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<h3 class="fs-h3 fw-semi-bold">CRISPR/Cas Staples</h3>
<ul>
<li class="fs-400 fw-medium mb-2">Highly <b>specific</b>, easily <b>programmable</b> tool, based on the CRISPR/Cas system, that allows targeting of <b>any location</b> within the genome</li>
- <li class="fs-400 fw-medium mb-2">Multiple Cas staples with <b>fusion guide RNAs and fusion Cas proteins</b> tested for their abillity to induce <b>proximity of genetic elements</b></li>
+ <li class="fs-400 fw-medium mb-2">Multiple Cas staples with <b>fusion guide RNAs and fusion Cas proteins</b> tested for their ability to induce <b>proximity of genetic elements</b></li>
<li class="fs-400 fw-medium mb-2">We show <b>successful engineering</b> of the <b>3D genome structure</b> with Cas staples</li>
</ul>
</div>
@@ -265,15 +265,12 @@
based on smaller proteins like zinc finger domains, remain difficult to adapt to any genomic locus of
choice (Kim and Kini, 2017).<br>
Here we present the usage of CRISPR/Cas proteins to reliably connect or "staple" two DNA strands.
- We create fusion guide RNAs (fgRNAs) that are composed of a Cas12a and Cas9 gRNA, connected via their
- spacers (Kweon <i class="italic">et al.</i>, 2017). This system solely
- requires the two Cas proteins dCas9 and dCas12a and an fgRNA to bring two elements
- close together. This allows for precise, adjustable targeting while keeping the system simple in general.
- By introducing several subsequent fgRNAs, this may also allow for multiplexing. By harnessing the Cas12a’s
- ability to process chimeras of Cas9 and Cas12a into individual units we can decide exactly which genes are
- brought into proximity and which not (Gonatopoulos-Pournatzis <i class="italic">et al.</i>, 2020).<br>
- The first part of this project focuses on establishing functional fgRNAs. We then continue to show that
- these fgRNAs can be used in combination with Cas12a and Cas9 to form a complex that brings two separate
+ We created fusion guide RNAs (fgRNAs) that are composed of Cas12a and Cas9 gRNAs, connected via their
+ spacers (Kweon <i class="italic">et al.</i>, 2017). These Cas staples solely
+ require the two Cas proteins dCas9 and dCas12a and an fgRNA to bridge two separate DNA strands. This allows for precise and adjustable genomic targeting while keeping the systems complexity to minimum.
+ Cas12a's ability to process concatenated crRNAs will allow for simple multiplexing by expressing several fgRNAs in repeat from a single promoter (Gonatopoulos-Pournatzis <i class="italic">et al.</i>, 2020).<br>
+ In the first part of this project we established functional fgRNAs. We then continued to show that
+ these fgRNAs can be used in combination with Cas12a and Cas9 to form complete Cas staples that bring two separate
DNA loci into proximity. </p>
</div>
<div class="here-are-results">
@@ -628,7 +625,7 @@
<div class="jumper" id="StapleFunctionalization"></div>
<div class="heading-wrapper">
<h2 class="fs-h2 fw-semi-bold">Staple Functionalization</h2>
- <p class="mt-3 mb-3">To enable the <b>functionalization</b> of our PICasSO toolbox for a wide range of therapeutic and synthetic biology applications, we designed cathepsin B-responsive peptide linkers. Cathepsin B is a <b>lysosomal protease</b> present in the cytosol of various cancer types. We overexpressed cathepsin B in HEK293T cells to investigate its ability to cleave <b>different peptide linkers</b> using a fluorescence readout assay. We successfully demonstrated that the <b>GFLG linker, which can be incorporated into our staple constructs, was efficiently cleaved</b> by cathepsin B <i class="italic">in vivo</i> when cells were treated with doxorubicin. Furthermore, we showed that wild-type cathepsin B matured into its <b>active forms</b> under these conditions.</p>
+ <p class="mt-3 mb-3">To enable the <b>functionalization</b> of our PICasSO toolbox for a wide range of therapeutic and synthetic biology applications, we designed cathepsin B-responsive peptide linkers to selectively control the connection of our protein staples. Cathepsin B is a <b>lysosomal protease</b> enriched in various cancer types. We overexpressed cathepsin B in HEK293T cells to investigate its ability to cleave <b>different peptide linkers</b> using a fluorescence readout assay. We successfully demonstrated that the <b>GFLG linker, which can be incorporated into our staple constructs, was efficiently cleaved</b> by cathepsin B <i class="italic">in vivo</i> when cells were treated with doxorubicin. Furthermore, we showed that wild-type cathepsin B matured into its <b>active forms</b> under these conditions.</p>
