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Generation and validation of homozygous fluorescent knock-in cells using CRISPR/Cas9 genome editing

Birgit Koch, Bianca Nijmeijer, Moritz Kueblbeck, Yin Cai, Nike Walther, View ORCID ProfileJan Ellenberg
doi: https://doi.org/10.1101/188847
Birgit Koch
1EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
2Max Planck Insitute for Medical Research, Jahnstraße 29, 69120 Heidelberg, Germany
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Bianca Nijmeijer
1EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
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Moritz Kueblbeck
1EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
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Yin Cai
1EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
3Roche SIS, Maybachstr. 30, 71332 Waiblingen, Germany
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Nike Walther
1EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
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Jan Ellenberg
1EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
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  • ORCID record for Jan Ellenberg
  • For correspondence: jan.ellenberg@embl.de
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Abstract

Gene tagging with fluorescent proteins is essential to investigate the dynamic properties of cellular proteins. CRISPR/Cas9 technology is a powerful tool for inserting fluorescent markers into all alleles of the gene of interest (GOI) and permits functionality and physiological expression of the fusion protein. It is essential to evaluate such genome-edited cell lines carefully in order to preclude off-target effects caused by either (i) incorrect insertion of the fluorescent protein, (ii) perturbation of the fusion protein by the fluorescent proteins or (iii) non-specific genomic DNA damage by CRISPR/Cas9. In this protocol1, we provide a step-by-step description of our systematic pipeline to generate and validate homozygous fluorescent knock-in cell lines.

We have used the paired Cas9D10A nickase approach to efficiently insert tags into specific genomic loci via homology-directed repair with minimal off-target effects. It is time- and cost-consuming to perform whole genome sequencing of each cell clone. Therefore, we have developed an efficient validation pipeline of the generated cell lines consisting of junction PCR, Southern Blot analysis, Sanger sequencing, microscopy, Western blot analysis and live cell imaging for cell cycle dynamics. This protocol takes between 6-9 weeks. Using this protocol, up to 70% of the targeted genes can be tagged homozygously with fluorescent proteins and result in physiological levels and phenotypically functional expression of the fusion proteins.

Editorial Summary This protocol provides a detailed workflow describing how to insert fluorescent markers into all alleles of a gene of interest using CRISPR/Cas 9 technology and how to generate and validate homozygous fluorescent knock-in cell lines.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted March 07, 2018.
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Generation and validation of homozygous fluorescent knock-in cells using CRISPR/Cas9 genome editing
Birgit Koch, Bianca Nijmeijer, Moritz Kueblbeck, Yin Cai, Nike Walther, Jan Ellenberg
bioRxiv 188847; doi: https://doi.org/10.1101/188847
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Generation and validation of homozygous fluorescent knock-in cells using CRISPR/Cas9 genome editing
Birgit Koch, Bianca Nijmeijer, Moritz Kueblbeck, Yin Cai, Nike Walther, Jan Ellenberg
bioRxiv 188847; doi: https://doi.org/10.1101/188847

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