Difference between revisions of "Part:BBa K5237001"
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<partinfo>BBa_K5237001</partinfo> | <partinfo>BBa_K5237001</partinfo> | ||
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<!-- Part summary --> | <!-- Part summary --> | ||
<section id="1"> | <section id="1"> | ||
− | <h1>Staple | + | <h1>Staple Subunit: dMbCas12a-Nucleoplasmin NLS</h1> |
<p>dMbCas12a is one crucial part of our PICasSO tool box, being able to function as a Cas staple with the fgRNA | <p>dMbCas12a is one crucial part of our PICasSO tool box, being able to function as a Cas staple with the fgRNA | ||
− | (BBa_K5237000) and the dSpCas9 (BBa_K5237002). Transactivation has been shown using this part proving the proper | + | (<a href="https://parts.igem.org/Part:BBa_K5237000">BBa_K5237000</a>) and the dSpCas9 (<a href="https://parts.igem.org/Part:BBa_K5237002">BBa_K5237002</a>). Transactivation has been shown using this part proving the proper |
formation of our Cas staple. Easy employment through swift reprogramming of the fgRNA.</p> | formation of our Cas staple. Easy employment through swift reprogramming of the fgRNA.</p> | ||
<p> </p> | <p> </p> | ||
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</li> | </li> | ||
<li class="toclevel-2 tocsection-3.6"><a href="#3.6"><span class="tocnumber">3.6</span> <span class="toctext">MbCas12a fused to SpCas9 editing utilizing a fgRNA</span></a> | <li class="toclevel-2 tocsection-3.6"><a href="#3.6"><span class="tocnumber">3.6</span> <span class="toctext">MbCas12a fused to SpCas9 editing utilizing a fgRNA</span></a> | ||
− | </li></ul> | + | </li> |
+ | </ul> | ||
</li> | </li> | ||
<li class="toclevel-1 tocsection-5"><a href="#4"><span class="tocnumber">4</span> <span class="toctext">Results</span></a> | <li class="toclevel-1 tocsection-5"><a href="#4"><span class="tocnumber">4</span> <span class="toctext">Results</span></a> | ||
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</div> | </div> | ||
</div> | </div> | ||
− | |||
<p> | <p> | ||
<br/> | <br/> | ||
− | + | While synthetic biology has in the past focused on engineering the genomic sequence of organisms, the <b>3D | |
− | cell fate, disease development and more. However, | + | spatial organization</b> of DNA is well-known to be an important layer of information encoding in |
− | 3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a | + | particular in eukaryotes, playing a crucial role in |
− | toolbox based on various DNA-binding proteins | + | gene regulation and hence |
+ | cell fate, disease development, evolution, and more. However, tools to precisely manipulate and control the | ||
+ | genomic spatial | ||
+ | architecture are limited, hampering the exploration of | ||
+ | 3D genome engineering in synthetic biology. We - the iGEM Team Heidelberg 2024 - have developed PICasSO, a | ||
+ | <b>powerful | ||
+ | molecular toolbox for rationally engineering genome 3D architectures</b> in living cells, based on | ||
+ | various DNA-binding proteins. | ||
</p> | </p> | ||
<p> | <p> | ||
The <b>PICasSO</b> part collection offers a comprehensive, modular platform for precise manipulation and | The <b>PICasSO</b> part collection offers a comprehensive, modular platform for precise manipulation and | ||
− | re-programming | + | <b>re-programming |
− | + | of DNA-DNA interactions</b> using engineered "protein staples" in living cells. This enables | |
− | interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation. | + | researchers to recreate naturally occurring alterations of 3D genomic |
− | Beyond its versatility, PICasSO includes robust assay systems to support the engineering, optimization, and | + | interactions, such as enhancer hijacking in cancer, or to design entirely new spatial architectures for |
− | testing of new staples | + | 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 <b>chimeric CRISPR/Cas complexes</b>, | ||
+ | connected either at | ||
+ | the protein or - in the case of CRISPR/Cas-based DNA binding moieties - the guide RNA level. These complexes are | ||
+ | referred to as protein- or Cas staples, respectively. Beyond its | ||
+ | versatility with regard to the staple constructs themselves, PICasSO includes <b>robust assay</b> systems to | ||
+ | support the engineering, optimization, and | ||
+ | testing of new staples <i>in vitro</i> and <i>in vivo</i>. Notably, the PICasSO toolbox was developed in a | ||
+ | design-build-test-learn <b>engineering cycle closely intertwining wet lab experiments and computational | ||
+ | modeling</b> and iterated several times, yielding a collection of well-functioning and -characterized | ||
+ | parts. | ||
</p> | </p> | ||
− | <p>At its heart, the PICasSO part collection consists of three categories. <br/><b>(i)</b> Our <b>DNA-binding proteins</b> | + | <p>At its heart, the PICasSO part collection consists of three categories. <br/><b>(i)</b> Our <b>DNA-binding |
+ | proteins</b> | ||
include our | include our | ||
− | finalized | + | 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 | + | "half staples" that can be combined by scientists to compose entirely |
− | and can be further engineered to create alternative, simpler and more compact staples. <br/> | + | new Cas staples in the future. We also include our Simple staples comprised of particularly small, simple |
− | <b>(ii)</b> As <b>functional elements</b>, we list additional parts that enhance the functionality of our Cas and Basic staples. These | + | and robust DNA binding domains well-known to the synthetic biology community, which serve as controls for |
− | consist of | + | successful stapling |
− | + | 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 | ||
+ | consist of staples dependent on | ||
+ | cleavable peptide linkers targeted by cancer-specific proteases or inteins that allow condition-specific, | ||
+ | dynamic stapling <i>in vivo</i>. | ||
+ | We also include several engineered parts that enable the efficient delivery of PICasSO's constructs into | ||
+ | target cells, including mammalian cells, | ||
+ | with our new | ||
interkingdom conjugation system. <br/> | interkingdom conjugation system. <br/> | ||
