Difference between revisions of "Team:Munich/Applied Design"

 
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<tr><td colspan=6 align=left valign=center>
<font size=7 color=#51a7f9><b style="color: #51a7f9">Application</b></font>
+
<font size=7 color=#51a7f9><b style="color: #51a7f9; margin-top: 40px;">Applied Design</b></font>
 
</td>
 
</td>
 
</tr>
 
</tr>
<tr>
 
<td  colspan = 6 align="left">
 
<p class="introduction">
 
Thanks to advances in molecular biology and biochemistry, scientists have been able to consistently detect lower and lower concentration of molecules<sup><a class="myLink" href="#ref_1">1</a></sup>, to the point that single molecules can be reliably recognized with methods such as polymerase chain reaction (PCR)<sup><a class="myLink" href="#ref_2">2</a></sup>, fluorescence in situ hybridization (FISH)<sup><a class="myLink" href="#ref_3">3</a></sup> and enzyme-linked immunosorbent assays (ELISA)<sup><a class="myLink" href="#ref_4">4</a></sup>. This has opened doors for synthetic biology to create better and more accurate diagnostic tests that use biomarkers like nucleic acids and proteins as targets<sup><a class="myLink" href="#ref_5">5</a>,<a class="myLink" href="#ref_6">6</a></sup>. Through such advances, the field of molecular diagnostics developed. Unfortunately, current standard methods require expensive equipment or trained personnel, which generally limits their usability to hospitals or laboratories. Recently, there has been a push to develop new tests that fuse the reliability of standard methods with affordable platforms such as lab-on-a-chip or paper strips  to overcome this restrictions<sup><a class="myLink" href="#ref_7">7-9</a></sup>. We wanted to help close this gap and set out to engineer a diagnosis principle for the detection of a wide array of targets that could be used without difficult-to-meet technical requirements.
 
                </p>
 
 
</td>
 
</tr>
 
 
 
 
 
  
  
 
<tr><td colspan=6 align=center valign=center>
 
<tr><td colspan=6 align=center valign=center>
<h3>CascAID</h3>
+
<h1>Diagnosis of infectious diseases and public health</h1>
 
<p>   
 
<p>   
Our project, which we named Cas13a controlled assay for infectious diseases (CascAID), features the recently identified CRISPR/Cas effector Cas13a<sup><a class="myLink" href="#ref_10">10</a></sup>. Unlike other proteins in the familiy, Cas13a has the unique ability to bind and cleave specific RNA targets rather than DNA ones. Moreover, after cleaving its target, Cas13a is able to unspecifically cleave RNA molecules. By using this collateral activity from Cas13a, our system is capable of detecting virtually any RNA target. This is done by changing the crRNA in the protein, that is a short RNA sequence that determines what is recognized as target.</p>
+
Traditionally, infectious diseases are diagnosed by cell culture or PCR-based methods. As these techniques require expensive infrastructure, trained personal, and time, the current practice suffers from three main problems. First, diagnosis is not available everywhere, and therefore pathogens are usually detected in central clinics<sup><a class="myLink" href="#ref_2">2</a></sup>, rather than at the point-of-care (POC). Second, diagnosis is not accessible to everyone. Especially in developing countries proper medical supply is often lacking, due to the high costs. And third, diagnosis is not available within a few hours, which can lead to negligence of laboratory tests, resulting in premature prescription of antibiotics, the primary reason for the recrudescence of resistant bacteria strains<sup><a class="myLink" href="#ref_3">3</a></sup>.</p>
 
</td>
 
</td>
 
</tr>
 
</tr>
<tr>
 
<td colspan=6 align=center valign=center>
 
<div class="captionPicture">
 
<img src="https://static.igem.org/mediawiki/2017/0/04/T--Munich--Description_Cas13a_Mechanism.svg" alt="Diagram for Cas13a's function">
 
<p>Cas13a binds specific target RNA depending on the crRNA sequence. After activation, Cas13a cleaves RNA indiscriminately.</p>
 
</div>
 
</td>
 
  
</tr>
+
<tr><td colspan=6 align=center valign=center>
 
+
<h1>Current solutions for rapid testing</h1>
<tr><td align=center valign=center colspan=3>
+
 
