Difference between revisions of "Part:BBa K5237996"

Revision as of 12:16, 30 September 2024

SV40 NLS-dSpCas9-SV40 NLS

dSpCas9 is one crucial part of our PICasSO tool box, being able to function as a Cas staple with the fgRNA (BBa_K5237000) and the dMbCas12a (BBa_K5237001). Transactivation has been shown using this part proving the proper formation of our Cas staple. Easy employment through swift reprogramming of the fgRNA.

1 Sequence overview
2 Usage and Biology
3 Assembly and part evolution
4 Results
- 4.1dSpCas9 transactivation as part of a Cas staple
- 4.2SpCas9 fused to dMbCas12a form the Cas staple
5 References

The PICasSO Toolbox

Figure 1: Example how the part collection can be used to engineer new staples

The 3D organization of the genome plays a crucial role in regulating gene expression in eukaryotic cells, impacting cellular behavior, evolution, and disease. Beyond the linear DNA sequence, the spatial arrangement of chromatin, influenced by DNA-DNA interactions, shapes pathways of gene regulation. 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. 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 component 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 readout via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking.

The following table gives a complete 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 Entryvector MbCas12a-SpCas9	Entryvector for simple fgRNA cloning via SapI
BBa_K5237001	Staple subunit: dMbCas12a-Nucleoplasmin NLS	Staple subunit that can be combined to form a functional staple, for example with fgRNA and dCas9
BBa_K5237002	Staple subunit: SV40 NLS-dSpCas9-SV40 NLS	Staple subunit that can be combined to form a functional staple, for example with our fgRNA or dCas12a
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 in 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 taple: 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	Cathepsin B which can be selectively express to cut the cleavable linker
BBa_K5237012	Caged NpuN Intein	Undergoes protein transsplicing after protease activation, can be used to create functionalized staple units
BBa_K5237013	Caged NpuC Intein	Undergoes protein transsplicing after protease activation, can be used to create functionalized staple units
BBa_K5237014	fgRNA processing casette	Processing casette to produce multiple fgRNAs from one transcript, can be used for multiplexing
BBa_K5237015	Intimin anti-EGFR Nanobody	Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large constructs
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	Donor part for the FRET assay binding 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, can be used for different 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

Sequence and Features

Assembly Compatibility:

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2. Usage and Biology

In 2012, Jinek et al. discovered the use of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system to induce double-strand breaks in DNA. Since then, the system has been well established as a tool for genome editing. The CRISPR/Cas system, which originates from the bacterial immune system, is constituted by a ribonucleoprotein complex. For class 1 CRISPR systems, the RNA is complexed by multiple Cas proteins, whereas class 2 systems consist of a singular protein and RNA. The class 2 type II system describes all ribonucleoprotein complexes with Cas9 (Pacesa et al., 2024). They include a CRISPR RNA (crRNA), which specifies the target with a 20 nucleotide (nt) spacer sequence, and a transactivating CRISPR RNA (tracrRNA), which induces the processing by the Cas protein (Jinek et al., 2012) (see FIGURE background Cas9 cas12 panel A). Furthermore, a specific three nucleotide sequence (NGG) on the 3' end in the targeted DNA is needed for binding and cleavage. This is referred to as the protospacer adjacent motif (PAM) (Sternberg et al., 2014). The most commonly used Cas9 protein is SpCas9 or SpyCas9, which originates from Streptococcus pyogenes (Pacesa et al., 2024).

Figure 2: The CRISPR/Cas system (adapted from Pacesa et al. (2024)) A and B, schematic structure of Cas9 and Cas12a with their sgRNA/crRNA, sitting on a DNA strand with the PAM. The spacer sequence forms base pairings with the dsDNA. In case of Cas9 the spacer is located at the 5' prime end, for Cas12a at the 3' end of the gRNA. The scaffold of the gRNA forms a specific secondary structure enabling it to bind to the Cas protein. The cut sites by the cleaving domains, RuvC and HNH, are symbolized by the scissors

A significant enhancement of this system was the introduction of single guide RNA (sgRNA)s, which combine the functions of a tracrRNA and crRNA (Mali et al., 2013). Moreover, Cong et al. (2013) established precise targeting of human endogenous loci by designing the 20 nt spacer sequence accordingly.

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). 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. 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

Before working with the dSpCas9 the catalytically active version was extensively tested to assess the SpCas9 to be fit for being part of a Cas staple. The SpCas9 plasmid was provided by our PI.

3.1 SpCas9 can be Co-Transfected wWth other Cas Proteins

Wanting to employ the SpCas9 as part of a Cas staple, our goal was to find out how well SpCas9 can stay active while being transfected together with MbCas12a. Therefore we engineered the dual luciferase assay to allow us to test 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. 3).

Figure 3: Double cut dual luciferase assay testing Fusion Cas simultaneous editing. Firefly and renilla luminescence intensity is plotted, both measured 48 h after transfection. On the x-axis the negative control, single edit and simultaneous edit, are grouped by the two Cas proteins in the fusion Cas. The fusion Cas contains MbCas12a and SpCas9. MbCas12a are the Firefly relative luminescence units (RLUs), while Cas9 are the Renilla RLUs. Data is depicted as the mean +/- SD (n=3). Statistical analysis was performed using 2way ANOVA with Tukey's multiple comparisons test. For better clarity, only significant differences within a group are shown.*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

For the SpCas9 we have a highly significant editing rate in the single cut, meaning both Cas proteins are transfected, but only SpCas9 has a targeting gRNA. When introducing a targeting gRNA for MbCas12a we see no reduction in the highly significant editing of SpCas9, strongly suggesting both Cas proteins to be able to edit simultaneously.

