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

Drug Testing and Analysis
Joon-Yeop YiChangmin Sung

Abstract

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.

References

Jun 4, 2011·Analytical and Bioanalytical Chemistry·Christian Reichel
Oct 26, 2013·Nature Protocols·F Ann RanFeng Zhang
Jan 31, 2014·Nature·Samuel H SternbergJennifer A Doudna
Sep 1, 2015·Nature Methods·Miguel A Moreno-MateosAntonio J Giraldez
Jan 19, 2016·Nature Biotechnology·John G DoenchDavid E Root
Jan 31, 2019·Cell Reports·Robin GrafKlaus Rajewsky

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Citations

Nov 18, 2021·Drug Testing and Analysis·Mario ThevisHans Geyer

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