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. 2025:2965:455-466.
doi: 10.1007/978-1-0716-4742-4_23.

A Facile Method for Assessing Intra Cellular Stability and Co-localization of Cas9 mRNA and sgRNA Using Confocal Microscopy

Siyu Chen  1 , Simone Pinto Carneiro  1 , Olivia M Merkel  2
Affiliations

A Facile Method for Assessing Intra Cellular Stability and Co-localization of Cas9 mRNA and sgRNA Using Confocal Microscopy

Siyu Chen et al. Methods Mol Biol. 2025.

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.

Keywords: CRISPR-Cas9; Confocal microscopy; Fluorescence labeling; Gene editing; Single guide RNA; mRNA delivery; mRNA localization.

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