一种基于定量PCR的CRISPR/Cas9基因编辑作物快速检测方法的研究

doi:10.19586/j.2095-2341.2023.0080

生物技术进展 ›› 2023, Vol. 13 ›› Issue (6): 907-912.DOI: 10.19586/j.2095-2341.2023.0080

• 研究论文 • 上一篇

一种基于定量PCR的CRISPR/Cas9基因编辑作物快速检测方法的研究

张笑天¹^,²(), 王智², 朱鹏宇², 魏霜³, 付伟¹^,², 黄春蒙², 李志红¹, 王慧煜², 焦悦⁴()

^1.中国农业大学植物保护学院，北京 100193
^2.中国检验检疫科学研究院，北京 100176
^3.广州海关技术中心，广州 510623
^4.农业农村部科技发展中心，北京 100122

收稿日期:2023-06-07 接受日期:2023-09-20 出版日期:2023-11-25 发布日期:2023-12-12
通讯作者: 焦悦
作者简介:张笑天 E-mail: Zhangxiaotian1229@163.com；
基金资助:
农业生物育种重大项目(2022ZD040200801)

A Rapid Detection Method Based on qPCR for CRISPR/Cas9 Edited Crops

Xiaotian ZHANG¹^,²(), Zhi WANG², Pengyu ZHU², Shuang WEI³, Wei FU¹^,², Chunmeng HUANG², Zhihong LI¹, Huiyu WANG², Yue JIAO⁴()

^1.College of Plant Protection，China Agricultural University，Beijing 100193，China
^2.Chinese Academy of Inspection and Quarantine，Beijing 100176，China
^3.Guangzhou Customs Technology Center，Guangzhou 510623，China
^4.Development Center for Science and Technology，Ministry of Agriculture and Rural Affairs，Beijing 100176，China

Received:2023-06-07 Accepted:2023-09-20 Online:2023-11-25 Published:2023-12-12
Contact: Yue JIAO

摘要/Abstract

摘要：

基因编辑技术自诞生以来，在作物育种、基因功能分析中得到了广泛的应用，其中CRISPR/Cas9系统是目前使用最多的基因编辑技术。基于荧光定量PCR技术，针对编辑位点设计特异性探针，结合基因组快速提取方法和成熟的水稻内参基因PLD引物，对自行研发的CRISPR/Cas9系统编辑水稻的Os11N3基因进行检测，并通过合成质粒模拟不同突变情况对该方法的特异性进行了验证。研究所建立的体系可在节约时间和成本的同时获取满足qPCR检测所需的基因组，能够区分1 bp基因编辑突变体与野生型，具有良好的特异性和灵敏度。检测方法可为作物育种筛选节省时间和成本，为基因编辑产品检测监管提供技术支持。

关键词: 基因编辑水稻, qPCR, 分子检测, CRISPR/Cas9

Abstract:

Gene editing technology has been widely used in crop breeding and gene function analysis since its birth. CRISPR/Cas9 system has been the most used gene editing technology at present. For detecting the Os11N3 gene of rice edited by the self-developed CRISPR/Cas9 system， this study designed specific Taqman probes for editing sites， combined with rapid genome extraction technology and mature rice PLD gene primer probes. The specificity of the method was verified by simulating different mutations with synthetic plasmids. The system established in this study could save time and cost while obtaining the genome that met the requirements of qPCR detection. It could distinguish at least 1 bp the gene-edited mutant from the wild type， with good specificity and sensitivity. This system can save time and cost for crop breeding and screening， and provide some technical support for gene editing product testing and supervision.

Key words: gene-edited rice, qPCR, molecular detection, CRISPR/Cas9

中图分类号:

张笑天, 王智, 朱鹏宇, 魏霜, 付伟, 黄春蒙, 李志红, 王慧煜, 焦悦. 一种基于定量PCR的CRISPR/Cas9基因编辑作物快速检测方法的研究[J]. 生物技术进展, 2023, 13(6): 907-912.

Xiaotian ZHANG, Zhi WANG, Pengyu ZHU, Shuang WEI, Wei FU, Chunmeng HUANG, Zhihong LI, Huiyu WANG, Yue JIAO. A Rapid Detection Method Based on qPCR for CRISPR/Cas9 Edited Crops[J]. Current Biotechnology, 2023, 13(6): 907-912.

图/表 8

图1 靶位点探针设计位置注：绿色部分为PAM序列，黄色部分为靶位点，蓝色划线部分为探针设计位置。

Fig. 1 Position of the target site probe

表1 荧光定量PCR所用引物探针

Table 1 Primers and probes used in fluorescent quantitative PCR

名称	引物/探针序列(5'→3')	T_m/℃	产物长度/bp
OS11N3-2T-F1	5'-CCCAAGAAGGCCAAGATGT-3'	62	142
OS11N3-2T-R1	5'-AGAAGCCAACGCAGACC-3'	62	142
OS11N3-2T-P6	5'-FAM-CCGCATCGTGGTTCTTGGTT-BHQ1-3'	69
PLD-F	5'-TGGTGAGCGTTTTGCAGTCT-3'	65	64
PLD-R	5'-CTGATCCACTAGCAGGAGGTCC-3'	65	64
PLD-P	5'-FAM-TGTTGTGCTGCCAATGTGGCCTG-BHQ1-3'	70

图2 质粒图谱及插入序列A：质粒图谱，红色代表插入序列；B：靶位点处差异序列，其中-代表缺失，红色部分代表替换或插入；C：PLD插入序列全长

Fig. 2 Plasmid map and insertion sequence

表2 3种基因组提取方法耗时、浓度、质量

Table 2 Time-consuming, concentration and quality of three genome extraction methods

提取方法	提取时间	样品	ND-1000/(ng·μL^-1)	基因组质量/ng	A₂₆₀/A₂₈₀	A₂₆₀/A₂₃₀
CTAB法	146 min	1	126.5	10 120	2.02	1.56
		2	176.7	14 136	1.96	1.89
		3	154.8	12 384	1.96	1.86
试剂盒	92 min	1	7.2	360	1.54	1.48
		2	8.6	430	1.52	1.51
		3	18.5	925	1.52	1.50
直提法	35 min	1	77.9	3 895	0.85	0.37
		2	45.0	2 250	0.77	0.29
		3	21.6	1 080	0.51	0.15

图3 3种提取方法所得基因组的PLD扩增曲线注：黄色曲线为CTAB法，红色曲线为直提法，草绿色曲线为试剂盒法。

Fig. 3 PLD amplification curve of the genome obtained from three extraction methods

图4 特异性评价荧光扩增曲线注：绿色为PLD扩增曲线，蓝色为靶位点扩增曲线，WT为亲本日本晴，M为19OSH-24、19OSH-32、19OSH-110、19OSH-105。

Fig. 4 Fluorescence amplification curve of specific evaluation

图5 水稻基因组测序结果注：-代表缺失，红色部分代表替换或插入。

Fig. 5 Sequencing results of rice genome

图6 灵敏度评价荧光扩增曲线注：绿色为PLD扩增曲线；蓝色为靶位点扩增曲线。

Fig. 6 Fluorescence amplification curve of sensitivity evaluation

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一种基于定量PCR的CRISPR/Cas9基因编辑作物快速检测方法的研究

A Rapid Detection Method Based on qPCR for CRISPR/Cas9 Edited Crops

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