3.2 SpCas9 shows editing with fgRNA

Wanting to test if the different parts of our Cas staples will work together, we tested editing rates of SpCas9 using fgRNA. Two spacers were tested: FANCF and VEGFA. To better assess the impact that the utilization of a fgRNA has on the editing rates, the sgRNAs were tested separately and in one sample.
Having the sgRNA with single Cas proteins in the same sample resulted in no clear difference in the editing rates (Fig. 4). 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 SpCas9 editing utilizing a fgRNA.

Figure 4: Fusion gRNA Editing Rates In Combination with MbCas12a. In A and B the editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band))^1/2. The schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts are included in each sample. A and B display both orientations of the two spacers for VEGFA and FANCF.

To further assess the effect of the genomic locus on the editing rate, we included CCR5 as an additional gene target. For this assay, a fgRNA with a 20 nt long linker was included between the two spacers. The editing rate for VEGFA was again relatively consistent throughout the samples (Fig. 5).

Figure 5: Fusion gRNA Editing Rates for Multiplexing CCR5 and VEGFA. The editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band)) ^1/2. The schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts are included in each sample. Cas12a targets VEGFA and Cas9 targets CCR5.

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.

3.3 SpCas9 can be fused to MbCas12a while maintaining functionality

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 SpCas9 can stay active while being fused to MbCas12a. 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. 6).

Figure 6: Double cut dual luciferase assay testing Fusion Cas simultaneous editing. Firefly and renilla luminescence intensity is plotted, both measured 48 h after transfection. On the x-axis the negative control, single edit and simultaneous edit, are grouped by the two Cas proteins in the fusion Cas. The fusion Cas contains MbCas12a and SpCas9. MbCas12a are the Firefly relative luminescence units (RLUs), while Cas9 are the Renilla RLUs. Data is depicted as the mean +/- SD (n=3). Statistical analysis was performed using 2way ANOVA with Tukey's multiple comparisons test. For better clarity, only significant differences within a group are shown.*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

MbCas12a showed highly significant editing rate in the single cut experiment (only MbCas12a has a targeting gRNA). When introducing a targeting gRNA for SpCas9 no significant change could be detected, strongly suggesting both Cas proteins in the fusion Cas to be able to edit simultaneously.

3.4 SpCas9 fused to MbCas12a shows editing with fgRNA

The capability of SpCas9 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. Biological duplicates were done for this assay.
SpCas9 editing rates were higher overall. At the same time, when targeting VEGFA they resulted in a higher editing efficiency than FANCF.

Figure 7: Editing rates for fusion guide RNAs with fusion Cas proteins. the editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band)) ^1/2. The schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts are included in each sample. Cas proteins linked by a dash ("-") were fused to each other. Biological replicates are marked as individual dots

4. Results

We extensively characterized the catalytically active SpCas9 in several assays asserting functional editing with a fusion guide RNA (BBa_K5237000), showing high activity while being co-transfected with MbCas9, our second staple protein's active version, and lastly a functioning fusion to MbCas12a.
After all these successful test we were confident to test the Cas staples in action.

4.1 dSpCas9 transactivation as part of a Cas staple

The next step was to use the SpCas9 as part of a Cas staple to staple two DNA loci together, and thereby induce proximity between two separate functional elements. For this, an enhancer plasmid and a reporter plasmid was used. The reporter plasmid has firefly luciferase behind several repeats of a Cas9 targeted sequence. The enhancer plasmid has a Gal4 binding site behind several repeats of a Cas12a targeted sequence. By introducing a fgRNA staple and a Gal4-VP64, expression of the luciferase is induced (Fig. 8, 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. 8, B). An extension of the linker from 20 nt up to 40 nt resulted in an increasingly higher expression of the reporter gene.

Figure 8: Applying Fusion Guide RNAs for Cas staples. A, schematic overview of the assay. An enhancer plasmid and a reporter plasmid are brought into proximity by a fgRNA Cas staple complex binding both plasmids. Target sequences were included in multiple repeats prior to the functional elements. Firefly luciferase serves as the reporter gene, the enhancer is constituted by multiple Gal4 repeats that are bound by a Gal4-VP64 fusion. B, results of using a fgRNA Cas staple for trans activation of firefly luciferase. Firefly luciferase activity was measured 48h after transfection. Normalized against ubiquitously expressed Renilla luciferase. Statistical significance was calculated with ordinary One-way ANOVA with Dunn's method for multiple comparisons (*p < 0.05; **p < 0.01; ***p < 0.001; mean +/- SD). The assay included sgRNAs and fgRNAs with linker lengths from 0 nt to 40 nt.

4.2 SpCas9 fused to dMbCas12a form the Cas staple

Continuing the procedure in a similar manner, we focused on inducing proximity between genetic loci using the fusion dCas next, consisting of dMbCas12a fused to dSpCas9. The same assay was used, with one enhancer plasmid and one reporter plasmid. Though less distinct than the results for not using a protein fusion, the fusion Cas proteins can be used to increase expression levels of the reporter firefly luciferase (see figure 13). While using sgRNAs results in similar relative luciferase activity as for the negative control between 0.1 and 0.2., using a fgRNA with a 20 to 30 nt linker consistently resulted in activities at 0.25. Fusion guide RNAs without a linker and with a 40 nt linker had on average about the same activity, but with a higher spread over the biological replicates. Nonetheless this showed the dMbCas12a fused to the dSpCas9 to be working as a Cas staple. Further tweaking is needed to get better results

Figure 9: Firefly luciferase trans activation through fusion Cas staple 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). Fusion Cas proteins were paired with sgRNAs and fgRNAs with linker lengths from 0 nt to 40 nt

5. References

Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., and 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., 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.

Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E., and 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., and 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., 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.

Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C., and Doudna, J. A. (2014). DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature, 507, 62–67. https://doi.org/10.1038/nature13011.

