title: CRISPR/Cas9-mediated endogenous protein tagging for RESOLFT super-resolution microscopy of living human cells.
abstract: Overexpression is a notorious concern in conventional and especially in super-resolution fluorescence light microscopy studies because it may cause numerous artifacts including ectopic sub-cellular localizations, erroneous formation of protein complexes, and others. Nonetheless, current live cell super-resolution microscopy studies generally rely on the overexpression of a host protein fused to a fluorescent protein. Here, we establish CRISPR/Cas9-mediated generation of heterozygous and homozygous human knockin cell lines expressing fluorescently tagged proteins from their respective native genomic loci at close to endogenous levels. We tagged three different proteins, exhibiting various localizations and expression levels, with the reversibly switchable fluorescent protein rsEGFP2. We demonstrate the benefit of endogenous expression levels compared to overexpression and show that typical overexpression-induced artefacts were avoided in genome-edited cells. Fluorescence activated cell sorting analysis revealed a narrow distribution of fusion protein expression levels in genome-edited cells, compared to a pronounced variability in transiently transfected cells. Using low light intensity RESOLFT (reversible saturable optical fluorescence transitions) nanoscopy we show sub-diffraction resolution imaging of living human knockin cells. Our strategy to generate human cell lines expressing fluorescent fusion proteins at endogenous levels for RESOLFT nanoscopy can be extended to other fluorescent tags and super-resolution approaches.
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077 Göttingen, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Ratz
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077 Göttingen, Germany.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Testa
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077 Göttingen, Germany.
authors.forename: Stefan W
authors.initials: SW
authors.lastname: Hell
authors.affiliation: 1] Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077 Göttingen, Germany [2] University of Göttingen Medical Faculty, Dept. of Neurology, Robert-Koch-Str. 40, 37075 Göttingen, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Jakobs
mesh_terms: D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D055879:Gene Knock-In Techniques; D056426:Genetic Loci; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D025741:HMGA1a Protein / Q000235:genetics; D006801:Humans; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014746:Vimentin / Q000235:genetics; D060589:Zyxin / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D014746:Vimentin; C092842:ZYX protein, human; D060589:Zyxin; C485184:enhanced green fluorescent protein; D025741:HMGA1a Protein; D049452:Green Fluorescent Proteins