2 tocsection-4.4">
<a href="#4.4"><span class="tocnumber">4.4</span> <span class="toctext">fgRNAs can be Used for
+
<a href="#4.4"><span class="tocnumber">4.4</span> <span class="toctext">Fusion Guide RNAs Enable Efficient Activation of Gene Expression via CRISPRa</span></a>
                CRISPRa</span></a>
+
 
</li>
 
</li>
 
<li class="toclevel-2 tocsection-4.5">
 
<li class="toclevel-2 tocsection-4.5">
<a href="#4.5"><span class="tocnumber">4.5</span> <span class="toctext">Stapling Two DNA Strands Together
+
<a href="#4.5"><span class="tocnumber">4.5</span> <span class="toctext">A Proof-Of-Concept for 3D Genome Engineering: Stapling Two DNA Strands Together In Human Cells Using fgRNAs</span></a>
                Using fgRNAs</span></a>
+
 
</li>
 
</li>
 
</ul>
 
</ul>
 +
<li class="toclevel-1 tocsection-5"><a href="#5"><span class="tocnumber">5</span> <span class="toctext"><i>In Silico</i> Characterization using DaVinci</span></a>
 
</li>
 
</li>
<li class="toclevel-1 tocsection-8"><a href="#5"><span class="tocnumber">5</span> <span class="toctext">References</span></a>
+
<ul>
 +
<li class="toclevel-2 tocsection-5.1">
 +
  <a href="#5.1"><span class="tocnumber">5.1</span> <span class="toctext">Enhancer Hijacking is Successfully Studied <i>In Silico</i></span></a>
 +
</li>
 +
<li class="toclevel-2 tocsection-5.2">
 +
  <a href="#5.2"><span class="tocnumber">5.2</span> <span class="toctext">Cas Staple Forces do not Disturb DNA Strand Integrity</span></a>
 +
</li>
 +
<li class="toclevel-2 tocsection-5.3">
 +
  <a href="#5.3"><span class="tocnumber">5.3</span> <span class="toctext">DaVinci Helps to Design Multi-Staple Arrangements</span></a>
 +
</li>
 +
</ul>
 +
<li class="toclevel-1 tocsection-6"><a href="#6"><span class="tocnumber">6</span> <span class="toctext">References</span></a>
 
</li>
 
</li>
 
</ul>
 
</ul>
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</div>
 
</div>
 
</div>
 
</div>
 
 
<p>
 
<p>
 
<br/>
 
<br/>
       Next to the well-studied linear DNA sequence, the <b>3D spatial organization</b> of DNA plays a crucial role in
+
       While synthetic biology has in the past focused on engineering the genomic sequence of organisms, the <b>3D
       gene regulation,
+
        spatial organization</b> of DNA is well-known to be an important layer of information encoding in
       cell fate, disease development and more. However, the tools to precisely manipulate this genomic
+
      particular in eukaryotes, playing a crucial role in
       architecture remain limited, rendering it challenging to explore the full potential of the
+
       gene regulation and hence
       3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a <b>powerful
+
       cell fate, disease development, evolution, and more. However, tools to precisely manipulate and control the
      molecular toolbox</b> based on various DNA-binding proteins to address this issue.
+
      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
 
       <b>re-programming
 
       <b>re-programming
      of DNA-DNA interactions</b> 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
       Specifically, the fusion of two DNA binding proteins enables to artifically bring distant genomic loci into
+
       interactions, such as enhancer hijacking in cancer, or to design entirely new spatial architectures for
       proximty.
+
      artificial gene regulation and cell function control.
       To unlock the system's full potential, we introduce versatile <b>chimeric CRISPR/Cas complexes</b>, connected either on
+
       Specifically, the fusion of two DNA binding proteins enables to artificially bring otherwise distant genomic
       the protein or the guide RNA level. These1 complexes are reffered to as protein- or Cas staples. Beyond its
+
      loci into
       versatility, PICasSO includes <b>robust assay</b> systems to support the engineering, optimization, and
+
       spatial proximity.
       testing of new staples, ensuring functionality <i>in vitro</i> and <i>in vivo</i>. We took special care to include
+
       To unlock the system's full potential, we introduce versatile <b>chimeric CRISPR/Cas complexes</b>,
       parts crucial for testing every step of the cycle (design, build, test, learn) when <b>engineering new parts</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
 
<p>At its heart, the PICasSO part collection consists of three categories. <br/><b>(i)</b> Our <b>DNA-binding
 
         proteins</b>
 
         proteins</b>
 
       include our
 
       include our
       finalized enhancer hijacking Cas staple as well as half staples that can be used by scientists to compose entirely
+
       finalized 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
+
      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. <br/>
 +
<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
 
       Basic staples. These
       consist of
+
       consist of staples dependent on
       protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling <i>in vivo</i>.
+
       cleavable peptide linkers targeted by cancer-specific proteases or inteins that allow condition-specific,
       Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs
+
      dynamic stapling <i>in vivo</i>.
       with our
+
       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
+
<b>(iii)</b> As the final category of our collection, we provide parts that underlie our <b>custom
 
         readout
 
         readout
         systems</b>. These include components of our established FRET-based proximity assay system, enabling users to
+
         systems</b>. These include components of our established FRET-based proximity assay system, enabling
 +
      users to
 
       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
+
       luciferase reporter, which allows for straightforward experimental assessment of functional enhancer
 +
      hijacking events
 
       in mammalian cells.
 
       in mammalian cells.
 
     </p>
 
     </p>
 
<p>
 
<p>
 
       The following table gives a comprehensive overview of all parts in our PICasSO toolbox. <mark 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
+
         exceptional performance as described on our iGEM wiki and can serve as a reference.</mark> The other
 +
      parts in
 
       the
 
       the
       collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their
+
       collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer
       own custom Cas staples, enabling further optimization and innovation.<br/>
+
      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>
Line 198: Line 235:
 
<td><a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a></td>
 
<td><a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a></td>
 
<td>Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS</td>
 
<td>Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS</td>
<td>Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands into close
+
<td>Functional Cas staple that can be combined with sgRNA and crRNA or fgRNA to bring two DNA strands into
 +
            close
 
             proximity
 
             proximity
 
           </td>
 
           </td>
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<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>
Line 243: Line 282:
 
<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>
Line 249: Line 289:
 
<td><a href="https://parts.igem.org/Part:BBa_K5237011" target="_blank">BBa_K5237011</a></td>
 
<td><a href="https://parts.igem.org/Part:BBa_K5237011" target="_blank">BBa_K5237011</a></td>
 
<td>Cathepsin B Expression Cassette</td>
 
<td>Cathepsin B Expression Cassette</td>
<td>Expression Cassette for the overexpression of cathepsin B</td>
+
<td>Expression cassette for the overexpression of cathepsin B</td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
 
<td><a href="https://parts.igem.org/Part:BBa_K5237012" target="_blank">BBa_K5237012</a></td>
 
<td><a href="https://parts.igem.org/Part:BBa_K5237012" target="_blank">BBa_K5237012</a></td>
 
<td>Caged NpuN Intein</td>
 
<td>Caged NpuN Intein</td>
<td>A caged NpuN split intein fragment that undergoes protein <i>trans</i>-splicing after protease activation.
+
<td>A caged NpuN split intein fragment that undergoes protein <i>trans</i>-splicing after protease
             Can be used to create functionalized staples
+
             activation, which can be used to create functionalized staple
             units</td>
+
             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.
+
<td>A caged NpuC split intein fragment that undergoes protein <i>trans</i>-splicing after protease
             Can be used to create functionalized staples
+
             activation, which can be used to create functionalized staple
             units</td>
+
             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
+
<td>Processing cassette to produce multiple fgRNAs from one transcript, that can be used for
             genome reprograming</td>
+
            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
+
<td>Origin of transfer that can be cloned into the plasmid vector and used for conjugation as a
 +
            means of
 
             delivery</td>
 
             delivery</td>
 
</tr>
 
</tr>
 
</tbody>
 
</tbody>
 
<td align="left" colspan="3"><b>Readout Systems: </b>
 
<td align="left" colspan="3"><b>Readout Systems: </b>
         FRET and enhancer recruitment to measure proximity of stapled DNA in bacterial and mammalian living cells
+
         FRET and enhancer recruitment 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
+
<td>FRET donor-fluorophore fused to Oct1-DBD that binds to the Oct1 binding cassette, which can be used to
 +
            visualize
 
             DNA-DNA
 
             DNA-DNA
 
             proximity</td>
 
             proximity</td>
Line 298: Line 343:
 
<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 310: Line 356:
 
<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 <i>Trans</i>-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>
 
<td>NLS-Gal4-VP64</td>
 
<td>NLS-Gal4-VP64</td>
<td>Trans-activating enhancer, that can be used to simulate enhancer hijacking</td>
+
<td><i>Trans</i>-activating enhancer, that can be used to simulate enhancer hijacking</td>
 
</tr>
 
</tr>
 
<td><a href="https://parts.igem.org/Part:BBa_K5237022" target="_blank">BBa_K5237022</a></td>
 
<td><a href="https://parts.igem.org/Part:BBa_K5237022" target="_blank">BBa_K5237022</a></td>
<td>mCherry Expression Cassette: UAS, minimal Promotor, mCherry</td>
+
<td>mCherry Expression Cassette: UAS, minimal 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>
 
</section>
 
</section>
 
</body>
 
</body>
Line 358: Line 404:
 
<div class="thumbcaption">
 
<div class="thumbcaption">
 
<i>
 
<i>
<b>Figure 2: The CRISPR/Cas system </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
+
                            A and B, schematic structure of Cas9 and Cas12a with their sgRNA/crRNA, sitting on a DNA
              PAM.
+
                            strand with their respective PAMs.
              The spacer sequence forms base pairings with the dsDNA. In case of Cas9 the
+
                            The sgRNA/crRNA spacer sequence binds the DNA target strand via complementary base pairing.
              spacer is located at the 5' prime end, for Cas12a at the 3' end of the gRNA. The scaffold of the gRNA
+
                            In case of Cas9 the
              forms a specific
+
                            spacer is located at the 5' prime end, for Cas12a at the 3' end of the gRNA. The scaffold of
              secondary structure enabling it to be bound by the Cas protein. DNA cleavage sites are indicated by the scissors.
+
                            the gRNA
            </i>
+
                            forms a specific
 +
                            secondary structure enabling it to be bound by the Cas protein. DNA cleavage sites are
 +
                            indicated by the scissors.
 +
                        </i>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
<p>
 
<p>
        In 2012, Jinek <i>et al.</i> discovered the use of the Clustered Regularly Interspaced Short Palindromic Repeats
+
                In 2012, Jinek <i>et al.</i> discovered the use of the Clustered Regularly Interspaced Short Palindromic
        (CRISPR)/Cas system to induce double-strand breaks in DNA. Since then, the system has been well established as a
+
                Repeats
        tool for genome editing. The CRISPR/Cas system, which originates from the bacterial immune system, is
+
                (CRISPR)/Cas system to induce double-strand breaks in DNA in a programmable manner. Since then, the
        constituted
+
                system has been well established as a
        by a ribonucleoprotein complex. For class 1 CRISPR systems, the RNA is complexed by multiple Cas proteins,
+
                tool for genome editing. The CRISPR/Cas system, which originates from the bacterial immune system, is
        whereas
+
                constituted
        class 2 systems consist of a singular protein and RNA. The class 2 type II system describes all
+
                by a ribonucleoprotein complex. For class 1 CRISPR systems, an RNA guide is complexed by multiple Cas
        ribonucleoprotein
+
                proteins,
        complexes with Cas9 (Pacesa <i>et al.</i>, 2024). They include a CRISPR RNA (crRNA), which specifies the target
+
                whereas
        with a 20
+
                class 2 systems consist of a singular protein binding RNA. The class 2 type II system describes all
        nucleotide (nt) spacer sequence, and a transactivating CRISPR RNA (tracrRNA), which induces the processing by
+
                ribonucleoprotein
        the
+
                complexes with Cas9 (Pacesa <i>et al.</i>, 2024). They include a CRISPR RNA (crRNA), which specifies the
        Cas protein (Jinek <i>et al.</i>, 2012) (Fig. 2 A). Furthermore, a specific three
+
                target sequence
        nucleotide sequence (NGG) at the 3' end in the targeted DNA is needed for binding and cleavage. This is referred
+
                with a ~20
        to as the protospacer adjacent motif (PAM) (Sternberg <i>et al.</i>, 2014). The most commonly used Cas9 protein
+
                nucleotide (nt) spacer sequence, and a transactivating CRISPR RNA (tracrRNA), which induces the
        is SpCas9
+
                processing by
        or SpyCas9, which originates from Streptococcus pyogenes (Pacesa <i>et al.</i>, 2024).
+
                the
      </p>
+
                Cas protein (Jinek <i>et al.</i>, 2012) (Fig. 2 A). Furthermore, a specific three
 +
                nucleotide sequence (NGG) at the 3' end in the targeted DNA is needed for Cas9 DNA binding and cleavage.
 +
                This is referred
 +
                to as the protospacer adjacent motif (PAM) (Sternberg <i>et al.</i>, 2014). The most commonly used Cas9
 +
                protein
 +
                is SpCas9
 +
                or SpyCas9, which originates from Streptococcus pyogenes (Pacesa <i>et al.</i>, 2024).
 +
            </p>
 