@@ Line 1: / Line 1: @@
 __NOTOC__
-<partinfo>BBa_K5237001</partinfo>
+<partinfo>BBa_K5237002</partinfo>
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@@ Line 38: / Line 37: @@
    <!-- Part summary -->
    <section id="1">
-     <h1>Staple subunit: dMbCas12a-Nucleoplasmin NLS</h1>
+     <h1>SV40 NLS-dSpCas9-SV40 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>dSpCas9 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_K523700">BBa_K5237000</a>) and the dMbCas12a (<a
+        href="https://parts.igem.org/Part:BBa_K523701">BBa_K5237001</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>
      <p>&nbsp;</p>
    </section>
-   <div id="toc" class="toc" style="width:30%;">
+   <div id="toc" class="toc">
      <div id="toctitle">
        <h1>Contents</h1>
@@ Line 59: / Line 60: @@
          <ul>
            <li class="toclevel-2 tocsection-3.1"><a href="#3.1"><span class="tocnumber">3.1</span> <span
-                class="toctext">Qualtitative assesment of Cas12a orthologs</span></a>
+              class="toctext">SpCas9 can be Co-Transfected wWth other Cas Proteins</span></a>
            </li>
            <li class="toclevel-2 tocsection-3.2"><a href="#3.2"><span class="tocnumber">3.2</span> <span
-                class="toctext">Quantitative comparison between AsCas12a and MbCas12a</span></a>
+              class="toctext">SpCas9 shows editing with fgRNA</span></a>
            </li>
            <li class="toclevel-2 tocsection-3.3"><a href="#3.3"><span class="tocnumber">3.3</span> <span
-                class="toctext">MbCas12a tolerates co-transfection and
+              class="toctext">SpCas9 can be fused to MbCas12a while maintaining functionality</span></a>
-                simultaneous editing of different Cas proteins</span></a>
            </li>
            <li class="toclevel-2 tocsection-3.4"><a href="#3.4"><span class="tocnumber">3.4</span> <span
-                class="toctext"></span>MbCas12a shows editing with fgRNA</a>
+              class="toctext">SpCas9 fused to MbCas12a shows editing with fgRNA</span></a>
            </li>
-          <li class="toclevel-2 tocsection-3.5"><a href="#3.5"><span class="tocnumber">3.5</span> <span
-                class="toctext">MbCas12a withstanding fusion to SpCas9 while staying functional</span></a>
-          </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>
          </ul>
        </li>
-       <li class="toclevel-1 tocsection-5"><a href="#4"><span class="tocnumber">4</span> <span
+       <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">dSpCas9 transactivation as part of a Cas staple</span> </a>
+          </li>
+          <li class="toclevel-2 tocsection-4.2"><a href="#4.2"><span class="tocnumber">4.2</span><span
+            class="toctext">SpCas9 fused to dMbCas12a form the Cas staple</span> </a>
+        </ul>
        </li>
        <li class="toclevel-1 tocsection-8"><a href="#5"><span class="tocnumber">5</span> <span
@@ Line 90: / Line 92: @@
      <p><br></p>
      <div class="thumb"></div>
-      <div class="thumbinner" style="width:550px"><img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/registry-part-collection-engineering-cycle-example-overview.svg" style="width:99%;" class="thumbimage">
+    <div class="thumbinner" style="width:550px"><img alt=""
-        <div class="thumbcaption">
+        src="https://static.igem.wiki/teams/5237/wetlab-results/registry-part-collection-engineering-cycle-example-overview.svg"
-          <i><b>Figure 1: Example how the part collection can be used to engineer new staples</b></i>
+        style="width:99%;" class="thumbimage">
-        </div>
+      <div class="thumbcaption">
+        <i><b>Figure 1: Example how the part collection can be used to engineer new staples</b></i>
        </div>
      </div>
+     </div>
      <p>
@@ Line 118: / Line 122: @@
      </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
        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. <br>
-       <b>(ii)</b> As <b>functional elements</b>, we list additional parts that enhance the functionality of our Cas and Basic staples. These
+       <b>(ii)</b> As <b>functional elements</b>, 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 <i>in vivo</i>.
-       Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs with our
+       Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs
+      with our
        interkingdom conjugation system. <br>
-       <b>(iii)</b> As the final component of our collection, we provide parts that support the use of our <b>custom readout
+       <b>(iii)</b> As the final component of our collection, we provide parts that support the use of our <b>custom
+        readout
          systems</b>. These include components of our established FRET-based proximity assay system, enabling users to
        confirm
@@ Line 309: / Line 317: @@
 <!--################################-->
 <span class='h3bb'>Sequence and Features</span>
-<partinfo>BBa_K5237001 SequenceAndFeatures</partinfo>
+<partinfo>BBa_K5237002 SequenceAndFeatures</partinfo>
 <!--################################-->
@@ Line 316: / Line 324: @@
 <section id="2">
-    <h1>2. Usage and Biology</h1>
+  <h1>2. Usage and Biology</h1>
+  <p>
+    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
+    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) (see FIGURE background Cas9 cas12 panel 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).
+  </p>
+  <div class="thumb">
+    <div class="thumbinner" style="width:60%;">
+      <img alt=""
+        src="https://static.igem.wiki/teams/5237/wetlab-results/cas-staple-svg/background-cas9-cas12a-principle.svg"
+        style="width:99%;" class="thumbimage">
+      <div class="thumbcaption">
+        <i>
+          <b>Figure 2: The CRISPR/Cas system (adapted from Pacesa <i>et al.</i> (2024))</b>
+          A and B, schematic structure of Cas9 and Cas12a with their sgRNA/crRNA, sitting on a DNA strand with the PAM.
+          The spacer sequence forms base pairings with the dsDNA. In case of Cas9 the
+          spacer is located at the 5' prime end, for Cas12a at the 3' end of the gRNA. The scaffold of the gRNA forms a
+          specific
+          secondary structure enabling it to bind to the Cas protein. The cut sites by the cleaving domains, RuvC and
+          HNH, are
+          symbolized by the scissors
