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

  • Solmaz Khosravi
  • Takayoshi Ishii
  • Steven Dreissig
  • Andreas HoubenEmail author
Review
First Online:

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.

Keywords

chromatin dynamics CRISPR/Cas9 FISH live cell imaging RGEN-ISL telomere 

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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Notes

Funding information

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

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© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Leibniz Institute of Plant Genetics and Crop Plant Research (IPK)SeelandGermany
  2. 2.Arid Land Research Center (ALRC)Tottori UniversityTottoriJapan
  3. 3.Institute of Agricultural and Nutritional SciencesMartin Luther University Halle-WittenbergHalleGermany

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