CRISPR/Cas系统在基因修饰植物及其产品检测应用中的原理和进展

doi:10.3969/j.issn.1006-8082.2023.06.005

摘要/Abstract

摘要：

转基因及基因编辑的基因修饰植物发展迅猛，相关的检测技术也面临更多挑战，尤其是对于无外源DNA引入的基因编辑产品，其检测难度更高，传统的检测技术已无法满足。基于CRISPR/Cas检测系统中不同Cas蛋白所具有的附属非特异切割活性被广泛应用于分子检测领域，它与LAMP和RPA等温扩增相结合可实现准确快速的现场检测，甚至是对单核苷酸突变的基因编辑产品识别和检测。但是，该检测系统在植物基因修饰产品检测中的应用才刚刚起步，本文对该检测系统的原理及其在基因修饰的植物及其产品中的应用进行了评述。

关键词: CRISPR/Cas系统, 分子检测, 转基因, 基因编辑, 基因修饰植物

Abstract:

With the rapid development of genetically modified plants including transgenic and gene-edited plants, the related detection technologies are also facing more challenges, especially for gene editing products without the introduction of exogenous DNA, its detection is more difficult, and the traditional detection technology cannot be satisfied. The accessory nonspecific cleavage activity of different Cas proteins in the CRISPR-Cas-based detection system is widely used in the field of molecular detection. It can be combined with LAMP and RPA isothermal amplification to achieve accurate and rapid on-site detection, even for single gene editing products for nucleotide mutations. However, the application of the detection system in the detection of plant genetically modified products has just started. This paper reviews the principle of the detection system and its application in genetically modified plants and their products.

Key words: CRISPR/Cas system, molecular detection, transgenic oranginism, gene-edting, genetically modified plants

中图分类号:

Q789
S511

王渭霞, 朱廷恒, 赖凤香, 万品俊, 魏琪, 傅强. CRISPR/Cas系统在基因修饰植物及其产品检测应用中的原理和进展[J]. 中国稻米, 2023, 29(6): 21-27.

WANG Weixia, ZHU Tingheng, LAI Fengxiang, WAN Pingjun, WEI Qi, FU Qiang. Principle and Progress of CRISPR/Cas System in the Detection of Genetically Modified Plants and Their Products[J]. China Rice, 2023, 29(6): 21-27.

图/表 5

图1 三种Cas蛋白附属切割活性进行分子诊断的示意图[9] 用于 SHERLOCK (Cas13a)、DETECTR (Cas12a) 或 Cas4-DETECTR (Cas14a) 中核酸检测的 CRISPR 效应子核酸酶示意图。在不存在其靶标核酸的情况下，Cas 核酸酶是无活性的。当有靶标核酸时， crRNA 与同源靶标（Cas13a 的 ssRNA、Cas12a 的 dsDNA、Cas14a 的 ssDNA）结合，核酸酶被激活，导致附近核酸（Cas13a 的 ssRNA、Cas12a 和 Cas14a 的 ssDNA）的靶标切割和附属切割。这种“附属”核酸酶活性通过与报告探针提供荧光团（通常是 FAM），寡核苷酸与猝灭剂（或生物素，用于后续流动分析）相连，转化为放大的信号。附属切割活性启动切割报告基因后，报告基因核苷酸会断裂，从而发出明亮的荧光或侧向流动试纸条检测线处出现条带。在 SHERLOCK 和 DETECTR 中，使用 RPA等进行等温预扩增可以提高目标丰度。

图2 利用Cas14系统检测人类HERC2基因单碱基突变的示意图 HERC2基因褐色眼睛和蓝色眼睛的单碱基突变位点分别用黑色斜体下划线和黑色斜体显示。

图3 基于Cas12a的侧向流动检测示意图[33] FITC/FAM：荧光素标记FITC（异硫氰酸荧光素）最大吸收光波长为490~495 nm，最大发射光波长为520~530 nm。FAM （羧基荧光素）吸收波长492 nm ，发射波长518 nm；Anti-FITC/FAM Antibody：可以特异性结合FITC/FAM的单克隆抗体，与金颗粒结合；Biotin ligand，生物素配体；Reporter：报告子， 5’端用荧光素标记、3’ 端用生物素标记的ssDNA（5-6-FAM-ttattttattttatt-Biotin-3）；当RPA反应产物中有目标序列扩增， sgRNA与目标序列的配对会引导Cas12a蛋白切割目标序列并激活附属切割活性，使得FITC/FAM的单克隆抗体+金颗粒结合+报告DNA分子复合物中的荧光素段与生物素段发生分离，随着毛细电泳各自上移，带有生物素标记单链段被控制线处生物素配体拦截，显黑色。金颗粒携带的荧光素标记报告子+荧光素FITC/FAM的单克隆抗体继续上移与检测线处二抗抗体结合，检测线处显黑色，检测结果阳性；如果样品中没有目标序列，则crRNA不能与目标序列配对，Cas12a不会产生切割活性，报告子的荧光素和生物素不发生分离，则金颗粒携带的荧光素标记抗体和荧光素及生物素标记报告子上移到控制线生物素配体处就与生物素配体结合，报告子拦截在此，检测线处二抗抗体则没有机会与荧光素FITC/FAM单克隆抗体发生结合，而不显色，结果为阴性。

图4 Cry1Ac的sgRNA1序列合成示意图(A)和二级结构（B）阴影部分为PAM序列；小写字母为靶标序列（227~250 bp）；小写下划线字母为与靶标序列互补的引导序列；黑色大写字母为sgRNA的保守茎环序列，大写斜体字母为T7启动子序列。

图5 CaMV35S的sgRNA序列二级结构和CaMV35S扩增子的对应靶标DNA序列示意图 sgRNA序列中与CaMV35S目标序列对应的序列标记为小写字母，形成sgRNA茎环结构的核心序列标记为黑色大写字母；与sgRNA序列中CaMV35S目标序列对应的序列标记为小写下划线， PAM序列用阴影标记。

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