Revealing gene doping with CRISPR/Cas through the detection of sgRNA

Code: 21E06MT

The rapid evolution of new medical/pharmaceutical methods extremely promotes the probability that those scientific progresses are illicitly used in order to achieve performing enhancing effects in sporting competitions. Especially the development of the CRISPR/Cas (Clustered regularly interspaced short palindromic repeat/CRISPR associated) technique for simple and precise gene editing of specific DNA sequences has drastically increased the potential of misuse in recent years. The application of the CRISPR/Cas method is classified as gene doping in the Prohibited List and is banned at all times (inand out- of competition) by the World Anti-Doping Agency. 

Therefore, the aim of this research project is the development of complementary detection methods to uncover the illicit attempt of gene doping with CRISPR/Cas by targeting the so called single guide RNA (sgRNA) as marker analyte in plasma/serum samples. Two different approaches are planned to be utilized for the detection of sgRNA, namely a direct approach employing either a gel-based detection or an identification procedure of specific xenobiotic RNA sequence segments (which will be assessed by means of SHERLOCK / specific high-sensitive enzymatic reporter unlocking), along with an indirect approach, which will be accomplished via the analysis of distinct chemical RNA modifications by means of HPLC-MS/MS. Furthermore, an administration study employing a mouse model will be conducted in order to demonstrate the proof-of-concept.