Genome-wide detection of DNA double-strand breaks by in-suspension BLISS.
Journal
Nature protocols
ISSN: 1750-2799
Titre abrégé: Nat Protoc
Pays: England
ID NLM: 101284307
Informations de publication
Date de publication:
12 2020
12 2020
Historique:
received:
21
02
2020
accepted:
04
08
2020
pubmed:
4
11
2020
medline:
2
2
2021
entrez:
3
11
2020
Statut:
ppublish
Résumé
sBLISS (in-suspension breaks labeling in situ and sequencing) is a versatile and widely applicable method for identification of endogenous and induced DNA double-strand breaks (DSBs) in any cell type that can be brought into suspension. sBLISS provides genome-wide profiles of the most consequential DNA lesion implicated in a variety of pathological, but also physiological, processes. In sBLISS, after in situ labeling, DSB ends are linearly amplified, followed by next-generation sequencing and DSB landscape analysis. Here, we present a step-by-step experimental protocol for sBLISS, as well as a basic computational analysis. The main advantages of sBLISS are (i) the suspension setup, which renders the protocol user-friendly and easily scalable; (ii) the possibility of adapting it to a high-throughput or single-cell workflow; and (iii) its flexibility and its applicability to virtually every cell type, including patient-derived cells, organoids, and isolated nuclei. The wet-lab protocol can be completed in 1.5 weeks and is suitable for researchers with intermediate expertise in molecular biology and genomics. For the computational analyses, basic-to-intermediate bioinformatics expertise is required.
Identifiants
pubmed: 33139954
doi: 10.1038/s41596-020-0397-2
pii: 10.1038/s41596-020-0397-2
doi:
Substances chimiques
Suspensions
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
3894-3941Références
McKinnon, P. J. & Caldecott, K. W. DNA strand break repair and human genetic disease. Annu. Rev. Genomics Hum. Genet. 8, 37–55 (2007).
pubmed: 17887919
doi: 10.1146/annurev.genom.7.080505.115648
Mills, K. D., Ferguson, D. O. & Alt, F. W. The role of DNA breaks in genomic instability and tumorigenesis. Immunol. Rev. 194, 77–95 (2003).
pubmed: 12846809
doi: 10.1034/j.1600-065X.2003.00060.x
Roukos, V. & Misteli, T. The biogenesis of chromosome translocations. Nat. Cell Biol. 16, 293–300 (2014).
pubmed: 24691255
pmcid: 6337718
doi: 10.1038/ncb2941
Tubbs, A. & Nussenzweig, A. Endogenous DNA damage as a source of genomic instability in cancer. Cell 168, 644–656 (2017).
pubmed: 28187286
pmcid: 6591730
doi: 10.1016/j.cell.2017.01.002
Cannan, W. J. & Pederson, D. S. Mechanisms and consequences of double-strand DNA break formation in chromatin. J. Cell. Physiol. 231, 3–14 (2016).
pubmed: 26040249
pmcid: 4994891
doi: 10.1002/jcp.25048
van Gent, D. C., Hoeijmakers, J. H. & Kanaar, R. Chromosomal stability and the DNA double-stranded break connection. Nat. Rev. Genet. 2, 196–206 (2001).
pubmed: 11256071
doi: 10.1038/35056049
Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).
pubmed: 24906146
pmcid: 4343198
doi: 10.1016/j.cell.2014.05.010
Tsai, S. Q. & Joung, J. K. Defining and improving the genome-wide specificities of CRISPR-Cas9 nucleases. Nat. Rev. Genet. 17, 300–312 (2016).
pubmed: 27087594
pmcid: 7225572
doi: 10.1038/nrg.2016.28
Sakuma, T. & Yamamoto, T. Acceleration of cancer science with genome editing and related technologies. Cancer Sci. 109, 3679–3685 (2018).
pubmed: 30315615
pmcid: 6272086
doi: 10.1111/cas.13832
Crosetto, N. et al. Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing. Nat. Methods 10, 361–365 (2013).
pubmed: 23503052
pmcid: 3651036
doi: 10.1038/nmeth.2408
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).
pubmed: 28497783
pmcid: 5437291
doi: 10.1038/ncomms15058
Mirzazadeh, R., Kallas, T., Bienko, M. & Crosetto, N. Genome-wide profiling of DNA double-strand breaks by the BLESS and BLISS methods. Methods Mol. Biol. 1672, 167–194 (2018).
