CRISPR-Cas反式切割技术在动物病原体多重核酸检测中的应用进展

doi:10.13560/j.cnki.biotech.bull.1985.2025-1413

摘要/Abstract

摘要：

病原体感染呈现多样性和复杂性的趋势，给动物疾病诊断和防治带来了严峻挑战。在病原体多重检测的过程中，传统核酸检测方法如聚合酶链式反应、等温扩增技术和全基因组测序存在操作复杂、成本高昂、依赖专业设备和人员等局限，因而迫切需要一种准确、快捷、便携的病原体多重检测技术。CRISPR-Cas系统是存在于许多原核生物中的适应性免疫系统。而基于CRISPR-Cas的反式切割技术，是一种能精准靶向和切割核酸的新型检测技术，该技术无需专业设备，即可在现场实现amol/L水平的检测。本文通过梳理近5年来CRISPR-Cas系统相关的研究进展，重点对CRISPR-Cas反式切割系统的分类，CRISPR-Cas反式切割技术在动物病原检测中的研究成果和应用现状，以及CRISPR-Cas反式切割系统的信号富集策略进行综述，并进一步阐述CRISPR-Cas技术在动物多重病原检测中的优势和发展前景，以期为进一步发展基于CRISPR-Cas反式切割系统的多重核酸检测技术提供参考。

关键词: CRISPR-Cas技术, 动物病原体, 多重检测, 反式切割活性

Abstract:

The increasing diversity and complexity of pathogen infections pose a serious challenge to the diagnosis, prevention, and control of animal diseases. Traditional nucleic acid detection methods, such as polymerase chain reaction (PCR), isothermal amplification techniques, and whole-genome sequencing, are limited by complex operation, high cost, and reliance on specialized equipment and personnel. Therefore, there is an urgent need for an accurate, rapid, and portable multiplex pathogen detection technology. The CRISPR-Cas system, an adaptive immune mechanism found in many prokaryotes, has inspired a new generation of detection platforms. The CRISPR-Cas-based trans-cleavage technology is a novel detection method that enables precise targeting and cleavage of nucleic acids. This technology allows for on-site detection at the attomolar (amol/L) level without the need for specialized equipment. In this review, we summarize recent advances in CRISPR-Cas systems over the past five years. We focus specifically on the classification of CRISPR-Cas trans‑cleavage systems, their research achievements and current application status in animal pathogen detection, and the signal amplification strategies employed in these systems. We also discuss the advantages and future prospects of CRISPR-Cas technology for multiplex pathogen detection in animals. This review aims to provide a reference for the further development of multiplex nucleic acid detection technologies based on CRISPR-Cas trans‑cleavage systems.

Key words: CRISPR-Cas technology, animal pathogens, multiplex detection, trans-cleavage activity

张津慎, 余永涛, 戚姣姣, 何海洋. CRISPR-Cas反式切割技术在动物病原体多重核酸检测中的应用进展[J]. 生物技术通报, 2026, 42(11): 81-91.

ZHANG Jin-shen, YU Yong-tao, QI Jiao-jiao, HE Hai-yang. Application Advances of CRISPR-Cas Trans-cleavage Technology in Multiplex Nucleic Acid Detection of Animal Pathogens[J]. Biotechnology Bulletin, 2026, 42(11): 81-91.

图/表 4

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效应蛋白类型 Effector protein type	基因命名法 Gene nomenclature	分子量大小 Mw （aa）	最佳作用温度 Optimum temperature （℃）	核酸内切酶活性结构域 Endonuclease active domain	引导RNA Guide RNA	靶基因 Target gene	靶基因识别位点 Target gene recognition site	反式切割底物 Trans-cleavage substrate	参考文献 References
Ⅴ型效应蛋白	Cas12a （Cpf1）	1 223	37	RuvC	crRNA	（ds/ss）DNA	PAM	ssDNA	［27-31］
	Cas12b （C2c1）	1 129	48	RuvC	tracrRNA+ crRNA	（ds/ss）DNA	PAM	ssDNA
	Cas12f1 （Cas14a）	422	45-55	RuvC	tracrRNA+ crRNA	dsDNA	PAM	ssRNA
Ⅵ型效应蛋白	Cas13a （C2c2）	1 250	37	HEPN	crRNA	ssRNA	PFS	ssRNA	［32-34］
Ⅵ型效应蛋白	Cas13b	1 150	55	HEPN	crRNA	ssRNA	PFS	ssRNA	［32-34］

效应蛋白类型 Effector protein type	基因命名法 Gene nomenclature	分子量大小 Mw （aa）	最佳作用温度 Optimum temperature （℃）	核酸内切酶活性结构域 Endonuclease active domain	引导RNA Guide RNA	靶基因 Target gene	靶基因识别位点 Target gene recognition site	反式切割底物 Trans-cleavage substrate	参考文献 References
Ⅴ型效应蛋白	Cas12a （Cpf1）	1 223	37	RuvC	crRNA	（ds/ss）DNA	PAM	ssDNA	［27-31］
	Cas12b （C2c1）	1 129	48	RuvC	tracrRNA+ crRNA	（ds/ss）DNA	PAM	ssDNA
	Cas12f1 （Cas14a）	422	45-55	RuvC	tracrRNA+ crRNA	dsDNA	PAM	ssRNA
Ⅵ型效应蛋白	Cas13a （C2c2）	1 250	37	HEPN	crRNA	ssRNA	PFS	ssRNA	［32-34］
Ⅵ型效应蛋白	Cas13b	1 150	55	HEPN	crRNA	ssRNA	PFS	ssRNA	［32-34］

特性 Characteristics	扩增目的基因 Target gene amplification	构建级联放大反应 Cascade amplification system	改造反式切割底物 Engineering of trans-cleavage substrate
基本原理	通过复制实现靶基因数量增加	通过一系列串联反应，实现信号逐级放大	通过设计新型底物，实现高强度信号释放
操作便捷性	高	高	高
设备依赖性	较高	较低	低
灵敏度	极高	极高	高
检测限	aM	aM	fM
成本	高	较高	中
优点	灵敏度高，技术成熟	操作简单且有较高灵敏度	操作简单
局限性	需要精确的温度控制	体系设计难度大	易受样品杂质干扰，影响实验的准确性

特性 Characteristics	扩增目的基因 Target gene amplification	构建级联放大反应 Cascade amplification system	改造反式切割底物 Engineering of trans-cleavage substrate
基本原理	通过复制实现靶基因数量增加	通过一系列串联反应，实现信号逐级放大	通过设计新型底物，实现高强度信号释放
操作便捷性	高	高	高
设备依赖性	较高	较低	低
灵敏度	极高	极高	高
检测限	aM	aM	fM
成本	高	较高	中
优点	灵敏度高，技术成熟	操作简单且有较高灵敏度	操作简单
局限性	需要精确的温度控制	体系设计难度大	易受样品杂质干扰，影响实验的准确性