Abstract Gene-editing techniques, such as TALEN and CRISPR/Cas9, have been widely used for target DNA editing in many research fields. However, how to rapidly screen and identify the expected gene-edited products with high efficiency and low cost is still a difficult task. Here, we report the development and optimization of one such method that combines quantitative PCR with high-resolution melting analysis (qPCR-HRM) to screen and identify CRISPR/Cas9-edited rice plants. The results showed that gene-edited rice plants with small target DNA in/dels or even single base pair insertion/deletions could be successfully identified. The sensitivity of the qPCR-HRM method is as low as 1%, which is satisfying to be used for quantitative evaluation of gene-editing efficiency. Sanger sequencing results confirmed that the qPCR-HRM method also has high accuracy. It is concluded that the developed qPCR-HRM method is reproducible, accurate, and efficient for quick screening and identification of gene-edited rice plants.

中文翻译：

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使用定量实时 PCR 结合高分辨率熔解分析快速、灵敏地筛选和鉴定 CRISPR/Cas9 编辑的水稻植株

摘要 TALEN 和 CRISPR/Cas9 等基因编辑技术已被广泛应用于许多研究领域的目标 DNA 编辑。然而，如何快速、高效、低成本地筛选和识别预期的基因编辑产品仍然是一项艰巨的任务。在这里，我们报告了一种这样的方法的开发和优化，该方法将定量 PCR 与高分辨率熔解分析 (qPCR-HRM) 相结合，以筛选和识别 CRISPR/Cas9 编辑的水稻植株。结果表明，基因编辑的水稻植株在/dels 中的小目标DNA 甚至单个碱基对插入/缺失都可以成功识别。qPCR-HRM 方法的灵敏度低至 1%，可满足用于基因编辑效率的定量评估。Sanger测序结果证实qPCR-HRM方法也具有较高的准确度。结论是，所开发的 qPCR-HRM 方法可重现、准确且高效，可用于基因编辑水稻植株的快速筛选和鉴定。