Harnessing CRISPR/Cas12a Activity and DNA-Based Ultrabright FluoroCube for <i>In Situ</i> Imaging of Metabolically Labeled Cell Membrane Glycoproteins.
basic_science · Level V
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- Record sourced from PubMed, PMID 39470128.
- Also identified by DOI 10.1021/acs.nanolett.4c03605.
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Abstract
Fluorescence imaging of cell membrane glycoproteins based on metabolic labeling faces challenges including the sensitivity and spatial specificity and the use of a high concentration of unnatural sugars. To overcome these limitations, we developed a method for <i>in situ</i> imaging of cell membrane glycoproteins by operating Cas12a activity, and employing the ultrabright DNA nanostructure, FluoroCube (FC), as a signal reporter. Following Cas12a activation, we observed stable and intense fluorescence signals within 15 min. The combination of bright FC and Cas12a's amplification capability allows for effective imaging with only 5 μM of unnatural sugars and a brief 24-h incubation. Computational modeling demonstrates that Cas12a specifically cleaves FC in the 11-17 nm range of the glycosylation site, enabling spatially precise imaging. This approach successfully enabled fluorescence imaging of glycoproteins across various cell lines and the detection of changes in glycoprotein levels induced by drugs.
Medical subject headings
- CRISPR-Cas Systems
- DNA
- Glycoproteins