Akiyama Kentaro, Momobayashi Atsushi, Okano Masato
Anti-Doping Laboratory, LSI Medience Corporation, 1-15-10 Shimura, Itabashi-Ku, Tokyo, 174-0056, Japan.
Anal Bioanal Chem. 2025 Jun 16. doi: 10.1007/s00216-025-05959-0.
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.
基因兴奋剂，如使用CRISPR - Cas9等技术，对体育的公正性构成了相当大的威胁。2018年，世界反兴奋剂机构实施了对基因组编辑的禁令，这凸显了对灵敏且特异的检测方法的需求。目前可用的检测技术在特定情况下已显示出有效性，但受限于低灵敏度和短检测窗口。为克服这些限制，本研究提出了一种针对CRISPR - Cas9核糖核蛋白（RNP）复合物的新检测方法，即RNA免疫沉淀后进行定量PCR（RIP - qPCR）。本研究的主要重点是在体外开发一种针对兴奋剂关键基因的检测方法，这些基因包括肌肉生长抑制素（MSTN）、α -辅肌动蛋白3（ACTN3）、促红细胞生成素受体（EPOR）和促红细胞生成素（EPO），并使用MSTN在体内进行了概念验证。RIP - qPCR方法表现出灵敏的性能，血浆中的定量限为0.1 ng/mL。该方法在体外成功检测到靶向MSTN、ACTN3、EPOR和EPO的单向导RNA以及RNP复合物中的两种Cas9蛋白类型。此外，当血浆样本在4℃保存时，RIP - qPCR的检测能力可维持长达30天。在体内实验中，通过肌肉注射和静脉注射给予RNP以靶向小鼠Mstn基因。肌肉注射后CRISPR - Cas9 RNP在长达24小时内仍可检测到，静脉注射后12小时内仍可检测到。本研究强调了RIP - qPCR作为反兴奋剂分析有力工具的潜力，未来将努力扩大目标基因库以加强对体育兴奋剂中基因编辑的检测。
