Difference between revisions of "Team:Munich/Description"

Thanks to advances in molecular biology and biochemistry, scientists have been able to consistently detect lower and lower concentration of molecules¹, to the point that single molecules can be reliably recognized with methods such as polymerase chain reaction (PCR)², fluorescence in situ hybridization (FISH)³ and enzyme-linked immunosorbent assays (ELISA)⁴. 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^5,6. 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^7-9. 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..

The ongoing crisis of increasing antibiotic resistance demands innovative preventive strategies. Recently, the RNA-targeting protein CRISPR-Cas13a has been used for highly sensitive DNA and RNA detection, promising diverse applications in point-of-care diagnostics. We integrated Cas13a in the detection unit of CascAID, our GMO-free diagnostic platform. CascAID combines an automated microfluidic device for rapid lysis and extraction of nucleic acids with a paper-based readout system. We demonstrated the performance of our device by targeting the 16S RNA from E. coli. We improved the detection limit of our platform, using simulations to optimize our amplification scheme and the final readout.

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

Picture of the Thermocycler

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.