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

Figure 1. Labeling genomic loci with specific dCas9-eGFP/gRNA complexes. (A) Schematic representation of the dCas9-eGFP expression construct. A chicken β-actin promoter with a CMV enhancer (CAG, blue triangle) drives the expression of dCas9-eGFP. Inactivation of RuvC1 and HNH (red crosses) by amino acid substitutions (D10A and H840A within RuvC1 and HNH, respectively) is indicated. A second nuclear localization signal (NLS) (beige) is introduced upstream of the eGFP coding sequence (green). (B) Outline of the experimental design. dCas9-eGFP interacts with a co-expressed gRNA and is thereby guided to the genomic target sequence. Note that the presence of a protospacer adjacent motif (PAM) is a prerequisite for dCas9 binding. (C) Schematic representation of a mouse acrocentric chromosome. gRNAs were designed to target 20 bp protospacer sequences of telomeres (Tel, green), major satellites (MaS, yellow) and minor satellites (MiS, red) as indicated. (D, E, F) Co-expression of dCas9-eGFP and gRNAs complementary to MaS repeats (MaSgRNA, D), MiS (MiSgRNA, E) and Tel gRNAs (TelgRNA, F) in J1 mouse embryonic stem cells. MaSgRNA recruits dCas9-eGFP to chromocenters (CCs), MiSgRNA/dCas9-eGFP signals are observed in the periphery of CCs, while targeting of TelgRNA/dCas9-eGFP to telomeres results in distinct dCas9-eGFP foci, which can be detected throughout the nucleoplasm. Bar: 5 µm.

Figure 2. 3D-FISH shows precise targeting of dCas9-eGFP. (A, B, C, D) Immuno-FISH experiments in gRNA/dCas9-eGFP expressing cells. Hybridization of probes designed to target MaS (A, red and C, blue), MiS (B-C, red), Tel (D, red) demonstrate that dCas9-eGFP co-localizes with the respective sequences. Multicolor immuno-FISH in C (MiS, red; MaS, blue) highlights the restricted targeting of MiSgRNA/dCas9-eGFP and demonstrates that non-targeted neighboring nuclear structures maintain their integrity. Bars, 5 µm; insets, 1 µm.

Figure 3. Association of gRNA/dCas9-eGFP to chromatin in mitotic cells. (A) Confocal optical section of a metaphase plate shows successful targeting of MaSgRNA/dCas9-eGFP. Note the robust eGFP signals at the CCs (DAPI bright regions, middle panel). Bar, 5 µm. (B) Two confocal optical sections (z1, z2) of a multicolor immuno-FISH stained metaphase show TelgRNA/dCas9-eGFP signals at the ends of chromosomes. The Integrity of (peri-) centromeric chromatin (MaS, MiS) is not compromised. Bar, 5 µm; insets, 1 µm. (C) Metaphase plate of a MiSgRNA/dCas9-eGFP expressing cell after immuno-FISH with MiS probe (red) acquired via 3D-SIM. Note the overlap between MiS probe and dCas9-eGFP. Bar, 5 μm; insets, 1 µm.

Figure 4. 3D-SIM highlights the ultrastructure of MiS and Tel repeats. (A) Left panel depicts a mid z-section of a DAPI stained nucleus (gray) in MiSgRNA/dCas9-eGFP (green) expressing cells immunolabeled with anti-CENP-B antibodies (red). Four × magnifications of boxed areas (mid gallery) show the spatial association of dCas9-eGFP decorated domains to CENP-B assemblies. Wide-field (wf) deconvolved simulations of the corresponding 3D-SIM magnifications are shown for comparison (far right gallery). Bar, 5 µm; magnifications, 500 nm. (B) Left panel depicts a mid z-section of a DAPI stained nucleus (gray) in TelgRNA/dCas9-eGFP (green) expressing cells immunolabeled with anti-TRF2 antibodies (red). Four × magnifications of boxed areas (mid gallery) show overlapping pattern of dCas9-eGFP signals with TRF2. Wide-field (wf) deconvolved simulations of the corresponding 3D-SIM magnifications are shown for comparison (far right gallery). Note the elucidation of interconnected ovoid intensities within the telomere-cluster (mid-gallery, lower panel, and dCas9-eGFP). Bar, 5 µm; magnifications, 500 nm.