− | <b>(iii)</b> As the final category of our collection, we provide parts that | + | <b>(iii)</b> As the final category of our collection, we provide parts that underlie our <b>custom |
− | systems</b>. These include components of our established FRET-based proximity assay system, enabling users to | + | readout |
+ | systems</b>. These include components of our established FRET-based proximity assay system, enabling | ||
+ | users to | ||
confirm | confirm | ||
− | accurate stapling. Additionally, we offer a complementary, application-oriented testing system | + | 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. | ||
</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 | |
− | collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their | + | parts in |
− | own custom Cas staples, enabling further optimization and innovation.<br/> | + | the |
+ | collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer | ||
+ | their | ||
+ | own custom Cas staples, enabling further optimization and innovation in the new field of 3D genome | ||
+ | engineering.<br/> | ||
</p> | </p> | ||
<p> | <p> | ||
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</p> | </p> | ||
<table style="width: 90%; padding-right:10px;"> | <table style="width: 90%; padding-right:10px;"> | ||
− | <td align="left" colspan="3"><b>DNA- | + | <td align="left" colspan="3"><b>DNA-Binding Proteins: </b> |
− | + | Modular building blocks for engineering of custom staples to mediate defined DNA-DNA interactions <i>in vivo</i></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> | + | <td>Fusion Guide RNA Entry Vector MbCas12a-SpCas9</td> |
− | <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 | + | <td>Staple Subunit: dMbCas12a-Nucleoplasmin NLS</td> |
− | <td>Staple subunit that can be combined with | + | <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 | + | <td>Staple Subunit: SV40 NLS-dSpCas9-SV40 NLS</td> |
− | <td>Staple subunit that can be combined | + | <td>Staple subunit that can be combined with a sgRNA or fgRNA and dMbCas12a to form a functional staple |
</td> | </td> | ||
</tr> | </tr> | ||
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<td><a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a></td> | ||
<td>Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS</td> | <td>Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS</td> | ||
− | <td>Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands into | + | <td>Functional Cas staple that can be combined with sgRNA and crRNA or fgRNA to bring two DNA strands into |
+ | close | ||
+ | proximity | ||
</td> | </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237004" target="_blank">BBa_K5237004</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237004" target="_blank">BBa_K5237004</a></td> | ||
− | <td>Staple | + | <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 | + | <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 | + | <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 | + | <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 | + | <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 | + | <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 | + | <td align="left" colspan="3"><b>Functional Elements: </b> |
− | Protease-cleavable peptide linkers and inteins are used to control and modify staples for further optimization | + | Protease-cleavable peptide linkers and inteins are used to control and modify staples for further |
+ | optimization | ||
for custom applications</td> | for custom applications</td> | ||
<tbody> | <tbody> | ||
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<td><a href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a></td> | ||
<td>Cathepsin B-cleavable Linker: GFLG</td> | <td>Cathepsin B-cleavable Linker: GFLG</td> | ||
− | <td>Cathepsin B-cleavable peptide linker that can be used to combine two staple subunits to make responsive | + | <td>Cathepsin B-cleavable peptide linker that can be used to combine two staple subunits to make |
+ | responsive | ||
staples</td> | staples</td> | ||
</tr> | </tr> | ||
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<td><a href="https://parts.igem.org/Part:BBa_K5237011" target="_blank">BBa_K5237011</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237011" target="_blank">BBa_K5237011</a></td> | ||
<td>Cathepsin B Expression Cassette</td> | <td>Cathepsin B Expression Cassette</td> | ||
− | <td>Expression | + | <td>Expression cassette for the overexpression of cathepsin B</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237012" target="_blank">BBa_K5237012</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237012" target="_blank">BBa_K5237012</a></td> | ||
<td>Caged NpuN Intein</td> | <td>Caged NpuN Intein</td> | ||
− | <td>A caged NpuN split intein fragment that undergoes protein <i>trans</i>-splicing after protease activation | + | <td>A caged NpuN split intein fragment that undergoes protein <i>trans</i>-splicing after protease |
− | + | activation, which can be used to create functionalized staple | |
+ | subunits</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237013" target="_blank">BBa_K5237013</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237013" target="_blank">BBa_K5237013</a></td> | ||
<td>Caged NpuC Intein</td> | <td>Caged NpuC Intein</td> | ||
− | <td>A caged NpuC split intein fragment that undergoes protein <i>trans</i>-splicing after protease activation | + | <td>A caged NpuC split intein fragment that undergoes protein <i>trans</i>-splicing after protease |
− | + | activation, which can be used to create functionalized staple | |
+ | subunits</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237014" target="_blank">BBa_K5237014</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237014" target="_blank">BBa_K5237014</a></td> | ||
− | <td> | + | <td>Fusion Guide RNA Processing Casette</td> |
− | <td>Processing | + | <td>Processing cassette to produce multiple fgRNAs from one transcript, that can be used for |
+ | multiplexed 3D | ||
+ | genome reprogramming</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237015" target="_blank">BBa_K5237015</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237015" target="_blank">BBa_K5237015</a></td> | ||
<td>Intimin anti-EGFR Nanobody</td> | <td>Intimin anti-EGFR Nanobody</td> | ||
