数字农科院2.0

Rapid and sensitive screening and identification of CRISPR/Cas9 edited rice plants using quantitative real-time PCR coupled with high resolution melting analysis

文献类型： 外文期刊

作者： Yuan, Zheng;Cui, Jinjie;Zhang, Xiujie;Yang, Litao;Yang, Litao;Ba, Yan;Li, Rong;Zhang, Dabing;Song, Yu

作者机构：

关键词： Quantitative Real-Time Pcr; High-Resolution Melting Analysis; In/Del; Gene Editing; Crispr/Cas9

期刊名称： FOOD CONTROL

ISSN： 0956-7135

年卷期： 2020 年 112 卷

页码：

收录情况： JCR（2021版）

摘要： 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 qPCRHRM 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.

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