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. 2016 May 19;44(9):e86.
doi: 10.1093/nar/gkw066. Epub 2016 Feb 4.

Long-term dual-color tracking of genomic loci by modified sgRNAs of the CRISPR/Cas9 system

Shipeng Shao  1 , Weiwei Zhang  1 , Huan Hu  2 , Boxin Xue  1 , Jinshan Qin  1 , Chaoying Sun  1 , Yuao Sun  1 , Wensheng Wei  3 , Yujie Sun  4
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Long-term dual-color tracking of genomic loci by modified sgRNAs of the CRISPR/Cas9 system

Shipeng Shao et al. Nucleic Acids Res. .
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Abstract

Visualization of chromosomal dynamics is important for understanding many fundamental intra-nuclear processes. Efficient and reliable live-cell multicolor labeling of chromosomal loci can realize this goal. However, the current methods are constrained mainly by insufficient labeling throughput, efficiency, flexibility as well as photostability. Here we have developed a new approach to realize dual-color chromosomal loci imaging based on a modified single-guide RNA (sgRNA) of the CRISPR/Cas9 system. The modification of sgRNA was optimized by structure-guided engineering of the original sgRNA, consisting of RNA aptamer insertions that bind fluorescent protein-tagged effectors. By labeling and tracking telomeres, centromeres and genomic loci, we demonstrate that the new approach is easy to implement and enables robust dual-color imaging of genomic elements. Importantly, our data also indicate that the fast exchange rate of RNA aptamer binding effectors makes our sgRNA-based labeling method much more tolerant to photobleaching than the Cas9-based labeling method. This is crucial for continuous, long-term tracking of chromosomal dynamics. Lastly, as our method is complementary to other live-cell genomic labeling systems, it is therefore possible to combine them into a plentiful palette for the study of native chromatin organization and genome ultrastructure dynamics in living cells.

