Method for detecting single-strand breaks in nucleic acids

Abstract

The present invention relates to a method for detecting single-strand breaks (SSBs) in double-stranded (ds) DNA. A sample of dsDNA is brought into contact with an oligonucleotide probe containing a first sequencing adapter and tag, and then into contact with a DNA ligase. The DNA ligase covalently binds the oligonucleotide probe to the dsDNA strand at the single-strand break sites in the dsDNA. The dsDNA sample is then brought into contact with a transposase loaded with a second sequencing adapter, resulting in the cleavage of the dsDNA by the transposase and the generation of a population of DNA fragments containing the second sequencing adapter. DNA strands containing the first and second sequencing adapters are isolated from the population, and the sequences of the isolated DNA strands are determined. A method and a kit for carrying out the method are provided.

Description

[Technical Field]

[0001] The present invention relates to a method for detecting and mapping single-strand breaks (SSBs) in double-stranded nucleic acids such as double-stranded deoxyribonucleic acid (dsDNA). [Background technology]

[0002] Shortly after the DNA structure was elucidated, it became clear that DNA undergoes endogenous gaps (Non-Patent Literature 1) resulting from various processes including oxidation, hydrolysis, alkylation, and DNA base mismatches, thereby mediating changes in cellular responses. It is estimated that each cell generates as many as 70,000 gaps per day (Non-Patent Literature 2). While such DNA gaps have previously been thought to be involved in mutations and various diseases, including cancer (Non-Patent Literature 3), in recent years, there has been growing recognition of the role of endogenous DNA gaps in normal cellular function (Non-Patent Literature 4).

[0003] Therefore, several techniques have been developed to measure the genomic distribution and frequency of DNA gaps. Most of these techniques can measure the genomic landscape of double-stranded DNA gaps with reasonable accuracy (Non-Patent Documents 5 and 6). Despite the widespread presence of single-stranded DNA gaps (Non-Patent Document 2), there are fewer than a handful of methods that enable genome-wide detection of single-stranded DNA gaps. These methods include SSiNGLe (single-strand break mapping at the nucleotide genome level; Non-Patent Document 7), SSB-Seq (Non-Patent Documents 8 and 9), XR-Seq (Non-Patent Document 10), and GLOE-Seq (Non-Patent Document 11). These methods always rely on capturing the 3'OH ends of DNA gaps and mapping their locations, and since 3'OH ends are also present in double-stranded DNA gaps, the resulting DNA gap measurements may profile nicks in the DNA rather than representing single-stranded DNA gaps.

[0004] There is still a need for simple techniques for rapid, high-resolution mapping of SSB. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Lindahl and Nyberg, 1972 [Non-Patent Document 2] Lindahl and Barnes, 2000 [Non-Patent Document 3] Vilenchik et al., 2003 [Non-Patent Document 4] Wu et al., 2021 [Non-Patent Document 5] Yan et al., 2017 [Non-Patent Document 6] Amente et al., 2021 [Non-Patent Document 7] Cao et al (2019) [Non-Patent Document 8] Baranello et al (2014) [Non-Patent Document 9] Baranello et al (2018) [Non-Patent Document 10] Hu et al (2015) [Non-Patent Document 11] Sriramachandran et al (2020) [Overview of the Initiative]

[0006] The inventors have developed a method (referred to herein as "DNA GAPS sequencing" or "GAPS-seq") that enables rapid, high-resolution detection of single-strand breaks (SSBs) in double-stranded (ds) DNA and may be useful, for example, for mapping or evaluating SSBs in genomic DNA in a strand-specific manner.

[0007] A first aspect of the present invention is a method for detecting single-strand break (SSB) sites in double-stranded (ds) DNA, (i) Contacting the dsDNA sample with an oligonucleotide probe containing a first sequencing adapter and tag, (ii) Contacting a dsDNA sample with a DNA ligase, wherein the DNA ligase covalently attaches oligonucleotide probes to the dsDNA strands at single-strand break sites in the dsDNA. (iii) Contacting a dsDNA sample with a transposase loaded with a second sequencing adapter, thereby cleaving the dsDNA by the transposase and generating a collection of DNA fragments containing the second sequencing adapter, (iv) Isolating DNA strands containing the first sequencing adapter and the second sequencing adapter from the above population, (v) Determining the sequence of the isolated DNA strand, This provides a method that includes [something].

[0008] The sequence of the isolated strand may indicate single-strand break (SSB) sites within the dsDNA.

[0009] The method of the first embodiment may include determining the location of SSBs and / or the frequency of SSBs at a location in a first and second sample of dsDNA. The difference in SSB locations or the difference in the frequency of SSBs at a location between the first and second samples can be determined.

[0010] One of the first and second samples may have been subjected to treatment, or may be obtained from diseased cells or cells of a defined age or developmental stage. The other sample may be a reference or control. The effects of treatment, disease, developmental stage, or age on the location or frequency of SSBs can be determined.

[0011] A second aspect of the present invention is a kit for detecting sites of single-strand breaks (SSBs) in double-stranded (ds) DNA, the kit comprising: an oligonucleotide probe comprising a first sequencing adapter and a tag; DNA ligase; transposase; a second sequencing adapter loaded or loadable on the transposase; and providing a kit.

[0012] The kit may further comprise a binding member that specifically binds to the tag, a sequencing primer, a solid support such as beads, PCR reagents and / or other reagents.

[0013] Other aspects and embodiments of the present invention are described in more detail below.

Brief Description of the Drawings

[0014] [Figure 1] It is a figure which shows the example of the strand-specific measurement method (GAPS-Seq) of a genome-wide DNA gap. Nuclei derived from cells or tissues are fixed in situ and then ligated with a biotinylated DNA adapter bound to a hexamer (random sequence). Next, the nuclei are lysed, the genomic DNA is fragmented and bound to a second (R2) adapter, and then the biotinylated fragment is captured with streptavidin and the strands are separated. These are then PCR amplified, sequenced and mapped to reveal genomic loci with DNA gaps. [Figure 2] It is a figure which shows the measurement of the ssDNA gap using GAPS-seq. GAPS-seq reads in HEK293 cells are shown normalized to library size and then averaged in 40bp tiles. Plotted is the average signal of all protein-coding genes per 40bp tile relative to the transcription start site. The line represents the average of 4 replicates and the shading is the standard deviation. [Figure 3]This figure shows examples of ssDNA gap profiles in etoposide-treated and untreated HEK293 cells. Etoposide treatment is known to increase endogenous DNA gaps by inhibiting topoisomerase. As predicted, GAPS-seq detected more ssDNA gaps in etoposide-treated cells compared to controls treated with DMSO alone. Interestingly, GAPS-seq also showed more ssDNA gaps in the enhancer sequence compared to the surrounding region, suggesting that the regulatory region is undergoing a dynamic cycle of ssDNA gap and repair. [Figure 4] This figure shows tapestation traces of DNA GAP-seq libraries obtained from various input samples. The inputs range from 1 million to 100 cells. [Figure 5] This figure illustrates the validation of GAPS-seq sensitivity and specificity using restriction enzymes. It shows GAPS-seq reads from HEK293 cells treated with restriction enzymes prior to GAPS-seq. Reads are normalized to library size and then averaged across 40 bp tiles. Plotted are the average signal per 40 bp tile relative to the central position of each restriction enzyme site in the human genome. Each panel represents a different restriction enzyme site, and each colored line represents a different sample, each sample treated with a different enzyme. [Figure 6] This figure demonstrates the validation of GAPS-seq specificity for single-strand gaps rather than nicks in DNA. It shows GAPS-seq reads from BJF cells treated with nickase enzyme and exonuclease III prior to GAPS-seq. Reads are normalized to library size and then averaged at 40 bp tiles. Plotted are the average signal per 40 bp tile relative to the central position of each site for all nickase motif examples in the human genome. The left panel shows control cells not treated with either enzyme. The center panel shows cells treated with nickase only. The right panel shows cells treated with both nickase and exonuclease. [Figure 7] This figure shows the measurement of DNA gaps using GAPS-seq in the promoters of genes with different expression levels. GAPS-seq reads in HEK293 cells were normalized to library size and then averaged at 400 bp tiles. The line represents the average of three repeats. This result demonstrates that GAPS-seq can detect known relationships between DNA breaks and transcription. [Figure 8] This diagram shows a hierarchical clustering of various samples profiled using GAPS-seq and quantified by gene promoters (±2kb from TSS). NPCs are neural progenitor cells derived from in vitro differentiation of human embryonic stem cells. hESCs are human embryonic stem cells. HEKs are HEK293 cells. BJFs are primary human foreskin fibroblasts. [Figure 9] The scatter plots of the first two principal components, quantified at the gene promoter and derived from GAPS-seq or sBLISS performed on HEK293 cells, are shown. This plot clearly demonstrates the separation of samples by the method used to prepare the libraries, thus demonstrating that GAPS-seq measures a different aspect of the genome than methods profiling double-strand breaks such as sBLISS. [Modes for carrying out the invention]

[0015] The present invention relates to a method for detecting single-strand breaks (SSBs) in ds nucleic acids such as dsDNA. A sample of dsDNA is contacted with an oligonucleotide probe containing a first sequencing adapter and tag, and then contacted with a DNA ligase. The oligonucleotide probe is covalently bound to the dsDNA strands by the DNA ligase at sites of single-strand breaks or gaps in the dsDNA. The dsDNA is then contacted with a transposase loaded with a second sequencing adapter. The transposase cleaves the dsDNA, generating a population of fragments having a second sequencing adapter at one or both ends. DNA strands are then isolated from this population of fragments containing a second sequencing adapter at one end and a first sequencing adapter at the other end, and the sequence of the isolated DNA strands is determined, for example, by sequencing. The sequence of the isolated strands may indicate sites of single-strand breaks (SSBs) in the dsDNA. Strand-specific SSB sites in the dsDNA in the sample can be identified or mapped from the sequence of the isolated strands.

[0016] The methods described herein may be useful, for example, for distinguishing SSBs from other genetic lesions, determining the location and / or frequency of SSBs in dsDNA samples such as genomic DNA, and enabling high-resolution strand-specific mapping of SSBs.

[0017] The method described herein may also be useful for detecting SSBs in low-input samples, from cells to tissue sections. Since cells or tissues do not need to be labeled before fixation, the method described herein directly detects nascent gaps. Furthermore, sample fixation allows for long-term storage of samples after initial collection and enables the method described herein to be performed without affecting the number of DNA SSBs or gaps detected. The sensitivity of the method described herein makes it possible to track the development of DNA SSBs or gaps over treatment (e.g., chemotherapy), developmental stage, and / or aging.

