CRISPR-Cas系统在病原核酸检测中的研究进展

doi:10.12211/2096-8280.2022-068

合成生物学

• 特约评述 •

CRISPR-Cas系统在病原核酸检测中的研究进展

杜瑶¹^,², 高宏丹¹^,², 刘家坤³^,⁴, 刘孝荣², 邢志浩², 张涛⁵, 马东礼²

^1. 蚌埠医学院检验医学院，安徽蚌埠 233030
^2. 深圳市儿童医院儿科研究所，广东深圳 518034
^3. 中国科学院深圳先进技术研究所深圳合成生物研究所，广东深圳 518055
^4. 深圳市海微生物科技有限公司，广东深圳 518057
^5. 蚌埠医学院病原生物学教研室，安徽蚌埠 233030

收稿日期:2022-11-26 修回日期:2023-02-15 出版日期:2023-02-16
通讯作者: 马东礼
作者简介:杜瑶（1999-），女，硕士研究生，研究方向：病原生物学及分子生物学。 E-mail: dy852578@163.com.
马东礼（1967-），男，硕士生导师，主任技师，研究方向：病原微生物致病机制及基因精准诊断。 E-mail: madl1234@126.com.
基金资助:
深圳市高水平医院建设基金;广东省高水平医院建设基金资助;深圳市发展和改革委员会赠款（2019）986

Research progress of CRISPR-Cas system in pathogen nucleic acid detection

Yao DU¹^,², Hongdan GAO¹^,², Jiakun LIU³^,⁴, Xiaorong LIU², Zhihao XING², Tao ZHANG⁵, Dongli MA²

^1. School of Laboratory Medicine，Bengbu Medical College，Bengbu Anhui 233030，China
^2. Pediatric Research Institute，Shenzhen Children’s Hospital，Shenzhen Guangdong 518034，China
^3. Shenzhen Institute of Synthetic Biology，Shenzhen Institute of Advanced Technology，Chinese Academy of Sciences，Shenzhen Guangdong 518055，China
^4. Shenzhen Hapmic Biotechnology Company limited，Shenzhen Guangdong 518057，China
^5. Department of Pathogenic Biology，Bengbu Medical College，Bengbu Anhui 233030，China.

Received:2022-11-26 Revised:2023-02-15 Online:2023-02-16
Contact: Dongli MA

摘要/Abstract

摘要：

CRISPR-Cas系统作为原核生物获得性免疫系统，由簇状规则间隔短回文重复序列（Clustered Regularly Interspaced Short Palindromic Repeats， CRISPR）和CRISPR相关蛋白（CRISPR-associated proteins， Cas）构成，因其识别和切割特定DNA或RNA序列，而成为分子诊断领域研究的热点。研究人员利用Cas蛋白（Cas12、Cas13、Cas14、Cas3等）结合信号放大和转化技术（荧光法、电位法、比色法、侧向流动技术等），开发了许多高灵敏度、高特异性、低成本的诊断平台，为病原核酸检测提供了新途径。本文介绍了CRISPR-Cas系统的生物学机制及分类，总结现有的基于Cas蛋白反式切割活性开发的病原核酸检测技术，描述其特点、功能和应用场景，并对该系统的未来应用前景进行展望，期望CRISPR-Cas系统成为包括核酸检测在内的多靶标的理想检测平台。

关键词: CRISPR-Cas系统, Cas12和Cas13, 病原体, 核酸检测, 生物传感

Abstract:

CRISPR-Cas system consists of clustered regularly interspaced short palindromic repeats(CRISPR) and CRISPR-associated (Cas) proteins, which have become the focus of molecular diagnosis because it recognizes and cleaves specific DNA or RNA sequences. Using Cas proteins (Cas12, Cas13, Cas14, Cas3 etc.) combined with signal amplification and transformation techniques (fluorescence, potentiometric, colorimetric, lateral flow assay etc.), researchers have developed many diagnostic platforms with high sensitivity, good specificity and low cost, which provide a new way for pathogen nucleic acid detection. This review presents the biological mechanism, classification of CRISPR-Cas system and summarizes the existing pathogenic nucleic acid detection technologies based on the trans-cleavage activity of Cas protein, and describes their properties, functions and application scenarios, and provides the future application prospects according to the functional characteristics of CRISPR-Cas system. The CRISPR-Cas system is expected to become the ideal detection platform for multiple targets.

Key words: CRISPR-Cas system, Cas12 and Cas13, pathogen, nucleic acid detection, biosensor

中图分类号:

R446.5

杜瑶, 高宏丹, 刘家坤, 刘孝荣, 邢志浩, 张涛, 马东礼. CRISPR-Cas系统在病原核酸检测中的研究进展[J]. 合成生物学 : 1-14, doi: 10.12211/2096-8280.2022-068.

