Difference between revisions of "Team:Munich/Hardware"

For the detection of fluorescence light we used an light depending resistor (LDR). An LDR is a resistor which decreases its resistance RLDR with increasing light intensity I. The dependence of the resistance RLDR on the light intensity I is RLDR / I− , (3.1) where is a parameter depending on the type of resistor being used and can even differ for LDRs with the same type designation. Equation 3.1 is motivated from the equation = lg � R10 R100 � lg 􀀀 100 Lx 10 Lx � , (3.2) which is given in the data sheet of the LDR. The dominator is the decadic logarithm of the fraction of two light intensities of 100 Lx and 10 Lx. R10 and R100 are the corresponding resistances at these light intensities. The used resistor with the type designation GL5516 NT00183 has a parameter of 0.8. The response of a LDR depends on the wavelength � of the incoming light. The data sheet provides information on the relative response normalized to the maximal response. The relative response is maximum for a wavelength of 540 nm and is therefore appropriate for detection of green fluorophores.

CascAID

Our project, which we named Cas13a controlled assay for infectious diseases (CascAID), features the recently identified CRISPR/Cas effector Cas13a¹⁰. 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.

Cas13a binds specific target RNA depending on the crRNA sequence. After activation, Cas13a cleaves RNA indiscriminately.

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.

Cas13a can be used to detect specific RNA sequences

Colorimetric read-outs

To couple CascAID with an easy read-out method we explored three colorimetric read-outs:

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.

Diagram of aeBlue

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.

Diagram of Intein-Extein

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

References

1. Cohen, Limor, and David R. Walt. "Single-Molecule Arrays for Protein and Nucleic Acid Analysis." Annual Review of Analytical Chemistry 0 (2017).
2. Nakano, Michihiko, et al. "Single-molecule PCR using water-in-oil emulsion." Journal of biotechnology 102.2 (2003): 117-124.
3. Taniguchi, Yuichi, et al. "Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells." science 329.5991 (2010): 533-538.
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.
5. Pardee, Keith, et al. "Rapid, low-cost detection of Zika virus using programmable biomolecular components." Cell 165.5 (2016): 1255-1266.
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.
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.
8. Vashist, Sandeep Kumar, et al. "Emerging technologies for next-generation point-of-care testing." Trends in biotechnology 33.11 (2015): 692-705.
9. Gubala, Vladimir, et al. "Point of care diagnostics: status and future." Analytical chemistry 84.2 (2011): 487-515.
10. Abudayyeh, Omar O., et al. "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector." Science 353.6299 (2016): aaf5573.
11. Gootenberg, Jonathan S., et al. "Nucleic acid detection with CRISPR-Cas13a/C2c2." Science (2017): eaam9321.
12. https://www.idtdna.com/pages/docs/technical-reports/in_vitro_nuclease_detectionD325FDB69855.pdf (retrieved: 13.10.17)
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.
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.