[0018] The methods described herein can be used with dsDNA in solution, cell-free dsDNA, or any cells or tissues obtainable in suspension, enabling the rapid generation of sequencing libraries and the provision of DNA SSB or gap information, making the methods suitable for clinical application. The methods described herein can also be used with samples of dsDNA obtained from cells, organoids or tissues, or, for example, cell-free DNA.

[0019] The method described herein is also compatible with bisulfite conversion and allows for the measurement of DNA methylation at sites adjacent to the gap. This makes it possible to assess the presence or absence of gaps and DNA methylation, and to evaluate the spatial relationship between the location of the gap and DNA methylation.

[0020] SSBs are a common form of DNA damage or genetic lesion. An SSB (also known as a single-strand gap) is a discontinuity in one strand of dsDNA ("the cleaved strand") while the other strand ("the intact strand") remains undamaged. In an SSB, one or more phosphodiester bonds are missing from the cleaved strand, resulting in a discontinuity or break in the nucleotide chain that makes up the strand. One or more nucleotides may be missing from the cleaved strand at the SSB site, creating a gap in the nucleotide sequence of the cleaved strand relative to the intact strand.

[0021] Examples of dsDNA include any dsDNA, such as genomic DNA, plasmid DNA, or viral DNA. Preferably, the dsDNA is genomic DNA. For example, the dsDNA may be a part of a chromosome, a minichromosome, an entire chromosome, or two or more chromosomes, and may be derived from circulating DNA. Other suitable dsDNAs may include mitochondrial DNA, plasmid DNA, or viral DNA inserted into a chromosome or genome.

[0022] Suitable dsDNAs include prokaryotic dsDNAs such as bacterial dsDNA. For example, the dsDNA used as described herein may include part or all of the genome of a prokaryotic cell such as a bacterial cell, or more preferably the entire genome of a prokaryotic cell.

[0023] Preferably, the dsDNA is eukaryotic dsDNA, such as mammalian dsDNA, including human DNA. For example, the dsDNA used as described herein may include part or all of the nuclear genome and / or mitochondrial genome of a eukaryotic cell, or more preferably the entire genome of a eukaryotic cell.

[0024] The dsDNA samples used as described herein may contain 1 or more, 10 or more, 100 or more, or 1000 or more cell genomes. The dsDNA samples used as described herein may contain 3 pg, 6 pg or more, 60 pg or more, 600 pg or more, or 6 ng or more of DNA.

[0025] In some embodiments, dsDNA samples can be obtained from tissues, organoids, cells, cell extracts, cell fractions, such as organelles like the nucleus or mitochondria, or from biological fluids such as blood, sperm, cerebrospinal fluid, amniotic fluid, pleural fluid, ascites, or saliva. dsDNA may be cellular DNA or cell-free DNA (cfDNA).

[0026] In some embodiments, dsDNA can be obtained from frozen cell or tissue samples.

[0027] In some embodiments, dsDNA may be present within cells, in the cell nucleus, or in nuclear extracts such as isolated genomic DNA (gDNA). Cell nuclei or nuclear extracts can be obtained from cells using standard techniques. In some preferred embodiments, dsDNA may be present within the cell nucleus. A sample containing one or more cell nuclei can be contacted with the oligonucleotide probe described herein, resulting in the probe contacting the dsDNA within the nucleus. This makes it possible to map the location of SSBs within the cell genome. For example, a method for mapping the location of SSBs within the cell genome is: (i) Contacting the cell nucleus with an oligonucleotide probe containing a first sequencing adapter and tag, (ii) Contacting the cell nucleus with DNA ligase, thereby causing the DNA ligase to covalently bond oligonucleotide probes to the dsDNA strands at single-strand break sites in the dsDNA, (iii) The cell nucleus is brought into contact with a transposase loaded with a second sequencing adapter, the transposase cleaving the dsDNA and generating a collection of fragments containing the terminal second sequencing adapter, (iv) Isolating DNA strands containing the first sequencing adapter and the second sequencing adapter from the above population, (v) Determining the sequence of the isolated DNA strand, It may include.

[0028] The sequence of isolated DNA strands may indicate the location of SSBs within the cellular genome.

[0029] In some embodiments, the dsDNA used as described herein can be obtained from eukaryotic cells. Eukaryotic cells may be, for example, isolated as primary cells from an immortalized cell line or an individual (e.g., a human subject), or they may be in the form of tissue or organoids. For example, cell nuclei can be obtained from eukaryotic cells in a sample obtained from an individual, such as a biopsy sample or xenograft sample.

[0030] Suitable eukaryotic cells include mammalian cells, preferably human cells. For example, eukaryotic cells may include somatic cells and germline cells, and may be at any developmental stage, including fully or partially differentiated cells, undifferentiated cells or pluripotent cells, including stem cells such as adult stem cells or somatic stem cells, fetal stem cells or embryonic stem cells. Suitable eukaryotic cells also include induced pluripotent stem cells (iPSCs), which can be obtained from any type of somatic cell according to standard techniques. Eukaryotic cells may also include nervous system cells, including neurons and glial cells, tyrosinocytes, smooth muscle cells, hepatocytes, hormone-synthetic cells, sebaceous gland cells, pancreatic islet cells, adrenal cortical cells, fibroblasts, mesenchymal cells, epithelial cells, keratinocytes, endothelial cells, urothelial cells, osteocytes, chondrocytes, immune cells such as leukocytes, mesothelial cells, adipocytes, bone marrow cells, or samples.

[0031] Appropriate eukaryotic cells include normal cells, cancer cells such as carcinoma cells, sarcoma cells, lymphoma cells, blastoma cells, or germline tumor cells, and disease cells, such as disease-associated cells, including cells with genotypes of genetic disorders such as Huntington's disease, cystic fibrosis, sickle cell disease, phenylketonuria, Down syndrome, or Marfan syndrome.

[0032] Other suitable eukaryotic cells include embryonic cells, extraembryonic cells, and cell culture models.

[0033] In some embodiments, the dsDNA used as described herein may be derived from diseased cells, such as cancer cells, or from cells obtained from an individual with a disease, such as a human subject. The frequency or distribution of SSBs in the dsDNA may indicate the diagnosis, severity, penetrant, or prognosis of a disease such as cancer in the individual from which the dsDNA was obtained. The disease or cancer may be caused by, for example, a defect in the DNA repair pathway, or may be characterized by a defect in the DNA repair pathway.

[0034] In other embodiments, dsDNA may be derived from cells at a specific developmental stage, such as cells obtained from an embryo; aging-related cells, such as cells obtained from an individual of a specific age, such as an elderly person; or treated cells. Examples of treated cells include cells treated with reprogramming factors such as Oct3 / 4, Sox2, Klf4, and c-Myc ("Yamanaka factor" or "OSKM"), radiation, compounds, and drugs such as chemotherapeutic agents.

[0035] For example, the methods described herein may be useful in cytotoxicity assays. A cytotoxicity assay may include stimulating cells and determining the frequency or distribution of gaps or SSBs in the cell-derived dsDNA. The frequency or distribution of gaps or SSBs in the cell-derived dsDNA may indicate the cytotoxicity of the stimulus. Suitable stimuli include exposure to compounds and radiation.

[0036] In other embodiments, the dsDNA may be derived from cells treated with a nuclease such as a CRISPR nuclease (e.g., Cas9 or Cpf1). The frequency or distribution of SSBs within the dsDNA may indicate off-target effects of the nuclease in the treated cells.

[0037] In other embodiments, the dsDNA may be derived from oocytes or spermatids obtained from a donor, such as a human donor. The frequency or distribution of SSBs within the dsDNA may indicate the quality of the donor's oocytes or spermatids. This may be useful, for example, in assisted reproductive technology (ART) procedures.

[0038] In some embodiments, cells can be immobilized and permeabilized prior to step (i) of the method described herein.

[0039] Fixation can be useful in reducing background noise, for example, by mitigating SSB generated during the handling of the sample before the addition of ligase. Appropriate methods for fixing cells are known in the art, and these include contacting cells with an aldehyde fixative, such as formaldehyde, formalin, or glutaraldehyde, or with an alcohol fixative, such as methanol, ethanol, or acetone. For example, cells can be fixed by exposing them to 2% formaldehyde.

[0040] Permeabilization can be useful, for example, for contacting oligonucleotide probes and DNA ligases with intracellular dsDNA, or for extracting the nucleus or dsDNA from cells. Appropriate methods for permeabilizing cells are known in the art and include contacting cells with surfactants, such as 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (e.g., Triton X-100™), nonylphenoxypolyethoxyethanol (e.g., NP-40™), polyoxyethylene sorbitan monolaurate (e.g., Tween™), saponins, or digitonin. For example, cells can be permeabilized by exposing them to 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, for example, 0.2% 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol.

[0041] The cell nucleus can be isolated or extracted from cells that have been fixed and permeabilized prior to step (i). For example, the method described may include preparing a cell sample and extracting the cell nucleus from the cell sample. The cell nucleus or the genomic DNA contained therein can then be used in this method.

[0042] In some embodiments, the dsDNA sample may be in solution in the method described herein. For example, cells containing dsDNA or nucleic acid can be brought into contact with the probe, DNA ligase and / or transposase in solution. Between steps, the dsDNA, the nucleic acid containing dsDNA, or the cells containing dsDNA can be washed, for example, by centrifugation and resuspension.

[0043] In other embodiments, the dsDNA sample may be immobilized on a solid support in the method described herein. The solid support is an insoluble, non-gelatinous material that provides a surface on which dsDNA or capture molecules that capture cells containing dsDNA can be immobilized. Examples of suitable supports include glass slides, microwells, membranes, or microbeads. The support may be in particulate or solid form, including, for example, plates, test tubes, beads, balls, filters, fabrics, polymers, or membranes. The capture molecules may bind to proteins, glycoproteins, or other molecules on the surface of cells. Capturing molecules suitable for cells are known in the art and include lectins that bind to extracellular glycoproteins on cells, such as concanavalin A.

[0044] The sequences of DNA strands isolated from a population of generated fragments may represent the sequences of strands cleaved at SSB sites in dsDNA. Multiple DNA strands may be isolated from the population. The sequences of multiple isolated DNA strands may represent the sequences of strands cleaved at multiple SSB sites in dsDNA.

[0045] In the method described herein, a sample of nucleic acid, such as dsDNA, is brought into contact with an oligonucleotide probe. The oligonucleotide probe includes a first sequencing adapter and a tag.

[0046] A sequencing adapter is a double-stranded oligonucleotide bound to a nucleic acid molecule so that sequencing is possible, for example, by promoting the amplification of the nucleic acid molecule using a sequencing primer. Sequencing adapters suitable for any sequencing method are available in the art. In some embodiments, the sequencing adapter may include a primer recognition sequence. The primer recognition sequence is a nucleotide sequence complementary to the sequence of the amplification primer. The presence of the primer recognition sequence makes it possible to amplify the DNA strand tagged with the adapter by PCR, for example, using an amplification primer that targets the primer recognition sequence. The primer recognition sequence may be a heterologous sequence that is not naturally present in dsDNA. Suitable primer recognition sequences are known in the art and include Illumina-compatible barcoded i7 / i5 primers. The first sequencing adapter may include the first primer recognition sequence.

