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High-resolution, ultrasensitive and quantitative DNA double-strand break labeling in eukaryotic cells using i-BLESS


DNA double-strand breaks (DSBs) are implicated in various physiological processes, such as class-switch recombination or crossing-over during meiosis, but also present a threat to genome stability. Extensive evidence shows that DSBs are a primary source of chromosome translocations or deletions, making them a major cause of genomic instability, a driving force of many diseases of civilization, such as cancer. Therefore, there is a great need for a precise, sensitive, and universal method for DSB detection, to enable both the study of their mechanisms of formation and repair as well as to explore their therapeutic potential. We provide a detailed protocol for our recently developed ultrasensitive and genome-wide DSB detection method: immobilized direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing (i-BLESS), which relies on the encapsulation of cells in agarose beads and labeling breaks directly and specifically with biotinylated linkers. i-BLESS labels DSBs with single-nucleotide resolution, allows detection of ultrarare breaks, takes 5 d to complete, and can be applied to samples from any organism, as long as a sufficient amount of starting material can be obtained. We also describe how to combine i-BLESS with our qDSB-Seq approach to enable the measurement of absolute DSB frequencies per cell and their precise genomic coordinates at the same time. Such normalization using qDSB-Seq is especially useful for the evaluation of spontaneous DSB levels and the estimation of DNA damage induced rather uniformly in the genome (e.g., by irradiation or radiomimetic chemotherapeutics).

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Fig. 1: Overview of the i-BLESS method.
Fig. 2: Encapsulation of cells in agarose beads.
Fig. 3: Spheroplasting and lysis.
Fig. 4: Anticipated results.

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Data availability

The raw data underlying Fig. 4b–d are deposited to the NCBI Sequence Read Archive (SRA) with accession codes SRP125409 and SRP189465.


  1. Alt, F. W. & Schwer, B. DNA double-strand breaks as drivers of neural genomic change, function, and disease. DNA Repair 71, 158–163 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Gothe, H. J., Minneker, V. & Roukos, V. Dynamics of double-strand breaks: implications for the formation of chromosome translocations. Adv. Exp. Med. Biol. 1044, 27–38 (2018).

    CAS  PubMed  Google Scholar 

  3. Mitelman, F., Johansson, B. & Mertens, F. The impact of translocations and gene fusions on cancer causation. Nat. Rev. Cancer 7, 233–245 (2007).

    CAS  PubMed  Google Scholar 

  4. O’Driscoll, M. Diseases associated with defective responses to DNA damage. Cold Spring Harb. Perspect. Biol. 4, a012773 (2012).

  5. White, R. R. & Vijg, J. Do DNA double-strand breaks drive aging? Mol. Cell 63, 729–738 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Adli, M. The CRISPR tool kit for genome editing and beyond. Nat. Commun. 9, 1911 (2018).

    PubMed  PubMed Central  Google Scholar 

  7. Carroll, D. Genome engineering with zinc-finger nucleases. Genetics 188, 773–782 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Ma, A. C., Chen, Y., Blackburn, P. R. & Ekker, S. C. TALEN-mediated mutagenesis and genome editing. Methods Mol. Biol. 1451, 17–30 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Tian, X. et al. CRISPR/Cas9—An evolving biological tool kit for cancer biology and oncology. NPJ Precis. Oncol. 3, 8 (2019).

    PubMed  PubMed Central  Google Scholar 

  10. Martin, S. A., Lord, C. J. & Ashworth, A. DNA repair deficiency as a therapeutic target in cancer. Curr. Opin. Genet. Dev. 18, 80–86 (2008).

    CAS  PubMed  Google Scholar 

  11. Turinetto, V. & Giachino, C. Multiple facets of histone variant H2AX: a DNA double-strand-break marker with several biological functions. Nucleic Acids Res. 43, 2489–2498 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. McManus, K. J. & Hendzel, M. J. ATM-dependent DNA damage-independent mitotic phosphorylation of H2AX in normally growing mammalian cells. Mol. Biol. Cell 16, 5013–5025 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Wang, F. & Higgins, J. M. Histone modifications and mitosis: countermarks, landmarks, and bookmarks. Trends Cell Biol. 23, 175–184 (2013).

