Visualization of specific DNA sequences in living mouse embryonic stem cells with a programmable fluorescent CRISPR/Cas system

Abstract

Labeling and tracing of specific sequences in living cells has been a major challenge in studying the spatiotemporal dynamics of native chromatin. Here we repurposed the prokaryotic CRISPR/Cas adaptive immunity system to specifically detect endogenous genomic loci in mouse embryonic stem cells. We constructed a catalytically inactive version of the Cas9 endonuclease, fused it with eGFP (dCas9-eGFP) and co-expressed small guide RNAs (gRNAs) to target pericentric, centric, and telomeric repeats, which are enriched in distinct nuclear structures. With major satellite specific gRNAs we obtained a characteristic chromocenter (CC) pattern, while gRNAs targeting minor satellites and telomeres highlighted smaller foci coinciding with centromere protein B (CENP-B) and telomeric repeat-binding factor 2 (TRF2), respectively. DNA sequence specific labeling by gRNA/dCas9-eGFP complexes was directly shown with 3D-fluorescent in situ hybridization (3D-FISH). Structured illumination microscopy (3D-SIM) of gRNA/dCas9-eGFP expressing cells revealed chromatin ultrastructures and demonstrated the potential of this approach for chromatin conformation studies by super resolution microscopy. This programmable dCas9 labeling system opens new perspectives to study functional nuclear architecture.

Keywords: :

• CRISPR/Cas9
• embryonic stem cells
• DNA labeling
• 3D-fluorescent in situ hybridization (3D-FISH)
• major satellite repeats
• minor satellite repeats
• telomeres
• CENP-B
• TRF2
• 3D-SIM

10.4161/nucl.2848

Disclosure of Potential Conflicts of Interest

No potential conflict of interest was disclosed.

Acknowledgments

The authors thank Katharina Thanisch (LMU Munich) for kindly providing the pEX-U6-gRNA cloning vector, George Church (Harvard Medical School) for the hCas9_D10A construct and En Li (China Novartis Institutes for BioMedical Research) for the J1 cell line. We also thank Irina Solovei (LMU Munich) for advice with FISH protocols. This work was supported by the Deutsche Forschungsgemeinschaft (DFG, SFB 1064, Nanosystems Initiative Munich, NIM) and T.A. is a fellow of the Graduiertenkolleg GRK1721.