CRISPR/Cas12a-mediated labeling of MET receptor enables quantitative single-molecule imaging of endogenous protein organization and dynamics

•CRISPR/Cas12a enables endogenous protein labeling for super-resolution microscopy•HEK293T cells were generated with MET endogenously labeled mEos4b•Quantitative PALM microscopy reports efficient dimerization of receptor•Single-particle tracking shows increased immobilization upon ligand treatment Single-molecule localization (SMLM) on organization in near-molecular resolution and combination stoichiometric counting. Fluorescent proteins allow cellular proteins; however, most methods either lead to overexpression or are complex time demanding. We introduce CRISPR/Cas12a simple tagging a photoactivatable quantitative SMLM single-particle tracking. constructed HEK293T cell line the receptor tyrosine kinase tagged mEos4b demonstrate full functionality. determine oligomeric state find reorganization from monomeric dimeric stimulation. In addition, we measured mobility single receptors vivo resting ligand-treated cells. 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Sun Shtengel Viswanathan Baird Macklin J.J. Patel Allen J.R. Howe E.S. Piszczek al.Fixation-resistant CLEM.Nat. 12: 215-218Crossref (112) confirmed specific functionality PCR-tagged MET-mEos4b biochemical methods. microscopy, largely present monomer, whereas treated ligands hepatocyte growth (HGF) internalin B (InlB), dimers found. live-cell (SPT) experiments analyzed untreated HGF. summary, propose suitable endogenous, stoichiometric, labeling, SPT nanoscopic spatial resolution, assemblies, native dynamics. A recent study promising tagging, stable fused C-terminally (Figure 1A). two helper After selection CRISPR/Cas12a-tagged via puromycin, individual clones isolated verified fluorescence signals. Pronounced observed 10 16 tested. These divided five very moderate after 405 nm. clone randomly selected further PCRs amplified both inserted region (outside arms expected size found showing 1B; C6; duplicate), while other (high fluorescence) positive (data shown). Sequencing purified fragment C6 correct mEos4b, hereafter referred MET-mEos4b. Multiple copies chromosome polyploid mixed population untagged receptors, decreases efficiency chromosomal sites targeted. contain each (in total 64–70 chromosomes (Stepanenko 2015Stepanenko Andreieva Korets Mykytenko Huleyuk Vassetzky Kavsan Step-wise punctuated evolution drive phenotype changes tumor cells.Mutat. Res. 771: 56-69Crossref (24) Scholar)), still number compared lines (e.g., HeLa, 76–80 (Macville 1999Macville Schröck Padilla-Nash Keck Ghadimi B.M. Zimonjic Popescu Ried Comprehensive definitive cytogenetic characterization HeLa spectral karyotyping.Cancer 1999; 59: 141-150PubMed Scholar)). determined western blot shift band about 25–30 kDa wild-type 1C). reflects receptor. order fraction versus clone, quantified intensities 140 170 contains approximately 81% mEos4b-tagged 19% unlabeled MET. 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