基于高分辨率熔解曲线技术的CRISPR/Cas9介导的细胞基因突变快速检测研究

Methodological Research on Rapid Detection of CRISPR/Cas9-Mediated Gene Mutations in Cells Based on High-resolution Melting Technique

旨在建立一种通过高分辨率熔解曲线(high-resolution melting,HRM)分析技术快速检测CRISPR/Cas9介导的细胞基因突变的方法。采用野生型、纯合突变及杂合突变小鼠胚胎干细胞优化和完善HRM检测条件,然后应用建立的HRM方法对30个测序结果已知的CRISPR/Cas9技术编辑过的小鼠胚胎干细胞单克隆进行检测,根据熔解温度与熔解曲线的差异区分野生型、纯合突变型及杂合突变型,以验证HRM方法的可行性和准确性。结果表明,优化的HRM检测方法能够鉴别野生型、纯合突变型及杂合突变型小鼠胚胎干细胞。应用建立的HRM方法分析30个CRISPR/Cas9技术编辑过的小鼠胚胎干细胞单克隆,结果显示,14个单克隆为杂合突变型、2个单克隆为纯合突变型、14个单克隆为野生型,与测序结果一致,准确率为100%。本研究建立的高分辨率熔解曲线法能对CRISPR/Cas9介导的细胞基因突变进行快速筛选,是一种灵敏、准确、简便、高通量的方法。

This study is aimed to establish a method for rapid detection of CRISPR/Cas9-mediated gene mutations in cells using highresolution melting(HRM)assay.The condition of HRM analysis was optimized and perfected using wild-type mouse embryonic stem cells(mESCs),homozygous mutation mESCs and heterozygous mutation mESCs.In order to verify the feasibility and accuracy of HRM method,the established HRM analysis was used to detect 30 monoclones of CRISPR/Cas9-edited mESCs with known their sequencing results.According to the difference of melting temperature and melting curve,wild-type mESCs,homozygous mutation mESCs and heterozygous mutation mESCs were distinguished.The results showed that the optimized HRM assay identified wild-type mESCs,homozygous mutation mESCs and heterozygous mutation mESCs.Thirty monoclones of mESCs edited by CRISPR/Cas9 technique were analyzed using the established HRM method.The results indicated that 14 monoclones were heterozygous mutation mESCs,2 monoclones were homozygous mutation mESCs and 14 monoclones were wild-type mESCs.These results were consistent with the sequencing results at accuracy of 100%.The high-resolution melting assay established in this study is a sensitive,accurate,simple and high-throughput method,and may rapidly scan CRISPR/Cas9-mediated gene mutations in cells.