Chapter 20
Live-Cell CRISPR Imaging in Plant Cells
with a Telomere-Specific Guide RNA
Solmaz Khosravi, Steven Dreissig, Patrick Schindele, Felix Wolter,
Twan Rutten, Holger Puchta, and Andreas Houben
Abstract
Chromatin organization is highly dynamic in living cells. Therefore, it might have a regulatory role over
biological mechanisms like transcription, replication, and DNA repair. To elucidate how these mechanisms
are regulated, it is required to establish imaging methods to visualize the chromatin dynamic in living cells.
Here, we provide a protocol for a live plant cell imaging technique based on application of two orthologs of
the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein
9 (Cas9) from Streptococcus pyogenes and Staphylococcus aureus. This technique uses the inactive variants
of Cas9 combined with different fluorescent proteins (GFP and mRuby) and telomere-specific guide RNA
to target telomeric repeats in Nicotiana benthamiana. Our immuno-FISH data revealed that signals arising
from the CRISPR/dCas9 method are specifically belonging to telomeric regions.
Key words Chromatin organization, Live-cell imaging, CRISPR/dCas9, Telomere, Nicotiana
benthamiana, Guide RNA
1 Introduction
Structural and spatial organization of chromatin affects gene regulation and pivotal processes like recombination and DNA repair
mechanisms. Eventually, studying chromatin structural changes in
different tissues over time has always been of interest to decipher
these regulatory mechanisms [1]. Our knowledge about the 3D
organization of chromatin is mainly based on fixed specimens.
Although imaging methods including fluorescence in situ hybridization (FISH) have been well applied to study subnuclear dynamics, harsh treatment of cells during FISH like heat-based
denaturation could provide data only from perturbed chromatin
structure which is not indicative of its structure in living cells
[2]. To overcome this problem, live chromatin imaging techniques
were developed to enable studying of biological compartments in
their native context [3].
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_20, © Springer Science+Business Media, LLC, part of Springer Nature 2020
343
Live-Cell CRISPR Imaging in Plant Cells
with a Telomere-Specific Guide RNA
Solmaz Khosravi, Steven Dreissig, Patrick Schindele, Felix Wolter,
Twan Rutten, Holger Puchta, and Andreas Houben
Abstract
Chromatin organization is highly dynamic in living cells. Therefore, it might have a regulatory role over
biological mechanisms like transcription, replication, and DNA repair. To elucidate how these mechanisms
are regulated, it is required to establish imaging methods to visualize the chromatin dynamic in living cells.
Here, we provide a protocol for a live plant cell imaging technique based on application of two orthologs of
the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein
9 (Cas9) from Streptococcus pyogenes and Staphylococcus aureus. This technique uses the inactive variants
of Cas9 combined with different fluorescent proteins (GFP and mRuby) and telomere-specific guide RNA
to target telomeric repeats in Nicotiana benthamiana. Our immuno-FISH data revealed that signals arising
from the CRISPR/dCas9 method are specifically belonging to telomeric regions.
Key words Chromatin organization, Live-cell imaging, CRISPR/dCas9, Telomere, Nicotiana
benthamiana, Guide RNA
1 Introduction
Structural and spatial organization of chromatin affects gene regulation and pivotal processes like recombination and DNA repair
mechanisms. Eventually, studying chromatin structural changes in
different tissues over time has always been of interest to decipher
these regulatory mechanisms [1]. Our knowledge about the 3D
organization of chromatin is mainly based on fixed specimens.
Although imaging methods including fluorescence in situ hybridization (FISH) have been well applied to study subnuclear dynamics, harsh treatment of cells during FISH like heat-based
denaturation could provide data only from perturbed chromatin
structure which is not indicative of its structure in living cells
[2]. To overcome this problem, live chromatin imaging techniques
were developed to enable studying of biological compartments in
their native context [3].
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_20, © Springer Science+Business Media, LLC, part of Springer Nature 2020
343