Difference between revisions of "Part:BBa K5237000"
Line 26: | Line 26: | ||
padding: 5px; | padding: 5px; | ||
} | } | ||
− | |||
.thumbcaption { | .thumbcaption { | ||
− | + | text-align:justify !important; | |
− | + | } | |
− | a[href ^="https://"], | + | a[href ^="https://"],.link-https { |
− | + | ||
background: none !important; | background: none !important; | ||
− | padding-right: 0px !important; | + | padding-right:0px !important; |
− | + | } | |
+ | |||
</style> | </style> | ||
− | |||
<body> | <body> | ||
− | + | <!-- Part summary --> | |
− | + | <section id="1"> | |
− | + | <h1>fgRNA Entry Vector MbCas12a-SpCas9</h1> | |
− | + | <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 | + | guide RNA (fgRNA). The fgRNA is functional, meaning that the 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 | + | can utilize the fgRNA to target two loci simultaneously. The fgRNA also works in combination with the catalyitcally inactive Cas |
− | + | ||
− | can utilize the fgRNA to target two loci simultaneously. The fgRNA also works with the catalyitcally inactive | + | |
versions. | versions. | ||
− | We | + | We successfully showed genome editing using active SpCas9 and Cas12a and induced proximity of two loci with the inactive dSpCas9 and dMbCas12a.<br/> |
+ | For our part collection, the PICasSO toolbox, this part has a crucial role in formation of our CRISPR/Cas staples. | ||
</p> | </p> | ||
− | + | <p> </p> | |
− | + | </section> | |
− | + | <div class="toc" id="toc"> | |
− | + | <div id="toctitle"> | |
− | + | <h1>Contents</h1> | |
− | + | </div> | |
− | + | <ul> | |
− | + | <li class="toclevel-1 tocsection-1"><a href="#1"><span class="tocnumber">1</span> <span class="toctext">Sequence | |
overview</span></a> | overview</span></a> | ||
− | + | </li> | |
− | + | <li class="toclevel-1 tocsection-2"><a href="#2"><span class="tocnumber">2</span> <span class="toctext">Usage and | |
Biology</span></a> | Biology</span></a> | ||
− | + | <ul> | |
− | + | <li class="toclevel-2 tocsection-2.1"> | |
− | + | <a href="#2.1"><span class="tocnumber">2.1</span> <span class="toctext">Discovery and Mechanism of | |
CRISPR/Cas9</span></a> | CRISPR/Cas9</span></a> | ||
− | + | </li> | |
− | + | <li class="toclevel-2 tocsection-2.2"> | |
− | + | <a href="#2.2"><span class="tocnumber">2.2</span> <span class="toctext">Differences between Cas9 and | |
Cas12a</span></a> | Cas12a</span></a> | ||
− | + | </li> | |
− | + | <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 | |
Application</span></a> | Application</span></a> | ||
− | + | </li> | |
− | + | <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> | |
− | + | </ul> | |
− | + | </li> | |
− | + | <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 class="toclevel-1 tocsection-4"><a href="#4"><span class="tocnumber">4</span> <span class="toctext">Results</span></a> | |
− | + | <ul> | |
− | + | <li class="toclevel-2 tocsection-4.1"> | |
− | + | <a href="#4.1"><span class="tocnumber">4.1</span> <span class="toctext">Editing endogenous loci with | |
− | + | ||
fgRNAs</span></a> | fgRNAs</span></a> | ||
− | + | </li> | |
− | + | <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 | |
proteins</span></a> | proteins</span></a> | ||
− | + | </li> | |
− | + | <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 | |
Lower Editing Rates</span></a> | Lower Editing Rates</span></a> | ||
− | + | </li> | |
− | + | <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 | |
CRISPRa</span></a> | CRISPRa</span></a> | ||
− | + | </li> | |
− | + | <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 | |
Using fgRNAs</span></a> | Using fgRNAs</span></a> | ||
− | + | </li> | |
− | + | </ul> | |
− | + | </li> | |
− | + | <li class="toclevel-1 tocsection-8"><a href="#5"><span class="tocnumber">5</span> <span class="toctext">References</span></a> | |
− | + | </li> | |
− | + | </ul> | |
− | + | </div> | |
− | + | <section><p><br/><br/></p> | |
− | + | <font size="5"><b>The PICasSO Toolbox </b> </font> | |
− | + | <div class="thumb" style="margin-top:10px;"></div> | |
− | + | <div class="thumbinner" style="width:550px"><img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/registry-part-collection-engineering-cycle-example-overview.svg" style="width:99%;"/> | |
− | + | <div class="thumbcaption"> | |
− | + | <i><b>Figure 1: How our part collection can be used to engineer new staples</b></i> | |
− | + | </div> | |
− | + | </div> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | <p> | |
− | + | <br/> | |
− | Next to the well-studied linear DNA sequence, the 3D spatial organization of DNA plays a crucial role in | + | Next to the well-studied linear DNA sequence, the <b>3D spatial organization</b> of DNA plays a crucial role in |
− | regulation, | + | gene regulation, |
− | cell fate, disease development and more. However, the tools to precisely manipulate this genomic | + | cell fate, disease development and more. However, the tools to precisely manipulate this genomic |
− | remain limited, rendering it challenging to explore the full potential of the | + | architecture remain limited, rendering it challenging to explore the full potential of the |
− | 3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful | + | 3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a <b>powerful |
− | toolbox based on various DNA-binding proteins to address this issue. | + | molecular toolbox</b> based on various DNA-binding proteins to address this issue. |
</p> | </p> | ||
− | + | <p> | |
The <b>PICasSO</b> part collection offers a comprehensive, modular platform for precise manipulation and | The <b>PICasSO</b> part collection offers a comprehensive, modular platform for precise manipulation and | ||
− | re-programming | + | <b>re-programming |
− | of DNA-DNA interactions using protein staples in living cells, enabling researchers to recreate natural 3D genomic | + | of DNA-DNA interactions</b> using protein staples in living cells, enabling researchers to recreate natural 3D genomic |
interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation. | interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation. | ||
− | Beyond its versatility, PICasSO includes robust assay systems to support the engineering, optimization, and | + | Specifically, the fusion of two DNA binding proteins enables to artifically bring distant genomic loci into |
+ | proximty. | ||
+ | To unlock the system's full potential, we introduce versatile <b>chimeric CRISPR/Cas complexes</b>, connected either on | ||
+ | the protein or the guide RNA level. These1 complexes are reffered to as protein- or Cas staples. Beyond its | ||
+ | versatility, PICasSO includes <b>robust assay</b> systems to support the engineering, optimization, and | ||
testing of new staples, ensuring functionality <i>in vitro</i> and <i>in vivo</i>. We took special care to include | testing of new staples, ensuring functionality <i>in vitro</i> and <i>in vivo</i>. We took special care to include | ||
− | parts crucial for testing every step of the cycle (design, build, test, learn) when engineering new parts. | + | parts crucial for testing every step of the cycle (design, build, test, learn) when <b>engineering new parts</b>. |