    CAS  PubMed  Google Scholar 

  14. Leduc, F. et al. Genome-wide mapping of DNA strand breaks. PLoS ONE 6, e17353 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Baranello, L. et al. DNA break mapping reveals topoisomerase II activity genome-wide. Int. J. Mol. Sci. 15, 13111–13122 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Shastri, N. et al. Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition. Mol. Cell 72, 222–238 e211 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Gittens, W. H. et al. A nucleotide resolution map of Top2-linked DNA breaks in the yeast and human genome. Nat. Commun. 10, 4846 (2019).

    PubMed  PubMed Central  Google Scholar 

  18. Pratto, F. et al. DNA recombination. Recombination initiation maps of individual human genomes. Science 346, 1256442 (2014).

    PubMed  PubMed Central  Google Scholar 

  19. Khil, P. P., Smagulova, F., Brick, K. M., Camerini-Otero, R. D. & Petukhova, G. V. Sensitive mapping of recombination hotspots using sequencing-based detection of ssDNA. Genome Res. 22, 957–965 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Biernacka, A. et al. i-BLESS is an ultra-sensitive method for detection of DNA double-strand breaks. Commun. Biol. 1, 181 (2018).

    PubMed  PubMed Central  Google Scholar 

  21. Hoffman, E. A., McCulley, A., Haarer, B., Arnak, R. & Feng, W. Break-seq reveals hydroxyurea-induced chromosome fragility as a result of unscheduled conflict between DNA replication and transcription. Genome Res. 25, 402–412 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Crosetto, N. et al. Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing. Nat. Methods 10, 361–365 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Aymard, F. et al. Genome-wide mapping of long-range contacts unveils clustering of DNA double-strand breaks at damaged active genes. Nat. Struct. Mol. Biol. 24, 353–361 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Shi, W. et al. Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress. Blood 129, 2479–2492 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Hu, J. et al. Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing. Nat. Protoc. 11, 853–871 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Lensing, S. V. et al. DSBCapture: in situ capture and sequencing of DNA breaks. Nat. Methods 13, 855–857 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Canela, A. et al. DNA breaks and end resection measured genome-wide by end sequencing. Mol. Cell 63, 898–911 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Mimitou, E. P., Yamada, S. & Keeney, S. A global view of meiotic double-strand break end resection. Science 355, 40–45 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Yamada, S. et al. Molecular structures and mechanisms of DNA break processing in mouse meiosis. Genes Dev. 34, 806–818 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Wiegand, R. C., Godson, G. N. & Radding, C. M. Specificity of the S1 nuclease from Aspergillus oryzae. J. Biol. Chem. 250, 8848–8855 (1975).

    CAS  PubMed  Google Scholar 

  32. Yan, W. X. et al. BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks. Nat. Commun. 8, 15058 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Promonet, A. et al. Topoisomerase 1 prevents replication stress at R-loop-enriched transcription termination sites. Nat. Commun. 11, 3940 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Zhu, Y. et al. qDSB-Seq is a general method for genome-wide quantification of DNA double-strand breaks using sequencing. Nat. Commun. 10, 2313 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Petek, L. M., Russell, D. W. & Miller, D. G. Frequent endonuclease cleavage at off-target locations in vivo. Mol. Ther. 18, 983–986 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).

    CAS  PubMed  Google Scholar 

  37. Gabriel, R. et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat. Biotechnol. 29, 816–823 (2011).

    CAS  PubMed  Google Scholar 

  38. Botstein, D., Chervitz, S. A. & Cherry, J. M. Yeast as a model organism. Science 277, 1259–1260 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Bassett, D. E. Jr., Boguski, M. S. & Hieter, P. Yeast genes and human disease. Nature 379, 589–590 (1996).

    CAS  PubMed  Google Scholar 

  40. Zhu, Y. et al. Integrated analysis of patterns of DNA breaks reveals break formation mechanisms and their population distribution during replication stress. Preprint at (2017).

  41. Sriramachandran, A. M. et al. Genome-wide nucleotide-resolution mapping of DNA replication patterns, single-strand breaks, and lesions by GLOE-Seq. Mol. Cell 78, 975–985 e977 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Chung, W. H., Zhu, Z., Papusha, A., Malkova, A. & Ira, G. Defective resection at DNA double-strand breaks leads to de novo telomere formation and enhances gene targeting. PLoS Genet. 6, e1000948 (2010).

