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CRISPR/Cas9-mediated Knock-in of an Optimized TetO Repeat for Live Cell Imaging of Endogenous Loci

Ipek Tasan, Gabriela Sustackova, Liguo Zhang, Jiah Kim, Mayandi Sivaguru, Mohammad HamediRad, Yuchuan Wang, Justin Genova, Jian Ma, Andrew S. Belmont, View ORCID ProfileHuimin Zhao
doi: https://doi.org/10.1101/162156
Ipek Tasan
1Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
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Gabriela Sustackova
2Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
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Liguo Zhang
2Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
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Jiah Kim
2Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
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Mayandi Sivaguru
3Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
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Mohammad HamediRad
3Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
4Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
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Yuchuan Wang
5Computational Biology Department, School of Computer Science, Carnegie Mellon University, Pittsburgh, PA 15213, USA
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Justin Genova
4Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
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Jian Ma
5Computational Biology Department, School of Computer Science, Carnegie Mellon University, Pittsburgh, PA 15213, USA
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Andrew S. Belmont
2Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
3Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
6Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
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Huimin Zhao
1Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
3Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
4Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
6Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
7Department of Chemistry, University of Illinois at Urbana- Champaign, Urbana, IL 61801, USA
8Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
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  • ORCID record for Huimin Zhao
  • For correspondence: zhao5@illinois.edu
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Abstract

Nuclear organization has an important role in determining genome function; however, it is not clear how spatiotemporal organization of the genome relates to functionality. To elucidate this relationship, a high-throughput method for tracking any locus of interest is desirable. Here, we report an efficient and scalable method named SHACKTeR (Short Homology and CRISPR/Cas9-mediated Knock-in of a TetO Repeat) for live cell imaging of specific chromosomal regions. Compared to alternatives, our method does not require a nearby repetitive sequence and it requires only two modifications to the genome: CRISPR/Cas9-mediated knock-in of an optimized TetO repeat and its visualization by TetR-EGFP expression. Our simplified knock-in protocol, utilizing short homology arms integrated by PCR, was successful at labeling 9 different loci in HCT116 cells with up to 20% efficiency. These loci included both nuclear speckle-associated, euchromatin regions and nuclear lamina-associated, heterochromatin regions. We anticipate the general applicability and scalability of our method will enhance causative analyses between gene function and compartmentalization in a high-throughput manner.

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Posted July 12, 2017.
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CRISPR/Cas9-mediated Knock-in of an Optimized TetO Repeat for Live Cell Imaging of Endogenous Loci
Ipek Tasan, Gabriela Sustackova, Liguo Zhang, Jiah Kim, Mayandi Sivaguru, Mohammad HamediRad, Yuchuan Wang, Justin Genova, Jian Ma, Andrew S. Belmont, Huimin Zhao
bioRxiv 162156; doi: https://doi.org/10.1101/162156
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CRISPR/Cas9-mediated Knock-in of an Optimized TetO Repeat for Live Cell Imaging of Endogenous Loci
Ipek Tasan, Gabriela Sustackova, Liguo Zhang, Jiah Kim, Mayandi Sivaguru, Mohammad HamediRad, Yuchuan Wang, Justin Genova, Jian Ma, Andrew S. Belmont, Huimin Zhao
bioRxiv 162156; doi: https://doi.org/10.1101/162156

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