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

Ratz, M., Testa, I., Hell, S. W., & Jakobs, S. (2015). CRISPR/Cas9-mediated endogenous protein tagging for RESOLFT super-resolution microscopy of living human cells. Scientific Reports, 5: 9592. doi:10.1038/srep09592.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0026-C081-C Version Permalink: http://hdl.handle.net/11858/00-001M-0000-002A-19BC-0
Genre: Journal Article

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 Creators:
Ratz, M.1, Author              
Testa, I.2, Author              
Hell, S. W.2, Author              
Jakobs, S.1, Author              
Affiliations:
1Research Group of Mitochondrial Structure and Dynamics, MPI for Biophysical Chemistry, Max Planck Society, ou_578566              
2Department of NanoBiophotonics, MPI for biophysical chemistry, Max Planck Society, ou_578627              

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 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.

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Language(s): eng - English
 Dates: 2015-04-22
 Publication Status: Published online
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 Rev. Method: Peer
 Identifiers: DOI: 10.1038/srep09592
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Title: Scientific Reports
Source Genre: Journal
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Pages: 6 Volume / Issue: 5 Sequence Number: 9592 Start / End Page: - Identifier: -