U2OS-CRISPR-SNAPf-Nup358/RanBP2 Cells

U2OS-CRISPR-SNAPf-Nup358/RanBP2 is a genome-edited human osteosarcoma cell line derived from U2OS cells in which the endogenous RANBP2 (also known as NUP358) locus has been modified by CRISPR/Cas9 to encode a SNAPf tag in-frame with the native protein. Nup358/RanBP2 is a large nucleoporin localized to the cytoplasmic filaments of the nuclear pore complex (NPC) and plays critical roles in nucleocytoplasmic transport, SUMOylation, and mitotic processes. Endogenous tagging ensures that SNAPf-Nup358 is expressed under physiological promoter control, maintaining native expression levels and minimizing artifacts associated with overexpression systems.

The SNAPf tag is a fast-labeling variant of the SNAP-tag that covalently binds benzylguanine-conjugated substrates, enabling selective and stable fluorescent labeling of Nup358 in live or fixed cells. In U2OS-CRISPR-SNAPf-Nup358/RanBP2 cells, the fusion protein localizes to the nuclear envelope in a punctate distribution characteristic of cytoplasmic NPC filaments. This configuration supports high-resolution fluorescence imaging, super-resolution microscopy, pulse-chase labeling, and single-molecule tracking approaches to study NPC architecture and dynamics. The flat morphology and large nuclei of U2OS cells further facilitate quantitative imaging of nuclear envelope structures.

This model enables investigation of Nup358-specific roles in CRM1/exportin-dependent nuclear export, Ran GTPase cycle regulation, and the spatial organization of cytoplasmic transport platforms. Given the involvement of Nup358 in mitotic spindle assembly and kinetochore function, the cell line is also suitable for studying cell cycle-dependent redistribution of nucleoporins and NPC disassembly/reassembly during mitosis. U2OS-CRISPR-SNAPf-Nup358/RanBP2 provides a physiologically relevant platform for dissecting structural and functional aspects of the cytoplasmic face of the nuclear pore complex in human cells.