基于CRISPR-Cas系统的病原体检测研究进展

Recent Advances of Pathogens Detection Based on CRISPR-Cas System

开发快速、灵敏、特异的检测方法对于新发突发人兽共患病、动物疫病的监测及防控至关重要。规律成簇的间隔短回文重复序列及相关蛋白(CRISPR-Cas)系统以高敏感性、特异性及可扩展性的DNA和RNA识别为特点,已成为新一代的分子诊断工具,在病原体快速检测技术研发中得到广泛应用。在新型冠状病毒肺炎疫情中,基于CRISPR-Cas系统开发的新型冠状病毒(SARS-CoV-2)检测试剂盒(SHERLOCK和DETECTR)在美国相继获批,中国研发的基于CRISPR-Cas12系统的SARS-CoV-2检测试剂盒也已获批上市,这表明基于CRISPR-Cas系统的病原体检测方法在未来的应用前景非常可观。文章首先简述了CRISPR-Cas系统类型,尤其是常用于病原体检测的Cas9、Cas12和Cas13;介绍了CRISPR-Cas系统的作用机理以及广泛应用。同时分析了CRISPR-Cas检测平台研发的关键技术问题,包括靶标基因的序列保守性、靶标基因的富集与扩增、终信号不同的读取方式等,并对CRISPR-Cas系统的未来发展及应用前景进行了展望。

Development of rapid,specific and sensitive detection method for emerging and re-emerging zoonosis and animal disease is critical for disease surveillance,prevention and control.CRISPR-Cas system,characterized by their sensitivity,specificity,and scalability upon DNA and RNA recognition,have been repurposed for the new generation of molecular diagnostic tools and are highly applied for pathogens detection.In the COVID-19 pandemic,CRISPR-Cas based SARS-CoV-2 detection kits(SHERLOCK and DETECTR)have been approved by USA,and CRISPR-Cas12 based SARS-CoV-2 detection kit developed in China has also been approved for marketing,implying that CRISPR-Cas assisted detection method could have a lot of uses in the future.This review firstly introduces the classification of CRISPR-Cas systems,especially CRISPR-Cas9/Cas12/Cas13,which are commonly used for pathogen detection.It also introduces the mechanism and the wider applications of CRISPR-Cas system.Meanwhile,the paper analyses the key technical issues in the development of CRISPR-Cas detection platform including sequence conservation of target genes,target enrichment and amplification,pathogens sensing with different signal readouts,and discusses the future development and application prospect of this method.