− | <td> | + | <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> | + | <td>IncP Origin of Transfer</td> |
− | <td>Origin of transfer that can be cloned into the plasmid vector and used for conjugation as a means of delivery</td> | + | <td>Origin of transfer that can be cloned into the plasmid vector and used for conjugation as a |
+ | means of | ||
+ | delivery</td> | ||
</tr> | </tr> | ||
</tbody> | </tbody> | ||
<td align="left" colspan="3"><b>Readout Systems: </b> | <td align="left" colspan="3"><b>Readout Systems: </b> | ||
− | FRET and enhancer recruitment to | + | FRET and enhancer recruitment readout systems to rapidly assess successful DNA stapling in bacterial and |
− | + | 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 | + | <td>FRET donor-fluorophore fused to Oct1-DBD that binds to the Oct1 binding cassette, which can be used to |
+ | visualize | ||
+ | DNA-DNA | ||
proximity</td> | proximity</td> | ||
</tr> | </tr> | ||
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<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 | + | <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> | ||
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<td><a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a></td> | ||
<td>TetR Binding Cassette</td> | <td>TetR Binding Cassette</td> | ||
− | <td>DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET | + | <td>DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the |
+ | FRET | ||
proximity assay</td> | proximity assay</td> | ||
</tr> | </tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237020" target="_blank">BBa_K5237020</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237020" target="_blank">BBa_K5237020</a></td> | ||
<td>Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64</td> | <td>Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64</td> | ||
− | <td>Readout system that responds to protease activity | + | <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> | ||
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</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 | + | <td>mCherry Expression Cassette: UAS, minimal Promoter, mCherry</td> |
− | <td>Readout system for enhancer binding | + | <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 | + | <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 | + | <td>TRE-minimal Promoter- Firefly Luciferase</td> |
− | <td>Contains | + | <td>Contains firefly luciferase controlled by a minimal promoter, which was used as a luminescence |
+ | readout for | ||
simulated enhancer hijacking</td> | simulated enhancer hijacking</td> | ||
</tr> | </tr> | ||
</tbody> | </tbody> | ||
− | </table> | + | </table></section> |
− | </section> | + | |
<section id="1"> | <section id="1"> | ||
<h1>1. Sequence overview</h1> | <h1>1. Sequence overview</h1> | ||
Line 304: | Line 356: | ||
<h1>2. Usage and Biology</h1> | <h1>2. Usage and Biology</h1> | ||
<p> | <p> | ||
− | + | In 2012, Jinek <i>et al.</i> discovered the use of the Clustered Regularly Interspaced Short Palindromic Repeats | |
− | + | (CRISPR)/Cas system to induce double-strand breaks in DNA. Since then, the system has been well established as a | |
− | + | tool for genome editing. The CRISPR/Cas system, which originates from the bacterial immune system, is constituted | |
− | + | by a ribonucleoprotein complex. For class 1 CRISPR systems, the RNA is complexed by multiple Cas proteins, whereas | |
− | + | class 2 systems consist of a singular protein and RNA. The class 2 type II system describes all ribonucleoprotein | |
− | + | complexes with Cas9 (Pacesa <i>et al.</i>, 2024). They include a CRISPR RNA (crRNA), which specifies the target with | |
− | + | a 20 | |
− | + | nucleotide (nt) spacer sequence, and a transactivating CRISPR RNA (tracrRNA), which induces the processing by the | |
− | + | Cas protein (Jinek <i>et al.</i>, 2012) (Figure 2 A). Furthermore, a specific three | |
− | + | nucleotide sequence (NGG) on the 3' end in the targeted DNA is needed for binding and cleavage. This is referred | |
− | + | to as the protospacer adjacent motif (PAM) (Sternberg <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> | ||
<div class="thumb"> | <div class="thumb"> | ||
<div class="thumbinner" style="width:60%;"> | <div class="thumbinner" style="width:60%;"> | ||
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<div class="thumbcaption"> | <div class="thumbcaption"> | ||
<i> | <i> | ||
− | <b>Figure 2: The CRISPR/Cas | + | <b>Figure 2: The CRISPR/Cas System.</b> |
− | + | A and B, schematic structure of Cas9 and Cas12a with their sgRNA/crRNA, sitting on a DNA strand with the PAM. | |
− | + | The spacer sequence forms base pairings with the dsDNA. In case of Cas9 the | |
− | + | spacer is located at the 5' prime end, for Cas12a at the 3' end of the gRNA. The scaffold of the gRNA forms a | |
− | + | specific | |
− | + | secondary structure enabling it to bind to the Cas protein. The cut sites by the cleaving domains, RuvC and | |
− | + | HNH, are | |
+ | symbolized by the scissors | ||
+ | </i> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
<p> | <p> | ||
− | + | A significant enhancement of this system was the introduction of single guide RNA (sgRNA)s, which combine the | |
− | + | functions of a tracrRNA and crRNA (Mali <i>et al.</i>, 2013). | |
− | + | Moreover, Cong (2013) established precise targeting of human endogenous loci by designing the 20 nt spacer sequence | |
− | + | accordingly. | |
+ | </p> | ||
</section> | </section> | ||
<section id="3"> | <section id="3"> | ||
− | <h1>3. Assembly and | + | <h1>3. Assembly and Part Evolution</h1> |
<section id="3.1"> | <section id="3.1"> | ||
− | <h2>3.1 | + | <h2>3.1 Qualitatively Assessing Gene Editing of Cas12a Orthologs</h2> |
<p> | <p> | ||
− | + | To select a suitable Cas12a ortholog for constructing the Cas staples, three different orthologs were ordered | |
− | + | from Addgene: | |
− | + | AsCas12a (<a href="https://www.addgene.org/69982/" target="_blank">#69982</a>), LbCas12a (<a href="https://www.addgene.org/69988/" target="_blank">#69988</a>), | |
− | + | and MbCas12a (<a href="https://www.addgene.org/115142/" target="_blank">#115142</a>). | |
− | + | <br/><br/> | |
