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The telomere exists in a unique state protected by shelterin complexes at the ends of each chromosome to protect genetic information. The most widely used method for imaging the telomere is to use TRF1, one of the shelterin proteins. However, the existing TRF1 method for telomere detection has limitations, such as differences due to changes in the expression level of TRF1, background signal of TRF1, and the inability to use wild-type cells.

Therefore, methods such as FISH, CRISPR transfection, and CASFISH were applied to telomere imaging to address this issue by directly binding fluorescence to the telomeric repeat sequence. However, in the case of CRISPR transfection, the transfection efficiency in the primarily used U2-OS cell line was too low, which makes it difficult to use the CRISPR system that requires transfection of multiple plasmids. During CASFISH, there were problems such as instability of manually synthesized RNA and degradation by RNase in the cell, which have not been well reproduced since the original paper, but this was solved by purchasing RNA that had been chemically treated.

As a result of CASFISH imaging, the number and size of telomeric foci were found to be significantly altered by DNA damage. This can be interpreted as the fact that, like the phenomenon of homologous recombination known as the phenomenon in ALT, the distance to the telomeres of other chromosomes becomes closer and the number of foci can increase due to DNA breakage. They surround the telomeres at the end of the chromosome and protect the telomere sequence from external damage.

Oligopaint DNA FISH used two kinds of probes, and PNA FISH only needs one probe to image telomeres.

