基于CRISPR/Cas原理的转基因产品检测技术研究进展

doi:10.19586/j.2095-2341.2021.0083

生物技术进展 ›› 2021, Vol. 11 ›› Issue (4): 430-437.DOI: 10.19586/j.2095-2341.2021.0083

基于CRISPR/Cas原理的转基因产品检测技术研究进展

谢宇宙¹(), 付伟¹, 闫超杰², 王智¹, 朱鹏宇¹, 任永超³, 张永江¹, 相宁¹()

^1.中国检验检疫科学研究院，北京 100176
^2.中华人民共和国锦州海关，辽宁锦州 121013
^3.贵州省黔南布依族苗族自治州检验检测院，贵州都匀 558000

收稿日期:2021-05-11 接受日期:2021-06-07 出版日期:2021-07-25 发布日期:2021-08-02
通讯作者: 相宁
作者简介:谢宇宙 E-mail：18811582786@163.com；
基金资助:
海南省重大科技计划项目(ZDKJ202002);中国检科院基本科研业务费项目(2020JK016)

Research Progress of Detection Technology of Genetically Modified Products Based on CRISPR/Cas Principle

Yuzhou XIE¹(), Wei FU¹, Chaojie YAN², Zhi WANG¹, Pengyu ZHU¹, Yongchao REN³, Yongjiang ZHANG¹, Ning XIANG¹()

^1.Chinese Academy of Inspection and Quarantine，Beijing 100176，China
^2.Jinzhou Customs District People’s Republic of China，Liaoning Jinzhou 121013，China
^3.Qiannan Inspection and Testing Institute，Guizhou Duyun 558000，China

Received:2021-05-11 Accepted:2021-06-07 Online:2021-07-25 Published:2021-08-02
Contact: Ning XIANG

摘要/Abstract

摘要：

随着转基因产品商业化种植面积不断增加、国际贸易日趋频繁，对转基因生物安全管理提出了更高的要求。转基因产品检测技术作为安全评价的关键环节，逐渐引起了各国政府的关注。目前，针对转基因产品的快速检测方法层出不穷，但这些检测方法对于设备、试剂和专业的实验人员均有较高的要求。因此，为了有效支撑转基因相关产业的发展和管理，亟需建立一种高灵敏度、高特异性及高效的转基因检测技术。基因组编辑技术是近年来迅速发展的一类遗传修饰技术，其代表技术——CRISPR/Cas技术，更是极大地推动了生物技术的发展。CRISPR/Cas技术除了被应用于基因编辑领域，也逐渐被应用于核酸分子检测领域。基于此，以转基因产品检测技术为立足点，从CRISPR/Cas的检测原理、检测效果等技术层面分析了CRISPR/Cas检测技术发展的必然性，并对其在转基因产品检测上的应用前景进行展望，旨在为我国转基因产品快速检测和有效监管工作提供资料，对于保障我国转基因产品贸易的顺利进行具有重要意义。

关键词: CRISPR/Cas, 转基因产品, 检测技术, 监管

Abstract:

With the increasing commercial planting area of genetically modified products and the increasing international trade， higher requirements are put forward for the safety management of genetically modified organisms. As the key link of safety evaluation， the detection technology of genetically modified products has gradually attracted the attention of governments all over the world. At present， there are many rapid detection methods for genetically modified products， but these detection methods have higher requirements for equipment， reagents and professional laboratory personnel. Therefore， in order to effectively support the development and management of transgenic related industries， it is urgent to establish a highly sensitive， specific and efficient transgenic detection technology. Genome editing technology is a kind of genetic modification technology developed rapidly in recent years， and CRISPR/Cas technology， as a representative technology， has greatly promoted the development of biotechnology. CRISPR/Cas technology has been applied not only in the field of gene editing but also in the field of nucleic acid molecular detection. Therefore， based on the detection technology of genetically modified products， the inevitability of the development of CRISPR/Cas detection technology from the detection principle， detection effect and other technical aspects was analyzed， and looked forward to its application prospect in the detection of genetically modified products， in order to provide information for the rapid detection and effective supervision of genetically modified products in China， which is of great significance for ensuring the smooth progress of the trade of genetically modified products in China.

Key words: CRISPR/Cas, genetically modified products, detection technology, supervision

中图分类号:

谢宇宙, 付伟, 闫超杰, 王智, 朱鹏宇, 任永超, 张永江, 相宁. 基于CRISPR/Cas原理的转基因产品检测技术研究进展[J]. 生物技术进展, 2021, 11(4): 430-437.

