Department of Analytical Chemistry, Faculty of Pharmacy, Ege University, Izmir 35100, Turkey.
Department of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster LA1 4YQ, UK.
Biosensors (Basel). 2021 Jan 8;11(1):17. doi: 10.3390/bios11010017.
The CRISPR-Cas9 system has facilitated the genetic modification of various model organisms and cell lines. The outcomes of any CRISPR-Cas9 assay should be investigated to ensure/improve the precision of genome engineering. In this study, carbon nanotube-modified disposable pencil graphite electrodes (CNT/PGEs) were used to develop a label-free electrochemical nanogenosensor for the detection of point mutations generated in the genome by using the CRISPR-Cas9 system. Carbodiimide chemistry was used to immobilize the 5'-aminohexyl-linked inosine-substituted probe on the surface of the sensor. After hybridization between the target sequence and probe at the sensor surface, guanine oxidation signals were monitored using differential pulse voltammetry (DPV). Optimization of the sensitivity of the nanogenoassay resulted in a lower detection limit of 213.7 nM. The nanogenosensor was highly specific for the detection of the precisely edited DNA sequence. This method allows for a rapid and easy investigation of the products of CRISPR-based gene editing and can be further developed to an array system for multiplex detection of different-gene editing outcomes.
CRISPR-Cas9 系统促进了各种模式生物和细胞系的基因修饰。应调查任何 CRISPR-Cas9 测定的结果，以确保/提高基因组工程的精确性。在这项研究中，使用碳纳米管修饰的一次性铅笔石墨电极 (CNT/PGE) 开发了一种无标记的电化学纳米基因传感器，用于检测由 CRISPR-Cas9 系统在基因组中产生的点突变。碳二亚胺化学用于将 5'-氨己基连接的肌苷取代探针固定在传感器表面。在传感器表面的目标序列和探针之间进行杂交后，使用差分脉冲伏安法 (DPV) 监测鸟嘌呤氧化信号。纳米基因测定的灵敏度的优化导致更低的检测限为 213.7 nM。纳米基因传感器对精确编辑的 DNA 序列的检测具有高度特异性。该方法允许快速简便地研究基于 CRISPR 的基因编辑的产物，并可进一步开发为用于多重检测不同基因编辑结果的阵列系统。
