|
|
| Line 59: |
Line 59: |
| | </tr> | | </tr> |
| | <tr><td colspan=6 align=left valign=center> | | <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">Applied Design</b></font> |
| | </td> | | </td> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| | <td colspan = 6 align="left"> | | <td colspan = 6 align="left"> |
| − | <p class="introduction"> | + | <p> |
| − | 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.
| + | CascAID combines portability, affordability, and usability of point-of-care tests with the universality and sensitivity of PCR-based nucleic acid detection. Also, the Cas13a enzyme provides a superior, single-nucleotide specificity, so it is no wonder that the range of application possibilities is wide. It can help differentiate between viral and bacterial infections, which is of great importance for lowering antibiotics over-prescription. This way a misuse of antibiotics as a leading reason for resistant bacteria strains would be significantly reduced. Currently, pathogens are discriminated by cell culture or PCR-based methods, requiring expensive equipment, trained personal, and time. Because of that, it is not rarely a case that doctors, facing the pressure of sick patient, prescribe antibiotics prematurely. Furthermore since our device is a point-of-care device optimised to be used by anyone regardless on education background, people would be able to spare themselves a visit to hospital, where they are likely to catch some other infection because of already weakened immune system. </p> |
| − | </p>
| + | <p> |
| − | | + | Other point-of-care tests on the market, like pregnancy tests, target certain metabolites and are therefore restricted to one specific application. CascAID on the other hand, can be easily adapted to variety of targets - from bacterial infections and rapidly evolving viral epidemics to a cancer-associated mutations. |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <tr><td colspan=6 align=center valign=center>
| + | |
| − | <h3>CascAID</h3>
| + | |
| − | <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>
| + | |
| − | </td> | + | |
| − | </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 align=center valign=center colspan=3>
| + | |
| − | <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.
| + | |
| | </p> | | </p> |
| − | </td> | + | <p> |
| − | <td align=center valing=center colspan=3>
| + | Since it is possible to adapt the device to detect practically any pathogen, it can be customized to fit the planet region where it is used. There are many populated regions that are far away from health stations, so mobile health-stations visiting these areas could use a device like ours. For instance in Africa where some diseases are very common for specific areas, our device could serve as a quick test that would tell a person if they should go and see a doctor as soon as possible. Unfortunately lot of people, because of the distance to hospitals, tend to wait for too long for symptoms to go away. This way a curable disease can be lethal. |
| − | <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>
| + | <p> |
| − | </tr>
| + | However with one highly modularised construct as ours, possibilities don’t end here. Each module can find its use separately or be further developed and optimised. For instance, led with an open-source philosophy, we provided detailed documentations and CAD-drawings of our fluorescence detector for anyone wanting to improve its design. The detector can be used to measure kinetics of any biological or chemical reactions on paper and the assemble costs are such that it can compete with commercially available fluorescence readers. Also because of its scale it is highly practical for in-field application in contrast to current detectors on market. |
| − | | + | |
| − | <tr class="lastRow">
| + | |
| − | <td align=center valign=center colspan=2>
| + | |
| − | <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>
| + | |
| − | 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.
| + | |
| − | </p>
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr><td colspan=6 align=center valign=center>
| + | |
| − | <h3>Colorimetric read-outs</h3>
| + | |
| − | <p>
| + | |
| − | To couple CascAID with an easy read-out method we explored three colorimetric read-outs:
| + | |
| − | </p>
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr><td colspan=3 align=center valign=center>
| + | |
| − | <p>
| + | |
| − | <b>AeBlue</b>: The RNA strand in a specially designed RNA/DNA dimer is cut by Cas13a's collateral
| + | |
| − | 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>
| + | |
| − | </td>
| + | |
| − | <td colspan=3 align=center valign=center>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/9/90/T--Munich--Description_aeBlue.svg" width=360>
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td colspan=3 align=center valign=center>
| + | |
| − | <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>
| + | |
| − | <b>Intein-Extein</b>: By binding TEV-protease with a RNA-linker we can use Cas13a's collateral activity
| + | |
| − | 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>
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <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> | | <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.
| + | Due to its speed, our device can already find its use in different laboratories, where it can enable quick sample testing essential for fast prototyping and contribute this way to many new amazing findings to come. |
| | </p> | | </p> |
| − | </td>
| + | </td> |
| − | <td colspan=3 align=center valign=center>
| + | |
| − | <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>
| + | |
| − | <h3>References</h3>
| + | |
| − | <p>
| + | |
| − | <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_2">Nakano, Michihiko, et al. "Single-molecule PCR using water-in-oil emulsion." Journal of biotechnology 102.2 (2003): 117-124.</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_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 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_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_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>
| + | |
| − | </p>
| + | |
| − | </td> | + | |
| | </tr> | | </tr> |
| − |
| |
| − |
| |
| | | | |
| | | | |
