We aim to create an in vitro DNA detection system that could be used for different molecular diagnostics purposes. A novel tool of this type will have the potential to simplify the detection of viral and microbial pathogens.Moreover, it will facilitate a fast and cheap identification of disease causing genetic variants in our genomes without the need of sequencing. Our DNA detection system relies on a pair of dCas9 proteins, linked to split halves of horseradish peroxidase (sHRP).If the target sequence is present, the enzyme is restored and its activity can be detected with a simple colorimetric reaction. This system has the full potential to be flexible, cheap and time-efficient, with no requirements for specialized and complex equipment. To ensure the correct identification of specific pathogens, we will concentrate on a specific DNA sequence, like CDS for unique viral proteins (viruses), or regions in the 16s rRNA locus (bacterial pathogens).Our system can also be used for the identification of diseases causing single nucleotide variants in the human genome. Having in mind the small differences between the two sequences, we will have to concentrate on variants located in the PAM region or affecting some of the bases in close proximity. This scenario will guarantee that the wild type sequence will not activate our sensor. Another group of potentially interesting targets consists of variants in genes with a well-known role in drug metabolism and transport. At the present time, a large number of medicaments requires molecular genotyping before initial use in order to adjust the right dose and/or to prevent serious (potentially lethal) side effects. To increase the number of potentially detectable mutations, we will include in our system not only the wild type dCAs9, but also evolved variants with different PAM preferences, as well as Cas9 analogs from different species.
Selected diagnostic targets: Cystic fibrosis (mutation F508del that affects approximately 70% of the patients worldwide), Borrelia (genus of bacteria that causes borreliosis, disease transmitted primarily by ticks), Human immunodeficiency virus (HIV, causes acquired immunodeficiency syndrome) and HLA-B*5701 (genetic variant HLA-B that is associated with an extreme sensitivity to the drug abacavir).
Our project has enormous potential to influence many fields in our country. Cystic fibrosis is the most common rare disease in Bulgaria. In large number of the cases, the patient is initially misdiagnosed at clinics when sweat chloride level is used as indicator. Having a cheap and reliable genetic test will allow easy detection of the disease and adequate treatment at an earlier time point. Moreover, our system can be applied in all local hospitals in many small townssince it does not require expensive and sophisticated equipment, for instance - DNA sequencing devices.
A novel, simple tool for nucleic acid detection will also have a grand impact on the detection of different bacterial and viral pathogens. Our country, Bulgaria, is at the border of the European Union. In this region, significant climate changes and large groups of migrating people from Asia and Africa are considered to be typical events. This creates the perfect environment for various pathogens that are not well known by the local health authorities. Having a simple and fast system for their detection and identification available on the field, will prove very helpful to the local health personnel, especially in remote regions. Last but not least, our system will allow an easy, quick and cheap detection of variants in genes playing a role in the metabolism of different drugs. Such knowledge will improve the empirical dose adjustment processes, leading to the reduction of potential side-effects and an overall better therapeutic outcome.