+        </i>
+      </div>
+    </div>
+  </div>
+  <p>
+    A significant enhancement of this system was the introduction of single guide RNA (sgRNA)s, which combine the
+    functions
+    of a tracrRNA and crRNA (Mali et al., 2013). Moreover, Cong et al. (2013) established precise targeting of human
+    endogenous loci by designing the 20 nt spacer sequence accordingly.
+    <br><br>
+    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). 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. 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).
+  </p>
+</section>
+<section id="3">
+  <h1>3. Assembly and part evolution</h1>
+  <p>
+    Before working with the dSpCas9 the catalytically active version was extensively tested to assess the SpCas9 to be
+    fit for being part of a Cas staple. The SpCas9 plasmid was provided by our PI.
+  </p>
+  <section id="3.1">
+    <h2>3.1 SpCas9 can be Co-Transfected wWth other Cas Proteins</h2>
      <p>
-       In 2012, Jinek <i>et al.</i> discovered the use of the Clustered Regularly Interspaced Short Palindromic Repeats
+       Wanting to employ the SpCas9 as part of a Cas staple, our goal was to find out how well SpCas9 can stay active
-      (CRISPR)/Cas system to induce double-strand breaks in DNA. Since then, the system has been well established as a
+       while being transfected together with MbCas12a. Therefore we engineered the dual luciferase assay to allow us to test
-       tool for genome editing. The CRISPR/Cas system, which originates from the bacterial immune system, is constituted
+       two catalytically active Cas proteins at once. Including a well established protospacer, CCR5, into the renilla
-       by a ribonucleoprotein complex. For class 1 CRISPR systems, the RNA is complexed by multiple Cas proteins, whereas
+       luciferase gene enables us to test the editing rates of two Cas proteins simultaneously (Fig. 3).
-      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="thumbinner" style="width:60%;">
-         <img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/cas-staple-svg/background-cas9-cas12a-principle.svg"
+         <img alt="" src="https://static.igem.wiki/teams/5237/engineering/fusioncas-registry.svg" style="width:99%;"
-          style="width:99%;" class="thumbimage">
+          class="thumbimage">
          <div class="thumbcaption">
            <i>
-             <b>Figure 2: The CRISPR/Cas system (adapted from Pacesa <i>et al.</i> (2024))</b>
+             <b>Figure 3: Double cut dual luciferase assay testing Fusion Cas simultaneous editing.</b>
-             A and B, schematic structure of Cas9 and Cas12a with their sgRNA/crRNA, sitting on a DNA strand with the PAM.
+             Firefly and renilla luminescence intensity is plotted, both measured 48 h after transfection. On the x-axis
-             The spacer sequence forms base pairings with the dsDNA. In case of Cas9 the
+            the negative
-             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
+            control, single edit and simultaneous edit, are grouped by the two Cas proteins in the fusion Cas. The
-             secondary structure enabling it to bind to the Cas protein. The cut sites by the cleaving domains, RuvC and HNH, are
+             fusion Cas
-             symbolized by the scissors
+            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&lt;0.05,
+             **p&lt;0.01, ***p&lt;0.001, ****p&lt;0.0001
            </i>
          </div>
@@ Line 347: / Line 415: @@
      </div>
      <p>
-       A significant enhancement of this system was the introduction of single guide RNA (sgRNA)s, which combine the
+       For the SpCas9 we have a highly significant editing rate in the single cut, meaning both Cas proteins are transfected, but only SpCas9 has a targeting gRNA. When introducing a targeting gRNA for MbCas12a we see no reduction in the highly significant editing of SpCas9, strongly suggesting both Cas proteins to be able to edit simultaneously.
-      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">
+     <h2 id="3.2">3.2 SpCas9 shows editing with fgRNA</h2>
-     <h1>3. Assembly and part evolution</h1>
+    <p>
-    <section id="3.1">
+      Wanting to test if the different parts of our Cas staples will work together, we tested editing rates of SpCas9
-      <h2>3.1 Qualtitative assesment of Cas12a orthologs</h2>
+      using
-      <p>
+      fgRNA. Two spacers were tested: FANCF and VEGFA. To better assess the impact that the utilization of a fgRNA has
-        To select a suitable Cas12a ortholog for cronstructing the Cas sstaple, three different orhtologs were ordered from Addgene:
+      on the
-        AsCas12a (<a href="https://www.addgene.org/69982/" target="_blank">#69982</a>), LbCas12a (<a href="https://www.addgene.org/69988/" target="_blank">#69988</a>),
+      editing rates, the sgRNAs were tested separately and in one sample. <br>
-        and MbCas12a (<a href="https://www.addgene.org/115142/" target="_blank">#115142</a>).
+      Having the sgRNA with single Cas proteins in the same sample resulted in no clear
-        <br><br>
+      difference in the editing rates (Fig. 4). The fusion of the gRNAs resulted in a lower editing rate
-        We assessed their performance using a T7 Endonuclease I (T7EI) assay on three genomic loci: RUNX1, DNMT1, and FANCF. For
+      overall. While the editing for VEGFA stayed at about 20% in all
-        comparison, we also included SpCas9 editing the CCR5 locus (Fig. 3). Editing was only observed at the
+      cases, the editing for FANCF dropped significantly. Nonetheless we were able to show SpCas9 editing utilizing a
-        RUNX1 locus, where we used the qualitative assay to compare the editing efficiencies of the Cas12a orthologs.
+       fgRNA.
-       </p>
+    </p>
-      <div class="thumb">
+    <div class="thumb">
-        <div class="thumbinner" style="width:60%;">
+      <div class="thumbinner" style="width:60%;">
-          <img alt="" src="https://static.igem.wiki/teams/5237/engineering/fuscas-engi-fig1-1.svg"
+        <img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/results-mbcas-2.svg" style="width:99%;"
-            style="width:99%; border:none;" class="thumbimage">
+          class="thumbimage">
-            <img alt="" src="https://static.igem.wiki/teams/5237/engineering/fuscas-engi-fig1-2.svg"