<p>   
 
<p>   
We wanted to start our project by showing that Cas13a's collateral activity could be used to detect the presence of specific RNA. For this, we used the RNAse alert system, as done in a recent publication<sup><a class="myLink" href="#ref_11">11</a></sup>, to detect RNA digestion. In this assay, the presence of RNAse-like activity is detected by an increase in green fluorescence. Our experiments yielded a convincing proof-of-principle which we went on to <a class=myLink" href="/Team:Munich/Model">model to determine the theoretical detection limit of our system</a>. Moreover, CascAID can be used to detect a wide spectrum of pathogens, as our experiments with gram-positive and viral targets suggested.  
+
Most point-of-care tests that are currently established on the market, like pregnancy tests, are based on antibodies targeting certain metabolites<sup><a class="myLink" href="#ref_4">4</a></sup>. These tests are therefore restricted to one specific application and require long and expensive design cycles for the development of new tests for other applications or changing pathogen epitopes as in case of endemic virus strains with high mutation rate.
 +
<p>
 +
According to our contact person at Médecins Sans Frontières (<a class="myLink" href="http://msf.org">MSF</a>), already existing low-cost POC-test for infectious diseases are mostly based on antibody-based lateral flow tests such as  <a class="myLink" href="http://www.biomerieux-diagnostics.com/bionexia-rota-adeno-rapid-test">BIONEXIA® Rota Adeno</a> or <a class="myLink" href="http://nhdiag.com/cholera_bt.shtml">SMART™II Cholera O1</a>. Recently, qPCR-based systems were developed that provide a more universal solution for highly automated nucleic-acid detection. According to our contact person at MSF, also cartridge-based systems  are currently employed as POC diagnostics for in-field applications. <a class="myLink" href="http://www.cepheid.com/us/cepheid-solutions/systems/genexpert-systems/genexpert-iv">GeneXpert</a> allows the  detection of MRSA in patient samples, and <a class="myLink" href="https://www.alere.com/en/home/search.html?searchtext=alereq&c=WW&%3Acq_csrf_token=undefined">Alere™ q</a> provides an automated bench top platform for nucleic acid testing in any healthcare setting.  
 
</p>
 
</p>
</td>
+
<p>
<td align=center valing=center colspan=3>
+
These qPCR- and ELISA-based methods represent a significant advance in the portability and usability of point-of-care testing. However, these tests include plenty of plastic waste and cost around 10$ for consumables and several thousands of dollars for the main device. This is too high for applications in developing countries for which doctors without borders calls a price of less than 1$ per test.
<img width=440  src="https://static.igem.org/mediawiki/2017/7/7f/T--Munich--Description_Cas13a_Readout_Comparision.svg">
+
<p style="color: #989898; font-size: small">
+
Cas13a can be used to detect specific RNA sequences.
+
 
</p>
 
</p>
 
</td>
 
</td>
</tr>  
+
</tr>
  
<tr class="lastRow">
+
<tr><td colspan=6 align=center valign=center>
<td align=center valign=center colspan=2>
+
<h1>CascAID as an ultimate solution</h1>
<a href="http://www.uni-muenchen.de/studium/lehre_at_lmu/index.html"><img src="https://static.igem.org/mediawiki/2017/9/9a/T--Munich--Logo_LehreLMU.gif" width="200"></a>
+
<p>Picture of the Thermocycler</p>
+
</td>
+
<td align=center valign=center colspan=4>
+
 
<p>   
 
<p>   
For RNA extraction from the samples we tested three methods: extraction with silica beads, extraction with silica membrane and heat lysis. We custom-built an affordable thermocycler for signal amplification by RT-PCR to improve the detection limit. We explored recombinase polymerase amplification (RPA), an isothermal amplification procedure, to use over more conventional PCR methods as its simplicity makes it the more attractive option.
+
We developed CascAID to combine the portability, affordability, and usability of point-of-care tests with the universality and sensitivity of PCR-based nucleic acid detection. We achieve this by using the tools of synthetic biology to minimize hardware requirements and by supplying CascAID in a low-cost paper-based format.
 +
Due to the rapid, software-aided design of crRNA, CascAID can be easily adapted to a variety of targets - from bacterial infections and rapidly evolving viral epidemics to cancer-associated mutations.  
 +
Additionally, the Cas13a enzyme was shown to find targets with a single-nucleotide specificity, superior to PCR-based methods<sup><a class="myLink" href="#ref_1">1</a></sup>. CascAID can be entirely conducted on-site by the doctor or patient and therefore reduces the logistic complexity, drastically hastening the diagnostic process.
 