Yuzhou XIE, Wei FU, Chaojie YAN, Zhi WANG, Pengyu ZHU, Yongchao REN, Yongjiang ZHANG, Ning XIANG. Research Progress of Detection Technology of Genetically Modified Products Based on CRISPR/Cas Principle[J]. Current Biotechnology, 2021, 11(4): 430-437.

图/表 5

图1 CRISPR/Cas9体外分型寨卡病毒流程图[9]

Fig.1 Flow chart of CRISPR/Cas9 in vitro typing of Zika virus[9]

图2 Cas13a蛋白非特异性切割RNA模型[8]

Fig.2 Cas13a protein non?specific cleavage of RNA model[8]

图3 SHERLOCK检测原理[17]

Fig.3 SHERLOCK detection principle[17]

图4 DETECTR检测原理[17]

Fig.4 DETECTR detection principle[17]

表1 CRISPR/Cas检测技术比较分析

Table 1 Comparative analysis of CRISPR/Cas detection technologies

	检测技术	靶分子	非特异性切割	灵敏度	特异性	仪器要求	等温扩增	PAM序列要求	检测时间	预处理
Cas9	CRISDA	dsDNA	否	aM水平	单碱基	无	是	高	90 min内	无
Cas12	DETECTR	dsDNA/ssDNA	ssDNA	aM水平	单碱基	无	是	中	1 h内	无
Cas13	HUDSON	ssRNA	ssRNA	aM水平	单碱基	无	是	低	1 h内	预转录DNA扩增产物
Cas14	Cas14‑DETECTR	ssDNA	ssDNA	aM水平	单碱基	无	是	无	1 h内	预处理dsDNA为ssDNA