<p>
 
<p>
        A significant enhancement of this system was the introduction of single guide RNAs (sgRNA[s]), which combine the
+
                A significant enhancement of the CRISPR/Cas9 system was the introduction of single guide RNAs
        functions of a tracrRNA and crRNA (Mali <i>et al.</i>, 2013).
+
                (sgRNA[s]), which combine the
        Moreover, Cong (2013) established precise targeting of human endogenous loci by designing the 20 nt spacer
+
                functions of a tracrRNA and crRNA (Jinek <i>et al.</i>, 2012; Mali <i>et al.</i>, 2013).
        sequence accordingly.
+
                Moreover, Cong <i>et al.</i> (2013) established precise targeting of human endogenous loci by designing
      </p>
+
                the 20 nt spacer
 +
                sequence accordingly.
 +
            </p>
 
</section>
 
</section>
 
<section id="2.2">
 
<section id="2.2">
 
<h2>2.2 Differences between Cas9 and Cas12a</h2>
 
<h2>2.2 Differences between Cas9 and Cas12a</h2>
 
<p>
 
<p>
        Over the following years, further CRISPR/Cas systems have been discovered, including the Cpf1 system, which has
+
                Over the following years, several additional class 2 CRISPR/Cas systems have been discovered, including
        been
+
                the Cpf1 system, which has
        classified as Cas12a since then (Zetsche <i>et al.</i>, 2015).
+
                been
        Cas12a forms a class 2 type V system with its RNA, that in comparison to the type II
+
                classified as Cas12a since then (Zetsche <i>et al.</i>, 2015).
        systems, only requires a crRNA for targeting and activation. Cas12a is capable of processing the precursor crRNA
+
                Cas12a forms a class 2 type V system. In contrast to the type II
        into crRNA independently, whereas Cas9 requires the RNase III enzyme and tracrRNA for this process (Paul and
+
                systems, the Cas12a RNA guide only requires a crRNA to mediate Cas12a DNA targeting. Moreover, Cas12a is
        Montoya, 2020). This crRNA is often also referred to as a guide RNA (gRNA). However, the stem loop that is
+
                capable of processing long precursor crRNA transcripts
        formed
+
                into several, single/independent crRNAs, whereas Cas9 requires the RNase III enzyme and tracrRNA for
        when binding the Cas protein is structurally distinct to the Cas9 gRNA and positioned on the 5' side of the
+
                this process (Paul and
        crRNA
+
                Montoya, 2020). This crRNA is often also referred to as a guide RNA (gRNA). However, the stem loop that
        (Fig. 2 B). Similarly, the PAM (TTTV) is also on the 5' side (Pacesa <i>et al.</i>,
+
                is
        2024). Cas9 possesses RuvC and HNH domains that are catalytically active, each of which cleaves one of the DNA
+
                formed
        strands at the same site, resulting in the formation of blunt end cuts (Nishimasu <i>et al.</i>, 2014). Cas12a
+
                when binding the Cas protein is structurally distinct to the Cas9 gRNA and positioned on the 5' side of
        possesses
+
                the
        one RuvC-like domain that creates staggered cuts with overhangs that are about 5nt long (Paul and Montoya,
+
                crRNA
        2020).
+
                (Fig. 2 B). Similarly, the PAM (TTTV) is also on the 5' side (Pacesa <i>et al.</i>,
      </p>
+
                2024). Cas9 possesses RuvC and HNH domains that are catalytically active, each of which cleaves one of
 +
                the DNA
 +
                strands at the same site, resulting in the formation of blunt end cuts (Nishimasu <i>et al.</i>, 2014).
 +
                Cas12a
 +
                possesses
 +
                one RuvC-like domain that creates staggered cuts with overhangs that are about 5nt long (Paul and
 +
                Montoya,
 +
                2020).
 +
            </p>
 
</section>
 
</section>
 
<section id="2.3">
 
<section id="2.3">
 
<h2>2.3 Dead Cas Proteins and their Application</h2>
 
<h2>2.3 Dead Cas Proteins and their Application</h2>
 
<p>
 
<p>
        Specific mutations of these domains result in catalytic inactivity and therefore allow for the creation of
+
                Specific mutations of these domains result in catalytic inactivity and therefore allow for the creation
        nickases that only cut one of the DNA strands, or completely inactive Cas proteins (Koonin <i>et al.</i>, 2023)
+
                of
        (Kleinstiver <i>et al.</i>, 2019). These are referred to as dead Cas proteins or dCas9 and dCas12a. These Cas
+
                nickases that only cut one of the DNA strands, or Cas protein mutants that retain their DNA binding
        proteins
+
                capability, but have no catalytic activity (Koonin <i>et al.</i>, 2023)
        can be used to activate (CRISPRa) or inhibit (CRISPRi) the expression of genes by fusing them to effector
+
                (Kleinstiver <i>et al.</i>, 2019). The latter are referred to as dead Cas proteins or dCas9 and dCas12a.
        domains
+
                These Cas
        and targeting the respective gene with the spacer sequence (Kampmann, 2017). A common approach for CRISPRa
+
                proteins
        involves fusing Cas9 with the transcriptional activator VP64 (Kampmann, 2017).
+
                can be used to activate (CRISPRa) or inhibit (CRISPRi) the expression of genes by fusing them to
      </p>
+
                effector
 +
                domains
 +
                and targeting the respective genes via complementary spacer sequences (Kampmann, 2017). A common
 +
                approach for CRISPRa
 +
                involves fusing Cas9 with the transcriptional activator, such as VP64 or VPR (Kampmann, 2017).
 +
            </p>
 
</section>
 
</section>
 
</section>
 
</section>
 
<section id="3" style="clear:both;">
 
<section id="3" style="clear:both;">
<h1>3. Assembly and part evolution</h1>
+
<h1>3. Assembly and Part Evolution</h1>
 
<p>
 
<p>
      Building on insights of our fusion Cas engineering cycle and findings from Kweon (2017), fgRNAs were designed by
+
            Building on insights of our fusion Cas engineering cycle and findings from Kweon (2017), fgRNAs were
      combining the sgRNA from SpCas9 with the crRNA from MbCas12a. Specifically the 3'-end of the MbCas12a gRNA was
+
            designed by
      linked to the 5'-end of the SpCas9 gRNA. Via this approach, the two spacer sequences are fused directly, ensuring a
+
            combining the sgRNA from SpCas9 with the crRNA from MbCas12a. Specifically the 3'-end of the MbCas12a gRNA
      minimal distance between the two DNA strands.This also facilitates efficient cloning of different spacer
+
            was
      sequences, as both spacers can be exchangeed as one consecutive sequence. Linking the crRNA and sgRNA further enables  
+
            linked to the 5'-end of the SpCas9 gRNA (through genetic fusion). With this approach, the two spacer
      multiplexing as Cas12a can inherently process gRNA repeats that are expressed from one single transcript enabling multiplexing. The entry vector includes a U6 promoter, the
+
            sequences are fused directly, ensuring a
      MbCas12a scaffold, a bacterial promoter driving <b>ccdB</b> expression, and the SpCas9 scaffold. Successful spacer
+
            minimal distance between the two DNA strands to be co-bound by the Cas staple complex. This design also facilitates
      integration leads to the removal of the <b>ccdB</b> gene, allowing bacterial growth to be used as an indicator for
+
            efficient cloning of different spacer
      cloning success.<br/>
+
            sequences, as both spacers can be obtained as one consecutive sequence encoded on a single oligo pair. Linking
      A conventional gRNA expression vector containing an MbCas12a crRNA scoffold under the control of an U6 promoter was selected as the basis
+
            the crRNA and sgRNA further enables
      for entry vector cloning. The vector and a ccdB-SpCas9 scaffold construct were PCR amplified and fitting overhangs
+
            multiplexing, as Cas12a can inherently process crRNA repeats that are expressed from one single transcript,
      for SapI were introduced (Fig. 3). Golden Gate assembly (GGA) with Esp3I was used to create the final plasmid. The
+
            enabling multiplexing. The entry vector includes a U6 promoter, the
      transformation was carried out in the ccdB-resistant XL1 Blue <i>E. Coli</i> strain.
+
            MbCas12a scaffold, a bacterial promoter driving <b>ccdB</b> expression, and the SpCas9 scaffold. Successful
    </p>
+
            spacer
 +
            integration leads to the removal of the <b>ccdB</b> gene, allowing bacterial growth to be used as an
 +
            indicator for
 +
            cloning success.<br/>
 +
            A conventional gRNA expression vector containing an MbCas12a crRNA scaffold under the control of an U6
 +
            promoter was selected as the basis
 +
            for entry vector cloning. The vector and a ccdB-SpCas9 scaffold construct were PCR amplified and fitting
 +
            overhangs
 +
            for SapI were introduced (Fig. 3). Golden Gate assembly (GGA) with Esp3I was used to create the final
 +
            plasmid. The
 +
            transformation was carried out in the ccdB-resistant XL1 Blue <i>E. Coli</i> strain.
 +
        </p>
 
<div class="thumb">
 
<div class="thumb">
 
<div class="thumbinner" style="width:80%;">
 
<div class="thumbinner" style="width:80%;">
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<div class="thumbcaption">
 
<div class="thumbcaption">
 
<i>
 
<i>
<b>Figure 3: Construction process of fgRNAs using the entry vector.</b> The ccdB gene excised using
+
<b>Figure 3: Construction Process of fgRNAs Using the Entry Vector.</b> The ccdB gene is excised
            SapI in a Golden Gate
+
                        using
            assembly. By inserting oligonucleotides with the desired spacer sequences and matching overhangs, the
+
                        SapI in a Golden Gate
            complete fgRNA
+
                        assembly reaction. Desired Cas12a and Cas9 spacer sequence combinations can be easily inserted using annealed oligonucleotides with matching
            can be assembled into the entry vector. Due to the cytotoxic nature of ccdB, only cells with the oligonucleotides as inserts
+
                        overhangs, resulting in a functional, complete fgRNA.
            survive.
+
                  Due to the cytotoxic nature of ccdB, only transformants carrying a correctly assembled fgRNA construct
          </i>
+
                        can survive, streamlining the cloning process.
 +
                    </i>
 
</div>
 
</div>
 
</div>
 
</div>
 
<p>
 
<p>
        The first goal after assembly was to prove the editing activity of both proteins using fgRNA. The genes
+
                As a first step to characterize the functionality of fgRNAs, we performed an experiment in which we simultaneously edited two fgRNA-targeted genomic sites in mammalian cells (HEK239T). The genes
        VEGFA and FANCF were selected as targets for Cas12a and Cas9, each target was tested with each Cas protein.
+
                VEGFA and FANCF were selected as targets for Cas12a and Cas9 and each target was tested with each Cas
        Editing efficiency was analyzed with the T7 Endonuclease I (T7EI) assay. Controls included crRNAs and
+
                protein using corresponding fgRNA designs.
        sgRNAs as positive controls, and non-targeting guides as negative controls. Desired spacer sequences were
+
                Editing efficiency was analyzed with the T7 Endonuclease I (T7EI) assay widely used in the CRISPR field.
        ordered as oligos, annealed, and cloned in via GGA utilizing SapI.
+
                Controls included the use of conventional crRNAs and
      </p>
+
                sgRNAs with their cognate Cas effectors as positive controls, and non-targeting guides as negative
 +
                controls. Desired spacer sequences were
 +
                ordered as synthetic oligos, annealed, and cloned in via GGA utilizing SapI.
 +
            </p>
 
<div class="thumb tright" style="margin:0;"></div>
 
<div class="thumb tright" style="margin:0;"></div>
 
<div class="thumbinner" style="width:400px;">
 
<div class="thumbinner" style="width:400px;">
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<div class="thumbcaption">
 