</p>
 
</p>
 
</td>
 
</td>
</tr>  
+
</tr>
  
 
<tr><td colspan=6 align=center valign=center>
 
<tr><td colspan=6 align=center valign=center>
<h3>Colorimetric read-outs</h3>
+
<h1>Impact on lives</h1>
 
<p>   
 
<p>   
To couple CascAID with an easy read-out method we explored three colorimetric read-outs:
+
With CascAID we venture to solve global health challenges and therefore impact the lives of people living in the developed world, as well as developing countries.
 
</p>
 
</p>
 
</td>
 
</td>
 
</tr>
 
</tr>
  
<tr><td colspan=3 align=center valign=center>
+
<tr><td class="verticalColumn" colspan=3 align=center valign=center>
 +
<h3>Global epidemics</h3>
 
<p>   
 
<p>   
<b>AeBlue</b>: The RNA strand in a specially designed RNA/DNA dimer is cut by Cas13a's collateral
+
CascAID can be made available everywhere. Due to its small size and little infrastructure requirements, CascAID can be used in mobile health-stations visiting remote areas, lacking a centralized healthcare system. This minimizes the travel expenses of patients living far from hospitals and therefore aids to receive a timely cure. As CascAID can be rapidly customized to fit local variants of pathogens, it is applicable in any region on the globe. By employing it as a safety test at airports, it would assist in containing the spread of potential global epidemics like Ebola, Zika, or the plague.  
activity. After digestion, the interaction between the two strands is too weak to hold the dimer and it
+
decays. We can then use the DNA-strand as template to translate the chromoprotein <a href="http://parts.igem.org/Part:BBa_K864401">aeBlue</a>.
+
 
</p>
 
</p>
 
</td>
 
</td>
<td colspan=3 align=center valign=center>
+
<td class="verticalColumn" colspan=3 align=center valign=center>
<img src="https://static.igem.org/mediawiki/2017/9/90/T--Munich--Description_aeBlue.svg" width=360>
+
<h3>At home testing</h3>
 +
<p>
 +
CascAID is accessible to everyone. Designed with an open-source philosophy, the hardware, like our fluorescence detector Lightbringer, can be assembled from common low-cost components by anyone. We did a poll, to address the question of whether people would use CascAID to diagnose themselves for infectious diseases at home, without going to a doctor. Although we received a good resonance from the public, we think that a universal tool for nucleic acid testing is very powerful and therefore raises ethical issues. If applied for detection of, e.g., severe genetic diseases or HIV, the presence of a doctor is also essential for guidance and psychological aid, which is why usage of such tests must be debated and regulated appropriately. More information on risks of self-tests can be found on our <a class="myLink" href="https://2017.igem.org/Team:Munich/Safety">Safety page</a>.
 +
</p>
 
</td>
 
</td>
</tr>  
+
</tr>
  
<tr>
+
<tr><td class="verticalColumn" colspan=3 align=center valign=center>
<td colspan=3 align=center valign=center>
+
<h3>Antibiotics resistance</h3>
<img src="https://static.igem.org/mediawiki/2017/6/64/T--Munich--Description_Intein_Extein.svg" width=360>
+
</td>
+
<td colspan=3 align=center valign=center>
+
 
<p>   
 
<p>   
<b>Intein-Extein</b>: By binding TEV-protease with a RNA-linker we can use Cas13a's collateral activity
+
CascAID is fast and high-sensitive. This is crucial to enable a timely and appropriate therapy. For instance, the ability to distinguish between viral and bacterial infections would be of great importance for lowering antibiotics over-prescription. This way a misuse of antibiotics as a leading reason for multi-resistant bacteria strains would be significantly reduced.
to regulate the protease's diffusion and use it to cleave a TEV tag separating the intein regions of a
+
modified chromophore. After the first cleavage, the intein segment excises itself<sup><a class="myLink" href="#ref_13">13</a></sup>, bringing together the
+
halves of the chromophore. Only then is the chromophore functional and produces the colorimetric
+
read-out.
+
 
