Format

Send to

Choose Destination
Methods Mol Biol. 2018;1767:395-415. doi: 10.1007/978-1-4939-7774-1_22.

Establishment of Cell Lines Stably Expressing dCas9-Fusions to Address Kinetics of Epigenetic Editing.

Author information

1
Epigenetic Editing, Department of Pathology and Medical Biology, University of Groningen, University Medical CenterGroningen, Groningen, The Netherlands.
2
Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary.
3
Doctoral School in Biology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary.
4
Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands. m.g.rots@umcg.nl.

Abstract

Epigenetic editing is a promising approach to modulate the local chromatin environment of target genes with the ultimate goal of stable gene expression reprogramming. Epigenetic editing tools minimally consist of a DNA-binding domain and an effector domain. The CRISPR/dCas9 platform, where mutations in the nuclease domains render the Cas9 protein inactive, is widely used to guide epigenetic effectors to their intended genomic loci. Its flexible nature, simple use, and relatively low cost have revolutionized the research field of epigenetic editing. Although effective expression modulation is readily achieved, only a few studies have addressed the maintenance of the induced effects on endogenous loci. Here, we describe a detailed protocol to engineer cells that stably express the CRISPR/dCas9-effectors. The protocol involves modification of published dCas9-based plasmid vectors for easy transfer of the effector domain between the vector designed for transient transfection and the vector used for establishing cell lines stably expressing the dCas9-effector fusion protein. Transient transfection of the dCas9-effector-producing cells with sgRNA-expressing plasmids allows studying of the maintenance of epigenetic editing. Targeting various genes in different chromatin contexts and/or co-targeting multiple CRISPR/dCas9-effectors can be used to unravel rules underlying maintained gene expression reprogramming.

KEYWORDS:

CRISPR/dCas9; Epigenetic memory; Epigenetic reprogramming; Fusion proteins

PMID:
29524148
DOI:
10.1007/978-1-4939-7774-1_22
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Springer
Loading ...
Support Center