+        <div class="thumbcaption">
-            style="width:99%; border:none;" class="thumbimage">
+          <i>
-          <div class="thumbcaption">
+            <b>Figure 4: Fusion gRNA Editing Rates In Combination with MbCas12a.</b>
-            <i>
+            In <b>A</b> and <b>B</b> the editing rates were determined 72h after transfection via T7EI assay. Editing %
-              <b>Figure 3: Preliminary T7 Endonuclease I testing of Cas12a orthologs.</b>
+            was
-              T7EI samples were run on a 2% agarose gel in 1x TBE buffer dyed with gel red. SpCas9 targeting CCR5 functions as a
+            determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved
-              benchmark for proper editing. For the Cas12a orthologs, LbCas12a, AsCas12a and MbCas12a, the three loci RUNX1, DNMT1 and
+            band))<sup>1/2</sup>. The
-              FANCF were targeted. Editing is indicated by an extra band compared to the negative control.
+            schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols
-             </i>
+             below
-           </div>
+            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>
-     </section>
+     </div>
-     <section id="3.2"><h2>3.2 Quantitative comparison between AsCas12a and MbCas12a</h2>
+     <p>
-       <p>
+       To further assess the effect of the genomic locus on the editing rate, we included CCR5 as an additional gene
-        Since the T7EI assay can only be used to qualitatively compare editing efficiencies, it was hard to distinguish
+      target.
-        between the better editing ortholog.<br>
+      For this assay, a fgRNA with a 20 nt long linker was included between the two spacers.
-        To accurately quantify the editing efficiency, we concted a dual luciferase assay. This assay measures the luminescence
+      The editing rate for VEGFA was again relatively consistent throughout the samples (Fig. 5).
-        of Firefly luciferase, which decreases proportionally to the editing efficiency at the target site.
+    </p>
-        To account for variations in cell count and transfection efficiency, the luminescence is normalized
+    <div class="thumb">
-        to Renilla luciferase, which acts as an internal control (Fig. 4).
+      <div class="thumbinner" style="width:60%;">
-        The results revealed a clear difference, with MbCas12a demonstrating significantly higher editing efficiency compared to
+        <img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/results-ccr5-2.svg"
-        AsCas12a (p=0.005). Based on these findings, we decided to proceed with MbCas12a.
+        style="width:99%;" class="thumbimage">
-      </p>
+        <div class="thumbcaption">
-      <div class="thumb">
+          <i>
-        <div class="thumbinner" style="width:60%;">
+            <b>Figure 5: Fusion gRNA Editing Rates for Multiplexing CCR5 and VEGFA.</b>
-          <img alt="" src="https://static.igem.wiki/teams/5237/engineering/cas12-decision.svg"
+            The editing rates were determined 72h after
-            style="width:99%;" class="thumbimage">
+            transfection via T7EI assay. Editing % was determined by measuring band intensities; Editing % = 100 x (1 -
-          <div class="thumbcaption">
+            (1- cleaved
-            <i>
+            band/uncleaved band)) <sup>1/2</sup>. The schematic at the top shows the composition of the fgRNA. Below
-              <b>Figure 4: Comparison of AsCas12a and MbCas12a with a dual luciferae assay.</b>
+            each spacer is
-              Firefly luminescence intensity measured 48 h after transfection. Normalized against renilla luminescence. On the x-axis
+            the targeted gene. The symbols below indicate which parts are included in each sample. Cas12a targets VEGFA
-              the samples Cas9 + AsCas12a , Cas9 + MbCas12a, AsCas12a and MbCas12a are depicted.
+            and Cas9
-              Data is depicted as the mean +/- SD (n=3).
+             targets CCR5.
-              Statistical analysis was performed using 1way ANOVA with Tukey's multiple comparisons test. For better clarity, only
+           </i>
-              significant differences within a group between the same Cas proteins are shown.*p&lt;0.05, **p&lt;0.01, ***p&lt;0.001,
-                ****p&lt;0.0001
-             </i>
-           </div>
          </div>
        </div>
-     </section>
+     </div>
+    <p>
-     <section id="3.3"><h2>3.3 MbCas12a tolerates co-transfection and simultaneous editing of different Cas proteins</h2>
+      For CCR5, the editing rate with sgRNAs was approximately the same at about 30%. However, it dropped below 10% for
-      <p>
+      the
-        Wanting to employ the MbCas12a as part of a Cas staple, our goal was to find out how well MbCas12a can stay active while
+      fgRNA. The addition of the 20 nt linker had no effect on the editing rates compared to no linker.
-        being transfected together with SpCas9. Therefore we engineered the dual luciferase assay to allow us for testing two
+     </p>
-        catalytically active Cas proteins at once. Including a well established protospacer, CCR5, into the renilla luciferase
+  </section>
-        gene enables us to test the editing rates of two Cas proteins simultaneously (Fig.5 ).
+  <section id="3.3"><h2>3.3 SpCas9 can be fused to MbCas12a while maintaining functionality</h2>
-      </p>
+    <p>
-      <div class="thumb">
+      Testing showed simultaneous editing of MbCas12a and SpCas9. Therefore we wanted to test protein-protein fusion,
-        <div class="thumbinner"  style="width:60%">
+      potentially giving us another way of Cas stapling. Our goal was to find out how well SpCas9 can stay active while
-          <img alt="" src="https://static.igem.wiki/teams/5237/engineering/co-transf-registry.svg"
+      being
-            style="width:99%;" class="thumbimage">
+      fused to MbCas12a. Therefore we employed the previously engineered dual luciferase assay to allow us for testing
-            <div class="thumbcaption">
+      two
-              <i>
+      catalytically active Cas proteins at once, this time being fused to each other (Fig. 6).
-                <b>Figure 5: Testing for Simultaneous Editing with Double Cut Luciferae Assay</b>
+    </p>
