Integration of CRISPR/Cas12a and Multiplexed RPA for Fast Detection of Gene Doping

Fast and on-site detection is important for an effective antigene-doping strategy. However, the current gene doping (GD) evaluation methods require sophisticated instruments and laborious procedures, limiting their field applications. This study proposes a CRISPR/Cas12a-based detection platform (termed CasGDP) combining CRISPR/Cas12a and multiplexed Recombinase Polymerase Amplification (RPA) for rapid evaluation of GD. CasGDP showed high specificity for identifying the putative target genes such as EPO, IGF-1, and GH-1. By using fluorescence as the readout, the method achieved a limit-of-detection of 0.1 nM and 1 aM for unamplified and amplified target plasmids, respectively. Additionally, an in vitro GD cell model was successfully established with the human EPO gene (hEPO). The results indicated that the hEPO gene transfection promoted the hEPO protein expression. Furthermore, trace amounts of EPO transgene spiked in human serum were efficiently measured by CasGDP with fluorescence- and lateral flow device (LFD)-based readouts in 40 min. Finally, we designed a multiplexed microfluidic device and realized simultaneous detection of the three transgenes via LFD embedded in the device. To our knowledge, this is the first work that combines the CRISPR-based system and multiplexed RPA for GD detection. We anticipate CasGDP to be widely used as a rapid, sensitive, and robust tool for GD evaluation.