124 ICRAV 2026 1. Laboratory of RacingChemistry, Utsunomiya, TOCHIGI, Japan 2. Molecular Physiology Laboratory, Pioneering Research Institute, RIKEN, Wako, Saitama, Japan 3. Equine Research Institute, Japan Racing Association, Shimotsuke, Tochigi, Japan 4. School of Agriculture, Utsunomiya University, Utsunomiya, Tochigi, Japan ABSTRACT Current gene-doping detection strategies primarily focus on identifying transgenes introduced via viral or plasmid vectors, targeting exon–exon junctions using hydrolysis probe-based quantitative PCR. These DNA-centered methods exhibit high specificity and sensitivity. However, recent advances in biopharmaceuticals have accelerated the development of RNA-based therapeutics, which bypass transcription and initiate protein translation directly. This leads to enhanced expression efficiency and reduced risk of genomic integration, making mRNA drugs an attractive modality — including potential misuse in gene doping. In this study, we address the challenge of detecting RNA-based gene doping agents, which traditionally require labor-intensive RNA extraction and reverse transcription steps. We report the development of directSATORI, a novel amplification-free detection platform employing CRISPR-Cas13a in conjunction with a femtoliter-scale microchamber array. This system enables direct, single-molecule-level detection of target RNAs from biological matrices without prior nucleic acid extraction or cDNA synthesis. As a proof of concept, a synthetic equine erythropoietin mRNA construct was used to simulate RNA-based doping. Method validation was performed using plasma samples spiked with the equine erythropoietin N1-methylpseudouridine modified mRNA encapsulated within lipid nanoparticles. Furthermore, we successfully detected the administered modified mRNA/LNP in postinjection plasma samples from a treated horse, demonstrating the feasibility of genedoping detection. This platform, direct-SATORI, offers a promising tool for future antidoping efforts, particularly as RNA-based modalities become increasingly prevalent in both therapeutic and illicit contexts. In addition, we estimated the spontaneous mutation rate in Thoroughbred horses. Whole-genome sequencing of a Thoroughbred parent-offspring trio revealed 46 single nucleotide variants and 2 deletions violating Mendelian inheritance. This corresponds to a de novo mutation rate of 1.92 × 10-8 per generation. This estimate may provide a critical reference for distinguishing gene edits from spontaneous mutations in equine gene-editing test. Together, these findings contribute to the establishment of robust frameworks for gene-doping control in horse racing. Teruaki Tozaki1, Risako Furukawa1, Hajime Shinoda2, Asami Makino2, Mami Yoshimura2, Tatsuya Ida2, Koki Kawate1, Mio Kikuchi1, Taichiro Ishige1, Yuji Takahashi3, Tomohiro Kato3, Hironaga Kakoi1, Emiko Fukui4, Rikiya Watanabe2 Overview and Future Perspectives on Gene Doping and Genome Editing Detection in Japan
RkJQdWJsaXNoZXIy MTI3ODI1