摘要：SummarySingle-molecule localization microscopy (SMLM) reports on protein organization in cells with near-molecular resolution and in combination with stoichiometric labeling enables protein counting. Fluorescent proteins allow stoichiometric labeling of cellular proteins; however, most methods either lead to overexpression or are complex and time demanding. We introduce CRISPR/Cas12a for simple and efficient tagging of endogenous proteins with a photoactivatable protein for quantitative SMLM and single-particle tracking. We constructed a HEK293T cell line with the receptor tyrosine kinase MET tagged with mEos4b and demonstrate full functionality. We determine the oligomeric state of MET with quantitative SMLM and find a reorganization from monomeric to dimeric MET upon ligand stimulation. In addition, we measured the mobility of single MET receptorsin vivoin resting and ligand-treated cells. The combination of CRISPR/Cas12a-assisted endogenous protein labeling and super-resolution microscopy represents a powerful tool for cell biological research with molecular resolution.Graphical AbstractDisplay OmittedHighlights•CRISPR/Cas12a enables endogenous protein labeling for super-resolution microscopy•HEK293T cells were generated with MET endogenously labeled with mEos4b•Quantitative PALM microscopy reports efficient dimerization of MET receptor•Single-particle tracking shows increased MET immobilization upon ligand treatmentBiochemistry; Molecular Biology; Biophysics; Biological Sciences Research Methodologies
