Related Experiment Video
Updated: Sep 9, 2025
Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation
Published on: March 5, 2018
A Facile Method for Assessing Intra Cellular Stability and Co-localization of Cas9 mRNA and sgRNA Using Confocal
Siyu Chen1, Simone Pinto Carneiro1, Olivia M Merkel2
1Ludwig-Maximilians-University, Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Munich, Germany.
Abstract:
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.