[0047] In some embodiments, the first sequencing adapter may further include a barcode. The barcode is a unique nucleotide sequence for a sample from which a population of nucleic acids has been obtained. A suitable barcode sequence may be 6 to 10 nucleotides. The barcode makes it possible to clearly identify sequence reads from a specific sample in a pooled multiplex sequencing reaction. Each sample may have a unique barcode, and therefore all nucleic acids from the same sample receive the same barcode. Once prepared, populations of nucleic acids from different samples can be mixed into a single pool and sequenced. The sample from which the sequence reads from the pool originated can then be identified from the barcode. For example, a barcode suitable for multiplex sequencing of 24 samples (24plex reaction) may consist of at least 6 nucleotides, preferably 6 nucleotides (Craig DW et al. 2008. Nat Methods 5, 887, Cronn R et al. 2008. Nucleic Acids Res, 36, e122). The use of barcodes in sequencing reactions is known in the art. For example, Illumina sequencing 8mer barcodes i5 and i7 can be used. In other embodiments, one or both sequencing primers may include a barcode.

[0048] The tag enables the isolation of the DNA strand to which the tag is bound. Suitable tags include any label, molecule, or group that enables the specific binding of a binding member to the generated fragment or strand to which the tag is bound. The tag can enable covalent or, more preferably, non-covalent bonding of the binding member. Suitable tags include immunogens, e.g., digoxigenin; short-chain peptides, e.g., glutathione and FLAG®; or small-molecule organic compounds, e.g., biotin and trimethoprim (TMP).

[0049] Preferably, the tag is biotin, and the oligonucleotide probe is a biotinylated oligonucleotide.

[0050] The oligonucleotide probe may further include a hybridization region. The hybridization region may contain 2 to 10 nucleotides, preferably 4 to 8 nucleotides, more preferably 6 nucleotides, which anneal to the intact strand of dsDNA at the SSB site.

[0051] In some embodiments, the hybridization region may be nonspecific and can bind to an intact chain at any SSB site. For example, the hybridization region may have a random sequence. The nucleotides at each position in the hybridization region may be random ("N"; i.e., nucleotides randomly selected from G, C, T, or A), resulting in diverse hybridization regions for oligonucleotide probes.

[0052] In other embodiments, the hybridization region may be specific and capable of binding to an intact strand at a particular SSB site. For example, the hybridization region may be complementary to the sequence of an intact strand at a target site in the dsDNA, such as the site where the SSB is evaluated or detected. The target sequence may be the site of interest or a known site of the SSB, such as a promoter.

[0053] Examples of oligonucleotide probes are shown in Table 4, with appropriate ssBreak_3prime and ssBreak_5prime sequences indicated.

[0054] The oligonucleotide probe is ligated to a strand of dsDNA containing gaps or SSBs (i.e., cleaved strands) by a DNA ligase. Suitable DNA ligases are known in the art and are available from suppliers. Examples of DNA ligases suitable for use in the method described herein include T4 DNA ligase, T7 DNA ligase, ligase 3, and ligase 1. In one embodiment, the method described herein is carried out using T4 DNA ligase.

[0055] The dsDNA may be brought into contact with the oligonucleotide probe and the DNA ligase simultaneously, or more preferably, the dsDNA may be brought into contact with the oligonucleotide probe first, and then with the DNA ligase, in a sequential manner.

[0056] The oligonucleotide probe can be brought into contact with dsDNA, resulting in the oligonucleotide probe annealing to the intact strand of the dsDNA at one or more gaps or SSB sites. Then, the DNA ligase can be brought into contact with the dsDNA, resulting in the oligonucleotide probe covalently bonding to the cleaved ends of the strands at gaps or SSB sites in the dsDNA.

[0057] In some embodiments, the oligonucleotide probe is ligated to the 3' end of the cleaved strand of dsDNA at a gap or SSB site. Oligonucleotide probes suitable for 3' end binding may include a 5' phosphate group and a 3' tag. After tagmentation by transposase, a second sequencing adapter may be located at the 5' end of the isolated DNA strand, and the tag may be located at the 3' end of the isolated DNA strand.

[0058] In other embodiments, the oligonucleotide probe is ligated to the 5' end of a strand of dsDNA cleaved at a gap or SSB site. Oligonucleotide probes suitable for 5' end binding may include a 5' tag and a 3' hydroxyl group. After tagging by transposase, a second sequencing adapter may be located at the 3' end of the isolated DNA strand, with the tag located at the 5' end of the isolated DNA strand.

[0059] After ligating the oligonucleotide probe to the strand cleaved at the SSB site, the dsDNA is fragmented with a transposase to generate a population of dsDNA fragments tagged with a second sequencing adapter at the end.

[0060] In some embodiments, the transposase may be an activatable transposase. An activatable transposase can be converted from an inactive state to an active state by changing the conditions. For example, a transposase activated by magnesium may be activated by magnesium ions (Mg 2+ ) or manganese ions (Mn 2+ The active state can be achieved by increasing the concentration of the substance to, for example, a range of approximately 0.1 mM to approximately 10 mM.

[0061] Suitable activatable transposases are known in the art, including Tn5, Tn7, Mu, IS5, and IS91 (e.g., U.S. Patent No. 9005935, Mizuuchi, K., Cell, 35: 785, 1983, Savilahti, H, et al, EMBO J., 14: 4893, 1995, Goryshin and Reznikoff, J. Biol. Chem, 273:7367 (1998), and International Publication No. 2022056309). In some preferred embodiments, the transposase may be a Tn5 transposase. Tn5 transposases are available from suppliers (e.g., Tagmentase®, Diagenode®).

[0062] A transposase can be loaded with one or more second sequencing adapters. The transposase may be pre-loaded with a second sequencing primer, or the method may include loading the transposase with the second sequencing primer. The second sequencing adapter can be non-covalently bonded to the transposase to form a complex containing the transposase and one or more second sequencing adapters. Suitable methods for loading the transposase with a second sequencing adapter are known in the art. For example, the second sequencing adapter can be incubated with the transposase at room temperature for 1 hour.

[0063] The sequencing adapter is as described above. A second sequencing adapter suitable for binding to a transposase may be a double-stranded oligonucleotide containing a transposase recognition sequence and a primer recognition sequence. The transposase recognition sequence is the sequence targeted for transposition by the transposase. For example, the transposon sequence inserted by the transposase can be flanked by the reverse transposase recognition sequence. The sequence of the transposase recognition sequence may be transposase-dependent. Transposase recognition sequences suitable for various transposases are known in the art. For example, suitable transposase recognition sequences for Tn5 include 19mer terminal sequences, such as the outer end (OE) sequence (e.g., 5'-CTG ACT CTT ATA CAC AAG T-3') and the inner end (IE) sequence (e.g., 5'-CTG TCT CTT GAT CAG ATC T-3'), and the mosaic end (ME) sequence (e.g., 5'-CTG TCT CTT ATA CAC ATC T-3'). In some preferred embodiments, a 19-mer ME sequence can be used. The second sequencing adapter may include a transposase recognition site and a second primer recognition sequence. Suitable sequencing adapters are known in the art (e.g., the N7 Nextera® adapter).

[0064] Examples of suitable second sequence adapters are shown in Table 4, which details the N7_top and N7_bottom sequences.

[0065] The dsDNA sample is cleaved by a transposase, generating a population of DNA fragments. DNA fragments within the population are tagged with a second sequencing adapter at one or both ends. DNA fragments within the population that do not contain gaps or SSB regions are tagged with a second sequencing adapter at both ends. DNA strands from a population of fragments containing gaps or SSB regions are tagged with a second sequencing adapter at one end and with an oligonucleotide probe containing a first sequencing adapter at the other end.

[0066] DNA strands tagged with both the first sequencing adapter and the second sequencing adapter can be isolated from the population using the tags. The fragments can be denatured using any suitable technique, such as NaOH treatment, to generate DNA strands. Then, the DNA strands tagged with both the first sequencing adapter and the second sequencing adapter can be isolated using a binding member that binds to the tags of the oligonucleotide probes.

[0067] The binding member is a target molecule or ligand, for example, a molecule that specifically binds to the tag. The binding member can bind covalently or, more preferably, non-covalently to the tags of the oligonucleotide probes. For example, if the tag is biotin, the binding member can specifically bind to biotin. Examples of binding members suitable for binding to biotin tags include anti-biotin antibodies, avidin, and streptavidin. The binding member that specifically binds to the tag may not show any significant binding to molecules other than the tag. In particular, the binding member may not show any significant binding to proteins, DNA, or other antigens that may be present in cells or cell extracts. Generally, an antibody or other binding member that specifically binds to the tag has a binding affinity (Ka) of greater than about 10 5 mol / liter (e.g., 10 6 mol / liter or more, 10 7 mol / liter or more, 10 8 mol / liter or more, 10 9 mol / liter or more, 10 10 mol / liter or more, 10 11 mol / liter or more, or 10 12 mol / liter or more). Suitable binding members are known in the art and can be made using standard techniques or obtained from commercial sources.

[0068] In a preferred embodiment, the tag is biotin and the binding member is streptavidin. In some embodiments, the binding member may be immobilized on a solid support such as a bead.

[0069] During any step of the method described herein, a washing step may be performed as needed to remove unbound reagents. For example, oligonucleotide probes that are not bound to dsDNA after the contact step can be removed by washing. Similarly, DNA ligases and / or transposases can also be removed by washing. Suitable washing methods are known in the art. For example, washing can be performed using the buffers described herein, e.g., 20 mM HEPES (pH 7.5), 150 mM NaCl, and 0.5 mM spermidine.

[0070] As described above, the isolated DNA strands can be amplified to produce amplified products for sequencing. The isolated DNA strands can be amplified using primers that hybridize with the primer recognition sequences of the sequencing adapter. Appropriate amplification methods are well established in the art, including polymerase chain reaction (PCR) (for example, reviewed in "PCR protocols; A Guide to Methods and Applications", Eds. Innis et al, 1990, Academic Press, New York; Mullis et al, Cold Spring Harbor Symp. Quant. Biol., 51:263, (1987); Ehrlich (ed), PCR technology, Stockton Press, NY, 1989; and Ehrlich et al, Science, 252:1643-1650, (1991)).

[0071] Amplification can generate a population or library of amplified products for sequencing. Each amplified product in the population or library may contain a nucleic acid sequence at the SSB site in dsDNA. The population or library of amplified products generated as described herein can be purified before sequencing using standard techniques, including spin column chromatography (e.g., Ampure XP® beads).

