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Collaborations
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Collaborations play a very important role in terms of the development of project. Collaborations with other teams helps us to learn about better ways to handle a problem, to learn new ways of working, to perceive different ideologies and to develop the project in general. It provides us a better chance to get to know other teams and to learn to cooperate. In scientific fields, cooperation and collaborations play a major role for growth and discovery. We are highly encouraged to work with other teams since it increases our horizon of knowledge and we are happy that iGEM promotes the idea of sharing knowledge and scientific materials. The following are the teams whom we can proudly call the collaborators this year.
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Their iGEM project is called CASE13A. Both our projects are similar in terms of the use of Cas13a and paper microfluidics. Our collaboration started with our meeting in Delft. We were excited to see that TU Delft were also working with Cas13a as their major protein. We both are trying to work on different ways of tackling the problem of the antibiotic resistance using Cas13a. Therefore we decided to collaborate since it gave us the opportunity to discuss the challenges and also to try out new stuffs together. We started a collaboration for our software since we both were working on optimizing the crRNA for the different targets. In our team, we designed a software that could give us the best design and structure of the crRNA for different targets. For this we created a database of different possible sequences using NUPACK and other platforms. The team Delft had a similar project where they predict the part of the target that can best serve as a crRNA. We provided them with a list of possible targets and best crRNAs structures for their software. Also, the team Delft sent us the Tardigrade proteins(TDPs) to experiment them with the Cas13a and to check the activity and stability of the Cas13a when used together with TDPs. We did some cleavage assay of the Cas13a along with the TDPs, to see if it can create some difference in the reactivity.
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Michael and Julian from the iGEM BOKU Vienna came to do some experiments in our lab on 4th and 5th of October. Their iGEM project is called D.I.V.E.R.T. (Directed in vivo evolution via reverse transcription) and they are trying out new strategies for in vivo evolution which shows potential advantages over classical in vitro methods. For this they use yeast and E.coli to demonstrate their concept. They used the flow cytometer in our lab in Garching to better characterize their constructs from E.coli and S. cerevisiae. For the use one of our lab member explained them how to use the flow cytometer in the lab and also provided them the necessary help. It was a long day work for them but they got convincing results by the end. We also had a small gathering in the evening before they left together with the old igemers who came to meet us. We were very happy to have them in our lab and to get to know each other's team.
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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 publication11, 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 model. Moreover, CascAID can be used to detect a wide spectrum of pathogens, as our experiments with gram-positive and viral targets suggested. As we wanted to make CascAID available for everyone, we focused on building an inexpensive fluorescence detector to measure the presence of the target. Our detector “Lightbringer” was designed to be able to detect the fluorescence produced by the fluorescein in the Rnase alert system12, but we theorize that changing the filters allows detection of other fluorophores. In addition, we experimented with freeze-drying on paper to make CascAID durable and easily portable.
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Cas13a can be used to detect specific RNA sequences
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Picture of the Thermocycler
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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.
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Colorimetric read-outs
To couple CascAID with an easy read-out method we explored three colorimetric read-outs:
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AeBlue: 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 aeBlue.
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Diagram of aeBlue
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Intein-Extein: 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 itself13, bringing together the
halves of the chromophore. Only then is the chromophore functional and produces the colorimetric
read-out.
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Diagram of Intein-Extein
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Gold nanoparticles: Gold nanoparticles coated with short DNA sequences are held closely
together by a complementary linker RNA, which makes the solution intense blue14. Activated Cas13a cuts
the linker RNA, causing the nanoparticles to diffuse away from each other. This increase in distance
causes a color change to intense red.
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Gold nanoparticles
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Software
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
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References
- Cohen, Limor, and David R. Walt. "Single-Molecule Arrays for Protein and Nucleic Acid Analysis." Annual Review of Analytical Chemistry 0 (2017).
- Nakano, Michihiko, et al. "Single-molecule PCR using water-in-oil emulsion." Journal of biotechnology 102.2 (2003): 117-124.
- Taniguchi, Yuichi, et al. "Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells." science 329.5991 (2010): 533-538.
- Rissin, David M., et al. "Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations." Nature biotechnology 28.6 (2010): 595-599.
- Pardee, Keith, et al. "Rapid, low-cost detection of Zika virus using programmable biomolecular components." Cell 165.5 (2016): 1255-1266.
- 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.
- 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.
- Vashist, Sandeep Kumar, et al. "Emerging technologies for next-generation point-of-care testing." Trends in biotechnology 33.11 (2015): 692-705.
- Gubala, Vladimir, et al. "Point of care diagnostics: status and future." Analytical chemistry 84.2 (2011): 487-515.
- Abudayyeh, Omar O., et al. "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector." Science 353.6299 (2016): aaf5573.
- Gootenberg, Jonathan S., et al. "Nucleic acid detection with CRISPR-Cas13a/C2c2." Science (2017): eaam9321.
- https://www.idtdna.com/pages/docs/technical-reports/in_vitro_nuclease_detectionD325FDB69855.pdf (retrieved: 13.10.17)
- 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.
- 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.
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