</div>
<div class="here-is-Introduction">
<h3 class="fs-h3 fw-semi-bold mt-4">Introduction</h3>
@@ -654,9 +651,9 @@
</div>
</div>
<h4 class="fs-h4 fw-semi-bold mt-5">Aim of This Subproject</h4>
- <p class="mt-3"> We want to explore the potential of our PICasSO platform approach for therapeutic applications by designing protein-based DNA staples that are responsive to the overexpression of cathepsin B in cancerous tissues. Furthermore, we demonstrate doxorubicin-dependent cathepsin B cleavage of one out of five documented linkers (Jin <i class="italic">et al.</i>, 2022; Shim <i class="italic">et al.</i>, 2022; Wang <i class="italic">et al.</i>, 2024) in HEK293T cells.<br>
- Additionally, we develop a construct consisting of one dead Cas9 (dCas9) connected to two SV40 nuclear localization sequences and two caged intein fragments (NpuN, NpuC) (Gramespacher <i class="italic">et al.</i>, 2017). The cages are connected to the split intein via cathepsin B-responsive linkers, preventing fragment association and consequent protein <i class="italic">trans</i>-splicing. In cancer cells overexpressing cathepsin B, the linkers would be cleaved, thus uncaging the inteins and enabling association of NpuN and NpuC. In the subsequent protein <i class="italic">trans</i>-splicing reaction NpuN and NpuC cleave themselves out of the construct, linking two dCas9 proteins to each other.<br>
- With these approaches, we aim to induce structural and functional changes in protein-stapled DNA selectively in cancerous tissue.</p>
+ <p class="mt-3"> We wanted to explore the potential of our PICasSO platform approach for therapeutic applications by designing protein-based DNA staples that are cleaved in response to the overexpression of cathepsin B in cancerous tissues. Furthermore, we demonstrated doxorubicin-dependent cathepsin B cleavage of one out of five documented linkers (Jin <i class="italic">et al.</i>, 2022; Shim <i class="italic">et al.</i>, 2022; Wang <i class="italic">et al.</i>, 2024) in HEK293T cells.<br>
+ Additionally, we developed a construct consisting of a dead Cas9 (dCas9) connected to two SV40 nuclear localization sequences and two caged intein fragments (NpuN, NpuC) (Gramespacher <i class="italic">et al.</i>, 2017). The cages were connected to the split intein via cathepsin B-responsive linkers, preventing fragment association and consequent protein <i class="italic">trans</i>-splicing. In cancer cells overexpressing cathepsin B, the linkers would be cleaved, thus uncaging the inteins and resulting in association of NpuN and NpuC. In the subsequent protein <i class="italic">trans</i>-splicing reaction NpuN and NpuC cleave themselves out of the construct, linking dCas9 proteins to each other.<br>
+ With these approaches, we aimed to induce structural and functional changes in protein-stapled DNA selectively in cancerous tissue.</p>
</div>
<div class="here-are-results">
<h3 class="fs-h3 fw-semi-bold mt-5" id="results_cathepsinBliner">Results</h3>
@@ -674,7 +671,7 @@
<!-- _______________________________________ HALLO Marik HIER EINFÃœGEN _______________________ -->
<p>
- To achieve cathepsin B cleavage-induced Cas stapling, catalytically active cathepsin B needs to be expressed in the cytosol. Therefore, we investigated the expression of different cathepsin B constructs under different conditions in HEK293T cells. In addition to wild-type (wt) cathepsin B, we also cloned a truncated and mutated version of cathepsin B (Δ1-20, D22A, H110A, R116A) and compared protein expression of both constructs in doxorubicin-treated and untreated conditions.<br><b>Figure 13</b> shows a western blot of the wild-type (wt) version of cathepsin B as well as the <a class="underline--magical" href="{{ url_for('pages', page='engineering') }}">truncated and mutated version of cathepsin B</a> (Δ1-20, D22A, H110A, R116A). The truncated and mutated version of cathepsin B lacked an N-terminal signal peptide responsible for co-translational targeting to the rough endoplasmic reticulum. This would lead to the cytoplasmic expression of cathepsin B (Müntener <i class="italic">et al.</i>, 2005). Additionally, three point mutations would disrupt the conformation of an occluding loop increasing cathepsin B activity in the cytoplasm (Nägler <i class="italic">et al.