− | + | We assessed their performance using a T7 Endonuclease I (T7EI) assay on three genomic loci: RUNX1, DNMT1, and | |
− | + | FANCF. For | |
− | + | comparison, we also included SpCas9 editing the CCR5 locus (Fig. 3). Editing was only observed at the | |
+ | RUNX1 locus, where we used the qualitative assay to compare the editing efficiencies of the Cas12a orthologs. | ||
+ | </p> | ||
<div class="thumb"> | <div class="thumb"> | ||
<div class="thumbinner" style="width:60%;"> | <div class="thumbinner" style="width:60%;"> | ||
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<div class="thumbcaption"> | <div class="thumbcaption"> | ||
<i> | <i> | ||
− | <b>Figure 3: Preliminary T7 Endonuclease I | + | <b>Figure 3: Preliminary T7 Endonuclease I Testing of Cas12a Orthologs.</b> |
− | + | T7EI samples were run on a 2% agarose gel in 1x TBE buffer dyed with gel red. SpCas9 targeting CCR5 | |
− | + | functions as a | |
− | + | benchmark for proper editing. For the Cas12a orthologs, LbCas12a, AsCas12a and MbCas12a, the three loci | |
− | + | RUNX1, DNMT1 and | |
+ | FANCF were targeted. Editing is indicated by an extra band compared to the negative control. | ||
+ | </i> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
</section> | </section> | ||
− | <section id="3.2"><h2>3.2 Quantitative | + | <section id="3.2"> |
+ | <h2>3.2 Quantitative Comparison Between AsCas12a and MbCas12a</h2> | ||
<p> | <p> | ||
− | + | Since the T7EI assay can only be used to qualitatively compare editing efficiencies, it was hard to distinguish | |
− | + | between the better editing ortholog.<br/> | |
− | + | To accurately quantify the editing efficiency, we conducted a dual luciferase assay. This assay measures the | |
− | + | luminescence | |
− | + | of firefly luciferase, which decreases proportionally to the editing efficiency at the target site. | |
− | + | To account for variations in cell count and transfection efficiency, the luminescence is normalized | |
− | + | to Renilla luciferase, which acts as an internal control (Fig. 4). | |
− | + | The results revealed a clear difference, with MbCas12a demonstrating significantly higher editing efficiency | |
− | + | compared to | |
+ | AsCas12a (p=0.005). Based on these findings, we decided to proceed with MbCas12a. | ||
+ | </p> | ||
<div class="thumb"> | <div class="thumb"> | ||
<div class="thumbinner" style="width:60%;"> | <div class="thumbinner" style="width:60%;"> | ||
Line 381: | Line 445: | ||
<div class="thumbcaption"> | <div class="thumbcaption"> | ||
<i> | <i> | ||
− | <b>Figure 4: Comparison of AsCas12a and MbCas12a with a | + | <b>Figure 4: Comparison of AsCas12a and MbCas12a with a Dual Luciferase Assay.</b> |
− | + | Firefly luminescence intensity measured 48 h after transfection. Normalized against renilla luminescence. On | |
− | + | the x-axis | |
− | + | the samples Cas9 + AsCas12a , Cas9 + MbCas12a, AsCas12a and MbCas12a are depicted. | |
− | + | Data is depicted as the mean +/- SD (n=3). | |
− | + | Statistical analysis was performed using 1way ANOVA with Tukey's multiple comparisons test. For better | |
− | + | clarity, only | |
− | + | significant differences within a group between the same Cas proteins are shown.*p<0.05, **p<0.01, | |
+ | ***p<0.001, | ||
+ | ****p<0.0001 | ||
+ | </i> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
</section> | </section> | ||
− | <section id="3.3"><h2>3.3 MbCas12a | + | <section id="3.3"> |
+ | <h2>3.3 Multiplex Gene Editing Using MbCas12a and SpCas9</h2> | ||
<p> | <p> | ||
− | + | To employ the MbCas12a as part of a Cas staple, we sought to determine MbCas12a's activity while | |
− | + | being transfected together with SpCas9. Therefore we engineered the dual luciferase assay to allow us for testing | |
− | + | two | |
− | + | catalytically active Cas proteins at once. Including a well established protospacer, CCR5, into the renilla | |
− | + | luciferase | |
+ | gene enables us to test the editing rates of two Cas proteins simultaneously (Fig. 5). | ||
+ | </p> | ||
<div class="thumb"> | <div class="thumb"> | ||
<div class="thumbinner" style="width:60%"> | <div class="thumbinner" style="width:60%"> | ||
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<div class="thumbcaption"> | <div class="thumbcaption"> | ||
<i> | <i> | ||
− | <b>Figure 5: Testing for Simultaneous Editing with Double Cut | + | <b>Figure 5: Testing for Simultaneous Editing with Double Cut Luciferase Assay.</b> |
− | + | Firefly and renilla luminescence intensity is plotted, both measured 48 h after transfection. Co-transformed | |
− | + | contains | |
− | + | MbCas12a and SpCas9. Cas12a are the firefly relative luminescence units (RLUs), while Cas9 are the Renilla | |
− | + | RLUs. | |
− | + | Data is depicted as the mean +/- SD (n=3). | |
− | + | Statistical analysis was performed using 2way ANOVA with Tukey's multiple comparisons test. For better | |
+ | clarity, only | ||
+ | significant differences within a group are shown.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < | ||
+ | 0.0001 | ||
+ | </i> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
<p> | <p> | ||
− | + | For the MbCas12a we have a highly significant editing rate in the single cut, meaning both Cas proteins are | |
− | + | transfected, | |
− | + | but only MbCas12a has a targeting gRNA. When introducing a targeting gRNA for SpCas9 we did not observe reduction in the | |
− | + | highly | |
+ | significant editing of MbCas12a, strongly suggesting both Cas proteins to be able to edit simultaneously. | ||
+ | </p> | ||
</section> | </section> | ||
− | <section id="3.4"><h2>3.4 | + | <section id="3.4"> |
+ | <h2>3.4 Fusion Guide RNA Enabled Editing with MbCas12a</h2> | ||
<p> | <p> | ||
− | + | To further confirm that MbCas12a can be combined with fgRNAs in a functional Cas staple, editing rates were tested | |
− | + | using a fusion guide RNA | |
− | + | (fgRNA, <a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a>) | |
− | + | targeting two different loci: <i>FANCF</i> and <i>VEGFA</i>. To better assess the impact that the utilization of a | |
− | + | fgRNA has on the editing rates, the sgRNAs were tested separately and in one sample.<br/> | |
− | + | Having the sgRNA with single Cas proteins in the same sample resulted in no clear | |