<div class="thumbcaption">
 
<i>
 
<i>
<b>Figure 4: Applications of the Fusion Guide RNA</b>
+
<b>Figure 4: Initial Experimental Setups to Assess the Functionality of fgRNAs</b>
              Fusion Guide RNAs can be used for multiplex genome editing by guidingactive Cas12a and Cas9 to two
+
                        Fusion Guide RNAs can be used for multiplex genome editing by simultaneously guiding catalytically active Cas12a and Cas9 to
              distinct loci. Similarly, fgRNAs allow for CRISPRa, by guiding the Cas9-VP64 transcriptional activator
+
                        two
              towards a
+
                        distinct loci. Similarly, fgRNAs allow for CRISPRa by guiding the dCas9-VP64 transcriptional
              target locus.
+
                        activator
            </i>
+
                        to a minimal promoter. These figure shows the basic experiments used for fgRNA characterization before applying it for DNA-DNA stapling (see below).
 +
                    </i>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
<table style="width:40%; margin-top:20px; margin-bottom:20px;">
+
<div style="display: flex;justify-content: left;gap: 20px; margin-top:20px; margin-bottom:20px;">
 +
<table style="width:40%; margin:0px;border-collapse: collapse;">
 
<thead>
 
<thead>
<td align="left" colspan="2">
+
<tr>
 +
<td align="left" colspan="2" style="padding: 2px; height: 40px;">
 
<b>Table 1:</b> A list of all the different spacers we cloned and tested within the fgRNA
 
<b>Table 1:</b> A list of all the different spacers we cloned and tested within the fgRNA
          </td>
+
                        </td>
 +
</tr>
 
</thead>
 
</thead>
 
<tbody>
 
<tbody>
 
<tr>
 
<tr>
<td>VEGFA</td>
+
<td style="width: 100px; padding: 2px; height: 40px; vertical-align: top;">CCR5</td>
<td>ctaggaatattgaagggggc</td>
+
<td style="word-wrap: break-word; padding: 2px; height: 40px; vertical-align: top;">
 +
                            TGACATCAATTATTATACAT
 +
                        </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
<td>FANCF</td>
+
<td style="padding: 2px; height: 40px; vertical-align: top;">Dnmt1</td>
<td>ggcggggtccagttccggga</td>
+
<td style="word-wrap: break-word; padding: 2px; height: 40px; vertical-align: top;">
 +
                            GCTCAGCAGGCACCTGCCTC
 +
                        </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
<td>CCR5</td>
+
<td style="padding: 2px; height: 40px; vertical-align: top;">Fancf</td>
<td>tgacatcaattattatacat</td>
+
<td style="word-wrap: break-word; padding: 2px; height: 40px; vertical-align: top;">
 +
                            GGCGGGGTCCAGTTCCGGGA
 +
                        </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
<td>TetO (<a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a>)</td>
+
<td style="padding: 2px; height: 40px; vertical-align: top;">Oct1 (<a href="https://parts.igem.org/Part:BBa_K5237018" target="_blank">BBa_K5237018</a>)</td>
<td>tctctatcactgatagggag</td>
+
<td style="word-wrap: break-word; padding: 2px; height: 40px; vertical-align: top;">
 +
                            ATGCAAATACTGCACTAGTG
 +
                        </td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
<td>Oct1-B (<a href="https://parts.igem.org/Part:BBa_K5237018" target="_blank">BBa_K5237018</a>)</td>
+
<td style="padding: 2px; height: 40px; vertical-align: top;">Runx1</td>
<td>atgcaaatactgcactagtg</td>
+
<td style="word-wrap: break-word; padding: 2px; height: 40px; vertical-align: top;">
 +
                            CCTTCGGAGCGAAAACCAAG
 +
                        </td>
 +
</tr>
 +
<tr>
 +
<td style="padding: 2px; height: 40px; vertical-align: top;">TetO (<a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a>)</td>
 +
<td style="word-wrap: break-word; padding: 2px; height: 40px; vertical-align: top;">
 +
                            TCTCTATCACTGATAGGGAG
 +
                        </td>
 +
</tr>
 +
<tr>
 +
<td style="padding: 2px; height: 40px; vertical-align: top;">VEGFA</td>
 +
<td style="word-wrap: break-word; padding: 2px; height: 40px; vertical-align: top;">
 +
                            CTAGGAATATTGAAGGGGGC
 +
                        </td>
 
</tr>
 
</tr>
 
</tbody>
 
</tbody>
 
</table>
 
</table>
 +
<table style="width:40%; margin:0px; border-collapse: collapse;">
 +
<thead>
 +
<tr>
 +
<td align="left" colspan="2" style="padding: 2px; height: 40px;">
 +
<b>Table 2:</b> A list of all the different linkers we cloned and tested within the fgRNA design
 +
                        </td>
 +
</tr>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td style="width: 100px; padding: 2px; height: 40px; vertical-align: top;">5 nt linker</td>
 +
<td style="word-wrap: break-word; padding: 2px; height: 40px; vertical-align: top;">ATGCG</td>
 +
</tr>
 +
<tr>
 +
<td style="padding: 2px; height: 40px; vertical-align: top;">10 nt linker</td>
 +
<td style="word-wrap: break-word; padding: 2px; height: 40px; vertical-align: top;">ATGCGAGCTG
 +
                        </td>
 +
</tr>
 +
<tr>
 +
<td style="padding: 2px; height: 40px; vertical-align: top;">10 nt Poly A linker</td>
 +
<td style="word-wrap: break-word; padding: 2px; height: 40px; vertical-align: top;">CAAAACAACA
 +
                        </td>
 +
</tr>
 +
<tr>
 +
<td style="padding: 2px; height: 40px; vertical-align: top;">20 nt linker</td>
 +
<td style="word-wrap: break-word; padding: 2px; height: 40px; vertical-align: top;">
 +
                            TGGCGGCGTGCTGACCGCTA
 +
                        </td>
 +
</tr>
 +
<tr>
 +
<td style="padding: 2px; height: 40px; vertical-align: top;">20 nt Poly A linker</td>
 +
<td style="word-wrap: break-word; padding: 2px; height: 40px; vertical-align: top;">
 +
                            CAAAACAACAATCAAAACAA
 +
                        </td>
 +
</tr>
 +
<tr>
 +
<td style="padding: 2px; height: 40px; vertical-align: top;">30 nt Poly A linker</td>
 +
<td style="word-wrap: break-word; padding: 2px; height: 40px; vertical-align: top;">
 +
                            CAAAACAACAATCAAAACAA<br/>ATCAAAACAA</td>
 +
</tr>
 +
<tr>
 +
<td style="padding: 2px; height: 40px; vertical-align: top;">40 nt Poly A linker</td>
 +
<td style="word-wrap: break-word; padding: 2px; height: 40px; vertical-align: top;">
 +
                            CAAAACAACAATCAAAACAACAAAACAA<br/>CAATCAAAACAA</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 
<p>
 
<p>
        We constructed a second entry vector incorporating an AsCas12a scaffold (5' taatttctactcttgtagat 3') instead of
+
            We constructed a second entry vector incorporating an AsCas12a scaffold (5' taatttctactcttgtagat 3') instead
        MbCas12a.
+
            of
        The sequence of the AsCas12a scaffold was the only modification in the composite part. This vector was tested
+
            MbCas12a.
        on the
+
            The sequence of the AsCas12a scaffold was the only modification present in the resulting composite part. This vector was
        loci VEGFA and FANCF to assess its functionality.
+
            tested
      </p>
+
            on the
 
+
            VEGFA and FANCF loci to assess functionality of the encoded fgRNA.
 +
        </p>
 
</section>
 
</section>
 
<section id="4">
 
<section id="4">
 
<h1>4. Results</h1>
 
<h1>4. Results</h1>
 +
In the following section, we provide a detailed quantitative characterization of part BBa_K5237000, tested under various experimental conditions and use cases.
 +
 
<section id="4.1">
 
<section id="4.1">
<h2>4.1 Editing endogenous loci with fgRNAs</h2>
+
<h2>4.1 Editing Endogenous Loci With Fusion Guide RNAs</h2>
 
<p>
 
<p>
        To prove that our fusion gRNAs still result in active ribonucleoproteins, a series of different fgRNAs were
+
                To show that our fusion gRNA design results in an active CRISPR/Cas ribonucleoprotein complex, a series of different fgRNAs
        created, each carrying spacers specific to the VEGFA and FANCF genes.HEK293-T cells were transfected with the
+
                were
        Cas
+
              cloned, each carrying spacer sequences specific to the VEGFA and FANCF target genes. HEK293T cells were then co-transfected
        protein and gRNA constructs. The editing rate was tested 72h after transfection via a T7 endonuclease I
+
                with the
        assay.<br/>
+
                Cas
 +
                protein and (f)gRNA encoding constructs. The editing rate was tested 72h after transfection via a T7 endonuclease I
 +
                assay.<br>
  
        AsCas12a and SpCas9 were used. The AsCas12a spacer targets VEGFA, while the SpCas9 spacer targets FANCF. The
+
                Here, AsCas12a and SpCas9 were used. The AsCas12a spacer, in this case, targets VEGFA, while the SpCas9 spacer targets FANCF.
        samples included standard single gRNAs with the corresponding Cas protein, the fgRNA with only one of the two
+
                Samples included either (i) conventional single gRNAs co-expressed with the corresponding Cas proteins (positive controls), (ii) the fgRNA co-expressed with only one of
        Cas
+
                the two
        proteins and the fgRNA with both Cas proteins simultaneously (Fig. 5). The sgRNAs allowed for
+
                Cas
        the highest editing rates for both genes (45% for VEGFA and 15% for FANCF), while the editing rates for FANCF
+
                proteins (as control for Cas ortholog dependency) and (iii) the fgRNA with both Cas proteins simultaneously co-expressed (Fig. 5). The conventional sgRNAs resulted in
        were
+
                potent editing ("editing" refers to the observed indel frequency) for both target genes (45% for VEGFA and 15% for FANCF). Note that editing rates for
        consistently lower in all experiments. Importantly, targeting FANCF with fgRNAs resulted in noticeable editing
+
                FANCF
        of
+
                were
        about 10%, with just the SpCas9 and both Cas proteins in the sample. For VEGFA, the AsCas12a only sample
+
                consistently lower in all experiments, which likely is due to the specific properties of the FANCF-targeted locus. Importantly, as hoped, targeting FANCF with fgRNAs resulted in noticeable
        resulted
+
                editing
        in approximately 20% editing rate in combination with the fgRNA, while adding both Cas proteins led to
+
                of
        approximately 40%. These initial results confirmed our engineering approach proving efficient genome editing
+
                about 10%, when we added the SpCas9 alone or both Cas proteins into the sample. For VEGFA, the AsCas12a only sample
        with
+
                resulted
        fgRNAs.
+
                in approximately 20% editing in combination with the fgRNA, while adding both Cas proteins led to
      </p>
+
                approximately 40%. These initial results confirmed that our fgRNA design indeed functions, enabling simultaneous recuitment of two different Cas proteins to separate loci in human cells.
 +
             
 +
            </br></p>
 
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<div class="thumbinner" style="width:60%;">
 
<div class="thumbinner" style="width:60%;">
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<i>
 
<i>
 
<b>Figure 5: fgRNAs Enable Efficient Editing of Endogenous Loci.</b>
 
<b>Figure 5: fgRNAs Enable Efficient Editing of Endogenous Loci.</b>
              The editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by
+
                            Indel rates were assessed 72h post transfection via T7EI assay. Editing % was
              measuring band
+
                            determined by
              intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band))<sup>1/2</sup>. The schematic at the
+
                            measuring band
              top shows the
+
                            intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band))<sup>1/2</sup>. The
              composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts
+
                            schematic at the
              are included in
+
                            top shows the
              each sample.
+
                            composition of each fgRNA. The symbols indicate, which Cas variants and which (f)gRNA were present in each sample.
            </i>
+
                        </i>
 
</div>
 
</div>
 
</div>
 
</div>
Line 567: Line 734:
 
<h2>4.2 Efficient Fusion Guide RNA-Mediated Editing With Various Cas Orthologs</h2>
 
<h2>4.2 Efficient Fusion Guide RNA-Mediated Editing With Various Cas Orthologs</h2>
 