-                Firefly and renilla luminescence intensity is plotted, both measured 48 h after transfection. Co-transformed contains
+    <div class="thumb">
-                MbCas12a and SpCas9. Cas12a are the Firefly relative luminescence units (RLUs), while Cas9 are the Renilla RLUs.
+      <div class="thumbinner" style="width:60%;">
-                Data is depicted as the mean +/- SD (n=3).
+        <img alt="" src="https://static.igem.wiki/teams/5237/engineering/fusioncas-registry.svg" style="width:99%;"
-                Statistical analysis was performed using 2way ANOVA with Tukey's multiple comparisons test. For better clarity, only
+          class="thumbimage">
-                significant differences within a group are shown.*p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p  &lt; 0.0001
+        <div class="thumbcaption">
-              </i>
+          <i>
-            </div>
+            <b>Figure 6: 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&lt;0.05,
+            **p&lt;0.01, ***p&lt;0.001, ****p&lt;0.0001
+          </i>
          </div>
        </div>
-      <p>
+    </div>
-        For the MbCas12a we have a highly significant editing rate in the single cut, meaning both Cas proteins are transfected,
+    <p>
-        but only MbCas12a has a targeting gRNA. When introducing a targeting gRNA for SpCas9 we see no reduction in the highly
+      MbCas12a showed highly significant editing rate in the single cut experiment (only MbCas12a has a targeting gRNA).
-        significant editing of MbCas12a, strongly suggesting both Cas proteins to be able to edit simultaneously.
+      When introducing a targeting gRNA for SpCas9 no significant change could be detected,
-      </p>
+      strongly suggesting both Cas proteins in the fusion Cas to be able to edit simultaneously.
-    </section>
+    </p>
-    <section id="3.4"><h2>3.4 MbCas12a shows editing with fgRNA</h2>
+  </section>
-      <p>
+  <section id="3.4">
-        To further confirm if MbCas12a is compatible with our Cas staples, editing rates were tested using a fusion guide RNA
+    <h2>3.4 SpCas9 fused to MbCas12a shows editing with fgRNA</h2>
-        (fgRNA, <a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a>)
+    <p>
-        targeting two different loci: <i>FANCF</i> and <i>VEGFA</i>. To better assess the impact that the utilization of a
+      The capability of SpCas9 in the fusion Cas was tested by assessing the editing rates via a T7EI assay. For this,
-        fgRNA has on the editing rates, the sgRNAs were tested separately and in one sample.<br>
+      the
-        Having the sgRNA with single Cas proteins in the same sample resulted in no clear
+      same target sequences as before were used, namely FANCF and VEGFA in both configurations. Biological
-        difference in the editing rates (Fig. 6). The fusion of the gRNAs resulted in a lower editing rate
+      duplicates were done for this assay. <br>
-        overall. While the editing for VEGFA stayed at about 20% in all
+      SpCas9 editing rates were higher overall. At the same time, when targeting VEGFA they resulted in a higher editing
-        cases, the editing for FANCF dropped significantly. Nonetheless we were able to show MbCas12a editing utilizing a fgRNA.
+      efficiency than FANCF.
-      </p>
+    </p>
-      <div class="thumb">
+    <div class=thumb>
-        <div class="thumbinner" style="width:60%;">
+      <div class="thumbinner" style="width:60%;">
-          <img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/results-mbcas-2.svg"
+        <img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/results-fcas-2.svg" style="width:99%;"
-            style="width:99%;" class="thumbimage">
+          class="thumbimage">
-          <div class="thumbcaption">
+        <div class="thumbcaption">
-            <i>
+          <i>
-              <b>Figure 6: Fusion gRNA Editing Rates In Combination with MbCas12a.</b>
+            <b>Figure 7: 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
+            the editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by
-              determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band))<sup>1/2</sup>. The
+            measuring band
-              schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below
+            intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band)) <sup>1/2</sup>. The schematic at the
-              indicate which parts are included in each sample. <i class="italic">A</i> and <i class="italic">B</i> display both
+            top shows the
-              orientations of the two spacers for VEGFA and FANCF.
+            composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts are
-             </i>
+            included in
-           </div>
+            each sample. Cas proteins linked by a dash ("-") were fused to each other. Biological replicates are marked
+             as
+            individual dots
+           </i>
          </div>
        </div>
-      <p>
+  </section>
-        To further assess the effect of the genomic locus on the editing rate, we included CCR5 as an additional gene target.
+</section>
-        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>
+<section id="4">
-      <div class="thumb">
+  <h1>4. Results</h1>
-        <div class="thumbinner" style="width:60%;">
+  <p>
-          <img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/results-ccr5-2.svg"
+    We extensively characterized the catalytically active SpCas9 in several assays asserting functional editing with a
-            style="width:99%;" class="thumbimage">
+    fusion guide RNA (BBa_K5237000), showing high activity while being co-transfected with MbCas9, our second staple
-          <div class="thumbcaption">
+    protein's active version, and lastly a functioning fusion to MbCas12a.<br>
-            <i>
+    After all these successful test we were confident to test the Cas staples in action.
-              <b>Figure 7: Fusion gRNA Editing Rates for Multiplexing CCR5 and VEGFA</b>
+  </p>
-              The editing rates were determined 72h after transfection via T7EI assay. Editing % was determined by measuring band
+  <section id="4.1"><h2>4.1 dSpCas9 transactivation as part of a Cas staple</h2>
-              intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band)) <sup>1/2</sup>. The schematic at the top shows the
