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<font size=7 color=#51a7f9><b style="color: #51a7f9;">Description</b></font>
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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">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">2</a></sup>, fluorescence in situ hybridization (FISH)<sup><a class="myLink">3</a></sup> and enzyme-linked immunosorbent assays (ELISA)<sup><a class="myLink">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">5</a>,<a class="myLink">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">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>
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Conceived as a distributable platform for rapid point-of-care diagnostics, CascAID can be used to distinguish between bacterial and viral infections, thus minimizing the widespread use of antibiotics.
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Furthermore, Cas13a allows the fast design of target sequences, making our system adaptive to the emergence of new viral outbreaks or fast mutating pathogens.
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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">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">2</a></sup>, fluorescence in situ hybridization (FISH)<sup><a class="myLink">3</a></sup> and enzyme-linked immunosorbent assays (ELISA)<sup><a class="myLink">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">5</a>,<a class="myLink">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">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..               
 
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Our project, which we named Cas13a controlled assay for infectious diseases (CascAID), features the recently identified CRISPR/Cas effector Cas13a<sup><a class="myLink">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>
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<a href="https://www.uni-muenchen.de/index.html"><img src="https://static.igem.org/mediawiki/2017/0/0d/T--Munich--Logo_LMU.png" alt="Diagram for Cas13a's function" width="270"></a>
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<p>Cas13a binds specific target RNA depending on the crRNA sequence. After activation, Cas13a cleaves RNA indiscriminately.</p>
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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 publication<sup><a class="myLink">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 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 system<sup><a class="myLink">12</a></sup>, 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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<p>Cas13a can be used to detect specific RNA sequences</p>
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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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Revision as of 14:39, 13 October 2017

Description

Design

Proof of Concept

Demonstration

Application

Final Results

Entrepeneurship

Achievements

Protocols and
Methods

Materials

LabJournal

Safety

Parts

Interlab

Software

Hardware

Human Practices (Silver)

Human Practices (Gold)

Public Engagement

Collaborations

Team members

Supervisors

PIs

Institutions

Sponsor s

Attributions

Date

Abstract

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 targets5,6. 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 restrictions7-9. 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..

Conceived as a distributable platform for rapid point-of-care diagnostics, CascAID can be used to distinguish between bacterial and viral infections, thus minimizing the widespread use of antibiotics.

Furthermore, Cas13a allows the fast design of target sequences, making our system adaptive to the emergence of new viral outbreaks or fast mutating pathogens.

Authors
Date

Headline

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Authors
Date

Headline

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Authors
Date

Headline

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Authors