基于CRISPR/Cas系统的分子诊断技术原理及应用研究进展

Advances in the Principle and Application of Molecular Diagnostic Techniques Based on CRISPR/Cas System

如今,CRISPR/Cas系统已被应用于基因组编辑,主要是基于其具有准确识别和切割特定DNA和RNA序列的能力。此外,随着对目标序列的识别,某些CRISPR/Cas系统,包括Cas12、Cas13、Cas14蛋白在向导RNA的引导下,除了识别目标序列启动靶向切割活性后,还表现出对其他单链DNA或RNA分子间接的非特异性切割活性,如果在反应体系中加入带有荧光基团的报告核酸分子,使其释放出荧光信号,就可以指示目标核酸分子的存在。同时,通过利用具有不同切割偏好的Cas蛋白,还可以实现多种靶标分子的同时检测。本文综述了4种代表性Cas蛋白的核酸分子诊断技术原理及应用研究进展,并对CRISPR/Cas系统在该领域的应用前景进行了展望。

The CRISPR/Cas systems have been used for genome editing,largely based on their ability to accurately identify and cut specific DNA and RNA sequences.In addition,with the recognition of target sequences,in some CRISPR/Cas systems,including Cas12 and Cas13 proteins,guided by a guide RNA,in addition to the recognition of target sequences that initiate targeted cutting activity,indirect non-specific cleavage(also known as“incidental cleavage”)activity is also demonstrated.If reporter nucleic acid molecules with fluorescent groups are added to the reaction system,the fluorescence signals can be released to indicate the presence of the target nucleic acid molecules.At the same time,by using Cas proteins with different cutting preferences,multiple target molecules can be detected simultaneously.In this paper,the principle and application of nucleic acid molecular diagnosis of four representative Cas proteins are reviewed,and the application prospect of CRISPR/Cas system in this field is prospected.