+    <p>
-              composition of the fgRNA. Below each spacer is the targeted gene. The symbols below indicate which parts are included in
+      The next step was to use the SpCas9 as part of a Cas staple to staple two DNA loci together, and thereby induce
-              each sample. Cas12a targets VEGFA and Cas9 targets CCR5.
+      proximity between two separate functional elements. For this, an enhancer plasmid and a reporter plasmid was used. The
-             </i>
+      reporter plasmid has firefly luciferase behind several repeats of a Cas9 targeted sequence. The enhancer plasmid has a
-           </div>
+      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. 8, 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. 8, B).
+       An extension of the linker from 20 nt up to 40 nt resulted in an increasingly higher expression of the reporter gene.
+    </p>
+    <div class="thumb">
+      <div class="thumbinner" style="width:60%;">
+        <img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/results-eh-2.svg"
+          style="width:99%;" class="thumbimage">
+        <div class="thumbcaption">
+          <i>
+            <b>Figure 8: 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 &lt;
+.05; **p &lt; 0.01; ***p &lt; 0.001; mean +/- SD). The assay included sgRNAs and fgRNAs with linker lengths from 0 nt
+            to 40 nt.
+           </i>
          </div>
        </div>
-     </section>
+     </div>
-    <section id="3.5"><h2>3.5 MbCas12a tolerates fusion to SpCas9</h2>
-      <p>
-        Testing showed simultaneous editing of MbCas12a and SpCas9. Therefore we wanted to test protein-protein fusion,
-        potentially giving us another way of Cas stapling. Our goal was to find out how well MbCas12a can stay active while
-        being fused to SpCas9. Therefore we employed the previously engineered dual luciferase assay to allow us for testing two
-        catalytically active Cas proteins at once, this time being fused to each other (Fig. 8).
-      </p>
-      <div class="thumb">
-        <div class="thumbinner" style="width:60%;">
-          <img alt="" src="https://static.igem.wiki/teams/5237/engineering/fusioncas-registry.svg"
-            style="width:99%;" class="thumbimage">
-          <div class="thumbcaption">
-            <i>
-              <b>Figure 8: Double cut dual luciferase assay testing Fusion Cas simultaneous editing.</b>
-              Firefly and renilla luminescence intensity is plotted, both measured 48 h after transfection. On the x-axis the negative
-              control, single edit and simultaneous edit, are grouped by the two Cas proteins in the fusion Cas. The fusion Cas
-              contains MbCas12a and SpCas9. MbCas12a are the Firefly relative luminescence units (RLUs), while Cas9 are the Renilla
-              RLUs. Data is depicted as the mean +/- SD (n=3). Statistical analysis was performed using 2way ANOVA with Tukey's multiple
-              comparisons test. For better clarity, only significant differences within a group are shown.*p&lt;0.05, **p&lt;0.01, ***p&lt;0.001, ****p&lt;0.0001
-            </i>
-          </div>
-        </div>
-      </div>
-      <p>
-        For the MbCas12a we have a highly significant editing rate in the single cut, meaning only MbCas12a has a targeting
-        gRNA. When introducing a targeting gRNA for SpCas9 we see no reduction in the highly significant editing of MbCas12a,
-        strongly suggesting both Cas proteins in the fusion Cas to be able to edit simultaneously.
-      </p>
-    </section>
-    <section id="3.6"><h2>3.6 MbCas12a fused to SpCas9 editing utilizing a fgRNA</h2>
-      <p>
-        The capability of MbCas12 in the fusion Cas was tested by assessing the editing rates via a T7EI assay. For this, the
-        same target sequences as before were used, namely FANCF and VEGFA in both configurations. We included biological
-        duplicates in this assay.<br>
-        MbCas12a editing rates were significantly lower, dropping to about 1%. At the same time, when targeting VEGFA they
-        resulted in a higher editing efficiency than FANCF.
-      </p>
-      <div class="thumb">
-        <div class="thumbinner" style="width:60%;">
-          <img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/results-fcas-2.svg"
-            style="width:99%;" class="thumbimage">
-            <div>
-              <i>
-                <b>Figure 9: Editing rates for fusion guide RNAs with fusion Cas proteins.</b>
-                In <b>A</b> and <b>B</b> the editing rates were determined 72h after transfection via T7EI assay. Editing % was
-                determined by measuring band intensities; Editing % = 100 x (1 - (1- cleaved band/uncleaved band))<sup>1/2</sup>. The
-                schematic at the top shows the composition of the fgRNA. Below each spacer is the targeted gene. The symbols below
-                indicate which parts are included in each sample. Cas proteins linked by a dash ("-") were fused to each other.
-                Biological replicates are marked as individual dots.
-              </i>
-            </div>
-        </div>
-      </div>
-    </section>
    </section>
-   <section id="4">
+   <section id="4.2"><h2>4.2 SpCas9 fused to dMbCas12a form the Cas staple</h2>
-    <h1>4. Results</h1>
      <p>
-       We extensively characterized the catalytically active MbCas12a in several assays determining the best ortholog out of
+       Continuing the procedure in a similar manner, we focused on inducing proximity between genetic loci using the fusion
-       three for our Cas staples, assert functional editing with a fusion guide RNA (BBa_K5237000), showing high activity
+       dCas next, consisting of dMbCas12a fused to dSpCas9. The same assay was used, with one enhancer plasmid and one reporter
-       while being co-transfected with SpCas9, our second staple protein's active version, and lastly a functioning fusion to
+      plasmid. Though less distinct than the results for not using a protein fusion, the fusion Cas proteins can be used to
-       SpCas9.<br>
+      increase expression levels of the reporter firefly luciferase (see figure 13). While using sgRNAs results in similar
-       After all these successful test we were confident to test the Cas staples in action.