<p>
 
<p>
        After showing efficient editing, the next step was to evaluate the capabilities of the fgRNAs, we tested them in combination
+
                After showing efficient editing, the next step was to evaluate the capabilities of the fgRNAs. To this end, we tested
        with different Cas12a orthologs. After some initial testing, we decided on using MbCas12a together with SpCas9,
+
                fgRNAs in combination
        because we found AsCas12a to be less active in a dual luciferase assay when co-transfected with SpCas9 compared
+
                with different Cas12a orthologs. Following some initial testing, we decided to use MbCas12a together with
        to
+
                SpCas9 in subsequent experiments,
        MbCas12a (Fig. 6). Between these two co-transfections the
+
                since MbCas12a turned out to be more effective than AsCas12a in a dual luciferase assay when co-transfected with SpCas9 (Fig. 6). Of note, SpCas9 reproducibly showed high potency.
        SpCas9 editing has not been significantly different.
+
            </p>
      </p>
+
 
<div class="thumb">
 
<div class="thumb">
 
<div class="thumbinner" style="width:60%;">
 
<div class="thumbinner" style="width:60%;">
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/engineering/cas12-decision.svg" style="width:99%;"/>
+
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/engineering/cas12-decision-v2.svg" style="width:99%;"/>
 
<div class="thumbcaption">
 
<div class="thumbcaption">
 
<i>
 
<i>
<b>Figure 6: Comparison of AsCas12a and MbCas12a with a dual luciferase assay.</b>
+
<b>Figure 6: Comparison of AsCas12a and MbCas12a with a Dual Luciferase Reporter Assay.</b>
              Firefly luminescence intensity measured 48 h after transfection. Normalized against renilla luminescence.
+
                            HEK293T cells were co-transfected with the indicated constructs and firefly and Renilla luminescence intensity was measured 48 h after transfection. Relative luciferase units correspond to firefly luciferase photon counts normalized to Renilla luciferase photon counts (Renilla luciferase serves as transfection control). Potent activity of CRISPR-Cas effectors result in knockdown of the firefly luciferase and hence smaller values.
              On the x-axis
+
                            Samples correspond to cells co-transfected with a firefly luciferase targeting fgRNA and the constructs encoding the indicated CRISPR-Cas effector (see color legend).
              the samples Cas9 + AsCas12a , Cas9 + MbCas12a, AsCas12a and MbCas12a are depicted.
+
                            Data represent the mean +/- SD from n = 3 independent experiments.
              Data is depicted as the mean +/- SD (n=3).
+
                            Statistical analysis was performed using 1way ANOVA with Tukey's multiple comparisons test.
              Statistical analysis was performed using 1way ANOVA with Tukey's multiple comparisons test. For better
+
                            ***p&lt;0.001,
              clarity, only
+
                         
              significant differences within a group between the same Cas proteins are shown.*p&lt;0.05, **p&lt;0.01,
+
                        </i>
              ***p&lt;0.001,
+
              ****p&lt;0.0001
+
            </i>
+
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
<p>
 
<p>
        Additionally, to test if the differences in editing rates from the preliminary assay resulted from the targeted
+
                Additionally, to determine whether the differences in editing rates observed in the preliminary assays were due to the specific properties of the targeted loci or the distinct characteristics of the Cas orthologs used, the spacers were tested in two configurations. In one setup, the fgRNA-encoded spacer for Cas12a targeted FANCF, while the spacer for SpCas9 targeted VEGFA; in the other, the targets were reversed.
        loci or the different Cas orthologs, the spacers were tested in both arrangements. Once with Cas12a targeting
+
                To more precisely assess the impact that the utilization
        FANCF and SpCas9 targeting VEGFA and once vice versa. To better assess the impact that the utilization of a
+
                of a
        fgRNA
+
                fgRNA design
        has on the editing rates, the sgRNAs were tested separately and in one sample.<br/>
+
                has on the editing rates, conventional crRNAs/sgRNAs were tested separately or, alternatively, combined in one sample.<br>
        Having the sgRNA with single Cas
+
                Combining a conventional crRNA/sgRNAs with the individual Cas proteins in the same sample showed no significant difference in editing rates (Fig. 7) as compared to using them in separate samples. While the fgRNA led to an overall lower editing rate as compared to the conventional guide RNAs, editing was still clearly noticeable for both targeted loci, indicating that the fgRNA works in principle. While editing efficiency for VEGFA remained consistently around 20%, the editing rate for FANCF dropped significantly, but was still detecable. Under identical conditions, MbCas12a consistently exhibited lower editing rates compared to SpCas9 when targeting the same gene, which is expected based on experience with this Cas orthologs in the CRISPR field.
        proteins in the same sample resulted in no clear difference in the editing rates (Fig. 7). The fusion of the
+
            </br></p>
        gRNAs resulted in a lower editing rate overall. While the editing for VEGFA
+
        stayed at about 20% in all cases, the editing for FANCF dropped significantly. When targeting the same gene
+
        under
+
        the same conditions, the editing rates for MbCas12a were overall lower than the ones from SpCas9.
+
      </p>
+
 
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<div class="thumbcaption">
 
<i>
 
<i>
<b>Figure 7: Fusion gRNA Editing Rates In Combination with MbCas12a.</b>
+
<b>Figure 7: FgRNA-mediated dual genome editing with SpCas9 and MbCas12a.</b>
              In <b>A</b> and <b>B</b> the editing rates were determined 72h after transfection via T7EI
+
                            In <b>A</b> and <b>B</b> the editing rates were determined 72h post transfection via T7EI
              assay. Editing % was determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved
+
                            assay. Editing % was determined by measuring band T7 cleavage band intensities as explained above. The schematic at the top shows the composition of the fgRNA. Below each
              band/uncleaved band)
+
                            spacer, the target gene is indicated. The
              <sup>1/2</sup>). The schematic at the top shows the composition of the fgRNA. Below each spacer is the
+
                            symbols below indicate which parts are included in each sample. The fgRNA in <b>A</b> and <b>B</b>
              targeted gene. The
+
                            differs by the order of used spacer sequences (FANCF-VEGFA vs VEGFA-FANCF).
              symbols below indicate which parts are included in each sample. <b>A</b> and <b>B</b>
+
                        </i>
              display both orientations of the two spacers for VEGFA and FANCF.
+
            </i>
+
 
</div>
 
</div>
 
</div>
 
</div>
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</section>
 
</section>
 
<section id="4.3">
 
<section id="4.3">
<h2>4.3 The Inclusion of a Linker Does Not Lower Editing Rates</h2>
+
<h2>4.3 Fusion Guide RNAs are Compatible with Linkers of Various lengths</h2>
 
<p>
 
<p>
        To further assess the effect of the genomic locus on the editing rate, we included CCR5 as an additional
+
                To further assess the effect of the genomic locus on the editing rate, we included CCR5 as an additional
        gene target. For this assay, a fgRNA with a 20 nt long linker was included between the two spacers. The editing
+
                gene target. For this assay, a fgRNA with a 20 nt long linker sequence between the two spacers was included. As above, fgRNA-mediated editing was assessed via T7E1 assay in HEK293T cells. The
        rate for VEGFA was again relatively consistent throughout the samples (Fig. 8). For CCR5, the
+
                editing
        editing rate with sgRNAs was approximately the same at about 30%. However, it dropped below 10% for the fgRNA.
+
                rate for VEGFA was again relatively consistent throughout the samples (Fig. 8). For CCR5, the
        The
+
                editing rate with the conventional sgRNAs was in a similar range as that of VEGFA (at about 30%), but dropped to below 10% in the
        addition of the 20 nt linker had no effect on the editing rates compared to no linker.
+
                fgRNA sample.
      </p>
+
                The
 +
                addition of the 20 nt linker had no effect on the editing rates compared to no linker.
 +
            </p>
 
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<div class="thumbcaption">
 
<i>
 
<i>
<b>Figure 8: Fusion gRNA Editing Rates for Multiplexing CCR5 and VEGFA</b>
+
<b>Figure 8: Fusion gRNAs with A 20 Nucleotide Spacer Still Mediate Editing For CCR5 and VEGFA</b>
              The editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by
+
                            HEK293T cells were co-transfected with the indicated components followed by assessing editing rates 72h post transfection via T7EI assay. Editing % was
              measuring band
+
                            determined as described above. The
              intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band))<sup>1/2</sup>. The schematic at the
+
                            schematic at the
              top shows the
+
                            top shows the
              composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts
+
                            composition of the fgRNA. Below each spacer, the targeted gene is indicated. The symbols below indicate
              are included in
+
                            which parts
              each sample. Cas12a targets VEGFA and Cas9 targets CCR5.
+
                            are included in
            </i>
+
                            each sample. Cas12a targets VEGFA and Cas9 targets CCR5 in this case.
 +
                        </i>
 
</div>
 
</div>
 
</div>
 
</div>
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</section>
 
</section>
 
<section id="4.4">
 
<section id="4.4">
<h2>4.4 fgRNAs can be used for CRISPRa</h2>
+
<h2>4.4 Fusion Guide RNAs Enable Efficient Activation of Gene Expression via CRISPRa</h2>
 
<p>
 
<p>
        To establish the foundation for their use as protein scaffolds, we identified the next step as demonstrating the
+
The data above provide manifold evidence that fgRNAs can indeed be employed for simultaneous genome editing at two defined loci with catalytically active Cas9 and Cas12a.             
        use
+
Next, to establish the foundation for the use of fgRNAs as scaffold for Cas-staple protein assembly and hence 3D genome enineering, we next combined fgRNAs with catalytically dead Cas9 and -12a in an CRISPRa (CRISPR activation of gene expression) experiment. For this, we intended to recruit the transcriptional activator VP64 to a
        of fgRNAs for CRISPR activation. For this, we intend to recruit the transcriptional activator VP64 to a firefly
+
                firefly
        luciferase gene to induce expression. The VP64 protein is attached to the catalytically inactive Cas9 protein,
+
                luciferase reporter gene to induce expression. The VP64 protein is genetically fused to the catalytically inactive Cas9
        which is then guided by gRNAs to the luciferase gene. The gRNAs target a TetO sequence, which is positioned in
+
                protein,
        front of the luciferase gene in multiple repeats. The firefly luciferase activity was then quantified as photon
+
                which is then guided by gRNAs to the luciferase-driving minimal promoter. More specifically, the gRNAs here target a TetO repeat sequence, which is
        counts and normalized against Renilla luciferase, which is expressed on a separate plasmid under an ubiquitous
+
                positioned 5' of a minimal tata site. The luciferase reporter activity was then quantified and
        promoter. In two biological replicates we saw similar Relative luciferase activity with fgRNA as a guide
+
                photon
        compared
+
                counts for Firefly luciferase (to be activated by CRISPRa) were normalized to the photon counts of Renilla luciferase, which is expressed on a separate plasmid under an
        to a sgRNA (Fig. 9).
+
                ubiquitous
      </p>
+
                promoter (transfection control). In two biological replicates we saw similar relative luciferase activity with fgRNA as a guide
 +
                compared
 +
                to a sgRNA (Fig. 9).
 +
            </p>
 
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<div class="thumbcaption">
 
<i>
 
<i>
<b>Figure 9: CRISPRa Induced Luciferase Expression for sgRNAs and fgRNAs.</b>
+
<b>Figure 9: The Efficacy of CRISPRa is Comparable Between Our fgRNAs and Conventional sgRNAs.</b>
              Firefly luciferase activity was measured 48h after transfection. Normalized against ubiquitously expressed
+
The schematic at the top shows the composition
              Renilla
+
                            of the fgRNA. Symbols show which parts were co-expressed in each sample. The empty spacer control is a negative control with a gRNA lacking the TetO-targeting spacer, thus preventing binding of dCas9-VP64 to the Firefly-luciferase driving promoter.                           
              luciferase. The tetO repeats were targeted by Cas9-VP64, once with a sgRNA and once with a fgRNA that had
+
Firefly luciferase activity was measured 48h post transfection and, as above, normalized to an
              a
+
                            ubiquitously expressed
              non-targeting sequence for the Cas12a spacer. The schematic at the top shows the composition of the fgRNA.
+
                            Renilla
              Below each
+
                            luciferase (transfection control). The tetO repeats were targeted by Cas9-VP64, once with a sgRNA (center) and once with a
              spacer is the targeted gene. The symbols below indicate which parts are included in each sample.
+
                            fgRNA (right) that carried
            </i>
+
                            a
 +
                            non-targeting spacer sequence for Cas12a.  
 +
                           
 +
                        </i>
 
</div>
 
</div>
 
</div>
 
</div>
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</section>
 
</section>
 
<section id="4.5">
 
<section id="4.5">
<h2>4.5 Stapling Two DNA Strands Together Using fgRNAs</h2>
+
<h2>4.5 A Proof-Of-Concept for 3D Genome Engineering: Stapling Two DNA Strands Together In Human Cells Using fgRNAs</h2>
 