</p>
 
</p>
 
</td>
 
</td>
</tr>
+
<td class="verticalColumn" colspan=3 align=center valign=center>
 
+
<h3>Spin-offs</h3>
<tr class="lastRow"><td colspan=3 align=center valign=center>
+
<p>
+
<b>Gold nanoparticles</b>: Other than in the other two colorimetric readouts, aeBlue and Intein-Extein, the only protein involved in the gold nanoparticle (AuNP)-readout is Cas13a, like in our RNase Alert readout. This reduces the necessary fine tuning of the biochemical circuit to a minimum, favoring high robustness of the readout. Due to the phenomenon of Localized Surface Plasmon Resonance, AuNPs appear in a distinct color, ranging from intense red to blue, black and colorless. This property depends on particle size, shape, the immediate environment, and -most critical for our purpose- aggregation state<sup><a class="myLink" href="#ref_14">14</a></sup>.
+
</p>
+
<p> In our project we use AuNPs with a diameter of roughly 10 nm, giving them a bright red color in solution. Their small size and therefore high surface-to-volume ratio makes them ideal for functionalization with thiolated compounds, forming covalent Au-S bonds. The first step of our concept is to use these properties to functionalize AuNPs with either 5’- or 3’- thiolated DNA and, through addition of linker- RNA which hybridizes with both thiolated DNA strands, form aggregates, changing the color from red to blue. The design of the linker-RNA includes an uracil-rich, single-stranded segment between the DNA-complementary termini, making it prone to Cas13a-mediated promiscuous cleavage.
+
</p>
+
 
<p>
 
<p>
It has been shown that, for purely DNA-based hybridization, AuNP aggregates can be spotted on filter paper, dried and severed by addition of a nuclease-containing solution, visible through diffusion of red AuNPs on the paper. Thus, the second part of our concept is to spot RNA-linked AuNPs on paper, dry them alongside the Cas13a mixture and detect specific target RNAs and resulting Cas13a activity with a simple change from blue to red.
+
Finally, as CascAID is a modular platform, single parts can be used as a spin-off. For instance, our detector could serve as a tool to measure kinetics of biological or chemical reactions, that is available to other iGEM teams or research laboratories.</p>
</p>
+
 
</td>
 
</td>
<td colspan=3 align=center valign=center>
+
</tr>
<img src="https://static.igem.org/mediawiki/2017/b/b3/T--Munich--Description_Goldnanoparticles.svg" width=360>
+
  
</td>
 
</tr>
 
 
<tr><td colspan=6 align=center valign=center>
 
<h3>Software</h3>
 
<p> 
 
To help facilitate the design of crRNA, the sequences that give CascAID its specificity, we developed a
 
software tool that checks crRNA for unwanted secondary structures. This gives valuable insight on
 
whether the sequence is suited to use with Cas13a or whether some modifications are needed.
 
Together with Team Delft's software tool which designs the corresponding crRNA based on the target,
 
we collaborated to develop a powerful tool that suggests crRNA sequences and checks their usability
 
</p>
 
</td>
 
</tr>
 
  
 
<tr><td colspan=6 align=center valign=center>
 
<tr><td colspan=6 align=center valign=center>
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<p>  
 