</i>, 1997). Cells of both cathepsin B versions were treated with 500 nM doxorubicin (dox) 24 hours post-transfection and incubated for additional 24 hours. For each condition, three replicates were blotted. We observed no differences in protein expression levels between the dox-treated and untreated wt versions of cathepsin B. For the truncated and mutated version of cathepsin B, however, only the untreated samples showed the corresponding band at approximately 36 kDa expected for this version of cathepsin B. Additionally, the bands of the truncated and mutated version appeared much weaker than the ones of the wt, indicating poorer protein expression. The household protein β-tubulin is visible in all samples at approximately 55 kDa. The wt cathepsin B additionally showed bands for pro-cathepsin B at approximately 42 kDa, a mature single-chain version of cathepsin B at approximately 33 kDa and a mature double-chain version at approximately 26 kDa.</p>
+ To achieve cathepsin B cleavage-induced Cas stapling, catalytically active cathepsin B needs to be expressed in the cytosol. Therefore, we investigated the expression of different cathepsin B constructs under different conditions in HEK293T cells. In addition to wild-type (wt) cathepsin B, we also cloned a truncated and mutated version of cathepsin B (Δ1-20, D22A, H110A, R116A) and compared protein expression of both constructs in doxorubicin-treated and untreated conditions.<br><b>Figure 13</b> shows a Western blot of the wild-type (wt) version of cathepsin B as well as the <a class="underline--magical" href="{{ url_for('pages', page='engineering') }}">truncated and mutated version of cathepsin B</a> (Δ1-20, D22A, H110A, R116A). The truncated and mutated version of cathepsin B lacked an N-terminal signal peptide responsible for co-translational targeting to the rough endoplasmic reticulum. This would lead to the cytoplasmic expression of cathepsin B (Müntener <i class="italic">et al.</i>, 2005). Additionally, three point mutations would disrupt the conformation of an occluding loop increasing cathepsin B activity in the cytoplasm (Nägler <i class="italic">et al.</i>, 1997). Cells of both cathepsin B versions were treated with 500 nM doxorubicin (dox) 24 hours post-transfection and incubated for additional 24 hours. For each condition, three replicates were blotted. We observed no differences in protein expression levels between the dox-treated and untreated wt versions of cathepsin B. For the truncated and mutated version of cathepsin B, however, only the untreated samples showed the corresponding band at approximately 36 kDa expected for this version of cathepsin B. Additionally, the bands of the truncated and mutated version appeared much weaker than the ones of the wt, indicating poorer protein expression. The household protein β-tubulin is visible in all samples at approximately 55 kDa. The wt cathepsin B additionally showed bands for pro-cathepsin B at approximately 42 kDa, a mature single-chain version of cathepsin B at approximately 33 kDa and a mature double-chain version at approximately 26 kDa.</p>
<div class="image-caption-wrapper" style="width: 80%; margin-left:auto; margin-right:auto;"><img id="cas9-cas12" alt=""
src=" https://static.igem.wiki/teams/5237/wetlab-results/catb-wb-w.svg">
@@ -694,7 +691,7 @@
<div class="accordion-body">
<!-- _______________________________________ HALLO Mark HIER EINFÃœGEN _______________________ -->
- <p>Based on the results of the western blot, we decided to use wt cathepsin B to investigate the proteolytic cleavage of different peptide linkers. To this end, we used a fluorescence readout assay based on VP64-induced mCherry expression. We successfully identified one linker that could be cleaved efficiently by cathepsin B <i class="italic">in vivo</i>.<br>
+ <p>Based on the results of the Western blot, we decided to use wt cathepsin B to investigate the proteolytic cleavage of different peptide linkers. To this end, we used a fluorescence readout assay based on VP64-induced mCherry expression. We successfully identified one linker that could be cleaved efficiently by cathepsin B <i class="italic">in vivo</i>.<br>
