Yi, Joon-Yeop Kim, Minyoung Ahn, Jung Ho Kim, Byung-Gee Son, Junghyun Sung, Changmin 2024-01-19T09:32:03Z 2024-01-19T09:32:03Z 2023-04-20 2023-06 0039-9140 https://pubs.kist.re.kr/handle/201004/113709 A genetic approach targeted toward improving athletic performance is called gene doping and is prohibited by the World Anti-Doping Agency. Currently, the clustered regularly interspaced short palindromic repeats -associated protein (Cas)-related assays have been utilized to detect genetic deficiencies or mutations. Among the Cas proteins, deadCas9 (dCas9), a nuclease-deficient mutant of Cas9, acts as a DNA binding protein with a target-specific single guide RNA. On the basis of the principles, we developed a dCas9-based high-throughput gene doping analysis for exogenous gene detection. The assay comprises two distinctive dCas9s, a magnetic bead immobilized capture dCas9 for exogenous gene isolation and a biotinylated dCas9 with streptavidin-polyHRP that enables rapid signal amplification. For efficient biotin labeling via maleimide-thiol chemistry, two cysteine residues of dCas9 were structurally validated, and the Cys574 residue was identified as an essential labeling site. As a result, we succeeded in detecting the target gene in a concentration as low as 12.3 fM (7.41 x 105 copies) and up to 10 nM (6.07 x 1011 copies) in a whole blood sample within 1 h with HiGDA. Assuming an exogenous gene transfer scenario, we added a direct blood amplification step to establish a rapid analytical procedure while detecting target genes with high sensitivity. Finally, we detected the exogenous human erythropoietin gene at concentrations as low as 2.5 copies within 90 min in 5 mu L of the blood sample. Herein, we propose that HiGDA is a very fast, highly sensitive, and practical detection method for actual doping field in the future. English Elsevier BV CRISPR/deadCas9-based high-throughput gene doping analysis (HiGDA): A proof of concept for exogenous human erythropoietin gene doping detection Article 10.1016/j.talanta.2023.124455 1 Talanta, v.258 Talanta 258 Y scie scopus 000957472800001 2-s2.0-85150266360 Chemistry, Analytical Chemistry Article DNA THERAPY ASSAYS RNA High-throughput analysis Gene doping CRISPR dCas9 Human erythropoietin Sports