</p> | </p> | ||
− | + | <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 enhancer hijacking Cas staple as well as half staples that can be used by scientists to compose entirely | ||
new Cas staples in the future. We also include our Simple staples that serve as controls for successful stapling | new Cas staples in the future. We also include our Simple staples that serve as controls for successful stapling | ||
− | and can be further engineered to create alternative, simpler and more compact staples. <br /> | + | and can be further engineered to create alternative, simpler and more compact staples. <br/> |
− | + | <b>(ii)</b> As <b>functional elements</b>, we list additional parts that enhance the functionality of our Cas and | |
Basic staples. These | Basic staples. These | ||
consist of | consist of | ||
Line 159: | Line 155: | ||
Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs | Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs | ||
with our | with our | ||
− | interkingdom conjugation system. <br /> | + | interkingdom conjugation system. <br/> |
− | + | <b>(iii)</b> As the final category of our collection, we provide parts that support the use of our <b>custom | |
readout | 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 | ||
Line 168: | Line 164: | ||
in mammalian cells. | in mammalian cells. | ||
</p> | </p> | ||
− | + | <p> | |
− | The following table gives a comprehensive overview of all parts in our PICasSO toolbox. <mark | + | 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 their | ||
− | own custom Cas staples, enabling further optimization and innovation.<br /> | + | own custom Cas staples, enabling further optimization and innovation.<br/> |
− | + | </p> | |
− | + | <p> | |
− | + | <font size="4"><b>Our part collection includes:</b></font><br/> | |
− | + | </p> | |
− | + | <table style="width: 90%; padding-right:10px;"> | |
− | + | <td 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 | The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring | ||
easy assembly.</td> | easy assembly.</td> | ||
− | + | <tbody> | |
− | + | <tr bgcolor="#FFD700"> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a></td> | |
− | + | <td>fgRNA Entry vector MbCas12a-SpCas9</td> | |
− | + | <td>Entryvector for simple fgRNA cloning via SapI</td> | |
− | + | </tr> | |
− | + | <tr bgcolor="#FFD700"> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237001" target="_blank">BBa_K5237001</a></td> | |
− | + | <td>Staple subunit: dMbCas12a-Nucleoplasmin NLS</td> | |
− | + | <td>Staple subunit that can be combined with sgRNA or fgRNA and dCas9 to form a functional staple</td> | |
− | + | </tr> | |
− | + | <tr bgcolor="#FFD700"> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237002" target="_blank">BBa_K5237002</a></td> | |
− | + | <td>Staple subunit: SV40 NLS-dSpCas9-SV40 NLS</td> | |
− | + | <td>Staple subunit that can be combined witha sgRNA or fgRNA and dCas12avto form a functional staple | |
</td> | </td> | ||
− | + | </tr> | |
− | + | <tr> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a></td> | |
− | + | <td>Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS</td> | |
− | + | <td>Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands into close | |
proximity | proximity | ||
</td> | </td> | ||
− | + | </tr> | |
− | + | <tr> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237004" target="_blank">BBa_K5237004</a></td> | |
− | + | <td>Staple subunit: Oct1-DBD</td> | |
− | + | <td>Staple subunit that can be combined to form a functional staple, for example with TetR.<br/> | |
Can also be combined with a fluorescent protein as part of the FRET proximity assay</td> | Can also be combined with a fluorescent protein as part of the FRET proximity assay</td> | ||
− | + | </tr> | |
− | + | <tr> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237005" target="_blank">BBa_K5237005</a></td> | |
− | + | <td>Staple subunit: TetR</td> | |
− | + | <td>Staple subunit that can be combined to form a functional staple, for example with Oct1.<br/> | |
Can also be combined with a fluorescent protein as part of the FRET proximity assay</td> | Can also be combined with a fluorescent protein as part of the FRET proximity assay</td> | ||
− | + | </tr> | |
− | + | <tr> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237006" target="_blank">BBa_K5237006</a></td> | |
− | + | <td>Simple staple: TetR-Oct1</td> | |
− | + | <td>Functional staple that can be used to bring two DNA strands in close proximity</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237007" target="_blank">BBa_K5237007</a></td> | |
− | + | <td>Staple subunit: GCN4</td> | |
− | + | <td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237008" target="_blank">BBa_K5237008</a></td> | |
− | + | <td>Staple subunit: rGCN4</td> | |
− | + | <td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237009" target="_blank">BBa_K5237009</a></td> | |
− | + | <td>Mini staple: bGCN4</td> | |
− | + | <td> | |
Assembled staple with minimal size that can be further engineered</td> | Assembled staple with minimal size that can be further engineered</td> | ||
− | + | </tr> | |
− | + | </tbody> | |
− | + | <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> | |
− | + | <tr bgcolor="#FFD700"> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a></td> | |
− | + | <td>Cathepsin B-cleavable Linker: GFLG</td> | |
− | + | <td>Cathepsin B-cleavable peptide linker that can be used to combine two staple subunits to make responsive | |
staples</td> | staples</td> | ||
− | + | </tr> | |
− | + | <tr> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237011" target="_blank">BBa_K5237011</a></td> | |
− | + | <td>Cathepsin B Expression Cassette</td> | |
− | + | <td>Expression Cassette for the overexpression of cathepsin B</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237012" target="_blank">BBa_K5237012</a></td> | |
− | + | <td>Caged NpuN Intein</td> | |
− | + | <td>A caged NpuN split intein fragment that undergoes protein <i>trans</i>-splicing after protease activation. | |
Can be used to create functionalized staples | Can be used to create functionalized staples | ||
units</td> | units</td> | ||
− | + | </tr> | |
− | + | <tr> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237013" target="_blank">BBa_K5237013</a></td> | |
− | + | <td>Caged NpuC Intein</td> | |
− | + | <td>A caged NpuC split intein fragment that undergoes protein <i>trans</i>-splicing after protease activation. | |
Can be used to create functionalized staples | Can be used to create functionalized staples | ||
units</td> | units</td> | ||
− | + | </tr> | |
− | + | <tr> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237014" target="_blank">BBa_K5237014</a></td> | |
− | + | <td>fgRNA processing casette</td> | |
− | + | <td>Processing casette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D | |
genome reprograming</td> | genome reprograming</td> | ||
− | + | </tr> | |
− | + | <tr> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237015" target="_blank">BBa_K5237015</a></td> | |