<p>
 
<p>
        After showing the general capability of the fgRNA
+
                Having demonstrated the excellent compatibility of the fgRNA design with simultaneous genome editing at two distinct loci and CRISPR activation, we set out to provide a proof-of-concept for engineering 3D genome conformation in living cells. To achieve this, we designed an experiment to "staple together" two regulatory elements—a synthetic enhancer and a minimal promoter—located on different strands of DNA. We developed a two-plasmid system consisting of an enhancer plasmid and a reporter plasmid. The reporter plasmid encodes firefly luciferase driven by a minimal promoter containing several repeats of a Cas9-targeted sequence, while the enhancer plasmid includes a Gal4 binding site (UAS) positioned next to several repeats of a Cas12a-targeted sequence.
        to work for editing and for CRISPR activation, the next step was to use it to staple two DNA loci together, and
+
 
        thereby induce proximity between two separate functional elements. For this, an enhancer plasmid and a reporter
+
We co-expressed these two plasmids with dCas9, dCas12, an fgRNA simultaneously co-targeting (i.e., tethering) the reporter and enhancer plasmids, and Gal4-VP64. This resulted in the expression of luciferase (Fig. 10, Panel A). Different linker lengths (as mentioned above) were tested for the fgRNA. Firefly luciferase values were normalized against ubiquitous renilla expression as a transfection control.
        plasmid was used. The reporter plasmid has firefly luciferase behind several repeats of a Cas9 targeted
+
 
        sequence.
+
Using no linker between the two spacers produced luciferase activity values similar to the baseline control (i.e., no reporter activation) (Fig. 10, Panel B). Excitingly, however, the stepwise extension of the fgRNA linker (i.e., the linker separating the Cas12a and Cas9 spacer elements) from 20 nt to 40 nt led to progressively increasing, and ultimately potent, luciferase reporter activation. This experiment provided the first demonstration that an fgRNA with an optimized linker can effectively "staple" two otherwise separate pieces of DNA. As this experimental readout—mediated by the spatial proximity of two separate regulatory elements—mimics naturally occurring enhancer hijacking events, we refer to this setup as "synthetic enhancer hijacking."
        The enhancer plasmid has a Gal4 binding site behind several repeats of a Cas12a targeted sequence. By
+
 
        introducing
+
 
        a fgRNA staple and a Gal4-VP64, expression of the luciferase is induced (Fig. 10, Panel A).
+
            </p>
        Different linker lengths were tested. Cells were again normalized against ubiquitous renilla expression.<br/>
+
        Using no linker between the two spacers showed similar relative luciferase activity to the baseline control
+
        (Fig. 10, Panel B). An extension of the linker from 20 nt up to 40 nt resulted in an increasingly higher
+
        expression of the
+
        reporter gene. These results suggest an extension of the linker might lead to better transactivation when
+
        hijacking an enhancer/activator.
+
      </p>
+
 
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<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.
+
<b>Figure 10: Applying Fusion Guide RNAs for Cas staples: Proof-Of-Concept 3D Genome Engineering Via Synthetic Enhancer Hijacking.</b> <b>A</b>, schematic overview of the assay is shown. An enhancer plasmid and a reporter plasmid are brought into proximity by an fgRNA-Cas staple complex, which co-targets and tethers both plasmids. Target sequences were included in multiple repeats upstream of the functional elements. Firefly luciferase serves as the reporter gene, while the enhancer is composed of multiple Gal4 repeats bound by a Gal4-VP64 fusion protein.
              An enhancer
+
                            <b>B</b>, HEK293T cells were co-transfected with the reporter plasmid, the enhancer plasmid, a Gal4-VP64-encoding plasmid as well dCas9 and dCas12. Firefly luciferase activity was measured 48 hours post transfection and normalized to ubiquitously expressed Renilla luciferase. Observed differences were tested for statistical significance using ordinary one-way ANOVA with Dunn's method for multiple comparisons. (*p &lt;
              plasmid and a reporter plasmid are brought into proximity by a fgRNA Cas staple complex binding both
+
                            0.05; **p &lt; 0.01; ***p &lt; 0.001; mean +/- SD from n = 3 independent experiments). The assay included sgRNAs and fgRNAs
              plasmids. Target
+
                            with linker
              sequences were included in multiple repeats prior to the functional elements. Firefly luciferase serves as
+
                            lengths varying between 0 nt
              the reporter
+
                            to 40 nt as indicated.
              gene, the enhancer is constituted by multiple Gal4 repeats that are bound by a Gal4-VP64 fusion.
+
                        </i>
              <b>B</b>, results of using a fgRNA Cas staple for trans activation of firefly luciferase. Firefly
+
              luciferase activity was measured 48h after transfection. Normalized against ubiquitously expressed Renilla
+
              luciferase.
+
              Statistical significance was calculated with ordinary One-way ANOVA with Dunn's method for multiple
+
              comparisons (*p &lt;
+
              0.05; **p &lt; 0.01; ***p &lt; 0.001; mean +/- SD). The assay included sgRNAs and fgRNAs with linker
+
              lengths from 0 nt
+
              to 40 nt.
+
            </i>
+
 
</div>
 
</div>
 
</div>
 
</div>
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</section>
 
</section>
 
<section id="5">
 
<section id="5">
<h1>5. References</h1>
+
    <h1>5. <i>In Silico</i> Characterization using DaVinci</h1>
<p>Aregger, M., Xing, K., &amp; Gonatopoulos-Pournatzis, T. (2021). Application of CHyMErA Cas9-Cas12a combinatorial
+
            <p>
      genome-editing platform for genetic interaction mapping and gene fragment deletion screening. <i>Nature
+
                We developed the <i>in silico</i> model <a href="https://2024.igem.wiki/heidelberg/model" target="_blank">DaVinci</a> for rapid engineering and development of our PICasSO
        Protocols</i>, 16, 4722-4765. <a href="https://doi.org/10.1038/s41596-021-00595-1" target="_blank">https://doi.org/10.1038/s41596-021-00595-1</a></p>
+
                system. DaVinci acts as a digital twin to PICasSO, designed to understand the forces acting on our
<p>Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L.
+
                system, refine experimental parameters, and find optimal connections between protein staples and
      A., &amp; Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. <i>Science</i>, 339, 819-823.
+
                target DNA.<br>
      <a href="https://doi.org/10.1126/science.1231143" target="_blank">https://doi.org/10.1126/science.1231143</a></p>
+
                We calibrated DaVinci with literature and our own experimental affinity data obtained via EMSA
<p>Gonatopoulos-Pournatzis, T., Aregger, M., Brown, K. R., Farhangmehr, S., Braunschweig, U., Ward, H. N., Ha, K. C.
+
                assays and purified proteins. This enabled us to simulate enhancer hijacking <i>in silico</i>, providing
      H., Weiss, A., Billmann, M., Durbic, T., Myers, C. L., Blencowe, B. J., &amp; Moffat, J. (2020). Genetic
+
                valuable input for the design of further experiments. Additionally, we apply the same approach to
      interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform. <i>Nature
+
                our part collection. <br><br>
        Biotechnology</i>, 38, 638-648. <a href="https://doi.org/10.1038/s41587-020-0437-z" target="_blank">https://doi.org/10.1038/s41587-020-0437-z</a></p>
+
                DaVinci is divided into three phases: static structure prediction, all-atom dynamics simulation, and
<p>Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., &amp; Charpentier, E. (2012). A programmable
+
                long-ranged dna dynamics simulation. Here, we applied the last step to our parts, characterizing
      dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. <i>Science</i>, 337, 816-821. <a href="https://doi.org/10.1126/science.1225829" target="_blank">https://doi.org/10.1126/science.1225829</a></p>
+
                structure and dynamics of the dna-binding interaction.
<p>Kampmann, M. (2017). CRISPRi and CRISPRa screens in mammalian cells for precision biology and medicine. <i>ACS
+
            </p>
        Chemical Biology</i>, 13, 406-416. <a href="https://doi.org/10.1021/acschembio.7b00657" target="_blank">https://doi.org/10.1021/acschembio.7b00657</a></p>
+
            <section id="5.1">
<p>Kleinstiver, B. P., Sousa, A. A., Walton, R. T., Tak, Y. E., Hsu, J. Y., Clement, K., Welch, M. M., Horng, J. E.,
+
                <h2>5.1. Enhancer Hijacking is Successfully Studied <i>In Silico</i></h2>
      Malagon-Lopez, J., Scarfò, I., Maus, M. V., Pinello, L., Aryee, M. J., &amp; Joung, J. K. (2019). Engineered
+
                        <div class="thumb tright" style="margin:0;">
      CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base
+
                            <div class="thumbinner" style="width:300px;">
      editing. <i>Nature Biotechnology</i>, 37, 276-282. <a href="https://doi.org/10.1038/s41587-018-0011-0" target="_blank">https://doi.org/10.1038/s41587-018-0011-0</a></p>
+
                                <img src="https://static.igem.wiki/teams/5237/figures-corrected/drylab-plasmids-cas-staple-fgrna.svg"
<p>Koonin, E. V., Gootenberg, J. S., &amp; Abudayyeh, O. O. (2023). Discovery of diverse CRISPR-Cas systems and
+
                                    class="thumbimage" style="width:99%;">
      expansion of the genome engineering toolbox. <i>Biochemistry</i>, 62, 3465-3487. <a href="https://doi.org/10.1021/acs.biochem.3c00159" target="_blank">https://doi.org/10.1021/acs.biochem.3c00159</a></p>
+
                                <div class="thumbcaption">
<p>Kweon, J., Jang, A.-H., Kim, D.-e., Yang, J. W., Yoon, M., Rim Shin, H., Kim, J.-S., &amp; Kim, Y. (2017). Fusion
+
                                    <i>
      guide RNAs for orthogonal gene manipulation with Cas9 and Cpf1. <i>Nature Communications</i>, 8. <a href="https://doi.org/10.1038/s41467-017-01650-w" target="_blank">https://doi.org/10.1038/s41467-017-01650-w</a>
+
                                        <b>Figure 11: Cas stapled plasmids.</b>
 +
                                    </i>
 +
                                </div>
 +
                            </div>
 +
                        </div>
 +
                        <p>
 +
                            With the Cas staple, we aimed to simulate the principles of enhancer hijacking
 +
                            experiments we
 +
                            conducted in the lab. For these experiments, we modeled the two plasmids also used in
 +
                            the wet lab
 +
                            (<a href="https://parts.igem.org/Part:BBa_K5237023" target="_blank">BBa_K5237023</a> and
 +
                            <a href="https://parts.igem.org/Part:BBa_K5237024" target="_blank">BBa_K5237024</a>). On
 +
                            top of the
 +
                            two plasmids, we modeled the Cas staple by applying a “DNA-DNA tethering force"
 +
                            throughout our
 +
                            simulation, selectively on the regions targeted by the fgRNA. This force was based on
 +
                            simulation
 +
                            data acquired in earlier phases of DaVinci. As there is no suitable model available that
 +
                            also
 +
                            simulates proteins, this proved to be the most effective modeling strategy.
 +
                        </p>
 +
 
 +
                        <p>
 +
                            Our simulation showed the expected behavior, holding the target sequences of the Cas
 +
                            staple (Fig. 12).
 +
                            Overall, these exciting results demonstrate that we can successfully model the core
 +
                            principles of
 +
                            enhancer hijacking with a total of 20 thousand simulated nucleotides <i>in silico</i>.
 +
                        </p>
 +
 
 +
                        <div class="thumb" style="width:50%;">
 +
                            <div class="thumbinner" style="position:center; padding-bottom:56.25%; height:257px;">
 +
                                <iframe title="Heidelberg: predicted_fgRNA_long_slow (2024)"
 +
                                    src="https://video.igem.org/videos/embed/db213b54-039e-42ca-aa29-c70614172e49?title=0&amp;warningTitle=0"
 +
                                    frameborder="0" allowfullscreen="" sandbox="allow-same-origin allow-scripts allow-popups allow-forms"
 +
                                    style="width:100%; height:100%;"
 +
                                    class="thumbimage">
 +
                                </iframe>
 +
                            </div>
 +
                            <div class="thumbcaption">
 +
                            <i>
 +
                                <b>Figure 12: Fusion Cas staples hold stapled nucleotides in close proximity.</b>
 +
                            </i>
 +
                            </div>
 +
                        </div>
 +
            </section>
 +
 