[0072] The sequence of the isolated strand or amplified product can be determined by any suitable technique. Suitable techniques include sequencing and hybridization-based techniques, preferably sequence-specific amplification such as qPCR.

[0073] In a preferred embodiment, the sequence of the isolated chain or amplified product may be determined by sequencing the chain or product.

[0074] Isolated DNA strands or amplification products can be sequenced using standard sequencing techniques. Suitable techniques include Sanger sequencing, Solexa-Illumina sequencing, ligation-based sequencing (SOLiD®), pyrosequencing; single-molecule real-time sequencing (SMRT®); PacBioscience sequencing; and semiconductor array sequencing (Ion Torrent®), including any convenient low-throughput or high-throughput sequencing technique or platform. Sequencing is preferably performed using next-generation sequencing techniques. Protocols, reagents, and equipment suitable for nucleic acid sequencing are known and commercially available in the art.

[0075] The sequence of an isolated DNA strand or amplification product may indicate the nucleotide sequence of the SSB site in the dsDNA. For example, the sequence of an isolated DNA strand or amplification product may include the nucleotide sequence at the site of the strand where the SSB occurred. The location of the SSB in the dsDNA can be identified or mapped from the sequence of the isolated DNA strand or amplification product. For example, the location of the SSB in the cell genome of eukaryotic cells, mammalian cells, or human cells can be identified or mapped by the method described herein. The sequence of the isolated strand or amplification product may indicate the frequency of SSB occurrence at a site in the dsDNA, or the probability of SSB occurrence at a site.

[0076] In some embodiments, the method described herein may include mapping SSB sites in the first and second dsDNAs, and identifying SSB sites that are present in the first dsDNA but not in the second dsDNA, or present in the second dsDNA but not in the first dsDNA. The method described herein may also include mapping the frequency of SSBs at sites in the first and second dsDNAs, and identifying sites where the frequency of SSB occurrence is increased in the first dsDNA relative to the second dsDNA, or in the second dsDNA relative to the first dsDNA.

[0077] The first dsDNA may be a test sample, and the second dsDNA may be a reference sample or control sample. In some embodiments, the first dsDNA may be derived from diseased cells, such as cancer cells, obtained from an individual with a disease, and the second dsDNA may be derived from normal or healthy cells.

[0078] The frequency or distribution of SSBs in the first dsDNA relative to the second dsDNA may indicate the diagnosis, severity, penetrant, or prognosis of diseases such as cancer in the individual from which the first dsDNA was obtained. Diseases or cancers may be caused by or characterized by defects in DNA repair pathways.

[0079] In other embodiments, the first dsDNA may be derived from cells at a specific developmental stage, such as cells obtained from an embryo, and the second dsDNA may be derived from adult or mature cells. In other embodiments, the first dsDNA may be derived from age-related cells, such as cells obtained from an individual of a specific age, such as an elderly person, and the second dsDNA may be derived from a control individual. In other embodiments, the first dsDNA may be derived from treated cells, and the second dsDNA may be derived from untreated control or reference cells. For example, the first dsDNA may be derived from cells treated with reprogramming factors such as Oct3 / 4, Sox2, Klf4, and c-Myc ("Yamanaka factor" or "OSKM"), radiation, compounds, and drugs such as chemotherapeutic agents.

[0080] In some embodiments, cells can be stratified according to their age and / or disease state, for example, cells obtained from test subjects, such as human subjects, using the method described herein.

[0081] In some embodiments, the methods described herein can be used in assays to detect the therapeutic effect of agents used to treat infectious diseases in which subjects, such as human subjects, are infected with viruses or bacteria. In such assays, viral dsDNA or bacterial dsDNA is obtained from subjects treated with an antiviral or antibacterial agent, and the dsDNA is then subjected to the methods described herein to detect SSBs. The test results can be compared with a control sample containing the same viral or bacterial DNA that has not been exposed to any antiviral / antibacterial agent. Using such assays, the effectiveness of anti-infective agents such as antiviral or antibacterial agents (e.g., antibiotics) can be determined.

[0082] dsDNA can be prepared using standard methods known in the art.

[0083] In some embodiments, the preparation of dsDNA from any of the cell types described herein may include one or more of the following steps that can facilitate the introduction of oligonucleotide probes. (i) Cell samples containing dsDNA can be subjected to a washing and lysis process, which can be carried out in a small volume, for example, about 0.1 ml to about 2.0 ml, for example about 0.2 ml, or about 0.5 ml or about 1.5 ml, in a container, for example, a tube, vial or coverslip. (ii) The sample containing dsDNA can then be subjected to a buffer exchange step, for example, by centrifugation of the sample or by dilution.

[0084] The aforementioned buffer exchange can be achieved, for example, by using the following method. First, the cells are suspended in PBS (or a similar isotonic buffer). To isolate the nuclei from the cells, they are centrifuged to remove the PBS, and the cells are resuspended in a lysis (or nuclear isolation) buffer containing a hypotonic, mild surfactant, which solubilizes the cell membrane while leaving the nuclear membrane intact. Next, the cells are centrifuged again and resuspended in a second lysis buffer containing a stronger surfactant (SDS) that solubilizes proteins, thereby removing chromatin proteins from the DNA (a process known as nucleosome removal). After this, the nuclei are centrifuged again and resuspended in a buffer without SDS, and this step is repeated several times to wash the nuclei and ensure that SDS is completely removed (because the presence of SDS can inhibit downstream reactions).

[0085] In some embodiments, the preparation of dsDNA from cells may include the following steps to facilitate dsDNA purification. After contacting a dsDNA sample with DNA ligase according to the method detailed herein, the sample can then be purified, for example, by proteinase digestion, for example by proteinase K digestion, for example by incubating the sample with proteinase K for about 2 hours to about 30 minutes, for example by incubating for about 30 minutes.

[0086] In some embodiments, the isolation of DNA strands including a first sequencing adapter and a second sequencing adapter can be carried out using magnetic beads, where the sample containing dsDNA is mixed with magnetic beads and incubated for about 1 to 2 hours, for example, about 30 minutes.

[0087] In some embodiments, the Disclosure provides a method for detecting SSB cleavage in dsDNA obtained from cells (e.g., any of the cells detailed herein), the method comprising the following steps: (i) A step of selectively fixing the above cells, (ii) The above cells are subjected to a washing and dissolution process in a tube / vial / coverslip, for example, in a volume of about 0.1 ml to about 0.3 ml, for example, about 0.2 ml, and then, (iii) A step of subjecting a sample containing dsDNA to buffer exchange, for example by centrifugation of the sample or by dilution, (iv) A step of contacting a dsDNA sample with an oligonucleotide probe containing a first sequencing adapter and tag, (v) A step of contacting a dsDNA sample with a DNA ligase, wherein the DNA ligase covalently attaches oligonucleotide probes to the dsDNA strands at single-strand break sites in the dsDNA. (vi) A step to purify dsDNA, for example by proteinase digestion, for example by using proteinase K, for example by incubating the sample with proteinase K for about 2 hours to about 30 minutes. (vii) A step of contacting a dsDNA sample with a transposase loaded with a second sequencing adapter, wherein the dsDNA is cleaved by the transposase, and a collection of fragments containing the second sequencing adapter at the end is generated. (viii) From the above population, a step of isolating DNA strands containing the first sequencing adapter and the second sequencing adapter by incubation with magnetic beads, for example, for an incubation period of about 5 minutes to about 2 hours, for example, about 30 minutes, and (ix) The step of determining the sequence of the isolated DNA strand.

[0088] In some of the embodiments described above, the oligonucleotide incorporation and ligation steps can be carried out directly on purified DNA or intact nuclei.

[0089] The number of cells in step (i) or step (ii) described above may range from approximately 10,000 to approximately 1,000,000, for example, approximately 25,000, approximately 50,000, approximately 75,000, approximately 100,000, approximately 150,000, or approximately 200,000.

[0090] Proteinase digestion can be carried out at temperatures ranging from approximately 37°C to 60°C, for example, at approximately 37°C when using thermally unstable proteinase K.

[0091] The above steps (i) to (ix) allow for the detection of SSBs to be performed using fewer cells and in a shorter period of time.

[0092] High-throughput method: A high-throughput method can be obtained by adapting the SSB detection method described herein. To scale the method to a larger number of samples, cells can be collected and processed in 96-well or 384-well PCR plates (or by using a microfluidic device). Oligonucleotides can be redesigned to contain barcode sequences specific to each sample (i.e., 96 different barcodes used for samples in a 96w plate). After ligating the barcoded oligos, all samples can be pooled together (e.g., rapidly using a centrifuge in a V-block (clickbio CBVBLOK200)) for purification and all other downstream processing. After sequencing and data processing, samples can be identified via the barcode sequences. Processing can also be made more efficient by using an automated liquid processing device. The required pipetting steps can be reduced by discontinuing buffer exchange instead of diluting the previous components by adding new buffer to old buffer.

[0093] Single-cell method: The SSB detection method described herein can be performed on a single cell, including, for example, when dsDNA is obtained using the above method for introducing oligonucleotide probes and optimizing dsDNA purification, and can generally be performed as detailed below. Samples containing dissociated, fixed cells are flow-sorted to isolate single cells into individual wells of a 96-well or 384-well plate containing the lysis buffer necessary for nuclear isolation and nucleosome removal. Similar to the high-throughput method described above, subsequent steps are performed in a PCR plate, and sample pooling can be enabled using barcoded oligonucleotides. Since cell loss is observed during centrifugation / buffer exchange, buffer exchange can be discontinued and buffer addition can be optimally employed instead. If buffer addition is employed, cells are collected in the lysis buffer after lysis incubation, and then another buffer that can dilute or inactivate the lysis reagents is added. After this step, oligonucleotides and ligation reagents can be added, ensuring that the concentrations of salts and surfactants are appropriate for ligase activity.

[0094] The methods described herein may be useful, for example, in cytotoxicity assays. A cytotoxicity assay may include stimulating cells and determining the frequency or distribution of gaps or SSBs in the cell-derived dsDNA. An increase in the frequency or distribution of gaps or SSBs in the dsDNA of the stimulated cells compared to dsDNA of unstimulated control cells indicates that the stimulation is cytotoxic. Suitable stimuli include exposure to compounds and radiation.

[0095] In other embodiments, the first dsDNA may be derived from cells treated with a nuclease such as a CRISPR nuclease (e.g., Cas9 or Cpf1), and the second dsDNA may be derived from untreated control cells. The frequency or distribution of SSBs in the first dsDNA relative to the second dsDNA may indicate off-target effects of the nuclease in the treated cells.