pubmed: 29043625
doi: 10.1007/978-1-4939-7306-4_14
Marnef, A. et al. A cohesin/HUSH- and LINC-dependent pathway controls ribosomal DNA double-strand break repair. Genes Dev. 33, 1175–1190 (2019).
pubmed: 31395742
pmcid: 6719620
doi: 10.1101/gad.324012.119
Iannelli, F. et al. A damaged genome’s transcriptional landscape through multilayered expression profiling around in situ-mapped DNA double-strand breaks. Nat. Commun. 8, 15656 (2017).
pubmed: 28561034
pmcid: 5499205
doi: 10.1038/ncomms15656
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).
pubmed: 28270450
pmcid: 5418634
doi: 10.1182/blood-2016-06-725093
Clouaire, T. et al. Comprehensive mapping of histone modifications at DNA double-strand breaks deciphers repair pathway chromatin signatures. Mol. Cell 72, 250–262.e6 (2018).
pubmed: 30270107
pmcid: 6202423
doi: 10.1016/j.molcel.2018.08.020
Dellino, G. I. et al. Release of paused RNA polymerase II at specific loci favors DNA double-strand-break formation and promotes cancer translocations. Nat. Genet. 51, 1011–1023 (2019).
pubmed: 31110352
doi: 10.1038/s41588-019-0421-z
Gao, L. et al. Engineered Cpf1 variants with altered PAM specificities. Nat. Biotechnol. 35, 789–792 (2017).
pubmed: 28581492
pmcid: 5548640
doi: 10.1038/nbt.3900
Ballarino R., Bouwman B. A. M. & Crosetto N. Genome-wide CRISPR off-target DNA break detection by the BLISS Method. in CRISPR Guide RNA Design (eds Fulga T. A. et al.) 261–281 (Humana, 2020).
Figueroa-González, G. & Pérez-Plasencia, C. Strategies for the evaluation of DNA damage and repair mechanisms in cancer. Oncol. Lett. 13, 3982–3988 (2017).
pubmed: 28588692
pmcid: 5452911
doi: 10.3892/ol.2017.6002
Banerjee, U. & Soutoglou, E. Finding DNA ends within a haystack of chromatin. Mol. Cell 63, 726–728 (2016).
pubmed: 27588600
doi: 10.1016/j.molcel.2016.08.012
Bouwman, B. A. M. & Crosetto, N. Endogenous DNA double-strand breaks during DNA Transactions: emerging insights and methods for genome-wide profiling. Genes 9, (2018).
Martin, F., Sánchez-Hernández, S., Gutiérrez-Guerrero, A., Pinedo-Gomez, J. & Benabdellah, K. Biased and unbiased methods for the detection of off-target cleavage by CRISPR/Cas9: an overview. Int. J. Mol. Sci. 17, 1507 (2016).
Klein, I. A. et al. Translocation-capture sequencing reveals the extent and nature of chromosomal rearrangements in B lymphocytes. Cell 147, 95–106 (2011).
pubmed: 21962510
pmcid: 3190307
doi: 10.1016/j.cell.2011.07.048
Chiarle, R. et al. Genome-wide translocation sequencing reveals mechanisms of chromosome breaks and rearrangements in B cells. Cell 147, 107–119 (2011).
pubmed: 21962511
pmcid: 3186939
doi: 10.1016/j.cell.2011.07.049
Frock, R. L. et al. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat. Biotechnol. 33, 179–186 (2015).
pubmed: 25503383
doi: 10.1038/nbt.3101
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).
pubmed: 25513782
doi: 10.1038/nbt.3117
Gabriel, R. et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat. Biotechnol. 29, 816–823 (2011).
pubmed: 21822255
doi: 10.1038/nbt.1948
Breton, C., Clark, P. M., Wang, L., Greig, J. A. & Wilson, J. M. ITR-Seq, a next-generation sequencing assay, identifies genome-wide DNA editing sites in vivo following adeno-associated viral vector-mediated genome editing. BMC Genomics 21, 239 (2020).
pubmed: 32183699
pmcid: 7076944
doi: 10.1186/s12864-020-6655-4
Hanlon, K. S. et al. High levels of AAV vector integration into CRISPR-induced DNA breaks. Nat. Commun. 10, 4439 (2019).