<p>  
 
     <ol style="text-align: left">
 
     <ol style="text-align: left">
       <li id="ref_1">Cohen, Limor, and David R. Walt. "Single-Molecule Arrays for Protein and Nucleic Acid Analysis." Annual Review of Analytical Chemistry 0 (2017).</li>
+
       <li id="ref_1">Gootenberg, J. S., et al. (2017). "Nucleic acid detection with CRISPR-Cas13a/C2c2." <i>Science</i> 356(6336): 438-442.
      <li id="ref_2">Nakano, Michihiko, et al. "Single-molecule PCR using water-in-oil emulsion." Journal of biotechnology 102.2 (2003): 117-124.</li>
+
</li>
      <li id="ref_3">Taniguchi, Yuichi, et al. "Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells." science 329.5991 (2010): 533-538.</li>
+
       <li id="ref_2">St John, A. and C. P. Price (2014). "Existing and Emerging Technologies for Point-of-Care Testing." <i>Clin Biochem Rev</i> 35(3): 155-167.
      <li id="ref_4">Rissin, David M., et al. "Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations." Nature biotechnology 28.6 (2010): 595-599.</li>
+
</li>
      <li id="ref_5">Pardee, Keith, et al. "Rapid, low-cost detection of Zika virus using programmable biomolecular components." Cell 165.5 (2016): 1255-1266.</li>
+
       <li id="ref_3">Llor, C., & Bjerrum, L. (2014). "Antimicrobial resistance: risk associated with antibiotic overuse and initiatives to reduce the problem." <i>Therapeutic Advances in Drug Safety</i>, 5(6), 229–241.</li>
       <li id="ref_6">Slomovic, Shimyn, Keith Pardee, and James J. Collins. "Synthetic biology devices for in vitro and in vivo diagnostics." Proceedings of the National Academy of Sciences 112.47 (2015): 14429-14435.</li>
+
       <li id="ref_4">Peeling, R. W. and R. McNerney (2014). "Emerging technologies in point-of-care molecular diagnostics for resource-limited settings." <i>Expert Rev Mol Diagn</i> 14(5): 525-534.</li>
      <li id="ref_7">Tang, Ruihua, et al. "A fully disposable and integrated paper-based device for nucleic acid extraction, amplification and detection." Lab on a Chip 17.7 (2017): 1270-1279.</li>
+
 
      <li id="ref_8">Vashist, Sandeep Kumar, et al. "Emerging technologies for next-generation point-of-care testing." Trends in biotechnology 33.11 (2015): 692-705.</li>
+
 
      <li id="ref_9">Gubala, Vladimir, et al. "Point of care diagnostics: status and future." Analytical chemistry 84.2 (2011): 487-515.</li>
+
       <li id="ref_10">Abudayyeh, Omar O., et al. "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector." Science 353.6299 (2016): aaf5573.</li>
+
      <li id="ref_11">Gootenberg, Jonathan S., et al. "Nucleic acid detection with CRISPR-Cas13a/C2c2." Science (2017): eaam9321.</li>
+
      <li id="ref_12">https://www.idtdna.com/pages/docs/technical-reports/in_vitro_nuclease_detectionD325FDB69855.pdf (retrieved: 13.10.17)</li>
+
       <li id="ref_13"> Anraku, Yasuhiro, Ryuta Mizutani, and Yoshinori Satow. "Protein splicing: its discovery and structural insight into novel chemical mechanisms." IUBMB life 57.8 (2005): 563-574.</li>
+
      <li id="ref_14">Link, Stephan, and Mostafa A. El-Sayed. "Size and temperature dependence of the plasmon absorption of colloidal gold nanoparticles." The Journal of Physical Chemistry B 103.21 (1999): 4212-4217.</li>
+
      <li id="ref_15">Zhao, W., Ali, M.M., Aguirre, S.D., Brook, M.A., and Li, Y. (2008). "Paper-based bioassays using gold nanoparticle colorimetric probes." Analytical Chemistry 80, 8431–8437.</li>
+
 
     </ol>  
 
     </ol>  
 
</p>
 
</p>
 
</td>
 
</td>
 
</tr>
 
</tr>
 
  
  

Latest revision as of 00:43, 2 November 2017


Applied Design

Diagnosis of infectious diseases and public health

Traditionally, infectious diseases are diagnosed by cell culture or PCR-based methods. As these techniques require expensive infrastructure, trained personal, and time, the current practice suffers from three main problems. First, diagnosis is not available everywhere, and therefore pathogens are usually detected in central clinics2, rather than at the point-of-care (POC). Second, diagnosis is not accessible to everyone. Especially in developing countries proper medical supply is often lacking, due to the high costs. And third, diagnosis is not available within a few hours, which can lead to negligence of laboratory tests, resulting in premature prescription of antibiotics, the primary reason for the recrudescence of resistant bacteria strains3.

Current solutions for rapid testing

Most point-of-care tests that are currently established on the market, like pregnancy tests, are based on antibodies targeting certain metabolites4. These tests are therefore restricted to one specific application and require long and expensive design cycles for the development of new tests for other applications or changing pathogen epitopes as in case of endemic virus strains with high mutation rate.

