U2OS-CRISPR-SNAPf-Nup133 Cells

U2OS-CRISPR-SNAPf-Nup133 is a genetically engineered human osteosarcoma cell line derived from the parental U2OS background in which the endogenous NUP133 locus has been modified using CRISPR/Cas9-mediated genome editing to encode a C-terminal SNAPf tag. NUP133 is a core component of the Y-complex (NUP107-160 complex), a structural subcomplex essential for nuclear pore complex (NPC) assembly and maintenance. By introducing the SNAPf coding sequence in-frame at the endogenous locus, the fusion protein is expressed under native regulatory control, preserving physiological expression levels and subcellular localization.

The SNAPf tag is a fast-labeling variant of the SNAP-tag, an engineered O6-alkylguanine-DNA alkyltransferase that covalently reacts with benzylguanine-conjugated substrates. This enables highly specific and versatile fluorescent labeling of Nup133 in live or fixed cells using cell-permeable or impermeable SNAP substrates. In U2OS-CRISPR-SNAPf-Nup133 cells, the fusion protein localizes to the nuclear envelope in a punctate pattern characteristic of nuclear pore complexes. Because tagging occurs at the endogenous locus, NPC stoichiometry and architecture are minimally perturbed, making this model suitable for quantitative super-resolution microscopy, single-molecule tracking, and kinetic analyses of NPC assembly and turnover.

This cell line provides a robust platform for studying nuclear transport, nucleocytoplasmic trafficking dynamics, NPC biogenesis during interphase and post-mitotic nuclear reassembly, and structural organization of the Y-complex within the pore scaffold. The U2OS background offers flat morphology and large nuclei, facilitating high-resolution imaging. U2OS-CRISPR-SNAPf-Nup133 cells are particularly well suited for pulse-chase labeling experiments, correlative light and electron microscopy, and multicolor imaging approaches in combination with additional endogenously tagged nucleoporins or transport factors.