Introduction

3 GGCCAATCG / ACAGAAACTGAGGGCACAACTGTGATGTAGATCTTGAG / GCTATCGTTCGTTCGAGGCC / TCTCGCGATTTCCGCACAGG 4 TTGCTAGCGTGGGCCAATCG / AAAATGTAGAAGGAAGCACGCAACAGAGGAGTAGAAGG / GCTATCGTTCGTTCGAGGATTGCCGGTCGGTCGGTCGGTCGGTCGGTCGGTCGGTCGGTCGGTCGGTCGGTCGA AGGGCTGTAAGTGTGTGTAGAGAAATGTAAGCC / GCTATCGTCGTTCGAGGCC / TCTCGCGATTTCCGCACAGG 6 TTGCTAGCGTGGGCCAATCG / ATATTGGCATGGTTTTAGCTTCAAGAACCATGCATTGAAGG / GCTATCGTTCGTTCGAGGCC / TCTCGTAGCGATTTCCACCTG / TCATAACTGAGGCACAAACATCTGAATCATGGGTTCTGA / GCTATCGTTCGTTCGAGGCC / TCTCGCGATTTCCGCACAGG 8 TTGCTAGCGTGGGCCAATCG / AAAGTACTGAGAAAGAAAATCATATACCCGGTTTCAGCCCG / GCTATCGTTCGTTCGAGGCC / TCTCGCGATTTCCGCACAGG 9 TTGCTAGCGTGGGCCAATCG / GGCTTGTTAACATCAGAGCAGAATTTTGCTTGGCAGC / GCTATCGTTCGTTCGAGGCC / TCTCGCGATTTCCGCACAGG 10 TTGCTAGCGTGGGCCAATCG / AAGTAGAAAGCACTCTCTAACAGGGATGGTCAGCAAG / GCTATCGTTCGTTCGAGGCC / TCTCGCGATTTCCGCACAGG 11 TTGCTAGCGTGGGCCAATCG / GACTCTGGACATCTGAAAATGGCCCAAGGCACACTG / GCTATCGTTCGTTCGAGGCC / TCTCGCGATTTCCGCACAGG 12 TTGCTAGCGTGGGCCAATCG / AAAAGTCACAGGCTTGCAGCTTGGAGCTAAGAACCC / GCTATCGTTCGTTCGAGGCC / TCTCGCGATTTCCGCACAGG 13 TTGCTAGCGTGGGCCAATCG / TCTTTCTGTCTAGACTCATTTATTGGCCAGGCTCACTTTCT / GCTATCGTTCGTTCGAGGCC / TCTCGCGATTTCCGCACAGG 14 TTGCTAGCGTGGGCCAATCG / GGGATCCACCAGTGTGGACAAGCAAGCAGGCTTCTA / GCTATCGTTCGTTCGAGGCC / TCTCGCGATTTCCGCACAGG 15 TTGCTAGCGGGACCTGGGCCCAATCGCTGAATTCAAGTCCGTCCGTCCGT TCGAGGCC / TCTCGCGATTTCCGCACAGG 16 TTGCTAGCGTGGGCCAATCG / GAAATTGGACGAATTTTCAGCGTTTAGAAACGAGGAAGAGC / GCTATCGTTCGTTCGAGGCC. 17 TTGCTAGCGTGGGCCAATCG / GGAGAGGACACAAGACGATCAGAAACCGCAGGGTCT / GCTATCGTTCGTTCGAGGCC / TCTCGCGATTTCCGCACAGG 18 TTGCTAGCGTGGGCCAATCG / GCGCCAGGCTTTCTGTCAGCATTTAGGAGCACACCATA / GCTTCGATCGGTCGGTCGGTCGGTCGAGCAACACCATA / GCTTCGATCGGTC CGTGGGCCAATCG / AAAATGGAATCCCACCACAAACAGAATCGGATCTGGT / GCTATCGTTCGTTCGAGGCC / TCTCGCGATTTCCGCACAGG 20 TTGCTAGCGTGGGCCAATCG / TGACAAAACGAAAGAACAGAGAACAAAATCAGGTACCCGCG / GCTATCGTTCGTTC. 20 TAGCGTGGGCCAATCG / ATTTGCTCCCTGAGGATCCAAAGGCGGGAAGTTTAC / GCTATCGTTCGTTCGAGGCC / TCTCGCGATTTCCGCACAGG 24 TTGCTAGCGTGGGCCAATCG / CTGCTGTGAAGTCGTGGCCAGACTGGAGCTCAGAGATA / GCTATCGTTCCAGTTCGACCGCCGATTCGATTCGTTCCGCGCGCGCCTGCC CTTTGAGGCTTCTGGCTCCTTCATGGGTCCCCACTC / GCTATCGTTCGTTCGAGGCC / TCTCGCGATTTCCGCACAGG 26 TTGCTAGCGTGGGCCAATCG / GGTGGTCCAGTGCTTTCCCTGTTGGACAAGGTGTACT / GCTATCGTTTCGTTCGAGGCC / TCTCACGATTCGGGATTCCGGGATCCAGTGGTCCGTG / CCATGGCCACGAAGCAACCAGGGAAACTACAGTGAG / GCTATCGTTCGTTCGAGGCC / TCTCGCGATTTCCGCACAGG 28 TTGCTAGCGTGGGCCAATCG / GATGGTATGTAATCTAAGTTTTGAAACGCGGGGAGGTCG / GCTATCGTCGTTCGTTCGAGGCGCGCGCC / TCTCACGGGTAGGGTCCGAGTGGTCCGAGTGTCGA GGTGGGATTCTGTGCTGACTTCAGG / GCTATCGTTTCGTTCGAGGCC / TCTCGCGATTTCCGCACAGG 30 TTGCTAGCGTGGGCCAATCG / TCCAGACTTCTGACAACCCTGTGGAACAAGGGATAC / GCTATCGTCGTTCGTTCGAGGCC / TCTCGCCCGATTTCCGCACAGGATCGATTGATTGCCAGTGACTGATTGATTGCCAGTGAG TTCCT / GCTATCGTTCGTTCGAGGCC / TCTCGCGATTTCCGCACAGG 32 TTGCTAGCGTGGGCCAATCG / GCTCTGGCTCCTGCATTTCCCTGGAACCACCAAAAC / GCTATCGTTCGTTCGAGGCC / TCTCGCGATTTCCGCACAGG 33 TTGCTAGCGTGGGTACCATGATCGTCCATGTCCGT / GCTATCGTTCGTTCGAGGCC / TCTCGCGATTTCCGCACAGG 34 TTGCTAGCGTGGGCCAATCG / AATACATAGGGAGAGTAGTGAAAACAGGGGAAAATCCTCCA /GCTATCGTCGTTCGTTCGAGGCC / TCTCGCGATTTCCGCACAGG 35 TTGCTAGCCAGTGGGACCATCATCTCCTGCCATCCAT GTTCGAGGCC / TCTCGCGATTTCCGCACAGG 36 TTGCTAGCGTGGGCCAATCG / AGAGGATGGAAATGTTACCTGCATTCATCTCAAACACATGC / GCTATCGTTCGTTCGAGGCC / TCTCGCGATTTCCGCACAGG 37 TTGCTAGCGTGGGCCAATCG / GAGCCATAACCACACTGACTGATCGTGATCGTGATCGTGATC CGCGATTTCCGCACAGG 38 TTGCTAGCGTGGGCCAATCG / AGTGCAACAGGTCTTTCCAGCCCTGAGTACAAGGTGT / GCTATCGTTTCGTTCGAGGCC / TCTCGCGATTTCCGCACAGG 39 TTGCTAGCGTGGGCCAATCG / TCGCCAGAAGTGAGAAAGGTCCCAGAACTGTCC / TCTCGCGATTTCCGCACAGG 40 TTGCTAGCGTGGGCCAATCG / ACCTTATGCATAGACTAGGCTAGCTCTGAACTCTGATTCAC / GCTATCGTTCGTTCGAGGCC / TCTCGCGATTTCCGCACAGG 41 TTGCTAGCGTGGGCCAATCG / GCAGAAAGGAAGGAAGTAAGTGAAGTCGATTGATTGAGTGAGTGAGTGATCGGT CACAGG.

