Yi, Joon-Yeop Kim, Minyoung Min, Hophil Kim, Byung-Gee Son, Junghyun Kwon, Oh-Seung Sung, Changmin 2024-01-19T15:02:42Z 2024-01-19T15:02:42Z 2022-01-10 2021-04 1942-7603 https://pubs.kist.re.kr/handle/201004/117187 The increased potential for gene doping since the introduction of gene therapy presents the need to develop antidoping assays. We therefore aimed to develop a quick and simple method for the detection of specifically targeted exogenous doping genes utilizing an in vitro clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) system. A human erythropoietin (hEPO) is a drug frequently used for doping in athletes, and gene doping using gene transfer techniques may be attempted. Therefore, we selected hEPO gene as a model of exogenous doping gene, and complemental single guide RNA (sgRNA) was designed to specifically bind to the four exon-exon junctions in the hEPO cDNA. For the rapid reaction of CRISPR-Cas9, further optimization was performed using an open-source program (CRISPOR) that avoids TT and GCC motifs before the protospacer adjacent motif (PAM) domain and predicts the efficiency of the sgRNA. We optimized the in vitro Cas9 assay and dual use of sgRNA for double cleavage and identified the limit of detection (LOD) of the 1.25 nM of the double cleavage method. We expect that the improved CRISPR-Cas9 method can be used for antidoping analysis of gene doping. English WILEY New application of the CRISPR-Cas9 system for site-specific exogenous gene doping analysis Article 10.1002/dta.2980 1 DRUG TESTING AND ANALYSIS, v.13, no.4, pp.871 - 875 DRUG TESTING AND ANALYSIS 13 4 871 875 N scie scopus 000611033200001 2-s2.0-85099942163 Biochemical Research Methods Chemistry, Analytical Pharmacology & Pharmacy Biochemistry & Molecular Biology Chemistry Pharmacology & Pharmacy Article CRISPR-Cas9 erythropoietin gene doping sport