− | + | difference in the editing rates (Fig. 6). The fusion of the gRNAs resulted in a lower editing rate | |
− | + | overall. While the editing for VEGFA stayed at about 20% in all | |
− | + | cases, the editing for FANCF dropped significantly. Nonetheless we were able to show MbCas12a editing utilizing a | |
+ | fgRNA. | ||
+ | </p> | ||
<div class="thumb"> | <div class="thumb"> | ||
<div class="thumbinner" style="width:95%;"> | <div class="thumbinner" style="width:95%;"> | ||
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<div class="thumbcaption"> | <div class="thumbcaption"> | ||
<i> | <i> | ||
− | <b>Figure 6: Fusion gRNA Editing Rates In Combination with MbCas12a.</b> | + | <b>Figure 6: Fusion gRNA Editing Rates In Combination with MbCas12a.</b> |
− | + | In <b>A</b> and <b>B</b> the editing rates were determined 72h after transfection via T7EI assay. Editing % | |
− | + | was | |
− | + | determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved | |
− | + | band))<sup>1/2</sup>. The | |
− | + | schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols | |
− | + | below | |
+ | indicate which parts are included in each sample. <i class="italic">A</i> and <i class="italic">B</i> | ||
+ | display both | ||
+ | orientations of the two spacers for VEGFA and FANCF. | ||
+ | </i> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
<p> | <p> | ||
− | + | To further assess the effect of the genomic locus on the editing rate, we included CCR5 as an additional gene | |
− | + | target. | |
− | + | For this assay, a fgRNA with a 20 nt long linker was included between the two spacers. | |
− | + | The editing rate for VEGFA was again relatively consistent throughout the samples (Fig. 7). | |
+ | </p> | ||
<div class="thumb"> | <div class="thumb"> | ||
<div class="thumbinner" style="width:60%;"> | <div class="thumbinner" style="width:60%;"> | ||
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<div class="thumbcaption"> | <div class="thumbcaption"> | ||
<i> | <i> | ||
− | <b>Figure 7: Fusion gRNA Editing Rates for Multiplexing CCR5 and VEGFA</b> | + | <b>Figure 7: Fusion gRNA Editing Rates for Multiplexing CCR5 and VEGFA</b> |
− | + | The editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by | |
− | + | measuring band | |
− | + | intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band)) <sup>1/2</sup>. The schematic at the | |
− | + | top shows the | |
− | + | composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts are | |
+ | included in | ||
+ | each sample. Cas12a targets VEGFA and Cas9 targets CCR5. | ||
+ | </i> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
</section> | </section> | ||
− | <section id="3.5"><h2>3.5 MbCas12a | + | <section id="3.5"> |
+ | <h2>3.5 Establishing Functional Fusion Cas Proteins with MbCas12a and SpCas9</h2> | ||
<p> | <p> | ||
− | + | Testing showed simultaneous editing of MbCas12a and SpCas9. Therefore we wanted to test protein-protein fusion, | |
− | + | potentially giving us another way of Cas stapling. Our goal was to find out how well MbCas12a can stay active | |
− | + | while | |
− | + | being fused to SpCas9. Therefore we employed the previously engineered dual luciferase assay to allow us for | |
− | + | testing two | |
+ | catalytically active Cas proteins at once, this time being fused to each other (Fig. 8). | ||
+ | </p> | ||
<div class="thumb"> | <div class="thumb"> | ||
<div class="thumbinner" style="width:60%;"> | <div class="thumbinner" style="width:60%;"> | ||
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<div class="thumbcaption"> | <div class="thumbcaption"> | ||
<i> | <i> | ||
− | <b>Figure 8: Double | + | <b>Figure 8: Double Cut Dual Luciferase Assay Testing Fusion Cas Simultaneous Editing.</b> |
− | + | Firefly and renilla luminescence intensity is plotted, both measured 48 h after transfection. On the x-axis | |
− | + | the negative | |
− | + | control, single edit and simultaneous edit, are grouped by the two Cas proteins in the fusion Cas. The | |
− | + | fusion Cas | |
− | + | contains MbCas12a and SpCas9. MbCas12a are the firefly relative luminescence units (RLUs), while Cas9 are | |
− | + | the Renilla | |
+ | RLUs. Data is depicted as the mean +/- SD (n=3). Statistical analysis was performed using 2way ANOVA with | ||
+ | Tukey's multiple | ||
+ | comparisons test. For better clarity, only significant differences within a group are shown.*p<0.05, | ||
+ | **p<0.01, ***p<0.001, ****p<0.0001 | ||
+ | </i> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
<p> | <p> | ||
− | + | For the MbCas12a we have a highly significant editing rate in the single cut, meaning only MbCas12a has a | |
− | + | targeting | |
− | + | gRNA. When introducing a targeting gRNA for SpCas9 we see no reduction in the highly significant editing of | |
− | + | MbCas12a, | |
+ | strongly suggesting both Cas proteins in the fusion Cas to be able to edit simultaneously. | ||
+ | </p> | ||
</section> | </section> | ||
− | <section id="3.6"><h2>3.6 | + | <section id="3.6"> |
+ | <h2>3.6 The Combination of Fusion Guide RNAs and Fusion Cas Proteins</h2> | ||
<p> | <p> | ||
− | + | The gene editing efficiency of MbCas12a in the fusion Cas was tested by assessing the editing rates via a T7EI | |
− | + | assay. For this, the | |
− | + | same target sequences as before were used, namely FANCF and VEGFA in both configurations. We included biological | |
− | + | duplicates in this assay.<br/> | |
− | + | MbCas12a editing rates were significantly lower, dropping to about 1%. At the same time, when targeting VEGFA they | |
− | + | resulted in a higher editing efficiency than FANCF. | |
+ | </p> | ||
<div class="thumb"> | <div class="thumb"> | ||
<div class="thumbinner" style="width:60%;"> | <div class="thumbinner" style="width:60%;"> | ||
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/results-fcas-2.svg" style="width:99%;"/> | <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/results-fcas-2.svg" style="width:99%;"/> | ||
− | <div> | + | <div class="thumbcaption"> |
<i> | <i> | ||
− | <b>Figure 9: Editing | + | <b>Figure 9: Editing Rates for Fusion Guide RNAs with Fusion Cas Proteins.</b> |
− | + | In <b>A</b> and <b>B</b> the editing rates were determined 72h after transfection via T7EI assay. Editing % | |
− | + | was | |
− | + | determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved | |
− | + | band))<sup>1/2</sup>. The | |
− | + | schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols | |
− | + | below | |
+ | indicate which parts are included in each sample. Cas proteins linked by a dash ("-") were fused to each | ||
+ | other. | ||
+ | Biological replicates are marked as individual dots. | ||
+ | </i> | ||
</div> | </div> | ||
</div> | </div> | ||
Line 523: | Line 626: | ||
<h1>4. Results</h1> | <h1>4. Results</h1> | ||
<p> | <p> | ||
− | + | We extensively characterized the catalytically active MbCas12a in several assays determining the best ortholog out | |
− | + | of | |
− | + | three for our Cas staples, assert functional editing with a fusion guide RNA (BBa_K5237000), showing high activity | |
− | + | while being co-transfected with SpCas9, our second staple protein's active version, and lastly a functioning fusion | |
− | + | to | |
− | + | SpCas9.<br/> | |
− | <section><h2 id="4.1">4.1 dMbCas12a | + | After all these successful test we were confident to test the Cas staples in action. |
+ | </p> | ||
+ | <section> | ||
+ | <h2 id="4.1">4.1 dMbCas12a as Part of a Cas Staple Establishes DNA-DNA Proximity</h2> | ||
<p> | <p> | ||
− | + | The next step was to use the MbCas12a as part of a Cas staple to bring two DNA loci together, and thereby induce | |
− | + | proximity between two separate functional elements. Mimicking the phenomenon of enhancer hijacking, in which an enhancer | |
− | + | activates an otherwise distant promoter, we constructed artificial enhancer and reporter plasmids. The | |
− | + | reporter plasmid encodes a firefly luciferase downstream of several repeats of a Cas9 targeted sequence. The enhancer plasmid | |
− | + | has a | |
− | + | Gal4 binding site behind several repeats of a Cas12a targeted sequence. By introducing a fgRNA staple and a | |
− | + | Gal4-VP64, | |
− | + | expression of the luciferase is induced (Fig. 10A). | |
− | + | Different linker lengths were tested. Cells were again normalized against ubiquitous renilla expression. Further | |
− | + | information on our learnings from this assay can be found in the Cas staple engineering cycle iteration 2. | |
+ | Using no linker between the two spacers showed similar relative luciferase activity to the baseline control (Fig. | ||
+ | 10B). | ||
+ | An extension of the linker from 20 nt up to 40 nt resulted in an increasingly higher expression of the reporter | ||
+ | gene. | ||
+ | </p> | ||
<div class="thumb"> | <div class="thumb"> | ||
<div class="thumbinner" style="width:60%;"> | <div class="thumbinner" style="width:60%;"> | ||
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<div class="thumbcaption"> | <div class="thumbcaption"> | ||
<i> | <i> | ||
− | <b>Figure 10: Applying Fusion Guide RNAs for Cas staples.</b> <b>A</b>, schematic overview of the assay. An enhancer | + | <b>Figure 10: Applying Fusion Guide RNAs for Cas staples.</b> <b>A</b>, schematic overview of the assay. An |
− | + | enhancer | |
− | + | plasmid and a reporter plasmid are brought into proximity by a fgRNA Cas staple complex binding both | |
− | + | plasmids. Target | |
− | + | sequences were included in multiple repeats prior to the functional elements. Firefly luciferase serves as | |
− | + | the reporter | |
− | + | gene, the enhancer is constituted by multiple Gal4 repeats that are bound by a Gal4-VP64 fusion. | |
− | + | <b>B</b>, results of using a fgRNA Cas staple for trans activation of firefly luciferase. Firefly | |
− | + | luciferase activity was measured 48h after transfection. Normalized against ubiquitously expressed Renilla | |
− | + | luciferase. | |
+ | Statistical significance was calculated with ordinary One-way ANOVA with Dunn's method for multiple | ||
+ | comparisons (*p < | ||
+ | 0.05; **p < 0.01; ***p < 0.001; mean +/- SD). The assay included sgRNAs and fgRNAs with linker lengths | ||
+ | from 0 nt | ||
+ | to 40 nt. | ||
+ | </i> | ||
</div> | </div> | ||
</div> | </div> | ||
Line 563: | Line 680: | ||
<section id="5"> | <section id="5"> | ||
<h1>5. References</h1> | <h1>5. References</h1> | ||
− | <p>Kampmann, M. (2017). CRISPRi and CRISPRa Screens in Mammalian Cells for Precision Biology and Medicine. <i>ACS Chemical Biology, 13</i>, 406–416. <a href="https://doi.org/10.1021/acschembio.7b00657" target="_blank">https://doi.org/10.1021/acschembio.7b00657</a>.</p> | + | <p>Kampmann, M. (2017). CRISPRi and CRISPRa Screens in Mammalian Cells for Precision Biology and Medicine. <i>ACS |
− | <p>Kleinstiver, B. P., Sousa, A. A., Walton, R. T., Tak, Y. E., Hsu, J. Y., Clement, K., Welch, M. M., Horng, J. E., Malagon-Lopez, J., Scarfò, I., Maus, M. V., Pinello, L., Aryee, M. J., and Joung, J. K. (2019). Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. <i>Nature Biotechnology, 37</i>, 276–282. <a href="https://doi.org/10.1038/s41587-018-0011-0" target="_blank">https://doi.org/10.1038/s41587-018-0011-0</a>.</p> | + | Chemical Biology, 13</i>, 406–416. <a href="https://doi.org/10.1021/acschembio.7b00657" target="_blank">https://doi.org/10.1021/acschembio.7b00657</a>.</p> |
− | <p>Koonin, E. V., Gootenberg, J. S., and Abudayyeh, O. O. (2023). Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox. <i>Biochemistry, 62</i>, 3465–3487. <a href="https://doi.org/10.1021/acs.biochem.3c00159" target="_blank">https://doi.org/10.1021/acs.biochem.3c00159</a>.</p> | + | <p>Kleinstiver, B. P., Sousa, A. A., Walton, R. T., Tak, Y. E., Hsu, J. Y., Clement, K., Welch, M. M., Horng, J. E., |
− | <p>Pacesa, M., Pelea, O., and Jinek, M. (2024). Past, present, and future of CRISPR genome editing technologies. <i>Cell, 187</i>, 1076–1100. <a href="https://doi.org/10.1016/j.cell.2024.01.042" target="_blank">https://doi.org/10.1016/j.cell.2024.01.042</a>.</p> | + | Malagon-Lopez, J., Scarfò, I., Maus, M. V., Pinello, L., Aryee, M. J., and Joung, J. K. (2019). Engineered |
− | <p>Paul, B., and Montoya, G. (2020). CRISPR-Cas12a: Functional overview and applications. <i>Biomedical Journal, 43</i>, 8–17. <a href="https://doi.org/10.1016/j.bj.2019.10.005" target="_blank">https://doi.org/10.1016/j.bj.2019.10.005</a>.</p> | + | CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base |