To investigate cathepsin B cleavage of different linkers, we incorporated five peptide linkers from literature (GFLG, FFRG, FRRL, VA, FK) (Jin <i class="italic">et al.</i>, 2022; Shim <i class="italic">et al.</i>, 2022; Wang <i class="italic">et al.</i>, 2024) in between the DNA binding domain (DBD) of Gal4 and the transactivator domain VP64 (as previously described in Muench <i class="italic">et al.</i> (2023)). Binding of Gal4-DBD upstream of a gene encoding the fluorescence protein mCherry induces overexpression of mCherry by VP64. Consequently, separation of Gal4-DBD and VP64 by cathepsin B cleavage of the peptide linker reduces mCherry expression (see <b>Fig. 14</b>).</p>
<div class="image-caption-wrapper" style="width: 80%; margin-left:auto; margin-right:auto;"><img id="cas9-cas12" alt=""
src="https://static.igem.wiki/teams/5237/wetlab-results/catb-gal4-vp64-mechanism.svg">
@@ -774,11 +771,11 @@
<div id="disc-c-1" class="accordion-collapse collapse" aria-labelledby="panelsStayOpen-headingFour">
<div class="accordion-body">
<!-- _______________________________________ HALLO Marik HIER EINFÃœGEN _______________________ -->
- <p>We validated the cleavage of peptide linkers by cathepsin B using a fluorescence readout assay. This assay employed a Gal4-VP64 system, where cleavage of the peptide linker by cathepsin B reduced mCherry expression, providing a reliable measure of cleavage efficiency. Five different peptide linkers (GFLG, FFRG, FRRL, VA, FK) were tested. A two-way ANOVA revealed a significant reduction in fluorescence between the negative control and the two test conditions for the GFLG linker in the presence of cathepsin B, demonstrating efficient cleavage. A significant increase in fluorescence was observed between the negative control and 60 ng test condition for the FFRG linker. However, since the increase between the negative control and the 30 ng test condition of the same linker was not significant, this difference is likely due to biological variability between the samples.<br>
- Fluorescence microscopy further supported these findings, with reduced mCherry fluorescence intensity observed in cells overexpressing cathepsin B. It was also observed that not all cells were fluorescent in accordance with an expected transfection efficiency. We also noticed that some cells seemed to only express eGFP or mCherry. However, most cells seemed to express both eGFP and mCherry as indicated by their yellow fluorescence. <br>
- Furthermore, western blot analysis confirmed that overexpressed wild-type cathepsin B was processed into its mature single-chain and double-chain forms as previously reported in the literature (Mentlein, Hattermann, Held-Feindt, 2012). This confirms that active cathepsin B is present inside of the cells. However, since lysis of the cells also disrupts the lysosomes, we can not conclude whether this active cathepsin B is also present in the cytosol <i class="italic">in vivo</i>. For the truncated and mutated version of cathepsin B, we only observed protein bands in samples that were not treated with doxorubicin. The three samples incubated with doxorubicin for 24 hours showed only faint bands for the housekeeping protein β-tubulin, indicating generally low protein levels. This suggests that the cells were subjected to stress, possibly through doxorubicin or inadequate handling. Along with low transfection efficiency, these factors may have contributed to the low protein levels observed.<br>
-
- In conclusion, these findings demonstrate that our fluorescence-based readout assay can reliably detect cathepsin B-mediated cleavage of peptide linkers, with the GFLG linker showing particular susceptibility to cleavage. This makes GFLG a promising candidate for targeted applications in environments with upregulated cathepsin B activity, such as in cancerous tissues. Additionally, our cathepsin B-cleavable linker can be combined with caged inteins (Gramespacher <i class="italic">et al.</i>, 2017) conjugated to a dead Cas9 to selectively induce Cas-stapling in the presence of cathepsin B.</p>
+ <p>Initially, Western blot analysis confirmed that overexpressed wild-type cathepsin B was processed into its mature single-chain and double-chain forms as previously reported in the literature (Mentlein, Hattermann, Held-Feindt, 2012). This confirms that active cathepsin B is present inside of the cells. However, since lysis of the cells also disrupts the lysosomes, we can not conclude whether this active cathepsin B is also present in the cytosol <i class="italic">in vivo</i>. For the truncated and mutated version of cathepsin B, we only observed protein bands in samples that were not treated with doxorubicin. The three samples incubated with doxorubicin for 24 hours showed only faint bands for the housekeeping protein β-tubulin, indicating generally low protein levels. This suggests that the cells were subjected to stress, possibly through doxorubicin or inadequate handling. Along with low transfection efficiency, these factors may have contributed to the low protein levels observed.<br>