+       relative luciferase activity as for the negative control between 0.1 and 0.2., using a fgRNA with a 20 to 30 nt linker
+       consistently resulted in activities at 0.25. Fusion guide RNAs without a linker and with a 40 nt linker had on average
+       about the same activity, but with a higher spread over the biological replicates. Nonetheless this showed the dMbCas12a
+      fused to the dSpCas9 to be working as a Cas staple. Further tweaking is needed to get better results
      </p>
-     <section><h2 id="4.1">4.1 dMbCas12a Transactivation as Part of Cas Staple</h2>
+     <div class="thumb">
-      <p>
+      <div class="thumbinner" style="width:60%;">
-        The next step was to use the MbCas12a as part of a Cas staple to staple two DNA loci together, and thereby induce
+        <img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/results-fcas-eh-2.svg"
-        proximity between two separate functional elements. For this, an enhancer plasmid and a reporter plasmid was used. The
+        style="width:99%;" class="thumbimage">
-        reporter plasmid has firefly luciferase behind several repeats of a Cas9 targeted sequence. The enhancer plasmid has a
+        <div class="thumbcaption">
-        Gal4 binding site behind several repeats of a Cas12a targeted sequence. By introducing a fgRNA staple and a Gal4-VP64,
+          <i>
-        expression of the luciferase is induced (Fig. 10 A).
+            <b>Figure 9: Firefly luciferase trans activation through fusion Cas staple</b>
-        Different linker lengths were tested. Cells were again normalized against ubiquitous renilla expression. Further
+            Firefly luciferase activity was measured 48h after transfection. Normalized against ubiquitously expressed Renilla
-        information on our learnings from this assay can be found in the Cas staple engineering cycle iteration 2.
+            luciferase. Statistical significance was calculated with ordinary One-way ANOVA with Dunn's method for multiple
-        Using no linker between the two spacers showed similar relative luciferase activity to the baseline control (Fig. 10 B).
+            comparisons (*p &lt; 0.05; **p &lt; 0.01; ***p &lt; 0.001; mean +/- SD). Fusion Cas proteins were paired with sgRNAs and fgRNAs
-        An extension of the linker from 20 nt up to 40 nt resulted in an increasingly higher expression of the reporter gene.
+              with linker lengths from 0 nt to 40 nt
-      </p>
+          </i>
-      <div class="thumb">
-        <div class="thumbinner" style="width:60%;">
-          <img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/results-eh-2.svg"
-            style="width:99%;" class="thumbimage">
-          <div class="thumbcaption">
-            <i>
-              <b>Figure 10: Applying Fusion Guide RNAs for Cas staples.</b> <b>A</b>, schematic overview of the assay. An enhancer
-              plasmid and a reporter plasmid are brought into proximity by a fgRNA Cas staple complex binding both plasmids. Target
-              sequences were included in multiple repeats prior to the functional elements. Firefly luciferase serves as the reporter
-              gene, the enhancer is constituted by multiple Gal4 repeats that are bound by a Gal4-VP64 fusion.
-              <B>B</B>, results of using a fgRNA Cas staple for trans activation of firefly luciferase. Firefly
-              luciferase activity was measured 48h after transfection. Normalized against ubiquitously expressed Renilla luciferase.
-              Statistical significance was calculated with ordinary One-way ANOVA with Dunn's method for multiple comparisons (*p &lt;
-.05; **p &lt; 0.01; ***p &lt; 0.001; mean +/- SD). The assay included sgRNAs and fgRNAs with linker lengths from 0 nt
-              to 40 nt.
-            </i>
-          </div>
          </div>
        </div>
-     </section>
+     </div>
    </section>
-  <section id="5">
+</section>
-    <h1>5. References</h1>
+<section id="5">
-    <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>
+  <h1>5. References</h1>
+  <p>Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., and Charpentier, E. (2012). A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. <i>Science, 337</i>, 816–821. <a href="https://doi.org/10.1126/science.1225829" target="_blank">https://doi.org/10.1126/science.1225829</a>.</p>
-    <p>Kleinstiver, B. P., Sousa, A. A., Walton, R. T., Tak, Y. E., Hsu, J. Y., Clement, K., Welch, M. M., Horng, J. E., Malagon-Lopez, J., Scarfò, I., Maus, M. V., Pinello, L., Aryee, M. J., and Joung, J. K. (2019). Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. <i>Nature Biotechnology, 37</i>, 276–282. <a href="https://doi.org/10.1038/s41587-018-0011-0" target="_blank">https://doi.org/10.1038/s41587-018-0011-0</a>.</p>
+  <p>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>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., Malagon-Lopez, J., Scarfò, I., Maus, M. V., Pinello, L., Aryee, M. J., and Joung, J. K. (2019). Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. <i>Nature Biotechnology, 37</i>, 276–282. <a href="https://doi.org/10.1038/s41587-018-0011-0" target="_blank">https://doi.org/10.1038/s41587-018-0011-0</a>.</p>
-    <p>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>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>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>Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E., and Church, G. M. (2013). RNA-Guided Human Genome Engineering via Cas9. <i>Science, 339</i>, 823–826. <a href="https://doi.org/10.1126/science.1232033" target="_blank">https://doi.org/10.1126/science.1232033</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>
+  <p>Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I., Dohmae, N., Ishitani, R., Zhang, F., and Nureki, O. (2014). Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. <i>Cell, 156</i>, 935–949. <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>
-   </section>
+  <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>Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C., and Doudna, J. A. (2014). DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. <i>Nature, 507</i>, 62–67. <a href="https://doi.org/10.1038/nature13011" target="_blank">https://doi.org/10.1038/nature13011</a>.</p>
+</section>
 </body>
 </html>