 +
            <section id="5.2">
 +
                <h2>5.2. Cas Staple Forces do not Disturb DNA Strand Integrity</h2>
 +
                <div class="thumb tright">
 +
                    <div class="thumbinner" style="width:300px;">
 +
                        <img src="https://static.igem.wiki/teams/5237/figures-corrected/drylab-plasmids-cas-staple-fgrna.svg"
 +
                            class="thumbimage" style="width:99%;">
 +
                        <div class="thumbcaption">
 +
                            <i>
 +
                                <b>Figure 13: Cas stapled plasmids.</b>
 +
                            </i>
 +
                        </div>
 +
                    </div>
 +
                </div>
 +
                <p>
 +
                    Next, we aimed to stress test our system to determine the amount of force required to induce DNA
 +
                    double-strand breaks. To achieve this, we used an identical setup to the previous experiment but
 +
                    instead of
 +
                    experimentally determined forces, we used artificial forces of varying strength.
 +
                    It is important to know that our <i>in silico</i> model responds to forces that cause double-strand
 +
                    breaks by
 +
                    scattering the nucleotides across the simulation box. As the specified bonds cannot actually
 +
                    break within
 +
                    the simulation, the phosphate backbone bonds become severely distorted and exhibit erratic
 +
                    behavior in the
 +
                    simulation videos. From our extensive simulations, we concluded that the forces required to damage the DNA are approximately 320 times (Fig. 15) and 680 times greater (Fig. 16) than the forces exerted by the Cas staple, respectively.
 +
                    <br><br>
 +
                    This provides important evidence regarding
 +
                    the safety of
 +
                    our staple approach for 3D genome engineering: According to our model, DNA-DNA tethering with
 +
                    our Cas
 +
                    staples is not expected to have a negative effect on the DNA stability.
 +
                </p>
 +
               
 +
 
 +
                  <div style="display: grid; grid-template-columns: repeat(2, 1fr); gap: 10px; overflow: auto;">
 +
                      <!-- First Video -->
 +
                      <div class="thumb">
 +
                          <div class="thumbinner">
 +
                              <iframe title="Heidelberg: fgRNA_neg_272x_slow (2024)" src="https://video.igem.org/videos/embed/7ea11707-ace8-44b0-9b6a-6e623474bc0a?title=0&warningTitle=0" allowfullscreen="" sandbox="allow-same-origin allow-scripts allow-popups allow-forms" style="width: 100%; aspect-ratio: 16/9;"></iframe>
 +
                          </div>
 +
                          <div class="thumbcaption">
 +
                              <i><b>Figure 14: Applying a force that is 270 times greater <br> than the predicted force typically exerted by a Cas staple on DNA.</b></i>
 +
                          </div>
 +
                      </div>
 +
                 
 +
                      <!-- Second Video -->
 +
                      <div class="thumb">
 +
                          <div class="thumbinner">
 +
                              <iframe title="Heidelberg: fgRNA_neg_318x_slow (2024)" src="https://video.igem.org/videos/embed/66db402a-c94a-4aad-b30b-386b8ccc20b0?title=0&warningTitle=0" allowfullscreen="" sandbox="allow-same-origin allow-scripts allow-popups allow-forms" style="width: 100%; aspect-ratio: 16/9;"></iframe>
 +
                          </div>
 +
                          <div class="thumbcaption">
 +
                              <i><b>Figure 15: Applying a force that is 320 times greater <br> than the predicted force typically exerted by a Cas staple on DNA.</b></i>
 +
                          </div>
 +
                      </div>
 +
                 
 +
                      <!-- Third Video -->
 +
                      <div class="thumb">
 +
                          <div class="thumbinner">
 +
                              <iframe title="Heidelberg: fgRNA_neg_681x_slow (2024)" src="https://video.igem.org/videos/embed/5e6240ab-1057-4db6-a992-22a17d2fcc55?title=0&warningTitle=0" allowfullscreen="" sandbox="allow-same-origin allow-scripts allow-popups allow-forms" style="width: 100%; aspect-ratio: 16/9;"></iframe>
 +
                          </div>
 +
                          <div class="thumbcaption">
 +
                              <i><b>Figure 16: Applying a force that is 680 times greater <br> than the predicted force typically exerted by a Cas staple on DNA.</b></i>
 +
                          </div>
 +
                      </div>
 +
                 
 +
                      <!-- Fourth Video -->
 +
                      <div class="thumb">
 +
                          <div class="thumbinner">
 +
                              <iframe title="Heidelberg: fgRNA_neg_1000x_slow (2024)" src="https://video.igem.org/videos/embed/26f9ba1c-51b9-4931-b1d3-8129ff3a57a7?title=0&warningTitle=0" allowfullscreen="" sandbox="allow-same-origin allow-scripts allow-popups allow-forms" style="width: 100%; aspect-ratio: 16/9;"></iframe>
 +
                          </div>
 +
                          <div class="thumbcaption">
 +
                              <i><b>Figure 17: Applying a force that is more than 1000 times greater <br> than the predicted force typically exerted by a Cas staple on DNA.</b></i>
 +
                          </div>
 +
                      </div>
 +
                  </div>
 +
            </section>
 +
 
 +
            <section id="5.3">
 +
                <h2>5.3. DaVinci Helps to Design Multi-Staple Arrangements</h2>
 +
 
 +
                <div class="thumb tright">
 +
                    <div class="thumbinner" style="width:300px;">
 +
                        <img src="https://static.igem.wiki/teams/5237/figures-corrected/drylab-plasmids-multiplexing-broken-cas-staple-fgrna-correct.svg"
 +
                            class="thumbimage" style="width:99%;">
 +
                        <div class="thumbcaption">
 +
                            <i>
 +
                                <b>Figure 18: Double Cas stapling within 40 nucleotide distance induces
 +
                                    double-strand
 +
                                    breaks.</b>
 +
                            </i>
 +
                        </div>
 +
                    </div>
 +
                </div>
 +
                <p>
 +
                    Finally, we simulated the behavior of multiple staples at once. Therefore, we expanded our
 +
                    previously
 +
                    introduced experimental setup by a second Cas staple.<br>
 +
                    In a first approach, we targeted an additional region next to the original chosen one. This
 +
                    region is 40
 +
                    nucleotides away from the first target region on the plasmid displayed in blue connecting it to
 +
                    the opposite
 +
                    site of the orange plasmid, therefore bringing both ends of the orange plasmid together (Fig. 18).<br>
 +
                    Our simulation predicted that with these staple points, DNA double-strand breaks would occur as clearly visible by the scattered nucleotides (Fig. 19).
 +
                </p>
 +
 
 +
 
 +
 
 +
 
 +
                <div class="thumb" style="width:50%;">
 +
                    <div class="thumbinner" style="position:center; padding-bottom:56.25%; height:257px;">
 +
                        <iframe title="Heidelberg: multiplex_pos_40nt_slow (2024)"
 +
                            src="https://video.igem.org/videos/embed/2d153686-202b-414f-9abd-20835110953c?title=0&amp;warningTitle=0"
 +
                            frameborder="0" allowfullscreen="" sandbox="allow-same-origin allow-scripts allow-popups allow-forms"
 +
                            style="width:100%; height:100%;"
 +
                            class="thumbimage">
 +
                        </iframe>
 +
                    </div>
 +
                    <div class="thumbcaption">
 +
                    <i>
 +
                        <b>Figure 19: Double Cas stapling within 40 nucleotide distance induces
 +
                            double-strand
 +
                            breaks.</b>
 +
                    </i>
 +
                    </div>
 +
                </div>
 +
 
 +
 
 +
                <div class="thumb tright">
 +
                    <div class="thumbinner" style="width:300px;">
 +
                        <img  src="https://static.igem.wiki/teams/5237/figures-corrected/drylab-plasmids-multiplexing-notbroken-cas-staple-fgrna-correct2.svg"
 +
                            class="thumbimage" style="width:99%;">
 +
                        <div class="thumbcaption">
 +
                            <i>
 +
                                <b>Figure 20: Stable multiplexing with 2 Cas staples.</b> On the blue plasmid, the
 +
                                Cas binding
 +
                                sequences are 980 nucleotides apart.</b>
 +
                            </i>
 +
                        </div>
 +
                    </div>
 +
                </div>
 +
 
 +
                <p>
 +
                    To simulate a setup where we expect no double-strand breaks, we increased the distance between
 +
                    the stapling
 +
                    sites on the blue plasmid (<a href="https://parts.igem.org/Part:BBa_K5237023"
 +
                        target="_blank">BBa_K5237023</a>) from 40 to 980 nucleotides (Fig. 20).<br>
 +
                    With this increased distance between stapling sites, we observed a stabilized system. Most
 +
                    interestingly,
 +
                    the non-stapled regions showed maximum distances close to 500 nm, indicating that the two
 +
                    staples led to
 +
                    more compact plasmid structures.
 +
                    <br><br>
 +
                    In conclusion, we show that applying multiple staples on the same structures can lead to
 +
                    double-strand
 +
                    breaks if the staples are positioned closely to one another. However, increasing the separation
 +
                    of staples
 +
                    leads to a stable system without DNA damage. Thus, it is definitely possible to multiplex Cas
 +
                    staples,
 +
                    thereby increasing the compactness of DNA structures (Fig. 21). This shows the potential of creating
 +
                    complex
 +
                    regulatory networks by bringing genetic elements into spatial proximity with a multitude of Cas
 +
                    protein
 +
                    staples.
 +
                </p>
 +
 
 +
 
 +
                <div class="thumb" style="width:50%;">
 +
                    <div class="thumbinner" style="position:center; padding-bottom:56.25%; height:257px;">
 +
                        <iframe title="Heidelberg: multiplex_pos_980nt_slow (2024)"
 +
                            src="https://video.igem.org/videos/embed/777bd304-e6a0-4457-9f57-29637b7b5436?title=0&amp;warningTitle=0"
 +
                            frameborder="0" allowfullscreen="" sandbox="allow-same-origin allow-scripts allow-popups allow-forms"
 +
                            style="width:100%; height:100%;"
 +
                            class="thumbimage">
 +
                        </iframe>
 +
                    </div>
 +
                    <div class="thumbcaption">
 +
                    <i>
 +
                        <b>Figure 21: Increasing the distance between Cas staple targets to 980 nucleotides
 +
                            stabilizes
 +
                            multiplexing.</b>
 +
                    </i>
 +
                    </div>
 +
                </div>
 +
            </section>
 +
  </section>
 +
<section id="6">
 +
<h1>6. References</h1>
 +
<p>Aregger, M., Xing, K., &amp; Gonatopoulos-Pournatzis, T. (2021). Application of CHyMErA
 +
            Cas9-Cas12a
 +
            combinatorial
 +
            genome-editing platform for genetic interaction mapping and gene fragment deletion screening.
 +
            <i>Nature
 +
                Protocols</i>, 16, 4722-4765. <a href="https://doi.org/10.1038/s41596-021-00595-1" target="_blank">https://doi.org/10.1038/s41596-021-00595-1</a>
 +
</p>
 +
<p>Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W.,
 +
            Marraffini,
 +
            L.
 +
            A., &amp; Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems.
 +
            <i>Science</i>, 339,
 +
            819-823.
 +
            <a href="https://doi.org/10.1126/science.1231143" target="_blank">https://doi.org/10.1126/science.1231143</a>
 +
</p>
 +
<p>Gonatopoulos-Pournatzis, T., Aregger, M., Brown, K. R., Farhangmehr, S., Braunschweig, U., Ward,
 +
            H. N., Ha,
 +
            K. C.
 +
            H., Weiss, A., Billmann, M., Durbic, T., Myers, C. L., Blencowe, B. J., &amp; Moffat, J. (2020).
 +
            Genetic
 +
            interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform.
 +
            <i>Nature
 +
                Biotechnology</i>, 38, 638-648. <a href="https://doi.org/10.1038/s41587-020-0437-z" target="_blank">https://doi.org/10.1038/s41587-020-0437-z</a>
 +
</p>
 +
<p>Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., &amp; Charpentier, E. (2012). A
 +
            programmable
 +
            dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. <i>Science</i>, 337, 816-821.
 +
            <a href="https://doi.org/10.1126/science.1225829" target="_blank">https://doi.org/10.1126/science.1225829</a>
 +
</p>
 +
<p>Kampmann, M. (2017). CRISPRi and CRISPRa screens in mammalian cells for precision biology and
 +
            medicine.
 +
            <i>ACS
 +
                Chemical Biology</i>, 13, 406-416. <a href="https://doi.org/10.1021/acschembio.7b00657" target="_blank">https://doi.org/10.1021/acschembio.7b00657</a>
 +
</p>
 +
<p>Kleinstiver, B. P., Sousa, A. A., Walton, R. T., Tak, Y. E., Hsu, J. Y., Clement, K., Welch, M.
 +
            M., Horng, J.
 +
            E.,
 +
            Malagon-Lopez, J., Scarfò, I., Maus, M. V., Pinello, L., Aryee, M. J., &amp; Joung, J. K.
 +
            (2019). Engineered
 +
            CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene,
 +
            epigenetic and base
 +
            editing. <i>Nature Biotechnology</i>, 37, 276-282. <a href="https://doi.org/10.1038/s41587-018-0011-0" target="_blank">https://doi.org/10.1038/s41587-018-0011-0</a></p>
 +
<p>Koonin, E. V., Gootenberg, J. S., &amp; Abudayyeh, O. O. (2023). Discovery of diverse CRISPR-Cas
 +
            systems and
 +
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<p>Kweon, J., Jang, A.-H., Kim, D.-e., Yang, J. W., Yoon, M., Rim Shin, H., Kim, J.-S., &amp; Kim,
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            Y. (2017).
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            Fusion
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            guide RNAs for orthogonal gene manipulation with Cas9 and Cpf1. <i>Nature Communications</i>, 8.
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            <a href="https://doi.org/10.1038/s41467-017-01650-w" target="_blank">https://doi.org/10.1038/s41467-017-01650-w</a>
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</p>
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<p>Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E., &amp;
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            Church, G. M.
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 +
<p>Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I., Dohmae, N., Ishitani, R.,
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            Zhang, F.,
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            &amp;
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            Nureki, O. (2014). Crystal structure of Cas9 in complex with guide RNA and target DNA.
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            <i>Cell</i>, 156,
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<p>Pacesa, M., Pelea, O., &amp; Jinek, M. (2024). Past, present, and future of CRISPR genome editing
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            technologies.
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            <i>Cell</i>, 187, 1076-1100. <a href="https://doi.org/10.1016/j.cell.2024.01.042" target="_blank">https://doi.org/10.1016/j.cell.2024.01.042</a>
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<p>Paul, B., &amp; Montoya, G. (2020). CRISPR-Cas12a: Functional overview and applications.
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                Journal</i>, 43, 8-17. <a href="https://doi.org/10.1016/j.bj.2019.10.005" target="_blank">https://doi.org/10.1016/j.bj.2019.10.005</a>
 