Yao DU, Hongdan GAO, Jiakun LIU, Xiaorong LIU, Zhihao XING, Tao ZHANG, Dongli MA. Research progress of CRISPR-Cas system in pathogen nucleic acid detection[J]. Synthetic Biology Journal : 1-14, doi: 10.12211/2096-8280.2022-068.

图/表 6

表1

基于CRISPR-Cas系统开发的部分诊断技术

CRISPR类型	技术名称	效应蛋白	目标分子	扩增方式	检测线	病原体	检测技术
Type Ⅰ-E	CONAN^[24]	Cas3	DNA	RT-LAMP	1 copy	SARS-CoV-2 IAV	侧向流动分析
Type Ⅴ	DETECTR^[105]	Cas12a	DNA	RPA	aM	HPV16/18 SARS-CoV-2	荧光信号
	OR-DETECTR^[96]	Cas12a	RNA	RT-RPA	1-2,5 copies/uL	SARS-CoV-2 H1N1	荧光信号、侧向流动分析
	HOLMES^[57]	Cas12a	DNA RNA	PCR	aM	日本脑炎病毒伪狂犬病毒	荧光信号
	HOLMESv2^[59]	Cas12b	DNA RNA	LAMP	aM	日本脑炎病毒	荧光信号
	CDetection^[66]	AaCas12b	DNA	RPA	nM	HPV16/18、SNPs	荧光信号
	E-CRISPR^[68]	Cas12a	DNA	-	pM	HPV1、B19、TGFβ1	电化学
	MoECS^[69]	Cas12	DNA	-	fM	SARS-CoV-2 Delta变异株	电化学
	CRISPR-ENHANCE^[67]	LbCas12a	RNA	RT-LAMP		SARS-CoV-2、HIV、HCV	侧向流动分析
	AIOD-CRISPR^[74]	LbaCas12a	RNA	RPA	11 copies	SARS-CoV-2 HIV-1	荧光信号、视觉法
	SCAN^[73]	Cas12a	DNA RNA	RT-PCR	13.5 copies/uL	SARS-CoV-2 HIV-1	纳米孔传感器
	TB-QUICK^[61]	Cas12b	DNA	LAMP	1.3copies/uL	结核分枝杆菌	荧光信号
	STOPCovid^[78]	Cas12	DNA RNA	LAMP	100copies	SARS-CoV-2	荧光信号
	STOPCovid.v2^[79]	Cas12	DNA RNA	LAMP	2000 copies/mL	SARS-CoV-2	荧光信号
	sPAMC^[77]	LbCas12a	DNA RNA	RPA	1copy/uL	SARS-CoV-2	荧光信号、侧向流动分析
	Cas14-DETECTR^[98]	Cas14a	DNA	RPA	fM	HBoV-1、SNPs	荧光信号
	ACasB^[99]	Cas14a1	DNA	-	400 CFU/mL	金黄色葡萄球菌	荧光信号
Type Ⅵ	SHERLOCK^[82]	Cas13a	DNA RNA	RPA	aM	病毒、细菌、SNPs	荧光信号
	HUDSON^[78]	Cas13a	RNA	RT-RPA	1copy/uL	寨卡病毒、登革热病毒	荧光信号、侧向流动分析
	OR-SHERLOCK^[96]	Cas13a	RNA	RT-RPA	1-2,5 copies/uL	SARS-CoV-2 H1N1	荧光信号、侧向流动分析
	SHINE^[95]	Cas13	RNA	RT-RPA	10copies/uL	SARS-CoV-2	智能手机（管内荧光读数或侧向流动分析）
	FIND-IT^[86]	Lbu Cas13a	RNA	-	31copies/uL	SARS-CoV-2	荧光信号（集成检测器）
	CARMEN^[89]	LwCas13a	DNA RNA	PCR或RPA	aM	169种人类感染病毒	荧光信号
	mCARMEN^[92]	LwCas13a	DNA RNA	PCR或RPA	10²copies/uL	21种人类呼吸道病毒	荧光信号
Type Ⅴ Type Ⅵ Type Ⅲ	SHERLOCKv2^[85]	Cas13 Cas12a Csm6	DNA RNA	RPA	zM	病毒、细菌、SNPs	荧光信号、侧向流动分析

表1

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图5

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CRISPR-Cas系统在病原核酸检测中的研究进展

Research progress of CRISPR-Cas system in pathogen nucleic acid detection

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