+ Using fluorescence microscopy, we demonstrated cathepsin B-mediated linker cleavage in our reporter assay setup. This assay employed a Gal4-VP64 system, in which cleavage of the peptide linker by cathepsin B reduced mCherry expression, providing a reliable measure of cleavage efficiency. The successful cleavage of linkers was indicated by reduced mCherry fluorescence intensity in cells overexpressing cathepsin B. It was also observed that not all cells were fluorescent in accordance with an expected transfection efficiency. We also noticed that some cells seemed to only express eGFP or mCherry. However, most cells seemed to express both eGFP and mCherry as indicated by their yellow fluorescence. <br>
+ Finally, we investigated the cleavage of different peptide linker variants by cathepsin B with help of our fluorescence readout assay. Five different peptide linkers (GFLG, FFRG, FRRL, VA, FK) were tested. A two-way ANOVA revealed a significant reduction in fluorescence between the negative control and the two test conditions for the GFLG linker in the presence of cathepsin B, demonstrating efficient cleavage. Surprisingly, a significant increase in fluorescence was observed between the negative control and 60 ng test condition for the FFRG linker. However, since the increase between the negative control and the 30 ng test condition of the same linker was not significant, this difference is likely due to biological variability between the samples.<br>
+ Additionally, our cathepsin B-cleavable linker can be combined with caged inteins (Gramespacher <i class="italic">et al.</i>, 2017) conjugated to a dead Cas9 to selectively induce Cas-stapling in the presence of cathepsin B. Corresponding parts can be found in our part collection and in the iGEM registry, but still require characterization.<br>
+ In conclusion, these findings demonstrate that our fluorescence-based readout assay can reliably detect cathepsin B-mediated cleavage of peptide linkers, with the GFLG linker showing particular susceptibility to cleavage. This makes GFLG a promising candidate for targeted applications in environments with upregulated cathepsin B activity, such as cancer tissues. </p>
</div>
</div>
</div>
@@ -786,7 +783,7 @@
<h4 class="fs-h4 fw-bold mt-5">Outlook</h4>
<p class="mt-3">
- Future experiments could investigate the influence of different doxorubicin concentrations on the activity of cathepsin B in the cytosol. Different linker lengths or a repeat of the GFLG linker could also be tested. Additionally, this system could be used for other proteases that are involved in certain diseases, such as different caspases in neurodegenerative conditions (Espinosa-Oliva <i class="italic">et al.</i>, 2019).
+ Future experiments will investigate the influence of different doxorubicin concentrations on the activity of cathepsin B in the cytosol. Different linker lengths or a repeat of the GFLG linker could also be tested. Additionally, this system could be used for other proteases that are involved in certain diseases, such as different caspases in neurodegenerative conditions (Espinosa-Oliva <i class="italic">et al.</i>, 2019).
</p>
</div>
</div>
@@ -795,8 +792,8 @@
<div class="heading-wrapper">
<h2 class="fs-h2 fw-semi-bold">Readout Systems</h2>
<p class="mt-3 mb-3">By developing custom <b>EMSA and FRET</b> assays, we established <b>key tools</b> for the <b>validation
- and characterization of various protein staples</b>. Beginning with the successful construction of the <b>basic
- staples</b>, these assays provided <b>key insight</b> into DNA binding proteins and stapling <b>mechanisms</b>. Through <b>testing of our workflow,</b> we developed <b>foundational techniques</b> that future <b>iGEMers and researchers</b>
+ and characterization of various protein staples</b>. Beginning with the successful construction of <b>basic
+ staples</b>, these assays provided <b>key insights</b> into the characteristics of DNA binding proteins and stapling <b>mechanisms</b>. By rigorously <b>testing our workflow,</b> we developed <b>foundational techniques</b> that future <b>iGEMers and researchers</b>
can leverage to engineer and optimize <b>protein-based DNA-folding</b> systems.