− | + | <td>Intimin anti-EGFR Nanobody</td> | |
− | + | <td>Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large | |
constructs</td> | constructs</td> | ||
− | + | </tr> | |
− | + | <tr> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K4643003" target="_blank">BBa_K4643003</a></td> | |
− | + | <td>incP origin of transfer</td> | |
− | + | <td>Origin of transfer that can be cloned into the plasmid vector and used for conjugation as a means of | |
delivery</td> | delivery</td> | ||
− | + | </tr> | |
− | + | </tbody> | |
− | + | <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 to measure proximity of stapled DNA in bacterial and mammalian living cells | ||
enabling swift testing and easy development for new systems</td> | enabling swift testing and easy development for new systems</td> | ||
− | + | <tbody> | |
− | + | <tr bgcolor="#FFD700"> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237016" target="_blank">BBa_K5237016</a></td> | |
− | + | <td>FRET-Donor: mNeonGreen-Oct1</td> | |
− | + | <td>FRET Donor-Fluorpohore fused to Oct1-DBD that binds to the Oct1 binding cassette. Can be used to visualize | |
DNA-DNA | DNA-DNA | ||
proximity</td> | proximity</td> | ||
− | + | </tr> | |
− | + | <tr bgcolor="#FFD700"> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237017" target="_blank">BBa_K5237017</a></td> | |
− | + | <td>FRET-Acceptor: TetR-mScarlet-I</td> | |
− | + | <td>Acceptor part for the FRET assay binding the TetR binding cassette. Can be used to visualize DNA-DNA | |
proximity</td> | proximity</td> | ||
− | + | </tr> | |
− | + | <tr> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237018" target="_blank">BBa_K5237018</a></td> | |
− | + | <td>Oct1 Binding Casette</td> | |
− | + | <td>DNA sequence containing 12 Oct1 binding motifs, compatible with various assays such as the FRET | |
proximity assay</td> | proximity assay</td> | ||
− | + | </tr> | |
− | + | <tr> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a></td> | |
− | + | <td>TetR Binding Cassette</td> | |
− | + | <td>DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET | |
proximity assay</td> | proximity assay</td> | ||
− | + | </tr> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237020" target="_blank">BBa_K5237020</a></td> | |
− | + | <td>Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64</td> | |
− | + | <td>Readout system that responds to protease activity. It was used to test cathepsin B-cleavable linker</td> | |
− | + | <tr> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237021" target="_blank">BBa_K5237021</a></td> | |
− | + | <td>NLS-Gal4-VP64</td> | |
− | + | <td>Trans-activating enhancer, that can be used to simulate enhancer hijacking</td> | |
− | + | </tr> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237022" target="_blank">BBa_K5237022</a></td> | |
− | + | <td>mCherry Expression Cassette: UAS, minimal Promotor, mCherry</td> | |
− | + | <td>Readout system for enhancer binding. It was used to test cathepsin B-cleavable linker</td> | |
− | + | <tr> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237023" target="_blank">BBa_K5237023</a></td> | |
− | + | <td>Oct1 - 5x UAS binding casette</td> | |
− | + | <td>Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td><a href="https://parts.igem.org/Part:BBa_K5237024" target="_blank">BBa_K5237024</a></td> | |
− | + | <td>TRE-minimal promoter- firefly luciferase</td> | |
− | + | <td>Contains Firefly luciferase controlled by a minimal promoter. It was used as a luminescence readout for | |
simulated enhancer hijacking</td> | simulated enhancer hijacking</td> | ||
− | + | </tr> | |
− | + | </tbody> | |
− | + | </table> | |
− | + | </section> | |
− | + | <section id="1"> | |
− | + | <h1>1. Sequence overview</h1> | |
− | + | </section> | |
</body> | </body> | ||
− | |||
</html> | </html> | ||
<span class="h3bb">Sequence and Features</span> | <span class="h3bb">Sequence and Features</span> | ||
<partinfo>BBa_K5237000 SequenceAndFeatures</partinfo> | <partinfo>BBa_K5237000 SequenceAndFeatures</partinfo> | ||
<html> | <html> | ||
− | |||
<body> | <body> | ||
− | + | <section id="2"> | |
− | + | <h1>2. Usage and Biology</h1> | |
− | + | <section id="2.1"> | |
− | + | <h2>2.1 Discovery and Mechanism of CRISPR/Cas9</h2> | |
− | + | <div class="thumb tright" style="margin:0;"> | |
− | + | <div class="thumbinner" style="width:450px;"> | |
− | + | <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/cas-staple-svg/background-cas9-cas12a-principle.svg" style="width:99%;"/> | |
− | + | <div class="thumbcaption"> | |
− | + | <i> | |
− | + | <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 strand with the | ||
PAM. | PAM. | ||
Line 374: | Line 364: | ||
spacer is located at the 5' prime end, for Cas12a at the 3' end of the gRNA. The scaffold of the gRNA | 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 | forms a specific | ||
− | secondary structure enabling it to | + | secondary structure enabling it to be bound by the Cas protein. DNA cleavage sites are indicated by the scissors. |
− | + | ||
− | + | ||
</i> | </i> | ||
− | + | </div> | |
− | + | </div> | |
− | + | </div> | |
− | + | <p> | |
In 2012, Jinek <i>et al.</i> discovered the use of the Clustered Regularly Interspaced Short Palindromic Repeats | In 2012, Jinek <i>et al.</i> discovered the use of the Clustered Regularly Interspaced Short Palindromic Repeats | ||
(CRISPR)/Cas system to induce double-strand breaks in DNA. Since then, the system has been well established as a | (CRISPR)/Cas system to induce double-strand breaks in DNA. Since then, the system has been well established as a | ||
Line 395: | Line 383: | ||
the | the | ||
Cas protein (Jinek <i>et al.</i>, 2012) (Fig. 2 A). Furthermore, a specific three | Cas protein (Jinek <i>et al.</i>, 2012) (Fig. 2 A). Furthermore, a specific three | ||
− | nucleotide sequence (NGG) | + | nucleotide sequence (NGG) at 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 | to as the protospacer adjacent motif (PAM) (Sternberg <i>et al.</i>, 2014). The most commonly used Cas9 protein | ||
is SpCas9 | is SpCas9 | ||
or SpyCas9, which originates from Streptococcus pyogenes (Pacesa <i>et al.</i>, 2024). | 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 | + | A significant enhancement of this system was the introduction of single guide RNAs (sgRNA[s]), which combine the |
functions of a tracrRNA and crRNA (Mali <i>et al.</i>, 2013). | functions of a tracrRNA and crRNA (Mali <i>et al.</i>, 2013). | ||
Moreover, Cong (2013) established precise targeting of human endogenous loci by designing the 20 nt spacer | Moreover, Cong (2013) established precise targeting of human endogenous loci by designing the 20 nt spacer | ||
sequence accordingly. | sequence accordingly. | ||
</p> | </p> | ||
− | + | </section> | |
− | + | <section id="2.2"> | |
− | + | <h2>2.2 Differences between Cas9 and Cas12a</h2> | |
− | + | <p> | |
Over the following years, further CRISPR/Cas systems have been discovered, including the Cpf1 system, which has | Over the following years, further CRISPR/Cas systems have been discovered, including the Cpf1 system, which has | ||
