CRISPR/Cas9-mediated endogenous protein tagging for RESOLFT super-resolution microscopy of living human cells.

Michael Ratz,Stefan W Hell,Stefan Jakobs,Ilaria Testa

doi:10.1038/srep09592

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.

Highlights

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
We demonstrate the use of CRISPR/Cas[9] genome editing to generate heterozygous and homozygous human cell lines expressing rsEGFP2 fusion proteins
We found that single cell sorting by fluorescence activated cell sorting (FACS) allows a selection of positive clonal cell lines even at low integration efficiencies of less than 1%, rendering the generation of fluorescent protein knockin cell lines an accomplishable task even for non-specialized labs

Summary

Introduction

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. There is substantial evidence that transiently or constitutively overexpressed proteins may cause a multitude of artifacts including mislocalizations and protein aggregation[1], aberrant organelle morphology[2,3], violated balanced gene dosage[4], and others[5] These overexpression induced problems are presumably even more articulate in studies using diffraction-unlimited super-resolution microscopy, or nanoscopy, which facilitates the visualization of protein localizations and dynamics on a length scale inaccessible by conventional light microscopy. CRISPR/Cas[9] has so far not been used for locus-specific fluorescent protein integration into human cells Such knockin cell lines would be expected to alleviate most problems otherwise associated to plasmid-driven overexpression allowing quantitative studies of protein dynamics at native expression levels[7]

Methods

Results

Conclusion