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

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.