Table 1. mTALT oligopaint probe. Each probe consists of four parts. The parts are [5’primer site / target sequence / tail sequence / 3’primer site]

Result

First, the experiment was first performed in the AD293 cell line with relatively small telomere, and the protocol was not optimized. In the AD293 cell line, the DNA FISH probe was used and the conditions were to completely degrade the cell wall by providing strong fixation. In the case of PNA FISH, it obviously has an advantage in terms of permeability to the extent that the denaturation step can be omitted.

In the case of the split GFP model, the GFP protein was divided into two parts to reduce the background signal so that no signal was emitted by molecules floating in the nucleus (Fig. 4A). In contrast, the EGFP model is the basic model and the entire GFP is fused directly onto dCas9, which can have a relatively low SBR (Fig. 4B). However, the number of plasmids used in each model is 3 for cleaved GFP and 2 for EGFP, and EGFP as a primary model has the advantage in terms of system complexity.

A total of 10 cells were analyzed and the intensity and SBR of 201 and 198 foci from CASFISH and TRF1 GFP imaging were measured, respectively. In the case of GFP-tagged TRF1, since the overexpressed proteins are present in the nucleus, the background intensity is higher and the SBR is lower, while in the case of CASFISH, a washing step was performed after hybridization, resulting in a Higher SBR (fig. 5C, D). The first negative control was performed using a gRNA that does not have a target sequence in the human genome.

Since it is a labeled RNA probe, a signal should come out if it binds to the target, but no signal can be found in the image. Based on this, the foci appearing in the telomeric gRNA do not hybridize with the gRNA alone, but bind to the target through the transport of dCas9. Afterwards, we decide to analyze these images as the number and size of foci, because the foci were too small and the resolution was low to compare the detailed structure.

In the case of cisplatin treatment, the number of foci did not change significantly, but there were several large foci that appeared to have formed during the DNA damage repair process (fig. 7B). In the case of UV-damaged samples, the number of foci increased significantly compared to normal CASFISH samples, and more large foci were observed (fig. 8). The number of foci was counted manually after setting the intensity threshold, and the standard for large foci was set by selecting foci that appeared to exceed twice the size of other foci.

The total number of cells used in the assay was 30 cells for each condition (Fig. 8B,C). When cisplatin was treated under normal conditions, the number of foci remained the same, while the number of large foci increased slightly, and both increased when UV damage was applied.

Table 2. DNA and PNA FISH probe. The oligopaint DNA FISH used two kinds of probes and PNA FISH need only one probe to image telomere

Discussion

This was only a study of the trend of foci that appeared when the telomere was imaged. To study the structure, higher resolution imaging technology is required, and since it is a small area, it must be performed in 3D. Even in the case of the atto550 dye we used, it is possible to use STORM, and dyes more beneficial to STORM can be conveniently labeled to tracrRNA and used as CASFISH probes.

If 3D imaging is possible, it appears that more detailed images can be obtained to perform research on 3D structures.

Experimental Method & Materials

Fumagalli, M., et al., Telomeric DNA damage is irreversible and causes persistent DNA damage response activation. Avci-Adali, M., et al., Upgrading SELEX technology using lambda exonuclease digestion for single-stranded DNA generation. Kim, W.T., et al., Cancer-associated POT1 mutations lead to telomere lengthening without induction of a DNA damage response.

Deng, W., et al., CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells. Hu, J., et al., Cisplatin DNA damage and repair maps the human genome at single-nucleotide resolution. Yimit, A., et al., Differential damage and repair of DNA adducts induced by anticancer cisplatin across mouse organs.

Dinant, C., et al., Activation of multiple DNA repair pathways by sub-nuclear damage induction methods. Vancevska, A., et al., The telomeric DNA damage response occurs in the absence of chromatin decompaction. Bintu, B., et al., Super-resolution chromatin detection reveals domains and cooperative interactions in single cells.

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