pubmed: 31570731
pmcid: 6769011
doi: 10.1038/s41467-019-12449-2
Lensing, S. V. et al. DSBCapture: in situ capture and sequencing of DNA breaks. Nat. Methods 13, 855–857 (2016).
pubmed: 27525976
pmcid: 5045719
doi: 10.1038/nmeth.3960
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.e11 (2018).
pubmed: 30293786
pmcid: 6407864
doi: 10.1016/j.molcel.2018.08.047
Canela, A. et al. DNA breaks and end resection measured genome-wide by end sequencing. Mol. Cell 63, 898–911 (2016).
pubmed: 27477910
pmcid: 6299834
doi: 10.1016/j.molcel.2016.06.034
Biernacka, A. et al. i-BLESS is an ultra-sensitive method for detection of DNA double-strand breaks. Commun. Biol. 1, 181 (2018).
pubmed: 30393778
pmcid: 6208412
doi: 10.1038/s42003-018-0165-9
Baranello, L. et al. Mapping DNA breaks by next-generation sequencing. Methods Mol. Biol. 1672, 155–166 (2018).
pubmed: 29043624
doi: 10.1007/978-1-4939-7306-4_13
pmcid: 8057127
Leduc, F. et al. Genome-wide mapping of DNA strand breaks. PloS One 6, e17353 (2011).
pubmed: 21364894
pmcid: 3045442
doi: 10.1371/journal.pone.0017353
Grégoire, M.-C. et al. Quantification and genome-wide mapping of DNA double-strand breaks. DNA Repair 48, 63–68 (2016).
pubmed: 27825743
doi: 10.1016/j.dnarep.2016.10.005
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).
pubmed: 25609572
pmcid: 4352882
doi: 10.1101/gr.180497.114
Canela, A. et al. Topoisomerase II-induced chromosome breakage and translocation is determined by chromosome architecture and transcriptional activity. Mol. Cell 75, 252–266.e8 (2019).
pubmed: 31202577
pmcid: 8170508
doi: 10.1016/j.molcel.2019.04.030
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: 31649282
pmcid: 6813358
doi: 10.1038/s41467-019-12802-5
Dorsett, Y. et al. HCoDES reveals chromosomal DNA end structures with single-nucleotide resolution. Mol. Cell 56, 808–818 (2014).
pubmed: 25435138
pmcid: 4272619
doi: 10.1016/j.molcel.2014.10.024
Szlachta, K., Raimer, H. M., Comeau, L. D. & Wang, Y.-H. CNCC: an analysis tool to determine genome-wide DNA break end structure at single-nucleotide resolution. BMC Genomics 21, 25 (2020).
pubmed: 31914926
pmcid: 6950916
doi: 10.1186/s12864-019-6436-0
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).
pubmed: 31127121
pmcid: 6534554
doi: 10.1038/s41467-019-10332-8
Kim, D. et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat. Methods 12, 237–243 (2015).
pubmed: 25664545
doi: 10.1038/nmeth.3284
Kim, D. & Kim, J.-S. DIG-seq: a genome-wide CRISPR off-target profiling method using chromatin DNA. Genome Res. 28, 1894–1900 (2018).
pubmed: 30413470
pmcid: 6280750
doi: 10.1101/gr.236620.118
Cameron, P. et al. Mapping the genomic landscape of CRISPR-Cas9 cleavage. Nat. Methods 14, 600–606 (2017).
pubmed: 28459459
doi: 10.1038/nmeth.4284
Tsai, S. Q. et al. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets. Nat. Methods 14, 607–614 (2017).
pubmed: 28459458
pmcid: 5924695
doi: 10.1038/nmeth.4278
Gothe, H. J. et al. Spatial chromosome folding and active transcription drive DNA fragility and formation of oncogenic MLL translocations. Mol. Cell 75, 267–283.e12 (2019).
pubmed: 31202576
doi: 10.1016/j.molcel.2019.05.015
Dziubańska-Kusibab, P. J. et al. Colibactin DNA-damage signature indicates mutational impact in colorectal cancer. Nat. Med. 26, 1063–1069 (2020).
pubmed: 32483361
doi: 10.1038/s41591-020-0908-2
Lieberman-Aiden, E. et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326, 289–293 (2009).
pubmed: 19815776
pmcid: 2858594
doi: 10.1126/science.1181369
Belaghzal, H., Dekker, J. & Gibcus, J. H. Hi-C 2.0: an optimized Hi-C procedure for high-resolution genome-wide mapping of chromosome conformation. Methods 123, 56–65 (2017).
