高分辨率熔解曲线在CRISPR基因编辑检测和基因型鉴定中的应用

Application of high resolution melting curve in CRISPR gene editing and genotyping

目的建立一种基于PCR和在线高分辨率熔解曲线(high-resolution melting curve,HRM)分析工具的检测方法,实现对DNA序列微小差异的快速检测。方法在CRISPR基因编辑检测和基因型鉴定中,采用两步PCR,分别对目的片段进行扩增和富集,并通过免费在线软件对HRM进行分析。结果 HRM分析结果显示,本实验所设计的CRISPR慢病毒系统未能对COS7细胞中的RPE65基因进行有效的基因编辑;而在瘦素突变大鼠的子代基因型中,HRM可以明显区分杂合子和纯合子。结论 HRM能够快速鉴定CRISPR编辑和区分基因型。

Objective To establish a new method based on online high.resolution melting （HRM）analysis and PCR to promptly verify the DNA mismatches. Methods In detection of CRISPR genomeediting and genotyping, two.step PCR was used to amplify and enrich the target fragments. HRM wasanalyzed by free online software, then the mismatches caused by CRISPR were detected and thegenotypes were identified. Results HRM analysis showed that the CRISPR lentivirus system did notefficiently modified RPE65 gene in COS7 cells. In genotyping the progeny of leptin.mutant rats,HRM apparently distinguished heterozygotes from homozygotes. Conclusion HRM allows rapididentification of CRISPR editing and genotyping.