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
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-editedcells. Fluorescence activatedcell sortinganalysis revealed anarrowdistribution 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.