New application of the CRISPR-Cas9 system for site-specific exogenous gene doping analysis

New application of the CRISPR-Cas9 system for site-specific exogenous gene doping analysis / Joon-Yeop Yi, Minyoung Kim, Hophil Min, Byung-Gee Kim, Junghyun Son, Oh-Seung Kwon, Changmin Sung. - (Drug Testing and Analysis (2020) 17 November)

  • PMID: 33201595
  • DOI: 10.1002/dta.2980

Abstract

The increased potential for gene doping since the introduction of gene therapy presents the need to develop anti-doping 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 anti-doping analyses of gene doping.

Parameters

Science
Study
Date
17 November 2020
People
Kim, Byung-Gee
Kim, Minyoung
Kwon, Oh-Seung
Min, Hophil
Son, Junghyun
Sung, Changmin
Yi, Joon-Yeop
Country
Korea, Republic of
Language
English
Other organisations
Seoul National University (SNU)
Laboratories
Seoul, Korea: Doping Control Center
Analytical aspects
Testing method development
Doping classes
M3. Gene And Cell Doping
S2. Peptide Hormones, Growth Factors
Substances
Erythropoietin (EPO)
Medical terms
Gene Therapy
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Abstract
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14 January 2021
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  • Doping classes
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