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<p>Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E., &amp; Church, G. M.
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+
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<p>Pacesa, M., Pelea, O., &amp; Jinek, M. (2024). Past, present, and future of CRISPR genome editing technologies.
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+
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<p>Paul, B., &amp; Montoya, G. (2020). CRISPR-Cas12a: Functional overview and applications. <i>Biomedical
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+
            RNA-guided
<p>Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C., &amp; Doudna, J. A. (2014). DNA interrogation by the
+
            endonuclease of a class 2 CRISPR-Cas system. <i>Cell</i>, 163, 759-771. <a href="https://doi.org/10.1016/j.cell.2015.09.038" target="_blank">https://doi.org/10.1016/j.cell.2015.09.038</a>
      CRISPR RNA-guided endonuclease Cas9. <i>Nature</i>, 507, 62-67. <a href="https://doi.org/10.1038/nature13011" target="_blank">https://doi.org/10.1038/nature13011</a></p>
+
<p>Zetsche, B., Gootenberg, J. S., Abudayyeh, O. O., Slaymaker, I. M., Makarova, K. S., Essletzbichler, P., Volz, S.
+
      E., Joung, J., van der Oost, J., Regev, A., Koonin, E. V., &amp; Zhang, F. (2015). Cpf1 is a single RNA-guided
+
      endonuclease of a class 2 CRISPR-Cas system. <i>Cell</i>, 163, 759-771. <a href="https://doi.org/10.1016/j.cell.2015.09.038" target="_blank">https://doi.org/10.1016/j.cell.2015.09.038</a>
+
 
</p>
 
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Latest revision as of 13:23, 2 October 2024

BBa_K5237000

fgRNA Entry Vector MbCas12a-SpCas9

This part integrates the crRNA of MbCas12a (BBa_K5237206) and the sgRNA of SpCas9 (BBa_K5237209) into a single fusion guide RNA (fgRNA). This fgRNA was functionally validated (see detailed characterization data below). MbCas12a (BBa_K5237001), SpCas9 (BBa_K5237002) and the novel fusion MbdCas12a-SpdCas9 (BBa_K5237003) can all utilize the fgRNA to target/bind two different genomic loci simultaneously. The fgRNA works in combination with both, the catalytically active as well as inactive Cas9 and Cas12a versions, facilitating multiplexed genome editing (with catalytically active Cas) as well as DNA-DNA stapling and hence 3D genome engineering in eukaryotes (with catalytically inactive Cas). Employing the fgRNA design described here, we successfully showed simultaneous genome editing at two different loci in human cells. Furthermore, the fgRNA enabled us to induce spatial proximity of otherwise separate gene regulatory elements (enhancer and promoter) with the catalytically inactive dSpCas9 and dMbCas12a.
In context of our part collection, the PICasSO toolbox, part BBa_K5237000 is the core component, since it enables the creation and programming of our so-called CRISPR/Cas staples: An innovative, trimeric complex comprised of a fgRNA, dCas9 and dCas12a employed for tethering two distinct genomic loci (see section 4.5 below), hence enabling rational engineering of the 3D genome conformation in living cells.

 



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

2. Usage and Biology

2.1 Discovery and Mechanism of CRISPR/Cas9

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

In 2012, Jinek et al. discovered the use of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system to induce double-strand breaks in DNA in a programmable manner. 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, an RNA guide is complexed by multiple Cas proteins, whereas class 2 systems consist of a singular protein binding 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 sequence with a ~20 nucleotide (nt) spacer sequence, and a transactivating CRISPR RNA (tracrRNA), which induces the processing by the Cas protein (Jinek et al., 2012) (Fig. 2 A). Furthermore, a specific three nucleotide sequence (NGG) at the 3' end in the targeted DNA is needed for Cas9 DNA binding and cleavage. This is referred to as the protospacer adjacent motif (PAM) (Sternberg et al., 2014). The most commonly used Cas9 protein is SpCas9 or SpyCas9, which originates from Streptococcus pyogenes (Pacesa et al., 2024).

A significant enhancement of the CRISPR/Cas9 system was the introduction of single guide RNAs (sgRNA[s]), which combine the functions of a tracrRNA and crRNA (Jinek et al., 2012; Mali et al., 2013). Moreover, Cong et al. (2013) established precise targeting of human endogenous loci by designing the 20 nt spacer sequence accordingly.

2.2 Differences between Cas9 and Cas12a

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

2.3 Dead Cas Proteins and their Application

Specific mutations of these domains result in catalytic inactivity and therefore allow for the creation of nickases that only cut one of the DNA strands, or Cas protein mutants that retain their DNA binding capability, but have no catalytic activity (Koonin et al., 2023) (Kleinstiver et al., 2019). The latter are referred to as dead Cas proteins or dCas9 and dCas12a. These Cas proteins can be used to activate (CRISPRa) or inhibit (CRISPRi) the expression of genes by fusing them to effector domains and targeting the respective genes via complementary spacer sequences (Kampmann, 2017). A common approach for CRISPRa involves fusing Cas9 with the transcriptional activator, such as VP64 or VPR (Kampmann, 2017).

3. Assembly and Part Evolution

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

Figure 3: Construction Process of fgRNAs Using the Entry Vector. The ccdB gene is excised using SapI in a Golden Gate assembly reaction. Desired Cas12a and Cas9 spacer sequence combinations can be easily inserted using annealed oligonucleotides with matching overhangs, resulting in a functional, complete fgRNA. Due to the cytotoxic nature of ccdB, only transformants carrying a correctly assembled fgRNA construct can survive, streamlining the cloning process.

As a first step to characterize the functionality of fgRNAs, we performed an experiment in which we simultaneously edited two fgRNA-targeted genomic sites in mammalian cells (HEK239T). The genes VEGFA and FANCF were selected as targets for Cas12a and Cas9 and each target was tested with each Cas protein using corresponding fgRNA designs. Editing efficiency was analyzed with the T7 Endonuclease I (T7EI) assay widely used in the CRISPR field. Controls included the use of conventional crRNAs and sgRNAs with their cognate Cas effectors as positive controls, and non-targeting guides as negative controls. Desired spacer sequences were ordered as synthetic oligos, annealed, and cloned in via GGA utilizing SapI.

Figure 4: Initial Experimental Setups to Assess the Functionality of fgRNAs Fusion Guide RNAs can be used for multiplex genome editing by simultaneously guiding catalytically active Cas12a and Cas9 to two distinct loci. Similarly, fgRNAs allow for CRISPRa by guiding the dCas9-VP64 transcriptional activator to a minimal promoter. These figure shows the basic experiments used for fgRNA characterization before applying it for DNA-DNA stapling (see below).
Table 1: A list of all the different spacers we cloned and tested within the fgRNA
CCR5 TGACATCAATTATTATACAT
Dnmt1 GCTCAGCAGGCACCTGCCTC
Fancf GGCGGGGTCCAGTTCCGGGA
Oct1 (BBa_K5237018) ATGCAAATACTGCACTAGTG
Runx1 CCTTCGGAGCGAAAACCAAG
TetO (BBa_K5237019) TCTCTATCACTGATAGGGAG
VEGFA CTAGGAATATTGAAGGGGGC
Table 2: A list of all the different linkers we cloned and tested within the fgRNA design
5 nt linker ATGCG
10 nt linker ATGCGAGCTG
10 nt Poly A linker CAAAACAACA
20 nt linker TGGCGGCGTGCTGACCGCTA
20 nt Poly A linker CAAAACAACAATCAAAACAA
30 nt Poly A linker CAAAACAACAATCAAAACAA
ATCAAAACAA
40 nt Poly A linker CAAAACAACAATCAAAACAACAAAACAA
CAATCAAAACAA

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

4. Results

In the following section, we provide a detailed quantitative characterization of part BBa_K5237000, tested under various experimental conditions and use cases.

4.1 Editing Endogenous Loci With Fusion Guide RNAs

To show that our fusion gRNA design results in an active CRISPR/Cas ribonucleoprotein complex, a series of different fgRNAs were cloned, each carrying spacer sequences specific to the VEGFA and FANCF target genes. HEK293T cells were then co-transfected with the Cas protein and (f)gRNA encoding constructs. The editing rate was tested 72h after transfection via a T7 endonuclease I assay.
Here, AsCas12a and SpCas9 were used. The AsCas12a spacer, in this case, targets VEGFA, while the SpCas9 spacer targets FANCF. Samples included either (i) conventional single gRNAs co-expressed with the corresponding Cas proteins (positive controls), (ii) the fgRNA co-expressed with only one of the two Cas proteins (as control for Cas ortholog dependency) and (iii) the fgRNA with both Cas proteins simultaneously co-expressed (Fig. 5). The conventional sgRNAs resulted in potent editing ("editing" refers to the observed indel frequency) for both target genes (45% for VEGFA and 15% for FANCF). Note that editing rates for FANCF were consistently lower in all experiments, which likely is due to the specific properties of the FANCF-targeted locus. Importantly, as hoped, targeting FANCF with fgRNAs resulted in noticeable editing of about 10%, when we added the SpCas9 alone or both Cas proteins into the sample. For VEGFA, the AsCas12a only sample resulted in approximately 20% editing in combination with the fgRNA, while adding both Cas proteins led to approximately 40%. These initial results confirmed that our fgRNA design indeed functions, enabling simultaneous recuitment of two different Cas proteins to separate loci in human cells.

Figure 5: fgRNAs Enable Efficient Editing of Endogenous Loci. Indel rates were assessed 72h post 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 each fgRNA. The symbols indicate, which Cas variants and which (f)gRNA were present in each sample.

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

After showing efficient editing, the next step was to evaluate the capabilities of the fgRNAs. To this end, we tested fgRNAs in combination with different Cas12a orthologs. Following some initial testing, we decided to use MbCas12a together with SpCas9 in subsequent experiments, since MbCas12a turned out to be more effective than AsCas12a in a dual luciferase assay when co-transfected with SpCas9 (Fig. 6). Of note, SpCas9 reproducibly showed high potency.