[0096] In other embodiments, the first dsDNA may be derived from oocytes or spermatids obtained from a donor, and the second dsDNA may be derived from control oocytes or spermatids. The frequency or distribution of SSBs in the first dsDNA relative to the second dsDNA may indicate the quality of the donor's oocytes or spermatids. This may be useful, for example, in assisted reproductive technology (ART) methods.

[0097] A set of sequence reads from amplified or isolated DNA strands can be generated by sequencing. For example, more than 1,000, more than 10,000, more than 1,000,000, more than 1,000,000, more than 1,000,000, or more than 1,000,000,000 sequence reads can be generated. The sequence reads can be analyzed using conventional bioinformatics techniques. Appropriate techniques are known in the art. For example, duplicate reads, low-quality sequence reads, and reads resulting only from sequencing adapters can be removed.

[0098] In some embodiments, sequence reads within a set can be analyzed for the presence of SSB sites in dsDNA, the frequency of SSBs occurring at those sites, and / or the pattern of SSB sites. Sequence reads within a set can be further analyzed for other features associated with SSBs, such as mutations, epigenetic modifications, or sequence motifs.

[0099] Fragment sequence reads within the set can be mapped to one or more locations within a reference genome. Suitable reference genomes are available in the art. For example, human nucleic acid fragment sequence reads within the set can be mapped to locations within the human genome sequence. In some embodiments, the reference genome may be matched to the sex, ethnicity, and / or other characteristics of the individual from which the sample was obtained.

[0100] The fragment sequence reads within a set can be mapped by aligning them with the sequence of a reference genome, such as the human genome. The location of the sequence reads within the reference genome can then be identified. Suitable software tools for mapping populations of sequence reads within a genome are readily available in the art.

[0101] The distribution of fragment sequence reads within a set of genomes or within a set of sites or loci (i.e., the number of sequence reads mapped to each location within the set of genomes or sites or loci) can be determined from the locations of the sequence reads within the set. Optionally, the distribution of fragment sequence reads can be subjected to mathematical transformations.

[0102] A sample SSB profile can be generated from the distribution or transformed distribution of nucleic acid fragment sequence reads. The sample SSB profile may include a set of scores or values ​​indicating the number or density of nucleic acid fragment sequence reads mapped to each location or position within the genome (i.e., a plot across the entire genome) or to each location or position within a set of sites or loci within the genome. The number or density of fragment sequence reads mapped to a location or position within the genome may indicate the frequency of SSBs at that location or position. Therefore, the sample SSB profile may reflect the distribution of SSBs in the genome or at target loci within the genome. The sample SSB profile can be represented in any convenient format, for example, numerically or graphically.

[0103] In some embodiments, sample SSB profiles can be used to identify SSB sites associated with biological responses to drugs or other compounds, such as cytotoxic responses, which may be useful in determining or predicting an individual's response to treatment with drugs or other compounds. Sample SSB profiles can be used for therapeutic stratification, optimization of combination therapies, diagnosis of disease status, determining whether cells in a subject (e.g., a human subject) have reduced resilience, and whether treatments that can stimulate SSB repair and / or rejuvenate and / or reprogram cells can be selected by exposing them to drugs (chemical and / or biological) that can stimulate SSB repair, for example, determining side effects of treatments with drugs or other compounds, cytotoxic assays, determining off-target effects of nucleases, methods for assisted reproductive technologies, or studies of aging or development. In some embodiments, SSB profiles can be used to distinguish between different cell types, for example, for cell type identification. This method can be used to profile cells or in screening to discover cell type-specific differences in the genomic distribution of DNA gaps, and to investigate the effects of transcriptional perturbations on DNA gap formation dynamics.

[0104] A marker (target) site is a location within the genome where the presence or frequency of SSBs varies among different sources; that is, a location where SSBs at a marker site are specific to, for example, a source from a different individual, tissue, cell type, developmental stage, or age. A set of marker sites within a sample SSB profile can provide a signature characteristic of the source.

[0105] In some embodiments, suitable marker sites can be identified by determining the presence of SSBs at multiple candidate sites within a reference SSB profile derived from a set of reference sources. The reference SSB profile may include a set of scores or values ​​indicating SSBs at a set of marker sites within genomic DNA derived from a known source, e.g., a specific known tissue or cell type. The reference SSB profile may reflect the presence of SSBs at locations within genomic DNA derived from a known source. A suitable reference SSB profile may be obtained or generated by conventional experiments using known tissues or cell types, or it may be constructed from publicly available data sources such as genomic information databases.

[0106] A candidate site can be identified as an SSB marker site if the frequency of SSB occurrences at that site is higher or lower than that of other sources in the set for one reference source in the set. For example, the frequency of SSB occurrences at that site may be higher or lower than the average frequency of SSB occurrences in the other reference sources in the set. In some embodiments, the frequency of SSB occurrences at a site in one reference source may be above or below a predetermined threshold compared to the average frequency of SSB occurrences at corresponding sites in the other reference sources in the set.

[0107] In other embodiments, suitable marker sites may be identified by providing a first set of sample SSB profiles from control individuals, e.g., healthy individuals, and a second set of sample SSB profiles from test individuals, e.g., individuals with a disease condition such as a specific cancer, individuals known to be responsive or unresponsive to a drug, or individuals of a specific age. The frequencies of SSBs at multiple candidate sites within the first and second sets of sample SSB profiles can be compared. Candidate sites can be identified as marker sites if the frequency of SSB occurrence at that site is higher or lower in the first set of sample SSB profiles than in the second set. For example, the average frequency of SSB occurrence at that site may be higher or lower in the first set than in the second set. In some embodiments, the average frequency of SSB occurrence at sites in the first set may be above or below a predetermined threshold compared to the average frequency of SSB occurrence at sites in the second set.

[0108] Candidate sites can also be identified as marker sites if they are found to be in chain disequilibrium with a site that has a higher or lower frequency of SSB occurrences than other sources in the set for one reference source in the set.

[0109] Using sample SSB profiles derived from individual organisms, it is possible to determine the presence or frequency of SSB occurrence at a set of marker sites in an individual. This can be useful in determining the effect of a drug or other compound on an individual, or the individual's responsiveness to a drug or other compound, for example, to determine the effectiveness of a drug or other compound treatment in an individual, or to determine the individual's suitability for a drug or other compound treatment. As part of a clinical trial, or as evidence of off-target toxicity where toxicity is caused by SSBs at additional / alternative sites in dsDNA, sample SSB profiles derived from individual organisms can also be used to demonstrate the mechanism of action of a drug or other compound.

[0110] The methods described herein can be used in combination with other analytical techniques, for example, in multi-omics genome analysis. In some embodiments, the methods described herein may further include determining the presence of epigenetic marks at or near SSB sites in dsDNA. Suitable epigenetic marks include modified nucleic acid bases such as methylated DNA cytosines. For example, isolated DNA strands prepared as described herein may be treated with bisulfite or in combination with other chemical modifications (e.g., conversion of existing modifications to 5caC to identify 5hmC / 5mC) and then sequenced. The sequence of the isolated DNA strands may be compared with a reference genome sequence and the modified cytosine residues in the identified isolated DNA strands.

[0111] The methods described herein may use dsRNA or a DNA / RNA hybrid in which one strand is DNA and the other is RNA instead of dsDNA. When dsRNA is used in the methods described herein, the methods may be modified as follows: optionally, an RNA ligase (e.g., T4 RNA ligase) may be used instead of a DNA ligase, and optionally, an RNA oligonucleotide may be used instead of a DNA oligonucleotide. After the ligation step, the RNA may be transcribed to DNA using reverse transcriptase.

[0112] dsRNA may be, for example, siRNA, mRNA, or tRNA. The method described herein can be used, for example, as part of quality control to evaluate the stability of dsRNA, such as therapeutic siRNA (including the stability of such RNA molecules when stored under different storage conditions, such as when stored in different formulations, or when stored at different temperatures and / or for different periods). When applied to dsRNA, the method can also be used to identify RNA base modifications on modified RNA strands.

[0113] Screening Method: The methods described herein for detecting SSBs can be used, for example, in a screening method for identifying agents (e.g., chemical agents and / or biological agents, e.g., small molecule and / or peptide and / or protein therapeutics) that can enable the repair and / or reprogramming of SSBs in dsDNA.

[0114] Therefore, a method is also provided for screening such drugs, e.g., small molecule and / or peptide and / or protein therapeutics, that can enable the repair of SSBs in dsDNA and / or the reprogramming of cells, and this method is, (a) A step of detecting single-strand breaks (SSBs) in a control sample of double-stranded (ds) DNA that has not been exposed to a drug (e.g., a chemical drug and / or a biological drug, e.g., a small molecule and / or peptide and / or protein therapeutic agent), and detecting single-strand breaks (SSBs) in a test version of the same sample of dsDNA that has been exposed to a drug (e.g., a chemical drug and / or a biological drug), (i) Contact both the test dsDNA and control dsDNA samples with an oligonucleotide probe containing the first sequencing adapter and tag. (ii) Contacting the test dsDNA and control dsDNA samples with DNA ligase, such that the DNA ligase covalently attaches oligonucleotide probes to the dsDNA strands at single-strand break sites in the dsDNA, and (iii) Contacting samples of test dsDNA and control dsDNA with a transposase loaded with a second sequencing adapter, thereby causing the dsDNA to be cleaved by the transposase and generating a collection of fragments containing the second sequencing adapter at the end. The process and (b) A step of isolating DNA strands containing the first sequencing adapter and the second sequencing adapter from the above population in both the test sample and the control sample, (c) A step of determining the sequence of DNA strands isolated from the test sample and the control sample, (d) A step of comparing the SSB profiles in both the test sample and the control sample, (e) A step of isolating chemical and / or biological agents associated with the decrease in SSB in the test sample compared to a control sample, (f) A step of compounding the isolated chemical and / or biological agents into a pharmaceutical composition by adding, for example, appropriate pharmaceutically acceptable excipients (which may be more than one), Includes.

[0115] The above pharmaceutical composition can be used to treat a subject (e.g., a human subject) to promote the repair of SSBs in cells and / or to reprogram the cells in the subject.

[0116] The SSB screening method detailed above can be applied to cells such as human cells in test subjects, and may further include one or more of the following steps to facilitate the introduction of oligonucleotide probes, for example. (i) Cell samples containing dsDNA can be washed and dissolved in small volumes, for example, about 0.1 ml to about 0.3 ml, for example, about 0.2 ml, in a tube, vial, or coverslip. (ii) The sample containing dsDNA can then be subjected to buffer exchange, for example, by centrifugation of the sample or by dilution.

[0117] In some embodiments, the above screening method may include the following steps to facilitate dsDNA purification: After contacting the dsDNA sample with DNA ligase, the sample can be purified, for example, by proteinase digestion, for example by proteinase K digestion, or by incubating the sample with proteinase K for about 2 hours to about 30 minutes.