参考文献 39

1	KAMBUROVA V S, NIKITINA E V, SHERMATOV S E, et al.. Genome editing in plants: an overview of tools and applications [J/OL]. Int. J. Agron., 2017:7315351[2021-06-17]. .
2	JINEK M, CHYLINSKI K, FONFARA I, et al.. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J]. Science, 2012, 337(6096):816-821.
3	ABUDAYYEH O O, GOOTENBERG J S, KONERMANN S, et al.. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector[J/OL]. Science, 2016, 353(6299):aaf5573[2021-06-17]. . DOI:10.1126/science.aaf5573 .
4	HSU P D, LANDER E S, ZHANG F. Development and applications of CRISPR-Cas9 for genome engineering[J]. Cell, 2014, 157(6):1262-1278.
5	HILLE F, RICHTER H, WONG S P, et al.. The biology of CRISPR-Cas: backward and forward[J]. Cell, 2018, 172(6):1239-1259.
6	CHYLINSKI K, MAKAROVA K S, CHARPENTIER E, et al.. Classification and evolution of type II CRISPR-Cas systems[J]. Nucl. Acids Res., 2014, 42(10):6091-6105.
7	SHMAKOV S, ABUDAYYEH O O, MAKAROVA K S, et al.. Discovery and functional characterization of diverse class 2 CRISPR-Cas systems[J]. Mol. Cell, 2015, 60(3):385-397.
8	EAST-SELETSKY A, O'CONNELL M R, KNIGHT S C, et al.. Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection[J]. Nature, 2016, 538(7624):270-273.
9	PARDEE K, GREEN A, TAKAHASHI M, et al.. Rapid, low-cost detection of Zika virus using programmable biomolecular components[J]. Cell, 2016, 165(5):1255-1266.
10	ZHOU W, HU L, YING L, et al.. A CRISPR-Cas9-triggered strand displacement amplification method for ultrasensitive DNA detection[J/OL]. Nat. Commun., 2018, 9:5012[2021-06-17]. .
11	HUANG M, ZHOU X, WANG H, et al.. Clustered regularly interspaced short palindromic repeats/Cas9 triggered isothermal amplification for site-specific nucleic acid detection[J]. Anal. Chem., 2018, 90(3):2193-2200.
12	HAJIAN R, BALDERSTON S, TRAN T, et al.. Detection of unamplified target genes via CRISPR-Cas9 immobilized on a graphene field-effect transistor[J]. Nat. Biomed. Eng., 2019, 3(6):427-437.
13	GOOTENBERG J S, ABUDAYYEH O O, LEE J W, et al.. Nucleic acid detection with CRISPR-Cas13a/C2c2 [J]. Science, 2017, 356(6336):438-442.
14	GOOTENBERG J S, ABUDAYYEH O O, KELLNER M J, et al.. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6[J]. Science, 2018, 360(6387):439-444.
15	MYHRVOLD C, FREIJE C A, GOOTENBERG J S, et al.. Field-deployable viral diagnostics using CRISPR-Cas13[J]. Science, 2018, 360(6387):444-448.
16	JOUNG J, LADHA A, SAITO M, et al.. Detection of SARS-CoV-2 with SHERLOCK one-pot testing[J]. New Engl. J. Med., 2020, 383(15):1492-1494.
17	MUSTAFA M I, MAKHAWI A M. SHERLOCK and DETECTR: CRISPR-Cas systems as potential rapid diagnostic tools for emerging infectious diseases[J]. J. Clin. Microbiol., 2021, 59(3):720-745.
18	ZETSCHE B, GOOTENBERG J S, ABUDAYYEH O O, et al.. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system[J]. Cell, 2015, 163(3):759-771.
19	CHEN J S, MA E, HARRINGTON L B, et al.. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity[J]. Science, 2018, 360(6387):436-439.
20	LI S Y, CHENG Q X, WANG J M, et al.. CRISPR-Cas12a-assisted nucleic acid detection[J/OL]. Cell Discov., 2018, 4:20[2021-06-17]. .
21	LI L, LI S, WU N, et al.. HOLMESv2: a CRISPR-Cas12b-assisted platform for nucleic acid detection and DNA methylation quantitation[J]. ACS Synth. Biol., 2019, 8(10):2228-2237.
22	BROUGHTON J P, DENG X, YU G, et al.. CRISPR-Cas12-based detection of SARS-CoV-2[J]. Nat. Biotechnol., 2020, 38(7):870-874.
23	HARRINGTON L B, BURSTEIN D, CHEN J S, et al.. Programmed DNA destruction by miniature CRISPR-Cas14 enzymes[J]. Science, 2018, 362(6416):839-842.
24	国际农业生物技术应用服务组织. 2019年全球生物技术/转基因作物商业化发展态势[J]. 中国生物工程杂志, 2021, 41(1):114-119.
25	王根平, 杜文明, 夏兰琴. 植物安全转基因技术研究现状与展望[J]. 中国农业科学, 2014, 47(5):823-843.
26	郭斌, 祁洋, 尉亚辉. 转基因植物检测技术的研究进展[J] . 中国生物工程杂志, 2010, 30(2):120-126.
27	QUERCI M, BULCKE M V D, ZEL J, et al.. New approaches in GMOdetection[J]. Anal. Bioanal. Chem., 2010, 396(6):1991-2002.
28	邓汉超, 尹长城, 刘国振, 等. 转基因植物核酸成分检测技术研究进展[J]. 中国生物工程杂志, 2011, 31(1):86-95.
29	NOTOMI T, OKAYAMA H, MASUBUSHI H, et al.. Loop-mediated isothermal amplification of DNA[J/OL]. Nucl. Acids Res., 2000, 28(12):E63[2021-06-17]. . DOI: 10.1093/nar/28.12.e63 .
30	SALISU I B, SHAHID A A, YAQOOB A, et al.. Molecular approaches for high throughput detection and quantification of genetically modified crops: a review[J/OL]. Front. Plant Sci., 2017, 8:1670[2021-06-17]. .
31	王颢潜, 陈锐, 李夏莹, 等. 转基因产品成分检测技术研究进展[J]. 生物技术通报, 2018, 34(3):31-38.
32	LI R, WANG C, JI L L, et al.. Loop-mediated isothermal amplification (LAMP) assay for GMO on: recent progresses and future perspectives[J/OL]. OALib J., 2015, 2: e1264[2021-06-17]. .
33	WU H, HE J, ZHANG F, et al.. Contamination-free visual detection of CaMV35S promoter amplicon using CRISPR/Cas12a coupled with a designed reaction vessel: rapid, specific and sensitive[J]. Anal. Chim. Acta, 2020, 1096:130-137.
34	ZHANG Y, ZHANG Y, XIE K. Evaluation of CRISPR/Cas12a-based DNA detection for fast pathogen diagnosis and GMO test in rice[J/OL]. Mol. Breed., 2020, 40:11[2021-06-17]. .
35	GOOTENBERG J S, ABUDAYYEH O O, LEE J W, et al.. Nucleic acid detection with CRISPR-Cas13a/C2c2[J]. Science, 2017, 356(6336):438-442.
36	GAO L, COX D B T, YAN W X, et al.. Engineered Cpf1 variants with altered PAM specificities[J]. Nat. Biotechnol., 2017, 35(8):789-792.
37	KLEINSTIVER B P, SOUSA A A, WALTON R T, et al.. Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing[J]. Nat. Biotechnol., 2019, 37(3):276-282.
38	TENG F, GUO L, CUI T, et al.. CDetection: CRISPR-Cas12b-based DNA detection with sub-attomolar sensitivity and single-base specificity[J/OL]. Genome Biol., 2019, 20:132[2021-06-17]. .
39	BERENSMEIER S. Magnetic particles for the separation and purification of nucleic acids[J]. Appl. Microbiol. Biotechnol., 2006, 73(3):495-504.