Figures

Figure 1.
Schematic diagram of the modified sgRNA scaffolds for dual-color imaging of genomic sequences in living cells. (A) Overview of the dual-color imaging by modified sgRNA scaffolds. sgRNA molecules are extended with additional domains (MS2/PP7) to recruit RNA binding proteins that are fused with fluorescent pro3teins (tdMCP-mCherry, tdPCP-EGFP). The modified sgRNA can be classified into several motifs including the DNA targeting motif, the dCas9 binding motif and the effector recruitment motif. Enrichment of the fluorescence signal by multiple fluorescently labeled modified sgRNAs allows imaging of different genomic elements in living cells. The color annotations in the nucleus stand for different chromosomes. (B) Motif annotations of the modified sgRNA scaffolds. The original sgRNA can be classified into eight motifs. The RNA aptamers (MS2/PP7) can be inserted into the tail (named as sgRNA1.0), the tetraloop (sgRNA1.1), loop 2 (sgRNA1.2), or both tetraloop and loop2 (sgRNA2.0). sgRNAs with additional optimizations are named as sgRNA1.12, 1.22 and 2.02 (see Supplementary Figure S1). (C) Three components of the modified dual-color CRISPR imaging system: a doxycycline-inducible dCas9 expression plasmid, two doxycycline-inducible fluorescent protein-tagged tdMCP/tdPCP plasmids and target-specific modified sgRNAs expressed from a murine U6 promoter. NLS sequences were added to dCas9 as well as tdMCP and tdPCP to increase their nuclear localization.
Figure 2.
Comparison of the modified sgRNA method and the fluorescent dCas9 method by co-labeling human centromeric α-satellites. (A) Co-labeling of human centromeric α-satellites using sgRNA1.1 (green) and dCas9-mCherry (red). The nucleus was stained by DAPI (blue). The merged image shows the two types of labeling are well co-localized, but dCas9-mCherry concentrates in the nucleoli, compared with sgRNA1.1. The inset shows the magnified image of the boxed region. (B) Upper panel: fluorescence colocalization analysis of the two channels. The scatter plot shows the intensity correlation of the two channels for each individual pixel. Pearson's correlation Rr = 0.759, Overlap coefficient R = 0.997; Lower panel: a colocalization image reconstructed from the correlated pixels. It indicates that the large fraction of uncorrelated red pixels are from the nucleoli signal. The color bar denotes the colocalization percentage. (C) Signal-to-noise ratios (SNRs) of the two channels. The SNRs of the centromere signal relative to the nuclear background is slightly higher for the sgRNA1.1 (G/B) than that of the dCas9 labeling (R/B). The SNRs referred to the nucleoli regions is about 3-fold higher for the sgRNA1.1 (G/N) than that of the dCas9 labeling (R/N). N = 10 cells. (D) Normalized line intensity profiles along the white line in A inset. Both GFP and mCherry channels form two intensity peaks, where the DAPI channel forms valleys. (E) Representative images of all 12 modified sgRNAs that target centromeres. Among them, sgRNA1.12, 1.22 and 2.02 show notably less nucleoli localizations than sgRNA1.1, 1.2 and 2.0. (F) Representative images of a MDA-MB-231 cell line stably expressing dCas9, tdPCP-EGFP and tdMCP-mCherry. (G and H) Total number of centromeres detected per cell and SNRs for all 12 modified sgRNAs. Cyan color represents the MS2 insertion, blue color represents the PP7 insertion and gray color represents the stable cell line. Error bars are standard deviations. N = 10 cells. All scale bars are 5 μm.
Figure 3.
Targeting specificity of the modified sgRNA labeling method. (A) Co-labeling of centromeres using sgRNA2.02-PP7-EGFP (green) and anti-CREST-Cy5 (red). The nucleus was stained by DAPI (blue). Scale bar: 5 μm. (B) Three boxed regions in the lower right panel of A are magnified to display three typical colocalization scenarios. Scenario 1: apparent colocalization between sgRNA-labeled punctum and anti-CREST-Cy5 labeled punctum; Scenario 2: anti-CREST-Cy5 labeled punctum without apparent sgRNA-labeled punctum; Scenario 3: sgRNA-labeled punctum without apparent anti-CREST-Cy5 labeled punctum. Scale bar: 1 μm. (C) Quantification of centromere targeting specificity based on the three colocalization scenarios in 10 cells with Scenario 1 marked in yellow, Scenario 2 marked in red and Scenario 3 marked in green. (D) Co-labeling of telomeres using sgRNA2.02-PP7-EGFP (green) and mCherry-TRF2 (red). The nucleus was stained by DAPI (blue). Scale bar: 5 μm. (E) Three boxed regions in the lower right panel of D are magnified to display three typical colocalization scenarios. Scenario 1: apparent colocalization between sgRNA-labeled punctum and mCherry-TRF2 labeled punctum; Scenario 2: mCherry-TRF2 labeled punctum without apparent sgRNA-labeled punctum; Scenario 3: sgRNA-labeled punctum without apparent mCherry-TRF2 labeled punctum. Scale bar: 0.3 μm. (F) Quantification of telomere targeting specificity based on the three colocalization scenarios in 12 cells with Scenario 1 marked in yellow, Scenario 2 marked in red and Scenario 3 marked in green.
Figure 4.
FRAP analysis of the dCas9 channel and the modified sgRNA scaffold channel. (A and B) Snapshots of the FRAP image series of dCas9-mCherry (A) and sgRNA2.02-MS2-EGFP (B). Localized photobleaching was applied to the centromeres marked by the black and purple circles. The centromere marked by the red circle was used as the control. The time lapse snapshots are shown in the right panel. All scale bars are 5 μm. The lookup tables on the lower right corners indicate the fluorescence intensity. (C and D) Quantifications of the FRAP experiments (11 centromeres for the dCas9-mCherry channel and 26 centromeres for the sgRNA2.02-MS2-EGFP channel). The data are background-subtracted and photobleach-corrected by the whole cell fluorescence. The dCas9-mCherry recovers at a much slower speed (t1/2 = 12 min) than that of sgRNA2.02-MS2-EGFP channel (t1/2 = 19 s) and the immobile fraction (Fi = 0.55) is larger than that of sgRNA2.02-MS2-EGFP channel (Fi = 0.22). Error bars are standard deviations.
Figure 5.
The modified sgRNA labeling system allows continuous, long-term imaging and tracking of chromosomal loci. (A) Selected frames from a time-lapse series of telomeres labeled with dCas9-EGFP or sgRNA1.12-PP7-EGFP. Scale bars are 1 μm. (B) The fluorescence intensity of telomeres in A was normalized and averaged. The data are displayed as mean ± sem. N = 24 puncta (3 cells) for dCas9-EGFP and N = 26 puncta (3 cells) for sgRNA1.12-PP7-EGFP. (C) Time-lapse snapshots from live imaging of a MDA-MB-231 cell through mitosis. Telomeres and centromeres were dual-color labeled with sgRNA2.02-MS2-mCherry and sgRNA2.02-PP7-EGFP, respectively. In this case, dCas9 was transfected to bring sgRNAs to the target sites and all fluorescence signal was from the sgRNA channels. Scale bar is 5 μm.
Figure 6.
Dual-color continuous tracking of centromeres and telomeres in living cells. (A) A confocal section image of telomeres labeled with the sgRNA2.02-MS2-EGFP (green) and dCas9-mCherry (red), exhibiting a good level of colocalization with Pearson's correlation coefficient Rr = 0.741, overlap coefficient R = 1.0. Tracking of individual telomeres is performed by Gaussian fitting and marked by cyan circles (sgRNA) and red circles (dCas9), respectively. Scale bar: 5 μm. (B) The tracking trajectories of labeled telomeres. Red trajectories represent the dCas9-mCherry channel and green trajectories represent the sgRNA-MS2-EGFP channel. Scale bar: 5 μm. (C) Three telomere trajectories in B are magnified to display by the color-coded time points with green for the starting, blue for the intermediate and red for the ending frames. Scale bar: 0.5 μm. (D) The averaging MSD curves of telomere trajectories in both channels. The colored shade area represents the 95% fitting confidence interval. (E) Dual-color labeling of telomeres (sgRNA2.02-PP7-EGFP, green) and centromeres (sgRNA2.02-MS2-mCherry, red) simultaneously in living HeLa cells using modified sgRNAs. In this case, dCas9 was transfected to bring sgRNAs to the target sites and all fluorescence signal was from the sgRNA channels. Scale bar: 5 μm. (F) The averaging MSD curves of both telomeres (green) and centromeres (red). The colored shade area represents the 95% fitting confidence interval.
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