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Application and prospects of CRISPR/Cas9-based methods to trace defined genomic sequences in living and fixed plant cells

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Abstract

The 3D organization of chromatin plays an important role in genome stability and many other pivotal biological programs. Therefore, the establishment of imaging methods, which enable us to study the dynamics of chromatin in living cells, is necessary. Although primary live cell imaging methods were a breakthrough, there is a need to develop more specific labeling techniques. With the discovery of programmable DNA binding proteins, such zinc finger proteins (ZFP), transcription activator-like effectors (TALE), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), a major leap forward was made. Here, we review the applications and potential of fluorescent repressor-operator systems, programmable DNA binding proteins with an emphasis on CRISPR-based chromatin imaging in living and fixed cells, and their potential application in plant science.

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Abbreviations

BIFC:

Bimolecular fluorescence complementation

CENH3:

Centromere specific histone H3

CRISPR:

Clustered regularly interspaced short palindromic repeats

Cas9:

CRISPR-associated protein 9

Cys2-His2:

Cysteine cysteine-histidine histidine

crRNA:

crisper RNA

dCas9:

dead Cas9

EdU:

5-Ethynyl-2′-deoxyuridine

FISH:

Fluorescence in situ hybridization

FROS:

Fluorescent repressor operator system

GFP:

Green fluorescent protein

gRNA:

guide RNA

LiveFISH:

Live-cell fluorescent in situ hybridization

Nm:

Neisseria meningitides

TALE:

Transcription activator-like effector

PAM:

Protospacer adjacent motif

PBS:

PUF binding site

PUF:

Pumilio/fem-3 mRNA binding factor

RGEN-ISL:

RNA-guided endonuclease-in situ labeling

RVD:

Repeat variable di-residue

scFv:

Single-chain variable fragment antibody

scFv-GCN4:

GCN4 peptide binding single-chain variable fragment antibody

Sp:

Streptococcus pyogenes

St1:

Streptococcus thermophilus

sgRNA:

single guide RNA

tracr-RNA:

Trans-activating RNA

ZFP:

Zinc finger proteins

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Funding

Cytogenetic research in the author’s laboratory has been supported by Deutsche Forschungsgemeinschaft (DFG) grant HO1779/28-1.

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Authors and Affiliations

  1. Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, 06466, Seeland, Germany

    Solmaz Khosravi, Steven Dreissig & Andreas Houben

  2. Arid Land Research Center (ALRC), Tottori University, 1390 Hamasaka, Tottori, 680-0001, Japan

    Takayoshi Ishii

  3. Institute of Agricultural and Nutritional Sciences, Martin Luther University Halle-Wittenberg, 06120, Halle, Germany

    Steven Dreissig

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  1. Solmaz Khosravi
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  2. Takayoshi Ishii
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  3. Steven Dreissig
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  4. Andreas Houben
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Correspondence to Andreas Houben.

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Khosravi, S., Ishii, T., Dreissig, S. et al. Application and prospects of CRISPR/Cas9-based methods to trace defined genomic sequences in living and fixed plant cells. Chromosome Res 28, 7–17 (2020). https://doi.org/10.1007/s10577-019-09622-0

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  • DOI: https://doi.org/10.1007/s10577-019-09622-0

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