− | <p>Zetsche, B., Gootenberg, J. S., Abudayyeh, O. O., Slaymaker, I. M., Makarova, K. S., Essletzbichler, P., Volz, S. E., Joung, J., van der Oost, J., Regev, A., Koonin, E. V., and Zhang, F. (2015). Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. <i>Cell, 163</i>, 759–771. <a href="https://doi.org/10.1016/j.cell.2015.09.038" target="_blank">https://doi.org/10.1016/j.cell.2015.09.038</a>.</p> | + | editing. <i>Nature Biotechnology, 37</i>, 276–282. <a href="https://doi.org/10.1038/s41587-018-0011-0" target="_blank">https://doi.org/10.1038/s41587-018-0011-0</a>.</p> |
+ | <p>Koonin, E. V., Gootenberg, J. S., and Abudayyeh, O. O. (2023). Discovery of Diverse CRISPR-Cas Systems and | ||
+ | Expansion of the Genome Engineering Toolbox. <i>Biochemistry, 62</i>, 3465–3487. <a href="https://doi.org/10.1021/acs.biochem.3c00159" target="_blank">https://doi.org/10.1021/acs.biochem.3c00159</a>.</p> | ||
+ | <p>Pacesa, M., Pelea, O., and Jinek, M. (2024). Past, present, and future of CRISPR genome editing technologies. | ||
+ | <i>Cell, 187</i>, 1076–1100. <a href="https://doi.org/10.1016/j.cell.2024.01.042" target="_blank">https://doi.org/10.1016/j.cell.2024.01.042</a>.</p> | ||
+ | <p>Paul, B., and Montoya, G. (2020). CRISPR-Cas12a: Functional overview and applications. <i>Biomedical Journal, | ||
+ | 43</i>, 8–17. <a href="https://doi.org/10.1016/j.bj.2019.10.005" target="_blank">https://doi.org/10.1016/j.bj.2019.10.005</a>.</p> | ||
+ | <p>Zetsche, B., Gootenberg, J. S., Abudayyeh, O. O., Slaymaker, I. M., Makarova, K. S., Essletzbichler, P., Volz, S. | ||
+ | E., Joung, J., van der Oost, J., Regev, A., Koonin, E. V., and Zhang, F. (2015). Cpf1 Is a Single RNA-Guided | ||
+ | Endonuclease of a Class 2 CRISPR-Cas System. <i>Cell, 163</i>, 759–771. <a href="https://doi.org/10.1016/j.cell.2015.09.038" target="_blank">https://doi.org/10.1016/j.cell.2015.09.038</a>. | ||
+ | </p> | ||
</section> | </section> | ||
</html> | </html> |
Revision as of 06:51, 2 October 2024
Staple Subunit: dMbCas12a-Nucleoplasmin NLS
dMbCas12a is one crucial part of our PICasSO tool box, being able to function as a Cas staple with the fgRNA (BBa_K5237000) and the dSpCas9 (BBa_K5237002). Transactivation has been shown using this part proving the proper formation of our Cas staple. Easy employment through swift reprogramming of the fgRNA.
Contents
- 1 Sequence overview
- 2 Usage and Biology
- 3 Assembly
and part evolution
- 3.1 Qualtitative assesment of Cas12a orthologs
- 3.2 Quantitative comparison between AsCas12a and MbCas12a
- 3.3 MbCas12a tolerates co-transfection and simultaneous editing of different Cas proteins
- 3.4 MbCas12a shows editing with fgRNA
- 3.5 MbCas12a withstanding fusion to SpCas9 while staying functional
- 3.6 MbCas12a fused to SpCas9 editing utilizing a fgRNA
- 4 Results
- 5 References
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
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Illegal BglII site found at 3311 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 274
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Illegal PstI site found at 3324
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In 2012, Jinek et al. discovered the use of the Clustered Regularly Interspaced Short Palindromic Repeats
(CRISPR)/Cas system to induce double-strand breaks in DNA. Since then, the system has been well established as a
tool for genome editing. The CRISPR/Cas system, which originates from the bacterial immune system, is constituted
by a ribonucleoprotein complex. For class 1 CRISPR systems, the RNA is complexed by multiple Cas proteins, whereas
class 2 systems consist of a singular protein and RNA. The class 2 type II system describes all ribonucleoprotein
complexes with Cas9 (Pacesa et al., 2024). They include a CRISPR RNA (crRNA), which specifies the target with
a 20
nucleotide (nt) spacer sequence, and a transactivating CRISPR RNA (tracrRNA), which induces the processing by the
Cas protein (Jinek et al., 2012) (Figure 2 A). Furthermore, a specific three
nucleotide sequence (NGG) on the 3' end in the targeted DNA is needed for binding and cleavage. This is referred
to as the protospacer adjacent motif (PAM) (Sternberg et al., 2014). The most commonly used Cas9 protein is
SpCas9
or SpyCas9, which originates from Streptococcus pyogenes (Pacesa et al., 2024).
A significant enhancement of this system was the introduction of single guide RNA (sgRNA)s, which combine the
functions of a tracrRNA and crRNA (Mali et al., 2013).
Moreover, Cong (2013) established precise targeting of human endogenous loci by designing the 20 nt spacer sequence
accordingly.
To select a suitable Cas12a ortholog for constructing the Cas staples, three different orthologs were ordered
from Addgene:
AsCas12a (#69982), LbCas12a (#69988),
and MbCas12a (#115142).
Since the T7EI assay can only be used to qualitatively compare editing efficiencies, it was hard to distinguish
between the better editing ortholog.
To employ the MbCas12a as part of a Cas staple, we sought to determine MbCas12a's activity while
being transfected together with SpCas9. Therefore we engineered the dual luciferase assay to allow us for testing
two
catalytically active Cas proteins at once. Including a well established protospacer, CCR5, into the renilla
luciferase
gene enables us to test the editing rates of two Cas proteins simultaneously (Fig. 5).
For the MbCas12a we have a highly significant editing rate in the single cut, meaning both Cas proteins are
transfected,
but only MbCas12a has a targeting gRNA. When introducing a targeting gRNA for SpCas9 we did not observe reduction in the
highly
significant editing of MbCas12a, strongly suggesting both Cas proteins to be able to edit simultaneously.
To further confirm that MbCas12a can be combined with fgRNAs in a functional Cas staple, editing rates were tested
using a fusion guide RNA
(fgRNA, BBa_K5237000)
targeting two different loci: FANCF and VEGFA. To better assess the impact that the utilization of a
fgRNA has on the editing rates, the sgRNAs were tested separately and in one sample.
To further assess the effect of the genomic locus on the editing rate, we included CCR5 as an additional gene
target.
For this assay, a fgRNA with a 20 nt long linker was included between the two spacers.