been | been | ||
Line 427: | Line 415: | ||
2020). | 2020). | ||
</p> | </p> | ||
− | + | </section> | |
− | + | <section id="2.3"> | |
− | + | <h2>2.3 Dead Cas Proteins and their Application</h2> | |
− | + | <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 of | ||
nickases that only cut one of the DNA strands, or completely inactive Cas proteins (Koonin <i>et al.</i>, 2023) | nickases that only cut one of the DNA strands, or completely inactive Cas proteins (Koonin <i>et al.</i>, 2023) | ||
Line 440: | Line 428: | ||
involves fusing Cas9 with the transcriptional activator VP64 (Kampmann, 2017). | involves fusing Cas9 with the transcriptional activator VP64 (Kampmann, 2017). | ||
</p> | </p> | ||
− | + | </section> | |
− | + | </section> | |
− | + | <section id="3" style="clear:both;"> | |
− | + | <h1>3. Assembly and part evolution</h1> | |
− | + | <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 designed by | ||
− | combining the sgRNA from SpCas9 with the crRNA from MbCas12a | + | 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. Via this approach, the two spacer sequences are fused directly, ensuring a | |
minimal distance between the two DNA strands.This also facilitates efficient cloning of different spacer | minimal distance between the two DNA strands.This also facilitates efficient cloning of different spacer | ||
− | sequences. Linking the crRNA and sgRNA | + | sequences, as both spacers can be exchangeed as one consecutive sequence. Linking the crRNA and sgRNA further enables |
− | + | 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 | |
MbCas12a scaffold, a bacterial promoter driving <b>ccdB</b> expression, and the SpCas9 scaffold. Successful spacer | MbCas12a scaffold, a bacterial promoter driving <b>ccdB</b> expression, and the SpCas9 scaffold. Successful spacer | ||
integration leads to the removal of the <b>ccdB</b> gene, allowing bacterial growth to be used as an indicator for | integration leads to the removal of the <b>ccdB</b> gene, allowing bacterial growth to be used as an indicator for | ||
− | cloning success.<br /> | + | cloning success.<br/> |
− | + | A conventional gRNA expression vector containing an MbCas12a crRNA scoffold 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 entry vector cloning. The vector and a ccdB-SpCas9 scaffold construct were PCR amplified and fitting overhangs | ||
− | for SapI were introduced. Golden Gate assembly (GGA) with Esp3I was used to create the final plasmid. The | + | 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. | + | transformation was carried out in the ccdB-resistant XL1 Blue <i>E. Coli</i> strain. |
</p> | </p> | ||
− | + | <div class="thumb"> | |
− | + | <div class="thumbinner" style="width:80%;"> | |
− | + | <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/entry-vector.svg" style="width:99%;"/> | |
− | + | <div class="thumbcaption"> | |
− | + | <i> | |
− | + | <b>Figure 3: Construction process of fgRNAs using the entry vector.</b> The ccdB gene excised using | |
− | + | SapI in a Golden Gate | |
− | SapI in | + | |
assembly. By inserting oligonucleotides with the desired spacer sequences and matching overhangs, the | assembly. By inserting oligonucleotides with the desired spacer sequences and matching overhangs, the | ||
complete fgRNA | complete fgRNA | ||
− | can be | + | can be assembled into the entry vector. Due to the cytotoxic nature of ccdB, only cells with the oligonucleotides as inserts |
survive. | survive. | ||
</i> | </i> | ||
− | + | </div> | |
− | + | </div> | |
− | + | <p> | |
The first goal after assembly was to prove the editing activity of both proteins using fgRNA. The genes | The first goal after assembly was to prove the editing activity of both proteins using fgRNA. 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, each target was tested with each Cas protein. | ||
− | Editing efficiency | + | Editing efficiency was analyzed with the T7 Endonuclease I (T7EI) assay. Controls included crRNAs and |
− | sgRNAs as positive controls, and non-targeting guides as negative controls. Desired spacer sequences | + | sgRNAs as positive controls, and non-targeting guides as negative controls. Desired spacer sequences were |
ordered as oligos, annealed, and cloned in via GGA utilizing SapI. | ordered as oligos, annealed, and cloned in via GGA utilizing SapI. | ||
</p> | </p> | ||
− | + | <div class="thumb tright" style="margin:0;"></div> | |
− | + | <div class="thumbinner" style="width:400px;"> | |
− | + | <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/cas-staple-svg/background-crispr-cas-system-fgrna-past.svg" style="width:99%;"/> | |
− | + | <div class="thumbcaption"> | |
+ | <i> | ||
+ | <b>Figure 4: Applications of the Fusion Guide RNA</b> | ||
+ | Fusion Guide RNAs can be used for multiplex genome editing by guidingactive Cas12a and Cas9 to two | ||
+ | distinct loci. Similarly, fgRNAs allow for CRISPRa, by guiding the Cas9-VP64 transcriptional activator | ||
+ | towards a | ||
+ | target locus. | ||
+ | </i> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <table style="width:40%; margin-top:20px; margin-bottom:20px;"> | ||
+ | <thead> | ||
+ | <td align="left" colspan="2"> | ||
+ | <b>Table 1:</b> A list of all the different spacers we cloned and tested within the fgRNA | ||
</td> | </td> | ||
− | + | </thead> | |
− | + | <tbody> | |
− | + | <tr> | |
− | + | <td>VEGFA</td> | |
− | + | <td>ctaggaatattgaagggggc</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>FANCF</td> | |
− | + | <td>ggcggggtccagttccggga</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>CCR5</td> | |
− | + | <td>tgacatcaattattatacat</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>TetO (<a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a>)</td> | |
− | + | <td>tctctatcactgatagggag</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>Oct1-B (<a href="https://parts.igem.org/Part:BBa_K5237018" target="_blank">BBa_K5237018</a>)</td> | |
− | + | <td>atgcaaatactgcactagtg</td> | |
− | + | </tr> | |
− | + | </tbody> | |
− | + | </table> | |
− | + | <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 of | ||
MbCas12a. | MbCas12a. | ||
− | The sequence of the AsCas12a scaffold | + | The sequence of the AsCas12a scaffold was the only modification in the composite part. This vector was tested |
on the | on the | ||
loci VEGFA and FANCF to assess its functionality. | loci VEGFA and FANCF to assess its functionality. | ||
</p> | </p> | ||
− | + | ||
− | + | </section> | |
− | + | <section id="4"> | |
− | + | <h1>4. Results</h1> | |
− | + | <section id="4.1"> | |
− | + | <h2>4.1 Editing endogenous loci with fgRNAs</h2> | |
− | + | <p> | |
To prove that our fusion gRNAs still result in active ribonucleoproteins, a series of different fgRNAs were | To prove that our fusion gRNAs still result in active ribonucleoproteins, a series of different fgRNAs were | ||
created, each carrying spacers specific to the VEGFA and FANCF genes.HEK293-T cells were transfected with the | created, each carrying spacers specific to the VEGFA and FANCF genes.HEK293-T cells were transfected with the | ||
Cas | Cas | ||
