基于CRISPR/Cas系统的病原核酸检测技术研究进展

doi:10.3785/j.issn.1008-9209.2022.08.051

浙江大学学报（农业与生命科学版）

2023, Vol. 49

Issue (5): 618-632 DOI: 10.3785/j.issn.1008-9209.2022.08.051

青年科学家论坛

基于CRISPR/Cas系统的病原核酸检测技术研究进展

李红招^1,²(

),王浩²(

),尹睿²,乐敏^1,²,李艳^1,²

^1.浙江大学海南研究院，海南三亚 572025
^2.浙江大学动物科学学院动物预防医学研究所/ 浙江省动物预防医学重点实验室，浙江杭州 310058

Research advances on pathogenic nucleic acid detection technology based on CRIPSR/Cas system

Hongzhao LI^1,²(

),Hao WANG²(

),Rui YIN²,Min YUE^1,²,Yan LI^1,²

^1.Hainan Institute of Zhejiang University, Sanya 572025, Hainan, China
^2.Institute of Preventive Veterinary Sciences/Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China

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摘要：

作为一种古老的细菌和古菌免疫系统，规律成簇的间隔短回文重复序列及其相关蛋白［clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas), CRISPR/Cas］系统现已发展为新兴的热门基因编辑工具，极大地推动了其他多个生物学相关领域的发展。其中，通过结合CRISPR/Cas系统与核酸恒温扩增技术建立了一些新型的高效、灵敏、不依赖仪器设备的检测方法，如DNA内切酶靶向的CRISPR反式报告系统（DNA endonuclease-targeted CRISPR trans-reporter, DETECTR）、特异性高灵敏度的酶报告器系统（specific high-sensitivity enzymatic reporter unlocking, SHERLOCK）等，不但提高了CRISPR/Cas系统在不同应用场景下的检测性能，更进一步激发了其在现场检测中的应用潜力。本文基于近年来被广泛应用的3种CRISPR/Cas系统（CRISPR/Cas9、CRISPR/Cas12a以及CRISPR/Cas13）的生物核酸检测方法，阐述了CRISPR/Cas系统的生物学意义及一般作用原理，并通过回顾以往开展的CRISPR/Cas系统相关的病原检测研究，比较分析了不同检测系统的特点以及在实际应用过程中可能存在的不足，为针对不同病原在不同应用场景下建立更加高效、合理的CRISPR/Cas系统检测方法提供了有益参考。

关键词： CRISPR/Cas系统; Cas蛋白; 病原; 核酸; 检测方法

Abstract:

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) (CRISPR/Cas) system, an ancient bacterial and archaeal immune system, has rapidly developed into a popular gene-editing tool, which largely promotes the development of several biology-related fields. By combining the CRISPR/Cas systems with the isothermal amplification techniques, the novel and effective detection methods with high sensitivity and independence of equipment have been established, such as DNA endonuclease-targeted CRISPR trans-reporter (DETECTR) and specific high-sensitivity enzymatic reporter unlocking (SHERLOCK). These new technologies not only improve the performance of the CRISPR/Cas system in different situations, but also inspire its application potential in the on-site detection. In this review, we summarized the nucleic acid detection methods developed on the three widely-used CRISPR/Cas systems (CRISPR/Cas9, CRISPR/Cas12a, and CRISPR/Cas13), and elucidated their biological significance and the principles of action. We also reviewed the recent studies on the applications of CRISPR/Cas systems in pathogen detection, and analyzed the characteristics and possible defects of different detection systems in practical applications. This review aims to provide more constructive advice on developing adaptable and efficient CRISPR/Cas-based detection methods for different pathogens in various practical scenarios.

Key words: CRISPR/Cas system Cas protein pathogen nucleic acid detection method

收稿日期: 2022-08-05 出版日期: 2023-11-03

CLC:

S85

基金资助: 国家重点研发计划政府间国际科技创新合作项目(2019YFE0103900);国家自然科学基金项目(31872837);浙江省自然科学基金项目(LZ22C8004);海南省科技计划三亚崖州湾科技城联合项目(2021JJLH0083)

通讯作者: 李艳 E-mail: 22017116@zju.edu.cn;11917042@zju.edu.cn

作者简介: 李红招（https://orcid.org/0000-0002-0107-826X），E-mail：22017116@zju.edu.cn|王浩（https://orcid.org/0000-0001- 6985-4643)，E-mail：11917042@zju.edu.cn。|李艳，浙江大学动物科学学院“百人计划”研究员，博士生导师。中国科学院武汉病毒研究所和美国纽约州卫生部Wadsworth 中心联合培养博士，2010 年获理学博士学位。2011 年起师从美国科学院院士Nancy Speck 教授进行博士后研究。2017 年10 月加盟浙江大学动物科学学院动物医学系，研究方向为兽医病理与比较生物医学。近年来在SCI 收录期刊共发表论文30 余篇，其中在Blood、Genes & Development 等期刊以第一作者或者通信作者发表论文17 篇。作为主要完成人之一获得2018 年国家自然科学奖二等奖。主持国家重点研发计划政府间国际科技创新合作项目、国家自然科学基金面上项目和青年项目、浙江省自然科学基金重点项目、海南省自然科学基金面上项目和海南省科技计划三亚崖州湾科技城联合项目。https://orcid.org/0000-0003-4813-5783），E-mail：yanli3@zju.edu.cn

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引用本文:

李红招,王浩,尹睿,乐敏,李艳. 基于CRISPR/Cas系统的病原核酸检测技术研究进展[J]. 浙江大学学报（农业与生命科学版）, 2023, 49(5): 618-632.

Hongzhao LI,Hao WANG,Rui YIN,Min YUE,Yan LI. Research advances on pathogenic nucleic acid detection technology based on CRIPSR/Cas system. Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(5): 618-632.

链接本文:

https://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2022.08.051 或 https://www.zjujournals.com/agr/CN/Y2023/V49/I5/618

图1 第2类CRISPR/Cas系统的分类REC：识别叶；PI：PAM互作域；HEPN：高等真核生物和原核生物核苷酸结合域；TM：跨膜结构域。

图2 Cas9、Cas12a、Cas13a特异性识别并切割目的核酸和Cas12a、Cas13a非特异性剪切单链DNA/RNA

图3 RPA和HDA技术的扩增原理

图4 LAMP技术的扩增原理A～B.循环模板合成阶段；C.循环扩增和再循环阶段。

表1 CRISPR/Cas系统在RNA病原检测中的应用

表2 CRISPR/Cas系统在DNA病原检测中的应用

图5 基于CRISPR/Cas12a和CRISPR/Cas13a检测系统的操作流程

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