[0118] In some embodiments, the isolation of DNA strands including a first sequencing adapter and a second sequencing adapter can be performed using magnetic beads, for example, a sample containing dsDNA can be incubated with magnetic beads for about 5 minutes to about 2 hours, for example, about 30 minutes.

[0119] Proteinase digestion can be carried out at temperatures ranging from approximately 37°C to 60°C, for example, at approximately 37°C when using thermally unstable proteinase K.

[0120] The above screening method can be used to identify drugs / compounds that can reprogram cells and / or repair SSBs in dsDNA present within cells. Furthermore, the screening method can be used to track the changes in cell reprogramming and / or repair over time, for example, by performing the method on dsDNA obtained at different time points after incubation with the drug / compound. The above screening method can also be adapted to produce and isolate dsDNA with gaps introduced by design by substituting the drug capable of enabling SSB repair with a gap-forming drug, and then testing the dsDNA exposed to such a drug and comparing it to a control sample not exposed to the gap-forming drug to confirm the presence of gaps.

[0121] Kits used in the methods described herein and the use of such kits in such methods are also provided. A first sequencing adapter and an oligonucleotide probe including a tag, DNA ligase and, Transposase and, A second sequencing adapter that is loaded or loadable onto a transposase, It can include...

[0122] The kit may further include a binding member that specifically binds to the tag, a sequencing primer, and / or other reagents. The kit may further include appropriate buffers and washing solutions; fixatives and permeabilizers.

[0123] The kit may further include a solid support. A suitable solid support may include a capture molecule that binds to eukaryotic cells, such as a lectin, and / or a binding member.

[0124] The kit may further include nucleic acid extraction and purification reagents. Suitable reagents are known in the art and include spin chromatography columns.

[0125] The kit may further include amplification reagents. Suitable reagents are known in the art and include primers, dNTPs, and thermostable polymerases. In some embodiments, the kit may include amplification primers for amplifying one or more target regions or loci in the genome.

[0126] The kit may include instructions for use in the manner described herein.

[0127] Other aspects and embodiments of the present invention provide the aspects and embodiments described above with the terms "comprising" replaced by the term "consisting of", and the aspects and embodiments described above with the terms "comprising" replaced by the term "consisting essentially of".

[0128] It is understood that this application discloses all aspects and all combinations of the embodiments described above, unless the context should be interpreted otherwise. Similarly, this application discloses all combinations of preferred and / or optional features, either individually or with any other aspects, unless the context should be interpreted otherwise.

[0129] Modifications of the above embodiments, further embodiments, and variations thereof will become apparent to those skilled in the art by reading this disclosure, and they themselves fall within the scope of the present invention.

[0130] All documents and sequence database entries referenced herein constitute part of this specification by reference in their entirety for any purpose.

[0131] As used herein, “and / or” shall be understood as a specific disclosure that includes or excludes each of two expressed features or components. For example, “A and / or B” shall be understood as if each of (i) A, (ii) B, and (iii) A and B were described separately herein. [Examples]

[0132] experiment Example 1: Materials and methods An example of the GAPS-Seq method is schematically shown in Figure 1. These examples include: 1) crosslinking cells / tissues using a crosslinking agent to fix DNA containing single-stranded DNA gaps or SSBs; 2) permeabilizing cells using a surfactant to denature chromatin proteins, thereby allowing access to genomic DNA containing SSBs; 3) directly ligating a DNA oligonucleotide probe containing a random hybridization region, a first sequencing adapter, and a tag to the DNA gap site; 4) lysing the nucleus to isolate the genomic DNA; 5) attaching a second adapter to the isolated DNA using tagmentation; 6) capturing the DNA gap-binding tagged adapter; and 7) separating, amplifying, and sequencing the DNA strand containing the gap. In summary, cells were dissociated into single cells using TrypLE Express Enzyme and then fixed in 2% formaldehyde at room temperature for 10 minutes. The nuclei were isolated on ice for 15 minutes using 0.2% Triton-X100, and then the chromatin proteins / DNA-binding proteins were denatured using SDS. The nuclei were then pelleted using centrifugation and resuspended in biotinylated GAPS-seq oligos containing Illumina Read 1 sequences. T4 DNA ligase was then added, and the suspension was incubated overnight at 16°C with shaking (800 rpm). Reverse crosslinks were then performed using proteinase K, the proteins were digested, and then the DNA was purified by column. Approximately 40 ng of purified DNA was tagged with Illumina Read 2, and then the GAPS-seq fragments were captured on streptavidin beads before indexed PCR.

[0133] Sample preparation 1M cells were dissociated into single cells and fixed in 2% formaldehyde at room temperature for 10 minutes. The reaction was stopped by adding 2M glycine to a final concentration of 125mM, and the supernatant was removed by rotating at 100g for 5 minutes at 4°C. The cells were then resuspended twice in 1 ml of phosphate-buffered saline (PBS), and the supernatant was removed by rotating at 100g for 5 minutes at 4°C for washing. The cells were then resuspended in 1 mL of ice-cold LB1 (10mM Tris (pH 8.0), 10mM NaCl, 1mM EDTA, 0.2% Triton) and incubated on ice for 15 minutes. The supernatant was then removed by rotating at 300g for 5 minutes at room temperature. Next, the nuclei were resuspended in 1 ml of LB2 (10 mM Tris (pH 8.0), 150 mM NaCl, 0.3% SDS) preheated to 37°C, then incubated in a thermal mixer at 37°C for 60 minutes, gently shaken at 400 rpm, and rotated at 300 g for 5 minutes at room temperature, after which the supernatant was removed. Fresh CTSX buffer was prepared (100 μl 10x Cutsmart, 10 μl 10% Triton X-100, 890 μl nuclease-free water), warmed to 37°C, and used to resuspend the nuclei. The nuclei were confirmed to be 100% trypan blue positive, intact, and round in shape by trypan blue staining and microscopic observation. Next, the nuclei were rotated at 300 g for 5 minutes at room temperature, the supernatant was removed, and the CTSX washing was repeated.

[0134] Sample preparation for etoposide treatment Etoposide is known to induce both single-stranded and double-stranded DNA gaps (Muslimovic et al., 2009), so etoposide treatment was performed to verify this method. In this experiment, passage 14 HEK293 cells were treated with 30 μM etoposide added to cell culture medium and incubated at 37°C in a 5% CO2 incubator for 4 hours. Control samples were treated with DMSO at the same concentration as the etoposide-treated cells and incubated for 4 hours at 37°C in a 5% CO2 incubator. Dissociation and fixation were performed as described above.

[0135] Sample preparation for restriction enzyme experiments 1M cells (6 pg × 1M = 6 μg dsDNA) were fixed and processed in exactly the same manner as described above. After washing with CTSX buffer, the nuclei were resuspended in 100 μl of CTSX buffer, and 2 μl of the appropriate restriction enzyme was added. The samples were then incubated overnight at the recommended temperature (37°C for all enzymes except TspRI, which required 65°C) with shaking at 400 rpm. A control sample without any restriction enzyme was processed in parallel and incubated at 37°C with shaking at 400 rpm.

[0136] GAPS-seq oligo ligation Next, the nuclei were resuspended in 4 μl of 100 μM ssBreak oligosaccharide, and 96 μl of ligation mixture (formulated as shown in Table 1 below) was added on ice.

[0137] [Table 1]

[0138] The nuclei were incubated overnight in a shaker with T4 ligase at 16°C and 400 rpm.

[0139] Reverse crosslinking agents and DNA purification 10 μl of proteinase K (NEB) was added to the ligation reaction mixture and incubated at 55°C for 2 hours. Next, nuclear DNA was purified using the zymo Quick-DNA Microprep kit, 500 μl of DNA lysis buffer was added, and the nuclei were vortexed and rotated at 10000 g for 1 minute. The supernatant was transferred to a column and rotated at 10000 g for 1 minute, 200 μl of pre-wash buffer was added to the column, and the column was rotated at 10000 g for 1 minute, then 500 μl of gDNA wash buffer was added to the column, and the column was rotated at 10000 g for 1 minute.

[0140] Next, 20 μl of water was added to the column and placed in a 1.5 ml Eppendorf tube, where it was rotated at maximum speed for 5 minutes at room temperature. Then, the DNA was quantified using a Qubit and the results were recorded. The tagmentation reaction was then set up as shown in Table 2 below.

[0141] [Table 2]

[0142] The reaction mixture was incubated at 55°C for 10 minutes, and then 10.5 μl of 0.2% SDS was added.

[0143] Biotin pull-down of SSB samples Streptabidin beads were prepared. 5 μl of C1 beads per sample were washed twice in 2x B&W (10 mM Tris, 1 mM EDTA, 2 M NaCl) and resuspended in 50 μl of 2x B&W. 50 μl of sample was added to 50 μl of beads and mixed in a shaker at room temperature for 1 hour. The supernatant was removed using a magnet, and the beads were washed twice in 0.1 M NaOH and twice in water. The beads were resuspended in the PCR mixture as shown in Table 3 below.

[0144] [Table 3]

[0145] DNA on beads was amplified as follows: 65°C for 5 minutes; 98°C for 45 seconds, followed by 20 cycles of 98°C for 15 seconds, 60°C for 30 seconds, and finally 72°C for 1 minute. The PCR product was then purified with 0.65x AMPure XP beads (13 μl) and QC was performed using tapestation D5000 high-sensitivity tape.

[0146] Cell number dilution experiment To titrate the input requirements, dilution series containing 1 million, 500,000, 100,000, 10,000, 1,000, 500, and 100 cells were prepared. All steps remained the same except that the number of PCR cycles was increased by an additional 2 cycles (100,000 cells), 3 cycles (10,000 cells), 5 cycles (1,000 cells), or 8 cycles (500 and 100 cells).

[0147] Sequencing and data processing The library was pooled in equimolar quantities and then sequenced using 50nt paired-end reads and 8nt i7 and i5 index reads with an Illumina NextSeq2000 instrument and P1 flow cell according to the manufacturer's instructions. The sequenced reads were demultiplexed using bcl2fastq2 (Illumina) to generate two fastq files corresponding to read 1 and read 2. The fastq files were processed using the Nextflow chipseq pipeline (Ewels, et al. 2020) to generate a BAM file containing aligned reads. In summary, this required QC of raw reads using FastQC (v0.11.9) (Andrews 2010), followed by trimming using Trim Galore (v0.6.7) (Krueger et al 2023) and cutadept (v3.4) (Martin et al 2011) to remove Illumina primer sequences from the 3' end and the first 6nt of the 5' end corresponding to random priming segments. The trimmed reads were then aligned to the GRCh38 version of the human genome using BWA (v0.7.17-r1188) (Li et al 2010) and filtered using Samtools (v1.15.1) (Danecek et al 2021) and picard (v2.27.4) to remove reads mapped to duplicate and blacklisted regions.