According to our contact person at Médecins Sans Frontières (MSF), already existing low-cost POC-test for infectious diseases are mostly based on antibody-based lateral flow tests such as BIONEXIA® Rota Adeno or SMART™II Cholera O1. Recently, qPCR-based systems were developed that provide a more universal solution for highly automated nucleic-acid detection. According to our contact person at MSF, also cartridge-based systems are currently employed as POC diagnostics for in-field applications. GeneXpert allows the detection of MRSA in patient samples, and Alere™ q provides an automated bench top platform for nucleic acid testing in any healthcare setting.

These qPCR- and ELISA-based methods represent a significant advance in the portability and usability of point-of-care testing. However, these tests include plenty of plastic waste and cost around 10$ for consumables and several thousands of dollars for the main device. This is too high for applications in developing countries for which doctors without borders calls a price of less than 1$ per test.

CascAID as an ultimate solution

We developed CascAID to combine the portability, affordability, and usability of point-of-care tests with the universality and sensitivity of PCR-based nucleic acid detection. We achieve this by using the tools of synthetic biology to minimize hardware requirements and by supplying CascAID in a low-cost paper-based format. Due to the rapid, software-aided design of crRNA, CascAID can be easily adapted to a variety of targets - from bacterial infections and rapidly evolving viral epidemics to cancer-associated mutations. Additionally, the Cas13a enzyme was shown to find targets with a single-nucleotide specificity, superior to PCR-based methods1. CascAID can be entirely conducted on-site by the doctor or patient and therefore reduces the logistic complexity, drastically hastening the diagnostic process.

Impact on lives

With CascAID we venture to solve global health challenges and therefore impact the lives of people living in the developed world, as well as developing countries.

Global epidemics

CascAID can be made available everywhere. Due to its small size and little infrastructure requirements, CascAID can be used in mobile health-stations visiting remote areas, lacking a centralized healthcare system. This minimizes the travel expenses of patients living far from hospitals and therefore aids to receive a timely cure. As CascAID can be rapidly customized to fit local variants of pathogens, it is applicable in any region on the globe. By employing it as a safety test at airports, it would assist in containing the spread of potential global epidemics like Ebola, Zika, or the plague.

At home testing

CascAID is accessible to everyone. Designed with an open-source philosophy, the hardware, like our fluorescence detector Lightbringer, can be assembled from common low-cost components by anyone. We did a poll, to address the question of whether people would use CascAID to diagnose themselves for infectious diseases at home, without going to a doctor. Although we received a good resonance from the public, we think that a universal tool for nucleic acid testing is very powerful and therefore raises ethical issues. If applied for detection of, e.g., severe genetic diseases or HIV, the presence of a doctor is also essential for guidance and psychological aid, which is why usage of such tests must be debated and regulated appropriately. More information on risks of self-tests can be found on our Safety page.

Antibiotics resistance

CascAID is fast and high-sensitive. This is crucial to enable a timely and appropriate therapy. For instance, the ability to distinguish between viral and bacterial infections would be of great importance for lowering antibiotics over-prescription. This way a misuse of antibiotics as a leading reason for multi-resistant bacteria strains would be significantly reduced.

Spin-offs

Finally, as CascAID is a modular platform, single parts can be used as a spin-off. For instance, our detector could serve as a tool to measure kinetics of biological or chemical reactions, that is available to other iGEM teams or research laboratories.

References

  1. Gootenberg, J. S., et al. (2017). "Nucleic acid detection with CRISPR-Cas13a/C2c2." Science 356(6336): 438-442.
  2. St John, A. and C. P. Price (2014). "Existing and Emerging Technologies for Point-of-Care Testing." Clin Biochem Rev 35(3): 155-167.
  3. Llor, C., & Bjerrum, L. (2014). "Antimicrobial resistance: risk associated with antibiotic overuse and initiatives to reduce the problem." Therapeutic Advances in Drug Safety, 5(6), 229–241.
  4. Peeling, R. W. and R. McNerney (2014). "Emerging technologies in point-of-care molecular diagnostics for resource-limited settings." Expert Rev Mol Diagn 14(5): 525-534.