title: Dynamics of CDKN1A in Single Cells Defined by an Endogenous Fluorescent Tagging Toolkit.
abstract: Observing the endogenous abundance, localization, and dynamics of proteins in mammalian cells is crucial to understanding their function and behavior. Currently, there is no systematic approach for the fluorescent tagging of endogenous loci. Here, we used Cas9-catalyzed DNA breaks, short homology arms, and a family of donor plasmids to establish endogenous Fluorescent tagging (eFlut): a low-cost and efficient approach to generating endogenous proteins with fluorescent labels. We validated this protocol on multiple proteins in several cell lines and species and applied our tools to study the cell-cycle inhibitor CDKN1A in single cells. We uncover heterogeneity in the timing and rate of CDKN1A induction post-DNA damage and show that this variability is post-transcriptionally regulated, depends on cell-cycle position, and has long-term consequences for cellular proliferation. The tools developed in this study should support widespread study of the dynamics and localization of diverse proteins in mammalian cells.
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA. Electronic address: jacob_stewart-ornstein@hms.harvard.edu.
authors.forename: Jacob
authors.initials: J
authors.lastname: Stewart-Ornstein
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA. Electronic address: galit@hms.harvard.edu.
authors.forename: Galit
authors.initials: G
authors.lastname: Lahav
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D000076987:CRISPR-Associated Protein 9; D002374:Catalase / Q000235:genetics / Q000378:metabolism; D059447:Cell Cycle Checkpoints / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics* / Q000378:metabolism; D004249:DNA Damage; D004285:Dogs; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D005456:Fluorescent Dyes / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D057809:HEK293 Cells; D022781:Hepatocytes / Q000378:metabolism / Q000473:pathology; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D061985:Madin Darby Canine Kidney Cells; D051379:Mice; D041681:NIH 3T3 Cells; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D059010:Single-Cell Analysis; D013194:Staining and Labeling / Q000379:methods*; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; C102664:hydroperoxidase II; D002374:Catalase; D000076987:CRISPR-Associated Protein 9; C000600049:Cas9 protein, Francisella novicida; D004720:Endonucleases