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.

中文翻译：

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CRISPR-Cas9系统在位点特异性外源基因掺杂分析中的新应用

自从引入基因疗法以来，基因兴奋剂的可能性增加，因此需要开发反兴奋剂检测方法。因此，我们旨在开发一种快速简单的方法，利用体外检测特异性靶向外源兴奋剂基因。成簇的规则间隔短回文重复序列-CRISPR相关蛋白9（CRISPR-Cas9）系统。人类促红细胞生成素 (hEPO) 是一种经常用于运动员兴奋剂的药物，并且可以尝试使用基因转移技术进行基因兴奋。因此，我们选择 hEPO 基因作为外源兴奋剂基因的模型，并设计互补单向导 RNA (sgRNA) 以特异性结合 hEPO cDNA 中的四个外显子-外显子连接。对于 CRISPR-Cas9 的快速反应，使用开源程序 (CRISPOR) 进行了进一步优化，该程序避免了原型间隔区相邻基序 (PAM) 域之前的 TT 和 GCC 基序，并预测了 sgRNA 的效率。我们优化了体外Cas9 测定和 sgRNA 的双重使用进行双裂解，并确定了双裂解方法的 1.25 nM 的检测限 (LOD)。我们期待改进的 CRISPR-Cas9 方法可用于基因兴奋剂的反兴奋剂分析。