Chinese Academy of Inspection and Quarantine, Beijing 100176, People's Republic of China.
Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100081, People's Republic of China.
J AOAC Int. 2023 May 3;106(3):558-567. doi: 10.1093/jaoacint/qsad022.
In recent years, genome editing technology represented by clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 （CRISPR/Cas9） has been developed and applied in transgenic research and development, and transgenic products have been developed for a variety of applications. Gene editing products, unlike traditional genetically modified crops, which are generally obtained by target gene deletion, insertion, base mutation, etc., may not differ significantly at the gene level from conventional crops, which increases the complexity of testing.
We established a specific and sensitive CRISPR/Cas12a-mediated gene editing system to detect target fragments in a variety of transgenic rice lines and commercial rice-based processing products.
In this study, the CRISPR/Cas12a visible detection system was optimized for the visualization of nucleic acid detection in gene-edited rice. The fluorescence signals were detected by both gel electrophoresis and fluorescence-based methods.
The detection limit of the CRISPR/Cas12a detection system established in this study was more precise, especially for low-concentration samples. In addition to achieving single-base detection in gene-edited rice, we showed that different base mutations in the target sequence have different detection efficiencies by sitewise variant compact analysis. The CRISPR/Cas12a system was verified via a common transgenic rice strain and commercial rice sources. The results proved that the detection method could not only be tested in samples with multiple mutation types but could also effectively detect target fragments in commercial rice products.
We have developed a set of efficient detection methods with CRISPR/Cas12a for gene-edited rice detection to provide a new technical basis for rapid field detection of gene-edited rice.
The CRISPR/Cas12a-mediated visual detection method used to detect gene-edited rice was evaluated for its specificity, sensitivity, and robustness.
近年来，以 CRISPR 相关核酸酶 9（CRISPR/Cas9）为代表的基因组编辑技术在转基因研发中得到了发展和应用，并开发出了多种应用的转基因产品。基因编辑产品与传统的转基因作物不同，其通常通过靶基因缺失、插入、碱基突变等方式获得，在基因水平上与常规作物可能差异不大，这增加了检测的复杂性。
我们建立了一种特异且敏感的 CRISPR/Cas12a 介导的基因编辑系统，用于检测各种转基因水稻品系和商业稻米加工产品中的目标片段。
在本研究中，优化了 CRISPR/Cas12a 可视化检测系统，用于基因编辑水稻中的核酸检测可视化。通过凝胶电泳和基于荧光的方法检测荧光信号。
本研究中建立的 CRISPR/Cas12a 检测系统的检测限更加精确，特别是对于低浓度的样本。除了能够实现基因编辑水稻中的单碱基检测外，我们还通过位点变异紧凑分析表明，目标序列中的不同碱基突变具有不同的检测效率。通过常见的转基因水稻品系和商业稻米来源验证了 CRISPR/Cas12a 系统。结果证明，该检测方法不仅可以在具有多种突变类型的样本中进行测试，而且可以有效地检测商业稻米产品中的目标片段。
我们开发了一套基于 CRISPR/Cas12a 的高效基因编辑水稻检测方法，为基因编辑水稻的快速现场检测提供了新的技术基础。
用于检测基因编辑水稻的 CRISPR/Cas12a 介导的可视化检测方法的特异性、灵敏度和稳健性进行了评估。