[1]	唐广宣, 何莉薇, 陈泳妤, 张佳研, 戴雅琪, 吕辉雄, 刘丽辉. 植物内生菌定殖检测技术及其应用[J]. 生物技术进展, 2023, 13(1): 55-64.
[2]	李晶晶, 赵海波, 翟睿, 梁亮, 范培蕾, 赵雨佳. 糖化血红蛋白（HbA_1c）检测技术与标准化研究进展[J]. 生物技术进展, 2022, 12(6): 837-846.
[3]	于鲲, 薛佳琪, 王进宽, 余永涛. CRISPR/Cas9基因编辑技术在丝状真菌中的应用[J]. 生物技术进展, 2022, 12(5): 696-704.
[4]	黄耀辉, 樊殿峰, 焦悦, 吴小智, 叶纪明. 浅谈多国转基因产品标识制度对我国的启示[J]. 生物技术进展, 2022, 12(4): 516-522.
[5]	刘婷婷, 仝涛, 黄昆仑. 转基因玉米的研究进展和食用安全性评价[J]. 生物技术进展, 2022, 12(4): 523-531.
[6]	高维崧, 窦金萍, 韦双, 刘兴健, 张志芳, 李轶女. CRISPR/Cas系统的分类及研究现状[J]. 生物技术进展, 2022, 12(4): 532-538.
[7]	费云燕, 杨军, 景德道, 林添资, 李闯, 钱华飞, 曾生元, 韩华新, 龚红兵. CRISPR/Cas技术在抗除草剂作物育种中的研究与应用进展[J]. 生物技术进展, 2022, 12(2): 189-197.
[8]	余卉茹, 纪艺, 彭城, 徐晓丽, 汪小福, 孙梅好, 陈笑芸. 肉类掺假检测技术研究进展[J]. 生物技术进展, 2022, 12(2): 213-221.
[9]	曹巧, 史占良, 张国丛, 班进福, 郑树松, 傅晓艺, 张士昌, 何明琦, 韩然, 高振贤. CRISPR/Cas9技术在小麦育种中的应用进展[J]. 生物技术进展, 2021, 11(6): 661-667.
[10]	李梦杰, 贺晓云, 仝涛, 梁晋刚, 黄昆仑. 阿根廷转基因作物安全管理制度概况及进展[J]. 生物技术进展, 2021, 11(6): 676-687.
[11]	王梦雨, 王颢潜, 王旭静, 王志兴. 基因编辑产品检测技术研究进展[J]. 生物技术进展, 2021, 11(4): 438-445.
[12]	王子健, 陈颖钰, 胡长敏, 郭爱珍. 我国兽用诊断试剂产业现状与未来趋势[J]. 生物技术进展, 2021, 11(4): 471-475.
[13]	权春菊, 郑忠亮. CRISPR/Cas及其衍生编辑技术在基因治疗中的应用进展[J]. 生物技术进展, 2021, 11(4): 518-525.
[14]	焦悦,朱鹏宇,梁晋刚,付伟. 国际转基因食品安全评价政策及启示[J]. 生物技术进展, 2021, 11(2): 121-127.
[15]	刘肖静,王旭静,王志兴. CRISPR-Cas系统在植物中的研究进展与监管政策[J]. 生物技术进展, 2021, 11(1): 1-8.

基于CRISPR/Cas原理的转基因产品检测技术研究进展

Research Progress of Detection Technology of Genetically Modified Products Based on CRISPR/Cas Principle

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 5

参考文献 39

相关文章 15

编辑推荐

Metrics

本文评价