Chen Siyu, Pinto Carneiro Simone, Merkel Olivia M
Ludwig-Maximilians-University, Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Munich, Germany.
Methods Mol Biol. 2025;2965:455-466. doi: 10.1007/978-1-0716-4742-4_23.
Messenger RNA (mRNA)-based CRISPR-Cas9 delivery is considered an advanced gene-editing strategy due to its rapid onset, transient expression, and reduced off-target effects, building on the success of mRNA therapeutics. However, challenges remain, particularly in efficiently co-delivering both Cas9 mRNA and single guide RNA (sgRNA). Here, we describe a straightforward fluorescence-labeling method for tracking the co-localization and stability of Cas9 mRNA and sgRNA in cells using confocal microscopy. This approach provides critical insights into optimizing the ratios and amounts of Cas9 mRNA and sgRNA during co-delivery. Furthermore, it enables a more intuitive investigation of metabolism and the kinetics of these components in cells after transfection, aiding the development of more effective delivery strategies.
基于信使核糖核酸（mRNA）的CRISPR-Cas9递送技术，凭借其起效迅速、表达短暂且脱靶效应降低的特点，在mRNA疗法成功的基础上，被视为一种先进的基因编辑策略。然而，挑战依然存在，尤其是在高效共递送Cas9 mRNA和单导向RNA（sgRNA）方面。在此，我们描述了一种简单的荧光标记方法，用于使用共聚焦显微镜追踪细胞中Cas9 mRNA和sgRNA的共定位及稳定性。该方法为优化共递送过程中Cas9 mRNA和sgRNA的比例及数量提供了关键见解。此外，它还能更直观地研究转染后这些成分在细胞中的代谢和动力学，有助于开发更有效的递送策略。
