Harnessing CRISPR/Cas12a Activity and DNA-Based Ultrabright FluoroCube for In Situ Imaging of Metabolically Labeled Cell Membrane Glycoproteins

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 in situ 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.