protein and gRNA constructs. The editing rate was tested 72h after transfection via a T7 endonuclease I | protein and gRNA constructs. The editing rate was tested 72h after transfection via a T7 endonuclease I | ||
− | assay.<br /> | + | assay.<br/> |
AsCas12a and SpCas9 were used. The AsCas12a spacer targets VEGFA, while the SpCas9 spacer targets FANCF. The | AsCas12a and SpCas9 were used. The AsCas12a spacer targets VEGFA, while the SpCas9 spacer targets FANCF. The | ||
Line 582: | Line 546: | ||
fgRNAs. | fgRNAs. | ||
</p> | </p> | ||
− | + | <div class="thumb"> | |
− | + | <div class="thumbinner" style="width:60%;"> | |
− | + | <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/results-ascas-2.svg" style="width:99%;"/> | |
− | + | <div class="thumbcaption"> | |
− | + | <i> | |
− | + | <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 | The editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by | ||
measuring band | measuring band | ||
Line 597: | Line 560: | ||
each sample. | each sample. | ||
</i> | </i> | ||
− | + | </div> | |
− | + | </div> | |
− | + | </div> | |
− | + | </section> | |
− | + | <section id="4.2"> | |
− | + | <h2>4.2 Efficient Fusion Guide RNA-Mediated Editing With Various Cas Orthologs</h2> | |
− | + | <p> | |
− | + | After showing efficient editing, the next step was to evaluate the capabilities of the fgRNAs, we tested them in combination | |
with different Cas12a orthologs. After some initial testing, we decided on using MbCas12a together with SpCas9, | with different Cas12a orthologs. After some initial testing, we decided on using MbCas12a together with SpCas9, | ||
because we found AsCas12a to be less active in a dual luciferase assay when co-transfected with SpCas9 compared | because we found AsCas12a to be less active in a dual luciferase assay when co-transfected with SpCas9 compared | ||
Line 611: | Line 574: | ||
SpCas9 editing has not been significantly different. | SpCas9 editing has not been significantly different. | ||
</p> | </p> | ||
− | + | <div class="thumb"> | |
− | + | <div class="thumbinner" style="width:60%;"> | |
− | + | <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/engineering/cas12-decision.svg" style="width:99%;"/> | |
− | + | <div class="thumbcaption"> | |
− | + | <i> | |
− | + | <b>Figure 6: Comparison of AsCas12a and MbCas12a with a dual luciferase assay.</b> | |
− | + | ||
Firefly luminescence intensity measured 48 h after transfection. Normalized against renilla luminescence. | Firefly luminescence intensity measured 48 h after transfection. Normalized against renilla luminescence. | ||
On the x-axis | On the x-axis | ||
Line 628: | Line 590: | ||
****p<0.0001 | ****p<0.0001 | ||
</i> | </i> | ||
− | + | </div> | |
− | + | </div> | |
− | + | </div> | |
− | + | <p> | |
Additionally, to test if the differences in editing rates from the preliminary assay resulted from the targeted | Additionally, to test if the differences in editing rates from the preliminary assay resulted from the targeted | ||
loci or the different Cas orthologs, the spacers were tested in both arrangements. Once with Cas12a targeting | loci or the different Cas orthologs, the spacers were tested in both arrangements. Once with Cas12a targeting | ||
FANCF and SpCas9 targeting VEGFA and once vice versa. To better assess the impact that the utilization of a | FANCF and SpCas9 targeting VEGFA and once vice versa. To better assess the impact that the utilization of a | ||
fgRNA | fgRNA | ||
− | has on the editing rates, the sgRNAs were tested separately and in one sample.<br /> | + | has on the editing rates, the sgRNAs were tested separately and in one sample.<br/> |
Having the sgRNA with single Cas | Having the sgRNA with single Cas | ||
proteins in the same sample resulted in no clear difference in the editing rates (Fig. 7). The fusion of the | proteins in the same sample resulted in no clear difference in the editing rates (Fig. 7). The fusion of the | ||
Line 644: | Line 606: | ||
the same conditions, the editing rates for MbCas12a were overall lower than the ones from SpCas9. | the same conditions, the editing rates for MbCas12a were overall lower than the ones from SpCas9. | ||
</p> | </p> | ||
− | + | <div class="thumb"> | |
− | + | <div class="thumbinner" style="width:90%;"> | |
− | + | <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/results-mbcas-2.svg" style="width:99%;"/> | |
− | + | <div class="thumbcaption"> | |
− | + | <i> | |
− | + | <b>Figure 7: 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 | 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 | assay. Editing % was determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved | ||
Line 659: | Line 620: | ||
display both orientations of the two spacers for VEGFA and FANCF. | display both orientations of the two spacers for VEGFA and FANCF. | ||
</i> | </i> | ||
− | + | </div> | |
− | + | </div> | |
− | + | </div> | |
− | + | </section> | |
− | + | <section id="4.3"> | |
− | + | <h2>4.3 The Inclusion of a Linker Does Not Lower Editing Rates</h2> | |
− | + | <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 was included between the two spacers. The editing | ||
Line 673: | Line 634: | ||
addition of the 20 nt linker had no effect on the editing rates compared to no linker. | addition of the 20 nt linker had no effect on the editing rates compared to no linker. | ||
</p> | </p> | ||
− | + | <div class="thumb"> | |
− | + | <div class="thumbinner" style="width:60%;"> | |
− | + | <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/results-ccr5-2.svg" style="width:99%;"/> | |
− | + | <div class="thumbcaption"> | |
− | + | <i> | |
− | + | <b>Figure 8: 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 | The editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by | ||
measuring band | measuring band | ||
Line 688: | Line 648: | ||
each sample. Cas12a targets VEGFA and Cas9 targets CCR5. | each sample. Cas12a targets VEGFA and Cas9 targets CCR5. | ||
</i> | </i> | ||
− | + | </div> | |
− | + | </div> | |
− | + | </div> | |
− | + | </section> | |
− | + | <section id="4.4"> | |
− | + | <h2>4.4 fgRNAs can be used for CRISPRa</h2> | |
− | + | <p> | |
To establish the foundation for their use as protein scaffolds, we identified the next step as demonstrating the | To establish the foundation for their use as protein scaffolds, we identified the next step as demonstrating the | ||
use | use | ||
Line 706: | Line 666: | ||
to a sgRNA (Fig. 9). | to a sgRNA (Fig. 9). | ||
</p> | </p> | ||
− | + | <div class="thumb"> | |
− | + | <div class="thumbinner" style="width:40%;"> | |
− | + | <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/results-crispra-2.svg" style="width:99%;"/> | |
− | + | <div class="thumbcaption"> | |
− | + | <i> | |
− | + | <b>Figure 9: CRISPRa Induced Luciferase Expression for sgRNAs and fgRNAs.</b> | |
− | + | ||
Firefly luciferase activity was measured 48h after transfection. Normalized against ubiquitously expressed | Firefly luciferase activity was measured 48h after transfection. Normalized against ubiquitously expressed | ||
Renilla | Renilla | ||
Line 721: | Line 680: | ||