[0148] For promoter region analysis, the BAM file was loaded into Seqmonk v1.48.1, and probe trend plots were generated using default parameters. The data was exported as a text file and imported into R (v4.1.2) (R Core Team 2022) to enable plotting using ggplot2 (v3.4.2) (Wickham 2009). For restriction enzyme site analysis, we first identified all instances of a given restriction enzyme motif in the GRCh38 genome using the BSgenome.Hsapiens.UCSC.hg38 (v1.4.4) package and the matchPattern function of biostrings (v2.64.1) in R. This was imported into Seqmonk as an annotation track, and probe trend plots were generated in the same way as for the promoters described above.

[0149] result The relative frequency of single-strand gaps in gene promoters was determined using the GAP-seq method (Figure 2). This data is consistent with existing findings regarding the distribution of breaks in the genome, confirming that the GAP-seq method can successfully detect SSBs.

[0150] ssDNA gaps were profiled in etoposide-treated and untreated HEK293 cells. Etoposide treatment is known to increase endogenous DNA gaps by inhibiting topoisomerase. GAPS-seq was found to detect more ssDNA gaps in etoposide-treated cells compared to controls treated with DMSO alone (Figure 3). Interestingly, GAPS-seq also showed the presence of more ssDNA gaps in enhancer sequences compared to surrounding regions, indicating that regulatory regions are undergoing a dynamic cycle of ssDNA gap and repair.

[0151] DNA GAP-seq was found to detect ssDNA gaps in libraries obtained from various input samples ranging from 1 million to 100 cells (Figure 4).

[0152] HEK293 cells were treated with restriction enzymes to cleave dsDNA at different motifs, leaving either a 3' overhang or a 5' overhang. Using GAP-seq, which targets the 3' end of the cleaved strands, cleavage sites with a 5' overhang (BstEII-HF; EcoR1-HF) were successfully mapped, but blunt-end cleavage sites (SrfI) or cleavage sites with a 3' overhang (Apa1) were not mapped (Figure 5). The plot shows the mean signal of all known restriction sites in the human genome.

[0153] The GAPS-Seq method described herein has been found to be a highly versatile and quantitative method for genome-wide detection of DNA gaps at base resolution.

[0154] Example 2: Improved Protocol - GAPS-seq v2 Further optimization of the above protocol allows the GAPS-seq method to be completed in a shorter timeframe (24 hours instead of 48 hours) and with fewer steps. Furthermore, this updated protocol (GAPS-seq v2) allows for a reduction in the input in terms of cell count (for example, from approximately 1 million to approximately 100,000 cells).

[0155] The following describes the changes from GAPS-seq v1 (the example above) to v2. After fixation, transfer the cells to 0.2 ml PCR tubes for the subsequent washing and lysis steps. Using smaller tubes reduces cell loss due to nuclei "adhering" to the tube walls.

[0156] Gap-seqv2 also makes the following possible: All centrifugation times were reduced from 5 minutes to 2 minutes. Simplified gDNA purification after overnight ligation. Proteinase K digestion is reduced from 2 hours to 30 minutes, and the use of magnetic beads instead of a column allows for processing more samples with less input material.

[0157] Specifically, the protocol was implemented as follows: (i) After overnight ligation, the nuclei are pelletized by centrifugation at 500g for 2 minutes. (ii) Remove the supernatant by pipetting, leaving approximately 10 μl of liquid in the tube. (iii) Add 1 μl of 8 U / μl proteinase K (NEB P8107S). (iv) Incubate the sample at 55°C for 15 minutes, followed by 80°C for 15 minutes. (v) Add 35 μl of RLT plus buffer (Qiagen, 1053393). (vi) Add 45 μl of SPRI select beads (Beckman, B23318), vortex thoroughly, and incubate at room temperature for 10 minutes. (vii) Place the sample on a magnet and remove the supernatant once the beads have pelletized. (viii) Wash the beads twice with 200 μl of 80% ethanol while the tube is still on the magnet. (ix) To elute the gDNA, 25 μl of EB buffer (Qiagen, 19086) was added to the beads, which were removed from the magnet, and mixed by vortexing. (x) Place the tube on the thermomixer at 50°C and 2000 rpm for 10 minutes. (xi) Next, place the tube on the magnet, and once the beads have pelletized, transfer the liquid to a clean PCR tube. (xii) Optimized tagmentation reaction: Here, instead of 1 μl, 4 μl of 12.5 μM N7-loaded Tn5 (available from Diagenode, catalog number C01070010-10) is used. This increases the complexity of the library. (xiii) Conjugate the sample to streptavidin beads for approximately 15 minutes instead of 1 hour.

[0158] This optimized protocol described above has been implemented with multiple cell lines, examples of which are detailed in the following examples. The number of cells used ranged from 10,000 to 1,000,000.

[0159] Example 3: Experiments with nicasse and exonuclease GAPS-seq is designed to profile single-strand gaps in gDNA, not nicks, because nicks are far more numerous and have less physiological impact. To determine whether GAPS-seq is more sensitive to nicks than gaps, the following experiment was designed and performed. BJ cells [cell type: human precipitous fibroblasts, supplier: ATCC: catalog number: CRL-2522 lot number: 70027151] were prepared according to the GAPS-seq v2 cell fixation and lysis protocol described in Example 2. The nuclei were then treated with 0.1 U / μl of nicking endonuclease, Nt.BbvCI (NEB R0632S), in 1x rCutSmart Buffer (NEB, B6004) at 37°C for 1 hour. Next, the nuclei were pelleted by centrifugation (300g for 2 minutes, as detailed above), washed twice in CTSX buffer, and then resuspended in 1x rCutSmart Buffer (NEB, B6004) containing 1 U / μl exonuclease III (NEB, M0206S), and incubated at 37°C for 1 hour. The nuclei were washed twice again in CTSX buffer, and then the 5-prime GAPS-seq v2 protocol was continued from the step where oligonucleotides and then ligation master mix were added. Controls included (a) nuclei treated only with nickase and not with exonuclease, and (b) nuclei not treated with any enzyme. Plotting at the Nt.BbvCI recognition site (CCTCAGC) was performed in the same manner as in the restriction enzyme experiment described above. When the two enzymes are combined, a single-stranded gap with a 5' end at the Nt.BbvCI recognition site is formed, while treatment with nickase only forms a nick in the DNA, which GAPS-seq is designed to ignore. Therefore, this experiment demonstrates that GAPS-seq is specific to single-stranded DNA gaps and does not profile nicks in DNA (Figure 6).

[0160] Example 4: Transcription initiation site profiles stratified by gene expression level Both double-stranded and single-stranded gaps are known to be enriched at the transcription start site of a gene in a manner dependent on the expression level of the corresponding gene. To confirm that GAPS-seq can detect this phenomenon, GAPS-seq v2 was performed in HEK293 cells as described above. For plotting, the GAPS-seq BAM file (i.e., mapped sequencing reads) was loaded into R using the Rsubread package (https: / / bioconductor.org / packages / release / bioc / html / Rsubread.html) and overlapped with a 10kb region around the transcription start site of all genes. Next, these were stratified into three groups based on the expression levels of the corresponding genes derived from publicly available HEK293 cell RNA-seq (https: / / doi.org / 10.1093 / nar / gky861): 1) highly expressed genes (raw counts ≥ 500), 2) moderately expressed genes (raw counts 10–500), and 3) lowly expressed genes (raw counts < 10). Then, the GAPS-seq signals were averaged in a 400 bp window, then averaged across all genes (separately for each group), and plotted as line graphs (Figure 7). Since transcription levels have been found to correlate with the level of basal DNA breaks, this method suggests that it may be usable, for example, to investigate the effects of transcriptional perturbations on DNA gap formation dynamics, and possibly the reverse.

[0161] Example 5: GAPS-seq can identify cell types. GAPS-seq has the potential to reveal cell type-specific DNA gaps and be used for cell type identification. To demonstrate this, GAPS-seq was performed on (1) HEK293 cells, (2) BJ fibroblasts, and (3) human ESCs and NPCs cultured from ESCs. Library preparation, sequencing, and data processing were performed as described above using Gap seq version 2. To plot the dendrogram, GAPS-seq signals were quantified in the promoter region (±2kb from the TSS of all protein-coding genes), and then hierarchical clustering was performed using the dist and hclust functions in R, and plotted using the ggdendro package [available from https: / / cran.r-project.org / web / packages / ggdendro / index.html]. Hierarchical clustering clearly separated the samples by their originating cell lines, thereby demonstrating the practical applicability of GAPS-seq in cell type identification (Figure 8). The results demonstrate that GAPS-seq can clearly distinguish between different cell types, which may be useful for cell type identification and suggests that this method can be used to discover cell type-specific differences in the genomic distribution of DNA gaps.

[0162] Example 6: GAPS-seq profiling is different from sBLISS, which is a method for profiling double-strand breaks. One method for profiling double-strand breaks is sBLISS. To test whether GAPS-seq profiles a distinct genomic characteristic for double-strand breaks, sBLISS was performed on the same batch of HEK293 cells used for GAPS-seq. The sBLISS protocol was followed, as described in Bouwman et al., Nature Protocols 15, 3894-3941 (2020). To compare the signals generated by the two methods, the inventors overlapped the signals with the promoter region (±2kb from the TSS) and then performed principal component analysis using the prcomp function in R (v4.1.2) (R Core Team 2022) https: / / cran.r-project.org / . The results are shown in Figure 9, clearly demonstrating the separation of samples by the method used to prepare the library, thus demonstrating that the GAPS-seq method measures a different genomic aspect than methods profiling double-strand breaks such as sBLISS.