Figure 6: Comparison of AsCas12a and MbCas12a with a Dual Luciferase Reporter Assay. HEK293T cells were co-transfected with the indicated constructs and firefly and Renilla luminescence intensity was measured 48 h after transfection. Relative luciferase units correspond to firefly luciferase photon counts normalized to Renilla luciferase photon counts (Renilla luciferase serves as transfection control). Potent activity of CRISPR-Cas effectors result in knockdown of the firefly luciferase and hence smaller values. Samples correspond to cells co-transfected with a firefly luciferase targeting fgRNA and the constructs encoding the indicated CRISPR-Cas effector (see color legend). Data represent the mean +/- SD from n = 3 independent experiments. Statistical analysis was performed using 1way ANOVA with Tukey's multiple comparisons test. ***p<0.001,

Additionally, to determine whether the differences in editing rates observed in the preliminary assays were due to the specific properties of the targeted loci or the distinct characteristics of the Cas orthologs used, the spacers were tested in two configurations. In one setup, the fgRNA-encoded spacer for Cas12a targeted FANCF, while the spacer for SpCas9 targeted VEGFA; in the other, the targets were reversed. To more precisely assess the impact that the utilization of a fgRNA design has on the editing rates, conventional crRNAs/sgRNAs were tested separately or, alternatively, combined in one sample.
Combining a conventional crRNA/sgRNAs with the individual Cas proteins in the same sample showed no significant difference in editing rates (Fig. 7) as compared to using them in separate samples. While the fgRNA led to an overall lower editing rate as compared to the conventional guide RNAs, editing was still clearly noticeable for both targeted loci, indicating that the fgRNA works in principle. While editing efficiency for VEGFA remained consistently around 20%, the editing rate for FANCF dropped significantly, but was still detecable. Under identical conditions, MbCas12a consistently exhibited lower editing rates compared to SpCas9 when targeting the same gene, which is expected based on experience with this Cas orthologs in the CRISPR field.

Figure 7: FgRNA-mediated dual genome editing with SpCas9 and MbCas12a. In A and B the editing rates were determined 72h post transfection via T7EI assay. Editing % was determined by measuring band T7 cleavage band intensities as explained above. The schematic at the top shows the composition of the fgRNA. Below each spacer, the target gene is indicated. The symbols below indicate which parts are included in each sample. The fgRNA in A and B differs by the order of used spacer sequences (FANCF-VEGFA vs VEGFA-FANCF).

4.3 Fusion Guide RNAs are Compatible with Linkers of Various lengths

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 sequence between the two spacers was included. As above, fgRNA-mediated editing was assessed via T7E1 assay in HEK293T cells. The editing rate for VEGFA was again relatively consistent throughout the samples (Fig. 8). For CCR5, the editing rate with the conventional sgRNAs was in a similar range as that of VEGFA (at about 30%), but dropped to below 10% in the fgRNA sample. The addition of the 20 nt linker had no effect on the editing rates compared to no linker.

Figure 8: Fusion gRNAs with A 20 Nucleotide Spacer Still Mediate Editing For CCR5 and VEGFA HEK293T cells were co-transfected with the indicated components followed by assessing editing rates 72h post transfection via T7EI assay. Editing % was determined as described above. The schematic at the top shows the composition of the fgRNA. Below each spacer, the targeted gene is indicated. The symbols below indicate which parts are included in each sample. Cas12a targets VEGFA and Cas9 targets CCR5 in this case.

4.4 Fusion Guide RNAs Enable Efficient Activation of Gene Expression via CRISPRa

The data above provide manifold evidence that fgRNAs can indeed be employed for simultaneous genome editing at two defined loci with catalytically active Cas9 and Cas12a. Next, to establish the foundation for the use of fgRNAs as scaffold for Cas-staple protein assembly and hence 3D genome enineering, we next combined fgRNAs with catalytically dead Cas9 and -12a in an CRISPRa (CRISPR activation of gene expression) experiment. For this, we intended to recruit the transcriptional activator VP64 to a firefly luciferase reporter gene to induce expression. The VP64 protein is genetically fused to the catalytically inactive Cas9 protein, which is then guided by gRNAs to the luciferase-driving minimal promoter. More specifically, the gRNAs here target a TetO repeat sequence, which is positioned 5' of a minimal tata site. The luciferase reporter activity was then quantified and photon counts for Firefly luciferase (to be activated by CRISPRa) were normalized to the photon counts of Renilla luciferase, which is expressed on a separate plasmid under an ubiquitous promoter (transfection control). In two biological replicates we saw similar relative luciferase activity with fgRNA as a guide compared to a sgRNA (Fig. 9).

Figure 9: The Efficacy of CRISPRa is Comparable Between Our fgRNAs and Conventional sgRNAs. The schematic at the top shows the composition of the fgRNA. Symbols show which parts were co-expressed in each sample. The empty spacer control is a negative control with a gRNA lacking the TetO-targeting spacer, thus preventing binding of dCas9-VP64 to the Firefly-luciferase driving promoter. Firefly luciferase activity was measured 48h post transfection and, as above, normalized to an ubiquitously expressed Renilla luciferase (transfection control). The tetO repeats were targeted by Cas9-VP64, once with a sgRNA (center) and once with a fgRNA (right) that carried a non-targeting spacer sequence for Cas12a.

4.5 A Proof-Of-Concept for 3D Genome Engineering: Stapling Two DNA Strands Together In Human Cells Using fgRNAs

Having demonstrated the excellent compatibility of the fgRNA design with simultaneous genome editing at two distinct loci and CRISPR activation, we set out to provide a proof-of-concept for engineering 3D genome conformation in living cells. To achieve this, we designed an experiment to "staple together" two regulatory elements—a synthetic enhancer and a minimal promoter—located on different strands of DNA. We developed a two-plasmid system consisting of an enhancer plasmid and a reporter plasmid. The reporter plasmid encodes firefly luciferase driven by a minimal promoter containing several repeats of a Cas9-targeted sequence, while the enhancer plasmid includes a Gal4 binding site (UAS) positioned next to several repeats of a Cas12a-targeted sequence. We co-expressed these two plasmids with dCas9, dCas12, an fgRNA simultaneously co-targeting (i.e., tethering) the reporter and enhancer plasmids, and Gal4-VP64. This resulted in the expression of luciferase (Fig. 10, Panel A). Different linker lengths (as mentioned above) were tested for the fgRNA. Firefly luciferase values were normalized against ubiquitous renilla expression as a transfection control. Using no linker between the two spacers produced luciferase activity values similar to the baseline control (i.e., no reporter activation) (Fig. 10, Panel B). Excitingly, however, the stepwise extension of the fgRNA linker (i.e., the linker separating the Cas12a and Cas9 spacer elements) from 20 nt to 40 nt led to progressively increasing, and ultimately potent, luciferase reporter activation. This experiment provided the first demonstration that an fgRNA with an optimized linker can effectively "staple" two otherwise separate pieces of DNA. As this experimental readout—mediated by the spatial proximity of two separate regulatory elements—mimics naturally occurring enhancer hijacking events, we refer to this setup as "synthetic enhancer hijacking."

Figure 10: Applying Fusion Guide RNAs for Cas staples: Proof-Of-Concept 3D Genome Engineering Via Synthetic Enhancer Hijacking. A, schematic overview of the assay is shown. An enhancer plasmid and a reporter plasmid are brought into proximity by an fgRNA-Cas staple complex, which co-targets and tethers both plasmids. Target sequences were included in multiple repeats upstream of the functional elements. Firefly luciferase serves as the reporter gene, while the enhancer is composed of multiple Gal4 repeats bound by a Gal4-VP64 fusion protein. B, HEK293T cells were co-transfected with the reporter plasmid, the enhancer plasmid, a Gal4-VP64-encoding plasmid as well dCas9 and dCas12. Firefly luciferase activity was measured 48 hours post transfection and normalized to ubiquitously expressed Renilla luciferase. Observed differences were tested for statistical significance using ordinary one-way ANOVA with Dunn's method for multiple comparisons. (*p < 0.05; **p < 0.01; ***p < 0.001; mean +/- SD from n = 3 independent experiments). The assay included sgRNAs and fgRNAs with linker lengths varying between 0 nt to 40 nt as indicated.

5. In Silico Characterization using DaVinci

We developed the in silico model DaVinci for rapid engineering and development of our PICasSO system. DaVinci acts as a digital twin to PICasSO, designed to understand the forces acting on our system, refine experimental parameters, and find optimal connections between protein staples and target DNA.
We calibrated DaVinci with literature and our own experimental affinity data obtained via EMSA assays and purified proteins. This enabled us to simulate enhancer hijacking in silico, providing valuable input for the design of further experiments. Additionally, we apply the same approach to our part collection.

DaVinci is divided into three phases: static structure prediction, all-atom dynamics simulation, and long-ranged dna dynamics simulation. Here, we applied the last step to our parts, characterizing structure and dynamics of the dna-binding interaction.

5.1. Enhancer Hijacking is Successfully Studied In Silico

Figure 11: Cas stapled plasmids.

With the Cas staple, we aimed to simulate the principles of enhancer hijacking experiments we conducted in the lab. For these experiments, we modeled the two plasmids also used in the wet lab (BBa_K5237023 and BBa_K5237024). On top of the two plasmids, we modeled the Cas staple by applying a “DNA-DNA tethering force" throughout our simulation, selectively on the regions targeted by the fgRNA. This force was based on simulation data acquired in earlier phases of DaVinci. As there is no suitable model available that also simulates proteins, this proved to be the most effective modeling strategy.

Our simulation showed the expected behavior, holding the target sequences of the Cas staple (Fig. 12). Overall, these exciting results demonstrate that we can successfully model the core principles of enhancer hijacking with a total of 20 thousand simulated nucleotides in silico.

Figure 12: Fusion Cas staples hold stapled nucleotides in close proximity.

5.2. Cas Staple Forces do not Disturb DNA Strand Integrity

Figure 13: Cas stapled plasmids.

Next, we aimed to stress test our system to determine the amount of force required to induce DNA double-strand breaks. To achieve this, we used an identical setup to the previous experiment but instead of experimentally determined forces, we used artificial forces of varying strength. It is important to know that our in silico model responds to forces that cause double-strand breaks by scattering the nucleotides across the simulation box. As the specified bonds cannot actually break within the simulation, the phosphate backbone bonds become severely distorted and exhibit erratic behavior in the simulation videos. From our extensive simulations, we concluded that the forces required to damage the DNA are approximately 320 times (Fig. 15) and 680 times greater (Fig. 16) than the forces exerted by the Cas staple, respectively.

This provides important evidence regarding the safety of our staple approach for 3D genome engineering: According to our model, DNA-DNA tethering with our Cas staples is not expected to have a negative effect on the DNA stability.

Figure 14: Applying a force that is 270 times greater
than the predicted force typically exerted by a Cas staple on DNA.
Figure 15: Applying a force that is 320 times greater
than the predicted force typically exerted by a Cas staple on DNA.
Figure 16: Applying a force that is 680 times greater
than the predicted force typically exerted by a Cas staple on DNA.
Figure 17: Applying a force that is more than 1000 times greater
than the predicted force typically exerted by a Cas staple on DNA.

5.3. DaVinci Helps to Design Multi-Staple Arrangements

Figure 18: Double Cas stapling within 40 nucleotide distance induces double-strand breaks.

Finally, we simulated the behavior of multiple staples at once. Therefore, we expanded our previously introduced experimental setup by a second Cas staple.
In a first approach, we targeted an additional region next to the original chosen one. This region is 40 nucleotides away from the first target region on the plasmid displayed in blue connecting it to the opposite site of the orange plasmid, therefore bringing both ends of the orange plasmid together (Fig. 18).
Our simulation predicted that with these staple points, DNA double-strand breaks would occur as clearly visible by the scattered nucleotides (Fig. 19).

Figure 19: Double Cas stapling within 40 nucleotide distance induces double-strand breaks.
Figure 20: Stable multiplexing with 2 Cas staples. On the blue plasmid, the Cas binding sequences are 980 nucleotides apart.

To simulate a setup where we expect no double-strand breaks, we increased the distance between the stapling sites on the blue plasmid (BBa_K5237023) from 40 to 980 nucleotides (Fig. 20).
With this increased distance between stapling sites, we observed a stabilized system. Most interestingly, the non-stapled regions showed maximum distances close to 500 nm, indicating that the two staples led to more compact plasmid structures.

In conclusion, we show that applying multiple staples on the same structures can lead to double-strand breaks if the staples are positioned closely to one another. However, increasing the separation of staples leads to a stable system without DNA damage. Thus, it is definitely possible to multiplex Cas staples, thereby increasing the compactness of DNA structures (Fig. 21). This shows the potential of creating complex regulatory networks by bringing genetic elements into spatial proximity with a multitude of Cas protein staples.

Figure 21: Increasing the distance between Cas staple targets to 980 nucleotides stabilizes multiplexing.

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