</p>
</div>
@@ -838,7 +835,7 @@
<h4 class="fs-h4 fw-semi-bold mt-5">Aim of This Subproject</h4>
<p class="mt-3">
- Engineering a solid and versatile toolbox is a huge challenge, especially when working with complex systems such as DNA-bound protein staples. To systematically characterize these components, we first set out to develop a well-tested collection of assays that lay the foundation for the investigation of the more complex aspects of our PICasSO toolbox. These assays provide essential tools for studying protein-DNA interactions and proximity. We used electrophoretic mobility shift assays (EMSA) to analyze protein-DNA binding kinetics and sequence specificity <i class="italic">in vitro </i> and a Förster resonance energy transfer (FRET) assay to detect DNA-DNA proximity <i class="italic">in vivo</i>. Together, these assays form the backbone of our experimental approach and were utilized to systematically analyze and create new staples.
+ Engineering a solid and versatile toolbox is a huge challenge, especially when working with complex systems such as DNA-bound protein staples. To systematically characterize these components, we first set out to develop a well-tested collection of assays that lay the foundation for the investigation of the more complex aspects of our PICasSO toolbox. These assays provide essential tools for studying protein-DNA interactions and DNA-DNA proximity. We used electrophoretic mobility shift assays (EMSA) to analyze protein-DNA binding kinetics and sequence specificity <i class="italic">in vitro </i> and a Förster resonance energy transfer (FRET) assay to detect interactions <i class="italic">in vivo</i>. Together, these assays form the backbone of our experimental approach and were utilized to systematically analyze and create new staples.
</p>
</div>
<div class="here-are-results">
@@ -1024,7 +1021,7 @@
</div>
<h4 class="fs-h4 fw-bold mt-5">Outlook</h4>
<p class="mt-3">
- Future work will focus on quantifying FRET interaction <i class= italic>in vivo </i> to assess stapling efficiency and DNA-DNA proximity more accurately. Additionally, the development of mini staples can be further improved by testing alternative linkers predicted by our dry lab simulations. By systematically testing different linker sequences, we aim to create functional staples and furthermore lay the foundation for future engineering of small bZip DNA binding domains.<br>
+ Future work will focus on quantifying FRET interaction <i class= italic>in vivo </i> to assess stapling efficiency and DNA-DNA proximity more accurately. Additionally, the development of mini staples can be further improved by testing alternative linkers predicted by <a class="underline--magical" href="{{ url_for('pages', page='model') }}">our dry lab simulations.</a> By systematically assessing different linker sequences, we aim to create functional staples and furthermore lay the foundation for future engineering of small bZip DNA binding domains.<br>
Finally, we are also currently testing out our measurement system with the more complex Cas staples.
</p>
</div>
@@ -1080,7 +1077,7 @@
</div>
<h4 class="fs-h4 fw-semi-bold mt-5">Aim of This Subproject</h4>
<p class="mt-3">
- We sought to explore the potential of using the bacterial Type IV secretion system (T4SS) as a novel platform to deliver genetic payloads to mammalian cells. Intrigued by the work of VL Waters (2001), who showed conjugation-mediated DNA transfer between bacteria and mammalian cells, we brainstormed ways to engineer conjugation as a generalized DNA delivery tool. Recognizing the importance of cell-cell contact as an important parameter for efficienct conjugation between bacteria (Robledo et al., 2022), we were curious to test the effects of the same in promoting conjugation between bacteria and mammalian cells.</p>
+ We sought to explore the potential of using the bacterial Type IV secretion system (T4SS) as a novel platform to deliver genetic payloads to mammalian cells. Intrigued by the work of VL Waters (2001), who showed conjugation-mediated DNA transfer between bacteria and mammalian cells, we brainstormed ways to engineer conjugation as a generalized DNA delivery tool. Recognizing the importance of cell-cell contact as an important parameter for efficienct conjugation between bacteria (Robledo et al., 2022), we were curious to test the effects of the same in promoting conjugation between bacteria and mammalian cells. To this end, we planned to incorporate adhesins into our conjugation system that increase conjugation efficiency and direct plasmid transfer to specific cell types.</p>
</div>
<div class="here-are-results">
<h3 class="fs-h3 fw-semi-bold mt-5">Results</h3>