spacer is the targeted gene. The symbols below indicate which parts are included in each sample. | spacer is the targeted gene. The symbols below indicate which parts are included in each sample. | ||
</i> | </i> | ||
− | + | </div> | |
− | + | </div> | |
− | + | </div> | |
− | + | </section> | |
− | + | <section id="4.5"> | |
− | + | <h2>4.5 Stapling Two DNA Strands Together Using fgRNAs</h2> | |
− | + | <p> | |
After showing the general capability of the fgRNA | After showing the general capability of the fgRNA | ||
to work for editing and for CRISPR activation, the next step was to use it to staple two DNA loci together, and | to work for editing and for CRISPR activation, the next step was to use it to staple two DNA loci together, and | ||
Line 736: | Line 695: | ||
introducing | introducing | ||
a fgRNA staple and a Gal4-VP64, expression of the luciferase is induced (Fig. 10, Panel A). | a fgRNA staple and a Gal4-VP64, expression of the luciferase is induced (Fig. 10, Panel A). | ||
− | Different linker lengths were tested. Cells were again normalized against ubiquitous renilla expression.<br /> | + | 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 | 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 | (Fig. 10, Panel B). An extension of the linker from 20 nt up to 40 nt resulted in an increasingly higher | ||
Line 743: | Line 702: | ||
hijacking an enhancer/activator. | hijacking an enhancer/activator. | ||
</p> | </p> | ||
− | + | <div class="thumb"> | |
− | + | <div class="thumbinner" style="width:60%;"> | |
− | + | <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/results-eh-2.svg" style="width:99%;"/> | |
− | + | <div class="thumbcaption"> | |
− | + | <i> | |
− | + | <b>Figure 10: Applying Fusion Guide RNAs for Cas staples.</b> <b>A</b>, schematic overview of the assay. | |
− | + | ||
An enhancer | An enhancer | ||
plasmid and a reporter plasmid are brought into proximity by a fgRNA Cas staple complex binding both | plasmid and a reporter plasmid are brought into proximity by a fgRNA Cas staple complex binding both | ||
Line 765: | Line 723: | ||
to 40 nt. | to 40 nt. | ||
</i> | </i> | ||
− | + | </div> | |
− | + | </div> | |
− | + | </div> | |
− | + | </section> | |
− | + | </section> | |
− | + | <section id="5"> | |
− | + | <h1>5. References</h1> | |
− | + | <p>Aregger, M., Xing, K., & Gonatopoulos-Pournatzis, T. (2021). Application of CHyMErA Cas9-Cas12a combinatorial | |
genome-editing platform for genetic interaction mapping and gene fragment deletion screening. <i>Nature | 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" | + | 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., & Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. <i>Science</i>, 339, 819-823. | A., & 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> | <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., & Moffat, J. (2020). Genetic | H., Weiss, A., Billmann, M., Durbic, T., Myers, C. L., Blencowe, B. J., & Moffat, J. (2020). Genetic | ||
interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform. <i>Nature | 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" | + | 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., & 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> | |
− | dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. <i>Science</i>, 337, 816-821. <a | + | <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., | |
− | Chemical Biology</i>, 13, 406-416. <a href="https://doi.org/10.1021/acschembio.7b00657" | + | |
− | + | ||
− | + | ||
Malagon-Lopez, J., Scarfò, I., Maus, M. V., Pinello, L., Aryee, M. J., & Joung, J. K. (2019). Engineered | Malagon-Lopez, J., Scarfò, I., Maus, M. V., Pinello, L., Aryee, M. J., & Joung, J. K. (2019). Engineered | ||
CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base | 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" | + | 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., & Abudayyeh, O. O. (2023). Discovery of diverse CRISPR-Cas systems and | |
− | + | 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> | |
− | expansion of the genome engineering toolbox. <i>Biochemistry</i>, 62, 3465-3487. <a | + | <p>Kweon, J., Jang, A.-H., Kim, D.-e., Yang, J. W., Yoon, M., Rim Shin, H., Kim, J.-S., & Kim, Y. (2017). Fusion |
− | + | 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> | |
− | + | </p> | |
− | + | <p>Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E., & Church, G. M. | |
− | guide RNAs for orthogonal gene manipulation with Cas9 and Cpf1. <i>Nature Communications</i>, 8. <a | + | (2013). RNA-guided human genome engineering via Cas9. <i>Science</i>, 339, 823-826. <a href="https://doi.org/10.1126/science.1232033" target="_blank">https://doi.org/10.1126/science.1232033</a></p> |
− | + | <p>Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I., Dohmae, N., Ishitani, R., Zhang, F., & | |
− | + | ||
− | + | ||
− | (2013). RNA-guided human genome engineering via Cas9. <i>Science</i>, 339, 823-826. <a | + | |
− | + | ||
− | + | ||
Nureki, O. (2014). Crystal structure of Cas9 in complex with guide RNA and target DNA. <i>Cell</i>, 156, 935-949. | Nureki, O. (2014). Crystal structure of Cas9 in complex with guide RNA and target DNA. <i>Cell</i>, 156, 935-949. | ||
− | <a href="https://doi.org/10.1016/j.cell.2014.02.001" | + | <a href="https://doi.org/10.1016/j.cell.2014.02.001" target="_blank">https://doi.org/10.1016/j.cell.2014.02.001</a></p> |
− | + | <p>Pacesa, M., Pelea, O., & Jinek, M. (2024). Past, present, and future of CRISPR genome editing technologies. | |
− | + | <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></p> | |
− | <i>Cell</i>, 187, 1076-1100. <a href="https://doi.org/10.1016/j.cell.2024.01.042" | + | <p>Paul, B., & Montoya, G. (2020). CRISPR-Cas12a: Functional overview and applications. <i>Biomedical |
− | + | 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></p> | |
− | + | <p>Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C., & Doudna, J. A. (2014). DNA interrogation by the | |
− | Journal</i>, 43, 8-17. <a href="https://doi.org/10.1016/j.bj.2019.10.005" | + | 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. | |
− | + | ||
− | CRISPR RNA-guided endonuclease Cas9. <i>Nature</i>, 507, 62-67. <a href="https://doi.org/10.1038/nature13011" | + | |
− | + | ||
− | + | ||
E., Joung, J., van der Oost, J., Regev, A., Koonin, E. V., & Zhang, F. (2015). Cpf1 is a single RNA-guided | E., Joung, J., van der Oost, J., Regev, A., Koonin, E. V., & Zhang, F. (2015). Cpf1 is a single RNA-guided | ||
− | endonuclease of a class 2 CRISPR-Cas system. <i>Cell</i>, 163, 759-771. <a | + | 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> | |
− | + | </section> | |
− | + | ||
</body> | </body> | ||
− | |||
</html> | </html> |
Revision as of 21:42, 1 October 2024
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). The fgRNA is functional, meaning that the MbCas12a (BBa_K5237001),
SpCas9 (BBa_K5237002) and the fusion dCas (BBa_K5237003)
can utilize the fgRNA to target two loci simultaneously. The fgRNA also works in combination with the catalyitcally inactive Cas
versions.
