Direct detection of CRISPR-Cas9 ribonucleoprotein gene doping using RNA immunoprecipitation and quantitative PCR

Gene doping, using technologies such as CRISPR-Cas9, poses a considerable threat to the integrity of sports. In 2018, the World Anti-Doping Agency implemented a ban on genome editing, which highlighted the need for sensitive and specific detection methods. Detection techniques that are currently available have shown effectiveness in specific contexts, but are limited by low sensitivity and short detection windows. To overcome these limitations, this study presents a new detection method for CRISPR-Cas9 ribonucleoprotein (RNP) complexes, termed RNA immunoprecipitation followed by quantitative PCR (RIP-qPCR). The primary focus of this research was the in vitro development of a detection method targeting genes critical for doping, including myostatin (MSTN), α-actinin 3 (ACTN3), erythropoietin receptor (EPOR), and erythropoietin (EPO), with in vivo proof-of-concept demonstrated using MSTN. The RIP-qPCR method demonstrated sensitive performance, with a limit of quantification of 0.1 ng/mL in plasma. This method successfully detected single guide RNA targeting MSTN, ACTN3, EPOR, and EPO, along with two types of Cas9 proteins in RNP complexes in vitro. Additionally, the detection capabilities of RIP-qPCR were maintained for up to 30 days when plasma samples were stored at 4 °C. In vivo experiments were performed where RNPs were administered via intramuscular and intravenous injections to target the murine Mstn gene. CRISPR-Cas9 RNPs remained detectable for up to 24 h following intramuscular injection and 12 h after intravenous injection. This study underscores the potential of RIP-qPCR as a powerful tool for anti-doping analysis, with future efforts on expanding the target gene panel to enhance the detection of gene editing in sports doping.