[0163] References Lindahl T, Nyberg B. Rate of depurination of native deoxyribonucleic acid. Biochemistry. 1972 Sep 12;11(19):3610-8. doi: 10.1021 / bi00769a018. PMID: 4626532 Lindahl T, Barnes DE. Repair of endogenous DNA damage. Cold Spring Harb Symp Quant Biol. 2000;65:127-33. doi: 10.1101 / sqb.2000.65.127. PMID: 12760027. Amente S, Scala G, Majello B, Azmoun S, Tempest HG, Premi S, Cooke MS. Genome-wide mapping of genomic DNA damage: methods and implications. Cell Mol Life Sci. 2021 Nov;78(21-22):6745-6762. doi: 10.1007 / s00018-021-03923-6. Epub 2021 Aug 31. PMID: 34463773; PMCID: PMC8558167. Yan, W., Mirzazadeh, R., Garnerone, S. et al. BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks. Nat Commun 8, 15058 (2017). https: / / doi.org / 10.1038 / ncomms15058 Wu W, Hill SE, Nathan WJ, Paiano J, Callen E, Wang D, Shinoda K, van Wietmarschen N, Colon-Mercado JM, Zong D, De Pace R, Shih HY, Coon S, Parsadanian M, Pavani R, Hanzlikova H, Park S, Jung SK, McHugh PJ, Canela A, Chen C, Casellas R, Caldecott KW, Ward ME, Nussenzweig A. Neuronal enhancers are hotspots for DNA single-strand break repair. Nature. 2021 May;593(7859):440-444. doi: Vilenchik MM, Knudson AG. Endogenous DNA double-strand breaks: production, fidelity of repair, and induction of cancer. Proc Natl Acad Sci U S A. 2003 Oct 28;100(22):12871-6. doi: 10.1073 / pnas.2135498100. Epub 2003 Oct 17. PMID: 14566050; PMCID: PMC240711 Cao et al Nature Communications (2019) 10 5799 Muslimovic A, Nystroem S, Gao Y, Hammarsten O. Numerical analysis of etoposide induced DNA breaks. PLoS One. 2009 Jun 10;4(6):e5859. doi: 10.1371 / journal.pone.0005859. Erratum in: PLoS One. 2009;4(6). doi: 10.1371 / annotation / 290cebfd-d5dc-4bd2-99b4-f4cf0be6c838. PMID: 19516899; PMCID: PMC2689654. Baranello et al Int J Mol Sci (2014) 15 13111-1322 Baranello, L. et al. (2018). Mapping DNA Breaks by Next-Generation Sequencing. In: Muzi-Falconi, M., Brown, G. (eds) Genome Instability. Methods in Molecular Biology, vol 1672. Humana Press, New York, NY. https: / / doi.org / 10.1007 / 978-1-4939-7306-4_13 Hu et al Genes & Dev. 2015. 29: 948-960 Sriramachandran et al Mol Cell (2020)78 975-985 Ewels, P.A., Peltzer, A., Fillinger, S. et al. Nat Biotechnol 38, 276-278 (2020). https: / / doi.org / 10.1038 / s41587-020-0439-x Andrews, S. (2010). FastQC: A Quality Control Tool for High Throughput Sequence Data [Online]. Available online at: http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / Felix Krueger; Frankie James; Phil Ewels; Ebrahim Afyounian; Michael Weinstein; Benjamin Schuster-Boeckler; Gert Hulselmans; sclamons [Online] https: / / doi.org / 10.5281 / zenodo.7598955 MARTIN, Marcel. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal, [S.l.], v. 17, n. 1, p. pp. 10-12, may 2011. ISSN 2226-6089. doi:https: / / doi.org / 10.14806 / ej.17.1.200. Heng Li, Richard Durbin, Fast and accurate long-read alignment with Burrows-Wheeler transform, Bioinformatics, Volume 26, Issue 5, March 2010, Pages 589-595, https: / / doi.org / 10.1093 / bioinformatics / btp698 R Core Team (2022). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https: / / www.R-project.org / Wickham H (2016). ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York. ISBN 978-3-319-24277-4, https: / / ggplot2.tidyverse.org.

[0164]

Table 4

Claims

1. A method for detecting single-strand break (SSB) sites in double-stranded (ds) DNA, (i) Contacting a dsDNA sample with an oligonucleotide probe containing a first sequencing adapter and tag, (ii) Contacting the dsDNA sample with a DNA ligase, wherein the DNA ligase covalently attaches the oligonucleotide probe to the dsDNA strand at the site of the single-strand break in the dsDNA, (iii) The dsDNA sample is brought into contact with a transposase loaded with a second sequencing adapter, the dsDNA being cleaved by the transposase, and a collection of fragments containing the second sequencing adapter at the end is generated. (iv) Isolating DNA strands containing the first sequencing adapter and the second sequencing adapter from the population, (v) Determining the sequence of the isolated DNA strand, Methods that include...

2. The method according to claim 1, wherein the sequence of the isolated DNA strand indicates a single-strand break (SSB) site in the dsDNA.

3. The method according to claim 1 or 2, wherein the dsDNA is genomic DNA.

4. The method according to any one of claims 1 to 3, wherein the dsDNA is present in the cell nucleus or an extract thereof.

5. The method according to any one of claims 1 to 4, wherein the dsDNA is derived from diseased cells, embryonic cells, age-related cells, or treated cells.

6. The treated cells, Exposure to one or more compounds, Exposure to or irradiation of light, Exposure to cell culture conditions, Nuclease, optional exposure to Cas9 or Cpf1, or, Reprogramming factors, optional exposure to Oct3 / 4, Sox2, Klf4 and c-Myc, The method according to claim 5, wherein the process is selected from the following.

7. The method according to any one of claims 1 to 6, wherein the sample of dsDNA is prepared by a method comprising extracting a nucleus from a eukaryotic cell.

8. The method according to claim 7, wherein the eukaryotic cells are fixed and permeabilized.

9. The dsDNA sample undergoes the following steps before contacting the oligonucleotide probe: (i) A step of washing and lysing a sample containing dsDNA (e.g., cells), (ii) A step of subjecting the sample containing dsDNA to buffer exchange, for example by centrifugation of the sample or by dilution. The method according to any one of claims 1 to 8, which may include one or more of the following.

10. The method according to any one of claims 1 to 9, wherein the sample of dsDNA is purified by proteinase digestion after contacting the dsDNA with the ligase.

11. The method according to any one of claims 1 to 10, wherein the isolation of the DNA strand including the first sequencing adapter and the second sequencing adapter is carried out using magnetic beads.

12. The method according to any one of claims 1 to 11, wherein the tag is biotin.

13. The method according to any one of claims 1 to 12, wherein the probe further comprises a hybridization region that anneals to the dsDNA at the SSB site.

14. The method according to claim 13, wherein the hybridization sequence comprises 5 to 10 nucleotides.

15. The method according to claim 13 or 14, wherein the hybridization sequence is diverse.

16. The method according to claim 15, wherein the hybridization sequence is random.

17. The method according to claim 13 or 14, wherein the hybridization sequence is complementary to the target sequence in the dsDNA.

18. The method according to claim 14, wherein the target sequence is a promoter.

19. The method according to any one of claims 1 to 18, wherein the oligonucleotide probe is ligated to the 3' end of the cleaved strand of the dsDNA at the SSB site.

20. The method according to claim 19, wherein the tag is located at the 3' end of the oligonucleotide probe, and the oligonucleotide probe further comprises a 5' phosphate group.

21. The method according to any one of claims 1 to 18, wherein the oligonucleotide probe is ligated to the 5' end of the cleaved strand of the dsDNA at the SSB site.

22. The method according to claim 21, wherein the tag is located at the 5' end of the oligonucleotide probe, and the oligonucleotide probe further comprises a 3'OH group.

23. The method according to any one of claims 1 to 22, wherein the transposase is Tn5 transposase.

24. The method according to any one of claims 1 to 23, wherein the fragment comprising the first sequencing adapter and the second sequencing adapter is isolated by bringing the tag into contact with an immobilized bonding member.

25. The method according to claim 24, wherein the tag is biotin and the binding member is streptavidin.

26. The method according to claim 24 or 25, wherein the bonding member is fixed on a bead.

27. The method according to any one of claims 1 to 26, comprising treating the isolated DNA strand with bisulfite.

28. The method according to claim 27, wherein the sequence of the bisulfite-treated strand is compared with a reference genome to identify methylated cytosine residues in the isolated DNA strand.

29. The method according to any one of claims 1 to 28, comprising amplifying the isolated DNA strand using sequencing primers to produce an amplification product.

30. The method according to any one of claims 1 to 29, comprising sequencing the isolated DNA strand or amplification product.

31. The method according to any one of claims 1 to 30, further comprising generating a set of sequence reads of the isolated DNA strand or amplification product.

32. The method according to claim 31, comprising mapping the sequence reads in the set to one or more sites in a reference genome.

33. The method according to claim 32, comprising determining the frequency of SSBs at a site in the reference genome from the number of sequence reads mapped to the site.

34. The method according to claim 33, comprising mapping the frequency of SSBs at sites within a first dsDNA and a second dsDNA, and identifying sites in the first dsDNA relative to the second dsDNA, or in the second dsDNA relative to the first dsDNA, where the frequency of SSB occurrence is increased.

35. The method according to claim 34, wherein the first dsDNA is a test sample and the second dsDNA is a reference or control sample.

36. The method according to claim 35, wherein the first dsDNA is derived from disease cells, embryonic cells, age-related cells, or treated cells.

37. The treated cells, Exposure to one or more compounds, Exposure to or irradiation of light, Exposure to cell culture conditions, Nuclease, optional exposure to Cas9 or Cpf1, or, Exposure to Oct3 / 4, Sox2, Klf4 and c-Myc, The method according to claim 36, wherein the process is selected from the following.

38. A kit for detecting single-strand breaks (SSBs) in double-stranded (ds) DNA, wherein the kit comprises: A first sequencing adapter and an oligonucleotide probe including a tag, DNA ligase and, Transposase and, A second sequencing adapter that is loaded onto or can be loaded onto the transposase, A kit that includes this.

39. The kit according to claim 38, comprising instructions for use in the method according to any one of claims 1 to 37.

40. A method for screening for drugs that can enable the repair of SSBs in dsDNA, such as chemical drugs and / or biological drugs (e.g., small molecule and / or peptide and / or protein therapeutics), the screening method comprising the following steps: (a) A step of detecting single-strand breaks (SSBs) in a control sample of double-stranded (ds) DNA that has not been exposed to the drug, and detecting single-strand breaks (SSBs) in a test version of the same sample of dsDNA that has been exposed to the drug, (i) Contact the test dsDNA and control dsDNA samples with an oligonucleotide probe containing a first sequencing adapter and tag. (ii) Contacting the samples of test dsDNA and control dsDNA with DNA ligase, such that the DNA ligase covalently attaches the oligonucleotide probe to the dsDNA strand at the site of the single-strand break in the dsDNA, and (iii) The samples of the test dsDNA and the control dsDNA are brought into contact with a transposase loaded with a second sequencing adapter, wherein the dsDNA is cleaved by the transposase, and a collection of fragments containing the second sequencing adapter at the end is generated. The process, and then, (b) A step of isolating DNA strands including the first sequencing adapter and the second sequencing adapter from the population, (c) A step of determining the sequence of the isolated DNA strands from the test sample and the control sample, (d) A step of comparing the SSB profile in both the test sample and the control sample, (e) A step of isolating the agent, such as a chemical agent and / or a biological agent, that is associated with the decrease in SSB in the test sample compared to a control sample, (f) A step of compounding the isolated drug obtained from (e) with pharmaceutically acceptable excipients (which may be multiple), Methods that include...

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