    PubMed  PubMed Central  Google Scholar 

  43. Symington, L. S. Mechanism and regulation of DNA end resection in eukaryotes. Crit. Rev. Biochem. Mol. Biol. 51, 195–212 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Zhou, Y., Caron, P., Legube, G. & Paull, T. T. Quantitation of DNA double-strand break resection intermediates in human cells. Nucleic Acids Res. 42, e19 (2014).

    CAS  PubMed  Google Scholar 

  45. Aymard, F. et al. Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks. Nat. Struct. Mol. Biol. 21, 366–374 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Vieira Braga, F. A. & Miragaia, R. J. Tissue handling and dissociation for single-cell RNA-Seq. Methods Mol. Biol. 1979, 9–21 (2019).

    PubMed  Google Scholar 

  47. Reichard, A. & Asosingh, K. Best practices for preparing a single cell suspension from solid tissues for flow cytometry. Cytom. A 95, 219–226 (2019).

    CAS  Google Scholar 

  48. Leelatian, N. et al. Preparing viable single cells from human tissue and tumors for cytomic analysis. Curr. Protoc. Mol. Biol. 118, 25C 21 21–25C 21 23 (2017).

    Google Scholar 

  49. Cannan, W. J. & Pederson, D. S. Mechanisms and consequences of double-strand DNA break formation in chromatin. J. Cell Physiol. 231, 3–14 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Li, F. et al. Apn2 resolves blocked 3′ ends and suppresses Top1-induced mutagenesis at genomic rNMP sites. Nat. Struct. Mol. Biol. 26, 155–163 (2019).

  51. Mitra, A., Skrzypczak, M., Ginalski, K. & Rowicka, M. Strategies for achieving high sequencing accuracy for low diversity samples and avoiding sample bleeding using illumina platform. PLoS ONE 10, e0120520 (2015).

    PubMed  PubMed Central  Google Scholar 

  52. Mitra, A. et al. Analyzing and interpreting DNA double-strand break sequencing data. Preprint at (2020).

  53. Tiwari, S., Wang, S., Hagen, G. & Guilfoyle, T. J. Transfection assays with protoplasts containing integrated reporter genes. Methods Mol. Biol. 323, 237–244 (2006).

    PubMed  Google Scholar 

  54. Buenrostro, J. D., Wu, B., Chang, H. Y. & Greenleaf, W. J. ATAC-seq: a method for assaying chromatin accessibility genome-wide. Curr. Protoc. Mol. Biol. 109, 21 29 21–21 29 29 (2015).

    PubMed  Google Scholar 

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We thank K. Jodkowska and T. Biernacki for help with the preparation of Supplementary Video 1. We also thank A. Kudlicki for critical reading of the manuscript. This work was supported by the Foundation for Polish Science (TEAM to K.G.) and the Polish National Science Centre (2015/17/D/NZ2/03711 to M.S.) and an NIH grant R01GM112131 to M.R.

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Authors and Affiliations



K.G. supervised the study, A.B., M.S., P.P., M.R., and K.G. designed the experiments, A.B. and M.S. performed the experiments, Y.Z. and M.R. performed bioinformatic analysis, A.B., M.S., Y.Z., P.P., M.R., and K.G. analyzed the results, and A.B. and K.G. wrote the manuscript. All authors read and edited the manuscript.

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Correspondence to Krzysztof Ginalski.

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Peer review information Nature Protocols thanks Anna Malkova and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Biernacka, A. et al. Commun. Biol. 1, 181 (2018):

Zhu, Y. et al. Nat. Commun. 10, 2313 (2019):

Promonet, A. et al. Nat. Commun. 11, 3940 (2020):

Supplementary information

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Supplementary Video 1

Encapsulation of human GM19239 cells in agarose beads.

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Biernacka, A., Skrzypczak, M., Zhu, Y. et al. High-resolution, ultrasensitive and quantitative DNA double-strand break labeling in eukaryotic cells using i-BLESS. Nat Protoc 16, 1034–1061 (2021).

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