The editing rate for VEGFA was again relatively consistent throughout the samples (Fig. 7).
Testing showed simultaneous editing of MbCas12a and SpCas9. Therefore we wanted to test protein-protein fusion,
potentially giving us another way of Cas stapling. Our goal was to find out how well MbCas12a can stay active
while
being fused to SpCas9. Therefore we employed the previously engineered dual luciferase assay to allow us for
testing two
catalytically active Cas proteins at once, this time being fused to each other (Fig. 8).
For the MbCas12a we have a highly significant editing rate in the single cut, meaning only MbCas12a has a
targeting
gRNA. When introducing a targeting gRNA for SpCas9 we see no reduction in the highly significant editing of
MbCas12a,
strongly suggesting both Cas proteins in the fusion Cas to be able to edit simultaneously.
The gene editing efficiency of MbCas12a in the fusion Cas was tested by assessing the editing rates via a T7EI
assay. For this, the
same target sequences as before were used, namely FANCF and VEGFA in both configurations. We included biological
duplicates in this assay.
We extensively characterized the catalytically active MbCas12a in several assays determining the best ortholog out
of
three for our Cas staples, assert functional editing with a fusion guide RNA (BBa_K5237000), showing high activity
while being co-transfected with SpCas9, our second staple protein's active version, and lastly a functioning fusion
to
SpCas9.
The next step was to use the MbCas12a as part of a Cas staple to bring two DNA loci together, and thereby induce
proximity between two separate functional elements. Mimicking the phenomenon of enhancer hijacking, in which an enhancer
activates an otherwise distant promoter, we constructed artificial enhancer and reporter plasmids. The
reporter plasmid encodes a firefly luciferase downstream of several repeats of a Cas9 targeted sequence. The enhancer plasmid
has a
Gal4 binding site behind several repeats of a Cas12a targeted sequence. By introducing a fgRNA staple and a
Gal4-VP64,
expression of the luciferase is induced (Fig. 10A).
Different linker lengths were tested. Cells were again normalized against ubiquitous renilla expression. Further
information on our learnings from this assay can be found in the Cas staple engineering cycle iteration 2.
Using no linker between the two spacers showed similar relative luciferase activity to the baseline control (Fig.
10B).
An extension of the linker from 20 nt up to 40 nt resulted in an increasingly higher expression of the reporter
gene.
Kampmann, M. (2017). CRISPRi and CRISPRa Screens in Mammalian Cells for Precision Biology and Medicine. ACS
Chemical Biology, 13, 406–416. https://doi.org/10.1021/acschembio.7b00657. Kleinstiver, B. P., Sousa, A. A., Walton, R. T., Tak, Y. E., Hsu, J. Y., Clement, K., Welch, M. M., Horng, J. E.,
Malagon-Lopez, J., Scarfò, I., Maus, M. V., Pinello, L., Aryee, M. J., and Joung, J. K. (2019). Engineered
CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base
editing. Nature Biotechnology, 37, 276–282. https://doi.org/10.1038/s41587-018-0011-0. Koonin, E. V., Gootenberg, J. S., and Abudayyeh, O. O. (2023). Discovery of Diverse CRISPR-Cas Systems and
Expansion of the Genome Engineering Toolbox. Biochemistry, 62, 3465–3487. https://doi.org/10.1021/acs.biochem.3c00159. Pacesa, M., Pelea, O., and Jinek, M. (2024). Past, present, and future of CRISPR genome editing technologies.
Cell, 187, 1076–1100. https://doi.org/10.1016/j.cell.2024.01.042. Paul, B., and Montoya, G. (2020). CRISPR-Cas12a: Functional overview and applications. Biomedical Journal,
43, 8–17. https://doi.org/10.1016/j.bj.2019.10.005. Zetsche, B., Gootenberg, J. S., Abudayyeh, O. O., Slaymaker, I. M., Makarova, K. S., Essletzbichler, P., Volz, S.
E., Joung, J., van der Oost, J., Regev, A., Koonin, E. V., and Zhang, F. (2015). Cpf1 Is a Single RNA-Guided
Endonuclease of a Class 2 CRISPR-Cas System. Cell, 163, 759–771. https://doi.org/10.1016/j.cell.2015.09.038.
2. Usage and Biology
3. Assembly and Part Evolution
3.1 Qualitatively Assessing Gene Editing of Cas12a Orthologs
We assessed their performance using a T7 Endonuclease I (T7EI) assay on three genomic loci: RUNX1, DNMT1, and
FANCF. For
comparison, we also included SpCas9 editing the CCR5 locus (Fig. 3). Editing was only observed at the
RUNX1 locus, where we used the qualitative assay to compare the editing efficiencies of the Cas12a orthologs.
3.2 Quantitative Comparison Between AsCas12a and MbCas12a
To accurately quantify the editing efficiency, we conducted a dual luciferase assay. This assay measures the
luminescence
of firefly luciferase, which decreases proportionally to the editing efficiency at the target site.
To account for variations in cell count and transfection efficiency, the luminescence is normalized
to Renilla luciferase, which acts as an internal control (Fig. 4).
The results revealed a clear difference, with MbCas12a demonstrating significantly higher editing efficiency
compared to
AsCas12a (p=0.005). Based on these findings, we decided to proceed with MbCas12a.
3.3 Multiplex Gene Editing Using MbCas12a and SpCas9
3.4 Fusion Guide RNA Enabled Editing with MbCas12a
Having the sgRNA with single Cas proteins in the same sample resulted in no clear
difference in the editing rates (Fig. 6). The fusion of the gRNAs resulted in a lower editing rate
overall. While the editing for VEGFA stayed at about 20% in all
cases, the editing for FANCF dropped significantly. Nonetheless we were able to show MbCas12a editing utilizing a
fgRNA.
3.5 Establishing Functional Fusion Cas Proteins with MbCas12a and SpCas9
3.6 The Combination of Fusion Guide RNAs and Fusion Cas Proteins
MbCas12a editing rates were significantly lower, dropping to about 1%. At the same time, when targeting VEGFA they
resulted in a higher editing efficiency than FANCF.
4. Results
After all these successful test we were confident to test the Cas staples in action.
4.1 dMbCas12a as Part of a Cas Staple Establishes DNA-DNA Proximity
5. References