pubmed: 28435001
pmcid: 5522765
doi: 10.1016/j.ymeth.2017.04.004
Kordon, M. M. et al. STRIDE—a fluorescence method for direct, specific in situ detection of individual single- or double-strand DNA breaks in fixed cells. Nucleic Acids Res. 48, e14 (2020).
pubmed: 31832687
doi: 10.1093/nar/gkz1118
Orlitsky, A., Suresh, A. T. & Wu, Y. Optimal prediction of the number of unseen species. Proc. Natl Acad. Sci. 113, 13283–13288 (2016).
pubmed: 27830649
doi: 10.1073/pnas.1607774113
pmcid: 5127330
Dsouza, M., Larsen, N. & Overbeek, R. Searching for patterns in genomic data. Trends Genet. 13, 497–498 (1997).
pubmed: 9433140
doi: 10.1016/S0168-9525(97)01347-4
Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589–595 (2010).
pubmed: 20080505
pmcid: 2828108
Ballinger, T. J. et al. Modeling double strand break susceptibility to interrogate structural variation in cancer. Genome Biol. 20, 28 (2019).
pubmed: 30736820
pmcid: 6368699
doi: 10.1186/s13059-019-1635-1
Hoa, N. N. et al. Mre11 Is essential for the removal of lethal topoisomerase 2 covalent cleavage complexes. Mol. Cell 64, 580–592 (2016).
pubmed: 27814490
doi: 10.1016/j.molcel.2016.10.011
Moor, A. E. et al. Spatial reconstruction of single enterocytes uncovers broad zonation along the intestinal villus axis. Cell 175, 1156–1167.e15 (2018).
pubmed: 30270040
doi: 10.1016/j.cell.2018.08.063
Anaconda, Inc. Anaconda Software Distribution https://docs.conda.io/en/latest/miniconda.html (2017).
Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).
pubmed: 20110278
pmcid: 2832824
doi: 10.1093/bioinformatics/btq033
Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
pubmed: 19505943
pmcid: 2723002
Tange, O. GNU Parallel 2018 https://doi.org/10.5281/zenodo.1146014 (2018).
R Core Team. R: a language and environment for statistical computing (R Foundation for Statistical Computing, 2014).
Wang, X. et al. Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors. Nat. Biotechnol. 33, 175–178 (2015).
pubmed: 25599175
doi: 10.1038/nbt.3127
Baranello, L. et al. DNA break mapping reveals topoisomerase II activity genome-wide. Int. J. Mol. Sci. 15, 13111–13122 (2014).
pubmed: 25056547
pmcid: 4139894
doi: 10.3390/ijms150713111
Lawrence, M. et al. Software for computing and annotating genomic ranges. PLoS Comput. Biol. 9, e1003118 (2013).
pubmed: 23950696
pmcid: 3738458
doi: 10.1371/journal.pcbi.1003118
Morgan, M. & Shepherd, L. AnnotationHub: Client to Access AnnotationHub Resources (Bioconductor, 2020).
Durinck, S., Spellman, P. T., Birney, E. & Huber, W. Mapping identifiers for the integration of genomic datasets with the R/Bioconductor package biomaRt. Nat. Protoc. 4, 1184–1191 (2009).
pubmed: 19617889
pmcid: 3159387
doi: 10.1038/nprot.2009.97
Lawrence, M., Gentleman, R. & Carey, V. rtracklayer: an R package for interfacing with genome browsers. Bioinformatics 25, 1841–1842 (2009).
pubmed: 19468054
pmcid: 2705236
doi: 10.1093/bioinformatics/btp328
Xie Y. knitr: A General-Purpose Package for Dynamic Report Generation in R. https://rdrr.io/cran/knitr/ (2020).
Wickham, H. ggplot2: Elegant Graphics for Data Analysis (Springer, 2016).
Gu, Z., Gu, L., Eils, R., Schlesner, M. & Brors, B. circlize implements and enhances circular visualization in R. Bioinformatics 30, 2811–2812 (2014).
pubmed: 24930139
doi: 10.1093/bioinformatics/btu393
Gu, Z., Eils, R. & Schlesner, M. Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics 32, 2847–2849 (2016).
pubmed: 27207943
doi: 10.1093/bioinformatics/btw313