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. 2017 Apr 17;112(9):1773–1776. doi: 10.1016/j.bpj.2017.03.024

Figure 2.

Figure 2

Multiplexed telomere tracking and identification of chromosomes with the “track first and identify later” approach in mES cells. (A) Schematic of the approach applied to telomere in a mouse chromosome. Proximal and distal telomere were labeled by the CRISPR-Cas9 system whereas only the distal subtelomeric region was labeled by DNA seqFISH. In total, 12 distal subtelomeric regions in 12 chromosomes were robustly read out by DNA seqFISH. (B and C) Here, we show one-color telomere imaging in live cells at different time points (B) and after fixing cells (C), using the constructed mES cell line. (D and E) Composite digitized three-color (Alexa 647: red, Alexa 594: green and Cy3B: yellow) DNA seqFISH data for three rounds of hybridizations targeting subtelomeric regions (D), and one-color (Cy7) data for the fourth hybridization targeting telomeres (E) is given. Based on the barcode identities, chromosome numbers are assigned to each of the subtelomeric spots (D). Note that DNA seqFISH spots do not perfectly colocalize with CRISPR imaging spots because they target adjacent regions in the genome. Dots without colocalization between hybridizations are due to nonspecific binding of probes or mishybridization in the cells. Images are maximum intensity projections of a z-stack of fluorescence images and the boxed region of the cell is magnified (B–E). (F) Here, we compare the number of telomeric or subtelomeric spots detected per cell with the CRISPR labeling and DNA seqFISH methods. In total, 938 CRISPR spots in live cells (last frame of the movie), 1138 CRISPR spots in fixed cells, 909 telomeric spots by DNA FISH, and 628 subtelomeric spots by DNA seqFISH in 28 cells were analyzed. (G) Here, we compare colocalization percentage of spots detected per cell. (Red dashed lines) Expected colocalization percentage per cell is given. (H) Trajectories of telomeric loci in the magnified cell are shown. In this cell, 30 telomeric trajectories were detected from CRISPR imaging and 10 of these trajectories were uniquely assigned to particular chromosomes based on the subtelomere color barcodes. Trajectories of three loci in the magnified images (B)–(E) were also highlighted as xy projections (inset). Projected trajectories start from (0.0, 0.0). (I) Cumulative square displacement traces (n = 30) calculated with two adjacent frames as a function of time from the magnified cell are shown. Traces of three loci in the magnified images (B)–(E) were shown as colored traces. To see this figure in color, go online.