We successfully showed genome editing using active SpCas9 and Cas12a and induced proximity of two loci with the inactive dSpCas9 and dMbCas12a.
For our part collection, the PICasSO toolbox, this part has a crucial role in formation of our CRISPR/Cas staples.
Contents
Next to the well-studied linear DNA sequence, the 3D spatial organization of DNA plays a crucial role in
gene regulation,
cell fate, disease development and more. However, the tools to precisely manipulate this genomic
architecture remain limited, rendering it challenging to explore the full potential of the
3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful
molecular toolbox based on various DNA-binding proteins to address this issue.
The PICasSO part collection offers a comprehensive, modular platform for precise manipulation and re-programming of DNA-DNA interactions using protein staples in living cells, enabling researchers to recreate natural 3D genomic interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation. Specifically, the fusion of two DNA binding proteins enables to artifically bring distant genomic loci into proximty. To unlock the system's full potential, we introduce versatile chimeric CRISPR/Cas complexes, connected either on the protein or the guide RNA level. These1 complexes are reffered to as protein- or Cas staples. Beyond its versatility, PICasSO includes robust assay systems to support the engineering, optimization, and testing of new staples, ensuring functionality in vitro and in vivo. We took special care to include parts crucial for testing every step of the cycle (design, build, test, learn) when engineering new parts.
At its heart, the PICasSO part collection consists of three categories.
(i) Our DNA-binding
proteins
include our
finalized enhancer hijacking Cas staple as well as half staples that can be used by scientists to compose entirely
new Cas staples in the future. We also include our Simple staples that serve as controls for successful stapling
and can be further engineered to create alternative, simpler and more compact staples.
(ii) As functional elements, we list additional parts that enhance the functionality of our Cas and
Basic staples. These
consist of
protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling in vivo.
Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs
with our
interkingdom conjugation system.
(iii) As the final category of our collection, we provide parts that support the use of our custom
readout
systems. These include components of our established FRET-based proximity assay system, enabling users to
confirm
accurate stapling. Additionally, we offer a complementary, application-oriented testing system for functional
readouts via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking
in mammalian cells.
The following table gives a comprehensive overview of all parts in our PICasSO toolbox. The highlighted parts showed
exceptional performance as described on our iGEM wiki and can serve as a reference. The other parts in
the
collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their
own custom Cas staples, enabling further optimization and innovation.
Our part collection includes:
DNA-binding proteins: The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring easy assembly. | ||
BBa_K5237000 | fgRNA Entry vector MbCas12a-SpCas9 | Entryvector for simple fgRNA cloning via SapI |
BBa_K5237001 | Staple subunit: dMbCas12a-Nucleoplasmin NLS | Staple subunit that can be combined with sgRNA or fgRNA and dCas9 to form a functional staple |
BBa_K5237002 | Staple subunit: SV40 NLS-dSpCas9-SV40 NLS | Staple subunit that can be combined witha sgRNA or fgRNA and dCas12avto form a functional staple |
BBa_K5237003 | Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS | Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands into close proximity |
BBa_K5237004 | Staple subunit: Oct1-DBD | Staple subunit that can be combined to form a functional staple, for example with TetR. Can also be combined with a fluorescent protein as part of the FRET proximity assay |
BBa_K5237005 | Staple subunit: TetR | Staple subunit that can be combined to form a functional staple, for example with Oct1. Can also be combined with a fluorescent protein as part of the FRET proximity assay |
BBa_K5237006 | Simple staple: TetR-Oct1 | Functional staple that can be used to bring two DNA strands in close proximity |
BBa_K5237007 | Staple subunit: GCN4 | Staple subunit that can be combined to form a functional staple, for example with rGCN4 |
BBa_K5237008 | Staple subunit: rGCN4 | Staple subunit that can be combined to form a functional staple, for example with rGCN4 |
BBa_K5237009 | Mini staple: bGCN4 | Assembled staple with minimal size that can be further engineered | Functional elements: Protease-cleavable peptide linkers and inteins are used to control and modify staples for further optimization for custom applications |
BBa_K5237010 | Cathepsin B-cleavable Linker: GFLG | Cathepsin B-cleavable peptide linker that can be used to combine two staple subunits to make responsive staples |
BBa_K5237011 | Cathepsin B Expression Cassette | Expression Cassette for the overexpression of cathepsin B |
BBa_K5237012 | Caged NpuN Intein | A caged NpuN split intein fragment that undergoes protein trans-splicing after protease activation. Can be used to create functionalized staples units |
BBa_K5237013 | Caged NpuC Intein | A caged NpuC split intein fragment that undergoes protein trans-splicing after protease activation. Can be used to create functionalized staples units |
BBa_K5237014 | fgRNA processing casette | Processing casette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D genome reprograming |
BBa_K5237015 | Intimin anti-EGFR Nanobody | Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large constructs |
BBa_K4643003 | incP origin of transfer | Origin of transfer that can be cloned into the plasmid vector and used for conjugation as a means of delivery | Readout Systems: FRET and enhancer recruitment to measure proximity of stapled DNA in bacterial and mammalian living cells enabling swift testing and easy development for new systems |
BBa_K5237016 | FRET-Donor: mNeonGreen-Oct1 | FRET Donor-Fluorpohore fused to Oct1-DBD that binds to the Oct1 binding cassette. Can be used to visualize DNA-DNA proximity |
BBa_K5237017 | FRET-Acceptor: TetR-mScarlet-I | Acceptor part for the FRET assay binding the TetR binding cassette. Can be used to visualize DNA-DNA proximity |
BBa_K5237018 | Oct1 Binding Casette | DNA sequence containing 12 Oct1 binding motifs, compatible with various assays such as the FRET proximity assay |
BBa_K5237019 | TetR Binding Cassette | DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET proximity assay | BBa_K5237020 | Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64 | Readout system that responds to protease activity. It was used to test cathepsin B-cleavable linker |
BBa_K5237021 | NLS-Gal4-VP64 | Trans-activating enhancer, that can be used to simulate enhancer hijacking | BBa_K5237022 | mCherry Expression Cassette: UAS, minimal Promotor, mCherry | Readout system for enhancer binding. It was used to test cathepsin B-cleavable linker |
BBa_K5237023 | Oct1 - 5x UAS binding casette | Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay |
BBa_K5237024 | TRE-minimal promoter- firefly luciferase | Contains Firefly luciferase controlled by a minimal promoter. It was used as a luminescence readout for simulated enhancer hijacking |
1. Sequence overview
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 339
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE 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
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) (Fig. 2 A). Furthermore, a specific three nucleotide sequence (NGG) at 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 RNAs (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.
