Difference between revisions of "Team:Utrecht/Placeholder"

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iGEM UTRECHT Placeholder Page

Utrecht 2017

OUTCASST

Important note: This is a placeholder page and will be replaced in due time!

OUT-of-cell CRISPR Activated Sequence-specific Signal Transducer

This year, Utrecht University participates for the first time and we aim to build a biological DNA sensor that can recognise sequences of the user's choice. Further down, we propose some applications but first we will give a description of how our sensor will work.

The OUTCASST sensor consist of two proteins, both expressed to the membrane of a mammalian HEK 293 cell. The first protein connects dCas9, a catalytically dead Cas9 variant, to a transmembrane domain and an intracellular transcription factor. The second protein connects dCpf1 to an intracellular protease. When provided with appropriate guide RNA's, the two proteins can bind to a DNA sequence. When one sequence brings the two proteins together, the transcription factor is released into the cytoplasm, and can activate a reporter cascade. See the scheme below for details.

1. Guide RNA binding:

The two proteins float around freely in the membrane. Two types of guide RNA (gRNA) can be added to the cells. Due to the different recognition sequences for the two proteins, the gRNA can bind specifically to the appropriate protein.

2. DNA sample addition:

Now that the guide RNA's have been bound, both proteins are primed to bind to a specific sequence of a DNA strand. A DNA sample from any source can be added and will then be bound by the protein with the guide RNA that is complementary to one of the DNA strands.

3. Sequence recognition:

One of the proteins has bound a DNA stretch in the sample. If the other protein is bound to a guide RNA that is complementary to a sequence on the same stretch, it too will bind.

4. Co-localization & cleavage:

If the second protein also binds the DNA, close enough to the other protein, the protease at the end of one will be able to cleave the transcription factor from the other protein. The transcription factor is then free to induce a reporter mechanism. In our case, this will be a fluorescent marker.

Applications of the OUTCASST system:

So far, our team has not yet decided on a specific application of the system as there are many different fields wherein sequence-specific detection is of use. Right now, most DNA detection is done by Polymerase Chain Reaction (PCR) and sequencing techniques. These techniques rely on materials and machinery that are not available to everyone. The OUTCASST system, being cell-based, culturable and thus renewable, could provide a quick and easy detection tool that does not require an expensive lab-setup. It only requires medium to culture the cells and the gRNA that is specific to the sequence that you want to detect. Right now, we are interviewing potential end-users to figure out what type of applications they see for our concept and to assess what features they wish to see in our design. So far, we have identified four possible applications for our tool:

Prenatal Genotyping:

Small quantities of cell-free embryo DNA is present in the maternal peripheral blood, already early in pregnancy (Lo et al., 1989). The detection of child-mutations by taking a serum sample from its mother reduces the risk to the unborn child. Such mutation calling is currently done by sequencing, which faces technological limitations when confronted with low sample concentrations, and subsequent genotyping using bioinformatics pipelines, a process that takes days, if not weeks. We aim to create the possibility to detect specific mutations in a matter of hours, cheaply.

Pathogen Detection:

Cell-free pathogen DNA can also be sampled from a patient's serum (Gan et al., 1994). Again, the aim of our design is to speed up the process of diagnosis. With one batch of our organism, multiple tests can be done. By adding a small sample of our organism to each well in a 96-well plate and then adding a different subset of gRNA sequences to each well, you could test for 96 separate pathogens. Such tests could speed up the diagnostics time in medical labs. The sensor could even be shipped to general practitioners themselves, making more precise diagnoses in the doctor's office possible.

Cancer Screening

Early and accurate detection of cancer is very important for proper treatment. Luckily, it too produces cell-free DNA that can be found in the serum of a patient (Schwarzenbach et al., 2011). By designing gRNA, complementary to known oncogene mutations, the detection of these sequences in the serum sample thus means that the harmful mutation is present in the patient. Our system can be modified such that signal intensity depends on the amount of dimerization and thus on the cell-free DNA concentration. Since the concentration of cell-free DNA depends on the growth of the tumor, this concentration could be used for monitoring of the disease.

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