2.2 Differences between Cas9 and Cas12a
Over the following years, further 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 with its RNA, that in comparison to the type II systems, only requires a crRNA for targeting and activation. Cas12a is capable of processing the precursor crRNA into crRNA independently, 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 completely inactive Cas proteins (Koonin et al., 2023) (Kleinstiver et al., 2019). These 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 gene with the spacer sequence (Kampmann, 2017). A common approach for CRISPRa involves fusing Cas9 with the transcriptional activator VP64 (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. Via this approach, the two spacer sequences are fused directly, ensuring a
minimal distance between the two DNA strands.This also facilitates efficient cloning of different spacer
sequences, as both spacers can be exchangeed as one consecutive sequence. Linking the crRNA and sgRNA further enables
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
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 scoffold 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.
The first goal after assembly was to prove the editing activity of both proteins using fgRNA. The genes VEGFA and FANCF were selected as targets for Cas12a and Cas9, each target was tested with each Cas protein. Editing efficiency was analyzed with the T7 Endonuclease I (T7EI) assay. Controls included crRNAs and sgRNAs as positive controls, and non-targeting guides as negative controls. Desired spacer sequences were ordered as oligos, annealed, and cloned in via GGA utilizing SapI.
Table 1: A list of all the different spacers we cloned and tested within the fgRNA | |
VEGFA | ctaggaatattgaagggggc |
FANCF | ggcggggtccagttccggga |
CCR5 | tgacatcaattattatacat |
TetO (BBa_K5237019) | tctctatcactgatagggag |
Oct1-B (BBa_K5237018) | atgcaaatactgcactagtg |
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 in the composite part. This vector was tested on the loci VEGFA and FANCF to assess its functionality.
4. Results
4.1 Editing endogenous loci with fgRNAs
To prove that our fusion gRNAs still result in active ribonucleoproteins, a series of different fgRNAs were
created, each carrying spacers specific to the VEGFA and FANCF genes.HEK293-T cells were transfected with the
Cas
protein and gRNA constructs. The editing rate was tested 72h after transfection via a T7 endonuclease I
assay.
AsCas12a and SpCas9 were used. The AsCas12a spacer targets VEGFA, while the SpCas9 spacer targets FANCF. The
samples included standard single gRNAs with the corresponding Cas protein, the fgRNA with only one of the two
Cas
proteins and the fgRNA with both Cas proteins simultaneously (Fig. 5). The sgRNAs allowed for
the highest editing rates for both genes (45% for VEGFA and 15% for FANCF), while the editing rates for FANCF
were
consistently lower in all experiments. Importantly, targeting FANCF with fgRNAs resulted in noticeable editing
of
about 10%, with just the SpCas9 and both Cas proteins in the sample. For VEGFA, the AsCas12a only sample
resulted
in approximately 20% editing rate in combination with the fgRNA, while adding both Cas proteins led to
approximately 40%. These initial results confirmed our engineering approach proving efficient genome editing
with
fgRNAs.
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, we tested them in combination with different Cas12a orthologs. After some initial testing, we decided on using MbCas12a together with SpCas9, because we found AsCas12a to be less active in a dual luciferase assay when co-transfected with SpCas9 compared to MbCas12a (Fig. 6). Between these two co-transfections the SpCas9 editing has not been significantly different.
Additionally, to test if the differences in editing rates from the preliminary assay resulted from the targeted
loci or the different Cas orthologs, the spacers were tested in both arrangements. Once with Cas12a targeting
FANCF and SpCas9 targeting VEGFA and once vice versa. To better assess the impact that the utilization of a
fgRNA
has on the editing rates, the sgRNAs were tested separately and in one sample.
Having the sgRNA with single Cas
proteins in the same sample resulted in no clear difference in the editing rates (Fig. 7). 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. When targeting the same gene
under
the same conditions, the editing rates for MbCas12a were overall lower than the ones from SpCas9.
4.3 The Inclusion of a Linker Does Not Lower Editing Rates
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. 8). For CCR5, the editing rate with sgRNAs was approximately the same at about 30%. However, it dropped below 10% for the fgRNA. The addition of the 20 nt linker had no effect on the editing rates compared to no linker.
4.4 fgRNAs can be used for CRISPRa
To establish the foundation for their use as protein scaffolds, we identified the next step as demonstrating the use of fgRNAs for CRISPR activation. For this, we intend to recruit the transcriptional activator VP64 to a firefly luciferase gene to induce expression. The VP64 protein is attached to the catalytically inactive Cas9 protein, which is then guided by gRNAs to the luciferase gene. The gRNAs target a TetO sequence, which is positioned in front of the luciferase gene in multiple repeats. The firefly luciferase activity was then quantified as photon counts and normalized against Renilla luciferase, which is expressed on a separate plasmid under an ubiquitous promoter. In two biological replicates we saw similar Relative luciferase activity with fgRNA as a guide compared to a sgRNA (Fig. 9).
4.5 Stapling Two DNA Strands Together Using fgRNAs
After showing the general capability of the fgRNA
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
plasmid was used. The reporter plasmid has firefly luciferase behind several repeats of a Cas9 targeted
sequence.
The enhancer plasmid has a Gal4 binding site behind several repeats of a Cas12a targeted sequence. By
introducing
a fgRNA staple and a Gal4-VP64, expression of the luciferase is induced (Fig. 10, Panel A).
Different linker lengths were tested. Cells were again normalized against ubiquitous renilla expression.
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.
5. References
Aregger, M., Xing, K., & Gonatopoulos-Pournatzis, T. (2021). Application of CHyMErA Cas9-Cas12a combinatorial genome-editing platform for genetic interaction mapping and gene fragment deletion screening. Nature Protocols, 16, 4722-4765. https://doi.org/10.1038/s41596-021-00595-1
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science, 339, 819-823. https://doi.org/10.1126/science.1231143
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., & Moffat, J. (2020). Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform. Nature Biotechnology, 38, 638-648. https://doi.org/10.1038/s41587-020-0437-z
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337, 816-821. https://doi.org/10.1126/science.1225829
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., & 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., & 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
Kweon, J., Jang, A.-H., Kim, D.-e., Yang, J. W., Yoon, M., Rim Shin, H., Kim, J.-S., & Kim, Y. (2017). Fusion guide RNAs for orthogonal gene manipulation with Cas9 and Cpf1. Nature Communications, 8. https://doi.org/10.1038/s41467-017-01650-w
Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E., & Church, G. M. (2013). RNA-guided human genome engineering via Cas9. Science, 339, 823-826. https://doi.org/10.1126/science.1232033
Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I., Dohmae, N., Ishitani, R., Zhang, F., & Nureki, O. (2014). Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell, 156, 935-949. https://doi.org/10.1016/j.cell.2014.02.001
Pacesa, M., Pelea, O., & 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., & Montoya, G. (2020). CRISPR-Cas12a: Functional overview and applications. Biomedical Journal, 43, 8-17. https://doi.org/10.1016/j.bj.2019.10.005
Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C., & Doudna, J. A. (2014). DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature, 507, 62-67. https://doi.org/10.1038/nature13011
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., & 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